Effect of acetylation and permethylation on the conformation and candidacidal activity of salivary histatin-5

Summary Salivary histatins provide the non-immune defense against oral pathogens such as Candida albicans. The structural requirements of histatin-5 for anticandida activity were examined with respect to its ability to adopt helical structures, its electrostatic interactions and the hydrogen-bonding potency of its basic residues. For this purpose, the lysine and/or histidine residues of histatin-5 were chemically modified by acetylation and permethylation. Acetylated histatin-5 retained its ability to adopt helical structures in 2,2,2-trifluoroethanol, but completely lost its ability to kill yeast cells. In contrast, permethylated histatin-5 shows very little tendency to adopt a helical structure, but retained significant anticandida activity. The results suggest that the candidacidal activity can arise even when the histatin does not have the ability to adopt helical structures. The candidacidal activity of the derivatives is discussed in terms of electrostatic interactions and hydrogen-bonding potency.     

SARS coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mRNAs: viral mRNAs are resistant to nsp1-induced RNA cleavage

SARS coronavirus (SCoV) nonstructural protein (nsp) 1, a potent inhibitor of host gene expression, possesses a unique mode of action: it binds to 40S ribosomes to inactivate their translation functions and induces host mRNA degradation. Our previous study demonstrated that nsp1 induces RNA modification near the 5'-end of a reporter mRNA having a short 5' untranslated region and RNA cleavage in the encephalomyocarditis virus internal ribosome entry site (IRES) region of a dicistronic RNA template, but not in those IRES elements from hepatitis C or cricket paralysis viruses. By using primarily cell-free, in vitro translation systems, the present study revealed that the nsp1 induced endonucleolytic RNA cleavage mainly near the 5' untranslated region of capped mRNA templates. Experiments using dicistronic mRNAs carrying different IRESes showed that nsp1 induced endonucleolytic RNA cleavage within the ribosome loading region of type I and type II picornavirus IRES elements, but not that of classical swine fever virus IRES, which is characterized as a hepatitis C virus-like IRES. The nsp1-induced RNA cleavage of template mRNAs exhibited no apparent preference for a specific nucleotide sequence at the RNA cleavage sites. Remarkably, SCoV mRNAs, which have a 5' cap structure and 3' poly A tail like those of typical host mRNAs, were not susceptible to nsp1-mediated RNA cleavage and importantly, the presence of the 5'-end leader sequence protected the SCoV mRNAs from nsp1-induced endonucleolytic RNA cleavage. The escape of viral mRNAs from nsp1-induced RNA cleavage may be an important strategy by which the virus circumvents the action of nsp1 leading to the efficient accumulation of viral mRNAs and viral proteins during infection.     

Fate and Transport of Trichloroethylene in a Chamber with Alfalfa Plants

Experiments in a laboratory chamber were used to investigate the influence of alfalfa plants on the fate and transport of trichloroethylene (TCE) fed at a concentration of 200 μl/L (~290 mg/L) in the entering groundwater. The dimensions of the chamber were 180 cm in axial length, 35 cm in depth and 10 cm in width. Concentrations of TCE were monitored in the aqueous and gas phases. Evapotranspirational fluxes of TCE from the soil to the headspace of the chamber were also measured. TCE concentration in the solid phase was measured as a function of depth. Mathematical modeling of the fate of TCE was developed assuming rate-independent physical equilibrium partitioning between solid, aqueous, and gas phases. The model included volatilization across a thin atmospheric boundary layer near soil surface. Numerical results were first validated with analytical results for simple cases and then compared with experimental data in the chamber. Results indicated that the water content and air content distributions significantly impact the transport and concentration of TCE in soils.     

Electron microscopic studies on the retinal photoreceptors of the spotted snakehead, Channa punctatus

The retinal photoreceptors of Channa punctatus include rods, single cones and the double cones. The double cones have the outer segments having lamellae which are distorted into tubules and vesicles, and the inner segments with hypertrophied mitochondria.     

Abnormal differentiation of selected nuclear centers in the brain of a duck embryo associated with partial duplication of the primitive streak

In a control set of duck embryos, an abnormal duck embryo of 16 days incubation was found which had two beaks as the only outward sign of duplication. The beaks were of equal size, each with upper and lower bills. Bill-clapping movements were absent. The embryo had two normal eyes placed one on either side of the head, and the rest of the body was normal in external appearance. Sections through the head revealed further duplication of the fore-, mid-, and hindbrain divisions. The medial half of each of the embryonic brain divisions, however, was greatly reduced. Two additional optic primordia were also noticed in sections, each of which was reduced to a mass of tissue representing a lens and a much-folded pigment epithelium. The orbital tissues associated with the rudimentary eyes were greatly disorganized. Abnormal differentiation associated with duplication of the brain divisions was determined by cell counts in selected nuclear centers. Cell numbers in each case appeared to be remarkably proportional to the size of the innervation field. Our data, based on cell counts in the nuclear centers chosen for this study in the abnormal embryo and normal control embryos of the same age, are consistent with the hypothesis that cell survival in related parts of the nervous system may be regulated by their peripheral field of innervation.     

Expression of the insecticidal crystal protein gene from a Gram-positive Bacillus thuringiensis in a Gram-negative Pseudomonas fluorescens mediated by protoplast fusion

Abstract Protoplast fusion between a Gram-negative strain Pseudomonas fluorescens having plant growth promoting activities and a Gram-positive Bacillus thuringiensis var. kurstaki HD 73 possessing insecticidal activity, was carried out to generate P. fluorescens hybrids possessing insecticidal activity. The antibiotic resistance markers of P. fluorescens (rif^r, nal^r) and the immunoreactivity to the antiserum raised against the crystal proteins of B. thuringiensis var. galleriae were used as selection markers for the hybrids. The hybrids exhibited lethal but differential activity in Heliothis armigera and in Spodoptera litura when compared to the parenthal B. thuringiensis strain. The anti-feedant activity which is characteristic of B. thuringiensis toxin was not observed in the hybrids. Although the presence of sequences homologous to the cloned insecticidal gene of B. thuringiensis was demonstrated, the Western blot analysis of cell extract of the hybrid (PK 105) showed that only low molecular mass crystal proteins (less than 40 kDa) could be detected under denaturing conditions. It indicates that the high molecular mass toxin peptide may be degraded by proteolysis. Besides this, a clear separation of lethal and anti-feedant activity of the B. thuringiensis toxin has been observed by this study.