Fecal steroid metabolites and reproductive monitoring in a female Tsushima leopard cat (Prionailurus bengalensis euptilurus)

Although the Tsushima leopard cat (Prionailurus bengalensis euptilurus) is one of the most endangered mammals in Japan, its reproductive physiology and endocrinology have been not elucidated. The objective was to establish the non-invasive monitoring of reproductive endocrinology in a female Tsushima leopard cat and to identify the types of fecal reproductive steroid metabolites in this species. Fecal concentrations of estrogen and progestin were determined by enzyme immunoassays, from 60 d before to 60 d after the last copulation, during three pregnancies. Fecal estrogen metabolite concentrations were increased before/around the mating period and after mid-pregnancy. Fecal progestin metabolite concentrations increased after the last copulation and remained high during pregnancy. The gestation period was 65.0 ± 0.6 d (mean ± SD). Fecal extracts were separated by high-performance liquid chromatography for identification of fecal metabolites. Fecal estrogens were identified as estradiol-17β and estrone. Fecal progestins during pregnancy contained 5α-reduced pregnanes: 5α-pregnan-3α-ol-20-one, 5α-pregnan-3β-ol-20-one and 5α-pregnan-3,20-dione, and nonmetabolized progesterone was barely detected in feces. In conclusion, measurement of fecal estrogen and progestin metabolites was effective for noninvasive reproductive monitoring in the Tsushima leopard cat. An immunoassay for fecal estradiol-17β concentrations seemed useful to monitor follicular activity, whereas an immunoassay with high cross reactivity for 5α-reduced pregnanes was useful to monitor ovarian luteal activity and pregnancy.     

Synthesis and analytics of 2,2,3,4,4-d5-19-nor-5alpha-androsterone--an internal standard in doping analysis

A short and efficient synthesis of pentadeuterated 2,2,3,4,4-d5-19-nor-5alpha-androsterone 7 starting from 19-norandrost-4-ene-3,17-dione 1 by a d1-L-Selectride mediated stereo- and regioselective reduction of the 3-keto group is presented. The use of compound 7 as internal standard for the detection of anabolic steroids via mass spectrometric techniques such as gas chromatography-mass spectrometry (GC-MS) is discussed.     

Antisense inhibition of the plastidial glucose-6-phosphate/phosphate translocator in Vicia seeds shifts cellular differentiation and promotes protein storage

The glucose-6-phosphate/phosphate translocator (GPT) acts as an importer of carbon into the plastid. Despite the potential importance of GPT for storage in crop seeds, its regulatory role in biosynthetic pathways that are active during seed development is poorly understood. We have isolated GPT1 from Vicia narbonensis and studied its role in seed development using a transgenic approach based on the seed-specific legumin promoter LeB4. GPT1 is highly expressed in vegetative sink tissues, flowers and young seeds. In the embryo, localized upregulation of GPT1 at the onset of storage coincides with the onset of starch accumulation. Embryos of transgenic plants expressing antisense GPT1 showed a significant reduction (up to 55%) in the specific transport rate of glucose-6-phosphate as determined using proteoliposomes prepared from embryos. Furthermore, amyloplasts developed later and were smaller in size, while the expression of genes encoding plastid-specific translocators and proteins involved in starch biosynthesis was decreased. Metabolite analysis and stable isotope labelling demonstrated that starch biosynthesis was also reduced, although storage protein biosynthesis increased. This metabolic shift was characterized by upregulation of genes related to nitrogen uptake and protein storage, morphological variation of the protein-storing vacuoles, and a crude protein content of mature seeds of transgenics that was up to 30% higher than in wild-type. These findings provide evidence that (1) the prevailing level of GPT1 abundance/activity is rate-limiting for the synthesis of starch in developing seeds, (2) GPT1 exerts a controlling function on assimilate partitioning into storage protein, and (3) GPT1 is essential for the differentiation of embryonic plastids and seed maturation.     

Determination of free and total S-phenylmercapturic acid by HPLC/MS/MS in the biological monitoring of benzene exposure

Urinary S-phenylmercapturic acid (SPMA) is a biomarker suggested by the American Conference of Governmental Industrial Hygienists (ACGIH) for assessing occupational exposure to benzene. A possible cause of the miscorrelation between environmental monitoring and biological monitoring for benzene exposure, which many authors complain about, is the existence of a urinary metabolite that turns into SPMA by acid hydrolysis. Forty urine samples were tested to determine which concentration value would correspond to the ACGIH Biological Exposure Index (BEI) of 25 µg g^-1 creatinine if exposure assessment was based on the determination of SPMA after quantitative hydrolysis of its precursor. An aliquot of each sample was hydrolysed with 9 M H₂SO₄, a second one was brought to pH 2 and a third one was used as it was (free SPMA). SPMA was determined by high-performance liquid chromatography/tandem mass spectrometric technique (HPLC/MS/MS) using an internal standard. The analytical method was validated in the range 0.5-50 µg l^-1. The average SPMA in pH 2 samples is 45-60% of the total, while free SPMA varies from 1% to 66%. The hydrolysis of pre-SPMA reduces the likelihood of variability in the results by reducing pH differences in urine samples and increasing the amount of measured SPMA. The BEI limit value would be about 50 µg g^-1 creatinine.     

Isolation and characterization of antagonistic Bacillus subtilis strains from the avocado rhizoplane displaying biocontrol activity

AIM: This study was undertaken to isolate Bacillus subtilis strains with biological activity against soil-borne phytopathogenic fungi from the avocado rhizoplane. METHODS AND RESULTS: A collection of 905 bacterial isolates obtained from the rhizoplane of healthy avocado trees, contains 277 gram-positive isolates. From these gram-positive isolates, four strains, PCL1605, PCL1608, PCL1610 and PCL1612, identified as B. subtilis, were selected on the basis of their antifungal activity against diverse soil-borne phytopathogenic fungi. Analysis of the antifungal compounds involved in their antagonistic activity showed that these strains produced hydrolytic enzymes such as glucanases or proteases and the antibiotic lipopeptides surfactin, fengycin, and/or iturin A. In biocontrol trials using the pathosystems tomato/Fusarium oxysporum f.sp. radicis-lycopersici and avocado/Rosellinia necatrix, two B. subtilis strains, PCL1608 and PCL1612, both producing iturin A, exhibited the highest biocontrol and colonization capabilities. CONCLUSIONS: Diverse antagonistic B. subtilis strains isolated from healthy avocado rhizoplanes have shown promising biocontrol abilities, which are closely linked with the production of antifungal lipopeptides and good colonization aptitudes. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the few reports dealing with isolation and characterization of B. subtilis strains with biocontrol activity against the common soil-borne phytopathogenic fungi F. oxysporum f.sp. radicis-lycopersici and R. necatrix.     

Biosynthesis of antibiotic prodiginines in the marine bacterium Hahella chejuensis KCTC 2396

AIMS: Hahella chejuensis KCTC 2396 produces red pigments, showing antibacterial and algicidal activities. The main red-coloured metabolite of the pigments was identified as antibiotic prodigiosin. With the expectation that the red pigments are a mixture of a series of close relatives, the aim of the present study is to detect new antibiotic prodigiosin analogues and to analyse the biosynthetic pattern for prodiginines in KCTC 2396. METHODS AND RESULTS: Except prodigiosin, the other constituents in the red pigments were confirmed as well-known dipyrrolyldipyrromethene prodigiosin, norprodigiosin, and undecylprodiginine. Additionally, four new prodigiosin analogues, each of which was distinguished from prodigiosin (C(5)), according to differences in alkyl chain length (C(3)-C(7)), were detected in small quantities by liquid chromatography mass spectrometry/mass spectrometry spectroscopy. Owing to the presence of a cytotoxic methoxy group, it is expected that all the new prodigiosin analogues are bioactive. CONCLUSIONS: Four characterized prodiginines, including prodigiosin and four new prodigiosin analogues are produced in different ratio in KCTC 2396. All of the prodiginines possess a common linear tripyrrolyl structure and a cytotoxic methoxy group. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows for the first time that KCTC 2396 is able to produce antibiotic prodigiosin, undecylprodiginine and new prodigiosin analogues in a mixture of pigments. It is also shown that KCTC 2396 possesses a novel system for the simultaneous production of multiple prodiginines in a single micro-organism.