The effect of human amniotic fluid in supporting long-term survival of pancreatic islets in tissue culture

Pancreatic islets obtained from newborn Lewis rats were kept free-floating in TCM 199 either in the presence of 50% human amniotic fluid (HAF), 50% Hank's balanced salt solution (HBSS) or 10% fetal calf serum (FCS) for up to 14 days. The culture, in the presence of HAF, resulted in a good islet viability as demonstrated by islet number, islet insulin content and insulin release into the medium. Contradictory results were obtained when HBSS was used as the medium supplement. Moreover, the islets cultured in HAF-supplemented medium are characterized by a marked replicatory activity as reflected by the incorporation of labeled thymidine into islet DNA and gradual increase in DNA content with the progression of culture time. Even if compared to DNA synthesis of islets cultured under so called standard culture conditions as e.g. supplementation of 10% FCS to the medium, 50% HAF but not yet 10% HAF was found to stimulate the islet replication twofold already after 4 days of culture. It was also demonstrated that FCS has no additional effect on HAF-stimulated DNA synthesis. These findings support the view that HAF-enriched culture medium may have a use in supporting the long-term survival of well preserved endocrine pancreatic tissue.     

Efficient flower induction from cultured buckwheat (Fagopyrum esculentum L.) node segments in vitro

Flower induction from shoot segments of buckwheat seedlings was examinedin vitro. Cytokinin, (especially kinetin at 0.1M), short day conditions and a high concentration ofsolidifying agent improved the flower induction from node segments invitro, in up to about 50% of node segments. The use of anaeration membrane on bottle caps and a high content of sucrose in the mediumimproved flower induction in vitro considerably. In theimproved conditions, flowers were induced from 100% cultures and 10bloomed flowers per explant were induced in vitro in 8weeks. Both long and short types of stigmas, and normal set of flowers wereobserved under the microscope. When pollen produced invitro was cultured on an artificial medium, 70% of the pollengrains germinated, indicating normal viability of in vitropollen, and indicating the potential for artificial pollination invitro. All the varieties examined flowered at a similar percentage,suggesting that the process was independent of variety and that flowers couldbeproduced in vitro. Flower induction from buckwheat plantsin vitro and possible cross breeding invitro are also discussed.     

Glutathione Ethyl Ester Supplementation during Pancreatic Islet Isolation Improves Viability and Transplant Outcomes in a Murine Marginal Islet Mass Model

Background

The success of pancreatic islet transplantation still faces many challenges, mainly related to cell damage during islet isolation and early post-transplant. The increased generation of reactive oxygen species (ROS) during islet isolation and the consumption of antioxidant defenses appear to be an important pathway related to islet damage.

Methodology/Principal Findings

In the present study we evaluated whether supplementation of glutathione-ethyl-ester (GEE) during islet isolation could improve islet viability and transplant outcomes in a murine marginal islet mass model. We also cultured human islets for 24 hours in standard CMRL media with or without GEE supplementation. Supplementation of GEE decreased the content of ROS in isolated islets, leading to a decrease in apoptosis and maintenance of islet viability. A higher percentage of mice transplanted with a marginal mass of GEE treated islets became euglycemic after transplant. The supplementation of 20 mM GEE in cultured human islets significantly reduced the apoptosis rate in comparison to untreated islets.

Conclusions/Significance

GEE supplementation was able to decrease the apoptosis rate and intracellular content of ROS in isolated islets and might be considered a potential intervention to improve islet viability during the isolation process and maintenance in culture before islet transplantation.     

Chronic exposure to TPA depletes PKCalpha and augments Ca-dependent insulin secretion from cultured rat islets

The insulin secretory responses of rat islets to glucose (15 mM), 12-O-tetradecanoylphorbol13-acetate (TPA; 500 nM), and potassium (30 mM) were determined fromperifused islets cultured for 22-24 h in CMRL-1066 medium (controlcultured) or islets cultured in the additional presence of 500 nM TPA.Islet content of protein kinase C  (PKC) and serine and threoninephosphoprotein patterns were also monitored after the culture period.Compared with freshly isolated islets, culturing alone had no adverseeffect on the capacity of TPA or 30 mM potassium to stimulate secretionor on the islet content of PKC. In agreement with previous studies, culturing in TPA reduced the islet content of immunoreactive PKC by>95% and abolished the capacity of the phorbol ester to stimulate secretion during a subsequent dynamic perifusion. Culturing in TPAslightly improved the insulin secretory response to 15 mM glucosecompared with control-cultured islets; however, sustained rates of 15 mM glucose-induced secretion from these islets were significantly lessthan the responses of freshly isolated islets. Islets cultured in TPAresponded to 30 mM potassium with a markedly amplified insulinsecretory response that was abolished by nitrendipine. Enhancedphosphorylation of several islet proteins was also observed inTPA-cultured islets compared with control-cultured islets. Thesefindings demonstrate that culturing alone impairs glucose-induced secretion, a response that is improved but still subnormal compared with freshly isolated islet responses, if TPA is included in the culture medium. Sustained phosphorylation of several islet proteins inTPA-cultured islets may account, at least in part, for augmented calcium-dependent secretion.

     

TBARS,carnitine, and reduced glutathione levels in human bladder carcinoma

In this study, we investigated tissue levels of reduced glutathione (GSH) and carnitine as well as thiobarbituric acid reactive substances (TBARS, as a marker of lipid peroxidation) levels in bladder carcinoma and control group of patients. The average GSH, carnitine and TBARS levels for tumor group were respectively 7.11 ± 3.3 g/mg protein, 1.81 ± 0.39 nmol/mg protein, and 4.29 ± 3.2 mol/mg protein, versus 14.45 ± 4.11 g/mg protein, 2.14 ± 0.66 nmol/mg protein, and 2.3 ± 0.6 mol/mg protein for normal bladder tissues. Thus, tissue reduced glutathione levels (GSH) were significantly lower in patients as compared with the control group (p < 0.001) whereas average TBARS levels in the tumor group were found to be higher than those in control group. The average tissue carnitine levels in the patient group were found to be lower compared with the control group but the difference was not statistically significant (p > 0.05).     

University of Wisconsin Solution with Trypsin Inhibitor Pefabloc Improves Survival of Viable Human and Primate Impure Islets During Storage

Background. Recent studies suggest that impure islets (islets which have not been isolated from exocrine tissue and other parts of the pancreas) have great potential for successful transplantation. The evidence that supports this view includes findings that embedded islets (islets surrounded by exocrine tissue) undergo less apoptosis, peripancreatic lymph nodes prevent recurrence of IDDM (insulin dependent diabetes mellitus), and that islet yields and insulin content decrease during the purification process. Improved protocols have also been developed to prevent allorejection of impure islets. Despite these promising results, the storage of impure islets remains difficult, and was a method sought to decrease storage losses. Methods. Storage methods of impure human and non-human primate islets were compared, using either culture media or University of Wisconsin solution (UW). The effects of trypsin inhibition using Pefabloc (Roche Molecular Biochemicals, Indianapolis, IN) during storage period were also examined. Results. Low temperature and inhibition of trypsin activity during storage of impure islets improved both islet yield and viability. It was found that using UW solution and trypsin inhibition allowed perfect preservation of viable impure islets up to 48h. A functional assay by glucose stimulation test showed these impure islet responded to glucose stimulation after 24h. Conclusion. The benefits of storing impure islets using UW solution and Pefabloc at low temperature have been established. This improved method of preserving impure islets makes this model of transplantation even more viable.     

Evaluation of the glyoxal-bis-(2-hydroxyanil)-method for staining of calcium in model gelatin films and pancreatic islets

Summary The nature of tissue calcium, detectable with glyoxal-bis-(2-hydroxyanil), (GBHA), was investigated using gelatin films as model. The results indicate that in the films the procedure detects only the calcium fraction which was ionized in the original gelatin solution. The GBHA staining intensity (absorbance) appeared to be linear with the amount of ionized calcium in the range from 0 to 2 g/cm². The method allows detection of amounts of ionized calcium as low as 0.15 g/cm² or 0.0015 pg/².For the measurement of calcium in pancreatic tissue of fed rats, the tissue was subjected to freeze-substitution at –80°C in acetone containing 1% oxalic acid. Adjacent sections were stained with either GBHA or aldehyde-fuchsin (AF). Exocrine tissue hardly stained with GBHA whereas islet tissue stained intensely. For GBHA as well as for AF a variation in staining intensity (visual evaluation) between islets was observed. Islet GBHA- and AF-staining intensities did not correlate. The AF-staining intensity but not the GBHA-staining intensity decreased with increasing islet diameter. Also in pancreatic islet tissue the GBHA method appears to be very sensitive and reproducible and small differences in islet GBHA-staining intensity can be detected. The results indicate that between islets differences in ionized calcium content exist. These differences do not correlate with the degree of B-cell granulation.     

GABA-agonists induce the formation of low-affinity GABA-receptors on cultured cerebellar granule cells via preexisting high affinity GABA receptors

The kinetics of specific GABA-binding to membranes isolated from cerebellar granule cells, cultured for 12 days from dissociated cerebella of 7-day-old rats was studied using [³H]GABA as the ligand. The granule cells were cultured in the presence of the specific GABA receptor agonist 4, 5, 6, 7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 150 M) or THIP plus the antagonist bicuculline methobromide (150 M of each) or in the absence of the agonist or antagonist. Membranes isolated from granule cells cultured in a medium without the GABA agonist revealed a single binding site for GABA with a binding constant (K _D) of 7.9±0.4 nM and aB _max of 3.42±0.08 pmol×mg^–1 protein. Membranes from cells cultured in the presence of THIP had two binding sites for GABA withK _D-values of 6.8±0.9 nM and 476±311 nM, respectively. The correspondingB _max values were 4.41±0.42 pmol×mg^–1 and 5.81±1.20 pmol×mg^–1. The effect of culturing the cells in THIP was antagonized by the simultaneous presence of bicuculline in the culture media, i.e. no significant low-affinity binding for GABA was found on the membranes from granule cells cultured in both THIP and bicuculline. TheK _D value (14.3±1.4 nM) for the high affinity binding site was, however, slightly increased compared to the non-treated cells. These findings suggest that the ability of THIP to induce formation of low-affinity GABA receptors is mediated by preexisting high-affinity GABA-receptors on the granule cells.     

Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation

Background aims

Mesenchymal stromal cells (MSCs) enhance islet function both in vitro and in vivo, at least in part by secreting ligands that activate islet G-protein coupled receptors (GPCRs). We assessed whether pre-treatment with a defined “cocktail” of MSC-secreted GPCR ligands enhances islet functional survival in vitro and improves the outcomes of islet transplantation in an experimental model of diabetes.

Streptozotocin toxicity to cultured pancreatic islets of the Syrian hamster

Pancreatic islets of the Syrian golden hamster were maintained in culture for extended periods of time. Toxicity of streptozotocin in these cultures was evaluated by measurement of insulin secretion. Exposure of islets to 1 or 2 mM streptozotocin immediately following isolation resulted in a permanent and dose-related inhibition of insulin secretion. This was accompanied by islet disruption as observed by phase-contrast microscopy. Culture of islets for 24 hours before streptozotocin exposure afforded protection from toxicity. For example, exposure of freshly isolated islets to 2 mM streptozotocin resulted in complete destruction of beta cells, whereas islets similarly exposed after a 24 hr culture period continued to secrete insulin for many months. Islets maintained in culture for one week before exposure to 0.1–0.5 mM streptozotocin, however, became more sensitive than freshly isolated islets. Repeated weekly exposure of cultured islets to a non-toxic concentration (0.1 mM) resulted in sustained suppression of insulin secretion after 11 weeks.     

Immunocytochemical localization of muscarinic acetylcholine receptors in the rat endocrine pancreas

Summary Immunocytochemical application of the antimuscarinic acetylcholine receptor antibody M35 to pancreas tissue revealed the target areas for the parasympathetic nervous system. Immunoreactivity in the endocrine pancreas was much higher than that in the exocrine part. Moreover, the endocrine cells at the periphery of the islets of Langerhans displayed the highest level of immunoreactivity. Based on these findings in the mantle of the islets, two types of islets have been distinguished: type-I islets with intensely stained mantle cells, and type-II islets with a much lower concentration of these cells. On average, type-I islets were larger (244.8 m±6.1 SEM) than type-II islets (121.5 m±3.8 SEM). M35-immunoreactivity was present on the majority of D cells, which were characterized by their immunoreactivity to somatostatin [of 446 D cells 356 (79.8%) were M35-immunopositive]. However, only a small proportion of the intensely stained mantle cells belonged to the D cell population. Therefore, it is concluded that the majority of the intensely stained mantle cells represent glucagon-secreting A and/or pancreatic polypeptide-secreting F cells. The intensity of M35-immunoreactivity at the periphery and central core of the islets paralleled the density of cholinergic innervation, suggesting a positive correlation between the intensity of cholinergic transmission and the number of muscarinic acetylcholine receptors at the target structures. The present study further revealed some striking parallels for the muscarinic acetylcholine receptor characteristics between the (endocrine) pancreas and the central nervous system.     

Islet organ,blood glucose and glucose tolerance of lean and obese Mongolian gerbils

Summary Gerbils were divided, on the basis of body weight, into obese (>80 gms) and lean (<80 gms) groups. Fasting blood glucose estimations on all 31 gerbils, and glucose tolerance tests on 9 lean and 6 obese animals, were carried out. All lean and some obese gerbils were normoglycaemic and other obese were hyperglycaemic. All obese gerbils exhibited glucose intolerance. General morphological studies were untertaken as follows: (i) assessment of mesenteric fat deposits, (ii) measurement of anterior abdominal wall thickness, (iii) ratio of animal length to width at specified loci (index of shape). The degree of obesity was less than previously reported in this species though blood glucose abnormality was comparable. The index of animal shape showed a strong correlation with body weight. The following kinds of histological observation were made on pancreases from 4 lean and 4 obese gerbils: (i) % islet representation, (ii) islet size distribution, (iii) -cell granularity, (iv) islet vascularity, (v) islet/duct association, (vi) proportions of - and D-cells, (vii) glycogen deposition in islet and duct cells. The pancreases of obese gerbils contained a higher proportion of islet tissue than those of lean due to generally larger islets: this hyperplasia was mainly attributable to -cell proliferation. Many obese gerbil islets exhibited hyperaemia and -cell degranulation. There was no evidence of glycogen depositon.     

Warm blood cardioplegia reduces the fall in the intracellular concentration of taurine in the ischaemic/reperfused heart of patients undergoing aortic valve surgery

Summary The effect of cold and warm intermittent antegrade blood cardioplegia, on the intracellular concentration of taurine in the ischaemic/ reperfused heart of patients undergoing aortic valve surgery, was investigated. Intracellular taurine was measured in ventricular biopsies taken before institution of cardiopulmonary bypass, at the end of 30 min of ischaemic arrest and 20 min after reperfusion. There was no significant change in the intracellular concentration of taurine in ventricular biopsies taken after the period of myocardial ischaemia in the two groups of patients (from 10.1 ± 1.0 to 9.6 ±0.9mol/g wet weight for cold and from 9.3 ± 1.3 to 10.0 ± 1.3mol/g wet weight for warm cardioplegia, respectively). Upon reperfusion however, there was a fall in taurine in both groups but was only significant (P 0.05) in the group receiving cold blood cardioplegia (6.9 ± 0.8mol/g wet weight after cold blood cardioplegia versus 8.0± 0.8mol/g wet weight following warm blood cardioplegia). Like taurine, there were no significant changes in the intracellular concentration of ATP after ischaemia in the two groups of patients (from 3.2 ± 0.32 to 2.95 ± 0.43mol/g wet weight for cold and from 2.75 ± 0.17 to 2.62 ± 0.21mol/g wet weight for warm cardioplegia, respectively). However upon reperfusion there was a significant fall in ATP in both groups with the extent of the fall being less in the group receiving warm cardioplegia (1.79 ± 0.19mol/g wet weight for cold and 1.98 ± 0.27mol/g wet weight for warm cardioplegia, respectively). This work shows that reperfusion following ischaemic arrest with warm cardioplegia reduces the fall in tissue taurine seen after arrest with cold cardioplegia. Accumulation of intracellular sodium provoked by hypothermia and a fall in ATP, may be responsible for the fall in taurine by way of activating the sodium/taurine symport to efflux taurine.     

Influence of culture passages on growth kinetics and adenovirus vector production for gene therapy in monolayer and suspension cultures of HEK 293 cells

To characterize the changes in cell growth rate and adenovirus vector (AdV) production capability of 293 cells during culture passages, 293 cells obtained at the 31st culture passage from ATCC (293M #31) were maintained as a monolayer culture and 293 cells obtained at an unknown culture passage from Invitrogen (293S) were maintained as suspension culture. In monolayer culture, the specific growth rate () of 293M cells increased rapidly with culture passage up to passage 65 and thereafter became saturated. The of 293M passage 43 (#43) was 0.29 day^–1, while the average of 293M from #66 to #86 was 0.74±0.01 day^–1 (average ± standard deviation). It was also noted that the cells became smaller in size during early culture passages. AdV production was also influenced by the number of culture passages. The AdV titer in the culture of 293M #66 was ca. tenfold higher than that of 293M #44, resulting from both a higher cell concentration and a higher AdV titer per cell at #66. In contrast, the , cell size, and AdV production of 293S cells in suspension culture did not change significantly as the culture passage number increased up to #40. Taken together, the culture passage influenced cell growth and AdV production of 293M cells in monolayer culture, but not those of 293S cells in suspension culture.     

Generation of Nitric Oxide by Peripheral Blood Leukocytes and Platelets in Healthy Humans and Patients with Thermal Trauma

The generation of nitric oxide (NO) by human peripheral blood leukocytes and platelets has been studied in healthy subjects and patients with burns (with the affected area varying from 10 to 45% of the body surface). Differential centrifugation was used to isolate leukocytes and platelets from the blood. The leukocyte suspension was diluted with a complete medium to a concentration of 1 × 10⁷ cells/ml, and the platelet suspension, to 1 × 10⁸ cells/ml; the suspensions were then cultured for 15 h (37°C). The concentration of nitrite, an NO metabolite, was determined using the Griss reaction. The relative production of NO by leukocytes of healthy subjects and patients was 0.75 ± 0.06 and 2.93 ± 0.16 mol/l, respectively (p < 0.001), and its relative production by platelets of healthy subjects and patients was 2.15 ± 0.14 and 3.62 ± 0.13 mol/l, respectively (p < 0.01). The absolute generation of NO by leukocytes of healthy subjects and patients is 0.47 ± 0.05 and 3.02 ± 0.28 mol/l, respectively (p < 0.001), and its absolute generation by platelets of healthy subjects and patients was 7.70 ± 0.55 and 14.68 ± 0.84 mol/l, respectively (p < 0.001). Thus, the absolute production of NO by platelets is 16 times higher than the absolute production of NO by leukocytes of healthy subjects. Stress increases the generation of NO by both leukocytes and platelets. The absolute generation of NO by platelets in thermal trauma is positively correlated with the plasma content of fibrinogen in the patients.     

Impact of lead exposure on pituitary-thyroid axis in humans

Thyroid function tests (serum levels of thyroxine-T4, triiodothyronine-T3 and thyroid stimulating hormone-TSH) were performed in fifty-eight men (mean age: 31.7±10.6 years; mean duration of lead exposure: 156.9±122.7 months). These subjects were exposed to lead either as petrol pump workers or automobile mechanics. The mean whole blood lead (Pb-B) levels were 2.49±0.45 mole/l (51.90±9.40 g/dl) in the lead exposed workers and were approximately 5 times higher than in the control (n=35) subjects. No significant alteration was seen in their mean T3 and T4 levels as compared with the controls. Interestingly, T3 was significantly lower with the longer (210 months) exposure time in comparison with the group having shorter (29 months) exposure duration. The mean TSH levels were significantly (p<0.01) higher in workers exposed in comparison with the control group. This rise in TSH was independent of exposure time, but it was definitely associated with the Pb-B levels. The increase being more pronounced with mean Pb-B levels of 2.66±0.2 mole/l (55.4±4.25 g/dl) when compared with the group having mean levels of 1.51±0.30 mole/l (31.5±6.20 g/dl). The rise is TSH associated with Pb-B levels was only statistical valid, however, the levels fall within the normal laboratory range. We thus conclude that the Pb-B levels of 2.4 mole/l (50 g/dl) could enhance the pituitary release of TSH without having any significant alterations in the circulating levels of T3 and T4.     

The contribution of size-fractionated plankton to biomass and primary production of phytoplankton in saline–alkaline ponds

Primary productivity, biomass and chlorophyll-a of size fractionated phytoplankton (<0.22 m, <3 m, <8 m, <10 m, <40 m, <64 m, <112 m and <200 m) were estimated in 6 ponds and 5 experimental enclosures. The results showed that the planktonic algae less than 10 m are important in the biomass and production of phytoplankton in saline–alkaline ponds. The production of size fractionated phytoplankton corresponding to <112 m, <10 m and <3 m in saline–alkaline ponds were 10.5 ± 6.6 , 8.6 ± 5.4 and 0.33 ± 0.1 mgC l^–1 d^–1, respectively. Mean community respiration rate was 1.80 ± 0.73, 1.69 ± 0.90 and 1.38 ± 1.12 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) were 1.61, 8.30 and 0.33 mgC l^–1 d^–1, respectively. The ratio of those to the total phytoplankton production was 15%, 79% and 3%, respectively. The mean respiration rate of the different size groups was 0.11, 0.31 and 1.38 mgC l^–1 d^–1; the ratio of those to total respiration of phytoplankton was 6%, 17% and 77%, respectively. The production of size-fractionated phytoplankton corresponding to <200 m, <10 m and <3 m in enclosures was 2.19 ± 1.63, 2.08 ± 1.75 and 0.22 ± 0.08 mgC l^–1 d^-1, respectively. Mean community respiration rates were 1.25 ± 1.55, 1.17 ± 1.42 and 0.47 ± 0.32 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton was 0.11, 1.86 and 0.22 mgC l^–1 d^–1, respectively. The ratio of those to the total production of phytoplankton was 5%, 85% and 10%, respectively. The mean respiration rate of different size groups were 0.08, 0.72 and 0.46 mgC l^–1 d^–1, the ratio of those to total respiration of phytoplankton was 6%, 57% and 37%, respectively. The concentrations of chlorophyll-a of the phytoplankton in the corresponding size of micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) plankton in the experimental ponds were 19.3, 98.2 and 11. 9 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 15%, 76% and 9%, respectively. The concentrations of chlorophyll-a of phytoplankton micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton in enclosures were 1.7, 34.3 and 3.0 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 4%, 88% and 8%, respectively.     

Cryopreservation of mouse pancreatic islets: effects of different glucose concentrations in the post-thaw culture medium on islet recovery

It was the aim of this study to investigate the influence of the glucose concentration of the post-thaw culture medium on islet B-cell survival after cryopreservation by the combined assessments of islet recovery, islet DNA and insulin contents, and insulin release. Collagenase isolated mouse islets were kept in culture for 3 days in the presence of 11.1 mM glucose and then transferred to freezing ampoules containing Hanks' solution supplemented with 10% calf serum and 2 M dimethyl sulfoxide. After a 20-min incubation at 0 degrees C the islets were cooled at a rate of 25 degrees C/min to -70 degrees C and subsequently plunged into liquid nitrogen. After 2 hr the frozen islets were rapidly thawed at 37 degrees C, transferred to culture dishes, and cultured for another 3 days in the presence of 2.8, 5.6, 11.1, 16.7, or 28 mM glucose. Nonfrozen control islets were treated identically after a preceding 3-day culture at 11.1 mM glucose. The percentage recovery of cryopreserved islets was decreased compared to that of nonfrozen islets, but was increased when higher glucose concentrations were used in the post-thaw culture medium. Since the DNA content of the cryopreserved islets was slightly decreased, the overall survival rate of the cryopreserved B-cells, when cultured at the higher glucose concentrations after thawing, was found to be about 75%. The insulin content of the cryopreserved islets was decreased but the glucose-stimulated insulin release was essentially the same as that of the nonfrozen islets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of T3 administration on electrophysiological properties of lizard ventricular muscle fibres

We investigated the effects on the electrophysiological properties of ventricular muscle fibres from lizards kept at 20 °C of mild and severe hyperthyroidism. The hyperthyroidism was induced by a 4-day treatment with either 0.025 or 1.0 g triiodothyronine g^-1 body weight, documented by increased serum levels of thyroid hormone. Triiodothyronine treatment did not modify the duration of the action potential recorded in vitro at 25 °C from ventricular muscles stimulated at 1 Hz. Recordings at higher temperatures were associated with a faster repolarization phase and a decrease of action potential duration in both euthyroid and hyperthyroid animals. However, in lizards treated with 1.0 g triiodothyronine · g^-1 body weight, the 90% repolarization recovery times at 30 and 35 °C (95.6±14.9 ms and 53.0±6.0 ms, respectively), were significantly shorter than normal (177.6±29.2 and 107.2±18.1 ms, respectively). Action potential duration was also dependent on stimulation frequency of the preparations. Increased frequency led to significant decrease of the duration of action potentials recorded at 25 °C. In euthyroid preparations the reductions in 90% repolarization recovery time, owing to increases in stimulation frequency to 2.5 and 5 Hz, were 19.3±1.7 and 35.6±2.0 ms, respectively. In hyperthyroid preparations, the reductions in the 90% recovery time due to stimulus frequency increases varied from 35.4±1.9 and 58.1±2.1 ms at low hormone doses to 38.9±2.0 and 58.2±2.1 ms at high hormone doses. As a result of these differences, the action potential durations recorded from the two hyperthyroid preparations at high stimulation rates were shorter than from euthyroid preparations. The results obtained suggest that lizard cardiac tissue is responsive to hormone action at low environmental temperature, but the effects of such action become evident when the temperature and heart rate increase.Abbreviations A _20% integrated area above 20% depolarization - bw body weight - hw heart weight - FT ₃ free triiodothyronine - RT ₄₀ RT ₅₀ RT ₇₀ and RT ₉₀ recovery time at 40, 50, 70, and 90% of repolarization, respectively - T ₃ triiodothyronine - TT ₃ Total triiodothyronine     

A dual population of islets of Langerhans in bovine pancreas

Summary A morphological study of the bovine pancreas from fetus to adult documents the presence of two distinct types of pancreatic islets: large islets, 100 to 1600 m in diameter, enmeshed in interlobular connective tissue; small islets, 25–200 m in diameter, enmeshed in exocrine tissue. Large islets consisting primarily of well granulated B cells, decrease in relative volume with increasing age and in the adult are seldom seen. The overall relative volume of endocrine tissue is age dependent and ranges from 30% in the sixth month fetus to 10% in the neonate and 5% in the adult. Small islets contain B cells that increase their cytoplasmic secretory granularity with increasing fetal age, significantly degranulate just prior to birth and subsequently regranulate several weeks after birth. Beta cells of the small islets are uniquely characterized by junctional complexes in close association with large numbers of maculae adherentes (desmosomes). Using lanthanum-hydroxide and freeze-fracture techniques the junctional complexes are shown to consist of macula occludens (focal tight junctions) enclosing nexuses (gap junctions).The two types of islet differ in distribution, times of growth and times of B-cell granularity and may be indicative of functional differences yet to be elucidated.This work was presented in part at the Thirty-Fifth Annual Meeting of the American Diabetes Association, New York City, 1975