AFDX-116 discriminates between muscarinic M2 receptors of the heart and the iris smooth muscle

Summary Studies with the atypical muscarinic antagonist pirenzepine provide convincing evidence for the classification of muscarinic acetylcholine receptors (mAChRs) into two subtypes, M₁ and M₂. The present study examines the heterogeneity of the M₂ subtype employing the newly developed competitive muscarinic antagonist, AFDX-116. Comparison of the binding affinities of pirenzepine, atropine, and AFDX-116 to mAChRs in microsomes from the rabbit cerebral cortex, heart, and iris smooth muscle shows that iris mAChRs, which are pharmacologically of the M₂ subtype, can be distinguished from M₂ cardiac receptors based on their affinity for AFDX-116. These results are consistent with the hypothesis that the M₂ receptor subtype consists of a heterogeneous population of receptors.Abbreviations mAChRs Muscarinic Acetylcholine Receptors - CCh Carbachol - NMS N-Methylscopolamine - AFDX-116 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6Hpyrido[2,3-b][1,4]benzodiazepine-6-one     

Glucose deprivation induces the selective accumulation of hexose transporter protein GLUT-1 in the plasma membrane of normal rat kidney cells

Antibody to the carboxyl-terminal of hexose transporter protein GLUT-1 was used to localize this carrier in normal rat kidney (NRK) cells during D-glucose (Glc) deprivation. Glc-deprivation of NRK cells induces increased hexose transport, inhibits the glycosylation of GLUT-1, and increases the content of both native, 55,000 apparent mol wt (Mr) and aglyco, 38,000 Mr GLUT-1 polypeptides. The distribution of GLUT-1 protein in subcellular fractions isolated from Glc-fed NRK cells shows that the 55,000 Mr polypeptide is most abundant in intracellular membrane fractions. Glc-fed cells that have been tunicamycin treated contain principally the 38,000 Mr GLUT-1 polypeptide, which is found predominantly in intracellular membrane fractions. In Glc-deprived cells the 55,000 Mr GLUT-1 polypeptide localizes predominantly in the Golgi and plasma membrane fractions, whereas the more abundant 38,000 Mr GLUT-1 polypeptide is distributed throughout all membrane fractions. In Glc-deprived but fructose-fed cells only the 55,000 Mr GLUT-1 polypeptide is detected, and it is found predominantly in the plasma membrane and Golgi fractions. The localization of GLUT-1 protein was directly and specifically visualized in NRK cells by immunofluorescence microscopy. Glc-fed cells show little labeling of cell borders and a small punctate juxtanuclear pattern suggestive of localization to the Golgi and, perhaps, endoplasmic reticulum. Glc-fed cells that have been tunicamycin treated show large punctate intracellular accumulations suggestive of localization to distended Golgi and perhaps endoplasmic reticulum. Glc-deprived cells exhibited intense labeling of cell borders as well as intracellular accumulations. Glc-deprived but fructose-fed cells show fewer intracellular accumulations, and the labeling is, in general, limited to the cell borders. Our results suggest that Glc deprivation induces the selective accumulation of GLUT-1 in the plasma membrane of NRK cells.     

Genetic disruption of protein phosphatase 5 in mice prevents high-fat diet feeding-induced weight gain

The role of serine/threonine protein phosphatase 5 (PP5) in the development of obesity and insulin resistance associated with high-fat diet-feeding (HFD) was examined using PP5-deficient mice (Ppp5c^−/−). Despite similar caloric intake, Ppp5c^−/− mice on HFD gained markedly less weight and did not accumulate visceral fat compared to wild-type littermates (Ppp5c^+/+). On a control diet, Ppp5c^−/− mice had markedly improved glucose control compared to Ppp5c^+/+ mice, an effect diminished by HFD. However, even after 10 weeks of HFD glucose control in Ppp5c^−/− mice was similar to that observed in Ppp5c^+/+ mice on the control diet. Thus, PP5 deficiency confers protection against HFD-induced weight gain in mice.     

Site fertility drives temporal turnover of vegetation at high latitudes

Experimental evidence shows that site fertility is a key modulator underlying plant community changes under climate change. Communities on fertile sites, with species having fast dynamics, have been found to react more strongly to climate change than communities on infertile sites with slow dynamics. However, it is still unclear whether this generally applies to high‐latitude plant communities in natural environments at broad spatial scales. We tested a hypothesis that vegetation of fertile sites experiences greater changes over several decades and thus would be more responsive under contemporary climate change compared to infertile sites that are expected to show more resistance. We resurveyed understorey communities (vascular plants, bryophytes, and lichens) of four infertile and four fertile forest sites along a latitudinal bioclimatic gradient. Sites had remained outside direct human disturbance. We analyzed the magnitude of temporal community turnover, changes in the abundances of plant morphological groups and strategy classes, and changes in species diversity. In agreement with our hypothesis, temporal turnover of communities was consistently greater on fertile sites compared to infertile sites. However, our results suggest that the larger turnover of fertile communities is not primarily related to the direct effects of climatic warming. Furthermore, community changes in both fertile and infertile sites showed remarkable variation in terms of shares of plant functional groups and strategy classes and measures of species diversity. This further emphasizes the essential role of baseline environmental conditions and nonclimatic drivers underlying vegetation changes. Our results show that site fertility is a key determinant of the overall rate of high‐latitude vegetation changes but the composition of plant communities in different ecological contexts is variously impacted by nonclimatic drivers over time.     

Genetic characterization of the Guinea-Bissau family of Mycobacterium tuberculosis complex strains

In a previous study of Mycobacterium tuberculosis complex isolates from Guinea-Bissau in West Africa, we identified a unique group of strains, designated here as the Guinea-Bissau family of strains, which, although genotypically closely related, phenotypically demonstrated a considerable heterogeneity. We conducted here a detailed genotypic analysis of a subset (n = 35) of these isolates. Based on the data obtained, and by comparison of known corresponding genes in mycobacteria outside the M. tuberculosis complex, we propose that the Guinea-Bissau strains belong to a unique branch of the M. tuberculosis complex tree in between classical M. tuberculosis and classical M. bovis. These observations are discussed in their significance in M. tuberculosis complex classification.     

Cytochrome P450 enzyme activity in five herbivorous,non-passerine bird species

We examined hepatic cytochrome P450 activity in wild and hand-reared grey partridges (Perdix perdix), capercaillies (Tetrao urogallus) and ring-necked pheasants (Phasianus colchicus), as well as the enzyme activity in a variety of tissues of hand-reared Japanese quails (Coturnix coturnix japonica) and pigeons (Columba livia). Post-mortem decrease in hepatic enzyme activity in the grey partridge was measured. Hepatic 7-ethoxyresorufin-O-deethylase activity was similar in wild and hand-reared grey partridges and pheasants, but the activity was significantly lower in wild than in hand-reared capercaillies, probably resulting from their phenolic-rich diet. In the tissues of both quails and pigeons 7-ethoxycoumarin-O-deethylase exhibited the highest and 7-pentoxyresorufin-O-deethylase the lowest activity. Hepatic enzyme activity was significantly higher than that in other tissues. In the small intestine some activity could be found, reflecting some intestinal detoxication capacity. Enzyme activity decreased by 34-69% during the 30-min sampling period, which confirmed the importance of equalising sampling time to obtain comparable data. Because the hand-reared birds in this study were fed the same commercial diets, we assumed that the enzyme activity values detected reflect species differences without any induction by dietary secondary compounds.     

Cryopreservation of embryogenic cultures of Scots pine

The aim of the study was to develop an effective cryopreservation method for Scots pine (Pinus sylvestris L.) embryogenic cultures. Altogether nine cell lines derived from three mother trees were cryopreserved after cold hardening using dimethylsulfoxide or two different mixtures of polyethyleneglycol 6000, glucose and dimethylsulfoxide as cryoprotectants. Seventy-eight percent of the cell lines remained viable after cryostorage, the best cryoprotectant treatment being 10% polyethyleneglycol 6000, 10% glucose, and 10% dimethylsulfoxide in water. This treatment resulted in significantly better regrowth of the embryogenic cultures than with the other cryoprotectants or with the controls. According to microscopical observations, the cells that retained their viability and regrowth ability after cryopreservation were the embryonal head cells, as well as some elliptic suspensor cells close to the embryonal head cell area. When proliferation growth of the frozen cultures had started, their morphological appearance was the same as the non-frozen cultures. In addition, the RAPD assays suggested that the cryostorage treatment used here preserved the genetic fidelity of the Scots pine embryogenic cultures. This revised version was published online in June 2006 with corrections to the Cover Date.