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Overexpression of vascular endothelial growth factor (VEGF) in the testis of transgenic mice induces infertility, suggesting a potential role for VEGF in the process of spermatogenesis. Spermatogenesis occurs within the confines of the seminiferous tubules, and the seminiferous epithelium lining these tubules consists of Sertoli cells and germ cells in various stages of maturation. We investigated the source of VEGF and VEGF-target cells within the seminiferous tubules of the normal mouse testis. Sections of testes fixed in Bouin solution and embedded in paraffin were subjected to immunofluorescent staining with specific antibodies against VEGF, and its receptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1). Total RNA was extracted from isolated populations of Sertoli cells, type A spermatogonia, pachytene spermatocytes, and spermatids. Primer pairs specific for VEGF and its receptors were designed and reverse-transcriptase polymerase chain reaction (RT-PCR) was performed. Immunofluorescent studies indicated that VEGF is strongly expressed in the cytoplasm of Sertoli cells. VEGFR-1 and VEGFR-2 were not expressed by the Sertoli cell. In contrast, a differential expression of VEGF receptors was observed in germ cells. Although VEGFR-2 was expressed in the cytoplasm of type A spermatogonia, VEGFR-1 was expressed in the acrosomal region of spermatids and spermatozoa. Pachytene spermatocytes did not exhibit any staining. Further, we examined the transcription of VEGF and its receptors by RT-PCR. VEGF was actively transcribed only in Sertoli cells. The transcription of VEGFR-2 was confined to type A spermatogonia. Interestingly, VEGFR-1 was transcribed both in pachytene spermatocytes and round spermatids. The mRNA expression of VEGFR-1 and VEGFR-2 in germ cells was inversely correlated during postnatal development of the mouse testis. Thus, VEGF may play a potential role in regulating the initial stages of the process of spermatogonial proliferation through VEGFR-2 and spermiogenesis through VEGFR-1. Show less

A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein. The purified leptin-binding domain (LBD) exhibited the predicted beta structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins. The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k(on) and k(off) values (mean +/- S.E.) of 1.20 +/- 0.23 x 10(-5) mol(-1) s(-1) and 1.85 +/- 0.30 x 10(-3) s(-1) and a K(d) value of 1.54 x 10(-8) m. Similar results were achieved with conventional binding experiments. LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD.human leptin complex. Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding. Show less

The smoking habit is widely spread between pregnant women too. Tobacco smoke components have negative influence on infant's development. In human trans-3'-hydroxycotinine is besides cotinine, one of the main nicotine metabolites. The research covered 17 patients with physiological pregnancy, which ended with delivery through natural passages at term. All women were active smokers as we revealed before according to their serum cotinine concentrations. Assessment of cotinine and trans-3'-hydroxycotinine concentrations has been done by the use of very sensitive liquid chromatographic techniques in the mothers venous blood samples and in the mixed umbilical cord blood of the newborns. Obtained results of the research have been statistically analysed using the STATISTICA package. Significance level p < 0.05 has been adopted as statistically relevant. In conclusion, trans-3'-hydroxycotinine concentrations were significantly lower in the neonates then in their smoking mothers and there was strong positive linear relationship between maternal and neonatal trans-3'-hydroxycotinine concentrations. The value of linear correlation coefficient was 0,7 with a p-value of 0.05. Show less

Bronchial asthma belongs to the group of illnesses of allergic and inflammatory origin. Despite numerous research divergent results are described in literature, that is the reason why a decision was made to conduct a careful study of the influence of various factors on induction and clinical course of bronchial asthma among children. The research included 5945 children of 10-11 from 86 primary schools of the Katowice province (presently Silesian province). The factor evaluated was the influence of the exposure to tobacco smoke on asthma induction and its clinical course. The data concerning the exposure were obtained from the children's parents. The dependence analyses were conducted with the use of statistical packets Statistica and BUGS. Smoking was declared in 3594 cases (68.5%), and non-smoking were 1637 people (31.2%). The probability of developing asthma is constant within the range of 0-40 cigarettes smoked daily; then it grows quite rapidly (R-0.794, p-0.011). The clinical course of asthma was evaluated on the basis of appearing wheezing respiration, pressure in chest, dyspnoea and cough. In the investigated population/group only the probability of pressure in chest incidence correlated with the number of cigarettes smoked (R-0.86, p = 0.014), in case of the remaining symptoms the correlation was statistically insignificant. The obtained results have proved a substantial influence of tobacco smoke on the development of asthma. This indicates the necessity of taking sanitary measures in the habitats, and especially elimination of tobacco smoke. The results of the research concerning the influence of tobacco smoke on clinical course of asthma were less unequivocal. This might be caused by the evaluation method of the exposure (purposeful underrating, smoking away from home, or in another room). This points to the necessity to employ a more unbiased method of evaluation of the exposure to tobacco smoke. Show less

A flow controlling system for pulsed inhaled nitric oxide has been developed and tested, and here its features and initial animal experiments and clinical applications are described. The physical characteristic test indicates that the practical released dose of NO gas is very close to the theoretical flow of NO gas at variant pressures. Animal experiments demonstrate that inhaled NO gas concentration is lower than the concentration of theoretical inhalation, but the variance is not remarkable (p>0.05). When sixteen cases with CHD and PH were chosen to inhale NO gas (15 ppm, 15 min) PAP and PVR of all cases were reduced after inhalation of NO gas from 617 +/-51.3 dyn x s x cm(-5), 54.4+/-13.1 mmHg to 417+/-36.9 dym x s x cm(-5), 33.8+/-12.3 mmHg (PVR, p<0.01; PAP, p<0.01) respectively. When gas inhalation was stopped, these values returned to their base lines after a short period of time. All these show that the pulsed inhaled NO flow controlling instrument in accordance with the requirements of the designing, can be widely used in clinical diagnoses and treatments and will be a new tool offered for the treatments of the patients with PH. Show less

The exposition to tobacco smoke is overall: in home, work and public places. For the examination of the presence and concentration of environmental tobacco smoke (ETS) in indoor environments the nicotine and respirable suspended particulates (RSP) are determined. A variety of biomarkers (nicotine, cotinine, thiocyanate, carboxyhemoglobin, protein and DNA adducts) are propose for measurement of exposure to tobacco smoke. The most popular is measurement of cotinine concentration in body fluids (blood, urine, saliva). Plasma cotinine concentration correlated to numbers of cigarettes smoked and to various biological effects of cigarette smoking and exposure to ETS. Other biomarkers as carboxyhemoglobin, thiocyanate, and amines and polycyclic aromatic hydrocarbons adduct can be also use. Show less

Spermatogenesis is the process by which spermatogonial stem cells divide and differentiate to produce sperm. In vitro sperm production has been difficult to achieve because of the lack of a culture system to maintain viable spermatogonia for long periods of time. Here we report the in vitro generation of spermatocytes and spermatids from telomerase-immortalized mouse type A spermatogonial cells in the presence of stem cell factor. This differentiation can occur in the absence of supportive cells. The immortalized spermatogonial cell line may serve as a powerful tool in elucidating the molecular mechanisms of spermatogenesis. Furthermore, through genomic modification and transplantation techniques, this male germ cell line may be used to generate transgenic mice and to develop germ cell gene therapy. Show less

Using differential display reverse transcriptase-polymerase chain reaction (RT-PCR) we have cloned a cDNA that encodes a putative peptide with homology to a recently reported A-kinase anchoring protein-associated protein (ASP) in human sperm. The mouse cDNA was 864 bases in length and encoded for a putative protein of 230 amino acids that had 90% amino acid similarity with the human ASP. The N terminal amino acid sequence had 65% similarity to the rat, mouse, and human protein kinase A regulatory type II sequences. Expression of the gene encoding this ASP was specific to testicular germ cells. Northern blot analysis of testis RNA from 5-, 15-, 25-, and 40-day-old mice showed expression of the ASP gene, but similar analyses of busulfan-treated germ cell-deficient mice failed to detect its expression. In addition, Northern blot analysis did not detect expression of the ASP mRNA in cultured Sertoli cells or cultured interstitial cells. Northern blot and RT-PCR analyses did not detect the ASP mRNA in mouse spleen, brain, liver, lung, heart, kidney, skeletal muscle, ovary, or Sertoli cells. In situ hybridization analysis localized the ASP mRNA to the germ cell compartment of the seminiferous tubules in the testis. Show less

A 61-year-old woman who was a New York City hospital employee developed fatal inhalational anthrax, but with an unknown source of anthrax exposure. The patient presented with shortness of breath, malaise, and cough that had developed 3 days prior to admission. Within hours of presentation, she developed respiratory failure and septic shock and required mechanical ventilation and vasopressor therapy. Spiral contrast-enhanced computed tomography of the chest demonstrated large bilateral pleural effusions and hemorrhagic mediastinitis. Blood cultures, as well as DNA amplification by polymerase chain reaction of the blood, bronchial washings, and pleural fluid specimens, were positive for Bacillus anthracis. The clinical course was complicated by liver failure, renal failure, severe metabolic acidosis, disseminated intravascular coagulopathy, and cardiac tamponade, and the patient died on the fourth hospital day. The cause of death was inhalational anthrax. Despite epidemiologic investigation, including environmental samples from the patient's residence and workplace, no mechanism for anthrax exposure has been identified. Show less

Mammalian phosphodiesterases types 3 and 4 (PDE3 and PDE4) hydrolyze cAMP and are essential for the regulation of this intracellular second messenger. These enzymes share structural and biochemical similarities, but each can be distinguished by its sensitivity to isoenzyme-specific, substrate-competitive inhibitors. We present a model configuration for the PDE4 substrate (cAMP) and a PDE4-specific inhibitor (rolipram) within the active site of the enzyme. The docked models were also used to examine the structural consequences of mutations that confer resistance to rolipram and other PDE4-specific inhibitors. The proposed rolipram-binding configuration is consistent with the substrate-competitive nature of inhibition and also provides a structural basis for the observed specificity of binding to the R- versus S-enantiomer. For mutations that render the enzyme rolipram-insensitive, there was generally an inverse relationship between the magnitude of the drug resistance and the distance of the altered residue from the predicted binding site. We observed a direct correlation between the net loss of protein residue interactions (van der Waals contacts and hydrogen bond interactions) and the degree of rolipram resistance. The positions of several drug sensitivity-determinant residues define a surface leading to the substrate- and drug-binding sites, suggesting a possible approach channel leading to the enzyme active site. The binding of other PDE4 inhibitors (high- and low-affinity) was also modeled and used to predict the involvement of residues that were not previously implicated in pharmacological interactions. Show less

In the mammalian testis, type A spermatogonia proliferate and differentiate into sperm under the tight control of both endocrine and paracrine factors. In order to study the complex process of spermatogenesis at the molecular level, an in vitro system must be devised in which type A spermatogonia can be cultured for a prolonged period of time. Therefore, cocultures including type A spermatogonia and Sertoli cells, which act as nurse cells to the developing germ cells, are desirable. We have developed a method for the specific isolation of type A spermatogonia using magnetic beads and antibodies that recognize the c-kit receptor or the homophilic adhesion molecule, Ep-CAM. Purified spermatogonia could survive for a period of 25 days when cocultivated on Sertoli cell monolayers. Moreover, we recently established Sertoli cell lines that produce growth factors that are essential for the maintenance of spermatogonia in a proliferative state. Some of these Sertoli cell lines are able to reorganize into tubular structures when cultivated on a layer of Matrigel as extracellular matrix. We show here that type A spermatogonia associate specifically with the Sertoli cell tubules, and are able to replicate their DNA in this environment. Thus, these in vitro culture systems could be used for the long-term culture of primary, nonimmortalized type A spermatogonia. Show less

We have examined the effects of increasing doses of chloroquine (CQ), on transferrin secretion in primary cultures of immature rat Sertoli cells (SC) grown on a reconstituted basement membrane (Matrigel) in bicameral chambers. SC cells were seeded in serum-free defined medium at a density of 3 x 10(6) cells/0.64cm2/well on Matrigel covered Millicell-HA filters. CQ at concentrations ranging from 0.04-1.0 microM was added to the basal compartment of the bicameral system from day 7 of the culture. The formation of the tight junction was monitored by the measurement of the transepithelial resistance (TER) at 24 hr intervals using an impedance meter TER in untreated controls was 50 Ohms/cm2 on day 1, and increased progressively to 80 Ohms/cm2 by day 7 and plateaued until day 12. On the seventh day of culture, CQ was introduced into the basal chamber During the 4 days of the experiment, the secretion of transferrin decreased with time. Maximal transferrin secretion by SC was detected during the initial 2 day collection period. During the subsequent collection period, CQ (1 microM) decreased significantly transferrin secretion by SC, while 0.04 microM CQ did not affect transferrin secretion. The polarized secretion of transferrin in response to CQ was also studied. During both collection periods there was no significant difference between controls and 0.04 microM CQ cultures in the ratio of apical to basal transferrin secretion. In the 1 microM culture medium, CQ diminished significantly the ratio of apical to basal transferrin secretion. These observations demonstrate the heterogenous effects of lower doses of CQ on immature rat SC in cultures. Show less

Members of the Notch gene family have been shown to play an important role in the control of cell fate in many developmental systems. We hypothesized that the fate of the male germ line stem cells may also be mediated through the Notch signaling pathway. We therefore sought to determine whether the components of the Notch pathway are expressed in the mouse testis. Western blot analysis revealed the expression of three Notch receptors (Notch 1, Notch 2, and Notch 3), Notch ligands (Jagged 1, Jagged 2, and Delta 1), and presenilin 1 (PS1) in neonatal mouse testis. We then examined their cellular localization by immunohistochemical analysis of cocultures of spermatogonia and Sertoli cells. The 3 Notch receptors were found to be expressed in spermatogonia. Sertoli cells expressed only Notch 2 receptor. Among the Notch ligands, Delta 1 and Jagged 1 were localized exclusively in spermatogonia and Sertoli cells, respectively. PS1 was apparent in both spermatogonia and Sertoli cells. The presence of Notch receptors and Notch ligands in spermatogonia and Sertoli cells indicates that these cells are capable of responding to and eliciting Notch signaling during the process of spermatogenesis. Key words: Cell fate, delta, jagged, presenilin, spermatogenesis. Show less

The activity of telomerase, an enzyme that synthesizes telomeric repeats at the ends of chromosomes, is not detectable in normal human prostate. However, the majority of human prostate cancers exhibit telomerase activity. Since androgens play a major role in prostate tumorigenesis, we investigated the effect of androgen-depletion on the expression of telomerase activity in the prostate. Adult male rhesus monkeys were either bilaterally castrated or subjected to sham surgery (n = 5 each). Approximately 6 weeks later, the animals were killed and the different regions of the prostate gland were removed and frozen immediately. Telomerase activity was assayed using the telomeric repeat amplification protocol. All five regions of the prostate from sham operated control animals failed to exhibit telomerase activity. In the castrated monkey, all regions of the prostate, except for the anterior lobe, expressed high levels of telomerase activity. Our results indicate that in monkeys, androgen-ablation leads to up-regulation of telomerase activity. The negative-regulation of telomerase activity by androgens is probably lost during prostate tumorigenesis. Show less

We have analyzed structure-sequence relationships in 32 families of flavin adenine dinucleotide (FAD)-binding proteins, to prepare for genomic-scale analyses of this family. Four different FAD-family folds were identified, each containing at least two or more protein families. Three of these families, exemplified by glutathione reductase (GR), ferredoxin reductase (FR), and p-cresol methylhydroxylase (PCMH) were previously defined, and a family represented by pyruvate oxidase (PO) is newly defined. For each of the families, several conserved sequence motifs have been characterized. Several newly recognized sequence motifs are reported here for the PO, GR, and PCMH families. Each FAD fold can be uniquely identified by the presence of distinctive conserved sequence motifs. We also analyzed cofactor properties, some of which are conserved within a family fold while others display variability. Among the conserved properties is cofactor directionality: in some FAD-structural families, the adenine ring of the FAD points toward the FAD-binding domain, whereas in others the isoalloxazine ring points toward this domain. In contrast, the FAD conformation and orientation are conserved in some families while in others it displays some variability. Nevertheless, there are clear correlations among the FAD-family fold, the shape of the pocket, and the FAD conformation. Our general findings are as follows: (a) no single protein 'pharmacophore' exists for binding FAD; (b) in every FAD-binding family, the pyrophosphate moiety binds to the most strongly conserved sequence motif, suggesting that pyrophosphate binding is a significant component of molecular recognition; and (c) sequence motifs can identify proteins that bind phosphate-containing ligands. Show less

The skeletal muscle mitochondrial uncoupling protein-3 (UCP3) promotes substrate oxidation, but direct evidence for its metabolic role is lacking. Here, we show that UCP3 overexpression in cultured human muscle cells decreased mitochondrial membrane potential (DYm). Despite this, the ATP content was not significantly decreased compared with control cells, whereas ADP content was reduced and thus the ATP/ADP ratio raised. This finding was contrasts with the effect caused by the chemical protonophoric uncoupler, CCCP, which lowered DYm, ATP, and the ATP/ADP ratio. UCP3-overexpression enhanced oxidation of oleate, regardless of the presence of glucose, whereas etomoxir, which blocks fatty acid entry to mitochondria, suppressed the UCP3 effect. Glucose oxidation was stimulated in UCP3-overexpressing cells, but this effect was inhibited by oleate. UCP3 caused weak increase of both 2-Deoxyglucose uptake and glycolytic rate, which differed from the marked stimulation by CCCP. We concluded that UCP3 promoted nutrient oxidation by lowering DYm and enhanced fatty acid-dependent inhibition of glucose oxidation. Unlike the uncoupler CCCP, however, UCP3 raised the ATP/ADP ratio and modestly increased glucose uptake and glycolysis. We propose that this differential effect provides a biological significance to UCP3, which is up-regulated in metabolic stress situations where it could be involved in nutrient partitioning. Show less

An original experimental setup has been designed that allows evaluation of the free preference of space containing tobacco smoke by small laboratory animals. For the laboratory mice (C57B1/6 and BALB/c) and rats (MNRA and MR) placed every day into this box, no emotional-stress reaction (ESR) caused by the environment novelty was observed on the 19th day of experiment. A difference in the free preference of space containing tobacco smoke was observed between inbred animals with active and passive ESR phenotypes. Show less

Influence of environmental smoke exposure during pregnancy on umbilical blood flow velocity and newborns birthweight was assessed in prospective study among 116 pregnant women between 20 and 24 week of pregnancy. The main aim was to search for a possible correlation between cotinine, an effective marker of smoke exposure, and umbilical blood flow as measured by S/D, RI and PI ratios. This study shows a significant increase of systolic/diastolic velocity ratio of the umbilical artery according with increased cotinine levels, either for active or passive smokers. Increase of S/D ratio > 3.0 in umbilical artery in 20-24 week of pregnancy was negatively correlated with newborns birthweight. The results of this study suggest that active and passive smoking by pregnant women causes a direct increase in the vascular resistance of the placenta and contribute to the decreased of the newborns birthweight associated with smoking. Show less

Stem cell factor (SCF)/c-kit plays an important role in the regulation of hematopoiesis, melanogenesis, and spermatogenesis. In the testis, the SCF/c-kit system is believed to regulate germ cell proliferation, meiosis, and apoptosis. Studies with type A spermatogonia in vivo and in vitro have indicated that SCF induces DNA synthesis and proliferation. However, the signaling pathway for this function of SCF/c-kit has not been elucidated. We now demonstrate that SCF activates phosphoinositide 3-kinase (PI3-K) and p70 S6 kinase (p70S6K) and that rapamycin, a FRAP/mammalian target of rapamycin-dependent inhibitor of p70S6K, completely inhibited bromodeoxyuridine incorporation induced by SCF in primary cultures of spermatogonia. SCF induced cyclin D3 expression and phosphorylation of the retinoblastoma protein through a pathway that is sensitive to both wortmannin and rapamycin. Furthermore, AKT, but not protein kinase C-zeta, is used by SCF/c-kit/PI3-K to activate p70S6K. Dominant negative AKT-K179M completely abolished p70S6K phosphorylation induced by the constitutively active PI3-K catalytic subunit p110. Constitutively active v-AKT highly phosphorylated p70S6K, which was totally inhibited by rapamycin. Thus, SCF/c-kit uses a rapamycin-sensitive PI3-K/AKT/p70S6K/cyclin D3 pathway to promote spermatogonial cell proliferation. Show less

The main goal of the project was to estimate the effect of environmental tobacco smoke (ETS) exposure on the birthweight. In a cohort of 196 pregnant women in 20-24 week of pregnancy the serum and urine cotinine levels were determined. The cohort included randomly selected pregnant patients of a maternity units in Lodz, Poland. To assess a 24 h exposure to ETS preceding the day of examination, both serum and urine cotinine measurements were applied. A statistically significant relationship was found between serum cotinine concentration and brithweight. The newborns of nonsmoking mothers whose serum cotinine levels were characteristic for passive smoking (2-25 ng/ml) had their birthweight lower by an average of 30 g, compared to those of women who were not exposed to ETS (serum cotinine below 2 ng/ml). It was concluded that more effective public health measures should be undertaken to ensure a tobacco smoke-free environment for pregnant women. Until this goal is achieved, pregnant women should be informed about health risks from ETS exposure so that they would avoid it both at home and workplace. Show less

Leptin, a recently identified hormonal product of the ob gene, is known to regulate appetite, body metabolism, and reproductive functions. We investigated the expression of the leptin receptor (Ob-R) in testes from different age groups. The messenger RNA for Ob-R was found in testes from all age groups using RT-PCR. Using immunohistochemistry, we observed age- and stage-dependent distribution of the Ob-R in mouse testis. In testis of 5-day-old mice, its expression was mainly in type A spermatogonia. In the 20- and 30-day-old testis, Ob-R expression was in the spermatocytes; in the adult testis, it was specific to spermatocytes in stages IX and X of the cycle of the seminiferous epithelium. Five main immunoreactive proteins were detected using Western blot (220, 120, 90, 66, and 46 kDa). The 120-kDa protein was evident only in 20-day-old and older testes, whereas the 90-kDa band was present only in the 5- and 10-day-old testis. Leptin treatment induced phosphorylation of signal transducer and activator of transcription-3 in cultured seminiferous tubules from adult and 5-day-old testes. Our results show for the first time age- and stage-specific localization of a functional Ob-R in testicular germ cells. We hypothesize a direct role for leptin, through phosphorylation of signal transducer and activator of transcription-3, in proliferation and differentiation of germ cells, which may partially explain the infertility observed in leptin-deficient mice. Show less

A family-focused psychosocial intervention for stroke survivors is described and illustrated with case studies. It is designed to improve functional recovery through four specific pathways: increased knowledge, efficacy, and control through stroke education; optimized social support; increased network cohesion; and improved problem-solving abilities. Rationales for these pathways are presented and methods of implementing them discussed. Show less

Telomeres, the noncoding sequences at the ends of chromosomes, progressively shorten with each cellular division. Spermatozoa have very long telomeres but they lack telomerase enzymatic activity that is necessary for de novo synthesis and addition of telomeres. We performed a telomere restriction fragment analysis to compare the telomere lengths in immature rat testis (containing type A spermatogonia) with adult rat testis (containing more differentiated germ cells). Mean telomere length in the immature testis was significantly shorter in comparison to adult testis, suggesting that type A spermatogonia probably have shorter telomeres than more differentiated germ cells. Then, we isolated type A spermatogonia from immature testis, and pachytene spermatocytes and round spermatids from adult testis. Pachytene spermatocytes exhibited longer telomeres compared to type A spermatogonia. Surprisingly, although statistically not significant, round spermatids showed a decrease in telomere length. Epididymal spermatozoa exhibited the longest mean telomere length. In marked contrast, telomerase activity, measured by the telomeric repeat amplification protocol was very high in type A spermatogonia, decreased in pachytene spermatocytes and round spermatids, and was totally absent in epididymal spermatozoa. In summary, these results indicate that telomere length increases during the development of male germ cells from spermatogonia to spermatozoa and is inversely correlated with the expression of telomerase activity. Show less

d-Lactate dehydrogenase (d-LDH) of Escherichia coli is a peripheral membrane respiratory enzyme involved in electron transfer, located on the cytoplasmic side of the inner membrane. d-LDH catalyzes the oxidation of d-lactate to pyruvate, which is coupled to transmembrane transport of amino acids and sugars. Here we describe the crystal structure at 1.9 A resolution of the three domains of d-LDH: the flavin adenine dinucleotide (FAD)-binding domain, the cap domain, and the membrane-binding domain. The FAD-binding domain contains the site of d-lactate reduction by a noncovalently bound FAD cofactor and has an overall fold similar to other members of a recently discovered FAD-containing family of proteins. This structural similarity extends to the cap domain as well. The most prominent difference between d-LDH and the other members of the FAD-containing family is the membrane-binding domain, which is either absent in some of these proteins or differs significantly. The d-LDH membrane-binding domain presents an electropositive surface with six Arg and five Lys residues, which presumably interacts with the negatively charged phospholipid head groups of the membrane. Thus, d-LDH appears to bind the membrane through electrostatic rather than hydrophobic forces. Show less

We investigated the effect of CQ, an antimalarial drug with antiprotease activity, and NH4Cl, a related amines on the development of intercellular tight junctions in cultured immature rat Sertoli cells. Sertoli cells were seeded in serum-free defined medium at a density of 3 x 10(6) cells/0.64 cm2/well on Matrigel-covered Millicell-HA filters. CQ (1 microM and 2 microM) or NH4Cl (6.25 mM and 12 mM) was added to the outer (basal) compartment of the bicameral system either on day 1 or day 7 of the culture. Formation of tight junctions was monitored by measurement of the transepithelial resistance (TER) at 24 hr intervals using an impedance meter. TER in untreated controls was 50 omega/cm2 on day 1, increased progressively to 80 omega/cm2 by day 7 and plateaued until day 12. The cells treated from day 1 with CQ showed dose-dependent progressive increase in TER until day 12, reaching 191 omega/cm2 in cells treated with 1 microM concentration. In cells treated with CQ starting from day 7 of culture onwards, TER patterns were similar to those noted following exposure to chloroquine from day 1. Also in cultures containing NH4Cl, in comparison to the control, the increase in TER was significantly higher. These observations demonstrate that CQ and HN4Cl promote tight junction formation between immature rat Sertoli cells invitro suggesting that antiproteases may be involved in the formation of blood-testis barrier. Show less

Although total limb volume measurements are used to track the progress of lymphedema and its treatment, these measurements can be confounded by changes other than fluid excess namely muscle or fat gain. Bioelectrical impedance analysis (BIA) is a technique that specifically quantifies both total body fluid and extracellular fluid in extremities. Whereas BIA has potential as a quick, inexpensive, and quantitative technique to measure directly fluid gain or loss from lymphedema, it also has certain shortcomings that must be addressed before it can be validated. this paper examines the back-ground that explains why measuring total limb volume is insufficient to quantify the extent of peripheral lymphedema and explores the advantages and drawbacks of using BIA for this purpose. Show less

The prostatic epithelium consists principally of basal epithelial cells, luminal epithelial cells, and neuroendocrine cells. Several studies support the concept that among basal cells, a subpopulation of stem cells resides which is capable of giving rise to other stem cells, basal epithelial cells, and also luminal epithelial cells and neuroendocrine cells. Other investigators suggest that luminal epithelial cells can also regenerate prostatic epithelium. Availability of pure populations of basal and luminal epithelial cells will aid in studies on defining the cellular pathways of differentiation during normal and pathological conditions. This study was designed to isolate and characterize pure populations of basal and luminal epithelial cells from adult rat ventral prostates. Sequential enzymatic digestion and differential plating permitted the separation of glandular epithelial cells from stromal cells. The glandular epithelial cells were subjected to the STAPUT technique. Two types of cell populations, a large single-cell population and a small single-cell population, were obtained and characterized as basal and luminal epithelial cells by immunostaining for cytokeratin 5 and cytokeratin 8, respectively. Our results indicate that purified populations of prostatic basal and luminal epithelial cells can be isolated by the STAPUT technique. Show less