Cardiac mesenchymal stromal cells are a source of adipocytes in arrhythmogenic cardiomyopathy.

AIM: Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder mainly due to mutations in desmosomal genes, characterized by progressive fibro-adipose replacement of the myocardium, arrhythmias, and sudden death. It is still unclear which cell type is responsible for fibro-adipose substitution and which molecular mechanisms lead to this structural change. Cardiac mesenchymal stromal cells (C-MSC) are the most abundant cells in the heart, with propensity to differentiate into several cell types, including adipocytes, and their role in ACM is unknown. The aim of the present study was to investigate whether C-MSC contributed to excess adipocytes in patients with ACM. METHODS AND RESULTS: We found that, in ACM patients' explanted heart sections, cells actively differentiating into adipocytes are of mesenchymal origin. Therefore, we isolated C-MSC from endomyocardial biopsies of ACM and from not affected by arrhythmogenic cardiomyopathy (NON-ACM) (control) patients. We found that both ACM and control C-MSC express desmosomal genes, with ACM C-MSC showing lower expression of plakophilin (PKP2) protein vs. CONTROLS: Arrhythmogenic cardiomyopathy C-MSC cultured in adipogenic medium accumulated more lipid droplets than controls. Accordingly, the expression of adipogenic genes was higher in ACM vs. NON-ACM C-MSC, while expression of cell cycle and anti-adipogenic genes was lower. Both lipid accumulation and transcription reprogramming were dependent on PKP2 deficiency. CONCLUSIONS: Cardiac mesenchymal stromal cells contribute to the adipogenic substitution observed in ACM patients' hearts. Moreover, C-MSC from ACM patients recapitulate the features of ACM adipogenesis, representing a novel, scalable, patient-specific in vitro tool for future mechanistic studies.

Prosthetic reconstruction after surgical resection of fibrous dysplasia of the maxillary and palatine bone.

Fibrous dysplasia is a rare disorder of the bone. It is seen in two main forms of presentation: monostotic and the polyostotic. A case of monostotic fibrous dysplasia of the maxillary and palatine bones in a 22-year old man who received prosthetic reconstruction is presented with a review of the literature.

[Desmoid infantile mandibular fibromatosis]. / Fibromatose desmoïde infantile de la mandibule.

INTRODUCTION: A desmoid tumor, or aggressive fibromatosis, is a benign fibrous tumor with a high potential for locoregional extension. This tumor is very rarely located in the mandible. OBSERVATION: A 2-year-old boy presented with an extensive mandibular desmoid tumor. The diagnosis was proved on histological examination. Two years after surgery, there was no recurrence. DISCUSSION: Aggressive fibromatosis is rarely located in the mandible. The differential diagnosis with malignant tumors is difficult. Surgery is the first-line treatment. However, alternative therapies should be considered, especially in children, to avoid mutilating operations.

Prosthetic reconstruction after surgical resection of fibrous dysplasia of the maxillary and palatine bone / Reconstrucción prostética tras la resección quirúrgica de la displasia fibrosa del hueso maxilar y el palatino

Fibrous dysplasia is a rare disorder of the bone. It is seen in two main forms ofpresentation: monostotic and the polyostotic. A case of monostotic fibrous dysplasia of the maxillary and palatine bones in a 22-year old man who received prosthetic reconstruction is presented with a review of the literature.

La displasia fibrosa es un trastorno raro del hueso. Se le ve en dos formas principales: la monostótica y la poliostótica. Junto con la correspondiente revisión de la literatura, se presenta un caso de displasia fibrosa monostótica de los huesos maxilar y palatino en un hombre de 22 anos que recibió una reconstrucción prostética.

Facial palsy associated with leptospirosis.

INTRODUCTION: The authors report a rare case of facial palsy associated with leptospirosis. CASE REPORT: A 60-year-old man was admitted to ICU with severe leptospirosis. On the eighth day of hospitalisation, he developed left peripheral facial palsy with a favourable course in response to corticosteroids. DISCUSSION: Several types of neurological complications of leptospirosis have been reported: encephalitis, myelitis, stroke, cerebral arteritis, mononeuritis, polyradiculoneuropathy, and cranial nerve palsy. Peripheral facial palsy is a rare complication of leptospirosis. CONCLUSION: This case illustrates the possible association between leptospirosis and facial palsy.

Comparative cytotoxicity of C-1311 in colon cancer in vitro and in vivo using the hollow fiber assay.

The aim of this study was to investigate the antiproliferative effects of C-1311 (Symadex), a member of the imidazoacridinone family, in human colorectal cancer cells. In the in vitro screen, C-1311 led to the most prominent growth inhibition in HT29, HCT116, and COLO205 cell lines when compared to oxaliplatin, CPT-11, DFUR, 5-FU and capecitabine. The GI(50)values for C-1311 ranged from 0.12 to 0.83 microM and the TGI concentrations (resulting in total growth inhibition) were 6- to 13-fold lower than those of other agents. In the hollow fiber assay in vivo, C-1311 caused 77% growth inhibition of HT29 in the intraperitoneal site as compared to paclitaxel (17% growth inhibition). In the subcutaneous site, C-1311 produced 57% growth inhibition while paclitaxel showed no cell growth inhibition effects. This unique cytotoxicity profile of C-1311 warrants further investigation and supports its clinical development in colon cancer patients. Symadex (C-1311) is currently in phase 2 clinical trials.

Comparative analysis of xanafide cytotoxicity in breast cancer cell lines.

Xanafide, a DNA-intercalating agent and topoisomerase II inhibitor, has previously demonstrated comparable cytotoxicity to the parent drug amonafide (NSC 308847). The current study was conducted to investigate further the anti-proliferative effects of xanafide in human breast cancer cell lines, in vitro and in vivo. The in vitro activity of xanafide against MCF-7, MDA-MB-231, SKBR-3 and T47D cell lines was compared to that of paclitaxel, docetaxel, gemcitabine, vinorelbine and doxorubicin. In MCF-7, xanafide demonstrated comparable total growth inhibition (TGI) concentrations to the taxanes and lower TGI values than gemcitabine, vinorelbine and doxorubicin. MCF-7 (oestrogen receptor (ER)+/p53 wild-type) was the most sensitive cell line to xanafide. MDA-MB-231 and SKBR-3 exhibited similar sensitivity to xanafide. T47 D (ER+/p53 mutated), showed no response to this agent. The in vivo activity of xanafide was further compared to that of docetaxel in MCF-7 and MDA-MB-231 cell lines using the hollow fibre assay. Xanafide was slightly more potent than docetaxel, at its highest dose in MCF-7 cell line, whereas docetaxel was more effective than xanafide in MDA-MB-231 cell line. Our results show that there is no relationship between sensitivity of these cell lines to xanafide and cellular levels of both isoforms of topoisomerase II and suggest that ER and p53 status and their crosstalk may predict the responsiveness or resistance of breast cancer patients to xanafide.

[Apolipoprotein E polymorphism in a Moroccan population: allele frequency and relation to plasma lipid concentrations]. / Polymorphisme de l'apolipoprotéine E dans une population marocaine: fréquence allélique et relation avec les paramétres lipidiques plasmatiques.

To date, no data are available on relationship between apolipoprotein E (apo E) polymorphism and lipid levels in Moroccan population. The present work reports an apo E polymorphism repartition in Moroccan population and relationship between this polymorphism and the levels of plasma cholesterol, triglycerides, apo A1, B and E. Blood samples from 168 healthy Moroccan individuals from Rabat area (90 men and 78 women), aged from 20 to 50 years (32 9 years), were analysed for serum apo E, A1 and B, triglycerides, and total cholesterol. In parallel, genotyping by means of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) was performed. The apo E allelic frequencies were 11% for epsilon4, 84% for epsilon3 and 5% for epsilon2. There were correlation between apo E alleles and serum lipid concentrations, E2/E3 carriers had significantly higher level of apo E than E3/E3, and E4/E3 carriers had significantly higher total cholesterol apo B and triglycerides than E3/E3 and E2/E3 carriers. The total cholesterol and apo B concentrations are significantly higher in women than in men but the triglycerides are lower. The apo A1 concentration is independent of both sex and apo E genotype. Thus, the results demonstrate an influence of apo E alleles on serum cholesterol, triglycerides, apo E and apo B concentrations among healthy Moroccan.

[Wegener's granulomatosis. Apropos of a case]. / Granulomatose de Wegener. A propos d'un cas.

A rare case of granulomatosis of Wegener is rapported in this study. One patient presented with ENT and pulmonary symptoms. The differential diagnosis with tuberculosis was raised. Based on a literature review, the authors discuss clinical, pathological and imaging features of the condition and its management.

Cloning and characterization of a gene cluster, phsBCDEF, necessary for the production of hydrogen sulfide from thiosulfate by Salmonella typhimurium.

We cloned, by complementation of an H2S- mutant, a cluster of Salmonella typhimurium genes, phsBCDEF, that appears to be essential for the anaerobic production of hydrogen sulfide from thiosulfate. Tn5 mutagenesis and ExoIII deletion analysis showed that approx. the entire region of a 3.3-kb subclone was necessary for H2S production. Subsequent sequencing revealed the presence of five potential translationally coupled open reading frames (ORFs). Their putative protein products were confirmed by synthesis from a phage T7 expression system. Comparison of the encoded sequences with previously determined sequences suggests that these genes constitute part of a thiosulfate-reducing operon coding for a membrane-associated electron transport chain which contains proteins potentially capable of ligating iron-sulfur clusters and heme. Immediately upstream from these genes, a region encoding the C-terminal portion of an ORF (OrfA) was identified that showed a high degree of similarity to some other anaerobic terminal reductases, polysulfide reductase (PsrA) of Wolinella succinogenes and dimethylsulfoxide reductase (DmsA), formate dehydrogenase (formate-hydrogene-lyase linked) (FdhF) and nitrate reductase (NarG) of Escherichia coli.

Transcriptional modulation of platelet-activating factor receptor gene expression by cyclic AMP.

We examined the effects of increasing intracellular cyclic AMP levels on the expression of human PAF receptor (hPAF-R). Peripheral blood monocytes constitutively expressed hPAF-R mRNA transcripts. A transiently elevated intracellular concentration of AMP induced with prostaglandin E2, cholera toxin, or forskolin was a sufficient signal to inhibit PAF-R expression. To determine the mechanisms of this inhibition, human monocytes were treated with dibutyryl cAMP, a cell-permeable cAMP analogue. cAMP reduced the expression of hPAF-R in a concentration- and a time-dependent manner. The effect was seen as early as 1 h and was essentially total by 4 h. Stability of hPAF-R mRNA was not markedly decreased by cAMP, as assessed by measuring the half-lives of the transcripts. Moreover, the nuclear transcription rate of the hPAF-R gene was reduced as early as 30 min after stimulation with cAMP. The inhibition of hPAF-R mRNA accumulation was associated with diminished responsiveness to PAF, as assayed by intracellular Ca2+ fluxes, decreased number of binding sites, and decreased hPAF-R protein expression on the cell surface, as assessed by flow cytometry using a polyclonal anti-hPAF-R antibody. These data indicate that PAF-R expression can be regulated at the transcriptional and possibly post-transcriptional levels by elevation of intracellular cAMP.

Identification and functional characterization of platelet-activating factor receptors in human leukocyte populations using polyclonal anti-peptide antibody.

Recently, the successful cloning of a receptor for platelet-activating factor (PAF), a lipid mediator of inflammation, was reported. Here we investigated the distribution and potential diversity of human PAF receptors (hPAF-Rs) among individual leukocyte populations by (i) hPAF-R mRNA transcription studies and (ii) analysis of cell surface expression of hPAF-R protein using a polyclonal anti-peptide antibody (anti-hPAF-R164-173). Northern blot analysis, flow cytometry, and immunoblotting with anti-hPAF-R antibody indicated that monocytic, neutrophilic, and B-lymphocytic cell lines all shared a similar hPAF-R species, whereas resting T-cell and natural killer cell lines failed to express detectable levels of either hPAF-R protein or mRNA. Peripheral blood leukocyte populations showed a distribution of hPAF-R cell surface expression similar to that of the corresponding cell lines. Furthermore, binding of anti-hPAF-R164-173 antiserum, purified IgG, or Fab and F(ab')2 fragments to the receptor of all investigated PAF-R-positive cell lines induced an increase in intracellular free calcium concentration. The characterization of the expression of a lipid ligand receptor using antibodies against an intrinsic portion of the receptor protein has, to our knowledge, never been reported previously.

Mutations that affect the regulation of phs in Salmonella typhimurium.

The regulation of phs [production of hydrogen sulphide (H2S)] in Salmonella typhimurium is complex. Previous studies have shown that expression is dependent upon the presence of reduced sulphur and anaerobiosis and is modulated by carbon source and growth stage. Transposon mutagenesis failed to find any potential trans-acting factors effective in the regulation of phs in relation to oxygen. Spontaneous mutants capable of expressing phs-lac aerobically were isolated and characterized. These mutations are closely linked to phs and affect not only oxygen regulation but also the requirement for cyclic AMP and reduced sulphur. Analysis of merodiploid strains indicates that these mutations cis-acting and that phs is not subject to autoregulation.

Batch cultures of recombinant Lactococcus lactis subsp. lactis in a stirred fermentor. I. Effect of plasmid content on bacterial growth and on genetic stability in pure cultures.

The effect of plasmid introduction into Lactococcus lactis subsp. lactis IL2661 on the growth of this strain and on plasmid stability was studied in pure batch cultures. The plasmids used (coding for erythromycin or chloramphenicol resistance) were the following: pIL205 (42 kb), pIL252 (4.6 kb, 6-9 copies), pIL253 (4.8 kb, 45-85 copies) and pE194 (inserted in the chromosome). Growth and acidification of L. lactis subsp. lactis IL2661 were similar to those of the derived recombinant lactococci. The maximal population at the end of the fermentation (9 h) was about 1.1 +/- 0.3 x 10(10) cfu/ml, and maximal growth rate 0.92 +/- 0.07 h-1. Growth yield and lactic acid concentrations were 3.9 +/- 0.8 x 10(11) cfu/g lactose consumed and 25.6 +/- 2.3 g/l, respectively. Different levels of plasmid stability were detected. Plasmid pE194, and plasmids pIL252 and pIL253 in the absence of pIL205, were stable after 10 h of culture. A slight loss (1-2%) of pIL205 was observed in all strains. In the presence of pIL205, plasmids pIL252 and pIL253 were maintained in only 56-95% of the cells. This result suggested an incompatibility between pIL205 and pIL252 or pIL253.

Batch cultures of recombinant Lactococcus lactis subsp. lactis in a stirred fermentor. II. Plasmid transfer in mixed cultures.

The transfer of plasmids was studied in a stirred fermentor in the course of mixed batch cultures combining recombinant strains of Lactococcus lactis subsp. lactis (donor strains) with L. lactis subsp. lactis CNRZ 268M3 (recipient strain). Donor strains contained one or two of the following plasmids (coding for erythromycin or chloramphenicol resistance): pIL205 (self-transmissible), pIL252, pIL253 (non-transmissible but mobilizable by pIL205, respectively small and large copy number) and pE194 (inserted in the chromosome). Only self-transmissible plasmid pIL205 was transferred, with frequencies ranging from 10(-7) to 10(-8) after 12 h of fermentation. These frequencies were 60-400 times lower than in unstirred M17 broth and 100,000 times lower than on agar medium. In the latter case, non-transmissible plasmids pIL252 and pIL253 were mobilized by pIL205 with a frequency of about 10(-5) - 10(-6).

Genetic characterization of adenovirus transformed cell revertants resistant to methylglyoxal bis(guanylhydrazone): evidence for the involvement of three genetic loci.

Five new independent rat somatic cell mutants resistant to the antileukemic drug methylglyoxal bis(guanylhydrazone) (MGBG) were isolated after mutagen treatment. The mutants were 7- to 10-fold more resistant to MGBG than were the parental wild-type cells. When the MGBG-resistant (MGR) mutants were exposed to the drug in the presence of Tween-80, a nonionic detergent, they became as sensitive (MGS) to MGBG as the wild-type cells, indicating that they were probably permeability mutants. Genetic analysis of hybrids between MGR mutants and wild-type cells showed that MGR and the nontransformed alleles to be recessive to the MGS (wild-type) and transformed phenotype, respectively. Complementation analysis of the seven mutants revealed three functional genetic units or loci responsible not only for the MGR phenotype but also for tumorigenicity as determined in nude mice. Only the MGS hybrids produced tumors in the nude mice, whereas the MGR hybrids and mutants did not. Our results suggest the existence of cellular membrane components that are responsible both for cellular tumorigenicity and resistance to MGBG.

Characterization of activation and deactivation pathways of 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) in rat hepatocytes.

We have characterized the metabolism of 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) in cultured rat hepatocytes and have established the relationship between various metabolic pathways and single-strand breaks (SSB) in DNA. Metabolism of [5-3H]-NNK by carbonyl reduction, alpha-carbon hydroxylation and pyridine N-oxidation was linear from 0.5 to 6 h with 0.25-2 x 10(6) hepatocytes. Using an alkaline elution assay, we observed that NNK induces SSB in DNA in a dose- and time-dependent manner. SSB induced by NNK were rejoined partially within 2 h and totally by 12 h after exposure. NNK-N-oxide produced a smaller number of SSB than NNK, suggesting that pyridine N-oxidation of NNK is a deactivation pathway. Carbonyl reduction of NNK led to 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butan-1-ol (NNAl). Reaction of NNK with methyl magnesium iodide gave 4-(N-nitrosomethylamino)-1-(methyl)-1-(3- pyridyl)butanol-1-ol (1-MeNNAl) 82% yield. NNAl, but not 1-MeNNA1, can be reoxidized to NNK. Doses of 5 mM NNAl and 1-MeNNAl both induced SSB, indicating that NNAl does not require reconversion to NNK to be activated to DNA damaging intermediates. alpha-Methylene hydroxylation resulted in the formation of 4-oxo-4-(3- pyridyl)butanal. At equimolar concentration (5 mM), the aldehyde was more damaging than NNK to hepatocyte DNA. The results of this study demonstrate that NNK is activated by rat hepatocytes and that metabolites formed by alpha-carbon hydroxylation induce SSB.

Metabolism and DNA single strand breaks induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and its analogues in primary culture of rat hepatocytes.

Previous studies have shown that the tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is hepatocarcinogenic and results in alkylation of hepatic DNA in F344 rats. In this study, we have characterized the metabolism of NNK in cultured rat hepatocytes and have established the relationship between various metabolic pathways and the induction of DNA single strand breaks. DNA fragmentation by NNK and two other related N-nitrosamines, N'-nitrosonornicotine and nitrosodimethylamine, were compared. Metabolism of [5-3H]NNK (4.5 microM) by carbonyl reduction, alpha-carbon hydroxylation, and pyridine N-oxidation was linear from 0 to 6 h and with 0 to 2 x 10(6) hepatocytes. Using the alkaline elution assay, we observed that NNK induces DNA single strand breaks (SSB) in a dose (1-10 mM) and time (0.5-6 h) dependent manner. SSB induced by NNK (5 mM; rate of elution between 3 and 9 h, 0.117) are rejoined partially within 2 h (rate, 0.039) and totally 12 h after exposure. NNK N-oxide (5 mM) produces a smaller number of SSB (rate, 0.017) than NNK (rate, 0.105) suggesting that pyridine N-oxidation of NNK is a deactivation pathway. Hydrolysis of carbethoxy-nitrosaminomethane and 4-(N-carbethoxy-N-nitrosamino)-1-(3-pyridyl)butanone yields methyldiazohydroxide and 4-(3-pyridyl)-4-oxobutyl-diazohydroxide, respectively. These two alkylating intermediates are generated during alpha-carbon hydroxylation of NNK. After treatment of hepatocytes with 5 microM carbethoxynitrosaminomethane and 1 mM 4-(N-carbethoxyl-N-nitrosamino)-1-(3-pyridyl)butanone, the rates of DNA elution were 0.092 and 0.120, respectively. Carbonyl reduction of NNK leads to 4-(methylnitrosamino)-1-(3-pyridyl)butan-1-ol (NNAl). Reaction of NNK with methyl magnesium iodide gives 1-MeNNAl with 82% yield, NNAl but not 1-MeNNAl can be reoxidized to NNK. Both 5 mM NNAl (rate, 0.073) and 5 mM 1-MeNNAl (rate, 0.054) induce SSB indicating that NNAl does not require reconversion to NNK to be activated to DNA damaging intermediates. alpha-Methylene hydroxylation of NNK results in an equimolar formation of methyldiazohydroxide and 4-oxo-4-(3-pyridyl)-butanal. This aldehyde, at a concentration of 1 mM, induces the same frequency of SSB (rate, 0.116) as 5 mM NNK (0.105) and could possibly play a role in the carcinogenicity of NNK.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytotoxicity, sister-chromatid exchanges and DNA single-strand breaks induced by 4-oxo-4-(3-pyridyl)butanal, a metabolite of a tobacco-specific N-nitrosamine.

The tobacco-specific N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is metabolized by alpha-carbon hydroxylation to reactive diazohydroxides and aldehydes. The aim of this study was to determine the relative ability of one NNK-derived aldehyde, 4-oxo-4-(3-pyridyl)butanal, to induce cytotoxicity, sister-chromatid exchanges (SCEs) and DNA single-strand breaks (SSBs) in V79 cells. Our data demonstrate that this aldehyde is cytotoxic for V79 cells (IC50 = 0.4 mM) and induces SCEs at concentrations ranging from 0.01 to 0.5 mM. DNA SSBs were observed at concentrations ranging from 0.05 to 1 mM and were repaired within 8 h. When V79 cells were cultured with primary hepatocytes, there was a reduction in the frequency of SCEs induced by the aldehyde. This suggests that hepatocytes can partially deactivate the aldehyde. Our results suggest that this aldehyde is one of the reactive intermediates generated during NNK metabolism.