Effects of water deficit and salinity stress on late mandarin trees.

To maintain the viability of citrus farms in a scenario of scarce water supplies for irrigation purposes, such as in south-eastern Spain, it is necessary to detect the maximum threshold of crop water status and vegetative growth, both of which are vulnerable to the combined effect of water and salinity stress. With this aim, adult late mandarin trees were subjected to a gradual increase in salinity of the irrigation water for four consecutive growing seasons, accompanied by a water deficit for the first three seasons, while the fourth season counted as a recovery period. The treatments tested were: i) control (CTL), irrigated at 100% of the ETc in the first season followed by an additional 33% water leaching requirement in the second and third seasons; (ii) a regulated deficit irrigation (RDI100) treatment, irrigated as the CTL except in phase I and until mid-phase II, coinciding with the beginning of the slowdown in trunk growth, where irrigation was set at 65 and 50% CTL in the first and last two seasons, respectively, and 80% in phase III; (iii) RDI50, irrigated as the CTL during the first season, and from the second season onwards, at 50% RDI100 during the deficit period; (iv) FARMER, irrigated in accordance with the farmer's criteria. A moderate saline water stress combined with the application of RDI100 treatment did not reduce crop yield after one growing season, but did reduce plant growth. However, after two consecutive seasons (in both RDI strategies) water stress drastically reduced both yield and plant growth, mainly due to a high accumulation of sodium and chloride in the leaves. Gas exchange parameters were also greatly reduced. Furthermore, it was found that the water deficit could be prolonged until the trunk growth slowed down during the second fruit growth stage in mandarin trees, leading to a substantial increase in water productivity values.

Inhibitory effect of biofilm-forming Lactobacillus kunkeei strains against virulent Pseudomonas aeruginosa in vitro and in honeycomb moth (Galleria mellonella) infection model.

Biofilms correspond to complex communities of microorganisms embedded in an extracellular polymeric matrix. Biofilm lifestyle predominates in Pseudomonas aeruginosa, an opportunistic Gram negative pathogen responsible for a wide spectrum of infections in humans, plants and animals. In this context, anti-biofilm can be considered a key strategy to control P. aeruginosa infections, thereby more research in the field is required. On the other hand, Lactobacillus species have been described as beneficial due to their anti-biofilm properties and their consequent effect against a wide spectrum of pathogens. In fact, biofilm-forming Lactobacilli seem to be more efficient than their planktonic counterpart to antagonise pathogenic bacteria. In this work, we demonstrated that Lactobacillus kunkeei, a novel Lactobacillus species isolated from honeybee guts, can form biofilms in vitro. In addition, the L. kunkeei biofilm can, in turn, inhibit the formation of P. aeruginosa biofilms. Finally, we found that L. kunkeei strains attenuate infection of P. aeruginosa in the Galleria mellonella model, presumably by affecting P. aeruginosa biofilm formation and/or their stability. Since L. kunkeei presents characteristics of a probiotic, this work provides evidence arguing that the use of this Lactobacillus species in both animals (including insects) and humans could contribute to impair P. aeruginosa biofilm formation.

Signal transduction pathways underlying the expression of tissue factor and thrombomodulin in promyelocytic cells induced to differentiate by retinoid acid and dibutyryl cAMP.

Acute promyelocytic leukaemia (APL) may be associated with disseminated intravascular coagulation, as a result of increased tissue factor (TF) expression and reduced thrombomodulin (TM) expression by APL blast cells. During retinoid acid (RA)- and dibutyryl cAMP (dbcAMP)-induced differentiation of the APL cells, there is a marked up-modulation of both the protein kinase A (PKA) and C (PKC) activities. In order to further assess whether these kinases are intimately associated with both the differentiation process and the regulation of TF and TM expression, we have correlated the modulation of their respective pathways with the extent of differentiation and modulation of these cellular receptors. NB4 cells were incubated with all-trans-RA (ATRA) or dbcAMP for up to 48 h. The contribution of phospholipase C (PLC), inositol phosphate (IP), PKC and PKA in the expression of CD11b, TF and TM was studied by the use of specific inhibitors. Myo-inositol uptake and PKC activity increased in cells induced to differentiate by ATRA but the retinoid did not affect cAMP levels or PKA activity. Under treatment with dbcAMP, PKA activity was increased while inositol uptake and PKC activity remained unchanged. Our results show that the effects of ATRA and dbcAMP on promyelocytic cells are closely related, respectively, to the PLC/IP/PKC and the cAMP/PKA pathways. In cells induced to differentiate by ATRA, CD11b expression seems more closely related to inositol uptake than to PKC activity while the expression of TF and TM show the opposite pattern, which suggests cellular events regulated at a different level within a common signal transduction pathway.

Plasma thrombopoietin level after liver transplantation: relationship to cold ischemia time and coagulation parameters.

OBJECTIVE: To determine the relation between thrombopoietin (Tpo) levels following orthotopic liver transplantation (OLT), cold ischemia time and postoperative peripheral blood platelet count and prothrombin activity. DESIGN: Prospective clinical study. SETTING: Intensive care unit. PATIENTS: Fourteen patients with uncomplicated postoperative course after OLT. MEASUREMENTS AND RESULTS: Plasma Tpo, as quantified by enzyme immunoassay, rose significantly from 194.9 +/- 45.7 pg/ml on day 1 after OLT to a peak value of 500.7 +/- 94.1 pg/ml on day 5 while platelet count was below normal values. Then the platelet count increased and reached normal values while Tpo decreased to normal. The rise of Tpo levels was associated with normalization of prothrombin time but peak Tpo concentrations were in inverse correlation with cold ischemia times. CONCLUSION: The extent of production of Tpo in the liver graft following OLT is affected by cold ischemia time. This observation may be applicable in the prevention of bleeding complications associated with postoperative thrombocytopenia.

Increased levels of tissue factor mRNA in mononuclear blood cells of patients with primary antiphospholipid syndrome.

Antiphospholipid antibodies (aPL) may stimulate tissue factor (TF) expression in cultured endothelial cells and monocytes, but there are discrepancies as to the expression of TF in the patients with antiphospholipid syndrome (APS). By using reverse transcription and polymerase chain reaction amplification, we have analysed TF mRNA accumulation in freshly isolated mononuclear blood cells (MBC) of 14 patients with primary APS (PAPS) and six normal controls. TF mRNA accumulation was low or absent in uncultured MBC from all normal controls, but was elevated in uncultured MBC from nine of the patients as well as in normal MBC incubated with 100 ng/ml lipopolysaccharide (LPS). Mean levels of TF mRNA, as measured by densitometry, were higher in MBC from patients (N = 14) than in those from controls (N = 6, P = 0.009), and in MBC from patients with a history of thrombosis (N = 9) than in those from patients without thrombosis (N = 5, P = 0.02). Uncultured MBC of patients with thrombosis accumulated TF mRNA at similar levels to LPS-treated normal MBC. Increased levels of TF mRNA were found in eight of ten patients with conventional aPL (ie, anti-cardiolipin antibodies [aCL] and/or lupus anticoagulant [LA]) and little if any accumulation of TF mRNA was observed in three of four patients without aPL at the time of study. These data strongly suggest that circulating monocytes of many patients with PAPS are subjected to an up-regulated TF expression that may well explain their prothrombotic state. Although the presence or absence of TF mRNA in MBC was associated with, respectively, the presence or absence of conventional aPL in 11 of the 14 patients studied, our study cannot exclude the involvement of factors other than aCL or LA in inducing TF expression.

Age-related changes of plasma alkaline phosphatase and inorganic phosphorus, and late ossification of the cranial roof in the Spanish imperial eagle (Aquila adalberti C. L. Brehm, 1861).

Plasma alkaline phosphatase and inorganic phosphorus levels were determined for 52 nestling Spanish imperial eagles from two wild populations and 22 captive adults and subadults (10 adults and 12 subadults). The exact age was known for all birds. Mean alkaline phosphatase and inorganic phosphorus were higher in chicks than in the captive adults and subadults. Sex differences were not observed, and nestlings from different populations showed similar values. No significant regression described the relationship between age and alkaline phosphatase or inorganic phosphorus throughout the nestling period. However, alkaline phosphatase and inorganic phosphorus decreased significantly throughout the subadult period, with age explaining 98.2% and 50.5% of the variation in alkaline phosphatase and inorganic phosphorus levels, respectively. Non-fully-ossified zones were measured in frontal bones of another 12 subadult eagles that died at known ages. Ossification increased throughout the subadult period and was significantly correlated with expected levels of alkaline phosphatase or inorganic phosphorus (i.e., values predicted from the regression equations derived from the first analysis). Minimum alkaline phosphatase levels and full ossification of the cranial roof coincided with puberty onset. We conclude that, in subadult Spanish imperial eagles, decreasing alkaline phosphatase and inorganic phosphorus values are related to the ossification of frontal bones, although a contribution of other unknown processes of late ossification cannot be excluded, and alkaline phosphatase (but not inorganic phosphorus) may be a useful parameter for age-predicting purposes.

Heterogeneity of growth hormone (GH)-producing cells in aging male rats: in vitro GH releasing activity of somatotrope subpopulations.

Studies on the age-related decline of growth hormone (GH) release have ignored that the population of GH-producing cells (somatotropes) is heterogeneous. In aging male rats, centrifugation of dispersed pituitary cells in a density gradient yields two somatotrope subpopulations, i.e. low- (LD) and high-density (HD) cells. A previous analysis of ultrastructure and GH mRNA levels has shown that storage and biosynthetic features were inversely related in both subsets. Furthermore, ultrastructural and molecular differences between LD- and HD-cells were retained throughout the rat lifespan, suggesting that the heterogeneity of somatotropes may have a biological meaning. Accordingly, the main objective of the present study was to analyze the functional heterogeneity of the somatotrope population during the aging process in male rats. For this purpose, the response of LD- and HD-somatotropes from 5-, 19-, and 26-month-old male rats was analyzed with an optimized cell immunoblot assay both under basal conditions, and after GH-releasing factor (GRF) and/or somatostatin (SS) treatments. Simultaneous measurements of hormonal release, intracellular GH content, and cell size were performed at the single-somatotrope level. Average values for those parameters were significantly higher in HD- than in corresponding LD-cells, such differences being irrespective of age or treatment. Releasing activity and GH content were significantly reduced with age in both subpopulations. GRF stimulated GH release from LD- and HD-somatotropes, and the GRF responsiveness was similar in both subpopulations and in all ages. On the other hand, SS prevented GRF-stimulated GH release in most cases. At the level of single cells, both releasing activity and cell size showed a significant, linear dependence on intracellular GH content, correlations being irrespective of age, subpopulation, or treatment. Taken together, our results demonstrate that LD- and HD-somatotrope subpopulations display quantitative differences in releasing activity that are essentially retained through aging. This functional heterogeneity is more dependent on the basal GH release of these somatotrope subsets than in their responsiveness to GRF and SS. The present findings suggest that the reduction in secretory activity at the single somatotrope level observed in both subpopulations underlies the age-related decline of pituitary GH release. Finally, a theoretical model of secretory cycle is proposed which might contribute to the understanding of the biological meaning of the somatotrope subpopulations in aging male rats.

Expression of the pituitary transcription factor GHF-1/PIT-1 in cell types of the adult porcine adenohypophysis.

We describe the expression of the transcription factor GHF-1/PIT-1 in adult porcine adenohypophysis by a nonradioactive in situ hybridization (ISH) method using a digoxigenin-labeled cDNA probe corresponding to the entire coding region of rat GHF-1. GHF-1 transcripts were found in 71.7% of adenohypophyseal cells. We also report the simultaneous detection of GHF-1 mRNA and pituitary hormones by combined ISH and immunocytochemistry (IC) in dispersed adenohypophyseal cells, detected with an alkaline phosphatase-NBT/BCIP technique and with an immunoperoxidase-3-amino-9-ethylcarbazole (AEC) method, respectively. The combination of the two techniques neither abolished nor diminished their sensitivity or specificity. GHF-1 is expressed in all five of the cell types in the adult porcine adenohypophysis, showing that this method is suitable for simultaneous detection of transcripts and proteins at the single-cell level.

Heterogeneity of growth hormone (GH)-producing cells in aging male rats: ultrastructure and GH gene expression in somatotrope subpopulations.

Mammalian aging is characterized by a decline in the content and release of pituitary growth hormone (GH). However, few studies on the age-related changes in the population of GH-producing cells (somatotropes) have been carried out. We have investigated whether changes in number, ultrastructure and GH gene expression in subpopulations of somatotropes could explain the reduced GH release in aged rats. Three representative ages were studied: adult (5-month-old), old (19-month-old), and senescent (26-month-old) male rats. The total number of immunoreactive-GH cells per pituitary gland remained invariable to age. The separation of dispersed pituitary cells on a density gradient yielded two somatotrope subpopulations, of low density (LD) and high density (HD). Both subpopulations were equally represented in adults, whereas in old and senescent rats a predominance of LD-somatotropes was observed. Morphometric analysis showed that subpopulations exhibited storage and biosynthetic features inversely related. In LD-somatotropes, rough endoplasmic reticulum (RER) was more prominent but secretory granules (SG) were less abundant than in HD somatotropes. Concurrently, in situ hybridization for GH mRNA showed that GH gene expression was higher in LD-cells. Differences between subpopulations were essentially retained through the animals' lifespan, but small-sized SG, reduced RER, and low GH mRNA levels were inherent to aging both in LD- and in HD-somatotropes. The present findings demonstrate that the reduced content of pituitary GH in aged male rats is not due to a diminished number of GH-producing cells, but to the numerical predominance of scarcely granulated LD-somatotropes, combined with the decline in GH biosynthetic capacity observed in both subpopulations. In addition, age-related changes in ultrastructure and GH gene expression suggest a chronic inhibition of GH release and/or a weak stimulation of GH biosynthesis affecting both subpopulations.

Heterogeneous response of porcine gonadotrope subpopulations to gonadotropin releasing hormone (GnRH) during postnatal development.

In previous papers, we showed the porcine gonadotrope population to be composed of three GtH subpopulations that can be separated by density using a continuous Percoll density gradient. We also demonstrated that these subpopulations exhibited different hormonal storage patterns and morphological features during porcine postnatal development at three representative ages: neonates (30 days), prepubers (5-6 months) and matures (16-18 months). In this work, we investigated whether these morphologically heterogeneous subpopulations are also functionally different. Thus, the effect of the hypothalamic gonadotropic hormone-releasing factor (GnRH) on these subpopulations was assessed in order to ascertain whether a mutual relationship between the reported morphological features, hormonal storage patterns and physiological response to the stimulation can be established. For this purpose, gonadotropin secretion was measured by cell immunoblot assay and hormonal content by scanning cytophotometry. Low-density gonadotropes (1.049 g/cm3), present in the three age groups studied, were mainly composed of bihormonal LH/FSH cells in neonates and monohormonal LH cells in prepubers and matures. GnRH stimulation was found to increase both LH and FSH secretion, as well as the intracellular content. These results indicate that GnRH can stimulate both the synthesis and release of both gonadotropins in this subpopulation. Middle-density gonadotropes (1.062 g/cm3), present in prepubers and matures only, were composed of bihormonal cells. GnRH stimulated the secretion of LH and FSH in prepubers and matures, but decreased hormonal contents except that of LH in prepubers. However, GnRH stimulation increased the proportion of immunoreactive gonadotropes (particularly monohormonal cells). Finally, high-density cells (1.087 g/cm3), present in neonates and prepubers only, were mostly composed of bihormonal LH/FSH gonadotropes, and exhibited low (neonates) or no response (prepubers) in terms of LH release and content when treated with GnRH. In conclusion, these results indicate that porcine gonadotrope subpopulations are morphologically and physiologically heterogeneous. The heterogeneity remained through porcine postnatal development, thus suggesting that all the subpopulations are physiologically relevant. However, the different hormonal storage patterns between subsets of the same density suggest age-related differences within each subpopulation due, at least in part, to the different physiological condition of the animals during development.

A comparison of restrictive endonuclease sites of bovine herpesvirus type 1 isolates in Chile.

The Frutillar 82, F nasal 90, F vaginal 90, Puente Alto 77, VMLB 79, and Bio Bio 92 isolates of bovine herpesvirus 1 (BHV-1) from Chile were analysed by Eco RI, Bam HI and Hind III restriction enzymes (RE). The Los Angeles (LA) strain was used as reference. The results indicated that the fingerprints of F nasal 90 and F vaginal 90 were identical to the three RE used, but they were different from the other isolates. Major differences were found for Puente Alto 77 and VMLB 79 isolates with Eco RI, and for Puente Alto 77 with Hind III compared with the other Chilean isolates. It is concluded that Chilean BHV-1 isolates obtained from diverse lesions and geographic locations are genomically different from each other and from the other strains described in the literature.

Effects of morphine and naloxone on prolactin and growth hormone gene expression in the male rat pituitary gland.

It is generally admitted that opioids can stimulate the release of both prolactin (PRL) and growth hormone (GH). In order to investigate the role of opioids in the regulation of PRL and GH gene expression in the rat pituitary, we studied the effects of chronic administration of the opioid drug morphine and an opiate receptor antagonist naloxone on both PRL and GH gene expression as measured by in situ hybridization. Four-day treatment with morphine (40 mg/kg/day) produced a 12% increase in PRL mRNA levels. Conversely, naloxone (4 mg/kg/day) decreased the autoradiographic reaction by 10%. The concomitant administration of morphine and naloxone induced no significant changes in PRL gene expression. On the other hand, treatment with morphine produced a 22% decrease in GH mRNA levels, an effect which was prevented by the concomitant administration naloxone. When injected alone, naloxone did not modify the hybridization signal. These results clearly indicate that opioids are involved not only in the regulation of GH and PRL release but also in the gene expression of the two hormones. The discordance observed between the acute effects of morphine on GH release and the effect of the opioid drug on mRNA levels remains to be clarified.

Molecular characterization of South American bovine herpesvirus-1 isolates with monoclonal antibodies and SDS-PAGE.

Antigenic patterns of BHV-1 isolates from Argentina, Chile and Brazil were examined with a panel of monoclonal antibodies (MAbs) generated with BHV-1.1 and BHV-1.3 strains. Among the Argentinian isolates, 7/8 neurological cases showed BHV-1.3 characteristics and 10/12 non-neurological cases from all countries exhibited BHV-1.1/2 pattern. All viral isolates from genital infections showed BHV-1.1/2 characteristics. Chilean isolates exhibited BHV-1.1/2 pattern and one among 3 Brazilian viruses was defined as belonging to BHV-1.3. Results of the characterization using MAbs, were confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of representative isolates. A total correlation was observed between the reactivity with MAbs and the SDS-polyacrylamide analysis. Based on the data presented, it is concluded that the type 1.3, most frequently obtained from the brain of neurological cases, is present in Argentina and Brazil and coexists with BHV-1.1/2.

Application of an optimized immunostaining technique to evaluate the heterogeneous secretory response from porcine somatotropes by cell blotting.

The objective of the present study was to quantify the absolute hormone release from individual porcine pituitary cells incubated on polyvinylidene difluoride (PVDF) transfer membranes (cell-blot assay). After immunoperoxidase staining, growth hormone (GH) release from isolated somatotrope cells appeared like a colored zone of secretion surrounding the cell. Optical densities of these secretion zones were quantitated by computerized image analysis and translated into picograms by means of an appropriate standard curve. As a prior step, the staining method and the optimal immunocytochemical conditions were selected by applying purified porcine growth hormone (pGH) to the transfer membranes. The avidin-biotin-peroxidase nickel-intensified (ABC-Ni) method produced a better resolution than the peroxide-anti-peroxidase (PAP) method, although both techniques were similar with regard to background, sensitivity, and range of quantitation. The amount of GH released from single porcine somatotropes was highly heterogeneous, although the cells were treated under the same conditions. Moreover, this fact was consistent with the stimulation of the average release of GH by GH-releasing factor (GHRF) but not by GHRF+somatostatin (SRIF). Our results confirm the availability of the recently developed cell-blot assay and support the concept of functional heterogeneity in anterior pituitary cell populations.

Human pancreatic growth hormone-releasing factor (1-44) stimulates GH cells in an anuran amphibian (Rana perezi).

Subcellular responses of amphibian growth hormone (GH)-producing cells to in vivo administration of human pancreatic growth hormone-releasing factor (1-44) (hpGRF) were investigated. The volume density (Vv) of secretory granules (SG), rough endoplasmic reticulum (RER), and Golgi complex (GC) and the numerical density (Nv) of the granules were estimated by ultrastructural morphometry. Immunogold staining was applied to ultrathin sections using an antiserum to ovine GH to identify GH-producing cells. In vivo treatment with hpGRF significantly decreased the Vv of the SG and the Nv of medium SG after 6 hr. The peptide also stimulated development of the cellular biosynthetic machinery and increased the number of small SG 24 hr after stimulation. Four-day-stimulated GH cells did not recover control morphological appearances. These morphological results suggest that: (1) In vivo administered hpGRF stimulates GH cells in Rana perezi by inducing hormone release and enhances biosynthetic activity; (2) after four injections, the cellular response is more intense, indicating that GH cells remain hyperactive, probably because the exogenous hpGRF overcomes the endogenous inhibitory control of GH secretion.