Deletion of the Candida glabrata ERG3 and ERG11 genes: effect on cell viability, cell growth, sterol composition, and antifungal susceptibility

We have cloned and sequenced the structural genes encoding the delta 5,6 sterol desaturase (ERG3 gene) and the 14 alpha-methyl sterol demethylase (ERG11 gene) from Candida glabrata L5 (leu2). Single and double mutants of these genes were created by gene deletion. The phenotypes of these mutants, including sterol profiles, aerobic viabilities, antifungal susceptibilities, and generation times, were studied. Strain L5D (erg3 delta::LEU2) accumulated mainly ergosta-7,22-dien-3 beta-ol, was aerobically viable, and remained susceptible to antifungal agents but had a slower generation time than its parent strain. L5LUD (LEU2 erg11 delta::URA3) strains required medium supplemented with ergosterol and an anaerobic environment for growth. A spontaneous aerobically viable mutant, L5LUD40R (LEU erg11 delta::URA3), obtained from L5LUD (LEU2 erg11 delta::URA3), was found to accumulate lanosterol and obtusifoliol, was resistant to azole antifungal agents, demonstrated some increase in resistance to amphotericin B, and exhibited a 1.86-fold increase in generation time in comparison with L5 (leu2). The double-deletion mutant L5DUD61 (erg3 delta::LEU2 erg11 delta::URA3) was aerobically viable, produced mainly 14 alpha-methyl fecosterol, and had the same antifungal susceptibility pattern as L5LUD40R (LEU2 erg11 delta::URA3), and its generation time was threefold greater than that of L5 (leu2). Northern (RNA) analysis revealed that the single-deletion mutants had a marked increase in message for the undeleted ERG3 and ERG11 genes. These results indicate that differences in antifungal susceptibilities and the restoration of aerobic viability exist between the C. glabrata ergosterol mutants created in this study and those sterol mutants with similar genetic lesions previously reported for Saccharomyces cerevisiae.     

Regulation of neutrophil-derived IL-8: the role of prostaglandin E2, dexamethasone, and IL-4

Historically, the neutrophil has been perceived as a terminally differentiated leukocyte with limited ability to produce de novo proteins. Furthermore, in the context of acute inflammation the activated neutrophil has been appreciated only for its ability to release various proteases, reactive oxygen, and arachidonic acid metabolites. Recently, the neutrophil has been shown to have the capacity to produce a number of cytokines that may be instrumental in orchestrating the progression of acute inflammation to a more chronic and specific immune response. These cytokines include IFN-alpha, M-CSF, G-CSF, TNF, IL-1, and IL-6. Our laboratory and others have shown that neutrophils produce IL-8 in response to LPS or a phagocytic challenge. Although these studies have shown the induction of IL-8 from polymorphonuclear neutrophils (PMN), relatively little is known regarding the regulation of PMN-derived IL-8. Because PMN and monocytes share the same stem cell, and monocyte-derived IL-8 is regulated by prostaglandin E2 (PGE2), glucocorticoids (dexamethasone; DEX) and the T-Lymphocyte-derived IL-4, we postulated that PMN-derived IL-8 production may be regulated in a similar manner. To test this hypothesis, PMN were isolated (> 99% pure) from peripheral blood and cultured in media with 5% FCS in the presence or absence of LPS (10 ng/ml; a concentration of LPS that induced the half-maximal production of PMN-derived IL-8) and in the presence or absence of DEX (10(-6) M to 10(-10) M), PGE2 (10(-6) M to 10(-10) M), or IL-4 (100 ng/ml to 100 pg/ml). PMN-derived IL-8 was measured using a specific sandwich ELISA. DEX and IL-4 in the presence of LPS were found to inhibit PMN-derived IL-8 in both a dose- and time-dependent fashion. DEX and IL-4 in concentrations of 10(-6) M and 10 ng/ml resulted in maximal inhibition of LPS-induced PMN-derived IL-8, respectively. Moreover, both DEX and IL-4 administration could be delayed 4 hr post-stimulation with LPS and result in significant suppression of PMN-derived IL-8. Interestingly, in contrast to the regulation of monocyte-derived IL-8 by PGE2, PGE2 treatment of PMN failed to inhibit the generation of LPS-induced IL-8. Northern blot analysis of steady-state IL-8 mRNA demonstrated that both DEX and IL-4 treatment of PMN resulted in a 40 and 52% reduction in LPS-stimulated PMN-derived IL-8 mRNA, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Consensus functions and patterns in molecular sequences

In recent years, methods of consensus, developed for the solution of problems in the social sciences, have become widely used in molecular biology. We study a method of consensus originally due to Waterman et al. (Waterman, Galas and Arratis. 1984. Pattern recognition in several sequences: consensus and alignment. Bull. math. Biol. 46, 515-527) which is used to identify patterns or features in a molecular sequence where a pattern can vary in position within a given window. We show that some well-known consensus methods of the social sciences, the median and the mean, are special cases of this method for certain choices of the parameters used in it and give a precise account of the parameters for which these special cases arise. We also show that the specific parameters used in the method of Waterman et al. make their method equivalent to the media procedure which is widely used in the social sciences.     

Two color hybridization analysis using high density oligonucleotide arrays and energy transfer dyes

High density oligonucleotide arrays (DNA chips) have been used in two color mutational analysis of the 3.43 kb exon 11 of the hereditary breast and ovarian cancer gene BRCA1 . Two color analysis allows competitive hybridization between a reference standard and an unknown sample, improving the performance of the assay. Fluorescein and phycoerythrin dyes werepreviously used due to their compatibility with a single line 488 nm excitation source. Here we show that an alternative dye combination, containing the energy transfer dye system phycoerythrin*cy5 along with phycoerythrin, provides more evenly matched signal intensities and decreased spectral overlap between the two fluorophores, while maintaining compatibility with a 488 nm excitation source.     

Ultrasound backscatter microscope analysis of mouse melanoma progression

The incidence and mortality rate of cutaneous melanoma continue to increase throughout the world, making the study of melanoma biology an important area of current research. While recent breakthroughs in transgenic mouse technology have led to promising mouse skin models of melanoma, there is presently no technique available for quantitatively studying subsurface melanoma progression, in vivo. We demonstrate the first application of an imaging method called ultrasound backscatter microscopy (UBM) for imaging early murine melanomas with spatial resolution of 30 microns axial and 60 microns lateral. Murine B16 F10 melanomas have been imaged from their earliest detection, over several days, until they are 2 to 5 mm in diameter. Melanoma dimensions measured by UBM were found to be in excellent agreement with those determined histopathologically on the excised tumours. The relative rms errors in UBM-determined melanoma height and width were found to be 8.7% and 4.2%, respectively. The mean rate of increase in tumour height of early murine melanoma was found to be 0.37 +/- 0.06 mm/day. Computer-generated volumetric renderings of melanomas have been produced from three-dimensional image data, allowing quantitative comparisons of tumour volumes to be made. Using a priori assumptions of ellipsoid tumour shape, the relative error in UBM-determined volume was shown to be less than 17%. These results should be of considerable interest to investigators studying melanoma biology using mouse skin models, and have implications in the use of high frequency ultrasound imaging for the clinical assessment of cutaneous melanoma.     

Physiological response of Lactobacillus plantarum to salt and nonelectrolyte stress

In this report, we compared the effects on the growth of Lactobacillus plantarum of raising the medium molarity by high concentrations of KCl or NaCl and iso-osmotic concentrations of nonionic compounds. Analysis of cellular extracts for organic constituents by nuclear magnetic resonance spectroscopy showed that salt-stressed cells do not contain detectable amounts of organic osmolytes, whereas sugar-stressed cells contain sugar (and some sugar-derived) compounds. The cytoplasmic concentrations of lactose and sucrose in growing cells are always similar to the concentrations in the medium. By using the activity of the glycine betaine transport system as a measure of hyperosmotic conditions, we show that, in contrast to KCl and NaCl, high concentrations of sugars (lactose or sucrose) impose only a transient osmotic stress because external and internal sugars equilibrate after some time. Analysis of lactose (and sucrose) uptake also indicates that the corresponding transport systems are neither significantly induced nor activated directly by hyperosmotic conditions. The systems operate by facilitated diffusion and have very high apparent affinity constants for transport (>50 mM for lactose), which explains why low sugar concentrations do not protect against hyperosmotic conditions. We conclude that the more severe growth inhibition by salt stress than by equiosmolal concentrations of sugars reflects the inability of the cells to accumulate K+ (or Na+) to levels high enough to restore turgor as well as deleterious effects of the electrolytes intracellularly.