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This study evaluated perioperative and postoperative variations in serum CgA levels induced by radical retropubic prostatectomy (RRP) and their relationship with serum PSA levels in prostate cancer patients. Thirty consecutive patients with clinically localized adenocarcinoma of the prostate undergoing RRP were prospectively analyzed. Serum levels of CgA and total PSA were analyzed in each case preoperatively (time 0), at removal of the prostate (time 1), 1 h after the end of RRP (time 2) and then at regular postoperative intervals till 12 weeks (time 14). During the postoperative period no adjuvant therapies were performed and none of the 30 cases showed biochemical (PSA > 0.2 ng/mL) and/or clinical progression. Mean preoperative serum levels of CgA were 57 +/- 14 ng/mL. Immediately after the surgical removal of the prostate gland (time 1), in all 30 cases there was a significant (time 0-time 1: p = .001) increase in serum PSA, but a nonsignificant modification in serum CgA levels (60 +/- 15 ng/mL). After time 1, serum PSA levels progressively decreased to below the detection limit of 0.2 ng/mL. On the contrary, at time 2, serum CgA levels were postoperatively increased (time 2 = 145 +/- 47) and they remained significantly higher than preoperative values (time 0) till the 21-day postoperative interval (time 11). Moreover, at the last control (time 14) mean and median CgA levels were very similar to those shown preoperatively (time 14: 58 +/- 18 ng/mL). In patients with untreated clinically localized adenocarcinoma of the prostate submitted to RRP, surgical and postoperative stress, more than surgical manipulation of the prostate gland, could produce a significant increase in serum CgA levels maintained for a longer period when compared to the increase in serum PSA levels.

Chorea-Acanthocytosis (CHAC) is an autosomal recessive disease characterized by neurodegeneration and acanthocytosis. Enhanced creatine kinase concentration is a constant feature of the condition. The mechanism underlying CHAC is unknown. However, acanthocytosis and enhanced creatine kinase suggest a protein defect that deranges the membrane-cytoskeleton interface in erythrocytes and muscle, thereby resulting in neurodegeneration. Acanthocytes have been correlated with structural and functional changes in membrane protein band 3--a ubiquitous anion transporter. Residue Gln-30 of band 3 serves as a membrane substrate for tissue transglutaminase (tTGase), which belongs to a class of intra- and extra-cellular Ca2+-dependent cross-linking enzymes found in most vertebrate tissues. In an attempt to cast light on the pathophysiology of CHAC, we used reverse-phase HPLC and immunohistochemistry to evaluate the role of tTGase in this disorder. We found increased amounts of tTGase-derived N(epsilon)-(-gamma-glutamyl)lysine isopeptide cross-links in erythrocytes and muscle from CHAC patients. Furthermore, immunohistochemistry demonstrated abnormal accumulation of tTGase products as well as proteinaceous bodies in CHAC muscles. These findings could explain the mechanisms underlying the increased blood levels of creatine kinase and acanthocytosis, which are the most consistent features of this neurodegenerative disease.

We present data collected among men attending a free call service on information on erectile dysfunction (ED) activated in Italy during the period 1997-1999. Their attitudes towards discussion with their partner and physician about the condition are considered. Each subject, was asked if he was affected by ED (defined as inability to achieve and maintain an erection sufficient for satisfactory sexual performance). In the case of a positive answer, the subject was asked if he had ever discussed his condition with partner or a physician. A total of 12 761 subjects with ED called the service: 7265 (56.9%) reported to have discussed their condition with their partner. The proportion tended to increase with duration of ED, being 47.9% in subjects reporting ED lasting <6 months and 59.9% in those reporting ED lasting >3 y (w(2)(1) trend <0.05). Likewise, the proportion of subjects reporting to have discussed ED with a physician was 50.3% (6416 subjects), being 33.6% in subjects with ED lasting <6 months and 57.9% of those with ED lasting >3 y (w(2)(1) trend, P<0.01).

Objective: To underline the role of antegrade sclerotherapy as first choice treatment in recurrent varicocele., Methods: In 53 patients, aged 11-38 years, observed over a 30-month period, antegrade sclerotherapy was carried out for the treatment of recurrent varicocele. In seven of these patients, varicocele was bilateral. The right varicocele, however, had not been previously detected and these patients were, therefore, submitted to simultaneous bilateral antegrade sclerotherapy., Results: Antegrade sclerotherapy was feasible in all 53 patients (60 varicoceles) and no significant complications were observed. Unsuccessful results occurred in only two out of the 55 varicoceles observed over a minimum follow-up period of six months., Conclusions: Whilst antegrade sclerotherapy is recognized as a low-cost, safe and effective method in the management of varicocele, the success rate in recurrent varicocele is even greater than in primary varicocele. If, in the preceding treatment, the internal spermatic vein has presumably been occluded (percutaneous retrograde sclerotherapy, surgical or laparoscopic retroperitoneal ligation of the entire bundle), antegrade sclerotherapy should be considered the treatment of choice.

Penile metastases from bladder cancer are unusual. A case of a man, 50 years old, with undifferentiated bladder carcinoma submitted to radical cystoprostatectomy and ileal conduit is presented. Twelve months after the primary diagnosis the patient presented metastases of the penis.

Coeliac disease (CD) is one of the most common food intolerances described in the western population. The main food agent that provokes the strong and diffuse clinical symptoms has been known for several years to be gliadin, a protein present in a very large number of human foods derived from vegetables. Only recently, some biochemical and immunological aspects of this very common disease have been clarified, and tissue transglutaminase, a multifunctional and ubiquitous enzyme, has been identified as one of the major factors. This enzyme, through its catalytic activities, produces the main biochemical and immunological effects observed in patients affected by this disease. The aim of this review is to summarize the most recent findings concerning the relationships between the biochemical properties of tissue transglutaminase and the basic molecular mechanisms responsible for CD. In addition, we present some frequent clinical associations of CD with other human diseases, with particular reference to neuropsychiatric disorders. Possible molecular links between CD, neuropsychiatric disorders and biochemical activities of transglutaminase enzymes are discussed.

Protein aggregates are a hallmark of Huntington's disease (HD) and other inherited neurodegenerative diseases caused by an elongated (CAG)(n) repeat in the genome and to a corresponding increase in the size of the Q(n) domain in the expressed protein. When the protein associated with HD (huntingtin) contains <35 Q repeats disease does not occur. However, an n>/=40 leads to disease. Some investigators have proposed that aggregates in the nuclei of affected cells are toxic, but other workers have suggested that the aggregates may be neutral or even protective. Whether or not they are toxic, an understanding of the processes whereby the aggregates develop may shed light on the neuropathological processes involved in the (CAG)(n)/Q(n)-expansion disorders. Q(n) domains have a tendency to non-covalently self align as 'polar zippers' rendering them less soluble, but evidence that such polar zippers occur in the aggregates in intact HD brain has so far been limited. The human brain contains at least three Ca(2+)-dependent enzymes (transglutaminases, TGases) that catalyze protein cross-linking reactions, namely TGase 1, TGase 2 (tissue transglutaminase, tTGase) and TGase 3. Q(n) aggregates have been found by several groups to be excellent substrates of tTGase. Moreover, the activity toward the Q(n) domains increases greatly as n is increased to 40 or beyond. tTGase mRNA and total TGase activity are elevated in HD brain. Moreover, some evidence suggests that Ca(2+) homeostasis is disrupted in HD brain. We propose that the combination of increased huntingtin (or huntingtin fragment containing the Q(n) domain) in the nucleus, increased the ability of the Q(n) domains to act as substrate, increased Ca(2+) levels and increased inherent TGase activity all contribute to increased cross-linking of proteins in HD brain. At first the proteasome machinery can recognize and degrade the cross-linked proteins, but over time the proteasome machinery may be overwhelmed and protein aggregates will accumulate.

Transglutaminases (Enzyme Commission 2.3.2.13) are a large family of enzymes that show the common capacity to catalyze cross-linking of protein substrates. Some members of this family of enzymes are also capable of catalyzing other reactions important for the cell life. The distribution and the role of these enzymes have been widely studied in numerous cell types and tissues, but only recently their expression and functions started to be investigated in the central nervous system. One of the main biochemical properties of the transglutaminase enzymes is to form large protein aggregates that are insoluble in all known protein detergents, such as urea, guanidinium, and sodium dodecyl sulfate. Recently, the transglutaminase activity has been hypothesized to be involved in the pathogenetic mechanisms responsible for the formation of cellular inclusions present in Huntington disease and in all the other polyglutamine (polyQ) diseases hitherto identified, such as spinobulbar muscular atrophy or Kennedy disease, spinocerebellar ataxias (SCA-1, SCA-2, SCA-3 or Machado-Joseph disease, SCA-6 and SCA-7) and dentatorubropallidoluysian atrophy. In this review we describe the biochemical properties of the transglutaminase enzymes and some recent findings about the physiopathological roles played by these enzymes in the central nervous system.

The aim of the present study was to evaluate the influence of physical exercise on seminal parameters of male athletes with varicocele. Sixty healthy male volunteers (athletes and non-athletes, n = 30 + 30) and 60 volunteers affected by varicocele (athletes and non-athletes, n = 30 + 30) were randomly selected for a clinical study. All subjects provided at least two semen samples for routine microscopic analysis. Determinations for basal luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin, oestradiol, total and free testosterone under resting conditions were also performed. In both groups with varicocele the percentage of total and progressive forward sperm motility and the percentage of normal spermatozoa were significantly reduced. The percentage of both progressive forward motility and normal spermatozoa were significantly lower in athletes with varicocele compared with non-athletes with varicocele (P < 0.05). Only athletes with varicocele had mean left testis volume significantly lower than the contralateral testis (P < 0.05). No modifications of hormonal parameters at rest were observed in any groups. Physical activity might represent an aggravating factor for spermatogenesis in athletes with varicocele. In countries where sport eligibility is granted by an authoritative body, these results suggest the need to establish general medical criteria to guarantee the continuation of an athlete's training whilst at the same time taking care of his reproductive health.

Background: Erectile dysfunction is one of the factors influencing negatively the quality of life of patients in hemodialytic treatment. The international literature shows that erectile dysfunction is present in 30% of patients with chronic renal failure and in 50% of patients undergoing dialytic treatment. Fertility, libido and erectile dysfunction, suffer progressive worsening with time, in spite of hemodialysis. The availability of a drug like Sildenafil can improve the quality of life of the patient and give him a normal sexual activity., Methods: Twenty patients between 29 and 51 years, were selected; 2 of these had been subjected to renal transplant, with a dialytic treatment time varying from 3 to 13 years. Before the treatment all the patients have been subjected to an andrological screening (testosterone, prolactin, penile color Doppler ultrasound) and proposed the IIEF test. Therapeutic strategy included the assumption of the drug in the days in which the patients were not subjected to dialysis, with an interval from 1 to 3 weeks between assumption and another. The dose was 25-50 mg. At the end of three months of therapy the patients were again subjected to the IIEF test., Results: All patients reported an improvement in sexual activity and sexual desire with very good repercussions on general and psychophysical conditions., Conclusions: The results demonstrate at least that Sildenafil is also effective in uremic patients in dialytic treatment or after renal transplant and that it can therefore resolve one of the main problems for the normal development of the life of such patients.

A study of antibody levels (in saliva and blood) against common vaccine antigens was performed in a population of 32 children suffering from recurrent upper respiratory tract infections (URTI). None of the patients had primary or secondary immunodeficiency syndromes or other known predisposing factors for respiratory diseases. Titres of the isotype-specific antibodies immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin G (IgG) against two vaccine antigens--poliovirus type 3 (P3) and tetanus toxoid (TT), a viral antigen and a bacterial antigen, respectively--were measured in unstimulated saliva and serum, both in patients and in 24 healthy children (controls), by using a standard enzyme-linked immunosorbent assay (ELISA). In addition, levels of total IgA and avidity of IgA antibodies to both P3 and TT in saliva were evaluated. No difference was found between patients and controls as to levels of total IgA, or specific IgA and IgM antibodies against both P3 and TT in saliva. Furthermore, the avidity of salivary IgA antibodies against the two antigens did not differ between the two populations. However, the average concentrations of saliva-specific IgG antibodies to both the viral and the bacterial antigen were significantly lower (p <0.01 for P3 and p <0.05 for TT, respectively) in saliva of children with recurrent URTI, whereas no difference was found in serum for any immunoglobulin isotype determined compared with healthy individuals. The results of the present study provide suggestive evidence for the existence of subtle IgG-restricted defects in antibody responses at the mucosal level, but not at the serum level, in some children with undue susceptibility to URTI.

A review has been made on the role of nitric oxide in the physiology and pathophysiology of penis, bladder, prostate and the nervous structures involved in the urinary control. NO is an essential mediator in penile erection and his action can be modulated by sildenafil. Nitric oxide could be involved in bladder detrusor relaxation and in the development of interstitial cystitis. Little is known about the role of nitric oxide in the physiology and pathophysiology of the prostate: this molecule is released by the epithelial and stromal cells of the prostate, and by the prostatic nerves. Actually some studies hypothesize a role played by nitric oxide in benign prostatic hyperplasia development.

We treated primary epithelial cells from human normal prostate (NEPC) and prostate cancer (CEPC) with all-trans-retinoic acid (RA) to study whether it regulates the activity of tissue transglutaminase (tTGase), an enzyme that accumulates in cells undergoing apoptosis. tTGase activity was assessed by [14C]spermidine incorporation; tTGase, P53, Bcl-2, and p21 protein levels were evaluated by Western blotting; and RA receptors (RAR alpha, -beta, and -gamma), tTGase, retinol-binding protein (RBP), and cellular RBP type I transcripts were determined by semiquantitative RT-PCR. After 72-96 h of 10(-6) mol/L RA treatment, cell growth inhibition and apoptosis were associated with increased tTGase activity in both NEPC and CEPC, and with increased tTGase protein and messenger ribonucleic acid levels only in NEPC. Moreover, RA down-regulated RAR alpha and -beta and increased RBP messenger ribonucleic acid levels in NEPC, whereas it increased RAR beta gene expression and decreased Bcl-2 protein levels in CEPC. Our results suggest that RA induces tTGase gene expression and enzyme activity in normal prostate cells, and that RA-regulated pathways are impaired in cancer cells. Moreover, down-regulation of Bcl-2 protein and up-regulation of RAR beta suggest that retinoid may act on the genetic defect responsible for prostate cancer progression.

At least eight neurodegenerative diseases, including Huntington disease, are caused by expansions in (CAG)n repeats in the affected gene and by an increase in the size of the corresponding polyglutamine domain in the expressed protein. A hallmark of several of these diseases is the presence of aberrant, proteinaceous aggregates in the nuclei and cytosol of affected neurons. Recent studies have shown that expanded polyglutamine (Qn) repeats are excellent glutaminyl-donor substrates of tissue transglutaminase, and that the substrate activity increases with increasing size of the polyglutamine domain. Tissue transglutaminase is present in the cytosol and nuclear fractions of brain tissue. Thus, the nuclear and cytosolic inclusions in Huntington disease may contain tissue transglutaminase-catalyzed covalent aggregates. The (CAG)n/Qn-expansion diseases are classic examples of selective vulnerability in the nervous system, in which certain cells/structures are particularly susceptible to toxic insults. Quantitative differences in the distribution of the brain transglutaminase(s) and its substrates, and in the activation mechanism of the brain transglutaminase(s), may explain in part selective vulnerability in a subset of neurons in (CAG)n-expansion diseases, and possibly in other neurodegenerative disease. If tissue transglutaminase is found to be essential for development of pathogenesis, then inhibitors of this enzyme may be of therapeutic benefit.

Tissue transglutaminase (tTGase) is a GTP-binding Ca(2+)-dependent enzyme which catalyses the post-translational modification via epsilon(gamma-glutamyl)lysine bridges. The physiological role of tTGase is not fully understood. It has been shown that in cartilage the expression of tTGase correlates with terminal differentiation of chondrocytes. Recent evidence suggests that the GTP-binding activity of tTGase may play a role in the control of cell cycle progression thus explaining some of the suggested roles for the enzyme.tTGase activity is present in primary cultures of epiphyseal chondrocytes and increases transiently upon retinoic acid (RA) treatment. Increase in enzyme activity occurs upon RA addition and is accompanied by a parallel increase in protein and mRNA levels. Stimulation of tTGase expression by RA correlates with suppression of cell growth and occurs independently of cell adhesion and cell differentiation.tTGase expression is not observed in MC2, a permanent chondrocyte cell line derived from retrovirus infected chondrocytes. RA treatment fails to activate tTGase expression in MC2 cells and to completely suppress cell proliferation. Our findings lend support to the idea that tTGase might play a role in non-dividing cultured chondrocytes., (Copyright 1999 Academic Press.)

Background: Many pathogens may be responsible of Non Gonococcal Urethritis (NGU) with the possible occurrence of symptomatic and asymptomatic mixed viral and bacterial infections. In particular, genital papillomaviruses (HPVs) have been searched since they are linked to both benign and malignant lesions of the penis and urethra and the presence of a potential male carried state has received limited scrutiny while the screening of sexually active females has received substantial attention., Methods: In male patients affected by chronic NGU, the presence of DNA of Chlamydia trachomatis, herpes simplex virus (HSV) type 1 and 2 and human papillomaviruses by PCR and the occurrence of Gram positive and Gram negative micro-organisms, of Mycoplasma hominis and Ureaplasma urealyticum, by conventional cultural methods have been investigated., Results: Results obtained indicated a high percentage of mixed infections, up to 36%. Genital HPV DNA was detected in 31% of specimens positive for two or more agents, and HSV DNA was detected in 10% of studied population., Conclusions: The concomitant presence of different infectious agents could determine latent, sub-clinical or chronic infections with periodic reactivation. In particular results suggest that HPV and HSV may stimulate cytokine production which can up regulate the expression of other infectious agents and may be responsible for latent chlamydial infections characterised by the persistence of this micro-organism in an altered form, viable but in a culture negative state. Therefore an increased awareness of mixed infections is relevant to define the management and treatment of chronic urethritis.

Post-translational formation of hypusine in eukaryotic initiation factor 5A (eIF-5A) is essential for cell viability. Recently, we showed that hypusine protein is an in vitro substrate for transglutaminases (TGases). We report the effect of tissue TGase expression on the in vivo hypusine metabolic pathway. The stable expression of tTGase in BALB/c 3T3 cells induced a 100-fold reduction of hypusine levels and a 50% increase of gamma-glutamyl-omega-hypusine formation. Such changes were paralleled by a consistent decrease in the free polyamine pool and an enhancement of their excretion and of the formation of their gamma-glutamyl derivatives. These effects occurred together with a significant reduction of cell proliferation. In this report we suggest, for the first time, that tTGase affects hypusine metabolism, thus regulating the eIF-5A activity and cell proliferation.

To investigate possible biochemical mechanisms underlying the "toxic gain of function" associated with polyglutamine expansions, the ability of guinea pig liver tissue transglutaminase to catalyze covalent attachments of various polyamines to polyglutamine peptides was examined. Of the polyamines tested, spermine is the most active substrate, followed by spermidine and putrescine. Formation of covalent cross links between polyglutamine peptides and polyamines yields high-M(r) aggregates--a process that is favored with longer polyglutamines. In the presence of tissue transglutaminase, purified glyceraldehyde-3-phosphate dehydrogenase (a key glycolytic enzyme that binds tightly to the polyglutamine domains of both huntingtin and dentatorubral-pallidoluysian atrophy proteins) is covalently attached to polyglutamine peptides in vitro, resulting in the formation of high-M(r) aggregates. In addition, endogenous glyceraldehyde-3-phosphate dehydrogenase of a Balb-c 3T3 fibroblast cell line overexpressing human tissue transglutaminase forms cross-links with a Q60 polypeptide added to the cell homogenate. Possibly, expansion of polyglutamine domains (thus far known to occur in the gene products associated with at least seven neurodegenerative diseases) leads to increased/aberrant tissue transglutaminase-catalyzed cross-linking reactions with both polyamines and susceptible proteins, such as glyceraldehyde-3-phosphate dehydrogenase. Formation of cross-linked heteropolymers may lead to deposition of high-M(r) protein aggregates, thereby contributing to cell death.

Genetic defects of the CD95 (Fas/Apo-1) receptor/ligand system, has recently been involved in the development of human and murine autoimmunity. We investigated whether a deregulation of the ;tissue' transglutaminase (tTG), a multifunctional enzyme which is part of the molecular program of apoptosis, may act as a cofactor in the development of autoimmunity. We found that MRLlpr/lpr, which are characterized by a defect in the CD95 receptor and suffer of a severe systemic lupus erythematosus-like disease, produce large amounts of circulating tTG autoantibodies. This phenomenon is paralleled by an abnormal accumulation of an inactive enzyme protein in the accessory cells of lymphoid organs. To investigate the molecular mechanisms by which tTG inhibition may contribute to the development of autoimmunity we generated a cell culture model system consisting of L929 cells stably transfected with a full length tTG cDNA. When L929 cells were killed by Tumor Necrosis Factor alpha (TNFalpha) a pronounced release of DNA and Lactate Dehydrogenase (LDH) was observed. Overexpression of tTG in these cells largely prevented the leakage of macromolecules determined by TNFalpha treatment, an effect which is abolished by inactivating the enzyme cross-linking activity by a synthetic inhibitor. These in vitro observations provided the basis to explain the increased levels of plasmatic LDH we detected in MRLlpr/lpr mice. These data suggest that lack of an active tTG may represent a cofactor in the development of autoimmunity.

Stably transfected Balb-C 3T3 fibroblasts (clone 5), overexpressing a catalytically active tissue transglutaminase, showed a basal adenylate cyclase activity lower than control cells (clone 1). Several modulators of the adenylate cyclase activity (forskolin, Mn2+ and pertussis toxin) showed the existence of a marked negative control on the adenylate cyclase activity present in clone 5 cells. Very interestingly, this same marked negative control was also found in a Balb-C 3T3 fibroblast clone stably transfected with a mutagenized human tissue transglutaminase (mut277 cys > ser) virtually devoid of transglutaminase catalytic activity (clone Ser). Conversely, a significant increase of the adenylate cyclase activity was observed in bovine aortic endothelial cells after the lowering of tissue transglutaminase expression levels by the transfection of an eukaryotic expression vector containing the gene for tissue transglutaminase in antisense orientation. All these findings suggest a possible role for type II tissue transglutaminase as a negative modulator of the adenylate cyclase activity in different cell types, beside its transglutaminase enzyme activity.

It has been pediatric dogma that most children with cystic fibrosis (CF) have pansinusitis and few are symptomatic. To reassess this premise, we compared symptoms, clinical examinations, radiographic and surgical findings, and results of treatment in 19 children with cystic fibrosis who had undergone computed tomography at St. Christopher's Hospital (Philadelphia) from 1991 to 1994. We found two distinct patterns of sinus disease: chronic sinusitis (n = 3) and polyposis (n = 12). Two of the patients with polyposis had ethmoid mucoceles and 1 child had no sinus disease at all. Children with chronic sinusitis had headache as a major complaint, while those with polyposis suffered nasal obstruction alone unless a mucocele was present. Surgery provided marked and lasting improvement in the 14 patients who were operated on.

Sexually transmitted diseases (STDs) may recognize multiple etiological agents. Among them, Chlamydia trachomatis (Ct) and herpes simplex virus type 2 (HSV-2) cause symptomatic, subclinical and asymptomatic infections of the urogenital tract which can lead to serious sequelae. In the present study the coinfection and superinfection by Ct and HSV-2 in epithelial cultured cells from human cervix (HeLa 229) are described. A double infection, followed by the intracellular synthesis of chlamydial and viral antigens, was established. Both synergistic and interfering phenomena were recorded: viral antigen synthesis resulted increased whereas Ct inclusion bodies were produced to a lower extent.

The tunica albuginea and corpus cavernosum from patients with Peyronie's disease (PD), patients with veno-occlusive dysfunction (VOD), and those from normal control subjects were studied by transmission electron microscopy and immunohistochemical staining for type I, III, and V collagens, platelet-derived growth factor (PDGF) AA and BB homodimers, and PDGF alpha and beta receptors. Ultrastructural modifications resembling a fibrotic reaction were detected in the two pathological tunica albuginea, but not in those from control subjects. Ultrastructural data demonstrated a general increase in fibrous and amorphous extracellular matrix material in the pathological tunica albuginea. The amorphous material probably represents glycoproteins and proteoglycans. The fibrous material, representing collagen, appears disorganized in the tissue and does not display the typical and homogeneous diameter, size, and spatial arrangement. Large areas of extracellular and intracytoplasmic, partially degraded, fibers are visible. An increased type I/III collagen ratio was detected by immunohistochemistry in the two pathological tunica albuginea. Moreover, a strong expression of type V collagen, correlated to fibroblasts, was revealed. Fibroblasts from control tissues, on the other hand, were totally negative. Finally, PDGF AA and BB were positive in fibroblasts from pathological tunica albuginea but were negative in control tissues. PDGF beta receptor was positive in pathological and normal tissue fibroblasts. Tunica albuginea from PD and VOD show similar ultrastructural and immunohistochemical alterations, whereas the corpus cavemosum shows no visible modifications.

The main aim of this double-blind randomized crossover study was to compare the efficacy and safety of alprostadil sterile powder (Caverject) (PGE1) in a new sterile powder formulation versus placebo in producing erection in patients with erectile dysfunction. Each patient was treated with 5 or 10 micrograms PGE1 and placebo in random order. If the results of the three injections were unsatisfactory, 20 micrograms PGE1 was administered in an open fashion. A total of 45 patients were recruited and evaluated; 31/45 patients (68.8%) responded to at least one injection of alprostadil. A dose-response relation was observed during the double-blind phase; the 10-micrograms dose was effective in 55.5% of patients. The acceptability and tolerability of the preparation, evaluated both clinically and by laboratory tests, was very good. In particular, only four drug-related side effects were observed, three (penile burning, penile pain and pain after injection) after 10 micrograms and one (hematoma) after 5 micrograms PGE.

Tissue transglutaminase (tTgase) is a GTP-binding Ca(2+)-dependent enzyme which catalyses the post-translational modification of proteins via epsilon(gamma-glutamyl) lysine bridges. Recent evidence suggests that the GTP-binding activity of tTgase may be important in intracellular signaling thus explaining some of the diverse suggested roles for the enzyme. In the following work a malignant hamster fibrosarcoma (Met B) has been stably transfected with both the full length tTgase cDNA (wild type) and a mutant form of the cDNA whereby the active site cysteine (Cys 277) has been replaced by serine. Expression of this mutant cDNA leads to a protein with GTP binding activity which is deficient of protein crosslinking activity. When synchronised into S-phase and allowed to progress through the cell cycle tTgase transfected clones (both mutant and wild type), when compared to transfected controls, show a delayed progression from S-phase to G2/M when analysed by flow cytometry which appears to be elicited by the G-protein activity of the tTgase.

Tissue transglutaminase belongs to a family of calcium-dependent enzymes, the transglutaminases that catalyze the covalent cross-linking of specific proteins by the formation of epsilon (gamma-glutamyl)lysine isopeptide bonds. The goal of this study has been the isolation and characterization of the human tissue transglutaminase gene promoter. Genomic DNA clones, spanning the 5' region of the gene, were isolated and the structure of the 5'-end of the human tissue transglutaminase gene was determined. 1.74 kilobases of flanking DNA were sequenced and were found to contain a TATA box element (TATAA), a CAAT box element (GGACAAT), a series of potential transcription factor-binding sites (AP1, SP1, interleukin-6 response element), and a glucocorticoid response elements. Transient transfection experiments showed that this DNA fragment included a functional promoter, which is constitutively active in multiple cell types.

Reduced expression of the tissue transglutaminase in both murine and human tumours has been consistently associated with tumour growth and progression. To investigate the functional effects of transglutaminase expression we have transfected a constitutive human tissue transglutaminase expression construct into a highly malignant hamster fibrosarcoma cell line Met B. Met B clones expressing the exogenous tissue transglutaminase exhibited a reduced incidence of primary tumour formation and an increased adherence to tissue culture plastic and fibronectin coated surfaces when compared to transfected and non transfected control cells. Transglutaminase transfected clones exhibited no significant differences in their growth rates measured in vitro, cell morphology or levels of spontaneous apoptosis measured by the determination of detergent insoluble apoptotic envelopes. The data demonstrates a suppressive effect of tissue transglutaminase on tumour growth and confirms its importance in the phenotypic changes associated with the cancer process.

In this report, we show that the overexpression of tissue transglutaminase (tTG) in the human neuroblastoma cell line SK-N-BE(2) renders these neural crest-derived cells highly susceptible to death by apoptosis. Cells transfected with a full-length tTG cDNA, under the control of a constitutive promoter, show a drastic reduction in proliferative capacity paralleled by a large increase in cell death rate. The dying tTG-transfected cells exhibit both cytoplasmic and nuclear changes characteristic of cells undergoing apoptosis. The tTG-transfected cells express high Bcl-2 protein levels as well as phenotypic neural cell adhesion molecule markers (NCAM and neurofilaments) of cells differentiating along the neuronal pathway. In keeping with these findings, transfection of neuroblastoma cells with an expression vector containing segments of the human tTG cDNA in antisense orientation resulted in a pronounced decrease of both spontaneous and retinoic acid (RA)-induced apoptosis. We also present evidence that (i) the apoptotic program of these neuroectodermal cells is strictly regulated by RA and (ii) cell death by apoptosis in the human neuroblastoma SK-N-BE(2) cells preferentially occurs in the substrate-adherent phenotype. For the first time, we report here a direct effect of tTG in the phenotypic maturation toward apoptosis. These results indicate that the tTG-dependent irreversible cross-linking of intracellular protein represents an important biochemical event in the induction of the structural changes featuring cells dying by apoptosis.

Chlamydia trachomatis (C.t.) cell culture represents a sensitive method for the diagnosis of chlamydial infection and the only one which makes it possible to determine the susceptibility of an isolate to antibiotics so that an appropriate drug can be selected for individual treatment. In 11 patients, affected by urethroprostatitis and suspected of treatment failure with standard drug regimens, either due to lack of compliance with therapy or antibiotic resistance, C.t. was isolated in McCoy cell culture from urethral swabs, after prostatic massage. The in vitro activity of azithromycin against these isolates and the in vivo efficacy of the drug in the patients treated with a single 1 g dose have been evaluated. All the C.t. strains tested were susceptible to the action of azithromycin (MIC range 0.125-1.0 microgram/ml). Bactericidal values were one dilution higher (MBC range 0.25-2.0 microgram/ml). These in vitro results are consistent with clinical observations as all the patients treated had negative culture at a 4-week follow-up visit.

A cDNA encoding for the human tissue transglutaminase gene has been used to identify the chromosomal localization of the corresponding structural gene. The precise chromosomal and subregional localizations have been established by using in situ fluorescence mapping with a recombinant lambda-Zap phage containing the full cDNA coding sequence. The study showed that the human tissue transglutaminase gene is localized on chromosome 20 and, more precisely, within the band 20q12. To date, this is the third member of the transglutaminase gene family to be mapped. Human factor XIIIa (plasma transglutaminase), human keratinocyte transglutaminase (type I), and human tissue transglutaminase (type II) genes, although codifying for homologous enzymes, are localized on three different chromosomes.

Human immunodeficiency virus envelope glycoprotein gp120, but not its precursor gp160, covalently incorporates both spermidine and glycine ethyl ester in the presence of Ca2+ and transglutaminase purified from guinea pig liver. The examined ability to act as enzyme substrate of various glutamine-containing gp120 fragments, including the principal neutralizing determinant, the CD4 binding domain, and the sequence 254-274, suggested to be involved in post-binding events and in virus entry in the host cell, indicated the glutamine-265 as possible reactive acyl donor site of the protein.

Tissue transglutaminase is a cytosolic enzyme whose primary function is to catalyze the covalent cross-linking of proteins. To investigate the functions of this enzyme in physiological systems, we have established lines of Balb-C 3T3 fibroblasts stably transfected with a constitutive tissue transglutaminase expression plasmid. Several cell lines expressing high levels of catalytically active tissue transglutaminase have been isolated and characterized. Transglutaminase-transfected cells showed morphologic features quite distinct from their nontransfected counterparts. Many of the cells showed an extended and very flattened morphology that reflected increased adhesion of the cells to the substratum. Other cells, particularly those showing the highest levels of intracellular transglutaminase expression, showed extensive membrane blebbing and cellular fragmentation. The results of these experiments suggest that the induction and activation of tissue transglutaminase may contribute both to changes in cellular morphology and adhesiveness.

We report the results of a biological and molecular study carried out on 11 Italian families, with a total of 111 individuals in which adult dominant polycystic kidney disease segregates. A restriction fragment length polymorphism analysis was performed. Two families have shown a genetic heterogeneity even if not phenotypically different from the other ones: they resulted unlinked to 16p markers. A prenatal diagnosis has been performed in a family of the linked type.

The deduced amino acid sequences for tissue transglutaminases from human endothelial cells and mouse macrophages have been derived from cloned cDNAs. Northern blot analysis of both tissue transglutaminases shows a message size of approximately 3.6-3.7 kilobases. The molecular weights calculated from the deduced amino acid sequences were 77,253 for human endothelial tissue transglutaminase and 76,699 for mouse macrophage tissue transglutaminase. The deduced amino acid sequence for the human endothelial transglutaminase was confirmed by comparison with the amino acid sequence obtained by cyanogen bromide digestion of the human erythrocyte transglutaminase. The amino acid sequences of both human endothelial and mouse macrophage tissue transglutaminases were compared to other transglutaminases. A very high degree of homology was found between human endothelial, mouse macrophage, and guinea pig liver tissue transglutaminase (greater than 80%). Moreover, human endothelial tissue transglutaminase was compared with human Factor XIIIa and a very high degree of homology (75% identity) was found in the active site region.

Proteins and peptides responsible for the celiac small intestinal lesion inhibit both the enterocyte recovery of in vitro cultured flat celiac mucosa and the in vitro development of fetal rat intestine. They also agglutinate K 562 (S) cells. Using these three in vitro systems (cultured human celiac and rat fetal intestine and cell agglutination), it is shown that several small-molecular-weight amines, mostly the polyamines spermidine and spermine, prevent and reverse K 562 (S) cell agglutination induced by gliadin peptides, whereas they do not prevent cell agglutination induced by concanavalin A and wheat germ agglutinin. Some of these amines also protected in vitro developing fetal rat intestine and flat celiac mucosa from the damaging effect of gliadin peptides. This protective effect may be related to the trophic activity exerted by amines on the intestine and/or the effect of amines on the functions of intestinal brush border or intracellular membranes involved in the intestinal handling of gliadins.

One of the major proteins secreted from the rat seminal vesicle epithelium, namely SV-IV, was shown to act in vitro as acyl donor and acceptor substrate for transglutaminase from both guinea pig liver and rat anterior prostate secretory fluid. Electrophoretic and chromatographic experiments indicated that the enzyme catalyzed the formation of multiple modified forms of SV-IV. In the absence of small Mr amines, transglutaminase was able to produce at least six different molecular forms of the protein, half of which possessed an Mr higher than that of native SV-IV. These findings suggested that a variable number of intermolecular, and perhaps intramolecular, crosslinks were formed between one or both glutamine residues and one or more lysine residues occurring in the SV-IV polypeptide chain. In addition, at least three modified forms of the protein were produced by transglutaminase in the presence of high concentrations of spermidine, thus indicating the formation of different (gamma-glutamyl)polyamine derivatives of SV-IV. Rabbit uteroglobin and rat anterior prostate secretory protein(s) were also shown to be able to covalently bind spermidine in the presence of the enzyme. The possible biological significance of transglutaminase-mediated modifications of SV-IV, as well as of other proteins occurring in the mammal seminal fluid, are discussed.

The behaviour of N-acetyl-B-D-Glucosaminidase (NAG) was studied in the urine of patients with chronic pyelonephritis under pharmacological treatment for renewed attacks of acute renal inflammation. It was noted that by the end of target chemo-antibiotic treatment all patients presented a reduction in the inflammation and hence the tubular distress. Once treatment was suspended several patients showed an increase tendency in the activity of the enzyme followed by the reappearance of significant bacteriuria and/or leukocyturia. The acute attack of renal inflammation would not have been recognised if the enzymuria technique had been used to monitor the patients.

However adequately treated, surface carcinomas of the bladder recur sooner or later with infiltration found in about 10% of cases. Any development that may offer therapeutic progress and reduce the incidence of recurrences is therefore of primary importance. Two homogeneous groups of patients were selected for the present study: 35 were given Adriamycin and 25 Mitomycin C. In evaluating results, the incidence of recurrences in relation to the primary neoplasia and the existence of single or multiple tumors was analysed. It is concluded that Mitomycin C in association with either T.U.R. or T.V.R. is a particularly useful and indubitably effective form of topical chemotherapy in cases of single or multiple primary neoplasias.