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.     

Human P301L-Mutant Tau Expression in Mouse Entorhinal-Hippocampal Network Causes Tau Aggregation and Presynaptic Pathology but No Cognitive Deficits

Accumulation of hyperphosphorylated tau in the entorhinal cortex (EC) is one of the earliest pathological hallmarks in patients with Alzheimer’s disease (AD). It can occur before significant Aβ deposition and appears to “spread” into anatomically connected brain regions. To determine whether this early-stage pathology is sufficient to cause disease progression and cognitive decline in experimental models, we overexpressed mutant human tau (hTauP301L) predominantly in layer II/III neurons of the mouse EC. Cognitive functions remained normal in mice at 4, 8, 12 and 16 months of age, despite early and extensive tau accumulation in the EC. Perforant path (PP) axon terminals within the dentate gyrus (DG) contained abnormal conformations of tau even in young EC-hTau mice, and phosphorylated tau increased with age in both the EC and PP. In old mice, ultrastructural alterations in presynaptic terminals were observed at PP-to-granule cell synapses. Phosphorylated tau was more abundant in presynaptic than postsynaptic elements. Human and pathological tau was also detected within hippocampal neurons of this mouse model. Thus, hTauP301L accumulation predominantly in the EC and related presynaptic pathology in hippocampal circuits was not sufficient to cause robust cognitive deficits within the age range analyzed here.     

Chemical, physicochemical and spectrophotometric properties of crystalline chlorophyll-protein complexes from Lepidium virginicum L

Two kinds of water-soluble chlorophyll-protein complexes were prepared from leaves of Lepidium virginicum L., one (CP661) from the plant cultivated in a green house from seeds collected near Mono Lake, CA, and the other (CP-663) from a plant collected at Narashino, Chiba, Japan, by ammonium sulfate fractionation followed by column chromatography on DEAE-cellulose and Sephacryl S-200. The chlorophyll . proteins were further purified by crystallization. CP661 has absorption peaks at 661, 468, 439, 419, 380, 339 and 272 nm. CP663 had absorption peaks at 663, 469, 438, 419, 379, 338 and 272 nm. Estimated molecular weights were 78 000 for CP661 and 80 000 for CP663 by gel filtration chromatography and 83 000 for CP661 and 107 000 for CP663 by an equilibrium sedimentation method. 1 mol chlorophyll . protein contained 4 mol chlorophyll a and b with ratios of 1.0 in CP661 and 1.6 to 1.9 in CP663, but no carotenoids. These characters are different from those of chlorophyll-protein complexes which are prepared from the thylakoid membranes of chloroplasts with detergents.     

Fragile X expression in thymidine-prototrophic and auxotrophic human-mouse somatic cell hybrids under low and high thymidylate stress conditions

Expression of the fragile X site fra(X)(q27.3) was studied in thymidine-prototrophic and auxotrophic human-mouse somatic cell hybrids. In these cells, low thymidylate stress, achieved by 5-fluoro-2'-deoxyuridine (FdU) treatment and by limiting the exogenous supply of thymidine (dT), induced fragile X expression. High thymidylate stress, produced by supplying excess amounts of dT, was also effective in inducing fragile X expression, even in a hybrid clone that retained a fragile X chromosome as the only human chromosome; addition of deoxycytidine (dC) completely abolished this effect. In contrast, 5-bromo-2'-deoxyuridine (BrdU) did not induce fragile X expression. Cell-cycle analysis of BrdU-deprived thymidine-auxotrophic hybrid cells indicated that one round of DNA replication under thymidylate stress conditions is sufficient for fragile X expression. Our results suggest that the expression is an intrinsic property of the fragile site itself, which is believed to be composed of replicon clusters with pyrimidine-rich DNA sequence(s).     

Effect of molar ratio of fluorescent dye to IgG on the detection of lymphocyte surface immunoglobulins by immunofluorescence

Labeled antibodies with different F/P molar ratios of FITC to protein (F/P molar ratio) were used for the detection of surface immunoglobulin (S-Ig) of human and mouse lymphocytes by membrane immunofluorescence, and the following results were obtained. 1. The percentage of S-Ig bearing cells increased markedly when labeled anti-human H- or L-chains antibodies were used with higher F/P molar ratios. The investigation of frozen kidney sections of mice injected with human immunoglobulin revealed that such an increase of the positive ratio in S-Ig was caused by increased non-specific adsorption of the fraction of labeled antibody with a high F/P molar ratio. 2. This non-specific adsorption phenomenon was observed at various intensities in materials from different species; materials from mcie showed less non-specific adsorption than those from humans. 3. It was possible to exclude reactivity with an Fc receptor using the top one third of the supernatant of labeled antibody centrifuged at 150,000 for 30 min.     

Characterization of translucent segments observed in an smbA mutant of Escherichia coli

Abstract The smbA gene of Escherichia coli is essential for cell proliferation. The smbA2 mutant shows cold-sensitive colony formation at 22°C. A novel morphological phenotype, formation of a translucent segment at midcell or at a cell pole, was observed by phase-contrastt microscopy at a high frequency in the smbA2 mutant cells incubated in L medium lacking NaCl at 22°C, but not observed in L medium containing 1% NaCl or 20% sucrose at the same temperature. No translucent segment was observed in the wild-type cells in any of the media used. Electron microscopic observation revealed that the translucent segments resulted from the enlargement of a periplasmic space by separation of the inner membrane from the peptidoglycan layer and the outer membrane.     

Dermatan sulfate isomers in human articular cartilage characterized by high-performance liquid chromatography

The Constituents of dermatan sulfate isomers in human articular cartilage were studied at the disaccharide level by high-performance liquid chromatography. Appreciable amounts of dermatan sulfate components, i.e., dermatan sulfate, chondroitin sulfate types G and B, could be detected after digestion with chondroitinases-B or -ABC. The oversulfated dermatan sulfate isomers were isolated only after digestion with chondroitinase-ABC but not with the AC-lyase. The dermatan sulfate isomers were found to be markedly increased in weight loading parts of articular cartilage. It is postulated that the dermatan sulfate isomers are formed as a result of the weight loading reaction, which may be responsible for the fibrosis of articular cartilage.     

Genetic characterization of the mei-41 locus in Drosophila melanogaster

Summary The mutagen-sensitive mutant mus(1)104 ^D1 of Drosophila melanogaster maps to a position on the X chromosome very close to the meiotic mutant mei-41 ^D5 . Both mutants have been characterized as mutagen-sensitive and defective in post-replication repair. In the present report we show by complementation studies that mus(1)104 and mus(1)103 are allelic with mei-41. In addition, two reported alleles of mus(1)104 lie between the mei-41 alleles A10 and D5. The size of the mei-41 locus is estimated to be about 0.1 centimorgans (cM). Because several alleles of mei-41 have been shown to reduce recombination and increase meiotic chromosome loss and nondisjunction, mus(1)104 ^D1 females were examined for defects in meiosis. Although there was no evidence for reduced recombination on the second chromosome in homozygous mus(1)104 ^D1 females, heterozygous mus(1)104 ^D1 /mei-41 ^>D5 and mus(1)104 ^D1 /deficiency females showed reduced levels of recombination. However, there was no evidence of an increase in nondijunction in these females.We dedicate this article to the memory of Larry Sandler, who passed away suddenly on February 7, 1987     

Effects of auxin and cytokinin on induction of sister chromatid exchanges in cultured cells of wheat (Triticum aestivum L.)

Summary In order to know the mutagenic effects of synthetic auxins (NAA, 2,4-D, and 2,4,5-T) and a cytokinin (kinetin) in vitro, sister chromatid exchanges (SCEs) were analyzed in cultured cells of a hexaploid wheat (Triticum aestivum L.). In the MS medium supplemented with 2.0 mg/l 2,4-D, the mean number of SCEs per cell was 15.2, and per pg of DNA, 0.42. No significant effect was found in the treatments of NAA or 2,4-D at concentrations of 0.5–10.0 mg/l, whereas more than 2.0 mg/l of 2,4,5-T induced dramatic increases of SCEs. Kinetin itself had no significant effect on SCE induction, but there was a tendency that SCEs induced by 2,4,5-T were suppressed by kinetin.     

Cord transferrin and ferritin values for erythropoiesis in newborn infants of diabetic mothers

Neonatal polycythemia is a perinatal complication in infants of diabetic mothers. The cord CBC (complete blood counts), serum iron, transferrin and ferritin concentrations were studied in newborn infants of 9 GDM (gestational diabetes), 21 NIDDM (noninsulin-dependent diabetes mellitus), and 8 IDDM (insulin-dependent diabetes mellitus) mothers. The RBC (red blood cell) count, Hb (hemoglobin) and Hct (hematocrit) of these infants were higher than control infants. There was no difference between the serum iron concentration of the infants of each group diabetic mothers and the infants in the control group, but the transferrin concentration was significantly higher and the ferritin was significantly lower in the infants of diabetic mothers than in those of control mothers. There was a significant negative correlation between transferrin and ferritin (r = -0.491 p less than 0.001). Erythropoiesis is considered to be enhanced in the fetuses of diabetic mothers, and the iron needed for erythropoiesis is reportedly transported from the mother to the fetus according to the demands of the fetus, but the iron storage was shown to be reduced in the fetuses of diabetic mothers.