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Although de novo computational enzyme design has been shown to be feasible, the field is still in its infancy: the kinetic parameters of designed enzymes are still orders of magnitude lower than those of naturally occurring ones. Nonetheless, designed enzymes can be improved by directed evolution, as recently exemplified for the designed Kemp eliminase KE07. Random mutagenesis and screening resulted in variants with >200-fold higher catalytic efficiency and provided insights about features missing in the designed enzyme. Here we describe the optimization of KE70, another designed Kemp eliminase. Amino acid substitutions predicted to improve catalysis in design calculations involving extensive backbone sampling were individually tested. Those proven beneficial were combinatorially incorporated into the originally designed KE70 along with random mutations, and the resulting libraries were screened for improved eliminase activity. Nine rounds of mutation and selection resulted in >400-fold improvement in the catalytic efficiency of the original KE70 design, reflected in both higher k(cat) values and lower K(m) values, with the best variants exhibiting k(cat)/K(m) values of >5×10(4) s(-)(1) M(-1). The optimized KE70 variants were characterized structurally and biochemically, providing insights into the origins of the improvements in catalysis. Three primary contributions were identified: first, the reshaping of the active-site cavity to achieve tighter substrate binding; second, the fine-tuning of electrostatics around the catalytic His-Asp dyad; and, third, the stabilization of the active-site dyad in a conformation optimal for catalysis. Show less

Schizophrenia is a disorder that affects a significant portion of the population. This case report and review article discusses potential causes and pathophysiology. The treatment of schizophrenia is outlined in detail, followed by a discussion of the perioperative management of schizophrenic patients, highlighting important information to keep in mind when treating this patient population. Show less

Dyggve-Melchior-Clausen (DMC) syndrome is a rare autosomal recessive disorder characterized by the association of a progressive spondyloepimetaphyseal dysplasia and mental retardation ranging from mild to severe. The disorder results from mutations in the dymeclin (DYM) gene in the 18q12-12.1 chromosomal region. We report two siblings with classical clinical and radiological features of DMC and asymptomatic atlanto-axial dislocation. A novel homozygous splice-site mutation (IVS15+3G>T) was detected. Reverse transcriptase polymerase chain reaction (RT-PCR) confirmed that this mutation affects normal splicing. To the best of our knowledge, this is the first report of DMC from Saudi Arabia. The splice mutation noted in our patients was compared to the previously reported cases and supports the hypothesis that loss of DYM function is the likely mechanism of disease pathogenesis. In conclusion, distinction between this type of skeletal dysplasia and Morquio disease (MPS IV) is important for paediatricians and clinical geneticist in providing standard patient care and genetic counselling. Show less

Understanding enzyme catalysis through the analysis of natural enzymes is a daunting challenge-their active sites are complex and combine numerous interactions and catalytic forces that are finely coordinated. Study of more rudimentary (wo)man-made enzymes provides a unique opportunity for better understanding of enzymatic catalysis. KE07, a computationally designed Kemp eliminase that employs a glutamate side chain as the catalytic base for the critical proton abstraction step and an apolar binding site to guide substrate binding, was optimized by seven rounds of random mutagenesis and selection, resulting in a >200-fold increase in catalytic efficiency. Here, we describe the directed evolution process in detail and the biophysical and crystallographic studies of the designed KE07 and its evolved variants. The optimization of KE07's activity to give a k(cat)/K(M) value of approximately 2600 s(-1) M(-1) and an approximately 10(6)-fold rate acceleration (k(cat)/k(uncat)) involved the incorporation of up to eight mutations. These mutations led to a marked decrease in the overall thermodynamic stability of the evolved KE07s and in the configurational stability of their active sites. We identified two primary contributions of the mutations to KE07's improved activity: (i) the introduction of new salt bridges to correct a mistake in the original design that placed a lysine for leaving-group protonation without consideration of its "quenching" interactions with the catalytic glutamate, and (ii) the tuning of the environment, the pK(a) of the catalytic base, and its interactions with the substrate through the evolution of a network of hydrogen bonds consisting of several charged residues surrounding the active site. Show less

The aim of the study was to analyze the association between prenatal environmental tobacco smoke exposure (ETS) and child anthropometric parameters at birth such birth weight, length, head and chest circumference. The study population consisted of 107 pregnant women from Lodz district as the part of Polish Mother and Child Cohort study. The child prenatal ETS exposure was assessed based on questionnaire with mothers and cotinine level in saliva collected three times in pregnancy. The level of cotinine in biological samples was analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS ESI+). About 35% of the children were prenatally exposed to ETS. The birth weight of the children prenatally exposed to ETS was 335 g lower than the birth weight of non-exposed newborns p < 0.001 after adjustment for: maternal educational level, marital status, prepregnancy weight, child gender, and gestational age. The same refers to child length and chest circumference (coef. -1.1 cm; p = 0.03 and coef. -1.3 cm; p = 0.002 respectively). Taking into account the negative effect of prenatal exposure to ETS, more efforts need to be taken to eliminate child ETS exposure. Show less

Six independent studies have identified linkage to chromosome 18 for developmental dyslexia or general reading ability. Until now, no candidate genes have been identified to explain this linkage. Here, we set out to identify the gene(s) conferring susceptibility by a two stage strategy of linkage and association analysis. Linkage analysis: 264 UK families and 155 US families each containing at least one child diagnosed with dyslexia were genotyped with a dense set of microsatellite markers on chromosome 18. Association analysis: Using a discovery sample of 187 UK families, nearly 3000 SNPs were genotyped across the chromosome 18 dyslexia susceptibility candidate region. Following association analysis, the top ranking SNPs were then genotyped in the remaining samples. The linkage analysis revealed a broad signal that spans approximately 40 Mb from 18p11.2 to 18q12.2. Following the association analysis and subsequent replication attempts, we observed consistent association with the same SNPs in three genes; melanocortin 5 receptor (MC5R), dymeclin (DYM) and neural precursor cell expressed, developmentally down-regulated 4-like (NEDD4L). Along with already published biological evidence, MC5R, DYM and NEDD4L make attractive candidates for dyslexia susceptibility genes. However, further replication and functional studies are still required. Show less

Preference for and intake of solid and emulsified fat (intralipid) solutions vary across different mouse strains. Fat intake in rodents is inhibited by dopamine and opioid receptor antagonists, but any variation in these responses as a function of genetic background is unknown. Therefore, the present study compared the ability of dopamine D1-like (SCH23390) and general opioid (naltrexone) receptor antagonism to alter intake of fat emulsions (intralipid) in mice. Two-hour intakes of 5% intralipid were measured (5-120 min) in seven inbred (BALB/c, C57BL/6, C57BL/10, DBA/2, SJL, SWR, 129P3) and one outbred (CD-1) mouse strains following treatment with vehicle, SCH23390 (50-1600 nmol/kg, ip) and naltrexone (0.001-5 mg/kg, sc). SCH23390 significantly, dose-dependently and differentially reduced intralipid intake at all five (DBA/2, SWR, CD-1), four (SJL, C57BL/6), three (129P3) and one (C57BL/10) of the doses tested, but failed to affect intralipid intake in BALB/c mice. Naltrexone significantly, dose-dependently and differentially reduced intralipid intake at all four (DBA/2), three (SWR, SJL), two (CD-1, C57BL/10) and one (C57BL/6, 129P3) of the doses tested, and also failed to affect intralipid intake in BALB/cJ mice. SCH23390 and naltrexone were respectively 13.3-fold and 9.3-fold more potent in inhibiting intralipid intake in the most sensitive (DBA/2) relative to the least sensitive (BALB/c) mouse strains. A strong positive relationship (r=0.91) was observed for the abilities of SCH23390 and naltrexone to inhibit intralipid intake across strains. These findings indicate that dopaminergic and opioid signaling mechanisms differentially control intralipid intake across different mouse strains, suggesting important genetic and pharmacological interactions in the short-term control of rewarding and post-ingestive consequences of fat intake. Show less

This study was designed to isolate, characterize, and culture human spermatogonia. Using immunohistochemistry on tubule sections, we localized GPR125 to the plasma membrane of a subset of the spermatogonia. Immunohistochemistry also showed that MAGEA4 was expressed in all spermatogonia (A(dark), A(pale), and type B) and possibly preleptotene spermatocytes. Notably, KIT was expressed in late spermatocytes and round spermatids, but apparently not in human spermatogonia. UCHL1 was found in the cytoplasm of spermatogonia, whereas POU5F1 was not detected in any of the human germ cells. GFRA1 and ITGA6 were localized to the plasma membrane of the spermatogonia. Next, we isolated GPR125-positive spermatogonia from adult human testes using a two-step enzymatic digestion followed by magnetic-activated cell sorting. The isolated GPR125-positive cells coexpressed GPR125, ITGA6, THY1, and GFRA1, and they could be cultured for short periods of time and exhibited a marked increase in cell numbers as shown by a proliferation assay. Immunocytochemistry of putative stem cell genes after 2 wk in culture revealed that the cells were maintained in an undifferentiated state. MAPK1/3 phosphorylation was increased after 2 wk of culture of the GPR125-positive spermatogonia compared to the freshly isolated cells. Taken together, these results indicate that human spermatogonia share some but not all phenotypes with spermatogonial stem cells (SSCs) and progenitors from other species. GPR125-positive spermatogonia are phenotypically putative human SSCs and retain an undifferentiated status in vitro. This study provides novel insights into the molecular characteristics, isolation, and culture of human SSCs and/or progenitors and suggests that the MAPK1/3 pathway is involved in their proliferation. Show less

The etiology of growth impairment in Crohn's disease (CD) has been inadequately explained by nutritional, hormonal, and/or disease-related factors, suggesting that genetics may be an additional contributor. The aim of this cross-sectional study was to investigate genetic variants associated with linear growth in pediatric-onset CD. We genotyped 951 subjects (317 CD patient-parent trios) for 64 polymorphisms within 14 CD-susceptibility and 23 stature-associated loci. Patient height-for-age Z-score < -1.64 was used to dichotomize probands into growth-impaired and nongrowth-impaired groups. The transmission disequilibrium test (TDT) was used to study association to growth impairment. There was a significant association between growth impairment in CD (height-for-age Z-score < -1.64) and a stature-related polymorphism in the dymeclin gene DYM (rs8099594) (OR = 3.2, CI [1.57-6.51], p = 0.0007). In addition, there was nominal over-transmission of two CD-susceptibility alleles, 10q21.1 intergenic region (rs10761659) and ATG16L1 (rs10210302), in growth-impaired CD children (OR = 2.36, CI [1.26-4.41] p = 0.0056 and OR = 2.45, CI [1.22-4.95] p = 0.0094, respectively). Our data indicate that genetic influences due to stature-associated and possibly CD risk alleles may predispose CD patients to alterations in linear growth. This is the first report of a link between a stature-associated locus and growth impairment in CD. Show less

The identification of estrogen receptors alpha and beta and aromatase in the testis has highlighted the important role of estrogens in regulating spermatogenesis. There is a wealth of information on the deleterious effects of fetal and neonatal exposure of estrogens and xenoestrogens in the testis, including spermiation failure and germ cell apoptosis. However, very little is known about gene transcripts affected by exogenous estradiol exposure in the testis. The objective of the present study was to unveil global gene expression profiles and testicular cell number changes in rats after estradiol treatment. 17beta-estradiol was administered to adult male rats at a dose of 100 micrograms/kg body weight in saline daily for 10 days; male rats receiving only saline were used as controls. Microarray analysis was performed to examine global gene expression profiles with or without estradiol treatment. Real time RT-PCR was conducted to verify the microarray data. In silico promoter and estrogen responsive elements (EREs) analysis was carried out for the differentially expressed genes in response to estradiol. Quantitation of testicular cell number based on ploidy was also performed using flow cytometry in rats with or without estradiol treatment. We found that 221 genes and expressed sequence tags (ESTs) were differentially expressed in rat testes treated with estradiol compared to the control; the microarray data were confirmed by real time RT-PCR. Gene Ontology analysis revealed that a number of the differentially expressed genes are involved in androgen and xenobiotic metabolism, maintenance of cell cytoskeleton, endocytosis, and germ cell apoptosis. A total of 33 up-regulated genes and 67 down-regulated genes showed the presence of EREs. Flow cytometry showed that estradiol induced a significant decrease in 2n cells (somatic and germ cells) and 4n cells (pachytene spermatocytes) and a marked increase in the number of elongated and elongating spermatids. This study provides a novel insight into the molecular basis for spermiation failure and apoptosis caused by 17beta-estradiol and it also offers new mechanisms by which adult exposure to environmental estrogens can affect spermatogenesis and fertility. Show less

Many gene variants are involved in the susceptibility to schizophrenia and some of them are expected to be associated with other human characters. Recently reported meta-analysis of genetic associations revealed nucleotide variants in synaptic vesicular transport/Golgi apparatus genes with schizophrenia. In this study, we selected the dymeclin gene (DYM) as a candidate gene for schizophrenia. The DYM gene encodes dymeclin that has been identified to be associated with the Golgi apparatus and with transitional vesicles of the reticulum-Golgi interface. A three-step case-control study of total of 2105 Japanese cases of schizophrenia and 2087 Japanese control subjects was carried out for tag single-nucleotide polymorphisms (SNPs) in the DYM gene and an association between an SNP, rs833497, and schizophrenia was identified (allelic P=2 × 10(-5), in the total sample). DYM is the causal gene for Dyggve-Melchior-Clausen syndrome and this study shows the second neuropsychiatric disorder in which the DYM gene is involved. The present data support the involvement of Golgi function and vesicular transport in the presynapse in schizophrenia. Show less

Protein flexibility is thought to play key roles in numerous biological processes, including antibody affinity maturation, signal transduction, and enzyme catalysis, yet only limited information is available regarding the molecular details linking protein dynamics with function. A single point mutation at the distal site of the endogenous tissue inhibitor of metalloproteinase 1 (TIMP-1) enables this clinical target protein to tightly bind and inhibit membrane type 1 matrix metalloproteinase (MT1-MMP) by increasing only the association constant. The high-resolution X-ray structure of this complex determined at 2 A could not explain the mechanism of enhanced binding and pointed to a role for protein conformational dynamics. Molecular dynamics (MD) simulations reveal that the high-affinity TIMP-1 mutants exhibit significantly reduced binding interface flexibility and more stable hydrogen bond networks. This was accompanied by a redistribution of the ensemble of substrates to favorable binding conformations that fit the enzyme catalytic site. Apparently, the decrease in backbone flexibility led to a lower entropy cost upon formation of the complex. This work quantifies the effect of a single point mutation on the protein conformational dynamics and function of TIMP-1. Here we argue that controlling the intrinsic protein dynamics of MMP endogenous inhibitors may be utilized for rationalizing the design of selective novel protein inhibitors for this class of enzymes. Show less

Spermatogenesis in man starts with spermatogonial stem cells (SSCs), and leads to the production of sperm in approximately 64 days, common to old and young men. Sperm from elderly men are functional and able to fertilize eggs and produce offspring, even though daily sperm production is more than 50% lower and damage to sperm DNA is significantly higher in older men than in those who are younger. Our hypothesis is that the SSC/spermatogonial progenitors themselves age. To test this hypothesis, we studied the gene expression profile of mouse SSC/progenitor cells at several ages using microarrays. After sequential enzyme dispersion, we purified the SSC/progenitors with immunomagnetic cell sorting using an antibody to GFRA1, a known SSC/progenitor cell marker. RNA was isolated and used for the in vitro synthesis of amplified and labeled cRNAs that were hybridized to the Affymetrix mouse genome microarrays. The experiments were repeated twice with different cell preparations, and statistically significant results are presented. Quantitative RT-PCR analysis was used to confirm the microarray results. Comparison of four age groups (6 days, 21 days, 60 days, and 8 months old) showed a number of genes that were expressed specifically in the older mice. Two of them (i.e. Icam1 and Selp) have also been shown to mark aging hematopoietic stem cells. On the other hand, the expression levels of the genes encoding the SSC markers Gfra1 and Plzf did not seem to be significantly altered by age, indicating that age affects only certain SSC/progenitor properties. Show less

The primary sequence of proteins usually dictates a single tertiary and quaternary structure. However, certain proteins undergo reversible backbone rearrangements. Such metamorphic proteins provide a means of facilitating the evolution of new folds and architectures. However, because natural folds emerged at the early stages of evolution, the potential role of metamorphic intermediates in mediating evolutionary transitions of structure remains largely unexplored. We evolved a set of new proteins based on approximately 100 amino acid fragments derived from tachylectin-2--a monomeric, 236 amino acids, five-bladed beta-propeller. Their structures reveal a unique pentameric assembly and novel beta-propeller structures. Although identical in sequence, the oligomeric subunits adopt two, or even three, different structures that together enable the pentameric assembly of two propellers connected via a small linker. Most of the subunits adopt a wild-type-like structure within individual five-bladed propellers. However, the bridging subunits exhibit domain swaps and asymmetric strand exchanges that allow them to complete the two propellers and connect them. Thus, the modular and metamorphic nature of these subunits enabled dramatic changes in tertiary and quaternary structure, while maintaining the lectin function. These oligomers therefore comprise putative intermediates via which beta-propellers can evolve from smaller elements. Our data also suggest that the ability of one sequence to equilibrate between different structures can be evolutionary optimized, thus facilitating the emergence of new structures. Show less

Here we describe the crystal structure of the N-terminal domain of the FK506-binding protein (FKBP) from wheat (wFKBP73), which is the first structure presenting three FK domains (wFK73₁, wFK73₂ and wFK73₃₎. The crystal model includes wFK73₂ and wFK73₃ domains and only part of the wFK73₁ domain. The wFK73₁ domain is responsible for binding FK506 and for peptidyl prolyl cis/trans isomerase (PPIase) activity, while the wFK73₂ and wFK73₃ domains lack these activities. A structure-based sequence comparison demonstrated that the absence of a large enough hydrophobic pocket important for PPIase activity, and of the conserved residues necessary for drug binding in the wFK73₂ and wFK73₃ domains explains the lack of these activities in these domains. Sequence and structural comparison between the three wFKBP73 domains suggest that the wFK73₂ domain is the most divergent. A structural comparison of the FK domains of wFKBP73 with other FKBPs containing more than one FK domain, revealed that while the overall architecture of each of the three FK domains displays a typical FKBP fold, their relative arrangement in space is unique and may have important functional implications. We suggest that the existence of FKBPs with three FK domains offers additional interactive options for these plant proteins enlarging the overall regulatory functions of these proteins. Show less

The cofactor-binding domains (residues 153-295) of the alcohol dehydrogenases from the thermophile Thermoanaerobacter brockii (TbADH), the mesophilic bacterium Clostridium beijerinckii (CbADH), and the protozoan parasite Entamoeba histolytica (EhADH1) have been exchanged. Three chimeras have been constructed. In the first chimera, the cofactor-binding domain of thermophilic TbADH was replaced with the cofactor-binding domain of its mesophilic counterpart CbADH [chimera Chi21((TCT))]. This domain exchange significantly destabilized the parent thermophilic enzyme (DeltaT(1/2) = -18 degrees C). The reverse exchange in CbADH [chimera Chi22((CTC))], however, had little effect on the thermal stability of the parent mesophilic protein. Furthermore, substituting the cofactor-binding domain of TbADH with the homologous domain of EhADH1 [chimera Chi23((TET))] substantially reduced the thermal stability of the thermophilic ADH (DeltaT(1/2) = -51 degrees C) and impeded the oligomerization of the enzyme. All three chimeric proteins and one of their site-directed mutants were crystallized, and their three-dimensional (3D) structures were determined. Comparison of the 3D structures of the chimeras and the chimeric mutant with the structures of their parent ADHs showed no significant changes to their Calpha chains, suggesting that the difference in the thermal stability of the three parent ADHs and their chimeric mutants could be due to a limited number of substitutions located at strategic positions, mainly at the oligomerization interfaces. Indeed, stabilization of the chimeras was achieved, to a significant extent, either by introduction of a proline residue at a strategic position in the major horse liver ADH-type dimerization interface (DeltaT(1/2) = 35 degrees C) or by introduction of intersubunit electrostatic interactions (DeltaT(1/2) = 6 degrees C). Show less

The aim of the study was to evaluate smoking prevalence and exposure to environmental tobacco smoke among school administrators. The study population consisted of 320 administrative workers in schools from Lodz district. Among the study participants self-administrative questionnaire was conducted. The questionnaire focused on socio-demographic characteristics, detail information about active smoking and environmental tobacco smoke exposure. Current tobacco smoking was indicated by 19% of women and 28% of men (p = 0.06). Only 35% of the study subjects declared willingness to give up the habit. Significantly less men than women felt that they should quit smoking (36% vs. 11.4%, p < 0.05). Only small part of the study population expected the help in quitting smoking from specialists, physicians or school. It is crucial to increase awareness among the school administrators about negative effects of smoking and to motivate them to give up the habit. About 7% of study subjects (5% of women and 11% of men, p < 0.05) declared that smoking is allowed in school building and 13% of them indicated that there are no regulations on it or did not know such regulations. Show less

Cigarette smoking constitutes one of the main sources of exposure to cancerogenic and procancerogenic xenobiotics in the human population. Smoking is not only a bad habit among adults but also among young people. Despite many anti-tobacco campaigns undertaken by European Union countries still the smoking percentage among young people is high and does not decrease in the recent years. Cigarettes smoking induces formation of reactive oxygen species and increases lipids peroxidation, which ultimately results in the production of MDA (malondialdehyde)--one of the oxidative stress biomarkers. Exposure to oxidative stress and the consequences of its influence can be measured by redox parameters monitoring. One of these methods is determination of total antioxidant activity by means of FRAP test (Ferric Reducing Antioxidant Power) in biological samples. The aim of this work was the evaluation of free radical processes increase related to tobacco smoke exposure by measurement of total antioxidant activity FRAP and MDA concentration in urine samples of medical students. In our study the authors'--designed questionnaire regarding the assessment of exposure to tobacco smoke and chosen xenobiotics was used. The investigated group included 134 students of 1-st and 2-nd year of study at Faculty of Medicine and Dentistry Division in Zabrze, Silesian Medical University. 85 students provided urine samples for further analysis. In the study group 40 students were active smokers and the control group consisted of 45 nonsmokers. The mean MDA concentrations in smoking students group were higher in comparison to the control group of non-smoking students. In the group of students exposed to second-hand smoke MDA concentrations were significantly higher in comparison to the group of students non-exposed to second hand smoke. The values of FRAP parameter were significantly higher for students who smoked in comparison with students non-exposed to tobacco smoke. Students exposed to second hand smoke had slightly higher FRAP values in comparison to smoking students. Exposure to tobacco smoke increased the intensity of free radical processes. Our study confirmed the harmful influence of second hand smoke that resulted in observed higher concentrations of determined biomarkers in the group of passive smoking students. Show less

Respiratory diseases are the most common cause of the child and family practice physicians are one of the main reasons for referral to a specialist clinic and hospital pediatric wards. The severity of respiratory disease in adolescence influenced by various factors, endo- and exogenous. Some of them, especially environmental factors can be eliminated or reduced and thus reduce the risk of developing this disease. The most common source of pollutants in dwellings is tobacco smoke. The aim of this study was to assess exposure to tobacco smoke in three years old children of attending local kindergartens in Białystok and its influence on the type of recovery from acute respiratory infections by the respondents. The study included 313 children from among the 1,200 who attend the local 51-kindergartens in Bialystok. Information on the structure of tobacco use in three-years-old-children's families and respiratory illnesses among random children were obtained, based on anonymous questionnaires completed by their carers. Exposure to tobacco smoke was based on questionnaires and serum cotinine in relation to creatinine in the urine of patients (K/K). In the 150 families surveyed children found 210 smoking people. Every day smoked 37.3% of fathers and 23.6% of mothers. Of the children surveyed--34% of the houses which where there was a prohibition on tobacco use, 35% of the houses which were smoked in enclosed areas, in 31% of homes have not been established no-smoking rules. Children who during the six-month period to attend kindergarten gone lower respiratory tract infection had mean K/K (59.57 ng/mg) higher than the ones that were healthy and underwent upper respiratory tract infection. Used by the parents of the children tested in part to reduce the exposure to tobacco smoke in the home environment was ineffective and did not influence the decrease in the incidence of lower respiratory tract. Show less

Recent reports have demonstrated that adult cells can be reprogrammed to pluripotency, but mostly with genes delivered using retroviruses. Some of the genes are cancer causing; thus, these adult-derived embryonic stem (ES)-like cells cannot be used for therapy to cure human diseases. Remarkably, it has also been demonstrated recently by several groups that, in mice, spermatogonial stem cells (SSCs) can be reprogrammed to ES-like cells without the necessity of exogenously added genes. SSCs constitute one of the most important stem cell systems in the body, not only because they produce spermatozoa that transmit genetic information from generation to generation, but also because of the recent studies showing their remarkable plasticity. Very little is known about SSCs in humans, except for the earlier work of Clermont and colleagues who demonstrated that there are A(dark) and A(pale) spermatogonia, with the A(dark) referred to as the reserve stem cells and the A(pale) being the renewing stem cells. We now demonstrate that G protein-coupled receptor 125 (GPR125) may be a marker for human SSCs. Putative human SSCs can also be reprogrammed to pluripotency. We were able to achieve this result without the addition of genes, suggesting that human SSCs have considerable potential for cell-based, autologous organ regeneration therapy for various diseases. Show less

Spermatogenesis is the process that involves the division and differentiation of spermatogonial stem cells (SSCs) into mature spermatozoa. SSCs are a subpopulation of type A spermatogonia resting on the basement membrane in the mammalian testis. Self-renewal and differentiation of SSCs are the foundation of normal spermatogenesis, and thus a better understanding of molecular mechanisms and signaling pathways in the SSCs is of paramount importance for the regulation of spermatogenesis and may eventually lead to novel targets for male contraception as well as for gene therapy of male infertility and testicular cancer. Uncovering the molecular mechanisms is also of great interest to a better understanding of SSC aging and for developing novel therapeutic strategies for degenerative diseases in view of the recent work demonstrating the pluripotent potential of the SSC. Progress has recently been made in elucidating the signaling molecules and pathways that determine cell fate decisions of SSCs. In this review, we first address the morphological features, phenotypic characteristics, and the potential of SSCs, and then we focus on the recent advances in defining the key signaling molecules and crucial signaling pathways regulating self-renewal and differentiation of SSCs. The association of aberrant expression of signaling molecules and cascades with abnormal spermatogenesis and testicular cancer are also discussed. Finally, we point out potential future directions to pursue in research on signaling pathways of SSCs. Show less

Dyggve-Melchior-Clausen dysplasia (DMC) is a rare inherited dwarfism with severe mental retardation due to mutations in the DYM gene which encodes Dymeclin, a 669-amino acid protein of yet unknown function. Despite a high conservation across species and several predicted transmembrane domains, Dymeclin could not be ascribed to any family of proteins. Here we show, using in situ hybridization, that DYM is widely expressed in human embryos, especially in the cortex, the hippocampus and the cerebellum. Both the endogenous and the recombinant protein fused to green fluorescent protein co-localized with Golgi apparatus markers. Electron microscopy revealed that Dymeclin associates with the Golgi apparatus and with transitional vesicles of the reticulum-Golgi interface. Moreover, permeabilization assays revealed that Dymeclin is not a transmembrane but a peripheral protein of the Golgi apparatus as it can be completely released from the Golgi after permeabilization of the plasma membrane. Time lapse confocal microscopy experiments on living cells further showed that the protein shuttles between the cytosol and the Golgi apparatus in a highly dynamic manner and recognizes specifically a subset of mature Golgi membranes. Finally, we found that DYM mutations associated with DMC result in mis-localization and subsequent degradation of Dymeclin. These data indicate that DMC results from a loss-of-function of Dymeclin, a novel peripheral membrane protein which shuttles rapidly between the cytosol and mature Golgi membranes and point out a role of Dymeclin in cellular trafficking. Show less

To characterize the molecular phenotype of spermatogonial stem cells (SSCs), we examined genes that are differentially expressed in the stem/progenitor spermatogonia compared to nonstem spermatogonia. We isolated type A spermatogonia (stem and nonstem type A) from 6-day-old mice using sedimentation velocity at unit gravity and further selected the stem/progenitor cell subpopulation by magnetic activated cell sorting with an antibody to GDNF-receptor-alpha-1 (GFRA1). It has been previously shown that GFRA1 is expressed in SSCs and is required for their stemness. The purity of the isolated cells was approximately 95% to 99% as indicated by immunocytochemistry using anti-GFRA1. Comparison of GFRA1-positive and GFRA1-negative spermatogonia by microarray analysis revealed 99 known genes and 12 uncharacterized transcripts that are overexpressed in the former cell population with a >2-fold change. Interestingly, the highest level of overexpression was observed for Csf1r, encoding the receptor for macrophage colony-stimulating factor (M-CSF, official symbol CSF1), which has a well-established role in the regulation of myeloid progenitor cells. Analysis of our microarray data with a bioinformatics software program (Ingenuity Systems) revealed the potential role of various signaling pathways in stem/progenitor spermatogonia and suggested a common pathway for GFRA1 and CSF1R that may lead to their proliferation. Further investigation to test this hypothesis has shown that CSF1 promotes cell proliferation in primary cultures of the isolated type A spermatogonia and in the spermatogonial-derived stem cell line C18-4. Semiquantitative RT-PCR and immunohistochemistry confirmed the previously mentioned microarray data. Collectively, this study provides novel molecular signatures for stem/progenitor spermatogonia and demonstrates a role for CSF1/CSF1R signaling in regulating their proliferation. Show less

The aim of the study was to evaluate the prenatal and postnatal child exposure to environmental tobacco smoke. The study population consisted of 83 women from Lodz, Poland among which detail questionnaire was conducted three times during pregnancy period. From all women included into the study the saliva sample was collected to verify smoking status in pregnancy. One year after delivery the second questionnaire was performed and urine sample from all children was collected. The cotinine level in saliva and urine was analyzed using Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS ESI+). About 22% of the children were expose to ETS during prenatal and 46% in postnatal period. Cotinine level in children's urine statistically differentiated children from smoking mothers (Geometric Mean (GM) 11.4 ng/ml; 95% CI 7.1-18.3) and exposed homes (GM 7.3 ng/ml; 95% CI 3.6-15.0) compared with non-exposed homes (GM 1.3 ng/ml; 95% CI 0.9-1.7) (p < 0.001). Children should be more protected from ETS exposure in prenatal and postnatal period. Show less

It has been already proved in many experimental studies that tobacco smoke has multiple toxic effects on respiratory tract cells. Alterations in ciliary epithelium of rats trachea after short exposition to high tobacco smoke concentrations in inhaled air were been determinate in current study. Morphological evaluation revealed in lining epithelium voluminous exudate located between epithelium and lamina propria cells with evidently larger number of mast cells. Show less

Usually, alcohol addicted persons smokes cigarettes. In the study, the effect of combined exposure to alcohol and tobacco smoke in alcohol addicted rats on liver peroxidation was evaluated. Alcohol abuse and its presence in blood did not influence the cotinine level, what indicates the lack of the importance of this factor in nicotine metabolism. Similarly, enzymatic markers of liver damage (AspAT, AIAT, ALP) did not change, what showed lack of hepatotoxic effect studied compounds in applied model of alcohol addiction and tobacco smoke exposure. Combined exposure to alcohol and tobacco smoke increases the level of lipid peroxidation in brain, liver and lungs however decreases in serum. In kidneys the results are not unambiguous. Show less

Mitochondria play a central role in the intrinsic pathway of apoptosis. In response to many pro-apoptotic stimuli, mitochondria undergo an irreversible process called mitochondrial membrane permeabilization (MMP). The detection of MMP in isolated mitochondria is most often based on assays that monitor either the loss of the inner transmembrane potential (DYm; classically with Rhodamine 123), permeability transition (PT, cyclosporin A-sensitive matrix swelling), or the release of critical pro-apoptotic intermembrane space effectors. To gain complementary information on MMP mechanisms, we have systematically used three additional assays optimized for the 96-well microplate format: (1) inner membrane permeability, (2) VDAC-associated NADH reductase activity, and (3) ATP/ADP translocase activity. We report that ad hoc combinations of ANT and VDAC ligands, carbonyl cyanide m-chlorophenylhydrazone (CCCP), mastoparan and Vpr52-96 peptide and PT inhibitors, permit to explore relationships between enzymatic functions of sessile mitochondrial proteins (i.e. ANT, VDAC) and MMP. These assays should be useful tools to investigate mitochondrial apoptosis, decipher the implication of inner and outer membrane permeabilization and provide a multi-parametric approach for drug discovery. Show less

Initiation and progression of laryngeal cancer is associated with tobacco smoking and abusing of strong alcoholic beverages. A significance of genetic factor, although not defined sufficiently yet has been raised as well. The studies were focused on an influence of AHR gene polymorphism on survival of squamous cell carcinoma laryngeal subjects. The study material was 65 archival DNA samples analyzed by RLP-PCR. The samples varying with electrophoretic mobility were DNA sequenced. In the study group 9 heterozygotic variants Arg554Lys (codon 554) were detected. One case was a carrier of two other mutations in codon: 490 (1468 A > G) and 570 (1708 G > A). Survival time, metastasis and occurrence of second primary tumors were compared in carriers of wild type and Arg554Lys variant AHR. Preliminary results indicate for a necessity of further studies as until now the study group is too small to find a conclusive association. Show less

Dyggve-Melchior-Clausen (DMC) syndrome is a rare autosomal recessive type of skeletal dysplasia. It is characterized by the association of progressive spondyloepimetaphyseal dysplasia (SEMD), microcephaly, mental retardation (MR), and coarse facies. The radiographic appearance of generalized platyspondyly with double-humped end plates and the lace-like appearance of iliac crests are pathognomonic and distinctive of DMC syndrome. The disorder results from mutations in the DYM gene mapped in the 18q12-12.1 chromosomal region. In this report, we studied 15 Egyptian cases with DMC syndrome from nine unrelated families. We aimed to emphasize the characteristic clinical and radiological features in order to differentiate the condition from other SEMDs and mucopolysaccharidosis (MPS). Patients were subjected to detailed history taking, three-generation family pedigree analysis, complete physical examination, anthropometric measurements, quantitative estimation, and two-dimensional electrophoresis of glycosaminoglycans in the urine and measurement of α-l-iduronidase and galactose-6-sulfatase enzyme activities to exclude Hurler and Morquio diseases (MPS type I and MPS type IVA), respectively. Other investigations were carried out whenever indicated. All patients were the offspring of consanguineous apparently normal parents. Positive family history and similarly affected sibs were noted, confirming the autosomal recessive inheritance pattern of the syndrome. Short stature, microcephaly, variable degree of MR, and coarse facies were constant features. The frequency of characteristic orthopedic and radiological findings was reported. Orthopedic surgical intervention was carried out for two patients. The study concluded that DMC syndrome may be more frequent in Egypt than previously thought, especially due to misdiagnosis. Characteristic facial dysmorphism, body habitus, and pathognomonic radiological signs suggest the diagnosis and differentiate it from other types of SEMDs and MPS for proper genetic counseling and management. Show less