RAB23 mutations in Carpenter syndrome imply an unexpected role for hedgehog signaling in cranial-suture development and obesity

Carpenter syndrome is a pleiotropic disorder with autosomal recessive inheritance, the cardinal features of which include craniosynostosis, polysyndactyly, obesity, and cardiac defects. Using homozygosity mapping, we found linkage to chromosome 6p12.1-q12 and, in 15 independent families, identified five different mutations (four truncating and one missense) in RAB23, which encodes a member of the RAB guanosine triphosphatase (GTPase) family of vesicle transport proteins and acts as a negative regulator of hedgehog (HH) signaling. In 10 patients, the disease was caused by homozygosity for the same nonsense mutation, L145X, that resides on a common haplotype, indicative of a founder effect in patients of northern European descent. Surprisingly, nonsense mutations of Rab23 in open brain mice cause recessive embryonic lethality with neural-tube defects, suggesting a species difference in the requirement for RAB23 during early development. The discovery of RAB23 mutations in patients with Carpenter syndrome implicates HH signaling in cranial-suture biogenesis--an unexpected finding, given that craniosynostosis is not usually associated with mutations of other HH-pathway components--and provides a new molecular target for studies of obesity.     

Validation of single nucleotide polymorphism quantification in pooled DNA samples with SNaPIT. A glycosylase-mediated methods for polymorphism detection method

Association studies using genome scans to identify quantitative trait loci for multifactorial disorders, with anything approaching reasonable power, have been compromised by the need for a very dense array of genetic markers and large numbers of affected individuals. These requirements impose enormous burdens on the genotyping capacity for most laboratories. DNA pooling has been proposed as a possible approach to reduce genotyping costs and effort. We report on the application of the SNaPIT™ technology to evaluate allele frequencies in pooled DNA samples and conclude that it offers a cost effective, efficient and accurate estimator and provides several advantages over competing technologies in this regard.     

Functional annotation of class I lysyl-tRNA synthetase phylogeny indicates a limited role for gene transfer

Functional and comparative genomic studies have previously shown that the essential protein lysyl-tRNA synthetase (LysRS) exists in two unrelated forms. Most prokaryotes and all eukaryotes contain a class II LysRS, whereas most archaea and a few bacteria contain a less common class I LysRS. In bacteria the class I LysRS is only found in the alpha-proteobacteria and a scattering of other groups, including the spirochetes, while the class I protein is by far the most common form of LysRS in archaea. To investigate this unusual distribution we functionally annotated a representative phylogenetic sampling of LysRS proteins. Class I LysRS proteins from a variety of bacteria and archaea were characterized in vitro by their ability to recognize Escherichia coli tRNA(Lys) anticodon mutants. Class I LysRS proteins were found to fall into two distinct groups, those that preferentially recognize the third anticodon nucleotide of tRNA(Lys) (U36) and those that recognize both the second and third positions (U35 and U36). Strong recognition of U35 and U36 was confined to the pyrococcus-spirochete grouping within the archaeal branch of the class I LysRS phylogenetic tree, while U36 recognition was seen in other archaea and an example from the alpha-proteobacteria. Together with the corresponding phylogenetic relationships, these results suggest that despite its comparative rarity the distribution of class I LysRS conforms to the canonical archaeal-bacterial division. The only exception, suggested from both functional and phylogenetic data, appears to be the horizontal transfer of class I LysRS from a pyrococcal progenitor to a limited number of bacteria.     

Novel benzothiophene H1-antihistamines for the treatment of insomnia

SAR of lead benzothiophene H₁-antihistamine 2 was explored to identify backup candidates with suitable pharmacokinetic profiles for an insomnia program. Several potent and selective H₁-antihistamines with a range of projected half-lives in humans were identified. Compound 16d had a suitable human half-life as demonstrated in a human microdose study, but variability in pharmacokinetic profile, attributed to metabolic clearance, prevented further development of this compound. Compound 28b demonstrated lower predicted clearance in preclinical studies, and may represent a more suitable backup compound.     

Biochar,Bentonite and Zeolite Supplemented Feeding of Layer Chickens Alters Intestinal Microbiota and Reduces Campylobacter Load

A range of feed supplements, including antibiotics, have been commonly used in poultry production to improve health and productivity. Alternative methods are needed to suppress pathogen loads and maintain productivity. As an alternative to antibiotics use, we investigated the ability of biochar, bentonite and zeolite as separate 4% feed additives, to selectively remove pathogens without reducing microbial richness and diversity in the gut. Neither biochar, bentonite nor zeolite made any significant alterations to the overall richness and diversity of intestinal bacterial community. However, reduction of some bacterial species, including some potential pathogens was detected. The microbiota of bentonite fed animals were lacking all members of the order Campylobacterales. Specifically, the following operational taxonomic units (OTUs) were absent: an OTU 100% identical to Campylobacter jejuni; an OTU 99% identical to Helicobacter pullorum; multiple Gallibacterium anatis (>97%) related OTUs; Bacteroides dorei (99%) and Clostridium aldenense (95%) related OTUs. Biochar and zeolite treatments had similar but milder effects compared to bentonite. Zeolite amended feed was also associated with significant reduction in the phylum Proteobacteria. All three additives showed potential for the control of major poultry zoonotic pathogens.     

Den 1, Den2 and Den3, ATP-inhibited deoxyribonucleases from Dropsophila embryonic nuclei

Three Drosophila embryonic deoxyribonucleases, designated den1, den2 and den3, are identified in nuclear extracts separated by glycerol density gradient centrifugation. Den1, removes short products from the 5-ends of single-stranded DNA or double-stranded DNA with either blunt or 5-recessed termini. Den2 is inactive with single-stranded DNA and acts as 3-exonuclease with double-stranded DNA possessing either blunt or 3-recessed termini. Den3 preferentially uses partial duplex DNA containing single-stranded gap and it catalyzes hydrolysis, in 3-5 direction, of one of the shorter strands that flank the gap. Nucleolytic activities of den1, den2 and den3 are inhibited with ATP.     

Variations in Chemical Composition,Vasorelaxant and Angiotensin I‐Converting Enzyme Inhibitory Activities of Essential Oil from Aerial Parts of Seseli pallasii Besser (Apiaceae)

The present paper describes environmental and seasonal‐related chemical composition variations, vasorelaxant and angiotensin I‐converting enzyme (ACE) activities of essential oil from aerial parts of Seseli pallasii Besser . The composition was analyzed by GC and GC/MS. Monoterpenes were found to be the most abundant chemical class with α ‐pinene (42.7 – 48.2%) as the most prevalent component. Seseli pallasi essential oil relaxed isolated endothelium‐intact mesenteric arteries rings precontracted with phenylephrine with IC ₅₀ = 3.10 nl/ml (IC ₅₀ = 2.70 μg/ml). Also, S. pallasii essential oil was found to exhibit a dose‐dependent ACE inhibitory activity with an IC ₅₀ value of 0.33 mg/ml. In silico evaluation of ACE inhibitory activity of the individual components showed that spathulenol exhibited the best binding affinity with ACE, and the lowest binding energy of ?7.5 kcal/mol. The results suggested that combination of vasorelaxing and ACE inhibitory effects of the analyzed S. pallasii essential oil might have the potential therapeutic significance in hypertension.     

Insulin Mimetic Effect of Tungsten Compounds on Isolated Rat Adipocytes

Investigations of effective, orally active, and safe antidiabetic metallopharmaceuticals have been carried out during the last two decades. It has been reported that tungsten compounds mimic the action of insulin in intact cell systems. As insulin mimetics, the most investigated tungsten compound was sodium tungstate (ST), rarely investigated was tungstophosphoric acid (WPA), but never alanine complex of tungstophosphoric acid (WPA-A). In this study, the insulin mimetic activity of three different tungsten compounds, ST, WPA, and WPA-A, was evaluated by means of in vitro measurements of the glucose uptake and inhibition of free fatty acids release from epinephrine-treated isolated rat white adipocytes. We investigated the influence of concentration (lower and higher, 0.1 and 1.0 mM, respectively) and solvent: isotonic salt solution—saline (0.9% w/v of NaCl) and dimethyl sulfoxide (DMSO; 2% v/v), on the biological effect of tested compounds. Our experimental data showed that all of the three investigated tungsten compounds possess insulin mimetic activity in vitro on the isolated adipocytes. Influence of concentration and solvents on insulin mimetic effect for the certain tungsten compounds were: WPA was shown effect independently of concentration and solvents; higher concentration and DMSO were significant decreasing insulin mimetic effect of ST; lower concentration and saline led to decreasing effect of WPA-A. Generally, there were no differences in insulin mimetic effect of three tungsten compounds in lower concentration and dissolved in DMSO. When saline was used as solvent, it was needed higher concentration of investigated compounds to accomplish the same effect. In conclusion, our results suggest that low concentration (0.1 mM) of ST, WPA, and WPA-A dissolved in 2% DMSO could be the good candidates for in vivo investigation of their antidiabetic properties.     

Organization and nucleotide sequence of the Streptococcus mutans galactose operon

The galactose operon encoding a repressor and genes for the Leloir pathway for galactose metabolism (galactokinase, galactose-1-phosphate-uridyl transferase and UDP glucose-4-epimerase) was located adjacent to the multiple sugar metabolism (msm) operon on the chromosome of Streptococcus mutans Ingbritt (serotype c) and the complete nucleotide sequence of this 5-kilobase region was determined. The Leloir pathway was induced by the presence of galactose in the growth medium or following the release of intracellular galactose after uptake and cleavage of -galactosides by the multiple sugar metabolism system. Analysis of the mechanism of galactose transport confirmed the absence of a galactose-specific phosphotransferase system and suggested the presence of an inducible galactose permease. Evidence is presented that galactose transport is independent of the proton motive force and may be ATP-dependent.