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H Nelson

Publications and source records attributed to H Nelson.

At least 145 records · Page 8Linked to original sources
PubMed1992

A family of genes encoding neurotransmitter transporters.

The genomic and cDNA clones of the mouse gamma-aminobutyric acid transporter were sequenced and analyzed. The genomic clone contains 12 introns including 1 intron prior to the initiator methionine. The second intron comes immediately after the stretch of amino acids that is most conserved among the neurotransmitter transporters sequenced so far. By using a probe constructed according to this conserved region, several partial genomic clones were isolated. Sequence analysis of those clones reveals not only homology to the family of neurotransmitter transporters within the reading frame but also an identical location of an exon-intron junction after the conserved region. A search of the GenBank data base (April 1991) revealed that two invertebrate genes exhibit homology to the conserved sequence of the above family. One, a Drosophila melanogaster gene, encoded the N-terminal part of a protein homologous to neurotransmitter transporters and the second was in Caenorhabditis elegans. The Drosophila gene contains an intron that starts at a position identical to the corresponding positions of all the mammalian genes of the family.

Amino Acid Sequence↗
PubMed1992

Cloning and expression of a glycine transporter from mouse brain.

We have isolated a cDNA clone from a mouse brain library encoding the glycine transporter (GLYT). Xenopus oocytes injected with a synthetic mRNA accumulated [3h]glycine to levels of up to 80-fold above control values. The uptake was specific for glycine and dependent on the presence of Na+ and Cl- in the medium. The cDNA sequence predicts a highly hydrophobic protein of 633 amino acids with 12 potential transmembrane helices. The predicted amino acid sequence has 40-45% identity to the GABA, noradrenaline, serotonin and dopamine transporters. This implies that all of these neurotransmitter transporters may have evolved from a common ancestral gene that diverged into the GABA, glycine and catecholamine subfamilies at nearly the same time.

Amino Acid Sequence↗
PubMed1992

Compartment acidification is required for efficient sorting of proteins to the vacuole in Saccharomyces cerevisiae.

The vacuole of the yeast Saccharomyces cerevisiae contains a proton-translocating ATPase that acidifies the vacuolar lumen and generates a pH gradient across the vacuole membrane. We have investigated the role of compartment acidification of the vacuolar system in the sorting of vacuolar proteins. Strains with chromosomal disruptions of the genes encoding the A, B, or c subunit of the vacuolar ATPase are unable to acidify their vacuoles. These vat mutant strains accumulate and secrete precursor forms of the soluble vacuolar hydrolases carboxypeptidase Y and proteinase A. The kinetics of secretion suggests that missorting occurs in the Golgi complex or in post-Golgi vesicles. The presence of mature forms of the vacuolar proteins within the cell indicates that vat mutations do not cause defects in zymogen processing. Precursor forms of the membrane-associated vacuolar hydrolase alkaline phosphatase are also accumulated in vat mutant cells but to a lesser extent, suggesting that sorting of vacuolar membrane proteins is less sensitive to changes in the lumenal pH. A similar type of missorting defect can be induced in wild-type cells at pH 7.5. These results indicate that acidification of the vacuolar system is important for efficient sorting of proteins to the vacuole.

Aspartic Acid Endopeptidases↗
PubMed1992

Cloning and mutational analysis of the gene encoding subunit C of yeast vacuolar H(+)-ATPase.

A DNA fragment containing the gene encoding subunit C of vaculor H(+)-ATPase (V-ATPase) was cloned from a yeast library. The predicted amino acid sequence indicated that the C subunit consists of 373 amino acids with a calculated molecular mass of 42,287 Da. The protein from yeast is 37% identical in its amino acid sequence to the C subunit of bovine V-ATPase. The DNA fragment that was cloned in this study contained two additional reading frames. At the 5' end an amino acid sequence that is homologous to Artemia elongation factor 1 was detected. At the 3' end the N-terminal part of a kinesin-like protein was observed. The gene encoding subunit C of the V-ATPase was interrupted, and the resulting mutant could not grow at high pH and was sensitive to low and high Ca2+ concentrations in the growth medium. Transformation of the mutant by a plasmid containing the gene encoding subunit C repaired the phenotype of the mutant. Substitution of more than half of the coding region by a corresponding DNA fragment encoding the bovine subunit C resulted in a phenotype indistinguishable from wild type. Immunological studies with the disruptant mutant revealed that subunit C is necessary for the assembly of the catalytic sector of the enzyme.

Amino Acid Sequence↗
PubMed1992

Heteroconjugated antibodies enhance lymphocyte-mediated tumour cell lysis in vitro and in vivo.

Covalent linkage of an antitumour antibody specific for a tumour cell surface antigen to an antilymphocyte antibody specific for the T lymphocyte receptor complex produces a heteroconjugated antibody that can activate and redirect cytotoxic T lymphocytes to lyse tumour cells. The ability of an antilymphocyte-antitumour heteroconjugate (500A2 x 96.5) to direct the lysis of murine melanoma cells by cultured murine lymphocytes was tested in vitro using a 4-h chromium release assay and in vivo with a tumour neutralization assay. In vitro, the addition of heteroconjugated antibody significantly increased tumour lysis by murine C3H/HeN lymphocytes (median specific lysis 82.7 per cent with lymphocytes plus heteroconjugate versus 9.5 per cent for lymphocytes alone, P less than 0.001). In vivo, treatment with heteroconjugated antibody plus lymphocytes significantly reduced the development of pulmonary metastases after intravenous tumour administration (median number of pulmonary metastases 28.5 for combined treatment versus 250 for heteroconjugate or lymphocytes alone, P less than 0.001).

Animals↗
PubMed1992

Mutations in the yeast vacuolar ATPase result in the mislocalization of vacuolar proteins.

The vacuolar ATPase of the yeast Saccharomyces cerevisiae acidifies the vacuolar lumen and generates an electrochemical gradient across the vacuole membrane. We have investigated the role of compartment acidification of the vacuolar system in the sorting of vacuolar proteins. Strains with chromosomal disruptions of genes (delta vat) encoding the A (69 x 10(3) M(r)), B (57 x 10(3) M(r)) or c (16 x 10(3) M(r)) subunits of the vacuolar ATPase accumulate and secrete precursor forms of the soluble vacuolar hydrolases carboxypeptidase Y and proteinase A. A kinetic analysis suggests that these precursor proteins accumulate in, and are secreted from, the Golgi complex or post-Golgi vesicles. In addition, subcellular fractionation shows that vacuolar hydrolase-invertase hybrid proteins are inefficiently localized to the vacuole in delta vat strains. This result suggests that the vat mutations cause a steady-state defect in vacuolar protein sorting. The vat mutations also affect the sorting of vacuolar membrane proteins. Precursor forms of alkaline phosphatase are accumulated in vat mutant cells, but to a lesser extent than is seen for the soluble vacuolar hydrolases. This finding, coupled with the insensitivity of alkaline phosphatase to the ATPase inhibitor bafilomycin A1, suggests that vacuolar membrane protein sorting is less sensitive to changes in lumenal pH when compared with the targeting of soluble vacuolar proteins. These results indicate that acidification of the vacuolar system is important for efficient sorting of soluble proteins to the vacuole.

Adenosine Triphosphatases↗
PubMed1991

T cell recognition of transforming proteins encoded by mutated ras proto-oncogenes.

Activated ras proto-oncogenes contribute to the pathogenesis of many animal and human malignancies. ras proto-oncogenes are generally activated by point mutations within codons 12 or 61, which result in the expression of ras protein (p21) bearing characteristic single amino acid substitutions at the corresponding residues. The purpose of the current study was to determine whether the presence of single transforming amino acid substitutions can render normal ras protein immunogenic and, thus, a possible target for T cell-mediated tumor therapy. In initial experiments, C57BL/6 mice were immunized with a synthetic peptide corresponding to residues 5 through 16 of p21 containing the transforming substitution of arginine for normal glycine at residue 12. The results demonstrated that class II MHC-restricted T cells which were specific for the peptide could be elicited, and that the peptide-induced T cells could specifically recognize the corresponding intact p21 ras protein. Recognition of p21 ras protein by peptide-specific T cells implies that C57BL/6 APC can process the activated ras protein in a fashion that allows presentation of digested protein by class II MHC molecules in a configuration similar to the configuration with synthetic peptide. Evaluation of the immunogenicity of peptides containing alternative transforming amino acid substitutions of ras protein demonstrated that some, but not all, were immunogenic in individual strains of mice. Therefore, although ras protein-specific T cells can be elicited by immunization with synthetic peptides, not all of the potential ras mutations commonly associated with malignancy may be recognizable by T cells from all individuals.

Amino Acid Sequence↗
PubMed1991

Mutational analysis of yeast vacuolar H(+)-ATPase.

Yeast mutants in which genes encoding subunits of the vacuolar H(+)-ATPase were interrupted were assayed for their vacuolar ATPase and proton-uptake activities. The vacuoles from the mutants lacking subunits A (72 kDa), B (57 kDa), or c (proteolipid, 16 kDa) were completely inactive in these reactions. Immunological studies revealed that in the absence of each one of those subunits the catalytic sector was not assembled. Labeling with N,N'-[14C]dicyclohexylcarbodiimide showed the presence of the proteolipid in vacuoles of mutants in which genes encoding subunits of the catalytic sectors were interrupted. No labeling was detected in the mutant in which the gene encoding the proteolipid was interrupted. We conclude that of all the ATPase subunits only the proteolipid is assembled independently and it serves as a template for the assembly of the other subunits. Site-specific mutations were generated in the gene encoding the proteolipid. All of the drastic changes and replacements gave inactive proteins. About half of the single amino acid replacements gave active proteins. Replacing glutamic acid-137 by any of several amino acids, except for aspartic acid, abolished the activity of the enzyme. Other amino acids that may function in proton conductance were changed. It was found that glycine residues may replace amino acids with exchangeable protons.

Amino Acid Sequence↗
PubMed1991

Anti-tumor X anti-lymphocyte heteroconjugates augment colon tumor cell lysis in vitro and prevent tumor growth in vivo.

Cross-linking an anti-tumor antibody, specific for tumor cell surface antigens, and an anti-lymphocyte antibody, specific for the T lymphocyte receptor complex (TCR/CD3), produces a heteroconjugate that can direct T cells to lyse tumor cells. We tested the ability of anti-tumor X anti-lymphocyte (CD3) heteroconjugates to redirect human peripheral blood lymphocytes (PBLs) to lyse human colon cancer cells in cytotoxicity assays and in a murine colon tumor model. We demonstrated in vitro, that cultured human PBLs alone produced low levels of tumor lysis, but PBLs treated with anti-tumor X anti-CD3 heteroconjugates produced significantly greater tumor cell lysis (P less than 0.0025). Similarly, nude mice injected with LS174T human colon cancer cells and treated with cultured human PBLs and anti-tumor X anti-CD3 heteroconjugates survived significantly longer than saline control mice (P less than 0.01), or mice treated with PBLs alone (P less than 0.01), or heteroconjugates alone (P less than 0.05). F(ab')2 heteroconjugates were equally as effective in prolonging animal survival, but irrelevant heteroconjugates and monoclonal anti-tumor antibodies showed no therapeutic benefit. Anti-tumor X anti-CD3 heteroconjugates may represent an effective approach to tumor-specific cellular immunotherapy.

Animals↗
PubMed1991

Effect of PaCO2 on cerebral blood flow distribution during halothane compared with isoflurane anaesthesia in the rat.

In order to examine anaesthetic effects on the distribution of cerebral blood flow (CBF) during normo- and hypocapnia, male adult Sprague-Dawley rats were allocated randomly to four groups in a 2 x 2 factorial design, using PaCO2 value and anaesthetic agent as between-group factors. Animals were anesthetized with either 1.38% isoflurane (inspired) or 1.05% halothane (inspired) and the lungs ventilated mechanically at either normocapnia (PaCO2 5.1-5.6 kPa) or hypocapnia (PaCO2 3.1-3.3 kPa) for 1 h. CBF was measured using 14C-iodoantipyrine autoradiography. Local CBF in selected cortical and subcortical regions of interest and area-weighted mean global CBF were calculated. Data were compared by analysis of variance. Normocapnic (mean (SE] CBF for halothane (n = 6) and isoflurane (n = 7) was 120 (8) ml/100 g min-1 (PaCO2 5.6 (0.49) kPa) and 117 (9) ml/100 g min-1 (PaCO2 5.4 (0.5) kPa), respectively. Hypocapnic CBF for halothane (n = 6) and isoflurane (n = 6) was 82 (7) ml/100 g min-1 (PaCO2 3.3 (0.12) kPa) and 82 (6) ml/100 g min-1 (PaCO2 3.2 (0.12) kPa), respectively. Hypocapnia reduced global CBF for both groups by 30% (P less than 0.001), but there was no difference between anaesthetic agents (P greater than 0.8). Hypocapnia decreased CBF in all local structures examined. Although subcortical structures had similar CBF at both normocapnia and hypocapnia, CBF in three cortical samples was greater (P less than 0.05) in both the normocapnic and hypocapnic halothane groups than the corresponding isoflurane groups. The CBF reactivity to changes in PaCO2 was similar for both agents (approximately 2 ml/100 g min-1 mm Hg). (ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesia, Inhalation↗
PubMed1991

Targeted cellular immunotherapy with bifunctional antibodies.

Linking an antitumor cell antibody with an antilymphocyte antibody produces a bifunctional antibody that can redirect T lymphocytes to lyse tumor cells. Bifunctional antibodies that bypass the normal specificity of the T cell antigen receptor can, theoretically, retarget all of a cancer patient's cytotoxic T lymphocytes to kill tumor cells. Studies have demonstrated that bifunctional antibodies redirect effector cells to lyse human tumor cells in vitro and neutralize human tumor xenografts in animal models. Several different effector cell populations have been studied and many different human tumors have been targeted for lysis. Current research goals include the improvement of methods for the preparation of antibody reagents and the enhancement of cellular trafficking to tumors.

Animals↗
PubMed1991

Perineal hidradenitis suppurativa: presentation of two unusual complications and a review.

Two patients with advanced perineal hidradenitis suppurativa, complicated by fecal incontinence and squamous cell carcinoma, are presented. The first patient was a 58-year-old man who had a 30-year history of chronic recurring perianal abscesses and perineal sinuses. At the time of presentation, he had extensive perineal suppurative disease, and scarring and fixation of the anal sphincters with resultant fecal incontinence. He was treated with wide excision and skin graft closure. The second patient was a 27-year-old man with an 11-year history of recurrent gluteal abscesses and perineal sinuses. At the time of presentation, his inflammatory disease was only mildly active, but he had a nonhealing gluteal lesion. The nonhealing lesion was diagnosed as a squamous cell carcinoma and was managed with wide excision and primary closure. The inflammatory disease was excised and grafted. Complications of advanced hidradenitis suppurativa can be debilitating and life threatening. We review the etiology, pathophysiology, complications, and treatment options of hidradenitis suppurativa, including a literature review of the association with malignancy. We propose that the incidence of disabilities and complications may be reduced by early diagnosis and treatment, by emphasis on prevention of recurrence, and by more aggressive surgical intervention for recurrent and extensive disease.

Adult↗
PubMed1990

Molecular cloning of cDNA encoding the C subunit of H(+)-ATPase from bovine chromaffin granules.

A cDNA encoding subunit C of the V-ATPase from bovine chromaffin granules was cloned and sequenced. The gene encodes a hydrophilic protein of 382 amino acids with a calculated molecular weight of 43,989. Hydropathy plots revealed no apparent transmembrane segments and a rather high helix content was detected. A cDNA encoding most of the C subunit of the V-ATPase of human brain was also cloned and sequenced. The deduced amino acid sequence of this gene is almost identical to the bovine polypeptide with only one change of tyrosine 336 that was replaced by histidine in the human gene. Two polypeptide fragments derived from subunit E of V-ATPase from chromaffin granules were sequenced and found to be identical to the predicted amino acid sequence of this subunit from bovine kidney. These observations support the idea that the amino acid sequences of corresponding subunits from different V-ATPases are highly conserved. Unlike the A and B subunits of V-ATPases, that are homologous to the beta and alpha subunits of F-ATPases, subunits C and E showed no homology with analogous subunits of the F-ATPase family. It is proposed that the addition of the C and gamma subunits to the respective V- and F-ATPases during evolution defined them as two separate families of H(+)-ATPases.

Amino Acid Sequence↗
PubMed1990

Regional and systemic distribution of anti-tumor x anti-CD3 heteroaggregate antibodies and cultured human peripheral blood lymphocytes in a human colon cancer xenograft.

Anti-tumor antibody (317G5) covalently coupled to an anti-CD3 antibody (OKT3) produces a heteroaggregate (HA) antibody that can target PBL to lyse tumor cells expressing the appropriate tumor Ag. The i.v. and i.p. distribution of radiolabeled HA antibody 317G5 x OKT3 and of radiolabeled cultured human PBL were studied in athymic nude mice bearing solid intraperitoneal tumor established from the human colon tumor line, LS174T. Mice were injected with 125I-labeled HA antibody, 125I-labeled anti-tumor mAb, or 111In-labeled PBL, and at designated timepoints tissues were harvested and measured for radioactivity. 125I-317G5 x OKT3 localized specifically to tumor sites. Tumor radioactivity levels (percent injected dose/gram) were lower with 125I-317G5 x OKT3 HA antibody than with 125I-317G5 anti-tumor mAb, but were similar to levels reported for other anti-tumor mAb. The major difference in radioactivity levels observed between i.v. and i.p. administration of 125I-317G5 x OKT3 was an increase in hepatic radioactivity after i.v. HA antibody administration. HA antibodies produced from F(ab')2 fragments, which exhibit decreased m. w. and decreased Fc receptor-mediated binding, demonstrated improved tumor:tissue ratios as compared to intact antibody HA. 125I-317G5 F(ab')2 x OKT3 F(ab')2 antibody levels were equivalent to intact HA antibody levels in tumor, but were lower than intact HA antibody levels in the blood, bowel, and liver. Tumor:bowel ratios (20:1 at 48 h) were highest when 317G5 F(ab')2 x OKT3 F(ab')2 was injected i.p. Autoradiography confirmed that anti-tumor x anti-CD3 HA antibodies localized specifically to intraperitoneal tumor; that i.p. administered HA antibodies penetrated tumor directly; and that i.v. administered HA antibodies distributed along tumor vasculature. Cultured human PBL distributed in moderate concentrations to intraperitoneal tumor when administered i.p., but not when administered i.v. The poor localization of i.v. injected PBL to tumor may reflect species disparity in homing receptors and/or endothelial ligands, a problem which may be overcome with a syngeneic model. These results suggest that regional therapy with HA antibodies and PBL may offer advantages over systemic therapy for initial clinical trials.

Animals↗
PubMed1990

Cloning and expression of a rat brain GABA transporter.

A complementary DNA clone (designated GAT-1) encoding a transporter for the neurotransmitter gamma-aminobutyric acid (GABA) has been isolated from rat brain, and its functional properties have been examined in Xenopus oocytes. Oocytes injected with GAT-1 synthetic messenger RNA accumulated [3H]GABA to levels above control values. The transporter encoded by GAT-1 has a high affinity for GABA, is sodium-and chloride-dependent, and is pharmacologically similar to neuronal GABA transporters. The GAT-1 protein shares antigenic determinants with a native rat brain GABA transporter. The nucleotide sequence of GAT-1 predicts a protein of 599 amino acids with a molecular weight of 67 kilodaltons. Hydropathy analysis of the deduced protein suggests multiple transmembrane regions, a feature shared by several cloned transporters; however, database searches indicate that GAT-1 is not homologous to any previously identified proteins. Therefore, GAT-1 appears to be a member of a previously uncharacterized family of transport molecules.

Amino Acid Sequence↗
PubMed1990

Cloning of the human brain GABA transporter.

A cDNA clone encoding a transporter for the neurotransmitter gamma-aminobutyric acid in human brain was cloned and sequenced. The cDNA contains an open reading frame encoding a hydrophobic protein of 599 amino acids with a calculated molecular weight of 67022 Da. Hydropathy analysis revealed twelve potential transmembrane segments. The human protein is highly homologous to the protein from rat brain. Northern hybridization demonstrated a ubiquitous distribution of the transporter in various parts of the brain.

Amino Acid Sequence↗