Inductively coupled mass spectrometry analysis of biometals in conditional Hamp1 and Hamp1 and Hamp2 transgenic mouse models.

Hepcidin, a circulatory antimicrobial peptide, is involved in iron homeostasis, inflammation, infection and metabolic signals. Humans express one hepcidin gene, HAMP but mice express two hepcidin genes, Hamp1 and Hamp2. Consecutive gene targeting events were performed to produce transgenic mice expressing conditional alleles of either Hamp1 or both Hamp1 and Hamp2 (Hamp1/2). The deletion of Hamp1 alleles elevated Hamp2 expression, particularly in males, which was reduced by endotoxin treatment. The tissue levels of iron and other biometals were quantified by inductively coupled mass spectrometry. The ubiquitous or liver-specific deletion of Hamp1 alleles yielded similar quantitative changes in iron levels in the liver, duodenum, spleen, kidney, heart and brain. The introduction of Hamp2 null allele did not exacerbate the iron-related phenotype of Hamp1 null allele. Besides iron, Hamp1 null allele significantly elevated the levels of selenium in the liver, manganese in the liver and duodenum, and copper in the brain. Mice with conditional Hamp alleles will be useful to determine the tissue-specific regulation and functions of Hamp1 and Hamp2 in biometal homeostasis and other biological processes.

Surveillance of antimicrobial resistance in gram-negative isolates from intensive care units in Turkey: analysis of data from the last 5 years.

A multicenter antimicrobial surveillance program was established in Turkey in 1995 to monitor the predominant Gram-negative pathogens from intensive care units (ICUs) and antimicrobial resistance patterns of these isolates. Sixteen hospitals participated in the study and a total of 1479 isolates from 1,100 patients were collected. The isolates were tested for their susceptibility against 13 antibiotics by E-test method. Minimum inhibitory concentrations (MICs) for each isolate were determined for imipenem, ceftazidime, ceftazidime-clavulanate, cefoperazone-sulbactam, ceftriaxone, cefepime, cefuroxime, piperacillin-tazobactam, ticarcillin-clavulanate, gentamicin, amikacin and ciprofloxacin. The most common isolates were Pseudomonas spp. (28.2%), Escherichia coli (19.2%) and Klebsiella spp. (19.1%). We found very high resistance rates to all major antibiotics that are used to treat serious infections. Although imipenem is the most active agent, it had an overall susceptibility rate of 68%. Half of the tested Klebsiella spp. strains were found to produce ESBL. This is a very high rate when compared with the literature. Cross-resistance among species was also investigated. 52% of ciprofloxacin-resistant strains were also resistant to imipenem, 80% to ceftazidime, 97% to ceftriaxone, 86% to amikacin and 19% of imipenem-resistant strains were susceptible to ceftazidime and 18% to amikacin. When susceptibilities of the years 1995 and 1999 were compared, the most interesting finding was the decrease in resistance to 3rd generation cephalosporins. In conclusion, this national clinical isolate database shows that resistance rates are high, the change over years is not predictable and continuous surveillance is necessary to monitor antimicrobial resistance and to guide antibacterial therapy.

A novel epitope of CD59 expressed by primitive human hematopoietic progenitors.

OBJECTIVE: The aim of this study was to determine the identity of the cell surface molecule on primitive hematopoietic cells recognized by monoclonal antibody HCC-1. MATERIALS AND METHODS: Screening of a cDNA expression library prepared from human bone marrow stromal cells with HCC-1 yielded a single cDNA, which when expressed in FDCP-1 cells, resulted in the specific acquisition of HCC-1 binding. The cDNA demonstrated complete identity with CD59, a phosphoinositol glycan-linked membrane protein that protects cells against autologous complement attack. The ubiquitous expression of CD59 is in marked contrast to the restricted reactivity of HCC-1. Studies were performed to examine the basis for the novel specificity of HCC-1 for CD59. The epitope on CD59 identified by HCC-1 was mapped using a series of rat/human CD59 chimeric proteins. Immunoprecipitation analyses were performed to determine whether CD59 associates with other membrane proteins. RESULTS: Mutagenesis of Asn18 did not alter the binding of HCC-1 to CD59, suggesting that N-linked carbohydrates are not responsible for the binding specificity of HCC-1. The epitope for HCC-1 was shown to differ from that identified by previously described CD59 antibodies, encompassing residues A31, L33, R55, and L59. An 80 kDa protein co-immunoprecipitated with CD59 in the HCC-1(-) cell line HL-60 but not in HCC-1(+) K562 cells. CONCLUSION: Collectively, these data support the hypothesis that the unique specificity of HCC-1 for CD59 is due in part to recognition of a novel epitope, which is masked as a result of association with an as yet unidentified 80 kDa protein.

Dynamin inhibits phosphatidylinositol 3-kinase in hematopoietic cells.

Phosphatidylinositol 3-kinase (PI 3-kinase) plays a role in late stages of endocytosis as well as in cellular proliferation and transformation. The SH3 domain of its regulatory p85 subunit stimulates the GTPase activity of dynamin in vitro. Dynamin is a GTPase enzyme required for endocytosis of activated growth factor receptors. An interaction between these proteins has not been demonstrated in vivo. Here, we report that dynamin associates with PI 3-kinase in hematopoietic cells. We detected both p85 and PI 3-kinase activity in dynamin immune complexes from IL-3-dependent BaF3 cells. However, this association was significantly reduced in BaF3 cells transformed with the BCR/abl oncogene. After transformation only a 4-fold increase in PI 3-kinase activity was detected in dynamin immune complexes, whereas grb2 associated activity was elevated 20-fold. Furthermore, dynamin inhibited the activity of both purified recombinant and immunoprecipitated PI 3-kinase. In BaF3 cells expressing a temperature-sensitive mutant of BCR/abl, a significant decrease in p85 and dynamin association was observed 4 h after the induction of BCR/abl activity. In contrast, in IL-3-stimulated parental BaF3 cells, this association was increased. Our results demonstrate an in vivo association of PI 3-kinase with dynamin and this interaction regulates the activity of PI 3-kinase.

GM-CSF binding to its receptor induces oligomerisation of the common beta-subunit.

The stoichiometry of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor complex is still unresolved. We have utilised a sensitive, functional assay for receptor homodimerisation to show that GM-CSF induces dimerisation of the common signalling subunit, hbeta(c). We generated a chimeric cytokine receptor in which the extracellular and transmembrane domains of hbeta(c)are fused to the cytoplasmic domain of erythropoietin receptor (EPO-R). Given that to induce EPO-R activation and mitogenic signalling there is a requirement for formation of a specific homodimeric complex, we reasoned that the cytoplasmic domain of EPO-R could be utilised as a highly sensitive reporter for functional homodimer formation. We show that, in the presence of a cytoplasmically truncated GM-CSF alpha-subunit, the hbetac-EPO receptor chimera transduces a mitogenic signal in BaF-B03 in response to GM-CSF. This is consistent with formation of a hbeta(c)homodimer following GM-CSF binding and implies that ligand stimulation induces formation of a higher order complex that contains the hbeta(c)homodimer.

The response of isolated anti-HBc positive subjects to recombinant hepatitis B vaccine.

OBJECTIVE: The aim of this study was to evaluate the response to hepatitis B vaccination in isolated anti-HBc positive subjects. PATIENTS AND METHODS: Forty-eight subjects with persistent isolated core antibody were included in the study. Fifty healthy people who were negative for HBsAg, anti-HBs and anti-HBc were included in the study as a control group. They all were vaccinated with recombinant hepatitis B vaccine at 0, 1 and 2 months. RESULTS: Thirty days after each dose of vaccination, serum levels over 10IU/l of anti-HBs are found in 50% of the subjects with isolated anti-HBc after first; in 68.7% after second and in 89.6% after third vaccination. There were no statistical differences between the two groups (P > 0.05). Twenty subjects in isolated anti-HBc group (41.6%) but none of the subjects from the control group responded with a titer of > 50IU/l after 30 days, which suggested an anamnestic response due to prior infection and immunity. Furthermore, 23 subjects in isolated anti-HBc group (47.9%) finally responded after three doses of vaccination (anti-HBs titer > 10IU/l) thus excluding chronic infection and suggesting initial false positive results. CONCLUSIONS: In isolated anti-HBc subjects false positive results (primary response) or prior infection by HBV (anamnestic response) can be detected by anti-HBs response after HBV vaccination.

Surveillance of antimicrobial resistance among gram-negative isolates from intensive care units in eight hospitals in Turkey.

With the participation of eight major reference hospitals in Turkey, 749 aerobic Gram-negative isolates obtained from 473 intensive care patients in 1997 were tested for their susceptibility to 13 commonly employed antibacterial agents. The frequency with which species were isolated and resistance rates were compared with data from the previous 2 years. Imipenem was the most active agent against the majority of isolates (75%), followed by ciprofloxacin, cefepime and amikacin. The per cent susceptibility to all antibiotics declined from 1995 to 1996. With the exception of imipenem, for which there was no change in resistance, the per cent susceptibility somewhat increased in 1997. However, it was still lower than in 1995.

Dysregulated hematopoiesis and a progressive neurological disorder induced by expression of an activated form of the human common beta chain in transgenic mice.

Previously we described activating mutations of hbetac, the common signaling subunit of the receptors for the hematopoietic and inflammatory cytokines, GM-CSF, IL-3, and IL-5. The activated mutant, hbetacFIDelta, is able to confer growth factor-independent proliferation on the murine myeloid cell line FDC-P1, and on primary committed myeloid progenitors. We have used this activating mutation to study the effects of chronic cytokine receptor stimulation. Transgenic mice were produced carrying the hbetacFIDelta cDNA linked to the constitutive promoter derived from the phosphoglycerate kinase gene, PGK-1. Transgene expression was demonstrated in several tissues and functional activity of the mutant receptor was confirmed in hematopoietic tissues by the presence of granulocyte macrophage and macrophage colony-forming cells (CFU-GM and CFU-M) in the absence of added cytokines. All transgenic mice display a myeloproliferative disorder characterized by splenomegaly, erythrocytosis, and granulocytic and megakaryocytic hyperplasia. This disorder resembles the human disease polycythemia vera, suggesting that activating mutations in hbetac may play a role in the pathogenesis of this myeloproliferative disorder. In addition, these transgenic mice develop a sporadic, progressive neurological disease and display bilateral, symmetrical foci of necrosis in the white matter of brain stem associated with an accumulation of macrophages. Thus, chronic hbetac activation has the potential to contribute to pathological events in the central nervous system.

Helicobacter pylori and hypergastrinemia in children with recurrent abdominal pain.

Recurrent abdominal pain (RAP) is a significant problem in the pediatric population, and there has been much recent interest in the role that Helicobacter pylori (Hp) might play in this disorder. In this case control study, the authors aimed to determine whether Hp is an agent responsible for RAP, and to assess fasting gastrin concentrations in children with and without RAP in the Hp-positive and -negative groups. The study was conducted in 42 patients with RAP and 50 healthy children attending routine day-case surgery as a control group, aged 3 to 15 years, over a 12-month period. Of the 42 children with RAP, 30 were seropositive (71.4%) for Hp IgG, and of 50 children in the control group, 32 were seropositive (64%) for Hp IgG (P > 0.05). We found that Hp infection was as high in healthy children as in children with RAP. The mean fasting gastrin levels in 62 Hp-seropositive children (60.4 ng/l) were not different from those in 30 Hp-seronegative children (57.3 ng/l) and those in 42 children with RAP (58.2 ng/l) were also not significantly different from those in 50 healthy children (62.9 ng/l). Thus, no association between childhood Hp infection, hypergastrinemia, and RAP was found in our Turkish population.

Phorbol ester treatment inhibits phosphatidylinositol 3-kinase activation by, and association with, CD28, a T-lymphocyte surface receptor.

CD28 is a costimulatory receptor found on the surface of most T lymphocytes. Engagement of CD28 induces interleukin 2 (IL-2) production and cell proliferation when combined with an additional signal such as treatment with phorbol ester, an activator of protein kinase C. Recent studies have established that after CD28 ligation, the cytoplasmic domain of CD28 can bind to the 85-kDa subunit of phosphatidylinositol 3-kinase (PI3 kinase). There is a concomitant increase in PI3 lipid kinase activity that may be important in CD28 signaling. Despite the requirement of phorbol 12-myristate 13-acetate (PMA) for effector function, we have found, however, that treatment of Jurkat T cells with the phorbol ester PMA dramatically inhibits (i) the association of PI3 kinase with CD28, (ii) the ability of p85 PI3 kinase to be immunoprecipitated by anti-phosphotyrosine antibodies, and (iii) the induction of PI3 kinase activity after stimulation of the cells with the anti-CD28 monoclonal antibody 9.3. These changes occur within minutes of PMA treatment and are persistent. In addition, we have found that wortmannin, a potent inhibitor of PI3 kinase, does not interfere with the induction of IL-2 after stimulation of Jurkat T cells with anti-CD28 monoclonal antibody and PMA. We conclude that PI3 kinase activity may not be required for CD28-dependent IL-2 production from Jurkat T cells in the presence of PMA.

Association of phosphatidylinositol 3-kinase with SHC in chronic myelogeneous leukemia cells.

Expression of p210 BCR/abl oncoprotein transforms hematopoietic cells. P210 BCR/abl tyrosine kinase induces tyrosine phosphorylation of Shc, and activation of p21ras and PI 3-Kinase. Here we show that PI 3-Kinase associates with Shc in cells transformed by BCR/abl oncoprotein. Immunoprecipitation of Shc from cells expressing p210 BCR/abl had 7.5-fold increase in PI 3-Kinase activity compared to parental cells. Tyrosine phosphorylated Shc specifically bound to the C-SH2 domain of the p85 subunit of PI 3-Kinase. The p85 SH3 domain also interacted with Shc in cell lysates from parental and transformed cells. The binding of p85 SH3 domain to Shc was substantially higher in BCR/abl transformed than in parental cells. Phenylphosphate blocked p85 SH2 mediated association with Shc but enhanced the binding of the p85 SH3 domain to Shc. The N-terminal proline-rich region of Shc between A263 and N273 specifically blocked the interaction of p85 SH3 domain with Shc. Our results indicate that PI 3-Kinase interacts with Shc directly in hematopoietic cells which express p210 BCR/abl oncoprotein.

Protein kinase A inhibitors enhance radiation-induced apoptosis.

In addition to a role for de novo protein synthesis in apoptosis we have previously shown that activation of a protein phosphatase or loss of activity of a kinase is also important in radiation-induced apoptosis in human cells [Baxter, and Lavin (1992): J Immunol 148:149-1954]. We show here that some inhibitors of protein kinases exacerbate radiation-induced apoptosis in the human cell line BM13674. The specific protein kinase A inhibitor isoquinoline sulfonamide (20 microM) gave rise to significantly increased levels of apoptosis at 2-6 h postirradiation compared to values after radiation exposure only. The same concentration of isoquinolinesulfonamide, which was effective in increasing apoptosis, reduced activity markedly. A 66% inhibition of cyclic AMP-dependent protein kinase A activity occurred in unirradiated cells at this concentration of H89 and activity was reduced to 58% in irradiated cells. Calphostin C, a specific inhibitor of protein kinase C, at a concentration of 0.1 microM, which caused 68% inhibition of enzyme activity in irradiated cells, failed to enhance the level of radiation-induced apoptosis. Other kinase inhibitors did not lead to an additional increase in apoptosis over and above that observed after irradiation. The results obtained here provide further support for an important role for modification of existing proteins during radiation-induced apoptosis.

Inhibition of apoptosis in human tumour cells by okadaic acid.

Gamma-radiation, tetrandrine, bistratene A, and cisplatin were all found to induce pronounced morphological changes characteristic of apoptosis and extensive DNA fragmentation in the human BM13674 cell line 8 h after treatment. Apoptosis induced in BM13674 cells by these diverse agents was markedly inhibited by 1 microM okadaic acid, a tumour promoter that inhibits protein phosphatases 1 and 2A. This compound also inhibited the appearance of apoptosis in fresh human leukaemia cells that had been exposed to gamma-radiation. The inhibition of apoptosis was confirmed using fluorescence microscopy and DNA gel electrophoresis. Dephosphorylation of a limited number of proteins was shown to be associated with apoptosis and okadaic acid prevented these dephosphorylations. Previous studies on the BM13674 cell line showed that an inhibitor of protein synthesis failed to prevent apoptosis in these cells. The present data provides further support that posttranslational modification of proteins, in particular, phosphorylation/dephosphorylation status, plays an important role in inhibition/activation of programmed cell death in different human cells after exposure to several cytotoxic agents.

Tetanus toxin inhibits depolarization-stimulated protein phosphorylation in rat cortical synaptosomes: effect on synapsin I phosphorylation and translocation.

Synapsin I, a prominent phosphoprotein in nerve terminals, is proposed to modulate exocytosis by interaction with the cytoplasmic surface of small synaptic vesicles and cytoskeletal elements in a phosphorylation-dependent manner. Tetanus toxin (TeTx), a potent inhibitor of neurotransmitter release, attenuated the depolarization-stimulated increase in synapsin I phosphorylation in rat cortical particles and in synaptosomes. TeTx also markedly decreased the translocation of synapsin I from the small synaptic vesicles and the cytoskeleton into the cytosol, on depolarization of synaptosomes. The effect of TeTx on synapsin I phosphorylation was both time and TeTx concentration dependent and required active toxin. One- and two-dimensional peptide maps of synapsin I with V8 proteinase and trypsin, respectively, showed no differences in the relative phosphorylation of peptides for the control and TeTx-treated synaptosomes, suggesting that both the calmodulin- and the cyclic AMP-dependent kinases that label this protein are equally affected. Phosphorylation of synapsin IIb and the B-50 protein (GAP43), a known substrate of protein kinase C, was also inhibited by TeTx. TeTx affected only a limited number of phosphoproteins and the calcium-dependent decrease in dephosphin phosphorylation remained unaffected. In vitro phosphorylation of proteins in lysed synaptosomes was not influenced by prior TeTx treatment of the intact synaptosomes or by the addition of TeTx to lysates, suggesting that the effect of TeTx on protein phosphorylation was indirect. Our data demonstrate that TeTx inhibits neurotransmitter release, the phosphorylation of a select group of phosphoproteins in nerve terminals, and the translocation of synapsin I. These findings contribute to our understanding of the basic mechanism of TeTx action.