bluuebunny/crossref_metadata_2025 huggingface dataset:
Schwann cell proliferation and localized proteolysis: expression of plasminogen-activator activity predominates in the proliferating cell populations.
Authors: Kalderon N
Published: May 2006

The role of the serum proteolytic system plasminogen/plasminogen activator as a biochemical tool used by the glia or neurons, or both, for maintaining their temporary and flexible cellular interactions during histogenesis of the nervous system is under study. The present report identifies a glia cell type, the Schwann cell, as one of the cellular components of the nervous system that uses extracellular proteolysis at the time of the tissue construction. Purified dividing mouse Schwann cells in culture produce extracellular plasminogen activator. The levels of extracellular plasminogen-activator activity, as measured by the biochemical fibrinolytic assay, were directly related to the proliferation rates of the Schwann cells. The cellular plasminogen-activator specific activity at the maximal rate of cell proliferation was 3-4 times higher than that of the cells at low rate of mitosis. It is concluded that plasminogen-activator activity is expressed predominantly by the proliferating Schwann cell populations, suggesting that the extracellular proteolysis is used by the tissue at those stages when the cells divide.
Electrical stimulation increases phosphorylation of tyrosine hydroxylase in superior cervical ganglion of rat.
Authors: Cahill A L , Perlman R L
Published: May 2006

Electrical stimulation of the superior cervical ganglion of the rat increased the phosphorylation of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) in this tissue. Ganglia were incubated with [32P]Pi for 90 min and were then electrically stimulated via the preganglionic nerve. Tyrosine hydroxylase was isolated from homogenates of the ganglia by immunoprecipitation followed by polyacrylamide gel electrophoresis. 32P-labeled tyrosine hydroxylase was visualized by radioautography, and the incorporation of 32P into the enzyme was quantitated by densitometry of the radioautograms. Stimulation of ganglia at 20 Hz for 5 min increased the incorporation of 32P into tyrosine hydroxylase to a level 5-fold that found in unstimulated control ganglia. The increase in phosphorylation of tyrosine hydroxylase was dependent on the duration and frequency of stimulation. Preganglionic stimulation did not increase the phosphorylation of tyrosine hydroxylase in a medium that contained low Ca2+ and high Mg2+. Increases in phosphorylation were reversible; within 30 min after the cessation of stimulation, the incorporation of 32P into tyrosine hydroxylase decreased to the level found in unstimulated ganglia. The nicotinic antagonist hexamethonium reduced the increase in 32P incorporation into tyrosine hydroxylase by about 50%, while the muscarinic antagonist atropine had no effect. Thus, preganglionic stimulation appeared to increase the phosphorylation of tyrosine hydroxylase in part by a nicotinic mechanism and in part by a noncholinergic mechanism. Antidromic stimulation of ganglia also increased the phosphorylation of tyrosine hydroxylase. Two-dimensional gel electrophoresis revealed that electrical stimulation also increased the incorporation of 32P into at least six other phosphoproteins in the ganglion.
Subunit b of the membrane moiety (F0) of ATP synthase (F1F0) from Escherichia coli is indispensable for H+ translocation and binding of the water-soluble F1 moiety.
Authors: Schneider E , Altendorf K
Published: May 2006

The ATP synthase complex, designated F1F0, of Escherichia coli is composed of a water-soluble portion (F1; membrane-associated ATPase, EC 3.6.1.3) with ATP-hydrolyzing activity and a membrane-integrated part (F0) with H+-translocating activity. F0 is built up from three kinds of subunits (a, b, and c). We have isolated the F0 portion directly from membranes of an E. coli strain (KY 7485) that overproduces the enzyme several fold. Subunit b was extracted from purified F0 by two methods. One method included prolonged incubation of the F0 complex in the presence of trichloroacetate (2.5 M) and the separation of subunit b and an a-c complex by gel filtration. Alternatively, subunit b was extracted by deoxycholate and separated from the a-c complex by hydrophobic-interaction chromatography. Integrated into liposomes, the a-c complex exhibited neither H+ uptake nor binding of F1. However, a functional F0 complex was reconstituted by adding stoichiometric amounts of subunit b to the a-c complex.
Escherichia coli DNA photolyase stimulates uvrABC excision nuclease in vitro.
Authors: Sancar A , Franklin K A , Sancar G B
Published: May 2006

Pyrimidine dimers are the major photoproducts produced in cellular DNA upon UV irradiation. In Escherichia coli there are dark and photorepair mechanisms that eliminate the dimers from DNA and prevent their lethal and mutagenic effects. To determine whether these repair mechanisms act cooperatively or competitively in repairing DNA, we investigated the effects upon one another of DNA photolyase, which mediates photorepair, and uvrABC excision nuclease, an enzyme complex of the uvrABC gene products, which catalyzes nucleotide excision repair. We found that photolyase stimulates the removal of pyrimidine dimers but not other DNA adducts by uvrABC excision nuclease. The two subunits of uvrABC excision nuclease, the uvrA and uvrB proteins which together bind to the dimer region of DNA, had no effect on the activity of photolyase. T4 endonuclease V, which like photolyase is specific for pyrimidine dimers, was inhibited by photolyase, suggesting that these two proteins recognize the same or similar chemical structures in UV-irradiated DNA that are different from those recognized by uvrABC excision nuclease.
Retrovirus-induced expression of interleukin 2 receptors on cells of human B-cell lineage.
Authors: Sugamura K , Fujii M , Kobayashi N , Sakitani M , Hatanaka M , Hinuma Y
Published: May 2006

We provide direct evidence for interleukin 2 receptor (IL2R) induction by human T-cell leukemia/lymphoma virus (HTLV) in human B-cell lines. A lymphoblastoid B-cell line (LCL-Ter) was established by Epstein-Barr virus-induced transformation of peripheral blood lymphocytes derived from a healthy HTLV carrier and cloned in vitro. HTLV gp21 and/or p19 antigens were detected in eight LCL-Ter clones, all of which also expressed the IL2R antigens Tac and Hiei (defined by monoclonal antibodies). However, five other LCL-Ter clones, which were negative for the HTLV antigens, did not express the IL2R antigens. Furthermore, when the IL2R-negative B-cell line LCL-Kan, derived form a normal donor, was cocultured with HTLV-producer cells, three HTLV-carrying clones were obtained and found to constitutively express IL2R. IL2R induced by HTLV on these B-cell lines bound recombinant interleukin 2 and were similar in apparent molecular mass (approximately equal to 60 kDa) to the IL2R of peripheral blood lymphocytes stimulated with phytohemagglutinin.
Analysis of the insulin receptor by anti-receptor antibodies and flow cytometry.
Authors: Maron R , Jackson R A , Jacobs S , Eisenbarth G , Kahn C R
Published: May 2006

We characterized insulin receptors on a human lymphoblastoid cell line (IM-9) and studied their regulation using anti-receptor antibodies and fluorescence flow cytometry. The fluorescence intensity distribution of insulin receptors on cells was determined by incubating the cells with one of three different anti-receptor antisera (human serum B-9 containing polyclonal autoantibodies, serum from a rabbit with polyclonal antibodies, and a monoclonal antibody to the receptor produced in mouse hybridomas), followed by incubation with an appropriate fluorescein isothiocyanate-labeled second antibody and analysis on an Epics-V flow cytometer. All three anti-receptor antibodies specifically labeled the insulin receptors. The monoclonal antibody showed the highest level of labeling. Treatment of cells with proteolytic enzymes, such as trypsin or chymotrypsin, produced a dose-dependent loss of 125I-labeled insulin (125I-insulin) binding but a relatively small decrease in the binding of anti-receptor antibodies, suggesting that most antibody binding occurred in domains other than the insulin binding site. Treatment with glycosidic enzymes, such as neuraminidase and beta-galactosidase did not affect the binding of 125I-insulin, and fluorescence was actually enhanced by about 20% in the beta-galactosidase-treated cells. Exposure of IM-9 cells to insulin resulted in a reduction in the number of insulin receptors. Analysis of the down-regulated cells by immunofluorescence revealed a complete correlation between the percent binding of 125I-insulin and percent peak fluorescence. In all cases, receptors were lost proportionally from all cells, yielding a single symmetrical peak by fluorescence analysis. Exposure of IM-9 cells to anti-receptor antibodies at 37 degrees C for 16 hr also produced a down-regulation in the number of insulin receptors. Incubation with human antiserum B-9 caused a 95% loss of both 125I-insulin binding and peak fluorescence, while the monoclonal antibody resulted in a 50% loss of receptors. Incubation of cells with anti-receptor antibodies for 2 hr at 4 degrees C did not produce any receptor loss; however, the human anti-receptor antisera B-2 and B-9 inhibited the binding of the monoclonal anti-receptor antibody by about 50%, suggesting that these antisera contained autoantibodies directed at the monoclonal antibody binding site. These data indicate that insulin receptors can be regulated by both insulin and anti-receptor antibody and demonstrate the utility of immunofluorescence and flow cytometry as a tool for the study of the insulin receptor.
Involvement of the activated form of RecA protein in SOS mutagenesis and stable DNA replication in Escherichia coli.
Authors: Witkin E M , Kogoma T
Published: May 2006

DNA damage activates RecA protein of E. coli to a form (RecA*) that promotes proteolytic cleavage of LexA protein, the repressor of at least 17 DNA damage-inducible genes, resulting in expression of the SOS response. In addition to this known role, RecA* performs another function necessary for expression of SOS mutagenesis [Blanco, M., Herrera, G., Collado, P., Rebollo, J. & Botella, L. M. (1982) Biochimie 64, 633-636]. The additional role of RecA* could be (i) cleavage of another repressor, (ii) proteolytic processing of one or more proteins, or (iii) mechanistic interaction with DNA or with one or more other proteins. We describe experiments designed to test the first possibility. Our results suggest that neither SOS mutator activity nor ultraviolet mutagenesis requires induction by RecA* of any gene(s) outside the LexA regulon and that the additional role of RecA* is not cleavage of another repressor. We show that stable DNA replication, another DNA damage-inducible function [Kogoma, T., Torrey, T. A. & Connaughton, M. J. (1979) Mol. Gen. Genet. 176, 1-9], shares with SOS mutagenesis the requirement for RecA* activity, even in a strain constitutively expressing all LexA-controlled genes. In this strain, conditions that activate RecA initiate expression of stable DNA replication in the presence of chloramphenicol, without an intervening period of protein synthesis. We conclude that the additional function of RecA* in stable DNA replication is not another antirepressor activity.
Acetylcholine receptor-specific suppressive T-cell factor from a retrovirally transformed T-cell line.
Authors: Sinigaglia F , Gotti C , Castagnoli R , Clementi F
Published: May 2006

In both experimental and human myasthenia gravis an impairment in the immune regulation leads to an increased synthesis of antibodies against the nicotinic acetylcholine receptor (AcChoR). The present work reports the establishment of an AcChoR-specific suppressive T-cell line obtained by viral transformation of AcChoR-enriched murine T lymphocytes. Enriched T cells from Torpedo AcChoR-primed mice, prestimulated in vitro with antigen, were infected with radiation leukemia viruses and injected intravenously in congeneic recipient mice. Six months later lymphomas were observed in 20% of the injected mice and two of them, of donor origin, were established as permanent continuous cell lines in vitro. One of these lines, named LA41, expresses Thy-1.2, Lyt-2, and I-Jb surface markers. Culture supernatants of LA41 cells suppress the antigen-specific in vitro proliferation of Torpedo AcChoR-primed lymphocytes. This suppression is antigen-specific since the response induced by fetal calf AcChoR and by other antigens is not affected by addition of LA41 culture supernatant in the proliferative assay. LA41 culture supernatant injected in vivo at the time of antigen-priming suppresses also significantly the production of anti-AcChoR antibodies but not the synthesis of antibodies against other antigens--i.e., fetal calf AcChoR or alpha-bungarotoxin. These data show that LA41 cells constitutively produce Torpedo AcChoR-specific suppressor factor.
Synthesis in animal cells of hepatitis B surface antigen particles carrying a receptor for polymerized human serum albumin.
Authors: Michel M L , Pontisso P , Sobczak E , Malpièce Y , Streeck R E , Tiollais P
Published: May 2006

A recombinant plasmid (pSVS dhfr) encoding the pre-S region and the S gene of human hepatitis B virus (HBV) and murine dihydrofolate reductase (DHFR) cDNA has been used for the transfection of Chinese hamster ovary (CHO) DHFR- cells. Selection of clones resistant to methotrexate has permitted amplification of HBV sequences and an increase in production of hepatitis B surface antigen (HBsAg). HBV-specific transcripts have been characterized. The HBsAg 22-nm particles contain a receptor for polymerized human serum albumin (pHSA) and elicit in animals the synthesis of antireceptor antibodies. This property is ascribed to a 34,000-dalton polypeptide in the particles, which is most likely encoded by the S gene and part of the pre-S region. Especially because the pHSA receptor is most abundantly present on the virion and because, in hepatitis B infection, the appearance of anti-pHSA receptor antibodies seems to be a highly reliable criterion for viral clearance, the HBsAg particles obtained may constitute a particularly efficient vaccine.
Effects of pH on the structure and function of carboxypeptidase A: crystallographic studies.
Authors: Shoham G , Rees D C , Lipscomb W N
Published: May 2006

High-resolution crystal structures are described for carboxypeptidase A (EC 3.4.17.1) in crystals grown at pH 8.5, 9.0, and 9.5 and compared with the structure at pH 7.5. The comparison shows that in the pH range of 7.5-9.5 the enzyme structure is practically unchanged, and, most importantly, that the flexible side chain of Tyr-248 remains exclusively in the "up" position, away from the Zn atom, throughout the pH range. There is no evidence for binding of Tyr-248 to Zn at any of these pH values. We conclude that the interaction of Tyr-248 with Zn is not an essential part of the mechanism of carboxypeptidase A and that its occurrence is an artifact of chemical modification of Tyr-248. It is also suggested that Tyr-248 is not uniquely associated with the observed high pK of the enzymatic hydrolysis.
Some plant leaves have orientation-dependent EPR and NMR spectra
Authors: McCain Douglas C. , Selig Ted C. , Govindjee  , Markley John L.
Published: May 2006

Proton nuclear magnetic resonance (
1
H NMR) spectra of leaves from 50 plant species were obtained at a spectrometer frequency of 470 MHz. Water present in leaf samples gives rise to characteristic spectral patterns. Most species show only one broad
1
H NMR peak; however, the leaves of some plants display complex, orientation-dependent spectra in which a common three-line pattern is discerned. The pattern varies with the angle between the leaf surface and the external magnetic field. Proton relaxation measurements show the presence of at least two water compartments in the leaves. The compartments are responsible for different components of the spectral pattern. EPR spectra, obtained at 35 GHz and at a temperature of -180°C, of plant leaf sections are dominated by the strong signals of manganous ions. We find that most plant leaves have isotropic Mn
2+
EPR spectra. However, in some species (including ones that exhibit orientation-dependent
1
H NMR spectra) we detect orientation-dependent intensities in the forbidden lines; the spectra indicate that Mn
2+
ions occupy binding sites with axial or lower symmetry on nonrandomly oriented membranes. Both the NMR and the EPR results suggest that the chloroplasts of some plants are preferentially aligned with respect to the leaf surface.
A pheromone-induced developmental switch in Caenorhabditis elegans: Temperature-sensitive mutants reveal a wild-type temperature-dependent process.
Authors: Golden J W , Riddle D L
Published: May 2006

Formation of a developmentally arrested dispersal stage called the dauer larva is enhanced by a Caenorhabditis-specific pheromone and is inhibited by increasing amounts of food. Pheromone-induced dauer larva formation of three tested wild-type strains is temperature-dependent, so that an increased percentage of the population forms dauer larvae at 25 degrees C compared to lower temperatures. Dauer-defective mutants fail to respond to added pheromone, and some behavioral mutants affected in thermotaxis or egg-laying also exhibit abnormal responses. Temperature-sensitive (ts) dauer-constitutive mutants form dauer larvae at a restrictive temperature regardless of environmental stimuli. At the permissive temperature (17.5 degrees C), alleles of six out of seven dauer-constitutive genes tested overrespond to the dauer-inducing pheromone. All known mutations in daf-4 (eight alleles) and daf-7 (five alleles) produce a ts dauer-constitutive phenotype. One daf-4 and one daf-7 allele are suppressed by the amber nonsense suppressor, sup-7(st5). At least these two dauer-constitutive mutations are likely to cause production of nonfunctional rather than ts gene products. These mutations appear to indirectly result in a ts phenotype by enhancing the expression of a wild-type ts developmental process.
Liver disease associated with duck hepatitis B virus infection of domestic ducks.
Authors: Marion P L , Knight S S , Ho B K , Guo Y Y , Robinson W S , Popper H
Published: May 2006

The liver disease associated with duck hepatitis B viremia was investigated in naturally infected ducks from Chi-tung county in China and in both naturally and experimentally infected ducks from the United States. Liver and serum specimens of adult Chinese ducks were examined for duck hepatitis B virus (DHBV) DNA by dot and gel blot hybridization. DHBV was found in serum and (in episomal form only) in livers of 6 of 11 birds exhibiting various degrees of chronic hepatitis. In 1 bird with hepatocellular carcinoma, DHBV DNA was detected at the limit of assay sensitivity and in another not at all, contrasting with findings in humans and woodchucks. In work with California Pekin and Khaki Campbell ducks, known amounts of DHBV were injected into the egg 10 days before, or into ducklings 1 day after, hatching and the livers were examined 6 weeks later. The majority of the injected ducklings had viremia detectable by hybridization 1 or 2 weeks after injection. The presence but not the amount of viremia correlated with incidence and degree of hepatitis, determined under code. The most severe instances of hepatitis, all in Pekin ducks, resembled the hepatitis in adult Chinese ducks of Chi-tung county. Severe and moderate hepatitis were found only in indoor-caged injected animals with viremia and in some uninjected birds without viremia that had been kept in outdoor flocks. The latter hepatitis, as some hepatitis in adult Chinese ducks, may not be related to DHBV. Mild and insignificant hepatitis were also found in injected and noninjected ducklings, some of which had the vertically transmitted spontaneous viremia previously described. The good correlation of experimentally induced viremia with incidence and severity of hepatitis in the Pekin duckling provides a simple, rapid, and relatively inexpensive model to study the relation of lesions to hepatitis B family infection in nonprimates.
The goldfish nervus terminalis: a luteinizing hormone-releasing hormone and molluscan cardioexcitatory peptide immunoreactive olfactoretinal pathway.
Authors: Stell W K , Walker S E , Chohan K S , Ball A K
Published: May 2006

Antisera to two putative neurotransmitters, luteinizing hormone-releasing hormone (LHRH) and molluscan cardioexcitatory tetrapeptide (H-Phe-Met-Arg-Phe-NH2; FMRF-amide), bind specifically to neurites in the inner nuclear and inner plexiform layers of the goldfish retina. Retrograde labeling showed that intraocular axon terminals originate from the nervus terminalis, whose cell bodies are located in the olfactory nerves. Double immunocytochemical and retrograde labeling showed that some terminalis neurons project to the retina; others may project only within the brain. All terminalis neurons having proven retinal projections were both LHRH- and FMRF-amide-immunoreactive. The activity of retinal ganglion cells was recorded with microelectrodes in isolated superfused goldfish retinas. In ON- and OFF-center double-color-opponent cells, micromolar FMRF-amide and salmon brain gonadotropin-releasing factor ( [Trp7, Leu8] LHRH) caused increased spontaneous activity in the dark, loss of light-induced inhibition, and increased incidence of light-entrained pulsatile response. The nervus terminalis is therefore a putatively peptidergic retinopetal projection. Sex-related olfactory stimuli may act through it, thereby modulating the output of ganglion cells responsive to color contrast.
Motor deficits in patients with large-fiber sensory neuropathy.
Authors: Sanes J N , Mauritz K H , Evarts E V , Dalakas M C , Chu A
Published: May 2006

The issue of whether brain signals in the absence of peripheral feedback are sufficient to specify accurate movement was evaluated by studying motor performance in patients with loss of somesthetic afferent input as a result of acquired large-fiber sensory neuropathy. With visual guidance, movements and postures were impaired relatively little, but when visual guidance was unavailable, the patients exhibited postural drift and gross inaccuracy of movement. Impairments were more apparent for smaller (3 degrees) than for larger (15 degrees) movements. Previous studies that have failed to show major motor impairments in deafferented subjects examined movements involving rather large joint displacements, and this may have been a factor in the failure of these studies to reveal severe deficits. The present results demonstrate a critical role for somesthetic feedback in regulating centrally generated levels of motor output and show that central motor programs deprived of such feedback are unable to subserve accurate motor control.
Rearranged sequences of a human Kpn I element.
Authors: Potter S S
Published: May 2006

The complete nucleotide sequence of a human Kpn I element inserted into alpha-satellite DNA is presented. This sequence reveals several features of interest. First, a large block of DNA normally associated with Kpn I elements has been deleted. Second, the order of the remaining sequences is permuted in a manner that cannot be accounted for by simple inversion. Third, a significant open reading frame of 675 bases is detected.
Thymic T cells are driven to expand upon interaction with self-class II major histocompatibility complex gene products on accessory cells.
Authors: Rock K L , Benacerraf B
Published: May 2006

Murine thymocytes induce the monokine interleukin 1 upon in vitro coculture with a radioresistant Ia-bearing accessory cell [murine Ia molecule is a class II major histocompatibility complex (MHC) antigen]. The generation of interleukin 1 is critically dependent on the function of I-region gene products on accessory cells. The induced interleukin 1 appears to allow the activation and proliferation of self-MHC-specific thymocytes. Thus, in the absence of added exogenous factors, there is an Ia-dependent thymocyte proliferation. This selective activation of thymocytes is observed with both mature and immature thymic T cells. This in vitro response results in the selective amplification of developing T cells with self-MHC specificity and could be of importance to the in vivo commitment of T cells to MHC determinants that occurs in the thymus.
IgM in a human neuropathy related to paraproteinemia binds to a carbohydrate determinant in the myelin-associated glycoprotein and to a ganglioside.
Authors: Ilyas A A , Quarles R H , MacIntosh T D , Dobersen M J , Trapp B D , Dalakas M C , Brady R O
Published: May 2006

The IgM in three patients with paraproteinemia and peripheral neuropathy was shown to bind to human myelin-associated glycoprotein (MAG) that had been purified to homogeneity by gel filtration on Sepharose CL-6B. The antigenic determinant reacting with the IgM from all three patients was in the carbohydrate part of the MAG molecule. In addition, the IgM from the same three patients bound to a single ganglioside of human sciatic nerve. The results indicate that the IgM paraproteins in these patients react with a carbohydrate determinant that is shared between MAG and a peripheral nerve ganglioside.
Critical phenomena and thermodynamics of dilute aqueous sodium chloride to 823 K
Authors: Pitzer Kenneth S. , Li Yi-gui
Published: May 2006

Semiempirical equations are developed that represent the behavior of dilute solutions of NaCl in water (steam) in the range 723-823 K where ion pairing is extensive. This supplements the equations given earlier for more concentrated solutions. In this temperature range the system NaCl/H
2
O shows critical behavior with two phases below the critical pressure. The equations for the dilute solutions yield critical behavior. Though the equations for concentrated solutions do not yield critical behavior at the critical pressure, only a very small interpolation function is required to connect smoothly the two equations. The ion-pairing equilibrium constants are reported as well as the Gibbs energies of hydration for both ions and ion pairs.
Monensin prevents terminal glycosylation of the N- and O-linked oligosaccharides of the HLA-DR-associated invariant chain and inhibits its dissociation from the alpha-beta chain complex.
Authors: Machamer C E , Cresswell P
Published: May 2006

In B-lymphoblastoid cells, the HLA-DR-associated invariant chain is processed to a form containing O-linked as well as N-linked oligosaccharides. After neuraminidase treatment, the O-linked carbohydrate is susceptible to digestion with an endoglycosidase (endo-beta-N-acetylgalactosaminidase) that cleaves glycans with the structure Gal(beta 1----3)-GalNAc-Ser/Thr, and sialic acid can be added back to this core oligosaccharide by specific sialyltransferases. Treatment of cells with the sodium ionophore monensin markedly affects the post-translational processing of the invariant chain, although that of associated alpha and beta chains is minimally affected. Only a small portion of the N-linked carbohydrate on the invariant chain is processed to an endoglycosidase-H-resistant form. The sialic acid residues normally found on the O-linked glycans are not added, but at least the first residue, GalNAc, is added. In addition to the changes in glycosylation, an intracellular accumulation of HLA-DR antigens also occurs in monensin-treated cells. The accumulation of HLA-DR antigens and the overall slower turnover rates of the alpha, beta, and invariant polypeptides observed after monensin treatment probably reflects the build-up of newly synthesized proteins in Golgi apparatus-derived vacuoles coupled with a decrease in normal degradation in lysosomes.
beta-Lactamase-catalyzed hydrolysis of acyclic depsipeptides and acyl transfer to specific amino acid acceptors.
Authors: Pratt R F , Govardhan C P
Published: May 2006

beta-Lactamases from all three classes, A, B, and C, catalyze the hydrolysis of specific acyclic depsipeptide (PhCH2CONHCR1R2CO2CHR3CO2H) analogs of acyl-D-alanyl-D-alanine peptides. The depsipeptides investigated, which are chemically as reactive toward nucleophiles as penicillins, are in general poor substrates, although differences between the classes of beta-lactamases have been observed: the order of effectiveness seems to be C greater than B greater than A. Certain class A and C beta-lactamases also catalyze phenylacetylglycyl transfer between phenylacetylglycyl depsipeptides and specific amino acid acceptors, a type of reaction hitherto identified more closely with D-alanyl-D-alanine transpeptidases than with beta-lactamases. Preliminary indications of an acyl-enzyme intermediate in these reactions have been obtained. These results support the suggestion [Tipper, D.J. and Strominger, J.L. (1965) Proc. Natl. Acad. Sci. USA 54, 1133-1141] that beta-lactamases are evolutionary descendants of bacterial cell wall D-alanyl-D-alanine transpeptidases.
Biologically active synthetic fragments of epidermal growth factor: localization of a major receptor-binding region.
Authors: Komoriya A , Hortsch M , Meyers C , Smith M , Kanety H , Schlessinger J
Published: May 2006

A primary receptor-binding region of mouse epidermal growth factor (EGF) was identified by comparing the relative affinities of selected synthetic fragments with overlapping sequences in the EGF receptor-binding assay, using human foreskin fibroblasts. Only synthetic peptides containing the amino acid residues 20-31 in the mouse EGF sequence showed the ability to compete with 125I-labeled EGF in binding to EGF receptors. The affinities of the cyclic EGF fragment [Ala20]EGF-(14-31) and the linear [(S-acetamidomethyl)-Cys20,31]-EGF-(20-31) were approximately 1/10(4) of the affinity of EGF. Despite their reduced receptor affinities, these two peptides exhibited the in vitro biological activities of native EGF, while fragments from other regions of the EGF molecule were devoid of these biological properties. The peptides induced DNA synthesis in human foreskin fibroblasts as measured by [3H]thymidine incorporation into DNA. They also induced EGF receptor clustering and activated the EGF-sensitive kinase, enhancing the autophosphorylation of EGF receptors in a dose-related manner. Moreover, a major antigenic determinant of EGF for rabbit anti-EGF antibodies was identified within this same localized region of the EGF molecule by competition experiments utilizing the synthetic EGF fragments. The predominant EGF antigenic determinant(s) was also found within the fragment [(S-acetamidomethyl)Cys20,31]-EGF-(20-31). The accessibility of the residues in positions 20-31 for antibody recognition is consistent with the conclusion that these residues constitute or contain a major receptor-binding region for EGF.
Autodigestion of lexA and phage lambda repressors.
Authors: Little J W
Published: May 2006

Proteolytic cleavage of lexA repressor is an early step in derepression of the SOS regulatory system of Escherichia coli. In vivo and in vitro data have indicated a role for recA protein in this specific proteolytic reaction. I show here that, under certain conditions, specific in vitro cleavage of highly-purified lexA protein can take place in the absence of recA protein. This autodigestion reaction cleaved the same alanine-glycine bond as did the recA-dependent cleavage reaction. Several lines of evidence argued that it was not due to a contaminating protease activity. Autodigestion was stimulated by alkaline pH. It occurred in the presence of EDTA but was stimulated several fold by the presence of Ca2+, Co2+, or Mg2+. The reaction appeared to be first-order, and its rate was independent of protein concentration over a wide range, strongly suggesting that it is intramolecular. Purified phage lambda repressor also broke down under similar conditions to yield products like those resulting from recA protein action. Phage lambda repressor broke down at a far slower rate than did lexA, as previously observed in the recA-catalyzed in vitro reaction and in vivo. This correlation between the two types of cleavage also extended to the reactions with mutant repressor proteins; taken together with the site specificity, it suggests that autodigestion and recA-dependent cleavage follow, at least in part, a similar reaction pathway. These findings indicate that specific cleavage of lexA protein can be catalyzed by the protein itself and suggest that recA protein plays an indirect stimulatory role, perhaps as an allosteric effector, in the recA-dependent reaction, rather than acting directly as a protease. The protease active site and the recA-recognition site lie in the central or COOH-terminal portion of the lexA protein, since a tryptic fragment containing these portions of lexA protein could take part in both reactions.
Comparison of the complete nucleotide sequences of the genomes of the neurovirulent poliovirus P3/Leon/37 and its attenuated Sabin vaccine derivative P3/Leon 12a1b.
Authors: Stanway G , Hughes P J , Mountford R C , Reeve P , Minor P D , Schild G C , Almond J W
Published: May 2006

As part of a study into the molecular basis of attenuation and reversion to neurovirulence in the Sabin poliovirus vaccines, we have determined the complete nucleotide sequence of a cloned DNA copy of the genome of P3/Leon/37, the neurovirulent progenitor of the type 3 Sabin vaccine strain, P3/Leon 12a1b. Comparison of the sequence with that which we previously obtained for the vaccine strain [Stanway, G., Cann, A. J., Hauptmann, R., Hughes, P., Clarke, L. D., Mountford, R. C., Minor, P. D., Schild, G. C. & Almond, J. W. (1983) Nucleic Acids Res. 11, 5629-5643] indicates that attenuation has been brought about by a maximum of 10 point mutations, at least 5 of which are likely to be of minor significance. Predicted amino acid sequences of all the known virus-encoded proteins show that amino acid substitutions have occurred at only three positions. Two of these are in structural proteins (i.e., Ser----Phe in VP3 and Lys----Arg in VP1), and the third, Thr----Ala, is in the nonstructural protein P2-3b. The distribution and nature of nucleotide and amino acid sequence differences suggest that a single base substitution may be responsible for the attenuated phenotype of the vaccine strain.
Autoradiographic visualization of angiotensin-converting enzyme in rat brain with [3H]captopril: localization to a striatonigral pathway.
Authors: Strittmatter S M , Lo M M , Javitch J A , Snyder S H
Published: May 2006

We have visualized angiotensin-converting enzyme (ACE; dipeptidyl carboxypeptidase, peptidylpeptide hydrolase, EC 3.4.15.1) in rat brain by in vitro [3H]captopril autoradiography. [3H]Captopril binding to brain slices displays a high affinity (Kd = 1.8 X 10(-9) M) and a pharmacological profile similar to that of ACE activity. Very high densities of [3H]captopril binding were found in the choroid plexus and the subfornical organ. High densities were present in the caudate putamen and substantia nigra, zona reticulata. Moderate levels were found in the entopeduncular nucleus, globus pallidus, and median eminence of the hypothalamus. Lower levels were detectable in the supraoptic and paraventricular nuclei of the hypothalamus, the medial habenula, the median preoptic area, and the locus coeruleus. Injection of ibotenic acid or colchicine into the caudate putamen decreased [3H]captopril-associated autoradiographic grains by 85% in the ipsilateral caudate putamen and by greater than 50% in the ipsilateral substantia nigra. Thus, ACE in the substantia nigra is located on presynaptic terminals of axons originating from the caudate putamen, and ACE in the caudate putamen is situated in neuronal perikarya or at the terminals of striatal interneurons. The lack of effect of similar injections into the substantia nigra confirmed that the caudate putamen injections did not cause trans-synaptic changes. The presence of [3H]captopril binding is consistent with an ACE-mediated production of angiotensin II in some brain regions. Although [3H]captopril autoradiography reveals ACE in a striatonigral pathway, there is no evidence for angiotensin II involvement in such a neuronal pathway.
Mutagenicity of quinones: pathways of metabolic activation and detoxification.
Authors: Chesis P L , Levin D E , Smith M T , Ernster L , Ames B N
Published: May 2006

The mutagenicity of various quinones, a class of compounds widely distributed in nature, is demonstrated in the Salmonella TA104 tester strain. The metabolic pathways by which four quinones, menadione, benzo[a]pyrene 3,6-quinone, 9,10-phenanthrenequinone, and danthron, caused mutagenicity in this test system were investigated in detail as were the detoxification pathways. The two-electron reduction of these quinones by NAD(P)H-quinone oxidoreductase (DT-diaphorase) was not mutagenic, whereas the one-electron reduction, catalyzed by NADPH-cytochrome P-450 reductase, was mutagenic, except for danthron, which was only slightly mutagenic. The mutagenicity of the quinones via this pathway was found to be attributable to the generation of oxygen radicals. The cytochrome P-450 monooxygenase also played a significant role in the detoxification and bioactivation of these quinones. For example, phenanthrenequinone was converted to a nonmutagenic metabolite in a cytochrome P-450-dependent reaction, whereas danthron was converted to a highly mutagenic metabolite. These studies show the complexity of metabolic pathways involved in the mutagenicity of quinones.
Detection of erythrocyte membrane proteins, sialoglycoproteins, and lipids in the same polyacrylamide gel using a double-staining technique.
Authors: Dzandu J K , Deh M E , Barratt D L , Wise G E
Published: May 2006

A silver/Coomassie brilliant blue R-250 staining technique that permits a color-coded differentiation of erythrocyte membrane proteins, sialoglycoproteins, and lipids in a single one-dimensional NaDodSO4/polyacrylamide gel has been described. Gels stained first with silver stain and then with Coomassie blue (CB) showed the characteristic blue staining of all conventional CB-sensitive membrane polypeptides, whereas periodic acid-Schiff reagent-sensitive sialoglycoproteins and lipids stained yellow. Several yellow Ag-stained bands corresponding to major and minor sialoglycoproteins were detected at Mr X 10(-3) of 88, 72, 65, 41, 35, 31, 28, 24, and 20. Neuraminidase treatment of intact erythrocytes caused shifts in the electrophoretic mobilities of several yellow-stained bands without affecting the CB-stained polypeptide pattern. These observations afforded evidence that the yellow-staining bands were sialoglycoproteins and lipids. The double-staining technique was used in a topological analysis of the membrane surface of the erythrocyte using protease digestion and selective solubilization. Trypsin cleaved the yellow bands at Mr 88,000 and 41,000. Membrane-associated cleavage products were noted at Mr 58,000 and 38,000. Pronase treatment of intact cells gave membrane-associated cleavage products at Mr 38,000 (yellow) and two CB-stained bands at Mr 58,000 and 60,000. These results suggested that the double-staining technique may be applicable in compositional and topological analyses of other biological membranes.
Selective reduction of c-myc mRNA in Daudi cells by human beta interferon.
Authors: Jonak G J , Knight E
Published: May 2006

Under normal growth conditions, the human lymphoblastoid cell line Daudi expresses high levels of c-myc mRNA. These cells are also sensitive to growth inhibition by interferons. We have compared the levels of mRNA for the c-myc in untreated and human beta interferon (IFN-beta)-treated Daudi cells by RNA dot-blot and blot-hybridization analysis methods. Using a synthetic oligonucleotide complementary to the human c-myc mRNA as the probe, we detected a more than 75% reduction in the c-myc hybridizable poly(A)+ RNA in the IFN-beta-treated cells. This reduction in the c-myc mRNA appears to be selective because the level of actin mRNA is not significantly affected by the IFN-beta treatment. In addition, neither in vitro translation of mRNA extracted from IFN-beta-treated cells nor in vivo synthesis of cellular proteins in IFN-beta-treated cells are quantitatively affected. We surmise that the selective reduction in the amount of c-myc mRNA in IFN-beta-treated Daudi cells may be related to the IFN-induced inhibition of the Daudi tumor cell growth.
A carbohydrate side chain on hemagglutinins of Hong Kong influenza viruses inhibits recognition by a monoclonal antibody.
Authors: Skehel J J , Stevens D J , Daniels R S , Douglas A R , Knossow M , Wilson I A , Wiley D C
Published: May 2006

A single amino acid substitution, Asp-63 to Asn-63, was detected in the hemagglutinin of an antigenic variant of the 1968 Hong Kong (H3) influenza virus that was selected by growth of the wild-type virus in the presence of a monoclonal antibody. The mutation generates an oligosaccharide attachment site, Asn-Cys-Thr at residues 63-65, that is glycosylated. Immunoprecipitation experiments with extracts from variant virus-infected cells prepared in the presence or absence of tunicamycin, which inhibits glycosylation, demonstrate that addition of the new oligosaccharide side chain is required to prevent reaction with the monoclonal antibody. Similar experiments with the virus of the 1969 Hong Kong influenza epidemic, A/England/878/69, which also contains a hemagglutinin glycosylated at residue 63, support this conclusion and provide evidence for the epidemiological significance of carbohydrate-mediated modifications of hemagglutinin antigenicity.
Cloning and expression of cDNA encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation.
Authors: White P C , New M I , Dupont B
Published: May 2006

We isolated a cDNA clone encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation (P-450C21). Serum from rabbits immunized with purified P-450C21 precipitated a single protein from the products of an in vitro translation reaction using bovine adrenal mRNA. This protein migrated with P-450C21 on NaDodSO4/polyacrylamide gel electrophoresis. After sucrose gradient sedimentation, mRNA encoding P-450C21 was found in the 19S fraction. This fraction was reverse transcribed into double-stranded cDNA and inserted into the Pst I site of pBR322 by the dC X dG tailing procedure. Escherichia coli cells transformed with recombinant plasmids were screened with an in situ immunoassay using anti-P-450C21 serum and 125I-labeled staphylococcal protein A. Two colonies consistently bound anti-P-450C21 serum. They were identified as carrying the same plasmid by restriction mapping. This plasmid, pC21a, contains an insert of 520 base pairs. It hybridizes with mRNA encoding P-450C21. The peptide encoded by the insert in pC21a is highly homologous to two peptides isolated from porcine P-450C21 and shows limited homology to the P-450 induced by phenobarbital in rat liver. This clone may be useful in studying the molecular genetics of human congenital adrenal hyperplasia due to 21-hydroxylase deficiency.
Structure and comparison of the oxytocin and vasopressin genes from rat.
Authors: Ivell R , Richter D
Published: May 2006

The gene encoding the precursor protein to the hormone oxytocin and its associated neurophysin has been isolated from a rat genomic library, and its sequence has been determined. The small gene (approximately equal to 850 base pairs) predicts a mRNA of approximately equal to 500 bases [without the poly(A) tail]. The exon-intron organization is similar to that of the vasopressin gene, with two splice sites in the protein-coding region. The first exon (A) comprises the 5' noncoding promoter region, a putative signal peptide, the nonapeptide hormone oxytocin, and the NH2-terminal, variable region of neurophysin. The second exon (B) encodes the central, conserved region of neurophysin, and the third exon (C) encodes the remaining COOH terminus of neurophysin, with an additional arginine residue at its end, presumably cleaved off during post-translational processing. A stretch of 143 nucleotides within exon B, except for a single base change, is entirely homologous to the equivalent part of the rat vasopressin gene, offering support for a gene conversion event having recently affected the two genes.
Increase in the Bmax of gamma-aminobutyric acid-A recognition sites in brain regions of mice receiving diazepam.
Authors: Ferrero P , Guidotti A , Costa E
Published: May 2006

gamma-Aminobutyric acid (GABA) receptors were characterized in vivo by studying ex vivo the binding of [3H]muscimol to cerebellum, cortex, hippocampus, and corpus striatum of mice receiving intravenous injections of tracer doses of high-specific-activity (approximately equal to 30 Ci/mmol) [3H]muscimol. This ligand binds with high affinity (apparent Kd, 2-3 X 10(-9) M) to a single population of binding sites (apparent Bmax, 250-180 fmol per 10 mg of protein). Pharmacological studies using drugs that selectively bind to GABAA or GABAB receptors suggest that [3H]muscimol specifically labels a GABAA recognition site. Moreover, diazepam (1.5 mumol/kg, i.p.) increases the Bmax but fails to change the affinity of [3H]muscimol binding to different brain areas. This diazepam-elicited increase in Bmax is blocked in mice receiving the diazepam antagonist Ro 15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a]-[1,4] benzodiazepine-3-carboxylate). Since the diazepam-induced increase of [3H]muscimol binding is paralleled by a significant potentiation of the inhibitory effect of muscimol on locomotor activity, it is proposed that the facilitatory action on GABAergic transmission elicited in vivo by diazepam is mediated by an increase in the Bmax of the binding sites of GABAA receptors.
Effects of monocular deprivation on the lateral geniculate nucleus in a primate.
Authors: Sesma M A , Irvin G E , Kuyk T K , Norton T T , Casagrande V A
Published: May 2006

In many mammalian species, rearing with one eyelid closed produces a loss of vision in the deprived eye and a change in cell size in the lateral geniculate nucleus (LGN). In cats, the reduction in the size of deprived LGN cells has been correlated with a loss of one functional class of cells, Y cells. In primates, such as galago, LGN cells also exhibit marked changes in size with deprivation. In the present study we recorded from single cells in the LGN of monocularly deprived galagos to determine if such changes in cell size would be accompanied by changes in physiological properties. The results revealed no alterations in the distribution or functional properties of any cell class. The differences in the effects of monocular deprivation on the function of LGN cells in cats and primates are most easily explained by a fundamental difference in visual system anatomy. In cats, different classes of retinal afferents (X vs. Y) are in a position to compete for postsynaptic LGN neurons: in primates, segregation of cell classes into different layers may preclude such developmental interactions.
Transformation by Rous sarcoma virus prevents acetylcholine receptor clustering on cultured chicken muscle fibers.
Authors: Anthony D T , Schuetze S M , Rubin L L
Published: May 2006

Acetylcholine receptors aggregate in the membrane of cultured chicken myotubes; the process of receptor clustering can be stimulated by exogenous factors that we, among others, have begun to characterize. Chicken myoblasts transformed by temperature-sensitive mutants of Rous sarcoma virus, such as tsNY68, fuse to form multinucleated myotubes at 42 degrees C, the nonpermissive temperature for transformation. However, tsNY68-infected myotubes do not cluster acetylcholine receptors at 42 degrees C, even in the presence of active clustering agents. This phenomenon is not merely a result of viral infection, since myotubes infected with a transformation-deficient viral mutant, td107A, behave like noninfected myotubes with respect to receptor clustering; thus, the effects of tsNY68 on the clustering process must be mediated by the src gene product. These experiments may provide a method of identifying essential elements of acetylcholine receptor clusters.