Brain-specific Angiogenesis Inhibitor-1 Signaling,Regulation, and Enrichment in the Postsynaptic Density

Brain-specific angiogenesis inhibitor-1 (BAI1) is an adhesion G protein-coupled receptor that has been studied primarily for its anti-angiogenic and anti-tumorigenic properties. We found that overexpression of BAI1 results in activation of the Rho pathway via a Gα_12/13-dependent mechanism, with truncation of the BAI1 N terminus resulting in a dramatic enhancement in receptor signaling. This constitutive activity of the truncated BAI1 mutant also resulted in enhanced downstream phosphorylation of ERK as well as increased receptor association with β-arrestin2 and increased ubiquitination of the receptor. To gain insights into the regulation of BAI1 signaling, we screened the C terminus of BAI1 against a proteomic array of PDZ domains to identify novel interacting partners. These screens revealed that the BAI1 C terminus interacts with a variety of PDZ domains from synaptic proteins, including MAGI-3. Removal of the BAI1 PDZ-binding motif resulted in attenuation of receptor signaling to Rho but had no effect on ERK activation. Conversely, co-expression with MAGI-3 was found to potentiate signaling to ERK by constitutively active BAI1 in a manner that was dependent on the PDZ-binding motif of the receptor. Biochemical fractionation studies revealed that BAI1 is highly enriched in post-synaptic density fractions, a finding consistent with our observations that BAI1 can interact with PDZ proteins known to be concentrated in the post-synaptic density. These findings demonstrate that BAI1 is a synaptic receptor that can activate both the Rho and ERK pathways, with the N-terminal and C-terminal regions of the receptor playing key roles in the regulation of BAI1 signaling activity.     

Protection against experimental salmonellosis by recombinant 49 kDa OMP of Salmonella enterica serovar Typhi: biochemical aspects

Typhoid fever is systemic illness caused by Salmonella enterica serovar Typhi (S. Typhi) in humans. Increasing multidrug resistant strains of S. Typhi and limited effect of available vaccines has necessitated exploring of new immunogens for protection against it. Earlier studies have shown that a crude preparation of outer membrane proteins (OMPs) of S. Typhi evokes strong immune response and induces a protective immunity against infection caused by diverse Gram-negative bacteria. In the present study we have evaluated the protective effect of a purified recombinant 49 kDa (r49kDa) OMP of S. Typhi alone or along with alum or complete Freund’s adjuvant, against a challenge by S. Typhi (0.4 × 50% lethal dose) by biochemical estimation of serum enzymes and oxidative stress enzymes in Swiss albino mice. There was a decrease in activity of alanine aminotransferase by 14.28%, 38.09%, 23.80%; aspartate aminotransferase by 6.25%, 25%, 16.25%; lipid peroxidation by 4.34%, 18.84%, 11.59%; and catalase by 8%, 14%, 10%, respectively, whereas increase in activity of reduced glutathione by 33.33%, 61.11%, 44.44%; glutathione peroxidase by 7%, 16%, 10%; and glutathione reductase by 8%, 20%, 12%, respectively, as compared to control animals challenged with bacteria without pre-immunization. The results indicated that immunization of mice with r49kDa OMP alone or in combination with adjuvants protected and normalized the liver. It reduces the development of oxidative stress in mice against Salmonella infection and the risk of getting typhoid. These results represent an additional supplement to our earlier reported data on protective immunity evoked by this protein.     

Isostructural unbranched alkyl‐chains as tools for stabilizing β‐turn structure

To investigate the structural role played by isostructural unbranched alkyl‐chains on the conformational ensemble and stability of β‐turn structures, the conformational properties of a designed model peptide: Plm‐Pro‐Gly‐Pda ( 1 , Plm: H₃C—(CH₂)₁₄—CONH—; Pda: —CONH— (CH₂)₁₄—CH₃) have been examined and compared with the parent peptide: Boc‐Pro‐Gly‐NHMe ( 2 , Boc: tert‐butoxycarbonyl; NHMe: N‐methylamide). The characteristic ¹³C NMR chemical‐shifts of the Pro C^β and C^γ resonances ascertained the incidence of an all‐trans peptide‐bond in low polarity deuterochloroform solution. Using FTIR and ¹H NMR spectroscopy, we establish that apolar alkyl‐chains flanking a β‐turn promoting Pro‐Gly sequence impart definite incremental stability to the well‐defined hydrogen‐bonded structure. The assessment of ¹H NMR derived thermodynamic parameters of the hydrogen‐bonded amide‐NHs via variable temperature indicate that much weaker hydrophobic interactions do contribute to the stability of folded reverse turn structures. The far‐UV CD spectral patterns of 1 and 2 in 2,2,2‐trifluoroethanol are consistent with Pro‐Gly specific type II β‐turn structure, concomitantly substantiate that the flanking alkyl‐chains induce substantial bias in enhanced β‐turn populations. In view of structural as well as functional importance of the Pro‐Gly mediated secondary structures, besides biochemical and biological significance of proteins lipidation via myristoylation or palmytoilation, we highlight potential convenience of the unbranched Plm and Pda moieities not only as main‐chain N‐ and C‐terminal protecting groups but also to mimic and stabilize specific isolated secondary and supersecondary structural components frequently observed in proteins and polypeptides. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 419–426, 2013.     

Proteome Analysis of Peroxisomes from Etiolated Arabidopsis Seedlings Identifies a Peroxisomal Protease Involved in β-Oxidation and Development

Plant peroxisomes are highly dynamic organelles that mediate a suite of metabolic processes crucial to development. Peroxisomes in seeds/dark-grown seedlings and in photosynthetic tissues constitute two major subtypes of plant peroxisomes, which had been postulated to contain distinct primary biochemical properties. Multiple in-depth proteomic analyses had been performed on leaf peroxisomes, yet the major makeup of peroxisomes in seeds or dark-grown seedlings remained unclear. To compare the metabolic pathways of the two dominant plant peroxisomal subtypes and discover new peroxisomal proteins that function specifically during seed germination, we performed proteomic analysis of peroxisomes from etiolated Arabidopsis (Arabidopsis thaliana) seedlings. The detection of 77 peroxisomal proteins allowed us to perform comparative analysis with the peroxisomal proteome of green leaves, which revealed a large overlap between these two primary peroxisomal variants. Subcellular targeting analysis by fluorescence microscopy validated around 10 new peroxisomal proteins in Arabidopsis. Mutant analysis suggested the role of the cysteine protease RESPONSE TO DROUGHT21A-LIKE1 in β-oxidation, seed germination, and growth. This work provides a much-needed road map of a major type of plant peroxisome and has established a basis for future investigations of peroxisomal proteolytic processes to understand their roles in development and in plant interaction with the environment.Peroxisomes, originally known as microbodies, are small and single-membrane eukaryotic organelles that compartmentalize various oxidative metabolic functions. Most peroxisomal matrix proteins carry a C-terminal tripeptide named PEROXISOME TARGETING SIGNAL TYPE1 (PTS1), and fewer contain an N-terminal nonapeptide, PTS2 (Lanyon-Hogg et al., 2010). PTS1 is further divided into major and minor PTS1s. Major PTS1 tripeptides, such as SKL> and SRL> (> represents the stop codon), are by themselves sufficient to direct a protein to the peroxisome (Reumann, 2004), whereas minor PTS1s are usually found in low-abundance proteins and require additional upstream elements for peroxisomal targeting (Kaur et al., 2009). Peroxisomes are highly variable morphologically and metabolically, as their size, shape, abundance, and enzymatic content can differ depending on the species, tissue and cell type, and prevailing environmental conditions (Beevers, 1979; van den Bosch et al., 1992; Kaur et al., 2009; Hu et al., 2012; Schrader et al., 2012).Plant peroxisomes participate in a wide range of metabolic processes, such as lipid metabolism, photorespiration, detoxification, biosynthesis of jasmonic acid, and metabolism of indole-3-butyric acid (IBA), nitrogen, sulfite, and polyamine (Kaur et al., 2009; Hu et al., 2012). Specific names had been given to certain types of peroxisomes due to their unique metabolic properties. For example, the term glyoxysome was coined when a new type of organelle that contained enzymes of the glyoxylate cycle was identified from the endosperm of castor bean (Ricinus communis; Breidenbach et al., 1968). It was later realized that glyoxysomes are in fact a type of peroxisome, and Beevers (1979) subsequently classified plant peroxisomes into three subtypes based on their primary biochemical functions. Glyoxysomes are located in storage organs such as fatty seedling tissues and play a major role in converting fatty acids to sugar; leaf peroxisomes are involved in photorespiration; and nonspecialized peroxisomes exist in other plant tissues and perform unknown functions.The primary function of leaf peroxisomes is the recycling of phosphoglycolate during photorespiration, a process coordinated by chloroplasts, peroxisomes, mitochondria, and the cytosol. In this pathway, phosphoglycolate produced by the oxygenase activity of Rubisco is ultimately converted to glycerate, which reenters the chloroplastic Calvin-Benson cycle (Foyer et al., 2009; Peterhansel et al., 2010). The peroxisome-localized enzymes glycolate oxidase (GOX), catalase, aminotransferase (serine:glyoxylate aminotransferase [SGT] and glutamate-glyoxylate aminotransferase [GGT]), HYDROXYPYRUVATE REDUCTASE1 (HPR1), and peroxisomal malate dehydrogenase (PMDH) are involved in the process (Reumann and Weber, 2006). On the other hand, lipid mobilization through fatty acid β-oxidation and the glyoxylate cycle is the main function for peroxisomes in seeds and germinating seedlings. In this process, fatty acids are first activated into fatty acyl-CoA esters by the acyl-activating enzyme (AAE)/acyl-CoA synthetase before entering the β-oxidation cycle, during which an acetyl-CoA is cleaved in each cycle by the successive action of acyl-CoA oxidase (ACX), multifunctional protein (MFP), and 3-keto-acyl-CoA thiolase (KAT). Acetyl-CoA, an end product of β-oxidation, is further converted to four-carbon carbohydrates by the glyoxylate cycle, in which isocitrate lyase (ICL) and malate synthase (MLS) are two key enzymes that function exclusively in this pathway. Products of the glyoxylate cycle exit the peroxisome, enter gluconeogenesis, and are further converted to hexose and Suc to fuel the postgerminative development of seedlings (Penfield et al., 2006).Immunocytochemical studies of germinating seeds from pumpkin (Cucurbita pepo), watermelon (Citrullis vulgaris), and cucumber (Cucumis sativus) demonstrated that seed peroxisomes (glyoxysomes) are directly transformed into leaf peroxisomes during greening of the cotyledons without de novo biogenesis of leaf peroxisomes (Titus and Becker, 1985; Nishimura et al., 1986; Sautter, 1986). This conversion was illustrated by the import of photorespiratory enzymes and their concomitant presence with glyoxylate cycle enzymes within the same organelle. Furthermore, the increase in abundance of photorespiratory enzymes coincided with the marked decrease, and subsequently the absence, of glyoxylate cycle enzymes (ICL and/or MLS) at the culmination of this process (Titus and Becker, 1985; Nishimura et al., 1986; Sautter, 1986). It was suggested that the specific names for plant peroxisomal variants should be eliminated because protein composition between leaf peroxisomes and glyoxysomes may differ by only two proteins (i.e. ICL and MLS) out of the over 100 total proteins in the peroxisome (Pracharoenwattana and Smith, 2008). This prediction needed to be tested. In addition, mutants lacking core peroxisome biogenesis factors or major β-oxidation enzymes are nonviable, suggesting that peroxisomes are essential to embryogenesis and seed germination (Hu et al., 2012). However, how peroxisomes contribute to seed germination and seedling establishment is not completely understood. In the past, studies have been successfully undertaken to catalog the proteome of mitochondria and plastids isolated from different plant tissues, which uncovered unique facets of organelle metabolism in various tissues (van Wijk and Baginsky, 2011; Havelund et al., 2013; Lee et al., 2013). As such, it was necessary to establish a protein atlas for peroxisomes in dark-grown seedlings.Proteomic analyses of leaf peroxisomes and peroxisomes from suspension-cultured, leaf-derived cells followed by protein subcellular localization studies confirmed a total of over 30 new peroxisomal proteins, uncovering additional metabolic functions for leaf peroxisomes (Fukao et al., 2002; Reumann et al., 2007, 2009; Eubel et al., 2008; Babujee et al., 2010; Kataya and Reumann, 2010; Quan et al., 2010). For Arabidopsis (Arabidopsis thaliana), around 100 peroxisomal proteins were shown to be present in leaves or leaf-derived cells. Compared with the over 80 bona fide peroxisomal proteins detected by leaf peroxisomal proteomics (Reumann et al., 2007, 2009), the number of proteins identified from peroxisomal proteomic studies on etiolated seedlings was significantly smaller, with less than 10 known peroxisomal proteins from Arabidopsis (Fukao et al., 2003) and approximately 31 from soybean (Glycine max; Arai et al., 2008a, 2008b). Thus, a more in-depth analysis of the proteome of peroxisomes from these tissues was highly needed.Here, we performed proteomic analysis of peroxisomes isolated from etiolated Arabidopsis seedlings and detected peroxisomal proteins that encompass most of the known plant peroxisomal metabolic pathways. Fluorescence microscopy verified the peroxisomal localization of a number of proteins newly identified in this study or detected from previous proteomics that had not been verified by independent means. Reverse genetic analysis demonstrated the role for a Cys protease in germination, β-oxidation, and growth.     

Cytomorphological studies in some members of tribe Paniceae (Poaceae) from district Kangra of Himachal Pradesh (Western Himalayas)

The present paper deals with cytological studies on the population basis of 21 species belonging to 9 genera of tribe Paniceae of family Poaceae from cytologically unexplored area of Western Himalayas i.e. district Kangra of Himachal Pradesh for the assessment of genetic diversity of grass flora. On world-wide basis, the chromosome counts have been made for the first time for three species such as Brachiaria remota (n = 16), Digitaria granularis (n = 36) and Isachne albens (n = 5). Similarly, on India basis, altogether new records are made for two species such as Echinochloa cruspavonis (n = 27) and Paspalum distichum (2n = 50). A comparison of the different euploid cytotypes studied at present for Digitaria adscendens, D. setigera and Oplismenus compositus revealed significant variations in their morphology, depicting increase in some of the characters of polyploid cytotypes. The course of meiosis has been observed to be normal in all the studied populations with high pollen fertility except for two species such as Paspalum dilatatum and P. distichum marked with abnormal meiosis and reduced pollen fertility.     

Effect of Feeding Inorganic Chromium on Growth Performance, Endocrine Variables, and Energy Metabolites in Winter-Exposed Buffalo Calves (Bubalus bubalis)

We investigated the effect of chromium (Cr) supplementation on the growth performance, energy metabolites, and hormonal variation in winter-exposed buffalo calves. Twenty-four female buffalo calves were randomly allotted to four dietary treatments (n?=?6) for a period of 120 days. Feeding regimen was the same in all the groups, except the animals in the four respective groups were additionally supplemented with 0.0, 0.5, 1.0, and 1.5 mg of Cr/kg DM in the form of CrCl₃.6H₂O. Calves were monitored daily for physiological variables and dry matter intake (DMI). Blood samples were collected at fortnightly intervals from each buffalo calves to measure concentrations of hormones (insulin, cortisol, and growth hormone), energy metabolites (glucose and non-esterified fatty acids), and plasma mineral levels. After 120 days of feeding trial, buffalo calves fed with Cr had lower (P?<?0.05) circulating plasma concentrations of glucose, insulin, and cortisol hormones, whereas plasma thyroid hormone and non-esterified fatty acids concentrations were found similar (P?>?0.05) among all the treatments. The results suggested that dietary Cr supplementation influenced plasma Cr levels without affecting the plasma concentrations of other trace minerals. However, physiological variables, nutrient intake, and growth performance of buffalo calves did not differ among all treatments (P?>?005). In summary, the current study showed that supplementation of Cr at the level of 1.0 and 1.5 mg of Cr/kg DMI was more effective in improving glucose utilization by increasing potency of insulin hormone and reducing concentration of cortisol hormone. Results also suggested that supplemental Cr also improves blood plasma Cr levels.     

Nitric Oxide Modulates the Expression of Proteins and Promotes Epiphyllous Bud Differentiation in Kalanchoe pinnata

Nitric oxide (NO), a recent addition to the signaling molecules in plants, plays an important role in mediating both biotic and abiotic stress responses. The occurrence of reproductive/vegetative structures, known as epiphylly, on the surface of leaves is a stress survival mechanism exhibited by some plants, including Kalanchoe pinnata. In the present study the role of NO during epiphyllous bud differentiation was investigated. NO donors l-arginine, NaNO₂, and SNP promoted epiphyllous bud differentiation in a dose-dependent manner, whereas NO inhibitors reversed the effect, suggesting the involvement of NO in the process. Albeit numerous physiological processes are reported to be modulated by NO, but their correlation with NO-responsive genes and proteins needs to be established. To address this issue, NO-responsive proteins (NORPs) were identified using 2D-PAGE and MS analysis. Major NORPs that were identified belonged to photosynthesis, oxidative phosphorylation, signaling, and stress metabolism, aptly indicating their probable role in this stress-induced growth process. The present study clearly indicates the role of NO in this stress survival mechanism, thus strongly affirming its role in stress responses.     

L-Asparaginase: A Promising Chemotherapeutic Agent

ABSTRACT

This article comprises detailed information about L-asparaginase, encompassing topics such as microbial and plant sources of L-asparaginase, treatment with L-asparaginase, mechanism of action of L-asparaginase, production, purification, properties, expression and characteristics of l-asparaginase along with information about studies on the structure of L-asparaginase. Although L-asparaginase has been reviewed by , our effort has been to include recent and updated information about the enzyme covering new aspects such as structural modification and immobilization of L-asparaginase, recombinant L-asparaginase, resistance to L-asparaginase, methods of assay of L-asparagine and L-asparaginase activity using the biosensor approach, L-asparaginase activity in soil and the factors affecting it. Also, side-effects of L-asparaginase treatment in acute lymphoblastic leukemia (ALL) have been discussed in the current review. L-asparaginase has been and is still one of the most widely studied therapeutic enzymes by researchers and scientists worldwide.     

Experimental validation of DNA interactions with nanoparticles derived from metal coupled amphiphiles

In the present report, a facile strategy for the synthesis of copper nanoparticles utilizing copper@cetylpyridinium chloride as the metal precursor in combination with vitamin C, was been developed. Synthesized nanoparticles (NPs) were well characterized through UV-Vis spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, energy dispersive X-ray (EDX) spectroscopy, and powder X-ray diffraction (XRD). The as-obtained NPs were used for binding with deoxyribonucleic acid from calf thymus (CT-DNA). Binding potential of synthesized NPs towards DNA was checked by calculating apparent binding constant and various thermodynamic parameters, like ΔG, ΔH, ΔS and number of binding sites from UV-Vis, circular dichroism, and fluorescence spectroscopy. NPs lead to the change in conformation and mobility of the genomic DNA as notify by the circular dichroism and DNA gel electrophoresis. Synergistic effect of synthesized nanoparticles on DNA was also visualized by the tapping mode atomic force microscopy. Research findings of the present work are expected to have an impact on genomic activities.     

Identification of inhibitors against interaction between pro-inflammatory sPLA2-IIA protein and integrin αvβ3

Increased concentrations of secreted phospholipase A2 type IIA (sPLA2-IIA), have been found in the synovial fluid of patients with rheumatoid arthritis. It has been shown that sPLA2-IIA specifically binds to integrin αvβ3, and initiates a signaling pathway that leads to cell proliferation and inflammation. Therefore, the interaction between integrin and sPLA2-IIA could be a potential therapeutic target for the treatment of proliferation or inflammation-related diseases. Two one-bead-one-compound peptide libraries were constructed and screened, and seven target hits were identified. Herein we report the identification, synthesis, and biological testing of two pyrazolylthiazole-tethered peptide hits and their analogs. Biological assays showed that these compounds were able to suppress the sPLA2-IIA–integrin interaction and sPLA2-IIA-induced migration of monocytic cells and that the blockade of the sPLA2-IIA–integrin binding was specific to sPLA2-IIA and not to the integrin.