Inhibition of internalization of glucose transporters and IGF-II receptors. Mechanism of action of MHC class I-derived peptides which augment the insulin response in rat adipose cells.

Peptides from the alpha 1 domain of the major histocompatibility complex class I antigen (MHC class I), e.g. Dk-(61-85) and Dk-(62-85), have been shown previously to augment glucose uptake in insulin-stimulated cells and to inhibit insulin receptor internalization (Stagsted, J., Reaven, G. M., Hansen, T., Goldstein, A., and Olsson, L. (1990) Cell 62, 297-307). We now report that these peptides inhibit by 80-100% the internalization of glucose transporters (GLUT4) and insulin-like growth factor II (IGF-II) receptors in insulin-stimulated cells and correspondingly double insulin-stimulated glucose transport activity and the number of GLUT4 and IGF-II receptors on the cell surface. In addition, the peptides enhance the apparent affinity about 3-fold of IGF-II binding to its receptor. It is concluded that the effects of the peptides on glucose transport and IGF-II binding are a consequence of the peptide-mediated inhibition of internalization of GLUT4 and IGF-II receptor. The active peptides are derived from the alpha 1 domain of a MHC class I molecule, suggesting that the latter is involved in regulation of internalization of cell surface integral membrane proteins such as the GLUT4 and IGF-II and insulin receptors.

A new approach to the chemical synthesis of the trisaccharide, and the terminal di- and mono-saccharide units of the major, serologically active glycolipid from Mycobacterium leprae.

The key intermediates for the synthesis of p-trifluoroacetamidophenyl O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1-->4)-O-(2,3-di-O-methyl-alp ha-L- rhamnopyranosyl)-(1-->2)-3-O-methyl-alpha-L-rhamnopyranoside (15), as well as p-trifluoroacetamidophenyl O-(3,6-di-O-methyl-beta-D- glucopyranosyl)-(1-->4)-2,3-di-O-methyl-alpha-L-rhamnopyranoside (29), were the methyl and ethyl O-(2-O-benzyl-4,6-O-benzylidene-3-O-methyl-beta- D-glucopyranosyl)-(1-->4)-2,3-O-diphenylmethylene-1-thio-alpha-L- rhamnopyranosides (10 and 24). Dichloroalane treatment of 10 and 24 removed the diphenylmethylene group, liberating HO-2 and HO-3 of the rhamnopyranoside residue, and opened the benzylidene acetal regioselectively to give the 4-O-benzyl-glucopyranosyl disaccharides. Methylation of the free OH groups resulted in the tetra-O-methyl 1-thio disaccharides (12 and 26), useful as glycosyl donors. Introduction of these temporary blocking groups allowed a drastic reduction in the number of synthetic steps to the target compounds.

Insulinomimetic effect on glucose transport by epidermal growth factor when combined with a major histocompatibility complex class I-derived peptide.

Peptides derived from the alpha 1-region of the murine H-2Dk molecule enhance glucose uptake in rat adipose cells above the maximum obtained with insulin stimulation alone (Stagsted, J., Reaven, G. M., Hansen, T., Goldstein, A., and Olsson, L. (1990) Cell 62, 297-307). We now describe that epidermal growth factor (EGF) in combination with the same peptides, Dk-(61-85) and Dk-(62-85), stimulates cellular glucose uptake 5-7 times over the basal level, i.e. to 30-50% of the maximal insulin effect. EGF alone increased glucose uptake by only approximately 50% above basal and the peptide alone by 100% above basal. Maximal effect of EGF and peptide was reached in 10-20 min with 30 microM peptide (EC50 10-15 microM) and 50 nM EGF (EC50 1-2 nM). The effect of EGF and peptide on glucose uptake was additive to that of insulin and peptide until the maximal level attained with insulin and peptide was reached. The combined effect of EGF plus peptide on glucose transport was associated with a recruitment of GLUT4 molecules to the plasma membrane. However, the phosphatidylinositol (PI) kinase which is activated by insulin was not activated by EGF plus peptide. Thus, the effect of EGF plus peptide on glucose uptake seems independent of the activity status of the insulin receptor. 125I-Labeled EGF bound specifically to rat adipose cells with an apparent affinity of approximately 2 nM and Bmax approximately 5 x 10(3). However, the major histocompatibility complex (MHC) peptides did not affect EGF-stimulated internalization of EGF receptor, in contrast to their effect on the insulin receptors. Transforming growth factor alpha had an effect similar to EGF on glucose uptake. Three other peptides derived from other parts of murine MHC class I had no effect on glucose uptake in combination with EGF. Thus, EGF in combination with certain MHC class I-derived peptides is insulinomimetic concerning glucose transport and this effect is independent of the insulin receptor activity.

Microtiter particle agglutination test for diagnosis of leprosy.

The results of studying the microtiter particle agglutination (MPA) test for detecting anti-Mycobacterium leprae antibodies in blood sera are presented. The serodiagnostic test is based on the agglutination of colored polyacrolein latex microparticles (PAMP) conjugated with 3,6-di-O-methyl-D-glucose (DMG). Sera from 45 leprosy patients (LL, BL), 34 leprosy contacts, and 148 control subjects were investigated by the MPA test. A correlation between the anti-M. leprae antibodies and the bacterial load was found. In many long-treated leprosy patients increased titers of anti-DMG antibodies were observed, which might be due to specific polyneuritis in them. Four contacts of leprosy patients were also positive in the MPA test. "Nonleprosy" sera did not react in the test. The method proposed proved to be of high specificity and sensitivity for the serological diagnosis of leprosy. The rapidity, simplicity, and visual assessment of the results allow the method to be used in the field for epidemiological studies of leprosy contacts and the general population in leprosy-endemic areas.

A simplified serological test for leprosy based on a 3,6-di-O-methylglucose-containing synthetic antigen.

The recent advent of synthetic antigens containing the Mycobacterium leprae-specific epitope, 3,6-di-O-methyl-beta-D-glucopyranoside, has allowed the development of simple specific serological tests for leprosy. The incorporation of one such product, 8-carbonyloctyl O-[4-O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-alpha-L- rhamnopyranoside]-BSA, into a simple "spot" test, diffusion-in-gel enzyme-linked immunosorbent assay (ELISA), allowed an over 90% detection rate of untreated lepromatous leprosy, and the results showed good concordance with conventional ELISA based on the native phenolic glycolipid I.

Control of ribosome synthesis in Escherichia coli: analysis of an energy source shift-down.

The rate of ribosome synthesis and accumulation in Escherichia coli during the transition after an energy source shift-down was analyzed. The shift was imposed on cultures of stringent and relaxed strains growing in glucose minimal medium by the addition of the glucose analogue alpha-methylglucoside. In the stringent strain, ribosome synthesis was almost instantaneously reduced after the shift, whereas the relaxed strain exhibited a more gradual response. The rate of messenger ribonucleic acid (mRNA) synthesis was affected similarly, though to a smaller extent. A comparison of the rates of synthesis and accumulation of ribosomal RNA (rRNA) and ribosomal proteins showed that far more ribosomal components were synthesized after the shift than were accumulated, indicating that a substantial part of the rRNA made after the shift was unstable. A new method was used to measure relative rates of rRNA synthesis and to estimate the transcription time for the rRNA operon under different conditions. In steady states of growth with growth rates ranging from 0.75 to 2.3 doublings/h, as well as during the transition after a shift-down, the transcription time of the rRNA operon was constant. The rate of synthesis of rRNA correlated during this transition - in contrast to the rate of accumulation (M. T. Hansen et al., J. Bacteriol. 122: 585-591, 1975) - with the ppGpp pool in the same way as has been observed during partial amino acid starvation.