Gene-conversion tract directionality is influenced by the chromosome environment

Spontaneous and double-strand break (DSB)-induced gene conversion in Saccharomyces cerevisiae was assayed using non-tandem chromosomal direct repeat crosses and plasmid × chromosome crosses. Each cross involved identical ura3 alleles marked with phenotypically silent restriction fragment length polymorphic (RFLP) mutations at approximately 100-bp intervals. DSBs introduced in vivo at HO sites in one allele stimulated recombination to Ura⁺ by more than two orders of magnitude. Spontaneous gene-conversion products were isolated from a related strain lacking a functional HO nuclease gene. The multiple markers did not appear to influence the frequency of direct repeat deletions for spontaneous or DSB-induced events. DSB-induced conversion reflected efficient mismatch repair of heteroduplex DNA. Conversion frequencies of equidistant markers on opposites sides of the DSB were similar in the direct repeat cross. In contrast, markers 5′ of the DSB (promoter-proximal) converted more often than 3′ markers in plasmid × chromosome crosses, a possible consequence of crossing-over associated with long conversion tracts. With direct repeats, bidirectional tracts (extending 5′ and 3′ of the DSB) occurred twice as often as in a plasmid × chromosome cross in which DSBs were introduced into the plasmid-borne allele. A key difference between the direct-repeat and plasmid×chromosome crosses is that the ends of a broken plasmid are linked, whereas the ends of a broken chromosome are unlinked. We tested whether linkage of ends influenced tract directionality using a second plasmid × chromosome cross in which DSBs were introduced into the chromosomal allele and found few bidirectional tracts. Thus, chromosome environment, but not linkage of ends, influences tract directionality. The similar tract spectra of the two plasmid × chromosome crosses suggest that similar mechanisms are involved whether recombination is initiated by DSBs in plasmid or chromosomal alleles. Received: 11 May / 24 July 1998     

Multiple ectopic thyroid masses in a hypothyroid child

Pediatric Radiology -     

Effect of Quercetin on lipid peroxidation and changes in lung morphology in experimental influenza virus infection

Influenza virus infection, induced experimentally in mice, was associated with marked changes in lung morphology viz. epithelial damage with focal areas of reactive papillary hyperplasia, infiltration of leukocytes and development of oxidative stress, as evidenced by increased superoxide radical production and lipid peroxidation (LPO) products by alveolar macrophages. These effects were observed on the 5th day after virus instillation. The levels of superoxide and LPO were measured spectrophotometrically by the nitroblue tetrazolium (NBT) assay and thiobarbituric acid reactive species (TBARS) assay, respectively. The former increased by 1.5-2 fold and the latter was raised by 85% when compared with normal control. Supplementation of intranasal viral instillation with the anti-oxidant, Quercetin, given orally, resulted in a significant decrease in the levels of both superoxide radicals and LPO products. There was also a significant decrease in the number of infiltrating cells. A mild to moderate protective effect was observed in lung morphology. Thus, Quercetin may be useful as a drug in reducing the oxidative stress induced by influenza virus infection in the lung, and protect it from the toxic effects of the free radicals.     

New approaches in hormone refractory prostate cancer

Effective therapeutic options have not existed for prostate cancer progressing after androgen deprivation therapy until very recently. Docetaxel based chemotherapy has been demonstrated to extend survival in 2 large randomized trials. These studies have provided the impetus to combine docetaxel with novel biologic agents to further consolidate the gains in long-term outcome. With the arrival of exciting agents including vaccines, monoclonal antibodies, bone-targeted drugs, antisense oligonucleotides, antiangiogenic drugs, and small molecule receptor tyrosine kinase inhibitors, the future of prostate cancer therapy appears promising.     

Enhanced oral absorption of halofantrine enantiomers after encapsulation in a proliposomal formulation

In this study, we evaluated the ability of a coated, encapsulated formulation to increase the oral bioavailability of (+/-)-halofantrine (HF) enantiomers, a drug with low and erratic oral bioavailability. After encapsulation of HF in distearoylphosphatidylcholine, the dried particles were coated with cellulose acetate phthalate. A suspension of the product was made using methylcellulose as a dispersion agent, and the product was administered to Sprague-Dawley rats to provide a HF dose of 7 mg kg-1 as the HCl salt. HF HCl powder in 1% methylcellulose with or without liposomal product excipients was also administered to separate groups of rats, which served as control groups. Serial blood samples were obtained from the rats and plasma was assayed by stereospecific high-performance liquid chromatography. There were no significant differences in the area under the concentration-time curve (AUC) or maximum concentration (Cmax) between the two control groups. Plasma concentrations of both HF enantiomers were significantly higher in the rats given HF as an encapsulated proliposomal formulation compared with the control groups. Compared with methylcellulose control, the encapsulation product resulted in increases of 41 to 47% in the AUC of HF enantiomers, and 90 to 100% in Cmax. The ability of an encapsulated proliposomal product to significantly increase the oral absorption of HF was clearly demonstrated.     

Protein kinase C alpha and epsilon differentially modulate hepatocyte growth factor-induced epithelial proliferation and migration

Protein kinase C (PKC) isoenzymes require membrane translocation for physiological activation. We have recently shown that the growth factors such as epidermal growth factor and hepatocyte growth factor (HGF), but not keratinocyte growth factor (KGF), regulate PKCalpha activation to promote epithelial wound healing [Sharma, G.D., Ottino, P., Bazan, H.E.P., 2005. Epidermal and hepatocyte growth factors, but not keratinocyte growth factor, modulate protein kinase C alpha translocation to the plasma membrane through 15(S)-hydroxyeicosatetraenoic acid synthesis. J. Biol. Chem. 280, 7917--924]. Protein kinase C alpha (PKCalpha) and protein kinase C epsilon (PKCvarepsilon) are two differentially regulated isoenzymes. While PKCalpha requires Ca(2+) for its activation, PKEvarepsilon is Ca(2+) independent. However, growth factor-induced activation of these enzymes and their specific regulation of epithelial migration and proliferation have not been explored. In the present study, we overexpressed PKCvarepsilon fused to green fluorescent protein to examine its translocation in real-time to the plasma membrane in living human corneal epithelial cells. Stimulation with HGF and KGF demonstrated translocation of PKCvarepsilon to the plasma membrane. Because HGF activates both PKCs, this growth factor was used to stimulate wound healing. PKCalpha or PKCvarepsilon-genes were knocked down individually without affecting the basal expression of the other PKC isoforms. Gene knockdown of PKCalpha significantly inhibited HGF-stimulated proliferation of human corneal epithelial cells. In contrast, PKCvarepsilon-gene-silencing severely impaired the HGF-stimulated migratory ability of human corneal epithelial cells. When migrating epithelial cells in the cornea wound bed after injury were transfected with specific PKCalpha- or PKCvarepsilon-siRNA, there was a significant delay in wound healing. Corneal wound healing stimulated with HGF in similar conditions was also inhibited. On the other hand, overexpression of PKCalpha or PKCvarepsilon-genes fused with green fluorescent protein in migrating corneal epithelium accelerated repair of the epithelial defect. Our findings demonstrate that PKCalpha and PKCvarepsilon modulate different stages of wound healing stimulated by HGF and contribute to epithelial repair by playing selective regulatory roles in epithelial proliferation and migration, both crucial to corneal wound healing.