Mechanism of depsipeptide formation catalyzed by enniatin synthetase

Covalently bound intermediates of enniatin B synthesis could be isolated from enniatin synthetase by treatment with performic acid. By comparison with products of mild alkaline cleavage of authentic enniatin B they could be identified as the dipeptide D-2-hydroxyisovaleryl-N-methylvaline and the corresponding tetrapeptide. Synthesis of enniatins apparently proceeds via condensation of dipeptides. This was confirmed by the use of the substrate analogue isovaleric acid, which has shown to be a strong inhibitor for enniatin synthesis by formation of N-isovaleryl-N-methyl valine.     

Serum-induced inhibition of isotonic fluid absorption by the kidney proximal tubule I. Mechanism of inhibition

Isotonic reabsorption by the rat kidney proximal tubule was drastically inhibited after less than 2 min intraluminal perfusion with fresh sera from rat (both homologous and autologous), cat, rabbit and human, but not with sera from mouse and guinea pig. The inhibitory factor in serum in a heat (56° C for 30 min) and storage (4°C for 2–5 days) labile macromolecule (mol. wt 50 000) and requires Ca²⁺ for its effect. The cellular electrical potential difference of the proximal tubular cells was irreversively destroyed and intraluminally perfused trypan blue dye incorporated into the tubular cells after the intraluminal perfusion with serum for 2 min. These observations suggest that lysis of the proximal tubular cells is the mechanism for serum-induced inhibition of proximal tubular isotonic reabsorption.     

Protein kinase C: a new linkage marker for growth hormone and for COL1A1

An expanded linkage group on the long arm of human chromosome 17 is reported. Using the CEPH panel of DNAs and restriction fragment length polymorphism (RFLP) markers for the centromere locus (D17Z1), growth hormone (GH1), collagen type I alpha 1 (COL1A1), and protein kinase C-alpha polypeptide (PKCA) loci, theta values of 0.03, 0.11, and 0.23 were found between PKCA and GH1, PKCA and COL1A1, and PKCA and D17Z1, respectively. The theta values calculated for GH1 versus COL1A1 or D17Z1 were 0.11 and 0.23, respectively. Sex-specific recombination rates were calculated for the best likelihood order and demonstrate female recombination greater than male recombination. Therefore, the loci studied span a map region of approximately 30 cm between 17cen and 17q24, with the most likely gene order being D17Z1-COL1A1-PKCA-GH1.     

Receptor-mediated internalization of insulin requires a 12-amino acid sequence in the juxtamembrane region of the insulin receptor beta-subunit

The juxtamembrane region of the insulin receptor (IR) beta-subunit contains an unphosphorylated tyrosyl residue (Tyr960) that is essential for insulin-stimulated tyrosyl phosphorylation of some endogenous substrates and certain biological responses (White, M.F., Livingston, J.N., Backer, J.M., Lauris, V., Dull, T.J., Ullrich, A., and Kahn, C.R. (1988) Cell 54, 641-649). Tyrosyl residues in the juxtamembrane region of some plasma membrane receptors have been shown to be required for their internalization. In addition, a juxtamembrane tyrosine in the context of the sequence NPXY [corrected] is required for the coated pit-mediated internalization of the low density lipoprotein receptor. To examine the role of the juxtamembrane region of the insulin receptor during receptor-mediated endocytosis, we have studied the internalization of insulin by Chinese hamster ovary (CHO) cells expressing two mutant receptors: IRF960, in which Tyr960 has been substituted with phenylalanine, and IR delta 960, in which 12 amino acids (Ala954-Asp965), including the putative consensus sequence NPXY [corrected], were deleted. Although the in vivo autophosphorylation of IRF960 and IR delta 960 was similar to wild type, neither mutant could phosphorylate the endogenous substrate pp185. CHO/IRF960 cells internalized insulin normally whereas the intracellular accumulation of insulin by CHO/IR delta 960 cells was 20-30% of wild-type. However, insulin internalization in the CHO/IR delta 960 cells was consistently more rapid than that occurring in CHO cells expressing kinase-deficient receptors (CHO/IRA1018). The degradation of insulin was equally impaired in CHO/IR delta 960 and CHO/IRA1018 cells. These data show that the juxtamembrane region of the insulin receptor contains residues essential for insulin-stimulated internalization and suggest that the sequence NPXY [corrected] may play a general role in directing the internalization of cell surface receptors.     

Effects of growth and insulin treatment on the levels of insulin receptors and their mRNA in Hep G2 cells

We have studied the variations in the number of insulin receptor and insulin receptor mRNA levels in (Hep G2) cells in response to growth and insulin treatment. The levels of insulin receptors are relatively low in growing cells. After approximately 5 days in culture, if cells are not refed they cease to divide and the number of receptors/cell increases, reaching 4 times the initial values by the 9th day. Refeeding the cells completely prevented both growth arrest and the increase in insulin receptor number. Insulin added daily to cells at 0.33 microM caused receptor down-regulation but did not prevent a 3-fold increase in binding with growth arrest. Pulse-chase studies of metabolically labeled ([35S]methionine) cells showed that the receptor degradation rate (apparent t 1/2, 18-20 h) was comparable in rapidly growing versus growth-arrested cells. The increased receptor level in non-refed cells is not due to generation of a soluble factor by confluent cells, nor is it caused by depletion of insulin, glucose, or insulin-like growth factor I from the culture medium. The levels of insulin receptor mRNA measured on Northern blots increased in growth-arrested cells in parallel to the increase in receptor number. The mRNA value begins to increase from the 3rd day in culture and by the 9th day reaches a level 6.0 times that on the 3rd day. Chronic insulin-induced receptor down-regulation did not alter insulin receptor mRNA levels at any time point studied. These data demonstrate that the increase in insulin receptor number/cell in growth-arrested cells is paralleled by an increase in insulin receptor mRNA content with no change in the receptor degradation rates. This suggests that the increase in the number of insulin receptors is due to enhanced receptor synthesis due to increased receptor mRNA content. Conversely, down-regulation of the insulin receptor does not affect the level of insulin receptor mRNA and thus must be due to increased receptor degradation.     

Immunogold localization of chymotrypsin inhibitor-2, a lysine-rich protein,in developing barley endosperm

Chymotrypsin inhibitor-2, a lysine-rich protein in the barley endosperm, has been localized at the ultrastructural level by immunocytochemistry in developing barley endosperm cells 14 days post anthesis. The protein is deposited in the protein bodies. Two morphologically distinct types of protein bodies, small spherical and large irregularly shaped, are present. Golgi-apparatus-derived vesicles whose content is labelled by chymotrypsin inhibitor-2 antibody-gold particles are observed at the Golgi complex and around the vacuoles. These observations indicate that the transport of the protein to the site of deposition is mediated by the Golgi apparatus.Abbreviations CI chymotrypsin inhibitor - DPA days post anthesis - ER endoplasmic reticulum The authors wish to thank Dr. V.R. Franceschi (Department of Botany, Washington State University, Pullman, USA) for many helpful discussions and advice during the work, and the staff at the Electron Microscope Center at Washington State University for technical assistance.     

Kinase activity controls the sorting of the epidermal growth factor receptor within the multivesicular body

We compared the internalization and intracellular sorting of epidermal growth factor receptor (EGF-R) and point mutant kinase-negative EGF-R separately expressed in NIH 3T3 cells lacking endogenous receptor. Both EGF-Rs internalized rapidly, but kinase-negative receptor was surface down-regulated only with monensin or at 20 degrees C. Furthermore, EGF internalized by mutant receptor alone was, in significant proportion, returned to the cell surface undegraded. Hence unlike wild-type receptor, kinase-negative EGF-R recycles. By electron microscopy the early pathways of endocytosis for the two receptors were identical; however, after 10-20 min the pathways diverged at the multivesicular body (MVB). Wild-type EGF-R, destined for degradation, localized to internal vesicles, while kinase-negative EGF-R, destined for recycling, localized to surface membranes of the MVBs and moved to small tubulovesicles. We conclude that sorting of internalized receptor for degradation or recycling can occur through spatial segregation within the MVB, and sorting of EGF-R is controlled by tyrosine kinase activity.     

Use of the additive main effects and multiplicative interaction model in QTL mapping for adaptation in barley

The additive main effects and multiplicative interaction (AMMI) model has emerged as a powerful analytical tool for genotype x environment studies. The objective of the present study was to assess its value in quantitative trait locus (QTL) mapping. This was done through the analysis of a large two-way table of genotype-by-environment data of barley (Hordeum vulgare L.) grain yields, where the genotypes constituted a genetic population suitable for mapping studies. Grain yield data of 150 doubled haploid lines derived from the Steptoe x Morex cross, and the two parental lines, were taken by the North American Barley Genome Mapping Project (NABGMP) at 16 environments throughout the barley production areas of the USA and Canada. Four regions of the genome were responsible for most of the differential genotypic expression across environments. They accounted for approximately 50% of the genotypic main effect and 30% of the genotype x environment interaction (GE) sums of squares. The magnitude and sign of AMMI scores for genotypes and sites facilitate inferences about specific interactions. The parallel use of classification (cluster analysis of environments) and ordination (principal component analysis of GE matrix) techniques allowed most of the variation present in the genotype x environment matrix to be summarized in just a few dimensions, specifically four QTLs showing differential adaptation to four clusters of environments. Thus, AMMI genotypic scores, when the genotypes constituted a population suitable for QTL mapping, could provide an adequate way of resolving the magnitude and nature of QTL x environment interactions.Ignacio Romagosa was on sabbatical leave from the University of Lleida and the Institut de Recerca i Tecnologia Agroalimentàries, Lleida, Spain, when this study was conducted     

Distinct structural elements of rab5 define its functional specificity.

Members of the rab family of small GTPases are localized to distinct cellular compartments and function as specific regulators of vesicle transport between organelles. Overexpression of rab5, which is associated with early endosomes and the plasma membrane, increases the rate of endocytosis [Bucci et al. (1992) Cell, 70, 715-728]. From sequence alignments and molecular modelling we identified structural elements that might contribute to the definition of the functional specificity of rab5. To test the role of these elements experimentally, we transplanted them onto rab6, which is associated with the Golgi complex. The chimeric proteins were assayed for intracellular localization and stimulation of endocytosis. First, we found that the C-terminus of rab5 could target rab6 to the plasma membrane and early endosomes but it did not confer rab5-like stimulation of endocytosis. Further replacement of other regions revealed that the N-terminus, helix alpha 2/loop 5 and helix alpha 2/loop 7 were all required to functionally convert rab6 into rab5. Reciprocal hybrids of rab5 containing these regions replaced with those of rab6 were inactive, demonstrating that each region is essential for rab5 function. These results indicate that distinct structural elements specify the localization, membrane association and regulatory function of rab5.