Extracellular secretion of levansucrase from Zymomonas mobilis in Escherichia coli

Secretion of levansucrase from Zymomonas mobilis in Escherichiacoli by glycine supplement was investigated. A significant amount of levansucrase (about 25% of total activity) was found in intact whole-cells. Cell fractionation experiments showed that levansucrase was found both in the periplasmic space and in the cytoplasmic fraction of E. coli. None or only trace amounts of levansucrase was detected in the extracellular culture broth at 24 h of cultivation and it accrued with the increasing concentration of glycine in the culture medium and duration of the culture period. Optimal glycine concentration for the maximum secretion of levansucrase was in the range of 0.8-1%, in which approximately 20-50% of levansucrase was released into the extracellular fraction at 24 h of cultivation, although glycine retarded the bacterial growth.     

Enhancing the fungicidal activity of amphotericin B via vacuole disruption by benzyl isothiocyanate,a cruciferous plant constituent

Amphotericin B (AmB), a typical polyene macrolide antifungal agent, is widely used to treat systemic mycoses. In the present study, we show that the fungicidal activity of AmB was enhanced by benzyl isothiocyanate (BITC), a cruciferous plant-derived compound, in the budding yeast, Saccharomyces cerevisiae. In addition to forming a molecular complex with ergosterol present in fungal cell membranes to form K⁺-permeable ion channels, AmB has been recognized to mediate vacuolar membrane disruption resulting in lethal effects. BITC showed no effect on AmB-induced plasma membrane permeability; however, it amplified AmB-induced vacuolar membrane disruption in S. cerevisiae. Furthermore, the BITC-enhanced fungicidal effects of AmB significantly decreased cell viability due to the disruption of vacuoles in the pathogenic fungus Candida albicans. The application of the combinatorial antifungal effect of AmB and BITC may aid in dose reduction of AmB in clinical antifungal therapy and consequently decrease side effects in patients. These results also have significant implications for the development of vacuole-targeting chemotherapy against fungal infections.     

Functional and molecular characterization of the conserved Arabidopsis PUMILIO protein,APUM9

Plant Molecular Biology - Here we demonstrate that the APUM9 RNA-binding protein and its co-factors play a role in mRNA destabilization and how this activity might regulate early plant development....     

Use of restriction endonucleases and exonuclease III to expose halogenated pyrimidines for immunochemical staining

We describe an enzymatic procedure for exposure of single-stranded DNA (ssDNA) containing the halogenated pyrimidines (HdUrd) bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd) in single cells to antibodies that bind to HrdUrd only in ssDNA. Production of ssDNA was accomplished by digesting the DNA using either restriction endonucleases alone or endonucleases followed by exonuclease III. The enzymatic production of ssDNA was maximal when 0.1 N HCl or 0.1 M citric acid plus Triton X-100 was added to extract nuclear proteins prior to enzymatic denaturation. The restriction endonucleases Bam HI, Dde I, Eco RI, and Hind III produced significant ssDNA when used alone to allow binding of detectable amounts of the anti-HdUrd antibody IU-4 in Chinese hamster ovary cells labeled with 10 microM BrdUrd or 10 microM IdUrd. However, these treatments did not expose sufficient ssDNA to allow binding of IU-1, an anti-HdUrd antibody with lower binding affinity. IU-4 binding was most intense after treatment with Eco RI. Treatment with exonuclease III following endonuclease digestion allowed substantially more IU-4 binding.     

HDL-induced cardiac prostacyclin synthesis: relative contribution of HDL apoprotein and lipid

The objective of this study was to determine if the apoprotein or lipid constituents of high density lipoproteins (HDL) mediate HDL-induced prostacyclin synthesis in the Langendorff-perfused rabbit heart. Acetylation, acetoacetylation, or partial removal by trypsin digestion of HDL apoprotein did not reduce the ability of the lipoprotein to stimulate cardiac prostacyclin synthesis. Delipidated apoproteins were less effective in stimulating cardiac prostacyclin synthesis in comparison to intact HDL. In contrast, protein-free lipid vesicles, made from HDL lipids, caused a pronounced stimulation of cardiac prostacyclin synthesis. These results suggest that HDL apoproteins, in their native state, are not essential for HDL-induced cardiac prostacyclin synthesis. The stimulation of cardiac prostacyclin synthesis by HDL may depend on the lipoprotein's lipid rather than on its apoprotein constituents.     

Mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid to bovine serum albumin

The mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid (HABA) to bovine serum albumin was studied by relaxation methods as well as the binding isotherm using gel chromatography. A single relaxation was observed over a wide range of HABA concentration except at the extremes of high concentration where another slow process was observed. The concentration dependence of the reciprocal relaxation time of the fast process decreased monotonically with increase in concentration of HABA at constant polymer concentration. The data were analyzed on the basis of Brown's domain structure model and were found to be consistent with a sequential binding mechanism. The azohydrazon tautomerism of HABA was identified with the intramolecular step of the complex. The activation parameters of the step, determined from the temperature dependence of the relaxation time of the fast process, showed that this step is rate limited by an enthalpy barrier in both forward and backward directions. Comparison of the activation parameters with those of other serum albumin-ligand systems suggests that there is an enthalpy-entropy compensation in the activation process of the intramolecular step with the compensation temperature at about 270 K; the enthalpy-entropy compensation is thought to be related to the hydrophobic nature of the ligand.     

Morphological changes in the cochlear nuclear complex in primate phylogeny and development

The primate cochlear nuclear complex exhibits several characteristic morphological differences in the various primate families from Lorisidae through Hominidae. The most striking differences occur in the organization of the dorsal cochlear nucleus in which the laminar pattern becomes progressively obscured. Granule cells form an external granular layer as well as being intermixed within the molecular and pyramidal layers in slow lorises and squirrel and rhesus monkeys. Whereas a prominent external granular layer remains in chimpanzees, granule cells are scant in other portions of the nucleus. Human adults lack an external granular layer. A small number of granule cells occur but with inconstant distribution. Primates lack the linear array of pyramidal cells oriented perpendicularly to the epithelial surface as seen in cats. The granule cell layer exhibits similar regression in development of the human cochlear complex. The external granular layer is prominent in the fetus but rapidly decreases in size after birth. It achieves its adult form prior to 18 months. The data suggest that neuronal attrition, or programmed cell death, may be the major mechanism accounting for the alterations that occur in the human granule cell layer. Other differences in cytoarchitecture, within the great apes and humans, include decreases in the small and giant cell populations of the cochlear complex. These changes, in consort with the organizational changes and reduction of granule cells as noted above, suggest a trend towards reduced intranuclear integration at the level of the cochlear nucleus coupled with encephalization of the auditory system.