Hypoglycaemic effect of metformin in genetically obese (fa/fa) rats results from an increased utilization of blood glucose by intestine.

The insulin-resistant obese fa/fa rat is a convenient model in which to study a potential effect of metformin, a biguanide used in the treatment of non-insulin-dependent diabetes, on insulin-mediated glucose utilization. Female fa/fa rats were given metformin orally for 8 days. Studies were performed on anaesthetized post-absorptive rats 5 h after the last dose of metformin. Glucose production and utilization were enhanced 1.5-fold in metformin-treated rats. The enhanced glucose production was almost entirely due to increased glucose recycling. The digestive tract was the only tissue responsible for the enhanced glucose utilization.     

Stereological and functional investigations on isolated adrenocortical cells: Zona fasciculata/reticularis cells of chronically ACTH-treated rats

Summary The morphology and function of isolated inner (zona fasciculata/reticularis) adrenocortical cells of rats pretreated with ACTH for 3, 6, 9 or 12 days were investigated. ACTH treatment induced a notable time-dependent enhancement in the steroidogenic capacity (corticosterone production) and growth of inner cells. The volumes of cells, mitochondrial compartment, membrane space [the cellular space occupied by smooth endoplasmic reticulum (SER) membranes] and lipid-droplet compartment, as well as the surface area of mitochondrial cristae and SER tubules, were increased in relation to the duration of ACTH pretreatment, and showed a highly significant positive linear correlation with both basal and stimulated corticosterone production. The acute exposure of isolated cells to ACTH provoked a striking lipid-droplet depletion, the extent of which was linearly and positively correlated with stimulated corticosterone secretion. The hypertrophy of the mitochondrial compartment and SER are interpreted as the morphological counterpart of the enhanced steroidogenic capacity of inner adrenocortical cells, inasmuch as the enzymes of steroid synthesis are located in these two organelles, and it is well known that chronic ACTH exposure stimulates the de novo synthesis of many of them in vivo. The rise in the number of lipid droplets, in which cholesterol is stored, is interpreted as being due to the fact that, under chronic ACTH treatment, the processes leading to cholesterol accumulation in adrenocortical cells (exogenous uptake and endogenous synthesis) exceed those of its utilization in basal steroid secretion. Cholesterol accumulated in lipid droplets as a reserve material may be rapidly utilized after acute ACTH exposure to meet the needs of the enhanced steroidogenic capacity of adrenocortical cells.     

Subfamily of submaxillary gland-specific Mup genes: chromosomal linkage and sequence comparison with liver-specific Mup genes

Mouse major urinary proteins (MUPs) are encoded by a family of ca. 35 genes that are expressed in a tissue-specific manner in several secretory organs; in the liver, in the submaxillary, sublingual, parotid and lachrymal glands, and in the skin sebaceous glands. In this paper we describe the isolation of a Mup gene, Mup-1.5a, which is expressed predominantly in the submaxillary gland of BALB/c mice. We show that Mup-1.5a is a member of a subfamily consisting of two closely related genes, both of which are closely linked to the Mup-1 locus on mouse chromosome 4. Mup-1 is the locus of a class of Mup genes (Group 1) expressed in the liver. The complete nucleotide sequence of Mup-1.5a has been determined, and was compared to a previously sequenced Group 1 Mup gene. The comparison shows that the differentially expressed Mup genes are uniformly divergent in exons, introns and in their flanking sequences. The regions of homology extend at least 5 kb into the 5' flanking region of Mup genes.     

Purification and characterization of two serine isoacceptor tRNAs from bovine mitochondria by using a hybridization assay method.

For large scale preparation of mitochondrial tRNAs, a new hybridization assay method using synthetic oligodeoxyribonucleotide probes (16-17mer) complementary to individual tRNA sequences was developed and applied for the purification of two serine isoacceptor tRNAs (tRNASerAGY and tRNASerUCN) from bovine mitochondria. It is about 100 times more sensitive than the conventional aminoacylation assay method. 2-4 A260 units each of both tRNASer isoacceptors were purified from 17.5 kg of bovine liver, and they were characterized by means of nuclease digestion, melting profiles and aminoacylation activity. It is suggested that tRNASerUCN possesses the D loop/T loop interaction like usual L-shaped tRNAs, and that tRNASerAGY lacking almost an entire D arm is aminoacylated with an efficiency not very much lower than that of tRNASerUCN.     

Solid-phase synthesis of oligoribonucleotides using 9-fluorenylmethoxycarbonyl (Fmoc) for 5''-hydroxyl protection.

Efficient solid-phase synthesis of a series of oligoribonucleotides of up to 20 residues is described that utilises the 9-fluorenylmethoxycarbonyl group (Fmoc) for 5'-protection and 4-methoxytetrahydropyran-4-yl (Mthp) for 2'-protection of ribonucleotide monomers and a phosphoramidite coupling procedure. The Fmoc group is removed after each coupling step by treatment with 0.1M DBU in acetonitrile. Oligoribonucleotides are isolated in 2'-protected form in good yield and shown to be readily and efficiently deprotected by mild acidic treatment.     

Purification and characterization of a protein that binds to the recombination signal sequence of the immunoglobulin J kappa segment.

A protein that binds to the recombination signal sequence (RS) of the immunoglobulin J kappa segment was purified almost to homogeneity from the nuclear extract of a murine pre-B cell line 38B9. A similar binding protein was found in lymphoid cell lines but not in non-lymphoid cell lines. The binding activity was associated with a polypeptide with a molecular weight of 60,000. DNase I footprinting analysis demonstrated that this binding protein interacted with the heptamer and several 3' bases close to the heptamer. The Kd value of the J kappa RS binding protein to the J kappa RS was 1 nM. One base substitution in the heptamer of the J kappa RS greatly reduced the affinity of the J kappa RS binding protein. The high specificity of the binding site of the J kappa RS binding protein suggests that this protein may be involved in V-J recombination.     

Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers.

An efficient method for the construction of multiple mutations in a sequential manner is described. It is based on the gapped duplex DNA approach to oligonucleotide-directed mutagenesis (Kramer et al. 1984, Nucl. Acids Res. 12, 9441-9456) and a set of newly constructed phasmid vectors. These are characterized by the following features. Presence of the phage fl replication origin permits ready conversion to the single stranded DNA form. An amber mutation within, alternatively, the bla or cat gene provides a means for ready selection of the strand into which the mutagenic oligonucleotide has been incorporated. By means of the alternating antibiotic resistance markers any number of mutations can be constructed in consecutive rounds of mutagenesis. The optional presence of gene expression signals allows the direct overproduction of structurally altered proteins without re-cloning. Both the mutagenesis and expression aspects were tested using the lacZ gene as a model.