Immunohistochemistry and in situ hybridization of P-45017 alpha (17 alpha-hydroxylase/17,20-lyase).

Cytochrome P-45017 alpha catalyzes both 17 alpha-hydroxylation and 17,20-side-chain cleavage in steroidogenesis and lies at a key branch point in the pathways of steroid hormone biosynthesis. To obtain information on the precise localization of P-45017 alpha in swine testis, ovary, and adrenal, we undertook the simultaneous detection of P-45017 alpha mRNA and protein by combining immunohistochemistry with in situ hybridization. In situ hybridization was performed on 4% paraformaldehyde-fixed, paraffin-embedded sections by employing either a 39-base oligomer or a cDNA insert (1.7 KB) of porcine testis P-45017 alpha as DNA probe. Immunohistochemical study was performed by employing anti-P-45017 alpha. Hybridization signals were obtained in Leydig cells of the testis, theca interna of the ovarian follicle, and zona fasciculata reticularis cells of the adrenal cortex. Oligonucleotide probing yielded lower background signal than the cDNA probe. No specific signals were obtained in seminiferous tubules of the testis, medulla, and zona glomerulosa of the adrenal, and in membrana granulosa and interstitial cells of the ovary. Hybridization signals were obtained in the cells where immunoreactivity of the enzyme was observed by immunohistochemistry, except for some Leydig cells of the testis and theca interna cells of the ovary in which only immunoreactivity but not hybridization signal was observed. The present study provided detailed information about the precise cellular localization of P-45017 alpha expression at both the protein and mRNA levels in swine adrenal glands and gonads. This approach of simultaneous immunohistochemistry and in situ hybridization analysis of steroidogenic enzymes can be applied in the future to tissues exhibiting abnormal steroid metabolism and should contribute to a better understanding of steroidogenesis.     

Direct comparison of the pharmacodynamics of four antifungal drugs in a mouse model of disseminated candidiasis using microbiological assays of serum drug concentrations

The aim of this study was to compare the pharmacodynamics of the azole antifungal drugs fluconazole, itraconazole and ketoconazole, and the polyene antifungal amphotericin B, in a mouse model of disseminated Candida albicans infection. In order to directly compare effective serum concentrations of these antifungals, drug concentrations were assayed microbiologically by measuring inhibition of C. albicans mycelial growth (mMIC) in a mouse serum-based assay (serum antifungal titer). Efficacy in the mouse infection model was determined using an organ-based (kidney burden) endpoint. For all four drugs, the serum antifungal titers, 8 hr after administration of single doses of drugs at a range of drug concentrations, correlated closely with C. albicans kidney fungal burden in the mouse model. The results showed that determining serum antifungal titer may be used to accurately represent kidney fungal burden in a mouse model of disseminated candidiasis and allowed direct comparison of the pharmacodynamics of differing classes of antifungal drugs.     

A population genomics insight into the Mediterranean origins of wine yeast domestication

The domestication of the wine yeast Saccharomyces cerevisiae is thought to be contemporary with the development and expansion of viticulture along the Mediterranean basin. Until now, the unavailability of wild lineages prevented the identification of the closest wild relatives of wine yeasts. Here, we enlarge the collection of natural lineages and employ whole‐genome data of oak‐associated wild isolates to study a balanced number of anthropic and natural S. cerevisiae strains. We identified industrial variants and new geographically delimited populations, including a novel Mediterranean oak population. This population is the closest relative of the wine lineage as shown by a weak population structure and further supported by genomewide population analyses. A coalescent model considering partial isolation with asymmetrical migration, mostly from the wild group into the Wine group, and population growth, was found to be best supported by the data. Importantly, divergence time estimates between the two populations agree with historical evidence for winemaking. We show that three horizontally transmitted regions, previously described to contain genes relevant to wine fermentation, are present in the Wine group but not in the Mediterranean oak group. This represents a major discontinuity between the two populations and is likely to denote a domestication fingerprint in wine yeasts. Taken together, these results indicate that Mediterranean oaks harbour the wild genetic stock of domesticated wine yeasts.     

Distinct responses of cones and melanopsin-expressing retinal ganglion cells in the human electroretinogram

ABSTRACT: BACKGROUND: The discovery of the novel photoreceptor, melanopsin-expressing retinal ganglion cells (mRGCs), has raised researchers' interest in photoreceptive tasks performed by the mRGC, especially in non-image-forming visual functions. In a prior study, we investigated the mRGC response to light stimuli independent of rods and cones with the four-primary illumination system, which modulates stimulus levels to the mRGC and cones independently, and mRGC baseline responses were recorded in the electroretinogram (ERG). METHODS: In the present study, we used the same illumination system to compare independent responses of the mRGC and cones in five subjects (mean +/- SD age, 23.0 +/- 1.7 years). The ERG waveforms were examined as direct measurements of responses of the mRGCs and cones to stimulation (250 msec). Implicit times (the time taken to peaks) and peak values from 30 stimuli given to each subject were analyzed. RESULTS: Two distinct positive peaks appeared in the mRGC response, approximately 80 msec after the onset of the stimuli and 30 msec after their offset, while no such peaks appeared in the cone response. The response to the mRGC stimulus was significantly higher than that to the cone stimulus at ~80 msec (p < 0.05) and tended to be higher than the cone stimulus at ~280 msec (p = 0.08). CONCLUSIONS: Implicit time of the first peak was much longer than that to the b-wave and this delay might reflect mRGC's sluggish responses. This is the first report of amplitudes and implicit time in the ERG from the response of the mRGC that is independent of rods and cones and obtained using the four-primary illumination system.     

Efficient establishment of pure cultures of yellow chanterelle Cantharellus anzutake from ectomycorrhizal root tips,and morphological characteristics of ectomycorrhizae and cultured mycelium

Species of fleshy yellow Cantharellus are known as chanterelles, which are among the most popular wild edible mycorrhizal mushrooms in the world. However, pure culture isolates of Cantharellus are rare. We report an efficient isolation technique of the Japanese golden chanterelle, Cantharellus anzutake, from its ectomycorrhizal root tips. Field-sampled fresh ectomycorrhizal root tips of C. anzutake on various hosts such as pines, spruce, and oaks were vortexed with 0.005% Tween 80 solution, surface sterilized with 1% calcium hypochlorite solution, rinsed with sterilized distilled water, and placed on modified Norkrans’ C (MNC) agar plate medium. Most ectomycorrhizal root tips of C. anzutake produced yellowish mycelial colonies within a few months. In contrast, tissue isolation from basidiomata provided limited cultures of C. anzutake but much contamination of bacteria and molds, even on media that contained antibiotics. The established C. anzutake cultures had clamp connections on the hyphae and contained intracellular oily droplets. These cultured isolates were identified as C. anzutake by sequence analysis of the rRNA internal transcribed spacer (ITS) region and translation elongation factor EF1-alpha (tef-1) genes.     

Social relationship and hair cortisol level in captive male chimpanzees (<Emphasis Type="Italic">Pan troglodytes</Emphasis>)

Understanding how social relationships affect long-term stress is important because stress has a profound impact on the welfare of animals and social relationships often exert a strong influence on their stress responses. The purpose of this study was to investigate the relationship between social behaviors and long-term stress levels as assessed by hair cortisol (HC) concentration. The subjects were 11 chimpanzees living in an all-male group (divided into two sub-groups) in Kumamoto Sanctuary, Kyoto University, Japan. Behavioral data were collected between December 2014 and March 2015. The total observation time was 129 h. Hair samples were collected in late March and early April 2015, and cortisol was extracted from the hair and measured with enzyme immunoassay. The hair growth rate was estimated to be 1.33 ± 0.06 cm/month. The results revealed that there was a positive correlation between the rate of receiving aggression and HC levels. We also found a significant negative correlation between the balance between giving and receiving grooming (grooming balance index: GBI), which was calculated by subtracting the rate with which grooming is given from that with which it is received, and the rate of receiving aggression and between the GBI and HC levels. Thus, individuals receiving higher levels of aggression also tended to give grooming for relatively long periods compared to the time they were being groomed. In contrast, the rate of initiating aggression did not have a relationship with either HC levels or any measure of social grooming. We did not find social buffering effects, as there was no correlation between mutual social grooming and HC levels. These results show that not only aggressive interactions but also overall social situations in which animals do not have balanced relationships with others might result in the long-term elevation of cortisol levels in captive male chimpanzees.     

Electron microscopic examination of the orexin immunoreactivity in the dorsal raphe nucleus

The ultrastructure and the synaptic relationships of the orexin-A-like immunoreactive fibers in the dorsal raphe nucleus were examined with an immunoelectron microscopic method. At the electron microscopic level, most of the immunoreactive fibers, a varicosity appearance at the light microscopic level, were found as axon terminals. The large dense-cored vesicles contained in the immunoreactive axon terminals were the most intensely immunostained organellae. These axon terminals were often found to make synapses. While the axo-dendritic synapses were usually asymmetric in appearance, the axo-somatic synapses were symmetric. Orexin-A-like immunoreactive processes with no synaptic vesicles were also found. These processes often received asymmetric synapses. With less frequency, the synapses were found between the orexin-like immunoreactive processes. The results suggest that the orexin peptides are stored in the large dense-cored vesicles; the orexin-containing fibers may have influences on the physiological activities of the dorsal raphe nucleus through direct synaptic relationships.