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Biomedical subjects

N Nelson

Publications and source records attributed to N Nelson.

At least 91 records · Page 5Linked to original sources
PubMed1995

Cloning and expression of a yeast gene encoding a protein with ATPase activity and high identity to the subunit 4 of the human 26 S protease.

The cloning, expression, and biochemical characterization of an essential gene of Saccharomyces cerevisiae that encodes for a new member of the TBP1-like subfamily of putative ATPases are described. The protein is 72% identical at the amino acid level to subunit four (S4) of the human 26 S protease and 73% identical to Schizosaccharomyces pombe MTS2 gene product. The purified, recombinant protein, designated Yhs4p, has an estimated molecular mass of 49 kDa and exhibits a Mg(2+)-dependent ATPase activity with nucleotide specificity and Km for ATP similar to those exhibited by the human 26 S protease. The observed ATPase activity was reduced by 73% upon the introduction of point mutation K229Q in the "P-loop" domain of the ATP-binding site relative to the nonmutated form of the protein. This is the first direct biochemical evidence supporting the putative ATPase activity of a member of the TBP1-like subfamily. Furthermore, the experimental results demonstrate a regulatory function for the amino-terminal region of the molecule. The amino-terminal truncated form of Yhs4p lacking two clusters of positively charged amino acids exhibits a greater ATPase activity. The ATPase activity of both the truncated and complete forms of Yhs4p is stimulated by polyanions. Polylysine partially inhibits the ATPase activity of the amino-terminal truncated form having no observable effect on the complete protein. N-Ethylmaleimide inhibits the ATPase activity of both forms of Yhs4p. We propose that Yhs4p ATPase may play an essential role in the regulatory function of the proteolytic activity of the yeast 26 S protease.

Adenosine Triphosphatases↗
PubMed1995

A bovine cDNA and a yeast gene (VMA8) encoding the subunit D of the vacuolar H(+)-ATPase.

Subunit D of vacuolar H(+)-ATPase (V-ATPase) from bovine chromaffin granules was subjected to partial proteolysis and amino acid sequencing. A cDNA encoding this subunit was isolated and sequenced. The predicted open reading frame encodes a protein of 247 amino acids with a calculated molecular weight of 28,336. Northern blot analysis revealed an mRNA distribution with higher transcript amounts in tissues that are active in secretion. A homologous gene was identified as open reading frame 11 in chromosome V of Saccharomyces cerevisiae. The two proteins exhibit 55% identity with several conservative replacements. Interruption of the yeast gene, denoted as VMA8, resulted in the null mutant delta vma8::URA3 that, like all the other V-ATPase null mutants, did not grow on medium buffered at pH 7.5 and showed no accumulation of quinacrine into their vacuoles. Transformation of the null mutant with a plasmid containing the VMA8 gene restored the wild-type phenotype. This supports the conclusion that subunit D is an integral subunit of the catalytic sector of V-ATPase and its structural analysis suggests analogy to the gamma subunit of F-ATPases.

Adrenal Medulla↗
PubMed1995

Isolation of LERK-5: a ligand of the eph-related receptor tyrosine kinases.

Hek and elk are members of the eph-related family of receptor tyrosine kinases. Recently we isolated four cDNAs encoding membrane-bound ligands to hek and elk [Beckman et al. (1994) EMBO J. 13, 3757-3762; Kozlosky et al. (1995) Oncogene 10, 299-306]. Because of the promiscuous nature of their binding, we have termed these proteins ligands of the eph-related kinases or LERKs. A search of GenBank revealed an expressed sequence tag (EST) with homology to the LERKs. Using this EST as a probe, we have isolated human and murine cDNAs that encode a protein which we call LERK-5. The human and murine cDNAs encode proteins of 333 and 336 amino acids, respectively, with a 97% amino acid identity; LERK-5 has an amino acid identity of 27-59% with the other reported LERKs. LERK-5 is a ligand for both elk and hek and induces receptor phosphorylation. It is expressed in adult lung and kidney and the fetal tissues heart, lung, kidney, and brain. In addition, Southern blot analysis of DNA from interspecific backcross mice indicated that LERK-5 (Eplg5) maps to the proximal region of mouse chromosome 8.

Amino Acid Sequence↗
PubMed1995

Toxicity, biodistribution and radioprotective capacity of L-homocysteine thiolactone in CNS tissues and tumors in rodents: comparison with prior results with phosphorothioates.

L-Homocysteine thiolactone (L-HCTL) was evaluated for its potential as an intravenously-administered central nervous system (CNS) radioprotector in C3H mice and F344 rats. Toxicity assessments in the mouse yielded a LD50 of 297 mg/kg and in the rat 389 mg/kg. Biodistribution studies in tumor-bearing mice showed that brain specimens contained more label at 10 min than the tumors but less at 30 or 60 min. Brain uptake relative to the tumors, the brain/tumor ratio, ranged between 0.5 and 3.3. The cervical spinal cord of non-tumor-bearing rats was irradiated with 32 Gy 137Cs with or without prior treatment with L-HCTL following which the time to forelimb or hindlimb paralysis was measured to determine the relative protective factors (RPFs) for this radiation dose. For forelimb paralysis the RPF was 1.9 (+/- 1.0, SD) and for hindlimb it was 2.0 (+/- 1.1, SD). 36B-10 glioma cells irradiated in vitro with or without L-HCTL and assayed for colony forming capacity demonstrated a dose modifying factor (DMF) of only 1.15 (+/- 0.16, SE). Rats bearing intracerebral 36B-10 glioma received 137Cs irradiation with or without L-HCTL after which the tumors were similarly assayed in vitro. From this the glioma DMF was 1.2 (+/- 0.30, SE). Compared to prior results with phosphorothioates our data show that the toxicity of L-HCTL is roughly the same as WR2721, WR77913 and WR3689 and that it distributes at higher levels in the CNS after systemic administration. L-HCTL may well equal these phosphorothioates at protecting normal CNS tissue without requiring administration directly into the cerebrospinal fluid-containing spaces and it does not protect the 36B-10 glioma.

Amifostine↗
PubMed1995

Cellular expression of glycine transporter 2 messenger RNA exclusively in rat hindbrain and spinal cord.

High-affinity transporters mediate the removal of released neurotransmitters from synapses, thereby terminating their synaptic action. A novel glycine transporter has recently been cloned from a rat brain complementary DNA library. In this study we examined, by means of in situ hybridization with 35S-labelled oligodeoxynucleotide probes, the distribution of messenger RNAs encoding glycine transporter 2 in the rat CNS. Moreover, adjacent series of sections were labelled with [3H]strychnine to reveal the regional distribution of strychnine-sensitive glycine receptors. A very discrete pattern of distribution of the transcripts was found exclusively at the level of the brainstem/cerebellum and spinal cord. In the cerebellum, Golgi cells in the granule cell layer as well as a subpopulation of neurons in the interposed nuclei were consistently labelled. In the brainstem, where the bulk of the labelling was concentrated, several nuclei showed a high level of transcript expression, including the superior olivary complex, nucleus of the trapezoid body and the ventral nucleus of the lateral lemniscus. In the spinal cord, many neurons throughout all layers were labelled, including putative Renshaw cells and a few large neurons at the border of layers 7 and 9. No labelled cells were detected at the levels of the fore- and midbrain. The distribution of glycine transporter 2 messenger RNA-containing cell bodies was very different to that of other glycine transporter messenger RNAs (glycine transporter 1a and glycine transporter 1b), but similar to that of known glycine-immunoreactive neurons and correlated very well with that of strychnine-sensitive glycine receptors in most CNS regions except cerebellum. Our results show that glycine transporter 2 (but not glycine transporter 1) in the brainstem, spinal cord and cerebellum is probably involved in the reuptake of glycine from synapses containing classical strychnine-sensitive glycine receptors. Our findings also suggest that glycine acts as a neurotransmitter in cerebellar Golgi neurons. Whether the synaptic concentration of glycine, as co-agonist at NMDA receptors, is regulated (if at all) by transaminase activity or by a glycine transporter (GLYT1a?) distinct from that described here is not yet known.

Amino Acid Transport Systems, Neutral↗
PubMed1995

Localization of glycine neurotransmitter transporter (GLYT2) reveals correlation with the distribution of glycine receptor.

We studied by immunocytochemical localization, the glycine neurotransmitter transporter (GLYT2) in mouse brain, using polyclonal antibodies raised against recombinant N-terminus and loop fusion proteins. Western analysis and immunocytochemistry of mouse brain frozen sections revealed caudal-rostral gradient of GLYT2 distribution with massive accumulation in the spinal cord, brainstem, and less in the cerebellum. Immunoreactivity was detected in processes with varicosities but not cell bodies. A correlation was observed between the pattern we obtained and previously reported strychnine binding studies. The results indicate that GLYT2 is involved in the termination of glycine neurotransmission accompanying the glycine receptor at the classic inhibitory system in the hindbrain.

Amino Acid Transport Systems, Neutral↗
PubMed1995

Severe Rh(D) immunization: anti-D quantitation and treatment possibilities during pregnancy and after birth.

An extremely aggressive Rh(D), (C) and Kell alloimmunization during pregnancy is reported. Exceptionally high concentrations of anti-D were observed in the mother, in the fetus and in the amniotic fluid, indicating an active transport across the placenta and a passive excretion into the amniotic fluid. Treatment during pregnancy included maternal plasmapheresis and high-dose intravenous immunoglobulin. Intravascular transfusions were given to the fetus. Postpartum the newborn was given immunoglobulin, one exchange transfusion and four top-up transfusions. In the newborn the elimination rate of anti-D could be followed. Not until almost 4 months postpartum did the anti-D concentration drop below the level of detection. This coincided with an elevated reticulocyte production and appearance of the child's true blood group in parallel with ceasing need for blood transfusions. Elimination rate and absolute anti-D values can be used as a prognostic tool to predict the need of blood transfusions. Immunoglobulin treatment can also be considered as an optional form of treatment in newborns affected by alloimmunization.

Bilirubin↗
PubMed1995

Clinical and histologic appearance in enamel of primary teeth from children with neonatal hypocalcemia induced by blood exchange transfusion.

This investigation was undertaken to study the clinical and histologic appearance of deciduous enamel from 11 infants who were subjected to blood exchange transfusions (ET) during the first days of life. As a result of the treatment they had a mean of 3 consecutive hypocalcemic days. At the age of 5 years a dental examination of their primary teeth was performed. Four of the children had clinically recorded enamel aberrations correlated with the neonatal period. Exfoliated teeth were then examined histologically. All had a normal major enamel morphology, and the neonatal line was present in all teeth. Histologically investigated teeth with enamel hypoplasia had the aberrations located close to the neonatal line. The conclusions drawn from this study are that hypocalcemia caused by ET in the newborn period did not affect the width of the neonatal line or the major enamel morphology to any extent. Enamel aberrations occurred only when four or more ETs were performed.

Dental Enamel↗
PubMed1994

Purification of the synaptic vesicle-binding protein physophilin. Identification as 39-kDa subunit of the vacuolar H(+)-ATPase.

Physophilin is a 36-kDa polypeptide originally identified in synaptic plasma membrane fractions, which binds to synaptic vesicles and has been implicated in vesicle docking and/or exocytosis during neurotransmitter release. Here we report on the purification, amino acid sequence analysis, and subcellular localization of physophilin. Physophilin was enriched from detergent extracts of crude synaptic plasma membranes by a combination of cation exchange and lentil-lectin chromatography. Sequence analysis of peptides generated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that physophilin is identical to the 39-kDa subunit (Ac39) of the vacuolar H(+)-ATPase. This was confirmed further by Western blot analysis with an Ac39-specific antiserum and by vesicle binding assays with recombinant Ac39 protein. Subcellular fractionation showed that Ac39 is enriched in synaptic vesicles, with lesser amounts being present in synaptic plasma membrane fractions. These results argue against a docking role of physophilin/Ac39 in synaptic vesicle exocytosis.

Amino Acid Sequence↗
PubMed1994

Features of vacuolar H(+)-ATPase revealed by yeast suppressor mutants.

The yeast Saccharomyces cerevisiae serves as an excellent model for the study of the structure and function of proteins. Numerous amino acid substitutions in the proteolipid subunit of yeast vacuolar H(+)-ATPase have been reported. Suppressed variants for several of the inactive mutants were selected after subjecting them to chemical or polymerase chain reaction mutagenesis and screening for second site suppressors. Suppressors for the mutation Gln90 to Lys change were intragenic and resulted from the changes: Ala14 to Val, Val74 to Ile, Ile89 to Leu, and Ile89 to Met. These residues are found on three different transmembrane segments but presumably at the same surface of the membrane. A new inactive proteolipid mutation was constructed by changing Val138 to Leu. This residue is situated in the middle of the fourth transmembrane segment, neighboring Glu137 which is the potential dicyclohexylcarbodiimide-binding site. The intragenic suppressor mutations for the above amino acid replacement resulted in changes of Val55 to Ala, Met59 to Val, or Ile130 to Thr. These residues are found in the second and fourth transmembrane segments, presumably on the same interface. It seems as if all those internal suppressor mutations compensate for the volume changes caused by the original displacement of the given amino acid. Five glycine residues, situated on the same face of the third transmembrane helix, were changed to valine and all these mutants were inactive. A suppressor mutation to one of those mutants (Gly101 to Val) was identified as substitution of Ile134 to Val. The structural and functional implications of these findings are discussed.

Amino Acid Sequence↗
PubMed1994

A novel accessory subunit for vacuolar H(+)-ATPase from chromaffin granules.

Three subunits, Ac115, Ac39, and the proteolipid, were positively identified in the membrane sectors of V-ATPases from different sources. We searched for organelle-specific protein in purified preparations of V-ATPase from bovine chromaffin granules. A diffused protein band at a position of about 45 kDa was identified in SDS-polyacrylamide gels of the above preparation. Following digestion with endopeptidase Glu-C (V-8), a polypeptide of about 10 kDa was isolated and subjected to amino acid sequencing. Hence, the cDNA encoding the protein Ac45 was cloned from a bovine adrenal medulla library. The cDNA sequence contains an open reading frame encoding a protein of 468 amino acids with a calculated molecular mass of 51,786 daltons. A potential signal sequence comprised of the first 35 amino acids and a potential transmembrane domain at the C terminus of the protein were identified. There exist seven potential glycosylation sites between the aforementioned protein motifs. Experiments with a specific antibody against Ac45 demonstrated that it is copurifying with the V-ATPase from chromaffin granules. Immunological cross-reactivity was observed with purified V-ATPase from bovine kidney microsomes but not from plasma membranes of epithelial cells. Cell-free expression of the protein from synthetic mRNA produced a single protein band at about 50 kDa on SDS gels. Upon inclusion of dog pancreas microsomes in the reaction mixture, a slow migrating band sensitive to peptide:N-glycosidase F was observed.

Adrenal Medulla↗
PubMed1994

The Saccharomyces cerevisiae VMA7 gene encodes a 14-kDa subunit of the vacuolar H(+)-ATPase catalytic sector.

Yeast vacuoles contain an H(+)-ATPase that acidifies the vacuolar lumen and generates an electrochemical gradient of protons across their membranes. Five polypeptides, denoted as subunits A to E in the order of decreasing molecular masses from 69 to 27 kDa, were identified in the catalytic sector of the enzyme. However, other polypeptides may be necessary for the activity of the enzyme. When a 14-kDa polypeptide was suggested to be a subunit of a specialized V-ATPase in Manduca sexta, and a homologous short sequence was detected downstream of the UGA1 gene in yeast, we cloned this counterpart gene from yeast. The gene VMA7 encodes a protein Vma7p of about 14 kDa. The predicted protein is highly homologous to the above mentioned M. sexta protein. The delta vma7::URA3 null mutant exhibits growth characteristics typical of other VMA disruptant mutants in genes encoding subunits of the catalytic sector. The delta vma7::URA3 null mutants are not able to grow on a medium buffered at pH 7.5, they fail to accumulate quinacrine into their vacuoles and the other subunits of the catalytic sector are not assembled onto the vacuolar membrane in its absence. Epitope-tagged Vma7p was constructed and the proton uptake activity of isolated vacuoles from this yeast strain was markedly inhibited by a monoclonal antibody against the epitope-tag. A cold inactivation experiment demonstrated that Vma7p is a genuine subunit of the catalytic sector of V-ATPase and it is denoted as subunit F.

Amino Acid Sequence↗
PubMed1994

Functional analysis of conserved cysteine residues in the catalytic subunit of the yeast vacuolar H(+)-ATPase.

The A subunit of the yeast vacuolar ATPase contains three highly conserved cysteines: Cys-261, Cys-284, and Cys-538. Cys-261 is located within the nucleotide-binding P-loop. Each of the conserved cysteines, and one nonconserved cysteine, Cys-254, were altered to serine by site-directed mutagenesis, and the effects on growth at pH 7.5 were determined. The Cys-254-->Ser, Cys-261-->Ser and the double mutants all grew at pH 7.5 and contained nitrate- and bafilomycin-sensitive ATPase activity. However, the ATPase activities of the Cys-261-->Ser and the double mutants were insensitive to the sulfhydryl group inhibitor, N-ethylmaleimide, demonstrating that Cys-261 is the site of inhibition by N-ethylmaleimide. Changing either Cys-284 or Cys-538 to serine prevented growth at pH 7.5. Cys-284 and Cys-538 thus appear to be essential cysteine residues which are required either for assembly or catalysis.

Base Sequence↗
PubMed1994

Mutagenesis of the b'-subunit of Synechocystis sp. PCC 6803 ATP-synthase.

We investigated the F0F1 ATP synthase of the cyanobacterium, Synechocystis sp. PCC 6803. The gene for the F0-subunit b', a peptide probably located at the interface between F0 and F1, has been partially or completely evicted from the bacterial genome. We found that the complete deletion of the subunit was lethal to the cells. However, the subunit could be truncated down to its hydrophobic N-terminal stretch without much harm. Since the gene for b' probably shares a common ancestor with the gene for subunit b and emerged by gene duplication, we propose that b' gathered a new role during evolution, perhaps in the regulation of photophosphorylation.

Amino Acid Sequence↗