Antifungal susceptibility of epigallocatechin 3-O-gallate (EGCg) on clinical isolates of pathogenic yeasts

This is the first report to investigate the antifungal susceptibility of 21 clinical isolates of seven Candida species to epigallocatechin 3-O-gallate (EGCg) and to compare with six antifungal agents, amphotericin B (AMPH), fluconazole (FLCZ), flucytosin (5FC), itraconazole (ITCZ), micafungin (MCFG), and miconazole (MCZ), using a method following the National Committee for Clinical Laboratory Standards (NCCLS) M27-A guidelines. Among the tested species, Candida glabrata exhibited the highest susceptibility to EGCg (MIC50, 0.5-1 microg/ml and MIC90, 1-2 microg/ml) compared favorably with FLCZ, although they were slightly less susceptible than to AMPH, 5FC, MCFG, ITCZ, and MCZ. Candida guilliemondii and Candida parapsilosis (MIC50, 1-4 microg/ml and MIC90, 2-16 microg/ml) were also susceptible to EGCg, although they appear to be slightly less susceptible to EGCg than C. glabrata and the other antifungal agents tested. Moreover, the susceptibility of Candida krusei strains (MIC50, 2 microg/ml and MIC90, 4-8 microg/ml) to EGCg was approximately 2- to 8-fold higher than those of 5FC and FLCZ. Our data indicate that EGCg can inhibit clinically pathogenic Candida species, although the concentrations of EGCg for antifungal susceptibility were slightly higher than those of tested antifungal agents on the whole. Based on these results, we suggest that EGCg may be effectively used as a possible agent or adjuvant for antifungal therapy in Candidiasis.     

Plasmodium falciparum: the fungal metabolite gliotoxin inhibits proteasome proteolytic activity and exerts a plasmodicidal effect on P. falciparum

The in vitro antimalarial activity of the fungal metabolite gliotoxin (GTX) was evaluated, and its mechanism of action was studied. GTX showed plasmodicidal activity against both Plasmodium falciparum chloroquine-resistant strain K-1 and chloroquine-susceptible strain FCR-3. GTX cytotoxicity was significantly lower against a normal liver cell line (Chang Liver cells). The intracellular reduced glutathione level of parasitized and of normal red blood cells was not affected by GTX treatment. However, GTX decreased the chymotrypsin-like activity of parasite proteasomes in a time-dependent manner. The results of this study indicate that GTX possesses plasmodicidal activity and that this effect is due to inhibition of parasite proteasome activity, suggesting that GTX may constitute a useful antimalarial therapy.     

Nitric oxide modulates elicitation of reflex swallowing from the pharynx in rats

The pharynx is very important for elicitation of reflex swallowing. The region of the pharynx is innervated by the pharyngeal branch of the glossopharyngeal nerve (GPN-ph). Nitric oxide (NO) plays an important role in various physiological functions. The purpose of this study is to investigate the contribution of NO to reflex swallowing evoked by electrical stimulation of the GPN-ph. Swallowing was evoked in urethane-anesthetized rats by application of repetitive electrical stimulation (10- to 20-microA amplitude, 10- to 20-Hz frequency, 1.0-ms duration) to the central cut end of the GPN-ph or superior laryngeal nerve. Swallowing was identified by electromyographic activity of the mylohyoid muscle. Latency to the first swallow and the interval between swallows were measured. Intravenous administration of N(G)-nitro-L-arginine (L-NNA, 0.6 mg/kg), a nonselective inhibitor of NO synthase (NOS), extremely prolonged latency to the first swallow and the interval between swallows evoked by the GPN-ph. Intraperitoneal administration of 7-nitroindazole (5.0 mg/kg), a selective inhibitor of neuronal NOS, significantly prolonged latency to the first swallow and the interval between swallows evoked by the GPN-ph. Administration of L-arginine (an NO donor, 500 mg/kg) and sodium nitroprusside (an NO releaser, 0.6 mg/kg) restored the suppression of swallowing induced by the NOS inhibitor. Superior laryngeal nerve-evoked swallowing was suppressed by administration of a higher dose of L-NNA (6.0 mg/kg). Swallowing evoked by water stimulation of the pharynx was also suppressed by L-NNA (0.6 mg/kg). These results suggest that NO plays an important role in signal processing for initiation of reflex swallowing from the pharynx.     

Nonspecific hydrophobic interactions of a repressor protein, PhaR, with poly[(R)-3-hydroxybutyrate] film studied with a quartz crystal microbalance

The gene expression for phasins (PhaP), which are predominantly polyhydroxyalkanoates (PHAs) granule-associated proteins, is regulated by a repressor protein of PhaR through the dual binding abilities of PhaR to the target DNAs and the granules. In this study, the binding functions of PhaR to poly[(R)-3-hydroxybutyrate] (P(3HB)) were investigated quantitatively by using a quartz crystal microbalance (QCM) technique. Adsorption of PhaR onto a melt-crystallized film of P(3HB) (cr-P(3HB)) was detected as a negative frequency shift of the QCM. The time course of the frequency changes observed for PhaR adsorption was composed of a quick frequency decrease at an initial stage and a subsequent slower frequency decrease for several hours, indicating multilayered adsorption of PhaR molecules onto cr-P(3HB). The initial rapid adsorption, which corresponds to direct adsorption of PhaR molecules onto a bare surface of cr-P(3HB), was a diffusion-controlled process. Strong interactions between PhaR and cr-P(3HB) were also observed as apparently irreversible adsorption. The comparative QCM measurement of PhaR adsorption onto various types of polymers with different aliphatic chemical structures revealed that PhaR was adsorbed onto the surfaces of polymers, including cr-P(3HB), mainly by nonspecific hydrophobic interactions. These results illustrate the high affinity and low specificity for adsorption of PhaR to P(3HB).     

RGS2 promotes formation of neurites by stimulating microtubule polymerization

Regulator of G-protein signaling (RGS) proteins interact with subunits of heterotrimeric G-proteins via the RGS domain and attenuate their activity by accelerating GTPase activity. RGS2, a member of the RGS family, regulates synaptic development via hereto unknown mechanism. In this study, we found that RGS2 directly interacted with tubulin via a short region at the N-terminus: amino acids 41–60. RGS2 enhanced microtubule polymerization in vitro, and the tubulin binding region was necessary and sufficient for this activity. In Vero cells, polymerization of microtubule was stimulated when peptides containing the tubulin binding region were microinjected. Immunocytochemical analysis showed that endogenous RGS2 was localized at the termini of neurites in differentiated PC12 cells. Over-expression of RGS2 enhanced the nerve growth factor-induced neurite outgrowth in PC12 cells, while specific knock-down of endogenous RGS2 suppressed the neurite outgrowth. These findings demonstrate that RGS2 contributes to the neuronal cell differentiation via regulation of microtubule dynamics.     

Spectroscopic characterization of a higher plant heme oxygenase isoform-1 from Glycine max (soybean)--coordination structure of the heme complex and catabolism of heme

Heme oxygenase converts heme into biliverdin, CO, and free iron. In plants, as well as in cyanobacteria, heme oxygenase plays a particular role in the biosynthesis of photoreceptive pigments, such as phytochromobilins and phycobilins, supplying biliverdin IX(alpha) as a direct synthetic resource. In this study, a higher plant heme oxygenase, GmHO-1, of Glycine max (soybean), was prepared to evaluate the molecular features of its heme complex, the enzymatic activity, and the mechanism of heme conversion. The similarity in the amino acid sequence between GmHO-1 and heme oxygenases from other biological species is low, and GmHO-1 binds heme with 1 : 1 stoichiometry at His30; this position does not correspond to the proximal histidine of other heme oxygenases in their sequence alignments. The heme bound to GmHO-1, in the ferric high-spin state, exhibits an acid-base transition and is converted to biliverdin IX(alpha) in the presence of NADPH/ferredoxin reductase/ferredoxin, or ascorbate. During the heme conversion, an intermediate with an absorption maximum different from that of typical verdoheme-heme oxygenase or CO-verdoheme-heme oxygenase complexes was observed and was extracted as a bis-imidazole complex; it was identified as verdoheme. A myoglobin mutant, H64L, with high CO affinity trapped CO produced during the heme degradation. Thus, the mechanism of heme degradation by GmHO-1 appears to be similar to that of known heme oxygenases, despite the low sequence homology. The heme conversion by GmHO-1 is as fast as that by SynHO-1 in the presence of NADPH/ferredoxin reductase/ferredoxin, thereby suggesting that the latter is the physiologic electron-donating system.     

Purification and characterization of a hemolysin-like protein, Sll1951, a nontoxic member of the RTX protein family from the Cyanobacterium Synechocystis sp. strain PCC 6803

The hemolysin-like protein (HLP) Sll1951, characterized by the GGXGXDXUX nonapeptide motif implicated in Ca(2+) binding, was purified from the glucose-tolerant strain (GT) of Synechocystis sp. strain PCC 6803. HLP was eluted at 560 kDa after gel filtration chromatography. Atomic absorption spectroscopy indicated that the protein bound calcium. The bound Ca(2+) was not chelated with EGTA; however, it was released after being heated at 100 degrees C for 1 min, and it rebound to the Ca(2+)-depleted protein at room temperature. The apparent HLP molecular mass increased to 1,000 kDa and reverted to 560 kDa during the release and rebinding of Ca(2+), respectively. The monomers of the respective forms appeared at 90 and 200 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. HLP showed no apparent hemolytic activity against sheep erythrocytes; however, a slight hemolytic activity was detected during the conformational change caused by the rebinding of Ca(2+). Immunoelectron microscopy using polyclonal antibodies against the 200-kDa monomer revealed that HLP is located in the cell surface layer. The localization and Ca(2+)-induced reversible conformational change suggest that HLP is a member of the repeat in toxin (RTX) protein family despite its latent and low toxicity. In some other cyanobacteria, RTX proteins are reported to be necessary for cell motility. However, the GT was immotile. Moreover, the motile wild-type strain did not express any HLP, suggesting that HLP is one of the factors involved in the elimination of motility in the GT. We concluded that the involvement of RTX protein in cyanobacterial cell motility is not a general feature.     

Identification of a Sed5-like SNARE gene LjSYP32-1 that contributes to nodule tissue formation of Lotus japonicus

We identified a Sed5-like clone LjSYP32-1 which contributes to nodule tissue formation and plant growth in Lotus japonicus. In the L. japonicus expressed sequence tag (EST) clone databases of Kazusa DNA Research Institute, another syntaxin-related clone (LjSYP32-2) was also detected, and the nucleotide and amino acid sequences of these two clone are very similar to each other. Real-time PCR and promoter analysis indicated that expression of LjSYP32-1 was dominant compared with LjSYP32-2 in the various plant organs. Promoter analysis and in situ hybridization revealed that LjSYP32-1 was expressed significantly in the inner cortex cell layer surrounding the infected zone of young nodules and in the meristem area of developing lateral root. To explore the function and physiological role of LjSYP32-1 in nodules and other plant organs, stable transformation lines of L. japonicus expressing either sense or antisense LjSYP32-1 were prepared. The antisense plants showed a significantly retarded plant growth phenotype, suggesting a role for LjSYP32-1 in supporting plant growth. In the same transgenic lines, the plants were capable of forming nodules, but the acetylene reduction activity was reduced by around 50% per plant. The nodules were much smaller and some nodules were fused to each other by sharing the inner cortex. The rate of occurrence of such irregular nodules was twice that observed in wild-type plants. The data suggest that LjSYP32-1 contributes to the support of plant growth and normal nodule tissue differentiation.     

n-Butanol induces depolymerization of microtubules in vivo and in vitro

The effects of butanol on microtubules (MTs) were examined by immunofluorescence microscopy. Fragmentation of cortical MTs was induced by n-butanol, but not by s- and t-butanols, in cultured tobacco BY-2 cells. Taxol prevented n-butanol-induced MT fragmentation. Fragmented cortical MTs were still attached to the inner face of the plasma membrane when n-butanol-treated protoplasts were ruptured on the slide glass. Moreover, MTs were depolymerized in the presence of n-butanol in vitro. Therefore, n-butanol is not only an activator of phospholipase D but also an effective MT-depolymerizing agent.