Replacement of the antifreeze-like domain of human N-acetylneuraminic acid phosphate synthase with the mouse antifreeze-like domain impacts both N-acetylneuraminic acid 9-phosphate synthase and 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid 9-phosphate synthase activities

Human NeuNAc-9-P synthase is a two-domain protein with ability to synthesize both NeuNAc-9-P and KDN-9-P. Its mouse counterpart differs by only 20 out of 359 amino acids but does not produce KDN-9-P. By replacing the AFL domain of the human NeuNAc-9-P synthase which accommodates 12 of these differences, with the mouse AFL domain we examined its importance for the secondary KDN-9-P synthetic activity. The chimeric protein retained almost half of the ability of the human enzyme for KDN-9-P synthesis while the NeuNAc-9-P production was reduced to less than 10%. Data from the homology modeling and the effect of divalent ions and temperature on the enzyme activities suggest conformational differences between the human and mouse AFL domains that alter the shape of the cavity accommodating the substrates. Therefore, although the AFL domain itself does not define the ability of the human enzyme for KDN-9-P synthesis, it is important for both activities by aiding optimal positioning of the substrates.     

Identification of mammalian Vps24p as an effector of phosphatidylinositol 3,5-bisphosphate-dependent endosome compartmentalization

Phosphatidylinositol 3,5-bisphosphate is a membrane lipid found in all eukaryotes so far studied but downstream effector proteins of this lipid have yet to be identified. Here we report the use of cDNA phage libraries in conjunction with synthetic biotinylated derivatives of phosphatidylinositol 3,5-bisphosphate in the identification of a mammalian phosphatidylinositol 3,5-bisphosphate-binding protein, mVps24p. This protein is orthologous to the Saccharomyces cerevisiae protein, Vps24p, a class-E vacuolar protein-sorting protein. Using in vitro liposome binding and competition assays, we demonstrate that mVps24p selectively binds to phosphatidylinositol 3,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate in preference to other phosphoinositides tested. When expressed in cultured mammalian cells, full-length mVps24p is cytosolic. However, when cells expressing the full-length mVps24p are co-transfected with a mutated form of mVps4p (which is defective in ATP hydrolysis), or when a N-terminal construct of mVps24p is expressed, the class-E cellular phenotype with swollen vacuoles is induced and mVps24p is membrane-associated. Furthermore, the accumulation of the N-terminal mVps24p construct on the swollen endosomal membranes is abrogated when phosphatidylinositol 3,5-bisphosphate synthesis is blocked with wortmannin. These data provide the first direct link between phosphatidylinositol 3,5-bisphosphate and the protein machinery involved in the production of the class-E cellular phenotype. We hypothesize that accumulation of Vps24 on membranes occurs when membrane association (dependent on interaction of phosphatidylinositol 3,5-bisphosphate with the N-terminal domain of the protein) is uncoupled from membrane disassociation (driven by Vps4p).     

Interplay between plasmid partition and postsegregational killing systems

Active partition systems and postsegregational killing (PSK) systems are present together in naturally occurring low-copy-number plasmids. Theory suggests that PSK may act as the ultimate determinant of plasmid retention, whereas the partition system may minimize the growth penalty to the host, resulting in a near-ideal symbiosis when the systems combine. Here, we prove the validity of this principle for a specific case involving the P1par system and the mvp PSK system.     

Neutrophil transepithelial migration: regulation at the apical epithelial surface by Fc-mediated events

Neutrophil (PMN) transepithelial migration is a major effector of epithelial defense in inflammatory diseases involving mucosal surfaces. However, major receptor-ligand interactions between epithelial cells and PMN remain incompletely characterized. To better define the molecular events involved in PMN interactions with epithelial cells, we produced a monoclonal antibody called g82 that inhibited PMN transepithelial migration in the physiological basolateral-to-apical direction. The g82 antigen localized to the apical surface of human colonic epithelium and was significantly upregulated under inflammatory conditions. Immunoprecipitation revealed two polypeptides of M(r) 207 and 32 kDa. F(ab')(2) fragments from g82 IgG had no effect on transmigration, suggesting Fc dependence. Further experiments confirmed dependence on the PMN Fc receptor CD32A and that the observed effects were secondary to a failure of PMN to detach from the apical epithelial surface. These Fc-mediated events were epitope specific since binding, isotype-matched antibodies did not affect detachment. These results identify a new mechanism for retention of PMN at the apical epithelial surface following transepithelial migration. This pathway may be important in pathogen clearance and mucosal pathophysiology associated with autoimmunity.     

The Role of Lipid Raft Aggregation in the Infection of Type II Pneumocytes by Mycobacterium tuberculosis

Dynamic, cholesterol-dense regions of the plasma membrane, known as lipid rafts (LR), have been observed to develop during and may be directly involved in infection of host cells by various pathogens. This study focuses on LR aggregation induced in alveolar epithelial cells during infection with Mycobacterium tuberculosis (Mtb) bacilli. We report dose- and time-dependent increases in LR aggregation after infection with three different strains at multiplicities of infection of 1, 10 and 100 from 2–24 hr post infection (hpi). Specific strain-dependent variations were noted among H37Rv, HN878 and CDC1551 with H37Rv producing the most significant increase from 15 aggregates per cell (APC) to 27 APC at MOI 100 during the 24 hour infection period. Treatment of epithelial cells with Culture Filtrate Protein, Total Lipids and gamma-irradiated whole cells from each strain failed to induce the level of LR aggregation observed during infection with any of the live strains. However, filtered supernatants from infected epithelial cells did produce comparable LR aggregation, suggesting a secreted mycobacterial product produced during infection of host cells is responsible for LR aggregation. Disruption of lipid raft formation prior to infection indicates that Mtb bacilli utilize LR aggregates for internalization and survival in epithelial cells. Treatment of host cells with the LR-disruption agent Filipin III produced a nearly 22% reduction in viable bacteria for strains H37Rv and HN878, and a 7% reduction for strain CDC1551 after 6 hpi. This study provides evidence for significant mycobacterial-induced changes in the plasma membrane of alveolar epithelial cells and that Mtb strains vary in their ability to facilitate aggregation and utilization of LR.     

Influence of single-level lumbar degenerative disc disease on the behavior of the adjacent segments—A finite element model study

The current study investigated mechanical predictors for the development of adjacent disc degeneration. A 3-D finite element model of a lumbar spine was modified to simulate two grades of degeneration at the L4–L5 disc. Degeneration was modeled by changes in geometry and material properties. All models were subjected to follower preloads of 800 N and moment loads in the three principal directions of motion using a hybrid protocol. Degeneration caused changes in the loading and motion patterns of the segments above and below the degenerated disc. At the level (L3–L4) above the degenerated disc, the motion increased due to moderate degeneration by 21% under lateral bending, 26% under axial rotation and 28% under flexion/extension. At the level (L5-S1) below the degenerated disc, motion increased only during lateral bending by 20% due to moderate degeneration. Both the L3–L4 and L5-S1 segment showed a monotonic increase in both the maximum von Mises stress and shear stress in the annulus as degeneration progressed for all loading directions, expect extension at L3–L4. The most significant increase in stress was observed at the L5-S1 level during axial rotation with nearly a ten-fold increase in the maximum shear stress and 103% increase in the maximum von Mises stress. The L5-S1 segment also showed a progressive increase in facet contact force for all loading directions with degeneration. Nucleus pressure did not increase significantly for any loading direction at either the caudal or cephalic adjacent segment. Results suggest that single-level degeneration can increase the risk for injury at the adjacent levels.     

The secreted Salmonella dublin phosphoinositide phosphatase, SopB, localizes to PtdIns(3)P-containing endosomes and perturbs normal endosome to lysosome trafficking

Invasion and survival in mammalian cells by Salmonella enterica is mediated by bacterial proteins that are delivered to the host cell cytoplasm by type III secretion systems. One of these proteins, SopB/SigD, is a phosphoinositide phosphatase that can hydrolyse a number of substrates in vitro including PtdIns(3,5)P2. These substrates are, however, likely to be restricted in vivo by the localization of SopB, as different phosphoinositides have distinct spatial distributions in mammalian cells. In the present study, we show that heterologously expressed SopB localizes almost exclusively to endosomes containing the lipid PtdIns(3)P, and on which ESCRT (endosomal sorting complexes required for transport) proteins assemble. Furthermore, we present evidence that SopB can inhibit trafficking of activated epidermal growth factor receptor to the lysosome. These results provide further evidence that PtdIns(3,5)P2, a lipid involved in endosomal maturation, may be a relevant in vivo substrate of SopB. We hypothesize that reduction of PtdIns(3,5)P2 levels in cells by the action of SopB may perturb the function of a subset of ESCRT proteins that have previously been shown to bind to this lipid.     

Apolipoprotein A-I gene expression is regulated by cellular zinc status in Hep G2 cells

The influence of Zn on the expression of the apolipoprotein A-I(apoA-I) gene in Hep G2 cells was examined. Zn depletion was achievedwith a low-Zn (ZD) medium prepared from Zn-free growth medium(Opti), a ZD medium containing Chelex 100-extracted fetal bovine serum (CHE), and a medium containing chelator1,10-phenanthroline (OP). Compared with those for their respectivecontrols, cellular Zn levels were reduced by 55, 48, and 46% andapoA-I mRNA abundances were reduced by 20, 29, and 28% in Opti, CHE,and OP systems, respectively, after one passage in ZD media or 24 h inOP medium. To establish the specificity of Zn treatment, groups of ZDcells were treated with their respective control media for the last 24 h (ZDA) or normal cells were cultured with OP medium supplemented withZn (OP-Zn). ZDA treatments partially normalized cellular Zn levels inthe Opti system and restored or elevated apoA-I mRNA levels in the Optior CHE system, respectively. Similarly, the OP-Zn treatment restoredthe cellular Zn and apoA-I mRNA levels. Furthermore, one passage ofculture with Zn-supplemented media in both the Opti and CHE systemsresulted in higher cellular Zn and apoA-I mRNA levels than those forcontrols. Most significantly, short-term high-Zn induction to normalcells markedly elevated the cellular Zn (3-fold) and apoA-I mRNA(5-fold) levels. Data derived from this study strongly suggest that theexpression of apoA-I is regulated by cellular Zn status.

     

The addition of CO2 to traditional taste solutions alters taste quality

Previous studies of the effect of carbonation on taste perception have suggested that it may be negligible, manifesting primarily in increases in the perceived intensity of weak salt and sour stimuli. Assuming CO2 solutions in the mouth stimulate only trigeminal nerve endings, this result is not altogether surprising; however, there are neurophysiological data indicating that CO2 stimulates gustatory as well as trigeminal fibers. In that case, carbonation might alter the quality profile of a stimulus without producing substantial changes in overall taste intensity--much as occurs when qualitatively different taste stimuli are mixed. To address this possibility, subjects were asked to rate the total taste intensity of moderate concentrations of stimuli representing each of the basic tastes and their binary combinations, with an without added carbonation. They then subdivided total taste intensity into the proportions of sweetness, saltiness, sourness, bitterness and 'other taste qualities' they perceived. The addition of carbonation produced only small increases in ratings of total taste intensity. However, rather dramatic alterations in the quality profiles of stimuli were observed, particularly for sweet and salty tastes. The nature of the interaction is consistent with a direct effect of carbonation/CO2 on the gustatory system, although the possibility that at least some of the observed effects reflect trigeminal-gustatory interactions cannot be ruled out.