The Insertion/Deletion Polymorphism in the Angiotensin-converting Enzyme Gene and Hypoglycemia Awareness in Patients with Type 1 Diabetes.

Impaired hypoglycemia awareness affects approximately 25% of all patients with type 1 diabetes (T1DM). Duration of diabetes and tight glycemic control represent main risk factors of impaired hypoglycemia awareness. However, even among patients with good glycemic control and longstanding T1DM, awareness of hypoglycemia may be intact. Genetic factors might explain some of this remaining variability. Recently, the insertion/deletion ( I/ D) polymorphism in angiotensin converting enzyme gene ( ACE) was shown to be associated with significantly higher risk of hypoglycemic events in subjects with T1DM. Here, we studied the effects of genetic polymorphisms in the ACE on impaired hypoglycemia awareness in 231 Caucasian T1DM patients. Hypoglycemia awareness status was determined using standardized questionnaires (Clarke et al. and Edinburgh Hypoglycemia Scale). ACE I/ D genotype was determined by PCR amplification of the respective fragments from intron 16 of the ACE and size fractionation (I allele frequency=0.49; P=0.74 for Hardy-Weinberg equilibrium). In the logistic regression analysis, significant risk factors of impaired hypoglycemia awareness were duration of diabetes, C-peptide and HbA (1c) (all P<0.01). However, no significant effect of the I/ D polymorphism on impaired hypoglycemia awareness was observed with and without adjustment for age, diabetes duration, C-peptide and HbA (1c). Even though the study provides a relatively large dataset, it is possible that small differences may have been missed.     

The presence and actions of NPY in the canine endocrine pancreas

Immunofluorescent staining for neuropeptide Y (NPY) in canine pancreatic tissue was performed together with an evaluation of the effects of synthetic NPY on the release of insulin (IRI), glucagon (IRG) and somatostatin (SLI) from the duodenal lobe of the canine pancreas in situ. NPY-like immunoreactivity was localized in perivascular nerve fibers throughout the acinar tissue. NPY-immunoreactive fibers were also demonstrated in the islets, usually surrounding blood vessels but also occasionally in fibers associated with endocrine cells, primarily at the periphery of islets. In addition, the ganglia dispersed in the pancreatic parenchyma were densely innervated by NPY-immunoreactive fibers, and these ganglia regularly contained cell bodies staining for NPY. Direct infusion of NPY into the pancreatic artery (p.a.) produced a dose-dependent decrease of pancreatic SLI output and of pancreatic venous blood flow. Low-dose p.a. infusion of NPY (50 pmol/min) had no effect on basal IRI or IRG output or on the islet response to glucose (5-g bolus, i.v.). High-dose p.a. infusion of NPY (500 pmol/min) transiently stimulated IRI output and modestly increased IRG output. However, the comparatively sparse innervation of canine islets with NPY-like immunoreactive fibers and the relatively minor effects of large doses of synthetic NPY on pancreatic hormone release lead us to conclude that this peptide is not an important neuromodulator of islet function in the dog.     

New form of lignolytically active mycelium generated by immobilization of protoplasts isolated from the white rot fungi Heterobasidion annosum and Polyporus pinsitus

Summary Immobilized mycelia regenerated from immobilized protoplasts isolated from lignin-degrading Basiodiomycetes have been shown to be able to decompose specifically ¹⁴C-labelled dehydropolymers of coniferylalcohol (DHP-lignin) and monomeric lignin-related compounds more intensively than native mycelium, by decarboxylation, demethylation, ring and side chain cleavage. Protoplasts of two white rot fungi were immobilized by entrapment in Na- alginate gel and remained intact after the immobilization procedure. Within the first 3 days of incubation in culture medium, regeneration of hyphal cells occurred. Since hyphal cells regenerated from protoplasts within gel beads were hindered from stretching by the matrix, the microbial immobilized cells differed from native mycelium in terms of their morphology. The time course and extent of lignin degradation by native mycelium and regenerated mycelium of the examined white rot fungi also differed, a sign that there may also be differences between them in terms of the physiology of lignin degradation.     

Thermogenesis of white adipocytes: a novel method allowing long-term microcalorimetric investigations

A microcalorimetric method was developed to facilitate long-term assessment of energy balance in isolated fat cells. White rat adipocytes were primary cultured in glass ampoules with a matrix of agar-gel. Heat production was measured continuously over 3 days with an LKB BioActivityMonitor. In order to assist interpretation of the microcalorimetric measurements, glucose consumption and lactate and pyruvate production were determined in parallel cultures. Heat production, glucose consumption and lactate production were in an apparent steady state throughout the study whether employing aerobic (94 pW, 0.50 and 0.44 pmol/cell. h, respectively) or partial anaerobic experimental conditions (39 pW, 0.41 and 0.57 pmol/cell.h, respectively). However, oxygen availability influenced the apparent heat production and glucose and lactate metabolism. With partial anaerobiosis a 59% lower heat production, an 18% lower glucose consumption and a 30% higher lactate production than by employing aerobic experimental conditions were observed.     

Peptide YY-like immunoreactivity in the central nervous system of the rat

The concentration of peptide YY (PYY)-like immunoreactivity in rat brain and spinal cord was determined by radioimmunoassay. The highest concentrations were found in the cervical spinal cord (18.1 +/- 1.3 ng/g, mean +/- S.E.M.) and in the medulla oblongata (16.3 +/- 1.5 ng/g). Lower amounts were found in the pons and in the hypothalamus. Chromatographic analysis of the PYY-like immunoreactivity from various regions of the brain revealed 95% of the immunoreactive material to be indistinguishable from synthetic porcine PYY. PYY-immunoreactive nerve cell bodies could be demonstrated by immunocytochemistry in the medulla oblongata of colchicine-treated rats, the largest group of cells being found in the midline area between and partly in the raphe pontis and obscurus nuclei. Another large group of immunoreactive cells was detected more laterally in the medial parts of the gigantocellular reticular nucleus. A few cells, finally, were seen in the dorsal parts of the medulla, including the nucleus of the solitary tract. Varicose nerve fibers displaying PYY immunoreactivity were observed in many parts of the hypothalamus, pons, medulla and spinal cord.     

Microbial oxidation of ebastine

The microbial oxidation of ebastine to carebastine was investigated. Among the 15 micro-organisms examined, only theCunninghamella strains showed the desired biotransformation.Cunninghamella blakesleeana oxidised the substrate within 7 days, via the intermediates alcohol and aldehyde, mainly to carebastine, the corresponding carboxylic acid. Optimisation of the culture conditions increased the yield from initially 10% up to a reproducible 40%. For the synthesis of carebastine a substrate concentration of 200 mg/l, a starting pH of 5.0 and the addition of 1% poly(vinyl alcohol) is favourable. The results achieved in experiments with shaking flasks are transferable to the fermentation scale and yielded 270 mg carebastine in a 3-l fermentation of 600 mg ebastine. The progress of the reaction was detected by TLC and HPLC, the products were identified by mass spectrometry and NMR.     

The carbonic anhydrase of the Chinese crabEriocheir sinensis: Effects of adaption from tap to salt water

In the present investigation we studied the carbonic anhydrase (CA) in various tissues of Chinese crabEriocheir sinensis which were acclimated to different salinities (0, 10, 20, 30‰). We found only negligible CA activity in haemolymph, heart, hypodermis, antennal gland, leg muscle and digestive gland, irrespective of the acclimation medium. However, high amounts of CA activity were found in the gills. In the case of the posterior gills, a strong dependence on the acclimatization of the animals was demonstrated; the highest activities were found in those adapted to tap water. To investigate the cellular distribution of the CA in the posterior gills, the additional enzyme activities were measured in all fractions of a differential centrifugation of the gill homogenate: Na⁺/K⁺-ATP'ase (a marker for the plasmamembrane); lactate dehydrogenase (LDH; as marker for the cytosol); and succinate dehydrogenase (SDH; as marker for mitochondria). Independent of the acclimation salinity (0 or 36‰ salinity), we found about 70% of CA associated with the highest level of the Na⁺/K⁺-ATP'ase in the second 100 000 g pellet (membrane fraction), while only 15% were found in the cytosolic fractions (associated with highest levels of LDH). We conclude that the carbonic anhydrase of posterior gills of the Chinese crab is mainly membrane-bound. Furthermore, the activity of CA shows a strong dependence on the salinity of the water in which the crabs were kept.     

Molecular heterogeneity of canine cholecystokinin in portal and peripheral plasma

The release of molecular forms of cholecystokinin (CCK) into the portal and peripheral blood in response to an intraduodenal perfusion of sodium oleate (9 mmol X h-1) was studied in six conscious dogs with chronic portal vein catheters. Immunoreactive CCK as concentrated from 20 ml plasma by C18 SEP PAK cartridges and the pattern of molecular forms of CCK were studied by G50 gel filtration. CCK-like immunoreactivity (CCK-LI) was measured in the column eluates with antibody 5135, which measures gastrin and CCK equally and requires the intact carboxyl-terminus for full recognition. Gastrin was measured specifically with antibody 1611. Intraduodenal perfusion with oleate did not alter basal gastrin release. Release of CCK-LI by intraduodenal oleate was calculated by the increments of the integrated CCK-LI peaks over basal. Total CCK-like immunoreactivity (CCK-LI), calculated by integration of all CCK-LI peaks in gel filtration eluates, increased over basal by 12 fmol/ml in the portal and by 6 fmol/ml in the peripheral plasma after intraduodenal perfusion with sodium oleate. The main molecular forms eluted on gel filtration in positions of CCK33,39 and of CCK8. The pattern of CCK in the peripheral plasma was similar to that in the portal plasma except that in the peripheral plasma large molecular forms were more abundant than small forms. This finding was confirmed when CCK39 and CCK8 were infused either into the portal vein or into the peripheral vein and peripheral plasma CCK levels were measured. Elimination of CCK8 after portal vein infusion compared to peripheral vein infusion was about 3 times higher than that of CCK39. The abundance of large molecular forms of CCK in the circulating blood which are similar in potency to small forms, underlines their role in the physiology of CCK.     

Genetic analysis of the yeastCandida maltosa by means of induced parasexual processes

The usefulness of hybridization by protoplast fusion and mitotic segregation for the genetic analysis of the imperfect fodder yeastCandida maltosa was tested. Mitotically stable fusion hybrids were obtained with frequencies between 10^–6 and 10^–7. Complementation tests were performed by protoplast fusion. Substances that are known to induce frequent mitotic segregation in other yeast species such as benomyl, p-fluorophenylalanine, and acriflavine were ineffective inC. maltosa. UV irradiation induced mitotic segregation in up to 10%. This agent induced mainly mitotic crossing over inC. maltosa. Our data enabled the construction of the linkage group I with the sequenceCEN-ade-26-pro-1.