Biomedical subjects

Radhey S Gupta

Publications and source records attributed to Radhey S Gupta.

At least 19 recordsLinked to original sources

PubMed2007

Identification and biochemical studies on novel non-nucleoside inhibitors of the enzyme adenosine kinase.

The enzyme adenosine kinase (AK) plays a key role in the regulation of intracellular and extracellular concentration of adenosine (Ado), which exhibits potent hormonal activity in cardiovascular, nervous and immune systems. In view of the pharmacological effects of Ado, there is much interest in identifying inhibitors of AK, which can augment its tissue-protective effects. In this study, we have screened 1040 compounds from a chemical library of putative kinase inhibitors for their effect on purified human recombinant AK. These studies have identified 8 novel, non-nucleoside AK inhibitors. Four of these compounds (viz. 2-tert-butyl-4H-benzo[1,2,4]thiadiazine-3-thione (2759-0749); N-(5,6-diphenyl-furo[2,3-d]pyrimidin-4-yl)-propionamide (3998-0118); 3-[5,6-Bis-(4-methoxy-phenyl)-furo[2,3-d]pyrimidin-4-ylamino]-propan-1-ol (4072-2732); and 2-[2-(3,4-dihydroxy-phenyl)-5-phenyl-1H-imidazol-4-yl]-fluoren-9-one (8008-6198)), which inhibited human AK in a concentration-dependent manner in a low micromolar range (IC(50) = 0.38 approximately 1.98 microM) were further studied. Kinetic and structural studies on these compounds provide evidence that inhibition of AK by these compounds was competitive with respect to Ado and non-competitive for ATP. All of these compounds also inhibited uptake of Ado and its metabolism in cultured mammalian cells at comparable concentrations indicating their efficient cellular penetrability. These AK inhibitors, whose chemical structures differ significantly from all previously known inhibitors, provide useful lead compounds for identification of more potent but less toxic AK inhibitors that may prove useful for therapeutic purposes.

Adenosine↗

PubMed2006

Application of the character compatibility approach to generalized molecular sequence data: branching order of the proteobacterial subdivisions.

The character compatibility approach, which removes all homoplasic characters and involves finding the largest clique of compatible characters in a dataset, in principle, provides a powerful means for obtaining correct topology in difficult to resolve cases. However, the usefulness of this approach to generalized molecular sequence data for phylogeny determination has not been studied in the past. We have used this approach to determine the topology of 23 proteobacterial species (6 each of alpha-, beta- and gamma-, 3 delta-, and 2 epsilon-proteobacteria) using sequence data for 10 conserved proteins (Hsp60, Hsp70, EF-Tu, EF-G, alanyl-tRNA synthetase, RecA, GyrA, GyrB, RpoB and RpoC). All sites in the sequence alignments of these proteins where only two amino acids were found, with each amino acid present in at least two species, were selected. Mutual compatibility determination on these binary state sites was carried out by two means. In one case, all of these sites were combined into a large dataset (Set A; 957 characters) prior to compatibility analysis. In the second case, compatibility analysis was carried out on characters from individual proteins and all compatible sites were combined into a large dataset (Set B; 398 characters) for further studies. Upon compatibility analyses, the largest cliques that were obtained from Sets A and B consisted of 337 and 323 compatible characters, respectively. In these cliques, all proteobacterial subgroups were clearly distinguished and branching orders of most of the species were also resolved. The epsilon-proteobacteria exhibited the earliest branching, whereas the beta- and gamma-subgroups were found to have emerged last. The relative placement of the alpha- and delta-subgroups, however, was not resolved. The topology of these species was also determined based on 16S rRNA sequences and a concatenated dataset of sequences for all 10 proteins by means of neighbor-joining, maximum likelihood, and maximum parsimony methods. In the protein trees, all proteobacterial groups were reliably resolved and they branched in the following order: (epsilon(delta(alpha(beta,gamma)))). However, in the rRNA trees, the gamma- and beta-subgroups exhibited polyphyletic branching and many internal nodes were not resolved. These results indicate that the character compatibility analysis using generalized molecular sequence data provides a powerful means for evolutionary studies. Based on molecular sequences, it should be possible to obtain very large datasets of compatible characters that should prove very helpful in clarifying difficult to resolve phylogenetic relationships.

Amino Acid Sequence↗

PubMed2006

Subcellular localization of fumarase in mammalian cells and tissues.

Fumarase, a mitochondrial matrix protein, is previously indicated to be present in substantial amounts in the cytosol as well. However, recent studies show that newly synthesized human fumarase is efficiently imported into mitochondria with no detectable amount in the cytosol. To clarify its subcellular localization, the subcellular distribution of fumarase in mammalian cells/tissues was examined by a number of different methods. Cell fractionation using either a mitochondria fraction kit or extraction with low concentrations of digitonin, detected no fumarase in a 100,000 g supernatant fraction. Immunofluorescence labeling with an affinity-purified antibody to fumarase and an antibody to the mitochondrial Hsp60 protein showed identical labeling pattern with labeling seen mainly in mitochondria. Detailed studies were performed using high-resolution immunogold electron microscopy to determine the subcellular localization of fumarase in rat tissues, embedded in LR White resin. In thin sections from kidney, liver, heart, adrenal gland and anterior pituitary, strong and specific labeling due to fumarase antibody was only detected in mitochondria. However, in the pancreatic acinar cells, in addition to mitochondria, highly significant labeling was also observed in the zymogen granules and endoplasmic reticulum. The observed labeling in all cases was completely abolished upon omission of the primary antibody indicating that it was specific. In a western blot of purified zymogen granules, a fumarase-antibody cross-reactive protein of the same molecular mass as seen in the mitochondria was present. These results provide evidence that fumarase in mammalian cells/tissues is mainly localized in mitochondria and significant amounts of this protein are not present in the cytosol. However, these studies also reveal that in certain tissues, in addition to mitochondria, this protein is also present at specific extramitochondrial sites. Although the cellular function of fumarase at these extramitochondrial locations is not known, the appearance/localization of fumarase outside mitochondria may help explain how mutations in this mitochondrial protein can give rise to a number of different types of cancers.

Animals↗

PubMed2006

Genomics as a means to understand bacterial phylogeny and ecological adaptation: the case of bifidobacteria.

The field of microbiology has in recent years been transformed by the ever increasing number of publicly available whole-genome sequences. This sequence information has significantly enhanced our understanding of the physiology, genetics and evolutionary development of bacteria. Among the latter group of microorganisms, bifidobacteria represent important human commensals because of their perceived contribution to maintaining a balanced gastrointestinal tract microbiota. In recent years bifidobacteria have drawn much scientific attention because of their use as live bacteria in numerous food preparations with various health-related claims. For this reason, these bacteria constitute a growing area of interest with respect to genomics, molecular biology and genetics. Recent genome sequencing of a number of bifidobacterial species has allowed access to the complete genetic make-up of these bacteria. In this review we will discuss how genomic data has allowed us to understand bifidobacterial evolution, while also revealing genetic functions that explains their presence in the particular ecological environment of the gastrointestinal tract.

Acclimatization↗

PubMed2006

Chlamydiae-specific proteins and indels: novel tools for studies.

Chlamydiae species are important human and animal pathogens. Their obligate intracellular mode of replication has precluded the use of genetic and molecular biological approaches for understanding their biology. Comparative genomics have identified many rare genetic changes consisting of whole proteins and conserved indels (i.e. inserts or deletions) in widely distributed proteins that are distinctive characteristics of either all, or various subgroups within, chlamydiae. Additionally, several interesting cases of the lateral transfer of genes from free-living bacteria to a common ancestor of chlamydiae, and from chlamydiae to Trypanosoma/Leishmania, have been identified. These novel signatures have possible applications for advancing our understanding of the chlamydiae.

Bacterial Proteins↗

PubMed2006

Lateral transfers of serine hydroxymethyltransferase (glyA) and UDP-N-acetylglucosamine enolpyruvyl transferase (murA) genes from free-living Actinobacteria to the parasitic chlamydiae.

The chlamydiae are important human and animal pathogens which form a phylogentically distinct lineage within the Bacteria. There is evidence that some genes in these obligate intracellular parasites have undergone lateral exchange with other free-living organisms. In the present work, we describe two interesting cases of lateral gene transfer between chlamydiae and actinobacteria, which have been identified based on the shared presence of conserved inserts in two important proteins. In the enzyme serine hydroxymethyltransferase (SHMT or GlyA protein), which links amino acid and nucleotide metabolisms by generating the key intermediate for one-carbon transfer reactions, two conserved inserts of 3 and 31 amino acids (aa) are uniquely present in various chlamydiae species as well as in a subset of Actinobacteria and in the Treponema species. Similarly, in the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), which is involved in the synthesis of cell wall peptidoglycan, a 16-aa conserved insert is specifically present in various sequenced chlamydiae and a subset of actinobacteria (i.e., Streptomyces, Actinomyces, Tropheryma, Bifidobacterium, Leifsonia, Arthrobacter, and Brevibacterium). To determine the phylogenetic depths of the GlyA and MurA inserts, the fragments of these genes from two chlamydiae-like species, Simkania negevensis and Waddlia chondrophila, were PCR amplified and sequenced. The presence of the corresponding inserts in both these species strongly indicates that these inserts are distinctive characteristics of the Chlamydiales order. In phylogenetic trees based on GlyA and MurA protein sequences, the chlamydiae species (and also the Treponema species in the case of GlyA) branched with a high affinity with various insert-containing actinobacteria within a clade of other actinobacteria. These results provide strong evidence that the shared presence of these indels in these bacteria is very likely a consequence of ancient lateral gene transfers from actinobacteria to chlamydiae. Pairwise sequence identity and the branching pattern of the GlyA homologues in the phylogenetic tree indicates that the glyA gene was initially transferred from an actinobacteria to an ancestor of the Treponema genus and from there it was acquired by the common ancestor of the Chlamydiales.

Actinobacteria↗

PubMed2006

Molecular signatures (unique proteins and conserved indels) that are specific for the epsilon proteobacteria (Campylobacterales).

BACKGROUND: The epsilon proteobacteria, which include many important human pathogens, are presently recognized solely on the basis of their branching in rRNA trees. No unique molecular or biochemical characteristics specific for this group are known. RESULTS: Comparative analyses of proteins in the genomes of Wolinella succinogenes DSM 1740 and Campylobacter jejuni RM1221 against all available sequences have identified a large number of proteins that are unique to various epsilon proteobacteria (Campylobacterales), but whose homologs are not detected in other organisms. Of these proteins, 49 are uniquely found in nearly all sequenced epsilon-proteobacteria (viz. Helicobacter pylori (26695 and J99), H. hepaticus, C. jejuni (NCTC 11168, RM1221, HB93-13, 84-25, CF93-6, 260.94, 11168 and 81-176), C. lari, C. coli, C. upsaliensis, C. fetus, W. succinogenes DSM 1740 and Thiomicrospira denitrificans ATCC 33889), 11 are unique for the Wolinella and Helicobacter species (i.e. Helicobacteraceae family) and many others are specific for either some or all of the species within the Campylobacter genus. The primary sequences of many of these proteins are highly conserved and provide novel resources for diagnostics and therapeutics. We also report four conserved indels (i.e. inserts or deletions) in widely distributed proteins (viz. B subunit of exinuclease ABC, phenylalanyl-tRNA synthetase, RNA polymerase beta '-subunit and FtsH protein) that are specific for either all epsilon proteobacteria or different subgroups. In addition, a rare genetic event that caused fusion of the genes for the largest subunits of RNA polymerase (rpoB and rpoC) in Wolinella and Helicobacter is also described. The inter-relationships amongst Campylobacterales as deduced from these molecular signatures are in accordance with the phylogenetic trees based on the 16S rRNA and concatenated sequences for nine conserved proteins. CONCLUSION: These molecular signatures provide novel tools for identifying and circumscribing species from the Campylobacterales order and its subgroups in molecular terms. Although sequence information for these signatures is presently limited to Campylobacterales species, it is likely that many of them will also be found in other epsilon proteobacteria. Functional studies on these proteins and conserved indels should reveal novel biochemical or physiological characteristics that are unique to these groups of epsilon proteobacteria.

Bacterial Proteins↗

PubMed2006

Mitochondrial import of human and yeast fumarase in live mammalian cells: retrograde translocation of the yeast enzyme is mainly caused by its poor targeting sequence.

Studies on yeast fumarase provide the main evidence for dual localization of a protein in mitochondria and cytosol by means of retrograde translocation. We have examined the subcellular targeting of yeast and human fumarase in live cells to identify factors responsible for this. The cDNAs for mature yeast or human fumarase were fused to the gene for enhanced green fluorescent protein (eGFP) and they contained, at their N-terminus, a mitochondrial targeting sequence (MTS) derived from either yeast fumarase, human fumarase, or cytochrome c oxidase subunit VIII (COX) protein. Two nuclear localization sequences (2x NLS) were also added to these constructs to facilitate detection of any cytosolic protein by its targeting to nucleus. In Cos-1 cells transfected with these constructs, human fumarase with either the native or COX MTSs was detected exclusively in mitochondria in >98% of the cells, while the remainder 1-2% of the cells showed varying amounts of nuclear labeling. In contrast, when human fumarase was fused to the yeast MTS, >50% of the cells showed nuclear labeling. Similar studies with yeast fumarase showed that with its native MTS, nuclear labeling was seen in 80-85% of the cells, but upon fusion to either human or COX MTS, nuclear labeling was observed in only 10-15% of the cells. These results provide evidence that extramitochondrial presence of yeast fumarase is mainly caused by the poor mitochondrial targeting characteristics of its MTS (but also affected by its primary sequence), and that the retrograde translocation mechanism does not play a significant role in the extramitochondrial presence of mammalian fumarase.

Animals↗

PubMed2006

Signature proteins that are distinctive characteristics of Actinobacteria and their subgroups.

The Actinobacteria constitute one of the main phyla of Bacteria. Presently, no morphological and very few molecular characteristics are known which can distinguish species of this highly diverse group. In this work, we have analyzed the genomes of four actinobacteria (viz. Mycobacterium leprae TN, Leifsonia xyli subsp. xyli str. CTCB07, Bifidobacterium longum NCC2705 and Thermobifida fusca YX) to search for proteins that are unique to Actinobacteria. Our analyses have identified 233 actinobacteria-specific proteins, homologues of which are generally not present in any other bacteria. These proteins can be grouped as follows: (i) 29 proteins uniquely present in most sequenced actinobacterial genomes; (ii) 6 proteins present in almost all actinobacteria except Bifidobacterium longum and another 37 proteins absent in B. longum and few other species; (iii) 11 proteins which are mainly present in Corynebacterium, Mycobacterium and Nocardia (CMN) subgroup as well as Streptomyces, T. fusca and Frankia sp., but they are not found in Bifidobacterium and Micrococcineae; (iv) 8 proteins that are specific for T. fusca and Streptomyces species, plus 2 proteins also present in the Frankia species; (v) 13 proteins that are specific for the Corynebacterineae or the CMN group; (vi) 14 proteins only found in Mycobacterium and Nocardia; (vii) 24 proteins unique to different Mycobacterium species; (viii) 8 proteins specific to the Micrococcineae; (ix) 85 proteins which are distributed sporadically in actinobacterial species. Additionally, many examples of lateral gene transfer from Actinobacteria to Magnetospirillum magnetotacticum have also been identified. The identified proteins provide novel molecular means for defining and circumscribing the Actinobacteria phylum and a number of subgroups within it. The distribution of these proteins also provides useful information regarding interrelationships among the actinobacterial subgroups. Most of these proteins are of unknown function and studies aimed at understanding their cellular functions should reveal common biochemical and physiological characteristics unique to either all actinobacteria or particular subgroups of them. The identified proteins also provide potential targets for development of drugs that are specific for actinobacteria.

Actinobacteria↗

PubMed2006

BLAST screening of chlamydial genomes to identify signature proteins that are unique for the Chlamydiales, Chlamydiaceae, Chlamydophila and Chlamydia groups of species.

BACKGROUND: Chlamydiae species are of much importance from a clinical viewpoint. Their diversity both in terms of their numbers as well as clinical involvement are presently believed to be significantly underestimated. The obligate intracellular nature of chlamydiae has also limited their genetic and biochemical studies. Thus, it is of importance to develop additional means for their identification and characterization. RESULTS: We have carried out analyses of available chlamydiae genomes to identify sets of unique proteins that are either specific for all Chlamydiales genomes, or different Chlamydiaceae family members, or members of the Chlamydia and Chlamydophila genera, or those unique to Protochlamydia amoebophila, but which are not found in any other bacteria. In total, 59 Chlamydiales-specific proteins, 79 Chlamydiaceae-specific proteins, 20 proteins each that are specific for both Chlamydia and Chlamydophila and 445 ORFs that are Protochlamydia-specific were identified. Additionally, 33 cases of possible gene loss or lateral gene transfer were also detected. CONCLUSION: The identified chlamydiae-lineage specific proteins, many of which are highly conserved, provide novel biomarkers that should prove of much value in the diagnosis of these bacteria and in exploration of their prevalence and diversity. These conserved protein sequences (CPSs) also provide novel therapeutic targets for drugs that are specific for these bacteria. Lastly, functional studies on these chlamydiae or chlamydiae subgroup-specific proteins should lead to important insights into lineage-specific adaptations with regards to development, infectivity and pathogenicity.

Algorithms↗

PubMed2006

Inhibition of adenosine kinase by phosphonate and bisphosphonate derivatives.

The enzyme adenosine kinase (AK) plays a central role in regulating the intracellular and interstitial concentration of the purine nucleoside adenosine (Ado). In view of the beneficial effects of Ado in protecting tissues from ischemia and other stresses, there is much interest in developing AK inhibitors, which can regulate Ado concentration in a site- and event-specific manner. The catalytic activity of AK from different sources is dependent upon the presence of activators such as phosphate (Pi). In this work we describe several new phosphorylated compounds which either activate or inhibit AK. The compounds acetyl phosphate, carbamoyl phosphate, dihydroxyacetone phosphate and imidodiphosphate were found to stimulate AK activity in a dose-dependent manner comparable to that seen with Pi. In contrast, a number of phosphonate and bisphosphonate derivatives, which included clodronate and etidronate, were found to inhibit the activity of purified AK in the presence of Pi. These AK inhibitors (viz. clodronate, etidronate, phosphonoacetic acid, 2-carboxyethylphosphonic acid, N-(phosphonomethyl)-glycine and N-(phosphonomethyl)iminodiacetic acid), at concentrations at which they inhibited AK, were also shown to inhibit the uptake of (3)H-adenosine and its incorporation into macromolecules in cultured mammalian cells, indicating that they were also inhibiting AK in intact cells. The drug concentrations at which these effects were observed showed limited toxicity to the cultured cells, indicating that these effects are not caused by cellular toxicity. These results indicate that the enzyme AK provides an additional cellular target for the clinically widely used bisphosphonates and related compounds, which could possibly be exploited for a new therapeutic application. Our structure-activity studies on different AK activators and inhibitors also indicate that all of the AK activating compounds have a higher partial positive charge (delta(+)) on the central phosphorous atom in comparison to the inhibitors. This information should prove helpful in the design and synthesis of more potent inhibitors of AK.

Adenosine↗

PubMed2006

Molecular signatures in protein sequences that are characteristics of the phylum Aquificae.

Species of the phylum Aquificae are of great interest due to their strict extreme thermophilic growth characteristics. Presently, there is no known molecular characteristic which is unique to this group of bacteria. This work describes six conserved inserts and deletions (indels or signature sequences) in four widely distributed proteins that are distinctive features of species from the phylum Aquificae. These include three signatures consisting of a 2 aa insert, a 5-6 aa insert and a 6 aa deletion in DNA polymerase I (PolA), a 6-7 aa insert in glucose-inhibited protein A (GidA), a 52 aa insert in the RNA polymerase beta'-subunit (RpoC) and a 4 aa insert in elongation factor Tu (EF-Tu). Fragments of these genes were amplified in most cases from Hydrogenobacter hydrogenophilus, Hydrogenothermus marinus and Thermocrinis ruber and combined with available sequence data from 'Aquifex aeolicus' and Sulfurihydrogenibium azorense. The presence of the PolA, GidA and RpoC indels in all of the species sequenced provides evidence that they are probably distinctive characteristics of the entire phylum. The indel in EF-Tu, which is shared by Aquifex species and Hydrogenobacter but not Hydrogenothermus and Sulfurihydrogenibium, may provide a molecular marker for the family Aquificaceae. We have also identified a 51 aa insert in SecA preprotein translocase that is commonly shared by various species of the Aquificae as well as two Thermotoga species (Thermotoga maritima and Thermotoga neapolitana) which may be due to lateral gene transfer between these groups. In phylogenetic trees based on a concatenated dataset of fragments from eight different proteins as well as 16S rRNA, the observed branching pattern of these species was very similar and it was consistent with the relationships inferred from various indels. The identified indels provide a novel means for distinguishing species of the Aquificae from all other bacteria in molecular terms and may prove useful for functional studies aimed at understanding the unique biochemical and physiological characteristics of the Aquificae.

Adenosine Triphosphatases↗

PubMed2005

Localization of mitochondrial DNA encoded cytochrome c oxidase subunits I and II in rat pancreatic zymogen granules and pituitary growth hormone granules.

Cytochrome c oxidase (COX) complex is an integral part of the electron transport chain. Three subunits of this complex (COX I, COX II and COX III) are encoded by mitochondrial (mit-) DNA. High-resolution immunogold electron microscopy has been used to study the subcellular localization of COX I and COX II in rat tissue sections, embedded in LR Gold resin, using monoclonal antibodies for these proteins. Immunofluorescence labeling of BS-C-1 monkey kidney cells with these antibodies showed characteristic mitochondrial labeling. In immunogold labeling studies, the COX I and COX II antibodies showed strong and specific mitochondrial labeling in the liver, kidney, heart and pancreas. However, in rat pancreatic acinar tissue, in addition to mitochondrial labeling, strong and specific labeling was also observed in the zymogen granules (ZGs). In the anterior pituitary, strong labeling with these antibodies was seen in the growth hormone secretory granules. In contrast to these compartments, the COX I or COX II antibodies showed only minimal labeling (five- to tenfold lower) of the cytoplasm, endoplasmic reticulum and the nucleus. Strong labeling with the COX I or COX II antibodies was also observed in highly purified ZGs from bovine pancreas. The observed labeling, in all cases, was completely abolished upon omission of the primary antibodies. These results provide evidence that, similar to a number of other recently studied mit-proteins, COX I and COX II are also present outside the mitochondria. The presence of mit-DNA encoded COX I and COX II in extramitochondrial compartments, provides strong evidence that proteins can exit, or are exported, from the mitochondria. Although the mechanisms responsible for protein exit/export remain to be elucidated, these results raise fundamental questions concerning the roles of mitochondria and mitochondrial proteins in diverse cellular processes in different compartments.

Animals↗

PubMed2005

Signature proteins that are distinctive of alpha proteobacteria.

BACKGROUND: The alpha (alpha) proteobacteria, a very large and diverse group, are presently characterized solely on the basis of 16S rRNA trees, with no known molecular characteristic that is unique to this group. The genomes of three alpha-proteobacteria, Rickettsia prowazekii (RP), Caulobacter crescentus (CC) and Bartonella quintana (BQ), were analyzed in order to search for proteins that are unique to this group. RESULTS: Blast analyses of protein sequences from the above genomes have led to the identification of 61 proteins which are distinctive characteristics of alpha-proteobacteria and are generally not found in any other bacteria. These alpha-proteobacterial signature proteins are generally of hypothetical functions and they can be classified as follows: (i) Six proteins (CC2102, CC3292, CC3319, CC1887, CC1725 and CC1365) which are uniquely present in most sequenced alpha-proteobacterial genomes; (ii) Ten proteins (CC1211, CC1886, CC2245, CC3470, CC0520, CC0365, CC0366, CC1977, CC3010 and CC0100) which are present in all alpha-proteobacteria except the Rickettsiales; (iii) Five proteins (CC2345, CC3115, CC3401, CC3467 and CC1021) not found in the intracellular bacteria belonging to the order Rickettsiales and the Bartonellaceae family; (iv) Four proteins (CC1652, CC2247, CC3295 and CC1035) that are absent from various Rickettsiales as well as Rhodobacterales; (v) Three proteins (RP104, RP105 and RP106) that are unique to the order Rickettsiales and four proteins (RP766, RP192, RP030 and RP187) which are specific for the Rickettsiaceae family; (vi) Six proteins (BQ00140, BQ00720, BQ03880, BQ12030, BQ07670 and BQ11900) which are specific to the order Rhizobiales; (vii) Four proteins (BQ01660, BQ02450, BQ03770 and BQ13470) which are specific for the order Rhizobiales excluding the family Bradyrhizobiaceae; (viii) Nine proteins (BQ12190, BQ11460, BQ11450, BQ11430, BQ11380, BQ11160, BQ11120, BQ11100 and BQ11030 which are distinctive of the Bartonellaceae family;(ix) Six proteins (CC0189, CC0569, CC0331, CC0349, CC2323 and CC2637) which show sporadic distribution in alpha-proteobacteria, (x) Four proteins (CC2585, CC0226, CC2790 and RP382) in which lateral gene transfers are indicated to have occurred between alpha-proteobacteria and a limited number of other bacteria. CONCLUSION: The identified proteins provide novel means for defining and identifying the alpha-proteobacteria and many of its subgroups in clear molecular terms and in understanding the evolution of this group of species. These signature proteins, together with the large number of alpha-proteobacteria specific indels that have recently been identified http://www.bacterialphylogeny.com, provide evidence that all species from this diverse group share many unifying and distinctive characteristics. Functional studies on these proteins should prove very helpful in the identification of such characteristics.

Alphaproteobacteria↗

PubMed2005

Induction of mitochondrial fusion by cysteine-alkylators ethacrynic acid and N-ethylmaleimide.

Mitochondrial fusion and fission are important aspects of eukaryotic cell function that permit the adoption of varied mitochondrial morphologies depending upon cellular physiology. We previously observed that ethacrynic acid (EA) induced mitochondrial fusion in cultured BSC-1 and CHO/wt cells. However, the mechanism responsible for it was not clear since EA has a number of known cellular effects including glutathione (GSH) depletion and alkylation of cysteine residues. To gain insight, we have tested the effects of a variety of compounds on EA induced cellular toxicity and mitochondrial fusion. N-acetyl cysteine (NAC), a GSH precursor, was found to abrogate both the toxic and fusion-inductive effects, whereas diethylmaleate (dEM), a GSH depletor, potentiated both these effects in a dose-dependent manner. However, treatment with dEM alone, which depleted GSH to the same degree as EA, did not induce mitochondrial fusion. These results indicate that although detoxification of EA via formation of GSH conjugates is dependant upon GSH levels, the depletion of GSH by EA is not responsible for its effect on mitochondrial fusion. Dihydro-EA (DH-EA), a saturated EA analogue, lacked EA's toxicity and effect on fusion, indicating that the alpha,beta-unsaturated ketone is central to its observed effects. N-ethylmaleimide (NEM), another well-known cysteine-alkylator, also induced mitochondrial fusion at near toxic concentrations. These data suggests that cysteine-alkylation is the causative factor for fusion and toxicity. In live BSC-1 cells, EA induced fusion of mitochondria occurred very rapidly (<20 min), which suggests that it is inducing fusion by modifying certain critical cysteine residue(s) in proteins involved in the process.

Animals↗

PubMed2005

Protein signatures distinctive of alpha proteobacteria and its subgroups and a model for alpha-proteobacterial evolution.

Alpha (alpha) proteobacteria comprise a large and metabolically diverse group. No biochemical or molecular feature is presently known that can distinguish these bacteria from other groups. The evolutionary relationships among this group, which includes numerous pathogens and agriculturally important microbes, are also not understood. Shared conserved inserts and deletions (i.e., indels or signatures) in molecular sequences provide a powerful means for identification of different groups in clear terms, and for evolutionary studies (see www.bacterialphylogeny.com). This review describes, for the first time, a large number of conserved indels in broadly distributed proteins that are distinctive and unifying characteristics of either all alpha-proteobacteria, or many of its constituent subgroups (i.e., orders, families, etc.). These signatures were identified by systematic analyses of proteins found in the Rickettsia prowazekii (RP) genome. Conserved indels that are unique to alpha-proteobacteria are present in the following proteins: Cytochrome c oxidase assembly protein Ctag, PurC, DnaB, ATP synthase alpha-subunit, exonuclease VII, prolipoprotein phosphatidylglycerol transferase, RP-400, FtsK, puruvate phosphate dikinase, cytochrome b, MutY, and homoserine dehydrogenase. The signatures in succinyl-CoA synthetase, cytochrome oxidase I, alanyl-tRNA synthetase, and MutS proteins are found in all alpha-proteobacteria, except the Rickettsiales, indicating that this group has diverged prior to the introduction of these signatures. A number of proteins contain conserved indels that are specific for Rickettsiales (XerD integrase and leucine aminopeptidase), Rickettsiaceae (Mfd, ribosomal protein L19, FtsZ, Sigma 70 and exonuclease VII), or Anaplasmataceae (Tgt and RP-314), and they distinguish these groups from all others. Signatures in DnaA, RP-057, and DNA ligase A are commonly shared by various Rhizobiales, Rhodobacterales, and Caulobacter, suggesting that these groups shared a common ancestor exclusive of other alpha-proteobacteria. A specific relationship between Rhodobacterales and Caulobacter is indicated by a large insert in the Asn-Gln amidotransferase. The Rhizobiales group of species are distinguished from others by a large insert in the Trp-tRNA synthetase. Signature sequences in a number of other proteins (viz. oxoglutarate dehydogenase, succinyl-CoA synthase, LytB, DNA gyrase A, LepA, and Ser-tRNA synthetase) serve to distinguish the Rhizobiaceae, Brucellaceae, and Phyllobacteriaceae families from Bradyrhizobiaceae and Methylobacteriaceae. Based on the distribution patterns of these signatures, it is now possible to logically deduce a model for the branching order among alpha-proteobacteria, which is as follows: Rickettsiales --> Rhodospirillales-Sphingomonadales --> Rhodobacterales-Caulobacterales --> Rhizobiales (Rhizobiaceaea-Brucellaceae-Phyllobacteriaceae, and Bradyrhizobiaceae). The deduced branching order is also consistent with the topologies in the 16 rRNA and other phylogenetic trees. Signature sequences in a number of other proteins provide evidence that alpha-proteobacteria is a late branching taxa within Bacteria, which branched after the delta,epsilon-subdivisions but prior to the beta,gamma-proteobacteria. The shared presence of many of these signatures in the mitochondrial (eukaryotic) homologs also provides evidence of the alpha-proteobacterial ancestry of mitochondria.

Alphaproteobacteria↗

PubMed2005

Conserved indels in protein sequences that are characteristic of the phylum Actinobacteria.

Gram-positive bacteria with a high G+C content are currently recognized as a distinct phylum, Actinobacteria, on the basis of their branching in 16S rRNA trees. Except for an insert in the 23S rRNA, there are no unique biochemical or molecular characteristics known at present that can distinguish this group from all other bacteria. In this work, three conserved indels (i.e. inserts or deletions) are described in three widely distributed proteins that are distinctive characteristics of the Actinobacteria and are not found in any other groups of bacteria. The identified signatures are a 2 aa deletion in cytochrome-c oxidase subunit 1 (Cox1), a 4 aa insert in CTP synthetase and a 5 aa insert in glutamyl-tRNA synthetase (GluRS). Additionally, the actinobacterial specificity of the large insert in the 23S rRNA was also tested. Using primers designed for conserved regions flanking these signatures, fragments of most of these genes were amplified from 23 actinobacterial species, covering many different families and orders, for which no sequence information was previously available. All the 61 sequenced fragments, except two in GluRS, were found to contain the indicated signatures. The presence of these signatures in various species from 20 families within this phylum provides evidence that they are likely distinctive characteristics of the entire phylum, which were introduced in a common ancestor of this group. The absence of all four of these signatures in Symbiobacterium thermophilum suggests that this species, which is distantly related to other actinobacteria in 16S rRNA and CTP synthetase trees, may not be a part of the phylum Actinobacteria. The identified signatures provide novel molecular means for defining and circumscribing the phylum Actinobacteria. Functional studies on them should prove helpful in understanding novel biochemical and physiological characteristics of this group of bacteria.

Actinobacteria↗

PubMed2005

Conserved indels in essential proteins that are distinctive characteristics of Chlamydiales and provide novel means for their identification.

All known chlamydiae are either proven human or animal pathogens or possess such potential. Due to increasing reports of chlamydiae diversity in the environment, it is important to develop reliable means for identifying and characterizing Chlamydiales species. The identification of environmental chlamydiae at present relies on their branching pattern in 16S rRNA trees, as well as 16S/23S consensus motifs which display variability. At present, no reliable molecular signatures are known which are unique to all Chlamydiales species. Besides the rRNAs, sequence information for different Chlamydiales is not available for any other gene sequence. In this report, a number of molecular signatures are described that consist of conserved inserts and deletions (indels), in widely distributed proteins [RNA polymerase alpha subunit (RpoA), elongation factor (EF)-Tu, EF-P, DNA gyrase B subunit (GyrB) and lysyl-tRNA synthetase (LysRS)], that are distinctive characteristics of all available chlamydiae homologues (from Chlamydiaeceae species and Parachlamydiae sp. UWE25) and not found in any other bacteria. Using PCR primers for highly conserved regions in these proteins, the corresponding fragments of these genes from Simkania negevensis, Waddlia chondrophila, and in a number of cases for Neochlamydia hartmanellae, covering all families within the phylum Chlamydiae, have been cloned and sequenced. The shared presence of the identified signatures in these species provides strong evidence that these molecular signatures are distinctive characteristics of the entire order Chlamydiales and can be used to reliably determine the presence of chlamydiae or chlamydia-related organisms in environmental samples. The sequence information for these protein fragments was also used to determine the interrelationships among chlamydiae species. In a phylogenetic tree based on a combined dataset of sequences from RpoA, EF-Tu, EF-P and GyrB, the environmental chlamydiae (i.e. Simkania, Waddlia and Parachlamydia) and the traditional Chlamydiaceae (i.e. Chlamydophila and Chlamydia) formed two distinct clades. Similar relationships were also observed in individual protein phylogenies, as well as in a 16S rRNA tree for the same species. These results provide evidence that the divergence between the traditional Chlamydiaceae species and the other chlamydiae families occurred very early in the evolution of this group of bacteria.

Chlamydiales↗