A Non-Synonymous HMGA2 Variant Decreases Height in Shetland Ponies and Other Small Horses

The identification of quantitative trait loci (QTL) such as height and their underlying causative variants is still challenging and often requires large sample sizes. In humans hundreds of loci with small effects control the heritable portion of height variability. In domestic animals, typically only a few loci with comparatively large effects explain a major fraction of the heritability. We investigated height at withers in Shetland ponies and mapped a QTL to ECA 6 by genome-wide association (GWAS) using a small cohort of only 48 animals and the Illumina equine SNP70 BeadChip. Fine-mapping revealed a shared haplotype block of 793 kb in small Shetland ponies. The HMGA2 gene, known to be associated with height in horses and many other species, was located in the associated haplotype. After closing a gap in the equine reference genome we identified a non-synonymous variant in the first exon of HMGA2 in small Shetland ponies. The variant was predicted to affect the functionally important first AT-hook DNA binding domain of the HMGA2 protein (c.83G>A; p.G28E). We assessed the functional impact and found impaired DNA binding of a peptide with the mutant sequence in an electrophoretic mobility shift assay. This suggests that the HMGA2 variant also affects DNA binding in vivo and thus leads to reduced growth and a smaller stature in Shetland ponies. The identified HMGA2 variant also segregates in several other pony breeds but was not found in regular-sized horse breeds. We therefore conclude that we identified a quantitative trait nucleotide for height in horses.     

Alpha,beta-dehydrophenylalanine containing cecropin-melittin hybrid peptides: conformation and activity.

Synthesis and conformational studies of a cecropin-melittin hybrid pentadecapeptide CA(1-7)MEL(2-9), and its three alpha, beta-dehydrophenylalanine (DeltaPhe) containing analogs in water-TFE mixtures are described. DeltaPhe is placed at strategic positions in order to preserve the amphipathicity of the molecule. The wild type CAMEL0 and its three analogs, containing one, two and three DeltaPhe residues namely CAMELDeltaPhe1, CAMELDeltaPhe2 and CAMELDeltaPhe3 respectively were synthesized in solid phase and their conformation determined by CD and NMR. CAMELDeltaPhe2 and CAMELDeltaPhe3 peptides exhibit the presence of 3(10)-helix and beta-turns in the former and only turns in the latter. CAMELDeltaPhe1 peptide was found to have a largely extended conformation. Antibacterial and hemolytic activities of the peptides were also evaluated. CAMELDeltaPhe2 peptide is maximally potent against both Staphylococcus aureus ATCC 259230 and Escherichia coli ATCC 11303. CAMELDeltaPhe1 with a single DeltaPhe at the center shows minimal hemolysis.     

Plant regeneration from callus and protoplasts of Brassica nigra (IC 257) through somatic embryogenesis

A method to obtain plants from embryogenic callus of Brassica nigra and protoplasts of hypocotyl expiants is described. Callus was initiated on Murashige and Skoog medium containing kinetin (kn) and 2,4-dichlorophenoxy acetic acid (2,4-D). Lowering of auxin induced embryo formation. Supplementation with gibberellic acid (GA₃) enhanced embryogenic response tenfold. Passage through liquid medium devoid of growth regulators was essential for the growth of embryos. Secondary embryos were produced on transfer to solid basal medium. Embryogenic callus retained its morphogenic ability even after 12 subcultures. Both primary and secondary embryos produced fertile plants. Hypocotyl-derived protoplasts were also regenerated to plants following the same protocol. The survival of plants on transfer to soil was about 80%. The seeds from plants derived from callus and protoplasts were viable.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA naphthalene acetic acid - IAA indole acetic acid - kn kinetin - GA₃ gibberellic acid     

Calcium entry in rat parotid acinar cells

Conclusions While it is generally accepted that Ca²⁺ plays an important regulatory role in the physiology of a number of non-excitable cells, the mechanisms which regulate intracellular [Ca²⁺ are far from well established. Ca²⁺ transporting mechanisms which distribute Ca²⁺ intracellularly as well as those which allow influx of extracellular Ca²⁺ are involved in mediating intracellular Ca²⁺ homestasis. In this paper we have described recent studies on the regulation of the Ca²⁺ influx system in the data, it appears that the process of Ca²⁺ entry is extremely complex and may involve several levels of regulation. Understanding the molecular basis of these regulatory mechanisms presents a challeging problem for future studies.     

Activation of the inositol trisphosphate second messenger system by cAMP in a mouse fibroblast cell line

Intracellular Ca²⁺ mobilization events were assessed in mouse L cells, which contain native prostaglandin E₁ receptors and transfected human ₂ adrenergic receptors. Both Fura2 (single cell measurements) and Quin 2, (cuvette assays) were used to determine [Ca²⁺]_i levels. Our results demonstrate that in the transfected cells there is a dose-dependent increase in [Ca²⁺]_i in response to isoproterenol (0.1 nM–100 nM), which is inhibited by the -adrenergic antagonist, propranolol, and is a result of intracellular Ca²⁺ release. [Ca²⁺]₁ in these cells was also increased by prostaglandin E₁, 8 bromo cyclic AMP, and aluminum fluoride. Both 8 bromo cAMP and isoproterenol induced a rapid increase in the levels of IP₁, IP₂, and IP₃. The data presented demonstrate that the elevation of intracellular cyclic AMP induces an increase in IP₃ production which leads to an elevation in [Ca²⁺];. We propose that this cyclic AMP dependent activation of the IP₃ generating system occurs at a post-receptor site.Abbreviations cAMP Adenosine Cyclic 3-5-Monophosphate - [Ca²⁺]_i intracellular [Ca²⁺]_i - 8 Br cAMP 8 Bromo Adenosine Cyclic 3-5-Monophosphate - DAG Diacylglycerol - EGTA] [Ethylene Bis (oxyethylenenitrilo)] Tetracetic acid - BSA Bovine Serum Albumin - HBSS-H Hanks' Balanced Salt Solution buffered with HEPES to pH 7.4 - HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - PIP₂ Phosphatidylinositol 4,5-bisphosphate - IP₂ Inositol 4 Phosphate - IP₂ Inositol 4,5 Bisphosphate - IP₃ Inositol Trisphosphate - PGE₁ Prostaglandin E₁ - PBS Phosphate Buffered Saline Solution     

beta-Glucosidase of Penicillium funiculosum. I. Purification

A strain of Penicillium funiculosum, isolated in this laboratory, produced in high yield both endo- and exo-glucanases and beta-glucosidases, which were suitable for the saccharification of cellulosic materials. The isolation of the beta-glucosidase of this organism, which differs from other beta-glucosidases of fungi in its substrate specificity, by preparative electrophoresis, is described in this article. The organism was grown on a lactose-casein medium and the culture filtrate concentrated by ammonium sulfate precipitation and dialysis. Electrophoresis was carried out on large slabs of polyacrylamide gel in an anodicrun in the presence of borate at pH 7. After elution of active fractions, a cathodic run was made at pH 6.0. Two precipitations with ammonium sulfate resulted in a homogeneous enzyme (specific activity 174 IU/mg). A second isozyme was also produced by P. funiculosum on cellulose-wheat bran medium. This isozyme was purified by electrophoresis at pH 7.0 in the absence of borate and was obtained free from other isozymes of beta-glucosidase and cellulases.     

beta-Glucosidase of Penicillium funiculosum. II. Properties and mycelial binding

The properties of two isozymes of beta-glucosidase of Penicillium funiculosum (part I of this series) are described. The molecular weights of isozyme 1 was 2.3 x 10(5) by gel filtration and 1.2 x 10(5) by SDS gel electrophoresis, indicating two subunits. The molecular weight of isozyme 2 was unusually low for a fungal beta-glucosidase: 1.6 x 10(4) by gel filtration and 3.7 x 10(4) in the presence of isopropanol. The two enzymes differed from other fungal beta-glucosidases in their substrate specificities. They showed high activity with pNPG, cellobiose, cellotriose, cellotetraose, cellopentaose, gentiobiose, and laminarin, but were inactive with filter paper, CM cellulose, or derivatives or stabilized by bovine serum albumin and several alcohols such as butanol and propanol. It was inhibited by glucono-delta-lactone (K(i) = 0.67muM) and glucose (K(i) = 0.92mM).The enzyme was quantitatively adsorbed by P. funiculosum mycelium at pH 4 and the immobilized enzyme was as enzymically active as the free enzyme, but more heat stable. The binding efficiency was very high (5000 IU enzyme/g mycelium). It could be quantitatively eluted with buffers at pH 7 or by 0.02M Ca, Mg, or Al chlorides. The binding was selective, since mycelium grown on lactose could produce and also bind only beta-glucosidase isozyme 1, whereas mycelium grown on cellulose could produce as well as bind both beta-glucosidase isozymes as well as cellulases. Mycelial binding was unaffected by washing with EDTA or trypsinization, but was totally lost by washing with dilute KOH, HCl, or ethylenediamine.     

Minimum inhibitory concentration of drugs againstMycobacterium leprae as determined by anin vitro assay

The observations that liveMycobacterium leprae after entry into cultured peritoneal macrophages from mice, reduced the EA rosetting macrophages, have been exploited to determine the minimum inhibitory concentration of diamino diphenyl sulphone and rifampicin. Diamino diphenyl sulphone showed a minimum inhibitory concentration of 0.028 μg/ml and rifampicin 0.11μg/ml when given externally. However, there was accumulation of diamino diphenyl sulphone inside the macrophages. At an external concentration of 0.028 μg/ml the concentration inside the macrophage was 0.5μg/ml. The minimum inhibitory concentration for diamino diphenyl sulphone in this assay system is higher by several folds and that for rifampicin is slightly lower, than what is reported earlier with mice foot pad experiments. The minimum inhibitory concentration reported in this assay system is quite close to what is observed forin vitro inhibition ofMycobacterium lufu with both the drugs