A ketoreductase domain in the PksJ protein of the bacillaene assembly line carries out both alpha- and beta-ketone reduction during chain growth

The polyketide signaling metabolites bacillaene and dihydrobacillaene are biosynthesized in Bacillus subtilis on an enzymatic assembly line with both nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules acting along with catalytic domains servicing the assembly line in trans. These signaling metabolites possess the unusual starter unit α-hydroxyisocaproate (α-HIC). We show here that it arises from initial activation of α-ketoisocaproate (α-KIC) by the first adenylation domain of PksJ (a hybrid PKS/NRPS) and installation on the pantetheinyl arm of the adjacent thiolation (T) domain. The α-KIC unit is elongated to α-KIC-Gly by the second NRPS module in PksJ as demonstrated by mass spectrometric analysis. The third module of PksJ uses PKS logic and contains an embedded ketoreductase (KR) domain along with two adjacent T domains. We show that this KR domain reduces canonical 3-ketobutyryl chains but also the α-keto group of α-KIC-containing intermediates on the PksJ T-domain doublet. This KR activity accounts for the α-HIC moiety found in the dihydrobacillaene/bacillaene pair and represents an example of an assembly-line dual-function α- and β-KR acting on disparate positions of a growing chain intermediate.     

Immunoglobulin E levels following allogeneic, autologous, and syngeneic bone marrow transplantation: an indirect association between hyperproduction and acute graft-v-host disease in allogeneic BMT

Markedly elevated serum IgE levels have been noted following allogeneic bone marrow transplantation (BMT) and have been correlated with graft-v- host disease (GVHD) in several studies. To investigate this phenomenon, we measured serum IgE levels in 387 allogeneic, 143 autologous, and 21 syngeneic BMT recipients before and at intervals after BMT. As a population, allogeneic BMT recipients displayed a biphasic elevation in IgE levels, with peak levels occurring either early (days 15 to 19) or late (days 80 to 89) posttransplant. Only in individuals in whom peak levels occurred early did IgE level correlate with liver disease, histological changes, and overall clinical stage of GVHD. The association of IgE elevation and GVHD does not appear to be direct since recipients of syngeneic (monozygotic twin) grafts had the highest incidence of IgE hyperresponsiveness as well as the highest absolute IgE levels. Similarly, 22 recipients of autologous marrow not treated with 4-hydroperoxycyclophosphamide had elevated IgE levels comparable to those seen in allogeneic graft recipients. We hypothesize that augmented IgE synthesis and its subsequent resolution is the natural consequence of immune reconstitution in the presence of potentially reaginic agents such as antibiotics and infectious agents. As such, IgE hyperresponsiveness in syngeneic graft recipients may reflect the maturational sequence of IgE regulatory elements in the absence of interference by GVHD, GVHD therapy, or minor histocompatibility disparities. The cell populations required for IgE response (T cells, B cells, and antigen-presenting cells) may be reconstituted in advance of the regulatory elements that limit IgE production in healthy subjects. Although this temporal relationship does not appear to hold in allogeneic BMT, the balance between positive and negative factors, which determines the rates of IgE synthesis and catabolism, may be altered by GVHD, infection, and liver dysfunction acting alone or in combination.     

Downregulation of the platelet surface glycoprotein Ib-IX complex in whole blood stimulated by thrombin, adenosine diphosphate, or an in vivo wound [see comments]

In washed platelet systems, thrombin has been demonstrated to downregulate the platelet surface expression of glycoprotein (GP) Ib and GPIX. In the present study, we addressed the question as to whether, in the more physiologic milieu of whole blood, downregulation of platelet surface GPIb and GPIX can be induced by thrombin, adenosine diphosphate (ADP), and/or by an in vivo wound. Thrombin-induced downregulation of GPIb and GPIX on the surface of individual platelets in whole blood was demonstrated by the use of flow cytometry, a panel of monoclonal antibodies (MoAbs) and, to inhibit fibrin polymerization, the peptide glycyl-L-prolyl-L-arginyl-L-proline. Platelets were identified in whole blood by a GPIV-specific MoAb and exclusion of monocytes by light scattering properties. Flow cytometric analysis of whole blood emerging from a standardized bleeding-time wound established that downregulation of platelet surface GPIb and GPIX can occur in vivo. A GPIb-IX complex-specific antibody indicated that the GPIb and GPIX remaining on the surface of platelets activated in vivo or in vitro were fully complexed. Simultaneous analysis of individual platelets by two fluorophores demonstrated that thrombin-induced platelet surface exposure of GMP-140 (degranulation) was nearly complete at the time that downregulation of platelet surface GPIb-IX was initiated. However, degranulation was not a prerequisite because ADP downregulated platelet surface GPIb-IX without exposing GMP-140 on the platelet surface. Inhibitory effects of cytochalasins demonstrated that the activation-induced downregulation of both GPIX and GPIb are dependent on actin polymerization. In summary, downregulation of the platelet surface GPIb-IX complex occurs in whole blood stimulated by thrombin, ADP, or an in vivo wound, and is independent of alpha granule secretion.     

Inhibition of receptor binding and neutralization of bioactivity by anti-erythropoietin monoclonal antibodies

We have generated four high affinity monoclonal antibodies (MoAbs) to recombinant human erythropoietin (EPO). All four MoAbs immunoprecipitate radioiodinated native EPO, and the concentrations of MoAbs required for maximum binding range from 10 nmol/L to 100 nmol/L. Two MoAbs, designated Group I MoAbs, bind to an epitope within the N- terminal 20 amino acids of EPO and also immunoprecipitate sodium dodecyl sulfate (SDS)-denatured EPO. Two other MoAbs (Group II MoAbs) do not immunoprecipitate SDS-denatured EPO and do not bind to any of the eight endo C fragments of EPO. We first used murine erythroleukemia (MEL) cells to test the MoAbs for inhibition of EPO-receptor binding. MEL cells, although unresponsive to EPO, express 760 high affinity receptors for EPO per cell (Kd = 0.24 nmol/L). To assay our MoAbs, MEL cells were grown as monolayers on fibronectin-coated Petri dishes and incubated at 4 degrees C with radioiodinated EPO. Group I MoAbs do not inhibit binding of radioiodinated EPO to the MEL EPO-receptor, but Group II MoAbs do inhibit binding in a dose-dependent manner. We next examined the neutralization of EPO bioactivity by our MoAbs, using EPO- dependent cell line. Only Group II MoAbs inhibit a newly developed EPO- dependent cell growth, demonstrating that inhibition of EPO-receptor binding correlates with neutralization of EPO bioactivity.     

Differential effect of 4-hydroperoxycyclophosphamide and antimyeloid monoclonal antibodies on T and natural killer cells during bone marrow purging

Autologous bone marrow (BM) transplantation after high dose therapy is widely used to treat acute leukemia, lymphoma, and selected solid tumors. In studies of BM purging with chemical agents, monoclonal antibodies (MoAbs), or other agents, the emphasis has been on the efficacy of tumor cell removal and sparing of hematopoietic progenitor cells. Two commonly used methods of BM purging for patients with acute myeloid leukemia have been the drug 4-hydroperoxycyclophosphamide (4- HC) and (MoAbs) directed to myeloid antigens such as CD14, CD15, and CD33. Although both methods of BM purging have potent activity against leukemia cells, 4-HC is also quite toxic to normal hematopoietic progenitor cells in the same concentrations that are used to deplete leukemia cells. To further characterize the cellular composition of BM after purging, we examined the effects of MoAbs plus complement and 4- HC on cells of the lymphoid lineage in the BM. 4-HC exerted a concentration-dependent cytotoxicity on clonogenic T lymphocytes, natural killer (NK) cells, and lymphokine (interleukin-2)-activated killer (LAK) cells, whereas the anti-CD14 and anti-CD15 MoAbs had little effect. At a concentration of 4-HC commonly used for BM purging (60 micrograms/mL), there were 4 to 5 logs of T-cell depletion and almost complete elimination of NK- and LAK-cell activity. In contrast, 4-HC at low concentrations (eg, 3 micrograms/mL) spared the majority of lymphoid cells suggesting that low concentration 4-HC combined with MoAb purging may be a desirable alternative to higher concentration 4- HC. These data indicate that purging with antimyeloid MoAbs, but not with 4-HC, spares the function of mature graft lymphocytes. Infusion of viable lymphocytes may be important for the transfer of immune memory against microbial and neoplastic antigens and may hasten immune reconstitution. In addition, mature graft lymphocytes may also be selectively activated and expanded in conjunction with interleukin-2 administration after BM transplantation.     

Culture of autologous bone marrow in diffusion chambers: effect of host irradiation

Autologous bone marrow (BM) cells were cultured in diffusion chambers (DC) implanted into whole-body irradiated, non-irradiated, or sham- irradiated goats. Proliferation was apparent in DC implanted in both irradiated and nonirradiated goats. However, cells in DC cultured in irradiated hosts increased in number beginning earlier, proceeded at a faster rate, and reached higher numbers than in DC in nonirradiated hosts. Growth enhancement could not have occurred as a result of radiation-induced immunosuppression in autologous hosts. The nonirradiated "target cells" in the DC therefore constituted an indicator system for stimulatory or inhibitory substances in the host. The simultaneous increase in the number of granulocytes in peripheral blood and in DC of irradiated hosts was paralleled by an initial rise in serum colony-stimulating factor (CSF). A second, prolonged period of severe granulocytopenia following irradiation of the host correlated with high levels of serum CSF. Increased numbers of megakaryocytes were seen in DC as thrombocytopenia developed in the irradiated host. DC erythropoiesis disappeared rapidly in nonirradiated goats; however, in DC of irradiated goats, the number of erythrocytic precursors increased exponentially during ablation of host erythroid marrow. Anemia did not develop in the host during the culture period. Proliferation of mononuclear cells in DC was markedly stimulated by irradiation of the host. Proliferation of macrophages appeared independent of host treatment. These observations provide strong evidence for diffusion of specific and/or nonspecific humoral hematopoietic stimulators from the host into the DC. This stimulation appears to be elicited and/or intensified by irradiation of the host.     

Olsalazine is not superior to placebo in maintaining remission of inactive Crohn's colitis and ileocolitis: a double blind, parallel, randomised, multicentre study

BACKGROUND AND AIMS: The benefit of 5-aminosalicylic acid therapy for maintenance of remission in Crohn's disease is controversial. The primary aim of this study was to evaluate the prophylactic properties of olsalazine in comparison with placebo for maintenance of remission in quiescent Crohn's colitis and/or ileocolitis. METHODS: In this randomised, double blind, parallel group study of olsalazine versus placebo, 328 patients with quiescent Crohn's colitis and/or ileocolitis were recruited. Treatment consisted of olsalazine 2.0 g daily or placebo for 52 weeks. The primary end point of efficacy was relapse, as defined by the Crohn's disease activity index (CDAI) and by clinical relapse. Laboratory and clinical disease activity indicators were also measured. Safety analysis consisted of documentation of adverse events and laboratory values. RESULTS: No differences in the frequency of termination due to relapse or time to termination due to relapse were noted between the two treatment groups (olsalazine 48.5% v placebo 45%) for either colitis or ileocolitis. The failure rate, defined as not completing the study, was significantly higher in olsalazine treated patients compared with placebo treated patients for the overall population (colitis and/or ileocolitis: olsalazine 65.4% v 53.9%; p=0.038). Similar failure rates were seen for patients with colitis. A significantly higher percentage of olsalazine treated patients experienced adverse gastrointestinal events. Drug attributed adverse events were reported more frequently in the olsalazine treated group with gastrointestinal symptoms being causally related to olsalazine treatment (olsalazine 40.7% v placebo 26.9%; p=0.010). Back pain was reported significantly more often by the placebo treated group. However, serious medical events did not differ between the two groups. Adverse events led to more early withdrawals in the olsalazine treated group than in the placebo treated group; thus average time in the study for patients in the olsalazine treatment group was significantly shorter than that of patients in the placebo group. CONCLUSIONS: Patients treated with olsalazine were more likely to terminate their participation in the trial than those taking placebo. This difference was not related to relapse of disease, as measured by CDAI and clinical measures, but rather was due to the development of intolerable adverse medical events of a non-serious nature related to the gastrointestinal tract. The gastrointestinal related events in the olsalazine treated group may be due to the difference in gastrointestinal status at baseline which favoured the placebo treatment group.     

Substitutions and deletions in the cytoplasmic domain of the phagocytic receptor Fc gamma RIIA: effect on receptor tyrosine phosphorylation and phagocytosis [published erratum appears in Blood 1994 Nov 1;84(9):3252]

Fc gamma RIIA in the absence of other Fc receptors or receptor subunits induces the ingestion of IgG-coated cells. The cytoplasmic domain of Fc gamma RIIA contains two Y-x-x-L sequences similar to those in other Ig gene family receptors plus an additional tyrosine residue not in a Y-x- x-L motif. Upon cross-linking, Fc gamma RIIA is phosphorylated on tyrosine and the cytoplasmic tyrosines, Y275 (Y1), Y282 (Y2), and Y298 (Y3), may be important for its phagocytic activity. Because COS-1 cells can serve as a model for examining molecular structures involved in phagocytosis, substitutions and deletions were introduced into the cytoplasmic domain of Fc gamma RIIA and examined in COS-1 cell transfectants for their effects on phagocytosis and tyrosine phosphorylation. Disruption of a single cytoplasmic Y-x-x-L motif by substitution of tyrosine Y2 or Y3 by phenylalanine or by removing the threonine and leucine residues within the motif inhibited phagocytosis 50% to 65%. Tyrosine phosphorylation of Fc gamma RIIA also was inhibited, although to a greater extent by the substitution of Y3 than of Y2. Replacement of the N-terminal first cytoplasmic domain tyrosine, Y1, which is not within a typical Y-x-x-L, by itself did not inhibit phagocytosis, but replacement of Y1 in mutants lacking Y2 or Y3 virtually eliminated phagocytic activity and receptor tyrosine phosphorylation. Thus, at least two cytoplasmic tyrosines, including at least one typical single Y-x-x-L motif, are required for phagocytosis by Fc gamma RIIA. The data suggest that there is a close but not a simple relationship between phosphorylation of the Fc gamma RIIA cytoplasmic tyrosines and Fc gamma RIIA-mediated phagocytosis. Y3 appears to be particularly important because its removal by truncation or replacement with phenylalanine inhibits both tyrosine phosphorylation and phagocytosis in parallel. Alterations in the 12 residue proline-containing sequence between the two Y-x-x-L motifs also reduced phagocytic activity and tyrosine phosphorylation. Thus, the specific structure of the Fc gamma RIIA cytoplasmic domain accounts for its ability to stimulate phagocytosis in the absence of other subunits.     

Hepatolithiasis with biliary ascariasis – a case report

Background  

Biliary ascariasis is regarded as possible etiological factor for hepatolithiasis. Here we report one case of a patient with hepatolithiasis with biliary ascariasis who developed a liver abscess, which was treated with partial hepatectomy.