A beat-by-beat, on-line, cardiovascular index, CARDEAN, to assess circulatory responses to surgery: a randomized clinical trial during spine surgery.

Automated assessment of circulatory response to surgical stimuli is unsolved. Would detection of cardiac baroreflex inhibition assess adequacy of intra-operative anti-nociception upon incision, as performed on-line on a beat-by-beat basis by a cardiovascular index, CARDEAN™? 18 ASA I-II patients undergoing spinal disc repair were studied, in a prospective randomized single-blinded trial (observational study). During infusion of propofol to maintain bispectral index between 40 and 60, patients were allocated to receive an effect site target-controlled infusion of remifentanil at Ce = 2 or 4 ng ml(-1). Upon incision and during surgery, circulatory response was assessed using beat-by-beat measurements of minor hypertension and tachycardia to give a cardiovascular index, CARDEAN, scaled between 0 and 100. Upon skin incision, CARDEAN increased in the remifentanil Ce = 2 ng ml(-1) group (n = 7, P < 0.05), while it did not increase in the remifentanil Ce = 4 ng ml(-1) group (n = 7, P = 0.18). During surgery, retrospectively, CARDEAN > 60 was associated with tachycardia and hypertension (P (k) = 0.81 ± 0.10). Changes in CARDEAN appeared linked to adequacy of anti-nociception.

All-trans retinoic acid targets gastric cancer stem cells and inhibits patient-derived gastric carcinoma tumor growth.

Gastric carcinoma is the third leading cause of cancer-related death worldwide. This cancer, most of the time metastatic, is essentially treated by surgery associated with conventional chemotherapy, and has a poor prognosis. The existence of cancer stem cells (CSC) expressing CD44 and a high aldehyde dehydrogenase (ALDH) activity has recently been demonstrated in gastric carcinoma and has opened new perspectives to develop targeted therapy. In this study, we evaluated the effects of all-trans-retinoic acid (ATRA) on CSCs in human gastric carcinoma. ATRA effects were evaluated on the proliferation and tumorigenic properties of gastric carcinoma cells from patient-derived tumors and cell lines in conventional 2D cultures, in 3D culture systems (tumorsphere assay) and in mouse xenograft models. ATRA inhibited both tumorspheres initiation and growth in vitro, which was associated with a cell-cycle arrest through the upregulation of cyclin-dependent kinase (CDK) inhibitors and the downregulation of cell-cycle progression activators. More importantly, ATRA downregulated the expression of the CSC markers CD44 and ALDH as well as stemness genes such as Klf4 and Sox2 and induced differentiation of tumorspheres. Finally, 2 weeks of daily ATRA treatment were sufficient to inhibit gastric tumor progression in vivo, which was associated with a decrease in CD44, ALDH1, Ki67 and PCNA expression in the remaining tumor cells. Administration of ATRA appears to be a potent strategy to efficiently inhibit tumor growth and more importantly to target gastric CSCs in both intestinal and diffuse types of gastric carcinoma.

LH and melatonin secretion patterns in pubertal boys.

Four normal pubertal boys had plasma LH and melatonin measured at 20-minute intervals for 24-hours. All four subjects showed a significant augmentation of LH and melatonin during nocturnal sleep. There was also a significant correlation between the LH and melatonin levels (P less than 0.001). These data indicate that the peripheral concentrations of melatonin which occur during sleep are insufficient to prevent spontaneous LH secretion during puberty.

Luteinizing hormone and follicle-stimulating hormone secretory dynamics in Turner's syndrome.

In eight teenage patients with Turner's syndrome, LH and FSH were measured at 20-min intervals for 24 h. The 24-h mean LH and FSH levels ranged from 20.2-70.5 mIU/ml and 60.4-229 mIU/ml, respectively. There was a significant positive correlation between the individual LH and FSH levels in the eight patients; the common correlation coefficient was 0.449 (P less than 0.001). The 24-h mean estradiol level was measurable in only two of the patients and the 24-h mean testosterone level for the eight patients was 0.10 ng/ml. The mean LH concentration during sleep was significantly higher (P less than .01) than during waking. The mean FSH concentration during sleep was also significantly higher (P less than 0.05) than during waking. The LH and FSH peak levels after LRH were significantly correlated with the 24-h mean LH (r = 0.918; P less than 0.01) and FSH concentrations (r = 0.754; P less than 0.05), respectively.

Characterization of a SNF1 homologue from the phytopathogenic fungus Sclerotinia sclerotiorum.

In yeast, the SNF1 gene product is essential for the release of catabolic repression. We report the isolation and characterization of an SNF1 homologue from the necrotrophic pathogen Sclerotinia sclerotiorum. Ss snf1 encodes a 765-amino-acid protein in which the catalytic domain has an overall identity with the yeast proteins varying from 55 to 76% while the C-terminal half of Ss SNF1 has a weak homology of about 20% with the yeast sequences. Reverse transcription-polymerase chain reaction showed that its transcripts were weakly and constitutively expressed in planta and in vitro regardless of the nature of the carbon sources and of the presence or absence of glucose. Expression of Ss snf1 in yeast cells allowed the snf1 mutant cells to utilize sucrose, raffinose or glycerol for growth while expression of the Ss snf1 catalytic domain did not restore growth on raffinose or glycerol. Ss SNF1 is structurally homologous to Snf1p, suggesting that the interactions between the kinase and the accessory subunits to activate the enzymatic complex are conserved in fungi.

Cloning and sequence analysis of a polygalacturonase-encoding gene from the phytopathogenic fungus Sclerotinia sclerotiorum.

The phytopathogenic fungus Sclerotinia sclerotiorum produces a number of extra-cellular pectin-degrading enzymes. We have cloned and determined the complete sequence of a gene (pg1) encoding an endopolygalacturonase (PG1). The coding region consists of a non-interrupted 1143-bp open reading frame. S. sclerotiorum pg1 was compared to other fungal PG-encoding genes. Basic transcription control sequences were identified in the 5' non-coding region. The deduced amino acid (aa) sequence (380 aa) of the enzyme is compared to seven fungal PG sequences and shows a high level of identity (41.5 to 59.8%). Predicted secondary structures were compared, revealing a similar protein organization most probably in antiparallel beta sheets. Hybridization analysis using a pg1 0.65-kb BamHI fragment as a probe allowed the identification of seven different recombinant phages from a genomic library. Analysis of the hybridizing restriction fragments suggests that PG-encoding genes are organized as a family.

Sequence of the phosphoenolpyruvate carboxykinase-encoding cDNA from the rumen anaerobic fungus Neocallimastix frontalis: comparison of the amino acid sequence with animals and yeast.

The nucleotide sequence of the cDNA of the phosphoenolpyruvate carboxykinase-encoding gene from the fungus Neocallimastix frontalis, was determined. The deduced amino acid sequence (608 residues) and the predicted protein structure were compared to their counterparts in animals and yeast. Catalytic regions (substrate-binding site and nucleotide-binding domains) are highly conserved among fungal and animal organisms. The yeast sequence showed no similarity to the fungal sequence.

The glucose repressor CRE1 from Sclerotinia sclerotiorum is functionally related to CREA from Aspergillus nidulans but not to the Mig proteins from Saccharomyces cerevisiae.

We isolated the putative glucose repressor gene cre1 from the phytopathogenic fungus Sclerotinia sclerotiorum. cre1 encodes a 429 amino acid protein 59% similar to the carbon catabolite repressor CREA from Aspergillus nidulans. In addition to the overall amino acid sequence relatedness between CRE1 and CREA proteins, cre1 can functionally complement the A. nidulans creAd30 mutation as assessed by repression of the alcohol dehydrogenase I gene expression. The CREI region carrying the two zinc fingers is also very similar to the DNA binding domains of the Saccharomyces cerevisiae glucose repressors Mig1p and Mig2p. Despite the presence in the CRE1 protein of several motifs involved in the regulation of Miglp activity, cre1 cannot complement mig deficiencies in S. cerevisiae. These data suggest that glucose repression pathways may have evolved differently in yeasts and filamentous fungi.

Age- and mental health-related circadian rhythms of plasma levels of melatonin, prolactin, luteinizing hormone and follicle-stimulating hormone in man.

Circadian changes in plasma levels of melatonin, prolactin, LH and FSH were studied in four groups: seven healthy young men, six elderly men, six elderly women and six elderly demented patients (two men and four women). The daily activities of the subjects were synchronous and blood samples were taken every 4 h. The 24-h mean concentrations of prolactin in plasma were the same in all groups, whereas those of LH and FSh were twice as high in the elderly as in the young men and eight and 23 times higher respectively in the elderly women. The 24-h mean plasma levels of melatonin in the elderly were half those in the young, but were not influenced by the sex or mental condition of the subjects. A statistically significant circadian rhythm for melatonin was defined in the four groups, for prolactin in all groups except the elderly men and for LH only in the demented patients and in the young men. No circadian rhythm could be detected for FSH in any of the four groups. The acrophases of melatonin and prolactin ranged between 02.30 and 04.00 h, those of LH (when a rhythm was validated) clustered around 01.00 h. The circadian rhythms of plasma levels of melatonin, prolactin and LH are not modified in old age nor in dementia. A positive correlation has been demonstrated in young men between melatonin and LH and between melatonin and prolactin, but no such correlation could be found in the elderly.

Changes in smoking and alcohol consumption during pregnancy: a population-based study in a rural area.

Changes in smoking and drinking behavior during pregnancy and factors influencing these changes were studied in a typical rural county. Using birth certificates and mailed questionnaires, information was obtained from 255 married women residents of Callaway County, Missouri, who gave birth in a one-year period. The women were much more likely to drink alcohol than to smoke before pregnancy (48.6 versus 28.2%, P less than .01). There was a significant decrease in both smoking and drinking during pregnancy, though the women were much more likely to modify drinking behavior than smoking behavior. Of the women who drank alcohol before pregnancy, 53.2% stopped alcohol consumption completely during pregnancy, while only 16.7% of smokers stopped smoking during pregnancy (P less than .001). Women cited fear for the infant's health as an important factor underlying the decision to decrease these behaviors more often than they did advice from doctor, family, friends, or media, or adverse physical effects of tobacco or alcohol. It was very difficult to predict changes in smoking and drinking behavior during pregnancy on the basis of demographic and behavioral characteristics such as age, income, education, attendance of childbirth classes, desirability of pregnancy, and method of infant feeding.

Purification and characterization of an extracellular beta-xylosidase from the rumen anaerobic fungus Neocallimastix frontalis.

The purification of beta-xylosidase (beta-D-xyloside xylohydrolase, EC 3.2.1.37) from Neocallimastix frontalis was performed by ammonium sulphate precipitation, ion exchange chromatography, gel filtration and preparative isoelectric focusing. The enzyme had a molecular mass of 180,000 Da, an isoelectric point at pH 4.35 and catalysed the hydrolysis of p-nitrophenyl-beta-D-xylopyranoside optimally at pH 6.5 and 35 degrees C with a Km of 0.33 mg ml-1. The enzymatic activity was strongly increased by the presence of Ca2+, Mn2+, Zn2+, Co2+ or Mg2+ and completely inhibited by Hg2+ and p-chloromercuribenzoate. The purified protein also had a low level of xylanase activity.

A 34-kilodalton polypeptide is associated with 1,3-beta-glucan synthase activity from the fungus Saprolegnia monoica.

Three major polypeptides of 34, 48 and 50 kDa which appear to copurify with 1,3-beta-glucan synthase activity were isolated by glycerol gradient centrifugation of Chaps-solubilized proteins from the fungus Saprolegnia monoica. The antiserum produced against the 34-kDa polypeptide revealed by protein immunoblotting that this polypeptide copurified with 1,3-beta-glucan synthase during enzyme purification. This antiserum adsorbs the enzymatic activity as well as the 48- and 50-kDa polypeptides. These results indicate that the 34-kDa peptide is a component of the multisubunit protein complex involved in 1,3-beta-glucan synthase activity.

Isolation and expression of a nitrogen regulatory gene, nmc, of Penicillium roqueforti.

The nmc gene, encoding a global nitrogen regulator, has been cloned and characterized from Penicillium roqueforti, a fungus used in the dairy industry. The deduced amino acid sequence predicts a protein of 860 amino acids in length whose zinc finger DNA binding domain is at least 94% identical to those of the homologous fungal proteins. Northern blot analysis showed that nmc expression is induced by nitrogen starvation and not repressed by variation of the external pH.

Molecular cloning of genes from the rumen anaerobic fungus Neocallimastix frontalis: expression during hydrolase induction.

Glycoside and polysaccharide hydrolase production by the rumen anaerobic fungus, Neocalimastix frontalis is induced by the presence of crystalline cellulose. A differential screening of a cDNA library was used to isolate DNA sequences transcribed at high levels under growth conditions which induce enzyme production. Seven clones were isolated that preferentially hybridized to the induced cDNA probe versus the non-induced cDNA probe. Southern analysis showed that a cDNA clone (118) hybridized to a DNA probe encoding part of the exo-cellobiohydrolase I (CBH I) gene of Trichoderma reesei. Northern analysis demonstrated that the cDNA 118 was transcribed to yield a 2.1 kb RNA. This transcript was induced in the presence of cellulose.

Purification and characterization of two endopolygalacturonases secreted during the early stages of the saprophytic growth of Sclerotinia sclerotiorum.

Two endopolygalacturonases (endo-PGs) secreted at the early stage of cultures of Sclerotinia sclerotiorum grown on polygalacturonate medium, were purified to apparent homogeneity, using ion exchange chromatography. They are glycoproteins of an apparent weight of 42 and 41.5 kDa and a pI of 4.8. The two purified isoforms found in early cultures were not detected in late cultures. Purification of the isoforms secreted at different stages of growth revealed that the increase of polygalacturonase activity during the culture corresponds to a sequential production of enzymes and to the successive replacement of isoforms by new enzymes.

Multiplicity and expression of xylanases in the rumen fungus Neocallimastix frontalis.

The time course production of xylanolytic enzymes by the rumen anaerobic fungus Neocallimastix frontalis was studied during growth on different carbon sources and revealed using isoelectric focusing and immunoblotting. A constant low level of endoxylanase expression was observed in glucose medium. A high level of xylanase activity was detected in methyl glucoside medium corresponding to the induction of new isoforms which were repressed by the presence of glucose. beta-Xylosidases were constitutively produced at a high level and remained mainly associated to the fungal cells. Polyclonal antibodies raised against the endoxylanases XYLI and XYLII revealed that XYLI was secreted to the different culture media showing a characteristic pattern of constitutive expression, while anti-XYLII recognized several polypeptides larger than XYLII indicating the production of multiple antigenically related enzymes during growth on the inducing substrate.

aspS encoding an unusual aspartyl protease from Sclerotinia sclerotiorum is expressed during phytopathogenesis.

The gene aspS encoding an aspartyl protease has been cloned from Sclerotinia sclerotiorum by screening a genomic library with a PCR-amplified fragment of the gene. The open reading frame of 1368 bp interrupted by one intron would encode a preproprotein of 435 amino acids. The catalytic aspartyl residues characteristic of aspartyl proteases are conserved; however, the active-site motif (DSG) in the N-terminal lobe is unusual in that Ser replaced Thr used in the active-site motif (DTG) of the C-terminal lobe and in all other fungal aspartyl proteases. RT-PCR revealed that aspS expression in axenic culture is not subjected to catabolite repression and demonstrated that aspS is expressed from the beginning of infection of sunflower cotyledons.

Comparison of adiphenine and TRH effects on TSH release by rat pituitary in vitro.

The mechanism of action of adiphenine on in vitro rat anterior pituitary TSH release was compared to that of the physiological stimulator TRH. The comparative study showed that adiphenine and TRH were able to increase TSH release in a dose-dependent manner, had similar time courses of action for equipotent stimulating concentrations and produced similar aspects of stimulated TSH cells. However, there were several differences between the effects of adiphenine and TRH. Adiphenine action was inhibited by 20 mM K+; was not calcium dependent; was inhibited by neither thyroid hormones nor somatostatin; was little affected by energy depression. It is concluded that adiphenine probably acts near the ultimate steps of the TSH release pathway and could be a useful pharmacological tool for studying the mechanism of TSH release.

Characterization of a protease deficient strain of Penicillium roqueforti generated by heterologous plasmid integration: potential use for protein production.

A strain of Penicillium roqueforti was transformed with a plasmid which confers resistance to phleomycin. Stable transformants were selected. They all present a high resistance to the antibiotic. Their proteolytic activity was tested. One transformant is a proteolytic deficient strain unable to degrade casein. It is characterized by a tandem integration of the transformant vector in one site of the genome. The extracellular proteins profile of the strain reveals the absence of a 43 kDa polypeptide which probably corresponds to the aspartyl protease of Penicillium roqueforti. The aspA gene which encodes aspartyl protease is not expressed in the transformant and Southern analyses show that the aspA gene is not disrupted by the transformation vector.

Purification of Endo Polygalacturonases from Sclerotinia sclerotiorum: Multiplicity of the Complex Enzyme System

Fourteen forms of endopolygalacturonases were purified from the culture medium of Sclerotinia sclerotiorum with ion exchange chromatographies. Individual forms differ in their isoelectric point but exhibit similar molecular masses. On the basis of the cross-reactions to antibodies raised against a purified acidic endopolygalacturonase, these forms could be divided into two related groups. Polygalacturonase activities constitute a complex enzyme system in which the extensive multiplicity is due to the expression of a large gene family.