Monoclonal antibody against Mycoplasma fermentans-specific aminoglycoglycerolipid

Previously, we reported that Mycoplasma fermentans has specific antigens (phosphocholine-containing glycoglycerolipids: GGPL-I and GGPL-III) and discussed the possibility of their pathogenic role. In this paper, we report the characterization of a monoclonal antibody (MF-III-1) specific to GGPL-III (phosphocholine-containing aminoglycoglycerolipid) using methods of electron microscopy, immunofluorescence cell surface staining, laser scanning microscopy, immunoelectron microscopy, and thin-layer chromatography immunostaining. The MF-III-1 antibody specifically recognized M. fermentans attached to the surface of HTLV-I-infected human helper T-cells, and it did not cross-react with other lipids nor with human T-cell antigens. Since MF-III-1 distinguishes GGPL-III from GGPL-I, the binding site may include a serinol (2-amino-1,3-propanediol) residue of GGPL-III. MF-III-1 is useful for the in vitro study of M. fermentans, and may also be useful as a tool for the study of the involvement of M. fermentans in human diseases.     

Mycoplasma fermentans glycolipid-antigen as a pathogen of rheumatoid arthritis

Mycoplasma fermentans has been suspected as one of the causative pathogenic microorganisms of rheumatoid arthritis (RA) however, the pathogenic mechanism is still unclear. We, previously, reported that glycolipid-antigens (GGPL-I and III) are the major antigens of M. fermentans. Monoclonal antibody against the GGPL-III could detect the existence of the GGPL-III antigens in synovial tissues from RA patients. GGPL-III antigens were detected in 38.1% (32/84) of RA patient’s tissues, but not in osteoarthritis (OA) and normal synovial tissues. Immunoelectron microscopy revealed that a part of GGPL-III antigens are located at endoplasmic reticulum. GGPL-III significantly induced TNF-α and IL-6 production from peripheral blood mononulear cells, and also proliferation of synovial fibroblasts. Further study is necessary to prove that M. fermentans is a causative microorganism of RA; however, the new mechanisms of disease pathogenesis provides hope for the development of effective and safe immunotherapeutic strategies based on the lipid-antigen, GGPL-III, in the near future.     

Molecular Cloning and Expression of a Novel Cholinephosphotransferase Involved in Glycoglycerophospholipid Biosynthesis of <Emphasis Type="Italic">Mycoplasma fermentans</Emphasis>

A gene, mf1, encoding a novel cholinephosphotransferase in glycoglycerophospholipid (GGPL) biosynthesis of Mycoplasma fermentans PG18 was identified by genomic analysis, cloned, and expressed in Escherichia coli. The mf1 gene comprises an open reading frame of 777 bp encoding 258 amino acids. The mf1 gene product, Mf1, has 23% amino acid homology with LicD of Haemophilus influenzae but no homology with genes of other Mycoplasma species in the GenBank database. The reaction product of Mf1 using α-glucopyranosyl-1,2-dipalmitoilglycerol and cytidine 5′-diphosphocholine (CDP-choline) as substrates showed the specific protonated molecule at m/z 896, which corresponded to GGPL-I as determined by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, the product ions of choline, phosphocholine, and hexose-bound phosphocholine were detected by tandem mass spectrometry (MS) analysis of protonated molecules at m/z 896. These results identified mf1 as a novel cholinephosphotransferase and showed that the phosphocholine transfer step is involved in the GGPL biosynthesis pathway of M. fermentans. This is the first report of a GGPL biosynthesis enzyme.     

Effect of Mycoplasmas on Apoptosis of 32D Cells Is Species-Dependent

We previously showed that mycoplasmal infection effectively prevented apoptosis of infected cells, whereas other researchers have indicated that mycoplasmal infection promoted apoptosis. To understand the mechanism underlying this discrepancy, five different species of mycoplasmas were investigated for their effects on apoptosis of interleukin (IL)-3–dependent 32D cells. Results revealed that Mycoplasma fermentans and M. penetrans effectively supported continuous growth of 32D cells after IL-3 withdrawal. M. fermentans was more potent than M. penetrans. This effect was achieved by way of preventing apoptosis and stimulating cell proliferation. On the contrary, M. hominis and M. salivarium accelerated apoptosis of 32D cells. M. genitalium had no significant effect on apoptosis. The RNase protection assay indicated that the proapoptotic and antiapoptotic mycoplasmas altered the expression of major apoptosis regulatory genes differently. The difference in apoptosis regulatory gene expression induced by different species of mycoplasmas might be accountable for their effects on host cell apoptosis.     

Characterization of an IS Element in <Emphasis Type="Italic">Mycoplasma orale</Emphasis> That Is Highly Homologous to <Emphasis Type="Italic">M. fermentans</Emphasis> ISLE

In a previous study, using a primer set designed from Mycoplasma fermentans, we amplified a PCR fragment from Mycoplasma orale similar to the 206-bp DNA fragment amplified from M. fermentans insertion-sequence-like element (ISLE). The presence of this similar ISLE fragment has the potential to cause confusion in the PCR diagnosis of M. fermentans and M. orale, which have significantly different clinical scenarios. An ISLE from three different M. orale strains was amplified by using a primer set designed from sequence within the left and right terminal stem and loop (S&L) structures flanking the ISLE of M. fermentans. Sequence analysis showed that the M. orale ISLE is 93% identical to that of M. fermentans at the nucleotide level and codes for two open reading frames also found in the M. fermentans ISLE. This is the first finding that two different mycoplasma species harbor highly homologous IS elements. This finding has great significance in clinical diagnosis and suggests a possibility of horizontal transfer of an IS element between two different mycoplasma species. Received: 17 April 2002 / Accepted: 9 July 2002     

High-Frequency DNA Rearrangements in the Chromosomes of Clinically Isolated Mycoplasma fermentans

Mycoplasma fermentans is currently being examined as an agent potentially associated with human disease. Several strains of M. fermentans were isolated from patients with respiratory tract disease and AIDS. Two of these clinical strains, M64 and SK6, were triple-filter-cloned and designated as the parental clones in this study. Genomic DNA of randomly picked subclones in four and five subsequent generations passed from the parental M64 and SK6 clones were analyzed by using a radiolabeled M. fermentans-specific insertion sequence (IS)-like element as the probe. The hybridization patterns of DNA restriction fragments revealed high frequencies of chromosomal changes accompanied with excision or new insertion of the IS-like element in M. fermentans chromosome. The findings indicate M. fermentans has an effective mechanism(s) to produce a rapid gene rearrangement that may be mediated by one or more copies of the IS-like element. Received: 8 October 1997 / Accepted: 8 December 1997     

Mycoplasma fermentans Inhibits the Activity of Cellular DNA Topoisomerase I by Activation of PARP1 and Alters the Efficacy of Its Anti-Cancer Inhibitor

To understand the effects of the interaction between Mycoplasma and cells on the host cellular function, it is important to elucidate the influences of infection of cells with Mycoplasma on nuclear enzymes such as DNA Topoisomerase type I (Topo I). Human Topo I participates in DNA transaction processes and is the target of anti-cancer drugs, the camptothecins (CPTs). Here we investigated the mechanism by which infection of human tumor cells with Mycoplasma fermentans affects the activity and expression of cellular Topo I, and the anti-cancer efficacy of CPT. Human cancer cells were infected or treated with live or sonicated M. fermentans and the activity and expression of Topo I was determined. M. fermentans significantly reduced (by 80%) Topo I activity in the infected/treated tumor cells without affecting the level of Topo I protein. We demonstrate that this reduction in enzyme activity resulted from ADP-ribosylation of the Topo I protein by Poly-ADP-ribose polymerase (PARP-1). In addition, pERK was activated as a result of the induction of the MAPK signal transduction pathway by M. fermentans. Since PARP-1 was shown to be activated by pERK, we concluded that M. fermentans modified the cellular Topo I activity by activation of PARP-I via the induction of the MAPK signal transduction pathway. Moreover, the infection of tumor cells with M. fermentans diminished the inhibitory effect of CPT. The results of this study suggest that modification of Topo I activity by M. fermentans may alter cellular gene expression and the response of tumor cells to Topo I inhibitors, influencing the anti-cancer capacity of Topo I antagonists.     

MT-4 Suppresses Resistant Ovarian Cancer Growth through Targeting Tubulin and HSP27

Objective

In this study, the anticancer mechanisms of MT-4 were examined in A2780 and multidrug-resistant NCI-ADR/res human ovarian cancer cell lines.

Methods

To evaluate the activity of MT-4, we performed in vitro cell viability and cell cycle assays and in vivo xenograft assays. Immunoblotting analysis was carried out to evaluate the effect of MT-4 on ovarian cancer. Tubulin polymerization was determined using a tubulin binding assay.

Results

MT-4 (2-Methoxy-5-[2-(3,4,5-trimethoxy-phenyl)-ethyl]-phenol), a derivative of moscatilin, can inhibit both sensitive A2780 and multidrug-resistant NCI-ADR/res cell growth and viability. MT-4 inhibited tubulin polymerization to induce G2/M arrest followed by caspase-mediated apoptosis. Further studies indicated that MT-4 is not a substrate of P-glycoprotein (p-gp). MT-4 also caused G2/M cell cycle arrest, accompanied by the upregulation of cyclin B, p-Thr161 Cdc2/p34, polo-like kinase 1 (PLK1), Aurora kinase B, and phospho-Ser10-histone H3 protein levels. In addition, we found that p38 MAPK pathway activation was involved in MT-4-induced apoptosis. Most importantly, MT-4 also decreased heat shock protein 27 expression and reduced its interaction with caspase-3, which inured cancer cells to chemotherapy resistance. Treatment of cells with SB203580 or overexpression of dominant negative (DN)-p38 or wild-type HSP27 reduced PARP cleavage caused by MT-4. MT-4 induced apoptosis through regulation of p38 and HSP27. Our xenograft models also show the in vivo efficacy of MT-4. MT-4 inhibited both A2780 and NCI-ADR/res cell growth in vitro and in vivo.

Conclusion

These findings indicate that MT-4 could be a potential lead compound for the treatment of multidrug-resistant ovarian cancer.     

Immunocytochemical localization of two key enzymes of the 2-hydroxyglutarate pathway of glutamate fermentation in Acidaminococcus fermentans

We have investigated the in situ location of glutaconyl-CoA decarboxylase and 2-htdroxyglutaryl-CoA dehydratase in Acidaminococcus fermentans using the antibody-gold and protein A-gold techniques carried out as a post-embedding immunoelectron microscopic procedure. Polyclonal antisera were raised in rabbits against homogeneous fractions of the enzymes. Anaerobically grown cells of A. fermentans of the late exponential growth phase were fixed with 0.2% glutaraldehyde and 0.3% formaldehyde (final concentrations) in the growth medium. Dehydration of the cells was achieved with methanol. The cells were embedded in the low temperature embedding resin Lowicryl K4M. The markers indicative for antigenic sites of the two enzymes unequivocally demonstrate that the sodium pump glutaconyl-CoA decarboxylase is located at the cell periphery being a membrane-bound enzyme as expected whereas 2-hydroxyglutaryl-CoA dehydratase is a soluble cytoplasmic enzyme.Abbreviations PAG protein A gold complex - GARG antibodygold complex - PBS phosphate buffered saline (50 mM sodium phosphate, 0.9% NaCl, pH 6.9)     

酒醅来源酵母菌合成异戊醇能力与途径解析

【背景】异戊醇是酵母菌在白酒发酵过程中通过氨基酸合成代谢途径和氨基酸分解代谢途径合成的主要高级醇,其含量影响白酒饮用的舒适度。【目的】分析和比较分离自浓香型白酒酒醅中的酵母菌合成异戊醇的能力,揭示酵母菌合成异戊醇的途径。【方法】从酒醅中分离具有异戊醇合成能力的酵母菌株,比较不同生长时期酵母菌合成异戊醇的能力,通过前体物代谢分析它们合成异戊醇的途径。【结果】分离自酒醅的5株酵母的异戊醇合成能力从强到弱依次为Naumovozyma castellii JP3-1、Saccharomyces cerevisiae JP3、Pichia fermentans JP22、Pichia kudriavzevii JP1和Naumovozyma dairenensis CBS421。这些酵母合成异戊醇的时期主要在对数生长期,N. castellii JP3-1、P. fermentans JP22和N. dairenensis CBS421在稳定生长期也合成异戊醇。S. cerevisiae JP3、N. castellii JP3-1和N. dairenensis CBS421在整个生长时期主要通过Harris途径合成异戊醇;P. kudriavzevii JP1在整个时期主要通过Ehrlich途径合成异戊醇;P. fermentans JP22在对数生长期通过Harris途径和Ehrlich途径合成异戊醇的能力接近,在稳定生长期主要通过Harris途径合成异戊醇。【结论】本研究揭示了酒醅来源5个属种酵母合成异戊醇的途径、能力与其生长时期的关系,研究结果可为解析浓香型白酒发酵过程异戊醇合成、积累机制及实施白酒发酵过程异戊醇合成的精准调控提供理论依据。     

Evaluating the influence of inhibitors present in lignocellulosic hydrolysates on the cell membrane integrity of oleaginous yeast Trichosporon fermentans by flow cytometry

The effect of ten typical organic acids and five aldehydes present in lignocellulosic hydrolysates on the cell membrane integrity of oleaginous yeast Trichosporon fermentans was evaluated by flow cytometry. Overall, organic acids affected the cell membrane integrity of T. fermentans more significantly than that of aldehydes albeit aldehydes are more toxic to T. fermentans. The PI (Propidium Iodide) uptake rate of T. fermentans’ cells gradually decreased as fermentation going on, indicating that T. fermentans could overcome the inhibition of organic acids or aldehydes by adaption. Interestingly, in some cases, the effect of organic acids or aldehydes on the cell membrane integrity of T. fermentans was well related to their hydrophobicity. However, for the outliers, no obvious similar phenomena were observed. Thus, the attack on hydrophobic sites of cell membrane was not the only determinant for the damage of organic acids or aldehydes on cell membrane integrity of T. fermentans.     

Human chorion cells respond to growth factors but lose steroidogenic capacity in primary monolayer cell culture

Summary This study has defined a method for preparation and monolayer culture of cells fromchorion laeve. Cell number and cell protein content are table over 7 d in culture. The cells will divide in response to epidermal growth factor in the presence of a supplemented, enriched medium and a collagen matrix, but they lose steroidogenic activity over time in culture. This culture system can be used as the starting point for the development of a chemically defined hormone-supplemented, serum-free culture system for studies of chorion cell differentiation and fetal membrane cell interactions. This work was supported by the Medical Research Council of Canada through Grant MT-7867 (to A. K. T.), Grant MA-7731 (to B. F. M.), and a Summer Studentship (to K. A. F.).     

Comparison of two methods for detection of Mollicutes (Mycoplasmatales and Acholeplasmatales) in cell cultures in The Netherlands

A total of 1949 cell cultures was tested for contamination with mollicutes by cultivation on and in mycoplasma media, 25.7% of the cell cultures was positive, 243 strains of Mycoplasma hyorhinis were isolated. Furthermore, mainly M. arginini and M. orale were detected, less often Acholeplasma laidlawii, M. fermentans and M. pneumoniae. Optimal conditions for isolation were discussed. About one third of 217 hybridoma cultures and two third of 57 myeloma cultures proved to be contaminated, all with M. hyorhinis. A DNA fluorochrome staining method (DAPI-test) was compared to cultivation for testing 1039 cell cultures. The efficiency of the DAPI-test could be estimated to be about 96% that of cultivation about 89%, but cultivation is more specific. The highest assurance is obtained when both methods are applied.     

Involvement of IL-10 and Bcl-2 in resistance against an asbestos-induced apoptosis of T cells

To analyze the possibility that immunological alteration in asbestos-related diseases (ARDs) such as asbestosis (ASB) and malignant mesothelioma (MM) may affect the progression of cancers, a human adult T cell leukemia virus-immortalized T cell line (MT-2Org) was continuously exposed to 10 μg/ml of chrysotile-B (CB), an asbestos. After at least 8 months of exposure, the rate of apoptosis in the cells became very low and the resultant subline was designated MT-2Rst. The MT-2Rst cells were characterized by (i) enhanced expression of bcl-2, with regain of apoptosis-sensitivity by reduction of bcl-2 by siRNA, (ii) excess IL-10 secretion and expression, and (iii) activation of STAT3 that was inhibited by PP2, a specific inhibitor of Src family kinases. These results suggested that the contact between cells and asbestos may affect the human immune system and trigger a cascade of biological events such as activation of Src family kinases, enhancement of IL-10 expression, STAT3 activation and Bcl-2 overexpression. This speculation was partially confirmed by the detection of elevated bcl-2 expression levels in CD4 + peripheral blood T cells from patients with MM compared with those from patients with ASB or healthy donors. Further studies will be required to verify the role of T cells with enhanced bcl-2 expression in tumor progression induced by asbestos exposure.     

Trehalose phosphorylase from Pichia fermentans and its role in the metabolism of trehalose

During a screening for novel microbial trehalose phosphorylase three Pichia strains were identified as producers of this particular enzyme that have not yet been described. To our knowledge, this is the first time that this enzyme activity has been shown in yeasts. Pichia fermentans formed trehalose phosphorylase when cultivated on a growth medium containing easily metabolizable sugers such as glucose. Addition of NaCl (0.4 M) to the medium increased the synthesis of the enzyme significantly. Production of trehalose phosphorylase was found to be growth-associated with a maximum of activity formed at the transition of the exponential to the stationary phase of growth. Trehalose phosphorylase catalyzes the phosphorolytic cleavage of trehalose, yielding glucose 1-phosphate (glucose-1-P) and glucose as products. In vitro the enzyme readily catalyzes the reverse reaction, the synthesis of trehalose from glucose and glucose-1-P. For this reaction, the enzyme of P. fermentans was found to utilize -glucose-1-P preferentially. A partially purified enzyme preparation showed a pH optimum of 6.3 for the synthesis of trehalose. The enzyme was found to be rather unstable; it was easily inactivated by dilution unless Ca²⁺ or Mn²⁺ were added. This instability is presumably caused by dissociation of the enzyme. In contrast to other yeasts, P. fermentans rapidly degraded intracellularly accumulated trehalose when the carbon source in the medium was depleted. Trehalose phosphorylase seems to be a key enzyme in the degradative pathway of trehalose in P. fermentans. Additional enzymes in this catabolic pathway of trehalose include phosphoglucomutase, glucose-6-phosphate dehydrogenase, and gluconolactonase.This contribution is part of the Ph.D. thesis of Ingrid Schick     

Characterization of galactosyl glycerolipids in the HT29 human colon carcinoma cell line.

Glycoglycerolipids constitute a family of glycolipids with apparently very restricted expression in human tissues. They have previously been detected only in the testis and the nervous system. In the present study, two glycoglycerolipids were isolated from the HT29 human colon carcinoma cell line. The glycoglycerolipids were structurally characterized as a monogalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl)-sn-glycerol) and a digalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl(1-4)alpha-galactosyl)-sn-glycerol) using NMR and mass spectrometry. This digalactosylglycerolipid has not previously been structurally characterized. When HT29 cells were allowed to differentiate into more enterocyte-like cells by culture in glucose-free medium, expression of both of these glycoglycerolipids was greatly diminished. The presence of glycoglycerolipids in a human colon carcinoma cell line indicates that expression of this family of glycolipids may not be as restricted as previously thought. Instead this class of glycolipids may serve as differentiation antigens in various normal tissues and in tumor development. The Galalpha1-4Gal epitope was previously identified as a receptor for bacterial adhesins and toxins. The finding that this epitope is also linked to a glycerolipid moiety opens up new possible roles for this carbohydrate receptor in intracellular signaling.     

Genetic and serologic relatedness between Mycoplasma fermentans strains and a mycoplasma recently identified in tissues of AIDS and non-AIDS patients

A mycoplasma previously identified in the tissues of both AIDS and non-AIDS patients dying of an acute fatal disease was earlier shown to share some biologic and genetic properties with a strain of Mycoplasma fermentans, an organism occurring infrequently in the human lower urogenital tract. More extensive genetic and serologic comparisons using DNA/DNA hybridization, DNA base composition (guanine + cytosine), restriction endonuclease DNA analysis, cellular protein patterns and metabolism inhibition serologic procedures confirm that the organism previously designated as “Mycoplasma incognitus” (Mi) is indeed very closely related to strains of M. fermentans. While the genetic and serologic features observed among the newly isolated mycoplasma and two M. fermentans strains suggest a species relationship, it now seems useful to re-examine the biological activities of other freshly isolated M. fermentans strains from man.     

Reconstituted Proteolipid Vesicles Prepared from Mycoplasma fermentans Membranes Are Able to Bind and Fuse with Molt-3 Cells

We describe and characterize reconstituted proteolipid vesicles (rPLV) prepared from solubilized Mycoplasma fermentans membranes and studied their binding to and fusion with host Molt-3 cells. The rPLV were prepared following membrane solubilization by Triton X-100 and detergent removal by SM-2 resin beads. The vesicles thus obtained had a rather uniform diameter of about 1 μm and were sealed as monitored by measuring in an assay that measures the quenching by sodium dithionite of a hydrophobic fluorescent probe incorporated into the rPLV membrane. The rPLV adhered to Molt-3 cells and, based on measurements of lipid mixing, fused with the host cells at a similar rate and to about the same extent as intact M. fermentans. Preliminary experiments showed that a chimeric protein, GnRH-PE66, could be encapsulated within these rPLV, opening the way to develop a system for the transfer of high-molecular weight soluble molecules, encapsulated in the rPLV, to target eukaryotic cells.     

Resistance Profiles of (+) 2′-Deoxy-3′-oxa-4′-thiocytidine and (-) 2′-Deoxy-3′-oxa-4′-thio-5-fluorocytidine

Abstract

Resistant variants were selected in vitro against two novel nucleoside analogues, (+) dOTC and (-) dOTFC using the HIV-1 molecular clone HXB2D. The variants obtained displayed 6.5-fold and 10-fold resistance to these compounds, respectively. Cloning and sequencing of the RT genes of the selected viruses identified two mutations, M184I for (+) dOTC and M184V for (-) dOTFC. Results with mutated recombinant clones of HXB2D confirmed the importance of these mutations in MT-4 cells. The resistance profiles of clinical samples with wild-type or 3TC-resistant phenotypes were also studied; low to moderate levels of cross-resistance were observed against the novel compounds.     

Differential Responses of Single Cells and Aggregates of Mycoplasmas to Ultraviolet Irradiation

Except for Mycoplasma fermentans strain PG 18, single-cell suspensions of M. arthritidis, M. fermentans (ATCC 19989), M. hominis type 1, M. orale types 1 and 2, M. pneumoniae, and M. salivarium were inactivated exponentially by ultraviolet (UV) irradiation, in contrast to broth cultures containing clusters of elementary bodies. The susceptibility of the mycoplasmas was unaffected by storage at 2-4 C and at -70 C, by sonication, and by filtration. The rate of inactivation was dependent on the intensity of the radiations but independent of the concentration of the cells. Therefore, single-cell suspensions of these mycoplasmas could be differentiated from aggregates of cells by exponential inactivation of the colony-forming units (CFU). By this criterion, the CFU of M. arthritidis in the exponential phase of growth consisted of single cells, in contrast to the other species in which the CFU contained two or more elementary bodies. Even though the cultures of M. fermentans (PG 18) were grown from single cells, they were not homogeneous in their susceptibility to UV light. Neither were cultures of M. arthritidis and M. orale type 1 grown from single cells which had survived irradiation.