Identification of bacilysin, chlorotetaine, and iturin a produced by Bacillus sp. strain CS93 isolated from pozol, a Mexican fermented maize dough

Three antimicrobial compounds produced by Bacillus sp. strain CS93 isolated from pozol were identified by using high-performance liquid chromatography and mass spectrometry. The three compounds were iturin, bacilysin, and chlorotetaine. Production of these compounds by CS93 could account for the medicinal properties attributed to pozol.     

Green tea polyphenol (-)-epigallocatechin gallate blocks epithelial barrier dysfunction provoked by IFN-gamma but not by IL-4

A characteristic of many enteropathies is increased epithelial permeability, a potentially pathophysiological event that can be evoked by T helper (Th)-1 (i.e., IFN-gamma) and Th2 (i.e., IL-4) cytokines and bacterial infection [e.g., enteropathogenic Escherichia coli (EPEC)]. The green tea polyphenol (-)-epigallocatechin gallate (EGCG) has immunosuppressive properties, and we hypothesized that it would ameliorate the increased epithelial permeability induced by IFN-gamma, IL-4, and/or EPEC. EGCG, but not the related epigallocatechin, completely prevented the increase in epithelial (i.e., T84 cell monolayer) permeability caused by IFN-gamma exposure as gauged by transepithelial resistance and horseradish peroxidase flux; EGCG did not alleviate the barrier disruption induced by IL-4 or EPEC. IFN-gamma-treated T84 and THP-1 (monocytic cell line) cells displayed STAT1 activation (tyrosine phosphorylation on Western blot analysis, DNA binding on EMSA) and upregulation of interferon response factor-1 mRNA, a STAT1-dependent gene. All three events were inhibited by EGCG pretreatment. Aurintricarboxylic acid also blocked IFN-gamma-induced STAT1 activation, but it did not prevent the increase in epithelial permeability. Additionally, pharmacological blockade of MAPK signaling did not affect IFN-gamma-induced epithelial barrier dysfunction. Thus, as a potential adjunct anti-inflammatory agent, EGCG can block STAT1-dependent events in gut epithelia and monocytes and prevent IFN-gamma-induced increased epithelial permeability. The latter event is both a STAT1- and MAPK-independent event.     

Feasibility of a porcine adult intensive care model

A porcine adult ICU model would be useful for several avenues of investigation relevant to the care of critically ill patients. The purpose of the experiments reported here was to test the feasibility of such a model, using healthy swine. Swine (n = 4; body weight, 76 +/- 5 kg) were instrumented with endotracheal, bladder, and central arterial and venous catheters, and were admitted to the intensive care unit (ICU) while undergoing mechanical ventilation under the continuous care of nurses. Cardiopulmonary parameters were monitored continuously, and serum biochemical parameters were measured intermittently. Survival was seven days in subject 1 and five and a half days in subject 2. Subjects 3 and 4 survived an abbreviated protocol (44 and 41 h, respectively). Care of the subjects was complicated by iatrogenic hemorrhage (n = 3), pneumonia (n = 2), and acute respiratory distress syndrome (n = 1). One subject was free of complications. Critically ill swine > or = 70 kg can survive mechanical ventilation in the ICU for up to seven days. When iatrogenic injury occurs, swine respond well to clinical care protocols. Further testing is needed to develop a reproducible model and determine whether healthy swine can survive the ICU environment for longer than 41 h.     

Stem cell biology and neurodegenerative disease

The fundamental basis of our work is that organs are generated by multipotent stem cells, whose properties we must understand to control tissue assembly or repair. Central nervous system (CNS) stem cells are now recognized as a well-defined population of precursors that differentiate into cells that are indisputably neurons and glial cells. Work from our group played an important role in defining stem cells of the CNS. Embryonic stem (ES) cells also differentiate to specific neuron and glial types through defined intermediates that are similar to the cellular precursors that normally occur in brain development. There is convincing evidence that the differentiated progeny of ES cells and CNS stem cells show expected functions of neurons and glia. Recent progress has been made on three fundamental developmental processes: (i) cell cycle control; (ii) the control of cell fate; and (iii) early steps in neural differentiation. In addition, our work on CNS stem cells has developed to a stage where there are clinical implications for Parkinson's and other degenerative disorders. These advances establish that stem cell biology contributes to our understanding of brain development and has great clinical promise.     

Synthesis and X-ray crystal structure of [Ag(phendio)<Subscript>2</Subscript>]ClO<Subscript>4</Subscript> (phendio = 1,10-phenanthroline-5,6-dione) and its effects on fungal and mammalian cells

The Cu(II) and Ag(I) complexes, [Cu(phendio)₃](ClO₄)₂4H₂O and [Ag(phendio)₂]ClO₄ (phendio = 1,10-phenanthroline-5,6-dione), are prepared in good yield by reacting phendio with the appropriate metal perchlorate salt. The X-ray crystal structure of the Ag(I) complex shows it to have a pseudo tetrahedral structure. `Metal-free' phendio and the Cu(II) and Ag(I) phendio complexes strongly inhibit the growth of the fungal pathogen Candida albicans, and are more active than their 1,10-phenanthroline analogues. The simple Ag(I) salts, AgCH<sub>3</sub>CO<sub>2</sub>, AgNO<sub>3</sub> and AgClO<sub>4</sub>.H<sub>2</sub>O display superior anti-fungal properties compared to analogous simple Cu(II) and Mn(II) salts, suggesting that the nature of the metal ion strongly influences activity. Exposing C. albicans to `metal-free' phendio, simple Ag(I) salts and [Ag(phendio)₂]ClO₄ causes extensive, non-specific DNA cleavage. `Metal-free' phendio and [Ag(phendio)₂]ClO₄ induce gross distortions in fungal cell morphology and there is evidence for disruption of cell division. Both drugs also exhibit high anti-cancer activity when tested against cultured mammalian cells.     

Genetics of drought adaptation in Arabidopsis thaliana: I. Pleiotropy contributes to genetic correlations among ecological traits

We examined patterns of genetic variance and covariance in two traits (i) carbon stable isotope ratio delta13C (dehydration avoidance) and (ii) time to flowering (drought escape), both of which are putative adaptations to local water availability. Greenhouse screening of 39 genotypes of Arabidopsis thaliana native to habitats spanning a wide range of climatic conditions, revealed a highly significant positive genetic correlation between delta13C and flowering time. Studies in a range of C3 annuals have also reported large positive correlations, suggesting the presence of a genetically based trade-off between mechanisms of dehydration avoidance (delta13C) and drought escape (early flowering). We examined the contribution of pleiotropy by using a combination of mutant and near-isogenic lines to test for positive mutational covariance between delta13C and flowering time. Ecophysiological mutants generally showed variation in delta13C but not flowering time. However, flowering time mutants generally demonstrated pleiotropic effects consistent with natural variation. Mutations that caused later flowering also typically resulted in less negative delta13C and thus probably higher water use efficiency. We found strong evidence for pleiotropy using near-isogenic lines of Frigida and Flowering locus C, cloned loci known to be responsible for natural variation in flowering time. These data suggest the correlated evolution of delta13C and flowering time is explained in part by the fixation of pleiotropic alleles that alter both delta13C and time to flowering.     

Repertoire shift in the humoral response to phosphocholine-keyhole limpet hemocyanin: VH somatic mutation in germinal center B cells impairs T15 Ig function

Phosphocholine (PC) is a naturally occurring Ag common to many pathogenic microorganisms. Early in the primary response to PC conjugated to keyhole limpet hemocyanin (KLH), T15 Id(+) Abs constitute >90% of the serum Ig in BALB/c mice. During the late primary and memory response to PC-protein, a shift in the repertoire occurs and T15 Id(+) Abs lose dominance. In this study, we use immunohistochemistry and single germinal center microdissection to locate T15 Id(+) cells in the spleen in a primary response to PC-KLH. We demonstrate T15 Id(+) B cells and V(H)1-DFL16.1-JH1 and V kappa 22-J kappa 5 rearrangements in germinal centers early in the immune response; thus loss of T15 dominance is not due to lack of T15 cells within germinal centers. One-hundred thirty one V(H)1 and 57 V kappa 22 rearrangements were cloned and sequenced. Thirty four percent of the V(H)1 clones and 37% of the V kappa 22 clones contained somatic mutations indicating participation in the germinal center response. Six variant T15 H clones were expressed with wild-type T15 L chain in vitro. Two of these Abs were defective in secretion providing the first evidence that mutation occurring in vivo can disrupt Ig assembly and secretion. Of the four secretion-competent Abs, two failed to display binding to PC-protein, while the other two displayed altered carrier recognition. These results indicate that somatic mutation of T15 in vivo can result in the loss of binding and secretion, potentially leading to B cell wastage. The failure of T15 to gain affinity enhancing mutations in the face of these detrimental changes may contribute to repertoire shift.