Impact of the hypoxic microenvironment on spermatogonial stem cells in culture.

The stem cell niche plays a crucial role in the decision to either self-renew or differentiate. Recent observations lead to the hypothesis that O2 supply by blood and local O2 tension could be key components of the testicular niche of spermatogonial stem cells (SSCs). In this study, we investigated the impact of different hypoxic conditions (3.5%, 1%, and 0.1% O2 tension) on murine and human SSCs in culture. We observed a deleterious effect of severe hypoxia (1% O2 and 0.1% O2) on the capacity of murine SSCs to form germ cell clusters when plated at low density. Severe effects on SSCs proliferation occur at an O2 tension ≤1% and hypoxia was shown to induce a slight differentiation bias under 1% and 0.1% O2 conditions. Exposure to hypoxia did not appear to change the mitochondrial mass and the potential of membrane of mitochondria in SSCs, but induced the generation of mitochondrial ROS at 3.5% and 1% O2. In 3.5% O2 conditions, the capacity of SSCs to form colonies was maintained at the level of 21% O2 at low cell density, but it was impossible to amplify and maintain stem cell number in high cell density culture. In addition, we observed that 3.5% hypoxia did not improve the maintenance and propagation of human SSCs. Finally, our data tend to show that the transcription factors HIF-1α and HIF-2α are not involved in the SSCs cell autonomous response to hypoxia.

Loss of the histone chaperone ASF1B reduces female reproductive capacity in mice.

Anti-silencing function 1 (ASF1) is an evolutionarily conserved histone H3-H4 chaperone involved in the assembly/disassembly of nucleosome and histone modification. Two paralogous genes, Asf1a and Asf1b, exist in the mouse genome. Asf1a is ubiquitously expressed and its loss causes embryonic lethality. Conversely, Asf1b expression is more restricted and has been less studied. To determine the in vivo function of Asf1b, we generated a Asf1b-deficient mouse line (Asf1b(GT(ROSA-ßgeo)437)) in which expression of the lacZ reporter gene is driven by the Asf1b promoter. Analysis of ß-galactosidase activity at early embryonic stages indicated a correlation between Asf1b expression and cell differentiation potential. In the gonads of both male and female, Asf1b expression was specifically detected in the germ cell lineage with a peak expression correlated with meiosis. The viability of Asf1b-null mice suggests that Asf1b is dispensable for mouse development. However, these mice showed reduced reproductive capacity compared with wild-type controls. We present evidence that the timing of meiotic entry and the subsequent gonad development are affected more severely in Asf1b-null female mice than in male mice. In female mice, in addition to subfertility related to altered gamete formation, variable defects compromising the development and/or survival of their offspring were also observed. Altogether, our data indicate the importance of Asf1b expression at the time of meiotic entry, suggesting that chromatin modifications may play a central role in this process.

Caspase-independent death of meiotic and postmeiotic cells overexpressing p53: calpain involvement.

In a model of male sterility (MTp53) owing to enforced p53 expression in spermatocytes II and spermatids of transgenic mice, we focused on the role of caspases. Most of them are expressed in all differentiation stages, but only the transcriptional levels of caspase-2 and caspase-3 are modified in MTp53 germ cells. In normal testis, cleaved caspase-3 and caspase-9 are detected during the elongation of spermatids. Despite this constitutive presence of caspases during terminal differentiation, calpains are the main effectors of germ cell loss in MTp53 testes: calpain 1 RNA levels are increased, caspase-3-like activity is markedly decreased while calpain activity is higher and the calpain inhibitor E64d ((2S, 3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester) reduces TUNEL labeling in MTp53 testis, whereas pancaspase inhibitor zVADfmk (N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone) has no effect. Our work suggests that despite the presence, and potent involvement, of caspases in male haploid cell maturation, calpains are the executioners of the death of terminally differentiating germ cells.

Non-epileptic seizures: delayed diagnosis in patients presenting with electroencephalographic (EEG) or clinical signs of epileptic seizures.

The clinical differentiation between epileptic seizures (ES) and non-epileptic seizures (NES) is often difficult and mostly based on the presence or absence of widely recognized features of ES such as tongue biting, falling, incontinence or concomitant epileptic abnormalities in the electroencephalogram (EEG). We retrospectively analysed the records of all patients referred to our Epilepsy Centre for refractory epilepsy and finally diagnosed with NES between 1980 and 1999 ( n= 103), half of them also exhibiting ES. The mean time-lapse between first attack and NES diagnosis was 8.7 +/- 1.3 years and 16.5 +/- 1.4 years for the NES and NES + ES groups respectively. At least one of the usual signs associated with generalized tonic-clonic seizures (tongue biting, falling or incontinence) was reported by 66% and 60% of patients with NES or NES + ES respectively. Interictal EEG abnormalities were recorded in 16% of NES patients vs. 80% of NES + ES patients. In the NES group, delay before establishing the correct diagnosis was significantly longer when the patients exhibited > or =1 symptom(s) of generalized seizures, or when patients exhibited interictal EEG abnormalities. Upon admission, 72% of NES patients and all NES + ES patients were being treated with antiepileptic drugs (AEDs).We conclude that EEG or clinical abnormalities suggestive of epileptic seizures are common in undiagnosed NES patients. Such diagnostic pitfalls, besides considerably delaying NES diagnosis, also considerably delay appropriate treatment implementation.

Apoptosis inhibition mediated by medroxyprogesterone acetate treatment of breast cancer cell lines.

Several reports suggested that steroidogenic hormones could be directly involved in the regulation of apoptosis in vitro, but whether this is due to blocking or promoting mechanism of these hormones remains controversial. However, it was shown that progesterone exhibited a protective effect against the apoptotic process during mouse mammary gland involution in vivo. In this study, we analyzed the effect of medroxyprogesterone acetate (MPA) treatment, an agonist of progesterone, on serum starvation induced apoptosis on breast cancer cell lines. Positive and negative progesterone receptor (PgR+ and PgR-) breast cancer cell lines were treated with MPA (10 nM), either in standard culture conditions or in serum-free medium to induce apoptosis. Cell survival, proliferation and apoptosis were simultaneously analyzed with the expression of apoptosis-related genes measured by a real time quantitative RT-PCR. At non cytotoxic doses, MPA protected PgR+ T47-D, MCF-7 and H466-B cell lines against serum depletion-induced apoptosis, while MPA did not protect PgR-MDA-MB-231 cells against serum depletion induced apoptosis. In PgR+ cell lines and in concordance with the protective effect, the pro-apoptotic HRK and BAK1 mRNAs were up-regulated after apoptosis induction, while they were no more induced in condition of protection against apoptosis after MPA treatment. We also observed, specifically in PgR+ cells, an up-regulation of BCLX-L and BCLX-S and a down-regulation of BCL2 mRNAs, which are specific to the MPA response and unrelated to apoptotic process. Involvement of these genes with regard to the MPA-mediated protection against apoptosis is discussed.

Comparative karyotype using bidirectional chromosome painting: how and why?

Rat is widely used in biomedical and pharmaceutical research but its genome has been significantly less studied than that of the mouse. This represents a major limitation for studying cytogenetic and molecular mechanisms in the rat model. As Muridae species underwent an intense chromosome evolution it is not possible to directly transpose knowledge of the mouse genome to that of the rat. For establishing a comparative karyotype between rat and mouse, painting probes of both species were prepared by PARM-PCR (Priming Authorizing Random Mismatches PCR) from a low copy number of sorted chromosomes, the mouse and rat specific painting probes being then hybridized on rat and mouse metaphases, respectively. The availability of rodent species chromosome painting probes as well as the information obtained by the comparative karyotype and comparative gene mapping data are of great interest to improve knowledge on species evolution but also to better understand carcinogenesis process, as illustrated by our data concerning the cytogenetic characterization of radon-induced rat lung tumors. Detailed methods for obtaining painting probes by PARM-PCR from sorted mouse and rat chromosomes and for their hybridization in homologous or heterologous conditions are described. Usefulness of chromosome painting is illustrated by the characterization of chromosomal abnormalities in a radon-induced rat lung tumor. Advantages and limitations of this technique as compared to classical cytogenetics, FISH and CGH are discussed.

Comparative karyotype of rat and mouse using bidirectional chromosome painting.

A comparative karyotype of rat (Rattus norvegicus) and mouse (Mus musculus) based on chromosome G-banding morphology, heterologous chromosome painting results and available gene mapping data is proposed. Whole chromosome painting probes from both species were generated by PARM-PCR amplification of flow sorted chromosomes. Bidirectional chromosome painting identifies 36 segments of syntenic homology and allows us to propose a nearly complete comparative karyotype of mouse and rat (except for RNO 13 p and RNO 19 p12-13). Seven segments completely covered the RNO chromosomes 3, 5, 8, 11, 12, 15 and 18. Eight segments completely covered the MMU chromosomes 3, 4, 6, 7, 9, 12, 18 and 19. The RNO chromosomes 5, 8, 18 show complete homology with the MMU chromosomes 4, 9 and 18, respectively. Bidirectional hybridization results clearly assign 16 segments to subchromosomal regions in both species. Interpretation of the results allows subchromosomal assignment of all the remaining segments apart from seven distributed on chromosomes MMU 15, MMU 10 B2-D3 and MMU 17 E3-E5. The proposed comparative karyotype shows overall agreement with available comparative mapping data. The proposed repartition of syntenic homologous segments between the two species provides useful data for gene-mapping studies. In addition, these results will enable the reconstruction of the karyotype of the Cricetidae and Muridae common ancestor and the definition of the precise rearrangements which have occurred in both mouse and rat lineages during evolution.

Fine deletion mapping of chromosome 8p in non-small-cell lung carcinoma.

Several somatic genetic alterations have been described in non-small-cell lung carcinomas (NSCLC). Recurrent chromosomal deletions have suggested the presence of tumor-suppressor genes specifically involved in lung carcinogenesis. For one of these, 2 non-overlapping regions have been proposed on the short arm of chromosome 8, encompassing the LPL and NEFL genes. The LPL region has been extensively studied in NSCLC and other cancer types. Two genes, N33 and PRLTS, have been identified, but the small number of mutations excludes their involvement in the vast majority of tumors. In order to delineate a reliable region of deletional overlap on chromosome 8p in NSCLC, a series of 77 NSCLC was studied for 34 microsatellite polymorphisms distributed on chromosome 8p, using multiplex-PCR amplification. After purification of tumor nuclei by flow cytometry based on either the abnormal DNA index or the presence of a high expression of cytokeratin, allelic losses on chromosome 8p were observed in 39% of cases. Measurement of DNA index showed that 62% of tumors were hyperploid; allelic losses were more frequent in hyperploid than in diploid tumors (54% vs. 14%; p < 10(-4)). Deletions of part of the short arm were observed in 7 instances. Our data allow definition of an interval of common deletion, flanked by the loci D8S511 and D8S1992, where the putative tumor-suppressor gene might be localized.

Deletion mapping of the tumor suppressor locus involved in colorectal cancer on chromosome band 8p21.

Several somatic genetic alterations have been described in colorectal carcinoma (CRC). Recurrent chromosomal deletions have suggested the presence of tumor suppressor genes (TSG) specifically involved in colorectal carcinogenesis. For one of them, two non-overlapping regions have been proposed on the short arm of chromosome 8, encompassing the LPL and NEFL genes. The short arm of chromosome 8 has been extensively studied in colorectal cancer and in other cancer types. Both regions have been reported as candidate loci for a TSG. In order to delineate a reliable region of deletional overlap on chromosome arm 8p in CRC, a series of 365 CRC samples was selected for the absence of microsatellite instability (RER, replication error); tumor and normal matched DNAs were studied for 54 microsatellite polymorphisms distributed on 8p using multiplex-PCR amplification. After purification of tumor nuclei by flow cytometry based on either the abnormal DNA index or the presence of a high expression of cytokeratin, complete allelic losses on 8p were observed in 48% of cases. Measurement of the DNA index showed that 88% of RER tumors were hyperploid. Complete allelic losses of only part of the short arm were observed on 26 occasions. These data allowed us to define a 1 cM interval of common deletion, flanked by the loci D8S1771 and NEFL, where a putative TSG may be localized.

Concerted nonsyntenic allelic losses in hyperploid hepatocellular carcinoma as determined by a high-resolution allelotype.

Although the occurrence of loss of genetic material in hepatocellular carcinoma (HCC) has been documented both by cytogenetic analysis and by monitoring of allelic losses, a global overview of the extent and frequency of deletion occurring throughout the genome is not yet available. To contribute to this information, DNAs extracted from flow-sorted aneuploid nuclei from HCC and matched normal DNAs were typed for 275 microsatellite loci that were distributed along the autosomes. An average of 190 (69%) informative loci per case were generated on 48 HCC. Complete loss of heterozygozity in the tumor DNA was observed for 15.6% of the typed loci. The chromosome segments that were most frequently affected by deletion were: 8p (60%), 17p (48%), 1p (44%), 4q (42%), 16p (40%), 16q (39%), 6q (35%), 9p (30%), and 13q (29%). On average, 8 of the 39 chromosome segments studied per tumor carried at least one locus that demonstrated loss of heterozygosity (ie., the fractional allelic loss was 0.21). Groups of concerted nonsyntenic losses were observed for 16p and 1p and for 16p and 4q. The location of putative tumor suppressor genes on the most frequently deleted regions was confirmed and, in some cases, refined.

[Localization of a tumor suppressor gene distal to D22S270 in colorectal cancers]. / Localisation d'un gène suppresseur de tumeur distal à D22S270 dans les cancers colorectaux.

Recurrent allelic losses on chromosome 22q have been reported in colorectal cancer, distal to the NF2 gene, suggesting that another tumor suppressor gene might be involved. We report here the typing of 256 sporadic colorectal tumors and 18 colonic cancer cell lines using a set of chromosome 22 polymorphisms, ranging from 20 to 45. A panel of somatic cell hybrids, that allows to distinguish 11 bins in the 22q13 region, was used to localize 19 of the 45 selected markers and the putative tumor suppressor gene BZRP. Allelic-loss was observed in 43% of tumors. The minimal region of deletion that could be determined, telomeric to locus D22S270, refines significantly the position of the gene. The localization of the BZRP gene in this region led to a systematic screening for somatic point mutation. Direct sequencing of its coding sequence in 36 tumors hemizygous for chromosome 22 allowed the identification of three polymorphisms but failed to detect somatic mutation.

Interleukin 1 beta, interleukin 6, tumor necrosis factor alpha, and interleukin 10 responses in peripheral blood mononuclear cells of cynomolgus macaques during acute infection with SIVmac251.

HIV infection ultimately leads to AIDS, despite the immune responses elicited soon after infection. In addition to the observed changes in lymphoid cell subsets, alteration of the cytokine network most likely accompanies and/or contributes to the lack of protective immune responses. In an attempt to delineate the early events in the immune response to lentivirus infection, we have sequentially monitored levels of proinflammatory (IL-1 beta, IL-6, and TNF-alpha) and antiinflammatory (IL-10) cytokine mRNAs in PBMCs of cynomolgus macaques during primary SIVmac infection. Eight monkeys were infected i.v. with 4 AID50 of cell-free SIVmac251. All monkeys seroconverted between days 16 and 21 postinfection (p.i.), and had detectable peripheral viremia. The viral load peaked between days 12 and 16 p.i., and fell sharply thereafter. A marked increase in the expression of IL-6 mRNA was observed in all macaques during the first weeks following infection. An increase in the levels of expression of IL-1 beta, TNF-alpha, and IL-10 mRNA was also determined in six, six, and five of the eight monkeys, respectively. While IL-6, TNF-alpha, and IL-10 increased transiently, increased levels of IL-1 beta mRNA expression were sustained over 44 days in most monkeys.

Flow cytometry studies of recombinant Escherichia coli in batch and continuous cultures: DNA and RNA contents; light-scatter parameters.

Flow cytometry has been used to study the contents of macromolecular compounds and light-scatter parameters in batch and continuous cultures of a recombinant Escherichia coli strain that forms protein inclusion bodies. Changes in relative DNA and RNA contents and cell mass as estimated by forward-angle light scatter were detected and tightly correlated in batch culture. In addition, heterogeneity of wide-angle light scatter (WALS), which we related to the presence of cellular inclusion bodies, was observed. In contrast, the relative RNA content and cell mass did not change during continuous culture, and homogeneity of WALS was found. In addition, unexpected changes in relative DNA content were observed after 67 h of culture, indicating a change in bacterial physiology.

Recent advances of flow cytometry in fundamental and applied microbiology.

This review focuses on the recent applications of flow cytometry (FCM) in microbiological research (1987-mid 1992). It tries to give a scope of the important breakthroughs which occurred in this field during this period. The technical difficulties of microorganism analysis by flow cytometry is briefly appraised. The significance and the limits of the different microbial cell parameters attainable by flow analyses are systematically evaluated: light scatter for cell size and structure, fluorescence measurements for quantification of cellular components, microbial antigen detection and cell physiological activity estimation. Emphasis is given on the new technological advances which appeared in the last two years. The second part of the review is devoted to the analysis of the usefulness of flow cytometric approach in the different fields of microbiology: fundamental studies in microbial physiology, differentiation, microbial ecology and aquatic sciences, medical microbiology, parasitology, microbial pharmacology and biotechnology.

Swine chromosomal DNA quantification by bivariate flow karyotyping and karyotype interpretation.

Human and swine chromosomes were analyzed separately and as a mix to obtain bivariate flow karyotypes. They were normalized to each other in order to use the human chromosomal DNA content as standard. Our results led to the characterization of the "DNA line" in swine identical to the human "DNA line." Estimation of the DNA content in mega-base pairs of the swine chromosomes is proposed. Chromosomal assignment to the various resolved peaks on the bivariate swine flow karyotype is suggested from the relation between DNA content quantified by flow cytometry and chromosomal size. Swine chromosomes 1, 13, 6, 5, 10, 16, 11, 18, and Y were assigned to peaks A, B, C, K, L, N, O, Q, and Y, respectively. Peaks D and E were assumed to contain chromosomes 2 and 14, but without specific assignment. Similarly, P and M peaks were expected to correspond to chromosomes 12 and 17. Of the remaining chromosomes (3, 7, X, 8, 15, 9, and 4), chromosomes 3, 7, and X, which were assigned previously to peaks F, G, and H, respectively, led us to deduce that chromosomes 15 and 8 belonged to peaks I and J, and chromosomes 9, 4, and X to peak H.