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R Raju

Publications and source records attributed to R Raju.

At least 37 records · Page 2Linked to original sources
PubMed1997

Epitope repertoire of human CD4+ T cells on tetanus toxin: identification of immunodominant sequence segments.

Sequence regions of tetanus toxin-forming CD4+ cell epitopes in 8 HLA-disparate subjects were identified. Overlapping synthetic peptides corresponding to the complete tetanus toxin sequence were used to test, in a proliferation assay, unselected blood CD4+ cells or CD4+ cell lines propagated by stimulation with tetanus toxoid. The CD4+ cell lines recognized most peptides recognized by the blood CD4+ cells and they recognized additional peptides. Their responses were stronger than those of unselected blood CD4+ cells. Two peptides were recognized by all subjects: one largely overlapped a tetanus toxin sequence region previously identified as a "universal" T cell epitope. Thirteen other peptides elicited a CD4+ cell response in 6 or 7 of the 8 subjects, and another 10 elicited responses in 5 subjects.

Adult↗
PubMed1997

RNA-RNA recombination in Sindbis virus: roles of the 3' conserved motif, poly(A) tail, and nonviral sequences of template RNAs in polymerase recognition and template switching.

Sindbis virus (SIN), a mosquito-transmitted animal RNA virus, carries a 11.7-kb positive-sense RNA genome which is capped and polyadenylated. We recently reported that the SIN RNA-dependent RNA polymerase (RdRp) could initiate negative-strand RNA synthesis from a 0.3-kb 3'-coterminal SIN RNA fragment and undergo template switching in vivo (M. Hajjou, K. R. Hill, S. V. Subramaniam, J. Y. Hu, and R. Raju, J. Virol. 70:5153-5164, 1996). To identify and characterize the viral and nonviral sequences which regulate SIN RNA synthesis and recombination, a series of SIN RNAs carrying altered 3' ends were tested for the ability to produce infectious virus or to support recombination in BHK cells. The major findings of this report are as follows: (i) the 3'-terminal 20-nucleotides (nt) sequence along with the abutting poly(A) tail of the SIN genome fully supports negative-strand synthesis, genome replication, and template switching; (ii) a full-length SIN RNA carrying the 3'-terminal 24 nt but lacking the poly(A) tail is noninfectious; (iii) SIN RNAs which carry 3' 64 nt or more without the poly(A) tail are infectious and regain their poly(A) tail in vivo; (iv) donor templates lacking the poly(A) tail do not support template switching; (v) full-length SIN RNAs lacking the poly(A) tail but carrying 3' nonviral extensions, although debilitated to begin with, evolve into rapidly growing poly(A)-carrying mutants; (vi) poly(A) or poly(U) motifs positioned internally within the acceptor templates, in the absence of other promoter elements within the vicinity, do not induce the jumping polymerase to reinitiate at these sites; and (vii) the junction site selection on donor templates occurs independently of the sequences around the acceptor sites. In addition to furthering our understanding of RNA recombination, these studies give interesting clues as to how the alphavirus polymerase interacts with its 3' promoter elements of genomic RNA and nonreplicative RNAs. This is the first report that an in vitro-synthesized alphavirus RNA lacking a poly(A) tail can initiate infection and produce 3' polyadenylated viral genome in vivo.

Animals↗
PubMed1996

Epitope repertoire of human CD4+ lines propagated with tetanus toxoid or with synthetic tetanus toxin sequences.

The use of synthetic antigen sequences allows propagation in vitro of T cell lines and clones specific for rare antigens, or for individual epitopes. In the present study we investigated the extent of similarity of the epitope repertoire of CD4+ T cell line specific for the antigen tetanus toxin (TTX), propagated with the complete molecule of tetanus toxoid (TTD), and with the synthetic TTX peptides. We propagated from two healthy subjects CD4+ T cell lines specific for TTD, by cycles of stimulation in vitro with TTD or with pools of overlapping synthetic peptides, 20 residues long and overlapping by five residues, corresponding to all or part of the tetanus toxin (TTX) sequence. One pool corresponded to the complete TTX sequence (peptide pool). Two other pools corresponded to residues 1-305 of the TTX light chain and 476-780 of the TTX heavy chain (peptide minipools). The peptide pool-propagated lines recognized TTD vigorously, at levels comparable with those of the TTD-propagated lines. They recognized several peptides, most of which were also recognized by the TTD-propagated line from the same subject. They also recognized to a low extent a few peptides not recognized by the corresponding TTD-propagated line, which might contain cryptic epitopes. The TTD-propagated lines recognized also several peptides that did not elicit a detectable response by the lines propagated with the complete peptide pool. The peptide minipool propagated lines recognized most of the peptides recognized by the TTD-propagated lines. They also recognized several peptides that did not elicit a measurable response of the TTD-propagated line from the same subject, which might contain cryptic epitopes. Very few peptides recognized by the TTD-propagated line did not evoke a response from the peptide minipool propagated lines. To verify that the response to the TTD molecule of the lines propagated with the peptide pools reflected the response of clones recognizing different epitopes produced upon in vitro processing of the TTD molecule, we propagated from each of the two subjects CD4+ T cell lines by stimulation with individual peptide recognized by the TTD-specific lines of that subject (13 peptide-specific lines from subject #1, and 15 from subject #2). All lines responded to the presence of TTD to an extent comparable to the response induced by the same concentration of the relevant peptide, demonstrating that propagation by synthetic epitope sequences allows expansion of T cell clones specific for epitopes which result from processing of the complete TTD molecule. Therefore, whereas the use of very large pools of synthetic antigen peptides for propagation of antigen specific human CD4+ cell lines might lead to loss of clones recognizing less immunogenic sequence regions, peptide pools comprising a relatively limited number of synthetic sequences allow propagation of the majority of the antigen specific T cell clones. The use of peptide pools, and especially of limited peptide pools, results in propagation of polyclonal T cell lines having a more diverse repertoire than the lines propagated by stimulation with the complete antigen molecule. The T clones propagated by the use of the short peptide sequences, which are not expanded when the complete antigen molecule is used, may recognize poorly processed, cryptic epitopes. This approach may be adopted to propagate and detect minor clonal populations, recognizing less immunogenic parts of the antigen.

Adult↗
PubMed1996

Nonhomologous RNA-RNA recombination events at the 3' nontranslated region of the Sindbis virus genome: hot spots and utilization of nonviral sequences.

The mechanism of RNA-RNA recombination at the 3' nontranslated region (3'NTR) of the Sindbis virus (SIN) genome was studied by using nonreplicative RNA precursors. The 11.7-kb SIN genome was transcribed in vitro as two nonoverlapping RNA fragments. RNA-1 contained the entire 11.4-kb protein coding sequence of SIN and also carried an additional 1.8-kb nonviral sequence at its 3' end. RNA-2 carried the remaining 0.26 or 0.3 kb of the SIN genome containing the 3'NTR. Transfection of these two fragments into BHK cells resulted in vivo RNA-RNA recombination and release of infectious SIN recombinants. Eighteen plaque-purified recombinant viruses were sequenced to precisely map the RNA-RNA crossover sites at the 3'NTR. Sixteen of the 18 recombinants were found to be genetically heterogeneous at the 3'NTR. Two major clustered sites within the 3'NTR of RNA-2 were found to be fused to multiple locations on the nonviral sequence of RNA-1, resulting in insertions of 10 to 1,085 nucleotides at the 3'NTR. Sequence analysis of crossover sites suggested only limited homology and heteroduplex-forming capability between substrate RNAs. Analysis of additional 23 recombinant viruses generated by mutagenized donor and acceptor templates supports the occurrence of recombination hot spots on donor templates. Introduction of a 17-nucleotide rudimentary replicase recognition signal in the acceptor template alone did not induce the polymerase to reinitiate at the 17-nucleotide signal. Interestingly, deletion of a 24-nucleotide hot spot locus on the donor template abolished crossover events at one of the two sites and allowed the polymerase to reinitiate at the 17-nucleotide replicase recognition signal inserted at the acceptor template. The possible roles of RNA-protein and RNA-RNA interactions in the differential regulation of apparent pausing, template selection, and reinitiation are discussed.

Base Sequence↗
PubMed1996

The teleos virtual environment toolkit for simulation-based surgical education.

In the past, surgical simulations have largely been created through the development of dedicated applications that require considerable programming and computer graphics skills. Advances in simulation-based surgical education have been limited by this dependence on computer graphics programming expertise. To lower barriers to the adoption of simulation-based training, we are developing tools based on Teleos, software technology that allows a wide variety of medical content developers to author surgical training simulations without programming.

Computer Graphics↗
PubMed1995

Epitopes for human CD4+ cells on diphtheria toxin: structural features of sequence segments forming epitopes recognized by most subjects.

The sequence regions of diphtheria toxin (DTX) recognized by CD4+ T cells of seven healthy humans of different major histocompatibility complex haplotypes were identified. Overlapping synthetic peptides, screening the DTX sequence, were used to test in proliferation assays unselected blood CD4+ cells, or DTX-specific CD4+ lines propagated by stimulation with DTX of blood mononuclear cells. Blood CD4+ cells and DTX-specific CD4+ lines gave consistent results. Although each subject had an individual pattern of peptide recognition, six peptide sequences (residues 271-290, 321-340, 331-350, 351-370, 411-430 and 431-450) were recognized by all subjects. In the native DTX molecule, these sequence regions are flanked by sequence loops exposed on the DTX surface. They overlap uncharged segments of the DTX sequence. These structural properties may be general requirements for immunodominance in CD4+ cell sensitization in humans.

Adult↗
PubMed1995

Genesis of Sindbis virus by in vivo recombination of nonreplicative RNA precursors.

Genetically engineered RNA transcripts coding for various Sindbis virus (SIN) genes were used to study structure and sequence requirements of RNA recombination in BHK cells. Three different groups of RNA transcripts were made: (i) RNAs which retain the ability to replicate and which carry sequences coding for either viral polymerase or viral structural proteins; (ii) RNAs which lack the complete 3' end of the SIN genome and thus are incapable of replicating; and (iii) RNAs which lack the complete 5' end of the SIN genome and also are incapable of replicating. BHK cells were transfected with specific combinations of these precursor RNAs, and virus production and RNA synthetic abilities of the released virus were determined. We demonstrate in vivo generation of infectious SIN by fusion of (i) replicative RNAs to nonreplicative RNAs and (ii) two nonreplicative RNA precursors. Both homologous and nonhomologous types of recombinations were observed. In the homologous type of recombination, a 694-nucleotide overlap at the crossover region of the first pair of precursors resulted in the addition of an A residue converting the UAG stop codon of nonstructural protein P4 to a UAA stop codon. In the nonhomologous type of recombination, the crossover sites contained deletion of up to 76 nucleotides from one of the precursors and complete preservation of junction sequence from the other precursor. This is also the first report that a cytoplasmic RNA virus can be generated from biologically nonreplicative RNA precursors. These results have implications for initiation of viral RNA synthesis and recombination between RNA viral genomes in general. We favor template switching as a mechanism for the fusion events described here and suggest inclusion of polymerase scanning of diverse nonreplicative RNAs as an inherent feature of the copy choice model of RNA recombination. Very importantly, the facile nature of RNA recombination occurring between nonreplicative RNA precursors should speed up the production and analysis of targeted mutants of SIN and possibly other RNA viruses.

Animals↗
PubMed1995

I-45 islet cell antigen is a 68kD neuroendocrine protein.

A monoclonal antibody approach was used to characterize islet cell differentiation antigens involved in autoimmunity related diabetes mellitus. This procedure yielded islet cell monoclonal antibodies (ICMAbs) that demonstrated varying tissue/cellular distribution. The ICMAb I-45 showed a pan-islet reactivity similar to the reactivity of islet cell autoantibodies. The target antigen of the ICMAb I-45 demonstrated a neuroendocrine distribution. Single step immunoaffinity purification of I-45 antigen using I-45 monoclonal antibody immunoaffinity matrix yielded a 68kD protein. The specificity of the immunoaffinity purified 68kD protein was further demonstrated by the lack of binding of this protein to immunoaffinity columns of irrelevant monoclonal antibodies. The neuroendocrine distribution of the I-45 antigen, like that of other differentiation molecules like HISL-19, neuron specific enolase and chromogranin A strengthens the hypothesis of neuroectodermal origin of the islets of Langerhans.

APUD Cells↗
PubMed1995

A genetically engineered fusion protein M4/TNF with increased bifunctional activity refolded in the presence of protein disulfide isomerase.

Recombinant DNA techniques were used to clone, to construct and to express a fusion protein M4/TNF in Escherichia coli. The fusion protein includes the chimeric F(ab')2 fragment (M4) recognizing the human tumor-associated TAG72 antigen and the tumor necrosis factor alpha (TNF) moiety. The M4/TNF purified from inclusion bodies of the bacteria homogenates was further solubilized in a denaturing buffer containing 6 mol l-1 guanidine and refolded in a refolding buffer. Our results showed that the M4/TNF refolded in a buffer containing 6 mmol l-1 oxidized glutathione (GSSG), 0.2 mmol l-1 dithioerythione (DTE) and 0.5 mumol l-1 protein disulfide isomerase (PDI) displayed a 4-fold higher anti-TAG72 immunoreactivity and a 5-fold higher TNF activity than that refolded in the same refolding buffer but without PDI. Our data thus indicates that the protein disulfide isomerase not only facilitates the correct formation of disulfide-bonds of the antibody molecule, but also the correct refolding of the TNF moiety in vitro.

Amino Acid Sequence↗
PubMed1994

Percutaneous balloon valvuloplasty compared with open surgical commissurotomy for mitral stenosis.

BACKGROUND: Percutaneous balloon mitral valvuloplasty has been proposed as an alternative to open surgical commissurotomy for the treatment of rheumatic mitral-valve stenosis. METHODS: We enrolled 60 patients with severe mitral stenosis and favorable valvular anatomy in a prospective, randomized trial comparing the two procedures. All patients underwent cardiac catheterization before the procedure and one week, six months, and three years thereafter. Hemodynamic data were analyzed by investigators who were blinded to the patients' treatment assignments. RESULTS: Mitral-valve areas improved initially in both groups, from a mean (+/- SD) of 0.9 +/- 0.3 cm2 to 2.1 +/- 0.6 cm2 in the balloon-valvuloplasty group (30 patients; P < 0.01) and from 0.9 +/- 0.3 cm2 to 2.0 +/- 0.6 cm2 in the surgical group (30 patients; P < 0.001). Although improvement was maintained in both groups, mitral-valve areas were greater in the patients in the balloon-valvuloplasty group at three years (2.4 +/- 0.6 cm2, vs. 1.8 +/- 0.4 cm2 in the surgery group, P < 0.001). Restenosis occurred in three patients in the balloon-valvuloplasty group and four in the surgery group. One patient in the balloon-valvuloplasty group died of an apparent stroke after 2.5 years; four patients in the balloon-valvuloplasty group had residual atrial septal defects, and three patients (two in the balloon-valvuloplasty group and one in the surgery group) were judged to have severe mitral regurgitation. Seventy-two percent of the patients who underwent balloon valvuloplasty and 57 percent of the surgically treated patients were in New York Heart Association functional class I (i.e., they had no cardiovascular symptoms) at three years. No patient was lost to follow-up. CONCLUSIONS: In the treatment of mitral stenosis, balloon valvuloplasty and open surgical commissurotomy have comparable initial results and low rates of restenosis, and both produce good functional capacity for at least three years. The potential complications associated with balloon valvuloplasty should be noted. The better hemodynamic results at three years, lower cost, and elimination of the need for thoracotomy suggest that balloon valvuloplasty should be considered for all patients with favorable mitral-valve anatomy.

Adolescent↗