A versatile assay for total cellulase activity using U-[14C]-labelled bacterial cellulose

Summary A versatile and sensitive assay for total cellulase activity and not just carboxymethylcellulase activity, is described. The method is equally suitable for studying the kinetics of solubilization of cellulose by growing cells or isolated enzyme fractions.     

Reaction of methyl β-d-glycero-l-manno-heptopyranoside with cyclohexanone: Synthesis of the 4- and 6-methyl ethers of d-glycero-l-manno-heptitol

Acid-catalysed condensation of methyl β-d-glycero-l-manno-heptopyranoside with cyclohexanone yielded an approximately 3:1 mixture of the 2,3:6,7- and 2,3:4,7-di-O-cyclohexylideneheptosides (1 and 2), which could be separated either as their benzoates (3 and 4) or as their methyl ethers (5 and 6). The latter compounds afforded the 4- and 6-methyl ethers (7 and 8) of d-glycero-l-manno-heptitol.     

ATPase-defective derivatives of Escherichia coli DnaK that behave differently with respect to ATP-induced conformational change and peptide release.

We have characterized the effects of the T199S, T199A, and K70A mutations on the biochemical activity and in vivo functioning of Escherichia coli DnaK. Threonine-199 is the site of autophosphorylation of DnaK, and the lysine residue of bovine Hsc70 corresponding to K70 of DnaK has been shown to be essential for the hydrolysis of ATP. The dnaK alleles T199A and K70A are completely unable, and the T199S allele is only partially able, to complement the defects of a DeltadnaK mutant. The ATPase activities of the DnaK T199A and DnaK K70A proteins are nearly abolished, while the ATPase activity of the DnaK T199S protein has a steady-state rate similar to that of wild-type DnaK. The DnaK T199S protein also retains approximately 13% of the autophosphorylation activity of wild-type DnaK, while the autophosphorylation activities of the T199A and K70A derivatives are completely abolished. All four DnaK proteins bind a model peptide substrate, and the wild-type, T199A, and T199S DnaK proteins release the peptide with similar kinetics upon the addition of ATP. The DnaK K70A protein, in contrast, does not release the peptide upon the addition of ATP. ATP induces a conformational change in the wild-type, T199A, and T199S DnaK proteins but not in the DnaK K70A protein. The T199A and K70A mutations both disrupt the ATPase activity of DnaK but have profoundly different effects on the ATP-induced conformational change and peptide release activities of DnaK, implying that the two mutations affect different steps in the functional cycle of DnaK. The DnaK T199S protein represents a new class of DnaK mutant, one which has near-normal levels of ATPase activity and undergoes an ATP-induced conformational change that results in the release of peptide but which is not able to fully complement loss of DnaK function in the cell.     

Effect of chronic ethanol and food deprivation on intestinal villus morphology and brush border membrane content of lipid and marker enzymes

Brush border membranes (BBM) were isolated from the jejunum and ileum of control, ad libitum (CAL); control, food-restricted (CFR); control, weight gain (CWG); and ethanol-fed (EF) rabbits. Jejunal alkaline phosphatase activity was similar among control groups, but higher in CAL than EF animals. Sucrase activity was higher in EF and CWG animals than in CAL and CFR. The alkaline phosphatase/sucrase ratio was lower in EF than control animals. Ileal enzyme marker activity was similar among EF and control animals. Sucrase (S) activity was lower in the ileum than in the jejunum. Jejunal free fatty acid and phospholipid/cholesterol (PL/C) were lower in EF than control animals, whereas ileal lipid content was generally similar among all animal groups. Total phospholipid content was similar between sites, but the cholesterol and free fatty acid content were lower in the ileum than the jejunum. The phospholipid/cholesterol ratio was increased only in the ileum of EF animals. The amount of lecithin was decreased in the jejunal BBM of EF animals resulting in a decreased choline/amine phospholipid ratio as compared with control animals. The ileal phospholipid composition was similar among all groups. A large increase in villus height is observed in the jejunum of EF animals. Villus surface area and mucosal surface area are altered with ethanol feeding and food deprivation. Thus, (i) there is a gradient of S and cholesterol between the BBM of jejunum and ileum; (ii) changes in food intake are associated with changes in the morphology as well as the enzyme marker and lipid content of BBM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hierarchical down-modulation of hemopoietic growth factor receptors

Granulocytes and macrophages can be produced in vitro when progenitor cells from mouse bone marrow are stimulated by any of four distinct colony stimulating factors, Multi-CSF (IL-3), GM-CSF, G-CSF, and M-CSF (CSF-1). At 0 degrees C the four CSFs do not cross-compete for binding to bone marrow cells, indicating that each has a specific cell surface receptor. However, at 21 degrees C or 37 degrees C, Multi-CSF inhibits binding of the other three CSFs and GM-CSF inhibits binding of G-CSF and M-CSF. Rather than competing directly for receptor binding, the binding of Multi-CSF, GM-CSF, or G-CSF to their own receptor induces the down-modulation (and thus activation) of other CSF receptors at 37 degrees C. The pattern and potency of down-modulation activity exhibited by each type of CSF parallels the pattern and potency of its biological activity. We propose a model in which the biological interactions of the four CSFs are explained by their ability to down-modulate and activate lineage-specific receptors.     

GRF-induced feeding: Evidence for protein selectivity and opiate involvement

Growth hormone-releasing factor (GRF) is a hypothalamic peptide named for its ability to induce release of growth hormone from the anterior pituitary. GRF also acts as a neurotransmitter in the suprachiasmatic nucleus/medial preoptic area (SCN/MPOA) to stimulate food intake. The purpose of this series of experiments was to explore the nature of GRF-induced feeding, with a particular emphasis on macronutrient selectivity, and to examine the role of opiate activity in the paraventricular nucleus of the hypothalamus (PVN). Chow intake stimulated by GRF microinjection (1 pmol/0.5 μl) into the SCN/MPOA was blocked by injection of methyl-naltrexone (3 μg/0.5 μl) into the PVN. In animals habituated to macronutrient diets (Teklad, WI), GRF preferentially stimulated intake of protein at 2 and 4 h postinjection, whereas it had no effect on carbohydrate intake. Further, this effect was blocked by injection of naloxone (40 nmol/0.5 μl) into the PVN. Microinjection of morphine (0, 1, 10, and 17 μg/0.5 μl) into the PVN also specifically stimulated protein intake at 2 and 4 h postinjection. These results suggest that feeding derived from GRF actions in the SCN/MPOA is macronutrient selective, and is dependent on PVN opiate activity for expression.     

Stereotypes Possess Heterogeneous Directionality: A Theoretical and Empirical Exploration of Stereotype Structure and Content

We advance a theory-driven approach to stereotype structure, informed by connectionist theories of cognition. Whereas traditional models define or tacitly assume that stereotypes possess inherently Group → Attribute activation directionality (e.g., Black activates criminal), our model predicts heterogeneous stereotype directionality. Alongside the classically studied Group → Attribute stereotypes, some stereotypes should be bidirectional (i.e., Group ⇄ Attribute) and others should have Attribute → Group unidirectionality (e.g., fashionable activates gay). We tested this prediction in several large-scale studies with human participants (N_Combined = 4,817), assessing stereotypic inferences among various groups and attributes. Supporting predictions, we found heterogeneous directionality both among the stereotype links related to a given social group and also between the links of different social groups. These efforts yield rich datasets that map the networks of stereotype links related to several social groups. We make these datasets publicly available, enabling other researchers to explore a number of questions related to stereotypes and stereotyping. Stereotype directionality is an understudied feature of stereotypes and stereotyping with widespread implications for the development, measurement, maintenance, expression, and change of stereotypes, stereotyping, prejudice, and discrimination.