PURIFICATION OF SPECIES SPECIFIC ANTIBODIES TO CARBOHYDRATE COMPONENTS OF MACROCYSTIS PYRIFERA (PHAEOPHYTA)1

Polyclonal rabbit antibodies to cell wall components were produced against gametophytes of the giant kelp Macrocystis pyrifera (Linnaeus) C. Agardh. These antibodies were found to react with carbohydrates extracted from M. pyrifera and Pterygophora californica Ruprecht by carbohydrate based enzyme immunoassay (EIA). The antibodies reacted with carbohydrates from both species. After affinity purification on a column with M. pyrifera carbohydrate coupled to AH-Sepharose, the eluted antibody was specific for M. pyrifera carbohydrate with little cross reactivity to P. californica carbohydrate in the EIA test. In experiments carried out to characterize the antigenic specificity of unfractionated antibody using commercially prepared carbohydrates in the EIA, the antibodies were shown to react primarily with fucoidan and to a lesser degree, alginate. The unfractionated antibody was also shown to bind to proteins from both M. pyrifera and P. californica. These results indicate that species specific carbohydrate determinants may be present in the kelp cell wall.     

Probable male heterogamety in the deep-sea fish Bathylagus wesethi (Teleostei: Bathylagidae)

A presumed XY chromosome pair is described fromt estis squashes from the mesopelagic deep-sea fish Bathylagus wesethi, whose 2N chromosome number was determined as 34-XY. Although the metacentric X-chromosome is the largest in the entire compliment, the Y is the smallest and only acrocentric element. The positive heteropycnosis of the sex elements was not easily distinguishable in the nuclei of first meiotic prophase. Tetraploid nuclei were observed in peripheral supporting cells of the testis. Males of at least two other congeners have similar karyotypes.     

Kappa-chain constant-region gene sequences in genus Rattus: coding regions are diverging more rapidly than noncoding regions

We have determined the nucleotide sequence of a 1,200-base pair (bp) genomic fragment that includes the kappa-chain constant-region gene (C kappa) from two species of native Australian rodents, Rattus leucopus cooktownensis and Rattus colletti. Comparison of these sequences with each other and with other rodent C kappa genes shows three surprising features. First, the coding regions are diverging at a rate severalfold higher than that of the nearby noncoding regions. Second, replacement changes within the coding region are accumulating at a rate at least as great as that of silent changes. Third, most of the amino acid replacements are localized in one region of the C kappa domain--namely, the carboxy-terminal "bends" in the alpha-carbon backbone. These three features have previously been described from comparisons of the two allelic forms of C kappa genes in R. norvegicus. These data imply the existence of considerable evolutionary constraints on the noncoding regions (based on as yet undetermined functions) or powerful positive selection to diversify a portion of the constant-region domain (whose physiological significance is not known). These surprising features of C kappa evolution appear to be characteristic only of closely related C kappa genes, since comparison of rodent with human sequences shows the expected greater conservation of coding regions, as well as a predominance of silent nucleotide substitutions within the coding regions.      

Hypoxic regulation of angiotensin-converting enzyme 2 and Mas receptor in human CD34+ cells

CD34⁺ hematopoietic stem/progenitor cells (HSPCs) are vasculogenic and hypoxia is a strong stimulus for the vasoreparative functions of these cells. Angiotensin-converting enzyme 2 (ACE2)/angiotensin-(1–7)/Mas receptor (MasR) pathway stimulates vasoprotective functions of CD34⁺ cells. This study tested if ACE2 and MasR are involved in the hypoxic stimulation of CD34⁺ cells. Cells were isolated from circulating mononuclear cells derived from healthy subjects (n = 46) and were exposed to normoxia (20% O₂) or hypoxia (1% O₂). Luciferase reporter assays were carried out in cells transduced with lentivirus carrying ACE2- or MasR- or a scramble-3′-untranslated region gene with a firefly luciferase reporter. Expressions or activities of ACE, angiotensin receptor Type 1 (AT1R), ACE2, and MasR were determined. In vitro observations were verified in HSPCs derived from mice undergoing hindlimb ischemia (HLI). In vitro exposure to hypoxia-increased proliferation and migration of CD34⁺ cells in basal conditions or in response to vascular endothelial growth factor (VEGF) or stromal-derived factor 1α (SDF) compared with normoxia. Expression of ACE2 or MasR was increased relative to normoxia while ACE or AT1R expressions were unaltered. Luciferase activity was increased by hypoxia in cells transfected with the luciferase reporter plasmids coding for the ACE2- or MasR promoters relatively to the control. The effects of hypoxia were mimicked by VEGF or SDF under normoxia. Hypoxia-induced ADAM17-dependent shedding of functional ACE2 fragments. In mice undergoing HLI, increased expression/activity of ACE2 and MasR were observed in the circulating HSPCs. This study provides compelling evidence for the hypoxic upregulation of ACE2 and MasR in CD34⁺ cells, which likely contributes to vascular repair.     

SIMPLE: implementation of recommendations from international evidence-based guidelines on caesarean sections in the Netherlands. Protocol for a controlled before and after study

Background

Caesarean section (CS) rates are rising worldwide. In the Netherlands, the most significant rise is observed in healthy women with a singleton in vertex position between 37 and 42 weeks gestation, whereas it is doubtful whether an improved outcome for the mother or her child was obtained. It can be hypothesized that evidence-based guidelines on CS are not implemented sufficiently. Therefore, the present study has the following objectives: to develop quality indicators on the decision to perform a CS based on key recommendations from national and international guidelines; to use the quality indicators in order to gain insight into actual adherence of Dutch gynaecologists to guideline recommendations on the performance of a CS; to explore barriers and facilitators that have a direct effect on guideline application regarding CS; and to develop, execute, and evaluate a strategy in order to reduce the CS incidence for a similar neonatal outcome (based on the information gathered in the second and third objectives).

Methods

An independent expert panel of Dutch gynaecologists and midwives will develop a set of quality indicators on the decision to perform a CS. These indicators will be used to measure current care in 20 hospitals with a population of 1,000 women who delivered by CS, and a random selection of 1,000 women who delivered vaginally in the same period. Furthermore, by interviewing healthcare professionals and patients, the barriers and facilitators that may influence the decision to perform a CS will be measured. Based on the results, a tailor-made implementation strategy will be developed and tested in a controlled before-and-after study in 12 hospitals (six intervention, six control hospitals) with regard to effectiveness, experiences, and costs.

Discussion

This study will offer insight into the current CS care and into the hindering and facilitating factors influencing obstetrical policy on CS. Furthermore, it will allow definition of patient categories or situations in which a tailor-made implementation strategy will most likely be meaningful and cost effective, without negatively affecting the outcome for mother and child.

Trial registration

http://www.clinicaltrials.gov: NCT01261676     

Trace amine-associated receptors form structurally and functionally distinct subfamilies of novel G protein-coupled receptors

Trace amines are endogenous compounds structurally related to classical biogenic amines that have been studied for decades, triggered by their link to psychiatric conditions of high epidemiological and economical relevance. The understanding of their pharmacology on the molecular level was hampered until the recent discovery of trace-amine-specific receptors. We completed the identification of all members of this novel GPCR family in human, chimpanzee, rat, and mouse and observed remarkable interspecies differences, even between human and chimpanzee. The analysis of the chromosomal localizations, phylogenetic relationships, and ligand pocket vectors reveals three distinct receptor subfamilies. As most of these receptors do not respond to trace amines, each subfamily will presumably have a distinct pharmacological profile, which remains to be identified. We propose a uniform nomenclature describing this novel GPCR family in all mammalian species as trace-amine-associated receptors (TAARs), which resolves the ambiguities and contradictions of the previous naming.     

Effects of Combined Calcium and Vitamin D Supplementation on Insulin Secretion,Insulin Sensitivity and β-Cell Function in Multi-Ethnic Vitamin D-Deficient Adults at Risk for Type 2 Diabetes: A Pilot Randomized,Placebo-Controlled Trial

Objectives

To examine whether combined vitamin D and calcium supplementation improves insulin sensitivity, insulin secretion, β-cell function, inflammation and metabolic markers.

Design

6-month randomized, placebo-controlled trial.

Participants

Ninety-five adults with serum 25-hydroxyvitamin D [25(OH)D] ≤55 nmol/L at risk of type 2 diabetes (with prediabetes or an AUSDRISK score ≥15) were randomized. Analyses included participants who completed the baseline and final visits (treatment n = 35; placebo n = 45).

Intervention

Daily calcium carbonate (1,200 mg) and cholecalciferol [2,000–6,000 IU to target 25(OH)D >75 nmol/L] or matching placebos for 6 months.

Measurements

Insulin sensitivity (HOMA2%S, Matsuda index), insulin secretion (insulinogenic index, area under the curve (AUC) for C-peptide) and β-cell function (Matsuda index x AUC for C-peptide) derived from a 75 g 2-h OGTT; anthropometry; blood pressure; lipid profile; hs-CRP; TNF-α; IL-6; adiponectin; total and undercarboxylated osteocalcin.

Results

Participants were middle-aged adults (mean age 54 years; 69% Europid) at risk of type 2 diabetes (48% with prediabetes). Compliance was >80% for calcium and vitamin D. Mean serum 25(OH)D concentration increased from 48 to 95 nmol/L in the treatment group (91% achieved >75 nmol/L), but remained unchanged in controls. There were no significant changes in insulin sensitivity, insulin secretion and β-cell function, or in inflammatory and metabolic markers between or within the groups, before or after adjustment for potential confounders including waist circumference and season of recruitment. In a post hoc analysis restricted to participants with prediabetes, a significant beneficial effect of vitamin D and calcium supplementation on insulin sensitivity (HOMA%S and Matsuda) was observed.

Conclusions

Daily vitamin D and calcium supplementation for 6 months may not change OGTT-derived measures of insulin sensitivity, insulin secretion and β-cell function in multi-ethnic adults with low vitamin D status at risk of type 2 diabetes. However, in participants with prediabetes, supplementation with vitamin D and calcium may improve insulin sensitivity.

Trial Registration

Australian New Zealand Clinical Trials Registry ACTRN12609000043235     

Identification of nifE-, nifN- and nifS-like genes in Bradyrhizobium japonicum

Summary The 17 kb region between the Bradyrhizobium japonicum nitrogenase genes (nifDK and nifH) was investigated for the presence of further nif or fix genes by site-directed insertion or deletion/replacement mutagenesis and interspecies hybridization. Mutant strains were tested for their ability to reduce acetylene in free-living, microaerobic culture (Nif phenotype) and in soybean root nodules (Fix phenotype). The presence of a gene, previously identified by hybridization with the Klebsiella pneumoniae nifB gene, was proved by isolation of a nifB insertion mutant which was completely Nif^- and Fix^-. Three other regions were found to be homologous to the K. pneumoniae genes nifE, nifN, and nifS, NifE and nifN insertion mutants were completely Nif^-/Fix^- whereas nifS mutants were leaky with 30% residual Fix activity. Taken together, the data show that the B. japonicum genome harbours a cluster of closely adjacent genes which are directly concerned with nitrogenase function.