Cholera outbreak in rural areas of southern Rajasthan.
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Cholera↗
Biomedical subjects
R S Gupta
Publications and source records attributed to R S Gupta.
An epidemiological surveillance of acute flaccid paralysis in Alwar district.
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Case-Control Studies↗
Immunoelectron microscopy provides evidence for the presence of mitochondrial heat shock 10-kDa protein (chaperonin 10) in red blood cells and a variety of secretory granules.
Hsp10 (10-kDa heat shock protein, also known as chaperonin 10 or Cpn10) is a co-chaperone for Hsp60 in the protein folding process. This protein has also been shown to be identical to the early pregnancy factor, which is an immunosuppressive growth factor found in maternal serum. In this study we have used immunogold electron microscopy to study the subcellular localization of Hsp10 in rat tissues sections embedded in LR Gold resin employing polyclonal antibodies raised against different regions of human Hsp10. In all rat tissues examined including liver, heart, pancreas, kidney, anterior pituitary, salivary gland, thyroid, and adrenal gland, antibodies to Hsp10 showed strong labeling of mitochondria. However, in a number of tissues, in addition to the mitochondrial labeling, strong and highly specific labeling with the Hsp10 antibodies was also observed in several extramitochondrial compartments. These sites included zymogen granules in pancreatic acinar cells, growth hormone granules in anterior pituitary, and secretory granules in PP pancreatic islet cells. Additionally, the mature red blood cells which lack mitochondria, also showed strong reactivity with the Hsp10 antibodies. The observed labeling with the Hsp10 antibodies, both within mitochondria as well as in other compartments/cells, was abolished upon omission of the primary antibodies or upon preadsorption of the primary antibodies with the purified recombinant human Hsp10. These results provide evidence that similar to a number of other recently described mitochondrial proteins (viz., Hsp60, tumor necrosis factor receptor-associated protein-1, P32 (gC1q-R) protein, and cytochrome c), Hsp10 is also found at a variety of specific extramitochondrial sites in normal rat tissue. These results raise important questions as to how these mitochondrial proteins are translocated to other compartments and their possible function(s) at these sites. The presence of these proteins at extramitochondrial sites in normal tissues has important implications concerning the role of mitochondria in apoptosis and genetic diseases.
Animals↗
Induction of Hsp60 by Photofrin-mediated photodynamic therapy.
Photodynamic therapy (PDT) invokes a number of cellular responses. Other studies have shown that PDT induces transcription and translation of heat shock proteins (Hsps). The expression of mitochondrial heat shock protein, Hsp60, was measured following in vitro Photofrin-mediated PDT in the colon cancer cell line HT29 and its PDT-induced resistant variant HT29-P14 as well as the radiation-induced fibrosarcoma cells RIF-1 and its PDT-induced resistant variant, RIF-8A. Basal levels of Hsp60 were found to be similar in the two murine cell lines. In the human model, the resistant HT29-P14 cell line showed a small increase in basal levels relative to its parental population. Incubation with Photofrin (PII) alone or photosensitization caused a significant increase in Hsp60 levels in all cell lines as determined by flow cytometry. A dose-dependent and temporal relationship for PDT response was observed, maximum levels were detected 6-8 h post PDT, at which time, Hsp60 induction was found to be significantly greater in the two resistant variants. Induction in the RIF cells was also found to be greater after incubation with PII alone at the highest doses tested. These results indicate that the presence of PII and the subsequent oxidative stress of PDT can induce Hsp60 and implicated it as a common factor that may contribute to the resistance observed in the induced resistant cells.
Animals↗
Cytochrome-C localizes in secretory granules in pancreas and anterior pituitary.
We used quantitative immunogold electron microscopy to evaluate the subcellular distribution of cytochrome-c in normal rat tissues, employing a wide variety of monoclonal and polyclonal antibodies against mammalian cytochrome-c. Immunogold labeling of tissues embedded in the acrylic resin LR Gold shows highly specific labeling of mitochondria in all tissues examined, including adrenal gland, cerebellum, cerebral cortex, heart, kidney, liver, pituitary, pancreas, skeletal muscle, spleen and thyroid. In pancreatic acinar cells and anterior pituitary, however, there was also strong cytochrome-c reactivity in zymogen granules and growth hormone granules, respectively. In the pancreas, strong immunoreactivity is also detected in condensing vacuoles and in the acinar lumen. Immunocytochemical controls included (i) use of monoclonal antibodies to horse cytochrome-c which recognize an epitope not present in rat cytochrome-c, (ii) preadsorption of antibodies with purified cytochrome-c, and (iii) omission of the primary antibody. The indicated presence of cytochrome-c outside mitochondria in certain tissues under normal physiological conditions raises interesting questions concerning translocation mechanisms and the cellular functions of cytochrome-c.
Animals↗
The branching order and phylogenetic placement of species from completed bacterial genomes, based on conserved indels found in various proteins.
The presence of shared conserved inserts and deletions (indels or signature sequences) in proteins provides a powerful means for understanding the evolutionary relationships among the Bacteria. Using such indels, all of the main groups within the Bacteria can be defined in clear molecular terms and it has become possible to deduce that they branched from a common ancestor in the following order: Low G + C gram-positive --> High G+C gram-positive --> Deinococcus Thermus --> Cyanobacteria --> Spirochetes --> Aquifex-Chlamydia-Cytophaga --> Proteobacteria-1 (epsilon, delta) --> Proteobacteria-2 (alpha) --> Proteobacteria-3 (beta) --> Proteobacteria -4 (gamma). The usefulness of this approach for understanding bacterial phylogeny was examined here using sequence data from various completed bacterial genomes. By using 12 indels in highly conserved and widely represented proteins, the species from all 41 completed bacterial genomes were assigned to different groups; and the observed distribution of these indels in different species was then compared with that predicted by the signature sequence model. The presence or absence of these indels in various proteins in different bacteria followed the pattern exactly as predicted: and, in more than 450 observations, no exceptions or contradictions in the placement of indels were observed. These results provide strong evidence that lateral gene transfer events have not affected the genes containing these indels to any significant extent. The phylogenetic placement of bacteria into different groups based on signature sequences also showed an excellent correlation with the 16 S rRNA with 39 of the 41 species assigned to the same group by both methods. These results strongly vindicate the usefulness of the signature sequence approach to understanding phylogeny within the Bacteria and show that it provides a reliable and internally consistent means for the placement of bacterial species into different groups and for determining the relative branching order of the groups.
Amino Acid Sequence↗
Antifertility studies of Colebrookia oppositifolia leaf extract in male rats with special reference to testicular cell population dynamics.
Oral feeding of male rats with the ethanolic leaf extract of Colebrookia oppositifolia at dose levels of 100 and 200 mg/kg for 8-10 weeks did not cause body weight loss, while the weights of testes and epididymides were significantly decreased. Seminal vesicles and ventral prostate showed a significant reduction at the higher dose only. Treated animals showed a notable depression of spermatogenesis. Following 100 and 200 mg/kg extract feeding, the preleptotene spermatocytes were decreased by 46.5 and 39.8%, the secondary spermatocytes by 13.4 and 12.7%, the step-19 spermatids by 36.6 and 35.2%, and the mature Leydig cells by 31.2 and 39.5%, respectively. At both dose levels, the seminiferous tubule diameter, Leydig cells nuclear area and cytoplasmic area, as well as the cross-sectional surface area of Sertoli cells, were significantly reduced (P<0.001) when compared to controls. Reduced sperm count and motility resulted in 100% negative fertility at 200 mg/kg dose level. A significant fall in the total protein and sialic acid content and acid phosphatase enzyme activity of the testes, epididymides, seminal vesicle and ventral prostate, as well as in the glycogen content of testes, was also observed at both dose levels in comparison with controls.
Animals↗
The effect of inorganic phosphate on the activity of bacterial ribokinase.
Ribokinase and adenosine kinase are both members of the PfkB family of carbohydrate kinases. The activity of mammalian adenosine kinase was previously shown to be affected by pentavalent ions (PVI). We now present evidence that the catalytic activity of E. coli ribokinase is also affected by PVI, increasing both the velocity and affinity of the enzyme for D-ribose. The Km, for ribose decreased from 0.61 mM to 0.21, 0.25, and 0.33 mM in the presence of 20 mM phosphate, arsenate, and vanadate, respectively. The activity of ribokinase was stimulated in a hyperbolic fashion, with the maximum velocity increasing 23-fold, 13-fold, and 11-fold under the same conditions, respectively. Activity was also affected upon the addition of phosphoenolpyruvate, suggesting that phosphorylated metabolites could be involved in enzymatic control. The similar effect of PVI on distantly related enzymes suggests that a common mechanism for activity is shared among PfkB family members.
Adenosine Kinase↗
Sequential anastomosis of accessory renal artery to inferior epigastric artery in the management of multiple arteries in live related renal transplantation: a critical appraisal.
In live related renal transplant program, management of multiple renal arteries (MRA) is technically demanding and used to be considered a relative contraindication because of increased risk of vascular and urologic complications. We present a retrospective analysis of the outcome of grafts with MRA and suggest certain guidelines. Of the 680 live related kidney transplantations done, 53 allografts had MRA. Cases were grouped according to the reconstruction technique: group A, MRA reconstructed ex vivo into a single renal artery (n=27); group B, MRA with multiple anastomoses in vivo (n =13); group C, MRA with sequential revascularization using inferior epigastric artery (n=11). We compared serum creatinine, acute tubular necrosis, rejection rates and the rewarm ischemia time between the three groups. Overall patient survival and graft survival were excellent (100 and 96%). Mean serum creatinine at 1 yr did not differ significantly between the three groups. Rewarm ischemia time was significantly less in group C (p<0.01). Incidence of acute tubular necrosis and rejection episodes was also less in group C although the difference was statistically significant only between group C and group B. We conclude that allografts with MRA can be used successfully in a live related renal transplantation program. Bench reconstruction should be done whenever possible. For reconstruction of an accessory vessel, inferior epigastric artery with sequential revascularization is recommended.
Adolescent↗
Gene structure for adenosine kinase in Chinese hamster and human: high-frequency mutants of CHO cells involve deletions of several introns and exons.
The structure for the adenosine kinase (AK) gene has been determined from Chinese hamster (CH) and human cells. The AK gene in CH is comprised of 11 exons ranging in length from 36 to 765 nt, with the majority <100 nt. The exact lengths of the intervening introns have not been determined, but most of them are indicated to be very large (>15 kb). A 6.6-kb fragment from human cells was also sequenced, and it contained only a single exon corresponding to exon 10 in CH. The BLAST searches of the subsequently released draft human genome sequence have revealed that the AK gene structure in human is identical to that in CH. In the human genome, the AK exons are distributed over four genomic clones totaling 752 kb, providing direct evidence that the AK gene in mammalian species is unusually large. In contrast to CH and human, the AK genes from several other eukaryotic organisms whose complete genomes are now known are quite small (between 1.2 and 2.5 kb) and either contain no introns (Saccharomyces cerevisiae and Schizosaccharomyces pombe) or various numbers of introns (Drosophila melanogaster [2], Caenorhabditis elegans [4], Arabidopsis thaliana [10]). Some of the intron-exon junctions in these species are in the same positions as in mammals. The AK gene in CH and human, as well as mouse, is linked upstream in a head-to-head fashion with the gene for the clathrin adaptor mu3 protein (or beta 3A subunit of the AP-3 protein complex), which is affected in type 2 Hermansky-Pudlak syndrome. These two genes are separated by <200 nt, and it is possible that they have a common or overlapping promoter(s). We have also determined the nature of the genetic alterations in two of the class A AK(-) mutants of CHO cells, which are obtained at a very high spontaneous frequency (10(-3)-10(-4)) in this cell line. Both mutants contained large deletions within the AK gene and greatly shortened AK transcripts. The cloning and sequencing of the transcripts from these mutants showed that the deletion in one of them led to the loss of exons 5 through 8, whereas in the other, all exons from 2 through 8 are deleted. The endpoints of these deletions lie in the large introns within the AK gene.
Adenosine Kinase↗
Immunoelectron microscopy provides evidence that tumor necrosis factor receptor-associated protein 1 (TRAP-1) is a mitochondrial protein which also localizes at specific extramitochondrial sites.
The tumor necrosis factor receptor-associated protein 1 (TRAP-1) interacts with a variety of proteins involved in diverse functions. We have used quantitative immunogold electron microscopy and biochemical analysis to evaluate the subcellular distribution of TRAP-1 in rat tissues. Immunofluorescence employing a polyclonal antibody raised to human recombinant TRAP-1 reveals specific staining of mitochondria and nuclear region in mammalian cells. Western blot analysis of purified rat liver mitochondrial subfractions with the TRAP-1 antibody reveals that the cross-reactive protein (M(r) approximately 80 kDa) is mainly present in the matrix compartment. Immunogold labeling of rat tissue sections embedded in LR Gold resin shows strong labeling of mitochondria in all the tissues examined (viz., liver, heart, pancreas, kidney, spleen, anterior pituitary gland). Additionally, specific and significant labeling with TRAP-1 antibody was also observed in certain tissues in a number of nonmitochondrial locations, including pancreatic zymogen granules, insulin secretory granules, cardiac sarcomeres, and nuclei of pancreatic and heart cells, and on the cell surface of blood vessel endothelial cells. Western blot analysis showed that a cross-reactive protein of similar molecular mass as TRAP-1 is present in purified pancreatic zymogen granules. Immunogold labeling was prevented in all tissues by preadsorption of the TRAP-1 antibody with the purified recombinant TRAP-1 protein. These observations and the fact that TRAP-1 is synthesized with a typical mitochondrial targeting presequence strongly indicate that TRAP-1 is primarily a mitochondrial matrix protein. The localization of this protein at specific extramitochondrial sites raises interesting and fundamental questions regarding the possible mechanisms by which these proteins are translocated to such sites.
Animals↗
Structure-activity studies on mammalian adenosine kinase.
The structure-activity relationship for Chinese hamster adenosine kinase (AK) was examined by making systematic deletions from the N- and C-terminal ends. The first 16 a.a. residues from the N-terminal end, which likely form a random coil, can be deleted without any effect on AK activity or stability. The successive removal of the next 11 residues, which stabilize the first beta structure of the protein, leads to a progressive loss of AK activity from 100 to about 3%. The loss in activity is accompanied by increasing thermal instability and a slight increase in the K(m) for adenosine. All deletions beyond residue M28, which should cause disruption of the tertiary structure, are devoid of AK activity. The residues at the C-terminal end form a substructure involved in the stability of the "adenosine 2 binding site" and removal of any residues results in significant loss of activity. Successive removal of the first 10 residues from this end causes progressive decrease in AK activity to about the 2% level, accompanied by a five-fold increase in the K(m) for ATP, supporting the view that the adenosine 2 binding site located near the C-terminal end is the ATP binding site. All deletions beyond residue R348, which forms two salt bridges with the ATP binding site, are inactive. Site-directed replacement of an aspartic acid residue (D316), which is postulated to function in the transfer of phosphate from ATP to adenosine by either asparagine or glutamic acid, leads to complete loss of activity, supporting the proposed role of D316 as the catalytic base.
Adenosine Kinase↗
The influence of inorganic phosphate on the activity of adenosine kinase.
The enzyme adenosine kinase (AK; EC 2.7.1.20) shows a dependence upon inorganic phosphate (Pi) for activity. The degree of dependence varies among enzyme sources and the pH at which the activity is measured. At physiological pH, recombinant AK from Chinese hamster ovary (CHO) cells and AK from beef liver (BL) show higher affinities for the substrate adenosine (Ado), larger maximum velocities and lower sensitivities to substrate inhibition in the presence of Pi. At pH 6.2, both BL and CHO AK exhibit almost complete dependence on the presence of Pi for activity. The data show that both enzymes exhibit increasing relief from substrate inhibition upon increasing Pi and the inhibition of BL AK is almost completely alleviated by the addition of 50 mM Pi. The affinity of CHO AK for Ado increases asymptotically from K(m) 6.4 microM to a limit of 0.7 microM upon the addition of increasing Pi from 1 to 50 mM. The concentration of Ado necessary to invoke substrate inhibition also increases asymptotically from K(i) 32 microM to a limit of 69 microM at saturating concentrations of phosphate. In the presence of increasing amounts of Pi, the maximal velocity of activity increases hyperbolically. The effect that phosphate exerts on AK may be either to protect the enzyme from inactivation at high adenosine and H(+) concentrations or to stabilize substrate binding at the active site.
Adenosine↗
Localization of P32 protein (gC1q-R) in mitochondria and at specific extramitochondrial locations in normal tissues.
P32 protein, also known as the gC1q receptor for complement component C1q, is a binding protein for nuclear pre-mRNA splicing factor SF2/ASF and numerous other nuclear and cell surface proteins, yet is targeted to the mitochondrial matrix compartment where these proteins are not present. In the present study, we use immunogold electron microscopy to evaluate the subcellular distribution of P32 protein (gC1q-R) in cultured cell lines and in rat tissues embedded in the acrylic resin LR Gold. Immunogold labeling of Raji lymphoma, CHO, human fibroblasts, HeLa and B-SC-1 cells shows reactivity primarily within mitochondria. Highly specific labeling of mitochondria is also obtained in rat tissues, including adrenal gland, cerebellum, cerebral cortex, heart, kidney, liver, pituitary, pancreas, skeletal muscle, spleen, testes and thyroid. However, strong P32 (gClq-R) reactivity is also present in (i) zymogen granules, condensing vacuoles, endoplasmic reticulum, and on the cell surface of pancreatic acinar cells, (ii) on the cell surface of microvascular endothelial cells in pancreas and kidney, (iii) on the cell surface and in nuclei of splenic lymphocytes, and (iv) in the acrosome of developing spermatids in testes. Western immunoblots show that the polyclonal antibody to P32 (gC1q-R) used in this study reacts specifically with a 32-kDa protein in both purified pancreatic zymogen granules and in mitochondria, and no other proteins are reactive. These results provide evidence that P32 (gC1q-R) is a mitochondrial protein that also localizes outside mitochondria in certain cells and tissues under normal physiological conditions.
Animals↗
Mitochondrial proteins at unexpected cellular locations: export of proteins from mitochondria from an evolutionary perspective.
Researchers in a wide variety of unrelated areas studying functions of different proteins are unexpectedly finding that their proteins of interest are actually mitochondrial proteins, although functions would appear to be extramitochondrial. We review the leading current examples of mitochondrial macromolecules indicated to be also present outside of mitochondria that apparently exit from mitochondria to arrive at their destinations. Mitochondrial chaperones, which have been implicated in growth and development, autoimmune diseases, cell mortality, antigen presentation, apoptosis, and resistance to antimitotic drugs, provide some of the best studied examples pointing to roles for mitochondria and mitochondrial proteins in diverse cellular phenomena. To explain the observations, we propose that specific export mechanisms exist by which certain proteins exit mitochondria, allowing these proteins to have additional functions at specific extramitochondrial sites. Several possible mechanisms by which mitochondrial proteins could be exported are discussed. Gram-negative proteobacteria, from which mitochondria evolved, contain a number of different mechanisms for protein export. It is likely that mitochondria either retained or evolved export mechanisms for certain specific proteins.
Animals↗
Hypocholesterolemic activity of Marsilea minuta in gerbils.
Feeding of a Marsilea minuta leaf extract [Fr. I] reduced serum cholesterol and triglycerides by 31 and 63%, respectively, in athero diet fed gerbils. Liver cholesterol and triglycerides were also lowered by 71 and 27%, respectively, in comparison to athero fed controls. Moreover, treatment with Fr. I prevented the accumulation of cholesterol and triglycerides in liver and aorta and was able to dissolve atheromatous plaques of thoracic and abdominal aorta. Fecal excretions of cholesterol and triglycerides were significantly increased in Fr. I fed gerbils.
Animals↗
Antifertility studies of the root extract of the Barleria prionitis Linn in male albino rats with special reference to testicular cell population dynamics.
Oral administration of root extract of Barleria prionitis L. to male rats (100 mg/rat per day) for the period of 60 days did not cause body weight loss. The root extract brought about an interference with spermatogenesis. The round spermatids were decreased by 73.6% (P< or =0.001). No significant change was found in the population of secondary spermatocytes. However, the population of preleptotene spermatocytes were decreased by 41.9%. The extract reduced the fertility of male rats by 100%. Cross sectional surface area of Sertoli cells and mature Leydig cell numbers were significantly reduced (36.9%). The total protein, sialic acid contents of the testes, epididymides, seminal vesicle and prostate were reduced. Testicular glycogen contents were low. Antifertility effects of Barleria seemed to be mediated by disturbances in testicular somatic cells functions (Leydig and Sertoli cells) resulting in the physio-morphological events of spermatogenesis.
Animals↗
The natural evolutionary relationships among prokaryotes.
Two contrasting and very different proposals have been put forward to account for the evolutionary relationships among prokaryotes. The currently widely accepted three domain proposal by Woese et al. (Proc. Natl. Acad. Sci. USA (1990) 87: 4576-4579) calls for the division of prokaryotes into two primary groups or domains, termed archaebacteria (Archaea) and eubacteria (Bacteria), both of which are suggested to have originated independently from a universal ancestor. However, this proposal, which is based primarily on genes involved in the information transfer processes, is inconsistent with the ultrastructural characteristics of prokaryotes as well as with many gene phylogenies and provides no explanation as to how the structural and molecular differences seen between these groups arose and how other prokaryotic taxa are related or evolved from the common ancestor. It also postulates that the last common ancestor of all organisms was a hypothetical entity lacking a cell membrane, which is contrary to the basic requirement of a cell membrane to define and separate all forms of life from the surrounding environment. A second alternate proposal for the evolutionary relationships among prokaryotes has emerged from extensive analyses of numerous conserved inserts and deletions found in various proteins (Gupta, R. S., Microbiol. Mol. Biol. Rev. (1998)62: 1435-1491; FEMS Microbiol. Rev. (2000) 24: in press. This proposal points to a specific relationship between archaebacteria and gram-positive bacteria, both of which are prokaryotes bounded by a single cell membrane (monoderm prokaryotes). Gram-negative bacteria, which are bounded by two different membranes (diderm prokaryotes), are indicated to comprise a structurally and phylogenetically distinct taxa originating from gram-positive bacteria. This proposal postulates that the earliest prokaryote was a gram-positive bacteria from which both archaebacteria and diderm prokaryotes evolved by normal evolutionary mechanisms in response to the strong selection pressure exerted by antibiotics produced by certain groups of gram-positive bacteria. This proposal accounts for both the molecular as well structural differences seen among the main groups of prokaryotes by known evolutionary mechanisms without invoking any hypothetical process or entity and thus is a closer representation of the natural relationships among prokaryotes than the proposal for two distinct domains. Based on this new proposal, it is now possible to logically deduce the branching order of different prokaryotic taxa from the common ancestor, which is as follows: Gram-positive bacteria (Low G + C) (<=> Archaebacteria) => Gram-positive bacteria (High G + C) (<=> Archaebacteria)=> Deinococcus-Thermus => Green nonsulfur bacteria => Cyanobacteria => Spirochetes => Chlamydia- Cytophaga-Green sulfur bacteria => Proteobacteria-1 (epsilon, delta)=> Proteobacteria-2 (alpha) => Proteobacteria-3 (beta) => Proteobacteria-4 (gamma). A surprising but very important aspect of the relationship deduced here is that the main eubacterial phyla are related to each other linearly rather than in a tree-like manner, suggesting that the major evolutionary changes within prokaryotes (bacteria) have occurred in a directional manner.
Archaea↗