Cloning and characterization of seven cDNAs for hyperosmolarity-responsive (HOR) genes of Saccharomyces cerevisiae

Yeast cells can respond and adapt to osmotic stress. In our attempt to clarify the molecular mechanisms of cellular responses to osmotic stress, we cloned seven cDNAs for hyperosmolarity-responsive (HOR) genes from Saccharomyces cerevisiae by a differential screening method. Structural analysis of the clones revealed that those designated HOR1, HORS, HOR4, HOR5 and HOR6 encoded glycerol-3-phosphate dehydrogenase (Gpd1p), glucokinase (Glklp), hexose transporter (Hxtlp), heat-shock protein 12 (Hsp12p) and Na⁺, K⁺, Li⁺-ATPase (Enalp), respectively. HOR2 and HOR7 corresponded to novel genes. Gpdlp is a key enzyme in the synthesis of glycerol, which is a major osmoprotectant in S. cerevisiae. Cloning of HOR1/GPD1 as a HOR gene indicates that the accumulation of glycerol in yeast cells under hyperosmotic stress is, at least in part, caused by an increase in the level of GPDH protein. We performed a series of Northern blot analyses using HOR cDNAs as probes and RNAs prepared from cells grown under various conditions and from various mutant cells. The results suggested that all the HOR genes are regulated by common signal transduction pathways. However, the fact that they exhibited certain distinct responses indicated that they might also be regulated by specific pathways in addition to the common pathways. Ca²⁺ seemed to be involved in the signaling systems. In addition, Hog1p, one of the MAP kinases in yeast, appeared to be involved in the regulation of expression of HOR genes, although its function seemed to be insufficient for the overall regulation of expression of these genes.     

Non-invasive diagnosis of cutaneous leishmaniasis by the direct boil loop-mediated isothermal amplification method and MinION™ nanopore sequencing

Cutaneous leishmaniasis (CL) is gaining attention as a public health problem. We present two cases of CL imported from Syria and Venezuela in Japan. We diagnosed them as CL non-invasively by the direct boil loop-mediated isothermal amplification method and an innovative sequencing method using the MinION? sequencer. This report demonstrates that our procedure could be useful for the diagnosis of CL in both clinical and epidemiological settings.     

The Immunodetection of the Abnormal Isoform of Prion Protein

Transmissible spongiform encephalopathies such as scrapie in sheep and goats, Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cattle, are neurodegenerative disorders. A proposed causative agent for these diseases is an infectious protein, the so called prion. An abnormal isoform of prion protein (PrPSc) can be detected according to the prion propagation method used. As PrPSc appears to constitute the main, if not the only, infectious entity its detection for the diagnosis of prion diseases is important. Immunodetection methods for PrPSc analysis are popular tools for diagnosis and research studies. In this paper, a review of the present knowledge concerning immunodetection is presented and the enhancement of the immunoreactivity of antisera to mouse and hamster prion protein peptides using the techniques of Western blotting and immunohistochemistry is summarized.     

Toll-like Receptors as a Target of Food-derived Anti-inflammatory Compounds

Toll-like receptors (TLRs) play a key role in linking pathogen recognition with the induction of innate immunity. They have been implicated in the pathogenesis of chronic inflammatory diseases, representing potential targets for prevention/treatment. Vegetable-rich diets are associated with the reduced risk of several inflammatory disorders. In the present study, based on an extensive screening of vegetable extracts for TLR-inhibiting activity in HEK293 cells co-expressing TLR with the NF-κB reporter gene, we found cabbage and onion extracts to be the richest sources of a TLR signaling inhibitor. To identify the active substances, we performed activity-guiding separation of the principal inhibitors and identified 3-methylsulfinylpropyl isothiocyanate (iberin) from the cabbage and quercetin and quercetin 4′-O-β-glucoside from the onion, among which iberin showed the most potent inhibitory effect. It was revealed that iberin specifically acted on the dimerization step of TLRs in the TLR signaling pathway. To gain insight into the inhibitory mechanism of TLR dimerization, we developed a novel probe combining an isothiocyanate-reactive group and an alkyne functionality for click chemistry and detected the probe bound to the TLRs in living cells, suggesting that iberin disrupts dimerization of the TLRs via covalent binding. Furthermore, we designed a variety of iberin analogues and found that the inhibition potency was influenced by the oxidation state of the sulfur. Modeling studies of the iberin analogues showed that the oxidation state of sulfur might influence the global shape of the isothiocyanates. These findings establish the TLR dimerization step as a target of food-derived anti-inflammatory compounds.     

Effects of acorn masting on population dynamics of three forest-dwelling rodent species in Hokkaido,Japan

The effects of the abundance of acorns of the oak, Quercus crispula, on the population dynamics of three rodent species (Apodemus speciosus, A. argenteus, and Clethrionomys rufocanus) were analyzed using time series data (1992–2006). The data were obtained in a forest in northern Hokkaido, Japan, by live trapping rodents and directly counting acorns on the ground. Apodemus speciosus generally increased in abundance following acorn masting. However, the clear effect of acorn abundance was not detected for the other two rodent species. Acorns of Q. crispula contain tannins, which potentially have detrimental effects on herbivores. Apodemus speciosus may reduce the damage caused by acorn tannins with tannin-binding salivary proteins and tannase-producing bacteria, whereas such physiological tolerance to tannins is not known in the other two rodent species. The differences in the effects of acorns between the three species may be due to differences in their physiological tolerance to tannins.     

A new method for improving the enantioselectivity of lipase-catalyzed hydrolysis in organic solvent containing a small amount of water in the presence of metal ions

The hydrolysis of racemic butyl 2-(4-substituted phenoxy)propionates having various substituents catalyzed by lipase MY from Candida rugosa was achieved in di-isopropyl ether containing 0.75% (v/v) of 2.4 M LiCl or 1.2 M MgCl₂ aqueous solution. Water molecules hydrated to the metal ion in isopropyl ether acted as a nucleophile to cause the hydrolysis of these esters as with water alone. Metal ions used significantly enhanced their enantioselectivities by 100-fold or above, as compared with the ordinary reaction media.     

Photosynthetic activities of a thermophilic blue-green alga

Photosynthetic activities of a thermophilic blue-green alga,a species of Synechococcus, were studied with special referenceto its growth at high temperatures. A rapid algal growth occurredin the temperature range between 50 and 60?C, showing the maximumrate, six doublings per day, at about 57?C. Photosynthetic oxygenevolution and methyl viologen photoreduction in the cells werealso active at high temperatures and the optimum temperaturesfor these activities agreed with that of the algal growth. Thegrowth and photosynthetic activities were very low at room temperatureand irreversibly inactivated at temperatures above 60?C. The thylakoid membranes isolated from the alga were also photochemicallyactive at high temperatures. The membranes mediated ferricyanidephotoreduction coupled with a stoichiometric oxygen evolutionat a rate comparable to that of photosynthetic oxygen evolutionin the cells. The optimum temperature for the reaction was ashigh as 50?C. The membranes also showed a photosystem I-mediatedreaction at high temperatures. These observations indicate thatthe thylakoid membranes are intrinsically thermophilic in thisorganism. Thus the growth of the alga at high temperatures canbe well correlated to thermophilic properties of the photosyntheticapparatus. (Received February 20, 1978; )     

A novel strategy to design highly specific PCR primers based on the stability and uniqueness of 3'-end subsequences

MOTIVATION: In contrast with conventional PCR using a pair of specific primers, some applications utilize a single unique primer in combination with a common primer, thereby relying solely on the former for specificity. These applications include rapid amplification of cDNA ends (RACE), adaptor-tagged competitive PCR (ATAC-PCR), PCR-mediated genome walking and so forth. Since the primers designed by conventional methods often fail to work in these applications, an improved strategy is required, particularly, for a large-scale analysis. RESULTS: Based on the structure of 'off-target' products in the ATAC-PCR, we reasoned that the practical determinant of the specificity of primers may not be the uniqueness of entire sequence but that of the shortest 3'-end subsequence that exceeds a threshold of duplex stability. We termed such a subsequence as a 'specificity-determining subsequence' (SDSS) and developed a simple algorithm to predict the performance of the primer: the algorithm identifies the SDSS of each primer and examines its uniqueness in the target genome. The primers designed using this algorithm worked much better than those designed using a conventional method in both ATAC-PCR and 5'-RACE experiments. Thus, the algorithm will be generally useful for improving various PCR-based applications.     

Human recombinant stem cell factor promotes spermatogonial proliferation,but not meiosis initiation in organ culture of newt testis fragments

We previously showed that mammalian FSH stimulates the proliferation of newt spermatogonia and induces their differentiation into primary spermatocytes in vitro. In the current study, to examine a possibility that stem cell factor (SCF) is involved in the proliferation of newt spermatogonia and/or their differentiation into primary spermatocytes, human recombinant SCF (rhSCF) was added to organ culture of testicular fragments. rhSCF was found to stimulate the spermatogonial proliferation and the spermatogonia progressed to the seventh generation that is the penultimate stage before primary spermatocyte stage. However, the spermatogonia did not differentiate into primary spermatocytes, but instead died of apoptosis. These results indicate that rhSCF promotes the proliferation of newt spermatogonia, but not the initiation of meiosis.     

Time course alterations of myocardial endothelin-1 production during the formation of exercise training-induced cardiac hypertrophy

Endothelin (ET)-1 is produced by endothelial cells and cardiac myocytes. ET-1 has positive inotropic and chronotropic effects on the heart and causes myocardial cell hypertrophy. Exercise training induces a physiologic cardiac hypertrophy. To study whether myocardial ET-1 is involved in the formation of exercise training-induced cardiac hypertrophy, we investigated time-course alterations of myocardial ET-1 gene expression and ET-1 peptide level in the heart of rats during a formative process of exercise training-induced cardiac hypertrophy. We used the hearts of rats that had been exercise-trained for 4 weeks (4WT) or 8 weeks (8WT) and sedentary control rats for 4 weeks (4WC) or 8 weeks (8WC). Exercise-trained rats performed treadmill running for 5 days/week (60 mins/day). Left ventricular mass index and wall thickness and stroke volume index, measured using echocardiography, in the 8WT group were significantly greater than in the 8WC group, although there were no differences between the 4WC and 4WT groups in these parameters. These results indicated that the 8WT rats developed physiologic cardiac hypertrophy, whereas the 4WT rats did not yet have cardiac hypertrophy. Myocardial ET-1 gene expression and tissue ET-1 concentration in the heart were significantly higher in the 8WT group than in the 8WC group, whereas these values did not differ between the 4WC and 4WT groups. The present study suggests that an alternation of myocardial ET-1 production corresponds with the formation of exercise training-induced cardiac hypertrophy. Therefore, the exercise training-induced change in myocardial ET-1 production may participate in a mechanism of exercise training-induced cardiac adaptation (e.g., cardiac hypertrophy).