Purification and characterisation of bovine brain protein kinase C isotypes alpha, beta and gamma

Several polyclonal antisera specific for each of the protein kinase C isotypes alpha, beta 1, beta 2 and gamma have been generated and used to monitor the purification and subsequent separation of these polypeptides. A simple protocol has been developed for the efficient co-purification of these isotypes from bovine brain. The separation of the alpha, beta 1, beta 2 and gamma isotypes has been monitored using the antibodies and pools containing pure alpha, beta 1, and gamma forms have been produced. These isotypes have been characterised for activator dependence and substrate specificity. The results indicate that while the isotypes have similar requirements for magnesium, calcium, ATP and phosphatidylserine, they differ in their dependence on phorbol esters and diacylglycerols. The isotypes also differ in their range of substrate specificities. The implications of these results are discussed.     

Biosynthesis of Vitamins and Cofactors in Bacterium-Harbouring Trypanosomatids Depends on the Symbiotic Association as Revealed by Genomic Analyses

Some non-pathogenic trypanosomatids maintain a mutualistic relationship with a betaproteobacterium of the Alcaligenaceae family. Intensive nutritional exchanges have been reported between the two partners, indicating that these protozoa are excellent biological models to study metabolic co-evolution. We previously sequenced and herein investigate the entire genomes of five trypanosomatids which harbor a symbiotic bacterium (SHTs for Symbiont-Haboring Trypanosomatids) and the respective bacteria (TPEs for Trypanosomatid Proteobacterial Endosymbiont), as well as two trypanosomatids without symbionts (RTs for Regular Trypanosomatids), for the presence of genes of the classical pathways for vitamin biosynthesis. Our data show that genes for the biosynthetic pathways of thiamine, biotin, and nicotinic acid are absent from all trypanosomatid genomes. This is in agreement with the absolute growth requirement for these vitamins in all protozoa of the family. Also absent from the genomes of RTs are the genes for the synthesis of pantothenic acid, folic acid, riboflavin, and vitamin B_6. This is also in agreement with the available data showing that RTs are auxotrophic for these essential vitamins. On the other hand, SHTs are autotrophic for such vitamins. Indeed, all the genes of the corresponding biosynthetic pathways were identified, most of them in the symbiont genomes, while a few genes, mostly of eukaryotic origin, were found in the host genomes. The only exceptions to the latter are: the gene coding for the enzyme ketopantoate reductase (EC:1.1.1.169) which is related instead to the Firmicutes bacteria; and two other genes, one involved in the salvage pathway of pantothenic acid and the other in the synthesis of ubiquinone, that are related to Gammaproteobacteria. Their presence in trypanosomatids may result from lateral gene transfer. Taken together, our results reinforce the idea that the low nutritional requirement of SHTs is associated with the presence of the symbiotic bacterium, which contains most genes for vitamin production.     

Gestational diabetes mellitus epigenetically affects genes predominantly involved in metabolic diseases

Offspring exposed to gestational diabetes mellitus (GDM) have an increased risk for chronic diseases, and one promising mechanism for fetal metabolic programming is epigenetics. Therefore, we postulated that GDM exposure impacts the offspring’s methylome and used an epigenomic approach to explore this hypothesis. Placenta and cord blood samples were obtained from 44 newborns, including 30 exposed to GDM. Women were recruited at first trimester of pregnancy and followed until delivery. GDM was assessed after a 75-g oral glucose tolerance test at 24–28 weeks of pregnancy. DNA methylation was measured at > 485,000 CpG sites (Infinium HumanMethylation450 BeadChips). Ingenuity Pathway Analysis was conducted to identify metabolic pathways epigenetically affected by GDM. Our results showed that 3,271 and 3,758 genes in placenta and cord blood, respectively, were potentially differentially methylated between samples exposed or not to GDM (p-values down to 1 × 10⁻⁰⁶; none reached the genome-wide significance levels), with more than 25% (n = 1,029) being common to both tissues. Mean DNA methylation differences between groups were 5.7 ± 3.2% and 3.4 ± 1.9% for placenta and cord blood, respectively. These genes were likely involved in the metabolic diseases pathway (up to 115 genes (11%), p-values for pathways = 1.9 × 10⁻¹³ < p < 4.0 × 10⁻⁰³; including diabetes mellitus p = 4.3 × 10⁻¹¹). Among the differentially methylated genes, 326 in placenta and 117 in cord blood were also associated with newborn weight. Our results therefore suggest that GDM has epigenetic effects on genes preferentially involved in the metabolic diseases pathway, with consequences on fetal growth and development, and provide supportive evidence that DNA methylation is involved in fetal metabolic programming.     

Determination and quantification of sinigrin glucosinolates in Alternaria solani susceptible tomato (Solanum lycopersicum) leaves treated with moringa (Moringa oleifera) leaf extract

Abstract

The study investigates the presence and quantity of antimicrobial sinigrin glucosinolates in tomato leaves after spraying them with moringa (Moringa oleifera) leaf extract (MLAE). Moringa concentrates (0.5, 0.75, 1.00 and 1.5?kg?L^?1 (w v^?1)) were prepared. Distilled water was the control. Sampled tomato leaves were air-dried, freeze-dried and extracted firstly using pure methanol in a hot water bath and then pellet re-extracted using 5?mL of hot aqueous methanol (70% v v^?1). An ion exchange column, and sulphatase was used to achieve glucosiodesulphonation. High performance liquid chromatography (HPLC) was employed in the identification and quantitative analysis of the sinigrin glucosinolates. Tomato (Solanum lycopersicum) leaves treated with MLAE revealed highly significant (p?<?.001) content of sinigrin glucosinolates. The sinigrin standard and the desulphated sinigrin glucosinolates had a 7?s retention time difference; 5?kg?L^?1 (w v^?1) resulted in a superior amount of sinigrin in tomato leaves as compared to all the other MLAE concentrations. The study reveals that spraying MLAE on putatively diseased tomato leaves donates specific quantifiable glucosinolates like sinigrin, which may be involved in defense against tomato diseases and, hence, recommends use of 5?kg?L^?1 (w v^?1) for the highest sinigrin defense tag.     

Early Medieval Muslim Graves in France: First Archaeological,Anthropological and Palaeogenomic Evidence

The rapid Arab-Islamic conquest during the early Middle Ages led to major political and cultural changes in the Mediterranean world. Although the early medieval Muslim presence in the Iberian Peninsula is now well documented, based in the evaluation of archeological and historical sources, the Muslim expansion in the area north of the Pyrenees has only been documented so far through textual sources or rare archaeological data. Our study provides the first archaeo-anthropological testimony of the Muslim establishment in South of France through the multidisciplinary analysis of three graves excavated at Nimes. First, we argue in favor of burials that followed Islamic rites and then note the presence of a community practicing Muslim traditions in Nimes. Second, the radiometric dates obtained from all three human skeletons (between the 7th and the 9th centuries AD) echo historical sources documenting an early Muslim presence in southern Gaul (i.e., the first half of 8th century AD). Finally, palaeogenomic analyses conducted on the human remains provide arguments in favor of a North African ancestry of the three individuals, at least considering the paternal lineages. Given all of these data, we propose that the skeletons from the Nimes burials belonged to Berbers integrated into the Umayyad army during the Arab expansion in North Africa. Our discovery not only discusses the first anthropological and genetic data concerning the Muslim occupation of the Visigothic territory of Septimania but also highlights the complexity of the relationship between the two communities during this period.     

Identifying Likely Transmission Pathways within a 10-Year Community Outbreak of Tuberculosis by High-Depth Whole Genome Sequencing

Background

Improved tuberculosis control and the need to contain the spread of drug-resistant strains provide a strong rationale for exploring tuberculosis transmission dynamics at the population level. Whole-genome sequencing provides optimal strain resolution, facilitating detailed mapping of potential transmission pathways.

Methods

We sequenced 22 isolates from a Mycobacterium tuberculosis cluster in New South Wales, Australia, identified during routine 24-locus mycobacterial interspersed repetitive unit typing. Following high-depth paired-end sequencing using the Illumina HiSeq 2000 platform, two independent pipelines were employed for analysis, both employing read mapping onto reference genomes as well as de novo assembly, to control biases in variant detection. In addition to single-nucleotide polymorphisms, the analyses also sought to identify insertions, deletions and structural variants.

Results

Isolates were highly similar, with a distance of 13 variants between the most distant members of the cluster. The most sensitive analysis classified the 22 isolates into 18 groups. Four of the isolates did not appear to share a recent common ancestor with the largest clade; another four isolates had an uncertain ancestral relationship with the largest clade.

Conclusion

Whole genome sequencing, with analysis of single-nucleotide polymorphisms, insertions, deletions, structural variants and subpopulations, enabled the highest possible level of discrimination between cluster members, clarifying likely transmission pathways and exposing the complexity of strain origin. The analysis provides a basis for targeted public health intervention and enhanced classification of future isolates linked to the cluster.     

Evolution under reversals: parsimony and conservation of common intervals

In comparative genomics, gene order data is often modeled as signed permutations. A classical problem for genome comparison is to detect common intervals in permutations, that is, genes that are colocalized in several species, indicating that they remained grouped during evolution. A second largely studied problem related to gene order is to compute a minimum scenario of reversals that transforms a signed permutation into another. Several studies began to mix the two problems and it was observed that their results are not always compatible: Often, parsimonious scenarios of reversals break common intervals. If a scenario does not break any common interval, it is called perfect. In two recent studies, Berard et al. defined a class of permutations for which building a perfect scenario of reversals sorting a permutation was achieved in polynomial time and stated as an open question whether it is possible to decide, given a permutation, if there exists a minimum scenario of reversals that is perfect. In this paper, we give a solution to this problem and prove that this widens the class of permutations addressed by the aforementioned studies. We implemented and tested this algorithm on gene order data of chromosomes from several mammal species and we compared it to other methods. The algorithm helps to choose among several possible scenarios of reversals and indicates that the minimum scenario of reversals is not always the most plausible     

Two distinct HLA-A * 0101-specific submotifs illustrate alternative peptide binding modes

 Previous studies have defined two different peptide binding motifs specific for HLA-A ^* 0101. These motifs are characterized by the presence of tyrosine (Y) at the C-termini of 9-mer and 10-mer peptides, and either a small polar or hydrophobic (S, T, M) residue in position 2, or a negatively charged (D or E) residue in position 3. In this study, the structural requirements for peptide binding to A ^* 0101 have been further analyzed by examining the binding capacity of large sets of peptides corresponding to naturally occurring sequences which bore one or the other of these two A ^* 0101-specific motifs. By correlating the presence of specific residue types at each position along the peptide sequence with increased (or decreased) binding affinity, the prominent influence of secondary anchor residues was revealed. In most cases, the two anchors in positions 2 and 3 appear to act synergistically. With the exception of the DE₃ submotif in 9-mer peptides, a positive role for aromatic residues in position 1 and the center of the peptide (positions 4 or 5 of 9- or 10-mer peptides, respectively), and proline at C-3, were also consistently detected. However, secondary anchor residues also appear to differ significantly between the two different submotifs, demonstrating that A ^* 0101 can utilize alternative modes in binding its peptide ligands. According to these analyses, specific refined submotifs were also established, and their merit verified by independent sets of potential A ^* 0101 binding peptides. Besides providing useful insight into the nature of the interaction of the A ^* 0101 allele with its peptide ligands, such refined motifs should also facilitate accurate prediction of potential A ^* 0101-restricted peptide epitopes. Received: 16 July 1996 / Revised: 18 September 1996     

Chylomicron remnant metabolism in familial dyslipidemias studied with a remnant-like emulsion breath test

We have developed a stable isotope breath test for the assessment of chylomicron remnant metabolism and report the results from the breath test in human subjects selected for disorders of chylomicron or remnant metabolism. In type I hyperlipemia, the phenotype is extreme hypertriglyceridemia due to a lack of lipoprotein lipase activity, which causes the failure of remnant formation. The type III dyslipidemia phenotype is caused by the inefficient removal of chylomicron remnants from plasma, generally because of homozygosity for apolipoprotein E2 alleles. The breath test was predicted to be abnormal in type III hyperlipemia, whereas a priori in type I hyperlipemia defective remnant clearance was not anticipated. Subjects were injected with lipid emulsions prepared with a composition similar to normal chylomicron remnants. The emulsions contained cholesteryl ester incorporating the stable nonradioactive isotope (13)C in the fatty acid moiety. End exhalation breath was collected at intervals after intravenous injection of the remnant-like emulsions and analyzed for (13)C enrichment by isotope-ratio mass spectrometry. Compared with the group of normolipemic men, the fractional catabolic rate of remnants measured by the breath test was significantly decreased (P = 0.006) in subjects with type III dyslipidemia. In the group with type I hyperlipemia, the fractional catabolic rate was not different (P = 0.233) from the control group. Therefore, the underlying capacity for remnant catabolism was normal in this group of markedly hypertriglyceridemic subjects.By short-circuiting the step of lipolysis, the remnant-like emulsion breath test provides direct information about remnant clearance and metabolism, which should assist in investigations of postprandial lipid metabolism.