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8794697

The efficacy of doramectin on arrested larvae of Ancylostoma caninum in early pregnancy of bitches.

The efficacy of doramectin treatment on arrested A. caninum larvae during early pregnancy of bitches was examined. Four bitches were percutaneously infected with 20,000 third-stage larvae of A. caninum on the day of conception and treated subcutaneously with 1 mg doramectin per kg body weight on day 30 of pregnancy. Four infected untreated pregnant bitches served as controls. A single application of dormectin substantially reduced the number of somatic larvae in bitches and the number of intestinal stages in bitches and puppies. However, it did pletely prevent lactogenic transmission of A. caninum larvae because five out of 23 puppies from three litters of the treated bitches harboured adult worms in their intestines, two of them shed eggs with the faeces. Although clinical disease did not occur in puppies from treated bitches the efficacy of the treatment was not satisfactory from an epidemiological point of view. Despite the treatment puppies with patent infections contaminated their environment with high numbers of eggs thus producing an intolerable infection risk for dogs and humans. No fetotoxic side-effects of the early treatment with doramectin were seen.

8794699

Detection of Chlamydia in formalin-fixed and paraffin-embedded avian tissue by in situ hybridization. A comparison between in situ hybridization and peroxidase-antiperoxidase labelling.

In situ hybridization, (ISH) using a digoxigenin-antisense RNA-probe to detect chlamydial rRNA was applied to post mortem tissue of birds. The technique was optimized and validated using tissue from experimentally-infected chicken embryos. Tissue sections were also tested by immunohistochemistry (peroxidase-antiperoxidase reaction, PAP) for the presence of chlamydial antigen using a genus specific monoclonal antibody. In the chicken embryo tissue, ISH and PAP parably sensitive and specific (100% and 100%, respectively). ISH and PAP in general were correlated to microscopic lesions. For parison, ISH with PAP was applied retrospectively to tissues of 82 birds from which Chlamydia had been isolated, or which were suggestive of chlamydiosis. Using in situ hybridization 47 of 82 birds were found to be positive, and as were 23 of 82 birds with PAP. None of the ISH-only positive cases were found to be strongly positive. On the other hand, cases which were found positive with the ISH were also positive with other methods (PAP and isolation of Chlamydiae from chicken embryos). There was no close correlation between the positive cells and histological lesions. In spite of the higher sensitivity and specificity of the ISH, this technique is not suitable for routine diagnostic investigations. ISH is expensive, laborious, and time consuming.

8794698

Differentiation of Salmonella gallinarum and Salmonella pullorum by their whole-cell fatty acid methyl ester profiles.

The whole-cell fatty acid methyl ester (FAME) profiles of Salmonella (S.) gallinarum and Salmonella (S.) pullorum strains pared in puter-linked gas-liquid chromatography. The profiles of whole cellular FAMEs allow the separation and identification S. gallinarum and S. pullorum by the Microbial Identification System and so can be used for their differentiation.

8794700

Experimental melioidosis in hens.

Experimental intramuscular infection of hens with Pseudomonas pseudomallei, strain 2796 (1 x 10(9) CFU from a 24-h culture) was reproduced. Clinical, paraclinical and pathomorphological findings were followed from 1 to 30 days after challenge. Haemagglutinin titre, bacterial dissemination in the viscera, number of leucocytes, alveolar (aMa) and peritoneal (pMa) macrophages and their phagocytic activity in vitro were studied. During the course of infection a leucocytosis as well as an increased haemagglutinin titre (1:256) were established. The number of bacteria per gram tissue in the spleen and liver was highest at 1 day post-infection (p.i.). Melioidose bacteria from egg yolk were isolated at 15 and 30 days p.i. Leucocyte and pMa phagocytic activity was maximal at 3 days p.i. unlike the activity of aMa which increased gradually until the end of the study. Inflammatory-necrotic changes were found in the viscera and brain at 3 and 15 days p.i. The investigation of experimental melioidosis infection in hens showed that they are susceptible to P. pseudomallei and this disease takes a generalized subacute course.

8794701

Bacterial causes of bovine mastitis in Wondogenet, Ethiopia.

A survey of selected causative organisms of bacterial mastitis in Zebu-Holstein dairy cows was carried out on four herds in Wondogenet. Pathogens were found in 39% of udder quarters and mastitis in 16% of quarters. The main mastitis causing organisms isolated were Staphylococcus aureus (47%), Streptococcus agalactiae (15%) and Streptococcus uberis (31%). It was indicated also that udder quarters with micrococci would be less susceptible to infections by pathogenic organisms.

8794702

Macular coloboma-like lesions and pigment abnormalities as complications of cryotherapy for retinopathy of prematurity in very low birth-weight infants.

Cryotherapy for retinopathy of prematurity (ROP) is effective in reducing the incidence of blindness in premature infants. However, plications associated with successful treatment have not yet been well studied.

8794703

Eye manifestations of congenital toxoplasmosis.

To determine the natural history of treated and untreated congenital toxoplasmosis and impact of this infection on vision.

8794704

Horizontal transposition of the vertical rectus muscles for cyclotropia.

We studied the effect of horizontal transposition of the vertical rectus muscles on incyclotropia and excyclotropia in terms of the amount of correction obtained and the stability of the e.

8794705

Primary infratarsal lower eyelid retractor lysis to prevent eyelid retraction after inferior rectus muscle recession.

To evaluate a procedure to prevent lower eyelid retraction, which may occur after inferior rectus muscle recession surgery as a direct consequence of the intimate anatomic connections between the inferior rectus muscle and lower eyelid retractors.

8794706

Excimer laser phototherapeutic keratectomy before cataract extraction and intraocular lens implantation.

To determine whether sequential excimer laser phototherapeutic keratectomy (PTK) followed by cataract extraction and intraocular lens (IOL) implantation with power calculation based on the new corneal curvature is effective in managing superficial corneal disease.

8794707

Effect of topical anti-inflammatory treatment on the outcome of laser trabeculoplasty. The Fluorometholone-Laser Trabeculoplasty Study Group.

We investigated the effect of anti-inflammatory treatment on the e of argon laser trabeculoplasty.

8794708

Rate of progression in open-angle glaucoma estimated from cross-sectional prevalence of visual field damage.

To estimate the rate of visual field loss in persons with open-angle a.

8794709

Blood-flow velocities in the extraocular vessels in normal volunteers.

To determine normal values of blood-flow velocities in extraocular vessels.

8794710

Visual field defects after macular hole surgery.

To describe a group of patients with dense visual field defects following macular hole surgery.

8794711

Pattern dystrophy of the retinal pigment epithelium and geographic atrophy of the macula.

Little information is available on the long-term course of pattern dystrophies, although some older individuals have been observed with macular atrophy. We sought to evaluate the evolution of symptoms, fundus changes, and physiologic findings by re-examining a family with pattern dystrophy after 20 years.

8794712

Basic fibroblast growth factor and vascular endothelial growth factor are present in epiretinal and choroidal neovascular membranes.

To determine whether vascular endothelial growth factor and basic fibroblast growth factor, which may be critical mitogens for neovascularization, are present together in human retinal and choroidal neovascular membranes.

8794713

Detection of visual dysfunction in optic atrophy by functional magnetic resonance imaging during monocular visual stimulation.

To evaluate functional magnetic resonance imaging as an objective method for detecting visual dysfunction in various ophthalmologic disorders involving the optic nerve and the chiasm.

8794714

Vascular compressive abducens nerve palsy disclosed by magnetic resonance imaging.

To assess magnetic resonance imaging as a diagnostic tool of pression in a patient with abducens nerve palsy.

8794716

Clear corneal graft overlying the seton tube to facilitate laser suture lysis.

To reduce hypotony and shallowing of the anterior chamber after seton implantation.

8794718

Laser synechialysis to prevent membrane recurrence on silicone intraocular lenses.

To determine whether laser posterior synechialysis will prevent recurrence of pigmented membranes that can form on the anterior surface of silicone intraocular lenses.

8794717

Acute angle-closure glaucoma associated with intranasal cocaine abuse.

We report a case of acute angle-closure a associated with ipsilateral intranasal cocaine abuse.

8794719

Branch retinal artery occlusion as the initial sign of giant cell arteritis.

To describe a patient whose initial sign of giant cell arteritis was a branch retinal artery occlusion.

8794720

Multiple evanescent white dot syndrome after hepatitis B vaccine.

Hepatitis B vaccine has e an effective means of plications of hepatitis B. However, it occasionally induces serious side effects. We report a case of multiple evanescent white dot syndrome (MEWDS) that occurred following hepatitis B vaccination.

8794721

Bilateral exudative retinal detachment complicating systemic corticosteroid therapy in the presence of renal failure.

We describe an elderly patient with chronic renal failure who developed bilateral exudative retinal detachments after initiation of systemic corticosteroid therapy, followed by prompt resolution of the retinal detachments after the corticosteroids were discontinued.

8794722

Mushroom-shaped choroidal hemangioma.

We examined the clinicopathologic features of a mushroom-shaped choroidal tumor that originated in the right eye of a 13-year-old girl.

8794723

Pseudotumor cerebri associated with cyclosporine use.

An 11-year-old boy had a one-month history of horizontal diplopia. Three years earlier, he had undergone allogeneic bone marrow plicated by graft versus host disease.

8794724

Localization of traumatic oculomotor nerve palsy to the midbrain exit site by magnetic resonance imaging.

To present the magnetic resonance imaging findings for a patient with traumatic oculomotor nerve injury.

8794725

Testing for hepatitis B surface antigen in processing donor tissue for penetrating keratoplasty.

The Food and Drug Administration recently proposed a change in the screening of eye bank cornea donors for hepatitis B virus. Currently, most eye banks run confirmatory tests to reduce the frequency of false-positive hepatitis B virus surface antigen (HBsAg) tests. The Food and Drug Administration is considering a policy that would not allow confirmatory testing.

8794726

Specular microscopy before and after enucleation of live donor eyes.

pare the results of specular microscopic examination of corneal endothelium before and after enucleation of eyes from live donors.

8794727

Enchondroma of the orbit.

To report a documented case of orbital enchondroma.

8794728

Benign mixed tumor (pleomorphic adenoma) of the lacrimal gland in a 6-year-old boy.

To report a 6-year-old boy who had benign mixed tumor (pleomorphic adenoma) of the lacrimal gland.

8794729

Vertical Duane's retraction syndrome.

We report three patients with a rare variant of Duane's retraction syndrome.

8794730

Acquired lower eyelid epiblepharon.

Although congenital epiblepharon is a mon condition, particularly in Asians, acquired lower eyelid epiblepharon is rare.

8794732

Fhit, a putative tumor suppressor in humans, is a dinucleoside 5',5"'-P1,P3-triphosphate hydrolase.

Human Fhit (fragile histidine triad) protein, encoded by the FHIT putative tumor suppressor gene, is a typical dinucleoside 5',5"'-P1,P3-triphosphate (Ap3A) hydrolase (EC 3.6.1.29) on the basis of its enzymatic properties we report here. Ap3A is the preferred substrate among ApnA (n = 3-6), and AMP is always one of the reaction products. Mn2+ and Mg2+ are equally stimulatory, while Zn2+ is inhibitory with Ap3A as the substrate. Values of the K(m) for Ap3A and Ap4A are 1.3 and 4.6 microM, respectively. Values of the specificity constant, kcat/K(m), for Ap3A and Ap4A are 2.0 x 10(6) and 6.7 x 10(3) s-1 M-1, respectively, for a glutathione S-transferase (GST)-Fhit fusion protein. Site-directed mutagenesis of FHIT demonstrated that all four conserved histidines are required for full activity, and the central histidine of the triad is absolutely essential for Ap3A hydrolase activity. This putative tumor suppressor is the first evidence for a connection between dinucleotide oligophosphate metabolism and tumorigenesis. Also, Fhit is the first HIT protein in which the histidine residues have been demonstrated by mutagenesis to be critical for function.

8794733

Biochemical analysis of catalytically crucial aspartate mutants of human immunodeficiency virus type 1 reverse transcriptase.

In order to clarify the role(s) of the individual member of the carboxylate triad in the catalytic mechanism of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, we carried out site-directed mutagenesis of D185, D186, and D110, followed by the extensive characterization of the properties of the individual mutant enzymes. We find that all three residues participate at or prior to the chemical step of bond formation. The incorporation pattern seen with phosphorothioate analogs of dNTP on both RNA-DNA and DNA-DNA template-primers indicated that D186 may be the residue that coordinates with the alpha-phosphate group of dNTP in the transition-state plex. Further support for the role assigned to D186 was obtained by examination of the ability of the individual carboxylate mutants to catalyze the reverse of the polymerase reaction (pyrophosphorolysis). Mutants of D185 exhibited near-normal pyrophosphorolysis activity, while those of D186 pletely devoid of this activity. Thus, D185 appears to participate only in the forward reaction, probably required for the generation of nucleophile by interacting with the 3'-OH of the primer terminus, while D186 seems to be involved in both the forward and the reverse reactions, presumably by participating in the pentavalent intermediate transition state. Lack of any elemental effects during polymerization with mutant enzymes of residue D110, together with their inability to catalyze pyrophosphorolysis, suggest its probable participation in the metal-coordinated binding to the beta-gamma-phosphate of dNTP or PPi in the forward and reverse reactions, respectively. A molecular model of the plex based on these results is also presented.

8794735

The structure of nucleotidylated histidine-166 of galactose-1-phosphate uridylyltransferase provides insight into phosphoryl group transfer.

Galactose-1-phosphate uridylyltransferase catalyzes the reaction of UDP-glucose with galactose 1-phosphate to form UDP-galactose and glucose 1-phosphate during normal cellular metabolism. The reaction proceeds through a double displacement mechanism characterized by the formation of a stable nucleotidylated histidine intermediate. This paper describes the preparation of the plex on the crystalline enzyme from Escherichia coli and its subsequent structure determination by X-ray crystallography. The refined structure has an R-factor of 19.6% (data between 65 and 1.86 A resolution) and reveals modest conformational changes at the active pared to the inactive plex reported previously [Wedekind, J.E., Frey, P.A., & Rayment, I. (1995) Biochemistry 34, 11049-11061]. In particular, positions of the respective UMP alpha-phosphoryl groups differ by approximately 4 A. Well-defined electron density for the nucleotidylated imidazole supports the existence of a covalent bond between N epsilon 2 of the nucleophile and the alpha-phosphorus of UMP. A hydrogen bond that is conserved in plexes between His 166 N delta 1 and the carbonyl O of His 164 serves to properly orient the nucleophile and electrostatically stabilize the positively charged imidazolium that results from nucleotidylation. Hydrogen bonds from side-chain Gln 168 to the nonbridging phosphoryl oxygens of the nucleotidyl intermediate appear crucial for the formation and reaction of the plex as well. The significance of the latter interaction is underscored by the fact that the predominant cause of the metabolic disease galactosemia is the mutation of the corresponding Gln (Gln 188 in humans) to Arg. parison to other phosphohistidyl enzymes is described, as well as a revised model for the mechanism of the uridylyltransferase.

8794734

The relative orientation of Gla and EGF domains in coagulation factor X is altered by Ca2+ binding to the first EGF domain. A combined NMR-small angle X-ray scattering study.

Coagulation factor X is a serine protease containing three noncatalytic domains: an N-terminal gamma-carboxyglutamic acid (Gla)1 domain followed by two epidermal growth factor (EGF)-like domains. The isolated N-terminal EGF domain binds Ca2+ with a Kd of 10(-3) M. When linked to the Gla domain, however, its Ca2+ affinity is increased 10-fold. In this paper, we present the NMR solution structure of the factor X Gla-EGF domain pair with Ca2+ bound to the EGF domain, as well as small angle X-ray scattering (SAXS) data on the Gla-EGF domain pair with and without Ca2+. Our results show that Ca2+ binding to the EGF domain makes the Gla and EGF domains fold toward each other using the Ca2+ site as a hinge. Presumably, a similar mechanism may be responsible for alterations in the relative orientation of protein domains in many other extracellular proteins containing EGF domains with the consensus for Ca2+ binding. The results of the NMR and SAXS measurements reported in this paper confirm our previous result that the Gla domain is folded also in its apo state when linked to the EGF domain [Sunnerhagen, M., et al. (1995) Nat. Struct. Biol. 2, 504-509]. Finally, our study clearly demonstrates the bination of NMR and SAXS in the study of modular proteins, since this enables reliable evaluation of both short-range (NMR) and long-range interactions (SAXS).

8794736

Crystal structure of an elastase-specific inhibitor elafin complexed with porcine pancreatic elastase determined at 1.9 A resolution.

The crystal structure of a plex between an elastase-specific inhibitor elafin and porcine pancreatic elastase (PPE) has been determined and refined to a crystallographic R-factor of 19.7% at 1.9 A resolution. The polypeptide chain of elafin has a planar spiral shape with an exposed external part and an internal core part which resembles both the crystal structure of human seminal plasma inhibitor (HUSI-1) [Grütter, M. G., Fendrich, G., Huber, R., & Bode, W. (1988) EMBO J. 7, 345-351] and the solution structure of Na+,K(+)-ATPase inhibitor (SPAI-1) revealed by NMR analysis [Kozaki, T., Kawakami, Y., Tachibana, S., Hatanaka, H., & Inagaki, F. (1994) Pept. Chem., 405-408]. The external region containing the primary binding loop is interconnected by four disulfide bonds to the internal posed of a beta-sheet and a hairpin loop. The scissile peptide bond Ala24i(P1)-Met25i(P1') in the primary binding site is intact, and its carbonyl carbon is in van der Waals contact with O gamma of the active site Ser195 of PPE. The seven residues of Leu20i(P5)-Leu26i(P2') of the primary binding loop and the three residues of Ser48i, Cys49i, and Ala52i of the adjacent hairpin loop are in contact with PPE by hydrogen bonds and/or van der Waals interactions in a manner similar to that observed for other serine plexes. Electron densities of the N-terminal residues Ala1i-Ser10i which are not responsible for the elastase inhibitory activity were not visible, probably due to disordered conformation. The guanido group (N eta 1, N eta 2) of Arg61 in plex interacts with S delta of Met25i(P1') by possible hydrogen bonds between N and S atoms, panying a large positional shift of the side chain of Arg61-(S1') between plexed and free forms of PPE. The primary binding site is stabilized by hydrogen bonds between the guanido group (N eta 1, N eta 2) of Arg22i(P3) and the carbonyl group of Met25i(P1') across the scissile bond, as well as by a hydrogen bond between the amino group of Cys23i(P2) and the carbonyl group of Ser48i in the internal core. This intramolecular hydrogen bond network and the network of four disulfide bonds might play a significant role in stabilizing the conformation of the binding site for expressing the potent specific inhibitory activity.

8794737

Solution structure of rat apo-S100B(beta beta) as determined by NMR spectroscopy.

S100B(beta beta), a member of the S100 protein family, is a Ca(2+)-binding protein with noncovalent interactions at its dimer interface. Each apo-S100 beta subunit (91 residues) has four alpha-helices and a small antiparallel beta-sheet, consistent with two predicted helix-loop-helix Ca(2+)-binding domains known as EF-hands [Amburgey et al. (1995) J. Biomol. NMR 6, 171-179]. The three-dimensional solution structure of apo-S100B(beta beta) from rat has been determined using 2672 distance (14.7 per residue) and 88 dihedral angle restraints derived from multidimensional nuclear magnetic resonance spectroscopy. Apo-S100B (beta beta) is found to be globular pact with an extensive hydrophobic core and a highly charged surface, consistent with its high solubility. At the symmetric dimer interface, 172 intermolecular nuclear Overhauser effect correlations (NOEs) define the antiparallel alignment of helix I with I' and of helix IV with IV'. The perpendicular association of these pairs of antiparallel helices forms an X-type four-helical bundle at the dimer interface. Whereas, the four helices within each apo-S100 beta subunit adopt a unicornate-type four-helix bundle, with helix I protruding from the parallel bundle of helices II, III, and IV. Accordingly, the orientation of helix III relative to helices I, II, and IV in each subunit differs significantly from that known for other Ca(2+)-binding proteins. Indeed, the interhelical angle (omega) observed in the C-terminal EF-hand of apo-S100 beta is -142 degrees, whereas omega ranges from 118 degrees to 145 degrees in the apo state and from 84 degrees to 128 degrees in the Ca(2+)-bound state for the EF-hands of calbindin D9k, calcyclin, and calmodulin. Thus, a significant conformational change in the C-terminal EF-hand would be required for it to adopt a structure typical of the Ca(2+)-bound state, which could readily explain the dramatic spectral effects observed upon the addition of Ca2+ to apo-S100B(beta beta).

8794738

Structure of the B-DNA oligomers d(CGCTAGCG) and d(CGCTCTAGAGCG) in new crystal forms.

We present the structure of the dodecamer CGCTCTAGAGCG and the related octamer CGCTAGCG, both in the B form, determined by single crystal X-ray diffraction. Two different crystal forms of the octamer have been obtained, with either three or four duplexes in the asymmetric unit. The dodecamer crystallizes in the P2(1) space group with two duplexes in the asymmetric unit. Very few such structures have been previously reported, while the octamer structure is the first one determined with three duplexes in the asymmetric unit. It is also the first octamer with standard Watson-Crick base pairs to be crystallized in the B form. The crystal structure is stabilized in both cases by interactions between the guanines in the two terminal base pairs of each duplex. This interaction is similar to that found in most dodecamers which have been previously studied, but here it is found in a new unit cell (for the dodecamer) and in one octamer. In the dodecamer cytosine-stacking interactions between neighbor duplexes are also present. The two dodecamer duplexes in the asymmetric unit show different patterns of bending, while the octamer molecule has a rather straight helical axis. The results presented confirm the strong conformational variability of the TA pyrimidine-purine step and demonstrate a clear alternating structure for the (CT/GA)n sequence.

8794739

Structural consequences of heme removal: molecular dynamics simulations of rat and bovine apocytochrome b5.

Molecular dynamics simulations of rat and bovine apocytochrome b5 were performed to investigate the structural and dynamical consequences of heme removal. A crystal structure is available for the bovine holoprotein, while experimental studies of apocytochrome b5 have focused on the rat protein. The rat and bovine proteins are 93% homologous by sequence, and the sequence differences (six residues) appear to have no effect on the structure of the native holoprotein, as seen by the correlation of a bovine simulation with rat holocytochrome b5 experimental data (Storch & Daggett, 1995). There was a marked effect, however, on the structure and dynamics of the apo form. The bovine apocytochrome b5 simulation displayed subtle inconsistencies pared to the experimental results on the rat apoprotein. Therefore, the rat protein was constructed from the bovine crystal structure coordinates. The MD simulation of the rat apoprotein displayed greater deviations from the crystal structure, yet it was in much closer agreement to the experimental data for the apoprotein. Additionally, the six variant residues fall in the regions where the bovine protein deviated from experiment. The two hydrophobic cores of the rat protein behaved very differently. Core 2 was well maintained, retained native-like structure, and is in good agreement with NMR data (Moore & te, 1990). Conversely, core 1, which is normally constrained by the prosthetic heme group, exhibited conformational heterogeneity, increased mobility, and some loss of secondary structure. Thus, the model of rat apocytochrome plements past studies by providing structural information about core 1 that has proved difficult to obtain by experiment. The bovine simulation serves as a prediction, since little to no experimental data exist for this form of the apoprotein.

8794740

Structure-based design of an intramolecular proton transfer site in murine carbonic anhydrase V.

Carbonic anhydrase V (CA V) is a mitochondrial enzyme that catalyzes the hydration of CO2 to produce bicarbonate and a proton. The catalytic properties of wild-type murine CA V suggest the presence of a proton shuttle residue having pKa = 9.2, the role of which is to transfer a proton from zinc-bound water to solution in the hydration direction to regenerate the zinc hydroxide form of the enzyme. Two likely candidates for shuttle residues are the tyrosines at positions 64 and 131 in the active site cavity. The crystal structure of wild-type carbonic anhydrase V [Boriack-Sjodin et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10949-10953] shows that the side chain of Tyr 64 is forced into an orientation pointing away from the zinc by Phe 65, although Tyr 131 is oriented toward the zinc. We have prepared mutants of murine CA V replacing both Tyr 64 and Tyr 131 with His and Ala and investigated the proton shuttle mechanism using stopped-flow spectrophotometry and the depletion of 18O from CO2 measured by mass spectrometry. Experiments with both single and double mutations showed that neither position 64 nor position 131 was a prominent site for proton transfer. However, a double mutant of CA V containing the two replacements, Tyr 64-->His and Phe 65-->Ala, demonstrated enhanced proton transfer with an apparent pKa of 6.8 and maximal contribution to kcat of 2.2 x 10(5) s-1. In addition to the altered catalytic properties, the crystal structure of the His 64/Ala 65 double mutant strongly suggested proton transfer by His 64 after removal of the steric hindrance of Phe 65. This is the first structure-based design of an efficient proton transfer site in an enzyme.

8794741

Nuclear multicatalytic proteinase subunit RRC3 is important for growth regulation in hepatocytes.

Multicatalytic proteinases (MCPs) are macromolecular structures involved in the intracellular degradation of many types of proteins. MCPs posed of a 20S "core" of both structural (alpha) and presumed catalytic (beta) subunits, in association with regulatory proteins. They are characteristically found in both the nucleus and cytoplasm of cells, although mechanisms governing the subcellular distribution of MCPs are not known. RRC3, an alpha subunit of rat MCPs, contains both a putative nuclear localization signal (NLS) and a potential tyrosine phosphorylation site which could play a role in nuclear import, and the nuclear form of RRC3 appears to be involved in the regulation of cell growth. Here we have generated a variety of RRC3 expression constructs to study features of RRC3 important in nuclear localization and cell growth. PCR was utilized to develop constructs containing point mutations in either the putative NLS (K51 mutated to A) or at a potential tyrosine phosphorylation site (Y121 mutated to F), and an epitope from influenza hemagglutinin (HA) was added in triplicate to the C-terminus of the constructs as a means of identification. RRC3 constructs were then made in which the nucleotide sequence near the translation initiation site of RRC3 was modified in such a way that the amino acid sequence of the protein translated from the constructs is unchanged from that of normal RRC3, thus allowing differential modulation of endogenous RRC3 with antisense oligonucleotide treatment. These N-terminally modified constructs are designated mC3, mC3NLS, and mC3y. In vitro transcription/translation reactions with these constructs produced the expected products, which were immunoprecipitated with a mouse monoclonal anti-HA antibody. Immunohistochemical studies with hepatocyte cell lines transiently transfected with either mC3NLS or mC3y showed only cytoplasmic staining, whereas cells transfected with mC3 had a staining pattern typical of endogenous RRC3 (both cytoplasmic and nuclear) with strong staining of the nuclear perimeter. Immunoblot analyses of subcellular fractions from stably transfected CWSV1 cells showed mC3 product in both the cytosol and nucleus of cells, whereas mC3NLS or mC3y products were restricted to the cytosol. CWSV1 cells stably transfected with the pTet-Splice vector containing no insert (as a control) were markedly inhibited (80%) in cell growth and showed altered morphology when treated with antisense oligonucleotides targeted to endogenous RRC3, reproducing previous studies. Similarly, CWSV1 cells stably transfected with either mC3NLS or mC3y constructs showed analogous growth inhibition and morphologic alteration upon antisense treatment. In contrast, CWSV1 cells stably transfected with the mC3 construct showed normal growth and morphology following antisense oligonucleotide treatment, demonstrating that replenishment of nuclear RRC3 was necessary and sufficient to relieve growth inhibition. In 32P-metabolic labeling studies, mC3 was tyrosine-phosphorylated in cytosol as the full-length protein (M(r) 36000). mC3NLS was also phosphorylated in cytosol, whereas mC3y was not. Nuclear mC3 showed phosphorylation of a M(r) 27000 processed form while neither mC3NLS nor mC3y showed any phosphorylated nuclear products. Our results show that nuclear RRC3 is important in control of cell growth and that both the NLS and Y121 are important in nuclear localization of RRC3. Control of nuclear import by tyrosine phosphorylation may represent a novel regulatory mechanism, and our results further suggest that RRC3 may travel as a maverick subunit.

8794742

The expression of poly(ADP-ribose) polymerase during differentiation-linked DNA replication reveals that it is a component of the multiprotein DNA replication complex.

3T3-L1 preadipocytes have been shown to exhibit a transient increase in poly(ADP-ribose) polymerase (PARP) protein and activity, as well as an association of PARP with DNA polymerase alpha, within 12-24 h of exposure to inducers of differentiation, whereas 3T3-L1 cells expressing PARP antisense RNA showed no increase in PARP and are unable plete the round of DNA replication required for differentiation into adipocytes. The role of PARP in differentiation-linked DNA replication has now been further clarified at both the cellular and enzymological levels. Flow cytometric analysis revealed that control 3T3-L1 cells progressed through one round of DNA replication prior to the onset of terminal differentiation, whereas cells expressing PARP antisense RNA were blocked at the G0/G1 phase of the cell cycle. Confocal microscope image analysis of control S phase cells demonstrated that PARP was localized within distinct intranuclear granular foci associated with DNA replication centers. On the basis of these results, purified plexes from other cell types that had been characterized for their ability to catalyze viral DNA replication in vitro were analyzed for the presence of PARP. PARP exclusively copurified through a series of centrifugation and chromatography steps with core proteins of an 18-21S multiprotein plex (MRC) from human HeLa cells, as well as with the corresponding mouse MRC from FM3A cells. The MRC were shown to contain DNA polymerases alpha and delta, DNA primase, DNA helicase, DNA ligase, and topoisomerases I and II, as well as accessory proteins such as PCNA, RF-C, and RP-A. Finally, immunoblot analysis of MRCs from both cell types with monoclonal antibodies to poly (ADP-ribose) revealed the presence of approximately 15 poly(ADP-ribosyl)ated proteins, some of which were further confirmed to be DNA polymerase alpha, DNA topoisomerase I, and PCNA by immunoprecipitation experiments. These results suggest that PARP may play a regulatory role within the replicative apparatus as a molecular nick sensor controlling the progression of the replication fork or ponent replicative enzymes or factors in plex by directly associating with them or by catalyzing their poly(ADP-ribosyl)ation.

8794743

Deoxyadenosine-based DNA polymerase photoprobes: design, synthesis, and characterization as inhibitors of the Escherichia coli DNA polymerase I Klenow fragment.

DNA polymerase photoprobes 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate (1), 2-[(4-azidophenylsulfenyl)thio]-2'-deoxyadenosine 5'-triphosphate (2), and 2-[(4-azido-2-nitrophenyl)-thio]-2'-deoxyadenosine 5'-triphosphate (3) were designed from a thermodynamic model of DNA polymerase 1-substrate interactions such that the triphosphate would anchor the inhibitor and allow the phenyl azide to interact with plementary template binding site. Photoprobes 1-3 were synthesized by condensation of 2-thio-2'-deoxyadenosine or its phosphate with p-azidophenacyl bromide, N-(4-azidophenylsulfenyl)phthalimide, and 4-azido-1-fluoro-2-nitrobenzene, respectively, and characterized as reversible and photoinduced irreversible inhibitors of the DNA polymerase I Klenow fragment and HIV I reverse transcriptase. The aryl azides posed with irradiation at 300 and 350 nm with half-lives ranging from 0.98 to 2.33 min and 2.15 to 5.38 min, respectively, with quantum efficiencies ranging from 0.29 to 0.55 and no apparent position of the 2-thio-2'-deoxyadenosine nucleotide. Photoprobes 1-3 showed mixed petitive inhibition of the Klenow fragment polymerase activity versus poly(dA).(T)10 as variable substrate with petitive inhibition constants of 2.1, 36, and 29 microM, respectively, evidence suggesting that these photoprobes bind to both the free enzyme form and the enzyme-template-primer plex. Of the three photoprobes, only nucleotide 1 photoinactivates the Klenow fragment; in the presence of a 200-fold excess of nitrene scavenger, photoprobe 1 inactivates 92% of the Klenow fragment polymerase activity with saturation observed at 9.7 microM and an IC50 of about 2 microM. This evidence demonstrates that photoprobe 1 does bind to the Klenow fragment in the absence of template-primer and that it is an efficient photoprobe.

8794744

DNA polymerase photoprobe 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate labels an Escherichia coli DNA polymerase I Klenow fragment substrate binding site.

The nucleotide photoprobe 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate (1) was evaluated as a photoaffinity label of the DNA polymerase I Klenow fragment. Photolabel [3H]-1 covalently labeled the Klenow fragment with photolysis at 300 nm, reaching saturation at an approximate 1:1 mole ratio at 5.7 microM and with an EC50 (the effective concentration at 50% maximum photoincorporation) of about 0.74 microM. Saturating concentrations of poly(dA).(T)10 protect the Klenow fragment from [3H]-1 photoincorporation, and TTP at a concentration approximately equal to its KD for the free enzyme form shifts the dose-response curve for photoincorporation of [3H]-1 into the Klenow fragment by a factor of 2, indicating petitive relationship between TTP and 1. Additionally, the photoincorporation of [3H]-1 into the Klenow fragment has an absolute requirement for magnesium, with no significant photoincorporation observed at concentrations of 1 up to 10 microM in the absence of magnesium. These results demonstrate that, as designed, photoprobe 1 binds to both the dNTP and a portion of the template-primer binding sites on the Klenow fragment. Photoaffinity labeling of the Klenow fragment by 1 yielded a single radiolabeled tryptic fragment which was isolated by HPLC; sequence analysis identified Asp732 in the peptide fragment Asp732-Ile733-His734-Arg735 as the site of covalent modification. Molecular modeling plementary NMR analysis of the conformation of 1 indicated preferred C3'-exo and C2'-exo-C3'-endo symmetrical twist furanose ring puckers, with a high antibase conformation and a +sc C-5 torsional angle. Docking studies using Asp732 as an anchor point for the azide alpha-nitrogen on the photolabel indicate that the dNTP binding site is at the edge of the DNA binding cleft opposite the exonuclease site and that the template binding site includes helix O in the finger motif of the Klenow fragment.

8794745

Recognition of the T-arm of tRNA by tRNA (m5U54)-methyltransferase is not sequence specific.

tRNA (m5U54)-methyltransferase (RUMT) catalyzes the methylation of U54 of tRNAs. In contrast to enzymes which recognize a particular tRNA, RUMT recognizes mon to all tRNAs. We have shown that these features reside in the T-arm of tRNA and constructed a minimal consensus sequence for RUMT recognition and catalysis (Gu et al., 1991b). Here, we have mutated each conserved T-loop residue and conserved T-stem base pair to bases or base pairs which are not observed in Escherichia coli tRNA. The substrate specificity of RUMT for 30 in vitro synthesized T-arm mutants of tRNAPhe and 37 mutants of the 17-mer analog of the T-arm derived from tRNA1Val was investigated. A 2-5 base pair stem was essential for recognition of the T-arm by RUMT, but the position of the stem was unimportant. The 7-base size of the T-loop maintained by the stem was essential for RUMT recognition. For tRNA, most base substitutions in the 7-base loop did not eliminate RUMT activity, except for any mutation of the methyl acceptor U54 and the C56G mutation. The effect of base and base pair mutations on Kcat or the rate of methylation by RUMT was more striking than the effect on the Kd for binding to RUMT. parison with mutations in the T-loop of intact tRNA, base mutation in the T-loop of the 17-mer T-arm had a more deleterious effect on binding and methylation. Surprisingly, recognition of tRNA by RUMT appears to reside in the three-dimensional structure of the seven-member T-loop rather than in its primary structure.

8794746

Different effects of histone H1 on de novo DNA methylation in vitro depend on both the DNA base composition and the DNA methyltransferase.

We have characterized the inhibition exerted by histone H1 on the activity of human placenta DNA (cytosine-5-)-methyltransferase. Our experiments demonstrate that the extent of inhibition depends on the DNA position, AT-rich substrates being more severely affected than GC-rich substrates and CpG-rich islands. With bacterial SssI methylase, the effect pletely reversed since its activity on AT-rich substrates undergoes a 4-5-fold stimulation upon the addition of H1. Poly(L-lysine) mimicks H1 effects, suggesting an essential role of lysine residues in both the inhibitory and stimulatory effects of H1. parison of the different behaviors of the two enzymes, the inhibitory effect over the eukaryotic enzyme might be accounted for by hypothesizing petition between minor groove-binding motifs (SPKK-like) present in placenta methylase as well as in histone H1.

8794748

Sequence effects on RNA bulge-induced helix bending and a conserved five-nucleotide bulge from the group I introns.

Bulge loops introduce bends in RNA double helices. Thus, a role for bulge loops in the tertiary folding of RNA is to orient helical elements. The location, size, and sequence of a five-nucleotide bulge are conserved in many of the self-splicing group I introns. We have used gel electrophoretic analysis of helix bending to test the hypothesis that this bulge loop is conserved to control the angle between the flanking helices. Interruption of an RNA duplex by the five-nucleotide bulge of the group I intron from Tetrahymena thermophila results in an electrophoretically retarded species, indicative of bending by the bulge. However, mutation of conserved bases in the bulge has a small effect on the retardation, suggesting that the average induced bend angle is not strongly dependent on the conserved sequence. Electrophoretic analysis of a mixture of bulged duplexes containing all five-nucleotide bulges reveals that most five-nucleotide bulge sequences induce bends that are similar to the bend induced by the conserved bulge. We have calibrated relative electrophoretic mobilities with bends of known magnitude, and characterized the distribution of bulge sequences among bend angles. Though the entire range of bend angles induced by different five-nucleotide bulges is from approximately 45 degrees to 75 degrees, most ( > 85%) five-nucleotide bulge loops induce bends between 65 degrees and 75 degrees. We have identified several of the anomalous five-nucleotide bulge sequences that induce bends of magnitude smaller than 65 degrees. They are generally, though not universally, pyrimidine-rich.

8794749

Identification and characterization of an estrogen-responsive element binding protein repressed by estradiol.

Cytosolic proteins from uteri of 19-day-old rats were analyzed by an electrophoresis mobility shift assay (EMSA) using a 31 base pair DNA probe containing an estrogen-responsive element (ERE) from the vitellogenin A2 gene. EMSA identified three distinct cytosolic plexes that are separable by Q-Sepharose anion exchange chromatography into an estrogen receptor (ER)-containing fraction (150 mM NaCl eluate) and a non-ER-containing fraction (250 mM NaCl eluate). We thus refer to the non-ER fraction as the ERE binding protein (ERE-BP). The ERE-BP-containing fraction was repressed to 40-50% of its normal levels following a single injection of estradiol. In addition, ERE-BP levels were repressed to the same extent (greater than 50%) by day 20 of the rat's gestational period. Examination of the expression pattern of ERE-BP shows that this activity is differentially expressed in both estrogen-responsive and nonresponsive tissues, with the highest levels of expression occurring in the pituitary. We next examined the specificity of ERE-BP binding petition analysis using DNA sequences corresponding to binding sites of several known transcription factors. ERE-BP was found to be specific for both the ER binding site (ERE) and TATA binding protein binding sites. Furthermore, saturation analysis demonstrated that ERE-BP binds to the ERE and TATA binding protein sequences with an apparent Kd of 1.2 and 0.12 nM, respectively. Partial purification of ERE-BP using three chromatography steps (Q-Sepharose, hydroxyapatite, and Sephacryl S300) followed by sodium dodecyl sulfate analysis indicated the presence of three major protein bands (p102, p81, and p48) as judged by Coomassie staining. UV cross-linking of the plex followed by sodium dodecyl sulfate analysis-polyacrylamide gel electrophoresis analysis indicates that the 48 kDa band seen in the final, partially purified fraction correlates with the ERE-BP activity. Thus, this study has identified a unique uterine cytosolic protein that binds to the ER binding site and may influence ER binding.

8794747

Kinetic analysis of human flap endonuclease-1 by flow cytometry.

Human flap endonuclease-1 (FEN-1) is a structure-specific endonuclease and exonuclease which is essential for DNA replication and repair. We have cloned a human FEN-1 gene, overexpressed it in Escherichia coli, purified the binant protein to near homogeneity, and characterized its cleavage of a flap DNA structure using a novel analytical approach based on flow cytometry. With this approach, we were able to measure continuously the kinetics of DNA cleavage by FEN-1 and to separate experimentally the binding and catalysis functions of the enzyme. When the reaction was initiated by the addition of FEN-1, the cleavage kinetics were dependent on enzyme concentration and appeared to saturate at high concentrations. When enzyme and substrate were preincubated in the presence of EDTA and the reaction initiated by the addition of Mg2+, rapid kinetic flow cytometry measurements showed that cleavage is fast (t1/2 approximately 6 s, k = 0.10 s-1). Using the single-turnover kinetics as a measure of the amount of plex present, we estimated the Kd for the FEN-1-flap DNA substrate to be 7.5 nM in the absence of Mg2+ and the rate constant for dissociation of the plex to be 0.07 s-1. Computer fitting of the experimental data to a kinetic model confirms these estimates for the individual steps and suggests some interesting features of enzymology using a surface-bound substrate.

8794750

Targeting sites within HIV-1 cDNA with a DNA-cleaving ribozyme.

A variant of the Tetrahymena ribozyme that efficiently cleaves single-stranded DNA under simulated physiological conditions [Tsang, J., & Joyce, G. F. (1994) Biochemistry 33, 5966-5973] was evaluated as a potential therapeutic agent on the basis of its ability to cleave synthetic oligonucleotide substrates corresponding to conserved target sites within HIV-I cDNA. In order to increase the sequence selectivity of the ribozyme, its substrate recognition domain was extended from 6 to 12 nucleotides, allowing base pairing with substrate nucleotides that lie both upstream and downstream of the cleavage site. The sequence of the extended recognition domain could be changed to allow cleavage of a variety of different DNA targets. The ribozyme exhibited a high degree of sequence specificity, discriminating by a factor of 10(2) to more than 10(4) against substrates that form a single-base mismatch with the ribozyme's recognition domain. Mismatches that occurred close to the cleavage site led to a greater decrease in pared to those that occurred farther away.

8794751

The disulfide folding pathway of tick anticoagulant peptide (TAP), a Kunitz-type inhibitor structurally homologous to BPTI.

The pathway of oxidative folding of tick anticoagulant peptide (TAP, 60 amino acids and three disulfides) has been analyzed by characterization of the acid and iodoacetate trapped folding intermediates. The results reveal a high degree of heterogeneity of the one- and two-disulfide intermediates and the presence of three-disulfide scrambled species along the folding pathway. The picture of TAP folding differs significantly from the well-documented case of bovine pancreatic trypsin inhibitor (BPTI), despite the fact that both proteins share close structural homology in term of 3-D conformation and disulfide pattern.

8794752

4-Hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum: characterization of FAD and iron-sulfur clusters involved in an overall non-redox reaction.

4-Hydroxybutyryl-CoA dehydratase catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA, which involves cleavage of an unactivated beta-C-H bond. The enzyme also catalyzes the apparently irreversible isomerization of vinylacetyl-CoA to crotonyl-CoA. Addition of crotonyl-CoA to the dehydratase, which contains FAD as well as non-heme iron and acid labile sulfur, led to a decrease of the flavin absorbance at 438 nm and an increase in the region from 500 to 800 nm. The protein-bound FAD was easily reduced to the semiquinone (redox equilibration within seconds) and only slowly to the hydroquinone (redox equilibration minutes to hours): the redox potentials were not unusual for flavoproteins (Eox/sq = -140 +/- 15 mV and Esq/red = -240 +/- 15 mV; pH 7.0, 25 degrees C). There was no equilibration of electrons between the flavin and the Fe-S cluster, which was difficult to reduce. After extensive photoreduction, an EPR signal indicative of a [4Fe-4S]+ cluster was detected (g-values: 2.037, 1.895, 1.844). Upon exposure to air at 0 degrees C, the enzyme lost dehydration pletely within 40 min, but isomerase activity dropped to about 40% of the initial value and persisted for more than a day. The properties of the protein-bound FAD are consistent with a mechanism involving transient one-electron oxidation of the substrate to activate the the beta-C-H bond. The putative [4Fe-4S]2+ cluster could serve a structural role and/or as Lewis acid facilitating the leaving of the hydroxyl group.

8794753

Nature of the unfolded state of ribonuclease A: effect of cis-trans X-Pro peptide bond isomerization.

The equilibrium unfolded state of disulfide-intact bovine pancreatic ribonuclease A is a heterogeneous mixture of unfolded species. Previously, four unfolded species have been detected experimentally. They are Uvf, Uf, UsII, and UsI which have refolding time constants on the millisecond, millisecond to second, second to tens of seconds, and hundreds of seconds time scales, respectively. In the current study, the refolding pathway of the protein was investigated under favorable folding conditions of 0.58 M GdnHCl, pH 5.0, and 15 degrees C. In addition to the above four unfolded species, the presence of a fifth unfolded species was detected. It has a refolding time constant on the order of 2 s under the conditions employed. This new unfolded species is labeled Um, for medium-refolding species. Single-jump refolding experiments monitored by tyrosine burial and by cytidine 2'-monophosphate inhibitor binding indicate that the different unfolded species refold to the native state along independent refolding pathways. The buildup of the different unfolded species upon unfolding of the protein from the native state was monitored by absorbance using double-jump experiments. These experiments were carried out at 15 degrees C and consisted of an unfolding step at 4.2 M GdnHCl and pH 2.0, followed, after a variable delay time, by a refolding step at 0.58 M GdnHCl and pH 5.0. The results of these experiments support the conclusion that the different unfolded species arise from cis-trans isomerizations at the X-Pro peptide bonds of Pro 93, 114, and 117 in the unfolded state of the protein. The rates of these isomerizations were obtained for each of these three X-Pro peptide bonds at 15 degrees C.

8794754

Structure of a hydrophobically collapsed intermediate on the conformational folding pathway of ribonuclease A probed by hydrogen-deuterium exchange.

The unfolded state of disulfide-intact bovine pancreatic ribonuclease A is a heterogeneous mixture of unfolded species which have different X-Pro peptide bond conformations. One of these unfolded species, labeled Uvf, has all its X-Pro peptide bonds in the native conformation. Therefore, the refolding of Uvf is a purely conformational folding process which is plicated by cis-trans X-Pro peptide bond isomerization. There are two identifiable intermediates on the folding pathway of Uvf: one which is a largely unfolded intermediate (IU) and another which is a hydrophobically collapsed intermediate (I phi). An instrument was built, and experiments were designed to study the structure in IU and I phi by hydrogen-deuterium exchange. These experiments are bination of a double-jump experiment followed by a pulse-labeling experiment. The native protein was first unfolded to populate Uvf to more than 99%, and then Uvf was refolded for a specified period of time. After refolding, hydrogen-deuterium exchange of the backbone amides was initiated for a given time by raising the pH. Subsequently, the exchange was quenched and the protein was allowed to continue to fold to the native state. The extent of exchange was determined quantitatively by two-dimensional NMR spectroscopy. The data indicate that IU has no secondary structure that can protect the backbone amides from exchange under the conditions employed. On the other hand, in I phi, the second helix (residues 24-34) and a large part of the beta-sheet region of the protein are formed, while the rest of the protein molecule remains unstructured. In general, the protection factors in I phi are low, indicating that this intermediate has a dynamic structure. Our observations are consistent with I phi being a molten-globule-like intermediate. The regular structure formed in I phi is much less than that observed in a hydrogen-bonded intermediate (Ii) populated early on the major slow-refolding pathway of the protein [Udgaonkar, J. B., & Baldwin, R. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8197-8201]; in addition, the structure in I phi has much lower stability than that in Ii. This implies that a slower refolding rate allows for a higher cooperativity between the different structural elements of the protein, resulting in the formation of more stable (native-like) intermediates (as in Ii) during the folding process.

8794755

Characterization of SH2-ligand interactions via library affinity selection with mass spectrometric detection.

binatorial libraries have proven to be a valuable source of diverse structures useful for large-scale biochemical screening. Their use has greatly facilitated the study of protein-protein interactions. We have developed a practical technique for screening such libraries by integrating affinity chromatography selection with electrospray ionization mass spectrometric detection, referred to as library affinity selection-mass spectrometry (LAS-MS). The process allows for rapid and efficient screening of solution phase libraries and provides detailed information such as the relative affinities of substrates. The method is generally applicable to include nonpeptide libraries; moreover, electrospray tandem mass spectrometry (ES-MS/MS) yields sequence-specific identification of ponents without the need for chemical tags. This technique is demonstrated using the Src homology 2 (SH2) domain of phosphatidylinositol 3-kinase (PI 3-kinase). The critical importance of methionine in the position +3 (relative to the phosphotyrosine position) is demonstrated in a library built with a phosphotyrosine mimic, (phosphonodifluoromethyl)phenylalanine. The described method has broad applicability binatorial library screening.

8794756

Cloning and expression of salmon cardiac troponin C: titration of the low-affinity Ca(2+)-binding site using a tryptophan mutant.

Activation of cardiac actomyosin ATPase requires the occupation of the single low-affinity Ca(2+)-binding site of troponin C (cTnC). Previously, we demonstrated pronounced differences between mammals and cold-water salmonid fish in the Ca2+ sensitivity of cardiac preparations, particularly in relation to temperature [Churcotte, C., Moyes, C. D., Baldwin, K., Bressler, B., & Tibbits, G. F. (1994) Am. J. Physiol. 267, R62-R70]. In this study, we examine the extent to which cTnC structure could account for the observed differences in myofibrillar Ca2+ sensitivity. Salmonid (Oncorhynchus mykiss) cTnC was cloned, sequenced, and expressed in Escherichia coli as a maltose-binding protein fusion. The coding region has 87% homology with human cTnC cDNA and differs in 13 of 161 amino acid residues from the human/bovine/porcine isoform. The sequence corresponding to the single regulatory Ca(2+)-binding site II pletely homologous to that of mammals. The protein expressed exhibits optical properties similar (circular dichroism, intrinsic fluorescence) to those of cTnC purified from salmonid (Salmo salar) and bovine ventricle. A single tryptophan residue was introduced into the inactive Ca(2+)-binding site I (ScTnC-FW27) to facilitate Ca2+ titration. The Ca(2+)-binding constant (K1/2 = 5.33 pCa units) was within the range reported for the low-affinity sites of mammalian cTnC. Although differences in TnC primary structure are striking, Ca2+ affinity of intact cardiac myofibrils is likely influenced by interactions with other troponin proteins.

8794757

The behavior of the active site salt bridge of bovine neurophysins as monitored by 15N NMR spectroscopy and chemical substitution. Relationship to biochemical properties.

The active site of liganded neurophysin contains a salt bridge that involves the side chains of Arg-8 and Glu-47 of the protein and the alpha-amino group of bound hormone or related peptide. The extent to which the Arg-8-Glu-47 salt bridge persists in the absence of peptide, or to which the environment of Arg-8 in the unliganded state differs in monomers and dimers, is relevant to an understanding of allosteric mechanism in this system. In the present study, the behavior of the salt bridge was investigated by 15N NMR and chemical replacement of Arg-8. Bovine neurophysin-I was converted to its des 1-8 derivative, and Arg-8 was replaced by 15N-substituted Arg or by other residues using chemical semisynthesis. The relative abilities of different amino acids to restore peptide affinity to the des 1-8 protein were in good accord with the view of the salt bridge in the liganded state obtained from crystals of bovine plexes. In the unliganded parison of the 15N and proton NMR signals from Arg-8 with those in smaller arginine systems suggested the absence of significant interactions between the guanidinium of Arg-8 and Glu-47 or between the amino terminal region of Arg-8 and other elements of the protein. No evidence of a difference in Arg-8 environment between unliganded monomers and dimers was found. Marked spectral changes panying the binding of oxytocin indicated changes in the environment of both the side chain and amino terminal region of Arg-8. The NMR results were in good agreement with a recently parison of bovine neurophysin-II derivatives in the liganded and unliganded states, with the notable exception of the extent of salt bridge formation in the unliganded state. The results are shown to be consistent with, and to help explain, significant differences between the two bovine neurophysins in the susceptibility to tryptic cleavage at Arg-8 in the unliganded state and in the pH dependence of peptide binding and additionally constrain potential allosteric mechanisms underlying neurophysin ligand-facilitated dimerization.

8794758

Effects of antibody binding on structural transitions of the nicotinic acetylcholine receptor.

Patch-clamping and photoaffinity-labeling techniques were used to study the effects of binding of monoclonal antibodies (mAbs) on the function of Torpedo californica nicotinic acetylcholine receptor (nAChR). The rat anti-Torpedo nAChR mAbs examined here are known to inhibit ligand binding to either the high-affinity (mAb 247) or both the high- and low-affinity binding sites (mAb 370 and mAb 387) [Mihovilovic, M. & Richman, D. P. (1984) J. Biol. Chem. 259, 15051-15059; Mihovilovic, M., & Richman, D. P. (1987) J. Biol. Chem. 262, 4978-4986]. Single-channel analysis shows that mAb 247 and the Fab fragment of mAb 247 inhibit the opening of the nAChR ion channel, although they have no effects on the structural transition from the resting to desensitized state as monitored by the extent of decreased labeling by the photoreactive probe 3-(trifluoromethyl)-3-(m- [125I]iodophenyl)diazirine ([125I]-TID). In the presence of mAb 387, the nAChR single-channel amplitude was decreased by 20%, whereas Fab pletely inhibited channel opening. [125I-TID]-labeling studies suggest that the mAb 387-nAChR and Fab plexes are able to undergo the transition between resting and desensitized states. This result confirms that the nAChR can assume a desensitized state without prior channel opening. In addition, mAb 35 and mAb 132, which recognize the main immunogenic region (MIR) of the nAChR, and mAb 370 do not alter either single-channel behavior or labeling patterns. Combining the results from characterization with respect to their epitopes and their effects on agonist (carbamylcholine) and antagonist [alpha-bungarotoxin (alpha-BTX) and curare] binding, these results indicate that mAbs could be used to map functional and structural domains.

8794759

Effect of liposomal composition on photoactivated liposome fusion.

Bennett and O'Brien [(1995) Biochemistry 34, 3102] showed that the ultraviolet light exposure of ponent large unilamellar liposomes posed of a 3:1 molar mixture of dioleoylphosphatidylethanolamine (DOPE) and 1,2-bis[10-(2'-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphatidyl- choline (bis-SorbPC) facilitated liposome fusion. The rate and extent of liposome fusion was dependent on the extent of photopolymerization, the temperature, and the pH. Examination of the temperature dependence of fusion of photolyzed and unphotolyzed liposomes demonstrated that an enhancement of the rate of fusion occurred in the temperature range associated with the initial appearance of precursors to the inverted cubic (QII) phase [Barry et al. (1992) Biochemistry 31, 10114]. Here, the effect of the molar lipid ratio of the DOPE/bis-SorbPC liposomes on the temperature for the onset of fusion, i.e. the critical fusion temperature, was characterized by changing the relative amounts of unreactive polymorphic lipid and reactive lamellar lipid. In each case, photopolymerization of bis-SorbPC lowered the critical fusion temperature by ca. 15-20 degrees C. The photoreaction of the bis-SorbPC-containing LUV yields cross-linked poly-SorbPC, enhancing the lateral separation of the DOPE and the polylipid and causing isothermal induction of liposome fusion by lowering the temperature for the onset of fusion. Evidence is presented to support the hypothesis that the critical temperature for fusion of two LUV populations depends on the molar ratio of the monomeric lipids in heterodimers of the two LUV. This analysis indicates that the photopolymerization of appropriately designed LUV can decrease the critical fusion temperature from above to below 37 degrees C.

8794760

Tritium isotope effects in adenosylcobalamin-dependent methylmalonyl-CoA mutase.

Methylmalonyl-CoA mutase from Propionibacterium shermanii is an adenosylcobalamin-dependent enzyme which catalyzes the reversible isomerization of methylmalonyl-CoA and succinyl-CoA. The rate of tritium loss from 5'-[3H]adenosylcobalamin during the enzymic reaction and the relative rates of tritium appearance in substrate and product were examined. Upon the addition of methylmalonyl-CoA to a solution of holoenzyme, tritium pletely released from the cofactor within about 500 ms. No tritium was found either bound to the enzyme or released into the water. The radioactivity was found in methylmalonyl-CoA and succinyl-CoA in a constant ratio of 1 to 3, which did not change during the first 300 ms of the reaction. Upon the addition of succinyl-CoA to a solution of holoenzyme, tritium was released at essentially the same rate, and the radioactivity was found in methylmalonyl-CoA and succinyl-CoA in the identical constant ratio of 1 to 3. The tritium isotope effect on the enzyme-catalyzed hydrogen transfer, measured using 14C-labeled methylmalonyl-CoA as substrate, was kH/kT = 4.9. This low value shows that hydrogen transfer is only partly rate limiting and that at least one subsequent slow step, such as product release, contributes substantially to the overall reaction velocity. The identical partitioning of tritium, regardless of the substrate used, shows that the rearrangement of the substrate radical into the product radical is not rate limiting. The very low tritium isotope effect and the fact that all the tritium is found bound either to the CoA esters or to the cofactor make it very unlikely that a protein radical is an intermediate in the methylmalonyl-CoA mutase-catalyzed rearrangement.

8794761

Reactivity of alcohols toward the phosphoenzyme intermediate in the protein-tyrosine phosphatase-catalyzed reaction: probing the transition state of the dephosphorylation step.

In solution phosphate monoesters hydrolyze via a highly dissociative mechanism involving a "loose" or "exploded" metaphosphate-like transition state where bond formation to the ing nucleophile is minimal and bond breaking between phosphorus and the leaving group is substantial. To better understand how protein-tyrosine phosphatase (PTPase) effects catalysis, it is important to determine the nature of the enzymic transition state. PTPases catalyze the hydrolysis of phosphate monoesters by a two-step mechanism that proceeds through a phosphoenzyme intermediate (E-P). Extensive heavy atom kinetic isotope effect and leaving group dependency studies have provided insights into the nature of the transition state for the first step (E-P formation) of the PTPase reaction. In this paper we have probed the transition state for the low M(r) PTPase-catalyzed dephosphorylation step by studying the effect of changing the alcohol basicity on its reactivity toward E-P. The Brønsted beta nu value for the reactions of alcohols and E-P is determined to be 0.14, which indicates that the enzymic transition state is highly dissociative and similar to that in uncatalyzed solution reactions. We show that the conserved hydroxyl group in the PTPase signature motif is primarily involved in the E-P dephosphorylation step. We further demonstrate that elimination of the hydroxyl group renders the transition state for E-P dephosphorylation less dissociative, suggesting that the main function of the hydroxyl group in the PTPase active site is to promote the E-P going through a dissociative pathway and to stabilize the dissociative transition state.

8794762

Inactivation of vanadium bromoperoxidase: formation of 2-oxohistidine.

The basis of the irreversible inactivation of the vanadium bromoperoxidase (V-BrPO) isolated from the marine alga Ascophyllum nodosum under turnover conditions at low pH (i.e., 15 to 100 mM H2O2, 0.1 KBr, ca. 15 nM V-BrPO in 0.1 M citrate, pH 4) has been investigated. Inactivation under these conditions was found to produce 2-oxohistidine as identified by HPLC using electrochemical detection. Formation of 2-oxohistidine requires all ponents of turnover (i.e., bromide, hydrogen peroxide, and V-BrPO) as well as low pH; inactivation does not occur nor is significant 2-oxohistidine formed in the presence of hydrogen peroxide alone. The oxidation of histidine did not occur by singlet oxygen generated by V-BrPO, because neither 2-oxohistidine nor inactivation occur under the conditions in which singlet oxygen is produced quantitatively by V-BrPO. The addition of aqueous bromine to N alpha-benzoylhistidine at low pH formed N alpha-benzoyl-2-oxohistidine. cis-Dioxovanadium(V) (VO2+) in strong acid and MoO(O2)2(ox)2- (ox2- is oxalate) at pH 5, both of which are functional mimics of V-BrPO by oxidizing bromide by hydrogen peroxide, catalyzed the oxidation of N alpha-benzoylhistidine to N alpha-benzoyl-2-oxohistidine. Furthermore, when hypobromite was added to N alpha-benzoylhistidine in the presence of hydrogen peroxide at neutral pH, conditions under which HOBr would react first with H2O2 to produce singlet oxygen, no N alpha-benzoyl-2-oxohistidine was formed. Thus the oxidation of histidine in V-BrPO is proposed to occur via oxidized bromine species. Irreversible inactivation V-BrPO was also found to be panied by release of vanadium.

8794763

Chemistry of the lyxose-containing mycobacteriophage receptors of Mycobacterium phlei/Mycobacterium smegmatis.

Mycobacterium phlei (strain Timothy) (Mycobacterium smegmatis ATCC 19249) is characterized by the presence of a family of alkali-labile glycolipids, reminiscent of the trehalose-containing lipooligosaccharide class of antigens but lacking the nonreducing trehalose core. Through bination of methylation analyses, 1H and 13C NMR, two-dimensional 1H/1H and 1H/13C NMR, fast atom bombardment-mass spectrometry, gas chromatography-mass spectrometry, and other analytical techniques, these new structures were shown to possess three distinct features. Firstly, they contained the pentose D-lyxose (Lyx), rarely found in biology, but an epimer of D-arabinose, a ponent of the mycobacterial cell wall arabinogalactan and lipoarabinomannnan. Thus, it was apparent that these glycolipids are the same as those described by Bisso et al. and attributed with phage receptor properties [Bisso, G., Castelnuovo, G., Nardelli, M.-G., Orefici, G., Arancia, G., Lanéelle, G., Asselineau, C., & Asselineau, J. (1976) Biochemie 58, 87-97]. Secondly, plex oligosaccharides within the glycolipids contain the repeating units Lyxn(6-O-CH3-Glc)m and Lyxn(6-O-CH3-Glc)mMan1, where n+m equal to approximately 16 glycosyl residues. Thirdly, the M. phlei glycolipids were found to be heavily O-acylated, such that every D-Lyx residue invariably possesses an acyl function at position -2 and, in some instances, at both positions -2 and -4. The chemical characterization of these glycolipids, not feasible 20 years ago, clearly demonstrates that they are distinct from the type- and species-specific glycopeptidolipids, lipooligosaccharides, phenolic glycolipids, and the genus-specific phosphatidylinositol-based lipoglycans of mycobacteria. This present and previous studies begin to define the precise structural requirements responsible for the attachment of mycobacteriophage to the host cell wall.

8794764

Energetics and kinetics of radical pairs in reaction centers from Rhodobacter sphaeroides. A femtosecond transient absorption study.

Femtosecond transient absorption spectra on reaction centers from Rhodobacter sphaeroides wild type have been recorded with high time and wavelength resolution and a very high S/N ratio in the 500-940 nm range with a diode array system. The data have been analyzed by global analysis. Five ponents of 1.5, 3.1, 10.8, and 148 ps and long-lived (several nanoseconds) were required to fit the entire three-dimensional data surface adequately with a single set of lifetimes and decay-associated difference spectra (DADS). Up to 30 ps, there is little dispersion in the lifetimes, but in the longer time range (50-250 ps), a substantial variation in lifetime was observed, depending on detection wavelength. The data from the global analysis have been subjected to kinetic paring sequential kinetic schemes either including (reversible model) or excluding (forward model) back-reactions in the early electron transfer process(es). Thus, the molecular rate constants for the model(s) and the difference spectra of the pure intermediates [species-associated difference spectra (SADS)] were obtained. The data unequivocally confirm the necessity of an electron transfer intermediate with spectral characteristics of P+B-H prior to the formation of the P+BH- state (P is special pair, B is accessory chlorophyll, and H is pheophytin), irrespective of the model chosen. Besides being in much better agreement with the observation of long-lived fluorescence ponents, the reversible model results in SADS, in particular for the P+BH- state, that are in somewhat better agreement with expectations than for the pure forward model. For these and other reasons, the reversible model is preferred over the pure forward model. The electrochromic shifts of the H bands in the P+B- state and of the B bands in the P+H- state are revealed clearly in the spectra, thus supporting the assignments. Within the reversible model, the rate constant for the forward reaction in the first step P*-->P+B-H is slightly larger [k12 approximately (2.48 ps)-1] than for the second step P+B-H-->P+BH- [k23 approximately equal to (2.53 ps)-1], in contrast to the pure forward model. From the rate constants for the respective back-reactions, the free energy differences delta G relative to P* for the states P+B-H and P+BH- have been determined to be -41 and -91 meV, respectively. Thus, the free energy difference for the P+BH- state at early times after electron transfer is by a factor of 2-3 smaller than assumed so far. This has the important consequence that a quasi-equilibrium exists from about 10 ps until further electron transfer on the 200 ps time scale with a substantial percentage (approximately 16%) of the P+B-H state present. These results present the first direct evidence from transient absorption data, where the nature of the intermediate can be assigned, for the validity of the slow radical pair relaxation concept. The results have various consequences for understanding the mechanism of the overall electron transfer reaction and imply a much more active role of the protein in the early charge separation processes of the reaction center than assumed so far. The data are discussed in terms of current electron transfer theory. It is suggested that the two first-electron steps operate at a rate very close to the maximal possible rate.

8794765

Site-directed mutagenesis of the subunit PsaC establishes a surface-exposed domain interacting with the photosystem I core binding site.

We have postulated that the orientation of PsaC on the photosystem I core involves electrostatic interactions between charged residues on the core binding site and the subunit [Rodday, S. M., Jun, S.-S., & Biggins, J. (1993) Photosynth. Res. 36, 1-9]. We, therefore, changed eight acidic residues on PsaC to arginine and examined the efficiency of the mutant subunits in the reconstitution of P700-Fx cores in vitro. Reconstitution of the cores by the mutant subunits was determined by analysis of the kinetics of bination reactions between P700+ and reduced acceptors as measured optically. Restoration plete forward electron transfer, indicative of efficient subunit binding, was estimated from the ca. 30 ms ponent in the flash transients. Slightly reduced levels of reconstitution were observed for the mutants D24R, E46R/D47R. D61R, and E72R. In contrast, mutants D9R, E27R, and D32R showed significantly lower efficiencies. The presence of the iron-sulfur centers, FA and FB, in these three mutant subunits was confirmed by low-temperature EPR spectroscopy indicating that the polypeptides had folded correctly. We conclude that the introduction of positively charged side chains at positions 9, 27, and 32 seriously disrupts PsaC binding. However, when the wild-type acidic residues in these positions were changed to alanine, only mutant D9A showed a reduced level of reconstitution, suggesting that this aspartate is the most important residue participating in the electrostatic interaction with the core. The results are discussed in relation to the photosystem I crystal structure and support an orientation of PsaC on the core such that center FB is proximal to Fx.

8794766

Binding of intermediate, product, and substrate analogs to neuronal nitric oxide synthase: ferriheme is sensitive to ligand-specific effects in the L-arginine binding site.

The electron paramagnetic resonance spectra of purified neuronal nitric oxide synthase indicates that the binding of ligands to the arginine site perturbs the environment of the high-spin ferriheme in a highly ligand-specific manner. Four categories of plex can be distinguished; all are five-coordinate, and all retain the axial thiolate ligand, but they differ in their ligation geometries. These spectroscopic species reveal distinct local conformations which can be stabilized individually by the binding of L-arginine, N omega-hydroxy-L-arginine, N omega-methyl-L-arginine, and N omega-nitro-L-arginine. Other arginine analog inhibitors stabilize one or more of these states, revealing patterns based on the nature of substituents at the terminal amino group.

8794767

The activation volume of a DNA helix-coil transition.

The role of hydration in the kinetics of a DNA helix-coil equilibrium is investigated by studying the effect of hydrostatic pressure on the rate constants describing the reaction. The kinetics were measured using the thermal hysteresis between the denaturation and renaturation curves of the triplex-forming oligonucleotides: 5'd[AAA-GGAGGAGAAGAAGAAAAAA] (sequence of purine strand) and 5'd[TTTCCTCCTCTTCTTCTTTTTT] (third strand). The kinetics at atmosphere pressure for this system have been recently reported [Rougée et al. (1992) Biochemistry 31, 9269-9278]. At all pressures the data are consistent with a single-step bimolecular reaction under the conditions of our experiments (100 mM NaCl, 10 mM cacodylate, pH 6.5). The rate of formation of the triplex from the duplex + single strand is accelerated by pressure. At the midpoint of the helix-coil transition (32.5 degrees C), the activation volume for helix formation, V*1, equals -11.8 (+/- 2.4) cm3 mol-1 at atmospheric pressure. At the same temperature, the activation volume for helix dissociation, V*-1, equals +39.9 (+/- 5.0) cm3 mol-1; that is, the rate of strand separation is slowed by pressure. These findings emphasize the importance of solvent interactions in the stabilization and formation of DNA helices. It is proposed that the activation volume of the forward reaction may arise from the volume change due to charging the cytosine residues and the formation of base-stacking interactions in the third strand. The positive activation volume of strand separation may be a consequence of poor solvent packing of the DNA duplex major groove during dissociation of the third strand.

8794768

Nuclear magnetic resonance solution structure of the growth factor receptor-bound protein 2 Src homology 2 domain.

A family of NMR solution structures of the growth factor receptor-bound protein 2 (Grb2) SH2 domain has been determined by heteronuclear multidimensional NMR. Proton, nitrogen, and carbon chemical shift assignments have been made for the SH2 domain of Grb2. Assignments were made from bination of homonuclear two-dimensional and 15N- and 13C-edited three-dimensional spectra at pH 6.2 and 298 K. Structure-induced proton and carbon secondary shifts were calculated and used to facilitate the spectral assignment process. NOE, scalar coupling, secondary chemical shift, and amide proton exchange data were used to characterize the secondary structural elements and hydrogen-bonding network in the Grb2 SH2 domain. The three-dimensional structure of the Grb2 SH2 domain was calculated using 1112 restraints obtained from NOE, coupling constant, and amide proton exchange data. The rmsd for the 24 calculated structures to the mean structure of the Grb2 SH2 domain was 0.75 A for backbone and 1.28 A for all heavy atoms. The three-dimensional fold of the Grb2 SH2 domain is similar to that observed for other SH2 domains and consists of two alpha-helical segments and eight beta-strands, six strands that make up two contiguous antiparallel beta-sheets, and two strands that form two short parallel beta-sheets. The structure of the phosphotyrosine binding pocket of Grb2 is similar to that observed for other SH2 domains. The hydrophobic binding pocket of Grb2 is similar to that observed for Src with the exception that tryptophan 121 of Grb2 occupies part of the pY+3 binding pocket. Structural implications for the Grb2 SH2 domain selectivity at the pY+2 and pY+3 sites are discussed.

8794769

Site-directed fluorescence labeling of P-glycoprotein on cysteine residues in the nucleotide binding domains.

P-Glycoprotein is a member of the ABC superfamily of membrane transporters, and functions as an ATP-driven active efflux pump for natural products and chemotherapeutic drugs. Overexpression of P-glycoprotein is a major cause of multidrug resistance in human cancers. Sulfhydryl modification agents are known to inactivate both P-glycoprotein ATPase activity and transport function. In the present study, P-glycoprotein purified from CHRB30 cells was covalently labeled at two conserved Cys residues, one within each of the nucleotide binding domains, using 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS). MIANS modification inactivated P-glycoprotein ATPase function, in a concentration-dependent fashion. Increasing concentrations of ATP blocked MIANS labeling with an IC50 of 0.37 mM (similar to the KM for ATP hydrolysis), which suggests that the label is located close to the site of ATP binding within the nucleotide binding domain. A blue shift in the fluorescence spectrum of MIANS bound to P-glycoprotein indicated that the labeled Cys residues are situated in a nonpolar environment. MIANS-labeled P-glycoprotein was still able to bind ATP, as demonstrated by quenching of the fluorescence, with a Kd of 0.46 mM. Addition of a variety of drugs and chemosensitizers to MIANS-labeled P-glycoprotein led to substantial quenching of the probe fluorescence within the nucleotide binding domains. Dissociation constants for drug binding measured by fluorescence quenching were in the range of 0.77 microM for vinblastine to 158 microM for colchicine. Quenching by ATP and drugs was independent and additive, suggesting that each produces a defined change in the protein. The rate of MIANS labeling of Pgp was reduced in the presence of drugs and chemosensitizers, implying that a long-range conformational change arises from drug binding which alters the accessibility of the nucleotide binding domains to MIANS. These results suggest that there is munication between the drug binding site(s) of P-glycoprotein and the ATPase catalytic sites within the nucleotide binding domains.

8794770

Purification of bovine thymus cytosolic C-terminal Src kinase (CSK) and demonstration of differential efficiencies of phosphorylation and inactivation of p56lyn and pp60c-src by CSK.

The C-terminal src kinase (CSK) is a ubiquitously expressed, cytosolic enzyme capable of phosphorylating and inactivating several plasma membrane-bound src-family protein tyrosine kinases in vitro [Nada, S., Okada, M., MacAuley, A., Cooper, J.A., & Nakagawa, H. (1990) Nature 351, 69-72; Bergman, M., Mustelin, T., Oetken, C., Partanen, J., Flint, N.A., Amrein, K.E., Autero, M., Burn, P., & Alitalo, K. (1992) EMBO J. 11, 2919-2924]. We purified CSK to apparent homogeneity from bovine thymus cytosol to study in vitro how the purified enzyme recognizes the various src-family kinases as its substrates. A novel assay method was developed for assaying the ability of CSK to inactivate src-family tyrosine kinases. With this assay method, we demonstrated that CSK inactivated p56lyn with a significantly higher efficiency than pp60c-src. Phosphopeptide mapping of CSK-phosphorylated p56lyn and pp60c-src shows that the consensus tyrosine residue (also termed tail tyrosine) in the C-terminal regulatory domain of p56lyn was phosphorylated by CSK with an efficiency much higher than that of pp60c-src. Thus, the higher efficiency of inactivation of p56lyn by CSK is a result of the ability of p56lyn to serve as a better substrate of CSK. The synthetic peptides derived from the C-terminal portion of p56lyn and pp60c-src were much poorer substrates than the intact src-family kinases for CSK, indicating that the local structure around the tail tyrosine is not sufficient to direct efficient phosphorylation of p56lyn by CSK. Nevertheless, the slightly higher efficiency displayed by CSK in phosphorylating the peptide derived from the C-terminal portion of p56lyn than that from pp60c-src suggests that the structural differences between the C-terminal portions of p56lyn and pp60c-src contribute to the differential efficiencies displayed by CSK in phosphorylating the two kinases. Determination of the CSK-phosphorylation site in the src-C-terminal peptide by phosphopeptide mapping reveals that the whole C-terminal regulatory domain and an adjacent part of the protein kinase domain contain some of the structural determinants directing CSK to phosphorylate the consensus tail tyrosine of the src-family kinases.

8794771

Interactions of monomeric rabbit neutrophil defensins with bilayers: comparison with dimeric human defensin HNP-2.

Human antimicrobial neutrophil defensin HNP-2 has been shown to form large multimeric pores in pure 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG) bilayers that lead to all-or-none release of vesicle contents [Wimley et al. (1994) Protein Sci.3, 1362-1373]. Because human neutrophil defensins form natural dimers in solution, the question arises as to the role of dimerization in pore formation. However, the dimers are so stable that this question is not easily answered directly. Rabbit neutrophil defensins, whose three-dimensional structures are very similar to those of human defensins, are monomeric in aqueous solution and thus provide an opportunity to test the hypothesis that dimerization may play a role in multimeric pore formation. We therefore examined the interactions of the six known rabbit neutrophil defensins with large unilamellar vesicles (LUV) under the conditions known to lead to stable pore formation by HNP-2. We find that the rabbit defensins bind strongly to LUVs formed from pure POPG or mixtures of POPG with neutral (zwitterionic) phospholipid but induce leakage of vesicle contents only from pure POPG vesicles. Rabbit defensin NP-4 does not cause leakage under any conditions examined. The remaining defensins, NP-1, NP-2, NP-3A, NP-3B, and NP-5, cause graded release of the contents of pure POPG vesicles as does a mixture of the six defensins. The graded release indicates that the rabbit defensins do not form stable pores in the membrane. This result thus suggests that the structural features of human defensins that permit dimer formation in aqueous solution are likely to be important in the formation of multimeric pores.

8794773

Electrostatic modification of the active site of myoglobin: characterization of the proximal Ser92Asp variant.

The structural and functional consequences of the introduction of a negatively charged amino acid into the active site of horse heart myoglobin have been investigated by replacement of the proximal Ser92 residue (F7) with an aspartyl residue (Ser92Asp). UV-visible absorption maxima of various ferrous and ferric derivatives and low-temperature EPR spectra of the metaquo (metMb) derivative indicate that the active site coordination geometry has not been perturbed significantly in the variant. 1H-NMR spectroscopy provides direct evidence for the existence of a distal water molecule as the sixth ligand in the oxidized form of the variant at pD 5.7. Spectrophotometric pH titration of the Ser92Asp variant is consistent with this finding and with a pKa = 8.90 +/- 0.02 [25.0 degrees C, mu = 0.10 M (NaCl)] for titration of the distal water molecule, identical to the value reported for the wild-type protein. X-ray crystallography of the metMb derivative indicates that the heme substituents conserve their orientations in the variant protein, except for a slight reorientation of the pyrrole A propionate group to which Ser92 normally hydrogen bonds and reorientation of the carboxyl end of the pyrrole D propionate group. No change is observed in conformation of the proximal (His93) or distal (Wat156) heme ligands. 1H-NMR spectroscopy of the metMbCN form of the protein indicates that a slight rotation of the proximal His93 ligand has occurred in this derivative. Resonance Raman experiments indicate increased conformational heterogeneity in the proximal pocket of the variant. Failure to detect electron density for the Asp residue in the X-ray diffraction map of the variant protein and high average thermal factors for the pyrrole A propionate substituent are consistent with this observation. The variant exhibits novel pH-dependent behavior in the metMb form, as shown by 1H-NMR spectroscopy, and provides evidence for a heme-linked titratable group with a pKa of 5.4 in this derivative. The metMbCN and deoxyMb derivatives also exhibit pH-dependent behavior, with pKas of 5.60 +/- 0.07 and 6.60 +/- 0.07, pared to the wild-type values of 5.4 +/- 0.04 and 5.8 +/- 0.1. The heme-linked ionizable group is proposed to be His97 in all three derivatives. The reduction potential of the variant is 72 +/- 2 mV vs SHE [25.0 degrees C, mu = 0.10 M (phosphate), pH 6.0], an increase of 8 mV over the wild-type value. The possible influence of a number of variables on the magnitude of the reduction potential in myoglobin and other heme proteins is discussed.

8794775

Allosteric effects of carbamoyl phosphate synthetase from Escherichia coli are entropy-driven.

When catalyzing the formation of MgATP and carbamate from MgADP and carbamoyl phosphate, Escherichia coli carbamoyl phosphate synthetase (CPS) binds MgADP with a large negative change in heat capacity. The magnitude of this heat capacity change is not appreciably altered by the presence of a saturating concentration of either the allosteric activator ornithine or the inhibitor UMP despite the substantial and opposing effects these ligands have on the binding affinity for MgADP. By contrast, no detectable change in heat capacity is associated with the thermodynamic coupling between MgADP and either ornithine or UMP. The sign of the apparently constant enthalpic and entropic contributions to the coupling free energy for each of these ligands is opposite that of the coupling free energy, indicating that the observed allosteric phenomenology is in net opposed by the enthalpy of the interaction and instead arises from a change in entropy of the system. IMP produces only a very small allosteric effect as indicated by a near-zero value for the MgADP-IMP coupling free energy. However, the enthalpic and entropic contributions are individually larger in absolute value for the IMP coupling than for those pertaining to the other allosteric ligands, and entropy dominates the coupling free energy above 36 degrees C, causing IMP to e an activator at high temperature. In addition, the sign of the coupling enthalpy and entropy for IMP has the same sign as the coupling enthalpy and entropy produced by ornithine, suggesting that IMP and ornithine may similarly influence the enzyme at a molecular level despite binding to different allosteric sites on the enzyme. The data are consistent with a model in which the actions of the allosteric ligands arise primarily from changes in the conformational degeneracy introduced by each ligand. With this model, one can also rationalize the failure of these allosteric ligands to substantially influence kcat.

8794776

Protein stability: urea-induced versus guanidine-induced unfolding of metmyoglobin.

We have studied the denaturation of metmyoglobin at pH 6.0 and 25 degrees C by urea and guanidine hydrochloride (GdnHCl) which are known to unfold the protein to the same extent. It has been observed that estimates of protein stability (delta G0N-U) from urea-induced and GdnHCl-induced denaturations do not agree with one another; the linear extrapolation method gave delta G0N-U values of 7.59 +/- 0.33 and 5.35 +/- 0.10 kcal mol-1 for urea and GdnHCl denaturations, respectively. Measurements of the effect of the addition of KCl in the concentration range 0.1-1.0 M to urea denaturation have suggested that this disagreement is not due to the nonionic and ionic characters of urea and GdnHCl, respectively. The functional dependence of the free energy change of unfolding (delta GN-U) on [denaturant], the molar concentration of the denaturant, has been investigated for understanding the cause(s) of the disagreement between the two estimates of delta G0N-U of metmyoglobin. For this purpose, we have studied the GdnHCl-induced denaturation of the protein in the presence of different urea concentrations at pH 6.0 and 25 degrees C and vice versa. These measurements yield delta GN-U values in the full concentration range [Ahmad et al. (1994) J. Biochem. 115, 322-327], and these results provide strong evidence that the delta GN-U dependence on [urea] is linear (linear free energy model of denaturation) and the relation between delta GN-U and [GdnHCl] is curved (binding model of denaturation). It has been observed that the extrapolated value of delta GN-U in urea using the linear free energy model es identical to the extrapolated value of delta GN-U in GdnHCl using the binding model.

8794777

Social support, social networks and coronary artery disease rehabilitation: a review.

To provide information to the munity about the importance of social support and networks through examination and definition of social support and network variables, and by reviewing the evidence found in the literature showing a relationship among social support, social networks, coronary artery disease (CAD) and rehabilitation.

8794774

Stabilization of granulocyte colony-stimulating factor and structurally analogous growth factors by anionic phospholipids.

binant granulocyte colony-stimulating factor (rhG-CSF) interacts with posed of the anionic phospholipid dioleoylphosphatidylglycerol (DOPG), and this interaction enhances the stability of the protein [Collins, D., & Cha, Y. (1994) Biochemistry 33, 4521-4526]. In the present studies, we have examined the interaction of rhG-CSF with phospholipids other than DOPG. Fluorescence spectroscopy of rhG-CSF with a variety of lipid vesicles demonstrated that rhG-CSF inserts into bilayers of anionic, but not zwitterionic, phospholipids. Isothermal titration calorimetry of the interaction between DMPG and rhG-CSF indicates that the binding is saturable and involves 10 lipids/rhG-CSF. Studies of phosphatidylglycerols with varying alkyl chain lengths determined that the stabilization of rhG-CSF by anionic phospholipids required a certain alkyl chain length; no stabilization was observed with lipids of shorter chain length. Also investigated was the stabilization of other growth factors, which are structurally similar to rhG-CSF, by anionic phospholipids. These proteins include binant porcine somatotropin (rpSt), binant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), binant human interleukin 4 (rhIL-4), and binant human interleukin 2 (rhIL-2). The helical secondary structure of the proteins was recoverable after heating and cooling in the presence of anionic phospholipids as observed by circular dichroism; the presence of zwitterionic lipids did not induce this effect. Results of these investigations concluded that a group of structurally similar proteins interact preferentially with anionic phospholipids and that plexation of the growth factors with posed of anionic phospholipids improves the stability of the proteins under conditions where they normally denature.

8794778

Evaluation of body surface potential mapping changes after successful percutaneous transluminal coronary angioplasty.

To assess the progress of chronic myocardial ischemia after successful percutaneous transluminal coronary angioplasty (PTCA) using body surface potential mapping (BSPM).

8794779

Systemic lupus erythematosus and pulmonary hypertension during pregnancy: report of a case fatality.

The case of a 25-year-old pregnant woman with systemic lupus erythematosus and severe pulmonary hypertension is presented. The pregnancy plicated by worsening right heart failure and pre-eclampsia, requiring a caesarian section at 29 weeks' gestation. On the fourth day postpartum, the patient's respiratory status worsened and she was transferred to the coronary care unit where she soon died bined right heart failure and respiratory arrest. The presumed pathogenesis and etiology of lupus-related pulmonary hypertension are discussed, in addition to noninvasive tests and proposed management. Given that the mortality rate is very high during pregnancy and therapy is of limited value, women with lupus-associated pulmonary hypertension should avoid conceiving. Those who choose to e pregnant must be carefully managed by a multidisciplinary team.

8794780

Intravascular ultrasound for diagnosis of left main coronary artery stenosis.

Coronary angiography has many limitations for the assessment of coronary artery disease. Intracoronary ultrasound imaging may e some of these limitations by providing direct visualization of the luminal area. This report describes a case where intracoronary ultrasound imaging was useful for correct assessment of left main coronary artery disease which enabled avoidance of coronary artery bypass grafting in this patient. Intravascular ultrasound may be a plement to coronary angiography in selected cases of left main coronary artery lesion.

8794781

Gastric emptying of oil from solid and liquid meals. Effect of human pancreatic insufficiency.

Digestion of fat in pancreatic insufficiency (PI) is strongly affected by how rapidly fat enters the duodenum. We postulated that: (1) oil empties faster in PI than in normals and (2) in both, it empties in a load-dependent fashion. We used a gamma camera to test these ideas paring gastric emptying of iodine-123 iodinated oil in normal and pancreatic-insufficient subjects after 15 g of free oil were ingested in a small spaghetti meal and 60 g of oil were ingested in a large spaghetti meal and in a milk emulsion. Indium-113m marked gastric emptying of water in the milk. In both groups after all meals, oil emptied fastest initially, slowing later; and oil emptied three to four times faster when 60 g vs 15 g were ingested. There were no significant differences between the groups of subjects with respect to gastric emptying of the spaghetti meals, but the pancreatic-insufficient subjects emptied both oil and water faster from the milk emulsion than did the normal subjects. The slower emptying of oil in the normal subjects was associated with significantly more layering of oil to the top of the intragastric milk emulsion.

8794782

Electrogastroenterographic examination of 22 patients before and after cholecystectomy.

The object of this study was to define the pattern of gastrointestinal myoelectrical activity before and after cholecystectomy. After surgery, on the first postoperative day, the mean and maximal activities of the gastrointestinal tracts decreased significantly, but there was no significant change in the pattern and the duration of the nonreactive period. A dyskinetic effect and/or weakness of electrical activity was observed in all patients before operation, and the same pattern persisted after operation for one month. This suggests the future onset of the so-called postcholecystectomy syndrome, which may result from the fundamental pathological effect of gallstones.

8794784

Technique for orthotopic reduced-size hepatic transplantation combined with ex vivo liver cut down in the rat.

A technique is described for orthotopic reduced-size hepatic bined with ex vivo liver cut down in the rat. Following perfusion of the donor liver with cold heparinized saline, the portal veins, bile ducts, and hepatic arteries to the median and left lobes together were dissected in situ, encircled, and divided. After harvesting the donor liver, a hepatectomy was performed by ex vivo liver cut down of the median and left lobes. The remnant amounted to 32% of the whole liver. As a result, the suprahepatic vena cava could be well visualized with adequate exposure for vascular anastomosis. Orthotopic reduced-size hepatic transplantation was performed using the right and caudate lobes of the liver. The suprahepatic vena cava was anastomosed with a 7-0 silk running suture. A simplified cuff without processes was made with an obliquely cut polyethylene tube and used for the portal and infrahepatic caval anastomoses. A Teflon tube stent was used for the biliary anastomosis. The newly devised angled clamp and flexible arm were used for the cuff attachment and operative procedure. Transplant survival following ex vivo liver cut down was as good as that with whole liver transplantation. Reestablishment of the hepatic artery restores liver function following transplantation. The maximum hepatocyte labeling index (LI) occurs 24 hr after a 68% hepatectomy, and at 36 hr following a reduced-size hepatic transplantation with or without hepatic arterialization. Possible explanations for the slight delay in achieving the maximal LI may include damage that is induced by the operation itself, pregraft preservation, and reperfusion injuries. In conclusion, the anatomical features of the hepatic lobes in rats are well suited to pletion of ex vivo liver cut down.

8794783

Role of sham feeding in postprandial changes of gastric myoelectrical activity.

The aim of this study was to evaluate the role of sham feeding in postprandial changes of gastric myoelectrical activity. Eighteen asymptomatic healthy volunteers (10 men, 8 women; mean age: 31), with no history of gastrointestinal disease were studied. Gastric myoelectrical activity was recorded for 30 min at baseline, 30 min after sham feeding, and 1 hr after eating, using surface electrogastrography. The electrogastrogram (EGG) was analyzed by spectral analysis. It was found that the changes of postprandial EGG parameters were significantly correlated with those after sham feeding (EGG dominant power: r = 0.6, P < 0.01; dominant frequency: r = 0.8, P < 0.001; percentage of regular slow waves: r = 0.7, P < 0.003). We concluded that intrinsic gastric electrical activity can be altered by sham feeding and the cephalic phase of digestion plays an important role in the postprandial response of gastric myoelectrical activity.

8794785

Hemodynamic and metabolic effects of terlipressin in patients with cirrhosis receiving a nonselective beta-blocker.

Terlipressin (Glypressin), a vasopressin analog, may be administered to patients with cirrhosis receiving a beta-adrenergic antagonist. Since terlipressin alone and beta-blockers alone both decrease portal pressure, bination of these substances may have additional portal hypotensive effects. However, the negative side effects of terlipressin may be accentuated by long-term beta-blockade. Thus, the present study examined hemodynamic and metabolic responses to terlipressin in 12 patients receiving nonselective beta-blockers (propranolol or nadolol). Hemodynamics and oxygen (O2) -derived variables were measured prior to and 30 min after the administration (intravenous bolus) of terlipressin (1 to 2 mg, according to body weight). The hepatic venous pressure gradient and azygos blood flow significantly decreased (from 15.3 +/- 1.1 to 12.5 +/- 1.1 mm Hg, and from 0.6 +/- 0.1 to 0.5 +/- 0.1 liters/min, respectively). Arterial and pulmonary wedged pressures significantly increased. Heart rate, cardiac index, and O2 consumption were not significantly affected by terlipressin. In conclusion, in patients with cirrhosis being treated with a nonselective beta-blocker, terlipressin administration decreased portal pressure. Moreover, terlipressin induced only mild systemic hemodynamic effects in these patients. These results suggest that terlipressin can be administered in patients receiving a beta-adrenergic blocker.

8794786

Gastric mucus generation in cirrhotic patients with portal hypertension. Effects of tetraprenylacetone.

We have evaluated gastric mucus generation (study 1) and the effects of tetraprenylacetone on gastric mucus generation (study 2) in cirrhotic patients with portal hypertension. Study 1: Included were 50 noncirrhotics (group A), 25 cirrhotics without portal hypertension (group B), and 25 cirrhotics with portal hypertension (group C). The antrum, corpus, and fundus mucus generation was assessed by hexosamine concentration using biopsy specimens. In groups A and B, the antrum hexosamine concentration was significantly pared with the corpus (P < 0.01, P < 0.01) and the fundus (P < 0.01). In contrast, the hexosamine concentration at each location was similar in group C. Furthermore, the antrum hexosamine concentration of group C was significantly pared with that of group A (P < 0.05). In study 2, a double-blind design, 300 mg of tetraprenylacetone was administered for four weeks in 10 cirrhotics with portal hypertension and placebo in 10. The regional hexosamine concentrations were measured before and after drug administration. Placebo administration did not change hexosamine concentration at each location. In contrast, tetraprenylacetone increased the antrum and corpus hexosamine concentration (P < 0.01, P < 0.05), although the fundus concentration did not change. These data suggest that cirrhotics with portal hypertension have reduced gastric antral mucus generation and tetraprenylacetone normalizes this.

8794788

Localization of transforming growth factor-beta 1 precursor and latent TGF-beta 1 binding protein in colorectal adenomas.

Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional cytokine and is thought to be involved in colorectal tumorigenesis as a regulator of cell growth and differentiation. This role is mainly supported by in vitro studies while its role in vivo remains unclear. The aim of the present study was to investigate whether the TGF-beta 1 precursor (beta 1-LAP) and the latent TGF-beta 1 binding protein (LTBP) are expressed in colorectal adenomas, the presumed precursors of most of colorectal adenocarcinomas. TGF-beta 1 precursor and LTBP were examined in 35 adenomas and 10 normal colonic mucosa specimens by immunohistochemistry, using specific polyclonal antibodies. In normal colonic mucosa, beta 1-LAP was moderately expressed in epithelial crypt cells and in the stromal cells in the lamina propria. In adenomas, beta 1-LAP was localized in epithelial cells with an heterogeneous pattern and was also present in stromal cells around the adenomatous glands. LTBP was not detected in epithelial cells but was observed in stromal cells and in the extracellular matrix (ECM). beta 1-LAP expression in epithelial cells did not correlate with the grade of dysplasia, while LTBP localized in stromal cells and ECM appeared to be closely associated with areas of higher grade of dysplasia. This study is the first demonstration of both beta 1-LAP and LTBP in colorectal adenomas with different dysplasia grades. Our results suggest that TGF-beta 1 might be involved in the mechanisms controlling in vivo colorectal tumorigenesis and support a role for the stromal-associated TGF-beta 1.

8794789

Utility of clinical symptoms versus laboratory tests for evaluation of acute gastroenteritis.

Our purpose was to find the utility of laboratory tests in emergency ward evaluation of patients with gastroenteritis. Medical records of 163 adult cases were retrospectively reviewed. Blood laboratory tests were drawn in 116 cases, 78 had at least one abnormality. Urine tested ketone-positive in 15 of 116 cases. One hundred fifteen were treated with intravenous fluids, 20 with antibiotics, and 4 were admitted. Fifty-eight stool cultures were sent, and 13 yielded enteric pathogens. Cultures from patients with fever or symptoms of long duration had higher yields (57% vs 11.3% and 38.1% vs 0%, P < 0.001, respectively), and bined had sensitivity of 100% and specificity of 65%. There was no association between abnormal blood laboratory results, intravenous hydration, and antibiotic treatment with the stool culture being positive or with the patient being hospitalized. Laboratory tests are used often, but are very seldom contributory for evaluating domestically acquired gastroenteritis.

8794787

Frequency and significance of antibodies to asialoglycoprotein receptor in type 1 autoimmune hepatitis.

Antibodies to asialoglycoprotein receptor have diagnostic specificity for autoimmune hepatitis, but it is uncertain if they plementary or redundant markers of the disease. Our aims were to assess their frequency and significance in type 1 autoimmune hepatitis and determine their contribution to the evaluation of these patients. Sera from 54 well-characterized patients were evaluated for antibodies to asialoglycoprotein receptor by a radioimmunofiltration assay based on rabbit-derived protein. Forty-four patients (82%) were seropositive. Seropositive patients were distinguished from seronegative counterparts by having higher serum gamma globulin (3.7 +/- 0.2 g/dl vs 2.3 +/- 0.3 g/dl, P = 0.0007) and immunoglobulin G levels (3707 +/- 179 mg/dl vs 2203 +/- 263 mg/dl, P = 0.0005) at presentation and a greater frequency of relapse after drug withdrawal (88% vs 33%, P = 0.01). Seropositivity for smooth muscle and/or antinuclear antibodies did not define treatment es and antinuclear antibodies occurred less frequently than the other markers. Concurrent testing for antibodies to asialoglycoprotein receptor and smooth muscle identified all patients. We conclude that antibodies to asialoglycoprotein receptor mon in type 1 autoimmune hepatitis and they identify patients with a high frequency of relapse after corticosteroid withdrawal. Concurrent testing for these antibodies and smooth muscle antibodies has the same diagnostic sensitivity as testing for antinuclear and smooth muscle antibodies but a greater prognostic implication.

8794790

Increased energy expenditure in growing adolescents with Crohn's disease.

Undernutrition is considered to have a central role in the pathogenesis of growth retardation in Crohn's disease. This may occur as a consequence of inadequate food intake, increased energy expenditure, or both. Ten growing adolescents with inactive Crohn's disease were assessed with respect to anthropometric parameters and resting energy expenditure, measured by indirect calorimetry during remission, repeated in relapse (N = 5), pared to that predicted from the Harris-Benedict formula. Mean energy intake was assessed with seven-day diaries in five patients pared to mended intake for age, sex, weight, and physical activity. Ten healthy, growing, age- and sex-matched adolescents served as controls. Nine patients with inactive Crohn's disease, who had ceased growing, were matched for disease site and duration and acted as disease controls. Patients and disease controls had lower body mass index (19.2 +/- 0.6; 20.9 +/- 0.7) than healthy controls (23.7 +/- 0.6; P < 0.001). Percent body fat was lower in patients (13.2 +/- pared to healthy controls (20.5 +/- 2.4%; P < 0.05) but not to disease controls (17.0 +/- 2.6%). Patients had higher resting energy expenditure per kilogram of fat-free mass than disease or healthy controls (36.9 +/- 5.1; 32.9 +/- 2.6; 30.9 +/- 2.1 kcal; P < 0.02). Measured resting energy expenditure in patients, but not in disease or healthy controls, was higher than the predicted (measured: predicted 1.15, 1.03, 0.9, respectively; P < 0.03). Energy intake in patients was 97% of mended intake but the measured ratio of energy intake/resting energy expenditure was lower than the predicted ratio (1.49 vs 1.71; P < 0.05). During subsequent relapse in five patients resting energy expenditure was unchanged. In growing adolescents with inactive Crohn's disease, there is increased energy expenditure that is not panied by an increase in energy intake. Relapse of disease does not appear to increase resting energy expenditure further but may "divert" energy from growth to disease activity. This suggests that nutritional therapy should be directed towards increasing caloric intake to maximize growth potential.

8794791

Smooth muscle adaptation after intestinal transection and resection.

Changes in motor function occur in the intestinal remnant after intestinal resection. Smooth muscle adaptation also occurs, particularly after extensive resection. The time course of these changes and their interrelationship are unclear. Our aim was to evaluate changes in canine smooth muscle structure and function during intestinal adaptation after transection and resection. Twenty-five dogs underwent either transection (N = 10), 50% distal resection (N = 10), or 50% proximal resection (N = 5). Thickness and length of the circular (CM) and longitudinal (LM) muscle layers were measured four and 12 weeks after resection. In vitro length-tension properties and response to a cholinergic agonist were studied in mid-jejunum and mid-ileum. Transection alone caused increased CM length in the jejunum proximal to the transection but did not affect LM length or muscle thickness. A 50% resection resulted in increased length of CM throughout the intestine and thickening of CM and LM near the anastomosis. Active tension of jejunal CM increased transiently four weeks after resection. Active tension in jejunal LM was decreased 12 weeks after transection and resection. Sensitivity of CM to carbachol was similar after transection and resection. It is concluded that: (1) Structural adaptation of both circular and longitudinal muscle occurs after intestinal resection. (2) This process is influenced by the site of the intestinal remnant. (3) Only minor and transient changes occur in smooth muscle function after resection. (4) Factors other than muscle adaptation are likely involved in the changes in motor function seen following massive bowel resection.

8794792

Characterization of mucin in whole-gut lavage fluid obtained from patients with inflammatory bowel disease.

Whole-gut lavage fluid, collected by administering an electrolyte lavage solution orally, was found to be an excellent and easily collectable source of abundant mucin. Furthermore, the biochemical features of the mucin from patients with ulcerative colitis and Crohn's disease were investigated. The mucin was separated into four fractions by Sepharose CL-4B, Sepharose CL-2B, and DEAE Sephacel chromatography. Compared with healthy subjects, the total yields of mucin from ulcerative colitis patients were low due to a deficiency of neutral mucin, whereas those from Crohn's disease patients were high, which was attributable mainly to high-molecular-weight mucin. The fucose and sulfate contents were low in ulcerative colitis, but only the former was low in Crohn's disease. The different biochemical features of the mucin obtained from whole gut lavage fluid appear to reflect mucosal pathological changes associated with inflammatory bowel disease.

8794793

A simple filter-paper technique allows detection of mucosal cytokine levels in vivo in ulcerative colitis. Interleukin-1 and interleukin-1-receptor antagonist.

A simple filter-paper method for in vivo assessment of cytokines in intestinal mucosa of patients with ulcerative colitis (UC) was developed and evaluated. Twenty-eight patients were included. Twenty-three patients had ulcerative colitis (UC) and five irritable bowel syndrome (IBS). Inflammation was assessed endoscopically. Through a rectoscope, filter paper was applied to the macroscopically most inflamed area of the rectal mucosa until soaked. The filter paper was transferred to a buffer solution, and IL-1 beta and IL-1ra were assessed using ELISA. Positive correlations between endoscopic grading and In(IL-1 beta) (P < 0.0001) and In(IL-1ra) (P < 0.001) and a negative correlation between endoscopic grading and In(IL-1ra /IL-1 beta) (P < 0.02) were found. In measurements during and after a flare-up no significant change in IL-1ra but a significant decrease in IL-1 beta was detected, which is in agreement with investigations on biopsies. In conclusion, the filter-paper technique is an easily applicable, low-risk method that provides a means of monitoring cytokines in vivo in UC.

8794795

Involvement of interleukin-4 and -10 in inflammatory bowel disease.

The pathogenesis of ulcerative colitis (UC) and Crohn's disease (CD) may be associated with a decreased production of cytokines suppressing macrophage and T-cell functions: interleukins (IL) -4 and IL-10. Serum concentrations of IL-4 and IL-10 were measured using an ELISA technique, and intestinal IL-4 and IL-10 mRNA was detected by a reverse transcriptase polymerase chain reaction (RT-PCR) in 34 patients with inflammatory bowel disease (IBD) (20 with UC and 14 with CD) pared to 12 control subjects. The superoxide production was measured spectrophotometrically in activated PMNs initially incubated in the presence of IL-4 or IL-10. No differences were found in numbers of cells that might be potential IL-4 or IL-10 producers (T cells, macrophages, B cells, and mast cells) in biopsy specimens using immuno- and histochemistry. IL-4 mRNA was detectable in specimens from 77.8% of the UC patients (P > 0.05) and 0% of the CD patients (P < 0.05), pared to 81.8 in controls, and was significantly different (P < 0.0001) between UC and CD patients. The IL-10 amplification product was detectable in specimens from 30.0% UC patients (P < 0.003), but not in CD patients (78.6%, P > 0.05) pared to controls (91.7%). The circulating protein levels of IL-4 were below the detection limit in all groups (detection limit 4 pg/ml), while the median IL-10 concentration was 12.5 pg/ml in UC, 18.1 pg/ml in CD, and 19.5 pg/ml among controls (detection limit 3 pg/ml), which did not differ in any of the three groups (P > 0.05). Finally, the superoxide production was inhibited and delayed by the addition of IL-10 (P < 0.01), whereas IL-4 only delayed this parameter. In conclusion, apart from the well-known suppressive effect on proinflammatory cytokine production, IL-4 delays and IL-10 inhibits superoxide generation. IL-4 mRNA expression is decreased in intestinal tissue from CD patients, while IL-10 mRNA expression is decreased in majority of UC patients, suggesting different immunopathogenesis of the two diseases.

8794796

Diarrhea and quality of life in ambulatory HIV-infected patients.

Our objectives were to determine HIV-infected patients' awareness and recognition of diarrheal symptoms; and to assess the impact of diarrhea on quality of life. The design was a cross-sectional study utilizing a structured telephone interview. The setting was the HIV/AIDS outpatient clinic of a tertiary referral hospital. HIV-infected patients who attended the clinic in 1994 were interviewed. The main e measure was the quality-of-life score (QLS). Fifty percent of patients acknowledged having diarrhea in the previous month. All four categories of diarrhea (self-reported or elicited, within the preceding week or month) were significantly associated with decreased QLS. Patients with diarrhea who did not recognize their symptoms as diarrhea also had significantly lower QLS than patients without diarrhea. Diarrhea in all categories was independently predictive of decreased QLS by multivariable analysis. Chronic diarrhea (symptoms for more than one month) was significantly associated with decreased QLS in patients with high as well as low CD4 cell counts. Lack of recognition of diarrhea may result in significant underreporting of diarrhea by patients to physicians. Diarrhea is highly prevalent in the HIV-infected population and is strongly associated with diminished quality of life.

8794794

Increased mucosal concentrations of soluble intercellular adhesion molecule-1 (sICAM-1), sE-selectin, and interleukin-8 in active ulcerative colitis.

Cell surface adhesion molecules (CAM) are important promotors of the immunoinflammatory cascade. The circulating levels of soluble intercellular adhesion molecule 1 (ICAM-1) have previously been shown to correlate with disease activity in inflammatory bowel disease. The primary aim of this study was consequently to investigate if this also applies to mucosal levels of soluble ICAM-1. We measured soluble ICAM-1 levels in intestinal biopsy specimens and the endoscopic activity of 69 patients with ulcerative colitis (UC) and 14 controls and found that the median concentration of soluble ICAM-1 was significantly higher in patients with moderately or very active UC (15.0 ng/ml) pared to slightly active (9.8 ng/ml) and inactive UC (9.5 ng/ml) as well as controls (6.5 ng/ml) (P < 0.005). To further elucidate the interactions, two other CAM [E-selectin and vascular cellular adhesion molecule 1 (VCAM-1)], together with interleukin-8 (IL-8), IL-2 receptor (IL-2R) alpha and beta chains, were also measured. A significant trend towards higher soluble E-selectin levels in biopsies with active UC (1.8 pg/ml) pared to inactive UC (1.3 pg/ml) and to controls (< 1.0 pg/ml) (P < 0.01) was also found. In contrast, soluble VCAM-1 was barely detectable in biopsies from two UC patients. A significant correlation was found between soluble ICAM-1 and IL-8 concentrations (r = 0.46; P < 0.0001), and between sICAM-1 and sIL-2R alpha concentrations (r = 0.69; P < 0.0001), while sIL-2R beta was not detected. This study shows that intestinal ICAM-1 and E-selectin correlate with endoscopic activity of UC and with IL-8 and IL-2R alpha levels. These mediators may be useful in monitoring mucosal inflammation in studies exploring the therapeutical potential of targeting CAM. The lack of detectable VCAM-1, which is induced only in venous endothelium is interesting. It may suggest that intestinal inflammation mainly affects arterial endothelial cells and support the theory that intestinal vasculitis is involved in the pathogenesis of inflammatory bowel disease.

8794799

Dientamoeba fragilis. An unusual intestinal pathogen.

This is a case report of a gastrointestinal infection caused by Dientamoeba fragilis. It is a flagellate protozoan that is an mon etiology of gastrointestinal disease. Primarily characterized by diarrhea and abdominal pain, other symptoms such as flatulence, nausea, vomiting, fatigue, malaise, and weight loss occur. Diagnosis is made using multiple fresh stool samples that are preserved and permanently stained looking for the typical binucleate trophozoite. Since there is a distinct association with Enterobius vermicularis (possibly the mode of protozoan transmission), the human pinworm is also sought. Treatment of choice consists of diiodohydroxyquin in adults and metronidazole in children.

8794797

Distribution of alkaline sphingomyelinase activity in human beings and animals. Tissue and species differences.

The alkaline sphingomyelinase (SMase) was first found in rat intestinal brush border. The important roles of this enzyme in digestion of sphingomyelin and in mucosal cell proliferation have been suggested. In the present work, the distribution of the alkaline SMase in the tissues of human beings and animals have been studied. By assaying the enzyme activity in human biopsy samples, we found that the alkaline SMase activity was absent in the stomach, increased in the duodenum, present at high levels in the small intestine, and slightly declined in the colon and rectum. High activities were found similarly in the intestinal contents of the healthy adults and infants. The activities were also found in the intestinal mucosa of rats, normal and germ-free mice, and hamsters with the same distribution pattern as in humans, but not in the intestinal mucosa of guinea pigs. Apart from the intestinal tract, a SMase activity preferring alkaline pH was identified in human and guinea pig bile, but not in the bile of rat, pig, sheep, and cow. No activity was found in either pancreatic tissue or pancreatic juice in all species tested, and none was detected in human urine and milk. In conclusion, alkaline SMase exists predominantly in the digestive system with considerable tissue and species differences.

8794798

Symptomatic cerebral microangiopathy preceding initial manifestation of ulcerative colitis.

Focal white-matter lesions in the brain have been frequently found in patients with inflammatory bowel disease. These findings have been discussed as a novel extraintestinal manifestation of inflammatory bowel disease. In this case report we delineate the clinical relevance of such lesions by presenting a patient with ulcerative colitis and his associated neurological symptoms. The cerebral lesions were shown by magnetic resonance imaging, whereupon a successful treatment with corticosteroids was initiated.