Plasma leptin and mRNA expression of lipogenesis and lipolysis-related factors in bovine adipose tissue around parturition

The objective was to study changes in plasma leptin concentration parallel to changes in the gene expression of lipogenic- and lipolytic-related genes in adipose tissue of dairy cows around parturition. Subcutaneous fat biopsies were taken from 27 dairy cows in week 8 antepartum (a.p.), on day 1 postpartum (p.p.) and in week 5 p.p. Blood samples were assayed for concentrations of leptin and non-esterified fatty acids (NEFA). Subcutaneous adipose tissue was analysed for mRNA abundance by real-time qRT-PCR encoding for leptin, adiponectin receptor 1 (AdipoR1), adiponectin receptor 2 (AdipoR2), hormones-sensitive lipase (HSL), perilipin (PLIN), lipoprotein lipase (LPL), acyl-CoA synthase long-chain family member 1 (ACSL1), acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN) and glycerol-3-phosphate dehydrogenase 2 (GPD2). Body weight and body condition score of the cows were lower after parturition than before parturition. The calculated energy balance was negative in week 1 and 5 p.p., with higher negative energy balance in week 1 p.p. compared with that in week 5 p.p. On day 1 p.p., highest concentrations of NEFA (353.3 mumol/l) were detected compared with the other biopsy time-points (210.6 and 107.7 mumol/l, in week 8 a.p., and week 5 p.p. respectively). Reduced plasma concentrations of leptin during p.p. when compared with a.p. would favour increasing metabolic efficiency and energy conservation for mammary function and reconstitution of body reserves. Lower mRNA abundance of ACC and FASN expression on day 1 p.p. compared with other biopsy time-points suggests an attenuation of fatty acid synthesis in subcutaneous adipose tissue shortly after parturition. Gene expression of AdipoR1, AdipoR2, HSL, PLIN, LPL, ACSL1 and GPD2 was unchanged over time.

5-Hydroxytryptamine-4 receptor messenger ribonucleic acid levels and densities in gastrointestinal muscle layers from healthy dairy cows

Serotonin (5-hydroxytryptamine, 5-HT) is involved in gastrointestinal tract (GIT) motor functions through binding to specific receptors located in the GIT walls. The objectives of the current study were to compare mRNA levels and binding sites of 5-HT(4) receptors (5-HTR(4)) in smooth muscle layers from the fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of healthy dairy cows, and to verify whether mRNA and protein expression were correlated. Smooth muscle samples were prepared by scraping the mucosa and submucosa from full-thickness intestinal wall samples. The mRNA levels of 5-HTR(4) were measured by real-time PCR and expressed relative to those of the housekeeping gene glyceraldehyde phosphate dehydrogenase. Binding studies were performed using the 5-HTR(4) antagonist [(3)H]GR113808. The mRNA levels of 5-HTR(4) were affected (P < 0.05) by location along the GIT. The mRNA levels of 5-HTR(4) in the ELSC and the ileum were greater than in the PLAC (P = 0.05 and P = 0.07, respectively) but similar to those of all other locations. The competitive binding of [(3)H]GR113808 to suspended membranes from the fundus abomasi, pylorus, cecum, and ELSC was best fit by a 2-site receptor model, whereas it was best fit by a 1-site receptor model in the ileum and PLAC. The mRNA levels and numbers of 5-HTR(4) were not correlated (r = 0.14; P = 0.71). In conclusion, mRNA and binding sites for 5-HTR(4) are present in the smooth muscle layer of the entire GIT of dairy cows and may play a role with respect to motility. The effects of activation of this receptor subtype may be different among GIT locations due to differences in the amount of high- relative to low-affinity binding sites.

mRNA expression and binding sites for alpha2-adrenergic receptor subtypes in muscle layers of the ileum and spiral colon of dairy cows

OBJECTIVE: To measure maximum binding capacity (B(max)) and levels of mRNA expression for alpha(2)-adrenergic receptor (AR) subtypes in ileal and colonic muscle layers of healthy dairy cows. SAMPLE POPULATION: Ileal and colonic muscle specimens from 6 freshly slaughtered cows. PROCEDURES: Ileal and colonic muscle layers were obtained by scraping the mucosa and submucosa from full-thickness tissue specimens. Level of mRNA expression for alpha(2)-AR subtypes was measured by real-time reverse transcriptase-PCR analysis and expressed relative to the mean mRNA expression of glyceraldehyde phosphate dehydrogenase, ubiquitin, and 18S ribosomal RNA. Binding studies were performed with tritiated RX821002 ((3)H-RX821002) and subtype-selective ligands as competitors. RESULTS: mRNA expression for alpha(2AD)-, alpha(2B)-, and alpha(2C)-AR subtypes was similar in ileal and colonic muscle layers. The mRNA expression for alpha(2AD)-AR was significantly greater than that for alpha(2B)- and alpha(2C)-AR subtypes, representing 92%, 6%, and 2%, respectively, of the total mRNA. Binding competition of (3)H-RX821002 with BRL44408, imiloxan, and MK-912 was best fitted by a 1-site model. The B(max) of alpha(2AD)- and alpha(2C)-AR sub-types was greater than that of alpha(2B)-AR. The B(max) and level of mRNA expression were only correlated (r = 0.8) for alpha(2AD)-AR. Ratio of B(max) to mRNA expression for alpha(2C)-AR was similar to that for alpha(2B)-AR, but significantly greater than for alpha(2AD)-AR. CONCLUSIONS AND CLINICAL RELEVANCE: Subtypes of alpha(2)-AR in bovine intestinal muscle layers are represented by a mixture of alpha(2AD)- and alpha(2C)-ARs and of alpha(2B)-AR at a lower density. Information provided here may help in clarification of the role of AR subtypes in alpha(2)-adrenergic mechanisms regulating bovine intestinal motility.

Gene expression in cultured endometrium from women with different outcomes following IVF

Estradiol and progesterone are crucial for the acquisition of receptivity and the change in transcriptional activity of target genes in the implantation window. The aim of this study was to differentiate the regulation of genes in the endometrium of patients with recurrent implantation failure (IF) versus those who became pregnant after in vitro fertilization (IVF) treatment. Moreover, the effect of embryo-derived factors on endometrial transcriptional activity was studied. Nine women with known IVF outcome (IF, M, miscarriage, OP, ongoing pregnancy) and undergoing hysteroscopy with endometrial biopsy were enrolled. Biopsies were taken during the midluteal phase. After culture in the presence of embryo-conditioned IVF media, total RNA was extracted and submitted to reverse transcription, target cDNA synthesis, biotin labelling, fragmentation and hybridization using the Affymetrix Human Genome U133A 2.0 Chip. Differential expression of selected genes was re-analysed by quantitative PCR, in which the results were calculated as threshold cycle differences between the groups and normalized to Glyceraldehyde phosphate dehydrogenase and beta-actin. Differences were seen for several genes from endometrial tissue between the IF and the pregnancy groups, and when comparing OP with M, 1875 up- and 1807 down-regulated genes were returned. Real-time PCR analysis confirmed up-regulation for somatostatin, PLAP-2, mucin 4 and CD163, and down-regulation of glycodelin, IL-24, CD69, leukaemia inhibitory factor and prolactin receptor between Op and M. When the different embryo-conditioned media were compared, no significant differential regulation could be demonstrated. Although microarray profiling may currently not be sensitive enough for studying the effects of embryo-derived factors on the endometrium, the observed differences in gene expression between M and OP suggest that it will become an interesting tool for the identification of fertility-relevant markers produced by the endometrium.

Highly efficient ketone body treatment in multiple acyl-CoA dehydrogenase deficiency-related leukodystrophy.

BACKGROUND Multiple acyl-CoA dehydrogenase deficiency- (MADD-), also called glutaric aciduria type 2, associated leukodystrophy may be severe and progressive despite conventional treatment with protein- and fat-restricted diet, carnitine, riboflavin, and coenzyme Q10. Administration of ketone bodies was described as a promising adjunct, but has only been documented once. METHODS We describe a Portuguese boy of consanguineous parents who developed progressive muscle weakness at 2.5 y of age, followed by severe metabolic decompensation with hypoglycaemia and coma triggered by a viral infection. Magnetic resonance (MR) imaging showed diffuse leukodystrophy. MADD was diagnosed by biochemical and molecular analyses. Clinical deterioration continued despite conventional treatment. Enteral sodium D,L-3-hydroxybutyrate (NaHB) was progressively introduced and maintained at 600 mg/kg BW/d (≈3% caloric need). Follow up was 3 y and included regular clinical examinations, biochemical studies, and imaging. RESULTS During follow up, the initial GMFC-MLD (motor function classification system, 0 = normal, 6 = maximum impairment) level of 5-6 gradually improved to 1 after 5 mo. Social functioning and quality of life recovered remarkably. We found considerable improvement of MR imaging and spectroscopy during follow up, with a certain lag behind clinical recovery. There was some persistent residual developmental delay. CONCLUSION NaHB is a highly effective and safe treatment that needs further controlled studies.

Why is 11beta-hydroxysteroid dehydrogenase type 1 facing the endoplasmic reticulum lumen? Physiological relevance of the membrane topology of 11beta-HSD1

11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is essential for the local activation of glucocorticoid receptors (GR). Unlike unliganded cytoplasmic GR, 11beta-HSD1 is an endoplasmic reticulum (ER)-membrane protein with lumenal orientation. Cortisone might gain direct access to 11beta-HSD1 by free diffusion across membranes, indirectly via intracellular binding proteins or, alternatively, by insertion into membranes. Membranous cortisol, formed by 11beta-HSD1 at the ER-lumenal side, might then activate cytoplasmic GR or bind to ER-lumenal secretory proteins. Compartmentalization of 11beta-HSD1 is important for its regulation by hexose-6-phosphate dehydrogenase (H6PDH), which regenerates cofactor NADPH in the ER lumen and stimulates oxoreductase activity. ER-lumenal orientation of 11beta-HSD1 is also essential for the metabolism of the alternative substrate 7-ketocholesterol (7KC), a major cholesterol oxidation product found in atherosclerotic plaques and taken up from processed cholesterol-rich food. An 11beta-HSD1 mutant adopting cytoplasmic orientation efficiently catalyzed the oxoreduction of cortisone but not 7KC, indicating access to cortisone from both sides of the ER-membrane but to 7KC only from the lumenal side. These aspects may be relevant for understanding the physiological role of 11beta-HSD1 and for developing therapeutic interventions to control glucocorticoid reactivation.

Glycerol metabolism is important for cytotoxicity of Mycoplasma pneumoniae

Glycerol is one of the few carbon sources that can be utilized by Mycoplasma pneumoniae. Glycerol metabolism involves uptake by facilitated diffusion, phosphorylation, and the oxidation of glycerol 3-phosphate to dihydroxyacetone phosphate, a glycolytic intermediate. We have analyzed the expression of the genes involved in glycerol metabolism and observed constitutive expression irrespective of the presence of glycerol or preferred carbon sources. Similarly, the enzymatic activity of glycerol kinase is not modulated by HPr-dependent phosphorylation. This lack of regulation is unique among the bacteria for which glycerol metabolism has been studied so far. Two types of enzymes catalyze the oxidation of glycerol 3-phosphate: oxidases and dehydrogenases. Here, we demonstrate that the enzyme encoded by the M. pneumoniae glpD gene is a glycerol 3-phosphate oxidase that forms hydrogen peroxide rather than NADH(2). The formation of hydrogen peroxide by GlpD is crucial for cytotoxic effects of M. pneumoniae. A glpD mutant exhibited a significantly reduced formation of hydrogen peroxide and a severely reduced cytotoxicity. Attempts to isolate mutants affected in the genes of glycerol metabolism revealed that only the glpD gene, encoding the glycerol 3-phosphate oxidase, is dispensable. In contrast, the glpF and glpK genes, encoding the glycerol facilitator and the glycerol kinase, respectively, are essential in M. pneumoniae. Thus, the enzymes of glycerol metabolism are crucial for the pathogenicity of M. pneumoniae but also for other essential, yet-to-be-identified functions in the M. pneumoniae cell.

Tools for mass screening of G6PD deficiency: validation of the WST8/1-methoxy-PMS enzymatic assay in Uganda.

BACKGROUND The distribution of the enzymopathy glucose-6-phosphate dehydrogenase (G6PD) deficiency is linked to areas of high malaria endemicity due to its association with protection from disease. G6PD deficiency is also identified as the cause of severe haemolysis following administration of the anti-malarial drug primaquine and further use of this drug will likely require identification of G6PD deficiency on a population level. Current conventional methods for G6PD screening have various disadvantages for field use. METHODS The WST8/1-methoxy PMS method, recently adapted for field use, was validated using a gold standard enzymatic assay (R&D Diagnostics Ltd ®) in a study involving 235 children under five years of age, who were recruited by random selection from a cohort study in Tororo, Uganda. Blood spots were collected by finger-prick onto filter paper at routine visits, and G6PD activity was determined by both tests. Performance of the WST8/1-methoxy PMS test under various temperature, light, and storage conditions was evaluated. RESULTS The WST8/1-methoxy PMS assay was found to have 72% sensitivity and 98% specificity when compared to the commercial enzymatic assay and the AUC was 0.904, suggesting good agreement. Misclassifications were at borderline values of G6PD activity between mild and normal levels, or related to outlier haemoglobin values (<8.0 gHb/dl or >14 gHb/dl) associated with ongoing anaemia or recent haemolytic crises. Although severe G6PD deficiency was not found in the area, the test enabled identification of low G6PD activity. The assay was found to be highly robust for field use; showing less light sensitivity, good performance over a wide temperature range, and good capacity for medium-to-long term storage. CONCLUSIONS The WST8/1-methoxy PMS assay was comparable to the currently used standard enzymatic test, and offers advantages in terms of cost, storage, portability and use in resource-limited settings. Such features make this test a potential key tool for deployment in the field for point of care assessment prior to primaquine administration in malaria-endemic areas. As with other G6PD tests, outlier haemoglobin levels may confound G6PD level estimation.

Cytotoxic steroidal saponins from the rhizomes of Tacca integrifolia

Three new steroid saponins (3beta,25R)-spirost-5-en-3-yl 6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->4)-6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranoside (1), (3beta,22R,25R)-26-(beta-D-glucopyranosyloxy)-22-hydroxyfurost-5-en-3-yl 6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranoside (3), and (3beta,22R,25R)-26-(beta-D-glucopyranosyloxy)-22-hydroxyfurost-5-en-3-yl 6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->4)-6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranoside (5), as well as the new pregnane glycoside (3beta,16beta)-3-{[6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranosyl]oxy}-20-oxopregn-5-en-16-yl (4R)-5-(beta-D-glucopyranosyloxy)-4-methylpentanoate (6), were isolated from the rhizomes of Tacca integrifolia together with two known (25R) configurated steroid saponins (3beta,25R)-spirost-5-en-3-yl 6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranoside (2) and (3beta,22R,25R)-26-(beta-D-glucopyranosyloxy)-22-methoxyfurost-5-en-3-yl 6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranoside (4). The cytotoxic activity of the isolated compounds was evaluated in HeLa cells and showed the highest cytotoxicity value for compound 2 with an IC(50) of 1.2+/-0.4 muM. Intriguingly, while compounds 1-5 exhibited similar cytotoxic properties between 1.2+/-0.4 (2) and 4.0+/-0.6 muM (5), only compound 2 showed a significant microtubule-stabilizing activity in vitro.

Metformin inhibits human androgen production by regulating steroidogenic enzymes HSD3B2 and CYP17A1 and complex I activity of the respiratory chain

Metformin is treatment of choice for the metabolic consequences seen in polycystic ovary syndrome for its insulin-sensitizing and androgen-lowering properties. Yet, the mechanism of action remains unclear. Two potential targets for metformin regulating steroid and glucose metabolism are AMP-activated protein kinase (AMPK) signaling and the complex I of the mitochondrial respiratory chain. Androgen biosynthesis requires steroid enzymes 17α-Hydroxylase/17,20 lyase (CYP17A1) and 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2), which are overexpressed in ovarian cells of polycystic ovary syndrome women. Therefore, we aimed to understand how metformin modulates androgen production using NCI-H295R cells as an established model of steroidogenesis. Similar to in vivo situation, metformin inhibited androgen production in NCI cells by decreasing HSD3B2 expression and CYP17A1 and HSD3B2 activities. The effect of metformin on androgen production was dose dependent and subject to the presence of organic cation transporters, establishing an important role of organic cation transporters for metformin's action. Metformin did not affect AMPK, ERK1/2, or atypical protein kinase C signaling. By contrast, metformin inhibited complex I of the respiratory chain in mitochondria. Similar to metformin, direct inhibition of complex I by rotenone also inhibited HSD3B2 activity. In conclusion, metformin inhibits androgen production by mechanisms targeting HSD3B2 and CYP17-lyase. This regulation involves inhibition of mitochondrial complex I but appears to be independent of AMPK signaling.

Effects of tryptophan supplementation on plasma tryptophan and related hormone levels in heifers and dairy cows

This study was conducted to investigate the effects of rumen-protected tryptophan (125g tryptophan per day) in heifers and dairy cows. Blood samples from dairy cows and heifers were collected for 24h in 3-h intervals on the day before tryptophan supplementation, on day 2, 5 and 7 of tryptophan supplementation, and in heifers additionally on d 14 after tryptophan supplementation was ceased. Plasma tryptophan, melatonin, serotonin, and prolactin concentrations were determined. Tryptophan plasma concentrations on d 5 were augmented at day (11:00h) and nighttime (02:00h), (P<0.05) in response to tryptophan supplementation in heifers by 119% and in dairy cows by 47%, respectively, as compared with d 0. Melatonin increased (P<0.05) in response to tryptophan supplementation in heifers, but not in cows. The effect of tryptophan supplementation on plasma tryptophan and melatonin was reversible as demonstrated in heifers on d 14 after cessation of tryptophan supplementation. Serotonin and prolactin in plasma did not respond to tryptophan supplementation. However, milk yield during morning milking increased significantly in tryptophan supplemented cows on d 1, 3 and 4 as compared to the day before tryptophan supplementation. Additional blood samples were taken during afternoon milking in cows at 1-min intervals for the analyses of oxytocin and prolactin on the day before the start and on d 7 of tryptophan supplementation. Milk flow curves were recorded during milking. No effect of tryptophan supplementation on the milking related release of oxytocin and prolactin and on any characteristic of milk flow was observed. In conclusion, tryptophan supplementation caused increased plasma tryptophan in cows and heifers and plasma melatonin in heifers. However, plasma serotonin, prolactin and oxytocin release in cows remained unchanged by tryptophan supplementation. Milk yield at morning milking increased slightly and transiently in response to tryptophan supplementation.

Gingival fluid cytokine expression and subgingival bacterial counts during pregnancy and postpartum: a case series

OBJECTIVES The aim of this study was to assess gingival fluid (GCF) cytokine messenger RNA (mRNA) levels, subgingival bacteria, and clinical periodontal conditions during a normal pregnancy to postpartum. MATERIALS AND METHODS Subgingival bacterial samples were analyzed with the checkerboard DNA-DNA hybridization method. GCF samples were assessed with real-time PCR including five proinflammatory cytokines and secretory leukocyte protease inhibitor. RESULTS Nineteen pregnant women with a mean age of 32 years (S.D. ± 4 years, range 26-42) participated in the study. Full-mouth bleeding scores (BOP) decreased from an average of 41.2% (S.D. ± 18.6%) at the 12th week of pregnancy to 26.6% (S.D. ± 14.4%) at the 4-6 weeks postpartum (p < 0.001). Between week 12 and 4-6 weeks postpartum, the mean probing pocket depth changed from 2.4 mm (S.D. ± 0.4) to 2.3 mm (S.D. ± 0.3) (p = 0.34). Higher counts of Eubacterium saburreum, Parvimonas micra, Selenomonas noxia, and Staphylococcus aureus were found at week 12 of pregnancy than at the 4-6 weeks postpartum examinations (p < 0.001). During and after pregnancy, statistically significant correlations between BOP scores and bacterial counts were observed. BOP scores and GCF levels of selected cytokines were not related to each other and no differences in GCF levels of the cytokines were observed between samples from the 12th week of pregnancy to 4-6 weeks postpartum. Decreasing postpartum counts of Porphyromonas endodontalis and Pseudomonas aeruginosa were associated with decreasing levels of Il-8 and Il-1β. CONCLUSIONS BOP decreased after pregnancy without any active periodontal therapy. Associations between bacterial counts and cytokine levels varied greatly in pregnant women with gingivitis and a normal pregnancy outcome. Postpartum associations between GCF cytokines and bacterial counts were more consistent. CLINICAL RELEVANCE Combined assessments of gingival fluid cytokines and subgingival bacteria may provide important information on host response.

Discontinuous Galerkin finite element approximation of quasilinear elliptic boundary value problems II: Strongly monotone quasi-Newtonian flows

In this article, we develop the a priori and a posteriori error analysis of hp-version interior penalty discontinuous Galerkin finite element methods for strongly monotone quasi-Newtonian fluid flows in a bounded Lipschitz domain Ω ⊂ ℝd, d = 2, 3. In the latter case, computable upper and lower bounds on the error are derived in terms of a natural energy norm, which are explicit in the local mesh size and local polynomial degree of the approximating finite element method. A series of numerical experiments illustrate the performance of the proposed a posteriori error indicators within an automatic hp-adaptive refinement algorithm.