Beta amino acid-modified and fluorescently labelled kisspeptin analogues with potent KISS1R activity.

Kisspeptin analogues with improved metabolic stability may represent important ligands in the study of the kisspeptin/KISS1R system and have therapeutic potential. In this paper we assess the activity of known and novel kisspeptin analogues utilising a dual luciferase reporter assay in KISS1R-transfected HEK293T cells. In general terms the results reflect the outcomes of other assay formats and a number of potent agonists were identified among the analogues, including ß(2) -hTyr-modified and fluorescently labelled forms. We also showed, by assaying kisspeptin in the presence of protease inhibitors, that proteolysis of kisspeptin activity within the reporter assay itself may diminish the agonist outputs. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Cloning and functional expression of novel cholesterol transporters ABCG1 and ABCG4 in gonadotropin-releasing hormone neurons of the tilapia.

In addition to reproduction, gonadotropin-releasing hormone (GnRH) has been postulated to control cholesterol metabolism via cholesterol transport, which is carried out partly by the members of ATP-binding cassette (ABC) transporters G1 (ABCG1) and G4 (ABCG4). However, there is yet to be evidence demonstrating the relationship between these transporters with reference to GnRH neurons. In the present study, we cloned two ABCG1 messenger RNA (mRNA) variants and one ABCG4 mRNA and examined their expression in the brain including GnRH neurons (GnRH1, GnRH2, and GnRH3) in the cichlid tilapia (Oreochromis niloticus). Comparison of nucleotide sequences of the tilapia ABCG1 and ABCG4 with that of other fish species showed that both of these genes are evolutionarily conserved among fishes. ABCG1 and ABCG4 were shown to have high mRNA expressions in the CNS, pituitary, and gonads. In the brain, real-time polymerase chain reaction (PCR) showed that ABCG4 mRNA was higher than ABCG1a in all brain regions including the olfactory bulb (ABCG1=13.34, ABCG4=6796.35; P<0.001), dorsal telencephalon (ABCG1=8.64, ABCG4=10149.13; P=0.001), optic tectum (ABCG1=22.12, ABCG4=13931.04; P<0.01), cerebellum (ABCG1=8.68, ABCG4=12382.90; P<0.01), and preoptic area-midbrain-hypothalamus (ABCG1=21.36, ABCG4=13255.41; P=0.001). Similarly, although ABCG1 mRNA level is much higher in the pituitary compared with the brain, it was still significantly lower compared with ABCG4 (ABCG1=337.73, ABCG4=1157.87; P=0.01). The differential pattern of expression of ABCG1 and ABCG4 in the brain versus pituitary suggests that the two transporters are regulated by different mechanisms. Furthermore, ABCG1 and ABCG4 mRNA expressions were found in all three types of laser-captured GnRH neurons with highly similar percentage of expressions, suggesting that cholesterol efflux from GnRH neurons may require heterodimerization of both ABCG1 and ABCG4.

Prolactin receptor, growth hormone receptor, and putative somatolactin receptor in Mozambique tilapia: tissue specific expression and differential regulation by salinity and fasting.

In fish, pituitary growth hormone family peptide hormones (growth hormone, GH; prolactin, PRL; somatolactin, SL) regulate essential physiological functions including osmoregulation, growth, and metabolism. Teleost GH family hormones have both differential and overlapping effects, which are mediated by plasma membrane receptors. A PRL receptor (PRLR) and two putative GH receptors (GHR1 and GHR2) have been identified in several teleost species. Recent phylogenetic analyses and binding studies suggest that GHR1 is a receptor for SL. However, no studies have compared the tissue distribution and physiological regulation of all three receptors. We sequenced GHR2 from the liver of the Mozambique tilapia (Oreochromis mossambicus), developed quantitative real-time PCR assays for the three receptors, and assessed their tissue distribution and regulation by salinity and fasting. PRLR was highly expressed in the gill, kidney, and intestine, consistent with the osmoregulatory functions of PRL. PRLR expression was very low in the liver. GHR2 was most highly expressed in the muscle, followed by heart, testis, and liver, consistent with this being a GH receptor with functions in growth and metabolism. GHR1 was most highly expressed in fat, liver, and muscle, suggesting a metabolic function. GHR1 expression was also high in skin, consistent with a function of SL in chromatophore regulation. These findings support the hypothesis that GHR1 is a receptor for SL. In a comparison of freshwater (FW)- and seawater (SW)-adapted tilapia, plasma PRL was strongly elevated in FW, whereas plasma GH was slightly elevated in SW. PRLR expression was reduced in the gill in SW, consistent with PRL's function in freshwater adaptation. GHR2 was elevated in the kidney in FW, and correlated negatively with plasma GH, whereas GHR1 was elevated in the gill in SW. Plasma IGF-I, but not GH, was reduced by 4 weeks of fasting. Transcript levels of GHR1 and GHR2 were elevated by fasting in the muscle. However, liver levels of GHR1 and GHR2 transcripts, and liver and muscle levels of IGF-I transcripts were unaffected by fasting. These results clearly indicate tissue specific expression and differential physiological regulation of GH family receptors in the tilapia.

Determination of vancomycin in human serum by micellar electrokinetic capillary chromatography with direct sample injection.

BACKGROUND: Vancomycin (VCM) has a bacteriostatic effect on gram-positive bacteria such as the methicillin-resistant Staphylococcus aureus. METHODS: A new assay for measuring vancomycin concentration by micellar electrokinetic capillary chromatography using direct serum injection was developed. A borate buffer (pH 10.0) containing 100 mmol/l sodium dodecyl sulfate was used as an electrophoresis buffer, and the detection was at 210 nm. The migration time of vancomycin was approximately 7 min. RESULTS: The linearity was from 0 to 100 microg/ml, with the limit of detection of 1.0 microg/ml (S/N=3). The within-run CV was 3.99-5.53%, and the recovery rate was 91-103% for a concentration range of 6.5-45.5 microg/ml. The between-day CV was 6.76% at 22.2 microg/ml. There was no interference from 32 other antibiotics. The correlation coefficient between the assay and fluorescence polarization immunoassay and direct injection HPLC was 0.982 and 0.985, respectively. The assay required no sample preparation of serum and used only microquantities of an electrophoresis buffer and samples. CONCLUSIONS: This assay is cost-effective and suitable for routine clinical use.

[Antimicrobial susceptibility pattern and epidemiological investigation of the clinical isolates of Pseudomonas aeruginosa in 1999and 2000].

During 1999-2000, a total of 1,215 isolates of Pseudomonas aeruginosa (P. aeruginosa) were analyzed by wards, type of clinical specimens and serotypes. Antimicrobial susceptibility was also examined for 10 agents (PIPC, CAZ, LMOX, IPM-CS, GM, AMK, MINO, LVFX, NFLX, and ST) with criteria by NCCLS (M7-A5). Sputum (33%) was most common source of P. aeruginosa followed by urine (17) and wound discharge (14%). According to wards, the isolation rate at the critical care unit was the highest (40%) in 1999 but decreased to 14% in 2000. In the isolates from inpatients (1999, 2000) the resistance rate was 96.8-98.9% for MINO and ST, 23.5-30.8% for IPM-CS, 27.2-28.9% for LMOX, 12.5-20.1% for GM, and 6.7-11.8% for NFLX. Comparison between the two years showed increases in isolates resistant to GM (p<0.01) and NFLX (p<0.05) but a decrease in IPM-CS resistant isolates (p<0.01). According to serotypes, the E and G type were most frequently observed (242 isolates each, 21.8%) followed in order by F type (127 isolates, 11%) and the B type (124 isolates, 11%). The incidence of the F type was high compared with other medical institutions. There was a reduction in the rates of susceptibility of serotype E isolates to PIPC (74.4%) and GM (60.3%), and of serotype F isolates to IPM-CS (52%). Multidrug-resistant P. aeruginosa (MDR-PSA) that were resistant to beta-lactams, aminoglycosides, and new quinolons accounted for 3%.

[Detection of astrovirus RNA from sewage works, seawater and native oysters samples in Chiba City, Japan using reverse transcription-polymerase chain reaction].

Through a year from April, 1999 to March, 2000, 20 samples, which consisted of raw sewage (2), chlorine-treated sewage (2), seawater (10) and naturally grown oysters (6), were collected monthly both from the sewage works at Mihama-ku, Chiba City and at a yacht harbor in Chiba City Bay, Japan. Astrovirus RNA were detected by reverse transcription-polymerase chain reaction (RT-PCR) and was typed by direct sequencing. Astrovirus positive products were detected from 9 samples (raw sewage; 1/2, chlorine-treated sewage; 2/2, seawater; 5/10 and oysters; 1/6) collected in April, 1999. In May, positive products were detected from 4 samples (raw sewage; 2/2 and seawater; 2/10). In June, only 1 positive product was detected from raw sewage. The number of positive samples showed a tendency to decrease and no positive products were detected from samples collected in July, 1999 to January, 2000. After that period, positive products were again detected from 3 samples (raw sewage; 1/2, chlorine-treated sewage; 2/2) collected in February, 2000. In March, the number of positive samples showed the peak and positive products were detected from 12 samples (raw sewage; 2/2, chlorine-treated sewage; 2/2, seawater; 7/10 and oysters: 1/6). Astrovirus positive products detected in April, May, June, July, 1999 and February, 2000 were classified into type 1 or 2 by sequencing, whereas in March, 2000 were type 1, 2, 3, 6 and 7.

[Quantitative determination of lipoprotein by agarose gel electrophoresis and enzymatic staining of cholesterol and triglyceride: effects of age and gender].

290 normal duplicate samples (152 males and 138 females), were subjected to lipoprotein analysis by agarose gel electrophoresis to yield HDL, VLDL and LDL fractions which were respectively stained with Cholesterol and Triglyceride reagents. HDL-Cholesterol were significantly higher in female than male, and VLDL-TG were significantly higher in male than female. Assay C.V.'s varied from 0.96 to 5.75 for cholesterol fractions and 2.00 to 4.34 for triglyceride fractions. Comparison of electrophoretic HDL-Cholesterol and LDL-Cholesterol concentrations with the results of a direct method (HDL-EX and LDL-EX, Denka Seiken) gave correlation coefficients of 0.967 and 0.952 respectively. This method is simple, rapid and can provide the simultaneous assessment of the cholesterol and triglyceride component in each Lipoprotein fraction. Additionally, the method is useful for evaluating lipoprotein assays.

Quantitative analysis of fushi tarazu factor 1 homolog messenger ribonucleic acids in the pituitary of salmon at different prespawning stages.

Steroidogenic factor 1 (SF-1) or Ad4BP is a member of the fushi tarazu factor 1 (FTZ-F1) family and an orphan nuclear receptor that plays an important role in the hypothalamus-pituitary-gonadal axis and the adrenal cortex. Although its critical role in the differentiation of adrenals, gonads, and pituitary gonadotropes has been well demonstrated, regulatory function of SF-1 during sexual maturation is yet to be examined. To investigate the potential role of SF-1 in sexual maturation, expression of two salmon FTZ-F1 homolog genes, sFF1-I and sFF1-II, was examined in the pituitaries of chum and sockeye salmons, using specific and sensitive RNase protection assays. Only sFF1-I mRNA was found in the pituitary and other organs, such as the ovary, spleen, liver, brain, and skeletal muscle. In chum salmon during upstream migration from the bay to the hatchery, the level of sFF1-I mRNA in the male fish was increased on the midway in the river, where the levels of gonadotropin alpha- and II beta-subunit mRNAs were increased. In maturing sockeye salmon, the expression of the sFF1-I gene was elevated in the mature male fish, but the administration of GnRH analog did not further enhance the expression. These results indicate that sFF1-I gene expression in the pituitary is upregulated in maturing salmon, and this upregulation may not depend on GnRH.

Rapid serum vancomycin assay by high-performance liquid chromatography using a semipermeable surface packing material column.

A new isocratic high-performance liquid chromatographic (HPLC) assay has been developed for vancomycin that uses direct injection of microquantities of serum into a separation column filled with octyl-C(8) silica support that has a semipermeable surface. A mixture of disodium hydrogen phosphate buffer (pH 7.0) and acetonitrile is used as the mobile phase, and vancomycin is directly detected at 240 nm. The minimum limit of detection was 0.5 microg/ml at a signal-to-noise ratio of 3:1. Linearity was established from 0 to 100 microg/ml. The coefficient of variation for within-run reproducibility was 1.1-2.7% for a concentration range of 2.9-52.5 microg/ml; for day-to-day reproducibility it was 4.0% and 3.1% for a concentration range of 5.8-26.4 microg/ml, and the recovery rate was 94-105%. There was no interference from 41 antibiotics or other drugs currently in use. The correlation coefficient between the fluorescence polarization immunoassay (x) and this method (y) was 0.995 with a linear equation, y = 1.06x - 0.924. This method is simple, rapid, and provides an economical quantification of serum vancomycin.

Effects of gonadotropin-releasing hormone analog on expression of genes encoding the growth hormone/prolactin/somatolactin family and a pituitary-specific transcription factor in the pituitaries of prespawning sockeye salmon.

Gonadotropin-releasing hormone (GnRH) is a possible secretagogue of growth hormone (GH) and somatolactin (SL) in teleosts. Effects of GnRH on the levels of pituitary mRNAs encoding GH, prolactin (PRL), and SL were therefore examined in prespawning sockeye salmon (Oncorhynchus nerka). A capsule of GnRH analog (GnRHa) was implanted into the dorsal muscle of maturing sockeye salmon for 3 weeks. The levels of hormonal mRNAs were then determined by a quantitative dot blot analysis using single-stranded sense DNA of the same sequence of mRNA as the standard. Further, we analyzed effects of GnRHa on expression of the genes encoding pituitary-specific transcription factor (Pit-1/GHF-1). Relative levels of Pit-1/GHF-1 mRNAs were estimated by Northern blot analysis, which showed specific 2- and 3-kb bands of mRNAs. GnRHa significantly increased the level of SL mRNA in the males, but not in the females, compared to the control fish. It did not induce significant increases in the levels of GH and PRL mRNAs in both the males and the females. The levels of Pit-1/GHF-1 mRNAs in the control males tended to be higher than those in the initial controls, so that GnRHa might not be effective in enhancing expression of Pit-1/GHF-1 gene, except for the level of 3-kb Pit-1/GHF-1 mRNA in the females treated with 150 microg GnRHa. The pattern of changes in the levels of Pit-1/GHF-1 mRNAs were similar to those of GH and PRL mRNAs in both the males and the females and to that of SL mRNA in the females. These results indicate that, in prespawning sockeye salmon, GnRH can stimulate SL gene expression, but probably not through the Pit-1/GHF-1-dependent system.

Changes in the levels of mRNAs for GH/prolactin/somatolactin family and Pit-1/GHF-1 in the pituitaries of pre-spawning chum salmon.

Changes in the levels of pituitary mRNAs encoding GH, prolactin (PRL) and somatolactin (SL) were determined in pre-spawning chum salmon (Oncorhynchus keta) caught at a few key points along their homing pathway in 1994 and 1995. Furthermore, we analyzed relationships between expression of pituitary-specific POU homeodomain transcription factor (Pit-1/GHF-1) and GH/PRL/SL family genes. In 1994, seawater (SW) fish and matured fresh-water (FW) fish were sequentially captured at two points along their homing pathway, the coast and the hatchery. In addition to these two points, maturing FW fish were captured at the intermediate of the two points in 1995. The levels of hormonal mRNAs were determined by a quantitative dot blot analysis using single-stranded sense DNA as the standard. Relative levels of Pit-1/GHF-1 mRNAs were estimated by Northern blot analysis. In 1994, the levels of GH/PRL/SL family mRNAs except for PRL mRNA in the male FW fish were 1.8-4 times higher than those in the SW fish. In 1995, the level of PRL mRNA was somewhat sharply elevated in the maturing FW fish soon after entry into the FW environment, while that of SL mRNA was gradually increased during upstream migration from the coast to the hatchery. The levels of 3 kb Pit-1/GHF-1 mRNA in the FW fish were higher than those in the SW fish in both 1994 and 1995. The present results indicate that expression of genes for the GH/PRL/SL family and Pit-1/GHF-1 is coincidentally enhanced in homing chum salmon. Moreover, the present study suggests that expression of the SL gene is elevated with sexual maturation, whereas that of PRL gene is elevated with osmotic change during the final stages of spawning migration.

[Detection of HAV, SRSV and astrovirus genomes from native oysters in Chiba City, Japan].

SRSV, astrovirus in tvirus and HAV genomes were detected by RT-PCR in naturally grown oysters (total 112) collected in Chiba City bay, Japan, through a year from April, 1997 to March, 1998 and genogroup was typed by sequencing. SRSV positive products were detected by RT-PCR from 28 out of 112 oysters and all of them were grouped into G-II by sequencing. The highest incidence was observed in February, 1998. Furthermore, astrovirus positive products were detected sporadically from 15 out of 112 samples and the highest incidence was observed in January, 1998. On the other hand, HAV was detected from only 2 out of 112 and no adenovirus positive product was detected. The results indicated that both SRSV and astrovirus were predominantly distributed into naturally grown oysters in the winter season. SRSV and astrovirus seem to contribute mainly to the food-born outbreaks of gastroenteritis occurred by eating contaminated oysters as causative agents.

[An outbreak caused by hepatitis A virus in an institution for the mentally handicapped--detection of hepatitis A virus RNA using CTAB method].

The prevalence of hepatitis A virus (HAV) antibody in people has decreased from year to year in Japan. A sequential outbreak occurred in an institution for the mentally handicapped people in Chiba City in the summer of 1995. Eight people were infected including 7 residents and one staff member. We tested to detect antigen in fecal samples by ELISA and PCR for early diagnosis for hepatitis A infection. Four sera and 5 feces were obtained from 5 patients between 2 and 8 days after the onset of symptoms. The anti-HAV IgM was found to be positive in 4 sera examined. The HAV antigen was detected in 3 out of 5 feces using ELISA. An existence of inhibitor in 2 negative specimens against the ELISA was suggested by the recovery test of added antigen. HAV RNA was extracted by CTAB method from feces and detected in 4 our of 5 specimens in PCR amplification and in all of 5 specimens in nested PCR amplification. The sequence of PCR products in the P1/P2 junction of the HAV genome revealed that the virus associated with the outbreak belongs to HAV subgenotype IA. HAV RNA was detected in ELISA negative specimens and in the specimen from a patient 2 days after the onset of symptoms using PCR amplification by CTAB method. These results indicate that PCR amplification was useful for the early diagnosis of hepatitis A infection.

Studies on Methicillin-Resistant Staphylococcus aureus Bacteremia Due to Laboratory Medical Analysis.

We encountered 64 patients with methicillin-resistant Staphylococcus aureus (MRSA) bacteremia between April 1993 and March 1994. Mean patient age was 54 years. There were 46 males and 18 females. Underlying diseases mainly consisted of traffic accident (10 patients), valvular heart disease (5 patients), chronic renal failure (5 patients), leukemia (5 patients), pneumonia (3 patients), and malignant lymphoma (3 patients). The common clinical laboratory findings of MRSA bacteremia included decreses in total protein, albumin and hemoglobin as well as increases in white blood cells (neutrophils) and C reactive protein. In particular, an increase in C reactive protein by 10 mg/dl or more may be useful for diagnosing bacteremia. Laboratory findings were compared between surviving and non-surviving patients. There were significant differences in albumin, cholesterol, bilirubin, creatinine, and CRP. In 18 patients (28.1%), bacteremia was caused by infection due to contamination of central venous catheters. Since medical treatment with intra-vascular devices may cause bacteremia, sufficient caution is needed.

Distinct promoter sequences of two precursor genes for salmon gonadotropin-releasing hormone in masu salmon.

Two types of genes encode salmon gonadotropin-releasing hormone (sGnRH), which is thought to act on both sexual maturation and reproductive behavior, in salmonids. We characterized the two sGnRH genes (sGnRH-I and -II) and their upstream regions in masu salmon, Oncorhynchus masou, since such information is a prerequisite for molecular approaches to salmon reproduction. The two genes have similar exon-intron structures composed of four exons and three introns. Sequence analyses of the two genes showed that coding regions are highly conserved, but upstream regions are distinctively divergent. In the upstream regions, only the sGnRH-II gene has a large palindromic sequence, which has been proposed to be involved in control of transcription via estrogen receptors. In contrast, the sGnRH-I gene is missing the large palindromic sequence, but has three distinct palindromes in the upstream region. These results may suggest divergent transcription regulatory mechanisms between the two sGnRH genes in masu salmon. The differences in the upstream regions of sGnRH genes in Atlantic salmon (Salmo salar), sockeye salmon (Oncorhynchus nerka) and masu salmon are discussed with respect to the evolution of sGnRH genes in salmonid fish.

A self-setting TTCP-DCPD apatite cement for release of vancomycin.

Vancomycin (VCM), a methiciline-cefem resistant Staphylococcus aureus (MRSA)-specific antibiotic, was incorporated in a self-setting tetracalcium phosphate (TTCP)-dicalcium phosphate dihydrate (DCPD) apatite cement that hardened isothermally into a hydroxyapatite (HAP) phase with crystallinity similar to that of host bone. Effective release of VCM into PBS lasted for 2 weeks from cements containing 1% VCM and for longer than 9 weeks from cements containing 5% VCM. The rate of release of VCM differed between cements with different crystallinities as well as between the two dissolution media, PBS and simulated body fluid. Mean concentration of VCM in the bone marrow tissue released from cements containing 5% VCM was 20 times the minimum inhibitory concentration 3 weeks after implantation in bone. Direct contact with new bone was observed with the cements containing 1% VCM. Slow delivery of VCM from a self-setting TTCP-DCPD apatite cement with low crystallinity could be used to treat MRSA osteomyelitis.