Biomedical subjects

Radhey S Gupta

Publications and source records attributed to Radhey S Gupta.

30 records · Page 2Linked to original sources

PubMed2004

Genomic organization and linkage via a bidirectional promoter of the AP-3 (adaptor protein-3) mu3A and AK (adenosine kinase) genes: deletion mutants of AK in Chinese hamster cells extend into the AP-3 mu3A gene.

The cDNA and genomic DNA for the mu3A subunit of the AP-3 (adaptor protein-3) complex were cloned from Chinese hamster cells. The AP-3 mu3A genes in Chinese hamster, human and mouse each comprise nine exons and eight introns, with all introns located in identical positions in the species studied. The AP-3 mu3A genes in these species are linked in a head-to-head fashion with the gene for the purine salvage pathway enzyme AK (adenosine kinase). These genes share the first exon, and a 512 bp fragment covering the intervening untranslated sequence has the characteristic of a CpG island promoter, and it effectively carried out transcription in both directions. Deletion studies indicate that this region contains both positive and negative regulatory elements affecting transcription of these genes. In comparison with the AP-3 mu3A gene (27 kb), the AK gene in human is very large (558 kb), with average exon and intron lengths of approx. 100 bp and 55.7 kb respectively. The ratio of non-coding to coding sequence in the human AK gene is >550, which is the highest reported for any gene. We also present evidence that a number of AK- mutants of Chinese hamster ovary cells contain large deletions that affect both of these genes. In addition to lacking part of the AK gene, two of these mutants also lacked all of the exons and introns corresponding to the AP-3 mu3A gene. These mutants should prove useful in elucidating the role of AP-3 mu3A in vesicle-mediated protein sorting--a process that is altered in Hermansky-Pudlak syndrome. Detailed phylogenetic analysis of the micro family of proteins presented here also provides insight into how different AP complexes are related and may have evolved.

Adaptor Protein Complex 3↗

PubMed2004

The T cells specific for the carboxyl-terminal determinants of self (rat) heat-shock protein 65 escape tolerance induction and are involved in regulation of autoimmune arthritis.

Immunization of Lewis rats with heat-killed Mycobacterium tuberculosis H37Ra leads to development of polyarthritis (adjuvant-induced arthritis; AA) that shares several features with human rheumatoid arthritis (RA). Immune response to the 65-kDa mycobacterial heat-shock protein (Bhsp65) is believed to be involved in induction of AA as well as in experimental modulation of this disease. However, the understanding of several critical aspects of the pathogenesis of AA in the Lewis rat has severely been hampered by the lack of information both regarding the level as well as epitope specificity of tolerance to the mammalian self (rat) homologue of Bhsp65, 65-kDa rat heat-shock protein (Rhsp65), and about the functional attributes of the T cell repertoire specific for this self protein. In this study, we established that tolerance to Rhsp65 in the Lewis rat is incomplete, and that the residual T cells primed upon challenge with this self hsp65 are disease regulating in nature. We also have defined the T cell epitopes in the C-terminal region within Rhsp65 that contribute predominantly to the immune reactivity as well as the AA-protective effect of this self protein. Furthermore, the T cells primed by peptides comprising these C-terminal determinants can be efficiently restimulated by the naturally generated epitopes from endogenous Rhsp65, suggesting that self hsp65 might also be involved in natural remission from acute AA. These novel first experimental insights into the self hsp65-directed regulatory T cell repertoire in AA would help develop better immunotherapeutic approaches for autoimmune arthritis.

Adoptive Transfer↗

PubMed2004

Phosphorylated derivatives that activate or inhibit mammalian adenosine kinase provide insights into the role of pentavalent ions in AK catalysis.

The enzyme adenosine kinase (AK) exhibits a nearly complete dependency on the presence of pentavalent ions (PVI) such as phosphate, arsenate, and vanadate. To understand its basis, the effect of a large number of phosphorylated compounds on AK activity was examined. Several compounds, such as phosphoribosyl pyrophosphate, phosphoenol pyruvate, creatine phosphate, phosphorous acid, phosphonoformic acid, and inorganic pyrophosphate, were found to substitute for PVI in stimulating AK activity. Similar to PVI, these compounds lowered the Km of AK for adenosine. In contrast, many other structurally related compounds (i.e., phosphonoacetic acid, 2-carboxyethyl phosphonic acid, N-phosphonomethyl glycine, N-phosphonomethyl iminodiacetic acid) inhibited AK activity. These compounds seemed to compete with the activators for binding to AK. Structural comparisons of different compounds indicate that all activating compounds contain a net positive charge on the pentavalent atom (e.g., phosphorous), which should enable it to act as an acceptor for a nucleophilic group. We suggest that a phosphate (or other activator) bound near the active site participates in AK catalysis by forming a transient pentavalent intermediate with a nonbridging oxygen of the beta-phosphate in ATP. This interaction likely facilitates the transfer of gamma-phosphate from ATP to adenosine, thus accounting for the stimulating role of PVI in AK catalysis. The insight provided by these studies concerning the structural features of activators and inhibitors should also prove helpful in the design of more potent inhibitors of AK.

Adenosine Kinase↗

PubMed2004

The phylogeny and signature sequences characteristics of Fibrobacteres, Chlorobi, and Bacteroidetes.

Fibrobacteres, Chlorobi, and Bacteroidetes (FCB group) comprise three main bacterial phyla recognized on the basis of 16S rRNA trees. Presently, there are no distinctive biochemical or molecular characteristics known that can distinguish these bacteria from other bacterial phyla. The relationship of these bacteria to other phyla is also not known. This review describes many signatures, consisting of defined and conserved inserts in widely distributed proteins, that provide distinctive molecular markers for these groups of bacteria. These signatures serve to clarify the evolutionary relationship between members of the FCB group, and to other bacterial phyla. A 4 aa insert in DNA Gyrase B (GyrB) and a 45 aa insert in the SecA proteins are uniquely shared by various Bacteroidetes species. The insert in GyrB is present in all Bacteroidetes species (>100) covering different orders and families, indicating that it is a distinctive characteristic of the group. Three signatures consisting of an 18 aa insert in ATPase alpha-subunit, an 8-9 aa insert in the FtsK protein and a 1 aa insert in the UvrB protein are commonly shared only by the Bacteroidetes and Chlorobi homologs providing evidence that these two groups are specifically related to each other. Two additional inserts in the RNA polymerase beta'-subunit (5-7 aa) and Serine hydroxymethyl-transferase (14-16 aa), which are commonly present in various Bacteroidetes, Chlorobi, and Fibrobacteres homologs, but not any other bacteria, provide evidence that these groups shared a common ancestor exclusive of all other bacteria. The FCB groups of bacteria are indicated to have diverged from this common ancestor in the following order: Fibrobacteres --> Chlorobi --> Bacteriodetes. The inferences from signature sequences are strongly supported by phylogenetic analyses. These observations suggest that the FCB groups of bacteria should be placed in a single phylum rather than three distinct phyla. Signature sequences in a number of other proteins provide evidence that the FCB group of bacteria diverged at a similar time as the Chlamydiae group, and that the Spirochetes and Aquificales groups are its closest relatives.

Adenosine Triphosphatases↗

PubMed2004

Distinctive protein signatures provide molecular markers and evidence for the monophyletic nature of the deinococcus-thermus phylum.

The Deinococcus-Thermus group of species is currently recognized as a distinct phylum solely on the basis of their branching in 16S rRNA trees. No unique biochemical or molecular characteristics that can distinguish this group from all other bacteria are known at present. In this work, we describe eight conserved indels (viz., inserts or deletions) in seven widely distributed proteins that are distinctive characteristics of the Deinococcus-Thermus phylum but are not found in any other group of bacteria. The identified signatures include a 7-amino-acid (aa) insert in threonyl-tRNA synthetase, 1- and 3-aa inserts in the RNA polymerase beta' subunit, a 5-aa deletion in signal recognition particle (Ffh/SR54), a 2-aa insert in major sigma factor 70 (sigma70), a 2-aa insert in seryl-tRNA synthetase (SerRS), a 1-aa insert in ribosomal protein L1, and a 2-aa insert in UvrA homologs. By using PCR primers for conserved regions, fragments of these genes were amplified from a number of Deinococcus-Thermus species, and all such fragments (except SerRS in Deinococcus proteolyticus) were found to contain the indicated signatures. The presence of these signatures in various species from all three known genera within this phylum, viz., Deinococcus, Thermus, and Meiothermus, provide evidence that they are likely distinctive characteristics of the entire phylum which were introduced in a common ancestor of this group. The signature in SerRS, which is absent in D. proteolyticus, was likely introduced after the branching of this species. Phylogenetic studies as well as the nature of the inserts in some of these proteins (viz., sigma70 and SerRS) also support a sister group relationship between the Thermus and the Meiothermus genera. The identified signatures provide strong evidence for the monophyletic nature of the Deinococcus-Thermus phylum. These molecular markers should prove very useful in the identification of new species related to this group.

Amino Acid Sequence↗

PubMed2004

Signature sequences in diverse proteins provide evidence for the late divergence of the Order Aquificales.

The Aquificales species are presently believed to be the earliest branching lineage within Bacteria. However, the branching order of this group in different phylogenetic trees is highly variable and not resolved. In the present work, the phylogenetic placement of Aquificales was examined by means of a cladistic approach based on the shared presence or absence of definite signature sequences (consisting of conserved inserts or deletions) in many highly conserved and important proteins, e.g. RNA polymerase beta (RpoB), RNA polymerase beta (RpoC), alanyl-tRNA synthetase (AlaRS), CTP synthase, inorganic pyrophosphatase (PPase), Hsp70 and Hsp60. For this purpose, fragments of the above genes that contained the signature regions were cloned from different Aquificales species (Calderobacterium hydrogenophilum, Hydrogenobacter marinus, and Thermocrinis ruber) and the sequence data were compared with those available from all other species. The presence in Aquificales species of distinctive inserts in Hsp70 and Hsp60 that are not found in any Firmicutes, Actinobacteria, or Thermotoga-Clostridium species excluded them from these groups of Bacteria. The shared presence of prominent indels in the RpoB (>100 amino acids), RpoC (>100 amino acids) and AlaRS (4 amino acids) proteins, which are only found in the various Aquificales species, the Chlamydiae, the CFBG (Cytophaga-Flavobacteria-Bacteroides-green sulfur bacteria) group, and Proteobacteria, strongly suggests their placement within these groups of Bacteria. A specific relationship between Proteobacteria and Aquificales is suggested by the presence in inorganic pyrophosphatase of a 2-amino-acid insert that is uniquely found in these phyla. However, the Aquificales species lacked a number of other protein signatures (e.g. indels in CTP synthase and Hsp70) that are characteristic of Proteobacteria, indicating that they constitute a distinct phylum related to Proteobacteria. These results provide strong and consistent evidence that the Aquificales diverged after the branching of Firmicutes, Actinobacteria, Thermotoga, Deinococcus-Thermus, green nonsulfur bacteria, Cyanobacteria, Spirochetes, Chlamydiae, and CFBG group, but before the emergence of the Proteobacteria.

Alanine-tRNA Ligase↗

PubMed2003

Evolutionary relationships among photosynthetic bacteria.

To understand the evolution of photosynthetic bacteria it is necessary to understand how the main groups within Bacteria have evolved from a common ancestor, a critical issue that has not been resolved in the past. Recent analysis of shared conserved inserts or deletions (indels) in protein sequences has provided a powerful means to resolve this long-standing problem in microbiology. Based on a set of 25 indels in highly conserved and widely distributed proteins, all main groups within bacteria can now be defined in clear molecular terms and their relative branching orders logically deduced. For the 82 presently completed bacterial genomes, the presence or absence of these signatures in various proteins was found to be almost exactly as predicted by the indel model, with only 11 exceptions observed in 1842 observations. The branching order of different bacterial groups as deduced using this approach is as follows: low G+C Gram-positive (Heliobacterium chlorum) <--> high G+C Gram-positive <--> Clostridium-Fusobacterium-Thermotoga <--> Deinococcus-Thermus <--> green nonsulfur bacteria (Chloroflexus aurantiacus) <--> Cyanobacteria <--> Spirochetes <--> Chlamydia-Cytophaga-Flavobacteria-green sulfur bacteria (Chlorobium tepidum) <--> Aquifex <--> Proteobacteria (delta and in) <--> Proteobacteria (alpha) <--> Proteobacteria (beta) and <--> Proteobacteria (gamma). The Heliobacterium species, which contain an Fe-S type of reaction center (RC 1) and represent the sole photosynthetic phylum from the Gram-positive or monoderm bacteria (i.e., bounded by only a single membrane), is indicated to be the most ancestral of the photosynthetic lineages. Among the Gram-negative or diderm bacteria (containing both inner and outer cell membranes) the green nonsulfur bacteria, which contain a pheophytin-quinone type of reaction center (RC 2), are indicated to have evolved first. The later emerging photosynthetic groups which contain either one or both of these reaction centers could have acquired such genes from the earlier branching lineages by either direct descent or by means of lateral gene transfer.

Journal Article↗

PubMed2003

Molecular signatures in protein sequences that are characteristic of cyanobacteria and plastid homologues.

Fourteen conserved indels (i.e. inserts or deletions) have been identified in 10 widely distributed proteins that appear to be characteristic of cyanobacterial species and are not found in any other group of bacteria. These signatures include three inserts of 6, 7 and 28 aa in the DNA helicase II (UvrD) protein, an 18-21 aa insert in DNA polymerase I, a 14 aa insert in the enzyme ADP-glucose pyrophosphorylase, a 3 aa insert in the FtsH protein, an 11-13 aa insert in phytoene synthase, a 5 aa insert in elongation factor-Tu, two deletions of 2 and 7 aa in ribosomal S1 protein, a 2 aa insert in the SecA protein, a 1 aa deletion and a 6 aa insert in the enzyme inosine-5'-monophosphate dehydrogenase and a 1 aa deletion in the major sigma factor. These signatures, which are flanked by conserved regions, provide molecular markers for distinguishing cyanobacterial taxa from all other bacteria and they should prove helpful in the identification of cyanobacterial species, simply on the basis of the presence or absence of these markers in the corresponding proteins. The signatures in six of these proteins (SecA, elongation factor-Tu, ADP-glucose pyrophosphorylase, phytoene synthase, FtsH and ribosomal S1 protein) are also commonly present in plastid homologues from plants and algae (chlorophytes, chromophytes and rhodophytes), indicating their specific relationship to cyanobacteria and supporting their endosymbiotic origin from these bacteria. In phylogenetic trees based on a number of these proteins (SecA, UvrD, DNA polymerase I, elongation factor-Tu) that were investigated, the available cyanobacterial homologues grouped together with high affinity (>95 % bootstrap value), supporting the view that the cyanobacterial phylum is monophyletic and that the identified signatures were introduced in a common ancestor of this group.

Adenosine Triphosphatases↗

PubMed2002

Pentavalent ions dependency is a conserved property of adenosine kinase from diverse sources: identification of a novel motif implicated in phosphate and magnesium ion binding and substrate inhibition.

The catalytic activity of adenosine kinase (AK) from mammalian sources has previously been shown to exhibit a marked dependency upon the presence of pentavalent ions (PVI), such as phosphate (PO4), arsenate, or vanadate. We now show that the activity of AK from diverse sources, including plant, yeast, and protist species, is also markedly enhanced in the presence of PVI. In all cases, PO4 or other PVI exerted their effects primarily by decreasing the Km for adenosine and alleviating the inhibition caused by high concentrations of substrates. These results provide evidence that PVI dependency is a conserved property of AK and perhaps of the PfkB family of carbohydrate kinases which includes AK. On the basis of sequence alignments, we have identified a conserved motif NXXE within the PfkB family. The N and E of this motif make close contacts with Mg2+ and PO4 ions in the crystal structures of AK and bacterial ribokinase (another PfkB member which shows PVI dependency), implicating these residues in their binding. Site-directed mutagenesis of these residues in Chinese hamster AK have resulted in active proteins with greatly altered phosphate stimulation and substrate inhibition characteristics. The N239Q mutation leads to the formation of an active protein whose activity was not stimulated by PO4 or inhibited by high concentrations of adenosine or ATP. The activity of the E242D mutant protein was also not significantly altered in the presence of phosphate. Although PO4 had no effect on the KmAdenosine for this mutant, the KmATP, K(i)Adenosine, and K(i)ATP were significantly decreased. In contrast to these mutations, N239L or E242L mutant proteins showed greatly decreased activity with an altered Mg2+ requirement. These observations support the view that N239 and E242 play an important role in the binding of PO4 and Mg2+ ions required for the catalytic activity of adenosine kinase.

Adenosine Kinase↗

PubMed2002

Critical issues in bacterial phylogeny.

To understand bacterial phylogeny, it is essential that the following two critical issues be resolved: (i) development of well-defined (molecular) criteria for identifying the main groups within Bacteria, and (ii) to understand how the different main groups are related to each other and how they branched off from a common ancestor. These issues are not resolved at present. We have recently described a new approach, based on shared conserved inserts and deletions (indels or signature sequences) found in various proteins, that provides a reliable means for understanding these issues. A large number of conserved indels that are shared by different groups of bacteria have been identified. Using these indels, and based simply on their presence or absence, all of the main groups within Bacteria can be defined in clear molecular terms and new species could be assigned to them with minimal ambiguity. The analysis of these indels also permits one to logically deduce that the various main bacterial groups have branched off from a common ancestor in the following order: Low G+C Gram-positive ==> High G+C Gram-positive ==> Clostridium-Fusobacteria-Thermotoga ==> Deinococcus-Thermus-Green nonsulfur bacteria ==> Cyanobacteria ==> Spirochetes ==> Chlamydia-Cytophaga-Bacteroides-Green sulfur bacteria ==> Aquifex ==> Proteobacteria 1 (epsilon and delta) ==> Proteobacteria-2. (alpha) ==> Proteobacteria-3 (beta) and ==> Proteobacteria-4 (gamma). The validity of this approach was tested using sequence data from bacterial genomes. By making use of 18 conserved indels, species from all 60 completed bacterial genomes were assigned to different groups. The observed distribution of these indels in different species was then compared with that predicted by the model. Of the 936 observations concerning the placement of these indels in various species, all except one were in accordance with the model. The placement of bacteria into different groups using this approach also showed excellent correlation with the 16S rRNA phylogenies with nearly all of the species assigned to the same groups by both methods. These results provide strong evidence that the genes containing these indels have not been affected by factors such as lateral gene transfers. However, such events are readily detected by this means and some examples are provided. The approach described here thus provides a reliable and internally consistent means for understanding various critical and long outstanding issues in bacterial phylogeny.

Amino Acid Sequence↗

PubMed2002

Protein signatures distinctive of chlamydial species: horizontal transfers of cell wall biosynthesis genes glmU from archaea to chlamydiae and murA between chlamydiae and Streptomyces.

Chlamydiae are major human and animal pathogens. Based on alignments of different protein sequences, a number of conserved indels (insertion/deletions) were identified that appear to be unique and distinctive characteristics of the chlamydial species. The identified signatures include one 16 aa and two single aa inserts in the enzyme UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA), a 1 aa insert in protein synthesis elongation factor P (EF-P), a 1 aa insert in the Mg(2+) transport protein (MgtE), a 1 aa insert in the carboxy-terminal protease and a 1 aa deletion in the tRNA (guanine-N(1)-)-methyltransferase (TrmD) protein. The homologues of these proteins are found in all major groups of bacteria and the observed indels are present in all available chlamydial sequences but not in any other species (except for the large insert in MurA in Streptomyces). The validity of three of these signatures (MurA, EF-P and MgtE) was tested by PCR amplifying the signature regions from several chlamydial species for which no sequence information was available. All Chlamydiaceae species for which specific fragments could be amplified (Chlamydia suis, Chlamydophila abortus, Chlamydophila psittaci, Chlamydophila felis) contained the expected signatures. Additionally, a fragment of the murA gene from Waddlia chondrophila and the efp gene from Simkania negevensis, two chlamydia-like species, were also cloned and sequenced. The presence of respective indels in these species provides strong evidence that they are specifically related to the traditional chlamydial species, and that these signatures may be distinctive of the entire Chlamydiales order. A 17 aa conserved indel was also identified in the cell wall biosynthesis enzyme UDP-N-acetylglucosamine pyrophosphorylase (GlmU), which is shared by all archaeal and chlamydial homologues. The gene for this protein is indicated to have been horizontally transferred from an archaeon to a common ancestor of the chlamydiae. The results also support a lateral transfer of the murA gene between chlamydiae and STREPTOMYCES: The large inserts in these peptidoglycan synthesis related genes in chlamydiae could account for their unusual cell-wall characteristics. These signatures are also potentially useful for screening of the chlamydiae species.

Alkyl and Aryl Transferases↗

PubMed2001

The use of signature sequences in different proteins to determine the relative branching order of bacterial divisions: evidence that Fibrobacter diverged at a similar time to Chlamydia and the Cytophaga-Flavobacterium-Bacteroides division.

The phylogenetic placement of the rumen bacterium Fibrobacter succinogenes was determined using a signature sequence approach that allows determination of the relative branching order of the major divisions among Bacteria [Gupta, R. S. (2000) FEMS Microbiol Rev 24, 367-402]. For this purpose, segments of the Hsp60 (groEL), Hsp70 (dnaK), CTP synthase and alanyl-tRNA synthetase genes, which are known to contain signature sequences that are useful for phylogenetic deterministic purposes, were cloned. Using degenerate oligonucleotide primers for highly conserved regions in these proteins, 1.4 kb, 0.75 kb, 401 bp and 171 bp fragments of the Hsp70, Hsp60, CTP synthase and alanyl-tRNA synthetase genes respectively were amplified by PCR, and these fragments were cloned and sequenced. These primers, because of their high degree of conservation, could also be used for cloning these genes from other bacterial species. The Hsp70 homologues from different Gram-negative bacteria contain a 21-23 aa insert that is not found in any Gram-positive bacteria. The presence of this insert in the F. succinogenes Hsp70 supports its placement within the Gram-negative group of bacteria. A conserved insert in F. succinogenes Hsp60 that is commonly present in all bacterial species, except various Gram-positive bacteria, Deinococcus-Thermus groups and green non-sulphur bacteria, provides evidence that F. succinogenes does not belong to these taxa. A particularly useful signature consisting of a 4 aa insert is found in Ala-tRNA synthetase. This insert is present in all proteobacterial homologues as well as in homologues from species belonging to the Chlamydia and Cytophaga-Flavobacterium- Bacteroides (CFB) groups, but it is not found in homologues from any other groups of bacteria. The presence of this insert in F. succinogenes Ala-tRNA synthetase provides evidence that this species is related to these groups. However, two other signatures in CTP synthase and Hsp70 proteins, that are distinctive of the proteobacterial species, are not present in the F. succinogenes homologues. These results provide evidence that F. succinogenes does not belong to the proteobacterial division and thus should be placed in a similar position as the Chlamydia and CFB groups of species.

Alanine-tRNA Ligase↗