bluuebunny/crossref_metadata_2025 huggingface dataset:
Development and regional expression of beta nerve growth factor messenger RNA and protein in the rat central nervous system.
Authors: Whittemore S R , Ebendal T , Lärkfors L , Olson L , Seiger A , Strömberg I , Persson H
Published: May 2006

The presence of nerve growth factor (NGF) mRNA and protein in the rat central nervous system is documented. Blot-hybridization analysis showed an abundance of NGF mRNA in the hippocampus, cerebral cortex, and olfactory bulb. Enzyme immunoassay confirmed significant levels of a NGF-like protein in the hippocampus and cerebral cortex. Bioassay of a NGF-like immunoaffinity-purified protein from these regions was physiologically indistinguishable from NGF. Immunohistochemistry revealed a widespread distribution of NGF-like reactivity in the adult brain, preferentially in fiber tracts. NGF mRNA accumulation began at birth, with adult levels reached 3 weeks postnatally. Enzyme immunoassay detected the presence of a NGF-like protein in the embryonic rat brain. Postnatally, the level of NGF-like protein reached a maximum at 3 weeks. Additionally, a distinct fetal form of NGF may exist.
Growth and differentiation of cerebellar suspensions transplanted into the adult cerebellum of mice with heredodegenerative ataxia.
Authors: Sotelo C , Alvarado-Mallart R M
Published: May 2006

Cell suspensions from cerebellar primordia of 12-day mouse embryos were grafted into the cerebellum of 4-month-old Purkinje cell degeneration (pcd) mutant mice and examined 2-3 months later. In contrast to those of nontreated mutants, all of the grafted cerebella exhibited Purkinje cells that had migrated into the molecular layer, where they were clustered over its superficial two-thirds. These Purkinje cells develop flattened dendritic trees perpendicular to bundles of parallel fibers. Ultrastructural examination of their synaptic inputs and outputs disclosed that (i) as in normal cerebella, climbing fibers and axons from basket and stellate cells synapse on thick dendrites, whereas parallel fibers almost exclusively contact the distal spiny branchlets, and (ii) a substantial number of Purkinje cell axons reach their appropriate targets in the deep cerebellar nuclei, where they establish synaptic connections on large and small neurons. These results indicate that embryonic Purkinje cells grafted into the cerebellum of adult mice with heredodegenerative ataxia integrate themselves very specifically into the cerebellar circuitry of the recipient mouse, where they can replace the missing Purkinje cells. They also provide a morphological basis favoring the notion of functional restorative capabilities of neural grafts in systems in which neurons are connected in an almost point-to-point manner.
Plasma membrane calcium fluxes in intact rods are inconsistent with the "calcium hypothesis".
Authors: Gold G H
Published: May 2006

The temporal relationship between the extracellular rod photovoltage and light-induced net Ca fluxes across the rod plasma membrane is investigated. The net Ca flux measurements are derived from extracellular Ca concentration measurements at the receptor surface of the isolated bullfrog retina. As reported previously, illumination leads to a net Ca efflux, which is followed by a net influx, during which the released Ca is taken back up. However, the net Ca flux has two characteristics that are inconsistent with the hypothesis that intracellular free Ca is the intracellular messenger for phototransduction in rods. First, during maintained photovoltage saturation, the net Ca efflux is transient, declining with a stereotypic time course that is independent of stimulus intensity and duration. Second, the significant rate of net influx during Ca uptake has no correlate in the photovoltage waveform. These observations are not consistent with the "Ca hypothesis." Rather, these data corroborate recent findings suggesting that light causes a decrease rather than an increase in intracellular free Ca concentration.
Control of the light-regulated current in rod photoreceptors by cyclic GMP, calcium, and l-cis-diltiazem.
Authors: Stern J H , Kaupp U B , MacLeish P R
Published: May 2006

The effect of calcium ions on the cGMP-activated current of outer segment membrane was examined by the excised-patch technique. Changes in the extracellular calcium concentration had marked effects on the cGMP-activated current, while changes in intracellular calcium concentration were ineffective. Changes in calcium concentration in the absence of cGMP had little, if any, effect on membrane conductance. These results suggest that both intracellular cGMP and extracellular calcium can directly affect the conductance underlying the light response in rod cells. The pharmacological agent l-cis-diltiazem reversibly inhibited the cGMP-activated current when applied to the intracellular side of an excised patch. When superfused over intact rod cells, l-cis-diltiazem reversibly blocked much of the normal light response. The isomer, d-cis-diltiazem, did not significantly affect either patches or intact rod cells. Thus, the light-regulated conductance has binding sites for both calcium and cGMP that may interact during the normal light response in rod cells and a site specific for l-cis-diltiazem that can be used to identify and further study the conductance mechanism.
Zeatin biosynthesis from
N
6
-(Δ
2
-isopentenyl)adenine in
Actinidia
and other woody plants
Authors: Einset John W.
Published: May 2006

Tissue cultures of
Actinidia kolomikta
can be maintained as callus through repeated passages on a nutrient medium devoid of cytokinin but containing inorganic nutrients, sucrose, and other basal organics plus auxin. Under these conditions, actively growing callus contained 2 and 0.5 nmol of the cytokinins zeatin [io
6
Ade; 6-(4-hydroxy-3-methylbut-2-enylamino)purine] and
N
6
-(Δ
2
-isopentenyl)adenine (i
6
Ade), respectively, per gram (fresh weight). When tissues were transferred from cytokininless medium to 30 μM i
6
Ade, endogenous io
6
Ade increased linearly to 160 nmol/g (fresh weight) during 8 hr, and i
6
Ade increased to 5 nmol/g (fresh weight) in 2 hr and then declined. The apparent
K
m
for i
6
Ade in
A. kolomikta
and
Actinidia chinensis
×
Actinidia arguta
callus and in tissue slices of
A. arguta
stems was 12 μM. In addition, the reaction(s) converting i
6
Ade to io
6
Ade was O
2
-requiring and specific for i
6
Ade versus N
6
-(Δ
2
-isopentenyl)adenosine (i
6
A). When
A. kolomikta
callus was fed 30 μM i
6
A, io
6
Ade increased and reached a concentration corresponding to 6 nmol/g (fresh weight) in 8 hr. Ribosylzeatin (io
6
A) did not increase. Under N
2
during i
6
A feeds, i
6
A accumulated rather than being metabolized to i
6
Ade, suggesting that i
6
A normally may be metabolized via i
6
AMP and i
6
Ade to io
6
Ade. A survey of 30 species of woody plants in 20 families of dicotyledonous angiosperms indicated that the ability to accumulate io
6
Ade (≥10 nmol/g) in 24-hr feeds with 30 μM i
6
Ade was restricted to certain systematic groups—e.g., order Ericales, families Oleaceae and Rubiaceae. This suggests that plants may differ in their pathways for io
6
Ade biosynthesis and that cytokinin biochemistry has potential as a taxonomic character above the species and genus levels.
The nerve growth factor receptor gene is at human chromosome region 17q12-17q22, distal to the chromosome 17 breakpoint in acute leukemias.
Authors: Huebner K , Isobe M , Chao M , Bothwell M , Ross A H , Finan J , Hoxie J A , Sehgal A , Buck C R , Lanahan A
Published: May 2006

Genomic and cDNA clones for the human nerve growth factor receptor have been used in conjunction with somatic cell hybrid analysis and in situ hybridization to localize the nerve growth factor receptor locus to human chromosome region 17q12-q22. Additionally, part, if not all, of the nerve growth factor receptor locus is present on the translocated portion of 17q (17q21-qter) from a poorly differentiated acute leukemia in which the chromosome 17 breakpoint was indistinguishable cytogenetically from the 17 breakpoint observed in the t(15;17)(q22;q21) translocation associated with acute promyelocytic leukemia. Thus the nerve growth factor receptor locus may be closely distal to the acute promyelocytic leukemia-associated chromosome 17 breakpoint at 17q21.
Monoclonal antibodies specific for H-2K and H-2D antigens on cytotoxic T cells can inhibit their function.
Authors: O'Neill H C
Published: May 2006

Antibodies specific for murine major histocompatibility gene complex (MHC) class I H-2K and H-2D molecules present on cytotoxic T (Tc) cells have been shown to inhibit their function of target cell lysis. This could only be demonstrated by using a more sensitive assay for T-cell-mediated lysis, and many monoclonal antibodies of different Ig class, origin, and specificity can be shown to inhibit alloreactive as well as MHC-restricted Tc cells. These antibodies inhibit different activated T-cell populations to varying extents, and anti-H-2K but not anti-H-2D antibodies show a synergistic effect with anti-Lyt-2 antibodies. Data here suggest that MHC molecules may be located in or near the T-cell receptor complex on these cells.
Protein kinase C phosphorylates topoisomerase II: topoisomerase activation and its possible role in phorbol ester-induced differentiation of HL-60 cells.
Authors: Sahyoun N , Wolf M , Besterman J , Hsieh T , Sander M , LeVine H , Chang K J , Cuatrecasas P
Published: May 2006

DNA topoisomerase II from Drosophila was phosphorylated effectively by protein kinase C. With a Km of about 100 nM, the reaction was rapid, occurring at 4 degrees C as well as at 30 degrees C and requiring as little as 0.6 ng of the protein kinase per 170 ng of topoisomerase. About 0.85 mol of phosphate could be incorporated per mol of topoisomerase II, with phosphoserine as the only phospho amino acid produced. The reaction was dependent on Ca2+ and phosphatidylserine and was stimulated by phorbol esters. Calmodulin-dependent protein kinase II, but not cyclic AMP-dependent protein kinase, was also able to phosphorylate the topoisomerase. Phosphorylation of topoisomerase II by protein kinase C resulted in appreciable activation of the topoisomerase, suggesting that it may represent a possible target for the regulation of nuclear events by protein kinase C. This possibility is supported by the finding that the phorbol ester-induced differentiation of HL-60 cells was blocked by the topoisomerase II inhibitors novobiocin and 4'-(9-acridinylamino)methanesulfon-m-anisidide(m-AMSA), but not by the inactive analog o-AMSA.
Primary-structure requirements for inhibition by the heat-stable inhibitor of the cAMP-dependent protein kinase.
Authors: Scott J D , Glaccum M B , Fischer E H , Krebs E G
Published: May 2006

The amino acid sequence of the heat-stable inhibitor of the cAMP-dependent protein kinase (PKI) was determined recently [Scott, J. D., Fischer, E. H., Takio, K., Demaille, J. G. & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. USA 82, 5732-5736]. An earlier report [Scott, J. D., Fischer, E.H., Demaille, J. G. & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. USA 82, 4379-4383] showed that at least part of the inhibitory domain of PKI is located in a 20-residue segment extending from residue 11 to residue 30: Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-His-Asp-Ile-Leu-Val-Ser- Ser-Ala . In the present study, we further mapped the inhibitory region of PKI by addition or deletion of residues at both ends of this peptide and by substitutions for specific amino acids. The results show that (i) deletion of residues 25-30 did not change inhibitory activity but addition of residues toward the amino terminus increased the inhibitory potency up to 150-fold (Ki 4.8 nM), to a level approaching that of PKI; (ii) replacement of alanine-21 by serine converted the inhibitor into a substrate having a relatively low affinity (Km 280 microM) for the enzyme; (iii) replacement of alanine-21 by phosphoserine or alpha-aminobutyric acid decreased inhibitory activity by a factor of 120 and 20, respectively; (iv) replacement of serine-13 had essentially no effect, whereas substitution of threonine-16 decreased inhibitory activity. The greatest decreases of inhibitory potency occurred with replacements of the arginines in positions 18 and 19.
Synthesis and biological activity of highly potent octapeptide analogs of somatostatin.
Authors: Cai R Z , Szoke B , Lu R , Fu D , Redding T W , Schally A V
Published: May 2006

In the search for selective and long-acting analogs of somatostatin, nearly 200 compounds were synthesized by solid-phase methods, purified, and tested biologically. Among these octapeptides, some contained N-terminal (Formula: see text) were 177 times and 113 times more potent, respectively, than somatostatin in tests for inhibition of growth hormone release. These two octapeptides containing tyrosine and valine in positions 3 and 6, respectively, were more active and more selective than their Phe-3 and Thr-6 counterparts, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2 and D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Trp-NH2. D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 was also about 6 times more potent than its L-Trp-4 diastereoisomer. The analogs D-Phe-Cys-Tyr-Lys-Val-Cys-Thr-NH2 and D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 showed a prolonged duration of action and were able to inhibit growth hormone release for at least 3 hr. Analogs of both Phe-3/Thr-6 and Tyr-3/Val-6 classes also suppressed the release of insulin and glucagon in rats and pentagastrin-induced secretion of gastric acid in dogs, but their potencies in these tests were much smaller than the growth-hormone-release inhibitory activity. Some of these analogs possessed antitumor activities as shown by the inhibition of growth of animal models of prostate, mammary, and ductal pancreatic tumors.
Voltage-gated and synaptic currents in rat Purkinje cells in dissociated cell cultures.
Authors: Hirano T , Ohmori H
Published: May 2006

The electrical properties of rat Purkinje cells and synapses from granule cells were studied in dissociated cell cultures. To identify the cells we used an immunohistochemical method and recorded voltage-gated and synaptic currents with the patch-clamp technique (the whole-cell mode). Cultured Purkinje cells generated action potentials similar to those recorded from in vitro slices or in vivo preparations. Early Na, late Ca inward current, and K outward currents were distinguished by ion substitution under voltage clamp. We also recorded spontaneous synaptic currents in Purkinje cells cultured with granule cells. These synaptic currents reversed direction at the membrane potential of 2.5 mV, which was similar to currents induced by L-glutamate. Therefore, these are most likely excitatory synaptic currents from granule cells. Since these properties of Purkinje cells examined here are similar to those in situ, the cells in dissociated cell cultures offer a great opportunity to study biophysical properties of identified neurons in the central nervous system.
On the stereochemistry and biosynthesis of lipoxin B.
Authors: Serhan C N , Hamberg M , Samuelsson B , Morris J , Wishka D G
Published: May 2006

Lipoxin B (LXB) was prepared by incubation of (15S)-15-hydroperoxy-5,8,11-cis-13-trans-icosatetraenoic acid (15-HPETE) with human leukocytes. Comparison with a number of trihydroxyicosatetraenes prepared by total synthesis showed that biologically derived LXB is (5S,14R,15S)-5,14,15-trihydroxy-6,10,12-trans-8-cis-icosatetraenoi c acid. Two isomers of LXB were identified by using an improved isolation procedure. These compounds were shown to be (5S,14R,15S)-5,14,15-trihydroxy-6,8,10,12-trans-icosatetraenoic acid (8-trans-LXB) and (5S,14S,15S)-5,14,15-trihydroxy-6,8,10,12-trans-icosatetraenoic acid [(14S)-8-trans-LXB]. Experiments with 18O2 showed that formation of LXB and its two isomers occurred with incorporation of molecular oxygen at C-5 but not at C-14. These results together with the finding that (15S)-hydroxy-5,8,11-cis-13-trans-icosatetraenoic acid (15-HETE) is a precursor of LXB compounds in activated leukocytes suggest that 15-hydroxy-5,6-epoxy-7,9,13-trans-11-cis-icosatetraenoic acid or its equivalent is a common intermediate in the biosynthesis of LXB and its two isomers.
Predatory prokaryotes: Predation and primary consumption evolved in bacteria
Authors: Guerrero Ricardo , Pedrós-Alió Carlos , Esteve Isabel , Mas Jordi , Chase David , Margulis Lynn
Published: May 2006

Two kinds of predatory bacteria have been observed and characterized by light and electron microscopy in samples from freshwater sulfurous lakes in northeastern Spain. The first bacterium, named
Vampirococcus
, is Gram-negative and ovoidal (0.6 μm wide). An anaerobic epibiont, it adheres to the surface of phototrophic bacteria (
Chromatium
spp.) by specific attachment structures and, as it grows and divides by fission, destroys its prey. An important
in situ
predatory role can be inferred for
Vampirococcus
from direct counts in natural samples. The second bacterium, named
Daptobacter
, is a Gram-negative, facultatively anaerobic straight rod (0.5 × 1.5 μm) with a single polar flagellum, which collides, penetrates, and grows inside the cytoplasm of its prey (several genera of Chromatiaceae). Considering also the well-known case of
Bdellovibrio
, a Gram-negative, aerobic curved rod that penetrates and divides in the periplasmic space of many chemotrophic Gram-negative bacteria, there are three types of predatory prokaryotes presently known (epibiotic, cytoplasmic, and periplasmic). Thus, we conclude that antagonistic relationships such as primary consumption, predation, and scavenging had already evolved in microbial ecosystems prior to the appearance of eukaryotes. Furthermore, because they represent methods by which prokaryotes can penetrate other prokaryotes in the absence of phagocytosis, these associations can be considered preadaptations for the origin of intracellular organelles.
Stabilization of lipid bilayer vesicles by sucrose during freezing
Authors: Strauss G. , Hauser H.
Published: May 2006

The freeze-induced fusion and leakage of small unilamellar vesicles (SUV) of natural and synthetic phosphatidylcholines and the suppression of these processes by sucrose was studied by electron microscopy, by high-resolution NMR, and by ESR techniques. During slow freezing of SUV suspensions in water, the lipid was compressed into a small interstitial volume and transformed into a multilamellar aggregate without vesicular structure. When frozen in sucrose solution, the lipid also was compressed between the ice crystals but remained in the form of vesicles. The fractional amount of lipid remaining as SUV after freezing was found to increase significantly only at sucrose/lipid molar ratios above 0.4. Eu
3+
displaced sucrose from the lipid by competitive binding. During freezing in the absence of sucrose, the vesicles became transiently permeable to ions. ESR studies showed that fusion of vesicles in the absence of sucrose is far more extensive when they are frozen while above their phase-transition temperature (

t
c

) than when frozen while below their

t
c

. It is concluded that the extent of membrane disruption depends on the membrane mobility at the moment of freezing and that sucrose exerts its protective effect by binding to the membrane interface and/or by affecting the water structure.
Molecular cloning of the muscle gene unc-22 in Caenorhabditis elegans by Tc1 transposon tagging.
Authors: Moerman D G , Benian G M , Waterston R H
Published: May 2006

The previously described mutator system of Caenorhabditis elegans var. Bergerac has as one of its targets unc-22, a previously uncloned gene on chromosome IV important in assembly and function of the body wall musculature. By assuming that the mutator activity involved transposition of the repetitive element Tc1 into the unc-22 gene we have succeeded both in cloning the unc-22 gene and in demonstrating that Tc1 transposition is the principal basis of the mutator activity in the Bergerac strain. Although germ-line excision of Tc1 is sensitive to genetic background, somatic excision appears to be less so, suggesting that Tc1 movement is controlled differently in germ-line and somatic tissue. The availability of a transposon-based mutator system should aid in the cloning of additional genes in C. elegans, and the particular properties of this Tc1 system may provide information about the control of transposable element activity more generally.
Cholecystokinin-associated COOH-terminal peptides are fully sulfated in pig brain.
Authors: Eng J , Gubler U , Raufman J P , Chang M , Hulmes J D , Pan Y C , Yalow R S
Published: May 2006

A radioimmunoassay was developed to detect the cholecystokinin (CCK)-associated nonapeptide (CAP-9) that forms the COOH terminus of pig preproCCK. This peptide (Ser-Ala-Glu-Glu-Tyr-Glu-Tyr-Thr-Ser) is presumably produced at the time that the tyrosine-sulfated octapeptide CCK8(s) is cleaved from preproCCK. Radioimmunoassay of a dried methanol extract of pig brain revealed no detectable CAP-9 immunoreactivity, whereas acid desulfation of the dried methanol extract prior to radioimmunoassay resulted in easily measurable concentrations of CAP-9 immunoreactivity. Two peptides, CAP-9 and des-Ser9-CAP-9, were purified from a methanol extract of 8 kg of commercially obtained whole pig brains. Amino acid analysis showed that each peptide has both tyrosines sulfated. Thus, the likely sequence of CCK post-translational processing events is sulfation of the three tyrosines in the COOH terminus of preproCCK followed by peptide cleavage and appearance of CCK8(s) and CAP-9(s,s).
Testosterone allosterically regulates ethanol oxidation by homo- and heterodimeric gamma-subunit-containing isozymes of human alcohol dehydrogenase.
Authors: Mårdh G , Falchuk K H , Auld D S , Vallee B L
Published: May 2006

Testosterone and its physiologically active metabolite 5 alpha-dihydrotestosterone are selective, allosteric inhibitors of the gamma subunit-containing isozymes of class I human alcohol dehydrogenase (ADH) with apparent Ki values for testosterone at pH 7.4 between 3.5 and 16 X 10(-6) M. Testosterone inhibition is noncompetitive with respect to ethanol, NAD+, 1,10-phenanthroline, and 4-methylpyrazole, identifying a regulatory site distinct from the catalytic site. Testosterone does not inhibit the class I isozymes composed only of alpha and/or beta subunits and only weakly inhibits the class II and III isozymes. Importantly, none of these human ADH isozymes oxidize or reduce the steroids with the delta 4 double bond or 5 alpha configuration. The allosteric effect of testosterone, restricted to the gamma subunits of human ADH, suggests unique metabolic specificities and pathways for these isozymes, apart from all others. This inhibition may ultimately be critical to an identification of their function(s). Analogous considerations of other metabolic effectors might further lead to similar insights regarding the alpha and beta subunit-containing isozymes as well as the class II and III ADH.
Contact pressures in the human hip joint measured in vivo.
Authors: Hodge W A , Fijan R S , Carlson K L , Burgess R G , Harris W H , Mann R W
Published: May 2006

The pressures on human articular cartilage have been measured in vivo. An instrumented femoral head prosthesis that telemeters interarticular pressure at 10 discrete locations 253 times per second was implanted in apposition to natural acetabular cartilage. Data were acquired during surgery, recovery, rehabilitation, and normal activity, for longer than 1 year after surgery. Pressure magnitudes were synchronized with body-segment kinematic data and foot-floor force measurements so as to locate transduced pressure areas on the natural acetabulum and correlate movement kinematics and dynamics with local cartilage pressures. The data reveal very high local (up to 18 MPa) and nonuniform pressures, with abrupt spatial and temporal gradients, that correlate well both in magnitude and distribution with in vitro data and computer simulations of synovial joint mechanics. Peak pressures in vivo are, however, considerably higher than pressures measured in vitro under the putative forces experienced by the joint in life, particularly in normal movements where cocontraction occurs in agonist and antagonist muscles across the hip joint. Thus, extant gait-analysis studies which apply inverse Newtonian calculations to infer joint forces establish the lower limit on such forces, since such analyses include only the net muscular torques about the joint and cannot account for the contribution of the increment in joint force due to muscular cocontraction. Our data also contribute to the understanding of normal synovial joint tribology and the possible role of mechanical factors in the deterioration evident in osteoarthritis. Further, design criteria for both partial and total hip replacement prostheses and specific aspects of rehabilitation protocols following hip surgery (e.g., the extent to which crutches and canes unload the hip joint) warrant reconsideration in light of the extraordinary high pressures measured during the activities of daily living.
Human brain glial cells synthesize thrombospondin.
Authors: Asch A S , Leung L L , Shapiro J , Nachman R L
Published: May 2006

Thrombospondin, a 450-kDa multinodular glycoprotein with lectin-type activity, is found in human platelets, endothelial cells, fibroblasts, smooth muscle cells, monocytes, and granular pneumocytes. Thrombospondin interacts with heparin, fibrinogen, fibronectin, collagen, histidine-rich glycoprotein, and plasminogen. Recently, thrombospondin synthesis by smooth muscle cells has been reported to be augmented by platelet-derived growth factor. We present evidence that thrombospondin is present within and synthesized by astrocytic neuroglial cells. Heparin-Sepharose affinity chromatography of material derived from a human brain homogenate yielded a protein that, when reduced, had an apparent size of 180 kDa and comigrated with reduced platelet thrombospondin on NaDodSO4/PAGE. Immunoblot analysis with monospecific anti-thrombospondin confirmed the presence of immunoreactive thrombospondin. Indirect immunofluorescence of cultured human glial cells indicated the presence of thrombospondin. Metabolic labeling of glial cell cultures with [35S]methionine followed by immunoprecipitation with monospecific anti-thrombospondin revealed synthesis of a 180-kDa polypeptide that comigrated with platelet thrombospondin on NaDodSO4/PAGE. Cultured human glial cells were incubated for 48 hr in serum-free medium with purified platelet-derived growth factor at concentrations up to 50 ng/ml. Aliquots taken at intervals were analyzed by a quantitative double-antibody ELISA. The growth factor stimulated the release of thrombospondin into the culture medium by as much as 10-fold over control cultures. The presence of thrombospondin within glial cells of the central nervous system and the augmentation of its synthesis by platelet-derived growth factor suggest that thrombospondin may play an important role in regulating cell-cell and cell-matrix interactions during periods of cell division and growth.
Tree squirrel hepatitis B virus: antigenic and structural characterization.
Authors: Feitelson M A , Millman I , Blumberg B S
Published: May 2006

Tree squirrel hepatitis B virus (THBV)-associated particles isolated from the livers of naturally infected animals share one or more antigenic determinants with hepatitis B surface antigen in solid-phase immunoassays. Characterization of THBV-associated polypeptides by sodium dodecyl sulfate/polyacrylamide gel electrophoresis reproducibly demonstrated major components with apparent sizes of 15.5 and 17 kDa. Peptide mapping of these components shows that they are related to the peptide maps of the major surface antigen polypeptides associated with hepatitis B virus and like viruses. Sodium dodecyl sulfate/polyacrylamide gel analysis also demonstrated discrete bands at 14.5, 19, 20, and 35 kDa. Upon blotting of THBV-associated polypeptides with sera containing antibodies to hepatitis B core antigen or hepatitis B X antigen, only the 35-kDa band became detectable, suggesting that this component is core related. These results establish the presence of both surface and core antigen-related polypeptides associated with purified THBV and better define the relationship of THBV to the family of hepatitis B virus and like viruses.
Regulation of phosphatidylinositol turnover in brain synaptoneurosomes: stimulatory effects of agents that enhance influx of sodium ions.
Authors: Gusovsky F , Hollingsworth E B , Daly J W
Published: May 2006

Norepinephrine and carbamoylcholine stimulate accumulation of [3H]inositol phosphates from [3H]inositol-labeled guinea pig cerebral cortical synaptoneurosomes through interaction with alpha 1-adrenergic and muscarinic receptors, respectively. In addition to such agonist, a variety of natural products that affect voltage-dependent sodium channels can markedly stimulate accumulation of [3H]inositol phosphates. These include alkaloids that activate sodium channels, such as batrachotoxin, veratridine, and aconitine; peptide toxins that alter activation or slow inactivation of sodium channels, such as various scorpion toxins from Leiurus, Centruroides, and Tityus species; and agents that cause repetitive firing of sodium channel-dependent action potentials, such as pyrethroids and pumiliotoxin B. Ouabain, and agent that will increase accumulation of internal sodium by inhibition of Na+, K+-ATPase, also stimulates formation of [3H]inositol phosphates, as does monensin, a sodium ionophore. Tetrodotoxin and saxitoxin, specific blockers of voltage-dependent sodium channels, prevent or reduce the stimulatory effects of sodium channel agents and ouabain on phosphatidylinositol turnover, while having lesser or no effect, respectively, on receptor-mediated or monensin-mediated stimulation. Removal of extracellular sodium ions markedly reduces stimulatory effects of sodium channel agents, while removal of extracellular calcium ions with EGTA blocks both receptor-mediated and sodium channel agent-mediated phosphatidylinositol turnover. The results provide evidence for a hitherto unsuspected messenger role for sodium ions in excitable tissue, whereby neuronal activity and the resultant influx of sodium will cause activation of phospholipase systems involved in hydrolysis of phosphatidylinositols, thereby generating two second messengers, the inositol phosphates, which mobilize calcium from internal stores, and the diacylglycerols, which activate protein kinase C.
Isolation of a photosystem II reaction center consisting of D-1 and D-2 polypeptides and cytochrome
b
-559
Authors: Nanba Osamu , Satoh Kimiyuki
Published: May 2006

A photosystem II reaction center complex consisting of D-1 and D-2 polypeptides and cytochrome
b
-559 was isolated from spinach grana thylakoids, treated with 4% (wt/vol) Triton X-100, by ion-exchange chromatography using DEAE-Toyopearl 650S. The isolated complex appears to contain five chlorophyll a, two pheophytin a, one β-carotene, and one or two cytochrome
b
-559 heme(s) (molar ratio) and exhibits a reversible absorbance change attributable to the photochemical accumulation of reduced pheophytin typical for the intermediary electron acceptor of photosystem II reaction center. These results strongly suggest that the site of primary charge separation in photosystem II is located on the heterodimer composed of D-1 and D-2 subunits.
Modulation of 3-hydroxy-3-methylglutaryl-CoA reductase by changes in microsomal cholesterol content or phospholipid composition.
Authors: Davis P J , Poznansky M J
Published: May 2006

Microsomal preparations from normal human skin fibroblasts were used to investigate the role of free cholesterol in the endoplasmic reticulum in the control of cholesterol biosynthesis by regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (NADPH) [(S)-mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34]. Controlled changes in the cholesterol/phospholipid ratio of microsomes were introduced either in intact cells by incubating fibroblast cultures with whole serum or lipoprotein-deficient serum or by enrichment or depletion of cholesterol in microsomal preparations by incubating microsomes with cholesterol-rich or cholesterol-poor egg phosphatidylcholine unilamellar vesicles. Cholesterol enrichment resulted in suppression of reductase activity and increased ESR order parameters for 12-nitroxystearate in the microsomal preparations. Conversely, cholesterol depletion caused an activation of reductase and a decrease in order parameter. Enrichment of microsomal preparations with a "nonfluid" lipid, dipalmitoyl phosphatidylcholine, also suppressed enzyme activity and increased membrane order. The effect was reversed by subsequent enrichment of the microsomes with fluid egg phosphatidylcholine. Our findings suggest that cholesterol may regulate its own biosynthesis, at least in part, by suppression of hydroxymethylglutaryl-CoA reductase mediated through changes in membrane fluidity as measured by ESR order parameter.
Human B-cell differentiation factor defined by an anti-peptide antibody and its possible role in autoantibody production.
Authors: Hirano T , Taga T , Yasukawa K , Nakajima K , Nakano N , Takatsuki F , Shimizu M , Murashima A , Tsunasawa S , Sakiyama F
Published: May 2006

The partial amino acid sequence of the NH2 terminus of a factor named human B-cell differentiation factor or B-cell stimulatory factor 2 (BSF-2) has been determined. Antibodies raised against the synthetic peptide corresponding to residues 1-13 of the NH2-terminal sequence specifically react with BSF-2 generated by a T-cell line and by phytohemagglutinin-stimulated normal T cells. Furthermore, the antipeptide antibodies react with a BSF-2-like factor produced by cardiac myxoma as well as uterine cervical carcinoma cells. The results show that BSF-2 functions in vivo as well and suggest that the constitutive production of BSF-2 may be involved in autoantibody production, since patients with cardiac myxoma and uterine carcinoma showed autoantibody production.
(5Z,13E)-(15S)-9 alpha,11 beta,15-trihydroxyprosta-5,13-dien-1-oic acid (9 alpha,11 beta-prostaglandin F2): formation and metabolism by human lung and contractile effects on human bronchial smooth muscle.
Authors: Seibert K , Sheller J R , Roberts L J
Published: May 2006

Prostaglandin D2 (PGD2) was recently found to be stereospecifically converted to the compound (5Z,13E)-(15S)-9 alpha,11 beta,15-trihydroxyprosta-5,13-dien-1-oic acid (9 alpha,11 beta-PGF2) by a human liver cytosolic NADPH-dependent 11-ketoreductase enzyme. Because PGD2 is a potent bronchoconstrictor and is released into bronchoalveolar lavage fluid after allergen stimulation in patients with allergic asthma, the ability of human lung to metabolize PGD2 to 9 alpha,11 beta-PGF2 and the contractile effects of 9 alpha,11 beta-PGF2 on human bronchial smooth muscle were investigated. The 100,000 X g supernatant of human lung converted PGD2 in the presence of an NADPH-generating system stereospecifically to 9 alpha,11 beta-PGF2 at a rate of 3.46 +/- 0.94 pmol per min per mg of protein. 9 alpha,11 beta-PGF2 was found to contract human bronchial rings in a dose-dependent fashion with a potency virtually identical with that of both PGD2 and PGF2 alpha, known potent bronchial constrictors. PGD2 was found to be a very poor substrate for human lung 15-hydroxyprostaglandin dehydrogenases and to be preferentially metabolized by lung to 9 alpha,11 beta-PGF2. 9 alpha,11 beta-PGF2 was also found to be a very poor substrate for the lung 15-hydroxyprostaglandin dehydrogenases. Thus, once formed, 9 alpha,11 beta-PGF2 would not be expected to be rapidly inactivated in situ by these metabolic enzymes. These results suggest that 9 alpha,11 beta-PGF2 may participate along with other putative mediators in the pulmonary allergic response in humans.
Molecular basis for group B beta-hemolytic streptococcal disease.
Authors: Hellerqvist C G , Sundell H , Gettins P
Published: May 2006

Group B beta-hemolytic Streptococcus (GBS) is a major pathogen affecting newborns. We have investigated the molecular mechanism underlying the respiratory distress induced in sheep after intravenous injection of a toxin produced by this organism. The pathophysiological response is characterized by pulmonary hypertension, followed by granulocytopenia and increased pulmonary vascular permeability to protein. 31P NMR studies of GBS toxin and model components before and after reductive alkaline hydrolysis demonstrated that phosphodiester residues are an integral part of the GBS toxin. Reductive alkaline treatment cleaves phosphate esters from secondary and primary alcohols and renders GBS toxin nontoxic in the sheep model and inactive as a mediator of elastase release in vitro from isolated human granulocytes. We propose that the interaction of cellular receptors with mannosyl phosphodiester groups plays an essential role in the pathophysiological response to GBS toxin.
Specific inhibition of Trypanosoma cruzi neuraminidase by the human plasma glycoprotein "cruzin".
Authors: Prioli R P , Rosenberg I , Pereira M E
Published: May 2006

Plasma of normal human individuals was shown to contain an inhibitor of Trypanosoma cruzi neuraminidase (NAase; acylneuraminyl hydrolase, sialidase, EC 3.2.1.18). The inhibitor has been purified to homogeneity by PEG precipitation, CM Affi-Gel Blue Sepharose chromatography, and gel filtration. The purified preparation inhibits T. cruzi NAase at a concentration as low as 10(-9) M and has no effect at concentrations at least 100 times higher on any of the other NAases tested, including those from influenza virus, the closely related trypanosome Trypanosoma rangeli, and mammalian NAases. The inhibitor is unique in that it prevents T. cruzi desialylation of intact mammalian cells but does not prevent desialylation of soluble glycoconjugates. In addition, the isolated material is effective in inhibiting the T. cruzi NAase whether the enzyme is on the parasite outer membrane or in solution. Molecular characterization indicates that the inhibitor is a glycoprotein with a Mr of 246,000 +/- 20,000 composed of subunits of Mr 28,000 +/- 2000. Its plasma concentration is at least 60 micrograms/ml. The mechanism of action has not been fully elucidated, but it appears to be noncompetitive. Attempts to match the isolated NAase inhibitor with known plasma glycoproteins have not been successful. In view of this and of the specificity of the inhibitor for T. cruzi, we have named the inhibitor "cruzin." This finding suggests a different approach in investigating the role that NAase plays in host-parasite interaction.
Random cloning of genes from mouse chromosome 17.
Authors: Kasahara M , Figueroa F , Klein J
Published: May 2006

We describe a method for isolating cosmid clones randomly from mouse chromosome 17. A cosmid library was constructed from the mouse-Chinese hamster cell line R4 4-1 that contains a limited amount of mouse DNA (chromosomes 17 and 18 and some other unidentified material) on a Chinese hamster background. The library was screened with the murine repetitive sequence probe pMBA14, which selectively hybridizes with mouse DNA. The mouse-derived cosmid clones thus identified were individually hybridized with DNA from the mouse-Syrian hamster cell line JS17 containing all mouse chromosomes except chromosome 17 on a Syrian hamster background. We deduced that the cosmid clones that contained sequences absent in JS17 were derived from mouse chromosome 17. One of the chromosome 17-derived cosmid clones, 3-4-1 (located proximal to the T122/T66C segment) was found to be highly polymorphic among European wild-mouse populations and may be a useful probe to elucidate the evolution and migration of Mus species. The randomly isolated mouse-derived cosmid clones can also be screened for the presence of functional genes. Using testicular cDNA as a probe, a testis-specific gene was cloned from mouse chromosome 17.
Multiple neuropeptides in cholinergic motor neurons of Aplysia: evidence for modulation intrinsic to the motor circuit.
Authors: Cropper E C , Lloyd P E , Reed W , Tenenbaum R , Kupfermann I , Weiss K R
Published: May 2006

Changes in Aplysia biting responses during food arousal are partially mediated by the serotonergic metacerebral cells (MCCs). The MCCs potentiate contractions of a muscle utilized in biting, the accessory radula closer (ARCM), when contractions are elicited by stimulation of either of the two cholinergic motor neurons B15 or B16 that innervate the muscle. We have now shown that ARCM contractions may also be potentiated by peptide cotransmitters in the ARCM motor neurons. We found that motor neuron B15 contains small cardioactive peptides A and B (SCPA and SCPB)--i.e., whole B15 neurons were bioactive on the SCP-sensitive Helix heart, as were reverse-phase HPLC fractions of B15 neurons that eluted like synthetic SCPA and SCPB. Furthermore, [35S]methionine-labeled B15 peptides precisely coeluted with synthetic SCPA and SCPB. SCPB-like immunoreactivity was associated with dense-core vesicles in the soma of B15 and in neuritic varicosities and terminals in the ARCM. B16 motor neurons did not contain SCPA or SCPB but contained an unidentified bioactive peptide. RP-HPLC of [35S]methionine-labeled B16s resulted in one major peak of radioactivity that did not coelute with either SCP and which, when subject to Edman degradation, yielded [35S]methionine in positions where there is no methionine in the SCPs. Exogenously applied B16 peptide potentiated ARCM contractions elicited by stimulation of B15 or B16 neurons. Thus, in this system there appear to be two types of modulation; one type arises from the MCCs and is extrinsic to the motor system, whereas the second type arises from the motor neurons themselves and hence is intrinsic.
Insulin-like synergistic stimulation of DNA synthesis in Swiss 3T3 cells by the BSC-1 cell-derived growth inhibitor related to transforming growth factor type beta.
Authors: Brown K D , Holley R W
Published: May 2006

A cell growth inhibitor (GI), purified from BSC-1 cell-conditioned medium, has little if any effect on DNA synthesis when added alone to monolayer cultures of quiescent Swiss mouse 3T3 cells in serum-free medium. However, the inhibitor, which is closely related to transforming growth factor type beta (TGF-beta), exhibits a pronounced synergistic stimulation of DNA synthesis in combination with certain peptide (bombesin, vasopressin) or polypeptide (platelet-derived growth factor) mitogens. A similar synergistic response has been demonstrated for TGF-beta purified from human platelets. In the presence of 3 nM bombesin, a half-maximal stimulation of DNA synthesis was obtained at a GI concentration of approximately 60 pg/ml, with a maximal response at approximately 600 pg/ml. The synergistic interactions demonstrated by GI or TGF-beta in stimulating Swiss 3T3 cells closely resemble those previously shown for insulin, and we have observed that GI does not synergize with insulin to stimulate DNA synthesis in these cells. Like insulin, and in contrast to bombesin, vasopressin, and platelet-derived growth factor, GI does not activate cellular inositolphospholipid hydrolysis, calcium mobilization, or cross-regulation of epidermal growth factor receptor affinity. These results raise the possibility that the biochemical pathways activated by GI/TGF-beta and insulin converge at a post-receptor stage.
Photoproduct frequency is not the major determinant of UV base substitution hot spots or cold spots in human cells.
Authors: Brash D E , Seetharam S , Kraemer K H , Seidman M M , Bredberg A
Published: May 2006

The role of UV radiation-induced photoproducts in initiating base substitution mutations in human cells was examined by measuring photoproduct frequency distributions and mutations in a supF tRNA gene on a shuttle vector plasmid transfected into DNA repair-deficient cells (xeroderma pigmentosum, complementation group A) and into normal cells. Frequencies of cyclobutane dimers and pyrimidine-pyrimidone (6-4) photoproducts varied by as much as 80-fold at different dipyrimidine sites within the gene. All transition mutations occurred at dipyrimidine sites, predominantly at cytosine, with a 17-fold variation in mutation frequency between different sites. Removal of greater than 99% of the cyclobutane dimers by in vitro photoreactivation before transfection reduced the mutation frequency while preserving the mutation distribution, indicating that (i) cytosine-containing cyclobutane dimers were the major mutagenic lesions at these sites and (ii) cytosine-containing non-cyclobutane dimer photoproducts were also mutagenic lesions. However, at individual dipyrimidine sites neither the frequency of cyclobutane dimers nor the frequency of pyrimidine-pyrimidone (6-4) photoproducts correlated with the mutation frequency, even in the absence of excision repair. Mutation hot spots occurred at sites with low or high frequency of photoproduct formation and mutation cold spots occurred at sites with many photoproducts. These results suggest that although photoproducts are required for UV mutagenesis, the prominence of most mutation hot spots and cold spots is primarily determined by DNA structural features rather than by the frequency of DNA photoproducts.
5'-structural analysis of genes encoding polymorphic antigens of chemically induced tumors.
Authors: Srivastava P K , Chen Y T , Old L J
Published: May 2006

We have proposed that the distinct tumor rejection antigens of chemically induced sarcomas in inbred mice belong to a family of Mr 96,000 glycoproteins (gp96). An identical 14-amino acid sequence was found at the amino terminus of gp96 from two antigenically distinct BALB/c sarcomas. Oligonucleotide probes derived from this sequence permitted isolation of 5' cDNA and genomic fragments coding for gp96. Three short exons interrupted by relatively long introns were identified at the 5' terminus of the gp96 gene. The first exon encodes a signal peptide, which is consistent with gp96 being a cell surface antigen. Southern blot analysis indicated that the gp96 family is encoded by a single gene, and 3-kilobase transcripts were detected in all normal and tumor cells tested. Nucleotide and deduced amino acid sequences from 311 base paris at the 5' terminus showed no homology with any known protein. The availability of molecular probes for the gp96 system permits analysis of the structural polymorphism of these antigens.
DNA polymerase III of Escherichia coli is required for UV and ethyl methanesulfonate mutagenesis.
Authors: Hagensee M E , Timme T L , Bryan S K , Moses R E
Published: May 2006

Strains of Escherichia coli possessing the pcbA1 mutation, a functional DNA polymerase I, and a temperature-sensitive mutation in DNA polymerase III can survive at the restrictive temperature (43 degrees C) for DNA polymerase III. The mutation rate of the bacterial genome of such strains after exposure to either UV light or ethyl methanesulfonate was measured by its rifampicin resistance or amino acid requirements. In addition, Weigle mutagenesis of preirradiated lambda phage was also measured. In all cases, no increase in mutagenesis was noted at the restrictive temperature for DNA polymerase III. Introduction of a cloned DNA polymerase III gene returned the mutation rate of the bacterial genome as well as the Weigle mutagenesis to normal at 43 degrees C. Using a recA-lacZ fusion, the SOS response after UV irradiation was measured and found to be normal at the restrictive and permissive temperature for DNA polymerase III, as was induction of lambda prophage. Recombination was also normal at either temperature. Our studies demonstrate that a functional DNA polymerase III is strictly required for mutagenesis at a step other than SOS induction.