Identification of MAL2, a novel member of the mal proteolipid family, though interactions with TPD52-like proteins in the yeast two-hybrid system

The TPD52 (tumor protein D52)-like proteins are small coiled-coil motif-bearing proteins which were first identified though their expression in human breast carcinoma. TPD52-like proteins are known to interact in hetero-and homomeric fashions, but there are no known heterologous binding partners for these proteins. We now report the cloning of a novel member of the MAL proteolipid family, named MAL2, though its interaction with a TPD52L2 bait in a yeast two-hybrid screen. MAL2 is predicted to be 176 residues (19 kDa) with four transmembrane domains and is 35.8% identical to MAL, a proteolipid required in apical vesicle transport. The MAL2 prey bound all TPD52-like baits tested in the yeast two-hybrid system and in vitro translation of MAL2 produced a single 19-kDa (35)S-labeled protein which specifically bound full-length GST-Tpd52 in GST pull-down assays. The gene MAL2, which was localized to human chromosomal band 8q23 and shown to consist of four exons, is predominantly expressed in human kidney, lung, and liver. Our study has therefore identified a novel member of the MAL proteolipid family and potentially implicates TPD52-like proteins in vesicle transport.     

Acyl CoA profiles of transgenic plants that accumulate medium-chain fatty acids indicate inefficient storage lipid synthesis in developing oilseeds

Several Brassica napus lines transformed with genes responsible for the synthesis of medium- or long-chain fatty acids were examined to determine limiting factor(s) for the subsequent accumulation of these fatty acids in seed lipids. Examination of a decanoic acid (10:0) accumulating line revealed a disproportionately high concentration of 10:0 CoA during seed development compared to long-chain acyl CoAs isolated from the same tissues, suggesting that poor incorporation of 10:0 CoA into seed lipids limits 10:0 fatty acid accumulation. This relationship was also seen for dodecanoyl (12:0) CoA and fatty acid in a high 12:0 line, but not for octadecanoic (18:0) CoA and fatty acid in a high 18:0 line. Comparison of 10:0 CoA and fatty acid proportions from seeds at different developmental stages for transgenic B. napus and Cuphea hookeriana, the source plant for the medium-chain thioesterase and 3-ketoacyl-ACP synthase transgenes, revealed that C. hookeriana incorporates 10:0 CoA into seed lipids more efficiently than transgenic B. napus. Furthermore, beta-oxidation and glyoxylate cycle activities were not increased above wild type levels during seed development in the 8:0/10:0 line, suggesting that lipid catabolism was not being induced in response to the elevated 10:0 CoA concentrations. Taken together, these data suggest that transgenic plants that are engineered to synthesize medium-chain fatty acids may lack the necessary mechanisms, such as specific acyltransferases, to incorporate these fatty acids efficiently into seed lipids.     

Acute hyperglycemia alters the ability of the normal beta-cell to sense and respond to glucose

Impaired glucose tolerance (IGT) and non-insulin-dependent diabetes mellitus (NIDDM) are associated with an impaired ability of the beta-cell to sense and respond to small changes in plasma glucose. The aim of this study was to establish whether acute hyperglycemia per se plays a role in inducing this defect in beta-cell response. Seven healthy volunteers with no family history of NIDDM were studied on two occasions during a 12-h oscillatory glucose infusion with a periodicity of 144 min. Once, low-dose glucose was infused at a mean rate of 6 mg x kg(-1) x min(-1) and amplitude 33% above and below the mean rate, and, once, high-dose glucose was infused at 12 mg x kg(-1) x min(-1) and amplitude 16% above and below the mean rate. Mean glucose levels were significantly higher during the high-dose compared with the low-dose glucose infusion [9.5 +/- 0.8 vs. 6.8 +/- 0.2 mM (P < 0.01)], resulting in increased mean insulin secretion rates [ISRs; 469.1 +/- 43.8 vs. 268.4 +/- 29 pmol/min (P < 0.001)] and mean insulin levels [213.6 +/- 46 vs. 67.9 +/- 10.9 pmol/l (P < 0.008)]. Spectral analysis evaluates the regularity of oscillations in glucose, insulin secretion, and insulin at a predetermined frequency. Spectral power for glucose, ISR, and insulin was reduced during the high-dose glucose infusion [11.8 +/- 1.4 to 7.0 +/- 1.6 (P < 0.02), 7.6 +/- 1.5 to 3.2 +/- 0.5 (P < 0.04), and 10.5 +/- 1.6 to 4.6 +/- 0.7 (P < 0.01), respectively]. In conclusion, short-term infusion of high-dose glucose to obtain glucose levels similar to those previously seen in IGT subjects results in reduced spectral power for glucose, ISR, and insulin. The reduction in spectral power previously observed for ISR in IGT or NIDDM subjects may be due partly to hyperglycemia.     

Invasion of human epithelial cells by Campylobacter upsaliensis

Few data exist on the interaction of Campylobacter upsaliensis with host cells, and the potential for this emerging enteropathogen to invade epithelial cells has not been explored. We have characterized the ability of C. upsaliensis to invade both cultured epithelial cell lines and primary human small intestinal cells. Epithelial cell lines of intestinal origin appeared to be more susceptible to invasion than non-intestinal-derived cells. Of three bacterial isolates studied, a human clinical isolate, CU1887, entered cells most efficiently. Although there was a trend towards more efficient invasion of Caco-2 cells by C. upsaliensis CU1887 at lower initial inocula, actual numbers of intracellular organisms increased with increasing multiplicity of infection and with prolonged incubation period. Confocal microscopy revealed C. upsaliensis within primary human small intestinal cells. Both Caco-2 and primary cells in non-confluent areas of the infected monolayers were substantially more susceptible to infection than confluent cells. The specific cytoskeletal inhibitors cytochalasin B, cytochalasin D and vinblastine attenuated invasion of Caco-2 cells in a concentration-dependent manner, providing evidence for both microtubule- and microfilament-dependent uptake of C. upsaliensis. Electron microscopy revealed the presence of organisms within Caco-2 cell cytoplasmic vacuoles. C. upsaliensis is capable of invading epithelial cells and appears to interact with host cell cytoskeletal structures in order to gain entry to the intracellular environment. Entry into cultured primary intestinal cells ex vivo provides strong support for the role of host cell invasion during human enteric C. upsaliensis infection.     

Skin graft survival in genetically identical cloned pigs

Nuclear transfer technology allows for the reprogramming of somatic cells, and the production of embryonic stem cells and animals that are genetically identical in terms of nuclear DNA to the parental somatic cell. It is assumed that these products of nuclear transfer technology will be immunologically compatible to each other in spite of the fact that there are data that show differences in the expression patterns and phenotypes between animals produced by nuclear transfer. We have produced a series of cloned pigs from embryonic fibroblasts. Microsatellite analysis was used to confirm that the clones were genetically identical. Skin transplants were performed to assess immunological reactivity. Skin transplants between genetically identical cloned pigs were accepted, whereas third party grafts were rejected. Histological analysis of the grafts showed edema and mononuclear cell infiltrates in the recipient's skin in rejected grafts and not in grafts that were accepted. Our data supports the notion that genetically identical cloned pigs are immunologically compatible.     

Vestigial ophiopluteal structures in the lecithotrophic larvae of Ophionereis schayeri (Ophiuroidea)

Evolution of echinoderm development from a feeding to a non-feeding mode can be examined by studying non-feeding larvae with structures that appear to be vestiges derived from a feeding ancestral state. The lecithotrophic larvae of the Australian brittle star Ophionereis schayeri possess such features, and the early development of this species was documented by light and scanning electron microscopy. The embryos undergo irregular cleavage, resulting in the formation of different sized blastomeres, with subsequent development through a wrinkled blastula stage. The lecithotrophic larva of O. schayeri possesses several vestigial ophiopluteal structures, including a continuous ciliated band, a larval gut, and a larval skeleton. The ciliated band is a reduced expression of the continuous ciliated band typical of ophioplutei. The larval gut is a transiently complete system, but an esophageal plug and rapid closure of the blastopore renders it nonfunctional. The larval skeleton, though reduced, consists of four rods corresponding to the body, posterolateral, anterolateral, and postoral rods characteristic of an ophiopluteus. Due to a heterochrony in larval skeletogenesis, the postoral rods develop early and simultaneously with the other rods. Compared with the larvae of other lecithotrophic ophiuroids, the larva of O. schayeri is one of the most reduced ophiopluteal forms reported to date.     

Systemic activation and antigen-driven oligoclonal expansion of T cells in a mouse model of colitis

Transfer of CD4+CD45RBhigh T cells into immunodeficient mice results in both the expansion of the transferred T cells and colitis. Here we show that colitis pathogenesis requires expression of MHC class II molecules by the immune-deficient host. Analysis of the TCRbeta repertoire of the cells found in the large intestine of diseased mice revealed a population with restricted TCR diversity. Furthermore, nucleotide sequence analysis demonstrated the selection for particular CDR3beta amino acid sequence motifs. Collectively, these data indicate that the expansion of T cells in the intestine and colitis pathogenesis are likely to require the activation of Ag-specific T cells, as opposed to nonspecific or superantigen-mediated events. There is relatively little overlap, however, when the TCR repertoires of different individuals are compared, suggesting that a number of Ags can contribute to T cell expansion and the generation of a T cell population in the intestine. Surprisingly, many of the expanded clones found in the large intestine also were found in the spleen and elsewhere, although inflammation is localized to the colon. Additionally, donor-derived T cells appear to be activated in both the intestine and the spleen at early time points after cell transfer. Together, these results strongly suggest that disease induction in this model involves either the early and systemic activation of antigen-specific T cells or the rapid dispersal of T cells activated at a particular site.     

A new method to monitor airborne inoculum of the fungal plant pathogens Mycosphaerella brassicicola and Botrytis cinerea

We describe a new microtiter immunospore trapping device (MTIST device) that uses a suction system to directly trap air particulates by impaction in microtiter wells. This device can be used for rapid detection and immunoquantification of ascospores of Mycosphaerella brassicicola and conidia of Botrytis cinerea by an enzyme-linked immunosorbent assay (ELISA) under controlled environmental conditions. For ascospores of M. brassicicola correlation coefficients (r(2)) of 0.943 and 0.9514 were observed for the number of MTIST device-impacted ascospores per microtiter well and the absorbance values determined by ELISA, respectively. These values were not affected when a mixed fungal spore population was used. There was a relationship between the number of MTIST device-trapped ascospores of M. brassicicola per liter of air sampled and the amount of disease expressed on exposed plants of Brassica oleracea (Brussels sprouts). Similarly, when the MTIST device was used to trap conidia of B. cinerea, a correlation coefficient of 0.8797 was obtained for the absorbance values generated by the ELISA and the observed number of conidia per microtiter well. The relative collection efficiency of the MTIST device in controlled plant growth chambers with limited airflow was 1.7 times greater than the relative collection efficiency of a Burkard 7-day volumetric spore trap for collection of M. brassicicola ascospores. The MTIST device can be used to rapidly differentiate, determine, and accurately quantify target organisms in a microflora. The MTIST device is a portable, robust, inexpensive system that can be used to perform multiple tests in a single sampling period, and it should be useful for monitoring airborne particulates and microorganisms in a range of environments.