A radiation hybrid map of bovine X Chromosome (BTAX)

We present a comprehensive radiation hybrid map of the bovine X chromosome (Chr) containing 20 new markers, including both microsatellites and expressed genes. This study was conducted with a 5000-rad whole genome RH cell panel consisting of 90 hybrid cell lines. Retention frequencies of individual markers range from 7.8% for XIST to 31.1% for TGLA325. Statistical analysis with RHMAPPER placed all the loci into five linkage groups under a LOD score criterion of 6.0. These groups could be oriented relative to each other because they included multiple microsatellite loci from the consensus linkage map of the X Chr. Markers included in both this RH map and the bovine cytogenetic map were in a consistent order. The comparative bovine–human map thus generated consists of five blocks of genes, the order of which is conserved, although in the opposite direction when presented as ideograms with p and q arms. Inversions of three blocks account for the difference in gene order across the entirety of the two X Chrs.     

Novel TCAP Mutation c.32C>A Causing Limb Girdle Muscular Dystrophy 2G

TCAP encoded telethonin is a 19 kDa protein, which plays an important role in anchoring titin in Z disc of the sarcomere, and is known to cause LGMD2G, a rare muscle disorder characterised by proximal and distal lower limb weakness, calf hypertrophy and loss of ambulation. A total of 300 individuals with ARLGMD were recruited for this study. Among these we identified 8 clinically well characterised LGMD2G cases from 7 unrelated Dravidian families. Clinical examination revealed predominantly proximo - distal form of weakness, scapular winging, muscle atrophy, calf hypertrophy and foot drop, immunoblot showed either complete absence or severe reduction of telethonin. Genetic analysis revealed a novel nonsense homozygous mutation c.32C>A, p.(Ser11*) in three patients of a consanguineous family and an 8 bp homozygous duplication c.26_33dupAGGTGTCG, p.(Arg12fs31*) in another patient. Both mutations possibly lead to truncated protein or nonsense mediated decay. We could not find any functionally significant TCAP mutation in the remaining 6 samples, except for two other polymorphisms, c.453A>C, p.( = ) and c.-178G>T, which were found in cases and controls. This is the first report from India to demonstrate TCAP association with LGMD2G.     

Mechanism for the Selective Interaction of C-terminal Eps15 Homology Domain Proteins with Specific Asn-Pro-Phe-containing Partners

Epidermal growth factor receptor tyrosine kinase substrate 15 (Eps15) homology (EH)-domain proteins can be divided into two classes: those with an N-terminal EH-domain(s), and the C-terminal Eps15 homology domain-containing proteins (EHDs). Whereas many N-terminal EH-domain proteins regulate internalization events, the best characterized C-terminal EHD, EHD1, regulates endocytic recycling. Because EH-domains interact with the tripeptide Asn-Pro-Phe (NPF), it is of critical importance to elucidate the molecular mechanisms that allow EHD1 and its paralogs to interact selectively with a subset of the hundreds of NPF-containing proteins expressed in mammalian cells. Here, we capitalize on our findings that C-terminal EH-domains possess highly positively charged interaction surfaces and that many NPF-containing proteins that interact with C-terminal (but not N-terminal) EH-domains are followed by acidic residues. Using the recently identified EHD1 interaction partner molecule interacting with CasL (MICAL)-Like 1 (MICAL-L1) as a model, we have demonstrated that only the first of its two NPF motifs is required for EHD1 binding. Because only this first NPF is followed by acidic residues, we have utilized glutathione S-transferase pulldowns, two-hybrid analysis, and NMR to demonstrate that the flanking acidic residues “fine tune” the binding affinity to EHD1. Indeed, our NMR solution structure of the EHD1 EH-domain in complex with the MICAL-L1 NPFEEEEED peptide indicates that the first two flanking Glu residues lie in a position favorable to form salt bridges with Lys residues within the EH-domain. Our data provide a novel explanation for the selective interaction of C-terminal EH-domains with specific NPF-containing proteins and allow for the prediction of new interaction partners with C-terminal EHDs.     

Pulsatile LH secretion and ovarian follicular wave emergence and growth in anestrous ewes

The objective of this study was to determine if pulsatile LH secretion was needed for ovarian follicular wave emergence and growth in the anestrous ewe. In Experiment 1, ewes were either large or small (10 × 0.47 or 5 × 0.47 cm, respectively; n = 5/group) sc implants releasing estradiol-17 beta for 10 d (Day 0 = day of implant insertion), to suppress pulsed LH secretion, but not FSH secretion. Five sham-operated control ewes received no implants. In Experiment 2, 12 ewes received large estradiol-releasing implants for 12 d (Day 0 = day of implant insertion); six were given GnRH (200 ng IV) every 4 h for the last 6 d that the implants were in place (to reinitiate pulsed LH secretion) whereas six Control ewes were given saline. Ovarian ultrasonography and blood sampling were done daily; blood samples were also taken every 12 min for 6 h on Days 5 and 9, and on Days 6 and 12 of the treatment period in Experiments 1 and 2, respectively. Treatment with estradiol blocked pulsatile LH secretion (P < 0.001). In Experiment 1, implant treatment halted follicular wave emergence between Days 2 and 10. In Experiment 2, follicular waves were suppressed during treatment with estradiol, but resumed following GnRH treatment. In both experiments, the range of peaks in serum FSH concentrations that preceded and triggered follicular wave emergence was almost the same as control ewes and those given estradiol implants alone or with GnRH; mean concentrations did not differ (P < 0.05). We concluded that some level of pulsatile LH secretion was required for the emergence of follicular waves that were triggered by peaks in serum FSH concentrations in the anestrous ewe.     

A review of multifunctional nanoemulsion systems to overcome oral and CNS drug delivery barriers

The oral and central nervous systems (CNS) present a unique set of barriers to the delivery of important diagnostic and therapeutic agents. Extensive research over the past few years has enabled a better understanding of these physical and biological barriers based on tight cellular junctions and expression of active transporters and metabolizing enzymes at the luminal surfaces of the gastrointestinal (GI) tract and the blood-brain barrier (BBB). This review focuses on the recent understanding of transport across the GI tract and BBB and the development of nanotechnology-based delivery strategies that can enhance bioavailability of drugs. Multifunctional lipid nanosystems, such as oil-in-water nanoemulsions, that integrate enhancement in permeability, tissue and cell targeting, imaging, and therapeutic functions are especially promising. Based on strategic choice of edible oils, surfactants and additional surface modifiers, and different types of payloads, rationale design of multifunctional nanoemulsions can serve as a safe and effective delivery vehicle across oral and CNS barriers.     

Antiviral activity of 2,3′-anhydro and related pyrimidine nucleosides against hepatitis B virus

Various 2,3′-anhydro analogs of 5-substituted 1-(2-deoxy-β-d-lyxofuranosyl)uracils (10–15) and a related 1-(3-O-mesyl-2-deoxy-β-d-lyxofuranosyl) pyrimidine nucleoside analog (18) have been synthesized for evaluation as a new class of potential anti-HBV agents. The compounds 10, 12, and 15 demonstrated most potent anti-HBV activities against duck HBV (DHBV) and human HBV with EC₅₀ values in the range of 2.5–10 and 5–10 μg/mL, respectively, at non-toxic concentrations (CC₅₀ = >200 μg/mL). The nucleoside 18 also demonstrated significant anti-HBV activity against DHBV with an EC₅₀ value of 2.5 μg/mL, however, it was less active against HBV in 2.2.15 cells (EC₅₀ = >10 μg/mL).     

Synthesis of substituted aryloxy alkyl and aryloxy aryl alkyl imidazoles as antileishmanial agents

A series of aryloxy alkyl/aryl alkyl imidazoles were synthesized and evaluated in vitro as antileishmanials against Leishmania donovani. All the 19 compounds exhibited 94–100% inhibition at 10 μg/mL against promastigotes and 12 compounds exhibited high inhibition with an IC₅₀ in the range of 0.47–4.85 μg/mL against amastigotes. Promising compounds were tested further in vivo. Among all, compounds 4 and 23 with 4-CF₃ aryloxy moiety exhibited medium in vivo inhibition of 58–60%, thus providing new structural lead for antileishmanials.     

Characterisation of the dynamic behaviour of lipid droplets in the early mouse embryo using adaptive harmonic generation microscopy

Background  

Lipid droplets (LD) are organelles with an important role in normal metabolism and disease. The lipid content of embryos has a major impact on viability and development. LD in Drosophila embryos and cultured cell lines have been shown to move and fuse in a microtubule dependent manner. Due to limitations in current imaging technology, little is known about the behaviour of LD in the mammalian embryo. Harmonic generation microscopy (HGM) allows one to image LD without the use of exogenous labels. Adaptive optics can be used to correct aberrations that would otherwise degrade the quality and information content of images.