Biochemical diversity of Mycoplasma fermentans strains

In this study, the metabolism of a diverse range of Mycoplasma fermentans strains was investigated. It was shown that the ability to utilise glucose, fructose and N-acetylglucosamine differentiated strains, and that the patterns and kinetics of substrate utilisation were correlated with the site of isolation, i.e. joint fluid, respiratory tract, urinary tract or cell culture. Interestingly, isolates from the urogenital tract of AIDS patients used fructose in preference to glucose. There was also some correlation of fructose and N-acetylglucosamine utilisation of isolates with M. fermentans sub-groups, identified in an independent study, and based on the distribution of insertion sequence-like elements in the M. fermentans genome.     

MDHM, a macrophage-activating product of Mycoplasma fermentans, stimulates murine macrophages to synthesize nitric oxide and become tumoricidal

Abstract In continuation of previous work on macrophage activation by a Mycoplasma fermentans -derived product, originally named “mycoplasma-derived high mol. wt. material” (MDHM), we have investigated whether MDHM was capable of inducing synthesis of the reactive nitrogen intermediate nitric oxide (NO), thus rendering macrophages cytocidal. Mycoplasmas were first delipidated with acetone, and MDHM activity was then extracted with 50 mM 1-O-octyl-β- d -glucopyranoside to yield a particularly active new preparation of MDHM which we have named MDHM-D (D for detergent). In combination with IFN-γ, MDHM-D activated macrophages to produce reactive nitrogen intermediates and kill P815 mastocytoma cells in co-culture. P815 target cells were chosen because they are TNF-resistant. Macrophages from the LPS-low responder strain C3H/HeJ were used to minimize interference from possible LPS contamination. MDHM-D activity in this system was strictly IFN-γ-dependent. In the presence of 25 U/ml IFN-γ MDHM-D gave a half maximal response at a dilution of 1/100 000, showing a parallel concentration dependency for nitrite production and cytocidal activity.     

Contrasting effects of Mycoplasma fermentans and M. felis on the viability and chemiluminescence response of human polymorphonuclear leukocytes

Abstract Trypan blue exclusion was used to estimate the viability of human polymorphonuclear leukocytes (PMNL) in the presence of Mycoplasma felis and two strains of M. fermentans (PG18 and incognitus). The competence of PMNL to mount a respiratory burst when challenged with the mycoplasmas was also monitored by luminol-dependent chemiluminescence (CL). Both un-opsonised and non-immune human serum opsonised M. felis cells had little effect on PMNL viability. In contrast, PMNL viability was reduced markedly by un-opsonised cells of M. fermentans strain incognitus and, to a lesser extent, strain PG18, and opsonisation of these mycoplasmas further enhanced killing. Death of PMNL in the presence of M. fermentans was not associated with the autonomous production of active oxygen species during the respiratory burst as M. felis induced a high CL response from PMNL, whereas that induced by M. fermentans strain incognitus was significantly lower. M. fermentans may invade mammalian cells and it is suggested that the mechanism of PMNL death could be related to the ability of M. fermentans to penetrate host cell membranes.     

Binding of host extracellular matrix proteins to Mycoplasma fermentans and its effect on adherence to, and invasion of HeLa cells

In the present study, we show that intact Mycoplasma fermentans cells have a wealth of adhesive interactions with components of the extracellular matrix. Mycoplasma fermentans intensively bind plasminogen, and to a lesser extent, fibronectin, heparin, and laminin. The binding of collagen type III, IV, or V was low. The binding of plasminogen, collagen type III, or collagen type V markedly enhanced the adherence of M. fermentans to HeLa cells, whereas the binding of fibronectin, heparin, laminin, or collagen IV induced only a small effect on mycoplasma adherence. Utilizing plasminogen-treated M. fermentans preparations, we detected microorganisms within host HeLa cells by the gentamicin protection assay or by confocal laser scanning microscopy of immunofluorescent preparations. However, no intracellular M. fermentans was detected when M. fermentans preparations treated with fibronectin, heparin, laminin, or collagen type III, IV, or V were utilized.     

Relationship between phylogenetic groups, genotypic clusters, and virulence gene profiles of Escherichia coli strains from diverse human and animal sources

Escherichia coli strains in water may originate from various sources, including humans, farm and wild animals, waterfowl, and pets. However, potential human health hazards associated with E. coli strains present in various animal hosts are not well known. In this study, E. coli strains from diverse human and animal sources in Minnesota and western Wisconsin were analyzed for the presence of genes coding for virulence factors by using multiplex PCR and biochemical reactions. Of the 1,531 isolates examined, 31 (2%) were found to be Shiga toxin-producing E. coli (STEC) strains. The majority of these strains, which were initially isolated from the ruminants sheep, goats, and deer, carried the stx(1c) and/or stx(2d), ehxA, and saa genes and belonged to E. coli phylogenetic group B1, indicating that they most likely do not cause severe human diseases. All the STEC strains, however, lacked eae. In contrast, 26 (1.7%) of the E. coli isolates examined were found to be potential enteropathogenic E. coli (EPEC) strains and consisted of several intimin subtypes that were distributed among various human and animal hosts. The EPEC strains belonged to all four phylogenetic groups examined, suggesting that EPEC strains were relatively widespread in terms of host animals and genetic background. Atypical EPEC strains, which carried an EPEC adherence factor plasmid, were identified among E. coli strains from humans and deer. DNA fingerprint analyses, done using the horizontal, fluorophore-enhanced repetitive-element, palindromic PCR technique, indicated that the STEC, potential EPEC, and non-STEC ehxA-positive E. coli strains were genotypically distinct and clustered independently. However, some of the potential EPEC isolates were genotypically indistinguishable from nonpathogenic E. coli strains. Our results revealed that potential human health hazards associated with pathogenic E. coli strains varied among the animal hosts that we examined and that some animal species may harbor a greater number of potential pathogenic strains than other animal species.     

Frequency and distribution of tetracycline resistance genes in genetically diverse, nonselected, and nonclinical Escherichia coli strains isolated from diverse human and animal sources

Nonselected and natural populations of Escherichia coli from 12 animal sources and humans were examined for the presence and types of 14 tetracycline resistance determinants. Of 1,263 unique E. coli isolates from humans, pigs, chickens, turkeys, sheep, cows, goats, cats, dogs, horses, geese, ducks, and deer, 31% were highly resistant to tetracycline. More than 78, 47, and 41% of the E. coli isolates from pigs, chickens, and turkeys were resistant or highly resistant to tetracycline, respectively. Tetracycline MICs for 61, 29, and 29% of E. coli isolates from pig, chickens, and turkeys, respectively, were >/=233 micro g/ml. Muliplex PCR analyses indicated that 97% of these strains contained at least 1 of 14 tetracycline resistance genes [tetA, tetB, tetC, tetD, tetE, tetG, tetK, tetL, tetM, tetO, tetS, tetA(P), tetQ, and tetX] examined. While the most common genes found in these isolates were tetB (63%) and tetA (35%), tetC, tetD, and tetM were also found. E. coli isolates from pigs and chickens were the only strains to have tetM. To our knowledge, this represents the first report of tetM in E. coli.     

Morphometric and genetic variation of small dwarf honeybees Apis andreniformis Smith, 1858 in Thailand

The small dwarf honey bee, Apis andreniformis, is a rare and patchily distributed Apis spp. and is one of the native Thai honey bees, yet little is known about its biodiversity. Thirty （27 Thai and 3 Malaysian） and 37 （32 Thai and 5 Malaysian） colonies of A. andreniformis were sampled for morphometric and genetic analysis, respectively. For morphometric analysis, 20 informative characters were used to determine the variation. After plotting the factor scores, A. andreniformis from across Thailand were found to belong to one group, a notion further supported by a cluster analysis generated dendrogram. However, clinal pattems in groups of bee morphometric characters were revealed by linear regression analysis. The body size of bees increases from South to North but decreases from West to East, although this may reflect altitude rather than longitude. Genetic variation was determined by sequence analysis of a 520 bp fragment of the mitochondrial cytochrome oxidase subunit b （cytb）. DNA polymorphism among bees from the mainland of Thailand is lower than that from Phuket Island and Chiang Mal. Although two main different groups of bees were obtained from phylogenetic trees constructed by neighbor-joining and unweighted pair-group method using arithmetic averages programs, no clear geographic signal was present. Thus, while the minor group （B） contained all of the samples from the only island sampled （Phuket in the south）, but not the southem mainland colonies, it also contained samples from the far northem inland region of Chiang Mai, other samples of which were firmly rooted in the major group （A）.     

Accumulation of genetic alterations and their significance in each primary human cancer and cell line

Analyses of multiple genetic alterations accumulated in each cancer cell is expected to provide useful information to elucidate the molecular mechanisms involved in tumorigenesis. Here, we summarized the results of studies on aberrations of oncogenes and tumor suppressor genes by ourselves and other groups. DNAs analyzed were from particular sets of surgical specimens from human tumors and cancer cell lines derived from non-small cell lung cancers, pancreatic cancers, hepatocellular carcinomas and gliomas. Tumors could be grouped into two types based on the genetic alterations detected. Tumors in group 1 had mutations in genes encoding proteins involved in a limited number of signal transduction cascades such as p16-cyclin D1/CDK4-RB or MDM2-p53-p21, where the aberration of one component seems to be sufficient to cause dysfunction of the cascade. Group 2 contained a subset of tumors in which no alteration was detected in the genes analyzed, even in the advanced stage or established cancer cells, indicating the involvement of completely different oncogenic pathways.     

Evolutionary analysis and distribution of type III effector genes in pathogenic Escherichia coli from human,animal and food sources

Molecular analysis of Shiga toxin‐producing Escherichia coli (STEC) from different sources is considered as a major approach to assess their risk potential. However, only limited data are available about the correlation of evolutionary relationship, the presence of major virulence factor genes and the putative risk of an STEC strain for human infection. In this study, we analysed the evolutionary relationship of 136 pathogenic E. coli strains from human, animal and food sources by multi‐locus sequence typing (MLST) and molecular subtyping of their Shiga toxin (stx) and intimin (eae) genes. Moreover, the distribution of three type III effector genes, encoded within the locus of enterocyte effacement (LEE), and 16 effector genes, which are encoded outside the LEE, was analysed. One hundred and five strains from different sources harboured 5–15 of the analysed non‐LEE‐encoded effector genes. In 101 of these strains, the LEE genes eae, map, espF and espG were present simultaneously. Thirty‐one isolates deriving mainly from food and patients suffering from haemolytic uraemic syndrome (HUS) were eae‐negative and did not carry any of the analysed effector genes. By combination of MLST and virulence gene data, we defined five genetic clusters. Within these clusters a clear‐cut affiliation of particular sequence types and the occurrence of certain effector genes was observed. However, in contrast to other studies, a significant correlation between the amount and type of effector genes and the risk to cause HUS could not be demonstrated.     

Analysis of genetic variation in the Belgian Blue Cattle breed using DNA sequence polymorphism at the growth hormone, low density lipoprotein receptor, α-subunit of glycoprotein hormones and thyroglobulin loci

Summary. New DNA sequence polymorphisms were identified at four bovine autosomal loci: growth hormone, low density lipoprotein receptor, α-subunit of glycoprotein hormones and thyroglobulin. Assuming independent assortment between these polymorphisms, the probabilities to be heterozygous at these four loci are 0.48, 0.36, 0.10 and 0.77 respectively, within the Belgian Blue Cattle breed (BBCB). Nucleotide diversity was estimated, showing that animals from the BBCB are heterozygous for 1/1450 nucleotides, a value significantly lower than the 1/500 value found in man. Moreover, we have estimated that the mutation rate at the cytosines of CG dinucleotides is about 10 times higher than that for other nucleotides.     

Ribosomal DNA variation in foxtail millet, Setaria italica (L.) P. Beauv., and a survey of variation from Europe and Asia

 Restriction fragment length polymorphism (RFLP) and the structure of ribosomal RNA genes (rDNA) were investigated in 117 landraces of foxtail millet, Setaria italica (L.) P. Beauv. Five RFLP phenotypes were found when the genomic DNA was digested with BamHI; these were named types I–V. Of these types I, II and III were the most frequent. Type I was mainly distributed in the temperature zone, type II in the Taiwan-Philippines Islands and type III in South Asia. Restriction mapping of the cloned rDNA and comparison with RFLP phenotypes showed that the different types originated from a polymorphism in the length within the intergenic spacer (IGS) and BamHI site changes within the IGS. Received: 28 August 1996 / Accepted: 28 February 1997     

The gene mpn310 (hmw2) from Mycoplasma pneumoniae encodes two proteins, HMW2 and HMW2-s, which differ in size but use the same reading frame

The gene mpn310 from Mycoplasma pneumoniae encodes the proteins HMW2 with a molecular weight of 215 621 and the smaller P28, here called HMW2-s. Because HMW2-s is not well defined, it was isolated from protein extracts of M. pneumoniae cells and its N-terminal end was determined by MS. HMW2-s starts with the methionine at the amino acid position 1620 of HMW2 and its residual sequence is identical to the last 198 amino acids of HMW2, predicting a molecular weight of 23 204. These results were confirmed by the comparative MS analysis of HMW2-s that had been synthesized in Escherichia coli . A precursor–product relationship between HMW2 and HMW2-s could be excluded, because HMW2-s can be translated from a specific mRNA starting within mpn310 . The conservation of an HMW2-s like protein in M. pneumoniae and Mycoplasma genitalium emphasizes its possible functional importance.     

Rapid, complete and large-scale generation of post-mitotic neurons from the human LUHMES cell line

We characterized phenotype and function of a fetal human mesencephalic cell line (LUHMES, Lund human mesencephalic) as neuronal model system. Neurodevelopmental profiling of the proliferation stage (d0, day 0) of these conditionally-immortalized cells revealed neuronal features, expressed simultaneously with some early neuroblast and stem cell markers. An optimized 2-step differentiation procedure, triggered by shut-down of the myc transgene, resulted in uniformly post-mitotic neurons within 5 days (d5). This was associated with down-regulation of some precursor markers and further up-regulation of neuronal genes. Neurite network formation involved the outgrowth of 1-2, often > 500 μm long projections. They showed dynamic growth cone behavior, as evidenced by time-lapse imaging of stably GFP-over-expressing cells. Voltage-dependent sodium channels and spontaneous electrical activity of LUHMES continuously increased from d0 to d11, while levels of synaptic markers reached their maximum on d5. The developmental expression patterns of most genes and of the dopamine uptake- and release-machinery appeared to be intrinsically predetermined, as the differentiation proceeded similarly when external factors such as dibutyryl-cAMP and glial cell derived neurotrophic factor were omitted. Only tyrosine hydroxylase required the continuous presence of cAMP. In conclusion, LUHMES are a robust neuronal model with adaptable phenotype and high value for neurodevelopmental studies, disease modeling and neuropharmacology.     

Biochemical and morphological differentiation of the human colonic epithelial cell line SW620 in the presence of dimethylsulfoxide.

In vitro models of intestinal cell differentiation provide an important adjunct for studying normal and abnormal intestinal epithelial cell differentiation. The studies reported herein describe morphologic and biochemical changes in the colonic epithelial cell line SW620 following dimethylsulfoxide (DMSO) incubation. Cells cultured in the presence of DMSO showed striking changes in morphology characterized by enlargement, elongation, and formation of process-like structures by light microscopy and a propensity to form microvillus-like structures by electron microscopy. These changes were accompanied by significant differences in the expression of the cell surface markers CD4 (HIV gp120 receptor), CD44 (hyaluronate receptor), and KS1 (adenocarcinoma/epithelial specific antigen). There was a marked decrease in CD4 expression (38% to 2%), an increase in CD44 expression (4% to 50%) and a decrease in KS1 expression (98% to 66%) as detected by flow cytometry following incubation of SW620 cells in DMSO. Parallel changes in the expression of these markers were seen by metabolic and surface labeling studies. Although SW620 cells were infected by HIV-1, DMSO-treated SW620 cells could not be infected. DMSO-induced changes in surface expression of CD4, CD44, and KS-1 were reversible over time upon removal of DMSO from the culture medium. Secretory component, sucrase, neuron-specific enolase, chromogranin-A, and mucin were not detectable in SW620 cells with or without DMSO treatment. SW620 cells provide a useful model for studying specific biochemical and molecular events involved in intestinal epithelial cell differentiation and function.