Caspase 3 activation and PARP cleavage in lymphocytes from newborn babies of diabetic mothers with unbalanced glycaemic control

Several epidemiological studies showed that gestational diabetes mellitus is the most frequent metabolic disorder of pregnancy, the pathogenesis of which has yet to be completely clarified. The aim of this study was to investigate the presence and processing of caspase 3 (Casp3) and poly(ADP‐ribose) polymerase 1 (PARP1) in cord blood lymphocytes as markers of apoptosis in relation to glycaemic control during intrauterine life. Our results showed a specific positive correlation between the levels of active Casp3 (17–19 kDa) and the inactive form of PARP1 (89 kDa) in lymphocytes isolated from newborn babies of diabetic women with unbalanced glycaemic control, with a direct correlation between the activation of casp3 and the inactivation of PARP1, that makes lymphocytes unresponsive towards lipopolysaccharide stimulation, highlighting an altered functional response. Besides more studies are required to fully correlate the activation of the apoptotic process during the intrauterine life with the foetal health later in life, our study indicates that a cord blood lymphocyte, an easily accessible source, is informative about the activation of apoptotic stimuli in circulating cells of newborn babies in relation to the glycaemic control reached by the mother during pregnancy. Copyright © 2013 John Wiley & Sons, Ltd.     

New Type of Papillomavirus and Novel Circular Single Stranded DNA Virus Discovered in Urban Rattus norvegicus Using Circular DNA Enrichment and Metagenomics

Rattus norvegicus (R. norvegicus) are ubiquitous and their presence has several effects on the human populations in our urban areas on a global scale. Both historically and presently, this close interaction has facilitated the dissemination of many pathogens to humans, making screening for potentially zoonotic and emerging viruses in rats highly relevant. We have investigated faecal samples from R. norvegicus collected from urban areas using a protocol based on metagenomic enrichment of circular DNA genomes and subsequent sequencing. We found a new type of papillomavirus, with a L1 region 82% identical to that of the known R. norvegicus Papillomavirus 2. Additionally, we found 20 different circular replication associated protein (Rep)-encoding single stranded DNA (CRESS-DNA) virus-like genomes, one of which has homology to the replication-associated gene of Beak and feather disease virus. Papillomaviruses are a group of viruses known for their carcinogenic potential, and although they are known to infect several different vertebrates, they are mainly studied and characterised in humans. CRESS-DNA viruses are found in many different environments and tissue types. Both papillomaviruses and CRESS-DNA viruses are known to have pathogenic potential and screening for novel and known viruses in R. norvegicus could help identify viruses with pathogenic potential.     

Purification of the Chlorella HUP1 hexose—proton symporter to homogeneity and its reconstitution in vitro

A prokaryotic biotin acceptor domain was fused to the carboxy terminal end of the Chlorella hexose—proton sym- porter. The plant symporter is biotinylated in vivo when expressed in Schizosaccharomyces pombe. The extended biotinylated transport protein is fully active, catalyzes accumulation of d -glucose analogs and restores growth of a glucose-uptake-deficient yeast strain. Crude membranes were solubilized with octyl-β-d -glucoside in the presence of Escherichia colil -α-phosphatidylethanolamine. Biotinylated symporter was purified to homogeneity by biotinavidin affinity chromatography. The symporter protein was reconstituted together with cytochrome-c oxidase prepared from beef heart mitochondria into proteo-liposomes. Cytochrome-c oxidase is a redox-driven H⁺-pump generating a proton motive force (inside negative and alkaline) while transferring electrons from cytochrome-c to oxygen; this energy is used by the symporter to accumulate d -glucose at least 30-fold. In the absence of the driving force the transport protein facilitates diffusion of d -glucose until the concentration equilibrium is reached. It was shown that maximal transport activity depends highly on the amount of co-reconstituted cytochrome-c oxidase and that the symporter possesses 10% of its in vivo turnover number under optimized in vitro transport conditions.     

Computer-aided gene design.

A computer program, which runs on MS-DOS personal computers, is described that assists in the design of synthetic genes coding for proteins. The goal of the program is the design of a gene which (i) contains as many unique restriction sites as possible and (ii) uses a specific codon usage. The gene designed according to the criteria above is (i) suitable for 'modular mutagenesis' experiments and (ii) optimized for expression. The program 'reverse-translates' protein sequences into degenerated DNA sequences, generates a map of potential restriction sites and locates sequence positions where unique restriction sites can be accommodated. The nucleic acid sequence is then 'refined' according to a specific codon usage to remove any degeneration. Unique restriction sites, if potentially present, can be 'forced' into the degenerated nucleic acid sequence by using 'priority codes' assigned to different restriction sequences.     

Inactivation of pyruvate decarboxylase by 3-hydroxypyruvate.

Pyruvate decarboxylase from Zymomonas mobilis is inhibited by 3-hydroxypyruvate, which can also act as a poor substrate. While catalysing the decarboxylation of this alternative substrate, the enzyme undergoes a progressive but partial inactivation over several hours. The extent of inactivation depends upon the pH and upon the concentration of 3-hydroxypyruvate. After partial inactivation and removal of unchanged 3-hydroxypyruvate, enzymic activity recovers slowly. We suggest that inactivation results from accumulation of enzyme-bound glycollaldehyde, which is relatively stable, possibly because it is dehydrated to form an acetyl group.     

Intra- and inter-observer analysis in the morphological assessment of early-stage embryos

Background  

The aim of this study was to determine the intra- and inter-observer variability in the evaluation of embryo quality. Multilevel images of embryos on day 1, day 2 and day 3, were analysed using different morphological parameters.