Non-dioxin-like organic toxicant PCB153 modulates sphingolipid metabolism in liver progenitor cells: its role in Cx43-formed gap junction impairment.

The non-dioxin-like environmental toxicant 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153), member of a group of persistent organic pollutants wide-spread throughout the environment, reduces gap junction intercellular communication (GJIC), an event possibly associated with tumor promotion. Since very few studies have investigated the signaling effectors and mode(s) of action of PCB153, and it is known that the gap junction (GJ) protein Cx43 can be regulated by the bioactive sphingolipid (SL) sphingosine 1-phosphate (S1P), this in vitro study mainly addresses whether SL metabolism is affected by PCB153 in rat liver epithelial WB-F344 cells. PCB153 treatment obtained significant changes in the S1P/ceramide (Cer) ratio, known to be crucial in determining cell fate. In particular, an increase in S1P at 30 min and a decrease of the bioactive lipid at 3 h were observed, whereas Cer level increased at 1 h and 24 h. Notably, a time-dependent modulation of sphingosine kinase (SphK), the enzyme responsible for S1P synthesis, and of its regulators, ERK1/2 and protein phosphatase PP2A, supports the involvement of these signaling effectors in PCB153 toxicity. Electrophysiological analyses, furthermore, indicated that the lipophilic environmental toxicant significantly reduced GJ biophysical properties, affecting both voltage-dependent (such as those formed by Cx43 and/or Cx32) and voltage-independent channels, thereby demonstrating that PCB153 may act differently on GJs formed by distinct Cx isoforms. SphK down-regulation alone induced GJIC impairment, and, when combined with PCB153, the acute effect on GJ suppression was additive. Moreover, after enzyme-specific gene silencing, the SphK1 isoform appears to be responsible for down-regulating Cx43 expression, while being the target of PCB153 at short-term exposure. In conclusion, we provide the first evidence of novel effectors in PCB153 toxic action in rat liver stem-like cells, leading us to consider SLs as potential markers for preventing GJIC deregulation and, thus, the tumorigenic action elicited by this environmental toxicant.

Carbon-Nanohorn Based Nanofluids for a Direct Absorption Solar Collector for Civil Application.

Direct solar absorption has been often considered in the past as a possible solution for solar thermal collectors for residential and small commercial applications. A direct absorption could indeed improve the performance of solar collectors by skipping one step of the heat transfer mechanism in standard devices and having a more convenient temperature distribution inside the collector. Classical solar thermal collectors have a metal sheet as absorber, designed such that water has the minimum temperature in each transversal section, in order to collect as much solar thermal energy as possible. On the other hand, in a direct configuration, the hottest part of the system is the operating fluid and this allows to have a more efficient conversion. Nanofluids, i.e., fluids with a suspension of nanoparticles, such as carbon nanohorns, could be a good and innovative family of absorbing fluids owing to their higher absorption coefficient compared to the base fluid and stability under moderate temperature gradients. Moreover, carbon nanohorns offer the remarkable advantage of a reduced toxicity over other carbon nanoparticles. In this work, a three-dimensional model of the absorption phenomena in nanofluids within a cylindrical tube is coupled with a computational fluid dynamics (CFD) analysis of the flow and temperature field. Measured optical properties of nanofluids at different concentrations have been implemented in the model. Heat losses due to conduction, convection and radiation at the boundaries are considered as well.

Optical design of a light-emitting diode lamp for a maritime lighthouse.

Traffic signaling is an emerging field for light-emitting diode (LED) applications. This sustainable power-saving illumination technology can be used in maritime signaling thanks to the recently updated norms, where the possibility to utilize LED sources is explicitly cited, and to the availability of high-power white LEDs that, combined with suitable lenses, permit us to obtain well-collimated beams. This paper describes the optical design of a LED-based lamp that can replace a traditional lamp in an authentic marine lighthouse. This source recombines multiple separated LEDs realizing a quasi-punctual localized source. Advantages can be lower energy consumption, higher efficiency, longer life, fewer faults, slower aging, and minor maintenance costs. The proposed LED source allows us to keep and to utilize the old Fresnel lenses of the lighthouse, which very often have historical value.

Deletion of small ankyrin 1 (sAnk1) isoforms results in structural and functional alterations in aging skeletal muscle fibers.

Muscle-specific ankyrins 1 (sAnk1) are a group of small ankyrin 1 isoforms, of which sAnk1.5 is the most abundant. sAnk1 are localized in the sarcoplasmic reticulum (SR) membrane from where they interact with obscurin, a myofibrillar protein. This interaction appears to contribute to stabilize the SR close to the myofibrils. Here we report the structural and functional characterization of skeletal muscles from sAnk1 knockout mice (KO). Deletion of sAnk1 did not change the expression and localization of SR proteins in 4- to 6-mo-old sAnk1 KO mice. Structurally, the main modification observed in skeletal muscles of adult sAnk1 KO mice (4-6 mo of age) was the reduction of SR volume at the sarcomere A band level. With increasing age (at 12-15 mo of age) extensor digitorum longus (EDL) skeletal muscles of sAnk1 KO mice develop prematurely large tubular aggregates, whereas diaphragm undergoes significant structural damage. Parallel functional studies revealed specific changes in the contractile performance of muscles from sAnk1 KO mice and a reduced exercise tolerance in an endurance test on treadmill compared with control mice. Moreover, reduced Qγ charge and L-type Ca(2+) current, which are indexes of affected excitation-contraction coupling, were observed in diaphragm fibers from 12- to 15-mo-old mice, but not in other skeletal muscles from sAnk1 KO mice. Altogether, these findings show that the ablation of sAnk1, by altering the organization of the SR, renders skeletal muscles susceptible to undergo structural and functional alterations more evident with age, and point to an important contribution of sAnk1 to the maintenance of the longitudinal SR architecture.

Carbon nanohorns-based nanofluids as direct sunlight absorbers.

The optimization of the poor heat transfer characteristics of fluids conventionally employed in solar devices are at present one of the main topics for system efficiency and compactness. In the present work we investigated the optical and thermal properties of nanofluids consisting in aqueous suspensions of single wall carbon nanohorns. The characteristics of these nanofluids were evaluated in view of their use as sunlight absorber fluids in a solar device. The observed nanoparticle-induced differences in optical properties appeared promising, leading to a considerably higher sunlight absorption. We found that the thermal conductivity of the nanofluids was higher than pure water. Both these effects, together with the possible chemical functionalization of carbon nanohorns, make this new kind of nanofluids very interesting for increasing the overall efficiency of the sunlight exploiting device.

Sphingosine 1-phosphate induces differentiation of adipose tissue-derived mesenchymal stem cells towards smooth muscle cells.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid which regulates multiple biological parameters in a number of cell types, including stem cells. Here we report, for the first time, that S1P dose-dependently stimulates differentiation of adipose tissue-derived mesenchymal stem cells (ASMC) towards smooth muscle cells. Indeed, S1P not only induced the expression of smooth muscle cell-specific proteins such as alpha-smooth muscle actin (alpha SMA) and transgelin, but also profoundly affected ASMC morphology by enhancing cytoskeletal F-actin assembly, which incorporated alpha SMA. More importantly, S1P challenge was responsible for the functional appearance of Ca(2+) currents, characteristic of differentiated excitable cells such as smooth muscle cells. By employing various agonists and antagonists to inhibit S1P receptor subtypes, S1P(2) turned out to be critical for the pro-differentiating effect of S1P, while S1P(3) appeared to play a secondary role. This study individuates an important role of S1P in AMSC which can be exploited to favour vascular regeneration.

An efficient experimental strategy for mouse embryonic stem cell differentiation and separation of a cytokeratin-19-positive population of insulin-producing cells.

OBJECTIVES: Embryonic stem cells are a potential source for insulin-producing cells, but existing differentiation protocols are of limited efficiency. Here, the aim has been to develop a new one, which drives development of embryonic stem cells towards insulin-producing cells rather than to neuronal cell types, and to combine this with a strategy for their separation from insulin-negative cells. MATERIALS AND METHODS: The cytokeratin-19 (CK19) promoter was used to control the expression of enhanced yellow fluorescence protein in mouse embryonic stem cells during their differentiation towards insulin-producing cells, using a new optimized four-stage protocol. Two cell populations, CK19(+) and CK19(-) cells, were successfully fluorescence sorted and analysed. RESULTS: The new method reduced neuronal progeny and suppressed differentiation into glucagon- and somatostatin-producing cells. Concomitantly, beta-cell like characteristics of insulin-producing cells were strengthened, as documented by high gene expression of the Glut2 glucose transporter and the transcription factor Pdx1. This novel protocol was combined with a cell-sorting technique. Through the combined procedure, a fraction of glucose-responsive insulin-secreting CK19(+) cells was obtained with 40-fold higher insulin gene expression and 50-fold higher insulin content than CK19(-) cells. CK19(+) cells were immunoreactive for C-peptide and had ultrastructural characteristics of an insulin-secretory cell. CONCLUSION: Differentiated CK19(+) cells reflect an endocrine precursor cell type of ductal origin, potentially suitable for insulin replacement therapy in diabetes.

Thyroid hormones promote cell differentiation and up-regulate the expression of the seladin-1 gene in in vitro models of human neuronal precursors.

Thyroid hormones (TH) play an important role in the development of human brain, by regulating the expression of specific genes. Selective Alzheimer's disease indicator-1 (seladin-1) is a recently discovered gene with neuroprotective properties, which has been found to be down-regulated in brain regions affected by Alzheimer's disease. Seladin-1 has anti-apoptotic properties mainly due to the inhibition of the activation of caspase 3. The aim of this study was to determine whether seladin-1 may be regarded as a new mediator of the effects of TH in the developing brain. In order to demonstrate this hypothesis, the effects of TH both on cell differentiation and on the expression of seladin-1 were assessed in two different cell models, i.e. fetal human neuroepithelial cells (FNC) and human mesenchymal stem cells (hMSC), which can be differentiated into neurons. 3,3',5-Triiodothyronine (T3) determined different biological responses (inhibition of cell adhesion, induction of migration, and increase in the expression of the neuronal marker neurofilament-M and Na+ and Ca2+ channel functionality) in both FNC and hMSC, which express TH receptors. Then, we showed that TH significantly increase the expression levels of seladin-1, and that T3 effectively prevents camptothecin-induced apoptosis. However, in hMSC-derived neurons the expression of seladin-1 was not affected by TH. Our results demonstrated for the first time that seladin-1 is a novel TH-regulated gene in neuronal precursors. In view of its anti-apoptotic activity, it might be hypothesized that one of the functions of the increased seladin-1 levels in the developing brain may be to protect neuronal precursor cells from death.

Sphingosine 1-phosphate induces myoblast differentiation through Cx43 protein expression: a role for a gap junction-dependent and -independent function.

Although sphingosine 1-phosphate (S1P) has been considered a potent regulator of skeletal muscle biology, acting as a physiological anti-mitogenic and prodifferentiating agent, its downstream effectors are poorly known. In the present study, we provide experimental evidence for a novel mechanism by which S1P regulates skeletal muscle differentiation through the regulation of gap junctional protein connexin (Cx) 43. Indeed, the treatment with S1P greatly enhanced Cx43 expression and gap junctional intercellular communication during the early phases of myoblast differentiation, whereas the down-regulation of Cx43 by transfection with short interfering RNA blocked myogenesis elicited by S1P. Moreover, calcium and p38 MAPK-dependent pathways were required for S1P-induced increase in Cx43 expression. Interestingly, enforced expression of mutated Cx43(Delta130-136) reduced gap junction communication and totally inhibited S1P-induced expression of the myogenic markers, myogenin, myosin heavy chain, caveolin-3, and myotube formation. Notably, in S1P-stimulated myoblasts, endogenous or wild-type Cx43 protein, but not the mutated form, coimmunoprecipitated and colocalized with F-actin and cortactin in a p38 MAPK-dependent manner. These data, together with the known role of actin remodeling in cell differentiation, strongly support the important contribution of gap junctional communication, Cx43 expression and Cx43/cytoskeleton interaction in skeletal myogenesis elicited by S1P.

Sphingosine 1-phosphate induces cell contraction via calcium-independent/Rho-dependent pathways in undifferentiated skeletal muscle cells.

We have previously shown that sphingosine 1-phosphate (S1P) can induce intracellular Ca(2+) mobilization and cell contraction in C2C12 myoblasts and that the two phenomena are temporally unrelated. Although Ca(2+)-independent mechanisms of cell contraction have been the focus of numerous studies on Ca(2+) sensitization of smooth muscle, comparatively less studies have focused on the role that these mechanisms play in the regulation of skeletal muscle contractility. Phosphorylation and activation of myosin by Rho-dependent kinase mediate most of Ca(2+)-independent contractile responses. In the present study, we examined the potential role of Rho/Rho-kinase cascade activation in S1P-induced C2C12 cell contraction. First, we showed that depletion of Ca(2+), by pre-treatment with BAPTA, did not affect S1P-induced myoblastic contractility, whereas it abolished S1P-induced Ca(2+) transients. These results correlated with the absence of troponin C and with the immature cytoskeletal organization of these cells. Experimental evidence demonstrating the involvement of Rho pathway in S1P-stimulated myoblast contraction included: the activation/translocation of RhoA to the membrane in response to agonist-stimulation in cells depleted of Ca(2+) and the inhibition of dynamic changes of the actin cytoskeleton in cells where Rho functions had been inhibited either by overexpression of RhoGDI, a physiological inhibitor of GDP dissociation from Rho proteins, or by pretreatment with Y-27632, a specific Rho kinase inhibitor. Contribution of protein kinase C in this cytoskeletal rearrangement was also evaluated. However, the pretreatment with Gö6976 or rottlerin, specific inhibitors of PKC alpha and PKC delta, respectively, failed to inhibit the agonist-induced myoblastic contraction. Single particle tracking of G-actin fluorescent probe was performed to statistically evaluate actin cytoskeletal dynamics in response to S1P. Stimulation with S1P was also able to increase the phosphorylation level of myosin light chain II. In conclusion, our results strongly suggest that Ca(2+)-independent/Rho-Rho kinase-dependent pathways may exert an important role in S1P-induced myoblastic cell contraction.

Ca+2 homeostasis and cytoskeletal rearrangement operated by sphingosine 1-phosphate in C2C12 myoblastic cells.

In hypogravity conditions unloading of skeletal muscle fibres causes alterations in skeletal muscle structure and functions including growth, gene expression, cell differentiation, cytoskeletal organization, contractility and plasticity. Recent studies have identified sphingosine I -phosphate (SPP) as a lipid mediator capable of eliciting intracellular Ca2+ transients, cell proliferation, differentiation, suppression of apoptosis, as well as cell injury repair. The aim of this research is to evaluate a possible involvement of SPP in skeletal muscle cells differentiation and repair from space-flight damage. Particularly, we investigated the Ca2+ sources and the changes on the cytoskeletal rearrangement induced by SPP in a mouse skeletal (C2C12) myoblastic cell line. Confocal fluorescence imaging revealed that SPP elicited Ca2+ transients which propagated throughout the cytosol and nucleus. This response required extracellular and intracellular Ca2+ mobilization. SPP also induced cell contraction through a Ca2(+)- independent/Rho-dependent pathway. The nuclear Ca2+ transients are suggestive for an action of SPP in the differentiation program and damage repair.

Separation of charge movement components in mammalian skeletal muscle fibres.

1. Intramembrane charge movements, I(ICM), were measured in rat skeletal muscle fibres in response to voltage steps from a -90 mV holding potential to a wide test voltage range (-85 to 30 mV), using a double Vaseline-gap voltage-clamp technique. Solutions were designed to minimise ionic currents. Ca(2+) current was blocked by adding Cd(2+) (0.8 mM) to the external solution. In a subset of experiments Cd(2+) was omitted to determine which components of the charge movement best correlated with L-type Ca(2+) channel gating. 2. Detailed kinetic analysis of I(ICM) identified two major groups of charges. The first two components, designated Q(a) and Q(b), were the only charges moved by small depolarising steps. The second group of components, Q(c) and Q(d), showed a more positive voltage threshold, -35.6 +/- 2.0 mV, (n = 6) in external solution with Cd(2+), and -41.1 +/- 2.0 mV (n = 12) in external solution without Cd(2+). Notably, in external solution without Cd(2+) the voltage threshold of Ca(2+) current, I(Ca), activation had a similar value, being -38.1 +/- 2.4 mV. 3. The sum of three Boltzmann functions, Q(1), Q(2) and Q(3), showing progressively more positive transition voltages, could be fitted to charge versus voltage, Q(ICM)-V, plots. The three Boltzmann terms identified three charge components: Q(1) described the shallow voltage-dependent Q(a) and Q(b) charges, Q(2) and Q(3) described the steep voltage-dependent Q(c) and Q(d) charges. 4. In external solution without Cd(2+) the charge kinetics changed: a slow decaying phase was replaced by a pronounced delayed hump. Moreover, the transition voltages of the individual steady-state charge components were shifted towards negative potentials (from 6.3 to 8.2 mV). Nevertheless, the overall charge and steepness factors were conserved. 5. In conclusion, these experiments allowed a clear separation of four components of intramembrane charge movements in rat skeletal muscle, showing that there are no fundamental differences with respect to charge movement components between amphibian and mammalian twitch muscle. Moreover, Q(c) and Q(d) charge are correlated with L-type Ca(2+) channel gating.

Effect of an acute glucose overload on Islet cell morphology and secretory function in the toad.

The aim of this work was to study the effect of induced hyperglycemia on islet cell mass and insulin secretion in normal toads. Immunolabeled beta cell area, replication (bromodeoxyuridine) and apoptosis (propidium iodide) rate, islet neogenesis (cytokeratin), and insulin secretion in vitro were measured in adult male specimens of Bufo arenarum during and after interruption of the injection of either a 50% glucose solution (2 g/100 g) or its vehicle for 4 days. Glucose administration caused hyperglycemia (122.6 +/- 16.7 and 508.3 +/- 115.9 mg/dl vs 23.5 +/- 1.26 and 22.8 +/- 1.8 mg/dl, at days 3 and 5, respectively, P < 0.05) and a significant decrease in the number of islets/mm(2) (day 3: 9.7 +/- 0.9 vs 3.3 +/- 0.4, P < 0.05; day 5: 9.4 +/- 0.8 vs 7.4 +/- 0.6; day 9: 9.6 +/- 0.9 vs 6.2 +/- 0.4, P < 0.05) and in the percentage of immunolabeled beta cell area (day 3: 2.07 +/- 0.2 vs 0.5 +/- 0.1%, P < 0.05; day 5: 1.8 +/- 0.1 vs 0.6 +/- 0.1%; day 9: 1.7 +/- 0.1 vs 0.7 +/- 0.1%, P < 0.05). Glucose-injected animals had a simultaneous significantly higher percentage of BrdU-labeled beta cells (day 3: 0.46 +/- 0.02 vs 0.23 +/- 0.03%; day 5: 0.54 +/- 0.13 vs 0.22 +/- 0.02%; day 9: 0.61 +/- 0.0 vs 0.27 +/- 0.05%, P < 0.05) and cytokeratin-labeled endocrine cells (day 3: 0.21 +/- 0.06 vs 0.01 +/- 0.00%; day 5: 0.17 +/- 0.06 vs 0.01 +/- 0.01%; day 9: 1.25 +/- 0.2 vs 0.01 +/- 0.008%, P < 0.05) and a higher rate of apoptotic beta cells (day 3: 0.14 +/- 0.04 vs 0.05 +/- 0.02%; day 5: 0.4 +/- 0.06 vs 0.05 +/- 0.2, P < 0.05; day 9: 0.47 +/- 0.04 vs 0.06 +/- 0.03, P < 0.05). Comparable amounts of insulin were secreted in vitro by both groups in response to 2 mM glucose, whereas there was a significantly reduced response to 8 mM glucose in treated animals (day 3: 73 +/- 12 vs 165 +/- 20%; day 5: 74 +/- 11 vs 204 +/- 18%, P < 0.05). This decreased response to high glucose reverted to normal after removal of the glucose injection. These results show for the first time that short-term hyperglycemia triggers marked morphological and transient secretory changes in the toad pancreas similar in part to those elicited in the pancreas of several mammals. As with other results previously reported, these results support the usefulness of the toad as an alternative easily handled model to study the growth and secretory function of the endocrine pancreas.

A useful model to study the effect of high sugar concentrations upon growth and enzymic activities of toad embryos and larvae.

The aim of this study was to develop an oviparous model suitable for studying the differential effects and mechanisms by which a high concentration of extracellular glucose and other sugars produce diabetes complications, particularly body growth retardation during development. Hence, we studied the experimental conditions necessary to obtain measurable effects of high sugar concentrations (5-mM glucose, mannitol, fructose and galactose) upon body growth and development of Bufo arenarum embryos and larvae, and upon the activity of aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), and alkaline phosphatase (APP). Unfed animals kept in glucose showed lower body weight than controls at all stages, a condition only observed at stage 26 for animals kept in galactose and fructose. All animals reached the same stage of development regardless of the solution in which they were kept. Glucose and fructose significantly decreased the activity of all enzymes tested, while galactose only affected GGT activity. The model provides the first experimental evidence for the deleterious effect exerted in vivo by different sugars upon developing embryos and larvaes of Bufo arenarum. The results prove that this model might help to elucidate the effects and the pathogenic mechanisms of hyperglycemia upon growth and development of embryos exposed to environments with high sugar concentrations. It might also become a useful tool for testing the effectiveness of drugs designed to prevent the deleterious effect of such exposure.

Possible relationship between the B-cell threshold for glucose-induced insulin secretion and blood glucose concentrations in the normal toad.

The aim of this work was to gain information on the possible relationship between basal glycemia in the toad and the B-cell threshold for glucose-induced insulin release. Hence, pieces of pancreas from Bufo arenarum were incubated with 2 to 20 mM glucose or preincubated with 2 mM glucose plus the hexokinase and glucokinase inhibitors (50 mM of 2-deoxyglucose and mannoheptulose, respectively) followed by an incubation with different glucose concentrations. The maximal rate of insulin release occurred at 8 mM glucose, while 50% of the release (K(s50)) was observed at 7 mM glucose. Regardless of the glucose concentration employed, pancreas pieces preincubated with 2-deoxyglucose released less insulin than the corresponding controls. On the other hand, mannoheptulose significantly inhibited the release of insulin at high glucose concentrations, having no effect at low glucose concentrations. The blocking effect of these two inhibitors is the first indirect evidence of the existence of the hexokinase/glucokinase enzymic system in the toad pancreas. Since the activity ratio of this system determines the glucose sensitivity of the insulin secretory mechanism, it is concluded that the possible existence of a higher ratio of these enzymes in toad B cells could explain the particular characteristics of glucose sensitivity in this animal, which in turn may explain its low blood glucose concentration.

Hormone coexpression in the adult toad endocrine pancreas: double-label immunofluorescence under basal conditions and after glucose injection.

We have investigated the type and frequency of hormone coexpression in the endocrine pancreas of amphibians both under basal conditions and after sustained glucose loading. Adult male specimens of the wild toad Bufo arenarum were injected with a 50% (w/v) glucose solution (2 g/100 g) for 2 days, while control animals received an equal volume of the vehicle. Serum glucose levels were measured at the time of sacrifice and the pancreatic free lobe was processed for light microscopy. A double-labeling immunofluorescence study was performed for the detection of insulin (I), glucagon (G), somatostatin (S), and pancreatic polypeptide (PP). Heterospecific antisera against the following hormone combinations were used for their detection and immunocytochemical localization: I+G, I+PP, G+PP, S+G, and S+PP; visualization of the reacted IgG's was effected by fluorescein- and rhodamine-conjugated species-specific antibodies as fluorophores. Intracellular hormone coexpression was found to occur in the combinations G+PP, S+G, and S+PP. Moreover, glucose administration caused, together with a marked hyperglycemia (123 +/- 17 vs 23 +/- 1 mg/dl; P < 0.05), a decrease in the fraction of cells containing both G and PP together (from 106.3 +/- 8.1 to 26 +/- 4 cell/mm2) along with a reciprocal rise in the number of cells possessing G alone (from 128.7-152.3 to 235.9-274 cell/mm2). The fewer number of cells coexpressing either of the other two hormone combinations, however, were unaffected by glucose injection. With respect to the simultaneous measurement of I+G and I+PP, no cells were detected with both hormones of either pair, and the I-containing cells were more frequent in each instance in the control toads (264.8 +/- 22.3 to 269.2 +/- 27 cell/mm2). For both combinations, however, this value diminished significantly in the glucose-treated animals (108 +/- 2 cell/mm2 for I+G and 112.1 +/- 7. 8 cell/mm2 for I+PP). While the G-containing cells became more numerous (rising to 235.9 +/- 12.4, 274 +/- 26, and 250.4 +/- 23.7 cell/mm2 for I-G, G-PP, and G-S combinations, respectively), the PP- and S-containing cells remained unaffected. We conclude that the copresence of different hormones within the same cell is a relatively common finding in the non-I-secreting elements of the adult toad pancreas and that the proportions of specific cell types are affected by glucose administration. We thus propose that intracellular hormonal coexpression in this fashion may well represent a rapid and efficient regulatory mechanism for compensating for the metabolic stress imposed by glucose loading.

Modulatory effect of hormones on insulin secretion in vitro in the toad.

The aim of this work was to demonstrate the possible direct effect of several hormones upon glucose-induced insulin secretion in amphibians. Hence, pancreas pieces of Bufo arenarum were incubated for 60 min at 25 degrees with 2 and 8 mM glucose plus the addition of hormones known to affect insulin secretion in mammals, measuring the release of insulin by radioimmunoassay. Glucagon (1 microM), ACTH (2.5 microM), human and bovine growth hormone (4.6 and 2.1 microM), prolactin (0.27 microM), corticosterone (0.4 microM), androstanolone (10(-2) microM), estradiol and estrone (10 microM), triiodothyronine and thyroxine (1 microM) enhanced significantly the glucose-induced insulin secretion. Androstanolone, human and bovine growth hormone, triiodothyronine and thyroxine only exerted such effect in the presence of 8 mM glucose. Conversely, somatostatin (1 microM), adrenalin (1 microM), clonidine (2 microM), dexamethasone (0.4 microM), and 2-hydroxyestradiol (5 microM) decreased significantly the glucose-induced insulin release. However, the effect of somatostatin was only apparent in the presence of high glucose. The direct effect of all these hormones--tested for the first time in the amphibian pancreas--was similar to that described in the mammalian pancreas, thus suggesting that such hormones might participate, at least in vitro, in the fine-tuning of insulin secretion in amphibians.

Effect of carbohydrates upon insulin secretion in Bufo arenarum (Amphibia:Bufonidae).

Pancreas pieces of Bufo arenarum were incubated with several sugars at basal and stimulatory concentrations, and with inhibitors of their metabolism, measuring the insulin released by radioimmunoassay. Glucose, mannose, fructose, glyceraldehyde and dihydroxyacetone all at 8 mM, significantly enhanced the release of insulin elicited by basal concentrations of these carbohydrates (2 mM). The nonmetabolizable sugars galactose and 2-deoxyglucose failed to enhance insulin secretion. N-Acetyl-glucosamine at 8 mM did not significantly affect the release of insulin. D-Glucose, but not L-glucose, at 8 mM stimulated insulin secretion above baseline (2 mM glucose). At 8 mM, the D-glucose alpha-anomer significantly increased insulin release, while this effect was not observed using the beta-anomer. Insulin release elicited by 2 mM of the alpha-anomer was significantly higher than that triggered by the beta-anomer. Iodoacetate (5 mM), and dinitrophenol (0.3 mM) exerted an inhibitory effect upon glucose-induced insulin secretion. The effect of these carbohydrates and metabolic inhibitors--tested for the first time in amphibians--was similar to that described in the mammalian pancreas, thus suggesting that such compounds play an important role in the metabolic control of insulin secretion in amphibians.

Soil textural classification by a photosedimentation method.

A photosedimentation technique is used to analyze the size composition of soil samples. The number and size of the particles are determined, respectively, by the Stokes formula and the Beer-Lambert law, measuring time-of-flight and laser light attenuation simultaneously and hence evaluating solution turbidity. A simple software procedure has been developed to obtain fractional volume size distribution, taking into account the particle's optical properties depending mainly on its size and refractive index. Laboratory measurements on calibrated particulates, showing their reproducibility and validation as well as a classification of ground samples, are presented. Size distribution data can then be utilized to obtain a textural classification of the soil samples for agricultural applications.

Effect of activators and inhibitors of K+ channels on insulin secretion in the amphibian pancreas.

The aim of this study was to obtain pharmacological evidence for the presence and participation of K+ channels in amphibian pancreatic islets. Pancreases from the toad Bufo arenarum were thus incubated with activators or blockers of K+ channels and the immunoreactive insulin released into the medium was measured by radioimmunoassay. Two K(+)-ATP channel openers (diazoxide and BPDZ44) inhibited; while a K(+)-ATP channel blocker (tolbutamide) and metabolizable sugars (glucose, glyceraldehyde) significantly stimulated the output of insulin. Although a nonmetabolizable sugar (galactose) failed to increase insulin release, dinitrophenol decreased the secretagogue effect of glucose. By contrast, although somatostatin and clonidine blocked the release of insulin, tetraethylammonium significantly stimulated secretion. For each compound tested, the effects on both insulin secretion and B-cell K+ channel activity were similar to those observed in the mammalian pancreas. These findings point to the existence of mammalian-like K+ channels in the B-cells of some amphibians.