Optimization of a novel class of benzimidazole-based farnesoid X receptor (FXR) agonists to improve physicochemical and ADME properties.

Structure-guided lead optimization of recently described benzimidazolyl acetamides addressed the key liabilities of the previous lead compound 1. These efforts culminated in the discovery of 4-{(S)-2-[2-(4-chloro-phenyl)-5,6-difluoro-benzoimidazol-1-yl]-2-cyclohexyl-acetylamino}-3-fluoro-benzoic acid 7g, a highly potent and selective FXR agonist with excellent physicochemical and ADME properties and potent lipid lowering activity after oral administration to LDL receptor deficient mice.

[Screening domestic violence. A German-language screening instrument for domestic violence against women]. / Screening Partnergewalt. Ein deutschsprachiges Screeninginstrument für häusliche Gewalt gegen Frauen.

Domestic violence is frequent. But up to now there is no screening instrument available in German which would allow a simple assessment. In this article a short screening interview is published that allows the identification of women who became victims of domestic violence. Such a screening interview has not been published in German speaking countries until now. A screening interview originally developed in English speaking countries called "Partner Violence Screen" (PVS) was translated, modified and validated in a population of female patients of a crisis intervention ward. The original version of the PVS consisted of 3 items which have been extended by two items, so that the modified version of the PVS is composed of 5 items now. In a validation study this instrument was compared to a much more detailed, 30 item self rating scale "Index of Spouse Abuse" (ISA). In addition to the validation of the PVS a life-time version of the PVS was developed which refers to the entire life period since the 18th birthday. The original version of the PVS showed a sensitivity of 0.79 and a specificity of 0.70. In the modified version "Screening Partner Violence" the sensitivity was 0.80 and the specificity 0.78. This screening instrument, which was translated and further developed by the authors, proved to be helpful for identifying domestic violence. With this instrument a screening interview is available for the first time in German speaking countries that can be accomplished with women in different institutions without losing much time.

Modeling of Escherichia coli inactivation by UV irradiation at different pH values in apple cider.

This study examined the effects and interactions of UV light dose (1,800 to 20,331 microJ/cm2) and apple cider pH (2.99 to 4.41) on the inactivation of Escherichia coli ATCC 25922, a surrogate for E. coli O157:H7. A predictive model was developed to relate the log reduction factor of E. coli ATCC 25922 to the UV dose. Bacterial populations for treated and untreated samples were enumerated with the use of nonselective media. The results revealed that UV dose was highly significant in the inactivation of E. coli, whereas pH showed no significant effect at higher UV doses. Doses of 6,500 microJ/cm2 or more were sufficient to achieve a greater than 5-log reduction of E. coli. Experimental inactivation data were fitted adequately by a logistic regression model. UV irradiation is an attractive alternative to conventional methods for reducing bacteria in unpasteurized apple cider.

Enhancement of oxidative damage to cultured cells and Caenorhabditis elegans by mitochondrial electron transport inhibitors.

The mechanisms that lead to mitochondrial damage under oxidative stress conditions were examined in primary and cultured cells as well as in the nematode Caenorhabditis elegans (C. elegans) treated simultaneously with electron transport inhibitors and oxygen gas. Oxygen loading enhanced the damage of PC 12 cells by thenoyltrifluoroacetone (TTFA, a complex II inhibitor), but did not by rotenone (a complex I inhibitor), antimycin (a complex III inhibitor), and sodium azide (a complex IV inhibitor). In primary hepatocytes, the enhancement was observed with the addition of sodium azide and rotenone, but not by TTFA or antimycin. In the nematode, only rotenone and TTFA enhanced the sensitivity under hyperoxia. These results demonstrate that highly specific inhibitors of electron transport can induce oxygen hypersensitivity in cell levels such as PC 12 cells and primary hepatocytes, and animal level of C. elegans. In addition the cell damage is different dependent on cell type and organism.

A defect in the cytochrome b large subunit in complex II causes both superoxide anion overproduction and abnormal energy metabolism in Caenorhabditis elegans.

A mev-1(kn1) mutant of the nematode Caenorhabditis elegans is defective in the cytochrome b large subunit (Cyt-1/ceSDHC) in complex II of the mitochondrial electron transport chain. We have previously shown that a mutation in mev-1 causes shortened life span and rapid accumulation of aging markers such as fluorescent materials and protein carbonyls in an oxygen-dependent fashion. However, it remains unclear as to whether this hypersensitivity is caused by direct toxicity of the exogenous oxygen or by the damage of endogenous reactive oxygen species derived from mitochondria. Here we report important biochemical changes in mev-1 animals that serve to explain their abnormalities under normoxic conditions: (i) an overproduction of superoxide anion from mitochondria; and (ii) a reciprocal reduction in glutathione content even under atmospheric oxygen. In addition, unlike wild type, the levels of superoxide anion production from mev-1 mitochondria were significantly elevated under hyperoxia. Under normal circumstances, it is well known that superoxide anion is produced at complexes I and III in the electron transport system. Our data suggest that the mev-1(kn1) mutation increases superoxide anion production at complex II itself rather than at complexes I and III. The mev-1 mutant also had a lactate level 2-fold higher than wild type, indicative of lactic acidosis, a hallmark of human mitochondrial diseases. These data indicate that Cyt-1/ceSDHC plays an important role not only in energy metabolism but also in superoxide anion production that is critically involved in sensitivity to atmospheric oxygen.

Mitochondrial mutations differentially affect aging, mutability and anesthetic sensitivity in Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, mutations have been previously isolated that affect the activities of Complex I (gas-1) and Complex II (mev-1), two of the five membrane-bound complexes that control electron flow in mitochondrial respiration. We compared the effects of gas-1 and mev-1 mutations on different traits influenced by mitochondrial function. Mutations in Complex I and II both increased sensitivity to free radicals as measured during development and in aging animals. However, gas-1 and mev-1 mutations differentially affected mutability and anesthetic sensitivity. Specifically, gas-1 was anesthetic hypersensitive but not hypermutable while mev-1 was hypermutable but displayed normal responses to anesthetics. These results indicate that Complexes I and II may differ in their effects on behavior and development, and are consistent with the wide variation in phenotypes that result from mitochondrial changes in other organisms.

A comparison of mutations induced by accelerated iron particles versus those induced by low earth orbit space radiation in the FEM-3 gene of Caenorhabditis elegans.

The fem-3 gene of Caenorhabditis elegans was employed to determine the mutation frequency as well as the nature of mutations induced by low earth orbit space radiation ambient to Space Shuttle flight STS-76. Recovered mutations were compared to those induced by accelerated iron ions generated by the AGS synchrotron accelerator at Brookhaven National Laboratory. For logistical reasons, dauer larvae were prepared at TCU, transported to either Kennedy Space Center or Brookhaven National Laboratory, flown in space or irradiated, returned to TCU and screened for mutants. A total of 25 fem-3 mutants were recovered after the shuttle flight and yielded a mutation frequency of 2.1x10(-5), roughly 3.3-fold higher than the spontaneous rate of 6.3x10(-6). Four of the mutations were homozygous inviable, suggesting that they were large deletions encompassing fem-3 as well as neighboring, essential genes. Southern blot analyses revealed that one of the 25 contained a polymorphism in fem-3, further evidence that space radiation can induce deletions. While no polymorphisms were detected among the iron ion-induced mutations, three of the 15 mutants were homozygous inviable, which is in keeping with previous observations that high LET iron particles generate deficiencies. These data provide evidence, albeit indirect, that an important mutagenic component of ambient space radiation is high LET charged particles such as iron ions.

Effects of caffeine on sensorimotor gating of the startle reflex in normal control subjects: impact of caffeine intake and withdrawal.

RATIONALE: Prepulse inhibition (PPI), a cross-species measure of sensorimotor gating, is impaired in certain neuropsychiatric disorders. This study was designed to assess caffeine effects on PPI in normal humans, as part of an effort to understand cross-species differences and similarities in the neurochemical regulation of PPI. METHODS: Startle was measured during a screening session; 7 days later, subjects were retested after placebo or caffeine (200 mg; double-blind design). Subjects were characterized as low versus high caffeine drinkers based on established scales (range 11-628 mg/day), and either maintained ad libitum caffeine intake (Ad lib study; n=18) or refrained from caffeine consumption for > or =15 h prior to testing (Withdrawal study; n=12). Autonomic and self-rating measures, acoustic and tactile startle, and unimodal and cross-modal PPI, were measured in divided sessions for 3 h post-treatment. RESULTS: There were significant effects of caffeine and/or caffeine withdrawal on several self-rating and autonomic measures, and on startle reflex habituation, but not on acoustic or tactile startle magnitude or PPI. Difference scores of startle data from screening versus test days revealed no group effects on startle magnitude, but PPI difference scores revealed that caffeine had opposite effects on low versus high caffeine drinkers (means=57 versus 258 mg/day) in the two withdrawal states. In the absence of withdrawal, caffeine reduced PPI in heavy caffeine drinkers; during withdrawal, caffeine increased PPI in heavy caffeine drinkers. The opposite pattern was evident in low caffeine drinkers. CONCLUSIONS: While a physiologically active dose of caffeine has no simple effects on PPI in normal humans, both withdrawal states and normal levels of caffeine consumption may be important factors in understanding this drug's effects on sensorimotor gating.

Sex differences in sensorimotor gating of the human startle reflex: all smoke?

RATIONALE: A recent report described sex differences in the effects of nicotine use and withdrawal on prepulse inhibition of acoustic startle (PPI), but no sex differences in PPI in non-smokers. OBJECTIVE: To determine whether previously reported male>female acoustic PPI reflect sex differences in smoking effects on PPI, rather than simple sex differences in the regulation of PPI. A retrospective analyses of >600 carefully screened normals tested over the past 12 years was completed. RESULTS: Male>female acoustic PPI was detected in analyses that included: 1) all subjects; or 2) self-declared non-smokers. CONCLUSIONS: Sex differences in PPI cannot be accounted for by smoking history, because they are present across a large sample of non-smoking normal controls.

Oxidative stress pretreatment increases the X-radiation resistance of the nematode Caenorhabditis elegans.

Pre-exposure of wild-type Caenorhabditis elegans to oxygen conferred a protective effect against the lethality imposed by subsequent X-irradiation. In contrast, two mutants (rad-1 and rad-2) that are UV and ionizing radiation hypersensitive but not oxygen sensitive, did not exhibit this adaptive response. To explore the molecular basis of protection, the expression of several key genes was examined using Northern blot analyses to measure mRNA levels. In the wild-type, expression of the heat shock protein genes, hsp16-1 and hsp16-48, increased dramatically after incubation under high oxygen. Expression of two superoxide dismutase genes (sod-1 and sod-3) was relatively unaffected. Unlike the wild-type, the basal levels of these four genes were significantly lower in the rad-1 and rad-2 mutants under atmospheric conditions. These genes were partially induced in response to oxidative stress. These data suggest that at least a portion of the hypersensitive phenotype of rad-1 and rad-2 may be attributed to inappropriate gene expression.

Enhanced visual latent inhibition in obsessive-compulsive disorder.

BACKGROUND: Latent inhibition (LI) is the slowed acquisition of a learned response to a conditioned stimulus (CS), that occurs if that CS has previously been experienced in a noncontingent setting. This retarded acquisition is thought to occur because, due to the previous noncontingent experience, an individual must "unlearn the irrelevance" of the CS, before learning its new association to the unconditioned stimulus. A previous report using an auditory paradigm did not detect abnormal LI in obsessive-compulsive disorder (OCD) subjects; this auditory LI task included a difficult acquisition phase, which made it relatively insensitive to detecting abnormally elevated LI (i.e., slowed "unlearning"). METHODS: We assessed LI using a highly sensitive computerized visual LI paradigm in 63 carefully screened control subjects and in 48 patients with OCD. RESULTS: Compared to controls, OCD subjects exhibited significantly more LI; if "preexposed" to a to-be-CS, OCD subjects required significantly more trials to learn a new association to that CS, compared to control subjects. This pattern was particularly evident in unmedicated OCD subjects. CONCLUSIONS: The inflated impact of preexposure on LI response acquisition in OCD subjects may be a quantitative measure of their tendency to remain "stuck in set" in this cognitive task.

Lethal consequences of simulated solar radiation on the nematode Caenorhabditis elegans in the presence and absence of photosensitizers.

The partial destruction of the earth's protective ozone layer has raised concerns about the impact of increased UV radiation on the earth's biological systems. In this study, polychromatic light sources were employed to observe the biological responses of the soil nematode Caenorhabditis elegans to simulated solar UV. Using various filter combinations, action spectra were constructed that approximated those generated previously with mono-chromatic radiation. In both cases, a mutant strain (rad-3) progressively lost its hypersensitivity as shorter wave-lengths were filtered out. In addition, both wild type and radiation-sensitive (rad) mutants were irradiated with several combinations of filtered light sources in the presence and absence of two exogenous photosensitizers (ethidium bromide and bromodeoxyuridine). Treatment with either of the introduced photosensitizers increased photosensitivity to solar UV. Solar UV also induced a fluence-dependent reduction in fertility in wild-type animals. These experiments extend previous data and substantially expand our understanding of the biological responses of C. elegans to solar radiation.

A mutation in succinate dehydrogenase cytochrome b causes oxidative stress and ageing in nematodes.

Much attention has focused on the aetiology of oxidative damage in cellular and organismal ageing. Especially toxic are the reactive oxygen byproducts of respiration and other biological processes. A mev-1(kn1) mutant of Caenorhabditis elegans has been found to be hypersensitive to raised oxygen concentrations. Unlike the wild type, its lifespan decreases dramatically as oxygen concentrations are increased from 1 to 60%. Strains bearing this mutation accumulate markers of ageing (such as fluorescent materials and protein carbonyls) faster than the wild type. We show here that mev-1 encodes a subunit of the enzyme succinate dehydrogenase cytochrome b, which is a component of complex II of the mitochondrial electron transport chain. We found that the ability of complex II to catalyse electron transport from succinate to ubiquinone is compromised in mev-1 animals. This may cause an indirect increase in superoxide levels, which in turn leads to oxygen hypersensitivity and premature ageing. Our results indicate that mev-1 governs the rate of ageing by modulating the cellular response to oxidative stress.

The characterization and description of representatives of 'G' bacteria from activated sludge plants.

The name Tetracoccus cechii is proposed for two strains of the tetrad arranged cocci, previously known as 'G' bacteria, which were isolated from laboratory scale activated sludge plants in the Czech Republic and in Italy. They were morphologically, phenotypically and phylogenetically characterized and found to comprise a novel lineage in the alpha-3 group of the proteobacterial phylum in the domain Bacteria. The strains are Gram-negative and produce intracellular inclusions of poly-beta-hydroxybutyrate. Although commonly seen in activated sludge mixed liquor as cocci 1-2 microns in diameter, arranged in tetrads, in pure culture they can also grow in amorphous aggregations and the cells are generally more variable in their size and shape with coccobacilli as well as cocci being present. They are not able to grow phototrophically, nor can they reduce nitrate beyond nitrite nor grow anaerobically. The closest phylogenetic neighbours of T. cechii are Rhodobacter sphaeroides and R. capsulatus which are 93% similar by 16S rDNA comparison. Tetracoccus cechii is oxidase- and catalase-positive, non-motile and has an optimal growth temperature between 25 degrees and 35 degrees C. The 16S rRNA of T. cechii has a 21 nucleotide deletion in the V9 region (Escherichia coli positions 1258-1278) and this feature is a unique molecular synapomorphy in the alpha-3 group.

Changes in sensorimotor inhibition across the menstrual cycle: implications for neuropsychiatric disorders.

The startle reflex is inhibited when the starting noise is preceded 30-500 msec by a weak prepulse. "Prepulse inhibition" (PPI) is reduced in specific neuropsychiatric disorders characterized clinically by impaired inhibition of sensory, motor, or cognitive information. PPI is sexually dimorphic, with men exhibiting significantly more PPI than women. We examined possible neuroendocrine substrates for this sex difference in PPI. The startle reflex, and a measure of visuospatial priming, were measured in 10 men, and in 46 normal women at different points in their menstrual cycle. In women, PPI was significantly reduced in the luteal vs follicular phase of the menstrual cycle. This reduction in PPI was most notable during the period corresponding to midluteal elevations of both estrogen and progesterone. In a task of visuospatial priming, follicular-phase women demonstrated a predominance of inhibition over facilitation, but this pattern reversed across the menstrual cycle.

Morphological and compositional shifts in an experimental bacterial community influenced by protists with contrasting feeding modes.

In a two-stage continuous-flow system, we studied the impacts of different protozoan feeding modes on the morphology and taxonomic structure of mixed bacterial consortia, which were utilizing organic carbon released by a pure culture of a Rhodomonas sp. grown on inorganic medium in the first stage of the system. Two of three second stages operated in parallel were inoculated by a bacterivorous flagellate, Bodo saltans, and an algivorous ciliate, Urotricha furcata, respectively. The third vessel served as a control. In two experiments, where algal and bacterial populations grew at rates and densities typical for eutrophic waters, we compared community changes of bacteria, algae, and protozoa under quasi-steady-state conditions and during the transient stage after the protozoan inoculation. In situ hybridization with fluorescent oligonucleotide probes and cultivation-based approaches were used to tentatively analyze the bacterial community composition. Initially the cell size distribution and community structure of all cultivation vessels showed similar patterns, with a dominance of 1- to 2.5-(mu)m-long rods from the beta subdivision of the phylum Proteobacteria ((beta)-Proteobacteria). Inoculation with the ciliate increased bacterial growth in this substrate-controlled variant, seemingly via a recycling of nutrients and substrate released by grazing on algae, but without any detectable effect on the composition of bacterial assemblage. In contrast, an inoculation with the bacterivore, B. saltans, resulted in a decreased proportion of the (beta)-Proteobacteria. One part of the assemblage (<4% of total bacterial numbers), moreover, produced large grazing-resistant threadlike cells. As B. saltans ingested only cells of <3 (mu)m, this strategy yielded a refuge for (symbl)70% of total bacterial biomass from being grazed. Another consequence of the heavy predation in this variant was a shift to the numerical dominance of the (alpha)-Proteobacteria. The enhanced physiological status of the heavily grazed-upon segment of bacterial community resulted in a much higher proportion of CFU (mean, 88% of total bacterial counts) than with other variants, where CFU accounted for (symbl)30%. However, significant cultivation-dependent shifts of the bacterial community were observed toward (gamma)-Proteobacteria and members of the Cytophaga/Flavobacterium group, which demonstrated the rather poor agreement between cultivation-based approaches and oligonucleotide probing.

A single amino acid substitution in Staphylococcus aureus dihydrofolate reductase determines trimethoprim resistance.

A single amino acid substitution, Phe98 to Tyr98, in dihydrofolate reductase (DHFR) is the molecular origin of trimethoprim (TMP) resistance in Staphylococcus aureus. This active site amino acid substitution was found in all S. aureus TMP-resistant clinical isolates tested. In order to explore the structural role of Tyr98 in TMP-resistance the ternary complexes of the chromosomal S. aureus DHFR (SaDHFR) with methotrexate (MTX) and TMP in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) as well as that of mutant Phe98Tyr DHFR SaDHFR(F98Y) ternary folate-NADPH complex have been determined by X-ray crystallography. Critical evidence concerning the resistance mechanism has also been provided by NMR spectral analyses of 15N-labelled TMP in the ternary complexes of both wild-type and mutant enzyme. These studies show that the mutation results in loss of a hydrogen bond between the 4-amino group of TMP and the carbonyl oxygen of Leu5. This mechanism of resistance is predominant in both transferable plasmid-encoded and non-transferable chromosomally encoded resistance. Knowledge of the resistance mechanism at a molecular level could help in the design of antibacterials active against multi-resistant Staphylococcus aureus (MRSA), one of todays most serious problems in clinical infectology.