Biomedical subjects
K Gowda
Publications and source records attributed to K Gowda.
Multistage multiantigen heterologous prime boost vaccine for Plasmodium knowlesi malaria provides partial protection in rhesus macaques.
A nonhuman primate model for malaria vaccine development allowing reliable, stringent sporozoite challenge and evaluation of both cellular and antibody responses is needed. We therefore constructed a multicomponent, multistage DNA vaccine for the simian malaria species Plasmodium knowlesi including two preerythrocytic-stage antigens, the circumsporozoite protein (PkCSP) and sporozoite surface protein 2 (PkSSP2), and two blood stage antigens, apical merozoite antigen 1 (PkAMA1) and merozoite surface protein 1 (PkMSP1p42), as well as recombinant canarypox viruses encoding the four antigens (ALVAC-4). The DNA vaccine plasmids expressed the corresponding antigens in vitro and induced antiparasite antibodies in mice. Groups of four rhesus monkeys received three doses of a mixture of the four DNA vaccine plasmids and a plasmid encoding rhesus granulocyte-monocyte colony-stimulating factor, followed by boosting with a single dose of ALVAC-4. Three groups received the priming DNA doses by different routes, either by intramuscular needle injection, by intramuscular injection with a needleless injection device, the Biojector, or by a combination of intramuscular and intradermal routes by Biojector. Animals immunized by any route developed antibody responses against sporozoites and infected erythrocytes and against a recombinant PkCSP protein, as well as gamma interferon-secreting T-cell responses against peptides from PkCSP. Following challenge with 100 P. knowlesi sporozoites, 1 of 12 experimental monkeys was completely protected and the mean parasitemia in the remaining monkeys was significantly lower than that in 4 control monkeys. This model will be important in preclinical vaccine development.
Sequence analysis of VP4 and VP7 genes of nontypeable strains identifies a new pair of outer capsid proteins representing novel P and G genotypes in bovine rotaviruses.
During a limited epidemiological study, the serotype specificities of several isolates of bovine rotavirus, exhibiting identical electropherotypes, from a single cattle farm near Bangalore, India, could not be determined using a panel of serotyping monoclonal antibodies (MAbs) specific for G serotypes 1-6 and 10. To determine the genotypes of these isolates, the nucleotide sequences of the genes encoding the outer capsid proteins VP4 and VP7 of two representative isolates, Hg18 and Hg23, were determined. The corresponding gene sequences from the two isolates were identical, indicating that these isolates represented a single strain of bovine rotavirus. Comparison of the VP4 nucleotide (nt) and the deduced amino acid (aa) sequences with those of several human and animal rotavirus strains representing all of the currently recognized 20 different VP4 (P) genotypes revealed low nt and aa sequence identities of 61.0 to 74.2% and 57.9 to 78.2% for VP4. The percentages of amino acid homology for the VP8* and VP5* regions of VP4 were 37.7 to 67.9 and 68.1 to 84.2%, respectively. The nt and aa sequences of the VP7 gene were also distinct from those of human and animal strains belonging to the previously established 14 VP7(G) serotypes (65.9 to 75.5% nt and 59.5 to 77.6% aa identities). These findings suggest the classification of the VP4 and VP7 genes of the bovine isolates represented by Hg18 as new P and G genotypes and provide further evidence for the vast genetic/antigenic diversity of group A rotaviruses.
Improving protective immunity induced by DNA-based immunization: priming with antigen and GM-CSF-encoding plasmid DNA and boosting with antigen-expressing recombinant poxvirus.
Intramuscular immunization with a naked DNA plasmid expressing the Plasmodium yoelii circumsporozoite protein (pPyCSP) protects mice against challenge with P. yoelii sporozoites. This protection can be improved either by coadministration of a plasmid expressing murine GM-CSF (pGMCSF) or by boosting with recombinant poxvirus expressing the PyCSP. We now report that combining these two strategies, by first mixing the priming dose of pPyCSP with pGMCSF and then boosting with recombinant virus, can substantially increase vaccine effectiveness. Not only were immune responses and protection improved but the pPyCSP dose could be lowered from 100 microg to 1 microg with little loss of immunogenicity after boost with recombinant poxvirus. Comparing mice primed by the 1-microg doses of pPyCSP plus 1 microg pGMCSF with mice primed by 1-microg doses of pPyCSP alone, the former were better protected (60% vs 0) and had higher concentrations of Abs (titers of 163, 840 vs 5, 120 by indirect fluorescent Ab test against sporozoites), more ex vivo CTL activity (25% vs 7% specific lysis), and more IFN-gamma-secreting cells by enzyme-linked immunospot assay (1460 vs 280 IFN-gamma spot-forming cells/106 cells). Priming with plasmid vaccine plus pGMCSF and boosting with recombinant poxviruses strongly improves the immunogenicity and protective efficacy of DNA vaccination and allows for significant reduction of dose.
Assembly of archaeal signal recognition particle from recombinant components.
Signal recognition particle (SRP) takes part in protein targeting and secretion in all organisms. Searches for components of archaeal SRP in primary databases and completed genomes indicated that archaea possess only homologs of SRP RNA, and proteins SRP19 and SRP54. A recombinant SRP was assembled from cloned, expressed and purified components of the hyperthermophilic archaeon Archaeoglobus fulgidus. Recombinant Af-SRP54 associated with the signal peptide of bovine pre-prolactin translated in vitro. As in mammalian SRP, Af-SRP54 binding to Af-SRP RNA required protein Af-SRP19, although notable amounts bound in absence of Af-SRP19. Archaeoglobus fulgidus SRP proteins also bound to full-length SRP RNA of the archaeon Methanococcus jannaschii, to eukaryotic human SRP RNA, and to truncated versions which corresponded to the large domain of SRP. Dependence on SRP19 was most pronounced with components from the same species. Reconstitutions with heterologous components revealed a significant potential of human SRP proteins to bind to archaeal SRP RNAs. Surprisingly, M.jannaschii SRP RNA bound to human SRP54M quantitatively in the absence of SRP19. This is the first report of reconstitution of an archaeal SRP from recombinantly expressed purified components. The results highlight structural and functional conservation of SRP assembly between archaea and eucarya.
Signal recognition particle components in the nucleolus.
The signal recognition particle (SRP) is a ribonucleoprotein composed of an Alu domain and an S domain. The S domain contains unique sequence SRP RNA and four SRP proteins: SRP19, SRP54, SRP68, and SRP72. SRP interacts with ribosomes to bring translating membrane and secreted proteins to the endoplasmic reticulum (ER) for proper processing. Additionally, SRP RNA is a member of a family of small nonribosomal RNAs found recently in the nucleolus, suggesting that the nucleolus is more plurifunctional than previously realized. It was therefore of interest to determine whether other SRP components localize to this intranuclear site. In transfected rat fibroblasts, green fluorescent protein fusions of SRP19, SRP68, and SRP72 localized to the nucleolus, as well as to the cytoplasm, as expected. SRP68 also accumulated in the ER, consistent with its affinity for the ER-bound SRP receptor. SRP54 was detected in the cytoplasm as a green fluorescent protein fusion and in immunofluorescence studies, but was not detected in the nucleolus. In situ hybridization experiments also revealed endogenous SRP RNA in the nucleolus. These results demonstrate that SRP RNA and three SRP proteins visit the nucleolus, suggesting that partial SRP assembly, or another unidentified activity of the SRP components, occurs at the nucleolus. SRP54 apparently interacts with nascent SRP beyond the nucleolus, consistent with in vitro reconstitution experiments showing that SRP19 must bind to SRP RNA before SRP54 binds. Our findings support the notion that the nucleolus is the site of assembly and/or interaction between the family of ribonucleoproteins involved in protein synthesis, in addition to ribosomes themselves.
Crystal structure of the conserved subdomain of human protein SRP54M at 2.1 A resolution: evidence for the mechanism of signal peptide binding.
Protein SRP54 is an integral part of the mammalian signal recognition particle (SRP), a cytosolic ribonucleoprotein complex which associates with ribosomes and serves to recognize, bind, and transport proteins destined for the membrane or secretion. The methionine-rich M-domain of protein SRP54 (SRP54M) binds the SRP RNA and the signal peptide as the nascent protein emerges from the ribosome. A focal point of this critical cellular function is the detailed understanding of how different hydrophobic signal peptides are recognized efficiently and transported specifically, despite considerable variation in sequence. We have solved the crystal structure of a conserved functional subdomain of the human SRP54 protein (hSRP54m) at 2.1 A resolution showing a predominantly alpha helical protein with a large fraction of the structure available for binding. RNA binding is predicted to occur in the vicinity of helices 4 to 6. The N-terminal helix extends significantly from the core of the structure into a large but constricted hydrophobic groove of an adjacent molecule, thus revealing molecular details of possible interactions between alpha helical signal peptides and human SRP54.
Construction and immunogenicity of DNA vaccine plasmids encoding four Plasmodium vivax candidate vaccine antigens.
Plasmodium vivax is the second most common agent of human malaria. Although infection is rarely fatal, it nonetheless imposes a significant burden of illness in endemic areas. A successful vaccine against P. vivax will likely need to induce immune responses against both pre-erythrocytic and erythrocytic stage forms of the parasite. Accordingly, we constructed eight nucleic acid vaccines based on four antigens, the circumsporozoite protein (PvCSP) and sporozoite surface protein 2 (PvSSP2) from the pre-erythrocytic stage, and apical membrane antigen 1 (PvAMA1) and merozoite surface protein 1 (PvMSP1) from the erythrocytic stage. The constructs induced high levels of specific antibody in mice regardless of whether the antigen was expressed in native form or fused to a human tissue plasminogen activator leader peptide. High titer antibodies induced against PvCSP did not react with the protective AGDR epitope within the sequence of this antigen. These results support the immunogenicity of these four vaccine candidate antigens when delivered as nucleic acid vaccines.
Expression, purification, and crystallography of the conserved methionine-rich domain of human signal recognition particle 54 kDa protein.
Protein SRP54 is an essential component of eukaryotic signal recognition particle (SRP). The methionine-rich M-domain (SRP54M or 54M) interacts with the SRP RNA and is also involved in the binding to signal peptides of secretory proteins during their targeting to cellular membranes. To gain insight into the molecular details of SRP-mediated protein targeting, we studied the human 54M polypeptide. The recombinant human protein was expressed successfully in Escherichia coli and was purified to homogeneity. Our studies determined the sites that were susceptible to limited proteolysis, with the goal to design smaller functional mutant derivatives that lacked nonessential amino acid residues from both termini. Of the four polypeptides produced by V8 protease or chymotrypsin, 54MM-2 was the shortest (120 residues; Mr = 13,584.8), but still contained the conserved amino acids suggested to associate with the signal peptide or the SRP RNA. 54MM-2 was cloned, expressed, purified to homogeneity, and was shown to bind human SRP RNA in the presence of protein SRP19, indicating that it was functional. Highly reproducible conditions for the crystallization of 54MM-2 were established. Examination of the crystals by X-ray diffraction showed an orthorhombic unit cell of dimensions a = 29.127 A, b = 63.693 A, and c = 129.601 A, in space group P2(1)2(1)2(1), with reflections extending to at least 2.0 A.
Protein SRP54 of human signal recognition particle: cloning, expression, and comparative analysis of functional sites.
Signal recognition particle (SRP) plays a critical role in the targeting of secretory proteins to cellular membranes. An essential component of SRP is the protein SRP54, which interacts not only with the nascent signal peptide, but also with the SRP RNA. To understand better how protein targeting occurs in the human system, the human SRP54 gene was cloned, sequenced, and the protein was expressed in bacteria and insect cells. Recombinant SRP54 was purified from both sources. The protein bound to SRP RNA in the presence of protein SRP19, and associated with the signal peptide of in vitro translated pre-prolactin. Comparative sequence analysis of human SRP54 with homologs from all three phylogenetic domains was combined with high-stringency protein secondary structure prediction. A conserved RNA-binding loop was predicted in the largely helical M-domain of SRP54. Contrary to general belief, the unusually high number of methionine residues clustered outside the predicted helices, thus indicating a mechanism of signal peptide recognition that may involve methionine-rich loops.
Determinants of a protein-induced RNA switch in the large domain of signal recognition particle identified by systematic-site directed mutagenesis.
Signal recognition particle (SRP) is a ribonucleoprotein complex that associates with ribosomes to promote the co-translational translocation of proteins across biological membranes. Human SRP RNA molecules exist in two distinct conformations, SR-A and SR-B, which may exchange during the assembly of the particle or could play a functional role in the SRP cycle. We have used systematic site-directed mutagenesis of the SRP RNA to determine the electrophoretic mobilities of altered RNA molecules, and we have identified the nucleotides that avert the formation of the two conformers. The conformer behavior of the various RNAs was addressed quantitatively by calculating a value zeta as an indicator of conformational variability. Single loose A-like forms were induced by changes in helix 5 [nucleotides (nt) at positions 111-128 or 222-231], helix 6 (nt at positions 141-150) and helix 7 (nt at position 169 and 170), whereas other mutations in helix 6 and helix 8 preserved the conformational variability of the mutant RNA molecules. The more compact B-like form was induced only when a small region (129-CAAUAU-134), located in the 5'-proximal portion of helix 6, was altered. Since this region is part of the binding site for SRP19, we suggest that protein SRP19 uses nucleotides at 129-134 to trigger the formation of the favored SR-B-form, and thus directs an early step in the hierarchical assembly of the large SRP domain.
Identification of an RNA-binding-loop in the N-terminal region of signal-recognition-particle protein SRP19.
Protein SRP19 is a 144-amino-acid polypeptide that associates intimately with the signal-recognition particle RNA (SRP RNA) and serves as an important structural and functional component of the SRP. We investigated the structure and RNA-binding activity of the human SRP19 protein by the use of comparative sequence analysis, high-stringency structure prediction, proteolytic susceptibility, and site-directed mutagenesis. SRP19 was found to consist of two distinct regions (called N-terminal and C-terminal regions) that are separated by a boundary of approximately 12-15 amino acid residues. Both regions contain an alpha-helix and several beta-strands that are connected by loops or turns. In agreement with the hypothetical model, proteolytic susceptibility demonstrated the predominant accessibility of two sites: one in a surface loop of the N-terminal region (YLNNKKTIAEGR33), and another site in the C-terminal tail at residues L129 and E133. The RNA-binding activities of mutant polypeptides with changes of conserved lysines and arginines (mutants K27Q, R33Q and R34Q) demonstrated that the proteolytically accessible loop of the N-terminal region is in direct contact with the SRP RNA. In contrast, alteration of a certain basic amino acid residues in the C-terminal region (R83, K116 and R118), as well as a deletion of four amino acid residues located at the boundary between the two regions, had no effect on the RNA-binding ability. The structural model that emerges from our data is thematically similar to that of ribosomal protein S5, the N-domain of which contains a loop motif believed to interact with double-stranded RNA. The presence of a similar structural feature in protein SRP19 has significant implications for the structure and function of the SRP19-RNA complex.
Binding site of the M-domain of human protein SRP54 determined by systematic site-directed mutagenesis of signal recognition particle RNA.
The interaction of protein SRP54M from the human signal recognition particle with SRP RNA was studied by systematic site-directed mutagenesis of the RNA molecule. Protein binding sites were identified by the analysis of mutations that removed individual SRP RNA helices or disrupted helical sections in the large SRP domain. The strongest effects on the binding activity of a purified polypeptide that corresponds to the methionine-rich domain of SRP54 (SRP54M) were caused by changes in helix 8 of the SRP RNA. Binding of protein SRP19 was diminished significantly by mutations in helix 6 and was stringently required for SRP54M to associate. Unexpectedly, mutant RNA molecules that resembled bacterial SRP RNAs were incapable of interaction with SRP54M, showing that protein SRP19 has an essential and direct role in the formation of the ternary complex with SRP54 and SRP RNA. Our findings provide an example for how, in eukaryotes, an RNA function has become protein dependent.
Autologous lymphoblastoid cell lines stably transfected with Plasmodium falciparum circumsporozoite protein as targets in cytotoxic T-lymphocyte assays.
To produce cell lines that can be used as a continuous source of antigen presenting cells for stimulating T-cell lines and clones and as targets in cytotoxic T-lymphocyte (CTL) assays, we used a retroviral vector with a simian virus (SV40) early promotor to transfer a Plasmodium falciparum circumporozoite (PfCSP) gene into human EBV transformed B-lymphoblastoid cell lines (B-LCL). We herein report successful, stable transfection and cell surface expression of this gene, as confirmed by PCR, Western blot analysis and immunoelectron microscopy. One of three successfully transfected autologous cell lines expressed PfCSP on the cell surface and was lysed by CD8+ T-cell dependent CTL from a donor volunteer who had been immunized with irradiated P. falciparum sporozoites. Such cell lines should provide excellent tools for characterizing human CD8+ T-cell responses against Plasmodium sp. proteins.
Interaction of rice and human SRP19 polypeptides with signal recognition particle RNA.
The signal recognition particle (SRP) controls the transport of secretory proteins into and across lipid bilayers. SRP-like ribonucleoprotein complexes exist in all organisms, including plants. We characterized the rice SRP RNA and its primary RNA binding protein, SRP19. The secondary structure of the rice SRP RNA was similar to that found in other eukaryotes; however, as in other plant SRP RNAs, a GUUUCA hexamer sequence replaced the highly conserved GNRA-tetranucleotide loop motif at the apex of helix 8. The small domain of the rice SRP RNA was reduced considerably. Structurally, rice SRP19 lacked two small region that can be present in other SRP19 homologues. Conservative structure prediction and site-directed mutagenesis of rice and human SRP19 polypeptides indicated that binding to the SRP RNAs occurred via a loop that is present in the N-domain of both proteins. Rice SRP19 protein was able to form a stable complex with the rice SRP RNA in vitro. Furthermore, heterologous ribonucleoprotein complexes with components of the human SRP were assembled, thus confirming a high degree of structural and functional conservation between plant and mammalian SRP components.
Epidemiology of symptomatic human rotaviruses in Bangalore and Mysore, India, from 1988 to 1994 as determined by electropherotype, subgroup and serotype analysis.
Epidemiology of symptomatic rotaviruses from Bangalore and Mysore in Southern India was investigated. While serotype G3 predominated throughout the 7-year study period from 1988 to 1994 in Bangalore, serotype G1 was more predominant than serotype G3 in Mysore during 1993 and 1994. Serotype G2 strains were either not detected or infrequently observed in both the cities. However, several strains with subgroup I and 'short' RNA pattern that exhibited high reactivity with typing MAbs specific for serotype 2 as well as other serotypes were detected throughout the period. Among the nonserotypeable strains from both cities, several exhibited dual subgroup (SGI + II) or subgroup I specificity and 'long' RNA pattern indicating their probable animal origin. Notably, a gradual, yet highly significant reduction in rotavirus gastroenteritis, from 45.3% in 1988 to 1.8% during 1994, was observed in Bangalore in stark contrast to the consistently high (about 34%) incidence of asymptomatic infections among neonates by I321-like G10P11 type strains during the same period. Moreover, I321-like asymptomatic strains were not detected in children with diarrhea.
Anatomic thoracoscopic lobectomy (ATL) without minithoracotomy: preliminary experience.
UNLABELLED: The purpose of this review is to outline our experience with a completely thoracoscopic approach to major pulmonary resection (lobectomy). There were 23 patients in this study selected by the usual criteria of resectability. The preoperative workup included chest x-ray, pulmonary function studies, computed tomography (CT) of the chest and abdomen, bronchoscopy, mediastinoscopy, and brain and bone scans in the majority of patients. ATL was attempted in 23 patients. In four patients the procedure was converted to the "open" approach for the following reasons: inability to identify the location of the primary lesion (two patients), obscurative bleeding (one patient), and inability to diagnose the pathology (one patient). The underlying pathology of the ATL group was as follows: bronchiolitis obliterans-organizing pneumonia = 1, granuloma = 2, non-small cell lung cancer = 14, solitary colonic metastases = 2. The breakdown by site of the 19 ATL resections is as follows: right upper lobe = 5, right middle lobe = 1, right lower lobe = 1, right upper and right middle lobes = 1, left upper lobe = 6, left lower lobe = 5. Group A patients (n = 11) were those who had no postoperative complications. Length of stay was short, ranging from 4 to 8 days, median 5 days. Group B patients (n = 8) had complications (prolonged air leak = 4, supplemental postoperative oxygen requirement = 3, pain control = 1) and stayed longer (range 10-21 days, median 13). There were no deaths, no blood transfusions, no chest tube reinsertions and no reoperations. The most significant complication in the ATL group was prolonged air leak in one patient (21 days). CONCLUSIONS: (a) ATL without minithoracotomy is feasible; (b) ATL has potential benefits in terms of reduction of pain and hospital stay; (c) ATL had better cosmetic and functional results.
Nucleic acid malaria vaccines. Current status and potential.
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