Diacylglycerol-containing oleic acid induces increases in [Ca]i via TRPC3/6 channels in human T-cells

Though most of the studies have focused on the effects of free fatty acids on T-cell activation, fatty acids incorporated into plasma membrane phospholipids may also affect cell signaling via diacylglycerol (DAG), generally produced by phospholipid hydrolysis. In the present study, we have synthesized a DAG-containing oleic acid and studied its implication in the modulation of calcium signaling in human Jurkat T-cells. 1-palmitoyl-2-oleoyl-sn-glycerol (POG) induced a dose-dependent increase in [Ca²⁺]_i. This effect was due to the presence of oleic acid at the sn-2 position as no differences were observed between POG and 1-stearoly-2-oleoyl-sn-glycerol (SOG). However, the substitution of oleic acid with arachidonic acid at the sn-2 position of the DAG moiety exerted a different response on the increases in [Ca²⁺]_i in these cells. POG-evoked increases in [Ca²⁺]_i were not due to its metabolites. Furthermore, POG-induced increases in [Ca²⁺]_i were due to the opening of TRPC3/TRPC6 channels as silencing of TRPC3 and TRPC6 genes by shRNA abolished calcium entry. Moreover, disruption of lipid rafts with methyl-β-cyclodextrin completely abolished POG-evoked increases in [Ca²⁺]_i. In conclusion, our results demonstrate that oleic acid can influence T-lymphocyte functions, in the conjugated form of DAG, via opening TRPC3/6 channels.     

Exogeneous diacylglycerols downregulate the activity of Na-K pump in Xenopus laevis oocytes

In this study, cell permeable diacyglycerols, sn-1,2-dioctanoglycerol (DiC8), and sn-1-oleoyl-2-acetylglycerol (OAG) were found to downregulate the activity of Na⁺-K⁺ pump in Xenopus laevis oocytes. Both DiC8 and OAG decreased the binding of [³H]ouabain to intact oocytes while phorbol esters did not appreciably influence the same. These diacylglycerols inhibited the amiloride-sensitive ²²Na⁺ influx and ouabain-sensitive ⁸⁸Rb⁺ uptake in the oocytes. Furthermore, DiC8 prevented the ²²Na⁺ efflux from the oocytes preloaded with ²²Na⁺. Addition of H-7 to DiC8- and OAG-treated oocytes stimulated the pump activity curtailed by the two latters. The impairment of Na⁺-K⁺ pump activity by diacylglycerols suggests that protein kinase C activators may stimulate endocytosis of membrane-coupled Na⁺-K⁺ ATPase.     

A review of the genus Rusa in the indo-malayan archipelago and conservation efforts

Genus Rusa, belonging to the deer family Cervidae is native to the Indo-Malaya Archipelago (IMA). However, detailed information on the Rusa genus in the IMA is limited. This review provides comprehensive information on the Rusa genus in the IMA including, threats and conservation efforts. There are four species of deer in Rusa genus, which is Sambar deer (Rusa unicolor), Javan deer (Rusa timorensis), Visayan spotted deer (Rusa alfredi) and Philippine deer (Rusa marianna). Despite their wide distribution in the South Asian and Southeast Asian regions, they are under serious threats. Some conservation efforts that are being done to protect and conserve them among others are; (1) facilities protection, (2) habitat enrichment programme, (3) Ex-situ conservation, (4) legislations, and (5) captive breeding. Conservation through genetics is also an important step in conserving these species. Recommendations for conservation of the genus are also discussed; 1. maintenance of ecosystem. 2. more effective monitoring system on the existing protected area. 3. ex-situ conservation, and 4. habitat monitoring.     

Airspora Measured in a Paddy Field in West Bengal,India

Aeromycoflora studies above an “Aman” variety of paddy (Oryza saliva L.) were carried out for two consecutive seasons, in 1990 and 1991, in the vicinity of Barrackpore, West Bengal, by means of the culture plate exposure technique. A more or less uniform spore concentration was observed during the early part of the vegetative stages in 1990 with an abrupt increase and peak during the maximum vegetative growth period. In 1991, a uniform spore count was found up until the flowering stages. A gradual increase in spore count after flowering was recorded in both seasons with the highest peaks during harvesting, followed by a sudden decrease after the harvest. The dominant genera isolated were Aspergillus, Currularia, Cladosporium and Penicillium. Aspergillus appeared in high concentrations from the very beginning of the crop season up to the flowering stages, with a gradual fall after flowering, while Cladosporium showed the reverse pattern. Currularia and Penicillium occurred regularly throughout the crop season.

Phytopathogenic types were represented by Helminthosporium oryzae, Fusarium, Altemaria and Nigrospora. Helminthosporium oryzae appeared in the air when the plants attained appreciable vegetative growth, being seen as “brown spots” on the foliage, and reaching peak concentration during harvesting. Fusarium in contrast to Altemaria appeared regularly in the air except during the later part of vegetative growth. Nigrospora was recorded only occasionally. A considerable number of “Sterile Forms” were present throughout both seasons.     

A novel type of detergent-resistant membranes may contribute to an early protein sorting event in epithelial cells

One sorting mechanism of apical and basolateral proteins in epithelial cells is based on their solubility profiles with Triton X-100. Nevertheless, apical proteins themselves are also segregated beyond the trans-Golgi network by virtue of their association or nonassociation with cholesterol/sphingolipid-rich microdomains (Jacob, R., and Naim, H. Y. (2001) Curr. Biol. 11, 1444-1450). Therefore, extractability with Triton X-100 does not constitute an absolute criterion of protein sorting. Here, we investigate the solubility patterns of apical and basolateral proteins with other detergents and demonstrate that the mild detergent Tween 20 is adequate to discriminate between apical and basolateral proteins during early stages in their biosynthesis. Although the mannose-rich forms of the apical proteins sucrase-isomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV reveal similar solubility profiles comprising soluble and nonsoluble fractions, the basolateral proteins, vesicular stomatitis virus G protein, major histocompatibility complex class I, and CD46 are entirely soluble with this detergent. The insoluble Tween 20 membranes are enriched in phosphatidylinositol and phosphatidylglycerol compatible with their synthesis in the endoplasmic reticulum and the existence of a novel class of detergent-resistant membranes. The association of the mannose-rich biosynthetic forms of the apical proteins, sucraseisomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV with the Tween 20-resistant membranes suggests an early polarized sorting mechanism prior to maturation in the Golgi apparatus.     

Structure, biosynthesis, and glycosylation of human small intestinal maltase-glucoamylase

Maltase-glucoamylase (MGA) was immunoprecipitated from detergent extracts of brush border membranes of the human small intestinal mucosa. Electrophoretic analysis of the precipitates under denaturing conditions revealed a single polypeptide of Mr = 335,000 in the presence or absence of reducing agents. Cross-linking of brush border membranes with the homobifunctional reagent dithiobis(succinimidylpropionate) did not result in considerable changes in the electrophoretic pattern of MGA. In contrast, aminopeptidase N, used in these studies as a control glycoprotein of the brush border membrane revealed dimeric structures of its single subunit in the presence of dithiobis(succinimidylpropionate). These data suggest that MGA is expressed in the human small intestinal brush border as a monomeric polypeptide. The biosynthesis of MGA was studied by pulse-labeling of human intestinal biopsy specimens or mucosal explants in organ culture. Continuous labeling with [35S]methionine for 30 min revealed a single polypeptide high mannose precursor of Mr = 285,000 (MGAh) which matures after 4 h of labeling to the Mr = 335,000 as judged by the susceptibility of these two forms to endo-beta-N-acetylglucosaminidase H. Owing to the absence of pancreatic secretions in the culture medium and the isolation of an identical species from nonlabeled mucosa, this result indicates that the Mr = 335,000 does not undergo an in situ extracellular cleavage by intraluminal proteases. Further, biosynthetically labeled, intracellularly cleaved polypeptides corresponding to the high mannose precursor or mature forms of MGA were not detected. The mature form of MGA (MGAm) bears in addition to N-linked glycans also O-glycosidically linked oligosaccharides. In fact, endo-beta-N-acetylglucosaminidase F/glycopeptidase F treatment of MGAm followed by chemical deglycosylation with trifluoromethanesulfonic acid revealed approximately 35,000 daltons of O-linked sugars. Furthermore, MGAm as well as its N-linked sugars-depleted form bound to Helix pomatia lectin which has specificity toward Gal-GalNAc structures. In addition, the data were suggestive of a post-translational O-glycosylation of the molecule since (i) the high mannose precursor of MGA did not bind to H. pomatia lectin and (ii) its endo-beta-N-acetylglucosaminidase H or endo-beta-N-acetylglucosaminidase F/glycopeptidase F form displayed an apparent molecular weight similar to that obtained upon endo-beta-N-acetylglucosaminidase F/glycopeptidase F/trifluoromethanesulfonic acid deglycosylation. Finally, pulse-chase experiments revealed a relatively slow rate of post-translational processing of MGA in comparison to aminopeptidase N.(ABSTRACT TRUNCATED AT 400 WORDS)     

The KRAS-regulated kinome identifies WEE1 and ERK coinhibition as a potential therapeutic strategy in KRAS-mutant pancreatic cancer

Oncogenic KRAS drives cancer growth by activating diverse signaling networks, not all of which have been fully delineated. We set out to establish a system-wide profile of the KRAS-regulated kinase signaling network (kinome) in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). We knocked down KRAS expression in a panel of six cell lines and then applied multiplexed inhibitor bead/MS to monitor changes in kinase activity and/or expression. We hypothesized that depletion of KRAS would result in downregulation of kinases required for KRAS-mediated transformation and in upregulation of other kinases that could potentially compensate for the deleterious consequences of the loss of KRAS. We identified 15 upregulated and 13 downregulated kinases in common across the panel of cell lines. In agreement with our hypothesis, all 15 of the upregulated kinases have established roles as cancer drivers (e.g., SRC, TGF-β1, ILK), and pharmacological inhibition of one of these upregulated kinases, DDR1, suppressed PDAC growth. Interestingly, 11 of the 13 downregulated kinases have established driver roles in cell cycle progression, particularly in mitosis (e.g., WEE1, Aurora A, PLK1). Consistent with a crucial role for the downregulated kinases in promoting KRAS-driven proliferation, we found that pharmacological inhibition of WEE1 also suppressed PDAC growth. The unexpected paradoxical activation of ERK upon WEE1 inhibition led us to inhibit both WEE1 and ERK concurrently, which caused further potent growth suppression and enhanced apoptotic death compared with WEE1 inhibition alone. We conclude that system-wide delineation of the KRAS-regulated kinome can identify potential therapeutic targets for KRAS-mutant pancreatic cancer.     

Expression of a full-length cDNA coding for human intestinal lactase-phlorizin hydrolase reveals an uncleaved, enzymatically active, and transport-competent protein

Lactase-phlorizin hydrolase (LPH) (EC 3.2.1.23/62) is a major intestinal microvillar membrane glycoprotein that digests lactose, the main carbohydrate of milk. To investigate structure/function relationships of LPH and to assess the impact of intracellular processing on the function of LPH and on its transport to the cell surface, we have expressed a full-length cDNA encoding LPH in mammalian COS-1 cells. Analysis of the expressed protein by immunoprecipitation with monoclonal anti-LPH antibodies and treatments with endo-beta-N-acetylglucosaminidase H and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two polypeptides with apparent molecular masses of 215 and 230 kDa, representing the mannose-rich (pro-LPHh) and complex (pro-LPHc) glycosylated forms of the precursor. By contrast to pro-LPH in human enterocytes, the expressed pro-LPH in COS-1 cells does not undergo intracellular proteolytic cleavage to generate a form similar to the mature enzyme of the brush-border membrane. Intracellular cleavage, however, is not essential for the molecule to acquire its enzymatic activity since pro-LPH in COS-1 cells is enzymatically as active as LPH isolated from intestinal brush-border membranes. Indirect immunofluorescent staining of transfected cells demonstrated that pro-LPH is expressed at the cell surface. This was further corroborated by the sensitivity of the complex glycosylated form (pro-LPHc) to trypsin in the medium. Our results provide the first conclusive evidence that pro-LPH is an enzymatically active molecule and that the intracellular proteolysis of pro-LPH is not essential for the generation of transport-competent forms of LPH.     

Case of lung carcinoma revealed by vulvar metastasis associated with systemic scleroderma and literature review

Metastatic carcinoma to the vulva is rare, where the incidence is believed to be between 5% and 8%.However, malignant tumors have been described in 3–11% of systemic scleroderma (SSc) cases.We report the case of one patient, a 66-year-old postmenopausal woman, whose medical history was marked with rheumatic vascular disease (systemic scleroderma) since 1993 without muscular, renal, cardiac lesions or HTA (arterial hypertension) and without tobacco history.The woman presented with a new vulvar mass of the right labia in December 2011 that had progressively enlarged in size.CT scan of the abdominopelvic region demonstrated a lobular mass of the right labia with central necrosis, 7 cm on the wide axis, and the rectum and the vaginal wall were normal. No inguinal or iliac lymphadenopathy was noted.An outpatient excisional biopsy revealed a poorly differentiated malignant tumor suggestive of carcinoma.IHC: CK7+/CK20−, estrogen receptors−, AE 1 AE 3+, vimentine+, S100−, Desmina−, CD34−, KI 67: 20%.The thoracic scan revealed a large mass of 4 cm × 3 cm in the right lung base with right paratracheal lymphadenopathy 3 cm × 2 cm.A bronchoscopy revealed discrete stenosis of the mediastinal portion of the right bronchial tree.The bronchial biopsy also revealed poorly differentiated lung carcinoma, non-small cell, which was identical with the vulvar tumor.

Conclusion

The presence of the single lung lesion with only one lymphadenopathy paratracheal with pathological and immunohistochemical (IHC) profile similar to the vulvar lesion, and a particular IHC profile with CK7+ and CK20− was detected – that is more specific to the primitive pulmonary cancer, and the presence of only one sarcoma marker vementine+, desmine and actine−. Also the presence of KI 67: 20%, predicted the proliferative and great metastatic power of the lung tumor was observed.Additionally, lung cancer was the most frequent type and may develop in scleroderma as reported in most studies.This allows to conclude for primitive lung carcinoma revealed with vulvar metastasis after elimination of the possibility of vulvar sarcoma.The patient was treated by chemotherapy (Taxol/Platin) with partial response from the lung after 3 cycles and palliative radiotherapy in the vulva with a good response.This case described primary lung carcinoma associated with scleroderma, revealed by a vulvar metastasis, which may be related to the aggressiveness of lung cancer when the lung fibrosis follow-up is not performed well to detect early the development of lung tumors in the patient with systemic scleroderma.