Detection of heavy metals in bacterial biofilms and microbial flocs with the fluorescent complexing agent Newport Green

The complexing agent Newport Green fluoresces upon binding of nickel, zinc or cobalt. It was used to detect nickel or zinc in MOPS buffer, in gel-like matrices, and in natural biofilms and microbial flocs cultivated in the laboratory. The response curves for increasing nickel concentrations indicated an equimolar binding capacity of Newport Green for nickel in MOPS buffer, whereas zinc fluorescence reached saturation in the presence of a 10-fold excess of zinc ions relative to Newport Green molecules. The maximum fluorescence intensity as determined by luminometry was 8-fold and 4-fold above background for nickel and zinc, respectively. The response of Newport Green to either nickel or zinc in the presence of the other metal is consistent with a different binding affinity of Newport Green for the two metals. Zinc binds more strongly to the complexing agent than nickel but it leads to a weaker fluorescent signal which was detectable by luminometry but not by confocal laser scanning microscopy (CLSM). Newport Green was able to complex nickel in the presence of 1% gelatin or agarose as determined by CLSM and image processing. Its application to fully hydrated bacterial biofilms or microbial flocs revealed the presence of nickel outside of cells. The results suggest that in addition to cellular sorption, metals are bound extracellularly by extracellular polymeric substances in intact and undisturbed microbial aggregates. Journal of Industrial Microbiology & Biotechnology (2000) 24, 116–123. Received 11 June 1999/ Accepted in revised form 04 November 1999     

The interaction of biological and noxious transition metals with the zinc probes FluoZin-3 and Newport Green

Zinc-sensitive fluorescent probes have become increasingly important in the investigation of the cellular roles of zinc. There is, however, little information on how the other transition metals in cells may influence the measurement of zinc. We have characterized in vitro the interaction of the nominal zinc indicators FluoZin-3 and Newport Green with all the cationic transition metals found within cells, Cr, Mn, Fe, Co, and Cu, as well as Ni and Cd, by measuring their dissociation constants. In addition, we have shown how FluoZin-3 can be used to quantify the concentration of copper in a cell-free assay and report that the fluorescence of Newport Green is boosted by both Cu(I) and Fe(II). Furthermore, we have introduced diagnostics for detecting the interference of metals other than zinc with its measurement within cells.     

Comparison of cellular and medium insulin and GABA content as markers for living beta-cells

Experimental and therapeutic use of islet cell preparations could benefit from assays that measure variations in the mass of living beta-cells. Because processes of cell death can be followed by depletion and/or discharge of cell-specific substances, we examined whether in vitro conditions of beta-cell death resulted in changes in tissue and medium content of insulin and of gamma-aminobutyric acid (GABA), two beta-cell-specific compounds with different cellular localization and turnover. Exposure of rat purified beta-cells to streptozotocin (5 mM, 120 min) or to the nitric oxide donor GEA-3162 (GEA; 50 microM, 120 min) caused 80% necrosis within 24 h; at the end of this period, cellular insulin content was not significantly decreased, but cellular GABA content was reduced by 70%; when cultured at basal glucose (6 mM), the toxin-exposed cells did not discharge less insulin but released 80% less GABA in the period 8-24 h. As in rat beta-cell purification, GABA comigrated with insulin during human islet cell isolation. Twenty-four hours after GEA (500 microM, 120 min), human islet cell preparations exhibited 90% dead cells and a 45 and 90% reduction, respectively, in tissue insulin and GABA content; in the period 9-24 h, insulin discharge in the medium was not reduced, but GABA release was decreased by 90%. When rat beta-cells were cultured for 24 h with nontoxic interleukin (IL)-1beta concentrations that suppressed glucose-induced insulin release, cellular GABA content was not decreased and GABA release increased by 90% in the period 8-24 h. These data indicate that a reduction in cellular and medium GABA levels is more sensitive than insulin as a marker for the presence of dead beta-cells in isolated preparations. Pancreatic GABA content also rapidly decreased after streptozotocin injection and remained unaffected by 12 h of hyperglycemia. At further variance with insulin, GABA release from living beta-cells depends little on its cellular content but increases with IL-1beta-induced alterations in beta-cell phenotype.     

Receptors for insulin and insulin-like growth factor-1 and insulin receptor substrate-1 mediate pathways that regulate islet function

The insulin/insulin-like growth factor-1 (IGF-1) signalling pathways are present in most mammalian cells and play important roles in the growth and metabolism of tissues. Most proteins in these pathways have also been identified in the beta-cells of the pancreatic islets. Tissue-specific knockout of the insulin receptor (betaIRKO) or IGF-1 receptor (betaIGFRKO) in pancreatic beta-cells leads to altered glucose-sensing and glucose intolerance in adult mice, and betaIRKO mice show an age-dependent decrease in islet size and beta-cell mass. These data indicate that these receptors are important for differentiated function and are unlikely to play a major role in the early growth and/or development of the pancreatic islets. Conventional insulin receptor substrate-1 (IRS-1) knockouts manifest growth retardation and mild insulin resistance. The IRS-1 knockouts also display islet hyperplasia, defects in insulin secretory responses to multiple stimuli both in vivo and in vitro, reduced islet insulin content and an increased number of autophagic vacuoles in the beta-cells. Re-expression of IRS-1 in cultured beta-cells is able to partially restore the insulin content indicating that IRS-1 is involved in the regulation of insulin synthesis. Taken together, these data provide evidence that insulin and IGF-1 receptors and IRS-1, and potentially other proteins in the insulin/IGF-1 signalling pathway, contribute to the regulation of islet hormone secretion and synthesis and therefore in the maintenance of glucose homeostasis.     

Timing of Ca2+ response in pancreatic beta-cells is related to mitochondrial mass

The timing and magnitude of calcium response are cell-specific in individual beta-cells. This may indicate that the cells have different roles in the intact islet. It is unknown what mechanisms determine these characteristics. We previously found that the mechanisms setting cell-specific response timing are disturbed in beta-cells from hyperglycemic mice and one of the causes is likely to be an altered mitochondrial metabolism. Mitochondria play a key role in the control of nutrient-induced insulin secretion. Here, we used confocal microscopy with the fluorescent probe MitoTracker Red CMXRos and Fluo-3 to study how the amount of active mitochondria is related to the lag-time and the magnitude of calcium response to 20mM glucose in isolated beta-cells and in cells within intact lean and ob/ob mouse islets. Results show that the mitochondrial mass is inversely correlated with the lag-times for calcium response both in lean and ob/ob mouse beta-cells (r=-0.73 and r=-0.43, respectively, P<0.05). Thus, the state of mitochondria may determine the timing of calcium response.     

On-line analysis of gap junctions reveals more efficient electrical than dye coupling between islet cells

Pancreatic beta-cells constitute a well-communicating multicellular network that permits a coordinated and synchronized signal transmission within the islet of Langerhans that is necessary for proper insulin release. Gap junctions are the molecular keys that mediate functional cellular connections, which are responsible for electrical and metabolic coupling in the majority of cell types. Although the role of gap junctions in beta-cell electrical coupling is well documented, metabolic communication is still a matter of discussion. Here, we have addressed this issue by use of a fluorescence recovery after photobleaching (FRAP) approach. This technique has been validated as a reliable and noninvasive approach to monitor functional gap junctions in real time. We show that control pancreatic islet cells did not exchange a gap junction-permeant molecule in either clustered cells or intact islets of Langerhans under conditions that allowed cell-to-cell exchange of current-carrying ions. Conversely, we have detected that the same probe was extensively transferred between islet cells of transgenic mice expressing connexin 32 (Cx32) that have enhanced junctional coupling properties. The results indicate that the electrical coupling of native islet cells is more extensive than dye communication. Dye-coupling domains in islet cells appear more restricted than previously inferred with other methods.     

Cellular distribution and contribution of cyclooxygenase COX-2 to diabetogenesis in NOD mouse

Unlike most other mammalian cells, beta-cells of Langerhans constitutively express cyclooxygenase (COX)-2 rather than COX-1. COX-2 is also constitutively expressed in type 1 diabetes (T1D) patients' periphery blood monocytes and macrophage. To understand the role of COX-2 in the beta-cell, we investigated COX-2 expression in beta-cells and islet infiltrates of NOD and BALB/c mice using fluorescence immunohistochemistry and cytochemical confocal microscopy and Western blotting. Immunostaining showed that COX-2 is expressed in islet-infiltrating macrophages, and that the expression of insulin and COX-2 disappeared concomitantly from the beta-cells when NOD mice progressed toward overt diabetes. Also cultured INS-1E cells coexpressed insulin and COX-2 but clearly in different subcellular compartments. Treatment with celecoxib increased insulin release from these cells in a dose-dependent manner in glucose concentrations ranging from 5 to 17 mM. Excessive COX-2 expression by the islet-infiltrating macrophages may contribute to the beta-cell death during insulitis. The effects of celecoxib on INS-1E cells suggest that PGE(2) and other downstream products of COX-2 may contribute to the regulation of insulin release from the beta-cells.     

Excitation wave propagation as a possible mechanism for signal transmission in pancreatic islets of Langerhans

In response to glucose application, beta-cells forming pancreatic islets of Langerhans start bursting oscillations of the membrane potential and intracellular calcium concentration, inducing insulin secretion by the cells. Until recently, it has been assumed that the bursting activity of beta-cells in a single islet of Langerhans is synchronized across the whole islet due to coupling between the cells. However, time delays of several seconds in the activity of distant cells are usually observed in the islets of Langerhans, indicating that electrical/calcium wave propagation through the islets can occur. This work presents both experimental and theoretical evidence for wave propagation in the islets of Langerhans. Experiments with Fura-2 fluorescence monitoring of spatiotemporal calcium dynamics in the islets have clearly shown such wave propagation. Furthermore, numerical simulations of the model describing a cluster of electrically coupled beta-cells have supported our view that the experimentally observed calcium waves are due to electric pulses propagating through the cluster. This point of view is also supported by independent experimental results. Based on the model equations, an approximate analytical expression for the wave velocity is introduced, indicating which parameters can alter the velocity. We point to the possible role of the observed waves as signals controlling the insulin secretion inside the islets of Langerhans, in particular, in the regions that cannot be reached by any external stimuli such as high glucose concentration outside the islets.     

Growth hormone is a growth factor for the differentiated pancreatic beta-cell

The regulation of the growth of the pancreatic beta-cell is poorly understood. There are previous indications of a role of GH in the growth and insulin production of the pancreatic islets. In the present study we present evidence for a direct long-term effect of GH on proliferation and insulin biosynthesis of pancreatic beta-cells in monolayer culture. In culture medium RPMI 1640 supplemented with 2% normal human serum islets or dissociated islet cells from newborn rats maintained their insulin-producing capacity. When supplemented with 1-1000 ng/ml pituitary or recombinant human GH the islet cells attached, spread out, and proliferated into monolayers mainly consisting of insulin-containing cells. The number of beta-cells in S-phase was increased from 0.9-6.5% as determined by immunochemical staining of bromodeoxyuridine incorporated into insulin-positive cells. The increase in cell number was accompanied with a continuous increase in insulin release to the culture medium reaching a 10- 20-fold increase after 2-3 months with a half-maximal effect at about 10 ng/ml human GH. The biosynthesis of (pro)insulin was markedly increased with a normal rate of conversion of proinsulin to insulin. It is concluded that GH is a potent growth factor for the differentiated pancreatic beta-cell.     

Effect of intracellular alkalinization on pancreatic islet calcium uptake and insulin secretion.

Microdissected beta-cell-rich pancreatic islets of ob/ob mice were used in studies of the relationship between intracellular pH (pHi) and 45Ca2+ uptake and insulin release. Stepwise increases in extracellular pH (pHo) from 6.80 to 8.00 resulted in a parallel, although less pronounced, elevation of pHi from 7.24 to 7.69. Experimental conditions that alkalinize the islet cell interior, i.e. addition of 5 mM-NH4+, sudden withdrawal of extracellular bicarbonate buffer or increase in pHo, induced insulin secretion in the absence of other types of secretory stimulation (1 mM-D-glucose). Intracellular acidification by lowering pHo below 7.40 or sudden addition of bicarbonate buffer did not induce insulin secretion. The removal of extracellular bicarbonate buffer, increase in pHo from 7.40 to 8.00, or the addition of 5 mM-L-5-hydroxytryptophan or 5 mM-NH4+, which all alkalinize the islet cells and induce insulin secretion, also increased the La3+-non-displaceable 45Ca2+ uptake in the presence of 1 mM-D-glucose. The results suggest that intracellular alkalinization in beta-cells can trigger insulin secretion. Taken together with the fact that D-glucose increases pHi in the islet cells, the results also point to the possibility that alkalinization may be a link in the stimulus-secretion coupling sequence in beta-cells.     

大鼠胰腺发育阶段Mesothelin的表达

目的:研究Mesothenlin在大鼠胰腺发育阶段的表达和细胞定位。方法:运用RT-PCR和Western Blot技术分别检测Mesothenlin在大鼠胰腺发育阶段的mRNA和蛋白表达水平;运用免疫荧光检测不同时期Mesothenlin在胰腺的组织细胞学定位。结果:RT-PCR结果显示E18.5 Mesothelin mRNA的表达水平显著增高,至P14达到高峰,成年较低。Western Blot结果显示其蛋白表达趋势与mRNA完全相同。免疫荧光结果显示在不同发育时期Mesothenlin与胰岛β细胞和间充质细胞共表达。结论:Mesothenlin在大鼠胚胎胰岛形成及生后结构重塑中出现显著性高表达,并表达于胰岛β细胞和间充质细胞。     

Effect of calcitonin gene-related peptide on glucose and gastric inhibitory polypeptide-stimulated insulin release from cultured newborn and adult rat islet cells

Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide that is present in peripheral cells of islets and in nerves around and within islets. CGRP can inhibit insulin secretion in vitro and in vivo. Whether the inhibitory action of CGRP is mediated by somatostatin or by nerve terminals is, however, not known. The objective of this study was to examine the effect of CGRP on insulin secretion, using cultured newborn and adult rat islet cells which did not contain nerve terminals. In adult rat islet cells, CGRP (10(-10) to 10(-8) M) significantly inhibited glucose-stimulated and gastric inhibitory polypeptide (GIP)-potentiated insulin secretion, but in newborn rat islet cells, CGRP did not inhibit glucose-stimulated insulin secretion. Inhibition of glucose-stimulated and GIP-potentiated insulin release was dependent on the glucose concentration during the prestimulation period. CGRP did not stimulate release of somatostatin. These findings suggest that rat CGRP can act directly on beta-cells through a specific receptor that is absent in newborn rat beta-cells.     

Baculovirus p35 increases pancreatic beta-cell resistance to apoptosis

beta-cells die by apoptosis in type 1 diabetes as a result of autoimmune attack mediated by cytokines, and in type 2 diabetes by various perpetrators including human islet amyloid polypeptide (hIAPP). The cascade of apoptotic events induced by cytokines and hIAPP is mediated through caspases and reactive oxygen species. The baculovirus p35 protein is a potent anti-apoptotic agent shown to be effective in a variety of species and able to inhibit a number of apoptotic pathways. Here, we aimed at determining the protective potential of p35 in beta-cells exposed to cytokines and hIAPP, as well as the effects of p35 on beta-cell function. The p35 gene was introduced into betaTC-tet cells, a differentiated murine beta-cell line capable of undergoing inducible growth-arrest. Both proliferating and growth-arrested cells expressing p35 manifested increased resistance to cytokines and hIAPP, compared with control cells, as judged by cell viability, DNA fragmentation, and caspase-3 activity assays. p35 was significantly more protective in growth-arrested, compared with proliferating, cells. No significant differences were observed in proliferation and insulin content between cells expressing p35 and control cells. In contrast, p35 manifested a perturbing effect on glucose-induced insulin secretion. These findings suggest that p35 could be incorporated as part of a multi-pronged approach of immunoprotective strategies to provide protection from recurring autoimmunity for transplanted beta-cells, as well as in preventive gene therapy in type 1 diabetes. p35 may also be protective from beta-cell damage caused by hIAPP in type 2 diabetes.     

Aggregates of islet cells, their structure and insulin-producing activity during cultivation in vitro

Purified rat islets were dissociated into single-cell suspension with an EDTA-Trypsin treatment. During a stationary culture in vitro the islet cells reassociated forming aggregates (neoislets). Electron microscopy revealed that the aggregates consisted mostly of beta-cells and not numerous alpha-cells. They showed a good insulin-secreting capacity and were able to increase insulin release in response to glucose plus theophylline. The lack of passenger leukocytes makes the neoislets particularly suited for experimental and clinical transplantation.     

Regulated autophagy controls hormone content in secretory-deficient pancreatic endocrine beta-cells

Endocrine cells are continually regulating the balance between hormone biosynthesis, secretion, and intracellular degradation to ensure that cellular hormone stores are maintained at optimal levels. In pancreatic beta-cells, intracellular insulin stores in beta-granules are mostly upheld by efficiently up-regulating proinsulin biosynthesis at the translational level to rapidly replenish the insulin lost via exocytosis. Under normal circumstances, intracellular degradation of insulin plays a relatively minor janitorial role in retiring aged beta-granules, apparently via crinophagy. However, this mechanism alone is not sufficient to maintain optimal insulin storage in beta-cells when insulin secretion is dysfunctional. Here, we show that despite an abnormal imbalance of glucose/glucagon-like peptide 1 regulated insulin production over secretion in Rab3A(-/-) mice compared with control animals, insulin storage levels were maintained due to increased intracellular beta-granule degradation. Electron microscopy analysis indicated that this was mediated by a significant 12-fold up-regulation of multigranular degradation vacuoles in Rab3A(-/-) mouse islet beta-cells (P 相似文献   

Transgenic mice with green fluorescent protein-labeled pancreatic beta -cells

We have generated transgenic mice that express green fluorescent protein (GFP) under the control of the mouse insulin I gene promoter (MIP). The MIP-GFP mice develop normally and are indistinguishable from control animals with respect to glucose tolerance and pancreatic insulin content. Histological studies showed that the MIP-GFP mice had normal islet architecture with coexpression of insulin and GFP in the beta-cells of all islets. We observed GFP expression in islets from embryonic day E13.5 through adulthood. Studies of beta-cell function revealed no difference in glucose-induced intracellular calcium mobilization between islets from transgenic and control animals. We prepared single-cell suspensions from both isolated islets and whole pancreas from MIP-GFP-transgenic mice and sorted the beta-cells by fluorescence-activated cell sorting based on their green fluorescence. These studies showed that 2.4 +/- 0.2% (n = 6) of the cells in the pancreas of newborn (P1) and 0.9 +/- 0.1% (n = 5) of 8-wk-old mice were beta-cells. The MIP-GFP-transgenic mouse may be a useful tool for studying beta-cell biology in normal and diabetic animals.     

Chlorotetracycline as a fluorescent Ca2+ probe in pancreatic islet cells

Pancreatic islets, or suspensions of islet cells, from noninbred ob/ob-mice were incubated with chlorotetracycline and analyzed for Ca2+-dependent fluorescence in a microscope. Unless logarithmically transformed, signals from islets were asymmetrically distributed with unstable variance. Signals from cells pelleted in glass capillaries were more homogeneous and depended linearly on the thickness of the sample. The effect of sample thickness and a significant enhancement of fluorescence by alloxan suggest that beta-cells were involved in producing the signal from whole islets. The signal from dispersed cells was probably diagnostic of Ca2+ in beta-cell plasma membranes because it was suppressed by La3+ and had a spectrum indicative of an apolar micromilieu; fluorescent staining of cell surfaces was directly seen at high magnification. Fluorescence from cells was enhanced by 0.5-10 mM Ca2+ in a dose-dependent manner, whereas less than 0.5 mM Ca2+ saturated the probe alone in methanol. The signal from islets or dispersed cells was suppressed by 5 mM theophylline; that from cells was also suppressed by 0.5 mM 3-isobutyl-1-methylxanthine, 1.2 or 15 mM Mg2+, 3-20 mM D-glucose, and, to a lesser extent, 20 mM 3-O-methyl-D-glucose. D-glucose was more inhibitory in the absence than in the presence of Mg2+, as if Mg2+ and D-glucose influenced the same Ca2+ pool. L-glucose, D-mannopheptulose, or diazoxide had no noticeable effect and 20 mM bicarbonate was stimulatory. The results suggest that microscopy of chlorotetracycline-stained cells can aid in characterizing calcium pools of importance for secretion. Initiation of insulin release may be associated with an increas     

The aggregation potential of human amylin determines its cytotoxicity towards islet beta-cells

Human amylin is a small fibrillogenic protein that is the major constituent of pancreatic islet amyloid, which occurs in most subjects with type 2 diabetes. There is evidence that it can elicit in vitro apoptosis in islet beta-cells, but the physical properties that underpin its cytotoxicity have not been clearly elucidated. Here we employed electron microscopy, thioflavin T fluorescence and CD spectroscopy to analyze amylin preparations whose cytotoxic potential was established by live-dead assay in cultured beta-cells. Highly toxic amylin contained few preformed fibrils and initially showed little beta-sheet content, but underwent marked time-dependent aggregation and beta-conformer formation following dissolution. By contrast, low-toxicity amylin contained abundant preformed fibrils, and demonstrated high initial beta-sheet content but little propensity to aggregate further once dissolved. Thus, mature amylin fibrils are not toxic to beta-cells, and aggregates of fibrils such as occur in pancreatic islet amyloid in vivo are unlikely to contribute to beta-cell loss. Rather, the toxic molecular species is likely to comprise soluble oligomers with significant beta-sheet content. Attempts to find ways of protecting beta-cells from amylin-mediated death might profitably focus on preventing the conformational change from random coil to beta-sheet.     

Group VIA phospholipase A2 forms a signaling complex with the calcium/calmodulin-dependent protein kinase IIbeta expressed in pancreatic islet beta-cells

Insulin-secreting pancreatic islet beta-cells express a Group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)beta) that contains a calmodulin binding site and protein interaction domains. We identified Ca(2+)/calmodulin-dependent protein kinase IIbeta (CaMKIIbeta) as a potential iPLA(2)beta-interacting protein by yeast two-hybrid screening of a cDNA library using iPLA(2)beta cDNA as bait. Cloning CaMKIIbeta cDNA from a rat islet library revealed that one dominant CaMKIIbeta isoform mRNA is expressed by adult islets and is not observed in brain or neonatal islets and that there is high conservation of the isoform expressed by rat and human beta-cells. Binary two-hybrid assays using DNA encoding this isoform as bait and iPLA(2)beta DNA as prey confirmed interaction of the enzymes, as did assays with CaMKIIbeta as prey and iPLA(2)beta bait. His-tagged CaMKIIbeta immobilized on metal affinity matrices bound iPLA(2)beta, and this did not require exogenous calmodulin and was not prevented by a calmodulin antagonist or the Ca(2+) chelator EGTA. Activities of both enzymes increased upon their association, and iPLA(2)beta reaction products reduced CaMKIIbeta activity. Both the iPLA(2)beta inhibitor bromoenol lactone and the CaMKIIbeta inhibitor KN93 reduced arachidonate release from INS-1 insulinoma cells, and both inhibit insulin secretion. CaMKIIbeta and iPLA(2)beta can be coimmunoprecipitated from INS-1 cells, and forskolin, which amplifies glucose-induced insulin secretion, increases the abundance of the immunoprecipitatable complex. These findings suggest that iPLA(2)beta and CaMKIIbeta form a signaling complex in beta-cells, consistent with reports that both enzymes participate in insulin secretion and that their expression is coinduced upon differentiation of pancreatic progenitor to endocrine progenitor cells.