Stimulation of rat pituitary phospholipid methyltransferase by vasopressin but not oxytocin

Rat pituitary extracts contain two methyltransferases that catalyze stepwise methylation of phosphatidyl-ethanolamine to phosphatidylcholine using S-adenosylmethionine as the methyl donor. The activities of both of these enzymes were stimulated by 40 μM lysine or arginine vasopresin but not oxytocin, arginine vasotocin and Pro-Leu-Gly NH₂. The concentration of lysine-vasopressin required for the half-maximal stimulation of phospholipid methylation was 27 μM. A comparison of the chemical structure of different peptides with their ability to stimulate phospholipid methylation suggests that the stimulatory activity resides in the covalent ring structure (pressinoic acid) of the vasopressin molecule.     

Comparative proteomics and variations in extracellular matrix of Candida tropicalis biofilm in response to citral

Candida tropicalis is an opportunistic human pathogen with an ability to cause superficial as well as systemic infections in immunocompromised patients. The formation of biofilm by C. tropicalis can cause dreadful and persistent infections which are difficult to treat due to acquired resistance. Presently, available anti-Candida drugs exhibit a high frequency of resistance, low specificity and toxicity at a higher dosage. In addition, the discovery of natural or synthetic anti-Candida drugs is slow paced and often does not pass clinical trials. Citral, a monoterpene aldehyde, has shown effective antimicrobial activities against various microorganisms. However, only few studies have elaborated the action of citral against the biofilm of C. tropicalis. In the present work, the aim was to study the fungicidal effect, differential expression of proteome and changes in extracellular matrix in response to the sub-lethal concentration (16 µg/mL) of citral. The administration of citral on C. tropicalis biofilm leads to a fungicidal effect. Furthermore, the differential expression of proteome has revealed twenty-five proteins in C. tropicalis biofilm, which were differentially expressed in the presence of citral. Among these, amino acid biosynthesis (Met6p, Gln1p, Pha2p); nucleotide biosynthesis (Xpt1p); carbohydrate metabolism (Eno1p, Fba1p, Gpm1p); sterol biosynthesis (Mvd1p/Erg19p, Hem13p); energy metabolism (Dnm1p, Coa1p, Ndk1p, Atp2p, Atp4p, Hts1p); oxidative stress (Hda2p, Gre22p, Tsa1p, Pst2p, Sod2p) and biofilm-specific (Adh1p, Ape1p, Gsp1p) proteins were identified. The overexpression of oxidative stress–related proteins indicates the response of biofilm cell to combating oxidative stress during citral treatment. Moreover, the upregulation of Adh1p is of particular interest because it subsidizes the biofilm inhibition through ethanol production as a cellular response. The augmented expression of Mvd1p/Erg19p signifies the effect of citral on ergosterol biosynthesis. The presence of citral has also shown an increment in hexosamine and ergosterol component in extracellular matrix of C. tropicalis biofilm. Hence, it is indicated that the cellular response towards citral acts through multifactorial processes. This study will further help in the interpretation of the effect of citral on C. tropicalis biofilm and development of novel antifungal agents against these potential protein targets.

     

Identification of compendial nonionic detergents for the replacement of Triton X-100 in bioprocessing

We have systematically investigated six compendial nonionic detergents as potential replacements for Triton ×-100 in bioprocessing applications. Use of compendial raw materials in cGMP bioprocessing is advantageous for a variety of reasons including material specifications developed to meet stringent pharmaceutical product quality requirements, regulatory familiarity and comfort, and availability from vendors experienced supplying the biopharmaceutical industry. We first examine material properties of the detergents themselves including melting point and viscosity. Process performance and product contact in real-world bioprocess applications are then investigated. Lastly, we test the detergents in virus inactivation (VI) experiments with recombinant proteins and adeno-associated virus. Two of the detergents tested, PEG 9 Lauryl Ether and PEG 6 Caprylic/Capric Glycerides, showed favorable properties that make them attractive for use as potential Triton X-100 replacements. Process performance testing indicated negligible impact of the detergents on product yield, purity, and activity compared to a control with no detergent. Importantly, both PEG 9 Lauryl Ether and PEG 6 Caprylic/Capric Glycerides demonstrated very fast VI kinetics with complete inactivation of XMuLV observed in less than 1 min at a target 1% detergent concentration. Potential advantages and disadvantages of both candidate detergents for use in cGMP bioprocessing are summarized and discussed.     

Functional characterization of a high-affinity iron transporter (PiFTR) from the endophytic fungus Piriformospora indica and its role in plant growth and development

Iron (Fe) is a micronutrient required for plant growth and development; however, most Fe forms in soil are not readily available to plants, resulting in low Fe contents in plants and, thereby, causing Fe deficiency in humans. Biofortification through plant-fungal co-cultivation might be a sustainable approach to increase crop Fe contents. Therefore, we aimed to examine the role of a Piriformospora indica Fe transporter on rice Fe uptake under low Fe conditions. A high-affinity Fe transporter (PiFTR) from P. indica was identified and functionally characterized. PiFTR fulfilled all criteria expected of a functional Fe transporter under Fe-limited conditions. Additionally, PiFTR expression was induced when P. indica was grown under low Fe conditions, and PiFTR complemented a yeast mutant lacking Fe transport. A knockdown (KD) P. indica strain was created via RNA interference to understand the physiological role of PiFTR. We observed that the KD-PiFTR-P. indica strain transported a significantly lower amount of Fe to colonized rice (Oryza sativa) than the wild type (WT) P. indica. WT P. indica-colonized rice plants were healthier and performed significantly better than KD-PiFTR-P. indica-colonized rice plants. Our study offers potential avenues for an agronomically sound amelioration of plant growth in low Fe environments.     

Real‐time PCR assays for rapid detection of Zeugodacus cucumis and Bactrocera jarvisi (Diptera: Tephritidae) for quarantine application

Zeugodacus cucumis and Bactrocera jarvisi are pests of fruit and vegetable crops and cause damage to horticulture industries. Immature stages of these two fruit fly species have been intercepted in New Zealand a number of times. Identification to species was not possible using morphological characters; thus, it is important to develop an assay for their species‐level identification. Here, the real‐time PCR assays for rapid identification of Z. cucumis and B. jarvisi were developed and validated. The PCR protocols demonstrated their specificity by amplifying the two target species successfully, with no cross‐reactions observed in the tested tephritid species. The in silico test of the primer and probe binding sites of the two assays also demonstrated the assays’ specificity by no mismatches present in the binding regions of the target species, but 1–4 mismatches in the binding regions of the non‐target fruit fly species. The thresholds of detection for the two assays are as low as 10 copies/µl of the target DNA, indicating that the assays have a very high sensitivity. The application of the real‐time PCR assays has greatly assisted in routine pest identifications at the New Zealand border and surveillance programme. Therefore, the assays have the potential to be used by diagnostic agencies and research organizations worldwide.     

The Crohn’s Disease Risk Factor IRGM Limits NLRP3 Inflammasome Activation by Impeding Its Assembly and by Mediating Its Selective Autophagy

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Cellular and molecular mechanisms of border cell migration analyzed using time-lapse live-cell imaging

Border cells in the Drosophila ovary originate within an epithelium, detach from it, invade neighboring nurse cells, and migrate as a coherent cluster. This migration has served as a useful genetic model for understanding epithelial cell motility. The prevailing model of growth factor-mediated chemotaxis in general, and of border cells in particular, posits that receptor activation promotes cellular protrusion at the leading edge. Here we report the time-lapse video imaging of border cell migration, allowing us to test this model. Reducing the activities of the guidance receptors EGFR and PVR did not result in the expected inhibition of protrusion, but instead resulted in protrusion in all directions. In contrast, reduction in Notch activity resulted in failure of the cells to detach from the epithelium without affecting direction sensing. These observations provide new insight into the cellular dynamics and molecular mechanisms of cell migration in vivo.     

Comparative proteomics analysis of Barrett metaplasia and esophageal adenocarcinoma using two-dimensional liquid mass mapping

Esophageal adenocarcinoma, currently the seventh leading cause of cancer-related death, has been associated with the presence of Barrett metaplasia. The malignant potential of Barrett metaplasia is evidenced by ultimate progression of this condition to invasive adenocarcinoma. We utilized liquid phase separation of proteins with chromatofocusing in the first dimension and nonporous reverse phase HPLC in the second dimension followed by ESI-TOF mass spectrometry to identify proteins differentially expressed in six Barrett metaplasia samples as compared with six esophageal adenocarcinoma samples; all six Barrett samples were obtained from the identical six patients from whom we obtained the esophageal adenocarcinoma tissue. Approximately 300 protein bands were detected by mass mappings, and 38 differentially expressed proteins were identified by microLC-MS/MS. The false positive rates of the peptide identifications were evaluated by reversed database searching. Among the proteins that were identified, Rho GDP dissociation inhibitor 2, alpha-enolase, Lamin A/C, and nucleoside-diphosphate kinase A were demonstrated to be up-regulated in both mRNA and protein expression in esophageal adenocarcinomas relative to Barrett metaplasia. Candidate proteins were examined at the mRNA level using high density oligonucleotide microarrays. The cellular expression patterns were verified in both esophageal adenocarcinomas and in Barrett metaplasia by immunohistochemistry. These differentially expressed proteins may have utility as useful candidate markers of esophageal adenocarcinoma.     

Down-regulation of DNA polymerase beta accompanies somatic hypermutation in human BL2 cell lines

Somatic hypermutation (SHM) is a fundamental process in immunoglobulin gene maturation that results in increased affinity of antibodies toward antigens. In one hypothesis explaining SHM in human B cells, the process is initiated by enzymatic deamination of cytosine to uracil in the immunoglobulin gene V-region and this in turn triggers mutation-prone forms of uracil-DNA base excision repair (BER). Yet, an uncertainty with this model is that BER of uracil-DNA in mammalian cells is generally error-free, wherein DNA polymerase beta (pol beta) conducts gap-filling synthesis by insertion of bases according to Watson-Crick rules. To evaluate this inconsistency, we examined pol beta expression in various SHM proficient human BL2 cell line subclones. We report that expression of pol beta in SHM proficient cell lines was strongly down-regulated. In contrast, in other BL2 subclones, we found that SHM was deficient and that pol beta expression was much higher than in the SHM proficient subclones. We also found that overexpression of recombinant human pol beta in a SHM proficient subclone abrogated its capacity for SHM. These results suggest that down-regulation of the normal BER gap-filling DNA polymerase, pol beta, accompanies induced SHM in BL2 cells. This is consistent with the hypothesis that normal error-free BER must be silenced to make way for an error-prone BER process that may be required during somatic hypermutation.