PubMed Health⌕ Search

Biomedical subjects

N Ramesh

Publications and source records attributed to N Ramesh.

At least 73 records · Page 4Linked to original sources
PubMed1993

Chromosomal localization of the gene for human B-cell antigen CD40.

CD40 is a surface glycoprotein expressed on all human B lymphocytes and plays an important role in B-cell development, growth, and differentiation. Anti-CD40 monoclonal antibodies cause isotype switching in B cells treated with IL-4. CD40 is a member of a family of proteins that include low-affinity nerve growth factor receptor, TNF receptor, and the antigen Fas. The ligand for CD40 had been recently identified and has been assigned to the X chromosome. Using a panel of human-rodent somatic cell hybrids, we now show that CD40 maps to human chromosome 20.

Animals↗
PubMed1993

Role of CD40-CD40-ligand interaction in Ig-isotype switching.

Ig-isotype switching to IgE requires interleukin-4 and a contact-dependent T-cell signal delivered by the ligand for CD40. The recent discovery that defects in the gene encoding the CD40 ligand are the underlying cause of the X-linked hyper IgM syndrome highlights the role of CD40 and its ligand in Ig-isotype switching.

Animals↗
PubMed1993

High-level human adenosine deaminase expression in dog skin fibroblasts is not sustained following transplantation.

Primary skin fibroblasts are an attractive target tissue for retroviral-mediated gene therapy; however, transient expression of therapeutic genes has been a recurrent problem in several rodent models. The gradual decrease in gene expression could be tissue or species specific. To investigate the phenomenon further, human adenosine deaminase (ADA) expression was monitored in genetically modified skin fibroblasts transplanted in beagle dogs. In culture, transduced canine fibroblasts expressed high levels of human ADA activity (33.6 mumoles adenosine metabolized per hour per milligram of soluble protein) in comparison to canine ADA in untreated control cells (1.3 mumol/hr.mg protein) and for 2 weeks following transplantation, the graft contained up to four-fold more enzyme activity from human ADA than the endogenous canine enzyme. However, by 10 weeks, human ADA expression was undetectable. At the time when human ADA expression was greatly reduced, DNA analysis showed the presence of vector sequences. These results directly parallel those observed in rodent models and suggest retroviral vector inactivation is a tissue-specific rather than species-specific mechanism.

Adenosine Deaminase↗
PubMed1993

Deletions in the ligand for CD40 in X-linked immunoglobulin deficiency with normal or elevated IgM (HIGMX-1).

Patients with X-linked Ig deficiency with normal or elevated IgM (HIGMX-1) fail to switch from IgM/IgD to other Ig isotypes. Interaction between the B cell antigen CD40 and the CD40 ligand expressed on activated T cells is critical for T cell driven isotype switching. We have reported that T lymphocytes from three unrelated male patients with HIGMX-1 failed to express CD40 ligand on their surface, but the mRNA for CD40 ligand was of an apparently normal size and level. Analysis of CD40 ligand cDNA from two of the patients revealed deletions that alter the reading frame. Patient 1 displayed two mutations: a C-->A transversion at nucleotide 590 and the deletion of an adjacent C nucleotide. The second patient had a 58 bp deletion from nucleotides 289-346. Furthermore, neither patient expressed a protein product detectable by the CD40L mAb, 5c8.

Antibodies, Monoclonal↗
PubMed1992

A toxic shock syndrome toxin-1 peptide that shows homology to mycobacterial heat shock protein 18 is presented as conventional antigen to T cells by multiple HLA-DR alleles.

We observe that PBMC from most adults (16 of 18 subjects tested) show a small but significant in vitro proliferative response to a 30-amino acid-long peptide (peptide 2, amino acids 34-63) derived from toxic shock syndrome toxin. By contrast, PBMC from newborn blood and thymocytes do not proliferate to this peptide, and furthermore, peptide 2 did not displace the binding of radiolabeled TSST-1 to MHC class II positive cells, nor did it induce IL-1 beta mRNA in monocytes, indicating that this peptide does not behave as a superantigen. Proliferation of PBMC to peptide 2 could be blocked by anti-HLA-DR, but not by anti-HLA-DP or DQ mAb, suggesting that HLA-DR molecules are the restriction elements for the recognition of this peptide by T cells. This premise was further confirmed by demonstrating that mouse L cells transfected with human HLA-DR, but not HLA-DP or DQ molecules, supported the proliferation of purified T cells to peptide 2. Studies with subjects of known HLA-DR types showed that all types tested are capable of responding to this peptide, PBMC from adults exposed to mycobacterial Ag showed significantly better proliferative response to peptide 2 than unexposed adults. Studies with truncations of this peptide suggest that a "core" region of eight amino acids that is conserved between low m.w. heat shock proteins and peptide 2 may be critical to T cell recognition of this peptide. The universal presentation of peptide 2 by HLA-DR molecules may contribute to the widespread natural immunity observed against toxic shock syndrome toxin.

Adult↗
PubMed1991

Assay of neomycin phosphotransferase activity in cell extracts.

A simple method for the measurement of neomycin phosphotransferase (NPT) activity in crude extracts of eukaryotic cells is described. This method is based on the elimination of interfering phosphorylated proteins by using phenol-chloroform extraction. This solution phase assay allows the detection of greater than or equal to 0.01 ng of NPT in the crude cell extract. Rapid screening of a large number of cell cultures generated in gene-transfer experiments, using NPT as a selective marker, is made possible by this simple technique. Further, the promoter strength of vector constructs used in gene therapy may also be estimated by this procedure.

Cell Extracts↗
PubMed1991

Induction of human T cell proliferation by a monoclonal antibody to CD5.

A mouse mAb, TS 43, which recognized the human CD5 molecule, was found to induce the proliferation of human peripheral blood T cells. TS 43 mAb precipitated from 125I-radiolabeled T cells a 67-kDa band, which comigrated with the 67-kDa band precipitated by the anti-CD5 mAb OKT1. Preclearing of cell lysates with OKT1 mAb abolished the capacity of TS 43 mAb to precipitate radiolabeled material from T cell lysates. Furthermore, a mouse T cell hybridoma transfected with human CD5 was stained by TS 43 mAb. T cell proliferation mediated by TS 43 mAb was monocyte dependent, and was accompanied by IL-2R expression and by IL-2 synthesis. T cell activation by TS 43 mAb involved a rise in intracellular calcium level (CA2+)i and was dependent on the expression of the TCR/CD3 complex because no rise in (Ca2+)i was observed in a TCR-beta-deficient Jurkat T cell mutant. This study indicates that CD5 should be added to the list of surface molecules that can signal T cells to proliferate.

Antibodies, Monoclonal↗
PubMed1991

Internal initiation of translation in retroviral vectors carrying picornavirus 5' nontranslated regions.

Previous work has shown that picornavirus 5' nontranslated regions (NTRs) can initiate internal translation of downstream coding regions both in vitro and in transient in vivo assays. We have used 5' NTR sequences from encephalomyocarditis virus and poliovirus to construct retroviral vectors that are designed to express two proteins from a single mRNA. Inclusion of 5' NTR sequences did not adversely affect vector titer. Protein expression was studied with stable cell lines generated by vector infection of mouse NIH 3T3 cells and human and canine skin fibroblasts. Expression of a coding region in the downstream position was at levels from 25 to 100% of the same coding region in the upstream position. Expression of downstream coding regions in control vectors that did not contain the 5' NTR sequences was very low, in agreement with the predictions of the scanning model for eukaryotic translation. These experiments demonstrate coordinate expression of two coding regions from a single mRNA in stable cell lines and provide further support for the model of internal translation initiation by sequences in the 5' NTRs of picornaviruses.

Adenosine Deaminase↗
PubMed1990

Supercoil-induced unusual DNA structures as transcriptional block.

The transcriptional activity of pBR322 form V DNA template, a topologically unlinked, highly supercoiled molecule having unusual structures around or within coding regions was studied. Significant transcription was observed in vitro from this template despite high levels of supercoiling. An attenuated transcript, initiated accurately from the P4 promoter of rep gene, was observed which indicated pausing of E. coli RNA polymerase within the gene. This pausing could be removed by relieving the torsional stress implying that a supercoil induced structural alteration within the gene was acting as a transcriptional block. A stabilized unusual structure, most likely a cruciform, was found to be responsible for the elongation block. Absence of initiation from the tetR gene was correlated with the unusual structure present within its promoter region in form V DNA. These in vitro studies show that structural alterations within natural DNA could act as transcriptional blocks both at the level of initiation and elongation.

Base Sequence↗
PubMed1990

Probing of unusual DNA structures in topologically constrained form V DNA: use of restriction enzymes as structural probe.

The ability of DNA sequences to adopt unusual structures under the superhelical torsional stress has been studied. Sequences that are forced to adopt unusual conformation in topologically constrained pBR322 form V DNA (Lk = 0) were mapped using restriction enzymes as probes. Restriction enzymes such as BamHI, PstI, AvaI and HindIII could not cleave their recognition sequences. The removal of topological constraint relieved this inhibition. The influence of neighbouring sequences on the ability of a given sequence to adopt unusual DNA structure, presumably left handed Z conformation, was studied through single hit analysis. Using multiple cut restriction enzymes such as NarI and FspI, it could be shown that under identical topological strain, the extent of structural alteration is greatly influenced by the neighbouring sequences. In the light of the variety of sequences and locations that could be mapped to adopt non-B conformation in pBR322 form V DNA, restriction enzymes appear as potential structural probes for natural DNA sequences.

Base Sequence↗
PubMed1990

Type II hereditary angioneurotic edema that may result from a single nucleotide change in the codon for alanine-436 in the C1 inhibitor gene.

Identical single-base changes in the C1 inhibitor gene that may result in dysfunctional inhibitor proteins are described in two different families with type II hereditary angioneurotic edema. Initially, a restriction fragment length polymorphism was defined that resulted from loss of a Pst I site within exon VIII, which encodes the region containing the reactive center. Exon VIII from the normal and abnormal allelles was amplified by the polymerase chain reaction. Amplified DNA product was cloned into plasmid pUC18; clones representing normal and mutant allelles were distinguished by the presence and absence, respectively, of the Pst I restriction site. DNA sequence analysis revealed a G----A mutation in the codon for alanine-436, which would result in replacement with a threonine residue. This position is nine amino acid residues amino-terminal to the reactive-center arginylthreonine peptide bond. In contrast, previously defined mutations in type II hereditary angioneurotic edema result in replacement of the reactive-center arginine.

Alanine↗
PubMed1989

DNA duplex with the potential to change handedness after every half a turn.

Polymorphic forms of the DNA duplex with long stretches of structural monotony are known. Several alternating purine-pyrimidine sequences have been shown to adopt left-handed Z-conformation. We report a DNA sequence d(CGCGCGATCGAT)n exhibiting alternating right-handed B and left-handed Z helical conformation after every half a turn. Further, this unusual conformation with change in handedness after every six base pairs was induced at physiological superhelical density.

Base Sequence↗
PubMed1989

Zintrons in rat alpha-lactalbumin gene.

The eukaryotic genome is characterised by the occurrence of a large amount of repetitive DNA which exists within and around coding sequences. Random repeats of (TG)n sequences have been observed in several introns. In this paper we show that (TG)14 and (TG)24 sequences present in the third intron of rat alpha-lactalbumin gene adopt left-handed Z-helices under varying superhelical densities. The overall sequence of the parent plasmid further influenced the level of supercoiling at which the B to Z transition could be induced in (TG)n sequences. Such Z-potential intervening sequences (zintrons) could act as buffers to maintain desired levels of supercoiling near the transcribed sequences.

Animals↗
PubMed1989

Structural alteration from non-B to B-form could reflect DNase I hypersensitivity.

Preferential cleavage of active genes by DNase I has been correlated with a structurally altered conformation of DNA at the hypersensitive site in chromatin. To have a better understanding of the structural requirements for gene activation as probed by DNase I action, digestability by DNase I of synthetic polynucleotides having the ability to adopt B and non-B conformation (like Z-form) was studied which indicated a marked higher digestability of the B-form of DNA. Left handed Z form present within a natural sequence in supercoiled plasmid also showed marked resistance towards DNase I digestion. We show that alternating purine-pyrimidine sequences adopting Z-conformation exhibit DNAse I foot printing even in a protein free system. The logical deductions from the results indicate that 1) altered structure like Z-DNA is not a favourable substrate for DNase I, 2) both the ends of the alternating purine-pyrimidine insert showed hypersensitivity, 3) B-form with a minor groove of 12-13 A is a more favourable substrate for DNase I than an altered structure, 4) any structure of DNA deviating largely from B form with a capacity to flip over to the B-form are potential targets for the DNase I enzymic probes in naked DNA.

DNA↗
PubMed1989

Role of the environment in the interaction of nonintercalators with Z-DNA.

Interaction of the DNA binding nonintercalators Netropsin, Distamycin and the mPD derivative with Z-DNA has been studied. It has been found that environmental factors like the solvent and added cations significantly modulate the interaction of these ligands with Z-DNA. However no definite Z to B transition in presence of these ligands was found in any case, in contrast to previously reported results (Ch. Zimmer, C. Marck and W. Guschlbauer, FEBS Lett. 154, 156-160 (1983)).

Chemical Phenomena↗