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The phylogeny of the large bacterial class Gammaproteobacteria has been difficult to resolve. Here we apply a telescoping multiprotein approach to the problem for 104 diverse gammaproteobacterial genomes, based on a set of 356 protein families for the whole class and even larger sets for each of four cohesive subregions of the tree. Although the deepest divergences were resistant to full resolution, some surprising patterns were strongly supported. A representative of the Acidithiobacillales routinely appeared among the outgroup members, suggesting that in conflict with rRNA-based phylogenies this order does not belong to Gammaproteobacteria; instead, it (and, independently, "Mariprofundus") diverged after the establishment of the Alphaproteobacteria yet before the betaproteobacteria/gammaproteobacteria split. None of the orders Alteromonadales, Pseudomonadales, or Oceanospirillales were monophyletic; we obtained strong support for clades that contain some but exclude other members of all three orders. Extreme amino acid bias in the highly A+T-rich genome of Candidatus Carsonella prevented its reliable placement within Gammaproteobacteria, and high bias caused artifacts that limited the resolution of the relationships of other insect endosymbionts, which appear to have had multiple origins, although the unbiased genome of the endosymbiont Sodalis acted as an attractor for them. Instability was observed for the root of the Enterobacteriales, with nearly equal subsets of the protein families favoring one or the other of two alternative root positions; the nematode symbiont Photorhabdus was identified as a disruptor whose omission helped stabilize the Enterobacteriales root.

The facultative intracellular bacterial pathogen Brucella infects a wide range of warm-blooded land and marine vertebrates and causes brucellosis. Currently, there are nine recognized Brucella species based on host preferences and phenotypic differences. The availability of 10 different genomes consisting of two chromosomes and representing six of the species allowed for a detailed comparison among themselves and relatives in the order Rhizobiales. Phylogenomic analysis of ortholog families shows limited divergence but distinct radiations, producing four clades as follows: Brucella abortus-Brucella melitensis, Brucella suis-Brucella canis, Brucella ovis, and Brucella ceti. In addition, Brucella phylogeny does not appear to reflect the phylogeny of Brucella species' preferred hosts. About 4.6% of protein-coding genes seem to be pseudogenes, which is a relatively large fraction. Only B. suis 1330 appears to have an intact beta-ketoadipate pathway, responsible for utilization of plant-derived compounds. In contrast, this pathway in the other species is highly pseudogenized and consistent with the "domino theory" of gene death. There are distinct shared anomalous regions (SARs) found in both chromosomes as the result of horizontal gene transfer unique to Brucella and not shared with its closest relative Ochrobactrum, a soil bacterium, suggesting their acquisition occurred in spite of a predominantly intracellular lifestyle. In particular, SAR 2-5 appears to have been acquired by Brucella after it became intracellular. The SARs contain many genes, including those involved in O-polysaccharide synthesis and type IV secretion, which if mutated or absent significantly affect the ability of Brucella to survive intracellularly in the infected host.

Background: Completed genome sequences are rapidly increasing for Rickettsia, obligate intracellular alpha-proteobacteria responsible for various human diseases, including epidemic typhus and Rocky Mountain spotted fever. In light of phylogeny, the establishment of orthologous groups (OGs) of open reading frames (ORFs) will distinguish the core rickettsial genes and other group specific genes (class 1 OGs or C1OGs) from those distributed indiscriminately throughout the rickettsial tree (class 2 OG or C2OGs)., Methodology/principal Findings: We present 1823 representative (no gene duplications) and 259 non-representative (at least one gene duplication) rickettsial OGs. While the highly reductive (approximately 1.2 MB) Rickettsia genomes range in predicted ORFs from 872 to 1512, a core of 752 OGs was identified, depicting the essential Rickettsia genes. Unsurprisingly, this core lacks many metabolic genes, reflecting the dependence on host resources for growth and survival. Additionally, we bolster our recent reclassification of Rickettsia by identifying OGs that define the AG (ancestral group), TG (typhus group), TRG (transitional group), and SFG (spotted fever group) rickettsiae. OGs for insect-associated species, tick-associated species and species that harbor plasmids were also predicted. Through superimposition of all OGs over robust phylogeny estimation, we discern between C1OGs and C2OGs, the latter depicting genes either decaying from the conserved C1OGs or acquired laterally. Finally, scrutiny of non-representative OGs revealed high levels of split genes versus gene duplications, with both phenomena confounding gene orthology assignment. Interestingly, non-representative OGs, as well as OGs comprised of several gene families typically involved in microbial pathogenicity and/or the acquisition of virulence factors, fall predominantly within C2OG distributions., Conclusion/significance: Collectively, we determined the relative conservation and distribution of 14354 predicted ORFs from 10 rickettsial genomes across robust phylogeny estimation. The data, available at PATRIC (PathoSystems Resource Integration Center), provide novel information for unwinding the intricacies associated with Rickettsia pathogenesis, expanding the range of potential diagnostic, vaccine and therapeutic targets.

Background: The genome sequence of Rickettsia felis revealed a number of rickettsial genetic anomalies that likely contribute not only to a large genome size relative to other rickettsiae, but also to phenotypic oddities that have confounded the categorization of R. felis as either typhus group (TG) or spotted fever group (SFG) rickettsiae. Most intriguing was the first report from rickettsiae of a conjugative plasmid (pRF) that contains 68 putative open reading frames, several of which are predicted to encode proteins with high similarity to conjugative machinery in other plasmid-containing bacteria., Methodology/principal Findings: Using phylogeny estimation, we determined the mode of inheritance of pRF genes relative to conserved rickettsial chromosomal genes. Phylogenies of chromosomal genes were in agreement with other published rickettsial trees. However, phylogenies including pRF genes yielded different topologies and suggest a close relationship between pRF and ancestral group (AG) rickettsiae, including the recently completed genome of R. bellii str. RML369-C. This relatedness is further supported by the distribution of pRF genes across other rickettsiae, as 10 pRF genes (or inactive derivatives) also occur in AG (but not SFG) rickettsiae, with five of these genes characteristic of typical plasmids. Detailed characterization of pRF genes resulted in two novel findings: the identification of oriV and replication termination regions, and the likelihood that a second proposed plasmid, pRFdelta, is an artifact of the original genome assembly., Conclusion/significance: Altogether, we propose a new rickettsial classification scheme with the addition of a fourth lineage, transitional group (TRG) rickettsiae, that is unique from TG and SFG rickettsiae and harbors genes from possible exchanges with AG rickettsiae via conjugation. We offer insight into the evolution of a plastic plasmid system in rickettsiae, including the role plasmids may have played in the acquirement of virulence traits in pathogenic strains, and the likely origin of plasmids within the rickettsial tree.

To determine if microwave exposure could elicit a biological effect in the absence of thermal stress, studies were designed in which chick embryos were exposed to athermal microwave radiation (915 MHz) to look for induction of Hsp70, a protein produced during times of cellular stress that aids in the protection of cellular components. Levels of Hsp70 were found to increase within 2 h, with maximum expression ( approximately 30% higher than controls) typically occurring by 3 h from the start of exposure. Other embryos were exposed to microwave radiation prior to being subjected to hypoxic stress, and were found to have significantly higher survival (P < 0.05) following re-oxygenation than non-exposed controls. The results of these studies indicate that not only can athermal microwave exposures activate the stress protein response pathway; they can also enhance survivability following exposure to a subsequent, potentially lethal stress. From a public health standpoint, it is important that more studies be performed to determine if repeated exposures, a condition likely to be found in cell phone use, are still beneficial., (Copyright 2002 Wiley-Liss, Inc.)

The yeast PDR5 locus encodes a 160-kDa member of the ABC family of transport proteins. Strains bearing a deletion of this locus are drug hypersensitive. Resistant revertants arise when cells are plated on cycloheximide medium. About one-third of these are cross resistant to other agents, including oligomycin, fluconazole and sulfometuron methyl. Most of the revertants exhibit linkage to the PDR5 locus and map in three locations. Curiously, the multi-drug resistance is not due to a single mutation. Most of the revertants behave as though they contained several tightly linked resistance factors.

Coordinately unsaturated Cu(II) and Fe(III) complexes of the stoichiometry [Cu(L)Cl] and [Fe(L)Cl2], where L = tridentate anion of 2-hydroxy-1,4-naphthoquinone 1-thiosemicarbazone (2HNQTSC) and its 3-methyl derivative (3M2HNQTSC), were screened in vitro against P388 lymphocytic leukemia cells. Copper complexes were found to be more effective inhibitors of DNA synthesis than analogous Fe(III) compounds. The inhibitory activities are suggested to be related to Cu(II)-Cu(I) redox couple or nitrogen adduct formation.

The ability of bepridil, a calcium channel blocker, to potentiate the antitumor activity of mitoxantrone (MITO) in human chronic myeloid leukemia (CML) cells was evaluated. MITO and bepridil, when incubated alone with the CML cells for 4 h, indicated a dose-dependent increase in the inhibition of 3H-thymidine incorporation. Incorporation rate of the radiolabeled thymidine into DNA was used as a measure of cell growth. When the CML cells were exposed to MITO (1 microgram/ml) in the presence of bepridil (1 and 5 micrograms/ml), an enhancement in the inhibition of DNA biosynthesis was observed in 14 out of 17 human CML samples studied. This significant inhibition (p less than 0.001) of 3H-thymidine incorporation due to the combination was found to be completely irreversible. Bepridil was identified predominantly in the octanol phase in the octanol/water partitioning studies. This lipophilic property of drug response modulators was implicated in the observed increase in the intracellular uptake of anticancer drugs, which in turn led to an enhanced cytotoxicity correlating well with the MITO activity observed in this study. The results are suggestive of clinical utility of bepridil as an adjuvant to enhance the anticancer ability of MITO in the treatment of CML.

Turnover rates of cerebral proteins were examined in control adult rats and in those subjected to prolonged in vivo treatment with "low" (0.02 mg/ml) or "high" (0.04 mg/ml) doses of nicotine (added to drinking water), using [14C]bicarbonate as the label. It was found that the turnover of proteins in various subcellular fractions consisted of two distinct components turning over at a "fast" or a "slow" rate and having relatively short or long half-lives, respectively. Thus in control animals the half-lives of the protein components turning over at a fast rate ranged from 1.31 to 3.61 days whereas for those turning over at a slow rate the half-lives ranged from 8.56 to 24.28 days. Treatment with low doses of nicotine resulted in a more rapid turnover of nuclear fast turning over component with a concomitant decreased turnover of homogenate, cytosol, mitochondrial, and microsomal proteins; in the synaptosomal membranes this component disappeared altogether. The half-lives of the slow turning over components decreased in general from 14.3 to 33.3% with the exception of the nuclear proteins, where the half-live increased by 71.1%. Turnover of microsomal proteins was not affected. When the animals were given a high dose of nicotine, the turnover of fast components became even more rapid for nuclear, myelin, and microsomal proteins with a decrease in half-life from 26.6 to 32.3%. By contrast, half-lives of synaptosomal and mitochondrial proteins increased by 16.1-89.3%. These changes were not reflected in the turnover rate of whole homogenate proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of prolonged in vivo treatment with nicotine on synaptosomal ATPase activity in rat brain were examined by employing doses of nicotine (0.02 and 0.04 mg/ml in the drinking water) which simulated intake by moderate and heavy smokers. Under these conditions the "low" dose of nicotine resulted in increased body weight whereas "high" dose of nicotine inhibited weight gain. Examination of synaptosomal ATPase activities revealed a dose and time dependent stimulatory/inhibitory effect. With "low" dose of nicotine, maximum stimulatory effect on ATPase activity was seen at the end of the 3rd week, while "high" dose stimulated the enzyme activities maximally by the 2nd week itself; further treatment up to the 4th week caused inhibition of the ATPase activities (10% to 43% decrease).

Five new copper(II) complexes of 5-phenylazo-3-methoxy salicylidene thiosemicarbazone and N4 substituted thiosemicarbazones have been synthesized. They have been characterized by chemical analyses, magnetic, conductance data, and by ultraviolet (UV)--visible, infrared, and electron spin resonance spectra. The complexes have the general formula CuL2, where HL is the ligand. One representative complex has been screened in vitro and in vivo against P388 lymphocytic leukemia cells sensitive and resistant to adriamycin (P388/S and P388/R). It has shown promising growth inhibition activity. We are reporting here for the first time the antineoplastic activity of this complex against experimental tumor systems.