The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression.

The Snail family of transcription factors has previously been implicated in the differentiation of epithelial cells into mesenchymal cells (epithelial-mesenchymal transitions) during embryonic development. Epithelial-mesenchymal transitions are also determinants of the progression of carcinomas, occurring concomitantly with the cellular acquisition of migratory properties following downregulation of expression of the adhesion protein E-cadherin. Here we show that mouse Snail is a strong repressor of transcription of the E-cadherin gene. Epithelial cells that ectopically express Snail adopt a fibroblastoid phenotype and acquire tumorigenic and invasive properties. Endogenous Snail protein is present in invasive mouse and human carcinoma cell lines and tumours in which E-cadherin expression has been lost. Therefore, the same molecules are used to trigger epithelial-mesenchymal transitions during embryonic development and in tumour progression. Snail may thus be considered as a marker for malignancy, opening up new avenues for the design of specific anti-invasive drugs.

Targeting of Salmonella typhimurium to vesicles containing lysosomal membrane glycoproteins bypasses compartments with mannose 6-phosphate receptors.

Salmonella typhimurium is an intracellular bacterial pathogen that remains enclosed in vacuoles (SCV) upon entry into the host cell. In this study we have examined the intracellular trafficking route of S. typhimurium within epithelial cells. Indirect immunofluorescence analysis showed that bacteria initiated fusion with lysosomal membrane glycoprotein (lgp)-containing compartments approximately 15 min after bacterial internalization. This process was completed approximately 75 min later and did not require microtubules. Cation-independent (CI)- or cation-dependent (CD)-mannose 6-phosphate receptors (M6PRs) were not observed at detectable levels in SCV. Lysosomal enzymes showed a different distribution in SCV: lysosomal-acid phosphatase (LAP) was incorporated into these vacuoles with the same kinetics as lgps, while cathepsin D was present in a low proportion (approximately 30%) of SCV. Uptake experiments with fluid endocytic tracers such as fluorescein-dextran sulphate (F-DX) or horseradish-peroxidase (HRP) showed that after 2 h of uptake, F-DX was present in approximately 75% of lgp-containing vesicles in uninfected cells, while only approximately 15% of SCV contained small amounts of the tracer during the same uptake period. SCV also showed only partial fusion with HRP-preloaded secondary lysosomes, with approximately 30% of SCV having detectable amounts of HRP at 6 h after infection. These results indicate that SCV show limited accessibility to fluid endocytic tracers and mature lysosomes, and are therefore functionally separated from the endocytic route. Moreover, the unusual intracellular trafficking route of S. typhimurium inside epithelial cells has allowed us to establish the existence of two different lgp-containing vesicles in Salmonella-infected cells: one population is separated from the endocytic route, fusogenic with incoming SCV and may arise from a secretory pathway, while the second involves the classical secondary or mature lysosomes.

Comparative genomics of Listeria species.

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

Evidence for a detrimental role of nitric oxide synthesized by endothelial nitric oxide synthase after peripheral nerve injury.

Physical injury to a nerve is the most common cause of acquired peripheral neuropathy. Identification of molecules involved in degenerative and regenerative processes is a key step toward development of therapeutic tools in order to accelerate motor, sensory and/or autonomic function recovery. We have studied the role of nitric oxide (NO) using as a model the severe crushing of a motor nerve in adult rats. This type of injury up-regulates the three isoforms of nitric oxide synthase (NOS) in the affected nerve. Chronic systemic inhibition of NOS accelerated the onset of functional muscle reinnervation evaluated by the recording of compound muscle action potential evoked by electrical stimulation of the injured nerve. Besides, it increased the number of back-labeled motoneurons by application, 2 days after injury, of a retrograde marker 10 mm distal to the crushing site. These effects were mimicked by chronic specific inhibition of the endothelial isoform of nitric oxide synthase (eNOS), but not by specific inhibitors of the neuronal or inducible isoform. Next, we intraneurally injected a replication-deficient adenoviral vector directing the expression of a dominant negative mutant of eNOS (Ad-TeNOS). A single injection of Ad-TeNOS on the day of crushing significantly accelerated functional recovery of neuromuscular junction and increased axonal regeneration. Moreover, Ad-TeNOS did not compromise motoneuron viability or stability of reestablished neuromuscular junctions. Taken together, these results suggest that NO of endothelial origin slows down muscle reinnervation by means of detrimental actions on axonal regeneration after peripheral nerve injury. These experiments identify eNOS as a potential therapeutic target for treatment of traumatic nerve injuries and highlight the potential of gene therapy in treating injuries of this type using viral vectors to suppress the activity of eNOS.

Transformation and pH homeostasis of fibroblasts expressing yeast H(+)-ATPase containing site-directed mutations.

Mouse fibroblasts expressing a yeast proton-pumping ATPase show tumorigenic transformation (R. Perona, and R. Serrano, Nature (London) 334:438-440, 1988). By expressing site-directed mutations of the yeast ATPase with different levels of activity, a close correlation has been found between enzyme activity, tumorigenic transformation, and intracellular pH measured by weak-acid distribution. Fibroblasts expressing the yeast proton-pumping ATPase showed increased capability to grow at acidic pH and to resist lethal acidification mediated by reversal of the Na(+)-H+ antiporter. Measurements with microelectrodes in individual cells demonstrated electrical hyperpolarization and confirmed the increased pH of cells expressing yeast ATPase. These results indicate that the yeast enzyme expressed in mouse fibroblasts has electrogenic proton-pumping activity and that this activity deregulates fibroblast growth. This suggests a connection between the biophysical phenomena of proton transport, intracellular pH, and membrane potential and the biochemical regulatory circuits based on protein kinases and transcription factors.

Regulation of yeast H(+)-ATPase by protein kinases belonging to a family dedicated to activation of plasma membrane transporters.

The regulation of electrical membrane potential is a fundamental property of living cells. This biophysical parameter determines nutrient uptake, intracellular potassium and turgor, uptake of toxic cations, and stress responses. In fungi and plants, an important determinant of membrane potential is the electrogenic proton-pumping ATPase, but the systems that modulate its activity remain largely unknown. We have characterized two genes from Saccharomyces cerevisiae, PTK2 and HRK1 (YOR267c), that encode protein kinases implicated in activation of the yeast plasma membrane H(+)-ATPase (Pma1) in response to glucose metabolism. These kinases mediate, directly or indirectly, an increase in affinity of Pma1 for ATP, which probably involves Ser-899 phosphorylation. Ptk2 has the strongest effect on Pma1, and ptk2 mutants exhibit a pleiotropic phenotype of tolerance to toxic cations, including sodium, lithium, manganese, tetramethylammonium, hygromycin B, and norspermidine. A plausible interpretation is that ptk2 mutants have a decreased membrane potential and that diverse cation transporters are voltage dependent. Accordingly, ptk2 mutants exhibited reduced uptake of lithium and methylammonium. Ptk2 and Hrk1 belong to a subgroup of yeast protein kinases dedicated to the regulation of plasma membrane transporters, which include Npr1 (regulator of Gap1 and Tat2 amino acid transporters) and Hal4 and Hal5 (regulators of Trk1 and Trk2 potassium transporters).

Three singleton deliveries with healthy children from one couple after cryo-tESE and ICSI.

UNLABELLED: We report on a couple who delivered three healthy babies in three deliveries after cryo-TESE combined with ICSI. The male patient suffers from congenital bilateral absence of the vas deferens (CBAVD). METHODS: Three testicular sperm extraction (TESE) operations were performed in the male accompanied by six stimulated ICSI cycles in the female patient. Altogether, 59 oocytes were retrieved. Fifty-one oocytes (86%) were in metaphase II and 38 fertilized regularly (75%). Sixteen embryos, in the 3-6 cell stage, were transferred to the uterus. RESULTS: The first, fifth and sixth embryo transfers of fresh embryos led to intact intrauterine singleton pregnancies. The pregnancy and implantation rates with fresh embryos were 50% and 20%, respectively. CONCLUSIONS: TESE or microscopic epididymal sperm aspiration in patients with CBAVD in combination with a healthy female partner is likely to yield very good results in ICSI/ET. As azoospermia can be caused by cystic fibrosis and cystic fibrous transmembrane conductance regulator gene mutation range varies dramatically in patients of different ethnic groups.

[The influence of the T stage on the risk of metastasis of penis cancer: T1 vs. T2]. / Der Einfluss des T-Stadiums auf das Metastasierungsrisiko des Peniskarzinoms: T1 vs. T2.

BACKGROUND: Controversies persist over the therapeutic approach to T1 penile carcinoma, particularly in patients with negative inguinal lymph nodes. Available data on lymph nodes metastases (LNM) in T1 carcinoma are contradictory. The aim of this study was to evaluate the metastatic risk of T1 carcinoma and to compare it with that of T2 carcinoma. MATERIAL AND METHODS: A total of 37 patients (pts) with T1 or T2 tumors were reviewed. Assessment of the inguinal lymph node condition was based on node dissection in 29 pts and surveillance in eight pts (mean 62 months, range 22-162). RESULTS: Grading was classified as good (G1), moderate (G2) and poor (G3) in seven, 26 and four pts, respectively. Tumor stage was T1 in 21 and T2 in 16 pts. LNM were observed in eight of 21 T1 (38%) and six of 16 T2 tumors (38%). No G1 and all G3 tumors developed LNM independently of tumor stage. Ten of the 26 G2 carcinomas (38%) harboured LNM and seven of these pts (70%) had a T1 tumor. CONCLUSIONS: According to our data, the metastatic potential of T1 penile carcinoma has been underestimated in the recent literature. Tumor grading has a substantially stronger impact on the metastatic risk in T1 and T2 penile carcinoma than tumor stage, indicating a surgical lymph node staging starting at the pT1G2 stage.

[Induction of spermatogenesis in patients with non-obstructive azoospermia after antegrade sclerotherapy]. / Induktion der Spermiogenese nach antegrader Varikozelensklerosierung bei Patienten mit nicht-obstruktiver Azoospermie.

PURPOSE: The aim of this study was to prove the efficacy of antegrade sclerotherapy for varicocele testis in patients with azoospermia and in patients with cryptozoospermia (less than 0.1 million spermatozoons/mL ejaculate). We have investigated the induction of spermatogenesis in patients with non-obstructive azoospermia after antegrade sclerotherapy. MATERIALS AND METHODS: 20 consecutive patients who had been trying to beget a child over a period of one year or longer were chosen for this study. All patients suffered from non-obstructive azoospermia or from cryptozoospermia. We produced a control spermiogram for each patient before, 3 and 6 months after antegrade sclerotherapy. The postoperative spermiogram was done according to WHO criteria and was then compared to the preoperative data. RESULTS: 15 patients (75 %) were found to suffer from azoospermia preoperatively and 5 patients (25 %) from cryptozoospermia. Out of the 15 patients with initial azoospermia 8 (53 %) showed cryptozoospermia (OAT/OT syndrome) after antegrade sclerotherapy. Out of the 5 patients with the initial cryptozoospermia 3 (60 %) showed an improvement in the sperm count and motility criteria. CONCLUSIONS: Antegrade sclerotherapy for varicocele testis is a valid treatment option to isolate the spermatozoons from the ejaculate for extracorporeal fertilisation in patients with non-obstructive azoospermia. Complete normalisation of the spermiogram parameters, i.e., sufficient for natural child conception, cannot safely be achieved by this method.

The varied lifestyles of intracellular pathogens within eukaryotic vacuolar compartments.

Many bacterial pathogens and eukaryotic parasites can enter mammalian cells and live intracellularly inside membrane-bound vacuoles. The intravacuolar lifestyle of these pathogens plays a key role in pathogenesis. Understanding the molecular basis of the development of these specialized intracellular compartments is critical to understanding how these organisms cause disease.

Genetic characterization of the (534)DPPR motif of the yeast plasma membrane H(+)-ATPase.

The highly conserved motif +(534)DPPR of Saccharomyces cerevisiae H(+)-ATPase, located in the putative ATP binding site, has been mutagenized and the resulting 23 mutant genes conditionally expressed in secretory vesicles. Fourteen mutant ATPases (D534A, D534V, D534L, D534N, D534G, D534T, P535A, P535V, P535L, P535G, P535T, P535E, P535K and R537T) failed to reach the secretory vesicles. Of these mutants, nine (D534N, D534T, P535A, P535V, P535L, P535G, P535T, P535E and P535K) were not detected in total cellular membranes, and five (D534A, D534V, D534G, D534L and R537T) were retained at the endoplasmic reticulum and exhibited a dominant lethal phenotype. The remaining mutants (D534E, R537A, R537V, R537L, R537N, R537G, R537E, R537K and R537H) reached the secretory vesicles at levels similar to that of the wild type. Of these, six (R537A, R537V, R537L, R537N, R537G, and R537E) showed severely decreased ATPase activity compared to the wild type enzyme, and three (D534E, R537K and R537H) rendered an enzyme with an altered K(m) for ATP.

Regulation of plasma membrane H(+)-ATPase in fungi and plants.

The plasma membrane H+-ATPase from fungi and plants is a proton pump which plays a key role in the physiology of these organisms controlling essential functions such as nutrient uptake and intracellular pH regulation. In fungal and plant cells the activity of the proton pump is regulated by a large number of environmental factors at both transcriptional and post-translational levels. During the last years the powerful tools of molecular biology have been successfully used in fungi and plants allowing the cloning of a wide diversity of H+-ATPase genes and rapid progress on the molecular basis of reaction mechanism and regulation of the proton pump. This review focuses on recent results on regulation of plasma membrane H+-ATPase obtained by molecular approaches.

Catalytic and regulatory sites of yeast plasma membrane H(+)-ATPase studied by directed mutagenesis.

More than 35 site-directed mutants of the plasma membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae have been constructed and expressed to investigate the function of N- and C-termini and of conserved amino acids. Conserved motif TGES seems to form part of both the catalytic machinery for the hydrolysis of the phosphorylated intermediate and the vanadate binding site. In addition, it is involved in the coupling of ATP hydrolysis to H+ transport. The phosphorylated intermediate is also essential for this coupling, but not for ATP hydrolysis. The aspartate residues of conserved motifs DPPR, TGD and TGDGVND (the last one) seem to form part of the ATP binding site. The positive charge of the conserved motif KGAP is important for the kinase or phosphorylating activity. A conserved proline and a conserved aspartate predicted to have a transmembrane location are essential for activity. The N-terminus contains a conserved acidic region which may be involved in assembly into the plasma membrane. All the hydrophobic stretches at the C-terminus are also required for assembly. The last 11 amino acids constitute a non-essential inhibitory domain involved in regulation of the enzyme by glucose metabolism.

Purification and properties of three intracellular proteinases from Candida albicans.

Three intracellular proteinases termed A, B and C were purified to homogeneity from the unicellular form of the yeast Candida albicans. Enzyme A is an aspartic proteinase that acts on a variety of proteins. Its optimal pH is around 5 and it is displaced to 6.5 by KSCN. It is not significantly inhibited by PMSF, TLCK (Tos-Lys-CHCl2) or soybean trypsin inhibitor but it is inhibited by pepstatin. Its molecular weight is 60 000. Enzyme B is a dipeptidase that acts on esters or on dipeptides without blocks in either the carboxyl or amino ends. Its pH optimum is around 7.5 and the molecular weight is 57 000. It is inhibited by PMSF, TLCK and DANME (N2Ac-Nle-OMe). Proteinase C is an aminopeptidase with an optimum pH around 8. Its molecular weight was 67 000 when determined by SDS gel electrophoresis and 243 000 when determined by gel weight was 67 000 when determined by SDS gel electrophoresis and 243 000 when determined by gel filtration. It is active towards dipeptides in which at least one amino acid is apolar and is not active when the N-terminal amino acid is blocked. It is inhibited by EDTA or o-phenanthroline and activated by several divalent cations.

Characterization of non-dominant lethal mutations in the yeast plasma membrane H+-ATPase gene.

Site-directed mutants of yeast ATPase were previously studied after introduction of mutant alleles into a yeast strain where these alleles were constitutively expressed while the expression of the wild-type chromosomal ATPase gene was turned off. As a functional H+ pump is essential, strong selective pressure leads to the accumulation of revertants during growth of cells harboring variants with low activity. Thus, constitutive expression of the mutant gene can select phenotypes which reflect events such as gene conversion or reversion. We have therefore re-evaluated the phenotypes of non-dominant lethal alleles in an alternative set of conditional expression systems. We show that eight of 11 previously described site-directed mutations behave as recessive lethal alleles.

The cell wall integrity/remodeling MAPK cascade is involved in glucose activation of the yeast plasma membrane H(+)-ATPase.

Glucose triggers transcriptional and post-transcriptional mechanisms that increase the amount and the activity of Saccharomyces cerevisiae plasma membrane H(+)-ATPase. In a previous study, we found that a mutation in the Rsp5 ubiquitin-protein ligase enzyme affected the post-transcriptional activation of the enzyme by glucose. Mutations at the RSP5 locus alter the glucose-triggered K(m) decrease. In a genetic screening for multicopy suppressors of the rsp5 mutation, we identified the WSC2/YNL283c gene. Deletion of the WSC2 gene disturbs ATPase activation by glucose, abolishing the K(m) decrease that occurs during this process. Wsc2 is a component of the PKC1-MPK1 mitogen-activated protein kinase (MAPK) signaling pathway that controls the cell wall integrity. Deletion of the MPK1/SLT2 gene disturbs the glucose-triggered K(m) decrease in ATPase.

Sequencing and heterologous expression in Saccharomyces cerevisiae of a Cryptococcus neoformans cDNA encoding a plasma membrane H(+)-ATPase.

A cDNA containing an open reading frame encoding a putative plasma membrane H(+)-ATPase in the human pathogenic basidiomycetous yeast Cryptococcus neoformans was cloned and sequenced by means of PCR and cDNA library hybridization. The cloned cDNA is 3475 bp in length, containing a 2994 bp open reading frame encoding a polypeptide of 997 amino acids. As in the case of another basidiomycetous fungus (Uromyces fabae), the deduced amino acid sequence of CnPMA1 was found to be more homologous to those of P-type H(+)-ATPases from higher plants than to those from ascomycetous fungi. In order to prove the sequenced cDNA corresponds to a H(+)-ATPase, it was expressed in Saccharomyces cerevisiae and found to functionally replace its own H(+)-ATPase. Kinetic studies of CnPMA1 compared to ScPMA1 show differences in V(max) values and H(+)-pumping in reconstituted vesicles. The pH optimum and K(m) values are similar in both enzymes.

Characterization of an allele-nonspecific intragenic suppressor in the yeast plasma membrane H+-ATPase gene (Pma1).

We have analyzed the ability of A165V, V169I/D170N, and P536L mutations to suppress pma1 dominant lethal alleles and found that the P536L mutation is able to suppress the dominant lethality of the pma1-R271T, -D378N, -D378E, and -K474R mutant alleles. Genetic and biochemical analyses of site-directed mutants at Pro-536 suggest that this amino acid may not be essential for function but is important for biogenesis of the ATPase. Proteins encoded by dominant lethal pma1 alleles are retained in the endoplasmic reticulum, thus interfering with transport of wild-type Pma1. Immunofluorescence studies of yeast conditionally expressing revertant alleles show that the mutant enzymes are correctly located at the plasma membrane and do not disturb targeting of the wild-type enzyme. We propose that changes in Pro-536 may influence the folding of the protein encoded by a dominant negative allele so that it is no longer recognized and retained as a misfolded protein by the endoplasmic reticulum.

[Pathophysiology and rehabilitation of erectile dysfunction after nerve-sparing radical prostatectomy]. / Pathophysiologie und Rehabilitation der erektilen Dysfunktion nach nerverhaltender radikaler Prostatektomie.

Radical prostatectomy is the current standard procedure for locally confined prostate cancer and accounts for the largest portion of invasive therapies. However, a major drawback of this approach remains the frequently ensuing postoperative erectile dysfunction. This aspect represents a frequent cause of fear and concern both for the patients and their partners and has a significant impact on the choice of therapy.After bilateral sparing of the neurovascular bundles, an average of 50% of the patients is likely to complain of erectile dysfunction. It is only in the course of the first 2 years after prostatectomy that rehabilitation of erectile dysfunction can be expected. It is all the more crucial to begin with rehabilitation therapy of the erectile tissue at an early postoperative stage to the prevent an irretrievable loss of erectile function. Application of PDE-5 inhibitors as well as prostaglandins, phentolamine, or papaverine can help to induce and to support penile blood perfusion and oxygenation, thus preserving structure and function of the corpora cavernosa. All efforts must be directed towards keeping the erectile function at the level ascertained prior to the intervention.

[Erectile function after nerve-sparing radical prostatectomy. Nocturnal early erection as a parameter of postoperative organic erectile integrity]. / Erektionsstatus nach nervenerhaltender radikaler Prostatektomie. Nächtliche Früherektion als Parameter der postoperativen organisch-erektilen Integrität.

The time lapse before recovery of erectile function after nerve-sparing radical prostatectomy is still under debate. Several pathophysiologies are postulated for postoperative erectile function rehabilitation. In prospective studies we measured nocturnal penile tumescence (NPTR) in the acute phase during the first night after catheter removal subsequent to nerve-sparing radical prostatectomy to assess the neuronal organic erectile integrity. Eighteen sexually active patients suffering from local prostate cancer underwent bilateral and unilateral nerve-sparing retropubic radical prostatectomy. All patients completed an IIEF-5 questionnaire concerning erectile function preoperatively. The transurethral catheter was removed 14 days after surgery, and nocturnal penile tumescence was measured with an erectometer (Rigi-Scan) in each patient during the following night. None of these patients received any comedication interacting with erectile function. The preoperative IIEF score was >18 in all patients. After catheter removal, 17 of 18 patients (95%) had nocturnal penile radial rigidity >70% that persisted for >10 min during one night. In a control of four patients without a nerve-sparing procedure, no nocturnal erections were recorded. The measurement of NPTR in the acute phase after nerve-sparing radical prostatectomy showed retained erectile function even during the "first" night after catheter removal. Our findings are important for an appropriate choice of pharmacotherapy for optimal recovery of erectile function. In cases of early penile erections, the cavernous nerve had been well preserved during surgery providing good neuronal integrity, and PDE-5 inhibitors can support organic rehabilitation of the corpus cavernosum. In the absence of early penile erections, the neuronal integrity of the cavernous nerve is presumed to be impaired. In this case, additional injection therapy should be chosen to support recovery of spontaneous erectile function.