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.     

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.

     

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.     

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.     

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.

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).     

Factors affecting in vitro microrhizome production in turmeric

In vitro microrhizome production was obtained in turmeric (Curcuma longa Linn.). Freshly sprouted buds with small rhizome portions excised from stored mature rhizomes were cultured on semi-solid culture initiation medium –- MS basal medium + 0.88 M BAP (6-benzylaminopurine) + 0.92 M kinetin + 5% coconut water + 2% sucrose + 0.5% agar –- resulting in bud elongation. Multiple shoots were produced from these elongated buds by culturing in liquid shoot multiplication medium –- MS basal medium + 2.2 M BAP + 0.92 M kinetin + 5% coconut water + 2% sucrose –- at 25±1°C and 16-h light (at 11.7 mol m^–2 s^–1)/8-h dark cycles. Clumps of four to five multiple shoots/single shoots were used in various experiments. Cultures were incubated in the dark at 25±1°C. Half strength MS basal medium supplemented with 80 g l^–1 sucrose was found to be optimal for microrhizome production. Cytokinin BAP had an inhibitory effect on microrhizome production. At the highest concentration of BAP tried (35.2 M) microrhizome production was totally inhibited. Microrhizome production depended on the size of the multiple shoots used. Microrhizomes produced were of a wide range in size (0.1–2.0 g) and, readily regenerated when isolated and cultured in vitro on culture initiation medium or shoot multiplication medium. Under in vivo conditions, small (0.1–0.4 g), medium (0.41–0.8 g) and big (>0.81 g) microrhizomes regenerated. Plantlets developed from big microrhizomes grew faster.     

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.     

Method for isolation of pancreatic blood vessels,their culture and coculture with islets of langerhans

The only cure available for Type 1 diabetes involves the transplantation of islets of Langerhans isolated from donor organs. However, success rates are relatively low. Disconnection from vasculature upon isolation and insufficient rate of revascularization upon transplantation are thought to be a major cause, as islet survival and function depend on extensive vascularization. Research has thus turned toward the development of pretransplantation culture techniques to enhance revascularization of islets, so far with limited success. With the aim to develop a technique to enhance islet revascularization, this work proposes a method to isolate and culture pancreas-derived blood vessels. Using a mild multistep digestion method, pancreatic blood vessels were retrieved from whole murine pancreata and cultured in collagen Type 1. After 8 days, 50% of tissue explants had formed anastomosed microvessels which extended up to 300 μm from the explant tissue and expressed endothelial cell marker CD31 but not ductal marker CK19. Cocultures with islets of Langerhans revealed survival of both tissues and insulin expression by islets up to 8 days post-embedding. Microvessels were frequently found to encapsulate islets, however no islet penetration could be detected. This study reports for the first time the isolation and culture of pancreatic blood vessels. The methods and results presented in this work provide a novel explant culture model for angiogenesis and tissue engineering research with relevance to islet biology. It opens the door for in vivo validation of the potential of these pancreatic blood vessel explants to improve islet transplantation therapies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2745, 2019.     

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.     

Regeneration of the islets of Langerhans in the guinea pig

Summary The regeneration of the islets of Langerhans in the guinea pig was studied after intravenous injection of alloxan. A number of cells in the islets was destroyed within 24 h after alloxan, but after 48 h there was a rapid proliferation of the surviving cells of the islets. This depended on the dosage of the drug as well as the timing. Electron microscopy of the islet at 48 h showed that the dividing cells had small electron dense granules and resembled a subtype of normal A cells, whose function is not yet known. There were also many agranular cells in the islet. These two groups of cells seen in the regenerating islet could be precursor cells, which could differentiate into cells. There was no evidence for transformation of duct cells or acinar cells into islet cells. None of the guinea pigs became permanently diabetic. This was probably due to inadequate dosage which resulted in only partial degeneration of the cells followed by regeneration and recovery. There was also some regeneration of the liver, kidney and the adrenal cortex following alloxan.The author is grateful to Professor R. Barer for his guidance and for providing the facilities for this study. Thanks are also due to Mrs. D. Barraclough for technical assistance and to Mrs. M. Hollingsworth for assistance with the photographsThis work was financed by a grant from the Cystic Fibrosis Research Trust     

Mercury distribution in estuarine environments from Argentina: the detoxification and recovery of salt marshes after 15 years

Total Hg contents from abiotic (surface sediments and suspendedparticulate matter) and biological (crabs, fishes and halophytes)compartments from Bahía Blanca estuary and Mar Chiquita CoastalLagoon, Argentina, have been monitored since the 1980's. At BahíaBlanca estuary, high Hg concentrations were recorded during the early1980's in surface sediments (0.34 ± 0.22 g/g) andsuspended particulate matter (0.19 ± 0.10 g/g). Fishspecies, Mustelus schmitti (0.89 ± 0.29 g/g), Paralichthys brasiliensis (0.85 ± 0.18 g/g) and Micropogonias furnieri (0.37 ± 0.11 g/g) also presentedhigh Hg concentrations. The large industrial nucleus located within theestuary has been identified as the main metal source for this environment.Hg contents from the same area during 1996–1998 were significantlylower: surface sediments (0.164 ± 0.023 g/g), suspendedparticulate matter (0.048 ± 0.0017 g/g), fish Micropogonias furnieri (0.13 ± 0.02 g/g) and crab Chasmagnathus granulata (0.334 ± 0.071 g/g). This trendof environmental detoxification is probably related with (i) thetechnological changes incorporated by the local industry, (ii) a mostadequate management of industrial effluents, and (iii) the removal ofgreat sediment volume by dredging and refill.During the 1980's Mar Chiquita Lagoon Hg concentrations reached 0.08± 0.01 g/g in surface sediments and 0.09 ±0.025 g/g in suspended particulate matter, and 0.14 ±0.04 g/g in the fish Basilichthys bonariensis and 0.22 ±0.08 g/g in Paralichthys brasiliensis, and 0.08 ±0.01 g/g in the crab C. granulata, Hg concentrations werelower than at Bahía Blanca. Remote Hg sources for this Coastal Lagoonand atmospheric and stream transport of Hg is proposed as major Hgsources, since no Hg point sources exists nearby. Mercury concentrationsrecorded in the 1996–1998 period were lower than those recorded inthe previous decade: surface sediments (0.019 ± 0.004 g/g), suspended particulate matter (0.030 ± 0.008 g/g), halophyte Spartina densiflora (0.013 ± 0.008 g/g) or crab C. granulata (0.011 ± 0.009 g/g).Both Hg bioaccumulation and biomagnification processes were verified inBahía Blanca estuary and in Mar Chiquita Coastal Lagoon. This apparentrecovery of both estuarine environments deserves to be carefully analyzed,in order to fully understand the foundations of these processes.     

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)     

Plantlet regeneration through different morphogenic pathways in pommelo tissue culture

Pommelo (Citrus grandis Osbeck) plantlets were regenerated through different morphogenic pathways in culture. Multiple shoot regeneration through de novo organogenesis was obtained with epicotyl segments and root cultures. Shoot regeneration was observed in 84% of the midtal epicotyl segments cultured in Murashige and Skoog's medium (MS) with 2.2 M benzyladenine (BA) and 83% of the middle and proximal epicotyl segments cultured on basal medium. Isolated root segments cultured on medium containing 0.089 M BA showed best shoot regeneration at 71% with an average of 3.3 shoots per segment. Callus tissues derived from cotyledon and leaf explants regenerated shoots on BA-enriched medium. Shoots were also obtained at high frequencies from shoot-tip and nodal explants. Roots developed when regenerated shoots were excised and cultured on half strength MS medium with 2.5 M indolebutyric acid.Abbreviations BA 6-Benzyladenine - IBA Indole-3-butyric acid - MS Murashige and Skoog medium - NAA I-Naphthaleneacetic acid - 2,4-d 2,4-Dichlorophenoxyacetic acid     

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.     

Alloxan reversibly impairs glucagon release and glucose oxidation by pancreatic A2-cells

Alloxan is known as a selective B-cell cytotoxic substance, and there is so far little evidence for a direct toxic effect on the other islet cell types. To elucidate further whether such effects occur, the actions of alloxan on glucagon release and glucose oxidation were studied in isolated normal or A₂-cell-rich pancreatic islets of the guinea pig. The A₂-cell-rich islets were obtained from animals injected with streptozotocin 1–2 weeks before islet isolation. After exposure to alloxan (2 or 5mm) in vitro for 30min at 4°C, the islets were incubated in media containing either 1.7mm-glucose or 16.7mm-glucose plus 30m-i.u. of bovine insulin/ml. In both types of islet, alloxan abolished the ability of glucose and insulin both to decrease glucagon release and to increase the rate of glucose oxidation. A high concentration of glucose (28mm) during exposure to alloxan protected against these injurious effects. Tissue culture of alloxan-treated islets for 24h in 5.5mm-glucose restored neither the suppressive effect of glucose on glucagon release nor the inhibition of glucose oxidation of the A₂-cells. However, culture for 1 week completely normalized both the glucagon-secretory response and glucose oxidation by both kinds of islets. It is therefore concluded that alloxan affects the secretory mechanism of not only the B-cell but also of the islet A₂-cell, although this latter cell type is not primarily destroyed by the drug. The data furthermore support the concept of a relationship between glucose metabolism and the glucose-mediated glucagon release of the A₂-cell.     

Effects of precryopreservation culture on survival of rat islets transplanted after slow cooling and rapid thawing

Despite the frequent use of in vitro tissue culture before islet cryopreservation, no study has evaluated the ability of this procedure to improve the recovery or in vivo function of frozen-thawed islets. To evaluate this, quantities of 2500 Wistar-Furth (WF) rat islets were allocated to each of four groups (n = 8 each): group 1, freshly isolated; group 2, 48 hr in vitro culture; group 3, cryopreservation; group 4, cryopreservation after 48 hr in culture. Islets were frozen slowly at 0.25 degrees C/min and thawed rapidly at 200 degrees C/min. The number of islets recovered after culture or cryopreservation was determined and viability was assessed by measuring weekly indices of plasma glucose (PG), urine glucose (UG), urine volume (UV), and weight after implantation into the portal vein of streptozotocin-diabetic WF recipients. Islet recovery was 97% after culture, 95% after cryopreservation, and 94% after culture-then cryopreservation. After implantation of group 1 and 2 islets, PG was less than 150 mg/dl at 1 week and UG and UV were normal by 1-2 weeks. Group 3 islets restored normoglycemia at 3 weeks and other indices of diabetes were reversed by 4 weeks; group 4 islets restored normoglycemia at 4 weeks and indices returned to basal after 4 weeks. At intravenous glucose tolerance testing (ivGTT), the K values (mean decline in glucose, %/min, +/- SE) were group 1, 1.6 +/- 0.3; group 2, 1.5 +/- 0.3; group 3, 0.6 +/- 0.1; and group 4, 0.7 +/- 0.2. These data show that cryopreservation preserves freshly isolated or cultured islets that reverse the indices of diabetes after implantation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Continuous culture and synchronization ofHyphomicrobium sp. B-522

A technique was developed for synchronization ofHyphomicrobium sp. strain B-522. Bacteria were grown in continuous culture with methanol (0.1%; v/v) growth limiting. Vitamin B₁₂ (2.5 g/l) was necessary to obtain steady state growth. The critical dilution rate wasD _c =0.112; maximum cell output occurred atD=0.105 (Dx=30 mg l^-1 h^-1). Continuous cultures ofHyphomicrobium B-522 atD=0.110 were used to obtain cells for synchronization experiments. Synchronization was achieved by trapping young hyphal and budding cells in a glass wool column, while the initial swarmer cells were allowed to pass through. By semicontinuously rinsing the system, newly produced swarmers could be sampled in the effluent. The mean length of these synchronous swarmer cells was 1.25 m (s=±0.13 m; range 0,6 m) as compared to 1.40 m (s=±0.21 m; range 1.2 m) for swarmer cells of the continuous culture. Division of synchronous swarmer populations was completed after 7 h; the synchronization index was 0.76.