Consecutive five abortions in a case of septate uterus--ended in a term pregnancy following modified Jones' metroplasty operation.
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Abortion, Habitual↗
Biomedical subjects
D Chatterjee
Publications and source records attributed to D Chatterjee.
Monocytic differentiation of HL-60 promyelocytic leukemia cells correlates with the induction of Bcl-xL.
Treatment of human promyelocytic leukemia HL-60 cells with phorbol esters ultimately induces the differentiation of these cells along the monocyte/macrophage lineage, whereas treatment with retinoic acid or DMSO induces granulocytic/neutrophillic differentiation. In this study, we demonstrate the disparate fates of HL-60 cells treated with the phorbol ester 12,13-phorbol dibutyric acid (PDBu) or DMSO. After DMSO treatment, HL-60 cells eventually died via apoptosis, whereas the viability of PDBu-treated cells was not affected during the same interval. The levels of the apoptosis effector proteins Bak and Bad were enhanced, whereas there was a slight down-regulation of the apoptosis suppressor protein Bcl-2 after treatment of the cells with PDBu and DMSO. Treatment with DMSO resulted in the elevation of the apoptosis effector Bax, whereas treatment with PDBu did not significantly alter the levels of this protein. However, treatment of HL-60 cells with PDBu induced the rapid expression of the apoptosis suppressor protein Bcl-xL, whereas the expression of this protein remained unaltered in DMSO-treated cells. The generality of this finding was confirmed by the induction of Bcl-xL in human myeloid U-937 cells, human peripheral blood monocytes exposed to phorbol ester, and mouse thioglycollate-activated and resident peritoneal macrophages. PDBu-treated HL-60 cells remained viable for 7 days and thereafter began to die via apoptosis, with a concomitant down-regulation of Bcl-xL. In conclusion, we propose that Bcl-xL expression is associated with differentiation and survival of hematopoietic cells along the monocyte/macrophage lineage.
Apoptosis↗
Induction of apoptosis in malignant and camptothecin-resistant human cells.
Flow cytometry studies demonstrate that androgen-independent human prostate carcinoma DU-145 cells are arrested at the G1-phase of the cell cycle in the presence of suramin, but they die by apoptosis in the presence of 9-nitrocamptothecin (9NC). The addition of cytostatic concentrations of suramin increases the apoptotic action of 9NC on DU-145 cells, and induces apoptosis in 9NC-resistant DU-145/RC cells that were derived from the parental DU-145 cells by continuous exposure to progressively increased concentrations of 9NC. In addition, the topoisomerase II-directed drug etoposide exerts more extensive apoptotic action on DU-145/RC than DU-145 cells. Increased resistance of DU-145 cells to 9 NC and collaterally increased sensitivity to etoposide and suramin appear to correlate with alterations in the structure rather than synthesis of topoisomerases and possibly with specific cellular proteins that regulate apoptosis. The results suggest that etoposide and suramin may be successful alternative treatments for 9NC-resistant androgen-independent prostate cancer.
Androgens↗
Truncated structural variants of lipoarabinomannan in ethambutol drug-resistant strains of Mycobacterium smegmatis. Inhibition of arabinan biosynthesis by ethambutol.
The anti-tuberculosis drug, ethambutol (Emb), was previously shown to inhibit the synthesis of arabinans of both the cell wall arabinogalactan (AG) and lipoarabinomannan (LAM) of Mycobacterium tuberculosis and other mycobacteria. However, an Emb-resistant mutant, isolated by consecutive passage of the Mycobacterium smegmatis parent strain in media containing increasing concentrations of Emb, while synthesizing a normal version of AG, produced truncated forms of LAM when maintained on 10 microg/ml Emb (Mikusová, K., Slayden, R. A., Besra, G. S., and Brennan, P. J. (1995) Antimicrob. Agents Chemother. 39, 2482-2489). We have now isolated and characterized the truncated LAMs made by both the resistant mutant and a recombinant strain transfected with a plasmid containing the emb region from Mycobacterium avium which encodes for Emb resistance. By chemical analysis, endoarabinanase digestion, high pH anion exchange chromatography, and mass spectrometry analyses, truncation was demonstrated as primarily a consequence of selective and partial inhibition of the synthesis of the linear arabinan terminal motif, which constitutes a substantial portion of the arabinan termini in LAM but not of AG. However, at higher concentrations, Emb also affected the general biosynthesis of arabinan destined for both AG and LAM, resulting in severely truncated LAM as well as AG with a reduced Ara:Gal ratio. The results suggested that Emb exerts its antimycobacterial effect by inhibiting an array of arabinosyltransferases involved in the biosynthesis of arabinans unique to the mycobacterial cell wall. It was further concluded that the uniquely branched terminal Ara6 motif common to both AG and LAM is an essential structural entity for a functional cell wall and, consequently, that the biosynthetic machinery responsible for its synthesis is the effective target of Emb in its role as a potent anti-tuberculosis drug.
Antigens, Bacterial↗
CD1c restricts responses of mycobacteria-specific T cells. Evidence for antigen presentation by a second member of the human CD1 family.
Previous studies suggest that CD1 is a family of Ag-presenting molecules distantly related to those encoded by the MHC. However, of the four known human CD1 proteins, only CD1b has been shown to restrict Ag-specific T cell responses. In this study, we have shown that a second member of the human CD1 family, CD1c, could also mediate Ag presentation to T cells. Three T cell lines recognizing mycobacterial Ags in a CD1c-restricted manner were isolated from normal donor blood. These T cells were MHC unrestricted, and their recognition of Ag was independent of the products of the transporter associated with Ag presentation-1/2 and DMA/B genes that are generally required for Ag presentation by MHC-encoded Ag-presenting molecules. Furthermore, unlike MHC-restricted responses to peptides, the CD1c-restricted T cell lines recognized protease-resistant mycobacterial lipid Ags. These T cell lines also showed significant cytotoxicity toward CD1c-expressing target cells even in the absence of mycobacterial Ags, which was shown by clonal analysis to be mediated by a subpopulation of T cells directly reactive to CD1c molecules. Our findings establish the ability of a second member of the CD1 family to restrict responses of Ag-specific T cells, and thus support the general hypothesis that the CD1 family comprises a third lineage of Ag-presenting molecules that presents a novel class of foreign and self Ags to MHC-unrestricted T cells.
Antigen Presentation↗
Novel O-methylated terminal glucuronic acid characterizes the polar glycopeptidolipids of Mycobacterium habana strain TMC 5135.
Mycobacterium "habana" strain TMC 5135, which has been proposed as a vaccine against both leprosy and tuberculosis, is considered to be a strain of serotype I of the recognized species Mycobacterium simiae. We have now shown that each of these strains possesses characteristic polar glycopeptidolipids (GPL) which are sufficiently different to allow unequivocal strain identification. Thin layer chromatographic analysis demonstrated that M. habana synthesizes a family of apolar GPLs and three distinct polar GPLs (pGPL-I to -III) which exhibited migration patterns different from those of M. simiae serotype I (pGPL-Sim). Using a combination of chemical, mass spectrometric, and proton-NMR analyses, the GPLs from M. habana were determined to be based on the same generic structure as those from the M. avium complex, namely N-fatty acyl-D-Phe-(O-saccharide)-D-allo-Thr-D-Ala-L-alaninyl-O-m onosaccharide. The de-O-acetylated apolar GPLs contain a 3-O-Me-6-deoxy-Tal attached to the allo-Thr and either a 3-O-Me-Rha or a 3,4-di-O-Me-Rha attached to the alaninol. In the pGPLs, oligosaccharides were found to be attached to the allo-Thr. The oligoglycosyl alditol reductively released from the least polar pGPL-I was fully characterized as L-Fucp alpha 1 in --7 with 3-(6-O-Me)-D-Glcp beta 1 in --7 with 3-(4-O-Me)-L-Rhap alpha 1 in --7 with 3-L-Rhap alpha 1 in --7 with 2-(3-O-Me)-6-deoxy-Tal. In pGPl-II and -III, the terminal Fuc residue is further 3-O-methylated and 4-O-substituted with an additional 2,4-di-O-Me-D-GlcA and 4-O-Me-D-GlcA, respectively. The corresponding oligosaccharide from pGPL-Sim was shown to be of identical molecular weight to pGPL-II but terminating with a 3,4-di-O-Me-GlcA. Enzyme-linked immunosorbent assay-based serological studies using anti-M. habana and anti-M. simiae sera against whole cells and purified pGPLs firmly established the polar GPLs as important antigens and indicated that the terminal epitopes L-Fuc-, 2,4-di-O-Me-D-GlcA, and 4-O-Me-D-GlcA uniquely present in pGPL-I, -II, and -III, respectively, confer sufficient specificity for the identification of M. habana as a distinct serotype of M. simiae.
Carbohydrate Sequence↗
Chemotactic activity of mycobacterial lipoarabinomannans for human blood T lymphocytes in vitro.
A crucial early event in tuberculosis is the ingestion of Mycobacterium tuberculosis (Mtb) by alveolar macrophages. Chemotactic factors released by infected macrophages are likely to initiate a granulomatous response, a key feature of host resistance to tuberculosis. To date, the role of mycobacterial products in regulating the granulomatous response has not been clearly defined. Here we report that the mycobacterial cell wall glycophospholipid lipoarabinomannan (LAM) could specifically induce human peripheral blood T cell chemotaxis in vitro. Both terminally mannosylated LAM isolated from Mtb and LAM lacking the terminal mannosyl units isolated from an avirulent mycobacterium could induce T cell migration in the absence of serum. In contrast, terminally mannosylated LAM isolated from Mycobacterium bovis BCG failed to induce T cell chemotaxis. These observations represent the first report that LAM is capable of directly inducing biologic responses in human T cells. Flow cytometry analysis revealed that CD4+, CD8+, and CD45RO+ lymphocytes were present in the migrating cell populations at ratios similar to those found in nonmigrating cells. The chemotactic response was found to require new protein synthesis, and could be blocked by inhibitors of protein tyrosine kinases at concentrations that did not affect random migration. Acyl groups at the reducing terminus of LAM appear to be required for the chemotactic activity of this mycobacterial glycolipid. Lastly, culture supernatants from human alveolar macrophages infected in vitro with a virulent strain of Mtb could induce T cell migration. Much of the migratory activity present in these supernatants could be blocked using a mAb against LAM, suggesting that LAM is one of the chemotactic factors released by Mtb-infected alveolar macrophages.
Adult↗
Isolation and characterization of an apoptosis-resistant variant of human leukemia HL-60 cells that has switched expression from Bcl-2 to Bcl-xL.
Human promyelocytic leukemia HL-60 cells treated with 8-chloroadenosine-3',5'-cyclic monophosphate (8-Cl-cAMP) undergo growth arrest and subsequently die by apoptosis. We describe here the isolation of a variant of HL-60 cells, HCW-2, which was resistant to the cytotoxic effects of 8-Cl-cAMP, but still underwent growth arrest. Thus, HCW-2 cells appeared to be altered in their ability to undergo apoptosis. HCW-2 cells were also completely refractory to the apoptotic action of cycloheximide and staurosporine, two compounds which were very potent inducers of apoptosis in the parental HL-60 cells, suggesting that the resistance to apoptosis was not unique to 8-Cl-cAMP. Western blot analysis demonstrated that the parental HL-60 cells expressed both Bcl-2 and Bax, two factors known to be intimately involved in the control of apoptosis. Surprisingly, HCW-2 cells no longer expressed Bcl-2 protein and paradoxically contained Bax protein at a level that was approximately 50-fold higher than in HL-60 cells. However, Northern and Western analyses indicated that the apoptotic suppressor gene, bcl-xL, which is not expressed in the parental HL-60 cells, was expressed in HCW-2 cells. Thus, the Bcl-2-independent resistance of HCW-2 cells to apoptotic induction is discussed in terms of the expression of bcl-xL.
8-Bromo Cyclic Adenosine Monophosphate↗
Identification of a novel Bcl-2 related gene, BRAG-1, in human glioma.
Programmed cell death (apoptosis) plays a major role in embryogenesis, in mature organ homeostasis and in many disease states including cancer. Apoptosis occurs as an orderly cell-intrinsic suicide program regulated by a family of genes related to Bcl-2. Here, we describe the cloning and molecular characterization of a gene homologous to Bcl-2 from a human glioma. This gene named BRAG-1 (for brain-related apoptosis gene) has an open reading frame that encodes for a protein of 31 kDa sharing significant sequence homology with the Bcl-2 family of genes in the BH1 and BH2 regions. Northern blot analyses revealed that BRAG-1 is expressed in human gliomas as a 1.8 kb message. This gene, interestingly, was found to be expressed predominantly in normal human brain as a 4.5 kb transcript which is different in size from the message found in tumor tissues. These results suggest that BRAG-1 may be rearranged in human gliomas leading to its over-expression as a truncated transcript. Utilizing a bacterial expression vector, we produced BRAG-1 protein which was found to cross-react with a Bcl-2 monoclonal antibody, further suggesting structural and immunological similarity to Bcl-2.
Amino Acid Sequence↗
Monocytic differentiation and synthesis of proteins associated with apoptosis in human leukemia U-937 cells acquiring resistance to vincristine.
Human leukemia U-937/WT cells were exposed to stepwise increased concentrations of Vincristine so that Vincristine-resistant cell sublines (termed U-937/RV) were developed. Established U-937/RV cell sublines have continuously propagated over a year, both in absence and presence of VCR, and have demonstrated similar features. In contrast to U-937/WT cells, U-937/RV cells have longer doubling time, and are more differentiated as determined by appearance of distinct morphological features and synthesis of mRNA that codes for the monocyte colony-stimulating factor-1 receptor (c-fms). Both apoptosis-suppressing Bcl-2 and Bcl-XL proteins were undectable in U-937/WT cells, whereas Bcl-2 was nearly detectable and Bcl-XL readily detectable in U-937/RV cells. The apoptosis-promoting Bax protein was also absent in U-937/WT cells and readily detected in U-937/RV cells. Vincristine-resistant cells with different levels of resistance synthesize similar levels of c-fms mRNA and Bax protein. Finally, unlike U-937/WT cells, U-937/RV cells have no ability to induce tumors when xenografted in immunodeficient mice. The findings collectively suggest that development of resistance to Vincristine in U-937/WT cells may correlate with cell differentiation and synthesis of proteins that regulate apoptosis.
Animals↗
The evolutionary relationship among Caucasian MS patients and controls.
Previous observations suggest that the mitochondrial (mt)DNA may confer susceptibility to multiple sclerosis (MS). However, the proportion of affected individuals and the range of contributing mtDNA abnormalities are unknown. To help clarify this question, we analyzed the first hypervariable D-loop sequences of the mtDNA in a group of randomly selected Caucasian MS patients, in MS patients with prominent optic neuritis (PON) and in controls. Phylogenetic analysis of these D-loop sequences revealed that individuals in both groups of patients are generally scattered in the Caucasian phylogeny. However, a small cluster of unrelated MS patients identified by this analysis suggests that a maternal lineage with MS relevant mtDNA sequences may exist, and merits a more comprehensive study.
Base Sequence↗
Establishment of human prostate tumor xenografts in nude mice and response to 9-nitrocamptothecin in vivo and in vitro does not correlate with the expression of various apoptosis-regulating proteins.
Flow cytometry and microscopy analyses have demonstrated that 9-nitrocamptothecin (9NC) induces apoptosis in prostate carcinoma LNCaP, DU-145 and PC-3 cells grown in culture or as xenografts. 9NC induces apoptosis regardless of the ability of the cells to induce tumors following xenografting into nude mice. Detection of apoptosis by flow cytometry was preceded or accompanied by increased cell size, loss of nuclear structure and vacuolization, as the tumor regressed, but no visible chromatin fragmentation. This is the first demonstration that 9NC is curative for human prostate carcinoma xenografts in the nude mouse model in the absence of detectable drug-induced toxicity during and after tumor regression. These findings indicate that 9NC may develop into a chemotherapeutic drug for the effective treatment of prostate cancer patients. Further, there was no apparent correlation of the steady-state level of the apoptosis-regulating proteins, Bcl-2, Bcl-XL, Bax and Ich-1, with tumorigenicity of the prostate cells xenografted in nude mice, aggressiveness of tumors grown in nude mice, and induction of apoptosis by 9NC. However, the TIAR protein was present at markedly high levels in all prostate carcinoma cell lines and this may correlate with their susceptibility to 9NC-induced apoptosis.
Animals↗
CD1-restricted T cell recognition of microbial lipoglycan antigens.
It has long been the paradigm that T cells recognize peptide antigens presented by major histocompatibility complex (MHC) molecules. However, nonpeptide antigens can be presented to T cells by human CD1b molecules, which are not encoded by the MHC. A major class of microbial antigens associated with pathogenicity are lipoglycans. It is shown here that human CD1b presents the defined mycobacterial lipoglycan lipoarabinomannan (LAM) to alpha beta T cell receptor-bearing lymphocytes. Presentation of these lipoglycan antigens required internalization and endosomal acidification. The T cell recognition required mannosides with alpha(1-->2) linkages and a phosphotidylinositol unit. T cells activated by LAM produced interferon gamma and were cytolytic. Thus, an important class of microbial molecules, the lipoglycans, is a part of the universe of foreign antigens recognized by human T cells.
Antigen Presentation↗
Inositol phosphate capping of the nonreducing termini of lipoarabinomannan from rapidly growing strains of Mycobacterium.
Previous studies have demonstrated that the nonreducing termini of the lipoarabinomannan (LAM) from Mycobacterium tuberculosis are extensively capped with mannose residues, whereas those from a fast growing Mycobacterium sp., once thought to be an attenuated strain of M. tuberculosis, are not. The noncapped LAM, termed AraLAM, is known to be more potent than the mannose-capped LAM (ManLAM) in inducing functions associated with macrophage activation. Using a combination of chemical and enzymatic approaches coupled with fast atom bombardment-mass spectrometry analysis, we demonstrated that LAMs from all M. tuberculosis strains examined (Erdman, H37Ra, and H37Rv), as well as the attenuated Mycobacterium bovis BCG strain, are mannose-capped with the extent of capping varying between 40 and 70%. The nonreducing termini of LAM from Mycobacterium leprae were also found to be capped with mannoses but at a significantly lower level. A novel inositol phosphate capping motif was identified on a minor portion of the otherwise uncapped arabinan termini of LAMs from the fast growing Mycobacterium sp. and Mycobacterium smegmatis ATCC 14468 and mc(2)155. In addition, an inositol phosphate tetra-arabinoside was isolated from among endoarabinase digestion products of AraLAM and was shown to induce tumor necrosis factor-alpha production. Accordingly, we concluded that AraLAM is characteristic of some rapidly growing Mycobacterium spp. It is distinct from ManLAMs of M. tuberculosis, M. bovis BCG, and Mycobacterium leprae not only in the absence of mannose-capping but also in containing some terminal inositol phosphate substituents which may account for its particular potency in inducing macrophage activation.
Antigens, Bacterial↗
Analysis of six protein structures predicted by comparative modeling techniques.
The protein structures of six comparative modeling targets were predicted in a procedure that relied on improved energy minimization, without empirical rules, to position all new atoms. The structures of human nucleoside diphosphate kinase NM23-H2, HPr from Mycoplasma capricolum, 2Fe-2S ferredoxin from Haloarcula marismortui, eosinophil-derived neurotoxin (EDN), mouse cellular retinoic acid protein I (CRABP1), and P450eryf were predicted with root mean square deviations on C alpha atoms of 0.69, 0.73, 1.11, 1.48, 1.69, and 1.73 A, respectively, compared to the target crystal structures. These differences increased as the sequence similarity between the target and parent proteins decreased from about 60 to 20% identity. More residues were predicted than form the common region shared by the two crystal structures. In most cases insertions or deletions between the target and the related protein of known structure were not correctly positioned. One two residue insertion in CRABP1 was predicted in the correct conformation, while a nine residue insertion in EDN was predicted in the correct spatial region, although not in the correct conformation. The positions of common cofactors and their binding sites were predicted correctly, even when overall sequence similarity was low.
Amino Acid Sequence↗
Molecular characterization of the in vivo alkylating agent resistant murine EMT-6 mammary carcinoma tumors.
The expression of several early-response genes and genes associated with malignant disease was assessed in the EMT-6/parent tumor and the EMT-6/CTX and EMT-6/CDDP in vivo resistant tumor lines growing as tumors or as monolayers in culture. In the absence of treatment the levels of mRNA for the genes c-jun, c-fos, c-myc, Ha-ras and p53 were increased in the EMT-6/CTX and EMT-6/CDDP as compared with the EMT-6/parent tumor, whereas the expression of erb-2 was similar in all three tumors. Although the cells from each of the three tumors show increased expression of early response genes after exposure to cisplatin (CDDP; 100 microM, 2 h) or 4-Hydroxyperoxycyclophosphamide (4-HC; 100 microM, 2 h) in culture, in mRNA extracted from tumor tissue these changes are absent or very small. Both C-jun and erb-2 were detectable in liver. There was increased expression of both of these genes in the livers of tumor-bearing animals as compared with non-tumor-bearing animals. The highest expression of both c-jun and erb-2 occurred in the livers of animals bearing the EMT-6/CDDP tumor. Treatment of the animals with CDDP or cyclophosphamide, in general, resulted in increased expression of both genes at 6 h post treatment. The increased expression of these genes may impart metabolic changes in the tumors and/or hosts that contribute to the resistance of these tumors to specific antitumor alkylating agents.
Alkylating Agents↗
The variable surface glycolipids of mycobacteria: structures, synthesis of epitopes, and biological properties.
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Amino Acid Sequence↗
Structural definition of acylated phosphatidylinositol mannosides from Mycobacterium tuberculosis: definition of a common anchor for lipomannan and lipoarabinomannan.
Based on chemical analysis, we have previously concluded that the biologically important lipoarabinomannan (LAM) and lipomannan (LM) from Mycobacterium are multiglycosylated forms of the phosphatidylinositol mannosides (PIMs), the characteristic cell envelope mannophosphoinositides of mycobacteria. Using definitive analytical techniques, we have now re-examined the reported multiacylated nature of PIMs in order to gain a better insight into their possible roles as biosynthetic precursors of LM and LAM. High-sensitivity fast atom bombardment-mass spectrometry analyses of the perdeuteroacetyl and permethyl derivatives of PIMs from Mycobacterium tuberculosis and Mycobacterium leprae enabled us to define the exact fatty acyl compositions of the multiacylated, heterogeneous PIM families, notably the dimannoside (PIM2) and the hexamannoside (PIM6). Specifically, in conjunction with other chemical and gas chromatography-mass spectrometry (GC-MS) analyses, the additional C16 fatty acyl substituent on PIM2 and its lyso form were defined as attached to the C6 position of mannose. We also present evidence for triacylated mannophosphoinositide as a common lipid anchor for both LM and LAM, and further postulate that acylation of PIM2 may constitute a key regulatory step in their biosynthesis.
Acetylation↗