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In recent years zebrafish have become commonly used as a model for studying human traits and disorders. Their small size, high fecundity, and rapid development allow for more high-throughput experiments compared to other vertebrate models. Given that zebrafish share >70% gene homologs with humans and their genomes can be readily edited using highly efficient CRISPR methods, we are now able to rapidly generate mutations impacting practically any gene of interest. Unfortunately, our ability to phenotype mutant larvae has not kept pace. To address this challenge, we have developed a protocol that obtains multiple phenotypic measurements from individual zebrafish larvae in an automated and parallel fashion, including morphological features (i.e., body length, eye area, and head size) and movement/behavior. By assaying wild-type zebrafish in a variety of conditions, we determined optimal parameters that avoid significant developmental defects or physical damage; these include morphological imaging of larvae at two time points (3 days post fertilization (dpf) and 5 dpf) coupled with motion tracking of behavior at 5 dpf. As a proof-of-principle, we tested our approach on two novel CRISPR-generated mutant zebrafish lines carrying predicted null-alleles of syngap1b and slc7a5, orthologs to two human genes implicated in autism-spectrum disorder, intellectual disability, and epilepsy. Using our optimized high-throughput phenotyping protocol, we recapitulated previously published results from mouse and zebrafish models of these candidate genes. In summary, we describe a rapid parallel pipeline to characterize morphological and behavioral features of individual larvae in a robust and consistent fashion, thereby improving our ability to better identify genes important in human traits and disorders.AUTHOR SUMMARYZebrafish (Danio rerio) are a well-established model organism for the study of neurodevelopmental disorders. Due to their small size, fast reproduction, and genetic homology with humans, zebrafish have been widely used for characterizing and screening candidate genes for many disorders, including autism-spectrum disorder, intellectual disability, and epilepsy. Although several studies have described the use of high-throughput morphological and behavioral assays, few combine multiple assays in a single zebrafish larva. Here, we optimized a platform to characterize morphometric features at two developmental time points in addition to behavioral traits of zebrafish larvae. We then used this approach to characterize two autism candidate genes (SYNGAP1 and SLC7A5) in two CRISPR-generated zebrafish null mutant models we developed in house. These data recapitulate previously published results related to enhanced seizure activity, while identifying additional defects not previously reported. We propose that our phenotyping platform represents a feasible method for maximizing the use of single zebrafish larvae in the characterization of additional mutants relevant to neurodevelopmental disorders.

Fruits and vegetables are the primary part of healthy human diet. Spectroscopy in various forms has emerged as a reliable and efficient technique for processing, value addition and quality assessment of the fruits and vegetables.

Green synthesis of metal oxide nanoparticles using plant extract is a promising alternative to traditional method of chemical synthesis. In this paper, we report the synthesis of nanostructured zinc oxide particles by biological method. Highly stable and spherical zinc oxide nanoparticles are produced by using zinc acetate and Ixora coccinea leaf extract. Formation of zinc oxide nanoparticles has been confirmed by UV-Vis absorption spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Dynamic light scattering analysis (DLS), zetapotential study and Scanning Electron Microscope with the Energy Dispersive X-ray studies (EDX). Dynamic light scattering analysis shows average particle size of 145.1 nm whereas high zeta potential value confirms the stability of formed zinc oxide nanoparticles. The Scanning Electron Microscope reveals spherical morphology of nanoparticles and Energy Dispersive X-ray analysis confirms the formation of highly pure zinc oxide nanoparticles. The zinc oxide nanoparticles from Ixora coccinea leaves are expected to have applications in biomedical, cosmetic industries, biotechnology, sensors, medical, catalysis, optical device, coatings, drug delivery and water remediation, and also may be applied for electronic and magneto-electric devices. This new eco-friendly approach of synthesis is a novel, cheap, and convenient technique suitable for large scale commercial production.

Corbicula regularis is a freshwater mussel found in the Indian sub-continent. In the present study, phylogenetic characterization of this important bivalve was attempted using 18S ribosomal RNA gene markers. Genomic DNA was extracted and 18S rRNA gene was amplified by universal primers. The amplification product was sequenced and compared with the nucleotide databases available online to evaluate phylogenetic relationship of the animal under study. Results indicated that 18S rRNA gene sequences of C. regularis showed high degree of similarity to another freshwater mussel, C. fluminea. This work constitutes the first ever sequence deposition of the C. regularis in the nucleotide databases highlighting the usefulness of 18S ribosomal gene markers for phylogenetic analysis.

Healthy wheat kernels and wheat kernels damaged by the feeding of the insects, rice weevil (Sitophilus oryzae), lesser grain borer (Rhyzopertha dominica), rusty grain beetle (Cryptolestes ferrugineus), and red flour beetle (Tribolium castaneum) were scanned using a near-infrared (NIR) hyperspectral imaging system (700-1100 nm wavelength range) and a colour imaging system. Dimensionality of hyperspectral data was reduced and statistical and histogram features were extracted from NIR images of significant wavelengths and given as input to three statistical discriminant classifiers (linear, quadratric, and Mahalanobis) and a back propagation neural network (BPNN) classifier. A total of 230 features (colour, textural, and morphological) were extracted from the colour images and the most contributing features were selected and used as input to the statistical and BPNN classifiers The quadratic discriminant analysis (QDA) classifier gave the highest accuracy and correctly identified 96.4% healthy and 91.0-1000% insect-damaged wheat kernels using the top 10 features from 230 colour image features combined with hyperspectral image features. Refereed/Peer-reviewed

Daikon and radish sprouts contain high levels of glucoraphenin, a glucosinolate which hydrolyses to form sulphoraphene. Sulphoraphene, like sulphoraphane from broccoli, is a potent inducer of phase 2 detoxification enzymes and consequently has potential anti-cancer action. Unlike broccoli however, daikon and radish do not possess epithiospecifier protein, a protein that inhibits conversion of glucosinolates to isothiocyanates, and consequently they may represent more suitable sources of phyto-chemicals with anti-cancer potential. Concentrations of glucoraphenin were highest in the seed, declining exponentially with sprout development. The rate of decline was observed to vary considerably between varieties of daikon and radish, with some varieties maintaining significantly high levels of glucoraphenin. Varieties maintaining a high level of glucoraphenin included 'Cherry Belle' and 'French Breakfast'.

Semolina is used for the manufacture of pasta (long goods and short goods) and couscous and any contrasting colored specks adversely affect the appearance of the finished product. The specks result from wheat bran, diseased wheat, ergot or weed seeds. However, there is also the possibility that insect fragments will appear as specks. Specks are currently mostly determined by a manual process or by a speck counter in milling units. We compared the speck counts from an electronic speck counter (SPX Maztech Micrco Vision), acid hydrolysis and flotation (AOAC method 993.26), and near-infrared (NIR) hyperspectral imaging in semolina seeded with insect fragments (50-300 fragments/50 g) of Tribolium castaneum (Coleoptera: Tenebrionidae). There was a significant positive correlation between the number of insect fragments added and detected by all three methods. These results underline the importance of controlling insects in flour mills producing semolina, and also in plants producing pasta and couscous, to reduce speck counts in the finished products. Refereed/Peer-reviewed

Hydatid disease is a zoonosis caused by Taenia echinococcus. The three main varieties Echinococcus granulosus, E. multilocularis and E. vogeli are primarily found in dogs and are transmitted to man by fecal–oral route. Commonly affected organs are liver, lungs and spleen. Brain is involved only in 2-5% cases. The authors herein present two cases of giant intracranial hydatid cysts managed at department of neurosurgery, Rajendra Institute of Medical Sciences, Ranchi, India.

We used the water relations model, WINWAT, to model winter water relations of three conifer species-eastern hemlock (Tsuga canadensis (L.) Carr.), eastern white pine (Pinus strobus L.) and red pine (P. resinosa Ait.)-growing at their upper elevational limits on Mt. Ascutney, Vermont, USA, in the winters of 1997 and 1999. Modeled relative water contents remained above 60% in the two youngest foliar age classes of all three species during both winters, indicating that desiccation stress in winter is not responsible for setting the upper elevational limits of these species at this site under present climatic conditions. WINWAT indicated that winter water relations of these low-elevational species were sensitive to low relative humidity, which increased transpiration rates, and low temperatures, which inhibited recharge, but are much less sensitive to summer climate than in the case of subalpine conifers in Colorado. Our results indicate that summer and winter temperatures and relative humidities (or precipitation/potential evapotranspiration ratios) should be incorporated into climate change models designed to simulate future tree distributions.

The biochemical rationale for the inhibition of citric acid fermentation by Aspergillus niger in the presence of Mn2+ ions has been investigated using high citric acid-yielding, Mn2+ ion-sensitive as well as Mn2+ ion-tolerant mutant strains of A. niger. In the presence of Mn2+ (1.5 mg/l), citric acid production by the Mn2+ ion-sensitive strain (KCU 520) was reduced by about 75% with no apparent effect on citric acid yield by the Mn2+ ion-tolerant mutant strain (GS-III) of A. niger. The significantly increased level of the Mn2+ ion-requiring NADP+-isocitrate dehydrogenase activity in KCU 520 cells and the lack of effect on the activity level of the enzyme in GS-III mutant cells by Mn2+ ions during fermentation seem to be responsible for the Mn2+ ion inhibition of citric acid production by the KCU 520 strain and the high citric acid yield by the mutant strain GS-III of A. niger even in the presence of Mn2+.

A field survey in Prunus armeniaca growing areas of Garhwal Himalaya was conducted with reference to the germination and growth of some companion crops at various sites of different soil fertility. Retardation in germination, growth and yield were noted in nearby Triticum aestivum plants, particularly on those sites where the percentage of soil organic matter was low. It was also observed that the magnitude of interference gradually decreased as the distance from the tree increased. Aqueous and organic solvent extracts of the aerial parts of Prunus armeniaca were made and their residues were tested, after proper dilution, for their phytotoxic effect on germination and growth of the test crop. Residues of light petroleum and ethyl acetate extracts showed maximum inhibition in growth and germination of the test species. The isolated compounds were identified with the help of spectroscopic techniques and bioassays. β -Sito-sterol, 2,6-dihydroxy-4-methoxyacetophenone and kaempferol were isolated from a petroleum extract whereas aromadendrin, quercetin, β -sitosterol- β - d -glucoside, pleoside and tannins (four proanthocyanidins – A-type) were characterized from an ethyl acetate extract. Of these, proanthocyanidins showed maximum inhibition, whereas quercetin, aromadendrin, kaempferol and genin of pleoside inhibited to a lesser extent.

A new conducting polymer of biotinylated bis(2,2'-bithien-5-yl)methane was prepared and applied as the recognition unit of two different biosensors for selective oligonucleotide determination using either electrochemical impedance spectroscopy (EIS) or piezoelectric microgravimetry (PM) for label-free analytical signal transduction. For preparation of this unit, first, a biotinylated bis(2,2'-bithien-5-yl)methane functional monomer was designed and synthesized. Then, this monomer was potentiodynamically polymerized to form films on the surface of a glassy carbon electrode (GCE) and a Au electrode of a quartz crystal resonator (QCR) for the EIS and PM transduction, respectively. On top of these films, neutravidin was irreversibly immobilized by complexing the biotin moieties of the polymer. Finally, recognizing biotinylated oligonucleotide was attached by complexing the surface-immobilized neutravidin. This layer-by-layer assembling of the poly(thiophene-biotin)-neutravidin-(biotin-oligonucleotide) recognition film served to determine the target oligonucleotide via complementary nucleobase pairing. Under optimized determination conditions, the target oligonucleotide limit of detection (LOD) was 0.5 pM and 50 nM for the EIS and PM transduction, respectively. The sensor response to the target oligonucleotide was linear with respect to logarithm of the target oligonucleotide concentration in a wide range of 0.5 pM to 30 μM and with respect to its concentration in the range of 50 to 600 nM for the EIS and PM transduction, respectively. The biosensors were appreciably selective with respect to the nucleobase mismatched oligonucleotides.

A plasma membrane-localized low molecular mass (26.3 kDa) adenosine 5′-monophosphatase (PM-AMPase) and a Golgi apparatus-localized high molecular mass (52.4 kDa) adenosine 5′-monophosphatase (GA-AMPase), have been purified to apparent homogeneity as evidenced by SDS-PAGE and Western blotting. The purification protocol involved selective solubilization of the membrane-bound enzyme, followed by gel filtration on Sephadex G-150 and ion-exchange chromatography on DEAE-cellulose. The PM- and GA-AMPases are glycoproteins containing approximately 37% and 39% of sugars, respectively, and show pH optimum in the range of 5.0 to 5.5. The Km values for 5′-AMP for the PM-AMPase and GA-AMPase are 0.58 × 10−3 M and 1.02 × 10−3 M, respectively. The PM-AMPase is highly specific for 5′-AMP. Other nucleotides, phosphorylated sugars and p-nitrophenyl phosphate (p-NPP) were not hydrolyzed. The purified GA-AMPase showed much broader substrate specificity with significant hydrolytic activity towards 5′-GMP, 5′-UMP and p-NPP. The PM- and GA-AMPases, besides having different molecular masses, vary in their immunological response. The polyclonal monospecific antibodies raised against the purified PM-AMPase did not cross-react with the purified GA-AMPase as shown by Western blotting and immunoprecipitation experiments, indicating that PM-AMPase and GA-AMPase do not share common antigenic determinants and may be structurally unrelated. The purified enzymes were extremely labile, losing up to 90% of the initial activity in 24 h at −20°C. Role of phospholipids for stability and optimum activity of PM- and GA-AMPases is indicated.

In Escherichia coli O157:H7 foodborne infections of humans, the Shiga-like toxins (SLTs) are thought to be the cause of life-threatening vascular complications, including acute renal disease known as hemolytic uremic syndrome or HUS. As virtually all E. coli O157:H7 isolates from HUS patients produce SLT-II (vs. SLT-I), the possible preferential interaction of SLT-II with human renal microvascular endothelial cells (HRMEC), the putative target of the SLTs in the development of HUS, was studied. SLT-II was 1000 times more potent a cytotoxic agent than SLT-I toward HRMEC. Toxin binding studies showed that this occurred although HRMEC could bind 10 times more SLT-I than SLT-II. This preferential action of SLT-II was specific for renal endothelial cells, as human umbilical vein endothelial cells were almost equally affected by SLT-I and SLT-II.

Escherichia coli O157:H7-related vascular damage such as hemolytic uremic syndrome is believed to require the Shiga-like toxins. This study demonstrated that sodium butyrate sensitized human umbilical vein endothelial cells to Shiga toxin and increased the expression of Shiga toxin receptor, globotriaosylceramide (Gb3), on human umbilical vein endothelial cells.

A new mutant strain,Aspergillus niger GS-III, showing resistance to manganese ions inhibition of citric acid fermentation on a sugarcane molasses containing medium was induced fromAspergillus niger KCU 520, a high citric acid-yielding strain. In submerged, surface or continuous cultures in the presence of manganese ions concentration upto 1.5 ppm the mutant strain yielded citric acid about 90 KgM−3 . The citric acid yield was comparable to that obtained with the parental strain KCU 520 in the absence of manganese ions, but it was atleast 3-fold higher than that obtained by the latter in the presence of manganese ions. The mutant strain immobilized in calcium alginate beads was used in combination with surface-stabilized cultures for about 36-days in a continuous flow horizontal fermenter without any apparent loss in citric acid productivity. These results indicate that the manganese-resistant mutant is stable and may be used in the presence of sufficient manganese ions concentration (1.5 ppm) in the fermentation medium. This capability of the mutant strainA. niger GS-III has been correlated with greatly reduced levels (about one-thirds) of the NADP+ -isocitric dehydrogenase, one of the control points for citric acid accumulation.

Renal glomerular endothelial cell damage is a hallmark of Shiga toxin-associated hemolytic uremic syndrome (HUS) following bacillary dysentery in humans. This study examined the binding of [125I]Shiga-like toxin-1 (SLT-I) to human umbilical vein endothelial cells (HUVEC) and to human renal glomerular microvascular endothelial cells (HRMEC) in vitro, in relation to the sensitivity of these endothelial cells to Shiga toxin. HUVEC isolated from individual umbilical cords differed in their sensitivity to Shiga toxin. These HUVEC isolates also differed in their expression of the receptor for SLT-1 (3.6 x lo5 and 2.0 x 105 receptors/cell for sensitive and moderately sensitive HUVEC, respectively). Bacterial lipopolysaccharide (LPS) sensitized HUVEC to Shiga toxin and induced a corresponding 2.5-fold increase in SLT-1 receptor expression on HUVEC. HRMEC are the putative target of Shiga toxins during HUS and were much more sensitive to Shiga toxin than were HUVEC (LD,, = 0.1 pM vs. 10 nM, respectively). HRMEC also expressed at least 200- to 360-fold more SLT-1 receptors (27.2 x 107 receptors/cell) than did HUVEC. These results suggest that Shiga toxin receptor expression in different endothelial cells may determine the degree of sensitivity of different endothelial cells to Shiga toxin and may be important in determining the susceptibility of different vascular beds to Shiga toxin-mediated disease.

A high performance fermentation process for the continuous production of citric acid from sugarcane molasses by using the combination of submerged calcium alginate-immobilized and surface-stabilized cultures of Aspergillus niger KCU 520 in a continuous flow horizontal bioreactor is described. The citric acid productivity was dependent on the dilution rate with an optimum value of 0.015/h. Presaturation of fermentation medium with sterile air, in addition to surface aeration, before feeding into the bioreactor enhanced the citric acid productivity. The highest productivity, citric acid product concentration and yield obtained were 1.7 kg M−3h−1, 110kg M−3 and 91% respectively. The cultures were continuously used for 30 days without any apparent loss in citric acid productivity.

Renal glomerular microvascular endothelial cell damage is characteristic of Shiga toxin-associated hemolytic uremic syndrome (HUS). An impaired renal fibrinolysis may be responsible for renal microvascular fibrin accumulation during the course of HUS disease. This study examined the effect of Shiga toxin, bacterial lipopolysaccharide (LPS, endotoxin), and tumor necrosis factor (TNF) on the expression of fibrinolysis factors by human renal glomerular microvascular endothelial cells (HRMEC) in vitro. The results were compared to a previously better-characterized endothelial cell type, human umbilical vein endothelial cells (HUVEC). In HUVEC, the ratio of fibrinolysis antigens was antifibrinolytic, consisting of 55-fold more plasminogen activator inhibitor type 1 (PAI-1) than tissue-type plasminogen activator (tPA). Treatment of HUVEC with LPS or TNF accentuated this ratio by decreasing tPA and increasing PAI-1 expression. In contrast, HRMEC produced urokinase-type plasminogen activator (uPA) in a 24-fold excess to PAI-1 and were thereby profibrinolytic with regard to fibrinolysis antigen expression. LPS and TNF further decreased PAI-1 antigen expression by HRMEC. These results argue against a role for LPS or TNF in decreasing renal fibrinolysis at the level of fibrinolysis factor expression by renal endothelial cells. Nevertheless, HUVEC and HRMEC were responsive to the same LPS analogs in the same order of potency. Shiga toxin decreased fibrinolysis factor expression to a greater extent in HRMEC than in HUVEC. Since HRMEC fibrinolysis antigen expression was profibrinolytic, the Shiga toxin-mediated decrease in renal endothelial uPA synthesis may predispose renal microvasculature to thrombosis and may have implications for the development of HUS.

Development of hemolytic uremic syndrome (HUS) after infection by Shigella dysenteriae 1 or enterohemorrhagic Escherichia coli has been associated with the production of Shiga toxins (verotoxins). The putative target of Shiga toxins in HUS is the renal microvascular endothelium. This report shows that preincubation of human umbilical vein endothelial cells (HUVEC) with interleukin-1 beta (IL-1 beta) enhances the cytotoxic potency of Shiga toxin toward HUVEC. A preincubation of HUVEC with IL-1 beta is required for sensitization of HUVEC to Shiga toxin. Sensitization of HUVEC to Shiga toxin is IL-1 beta dose dependent. Development of the IL-1 beta response is time dependent, beginning within 2 h of IL-1 beta preincubation and increasing over the next 24 h. That these responses were due to IL-1 beta was demonstrated by heat inactivation of IL-1 beta, by neutralization of IL-1 beta by specific antibody, and by the ability of an IL-1 beta receptor antagonist to inhibit the effect of IL-1 beta. Shiga toxin-related inhibition of HUVEC protein synthesis preceded loss of cell viability. IL-1 beta incubation with HUVEC induced the receptor for Shiga toxin, globotriaosylceramide. Lipopolysaccharide included during IL-1 beta preincubation with HUVEC increased sensitivity to Shiga toxin in an additive manner. We conclude that IL-1 beta may induce Shiga toxin sensitivity in endothelial cells and contribute to the development of HUS.

This study addresses the basis for regional microvascular susceptibility to bacterial toxins implicated in hemolytic uremic syndrome. The results indicate a relationship between the degree of Shiga toxin sensitivity of human endothelial cells from different sources and the amount of globotriaosylceramide (Gb3) glycosphingolipid receptor for Shiga toxin expressed by these cells. Cell viability and protein synthesis of renal endothelial cells were reduced to 50% by 1 pM Shiga toxin, while umbilical vein cells were not affected by > 1 nM toxin. Similarly, basal levels of Gb3 were approximately 50 times higher in renal endothelial cells than in the umbilical endothelial cells. Pre-exposure of umbilical endothelial cells to tumor necrosis factor-alpha or bacterial lipopolysaccharide increased Gb3 content 4-6-fold coincident with increases in sensitivity to cytotoxic and protein synthesis inhibitory effects of Shiga toxin. Lipopolysaccharide induction of both Gb3 and sensitivity to Shiga toxin cytotoxic action in umbilical endothelial cells was dependent on the structure of lipopolysaccharide. Neither tumor necrosis factor-alpha nor lipopolysaccharide altered the Shiga toxin sensitivity or the Gb3 content of renal endothelial cells. These data indicate that differential endothelial expression of glycolipid receptors for Shiga toxins may be responsible for localized involvement of the kidney in hemolytic uremic syndrome.

A process of continuous citric acid production from sugarcane molasses by fermentation with immobilized whole cells of A. niger KCU 520 is described. Both calcium alginate beads and polyacrylamide gel (PAG) slab entrapment methods were used for immobilization of cells. The optimum fermentation conditions for citric acid fermentation by immobilized cells were sugars, 10%; pH, 4.0; inoculum size, 20%, and temperature, 32-35°C. With calcium-alginate and PAG-immobilized cells, an overall citric acid production rate of 8.5g and 12.0g/l/day (9.3% and 12% sugar conversion to citric acid per day), respectively was achieved in a single-stage bioreactor. When the PAG-immobilized cells were used in a two-stage bioreactor, approximately 20.0g citric acid/l/day (20% sugar conversion to citric acid per day) was produced and the PAG-immobilized cells were continuously used for at least 20 days without any significant loss of productivity.

The recycled solid-state surface fermentation (SSF) culture ofAspergillus niger KCU520 was used for repeated batch production of citric acid from sugarcane molasses. The rate of citric acid production was doubled, reducing the fermentation time to half, compared to the normal single cycle batch submerged or surface fermentation process. About 80% sugar was converted to citric acid in five-day batch fermentation and three batches were carried out with the same fungal mat without any significant loss of productivity.

Quinone-methide and phenolic triterpenoids of the root outer bark of Celastrus paniculatus have been identified as celastrol, pristimerin, zeylasterone and zeylasteral whereas those of Kokoona reflexa root outer bark were pristimerin, zeylasterone and zeylasteral. Celastranhydride, an unusual triterpene anhydride isolated from K. zeylanica was shown to be present in K. reflexa, Cassine balae and Reissantia indica . Details of the isolation and structure elucidation of celastranhydride are presented along with the analysis of 1 H-detected heteronuclear multiple-bond multi-quantum correlation (HMBC) NMR spectrum which aided the confirmation of its structure. Extracts of C. balae, Gymnosporia emarginata, Pleurostylia opposita and R. indica were found to contain quinone-methides but were devoid of phenolic triterpenoids. The biosynthetic and chemotaxonomic significance of the co-occurrence of these triterpenoids in Celastraceae is discussed.

The thiazole-diamide series (1) has been identified as highly potent gamma-secretase inhibitors. Several representative compounds showed IC(50) values of0.3 nM. The synthesis and SAR, as well as a radiolabeled synthesis of [(3)H]-2a, are described.

6-Hydroxydopamine (6-OHDA), a neurotoxic substrate of the dopamine transporter (DAT), is widely used in Parkinson's disease models. However, the molecular mechanisms underlying 6-OHDA's selectivity for dopamine neurons and the injurious sequelae that it triggers are not well understood. We tested whether ectopic expression of DAT induces sensitivity to 6-OHDA in non-dopaminergic rat cortical neurons and evaluated the contribution of voltage-dependent potassium channel (Kv)-dependent apoptosis to the toxicity of this compound in rat cortical and midbrain dopamine neurons. Cortical neurons expressing DAT accumulated dopamine and were highly vulnerable to 6-OHDA. Pharmacological inhibition of DAT completely blocked this toxicity. We also observed a p38-dependent Kv current surge in DAT-expressing cortical neurons exposed to 6-OHDA, and p38 antagonists and Kv channel blockers were neuroprotective in this model. Thus, DAT-mediated uptake of 6-OHDA recruited the oxidant-induced Kv channel dependent cell death pathway present in cortical neurons. Finally, we report that 6-OHDA also increased Kv currents in cultured midbrain dopamine neurons and this toxicity was blocked with Kv channel antagonists. We conclude that native DAT expression accounts for the dopamine neuron specific toxicity of 6-OHDA. Following uptake, 6-OHDA triggers the oxidant-associated Kv channel-dependent cell death pathway that is conserved in non-dopaminergic cortical neurons and midbrain dopamine neurons.

Pathological and physiological hypertrophy of the heart is associated with decreased expression of the Kv4.3 transient outward current ( I to ) channel. The downregulation of channel mRNA and protein, which may be proarrhythmic, is recapitulated with cultured neonatal rat ventricular myocytes treated with angiotensin II (Ang II). Here we show that the 4.9 kb 3′ untranslated region (3′ UTR) of the Kv4.3 channel transcript confers Ang II sensitivity to a promoter-reporter construct. In contrast, Kv4.2 and Kv1.5 3′-UTR sequences are insensitive to Ang II. Both Kv4.3 3′-UTR reporter mRNA and activity are decreased in Ang II–treated cardiac myocytes, in accordance with a decrease in mRNA stability. This regulation is mediated by Ang II type 1 (AT 1 ) receptors and abolished by NADPH oxidase inhibitors and dominant negative rac. The Ang II effect is also blocked by expression of superoxide dismutase (SOD), but not catalase, showing that superoxide is required. Dominant negative subunits, enzyme inhibitors and hydrogen peroxide experiments show that the apoptosis signal-regulating kinase 1 (ASK1)–p38 kinase pathway mediates downstream signaling from NADPH oxidase. Mechanical stretch also downregulates Kv4.3 3′-UTR reporter activity and this requires AT 1 receptors and NADPH oxidase. Thus, activation of AT 1 receptors by Ang II or stretch specifically destabilizes cardiac myocyte Kv4.3 channel mRNA by activating NADPH oxidase. These results link long-term control of cardiac K + channel gene expression to a physiological reactive oxygen species signaling pathway.

Infection of humans with Shiga toxin-producing Escherichia coli O157:H7 and Shigella dysenteriae 1 is strongly associated with vascular endothelial cell damage and the development of hemolytic-uremic syndrome. The cytotoxic effect of Shiga toxins on vascular endothelial cells in vitro is enhanced by prior exposure to bacterial lipopolysaccharide (LPS) or either of the host cytokines tumor necrosis factor alpha (TNF) and interleukin-1beta (IL-1). The purpose of this study was to examine individual signal transduction components involved in the sensitization of human umbilical vein endothelial cells (HUVEC) to Shiga toxin 1. The results demonstrate that class I and II protein kinase C (PKC) isozymes are required for sensitization of HUVEC to Shiga toxin by phorbol myristate acetate (PMA) or LPS but not by TNF or IL-1. Thus, the specific competitive inhibitor of class I/II PKC, 1-O-hexadecyl-2-O-methyl-rac-glycerol (AMG), prevented only the action of PMA and LPS on HUVEC. Additional data obtained with ATP binding site inhibitors which affect all PKCs (i.e., classes I, II, and III) suggest that TNF may utilize class III PKC isozymes in the Shiga toxin sensitization of HUVEC. Transcriptional activator NF-kappaB did not appear to be involved in the sensitization of HUVEC to Shiga toxin by LPS, TNF, IL-1, or PMA. Thus, the specific serine protease inhibitor L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) did not inhibit the sensitization of HUVEC to Shiga toxin by LPS, TNF, IL-1, or PMA despite its ability to inhibit NF-kappaB activation and the induction of the NF-kappaB-dependent tissue factor gene by these agents. Finally, all-trans retinoic acid partially inhibited the sensitization of HUVEC to Shiga toxin, by unknown mechanisms which also appeared to be independent of NF-kappaB activation. These results indicate that PKC plays a role in the sensitization of HUVEC to Shiga toxin in response to some, but not all, sensitizing agents. In contrast, NF-kappaB activation appears not to be involved in the sensitization of HUVEC to Shiga toxin by LPS, TNF, IL-1, or PMA.

This study explores the in vitro relationship between Shiga toxin-producing Shigella spp. and Escherichia coli and the development of vascular complications in humans following bacillary dysentery. We propose that lipopolysaccharide (LPS; endotoxin) may combine with Shiga toxin to facilitate vascular damage characteristic of hemolytic uremic syndrome. We have examined the direct cytotoxic effects of Shiga toxin and LPS on human umbilical vein endothelial cells (HUVEC) in culture. Shiga toxin alone was cytotoxic to HUVEC, whereas LPS was noncytotoxic at concentrations at or below 10 micrograms/ml. Combinations of LPS with Shiga toxin resulted in a synergistic cytotoxic effect. The synergistic cytotoxic response of HUVEC to Shiga toxin plus LPS was dose dependent for both agents and was maximal at 24 h of exposure. This synergistic response was enhanced by preincubation of HUVEC with LPS. LPS (1 micrograms/ml) alone depressed HUVEC protein synthesis in a transient manner and enhanced the protein synthesis-inhibiting activity of Shiga toxin. The synergistic cytotoxic activity of LPS analogs was as follows, in decreasing order: complete LPS = diphosphoryl lipid A greater than monophosphoryl lipid A greater than deacylated LPS. These results are consistent with a role for Shiga toxin and LPS in the development of hemolytic uremic syndrome at the level of the vascular endothelium in humans.

The three novel 19(10→9)abeo-8α,9β,10α-euphane (reissantane) triterpenoids isolated from Reissantia indica(Celastraceae) and related to reissantioloxide (reissantenol oxide)(1) have been shown to be 24-oxoreissant-5-ene-3β,25-diol (2), 3-oxoreissant-5-ene-24β,25-diol (3), and reissant-5-ene-3β,24β,25-triol (4). A possible biosynthetic relationship for reissantane, D : B-friedo-oleanane, D : A-friedo-oleanane, and quinone-methide triterpenoids which occur in R. indica is presented.

The structure of celastranhydride, a novel triterpene anhydride isolated from Kokoona zeylanica and present in Cassine balae and Reissantia indica , all of the family Celastraceae, has been deduced as 1 from its spectral data including 2-D heteronuclear 1 H 13 C shift correlated NMR spectra.

The enzyme transferring the oligosaccharide from DolPP-(GlcNAc)2(Glc)3 to asparagine residues of glycoproteins has been solubilized from yeast membranes by extraction with detergents. Enzyme activity was tested by measuring transfer of the glycosyl moiety from DolPP-[14C]saccharides to the hexapeptide Tyr-Asn-Leu-Thr-Ser-Val. The rate of transfer was linear for 20 min, with about 40% of dolichyl-diphosphate-bound radioactivity transferred to the peptide. The solubilized enzyme has been characterized as follows: 1. The enzyme is most efficiently solubilized (60% of the membrane-associated activity) by 0.5% Nonidet P40 at a protein/detergent ratio of 2. Octylglucoside solubilizes one third of the activity, but strongly inhibits the reaction if present in the test at a concentration of 1%. 2. Divalent cations are absolutely required. 1 mM Mn2+ is optimal; Mg2+ at a concentration of 10mM yields only one third the activity observed with Mn2+. 3. The enzyme transfers besides dolichyl-diphosphate-bound (GlcNAc)2(Man)9(Glc)3 also (GlcNAc)2(Man)1 and (GlcNAc)2; the rate decreases in this order. No transfer is observed from DolPP-(GlcNAc)2(Man)9 and from DolPP-GlcNAc. 4. The Km value for DolPP-(GlcNAc)2(Man)9(Glc)3 of 0.5 microM does not differ significantly from that for DolPP-(GlcNAc)2 of 1.2 microM. A broad pH-optimum for the reaction with both substrates was found between 6.5 and 7.7. 5. However, a clear difference in Km values for the hexapeptide was observed with difference dolichol-linked sugar derivatives. With DolPP-(GlcNAc)2 a peptide concentration of 0.6 mM resulted in half-maximal transfer rate, whereas 0.05 mM peptide were sufficient with DolPP-(GlcNAc)2(Man)9(Glc)3 as donor.

An adenosine 5′-monophosphatese (AMPase) has been purified from the Golgi-apparatus (GA) of peanut cotyledons by first selective solubilizing the enzyme with 0.5% non-specific detergent, n -octyl- β -D-glucopyranoside, at a protein-to-detergent ratio of 2:3 in the presence of 20 mM MgCl 2 and 5 mM EDTA, and then by ion-exchange chromatography on DEAE-cellulose. The GA-AMPase is a glycoprotein having 38.5% carbohydrate and apparent M r = 53.7 dkDa. It was inhibited competitively by ADP ( K i = 3.4 mM) and non-competitively by NaF ( K i = 41 mM). The values of optimum pH, K m and V max for the hydrolysis of AMP were 5.0–5.5, 0.9 mM and 0.4 mM/min per mg, respectively. The enzyme is highly specific for AMP with no hydrolysis of ATP, ADP, GTP, GDP, GMP, UTP, UDP, UMP, CTP and CMP. The purified AMPase from GA resembled the plasma membrane (PM) enzyme closely, except for a slightly lower carbohydrate content. It is suggested that GA-AMPase may be a precursor of the PM-AMPase in peanut cotyledon cells.

Two novel phenolic dinor -triterpenes isolated from Kokoona zeylanica have been identified as 23- nor -6-oxopristimerol and 23- nor -6-oxodemethylpristimerol on the basis of spectroscopic evidence and chemical interconversions. The biosynthetic significance of the co-occurrence of phenolic D:A- friedo -23, 24-dinoroleananes with phenolic and quinone-methide triterpenes in K. zeylanica is discussed.

An adenosine 5′-monophosphatase (AMPase) has been purified from the plasma membrane fraction of germinating cotyledons of peanut (Arachis hypogaea L.) by selective solubilization of the membrane-bound enzyme with 0.5% n-octyl β-glucoside at a protein-to-detergent ratio of 2:3 in the presence of Mg2+ and EDTA, followed by ion exchange chromatography on DEAE-cellulose. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis it showed a single protein band with a molecular weight of 55 000. The enzyme is a glycoprotein with 42.7% carbohydrate content. It had a broad pH optimum of 5.0–6.0. The Km and Vmax values were 1.08·10−3 M and 8.5 μmol/min per mg protein, respectively, with 5′-AMP as substrate. The enzyme is specific for 5′-AMP. Other nucleotides (GMP, UMP, CMP, ADP, GDP, ATP, GTP and UTP) as well as phosphorylated sugars were not hydrolyzed. p-Nitrophenyl phosphate was hydrolyzed at a relatively much lower rate (15%) and the substrate affinity (1/Km was only one-tenth that of AMP. The purified enzyme is competitively inhibited by ADP (Ki = 2.4 mM) and is also inhibited by NaF in a non-competitive manner with a Ki value of 35 mM. Divalent cations, Ca2+, Mg2+, Hg2+, Zn2+, Mn2+, Ni2+ and monovalent cations, K+, Li+ and Na+ had no effect on the enzyme activity. The purified enzyme was highly unstable, losing its total activity within 24 h at −20°C, or 0°C, while under these conditions the unpurified solubilized enzyme (octyl glucoside extract) was stable for several days, indicating that some stabilizing factors, most likely phospholipids, were lost during the enzyme purification.

The three new phenolic nortriterpenes, demethylzeylasteral, zeylasteral, and demethylzeylasterone, isolated from Kokoona zeylanica(Celastraceae), have been shown to be 2,3-dihydroxy-6,23-dioxo-D:A-friedo-24-noroleana-1,3,5(10),7-tetraen-29-oic acid (5), its methyl ester (6), and 2,3-dihydroxy-6-oxo-D:A-friedo-24-noroleana-1,3,5(10),7-tetraene-23,29-dioic acid (7), respectively. The structure of the new phenolic (9 → 8)-D:A-friedo-24-noroleanane triterpene obtained from ‘kokum soap’ has been established as 23-oxoisopristimerin III (11) on the basis of spectroscopic evidence. The biosynthetic significance of 23-oxoisopristimerin III is discussed.