mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Variation in the ribosomal internal transcribed spacers and 5.8S rDNA among five species of Acropora (Cnidaria; Scleractinia): patterns of variation consistent with reticulate evolution

The ITS sequences of Acropora spp. are the shortest so far identified in any metazoan and are among the shortest seen in eukaryotes; ITS1 was 70-80 bases, and ITS2 was 100-112 bases. The ITS sequences were also highly variable, but base composition and secondary structure prediction indicate that divergent sequence variants are unlikely to be pseudogenes. The pattern of variation was unusual in several other respects: (1) two distinct ITS2 types were detected in both A. hyacinthus and A. cytherea, species known to hybridize in vitro with high success rates, and a putative intermediate ITS2 form was also detected in A. cytherea; (2) A. valida was found to contain highly (29%) diverged ITS1 variants; and (3) A. longicyathus contained two distinct 5.8S rDNA types. These data are consistent with a reticulate evolutionary history for the genus Acropora.      

Proof-of-concept human laboratory study for protracted abstinence in alcohol dependence: effects of gabapentin

There is a need for safe medications that can effectively support recovery by treating symptoms of protracted abstinence that may precipitate relapse in alcoholics, e.g. craving and disturbances in sleep and mood. This proof-of-concept study reports on the effectiveness of gabapentin 1200 mg for attenuating these symptoms in a non-treatment-seeking sample of cue-reactive, alcohol-dependent individuals. Subjects were 33 paid volunteers with current Diagnostic and Statistical Manual of Mental Disorders-IV alcohol dependence and a strength of craving rating 1 SD or greater for alcohol than water cues. Subjects were randomly assigned to gabapentin or placebo for 1 week and then participated in a within-subjects trial where each was exposed to standardized sets of pleasant, neutral and unpleasant visual stimuli followed by alcohol or water cues. Gabapentin was associated with significantly greater reductions than placebo on several measures of subjective craving for alcohol as well as for affectively evoked craving. Gabapentin was also associated with significant improvement on several measures of sleep quality. Side effects were minimal, and gabapentin effects were not found to resemble any major classes of abused drugs. Results suggest that gabapentin may be effective for treating the protracted abstinence phase in alcohol dependence and that a randomized clinical trial would be an appropriate next step. The study also suggests the value of cue-reactivity studies as proof-of-concept screens for potential antirelapse drugs.     

Multiplexed expression and screening for recombinant protein production in mammalian cells

Background  

A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.     

p-Nitrophenol and glutathione response in medaka (Oryzias latipes) exposed to MX,a drinking water carcinogen

When chlorine is introduced into public drinking water for disinfection, it can react with organic compounds in surface waters to form toxic by-products such as 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX). We investigated the effect of exposure to MX on cytochrome P450 2E1 (CYP2E1)-like activity and total glutathione (GSH) in the liver of the small fish model, medaka (Oryzias latipes). The multi-site carcinogen methylazoxymethanol acetate (MAMAc) was the positive control compound. Both medaka liver microsome preparations and S-9 fractions catalyzed the hydroxylation of p-nitrophenol (PNP), suggesting CYP2E1-like activity in the medaka. Male medaka exposed for 96 h to the CYP2E1 inducers ethanol and acetone under fasted conditions showed significant increases in PNP-hydroxylation activity. Furthermore, total reduced hepatic GSH was reduced in fish fasted for 96 h, indicating that normal feeding is a factor in maintaining xenobiotic defenses. Exposure to MX and MAMAc induced significant increases in hepatic CYP2E1-like activity, however MX exposure did not alter hepatic GSH levels. These data strengthen the role of the medaka as a suitable species for examining cytochrome P450 and GSH detoxification processes and the role these systems play in chemical carcinogenesis.     

Analysis of in vivo and in vitro DNA strand breaks from trihalomethane exposure

BACKGROUND: Epidemiological studies have linked the consumption of chlorinated surface waters to an increased risk of two major causes of human mortality, colorectal and bladder cancer. Trihalomethanes (THMs) are by-products formed when chlorine is used to disinfect drinking water. The purpose of this study was to examine the ability of the THMs, trichloromethane (TCM), bromodichloromethane (BDCM), dibromochloromethane (DBCM), and tribromomethane (TBM), to induce DNA strand breaks (SB) in (1) CCRF-CEM human lymphoblastic leukemia cells, (2) primary rat hepatocytes (PRH) exposed in vitro, and (3) rats exposed by gavage or drinking water. METHODS: DNA SB were measured by the DNA alkaline unwinding assay (DAUA). CCRF-CEM cells were exposed to individual THMs for 2 hr. Half of the cells were immediately analyzed for DNA SB and half were transferred into fresh culture medium and incubated for an additional 22 hr before testing for DNA SB. PRH were exposed to individual THMs for 4 hr then assayed for DNA SB. F344/N rats were exposed to individual THMs for 4 hr, 2 weeks, and to BDCM for 5 wk then tested for DNA SB. RESULTS: CCRF-CEM cells exposed to 5- or 10-mM brominated THMs for 2 hr produced DNA SB. The order of activity was TBM>DBCM>BDCM; TCM was inactive. Following a 22-hr recovery period, all groups had fewer SB except 10-mM DBCM and 1-mM TBM. CCRF-CEM cells were found to be positive for the GSTT1-1 gene, however no activity was detected. No DNA SB, unassociated with cytotoxicity, were observed in PRH or F344/N rats exposed to individual THMs. CONCLUSION: CCRF-CEM cells exposed to the brominated THMs at 5 or 10 mM for 2 hr showed a significant increase in DNA SB when compared to control cells. Additionally, CCRF-CEM cells exposed to DBCM and TBM appeared to have compromised DNA repair capacity as demonstrated by an increased amount of DNA SB at 22 hr following exposure. CCRF-CEM cells were found to be positive for the GSTT1-1 gene, however no activity was detected. No DNA SB were observed in PRH or F344/N rats exposed to individual THMs.     

Altered gene expression in mouse livers after dichloroacetic acid exposure

Dichloroacetic acid (DCA) is a major by-product of water disinfection by chlorination. Several studies have demonstrated that DCA exhibits hepatocarcinogenic effects in rodents when administered in drinking water. This chemical does not appear to be highly mutagenic, and the mechanism(s) involved in DCA induction of cancer are not clear. The present work was aimed at identifying changes in gene expression which may indicate critical alterations/pathways involved in this chemical's carcinogenic activities. We used cDNA microarray methods for analyses of gene expression in livers of mice treated with the tumorigenic dose of 2 g/l DCA in drinking water for 4 weeks. Total RNA samples obtained from livers of the control and DCA-treated mice were evaluated for gene expression patterns with Clontech Atlas Mouse 1.2 cDNA and Atlas mouse stress/toxicology arrays, and the data analyzed with AtlasImage 2.01 and one-way ANOVA in JMP4 software. From replicate experiments, we identified 24 genes with altered expression, of which 15 were confirmed by Northern blot analysis. Of the 15 genes, 14 revealed expression suppressed two- to five-fold; they included the following: MHR 23A, cytochrome P450 (CYP) 2C29, CYP 3A11, serum paraoxonase/arylesterase 1 (PON 1), liver carboxylesterase, alpha-1 antitrypsin, ER p72, glutathione S-transferase (GST) Pi 1, angiogenin, vitronectin precursor, cathepsin D (CTSD), plasminogen precursor (contains angiostatin), prothrombin precursor and integrin alpha 3 precursor (ITGA 3). An additional gene, CYP 2A4/5, had a two-fold elevation in expression. Further, in ancillary Northern analyses of total RNA isolated from DCA-induced hepatocellular carcinomas (from earlier reported studies of mice treated with 3.5 g/l DCA for 93 weeks), many of the same genes (11 of 15) noted above showed a similar alteration in expression. In summary, we have identified specific genes involved in the functional categories of cell growth, tissue remodeling, apoptosis, cancer progression and xenobiotic metabolism that have altered levels of expression following exposures to DCA. These findings serve to highlight new pathways in which to further probe DCA effects that may be critical to its tumorigenic activity.     

Serosurvey of Brucella spp. infection in the Kafue lechwe (Kobus leche kafuensis) of the Kafue flats in Zambia

One of the diseases of veterinary and public health importance affecting the Kafue lechwe (Kobus leche kafuensis) on the Kafue flats is brucellosis, for which only scant information is available. During the 2003 (October), 2004 (December), and 2008 (July-December) hunting seasons in the Kafue flats, we conducted a study to determine the seroprevalence of Brucella spp. in the Kafue lechwe and to evaluate serologic tests for detection of Brucella spp. antibodies in lechwe. The Rose Bengal Test (RBT), competitive enzyme-linked immunosorbent assay (cELISA), and fluorescence polarization assay (FPA) were used. A total of 121 Kafue lechwe were hunted for disease investigations in 2003, 2004, and 2008 in the Kafue Flat Game Management Area. Of these, 21.6%, (95% confidence interval [CI]: 14.2-29.1%) had detectable antibodies to Brucella spp. The Kafue lechwe in Lochnivar National Park had higher antibody results than those in Blue Lagoon National Park (odds ratio=3.0; 95% CI: 0.94-9.4). Infection levels were similar in females (21.6%) and males (21.7%). Results were similar among RBT, FPA, cELISA tests, suggesting that these could effectively be used in diagnosing brucellosis in the Kafue lechwe. Our study demonstrates the presence of Brucella infections in the Kafue lechwe in two national parks located in the Kafue flats and further highlights the suitability of serologic assays for testing the Kafue lechwe. Because the Kafue lechwe is the most hunted wildlife species in Zambia, hunters need to be informed of the public health risk of Brucella spp. infection.     

SEPALLATA3: the 'glue' for MADS box transcription factor complex formation

Background  

Plant MADS box proteins play important roles in a plethora of developmental processes. In order to regulate specific sets of target genes, MADS box proteins dimerize and are thought to assemble into multimeric complexes. In this study a large-scale yeast three-hybrid screen is utilized to provide insight into the higher-order complex formation capacity of the Arabidopsis MADS box family. SEPALLATA3 (SEP3) has been shown to mediate complex formation and, therefore, special attention is paid to this factor in this study.