Characterisation of strain-specific sequences from an abortifacient strain of ovine Chlamydia psittaci using subtraction hybridisation

BACKGROUND: The antigens of the human Rh system are of great clinical significance in transfusion medicine and pregnancy. Of the Rh system antigens, D is clinically the most important, being one of the most immunogenic structures arising from human cells. The human D antigen represents a collection of epitopes expressed on a red cell membrane protein that is predicted to have 12 membrane-spanning segments giving rise to six exofacial domains. STUDY DESIGN AND METHODS: By site-directed mutagenesis using the method of inverse polymerase chain reaction, cE and D cDNA mutant constructs were generated with changes to the RHD-specific residues 350, 353, and 354 in the predicted sixth exofacial loop. Each mutant cDNA was subcloned into the pBabe puromycin retroviral vector, and supernatants were used to transduce K562 cells. Puromycin-resistant K562 clones were screened by flow cytometric analysis using a panel of monoclonal antibodies with specificities to ep (epitope) D1 through epD9. RESULTS: De novo expression of epD3 and epD9 was generated in the K562 cell lines expressing the mutated cE polypeptide (cE-Asp350His, Gly353Trp, Ala354Asn). Expression of c and E was unaffected. Conversely, the cells expressing the mutated D polypeptide demonstrated loss of expression of epD1, epD2, epD3, epD4, and epD9. CONCLUSION: The data provide strong evidence for the critical involvement of three amino acids, Asp350, Gly353, and Ala354, in the expression of epD3 and epD9 on the predicted sixth external domain of the D protein. This domain also appears to be essential for the expression of epD1, epD2, and epD4, as a loss of expression of these epitopes was observed in K562 cells transduced with the Dmut construct (encoding His350, Trp353, and Asn354). The K562/Dmut cell line has an identical molecular and serologic profile as the red cell D(IVb) phenotype, which confirms that retroviral gene transfer of Rh cDNA into K562 cells provides us with a powerful means by which to further map epitopes of D.     

Triple helix formation: binding avidity of acridine-conjugated AG motif third strands containing natural, modified and surrogate bases opposed to pyrimidine interruptions in a polypurine target

A critical issue for the general application of triple-helix-forming oligonucleotides (TFOs) as modulators of gene expression is the dramatically reduced binding of short TFOs to targets that contain one or two pyrimidines within an otherwise homopurine sequence. Such targets are often found in gene regulatory regions, which represent desirable sites for triple helix formation. Using intercalator-conjugated AG motif TFOs, we compared the efficacy and base selectivity of 13 different bases or base surrogates in opposition to pyrimidines and purines substituted into selected positions within a paradigm 15-base polypurine target sequence. We found that substitutions closer to the intercalator end of the TFO (positions 4-6) had a more deleterious effect on the dissociation constant (K d) than those farther away (position 11). Opposite T residues at position 11, 3-nitropyrrole or cytosine in the TFO provided adequate binding avidity for useful triplex formation (K ds of 55 and 110 nM, respectively). However, 3-nitropyrrole was more base selective than cytosine, binding to T >/=4 times better than to A, G or C. None of the TFOs tested showed avid binding when C residues were in position 11, although the 3-nitropyrrole-containing TFO bound with a K d of 200 nM, significantly better than the other designs. Molecular modeling showed that the 3-nitropyrrole.T:A triad is isomorphous with the A.A:T triad, and suggests novel parameters for evaluating new base triad designs.     

Cutting edge: protective effects of notch-1 on TCR-induced apoptosis

The Notch receptor protein was originally identified in Drosophila and is known to mediate cell to cell communication and influence cell fate decisions. Members of this family have been isolated from invertebrates as well as vertebrates. We isolated mouse Notch-1 in a yeast two-hybrid screen with Nur77, which is a protein that has been shown previously to be required for apoptosis in T cell lines. The data presented below indicate that Notch-1 expression provides significant protection to T cell lines from TCR-mediated apoptosis. These data demonstrate a new antiapoptotic role for Notch-1, providing evidence that, in addition to regulating cell fate decisions, Notch-1 can play a critical role in controlling levels of cell death in T cells.     

Amino-terminal sequencing of sheep CD1 antigens and identification of a sheep CD1D gene

The anti-CD1 monoclonal antibodies IAH-CC14 and SBU-T6 were used to immunopurify CD1 antigens from sheep thymocytes. The amino-terminal sequence of IAH-CC14 yielded 13 amino acids, and 29 amino acids were obtained from the SBU-T6 antigen. The sequence of the IAH-CC14 antigen was 100% identical to the predicted sequence of the sheep CD1B clone, SCD1B-42. The 29 amino acid sequence of the SBU-T6 antigen did not match identically with the derived amino acid sequence of any of the previously reported sheep CD1 genes but had closest similarity to the derived sequence of human CD1E. Degenerate polymerase chain reaction primers based on this sequence identified a group 2 sheep CD1 gene. The predicted amino acid sequence of this gene shows that it is not identical to the SBU-T6 peptide, indicating that a different, CD1D-like gene was cloned.     

Reactive oxygen species activate human peripheral blood dendritic cells

This study investigates the effects of hydrogen peroxide, a potent oxygen free radical donor, on the phenotype and function of dendritic cells differentiated from peripheral blood precursors. We report that hydrogen peroxide induces an up-regulation of several dendritic cell surface markers involved in interaction with T cells, including MHC Class II molecules (DQ and DR) and the co-stimulatory molecules CD40 and CD86. Moreover we have observed that H2O2-treated dendritic cells are more efficient in promoting T cell proliferation than normal dendritic cells and that this enhancement can be blocked using the free radical scavenger agent N-acetylcysteine. Oxygen free radicals are a common by-product of inflammation, and our results suggest they may play an important role in activation of sentinel dendritic cells, linking tissue damage to the initiation of an adaptive immune response.     

Leukosialin (CD43) is proteolytically cleaved from stimulated HMC-1 cells

Leukosialin (CD43), the major sialoprotein on circulating leukocytes, has been previously described to be down-regulated on neutrophils following activation with phorbol myristate acetate (PMA). The other single cells previously examined, blood lymphocytes, do not down-regulate CD43 when stimulated by PMA. Recently, we have characterized leukosialin on the human mast cell line HMC-1 and observed that leukosialin is down-regulated after stimulation with PMA. In the present study, we have investigated the mechanism of PMA-mediated down-regulation of CD43 on HMC-1 cells (subclone 5C6). PMA caused the release of soluble leukosialin (123 kD) during HMC-1 cell activation. The molecular weight of soluble leukosialin was nearly identical to that of the cell-membrane bound molecule, suggesting a cleavage proximal from the cell membrane. Inhibitors of serine proteases, like phenylmethylsulphonyl fluoride (PMSF), benzamidine and 3, 4-dichloroisocoumarin, blocked the PMA-mediated cleavage of CD43. In all experiments, the inhibition of CD43-down-regulation was dependent on the concentration of protease inhibitors. Treatment of HMC-1 cells with various proteases (trypsin, alpha-chymotrypsin, elastase, papain, nagarse) substantially decreased anti-CD43 binding capacity and caused the release of soluble leukosialin (116 kD) or its fragments into the supernatant. Pretreatment of HMC-1 cells with neuraminidases from Vibrio cholerae or Arthrobacter ureafaciens resulted in an increased sensitivity of CD43 against proteases, whereas the effects of PMA were not influenced. In conclusion, proteolytic cleavage of CD43 is described for the first time in a cell other than neutrophils, namely HMC-1 cells. Our results suggest that serine proteases are involved in the PMA-mediated down-regulation of leukosialin on HMC-1 cells.     

Changes in gene expression in the intact human heart. Downregulation of alpha-myosin heavy chain in hypertrophied, failing ventricular myocardium

Using quantitative RT-PCR in RNA from right ventricular (RV) endomyocardial biopsies from intact nonfailing hearts, and subjects with moderate RV failure from primary pulmonary hypertension (PPH) or idiopathic dilated cardiomyopathy (IDC), we measured expression of genes involved in regulation of contractility or hypertrophy. Gene expression was also assessed in LV (left ventricular) and RV free wall and RV endomyocardium of hearts from end-stage IDC subjects undergoing heart transplantation or from nonfailing donors. In intact failing hearts, downregulation of beta1-receptor mRNA and protein, upregulation of atrial natriuretic peptide mRNA expression, and increased myocyte diameter indicated similar degrees of failure and hypertrophy in the IDC and PPH phenotypes. The only molecular phenotypic difference between PPH and IDC RVs was upregulation of beta2-receptor gene expression in PPH but not IDC. The major new findings were that (a) both nonfailing intact and explanted human ventricular myocardium expressed substantial amounts of alpha-myosin heavy chain mRNA (alpha-MHC, 23-34% of total), and (b) in heart failure alpha-MHC was downregulated (by 67-84%) and beta-MHC gene expression was upregulated. We conclude that at the mRNA level nonfailing human heart expresses substantial alpha-MHC. In myocardial failure this alteration in gene expression of MHC isoforms, if translated into protein expression, would decrease myosin ATPase enzyme velocity and slow speed of contraction.     

T cell proliferation in response to interleukins 2 and 7 requires p38MAP kinase activation

Interleukin-2 (IL-2) is a potent T cell mitogen. However, the signaling pathways by which IL-2 mediates its mitogenic effect are not fully understood. One of the members of the mitogen-activated protein kinase (MAPK) family, p42/44MAPK (ERK2/1), is known to be activated by IL-2. We have now investigated the response to IL-2 of two other members of the MAP kinase family, p54MAP kinase (stress-activated protein kinase (SAPK)/Jun-N-terminal kinase (JNK)) and p38MAP kinase (p38/Mpk2/CSBP/RK), which respond primarily to stressful and inflammatory stimuli (e.g. tumor necrosis factor-alpha, IL-1, and lipopolysaccharide). Here we show that IL-2, and another T cell growth factor, IL-7, activate both SAPK/JNK and p38MAP kinase. Furthermore, inhibition of p38MAP kinase activity with a specific pyrinidyl imidazole inhibitor SB203580 that prevents activation of its downstream effector, MAPK-activating protein kinase-2, correlated with suppression of IL-2- and IL-7-driven T cell proliferation. These data indicate that in T cells p38MAP kinase has a role in transducing the mitogenic signal.     

Light and electron microscopic distribution of the AMPA receptor subunit, GluR2, in the spinal cord of control and G86R mutant superoxide dismutase transgenic mice

The NMR structure of the 98 residue beta-elicitin, cryptogein, which induces a defence response in tobacco, was determined using 15N and 13C/15N labelled protein samples. In aqueous solution conditions in the millimolar range, the protein forms a discrete homodimer where the N-terminal helices of each monomer form an interface. The structure was calculated with 1047 intrasubunit and 40 intersubunit NOE derived distance constraints and 236 dihedral angle constraints for each subunit using the molecular dynamics program DYANA. The twenty best conformers were energy-minimized in OPAL to give a root-mean-square deviation to the mean structure of 0.82 A for the backbone atoms and 1.03 A for all heavy atoms. The monomeric structure is nearly identical to the recently derived X-ray crystal structure (backbone rmsd 0.86 A for residues 2 to 97) and shows five helices, a two stranded antiparallel beta-sheet and an omega-loop. Using 1H,15N HSQC spectroscopy the pKa of the N- and C-termini, Tyr12, Asp21, Asp30, Asp72, and Tyr85 were determined and support the proposal of several stabilizing ionic interactions including a salt bridge between Asp21 and Lys62. The hydroxyl hydrogens of Tyr33 and Ser78 are clearly observed indicating that these residues are buried and hydrogen bonded. Two other tyrosines, Tyr47 and Tyr87, show pKa's > 12, however, there is no indication that their hydroxyls are hydrogen bonded. Calculations of theoretical pKa's show general agreement with the experimentally determined values and are similar for both the crystal and solution structures.     

Fetal growth retardation and increased placental weight in the spontaneously hypertensive rat

Epidemiological studies have linked low birth weight and increased placental weight with increased risk of hypertension in adult life. It has been proposed that the cardiovascular changes which lead to hypertension are initiated in utero by processes associated with intrauterine growth retardation. The alternative possibility, that hypertension may result from genetic influences which also determine fetal and placental size, has had less support because birth weight is not determined genetically in humans. However, in the spontaneously hypertensive rat (SHR) essential hypertension is known to be transmitted genetically. Fetal and placental weights were, therefore, measured at Day 20 gestation in SHRs and compared with those in the normotensive Wistar Kyoto (WKY) control strain. Fetal weight (1.93 +/- 0.04 g) was significantly (P < 0.001) reduced in SHRs compared with WKY fetuses (2.23 +/- 0.01 g) but placental weight was heavier (P < 0.001) in SHRs (0.347 +/- 0.005 g) than in WKY rats (0.300 +/- 0.006 g) although litter size was not different. As expected, maternal blood pressure recorded under 1% halothane anaesthesia was higher (126 +/- 2.7 mm Hg) in SHR than WKY rats (100 +/- 2.1 mm Hg; 1 mm Hg = 133 Pa). In addition the concentration of maternal blood glucose in SHR was significantly (P < 0.001) higher (4.8 +/- 0.32 mM v. 3.7 +/- 0.11 mM) and the concentration of plasma insulin was significantly (P < 0.05) lower in SHRs (18.8 +/- 3.0 ng mL-1) than in WKY dams (29.4 +/- 3.1 ng mL-1). Thus, the data support human population studies which show an association between adult hypertension and a reduced fetal:placental weight ratio at birth. However, because hypertension in the SHR is genetically determined, these data suggest that fetal growth retardation and increased placental weight may also be determined genetically.