Drosophila ventral furrow morphogenesis: a proteomic analysis

Ventral furrow formation is a key morphogenetic event during Drosophila gastrulation that leads to the internalization of mesodermal precursors. While genetic analysis has revealed the genes involved in the specification of ventral furrow cells, few of the structural proteins that act as mediators of ventral cell behavior have been identified. A comparative proteomics approach employing difference gel electrophoresis was used to identify more than fifty proteins with altered abundance levels or isoform changes in ventralized versus lateralized embryos. Curiously, the majority of protein differences between these embryos appeared well before gastrulation, only a few protein changes coincided with gastrulation, suggesting that the ventral cells are primed for cell shape change. Three proteasome subunits were found to differ between ventralized and lateralized embryos. RNAi knockdown of these proteasome subunits and time-dependent difference-proteins caused ventral furrow defects, validating the role of these proteins in ventral furrow morphogenesis.     

Involvement of extracellular Ca2+ influx through voltage-independent Ca2+ channels in endothelin-1 function

This article reviews the types and roles of voltage-independent Ca(2+) channels involved in the endothelin-1 (ET-1)-induced functional responses such as vascular contraction, cell proliferation, and intracellular Ca(2+)-dependent signaling pathways and discusses the molecular mechanisms for the activation of voltage-independent Ca(2+) channels by ET-1. ET-1 activates some types of voltage-independent Ca(2+) channels, such as Ca(2+)-permeable nonselective cation channels (NSCCs) and store-operated Ca(2+) channels (SOCC). Extracellular Ca(2+) influx through these voltage-independent Ca(2+) channels plays essential roles in ET-1-induced vascular contraction, cell proliferation, activation of epidermal growth factor receptor tyrosine kinase, regulation of proline-rich tyrosine kinase, and release of arachidonic acid. The experiments using various constructs of endothelin receptors reveal the importance of G(q) and G(12) families in activation of these Ca(2+) channels by ET-1. These findings provide a potential therapeutic mechanism of a functional interrelationship between G(q)/G(12) proteins and voltage-independent Ca(2+) channels in the pathophysiology of ET-1, such as in chronic heart failure, hypertension, and cerebral vasospasm.     

Protein fingerprinting profiles in different strains of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> isolated from diseased freshwater fish

Summary Aeromonas hydrophila(Ah) strains isolated from diseased fish in India were studied for protein profiling using the SDS-PAGE protein fingerprinting profile pattern of whole cells of 12 local strains of A. hydrophilaand one reference strain (MTCC 646). Variability among the strains was observed. The average similarity between the 12 strains of A. hydrophila ranged from 0.272 to 0.916. Proteins with molecular mass of 55.6 and 14.67 kDa in Ah1, Ah2 and Ah3, 28.5 and 27.9 kDa in Ah4, Ah5 and Ah6, 21.4 and 19.5 kDa in Ah7, Ah8, Ah9 and 72.9, 91.5 and 71.3 kDa in Ah10, Ah11 and Ah12 were common. The protein polypeptide bands from 19.5 to 86.2 kDa were common in both local strains and reference strain of A. hydrophila. The protein fingerprinting study showed that there is genetic similarity between strains of A. hydrophila and reference strain (MTCC 646). These protein markers may be useful for further strain differentiation in epidemiological study.     

Microbial production of 4-amino-1-butanol,a four-carbon amino alcohol

4-Amino-1-butanol (4AB) serves as an important intermediate compound for drugs and a precursor of biodegradable polymers used for gene delivery. Here, we report for the first time the fermentative production of 4AB from glucose by metabolically engineered Corynebacterium glutamicum harboring a newly designed pathway comprising a putrescine (PUT) aminotransferase (encoded by ygjG) and an aldehyde dehydrogenase (encoded by yqhD) from Escherichia coli, which convert PUT to 4AB. Application of several metabolic engineering strategies such as fine-tuning the expression levels of ygjG and yqhD, eliminating competing pathways, and optimizing culture condition further improved 4AB production. Fed-batch culture of the final metabolically engineered C. glutamicum strain produced 24.7 g/L of 4AB. The strategies reported here should be useful for the microbial production of primary amino alcohols from renewable resources.     

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single α-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured.       

Regulated Expression of a Cytokinin Biosynthesis Gene IPT Delays Leaf Senescence and Improves Yield under Rainfed and Irrigated Conditions in Canola (Brassica napus L.)

Delay of leaf senescence through genetic modification can potentially improve crop yield, through maintenance of photosynthetically active leaves for a longer period. Plant growth hormones such as cytokinin regulate and delay leaf senescence. Here, the structural gene (IPT) encoding the cytokinin biosynthetic enzyme isopentenyltransferase was fused to a functionally active fragment of the AtMYB32 promoter and was transformed into canola plants. Expression of the AtMYB32xs::IPT gene cassette delayed the leaf senescence in transgenic plants grown under controlled environment conditions and field experiments conducted for a single season at two geographic locations. The transgenic canola plants retained higher chlorophyll levels for an extended period and produced significantly higher seed yield with similar growth and phenology compared to wild type and null control plants under rainfed and irrigated treatments. The yield increase in transgenic plants was in the range of 16% to 23% and 7% to 16% under rainfed and irrigated conditions, respectively, compared to control plants. Most of the seed quality parameters in transgenic plants were similar, and with elevated oleic acid content in all transgenic lines and higher oil content and lower glucosinolate content in one specific transgenic line as compared to control plants. The results suggest that by delaying leaf senescence using the AtMYB32xs::IPT technology, productivity in crop plants can be improved under water stress and well-watered conditions.     

Solution NMR structure of CD1104B from pathogenic Clostridium difficile reveals a distinct α-helical architecture and provides first structural representative of protein domain family PF14203

A high-quality structure of the 68-residue protein CD1104B from Clostridium difficile strain 630 exhibits a distinct all α-helical fold. The structure presented here is the first representative of bacterial protein domain family PF14203 (currently 180 members) of unknown function (DUF4319) and reveals that the side-chains of the only two strictly conserved residues (Glu 8 and Lys 48) form a salt bridge. Moreover, these two residues are located in the vicinity of the largest surface cleft which is predicted to contribute to a surface area involved in protein–protein interactions. This, along with its coding in transposon CTn4, suggests that CD1104B (and very likely all members of Pfam 14203) functions by interacting with other proteins required for the transfer of transposons between different bacterial species.     

Maternal cholesterol levels during gestation: boon or bane for the offspring?

An increase in cholesterol levels is perceived during pregnancy and is considered as a normal adaptive response to the development of the fetus. In some pregnancies, excessive increase in total cholesterol with high levels of Low-Density Lipoprotein leads to maladaptation by the fetus to cholesterol demands, resulting in a pathological condition termed as maternal hypercholesterolemia (MH). MH is considered clinically irrelevant and therefore cholesterol levels are not routinely checked during pregnancy, as a consequence of which there is scarce information on its global prevalence in pregnant women. Studies have reported that MH during pregnancy can cause atherogenesis in adults emphasizing the concept of in utero programming of fetus. Moreover, Gestational Diabetes Mellitus, obesity and Polycystic Ovary Syndrome are potential risk factors which strengthen combined pathologies in placenta and fetuses of mothers with MH. However, lack of conclusive evidence on cholesterol transport and underlying programming demand substantial research to develop population-based life style strategies for women in their childbearing years. The current review focuses on the mechanisms and outcomes of MH from existing epidemiological as well as experimental data and presents a detailed insight on this novel risk factor of cardiovascular diseases.

     

Cloning and characterization of 2-C-methyl-D-erythritol-4-phosphate pathway genes for isoprenoid biosynthesis from Indian ginseng, Withania somnifera

Withania somnifera (L.) is one of the most valuable medicinal plants used in Ayurvedic and other indigenous medicines. Pharmaceutical activities of this herb are associated with presence of secondary metabolites known as withanolides, a class of phytosteroids synthesized via mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate pathways. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, not much is known about the genes responsible for biosynthesis of these compounds. In this study, we have characterized two genes encoding 1-deoxy-d-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7) and 1-deoxy-d-xylulose-5-phosphate reductase (DXR; EC 1.1.1.267) enzymes involved in the biosynthesis of isoprenoids. The full-length cDNAs of W. somnifera DXS (WsDXS) and DXR (WsDXR) of 2,154 and 1,428 bps encode polypeptides of 717 and 475 amino acids residues, respectively. The expression analysis suggests that WsDXS and WsDXR are differentially expressed in different tissues (with maximal expression in flower and young leaf), chemotypes of Withania, and in response to salicylic acid, methyl jasmonate, as well as in mechanical injury. Analysis of genomic organization of WsDXS shows close similarity with tomato DXS in terms of exon–intron arrangements. This is the first report on characterization of isoprenoid biosynthesis pathway genes from Withania.