Dynamical Generation of Elementary Fermion Mass: First Lattice Evidence.

Using lattice simulations we demonstrate from first principles the existence of a nonperturbative mechanism for elementary particle mass generation in models with gauge fields, fermions, and scalars, if an exact invariance forbids power divergent fermion masses and fermionic chiral symmetries broken at UV scale are maximally restored. We show that in the Nambu-Goldstone phase a fermion mass term, unrelated to the Yukawa operator, is dynamically generated. In models with electroweak interactions weak boson masses are also generated, opening new scenarios for beyond the standard model physics.

Inhibition of purified CD34+ hematopoietic progenitor cells by human immunodeficiency virus 1 or gp120 mediated by endogenous transforming growth factor beta 1.

Human CD34+ hematopoietic progenitor cells, stringently purified from the peripheral blood of 20 normal donors, showed an impaired survival and clonogenic capacity after exposure to either heat-inactivated human immunodeficiency virus (HIV) 1 (strain IIIB) or cross-linked envelope gp120. Cell cycle analysis, performed at different times in serum-free liquid culture, showed an accumulation in G0/G1 in HIV-1- or gp120-treated cells and a progressive increase of cells with subdiploid DNA content, characteristic of apoptosis. In blocking experiments with anti-transforming growth factor (TGF) beta 1 neutralizing serum or TGF-beta 1 oligonucleotides, we demonstrated that the HIV-1- or gp120-mediated suppression of CD34+ cell growth was almost entirely due to an upregulation of endogenous TGF-beta 1 produced by purified hematopoietic progenitors. Moreover, by using a sensitive assay on the CCL64 cell line, increased levels of bioactive TGF-beta 1 were recovered in the culture supernatant of HIV-1/gp120-treated CD34+ cells. Anti-TGF-beta 1 neutralizing serum or TGF-beta 1 oligonucleotides were also effective in inducing a significant increase of the plating efficiency of CD34+ cells, purified from the peripheral blood of three HIV-1-seropositive individuals, suggesting that a similar mechanism may be also operative in vivo. The relevance of these findings to a better understanding of the pathogenesis of HIV-1-related cytopenias is discussed.

Role of full-length osteoprotegerin in tumor cell biology.

Osteoprotegerin (OPG) is a soluble tumor necrosis factor receptor family member, which potently inhibits RANKL-mediated osteoclastogenesis. Numerous constructs have been created for therapeutic purposes in which the heparin-binding and death homology domains of OPG were removed and the remaining peptide (amino acids 22-194) was fused to the Fc domain of human IgG1 (OPG-Fc). The administration of OPG-Fc efficiently counteracted bone loss in a variety of preclinical models of cancers. However, several in vitro studies have shown that native or recombinant full-length OPG not only neuralizes RANKL, but also the death-inducing ligand TRAIL, suggesting that OPG might potentially counteract the anti-tumor activity of TRAIL. Additional evidence suggests that full-length OPG possesses RANKL- and TRAIL-independent biological properties, mainly related to the promotion of endothelial cell survival and angiogenesis. Finally, breast tumor cells overexpressing OPG have shown increased bone metastatic potential in vivo. The relevance of these apparently conflicting findings in tumor cell biology is highlighted.

Phytothermotherapy: a possible complementary therapy for fibromyalgia patients.

OBJECTIVE: It is a traditional practice in the Alpine region of Trentino and Alto Adige (Italy) to use phytothermotherapeutic treatment with fermenting grass ("hay baths") for rheumatic diseases. However, despite its long history and popularity, a clinical validation of the efficacy and tolerability of the treatment has yet to be found in current literature. Fibromyalgia syndrome (FMS) is characterised by generalised musculoskeletal pain, high tender point counts, sleep disturbance, fatigue, headaches, irritable bowel syndrome, frequent psychological distress and depressed mood. There is no standard therapy regime for FMS and the variety of medical treatments used have given limited benefits. The aim of this study was to assess the efficacy and tolerability of a cycle of phytothermotherapy through a single-blind, controlled, randomised trial, in patients with primary FMS. METHODS: Fifty-six patients with primary FMS according to the ACR criteria were randomly allocated to two groups: 30 were submitted to phytothermotherapy at the thermal resort of Garniga Terme (Trento, Italy) and the other 26 were considered as controls. All patients were evaluated by FIQ, Tender Points Count, HAQ and AIMS1 at baseline, after 10 days, then after 12 and 24 weeks. RESULTS: Patients submitted to phytothermotherapy showed visible and significant improvement of all evaluation parameters at the end of the treatment, which persisted during the follow-up period. No significant difference was found in the control group. Regarding the tolerability, none of the patients presented side effects. CONCLUSIONS: Our results suggest the efficacy and the tolerability of phytothermotherapy in patients with primary FMS.

Familial non-medullary thyroid carcinoma displays the features of clinical anticipation suggestive of a distinct biological entity.

Non-medullary thyroid carcinoma (NMTC) is mostly sporadic, but familial clustering is described. We aimed to compare the features of patients with sporadic and familial NMTC (FNMTC) patients and to assess whether FNMTC patients with parent-child relationship exhibit the 'anticipation' phenomenon (earlier age at disease onset and increased severity in successive generations). Among 300 NMTCs followed in the Section of Endocrinology (University of Siena, Italy), 34 (11.3%) patients, all with the papillary histotype, (16 kindred), met the criteria of FNMTC. Twenty-seven of them (79.4%) exhibited a parent-child relationship and seven (20.6%) a sibling relationship. These patients were compared with 235 patients with sporadic papillary thyroid cancer (PTCs). To analyze the features of FNMTC of the first and second generations, we cumulated the series of Siena with 32 additional FNMTC patients (15 kindred) from the Department of Endocrinology-Endocrine Oncology, Thessaloniki, Greece. Significant difference between sporadic PTC and FNMTC patients included more frequent tumor multifocality (P=0.001) and worse final outcome in FNMTC patients (P=0.001). Among 47 FNMTC with parent-child relationship, we found an earlier age at disease presentation (P<0.0001), diagnosis (P<0.0001), and disease onset (P=0.04) in the second generation when compared with the first generation. Patients in the second generation were more frequently males (P=0.02); their tumors were more frequently multifocal (P=0.003) and bilateral (P=0.01), had higher rate of lymph node metastases at surgery (P=0.02) and worse outcome (P=0.04) when compared with the first generation. In conclusion, FNMTC displays the features of clinical 'anticipation' with the second generation acquiring the disease at an earlier age and having more advanced disease at presentation.

Nuclear phosphatidylinositol 3,4,5-trisphosphate, phosphatidylinositol 3-kinase, Akt, and PTen: emerging key regulators of anti-apoptotic signaling and carcinogenesis.

Inositol lipid-derived second messengers have long been known to have an important regulatory role in cell physiology. Phosphatidylinositol 3-kinase (PI3K) synthesizes the second messenger 3,4,5'-phosphatidylinositol trisphosphate (Ptdlns 3,4,5P3) which controls a multitude of cell functions. Down-stream of PI3K/PtdIns 3,4,5P3 is the serine/threonine protein kinase Akt (protein kinase B, PKB). Since the PI3K/ PtdIns 3,4,5P3 /Akt pathway stimulates cell proliferation and suppresses apoptosis, it has been implicated in carcinogenesis. The lipid phosphatase PTEN is a negative regulator of this signaling network. Until recently, it was thought that this signal transduction cascade would promote its anti-apoptotic effects when activated in the cytoplasm. Several lines of evidence gathered over the past 20 years, have highlighted the existence of an autonomous nuclear inositol lipid cycle, strongly suggesting that lipids are important components of signaling pathways operating at the nuclear level. PI3K, PtdIns(3,4,5)P3, Akt, and PTEN have been identified within the nucleus and recent findings suggest that they are involved in cell survival also by operating in this organelle, through a block of caspase-activated DNase and inhibition of chromatin condensation. Here, we shall summarize the most updated and intriguing findings about nuclear PI3K/ PtdIns(3,4,5)P3/Akt/PTEN in relationship with carcinogenesis and suppression of apoptosis.

Effects of simulated microgravity on the morphology and function of neonatal porcine cell clusters cultured with and without Sertoli cells.

Human islet allografts are well known to induce full and sustained remission of hyperglycemia, with complete normalization of key metabolic parameters. Nevertheless, acquiring human islets, even from cadaveric human donor pancreases, remains a significant impediment to successful transplantation therapy for diabetes. To overcome this difficulty, neonatal porcine cell clusters (NPCCs) have been considered for human islet substitutes because they are easily obtained by collagenase digestion of the neonatal piglet pancreas. Currently, the major hurdle in using NPCCs for xenograft is the delay (time lag) in achieving the posttransplant normalization of blood glucose levels in animal diabetic recipients. The present work is the first attempt to evaluate whether incubation of NPCCs in simulated microgravity, in the presence or absence of Sertoli cells (SC), may reduce the maturation time lag of beta-cells by differentiation acceleration in vitro, thereby expediting production, viability, and acquisition of functional competence of pretransplantation beta-cell-enriched islets. Following a 3-day incubation period, NPCCs maintained in conventional culture, NPCCs incubated in simulated microgravity in the HARV biochamber, and NPCCs plus co-incubated SC in simulated microgravity were examined for viability, morphology, and insulin secretion. Results show that NPCCs grown alone in the HARV biochamber are superior in quality, both in terms of viability and functional competence, when compared to other culture pretreatment protocols. This finding strongly suggests that NPCC pretreatment in simulated microgravity may enhance the transplantation success of NPCCs in the diabetic recipient.

Ultrastructural sperm study in infertile males with microdeletions of Y chromosome.

A retrospective study to detect specific Y chromosome microdeletions and to evaluate sperm ultrastructural characteristics in infertile men was set up. We selected 219 infertile men referred to Regional Referral Center for Male Infertility, Siena, Italy for semen analysis from January 1999 to April 2004. Family history, lymphocyte karyotype determination, Y microdeletion screening, physical examination, hormonal assays, semen analysis were carried out. Sperm concentration and progressive motility, ultrastructural analysis of sperm organelles, PCR amplification of sequence tagged sites for Y microdeletion screening were performed. Different Y-chromosome deletions were found, mainly in the AZFb and AZFc regions. Severe alterations of sperm ultrastructure, affecting whole sperm population, were detected in carriers of Y-deletions. Our data confirms the highest frequency of Y deletions in azoospermic patients. In all other patients with Y microdeletions, sperm ultrastructural defects affected the whole sperm population and were mainly related to apoptosis or immaturity.

Phosphoinositide 3-kinase activity is essential for all-trans-retinoic acid-induced granulocytic differentiation of HL-60 cells.

Phosphoinositide 3-kinase (PI 3-K) activity increases in HL-60 cells that are induced to granulocytic differentiation by all-trans-retinoic acid. Immunochemical and immunocytochemical analyses by confocal microscopy also reveal an increase in the amount of the enzyme, which is particularly evident at the nuclear level. Inhibition of PI 3-K activity by nanomolar concentrations of wortmannin and of its expression by transfection with an antisense fragment of p85alpha prevented the differentiative process. The data obtained indicate that PI 3-K activity plays an essential role in promoting granulocytic differentiation.

Chromatin phospholipids in normal and chronic lymphocytic leukemia lymphocytes.

Certain phospholipids are associated with the nonhistone chromosomal proteins extracted from normal B- and chronic lymphocytic leukemia lymphocytes. The ratio of phospholipids to nonhistone chromosomal proteins was constant with the different methods used for isolating nuclei and extracting the chromatin, although the various methods allowed a different recovery of total lipids from chromatin. Three phospholipids were extractable from the nonhistone protein fraction, but their respective ratios varied in chronic lymphocytic leukemia compared to normal B-lymphocytes. The most significant variation concerns the reduction of sphingomyelin content in leukemic lymphocytes, since this prospholipid in vitro affects both DNA stability and transcription.

Human immunodeficiency virus type 1 Tat protein protects lymphoid, epithelial, and neuronal cell lines from death by apoptosis.

We report here that the tat gene product of human immunodeficiency virus type 1 was able to protect lymphoblastoid (Jurkat), epithelial (293) and neuronal (PC12) cell lines from apoptotic death induced by serum withdrawal. The rescue from apoptosis by Tat was reflected by an increased expression of Bcl-2 protein in tat-positive Jurkat cells with respect to mock-transfected Jurkat cells after 3-6 days of serum-free cultures. We propose that the ability of the regulatory human immunodeficiency virus type 1 Tat protein to suppress apoptosis might have important implications in understanding the pathogenesis of frequent neoplastic disorders observed in human immunodeficiency virus type 1-seropositive individuals.

Uptake and phosphorylation of phosphatidylinositol by rat liver nuclei. Role of phosphatidylinositol transfer protein.

The incorporation of phosphatidyl[2-3H]inositol ([3H]PI) from vesicles or microsomal membranes into rat liver nuclei is greatly stimulated by phosphatidylinositol transfer protein (PI-TP). The nuclei are able to phosphorylate [3H]PI, with the production of phosphatidylinositol 4-phosphate (PIP). Recovery of tritiated inositol trisphosphate, inositol phosphate, glycerophosphoinositol and inositol, suggests that in isolated nuclei a large set of enzymes of the PI cycle is present, similar to the enzymes involved in the plasma membrane PI cycle. Incubation with [gamma-32P]ATP shows that isolated nuclei are able to phosphorylate endogenous PI to PIP and phosphatidylinositol 4,5-bisphosphate (PIP2). In the presence of exogenous PI and detergent the synthesis of PIP is increased, indicating that in nuclei the PI pool is suboptimal for the PI-kinase activity. The present study suggests that PI-TP may be involved in providing substrates for PI metabolism at the nuclear level.

Identification of PI-PLC beta 1, gamma 1, and delta 1 in rat liver: subcellular distribution and relationship to inositol lipid nuclear signalling.

The subcellular distribution of PI-PLC beta 1, gamma 1, and delta 1 has been investigated in rat liver by western blot and immunohistochemical analysis with a panel of isoform-specific antibodies. The data obtained in situ on cryo-sectioned tissue indicate that PI-PLC beta 1 is predominantly nuclear, while gamma 1 is largely cytoplasmic and delta 1 is sharply restricted to the cytoplasm. In fractionation experiments, the Western blot analysis indicated that the recovery of the nuclear isoforms beta 1 and gamma 1 was not affected by the removal of the nuclear membrane, and that the two enzymes persisted in nuclear matrix and lamina, obtained after nuclease digestion and extraction with high salt and detergent. The assay of the phosphodiesterase activity in different cell fractions correlates with the observed relative abundance of the enzymes, and specific inhibition with neutralizing anti-beta 1 and -gamma 1 isoforms confirms that these are the enzymes active at the nuclear level. These results demonstrate that in rat liver cells, as in other cell types, different members of the PI-PLC family show a discrete intracellular distribution, and suggest that PI-PLC beta 1 and gamma 1 play a central role in modulating the nuclear phosphoinositide cycle.

Diacylglycerol kinase activity in rat liver nuclei.

Membrane-depleted rat liver nuclei contain diacylglycerol (DAG) kinase showing a specific activity which doubles that of the whole homogenate. In contrast, cytoplasmic and plasma membrane marker enzymes attain a specific activity of 0.4% at the most, when nuclear DAG kinase approaches 4.5% of the total tissue activity. The enzyme shows a Km of 161 and 200 microM for ATP in both nuclei and microsomes whereas the Km for DAG is 75 microM in nuclei and 658 microM in microsomes. Octylglucoside, CHAPS and Triton X-100 behave mainly as inhibitors, while deoxycholate stimulates the enzyme activity in both cellular fractions, increasing specific activity (3.2-fold in nuclei and 29.1-fold in microsomes) and decreasing Km for DAG (39 microM in nuclei and 237 microM in microsomes). Phospholipids and ceramide stimulate the enzyme activity in isolated nuclei, while no effect occurs in the microsomal fraction. At variance, sphingosine behaves as an inhibitor in both cellular fractions. DAG kinase also utilizes endogenous substrates mobilized by Bacillus cereus phospholipase C, which hydrolyses nuclear phosphatidylcholine and phosphatidylethanolamine and by phosphatidylinositol-specific phospholipase C, which hydrolyses nuclear PI and PIP. These data indicate that nuclear DAG can be controlled by converting it into phosphatidic acid by the action of a nuclear enzyme and support the contention that protein kinase C activity can be modulated at the nuclear level by a discrete system involving phospholipase C and DAG kinase that could operate independently from the cytoplasm.

Changes of nuclear PI-PLC gamma1 during rat liver regeneration.

We have previously demonstrated that rat liver nuclei contain PI-PLC beta1 and gamma1 in the inner nuclear matrix and lamina associated with specific phosphodiesterase activity (Bertagnolo et al., 1995, Cell Signall. 7, 669-678). Since compensatory hepatic growth is an informative and well characterized model for natural cell proliferation, the presence of specific PI-PLC isoforms and their activity as well as PIP2 recovery were studied at various regenerating times, ranging from 3 to 22 h after partial hepatectomy. Three PI-PLC isoforms (beta1, gamma1, delta1) were examined in control and regenerating liver cells by using specific antibodies. By means of in situ immunocytochemistry and confocal microscopy, PI-PLC beta1 was found mainly in the nucleoplasm and this pattern was not modified after hepatectomy. On the contrary, the nuclear gamma1 isoform showed a marked decrease at 3 and 16 h after hepatectomy, but a clear increase at 22 h covering with bright intensity the whole nucleus. The PI-PLC delta1 isoform, which is exclusively cytoplasmic, was not altered during rat liver regeneration. By western blotting analysis on whole cell homogenates, none of the PI-PLC isozymes under study showed proliferation-linked modification. However, analyses of isolated nuclei identified changes in the nucleus associated PI-PLC gamma1 that paralleled the in situ observation whereas the beta1 isoform was unmodified at all the times examined. Nuclear phosphodiesterase activity on PIP2 was lower at 3 and 16 h, in comparison with sham operated rats, increased at 6 h and reached the highest value after 22 h. Consistently, the recovery of PIP2, obtained in conditions that optimise PIP-kinase activity, showed a marked decrease at 3 h and an increase up to 16 h of liver regeneration, followed by a further decrease at 22 h. These data are consistent with a close relationship between cell proliferation and the nuclear inositide cycle, depending, in rat liver, predominantly on the modulation of the gamma1 isoform of PI-PLC.

Increased phosphorylation of nuclear substrates for rat brain protein kinase C in regenerating rat liver nuclei.

Protein phosphorylation catalysed by rat brain protein kinase C (PKC) has been studied in nuclei isolated from normal and regenerating rat liver. Histone H1 and a 40,000 molecular weight protein were hyperphosphorylated at all the explored regeneration times, ranging from 3 to 22 h after partial hepatectomy. Phosphorylation of the two substrates was totally dependent on calcium and lipids and was abolished by low concentration of staurosporine. The observed early change of phosphate content of histone H1 and of the 40,000 molecular weight protein on the time scale of liver regeneration suggests that PKC might be involved in the initial nuclear events leading to cell proliferation.

Stromal derived factor-1 alpha (SDF-1 alpha) induces CD4+ T cell apoptosis via the functional up-regulation of the Fas (CD95)/Fas ligand (CD95L) pathway.

Stromal-derived factor-1alpha (SDF-1alpha), the high-affinity ligand of CXC-chemokine receptor 4 (CXCR4), induced a progressive increase of apoptosis when added to the Jurkat CD4+/CXCR4+ T cell line. The SDF-1alpha-mediated Jurkat cell apoptosis was observed in serum-free or serum-containing cultures, peaked at SDF-1alpha concentrations of 10-100 ng/ml, required 3 days to take place, and was completely blocked by the z-VAD-fmk tripeptide caspase inhibitor. Although SDF-1alpha did not modify the expression of TNF-alpha or that of TNF-RI and TNF-RII, it increased the expression of surface Fas/APO-1 (CD95) and intracellular Fas ligand (CD95L) significantly. Moreover, the ability of SDF-1alpha to induce apoptosis was inhibited by an anti-CD95 Fab' neutralizing antibody. These findings suggest a role for SDF-1alpha in the homeostatic control of CD4+ T-cell survival/apoptosis mediated by the CD95-CD95L pathway.

Necrosis in human spermatozoa. I. Ultrastructural features and FISH study in semen from patients with uro-genital infections.

Ultrastructural characteristics and meiotic segregation in spermatozoa from twelve patients affected by uro-genital bacterial infections were investigated. The sperm quality was examined by light and transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH) analysis was performed in eight out of twelve individuals in order to investigate the meiotic behaviour of chromosomes namely gonosomes and chromosome 18. TEM analysis highlighted a severely altered sperm morphology, typical of apoptosis and in particular, necrosis. We define the ultrastructural characteristics of necrosis as involving the acrosome, chromatin, mitochondrial helix, axonemal structure and plasma membrane. Based on our observations, it is possible to hypothesize that infection acts at the testicular level causing sperm death, due to necrosis itself or by necrosis proposed as the final step of apoptosis. Moreover, FISH analysis revealed the presence of altered meiotic segregation in these patients. The high rate of diploidy and gonosomes disomy in our group of patients suggests the possibility of a negative effect of infection and/or inflammation on sperm morphogenesis.

Necrosis in human spermatozoa. II. Ultrastructural features and FISH study in semen from patients with recovered uro-genital infections.

Inflammation of the male genital tract is a potential cause of male sterility. The quality of spermatozoa from ten patients with recovered uro-genital infections was examined by transmission electron microscopy (TEM); fluorescence in situ hybridization (FISH) was performed on sperm nuclei in six our of ten patients to investigate the frequency of aneuploidies. TEM analysis demonstrated the presence of a high percentage of necrosis in all patients, whereas apoptosis was present in only five of them. Meiotic segregation was altered in all analysed semen samples. Recovery from infections does not seem to coincide with improved sperm quality, probably because a persistent inflammatory state demonstrated by a high percentage of sperm necrosis sometimes associated with the presence of white blood cells (WBC) in the seminal plasma, is present. The effects of infections of the male genital tract could proceed in the absence of microbial agents due to immunological mechanisms involving the pattern of chemical products typical of inflammation. Our results suggest that the presence of necrosis, sometimes associated with apoptosis, could be considered to be an indicator of male genital tract inflammation. However, further studies are necessary to test the correlation between biochemical parameters and ultrastructural and molecular markers of inflammation.

Extracellular HIV-1 Tat protein differentially activates the JNK and ERK/MAPK pathways in CD4 T cells.

OBJECTIVE: To investigate the intracellular signals elicited by extracellular HIV-1 Tat protein in lymphoid CD4 T cells. METHODS: CD4 Jurkat T cells were treated with a series of glutathione S-transferase (GST)-Tat fusion proteins: full-length two-exon GST-Tat (GST-Tat2E); one-exon Tat, in which the second exon of Tat was deleted (GST-Tat1E); two-exon Tat, in which the seven arginine residues have been changed to alanine residues (GST-TatArg(mut)), GST-TatdeltaN, which shows a deletion of the N-terminal 21 amino acids. The cells were either treated with soluble GST-Tat proteins or seeded on plates coated with GST-Tat proteins immobilized on plastic. At various time points, Jurkat cells were lysed and examined for c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) activity. RESULTS: Soluble and immobilized GST-Tat2E, but not GST-Tat1E, GST-TatArg(mut) and GST-TatdeltaN, activated JNK in a dose-dependent manner, induced a rapid phosphorylation of c-Jun on Ser63 and promoted the de novo synthesis of c-Jun protein. Moreover, both GST-Tat2E and GST-Tat1E also stimulated ERK/MAPK. However, the activation of JNK was maximal at concentrations of 100 nM of GST-Tat2E and was blocked by the S6-kinase inhibitor rapamycin, whereas the activation of ERK/MAPK was already maximal at 1 nM of GST-Tat2E and was enhanced by rapamycin. CONCLUSIONS: Tat-mediated activation of JNK requires the second exon of Tat, which is dispensable for the activation of ERK/MAPK. The ability to stimulate JNK and ERK/MAPK does not require Tat internalization.