Biochemical, molecular, and functional characterization of PISCF-allatostatin, a regulator of juvenile hormone biosynthesis in the mosquito Aedes aegypti

Aedes aegypti PISCF-allatostatin or allatostatin-C (Ae-AS-C) was isolated using a combination of high performance liquid chromatography and enzyme-linked immunosorbent assay (ELISA). The matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrum of positive ELISA fractions revealed a molecular mass of 1919.0 Da, in agreement with the sequence qIRYRQCYFNPISCF, with bridged cysteines. This sequence was confirmed by matrix-assisted laser desorption/ionization tandem TOF/TOF mass spectrometry analysis. The corresponding Ae-AS-C cDNA was amplified by PCR, and the sequence of the peptide was confirmed. An in vitro radiochemical assay was used to study the inhibitory effect of synthetic Ae-AS-C on juvenile hormone biosynthesis by the isolated corpora allata (CA) of adult female A. aegypti. The inhibitory action of synthetic Ae-AS-C was dose-dependent; with a maximum at 10(-9) m. Ae-AS-C showed no inhibitory activity in the presence of farnesoic acid, an immediate precursor of juvenile hormone, indicating that the Ae-AS-C target is located before the formation of farnesoic acid in the pathway. The sensitivity of the CA to inhibition by Ae-AS-C in the in vitro assay varied during the adult life; the CA was most sensitive during periods of low synthetic activity. In addition, the levels of Ae-AS-C in the brain were studied using ELISA and reached a maximum at 3 days after eclosion. These studies suggest that Ae-AS-C is an important regulator of CA activity in A. aegypti.     

A potentially insect-implantable trehalose electrooxidizing anode

The dominant sugar in the body fluids of many insects is not glucose, the sugar of the vertebrates, but trehalose. In a step toward a cell that would operate in insects, we describe here a trehalose electrooxidizing anode. The novel component of the anode is its engineered, trehalose oxidation catalyzing, FAD-glucose-3-dehydrogenase (G3DH). Screening for gene-sources of G3DH pointed to the G3DH of Agrobacterium tumefaciens. Sequencing of the A. tumefaciens genome revealed a 1.7 kb fragment which contained the G3DH coding gene. The fragment was isolated, cloned and expressed in E. coli strain BL-21, to yield the approximately 65 kDa his-tagged flavoenzyme, with a specific activity of approximately 2.5U/mg protein. Electrical wiring of its reaction center to a carbon electrode through a high apparent electron diffusion coefficient (5.8 x 10(-6)cm(2)/s) redox hydrogel with a -0.2V versus Ag/AgCl redox potential resulted in the trehalose electrooxidizing anode. Trehalose was electrooxidized at pH 7.2 already at -0.36 V versus Ag/AgCl. At 0 V versus Ag/AgCl the trehalose electrooxidation current density was 0.1 mA/cm(2).     

A novel antimicrobial peptide derived from modified N-terminal domain of bovine lactoferrin: Design,synthesis, activity against multidrug-resistant bacteria and Candida

Lactoferrin (LF) is believed to contribute to the host's defense against microbial infections. This work focuses on the antibacterial and antifungal activities of a designed peptide, L10 (WFRKQLKW) by modifying the first eight N-terminal residues of bovine LF by selective homologous substitution of amino acids on the basis of hydrophobicity, L10 has shown potent antibacterial and antifungal properties against clinically isolated extended spectrum beta lactamases (ESBL), producing gram-negative bacteria as well as Candida strains with minimal inhibitory concentrations (MIC) ranging from 1 to 8 μg/mL and 6.5 μg/mL, respectively. The peptide was found to be least hemolytic at a concentration of 800 μg/mL. Interaction with lipopolysaccharide (LPS) and lipid A (LA) suggests that the peptide targets the membrane of gram-negative bacteria. The membrane interactive nature of the peptide, both antibacterial and antifungal, was further confirmed by visual observations employing electron microscopy. Further analyses, by means of propidium iodide based flow cytometry, also supported the membrane permeabilization of Candida cells. The peptide was also found to possess anti-inflammatory properties, by virtue of its ability to inhibit cyclooxygenase-2 (COX-2). L10 therefore emerges as a potential therapeutic remedial solution for infections caused by ESBL positive, gram-negative bacteria and multidrug-resistant (MDR) fungal strains, on account of its multifunctional activities. This study may open up new approach to develop and design novel antimicrobials.     

Role of Secondary Metabolites and Brassinosteroids in Plant Defense Against Environmental Stresses

Being sessile, plants are subjected to a diverse array of environmental stresses during their life span. Exposure of plants to environmental stresses results in the generation of reactive oxygen species (ROS). These activated oxygen species tend to oxidize various cellular biomolecules like proteins, nucleic acids, and lipids, a process that challenges the core existence of the cell. To prevent the accumulation of these ROS and to sustain their own survival, plants have developed an intricate antioxidative defence system. The antioxidative defence system comprises various enzymatic and nonenzymatic molecules, produced to counter the adverse effect of environmental stresses. A sizable number of these molecules belong to the category of compounds called secondary metabolites. Secondary metabolites are organic compounds that are not directly involved in the growth and development of plants but perform specialized functions under a given set of conditions. Absence of secondary metabolites results in long-term impairment of the plant’s survivability. Such compounds generally include pigments, phenolics, and so on. Plant phenolic compounds such as flavonoids and lignin precursors have been reported to accumulate in response to various biotic and abiotic stresses and are regarded as crucial defence compounds that can scavenge harmful ROS. Another important category of plant metabolites, called brassinosteroids, exhibit stress regulatory and growth-promoting activity and are classified as phytohormones. Elucidation of the physiological and molecular effects of secondary metabolites and brassinosteroids have catapulted them as highly promising and environment-friendly natural substances, suitable for wider application in plant protection and crop yield promotion. The present review focuses on our current understanding of how plants respond to the generation of excessive ROS and the role of secondary metabolites and brassinosteroids in countering the adverse effects of environmental stresses.     

Casein kinase 1-dependent phosphorylation of familial advanced sleep phase syndrome-associated residues controls PERIOD 2 stability

The mammalian circadian clock component PERIOD2 (PER2) plays a critical role in circadian rhythm entrainment. Recently, a missense mutation at a putative phosphorylation site in hPER2, Ser-662, was identified in patients that suffer from familial advanced sleep phase syndrome (FASPS). Patients with FASPS display abnormal sleep-wake patterns characterized by a lifelong pattern of sleep onset in the early evening and offset in the early morning. Although the phosphorylation of PER2 is strongly implied from functional studies, it has not been possible to study the site-specific phosphorylation of PER2 on Ser-662, and the biochemical functions of this residue are unclear. Here, we used phospho-specific antibodies to show that PER2 is phosphorylated on Ser-662 and flanking casein kinase (CK) sites in vivo. The phosphorylation of PER2 was carried out by the combined activities of casein kinase 1δ (CK1 δ) and casein kinase 1ε (CK1ε) and was antagonized by protein phosphatase 1. PER2 phosphorylation was rapidly induced in response to circadian entrainment of mammalian cell lines and occurred in both cytosolic and nuclear compartments. Importantly, we found that the pool of Ser-662-phosphorylated PER2 proteins was more stable than the pool of total PER2 molecules, implying that the FASPS phosphorylation cluster antagonizes PER2 degradation. Consistent with this idea, a Ser-662→Ala mutation that abrogated PER2 phosphorylation significantly reduced its half-life, whereas a phosphomimetic Ser-662→Asp substitution led to an elevation in half-life. Our combined findings provide new insights into PER2 regulation and the biochemical basis of FASPS.     

An in vitro model of glucose and lipid metabolism in a multicompartmental bioreactor

The energy balance in vivo is maintained through inter-organ cross-talk involving several different tissues. As a first step towards recapitulating the metabolic circuitry, hepatocytes, endothelial cells and adipose tissue were connected in a multicompartmental modular bioreactor to reproduce salient aspects of glucose and lipid metabolism in vitro. We first examined how the two-way cellular interplay between adipose tissue and endothelial cells affects glucose and lipid metabolism. The hepatocyte cell line HepG2 was then added to the system, creating a three-way connected culture, to determine whether circulating metabolite concentrations were normalized, or whether metabolic shifts, which may arise when endothelial cells and adipose tissue are placed in connection, were corrected. The addition of hepatocytes to the system prevented the drop in the concentrations of glucose, L-alanine and lactate, and the rise in that of free fatty acids. There was no significant change in glycerol levels in either of the connected cultures. The results show that connected cultures recapitulate complex physiological systemic processes, such as glucose and lipid metabolism, and that the HepG2 hepatocytes normalize circulating metabolites in this in vitro environment in the presence of other cell types.     

Identification and Association Analysis of Castor Bean Orthologous Candidate Gene-Based Markers for High Oil Content in Jatropha curcas

Jatropha (Jatropha curcas) and castor bean (Ricinus communis) possess several taxonomic similarities, and their seeds contain a high proportion of oil (up to 40%) which has been used in various industrial products, including diesel oil. Thirty-two candidate genes responsible for fatty acid biosynthesis were identified in the castor bean genome sequence. Testing of 48 primer pairs from candidate gene regions, including 12 SSRs from castor bean on 54 genotypes of J. curcas, 65% amplified successfully on Jatropha out of which 20% showed polymorphisms. Jatropha genotypes, categorized for oil content, were used in association analysis of candidate gene regions with high oil content. One marker–trait association for the oil trait was identified. Stearoyl desaturase amplicon (700 bp) consisting of intron and exon (P?=?0.00013) showed association with high oil content in Jatropha genotypes. Sequencing of the 1.3-kb amplicon, including the 700-bp fragment of stearoyl desaturase, which had shown association with the high oil content, revealed SNPs in the exonic region. The SNPs resulted in substitution of leucine with glutamine in the open reading frame of stearoyl desaturase of low oil content genotypes. The molecular marker is expected to be useful in marker-assisted breeding of high oil content genotypes in Jatropha.     

Genetic divergence in wild population of <Emphasis Type="Italic">Labeo rohita</Emphasis> (Hamilton, 1822) from nine Indian rivers,analyzed through MtDNA cytochrome b region

The present study examined partial cytochrome b gene sequence of mitochondrial DNA for polymorphism and its suitability to determine the genetic differentiation in wild Labeo rohita. The 146 samples of L. rohita were collected from nine distant rivers; Satluj, Brahmaputra, Son, Chambal Mahanadi, Rapti, Chauka, Bhagirathi and Tons were analyzed. Sequencing of 307 bp of Cyto b gene revealed 35 haplotypes with haplotype diversity 0.751 and nucleotide diversity (π) 0.005. The within population variation accounted for 84.21% of total variation and 15.79% was found to among population. The total Fst value, 0.158 (P < 0.05) was found to be significant. The results concluded that the partial cyto b is polymorphic and can be a potential marker to determining genetic stock structure of L. rohita wild population.     

Matrix metalloproteinases (MMP-2 and MMP-9) activity in corneal ulcer and ocular surface disorders determined by gelatin zymography

The purpose of this paper is to determine the active form of matrix metalloproteinases (MMP-2 and MMP-9) in corneal ulcer and ocular surface disorder patients. A total of 35 patients of corneal ulcer, 20 patients of ocular surface disorders and 10 control subjects were included in this study and estimation of active form of MMP-2 and MMP-9 was done by gelatin zymography. Tear samples were collected by capillary tube method. Both pro- and active forms of MMP-9 were detected in 24 out of 35 patients with corneal ulcer and 15 out of 20 patients with ocular surface disorders. None of the patients were showing MMP-2 activity. Neither MMP-2 nor MMP-9 was detected in the control group. Active forms of MMP-9 are present in tears of severe ulcerative and ocular surface disorder patients. Thus, proteinase inhibitors have been recommended for the treatment of corneal ulcer and ocular surface disorders to reduced the progression of stromal ulcer and to minimize corneal scarring.