The detection of 20S-hydroxycholesterol in extracts of rat brains and human placenta by a gas chromatograph/mass spectrometry technique

The presence of 20(S)-hydroxycholesterol in rat brains and human placenta has been established using the gas chromatography/mass spectrometry (GC/MS) select ion monitoring (SIM) technique. Identification was ensured by three criteria: the specific retention time when the compound emerges from the gas chromatogram and the two m/z ions (201 and 461amu) which are characteristic of its mass spectrum. The possible role of 20(S)-hydroxycholesterol in steroid hormone biosynthesis and in other biological processes is discussed.     

Recent advances in the neurobiology of schizophrenia

Despite great progress in basic schizophrenia research, the conclusive identification of specific etiological factors or pathogenic processes in the illness has remained elusive. The convergence of modern neuroscientific studies in molecular genetics, molecular neuropathology, neurophysiology, in vivo brain imaging, and psychopharmacology, however, indicates that we may be coming much closer to understanding the molecular basis of schizophrenia. Schizophrenia may be a neurodevelopmental and progressive disorder with multiple biochemical abnormalities involving the dopaminergic, serotonin, glutamate, and gamma -aminobutyric acidergic systems. In the near future, biological markers for the illness may come from the combination of diverse assessment techniques. An understanding of the pathophysiology of schizophrenia will be essential to the discovery of preventive measures and therapeutic intervention. Rapidly advancing research into schizophrenia includes diverse etiological hypotheses, and offers directions for future research and treatments.     

SadB is required for the transition from reversible to irreversible attachment during biofilm formation by Pseudomonas aeruginosa PA14

Current models of biofilm formation by Pseudomonas aeruginosa propose that (i) planktonic cells become surface associated in a monolayer, (ii) surface-associated cells form microcolonies by clonal growth and/or aggregation, (iii) microcolonies transition to a mature biofilm comprised of exopolysaccharide-encased macrocolonies, and (iv) cells exit the mature biofilm and reenter the planktonic state. Here we report a new class of P. aeruginosa biofilm mutant that defines the transition from reversible to irreversible attachment and is thus required for monolayer formation. The transposon insertion carried by the sadB199 mutant was mapped to open reading frame PA5346 of P. aeruginosa PA14 and encodes a protein of unknown function. Complementation analysis and phage-mediated transduction demonstrated that the transposon insertion in PA5346 was the cause of the biofilm-defective phenotype. Examination of flow cell-grown biofilms showed that the sadB199 mutant could initiate surface attachment but failed to form microcolonies despite being proficient in both twitching and swimming motility. Closer examination of early attachment revealed an increased number of the sadB199 mutant cells arrested at reversible attachment, functionally defined as adherence via the cell pole. A positive correlation among biofilm formation, irreversible attachment, and SadB level was demonstrated, and furthermore, RpoN and FleR appear to negatively affect SadB levels. Fractionation studies showed that the SadB protein is localized to the cytoplasm, and with the use of GPS-linker scanning mutagenesis, the C-terminal portion of SadB was shown to be dispensable for function, whereas the two putative domains of unknown function and the linker region spanning these domains were required for function. We discuss the results presented here in the context of microbial development as it applies to biofilm formation.     

Xanthine dehydrogenase and aldehyde oxidase impact plant hormone homeostasis and affect fruit size in ‘Hass’ avocado

The contribution of xanthine dehydrogenase (XDH, EC 1.1.1.204) to fruit size was investigated using the normal and small-fruit variants of Persea americana Mill. cv. Hass. Inhibition of XDH by treatment of normal fruit, in the linear phase of growth (phase II), with allopurinol (Allo) arrested fruit growth. Adenine (Ade), a less effective inhibitor of this enzyme, also arrested fruit growth when applied in phase II and slowed fruit growth when applied in phase III. A time-course study on the activity of XDH in mesocarp tissue from normal and small fruit showed that maximum activity occurred late in phase II and that the peak in activity was absent in mesocarp of the small fruit. Feeding Ade to growing fruit in phase III caused a transient decline in fruit growth (measured as change in fruit length). Thereafter, growth resumed although fruit size was irreversibly affected. Treatment of fruit with Ade and Ade-containing cytokinins altered activity of another molybdenum enzyme, aldehyde oxidase (EC 1.2.3.1). Cytokinin oxidase was induced by cytokinin and auxin. Purine catabolism via hypoxanthine/xanthine was operative in normal fruit and in mesocarp from the small-fruit variant and as expected, Allo treatment caused accumulation of xanthine and adenine. In the absence of an increase in XDH during growth of the small-fruit phenotype, low levels of Ade were interpreted as resulting from respiration-enhanced adenylate depletion. Stress and/or pathogen induction of the alternative oxidase pathway is proposed as a possible cause.     

Rescuing the N-cadherin knockout by cardiac-specific expression of N- or E-cadherin

Cell-cell adhesion mediated by some members of the cadherin family is essential for embryonic survival. The N-cadherin-null embryo dies during mid-gestation, with multiple developmental defects. We show that N-cadherin-null embryos expressing cadherins using muscle-specific promoters, alpha- or beta-myosin heavy chain, are partially rescued. Somewhat surprisingly, either N-cadherin or E-cadherin was effective in rescuing the embryos. The rescued embryos exhibited an increased number of somites, branchial arches and the presence of forelimb buds; however, in contrast, brain development was severely impaired. In rescued animals, the aberrant yolk sac morphology seen in N-cadherin-null embryos was corrected, demonstrating that this phenotype was secondary to the cardiac defect. Dye injection studies and analysis of chimeric animals that have both wild-type and N-cadherin-null cells support the conclusion that obstruction of the cardiac outflow tract represents a major defect that is likely to be the primary cause of pericardial swelling seen in null embryos. Although rescued embryos were more developed than null embryos, they were smaller than wild-type embryos, even though the integrity of the cardiovascular system appeared normal. The smaller size of rescued embryos may be due, at least in part, to increased apoptosis observed in tissues not rescued by transgene expression, indicating that N-cadherin-mediated cell adhesion provides an essential survival signal for embryonic cells. Our data provide in vivo evidence that cadherin adhesion is essential for cell survival and for normal heart development. Our data also show that E-cadherin can functionally substitute for N-cadherin during cardiogenesis, suggesting a critical role for cadherin-mediated cell-cell adhesion, but not cadherin family member-specific signaling, at the looping stage of heart development.     

The MRP2/cMOAT transporter and arsenic-glutathione complex formation are required for biliary excretion of arsenic

Worldwide, millions of people are exposed to arsenic in drinking water that exceeds the World Health Organization standard of 10 microg/liter by as much as 50-300-fold, yet little is known about the molecular basis for arsenic excretion. Here we show that transport of arsenic into bile depends on the MRP2/cMOAT transporter and that glutathione is obligatory for such transport. Using reversed phase liquid chromatography/mass spectrometry, we demonstrate that two arsenic-glutathione complexes not previously identified in vivo, arsenic triglutathione and methylarsenic diglutathione, account for most of the arsenic in the bile. The structure of the compounds was also confirmed by nuclear magnetic resonance spectroscopy. Our findings may help explain the increased susceptibility of malnourished human populations to arsenic.