The cytoplasmic and the transmembrane domains are not sufficient for class I MHC signal transduction

Class I MHC molecules deliver activation signals to T cells. To analyze the role of the cytoplasmic and the transmembrane (TM) domains of class I MHC molecules in T cell activation, Jurkat cells were transfected with genes for truncated class I MHC molecules which had only four intracytoplasmic amino acids and no potential phosphorylation sites or native molecules or both. Cross-linking either the native or the truncated molecules induced IL-2 production even under limiting stimulation conditions of low engagement of the stimulating mAb. Moreover, direct comparison of transfected truncated and native class I MHC molecules expressed on the same cell revealed significant stimulation induced by cross-linking the truncated molecules, despite low expression. In addition, truncated class I MHC molecules were as able to synergize with CD3, CD2, or CD28 initiated IL-2 production as native molecules. In further experiments, hybrid constructs made of the extracellular portion of the murine CD8 alpha chain and of the TM and the intracytoplasmic domains of H-2Kk class I MHC molecule were transfected into Jurkat T cells. The expression of the transfected hybrid molecules was comparable to that of the native HLA-B7 molecules. Cross-linking the intact monomorphic HLA-A,B,C epitope or the polymorphic HLA-B7 epitope induced IL-2 production upon costimulation with PMA. In contrast, cross-linking the hybrid molecules generated neither an increase in intracellular calcium concentration ([Ca2+]i) nor stimulated IL-2 production. By contrast, cross-linking intact murine class I MHC molecules induced [Ca2+]i, signal and IL-2 production in transfected Jurkat cells. The data therefore indicate that unlike many other signaling molecules, signaling via class I MHC molecules does not involve the cytoplasmic and the TM portions of the molecule, but rather class I MHC signal transduction is likely to be mediated by the extracellular domain of the molecule.     

The matrix protein of HIV-1 is not sufficient for assembly and release of virus-like particles

There is an expectation for Nursing Development Units (NDUs) to explore and develop the clinical leadership role. This paper describes how 28 Department of Health funded NDUs in England sought to meet this objective. We describe the personal and professional characteristics of NDU clinical leaders (CLs) and their perceived responsibilities. We identify 10 elements which appear to be central to the CL role and together form a core role set of the leader which is applicable, to some extent, across clinical specialties and settings. The position of the CL in the organizational hierarchy emerges as crucial to his/her ability to act on these responsibilities and fulfil a leadership role.     

The FGF receptor-1 tyrosine kinase domain regulates myogenesis but is not sufficient to stimulate proliferation

STUDY OBJECTIVES: The aims of this study were: to evaluate the performance of a novel arterial biopsy catheter in obtaining pulmonary endovascular samples in hypertensive dogs; to compare the results of pulmonary endoarterial biopsy in hypertensive vs normotensive dogs; and to assess the histologic changes in the hypertensive model. DESIGN AND INTERVENTIONS: Thirty-four dogs (27 with normal pulmonary arterial pressures and seven with pulmonary hypertension) were catheterized through an external jugular vein to obtain endovascular biopsy samples from distal pulmonary arteries 2 to 3 mm in luminal diameter. To induce pulmonary hypertension, seven dogs were given repeated infusions of 0.6- to 0.9-mm ceramic microspheres into the superior vena cava. Endoarterial samples were obtained at pulmonary systolic arterial pressures ranging from 10 to 110 mm Hg. MEASUREMENTS AND RESULTS: Sixty-two biopsy catheterization procedures were performed in the 34 dogs. After 12 initial procedures of technique refinement, endoarterial samples were obtained in each of the last 50 procedures (21 in normotensive dogs and 29 in hypertensive dogs). The average number of endovascular biopsy samples retrieved was 7.1 (range, 2 to 12) from a mean of 8.6 (range, 2 to 15) biopsy attempts per catheterization (success rate=83%). The average biopsy piece measured 1.13 mm in length, 0.33 mm in depth, and up to 1.0 mm in width. The biopsy success rates and endoarterial sample sizes were similar in normotensive and hypertensive dogs. Smooth muscle cells and endothelial cells were grown from the biopsy samples. There were no significant procedural complications, except for one self-limited hemorrhage. Histologically, samples obtained from dogs with pulmonary hypertension showed characteristic changes when compared with biopsies from normotensive dogs. CONCLUSION: This new endoarterial biopsy catheter was safe and effective when used to obtain pulmonary endoarterial samples in dogs with normal and experimentally elevated pulmonary arterial pressures. The quality and quantity of the biopsy samples allowed identification of pathologic changes.     

The tyrosine-containing cytoplasmic tail of CD1b is essential for its efficient presentation of bacterial lipid antigens

OBJECTIVE: As many as 35 percent of Tourette's Syndrome patients do not acquire this disorder genetically. Since there has been little research conducted in this area, the purpose of this study was to compare the clinical differences between two groups of patients with Tourette's Syndrome (TS), one with family history of TS and one without. METHOD: Using data of eight previously diagnosed TS patients, the authors made comparisons of clinical and sociodemographic variables between a group of three patients with family history of TS and five with no family history. RESULTS: There were no differences in clinical presentation, current age, age at diagnosis, gender, and socioeconomic status. There were differences in birth history, developmental milestones, I.Q., and neurological findings between patients with family history and no family history of TS. CONCLUSIONS: Our findings support the need for testing the hypothesis of a multidetermined origin of TS, a disorder in which hereditary, neuropsychological, and environmental factors play a role.     

The cytoplasmic kinase domain of PhoR is sufficient for the low phosphate-inducible expression of pho regulon genes in Bacillus subtilis

PhoP-PhoR, one of three two-component systems known to be required to regulate the pho regulon in Bacillus subtilis, directly regulates the alkaline phosphatase genes that are used as pho reporters. Biochemical studies showed that B. subtilis PhoR, purified from Escherichia coli, was autophosphorylated in vitro in the presence of ATP. Phosphorylated PhoR showed stability under basic conditions but not acidic conditions, indicating that the phosphorylation probably occurs on a conserved histidine residue. Phospho-PhoR phosphorylated its cognate response regulator, PhoP in vitro. B. subtilis phoR was placed in the Bacillus chromosome under the control of the Pspac promoter, which is IPTG inducible. The wild-type phoR, under either native promoter or Pspac promoter with IPTG induction, resulted in a similar level of alkaline phosphatase production. Under high phosphate conditions, strains containing wild-type phoR, or phoR mutant gene products that lacked either the periplasmic domain, or both N-terminal transmembrane PhoR mutant gene products that lacked either the periplasmic domain, or both N-terminal transmembrane PhoR sequences or various extended N-terminal sequences, showed no significant APase production. Under phosphate starvation conditions, in the presence of IPTG, all strains containing mutated phoR genes showed alkaline phosphatase induction patterns similar to that of the wild-type strain, although the fully induced level was lower in the mutants. The decrease in total alkaline phosphatase production in these mutant strains can be compensated completely or partially by increasing the copy number of the mutant phoR gene. These in vivo results suggest that the C-terminal kinase domain of PhoR is sufficient for the induction of alkaline phosphatase expression under phosphate-limited conditions, and that the regulation for repression of APase under phosphate-replete conditions remains intact.     

The P2Y1 receptor, necessary but not sufficient to support full ADP-induced platelet aggregation, is not the target of the drug clopidogrel

Recently we showed that the P2Y1 receptor coupled to calcium mobilization is necessary to initiate ADP-induced human platelet aggregation. Since the thienopyridine compound clopidogrel specifically inhibits ADP-induced platelet aggregation, it was of interest to determine whether the P2Y1 receptor was the target of this drug. Therefore we studied the effects of clopidogrel and of the two specific P2Y1 antagonists A2P5P and A3P5P on ADP-induced platelet events in rats. Although clopidogrel treatment (50 mg/kg) greatly reduced platelet aggregation in response to ADP as compared to untreated platelets, some residual aggregation was still detectable. In contrast, A2P5P and A3P5P totally abolished ADP-induced shape change and aggregation in platelets from both control and clopidogrel-treated rats. A2P5P and A3P5P (100 microM) totally inhibited the [Ca2+]i rise induced by ADP (0.1 microM) in control and clopidogrel-treated platelets, whereas clopidogrel treatment had no effect. Conversely, the inhibition of adenylyl cyclase induced by ADP (5 microM) was completely blocked by clopidogrel but not modified by A2P5P or A3P5P (100 microM). A3P5P (1 mM) reduced the number of [33P]2MeSADP binding sites on control rat platelets from 907 +/- 50 to 611 +/- 25 per platelet. After clopidogrel treatment, binding of [33P]2MeSADP decreased to 505 +/- 68 sites per platelet and further decreased to 55 +/- 12 sites in the presence of A3P5P (1 mM). In summary, these results demonstrate that the platelet P2Y1 receptor responsible for the initiation of aggregation in response to ADP is not the target of clopidogrel. Platelets may express another, as yet unidentified, P2Y receptor, specifically coupled to the inhibition of adenylyl cyclase and necessary to induce full platelet aggregation, which could be the target of this drug.     

Up-regulation of hypoxia-inducible factor-1alpha is not sufficient for hypoxic/anoxic p53 induction

Oxygen-deprived regions of a solid tumor can induce tumor suppressor p53 expression and hence select for p53-mutant tumor cells with diminished apoptotic potential. It has been proposed that the hypoxia-inducible factor-1 (HIF-1) alpha subunit binds to p53 and protects it from proteasomal degradation. However, we found that hypoxic conditions that strongly induce HIF-1-dependent endogenous gene expression as well as HIF-1alpha protein neither induce p53-dependent gene expression nor p53 protein. The iron chelator deferoxamine induced both HIF-1alpha and p53, but p53 up-regulation could still be detected in HIF-1alpha-deficient cells, suggesting that mechanisms other than HIF-1alpha activation contribute to oxygen-regulated p53 induction.     

A homologue of Saccharomyces cerevisiae Dpm1p is not sufficient for synthesis of dolichol-phosphate-mannose in mammalian cells

Dolichol-phosphate-mannose (Dol-P-Man) serves as a donor of mannosyl residues in major eukaryotic glycoconjugates. It donates four mannosyl residues in the N-linked oligosaccharide precursor and all three mannosyl residues in the core of the glycosylphosphatidylinositol anchor. In yeasts it also donates one mannose to the O-linked oligosaccharide. The yeast DPM1 gene encodes a Dol-P-Man synthase that is a transmembrane protein expressed in the endoplasmic reticulum. We cloned human and mouse homologues of DPM1, termed hDPM1 and mDPM1, respectively, both of which encode proteins of 260 amino acids, having 30% amino acid identity with yeast Dpm1 protein but lacking a hydrophobic transmembrane domain, which exists in the yeast synthase. Human and mouse DPM1 cDNA restored Dol-P-Man synthesis in mouse Thy-1-deficient mutant class E cells. Mouse class E mutant cells had an inactivating mutation in the mDPM1 gene, indicating that mDPM1 is the gene for class E mutant. In contrast, hDPM1 and mDPM1 cDNA did not complement another Dol-P-Man synthesis mutant, hamster Lec15 cells, whereas yeast DPM1 restored both mutants. Therefore, in contrast to yeast, mammalian cells require hDPM1/mDPM1 protein and a product of another gene that is defective in Lec15 mutant cells for synthesis of Dol-P-Man.     

The presence of transthyretin in rat ependymal cells is due to endocytosis and not synthesis

The presence and synthesis of transthyretin, a major carrier protein of thyroxine in rat cerebrospinal fluid, was investigated in choroid plexus epithelial cells and ependymal cells by immunocytochemistry, in situ hybridization, and analysis by Northern and Western blot using a specific oligonucleotide probe and a specific polyclonal antibody to transthyretin. Choroid plexus epithelial cells expressed transthyretin at high levels in developing rat cerebral hemispheres and in cultured cells. These cells secreted transthyretin into the cerebrospinal fluid. In the developing rat brain transthyretin was present in the cytoplasm of ependymal cells, in vesicles in contact with the apical membrane and in cilia. In ependymal cell cultures this protein was particularly abundant in the cilia of these cells. In contrast, ependymal cells did not synthesize transthyretin. It is postulated that transthyretin is transported to ependymal cells from the cerebrospinal fluid by endocytosis.     

C3a is a chemotaxin for human eosinophils but not for neutrophils. I. C3a stimulation of neutrophils is secondary to eosinophil activation

Inflammatory action of the potent chemotaxin C5a has been well characterized on a variety of human cell types, including neutrophils, monocytes, basophils, and eosinophils. The cellular effects of C3a are less well defined. Contradictory reports have been published for C3a activation of neutrophils. Recent reports that C3a activates both basophils and eosinophils prompted us to reinvestigate the effects of C3a stimulation on eosinophils. We hypothesized that C3a activation of eosinophils, cells that are present in most neutrophil preparations, might lead to neutrophil activation. Using neutrophils of 98% purity, we observed no evidence of cellular activation after stimulation with either C3a, recombinant human C3a (rhC3a), or the synthetic C3a analogue C3a 57-77, Y57. Eosinophils purified to > 98% purity displayed concentration-dependent polarization, chemotaxis, and enzyme release by stimulation with C3a, rhC3a, and the synthetic C3a analogue. An inactive form of C3a, C3adesArg, failed to stimulate either eosinophils or neutrophils. Using neutrophil preparations containing 5-9% eosinophils, up to 20% of neutrophils became polarized after exposure to C3a. Likewise, we demonstrated that supernatant from C3a-stimulated eosinophils promotes neutrophil chemotaxis. Eosinophil polarization experiments were repeated in the presence of antibody to the C5a receptor (C5aR) to show that C3a and C5a interact with different receptors. C3a activates eosinophils in the presence of anti-C5aR antibody at concentrations that fully block C5a activation. We conclude that eosinophils are directly activated by either C3a or C5a, whereas C3a failed to activate neutrophils. C3a acts on eosinophils via a receptor that is distinct from C5aR. Since neutrophils are indirectly stimulated by C3a, eosinophils contaminating neutrophil preparations may explain earlier reports that C3a activates human neutrophils.     

Activation of microglia in HIV-1 infected brains is not dependent on the presence of HIV-1 antigens

The activation pattern of microglia in the cerebral cortex of AIDS patients with the neuropathological diagnosis of HIV-1 encephalitis was investigated by immunohistochemistry and morphometry. The number of activated microglial cells in the grey and white matter of five cortical regions was determined. In the grey and white matter of all cortical regions a significant increase in the number of microglial cells was demonstrated in HIV-1 infected brains. Moreover, the activation of microglia was not correlated with the presence of HIV-1 antigen in the brain region. The data show a significantly increased number of microglia in HIV-1 infected brains. These activated microglial cells could, among others, be those cells producing cytotoxic factors which, in turn, cause brain damage.     

Activation of p21ras is not sufficient to ensure a complete G1 phase of the cell division cycle in mouse fibroblasts

In the mouse BP-A31 fibroblasts, mRNAs coding the three isoforms (Ha, Ki, N) of ras are expressed, and there are no activating mutations in the codons 12, 13 or 61. We have produced a subline (Ras2) expressing an oestrogen-inducible v-Ha-ras gene. The contribution of v-Ha-ras to the overall p21ras-GTP content was evaluated by metabolic labelling with 32P. Surprisingly, p21ras-GTP complexes were predominant in the serum-deprived BP-A31 cells as well as in the Ras2 cells. The excess of p21ras-GTP was not due to the lack of the GTPase activating protein. In transient transfection experiments, the serum response element (SRE)-directed CAT was expressed in serum-deprived BP-A31 cells, and insulin caused a further two- to threefold increase in CAT activity. A dominant negative ras mutant (Ha-Ras Asn-17) cancelled both the basal and insulin-induced CAT expression in the BP-A31 but not in the Ras2 cells. Expression of v-Ha-ras in Ras2 cells did not relax their growth factor-dependence and oestradiol had only a minor mitogenic effect. We conclude that p21ras activation does not ensure a complete cell division cycle in these cells, and does not entirely account for the transduction of the mitogenic signal initiated by insulin.     

Expression of the poliovirus receptor in intestinal epithelial cells is not sufficient to permit poliovirus replication in the mouse gut

Although the initial site of poliovirus replication in humans is the intestine, previously isolated transgenic mice which carry the human poliovirus receptor (PVR) gene (TgPVR mice), which develop poliomyelitis after intracerebral inoculation, are not susceptible to infection by the oral route. The low levels of PVR expressed in the TgPVR mouse intestine might explain the absence of poliovirus replication at that site. To ascertain whether PVR is the sole determinant of poliovirus susceptibility of the mouse intestine, we have generated transgenic mice by using the promoter for rat intestine fatty acid binding protein to direct PVR expression in mouse gut. Pvr was detected by immunohistochemistry in the enterocytes and M cells of transgenic mouse (TgFABP-PVR) small intestine. Upon oral inoculation with poliovirus, no increase in virus titer was detected in the feces of TgFABP-PVR mice, and no virus replication was observed in the small intestine, although poliovirus replicated in the brain after intracerebral inoculation. The failure of poliovirus to replicate in the TgFABP-PVR mouse small intestine was not due to lack of virus binding sites, because poliovirus could attach to fragments of small intestine from these mice. These results indicate that the inability of poliovirus to replicate in the mouse alimentary tract is not solely due to the absence of virus receptor, and other factors are involved in determining the ability of poliovirus to replicate in the mouse gut.     

Afferent signals to the CNS appear not to condition the modulation of interleukin-1 receptors in the hippocampus

Conditioned alteration of natural killer (NK) cell activity was used as an indicator of the functional bidirectional communication between the immune and central nervous systems. Poly I:C and lipopolysaccharide (LPS) from Escherichia coli and Salmonella typhimurium were used as unconditioned stimuli and odor of camphor as the conditioned stimulus. An attempt was made to demonstrate the role of central interleukin (IL-1) receptors in this communication process. Brain IL-1 receptors were down-regulated by treatment with 50 microg/mouse of LPS from S. typhimurium, but not with the same dose of LPS from E. coli or poly I:C. A significant level of conditioned augmentation of NK cell activity was observed with poly I:C. Conditioned alteration in NK cell activity was also observed with LPS from E. coli, but at much lower level than poly I:C. NK cell activity was not conditioned with LPS from S. typhimurium at the same dose as E. coli LPS, but conditioned enhancement of NK cell activity was observed with a higher dose (100 microg) of S. typhimurium LPS. These results suggest that modulation of central IL-1 receptors do not seem to play a role in the conditioned augmentation of NK cell activity.     

The P-selectin glycoprotein ligand-1 is important for recruitment of neutrophils into inflamed mouse peritoneum

The P-selectin glycoprotein ligand-1 (PSGL-1) is a high-affinity ligand of P-selectin on myeloid cells and certain subsets of lymphoid cells. We generated the rat monoclonal antibody (MoAb) 2PH1 that recognizes an epitope within the first 19 amino acids at the N-terminus of the processed form of mouse PSGL-1. This antibody blocks attachment of mouse myeloid cells to P-selectin under both static and flow conditions. Intravenous administration of saturating amounts of 2PH1 reduced the number of rolling leukocytes in venules of the acutely exposed mouse cremaster muscle by 79% (+/-5.7%), whereas an anti-P-selectin MoAb reduced it completely. Examining the effect of the MoAb 2PH1 on the recruitment of neutrophils into chemically inflamed mouse peritoneum showed that blocking PSGL-1 inhibited neutrophil accumulation in the peritoneum by 82% (+/-7%) at 2 hours and by 59% (+/-7.9%) at 4 hours after stimulation. A similar effect was seen with the MoAb against P-selectin. Simultaneous administration of both antibodies at the 4-hour time point blocked neutrophil accumulation by 86% (+/-4.2%), arguing for an additional partner molecule for PSGL-1 besides P-selectin. This is the first demonstration of the importance of PSGL-1 in the recruitment of mouse neutrophils into inflamed tissue.     

The C-terminal half of the human immunodeficiency virus type 1 Gag precursor is sufficient for efficient particle assembly

Human immunodeficiency virus type 1 particle assembly is directed by the Gag polyprotein Pr55(gag), the precursor for the matrix (MA), capsid (CA), and nucleocapsid proteins of the mature virion. We now show that CA sequences N terminal to the major homology region (MHR), which form a distinct domain, are dispensable for particle formation. However, slightly larger deletions which extend into the MHR severely impair particle production. Remarkably, a deletion which removed essentially all MA and CA sequences between the N-terminal myristyl anchor and the MHR reduced the yield of extracellular particles only moderately. Particle formation even exceeded wild-type levels when additional MA sequences, either from the N or the C terminus of the domain, were retained. We conclude that no distinct region between the myristyl anchor and the MHR is required for efficient particle assembly or release.     

Sequences present in the cytoplasmic domain of CD4 are necessary and sufficient to confer sensitivity to the human immunodeficiency virus type 1 Vpu protein

CD4 is an integral membrane glycoprotein which functions as the human immunodeficiency virus receptor for infection of human host cells. We have recently demonstrated that Vpu, a human immunodeficiency virus type 1-encoded integral membrane phosphoprotein, induces rapid degradation of CD4 in the endoplasmic reticulum. Using an in vitro model system, we demonstrated that Vpu targets specific sequences in the cytoplasmic domain of CD4 to promote its degradation. In this report, we have further delineated regions within CD4 which are required for susceptibility to Vpu. Transfer of the CD4 cytoplasmic region into a heterologous protein, CD8, rendered the chimeric protein sensitive to Vpu-dependent degradation. In contrast, substitution of the CD8 transmembrane domain with the analogous region from CD4 did not confer sensitivity to Vpu. Finally, mutant forms of the CD4 protein containing the extracellular region alone or the extracellular and transmembrane regions linked to a heterologous cytoplasmic domain were not targeted by Vpu. Thus, sequences present in the cytoplasmic domain of CD4 are necessary and sufficient to confer sensitivity to Vpu.     

The membrane proximal cytokine receptor domain of the human interleukin-6 receptor is sufficient for ligand binding but not for gp130 association

Interleukin-6 (IL-6) belongs to the family of the "four-helix bundle" cytokines. The extracellular parts of their receptors consist of several Ig- and fibronectin type III-like domains. Characteristic of these receptors is a cytokine-binding module consisting of two such fibronectin domains defined by a set of four conserved cysteines and a tryptophan-serine-X-tryptophan-serine (WSXWS) sequence motif. On target cells, IL-6 binds to a specific IL-6 receptor (IL-6R), and the complex of IL-6.IL-6R associates with the signal transducing protein gp130. The IL-6R consists of three extracellular domains. The NH2-terminal Ig-like domain is not needed for ligand binding and signal initiation. Here we have investigated the properties and functional role of the third membrane proximal domain. The protein can be efficiently expressed in bacteria, and the refolded domain is shown to be sufficient for IL-6 binding. When complexed with IL-6, however, it fails to associate with the gp130 protein. Since the second and the third domain together with IL-6 can bind to gp130 and induce signaling, our data demonstrate the ligand binding function of the third domain and point to an important role of the second domain in complex formation with gp130 and signaling.