Type 2 diabetes progression: A regulatory network approach.

In order to elucidate central elements underlying type 2 diabetes, we constructed a regulatory network model involving 37 components (molecules, receptors, processes, etc.) associated to signaling pathways of pancreatic beta-cells. In a first approximation, the network topology was described by Boolean rules whose interacting dynamics predicted stationary patterns broadly classified as health, metabolic syndrome, and diabetes stages. A subsequent approximation based on a continuous logic analysis allowed us to characterize the progression of the disease as transitions between these states associated to alterations of cell homeostasis due to exhaustion or exacerbation of specific regulatory signals. The method allowed the identification of key transcription factors involved in metabolic stress as essential for the progression of the disease. Integration of the present analysis with existent mathematical models designed to yield accurate account of experimental data in human or animal essays leads to reliable predictions for beta-cell mass, insulinemia, glycemia, and glycosylated hemoglobin in diabetic fatty rats.

Assessing Tn5 and Sleeping Beauty for transpositional transgenesis by cytoplasmic injection into bovine and ovine zygotes.

Transgenic domestic animals represent an alternative to bioreactors for large-scale production of biopharmaceuticals and could also provide more accurate biomedical models than rodents. However, their generation remains inefficient. Recently, DNA transposons allowed improved transgenesis efficiencies in mice and pigs. In this work, Tn5 and Sleeping Beauty (SB) transposon systems were evaluated for transgenesis by simple cytoplasmic injection in livestock zygotes. In the case of Tn5, the transposome complex of transposon nucleic acid and Tn5 protein was injected. In the case of SB, the supercoiled plasmids encoding a transposon and the SB transposase were co-injected. In vitro produced bovine zygotes were used to establish the cytoplasmic injection conditions. The in vitro cultured blastocysts were evaluated for reporter gene expression and genotyped. Subsequently, both transposon systems were injected in seasonally available ovine zygotes, employing transposons carrying the recombinant human factor IX driven by the beta-lactoglobulin promoter. The Tn5 approach did not result in transgenic lambs. In contrast, the Sleeping Beauty injection resulted in 2 lambs (29%) carrying the transgene. Both animals exhibited cellular mosaicism of the transgene. The extraembryonic tissues (placenta or umbilical cord) of three additional animals were also transgenic. These results show that transpositional transgenesis by cytoplasmic injection of SB transposon components can be applied for the production of transgenic lambs of pharmaceutical interest.

Tiger, Bengal and Domestic Cat Embryos Produced by Homospecific and Interspecific Zona-Free Nuclear Transfer.

The aim of this study was to evaluate three different cloning strategies in the domestic cat (Felis silvestris) and to use the most efficient to generate wild felid embryos by interspecific cloning (iSCNT) using Bengal (a hybrid formed by the cross of Felis silvestris and Prionailurus bengalensis) and tiger (Panthera tigris) donor cells. In experiment 1, zona-free (ZP-free) cloning resulted in higher fusion and expanded blastocyst rates with respect to zona included cloning techniques that involved fusion or injection of the donor cell. In experiment 2, ZP-free iSCNT and embryo aggregation (2X) were assessed. Division velocity and blastocyst rates were increased by embryo aggregation in the three species. Despite fewer tiger embryos than Bengal and cat embryos reached the blastocyst stage, Tiger 2X group increased the percentage of blastocysts with respect to Tiger 1X group (3.2% vs 12.1%, respectively). Moreover, blastocyst cell number was almost duplicated in aggregated embryos with respect to non-aggregated ones within Bengal and tiger groups (278.3 ± 61.9 vs 516.8 ± 103.6 for Bengal 1X and Bengal 2X groups, respectively; 41 vs 220 ± 60 for Tiger 1X and Tiger 2X groups, respectively). OCT4 analysis also revealed that tiger blastocysts had higher proportion of OCT4-positive cells with respect to Bengal blastocysts and cat intracytoplasmic sperm injection blastocysts. In conclusion, ZP-free cloning has improved the quality of cat embryos with respect to the other cloning techniques evaluated and was successfully applied in iSCNT complemented with embryo aggregation.

Cheetah interspecific SCNT followed by embryo aggregation improves in vitro development but not pluripotent gene expression.

The aim of this study was to evaluate the capacity of domestic cat (Dc, Felis silvestris) oocytes to reprogram the nucleus of cheetah (Ch, Acinonyx jubatus) cells by interspecies SCNT (iSCNT), by using embryo aggregation. Dc oocytes were in vitro matured and subjected to zona pellucida free (ZP-free) SCNT or iSCNT, depending on whether the nucleus donor cell was of Dc or Ch respectively. ZP-free reconstructed embryos were then cultured in microwells individually (Dc1X and Ch1X groups) or in couples (Dc2X and Ch2X groups). Embryo aggregation improved in vitro development obtaining 27.4, 47.7, 16.7 and 28.3% of blastocyst rates in the Dc1X, Dc2X, Ch1X and Ch2X groups, respectively (P<0.05). Moreover, aggregation improved the morphological quality of blastocysts from the Dc2X over the Dc1X group. Gene expression analysis revealed that Ch1X and Ch2X blastocysts had significantly lower relative expression of OCT4, CDX2 and NANOG than the Dc1X, Dc2X and IVF control groups. The OCT4, NANOG, SOX2 and CDX2 genes were overexpressed in Dc1X blastocysts, but the relative expression of these four genes decreased in the Dc2X, reaching similar relative levels to those of Dc IVF blastocysts. In conclusion, Ch blastocysts were produced using Dc oocytes, but with lower relative expression of pluripotent and trophoblastic genes, indicating that nuclear reprogramming could be still incomplete. Despite this, embryo aggregation improved the development of Ch and Dc embryos, and normalized Dc gene expression, which suggests that this strategy could improve full-term developmental efficiency of cat and feline iSCNT embryos.

Removal of bacteria, protozoa and viruses through a multiple-barrier household water disinfection system.

Municipal water disinfection systems in some areas are not always able to meet water consumer needs, such as ensuring distributed water quality, because household water management can be a contributing factor in water re-contamination. This fact is related to the storage options that are common in places where water is scarce or is distributed over limited time periods. The aim of this study is to assess the removal capacity of a multiple-barrier water disinfection device for protozoa, bacteria, and viruses. Water samples were taken from households in Mexico City and spiked with a known amount of protozoa (Giardia cyst, Cryptosporidium oocyst), bacteria (Escherichia coli), and viruses (rotavirus, adenovirus, F-specific ribonucleic acid (FRNA) coliphage). Each inoculated sample was processed through a multiple-barrier device. The efficiency of the multiple-barrier device to remove E. coli was close to 100%, and more than 87% of Cryptosporidium oocysts and more than 98% of Giardia cysts were removed. Close to 100% of coliphages were removed, 99.6% of the adenovirus was removed, and the rotavirus was almost totally removed. An effect of site by zone was detected; this observation is important because the water characteristics could indicate the efficiency of the multiple-barrier disinfection device.

Production of chimeric embryos by aggregation of bovine egfp eight-cell stage blastomeres with two-cell fused and asynchronic embryos.

Embryo disaggregation allows the production of two to four identical offspring from a single cow embryo. In addition, embryo complementation has become the technique of choice to demonstrate the totipotency of embryonic stem cells and induced pluripotent stem cells. Therefore, the aim of this study was to generate a new and simple method by aggregation in the well-of-the-well system to direct each single enhanced green fluorescent protein (egfp) eight-cell blastomere derived from bovine in vitro fertilization embryos to the inner cell mass (ICM) of chimeras produced with fused and asynchronic embryos. To this end, the best conditions to generate in vitro fertilization-fused embryos were determined. Then, the fused (F) and nonfused (NF) embryos were aggregated in two distinct conditions: synchronically (S), with both transgenic and F embryos produced on the same day, and asynchronically (AS), with transgenic embryos produced one day before F embryos. The highest fusion and blastocysts rates were obtained with two pulses of 40 V. The 2ASF and 2ASNF groups showed the best number of blastocysts expressing the EGFP protein (48% and 41%, respectively). Furthermore, the 2ASF group induced the highest localization rates of the egfp-expressing blastomere in the ICM (6/13, 46% of ICM transgene-expressing blastocysts). This technique will have great application for multiplication of embryos of high genetic value or transgenic embryos and also with the generation of truly bovine embryonic stem cells and induced pluripotent stem cells.

Simple gene transfer technique based on I-SceI meganuclease and cytoplasmic injection in IVF bovine embryos.

Although transgenic methods in mammals are inefficient, an easy and highly efficient transgenesis system using I-SceI meganuclease (intron-encoded endonuclease from S. cerevisiae) was recently described in Xenopus. The method consisted of injection into fertilized eggs of an I-SceI reaction mixture with a plasmid DNA carrying the transgene, flanked by the meganuclease recognition sites (pIS). In the present study, the effects of I-SceI on gene transfer were tested apparently for the first time in mammals, in particular, in cattle. Various conditions were evaluated, including three concentrations of the plasmid pIS Pax6egfp, carrying I-SceI recognition sites flanking egfp under Pax6 promoter and two injection times (before IVM and after IVF) of pIS CAGegfp, carrying I-SceI sites fanking egfp under CAG promoter. In addition, the quantity of transgene was measured using quantitative polymerase chain reaction, and presence of transgene signals was evaluated using fluorescence in situ hybridization analysis. Transgene expression rates were higher (P < 0.05) for groups treated after IVF (79.1%, 91/115 and 63.0%, 75/119) than before IVM (32.6%, 31/95 and 34.7%, 33/95), with and without I-SceI, respectively. Interestingly, injection with pIS plus I-SceI after IVF increased frequency (P < 0.05) of nonmosaic transgene-expressing embryos (58.3%, 42/72 vs. 29.7%, 25/84) for pIS plus I-SceI and pIS alone. Based on fluorescence in situ hybridization analysis, injection with I-SceI increased (P < 0.05) the proportion of embryos with transgene signals in all blastomeres compared with pIS alone (44.0%, 11/25 vs. 6.9%, 2/29) for pIS plus I-SceI and pIS alone. In addition, transgene copy number was numerically higher for the group treated with pIS plus I-SceI compared with pIS alone. In conclusion, I-SceI gene transfer increased transgene signals in bovine embryos.

Toxins from Physalia physalis (Cnidaria) raise the intracellular Ca(2+) of beta-cells and promote insulin secretion.

Physalia physalis is a marine cnidarian from which high molecular weight toxins with hemolytic and neurotoxic effects have been isolated. In the present work, two novel toxins, PpV9.4 and PpV19.3 were purified from P. physalis by bioactive guideline isolation. It involved two steps of column chromatography, gel filtration and RP-HPLC. The molecular weights were 550.7 and 4720.9 Da for PpV9.4 and PpV19.3, respectively. In the light of the Edman sequencing results, the structure of these toxins included the presence of modified amino acids. Both toxins increased the percentage of insulin secreting beta-cells and induced cytosolic Ca2+ elevation. To date, this is the first report of low molecular weight toxins increasing insulin secretion purified from cnidarians, by constituting a new approach to the study of beta-cells physiology.

Production of IVF transgene-expressing bovine embryos using a novel strategy based on cell cycle inhibitors.

The objective was to evaluate the effects of cell cycle inhibitors (6-dimethylaminopurine [DMAP], and dehydroleukodine [DhL]) on transgene expression efficiency and on mosaic expression patterns of IVF bovine zygotes cytoplasmically injected with oolema vesicles coincubated with transgene. The DNA damage induced by the transgene or cell cycle inhibitors was measured by detection of phosphorylated histone H2AX foci presence (marker of DNA double-stranded breaks). Cloning of egfp blastomeres was included to determine continuity of expression after additional rounds of cellular division. The pCX-EGFP [enhanced green fluorescent protein gene (EGFP) under the chimeric cytomegalovirus IE-chicken-ß-actin enhancer promoter control] gene plasmid (50 ng/µL) was injected alone (linear or circular exogenous DNA, leDNA and ceDNA, respectively) or associated with ooplasmic vesicles (leDNA-v or ceDNA-v). The effects of 2 mm DMAP or 1 µm DhL for 6 h (from 15 to 21 h post IVF) was evaluated for groups injected with vesicles. The DMAP increased (P < 0.05) egfp homogenous expression relative to transgene alone (21%, 18%, and 11% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively) and also increased (P < 0.05) the phosphorylated histone H2AX foci area. Expression of egfp was higher (P < 0.05) for linear than for circular pCX-EGFP, and egfp blastocyst rates were higher (P < 0.05) for groups injected with linear transgene coincubated with vesicles than for linear transgene alone (95%, 77%, 84%, and 52% for leDNA-v + DMAP, leDNA-v + DhL, leDNA-v, and leDNA, respectively). Moreover, DMAP tended to improve egfp blastocysts rates for both circular and linear transgenes. Based on fluorescent in situ hybridization (FISH) analysis, there was evidence of integration in egfp embryos. Finally, clones derived from leDNA-v + DMAP had the highest egfp expression rates (96%, 65%, and 65% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively). Transgenesis by cytoplasmic injection of leDNA-v + DMAP is a promising alternative for transgenic animal production.

Potentially pathogenic nontuberculous mycobacteria found in aquatic systems. Analysis from a reclaimed water and water distribution system in Mexico City.

We investigated the presence of nontuberculous mycobacteria (NTM) in three Mexican aquatic systems to evaluate the prevalence with the distribution of NTM species. Key physicochemical parameters of the water samples were determined to find correlations with the species' distributions. We used multilocus sequence analysis (MLSA) based on hsp65, rpoB, and 16S rRNA fragments to determine their taxonomic affiliations. NTM were recovered from water distribution systems and reclaimed water from the Mexico City Metropolitan Area (MCMA). The isolated species were associated with a temperature of 21°C and pH >7.7. The phylogenetic analysis showed that eight of the 14 different NTM strains were unambiguously classifiable: Mycobacterium peregrinum, M. nonchromogenicum (2), M. smegmatis (2), M. fortuitum, M. avium ssp. hominissuis, M. arupense, M. gordonae, and M. chitae. One strain was tentatively identified as M. mantenni/ scrofulaceum and another strain was related to M. porcinum/M. septicum. All NTM species identified in the water distribution system were also detected in the reclaimed water, but some species from the reclaimed water were not found in the water distribution systems. Two of the identified species found in the reclaimed water, M. avium and M. fortuitum, are considered important human opportunistic pathogens.

Protective effect of testosterone on early apoptotic damage induced by streptozotocin in rat pancreas.

Beta-cell apoptosis is responsible for the development of insulin-dependent diabetes mellitus in the streptozotocin (STZ) rat model. It has been demonstrated that steroid hormones possess antioxidant and protective antiapoptotic effects in many tissues. The aim of the present study was to investigate the early apoptotic damage induced by STZ in rat pancreas, and the effect of testosterone in preventing apoptosis of pancreatic beta cells. Intact and castrated adult male Wistar rats were subjected to a unique injection of STZ 60 mg/kg (body weight) in citrate buffer, and the kinetics of apoptosis in beta cells was assessed. Insulin and glucose were measured by RIA and a glucometer respectively, and in pancreatic tissue by immunohistochemistry. At 6 h after STZ injection, a marked increase in apoptotic beta cells was detected; however, glucose and insulin serum levels were not significantly different from the controls. The castrated animals presented higher percentages of apoptotic beta cells (65.75 +/- 5.42%) than intact males (20.6 +/- 4.38%) and castrated, testosterone-substituted males (30.66 +/- 1.38%). The decrease in apoptotic beta cells induced by testosterone was reversed by the antiandrogen flutamide (67.69 +/- 3.45%). The overall results indicate that early apoptotic damage produced by STZ in castrated animals was reversed by testosterone, suggesting that this hormone exerts a natural protective effect in rat pancreas. This effect could help to explain some sexual differences in diabetes mellitus incidence in man, reinforcing the idea that new approaches in steroid hormone therapies should be considered for treatment of this disease.

Thapsigargin-sensitive cationic current leads to membrane depolarization, calcium entry, and insulin secretion in rat pancreatic beta-cells.

Glucose-induced insulin secretion by pancreatic beta-cells depends on membrane depolarization and [Ca2+]i increase. We correlated voltage- and current-clamp recordings, [Ca2+]i measurements, and insulin reverse hemolytic plaque assay to analyze the activity of a thapsigargin-sensitive cationic channel that can be important for membrane depolarization in single rat pancreatic beta-cells. We demonstrate the presence of a thapsigargin-sensitive cationic current, which is mainly carried by Na+. Moreover, in basal glucose concentration (5.6 mM), thapsigargin depolarizes the plasma membrane, producing electrical activity and increasing [Ca2+]i. The latter is prevented by nifedipine, indicating that Ca2+ enters the cell through L-type Ca2+ channels, which are activated by membrane depolarization. Thapsigargin also increased insulin secretion by increasing the percentage of cells secreting insulin and amplifying hormone secretion by individual beta-cells. Nifedipine blocked the increase completely in 5.6 mM glucose and partially in 15.6 mM glucose. We conclude that thapsigargin potentiates a cationic current that depolarizes the cell membrane. This, in turn, increases Ca2+ entry through L-type Ca2+ channels promoting insulin secretion.

Nerve growth factor increases L-type calcium current in pancreatic beta cells in culture.

We analyzed the effect of culturing adult rat beta cells with NGF2.5 S for 5 to 7 days on macroscopic barium current (I(Ba)), and determined the role of Na and Ca channels on neurite-like process extension induced by NGF and dbcAMP, and by KCI depolarization. After five days in culture with 2.5S NGF, beta cells exhibit a 102% increase in I(Ba) density. This effect is on L-type calcium channels because most of the current is blocked by nifedipine. The application of NGF for 5 minutes to the cells deprived of the trophic factor for 24 hr further increases I(Ba) current by 91%. These results suggest that the trophic factor regulates I(Ba) by two different mechanisms, a) an increase in channel density and b) a rapid modulation of the channels already present in the membrane. Finally, we found that ion-channel activity modifies the growth of neurite-like processes. After 2 weeks in culture with high KCl, almost 14% of beta cells extend neurite-like processes and the most impressive effect is observed in the presence of KCl, NGF, and dbcAMP simultaneously, where nearly 60% of the cells extend neurite-like processes. Tetrodotoxin and nifedipine reduce the morphological changes induced by these agents.

Biochemical characteristics of the gamma-aminobutyric acid system in the insulinoma cell lines HIT-T15, RIN-m5F, betaTC3, and comparison with rat brain.

BACKGROUND: gamma-aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the mammalian brain. Both GABA and its synthesizing enzyme, L-glutamate decarboxylase (GAD), are also present in the insulin-secreting pancreatic beta cells, in which its physiologic role is unclear. We have studied several aspects of the GABA system in the insulinoma cell lines HIT-T15, RIN-m5F, and betaTC3 in comparison with rat brain tissue. METHODS: Insulinoma cell lines and embryonic rat brain cortex neurons were cultured. GAD activity was determined by a radioenzymatic method and the presence of GAD(67) protein was assessed by immunocytochemistry. Amino acid content and the effect of different conditions on the release of endogenous GABA were measured by HPLC and fluorometric detection after o-phthaldialdehyde derivatization. [3H]GABA was used for measuring the uptake of the amino acid in the insulinoma cultures and in rat forebrain synaptosomes. RESULTS: The three insulinoma lines possess GABA and GAD activity at levels of approximately 20% compared with adult rat brain cortex. Dissimilar from the latter, in insulinoma cultures enzyme activity was not enhanced by addition of an excess of the coenzyme pyridoxal-5'-phosphate. Immunocytochemical visualization of GAD showed that the cells in both neuronal cultures and insulinoma lines were GAD(67)-positive, similar to Purkinje cell somata of adult rat cerebellar cortex. [3H]GABA uptake in the cell lines was approximately 10% of that in rat forebrain synaptosomes and showed less ionic and temperature dependence. In both cultured cerebral neurons and RINm5F cells, the addition of arginine induced the release of GABA, whereas neither high K(+) concentration nor glucose had any effect. CONCLUSIONS: The insulinoma cell lines studied possess the same GAD(67) form of the enzyme present in brain. RIN line cells are capable of transporting glutamate. In these cells as well as in cultured cortical neurons, arginine stimulates the release of GABA and glutamate probably as the result of its electrogenic transport. Insulinoma cell lines may therefore be useful to study GABA metabolism and function in pancreatic beta cells.

Helicobacter pylori and other enteric bacteria in freshwater environments in Mexico City.

BACKGROUND: Helicobacter pylori infection is common in the Mexican population; however, sources, routes, and risk factors for infection as well as mode of transmission remain unclear. METHODS: H. pylori was detected by polymerase chain reaction (PCR) technique in three aquatic systems located in the Mexico City area. In addition, microbiologic cultures and physicochemical parameters were measured. The systems were sampled over an 18-month period (1997-1999), resulting in a total of 212 samples for the different analyses. RESULTS: Twenty-one percent of the samples (16/77) were positive for H. pylori; of these, 42% (5/12) were confirmed for cagA gene detection by PCR hybridization. Microbiologic samples (n = 74) yielded Aeromonas hydrophila, Aeromonas caviae, Aeromonas veronii, and Vibrio fluvialis. In the samples for physicochemical analyses (n = 61), low concentrations of dissolved oxygen were detected and residual chlorine was less than the inactivation dose, both providing conditions for potential survival of H. pylori and other enteric pathogens in these environments. CONCLUSIONS: The results of this study suggest that, in Mexico City, water used for human consumption and irrigation may play an important role as a vehicle in the transmission of H. pylori as well as infection by other known enteric pathogens.

Ultrastructural changes in pancreatic beta cells treated with NGF and dbcAMP.

Nerve growth factor (NGF) induces morphological and physiological changes in cultured pancreatic beta-cells, including the extension of neurite-like processes. This latter effect is potentiated by dibutyryl cAMP (dbcAMP). beta-cells cultured under these conditions maintain their immunoreactivity to insulin and gamma-amino-butyric acid (GABA). NGF, dbcAMP, and high glucose concentrations also increase the expression of the GABA-synthesizing enzyme glutamic acid decarboxylase-65 in cultured beta-cells. The aim of this work was to study the effect of NGF alone or in combination with dbcAMP on pancreatic beta-cell ultrastructural morphology, after 10 days in culture. We used light microscopy, scanning electron microscopy, and transmission electron microscopy to analyze the modifications in cell surface and neurite-like projections. Morphometric analysis showed that NGF and/or dbcAMP treatment substantially increased the insulin and GABA content in granules and rough endoplasmic reticulum. Given that pancreatic beta-cells express NGF receptors and that NGF is synthesized and secreted by beta-cells, these results further suggest that NGF could have trophic actions on pancreatic hormone synthesis and/or storage.

Nerve growth factor increases insulin secretion and barium current in pancreatic beta-cells.

We analyzed the effect of a brief exposure to nerve growth factor (NGF) on insulin secretion and macroscopic barium currents of single adult rat pancreatic beta-cells. After a 1-h exposure to NGF (50 ng/ml), single beta-cells show a 2.5-fold increase in the insulin secretion index in 5.6 mmol/l glucose and a nearly twofold increase in 15.6 mmol/l glucose compared with control cells. We have recently demonstrated that pancreatic beta-cells synthesize and secrete NGF. We analyzed the effect of endogenous NGF on insulin secretion by incubating islet cells in the presence of an anti-NGF monoclonal antibody for 1 h in different glucose concentrations. Although the basal insulin secretion index (5.6 mmol/l glucose) is not affected, glucose-stimulated insulin secretion (15.6 mmol/l glucose) is decreased by 41% in the presence of the antibody. This effect is mediated by the activation of the NGF receptor TrkA because the specific inhibitor of Trk phosphorylation K252a also blocks NGF-induced increase in insulin secretion, both in the presence and absence of exogenous NGF. Using the whole-cell variation of the patch-clamp technique, we found that cells exposed to NGF for 5 min exhibit a 32% increase in the average barium current density. These results suggest that the effects of NGF on insulin secretion are partially mediated by an increase in calcium current through Ca channels. These results further suggest that NGF plays an important autoregulatory role in pancreatic beta-cell function. Two targets of short-term NGF-modulation are insulin secretion and calcium-channel activity.

Helicobacter pylori in water systems for human use in Mexico City.

Helicobacter pylori infection is associated with peptic ulcers and gastric cancer in humans. Transmission of H. pylori is still not certain with some epidemiological data suggesting water as a possible transmission route. The objective of this study was to detect H. pylori 16S rRNA gene in five water systems in the Mexico City area. Samples were taken between 1997 and 2000 from extraction wells (system 1), from dams used as water sources, both pre- and post-treatment (systems 2 and 3), treated wastewater (system 4) and non-treated wastewater (system 5). Detection of the H. pylori 16S rRNA gene in water samples was carried out using nested PCR in 139 water samples and confirmed by using cagA gene detection by PCR-hybridisation. The results showed the presence of H. pylori in 58 (42%) of the water samples in total with a prevalence of 68% in system 1, 100% in system 2, 0% in system 3, 17% in system 4 and 20% in system 5. This first stage showed the presence of H. pylori in the tested water systems; nevertheless, viability of the microorganism in water and vegetables needs to be confirmed as well as demonstration of a relationship between human and environmental strains.

Nerve and fibroblast growth factors as modulators of pancreatic beta cell plasticity and insulin secretion.

Trophic factors such as nerve and fibroblast growth factors are important modulators of beta cell physiology. These two factors induce the extension of neurite-like processes in primary cultures of adult rat beta cells. Moreover, both NGF and FGF enhance glucose-induced insulin secretion. Since beta cells synthesize NGF and pancreatic islet cells produce FGFs, it is possible that autocrine/paracrine interactions may be major regulators of insulin secretion, and impairment of these interactions could lead to pathological states such as diabetes mellitus.