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The identification and physical isolation of testis stem cells, a subset of type A spermatogonia, is critical to our understanding of their growth regulation during the first steps of spermatogenesis. These stem cells remain poorly characterized because of the paucity of specific molecular markers that permit us to distinguish them from other germ cells. Thus, the molecular mechanisms driving the first steps of spermatogenesis are still unknown. We show in the present study that GFR alpha-1, the receptor for GDNF (glial cell line-derived neurotrophic factor), is strongly expressed by a subset of type A spermatogonia in the basal part of the seminiferous epithelium. Using this characteristic, we devised a method to specifically isolate these GFR alpha-1-positive cells from immature mouse testes. The isolated cells express Ret, a tyrosine kinase transmembrane receptor that mediates the intracellular response to GDNF via GFR alpha-1. After stimulation with rGDNF, the isolated cells proliferated in culture and underwent the first steps of germ cell differentiation. Microarray analysis revealed that GDNF induces the differential expression of a total of 1124 genes. Among the genes upregulated by GDNF were many genes involved in early mammalian development, differentiation, and the cell cycle. This report describes the first isolation of a pure population of GFR alpha-1-positive cells in the testis and identifies signaling pathways that may play a crucial role in maintaining germ-line stem cell proliferation and/or renewal. Show less

In this paper review of chromatographic techniques and detectors used to analyse nicotine and its metabolites has been presented. The chromatographic determination of xenobiotics in biological material is usually proceeded by multistage isolation procedures, using liquid-liquid and liquid-solid extraction in various modifications. In our investigations, planar chromatography with densitometry has been used to identify and quantify nicotine and its metabolites in body fluids for assessment of tobacco smoke exposure. This technique does not require special cleanup techniques and as well is economic and environmental friendly. Show less

In order to estimate the exposure of passive and active smokers to tobacco smoke one can use the questionnaire method or laboratory examination of chemical compounds being widely accepted exposure biomarkers. Substances that make such biomarkers include some of the tobacco smoke components and its metabolites formed in the body. The study discusses two groups of biomarkers. First, includes substances that serve as exposure markers of carcinogenous properties (metabolites of polycyclic aromatic hydrocarbons, N-nitrosamines, trans,transmuconic acid, S-phenylmercapturic acid). Second group includes substances which role is limited to the evaluation of exposure to tobacco smoke (nicotine, cotinine, anatabine, anabasine, trans-3'-hydroxycotinine, thiocyanate, carboxyhemoglobin, carbon monoxide). Sensitivity and specificity of biomakers used were evaluated, their concentration ranges in physiological fluids in non-smokers, passive-, and active smokers. The simplicity of the examination method was evaluated. Articles published during last two decades indicate that the substance that have all features that make it the most appropriate biomarker is cotinine. It can be assessed in plasma and in urine of smokers and persons exposed to environmental tobacco smoke. Show less

Taking the tobacco plant will cause close 450 millions of deaths worldwide within 50 closest years. The numerous epidemiological research is pointing that exposing children to the tobacco smoke is involving with more frequent occurrence of lower airways and middle ear diseases, asthma or SIDS (sudden infant death syndrome). Exposing in the childhood can involve the development of cancer also in adult patients. Current epidemiological notifications are pointing that the 56% of children and the 24% of pregnant women are exposed to passive smoking. 27% of women breast-feeding and the 15-20% of pregnant women are smoking (35-40% of unemployed women with the low degree of education). 100,000 of Polish newborns are having the symptoms of poisoning with the tobacco smoke, and 4 millions are exposed to passive smoking. Exposure of the home environment is inversely proportional to child's age - the younger child the longer time of exposure to environmental factors that affecting state of health negatively. It is mentioning as main of home environment factors the exposure to the tobacco smoke (mothers made conditional on the nicotine). Show less

The aim in this survey was analysis of contribution of exposure to tobacco smoke and selected socioeconomic factors in occurrence of low birth weight. The research was done on the group of 1528 pregnant women. Smoking habit was declared by 18.2% of women and passive exposure to tobacco smoke was stated by 31% of respondents. Significant association between active and passive smoking and socioeconomic status of respondents was observed. Cigarette smoking during pregnancy was the main cause of lower birth weight of newborns. The newborns of actively smoking mothers were lighter of about 332 g according to those ones of mothers who did not smoke and were not exposed to passive smoking, and were lighter than newborns of mothers who were not exposed to passive smoking of about 342 g. Show less

Sudden infant death syndrome (SIDS) means the sudden death of an infant which is unexpected by history and in which an examination of the death scene and a thorough postmortem examination fails to reveal an adequate cause of death. Etiology of this syndrome is unknown but many risk factors were identified; the most important and preventable risk factors are prone sleeping position and influence of tobacco smoke (both smoking by pregnant woman and environmental tobacco smoke (ETS) near child's bed). The aim of this study was to assess the prevalence of smoking among women (also during pregnancy) and exposure of neonates and infants to ETS, to establish what circumstances are likely to make smoking woman stop smoking and to examine women's knowledge concerning risk factors of SIDS (mainly influence of tobacco smoke). The study comprised 61 women, aged 24 to 47, among them 35 nurses and 26 women without any medical education. The average percentage of smoking women was near 25% but among nurses was up to 30%. The prevalence of smoking during pregnancy was 6%. We identified high risk of ETS influence among both women and their children. The most important cause of smoking cessation was pregnancy. Among arguments for smoking cessation the risk of SIDS takes an important place. Women's knowledge about risk factors of SIDS (among them influence of tobacco smoke) and activity of physicians and midwifes to educate women as for this risk factors are unsatisfactory. Show less

In the mammalian testis, the germ line stem cells are a small subpopulation of type A spermatogonia that proliferate and ultimately differentiate into sperm under the control of both endocrine and paracrine factors. To study the early phases of spermatogenesis at the molecular level, an in vitro system must be devised whereby germ line stem cells can be either cultured for a prolonged period of time or expanded as cell lines. In the study reported here, we chose to immortalize type A spermatogonia using the Simian virus large T-antigen gene (LTAg) under the control of an ecdysone-inducible promoter. While the cells escaped the hormonal control after a finite number of generations and expressed the LTAg constitutively, their growth remained slow and the cells exhibited morphological features typical of spermatogonia at the light microscopic level. Moreover, the cells expressed detectable levels of protein markers specific for germ cells such as Dazl, and specific for germ line stem cells such as Oct-4, a transcription factor, and GFRalpha-1, the receptor for glial cell line-derived neurotrophic factor (GDNF). Further analysis confirmed the spermatogonial phenotype and also revealed the expression of markers expressed in stem cells such as Piwi12 and Prame11. Since the cells respond to GDNF by a marked increase in their rate of proliferation, this cell line represents a good in vitro model for studying aspects of mouse germ line stem cell biology. Show less

Protein-protein interactions are essential for life. Yet, our understanding of the general principles governing binding is not complete. In the present study, we show that the interface between proteins is built in a modular fashion; each module is comprised of a number of closely interacting residues, with few interactions between the modules. The boundaries between modules are defined by clustering the contact map of the interface. We show that mutations in one module do not affect residues located in a neighboring module. As a result, the structural and energetic consequences of the deletion of entire modules are surprisingly small. To the contrary, within their module, mutations cause complex energetic and structural consequences. Experimentally, this phenomenon is shown on the interaction between TEM1-beta-lactamase and beta-lactamase inhibitor protein (BLIP) by using multiple-mutant analysis and x-ray crystallography. Replacing an entire module of five interface residues with Ala created a large cavity in the interface, with no effect on the detailed structure of the remaining interface. The modular architecture of binding sites, which resembles human engineering design, greatly simplifies the design of new protein interactions and provides a feasible view of how these interactions evolved. Show less

The aim of this study was to find out if children's exposure to tobacco smoke in the apartments influences the lead and cadmium content and the values of Pb/Ca and Cd/Ca ratios in hard tissues of their deciduous teeth. Material consisted of 71 shed deciduous teeth; 37 from children in the age of 6 to 13 years additionally exposed to tobacco smoke in the apartments and 34 from children in the same age, whose apartments were free of tobacco smoke. Lead, cadmium and calcium content was determined by means of flame atomic absorption spectrometry. Deciduous teeth of children additionally exposed to tobacco smoke in the apartments exhibited higher lead and cadmium content and higher values of Pb/Ca and Cd/Ca ratios, which indicated significant accumulation of these metals, when compared to deciduous teeth of children whose apartments were free of tobacco smoke. Show less

Addiction to tobacco smoking is very popular among young and middle age people in Poland, who are parents of pre-school children as well. Respiratory tract diseases are the main reason of visits of pre-school children in General Practice surgery. Anonymous questionnaires were given to the random sample of mothers of three years old children. 311 respondents were chosen from whole population of 1200 children attending kindergartens in Białystok. The questionnaires showed that half of the examined children are involuntary exposed to tobacco smoke at home 20% also outside home (neighbors, homes and, cafeterias, cars etc). We did not find any relationship between exposure to smoke and occurrence of respiratory tract infection in children. We presume that this may be apparently the result of the fact that many smoking parents try to not to smoke in the presence of their children. Increasing awareness of harm resulting from passive smoking is the benefit of educational campaigns in Poland. Show less

The aim of the study was to estimate both active and passive pregnant women exposure to tobacco smoke. 154 women hospitalized at the Gynaecology and Obstetrics Department in Opole, Poland and at the Gynaecology and Obstetrics Department of Hospital Universitario Monterey in Mexico have undergone the examination. The patients have been surveyed in order to obtain a subjective evaluation of their exposure. Then the exhaled carbon monoxide (ExhCO) measurement used for objective monitoring tobacco smoking has been performed. According to the results of the questionnaire 40 women (26% of the examined patients) confirmed smoking in pregnancy. 24 women (60% of smoking ones) have been smoking for the whole period of pregnancy. Though the rate of smoking among Polish and Mexican women was comparable, Polish mothers were less likely to quit after confirmation of pregnancy. Both Polish and Mexican smoking mothers were much younger than their non-smoking counterparts. The babies of the smoking mothers have been noticed to obtain the lower count in Apgar scale. The exhaled carbon monoxide (ExhCO) measurement has revealed that 14 Polish and 10 Mexican patients have been exposed to tobacco smoke. The results of their questionnaires have been thoroughly examined. 71% of them confirmed active smoking in pregnancy, 8.3% pointed to second-hand smoke exposure, 20.7% lied by denying the exposure. Thus the need to verify the questionnaires results has been confirmed. The mean value of ExhCO of both non-smoking women and those, who quit smoking after confirmation of pregnancy was equal. This may suggest that quitting smoking in the early stages of pregnancy reduce the risk of the harmful smoke impact on the unborn baby. Nearly doubled average concentration of ExhCO among passive smoking mothers in comparison to the non-smoking ones arises a serious concern. Show less

To investigate the effect of curcumin on tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in rat cortical neurons and to explore the possible mechanism. Primary cultured rat cortical neurons were performed in vitro and cell viability was measured by MTT assay. DNA fragmentation was used to evaluate cell apoptosis. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (Deltapsim) was determined by flow cytometric assay. Cellular glutathione (GSH) content was measured by spectrophotometer. Bcl-2 family proteins, cytochrome c, cleaved caspase-3, and poly (ADP-ribose) polymerase (PARP) were detected by Western blot. Exposure of tBHP 100 micromol/L to neurons for 60 min resulted in DYm loss and cytochrome c release from mitochondria and subsequent activation of caspase-3 and PARP cleavation, and cell apoptosis. After removal of tBHP and then further treatment with curcumin (2.5-20 micromol/L) for 18 h, curcumin abrogated Deltapsim loss and cytochrome c release, blocked activation of caspase 3, and altered the expression of Bcl-2 family. Further curcumin treatment also prevented cellular GSH and decreased intracellular ROS generation markedly. Curcumin eventually attenuated tBHP-induced apoptosis in cortical neurons. Curcumin may attenuate oxidative damages in cortical neurons by reducing intracellular production of ROS and protecting mitochondria from oxidative damage. Show less

According to the current data over 12 million Polish citizens smoke tobacco, 40% of them are women. Maternal smoking in pregnancy significantly increases the rate of having a low-birthweight baby, the rate of perinatal mortality and several other adverse pregnancy outcomes. The aim of the study was to estimate the rate of smoking women during pregnancy and those who are exposed to secondhand smoke. 219 women hospitalized at Gynecology and Obstetrics Department of Wroclaw Medical University and Gynecology and Obstetrics Department in Opole have undergone the examination. The pregnant women have been interviewed and the results of questionnaire have been checked by the exhaled carbon monoxide (ExhCO) measurement. CO expresses the percentage of carboxyhemoglobin (HbCO) and is used for objective monitoring tobacco smoking. The results of the study suggest that regular exposure to secondhand smoke becomes a serious problem. 25.5% patients from Wrocław and 16.4% patients from Opole have declared passive smoking. The mean value of ExhCO in this group of women obtained the level of 3.3 ppm vs 1.95 ppm in the group of patients who have denied both active and passive exposure. Show less

A functionally important, interface domain between transmembrane segments (TMSs) IV and XI of the NhaA Na+/H+ antiporter of Escherichia coli has been unraveled. Scanning by single Cys replacements identified new mutations (F136C, G125C, and A137C) that cluster in one face of TMS IV and increase dramatically the Km of the antiporter. Whereas G125C, in addition, causes a drastic alkaline shift to the pH dependence of the antiporter, G338C alleviates the pH control of NhaA. Scanning by double Cys replacements (21 pairs of one replacement per TMS) identified genetically eight pairs of residues that showed very strong negative complementation. Cross-linking of the double mutants identified six double mutants (T132C/G338C, D133C/G338C, F136C/S342C, T132C/S342C, A137C/S342C, and A137C/G338C) of which pronounced intramolecular cross-linking defined an interface domain between the two TMSs. Remarkably, cross-linking by a short and rigid reagent (N,N'-o-phenylenedimaleimide) revived the Li+/H+ antiport activity, whereas a shorter reagent (1,2-ethanediyl bismethanethiosulfonate) revived both Na+/H+ and Li+/H+ antiporter activities and even the pH response of the dead mutant T132C/G338C. Hence, cross-linking at this position restores an active conformation of NhaA. Show less

Dyggve-Melchior-Clausen (DMC) is a rare autosomal-recessive disorder characterized by the association of a progressive spondylo-epi-metaphyseal dysplasia and mental retardation ranging from mild to severe. Electron microscopy studies of both DMC chondrocytes and fibroblasts reveal an enlarged endoplasmic reticulum network and a large number of intracytoplasmic membranous vesicles, suggesting that DMC syndrome may be a storage disorder. Indeed, DMC phenotype is often compared to that of type IV mucopolysaccharidosis (Morquio disease), a lysosomal disorder due to either N-acetylgalactosamine-6-sulphatase or beta-galactosidase deficiency. To date, however, the lysosomal pathway appears normal in DMC patients and biochemical analyses failed to reveal any enzymatic deficiency or accumulated substrate. Linkage studies using homozygosity mapping have led to the localization of the disease-causing gene on chromosome 18q21.1. The gene was recently identified as a novel transcript (Dym) encoding a 669-amino acid product (Dymeclin) with no known domains or function. Sixteen different Dym mutations have now been described in 21 unrelated families with at least five founder effects in Morocco, Lebanon, and Guam Island. Smith-MacCort syndrome (SMC), a rare variant of DMC syndrome without mental retardation, was shown to be allelic of DMC syndrome and to result from mutations in Dym that would be less deleterious to the brain. The present review focuses on clinical, radiological, and cellular features and evolution of DMC/SMC syndromes and discusses them with regard to identified Dym mutations and possible roles of the Dym gene product. Show less

Despite of significant progress in the area of perinatal care, there have been numerous perinatal complications still existing. The health status of mother and newborn is affected by many factors, namely: socio-economical; medical prior to pregnancy and medical through the pregnancy; dietary customs, substance use, including tobacco smoking; health service quality; and risk factors that may occur through fertilization. The active and passive exposure to tobacco smoke in utero have been linked to numerous gestation and delivery complications, including low birth weight, preterm delivery and low Apgar score. In this survey, the awareness of health consequences of active and passive smoking in the population of pregnant women and access to the information of this consequence was assessed. Among of women smoking through their pregnancies, the only 19.7% got the information from their doctors about harmful effect of active and passive smoking, and 16.5% of respondents declared self-awareness on health effects of exposure to the tobacco smoke. Show less

The transcriptomes of mouse type A spermatogonia (Spga), pachytene spermatocytes (Spcy), and round spermatids (Sptd) were determined by sequencing the respective SAGE (Serial Analysis of Gene Expression) libraries. A total of 444,015 tags derived from one Spga, two Spcy, and one Sptd library were analyzed, and 34,619 different species of transcripts were identified, 5279 of which were novel. Results indicated the germ-cell transcriptome comprises of more than 30,000 transcripts. Virtual subtraction showed that cell-specific transcripts constitute 12-19.5% of the transcriptome. Components of the protein biosynthetic machinery are highly expressed in Spga. In Spcy transcription factors are abundantly expressed while transcripts encoding proteins involved in chromosome remodeling and testis-specific transcripts are prominent in Sptd. The databases generated by this work provide very useful resources for cellular localization of genes in silico. They are also extremely useful as sources for identification of splice variants of genes in germ cells. Show less

Inflammatory myofibroblastic tumor is a rare clinical entity that can first present in the oral cavity. Though considered a benign lesion, it can behave very locally and aggressively, with significant bony destruction. A rare case of inflammatory myofibroblastic tumor is presented, along with the surgical treatment and an overview of this interesting clinical lesion. Show less

The incidence of laryngeal cancer is strongly connected with exposure to tobacco smoke containing at least 40 genotoxic carcinogens. DNA lesions induced by tobacco smoke carcinogens can be turned into stable mutations. Oncogenes and tumor suppressor genes are first of all responsible for initiation of carcinogenesis. The same time cells provide such self-protection processes as: carcinogen detoxication, DNA repair and apoptosis. The initiation of carcinogenesis followed by oncogenesis results from the competition between DNA lesions/mutations formation and the protection processes; the latter are genetically controlled. Show less

Among other toxic compounds, tobacco smoke contains also heavy metals, for example lead. Lead can cross the placenta barrier and have negative influences on fetus development. In the study compared urine lead concentration of pregnancy women exposed to tobacco smoke (active smokers and exposed to environmental tobacco smoke--ETS) with unexposed. There was find that mean urine lead concentrations in groups of pregnant women exposed and unexposed to tobacco smoke was no different (F2,189=0.2 p>0.8). However it was found that in urine of women exposed to tobacco smoke the concentration of lead increase with increase the concentration of cotinine (r2=0.14, t106=4.2, p<<0.0001). Show less

In this prospective study different semen parameters of fertile smokers were compared with fertile non-smokers. 58 men with proven fertility (17 smokers and 41 non-smokers) at the same age were evaluated. Smokers had significantly less spermatozoa with motility grade A (p<0.01), B (p<0.05), and C (p<0.05), and more abnormal forms in the ejaculate (p< 0.001). The percentage of sperm with head defects (p<0.05) and cytoplasmic droplets (p<0.001) were also significantly higher in smokers. No differences were found in the assessment of semen volume, total sperm count, hypoosmotic swelling test, induced acrosomal reaction and acidic aniline blue staining test between two groups (p>0.05). However the concentration of leukocytes in semen was significantly higher in smokers (p<0.001), which can suggests oxidative imbalance in the ejaculates of these men. Our data demonstrate that cigarette smoking is associated with reduced semen quality which could worsen fertilizing potential in fertile men. Show less

Passive smoking results from involuntary smoke inhalation in the environment where tobacco and other tobacco products are smoked. This is a common situation in numerous public objects, as restaurants, offices, dormitories and students' clubs. Non-smokers are exposed to very high concentrations of tobacco smoke in these places. Environmental exposure to tobacco smoke of the inhabitants of the Pharmacy School dormitory in Sosnowiec was studied. The evaluation was based on urine cotinine concentration measured with the HPLC method. Additionally, a Fagerström test was conducted among declared smokers in order to evaluate the degree of their pharmacologic addiction to nicotine. The obtained results indicate that non-smoking dormitory inhabitants are strongly exposed to environmental tobacco smoke, but active smokers did not presented with pharmacological addiction to nicotine. Show less

Complementary DNA microarray and quantitative polymerase chain reaction were used as tools for discovering genes that are differentially expressed in the mouse under normal physiological conditions at distinctive stages of male germ cell development, that is, type A spermatogonia, pachytene spermatocytes, and round spermatids. By using this strategy, we identified a set of genes exhibiting differential expression patterns in spermatogenesis, suggesting that specific functions of the encoded products occurred during the developmental process. Among them were several genes previously not known to be active in testis, which signified undiscovered functional roles of these genes during spermatogenesis. Many of the genes identified were not previously characterized. This study highlights new targets for manipulation to unravel the molecular mechanism of spermatogenesis. Show less

Buccal cellulitis resulting from Haemophilus Influenzae type B (Hlb) is an uncommon yet potentially life-threatening illness that afflicts the facial soft tissues of the very young. Early recognition is essential for the effective treatment of this illness. A clinical case of Haemophilus Influenzae buccal cellulitis is presented, accompanied by a discussion of the presenting symptoms, diagnosis and treatment of this unusual childhood infection. Show less

Dyggve-Melchior-Clausen syndrome (DMC) is a rare autosomal-recessive disorder, the gene for which maps to chromosome 18q21.1. DMC is characterized by the association of a spondylo-epi-metaphyseal dysplasia and mental retardation. Electron microscopic study of cutaneous cells of an affected child showed dilated rough endoplasmic reticulum, enlarged and aberrant vacuoles and numerous vesicles. As the etiology of the disorder is unknown, we have used a positional cloning strategy to identify the DMC gene. We detected seven deleterious mutations within a gene predicted from a human transcript (FLJ20071) in 10 DMC families. The mutations were nonsense mutations (R194X, R204X, L219X, Q483X), splice site or frameshift mutations (K626N+92aa to stop). The DMC gene transcript is widely distributed but appears abundant in chondrocytes and fetal brain. The predicted protein product of the DMC gene yields little insight into its likely function, showing no significant homology to any known protein family. However, the carboxy terminal end comprises a cluster of dileucine motifs, highly conserved across species. We conclude that DMC syndrome is consequent upon loss of function of a gene that we propose to name Dymeclin, which may have a role in process of intracellular digestion of proteins. Show less

Using differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) we identified transcripts encoding for the RNA helicase mDEAH9, Ran binding protein 5 (RanBP5), and 3 novel complementary DNAs designated GC3, GC12, and GC14 in developing testicular germ cells. Sources of RNA for the initial DDRT-PCR screen were purified mouse type A spermatogonia, adult mouse wild-type testis, and W/W(v) mutant mouse testis. We identified cDNA fragments for mDEAH9, RanBP5, GC3, GC12, and GC14 in testis and type A spermatogonia samples from wild-type mice, but not in samples from the W/W(v) mouse testis. These same transcripts were absent in Northern blots of testis RNA from mice treated with busulfan 30 days prior, but were present in testis RNA from wild-type mice at 5, 15, 25, and 40 days of age. The mDEAH9 gene was expressed in many tissues, whereas RanBP5 and GC12 genes were expressed predominantly in the testis with much lower expression in other tissues. The expression of GC3 and GC14 were limited to the testis as evidenced by Northern blot and RT-PCR analyses. The mDEAH9 transcript was not detected in cultured interstitial cells but was found at low levels in cultured immature Sertoli cells, whereas the RanBP5, GC3, GC12, and GC14 transcripts were not detected in either cultured testicular interstitial cells or cultured Sertoli cells. RT-PCR analyses of isolated spermatogonia, pachytene spermatocytes, and round spermatids revealed that mDEAH9, RanBP5, GC3, GC12, and GC14 genes were expressed in all 3 cellular populations. In situ hybridization analyses of testis samples from 40-day-old mice localized expression of mDEAH9, RanBP5, GC3, GC12, and GC14 to the seminiferous tubules. RanBP5 expression appeared to be regulated during the cycle of the seminiferous epithelium, with the highest expression in stages III through VII. Expression of GC14 was greatest in the meiotic germ cell populations. Show less

In this study, we examined possible mechanisms of caspase activation during carotenoid-induced apoptosis in tumor cells. We found that beta-Carotene induces apoptosis by the activation of caspase-3 in human leukemia (HL-60), colon adenocarcinoma (HT-29) as well as melanoma (SK-MEL-2) cell lines. This activation is dose dependent and follows that of caspase-8 and caspase-9. Although caspase-8 cleavage is an early event, reaching its maximum activation at 3 h, caspase-9 reaches its maximum activation only at 6 h. The addition of IETD-CHO, a caspase-8-specific inhibitor, completely prevents beta-Carotene-induced apoptosis, whereas only a partial prevention was observed in the presence of LEHD-CHO, a caspase-9-specific inhibitor. beta-Carotene activates caspase-9 via cytochrome c release from mitochondria and loss of mitochondrial membrane potential (Dym). Concomitantly, a dose-dependent decrease in the antiapoptotic protein Bcl-2 and a dose-dependent increase in the cleaved form of BID (t-BID) are observed. Moreover, NF-kB activation is involved in beta-Carotene-induced caspase cascade. These results support a pharmacological role for beta-Carotene as a candidate antitumor agent and show a possible sequence of molecular events by which this molecule may induce apoptosis in tumor cells. Show less

Spermatogenesis is the process of differentiation of diploid type A spermatogonia to haploid spermatozoa. Several subtypes of A spermatogonia have been characterized in the adult mouse testis. These include A-single (A(s)), A-paired (A(pr)), A-aligned (A(al)), and A1-A4. However, in the immature testis, very little information is available on subtypes and morphological features of type A spermatogonia. Six-day-old mouse testes, fixed either in Bouin solution or 5% glutaraldehyde, were embedded in paraffin and Epon, respectively. Thick sections (approximately 1 microm) of Epon-embedded tissue were stained with toluidine blue and revealed three subtypes of spermatogonia by light microscopy. The smallest spermatogonia (subtype I) appeared as single cells and exhibited a round or oval flattened nucleus with one or two prominent dense nucleoli and a characteristic unstained round and centrally located vacuole. These cells bound toluidine blue more avidly and appeared darker in comparison with the other cell types. Electron microscopy of thin sections (90 nm) revealed a finely granulated chromatin homogeneously distributed in the nucleus and sparse organelles in the cytoplasm. The second subtype of spermatogonia (subtype II) also displayed dark staining but was larger than subtype I; there was no central vacuole in the nucleus and heterochromatin clumps were observed. The largest subtype of spermatogonia (subtype III) showed large heterochromatin clumps and a pale staining nucleus. Intercellular bridges were noted between subtypes II and III. Based on the dye avidity, the three subtypes were classified as dark, transitional, and pale spermatogonia, respectively. Image analyses of 30 different cells of each subtype revealed a decline in gray-scale intensity from subtype I to III. Five-micrometer sections of paraffin-embedded tissue were immunoassayed with an antibody against the glial cell-derived neurotrophic factor family receptor alpha-1 (GFRalpha-1) receptor, a putative marker for undifferentiated spermatogonia, showing positive reaction only in germ cells. The pattern of GFRalpha-1 expression, coupled to the overall morphology of the cells, indicates that at this stage of development, mouse seminiferous tubules contain essentially A(s), A(pr), and possibly A(al) spermatogonia. Thus, the present study indicates the presence of subtypes of type A spermatogonia in the immature mouse testis similar to that described previously in adult monkey and man. Show less

SP1 (108 amino acids) is a boiling-stable stress-responsive protein. It has no significant sequence homology to other stress-related proteins or to small heat-shock proteins (sHsps). SP1 activity is ATP-independent, similar to other small heat-shock proteins. Based on these features, it is expected that the structure-function relationship of SP1 will be unique. In this work, the crystallization and preliminary crystallographic data of native SP1 and its selenomethionine derivative are described. Recombinant SP1 and its selenomethionine derivative were expressed in Escherichia coli and used for crystallization experiments. SP1 crystals were grown from 0.1 M HEPES pH 7.5, 20% PEG 3K, 0.2 M NaCl. One to four single crystals appeared in each droplet within a few Days and grew to dimensions of about 0.5 x 0.5 x 0.8 mm after about two weeks. Diffraction studies of these crystals at low temperature indicated that they belong to space group I422, with unit-cell parameters a = 89, b = 89, c = 187 A. Efforts to crystallize the selenomethionine derivative of SP1 are in progress. Show less

Sertoli cells isolated from 6-day postpartum mouse testes were conditionally immortalized with the simian virus 40 large tumor antigen gene (SV40-LTAg) under the control of a promoter inducible with ponasterone A, an analog of ecdysone. This strategy produced 2 cell lines, which exhibited mixed phenotypes. We first tested the conditional expression of the LTAg gene in the presence or absence of ponasterone A. The results showed that both cell lines expressed LTAg when the inducer was present in the culture media. When ponasterone A was removed, the majority of the cells died. After 60 generations, however, the continued expression of LTAg in the absence of the hormone indicated that unknown changes may have occurred in the genome of the cells. One of the cell lines was further subcloned, resulting in 7 new lines exhibiting a morphology resembling that of Sertoli cells in tissue culture. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on RNA collected from each cell line in order to determine which cells were phenotypically similar to Sertoli cells in vivo. All cell lines expressed the products of the Sertoli cell-specific genes stem cell factor (SCF) and sulfated glycoprotein-2 (SGP-2), in addition to alpha-inhibin, GATA-1, and steroidogenic factor-1. Further, the lines express growth and differentiation factors known to act upon germ cells in vivo and in vitro such as leukemia inhibitory factor (LIF), transforming growth factor beta (TGF-beta), and basic fibroblast growth factor (bFGF). Moreover, when used as feeder layers in cocultures, at least 2 of these lines are able to maintain the viability of type A spermatogonia for at least 7 days and to support the first steps of spermatogonial differentiation. Show less