Trial of support treatment with human chorionic gonadotrophin in the luteal phase after treatment with buserelin and human menopausal gonadotrophin in women taking part in an in vitro fertilisation programme.

OBJECTIVE--To evaluate the effect of support with human chorionic gonadotrophin in the luteal phase in women taking part in an in vitro fertilisation programme after buserelin and human menopausal gonadotrophin were used to hyperstimulate their ovaries. DESIGN--Controlled group comparison. SETTING--Outpatient department of a private hospital. PATIENTS--115 Women with indications for in vitro fertilisation, all of whom had at least one embryo transferred. INTERVENTIONS--After suppression of the pituitary with buserelin the ovaries of all the women were stimulated with human menopausal gonadotrophin on day 4 of the luteal phase. Human chorionic gonadotrophin (10,000 IU) was given to induce ovulation, and oocytes were recovered 34 hours later. Embryos were transferred 46 to 48 hours after insemination. Women who had received the 10,000 IU of human chorionic gonadotrophin on a date that was an uneven number (n = 61) were allocated to receive support doses of 2500 IU human chorionic gonadotrophin three and six days after that date. The remaining 54 women did not receive hormonal support. END POINT--Determination of the rates of pregnancy. MEASUREMENTS and main results--Support with human chorionic gonadotrophin did not significantly alter the progesterone or oestradiol concentrations in the early or mid-luteal phase. The mean (range) progesterone concentrations in the late luteal phase in women who did not become pregnant were, however, significantly higher in those who received support (16(9-110) nmol/l nu 8(4-46) nmol/l), and the luteal phase was significantly longer in this group (14 days nu 12 days). The rate of pregnancy was significantly higher in the women who received support than in those who did not (25/61 nu 8/54). CONCLUSIONS--When buserelin and human menopausal gonadotrophin are used to hyperstimulate ovaries support with human chorionic gonadotrophin in the luteal phase has a beneficial effect on in vitro fertilisation.     

Construction of Escherichia coli amber suppressor tRNA genes. III. Determination of tRNA specificity

Using synthetic oligonucleotides, we have constructed a collection of Escherichia coli amber suppressor tRNA genes. In order to determine their specificities, these tRNAs were each used to suppress an amber (UAG) nonsense mutation in the E. coli dihydrofolate reductase gene fol. The mutant proteins were purified and subjected to N-terminal sequence analysis to determine which amino acid had been inserted by the suppressor tRNAs at the position of the amber codon. The suppressors can be classified into three groups on the basis of the protein sequence information. Class I suppressors, tRNA(CUAAla2), tRNA(CUAGly1), tRNA(CUAHisA), tRNA(CUALys) and tRNA(CUAProH), inserted the predicted amino acid. The class II suppressors, tRNA(CUAGluA), tRNA(CUAGly2) and tRNA(CUAIle1) were either partially or predominantly mischarged by the glutamine aminoacyl tRNA synthetase. The class III suppressors, tRNA(CUAArg), tRNA(CUAAspM), tRNA(CUAIle2), tRNA(CUAThr2), tRNA(CUAMet(m)) and tRNA(CUAVal) inserted predominantly lysine.     

Monoclonal Antibodies Allow Precipitation of Esterasic but Not Peptidasic Activities Associated with Butyrylcholinesterase

Commercially available and affinity-purified butyrylcholinesterases isolated from human serum were examined for their esterasic activity and their ability to hydrolyze various neuropeptides, including neurotensin, substance P, and leucine-enkephalin. The three pools that displayed the lowest esterasic activities were shown to hydrolyze neurotensin with the same HPLC degradative pattern. By contrast, noticeable qualitative and quantitative discrepancies were observed when hydrolyses of substance P and leucine-enkephalin by these three butyrylcholinesterase pools were studied. The pool that exhibited the highest esterasic activity appeared to be homogeneously constituted by 90- and 180-kDa protein bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and was totally unable to hydrolyze these three neuropeptides. This suggested that the three other butyrylcholinesterase preparations could be contaminated by exogenous peptidases. This was confirmed by means of three distinct monoclonal antibodies directed toward human serum butyrylcholinesterase. The three IgG-purified fractions precipitated the esterasic activity, whereas they failed to precipitate the neuropeptide-hydrolyzing activities whatever the substrate examined. Altogether, these results demonstrate that peptidases associated with butyrylcholinesterase are contaminating enzymes that cannot be considered as intrinsic activities of this enzyme.     

The 31P NMR visibility of ATP in perfused rat liver remains about 90%, unaffected by changes of metabolic state.

The 31P NMR visibility of ATP of the perfused rat liver was tested over a wide range of metabolic conditions, including normoxic and hypoxic perfusions, fructose loads, and various intervals of normothermic ischemia, for both ad libitum fed and 24-h fasted rats. The 31P NMR signal of ATP was compared to the concentration of ATP determined by enzymatic assays on liver biopsies performed at the end of NMR acquisition. In a first series of experiments, the NMR resonance of intracellular ATP was quantitated in absolute terms by applying the 1H NMR water signal as internal reference: during normoxic and hypoxic perfusions, a constant amount of ATP (0.43 +/- 0.19 mM, mean +/- SD), approximately 12% of the cellular ATP, is not detected by NMR. Nevertheless, there is a high correlation (slope = 0.96 +/- 0.09; r2 = 0.93) between the measurements of ATP by 31P NMR spectroscopy and by biochemical analysis. In a second series of experiments, there was a highly significant correlation between the NMR and analytical biochemical measurements of ATP for whole range of metabolic states, i.e., fructose loads (1.0-10 mM) and various intervals of normothermic ischemia (ranging from 2 to 12 min), indicating unchanged ATP visibility. Thus, as opposed to the studies of Murphy et al. [Murphy, E., et al. (1988) Biochemistry 27, 526-528], it is concluded that ATP at 37 degrees C remains almost entirely visible in the perfused rat liver, also during ischemia.     

Polyamines and polyamino acids regulation of cytosolic tyrosine protein (Tyr-P) kinase from human erythrocytes.

In vitro regulation of cytosolic tyrosine protein (Tyr-P) kinase from human erythrocytes by polyamines, polyamino acids, negative charged compounds or by insulin using angiotensin II or poly (Glu-Tyr)4:1 as substrates was studied. All the three polyamines, putrescine (Put), spermidine (Spd) and spermine (Spm) stimulated the Tyr-P kinase activity in a dose dependent manner. Spm stimulated Tyr-P kinase activity higher than Put and Spd whether the substrate was angiotension II or poly (Glu-Tyr)4:1. Polyamino acids (polyornithine, polyarginine, polyglutamic acid and polyaspartic acid) did not affect significantly the Tyr-P kinase phosphorylation except polylysine which significantly stimulated the Tyr-P kinase activity. Negative charged compounds (chondroitin sulfate A, B and C) and heparin inhibited the Tyr-P kinase phosphorylation while insulin did not influence the enzyme activity in the presence of either substrates.     

Selection and biochemical studies of pyrimidine-requiring mutants of Lactobacillus plantarum

Mutagenesis and mutant enrichment in Lactobacillus plantarum, a lactic acid bacterium used in silage, sauerkraut and sausage fabrication, were studied. In optimal conditions, auxotrophic mutants were obtained that permitted investigation of the de novo pyrimidine biosynthesis. Uracil-requiring mutants were characterized for their enzymatic defect, in aspartate transcarbamylase (ATCase), dihydro-orotase (DHOase), dihydro-orotate dehydrogenase (DHOdehase), orotidine monophosphate pyrophosphorylase (OMPppase) or in orotidine monophosphate decarboxylase (OMPdecase). The five enzymatic activities are totally repressed by uracil in the growth medium.     

Arbitrary primer polymerase chain reaction, a powerful method to identify Bacillus thuringiensis serovars and strains.

Arbitrary primer polymerase chain reaction technology has been applied to the identification of commercial strains of Bacillus thuringiensis by using total DNAs extracted from single bacterial colonies as templates. Characteristic DNA banding patterns can be readily and reproducibly obtained by agarose gel electrophoresis. This method has been used to distinguish commercial products containing B. thuringiensis serovar kurstaki (3a3b). When a single primer was used this method was capable of producing discriminating DNA fingerprints for 33 known serovars. Differentiation from the closely related species Bacillus cereus is also readily achieved. This technique should prove to be a powerful tool for identification and discrimination of individual B. thuringiensis strains.     

Characterization of Xanthomonas campestris Pathovars by rRNA Gene Restriction Patterns

Genomic DNA of 191 strains of the family Pseudomonadaceae, including 187 strains of the genus Xanthomonas, was cleaved by EcoRI endonuclease. After hybridization of Southern transfer blots with 2-acetylamino-fluorene-labelled Escherichia coli 16+23S rRNA probe, 27 different patterns were obtained. The strains are clearly distinguishable at the genus, species, and pathovar levels. The variability of the rRNA gene restriction patterns was determined for four pathovars of Xanthomonas campestris species. The 16 strains of X. campestris pv. begoniae analyzed gave only one pattern. The variability of rRNA gene restriction patterns of X. campestris pv. manihotis strains could be related to ecotypes. In contrast, the variability of patterns observed for X. campestris pv. malvacearum was not correlated with pathogenicity or with the geographical origins of the strains. The highest degree of variability of DNA fingerprints was observed within X. campestris pv. dieffenbachiae, which is pathogenic to several hosts of the Araceae family. In this case, variability was related to both host plant and pathogenicity.     

The distribution of coastal fish eDNA sequences in the Anthropocene

Aim

Coastal fishes have a fundamental role in marine ecosystem functioning and contributions to people, but face increasing threats due to climate change, habitat degradation and overexploitation. The extent to which human pressures are impacting coastal fish biodiversity in comparison with geographic and environmental factors at large spatial scale is still under scrutiny. Here, we took advantage of environmental DNA (eDNA) metabarcoding to investigate the relationship between fish biodiversity, including taxonomic and genetic components, and environmental but also socio-economic factors.

Location

Tropical, temperate and polar coastal areas.

Time period

Present day.

Major taxa studied

Marine fishes.

Methods

We analysed fish eDNA in 263 stations (samples) in 68 sites distributed across polar, temperate and tropical regions. We modelled the effect of environmental, geographic and socio-economic factors on α- and β-diversity. We then computed the partial effect of each factor on several fish biodiversity components using taxonomic molecular units (MOTU) and genetic sequences. We also investigated the relationship between fish genetic α- and β-diversity measured from our barcodes, and phylogenetic but also functional diversity.

Results

We show that fish eDNA MOTU and sequence α- and β-diversity have the strongest correlation with environmental factors on coastal ecosystems worldwide. However, our models also reveal a negative correlation between biodiversity and human dependence on marine ecosystems. In areas with high dependence, diversity of all fish, cryptobenthic fish and large fish MOTUs declined steeply. Finally, we show that a sequence diversity index, accounting for genetic distance between pairs of MOTUs, within and between communities, is a reliable proxy of phylogenetic and functional diversity.

Main conclusions

Together, our results demonstrate that short eDNA sequences can be used to assess climate and direct human impacts on marine biodiversity at large scale in the Anthropocene and can further be extended to investigate biodiversity in its phylogenetic and functional dimensions.