Involvement of the galactosyl-1-phosphate transferase encoded by the Salmonella enterica rfbP gene in O-antigen subunit processing.

rfbT of Salmonella enterica LT2 was previously thought, together with rfaL, to be involved in the ligation of polymerized O antigen to core-lipid A, and three mutants were known. We report the mapping of the mutations to rfbP, the galactosyl-1-phosphate transferase gene, which is now shown to encode a bifunctional protein. The mutations which have the former rfbT phenotype are referred to as rfbP(T). We also show that rfbP(T) mutants are not blocked in the ligation step as previously believed but in an earlier step, possibly in flipping the O-antigen subunit on undecaprenyl pyrophosphate from the cytoplasmic to periplasmic face of the cytoplasmic membrane.     

Increased Expression of Apolipoprotein D Following Experimental Traumatic Brain Injury

Increasing evidence suggests that apolipoprotein D (apoD) could play a major role in mediating neuronal degeneration and regeneration in the CNS and the PNS. To investigate further the temporal pattern of apoD expression after experimental traumatic brain injury in the rat, male Sprague-Dawley rats were subjected to unilateral cortical impact injury. The animals were killed and examined for apoD mRNA and protein expression and for immunohistological analysis at intervals from 15 min to 14 days after injury. Increased apoD mRNA and protein levels were seen in the cortex and hippocampus ipsilateral to the injury site from 48 h to 14 days after the trauma. Immunohistological investigation demonstrated a differential pattern of apoD expression in the cortex and hippocampus, respectively: Increased apoD immunoreactivity in glial cells was detected from 2 to 3 days after the injury in cortex and hippocampus. In contrast, increased expression of apoD was seen in cortical and hippocampal neurons at later time points following impact injury. Concurrent histopathological examination using hematoxylin and eosin demonstrated dark, shrunken neurons in the cortex ipsilateral to the injury site. In contrast, no evidence of cell death was observed in the hippocampus ipsilateral to the injury site up to 14 days after the trauma. No evidence of increased apoD mRNA or protein expression or neuronal pathology by hematoxylin and eosin staining was detected in the contralateral cortex and hippocampus. Our results reveal induction of apoD expression in the cortex and hippocampus following traumatic brain injury in the rat. Our data also suggest that increased apoD expression may play an important role in cortical neuronal degeneration after brain injury in vivo. However, increased expression of apoD in the hippocampus may not necessarily be indicative of neuronal death.     

Phosphorylation of lens membranes with a cyclic AMP-dependent protein kinase purified from the bovine lens

We report the phosphorylation of lens membranes with a cAMP-dependent protein kinase isolated from bovine lenses. The holoenzyme was eluted from DEAE agarose at less than 100 mM NaCl and from gel filtration columns with a relative molecular weight of 180 000. The regulatory subunit was identified with the affinity label 8-azido-[32P]cAMP. Four focusing variants with relative molecular weights of 49 000 were seen on two-dimensional gels. The catalytic subunit was purified approx. 5000-fold and migrated at 42 000 Mr on SDS gels. Based on these observations, the enzyme is classified as a Type I cAMP-dependent protein kinase. Purified lens plasma membranes were incubated with the holoenzyme or its catalytic subunit in the presence of 32P-labeled ATP. Several membrane proteins, including the major lens membrane polypeptide, MP26, were shown to be substrates for the kinase in this reaction. MP26 appears to be the major component of intercellular junctions in the lens. Studies with protease treatments on labeled membranes appeared to localize the phosphorylation sites to the cytoplasmic side of the membrane.     

Immunosuppressive activity of T cell clones generated from human T cells stimulated with autologous TPHA cells

T cell clones were generated from human T cells stimulated with autologous phytohemagglutinin (PHA)-activated T (TPHA) cells. Characterization of three T cell clones originated from donor SF and one from donor JM showed that they proliferated when stimulated with autologous TPHA cells, non-T cells, and peripheral blood mononuclear cells, but did not proliferate when stimulated with allogeneic TPHA cells, non-T cells, and mononuclear cells, with autologous and allogeneic resting T cells, and with PHA. These results in conjunction with the blocking of the proliferation by anti-histocompatibility leukocyte antigen class II monoclonal antibodies indicate that these class II antigens are involved in the proliferation of T cell clones stimulated with autologous lymphoid cells. The four T cell clones are cytotoxic neither to autologous lymphoid cells nor to a panel of cultured human cell lines. The four T cell clones display immunosuppressive activity, since they inhibit the proliferation of autologous and allogeneic cells stimulated with antigens and mitogens and the secretion of immunoglobulin by B cells stimulated with pokeweed mitogen in presence of T cells. Furthermore, the four T cell clones display differential inhibitory activity on the proliferation of cultured human cell lines. The immunosuppressive activity is species-specific, since the T cell clones do not inhibit the proliferation of murine cells. The suppression is mediated by a factor(s) with an apparent m.w. of 13,000 to 16,000. The suppressor activity is labile at alkaline pH and is lost following incubation with pronase (100 U/ml) for 30 min at 37 degrees C.     

Effect of dolichyl monophosphate on the permeability properties of lipid membranes

The effect of dolichyl monophosphate on the permeability properties of dimyristoylphosphatidylcholine bilayers to alkaline cations, Ca²⁺ and glucose has been determined by stop-flow spectrophotometry. The results show that, in con trast to free dolichol effects, the monophosphate derivative increased the permeability following a decreasing order of the permeating particle size. Phase diagrams indicate that dolichyl monophosphate is fully incorporated into the phosphatidylcholine bilayer around 0.75% weight/weight ratio. For these ratios, the permeation of ions is higher in the gel than in the liquid crystalline state.     

Immunoaffinity purification of the lipid transfer protein complex directly from human plasma

The human cholesteryl ester (CE) and triglyceride (TG) exchange protein (denoted LTC or lipid transfer complex) was isolated in a single step from plasma using immunoaffinity batch extraction. Antibodies were raised against two preparations of conventionally purified LTC. LTC-I and LTC-II (purified 20,000-fold and 3500-fold, respectively) were used as immunogens. The antiLTC antibodies were isolated by anion-exchange chromatography and coupled to Affi-Gel 10. Chromatography of plasma on antiLTC Affi-Gel removed all of the CE and TG transfer activity. Moreover, LTC prepared from both antiLTC-I and antiLTC-II-Affi-Gel matrices were identical when analyzed by sodium dodecyl sulfate-polyacrylamide gel LTC electrophoresis. LTC exhibited two protein bands of Mr (apparent) 67,000 and 58,000 and a broad, faintly staining region at greater than 150,000. Analysis of LTC by immunoblotting indicated that both antiLTC-I and antiLTC-II antibodies recognized the same LTC proteins. Isoelectric focussing of LTC gave two pI values, 5.2 and 8.7. These data suggest that LTC is a complex of specific proteins and perhaps lipid. Specific CE and TG exchange activities of immunoaffinity-purified LTC were comparable, although the activities were low with respect to that of the antigen used to generate antiLTC-I. This is not due to contamination of LTC by albumin, lecithin:cholesterol acyltransferase, or apolipoproteins AI, AII, B, CIII, D, or E.