The three-dimensional structure of acarbose bound to glycogen phosphorylase

Acarbose, a pseudotetrasaccharide with a conduritol ring at the nonreducing terminus, is a naturally occurring inhibitor of amylases. It is shown here to be an inhibitor of glycogen phosphorylase and to bind more tightly to the enzyme than the equivalent malto-oligosaccharide substrate. X-ray crystallographic studies of the acarbose-phosphorylase a complex in the presence of glucose and caffeine reveal the structure of acarbose as bound to the storage site of phosphorylase. The acarbose binds in an orientation such that the conduritol ring makes no protein contacts. As with malto-oligosaccharides bound at this site, the observed conformation of acarbose is stabilized by O-2-O-3' hydrogen bonding and is similar to, but not identical with, that predicted by hard-sphere exo-anomeric effect calculations and justified by 1H nuclear magnetic resonance studies (Bock, K., and Pedersen, H. (1984) Carbohydr. Res. 132, 142-149). Intramolecular O2-O3' hydrogen bonds appear to play an important role in stabilizing the conformation observed in these studies, even for those residues closely associated with the protein.     

A freeze-preservation of synchronously dividing cultured cells of Acer pseudoplatanus L

A suspension culture of sycamore (Acer pseudoplatanus L.) was synchronised in division by release from nitrogen starvation. Cell samples, taken during the lag phase and synchronous growth, were examined cytologically and subjected to a freeze-preservation protocol. A high positive correlation was found between mitotic index (percentage of cells showing mitotic figures) and cell survival, as measured by fluorescein diacetate staining and reduction of 2,3,5-triphenyl tetrazolium chloride. Specific staining of lethally damaged cells and subsequent examination of the surviving cells demonstrated that the latter had a lower mitotic index and more consistent, low value of nuclear DNA than the total population. This indicated that it is cells, newly entered into G₁ phase of the cell cycle, which are particularly resistant to the stresses imposed by the freeze preservation protocol.     

Direct 1H n.m.r. determination of the stereochemical course of hydrolyses catalysed by glucanase components of the cellulase complex

The stereochemical courses of the hydrolyses catalysed by three glycosidases have been determined directly by 1H nmr. The anomeric configuration of the initially formed product was ascertained in each case by observation of the chemical shift and coupling constant of the anomeric proton at the new hemiacetal centre. Two of the enzymes investigated, an endo-glucanase and an exo-glucanase are components of the cellulase complex of Cellulomonas fimi. The third enzyme is the beta-glucosidase from almond emulsin. Two of these enzymes, the exo-glucanase and the almond beta-glucosidase catalysed hydrolysis with retention of anomeric configuration, in agreement with previous observations on the almond enzyme. The endo-glucanase catalysed hydrolysis with inversion of configuration, this result being confirmed by optical rotation measurements. This 1H nmr approach has several advantages over other techniques in that it is applicable to a wide variety of glycosidases and substrates and it is non-destructive, allowing recovery of the enzyme.     

1-Deoxy-d-galactonojirimycins with dansyl capped N-substituents as β-galactosidase inhibitors and potential probes for GM1 gangliosidosis affected cell lines

Two simple and reliably accessible intermediates, N-carboxypentyl- and N-aminohexyl-1-deoxy-d-galactonojirimycin were employed for the synthesis of a set of terminally N-dansyl substituted derivatives. Reaction of the terminal carboxylic acid of N-carboxypentyl-1-deoxy-d-galactonojirimycin with N-dansyl-1,6-diaminohexane provided the chain-extended fluorescent derivative. Employing bis(6-dansylaminohexyl)amine, the corresponding branched di-N-dansyl compound was obtained. Partially protected N-aminohexyl-1-deoxy-d-galactonojirimycin served as intermediate for two additional chain-extended fluorescent 1-deoxy-d-galactonojirimycin (1-DGJ) derivatives featuring terminal dansyl groups in the N-alkyl substituent. These new compounds are strong inhibitors of d-galactosidases and may serve as leads en route to pharmacological chaperones for GM1-gangliosidosis.     

Differential Pre-mRNA Splicing Regulates Nnat Isoforms in the Hypothalamus after Gastric Bypass Surgery in Mice

Background

Neuronatin (NNAT) is an endoplasmic reticulum proteolipid implicated in intracellular signalling. Nnat is highly-expressed in the hypothalamus, where it is acutely regulated by nutrients and leptin. Nnat pre-mRNA is differentially spliced to create Nnat-α and -β isoforms. Genetic variation of NNAT is associated with severe obesity. Currently, little is known about the long-term regulation of Nnat.

Methods

Expression of Nnat isoforms were examined in the hypothalamus of mice in response to acute fast/feed, chronic caloric restriction, diet-induced obesity and modified gastric bypass surgery. Nnat expression was assessed in the central nervous system and gastrointestinal tissues. RTqPCR was used to determine isoform-specific expression of Nnat mRNA.

Results

Hypothalamic expression of both Nnat isoforms was comparably decreased by overnight and 24-h fasting. Nnat expression was unaltered in diet-induced obesity, or subsequent switch to a calorie restricted diet. Nnat isoforms showed differential expression in the hypothalamus but not brainstem after bypass surgery. Hypothalamic Nnat-β expression was significantly reduced after bypass compared with sham surgery (P = 0.003), and was positively correlated with post-operative weight-loss (R² = 0.38, P = 0.01). In contrast, Nnat-α expression was not suppressed after bypass surgery (P = 0.19), and expression did not correlate with reduction in weight after surgery (R² = 0.06, P = 0.34). Hypothalamic expression of Nnat-β correlated weakly with circulating leptin, but neither isoform correlated with fasting gut hormone levels post- surgery. Nnat expression was detected in brainstem, brown-adipose tissue, stomach and small intestine.

Conclusions

Nnat expression in hypothalamus is regulated by short-term nutrient availability, but unaltered by diet-induced obesity or calorie restriction. While Nnat isoforms in the hypothalamus are co-ordinately regulated by acute nutrient supply, after modified gastric bypass surgery Nnat isoforms show differential expression. These results raise the possibility that in the radically altered nutrient and hormonal milieu created by bypass surgery, resultant differential splicing of Nnat pre-mRNA may contribute to weight-loss.     

Phylogenetic differences of mammalian basal metabolic rate are not explained by mitochondrial basal proton leak

Metabolic rates of mammals presumably increased during the evolution of endothermy, but molecular and cellular mechanisms underlying basal metabolic rate (BMR) are still not understood. It has been established that mitochondrial basal proton leak contributes significantly to BMR. Comparative studies among a diversity of eutherian mammals showed that BMR correlates with body mass and proton leak. Here, we studied BMR and mitochondrial basal proton leak in liver of various marsupial species. Surprisingly, we found that the mitochondrial proton leak was greater in marsupials than in eutherians, although marsupials have lower BMRs. To verify our finding, we kept similar-sized individuals of a marsupial opossum (Monodelphis domestica) and a eutherian rodent (Mesocricetus auratus) species under identical conditions, and directly compared BMR and basal proton leak. We confirmed an approximately 40 per cent lower mass specific BMR in the opossum although its proton leak was significantly higher (approx. 60%). We demonstrate that the increase in BMR during eutherian evolution is not based on a general increase in the mitochondrial proton leak, although there is a similar allometric relationship of proton leak and BMR within mammalian groups. The difference in proton leak between endothermic groups may assist in elucidating distinct metabolic and habitat requirements that have evolved during mammalian divergence.     

LKB1 is required for hepatic bile acid transport and canalicular membrane integrity in mice

LKB1 is a 'master' protein kinase implicated in the regulation of metabolism, cell proliferation, cell polarity and tumorigenesis. However, the long-term role of LKB1 in hepatic function is unknown. In the present study, it is shown that hepatic LKB1 plays a key role in liver cellular architecture and metabolism. We report that liver-specific deletion of LKB1 in mice leads to defective canaliculi and bile duct formation, causing impaired bile acid clearance and subsequent accumulation of bile acids in serum and liver. Concomitant with this, it was found that the majority of BSEP (bile salt export pump) was retained in intracellular pools rather than localized to the canalicular membrane in hepatocytes from LLKB1KO (liver-specific Lkb1-knockout) mice. Together, these changes resulted in toxic accumulation of bile salts, reduced liver function and failure to thrive. Additionally, circulating LDL (low-density lipoprotein)-cholesterol and non-esterified cholesterol levels were increased in LLKB1KO mice with an associated alteration in red blood cell morphology and development of hyperbilirubinaemia. These results indicate that LKB1 plays a critical role in bile acid homoeostasis and that lack of LKB1 in the liver results in cholestasis. These findings indicate a novel key role for LKB1 in the development of hepatic morphology and membrane targeting of canalicular proteins.     

Fibroblasts derived from long‐lived insulin receptor substrate 1 null mice are not resistant to multiple forms of stress

Reduced signalling through the insulin/insulin-like growth factor-1 signalling (IIS) pathway is a highly conserved lifespan determinant in model organisms. The precise mechanism underlying the effects of the IIS on lifespan and health is currently unclear, although cellular stress resistance may be important. We have previously demonstrated that mice globally lacking insulin receptor substrate 1 (Irs1^−/−) are long-lived and enjoy a greater period of their life free from age-related pathology compared with wild-type (WT) controls. In this study, we show that primary dermal fibroblasts and primary myoblasts derived from Irs1^−/− mice are no more resistant to a range of oxidant and nonoxidant chemical stressors than cells derived from WT mice.