Development of communication supporting device controlled by eye movements and voluntary eye blink.

A communication interface controlled by eye movements and voluntary eye blink has been developed for disabled individuals who have motor paralysis and therefore cannot speak. Horizontal and vertical electro-oculograms were measured using two surface electrodes referring to an earlobe electrode. Four directional cursor movements and one selection were realized by logically combining the detected two channel signals. Virtual input experiments were conducted on a virtual screen keyboard. Its usability and accuracy were improved using our proposed method.

Hydroxyurea induces site-specific DNA damage via formation of hydrogen peroxide and nitric oxide.

Hydroxyurea is a chemotherapeutic agent used for the treatment of myeloproliferative disorders (MPD) and solid tumors. The mutagenic and carcinogenic potential of hydroxyurea has not been established, although hydroxyurea has been associated with an increased risk of leukemia in MPD patients. To clarify whether hydroxyurea has potential carcinogenicity, we examined site-specific DNA damage induced by hydroxyurea using (32)P-5'-end-labeled DNA fragments obtained from the human p53 and p16 tumor suppressor genes and the c-Ha-ras-1 protooncogene. Hydroxyurea caused Cu(II)-mediated DNA damage especially at thymine and cytosine residues. NADH efficiently enhanced hydroxyurea-induced DNA damage. The DNA damage was almost entirely inhibited by catalase and bathocuproine, a Cu(I)-specific chelator, suggesting the involvement of hydrogen peroxide (H(2)O(2)) and Cu(I). Typical free hydroxyl radical scavengers did not inhibit DNA damage by hydroxyurea, but methional did. These results suggest that crypto-hydroxyl radicals such as Cu(I)-hydroperoxo complex (Cu(I)-OOH) cause DNA damage. Formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was induced by hydroxyurea in the presence of Cu(II). An electron spin resonance spectroscopic study using N-(dithiocarboxy)sarcosine as a nitric oxide (NO)-trapping reagent demonstrated that NO was generated from hydroxyurea in the presence and absence of catalase. In addition, the generation of formamide was detected by both gas chromatography-mass spectrometry (GC-MS) and time-of-flight-mass spectrometry (TOF-MS). A high concentration of hydroxyurea induced depurination at DNA bases in an H(2)O(2)-independent manner, and endonuclease IV treatment led to chain cleavages. These results suggest that hydroxyurea could induce base oxidation as the major pathway of DNA modification and depurination as a minor pathway. Therefore, it is considered that DNA damage by hydroxyurea participates in not only anti-cancer activity, but also carcinogenesis.

Mechanism of metal-mediated DNA damage induced by metabolites of carcinogenic 2-nitropropane.

2-Nitropropane (2-NP), a widely used industrial solvent, is carcinogenic to rats. To clarify the mechanism of carcinogenesis by 2-NP, we investigated DNA damage by 2-NP metabolites, N-isopropylhydroxylamine (IPHA) and hydroxylamine-O-sulfonic acid (HAS), using 32P-5'-end-labelled DNA fragments obtained from genes that are relevant to human cancer. In the presence of Fe(III) EDTA, both IPHA and HAS caused DNA damage at every nucleotide position without marked site preference. The damage was inhibited by free hydroxyl radical (-*OH) scavengers, catalase and deferoxamine mesilate, an iron chelating agent. These results suggest that the DNA damage was caused by -*OH generated via H(2)O(2) by both IPHA and HAS. In contrast, in the presence of Cu(II), IPHA frequently caused DNA damage at thymine. The Cu(II)-mediated DNA damage caused by IPHA was inhibited by catalase, methional and bathocuproine, a Cu(I)-specific chelator, suggesting the involvement of H(2)O(2) and Cu(I). These results suggest that the DNA damage induced by IPHA in the presence of Cu(II) was caused by a reactive oxygen species like the Cu(I)-hydroperoxo complex. On the other hand, HAS most frequently induced DNA damage at 5'-TG-3', 5'-GG-3' and 5'-GGG-3' sequences. Catalase and methional only partly inhibited the Cu(II)-mediated DNA damage caused by HAS, suggesting that the reactive oxygen species and another reactive species participate in this process. Formation of 8-oxodG by IPHA or HAS increased in the presence of metal ions. This study suggests that metal-mediated DNA damage caused by 2-NP metabolites plays an important role in the mutagenicity and the carcinogenicity of 2-NP.

Metabolic regulation of pH in plant cells: role of cytoplasmic pH in defense reaction and secondary metabolism.

A new biochemical pH-stat hypothesis that revised the classic hypothesis is presented to understand the metabolic regulation of intracellular pH in plant cells. Alternative pathway glycolysis, alternative pathway respiration and malate-derived lactic and alcoholic fermentation (alternative pathway fermentation), all unique to plants, are integrated into a regulatory mechanism of pH in the cytoplasm. Its uniqueness to plant kingdom is discussed from the evolutionary viewpoint: it is suggested that when the ancestors of extant terrestrial plants expanded their habitat from oceans to freshwater, they abandoned a "sodium system" and adopted a "proton system" for nutrient uptake. Validity of the new hypothesis is examined with available data on a secondary active transport, anoxia and other experimental evidence. The hypothesis predicts that biotic and abiotic stress-induced cytoplasmic acidification triggers synthesis of phytoalexins and other secondary metabolites. Possible roles of cyanide-resistant alternative pathway respiration in the secondary metabolite production, metabolic switching between primary and secondary metabolisms, and defense reactions are proposed.

Antimutagenic activity of isoflavone from Pueraria lobata.

A methanol extract from Pueraria lobata showed a suppressive effect on umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide). The methanol extract from P. lobata was re-extracted with hexane, dichloromethane, ethyl acetate, butanol, and water, respectively. A suppressive compound in the dichloromethane and ethyl acetate extract fractions was isolated by SiO(2) column chromatography and identified as tectorigenin (1) by EI-MS and (1)H and (13)C NMR spectroscopy. Compound 1 and its methylated derivative [7,4'-di-O-methyltectorigenin (2)] had the suppressive effects on umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against furylfuramide, 4-nitroquinoline-1-oxide, N-methyl-N'-nitrosoguanidine, and activated Trp-P-1, which do not require live metabolic activation by S9. These compounds also showed suppression of SOS-inducing activity against Trp-P-1 and AfB(1), which requires liver metabolizing enzymes. In addition to the antimutagenic activities of these compounds against furylfuramide, Trp-P-1 and activated Trp-P-1 were also assayed by an Ames test using S. typhimurium TA100.

Endomorphin-1 discriminates the mu-opioid receptor from the delta- and kappa-opioid receptors by recognizing the difference in multiple regions.

Endomorphin-1 is a novel endogenous peptide that is highly selective for the mu-opioid receptor over the delta- and kappa-opioid receptors. The structural basis of high selectivity of endomorphin-1 to the mu-opioid receptor was examined using chimeric receptors between mu- and delta-opioid receptors and those between mu- and kappa-opioid receptors. The chimeric receptors were constructed by using restriction enzyme sites intrinsically possessed by or introduced to the mu-, delta- and kappa-opioid receptor cDNAs. The junctions for the construction were located at the first intracellular loop (Bbs I site), third transmembrane domain (Afl III site) and fifth transmembrane domain (Bgl II site). The competitive binding assay using chimeric receptors revealed that the region from the Bbs I site to the Afl III site, including the first extracellular loop, contributes to the discrimination between mu- and delta-opioid receptors by endomorphin-1 more than any other regions. However, the region from the Afl III site to the Bgl II site and that from the Bgl II site to the carboxy terminal also somewhat contribute to the discrimination between mu- and delta-opioid receptors. For the discrimination between mu- and kappa-opioid receptors, two regions, that is, the region from the Bbs I site to the Afl III site and that from the Bgl II site to the carboxy terminal, were shown to be important. The present results show that endomorphin-1 discriminates the mu-opioid receptor from the other two types of opioid receptors by recognizing the differences in several amino acid residues widely distributed through the receptor structure. We previously reported that DAMGO, a synthetic highly mu-selective peptide, discriminates between mu- and delta-opioid receptors by recognizing the difference in only one amino acid residue and discriminates between mu- and kappa-opioid receptors by recognizing the difference in four residues localized in the restricted region. Although both endomorphin-1 and DAMGO are mu-opioid receptor selective peptides, molecular mechanisms for mu-selectivity are different between these peptides.

Regulation of cytoplasmic pH under extreme acid conditions in suspension cultured cells of Catharanthus roseus: a possible role of inorganic phosphate.

Changes in cytoplasmic pH of suspension-cultured cells of Catharanthus roseus under extreme acid conditions were measured with the pH-dependent fluorescence dye; 2',7'-bis-(2-carboxyethyl)-5 (and-6) carboxyfluorescein (-acetoxymethylester) (BCECF). When cells were treated with 1 mM HCl (pH 3 solution), the cytoplasmic pH first decreased then returned to the original level. Treatment with 10 mM HCl (pH 2 solution) acidified the cytoplasm to a greater extent, and the acidification continued at a constant level throughout the measurement. Treatment with a pH 2 solution resulted in a gradual decrease of the malate content, indicating the operation of biochemical pH regulation mechanism. The pH 2 treatment also caused a sudden decrease of the intracellular level of Pi. The cellular content of total phosphorus did not change during the acidification. The Pi was converted to the organic phosphate form. The ATP level was not increased by the pH 2 treatment, but slightly decreased. The role of Pi, which might be functioning as a regulatory factor of cytoplasmic pH, a non-competitive inhibitor of the H+-pumps of both the plasma membrane and tonoplast is discussed.

Cloning, sequencing, and expression of the gene encoding the Clostridium stercorarium xylanase C in Escherichia coli.

The nucleotide sequence of the Clostridium stercorarium F-9 xynC gene, encoding a xylanase XynC, consists of 3,093 bp and encodes a 1,031-amino acids with a molecular weight of 115,322. XynC is a multidomain enzyme composed of an N-terminal signal peptide and six domains in the following order: two thermostabilizing domains, a family 10 xylanase domain, a family IX cellulose-binding domain, and two S-layer homologous domains. Immunological analysis indicated the presence of XynC in the culture supernatant of C. stercorarium F-9 and in the cells, most likely on the cell surface. XynC purified from a recombinant E. coli was highly active toward xylan and slightly active toward p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-cellobioside, p-nitrophenyl-beta-D-glucopyranoside, and carboxymethylcellulose. XynC hydrolyzed xylan and xylooligosaccharides larger than xylotriose to produce xylose and xylobiose. This enzyme was optimally active at 85 degrees C and was stable up to 75 degrees C at pH 5.0 and over the pH range of 4 to 7 at 25 degrees C.

Antimutagenic activity of isoflavones from soybean seeds (Glycine max merrill).

Two isoflavones, daidzein (1) and genistein (2), were isolated from soybean hypocotyls. Daidzein and genistein showed a suppressive effect on umu gene expression of the SOS response in Salmonellatyphimurium TA1535/pSK1002 against the mutagen 3-amino-1, 4-dimethyl-5H-pyrido[4,3b]indole (Trp-P-1), which requires liver metabolizing enzymes. Compound 1 suppressed 73% of the SOS-inducing activity at concentrations <0.74 micromol/mL, and the ID(50) value was 0.37 micromol/mL. Compound 2 suppressed 95% of the SOS-inducing activity at concentrations <0.74 micromol/mL, and the ID(50) value was 0.17 micromol/mL. Compounds 1 and 2 were also assayed with the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) and activated Trp-P-1. In addition to the antimutagenic activities of daidzein and genistein against Trp-P-1, frylfuramide and activated Trp-P-1 were assayed by an Ames test using S. typhimurium TA100.

Pharmacological properties of TRK-820 on cloned mu-, delta- and kappa-opioid receptors and nociceptin receptor.

We analyzed the pharmacological properties of 17-cyclopropylmethyl-3,14beta-dihydroxy-4,5alpha-epoxy-6b eta-[N-methyl-trans-3-(3-furyl)acrylamido]morphinan hydrochloride (TRK-820) using Chinese hamster ovary (CHO) cells expressing cloned rat mu-, delta- and kappa-opioid receptors and human nociceptin receptor. TRK-820 showed high affinity for the kappa-opioid receptor, with a Ki value of 3.5 +/- 0.9 nM. In CHO cells expressing kappa-opioid receptors, TRK-820 inhibited forskolin-stimulated cAMP accumulation, and the maximal inhibitory effect was equivalent to that of (+)-(5alpha,7alpha,8beta)-N-methyl-N-[7-(1-pyrrolidiny l)-1-oxaspiro-(4,5)dec-8-yl]benzeneacetamide (U69,593), a full agonist of kappa-opioid receptor. In CHO cells expressing mu-opioid receptors, TRK-820 inhibited cAMP accumulation, but the maximal inhibitory effect was significantly smaller than that of [D-Ala2, N-MePhe4, Gly-ol5]enkephalin (DAMGO), a full agonist of mu-opioid receptor. In CHO cells expressing delta-opioid receptor, the inhibitory effect of TRK-820 on cAMP accumulation was very weak. Using site-directed mutagenesis, the high affinity of TRK-820 for the kappa-opioid receptor was revealed to require Glu297. TRK-820 bound to the nociceptin receptor with a Ki value of 380 +/- 50 nM. TRK-820 by itself had no effect on cAMP accumulation in CHO cells expressing nociceptin receptors, but significantly antagonized the nociceptin (10 nM)-mediated inhibition of cAMP accumulation at high concentrations. These results indicate that TRK-820 acts as a full agonist for the kappa-opioid receptor, a partial agonist for the mu-opioid receptor and a low-affinity antagonist for the nociceptin receptor.

Binding sites and binding properties of binary and ternary complexes of insulin-like growth factor-II (IGF-II), IGF-binding protein-3, and acid-labile subunit.

We have examined regions of rat IGF-binding protein-3 (IGFBP-3) important for complex formations using two kinds of deletion mutants, three kinds of chimera molecules between rat IGFBP-3 and rat IGFBP-2, and a synthetic peptide (41 residues, Glu52-Ala92) derived from rat IGFBP-3. Solid-phase binding assays using 96-well microtiter plates were designed to quantitate the relative binding affinities. It was found that not only the IGFBP-3 derivatives with the amino-terminal, cysteine-rich domain (N domain) but also the synthetic peptide maintained affinity for IGF-II. Ternary complex formation was observed with full-length IGFBP-3 and chimera IGFBP, the carboxyl-terminal cysteine-rich domain (C domain) of which was derived from IGFBP-3, unlike the mutants lacking the C domain and the chimera IGFBPs, the C domain of which was derived from IGFBP-2. These results were confirmed by affinity cross-linking experiments. Furthermore, the IGFBP-3 derivatives that possessed the C domain of IGFBP-3 bound to the acid-labile subunit, even in the absence of IGFs. Finally, we observed sites in IGF-II important for the ternary complex formation using various IGF-II mutants. These IGF-II mutants, which contained a substitution of Tyr27 for Leu, had extremely reduced activity. These results strongly suggest that: 1) the N domain, containing at least Glu52-Ala92, of rat IGFBP-3 is important for binding to IGF-II; 2) the C domain of IGFBP-3 is essential for binding to the acid-labile subunit both in the presence and absence of IGF-II; and 3) Tyr27 of IGF-II is important for the ternary complex formation.

A para-site-specific hydroxylation of aromatic compounds by Mycobacterium sp. strain 12523: stabilization of the hydroxylation activity.

Mycobacterium sp. strain 12523 has a para-site-specific hydroxylation activity, which produce para-substituted phenols from various aromatic compounds. However, the activity is unstable and the reactions are inactivated within 24 h. In order to extend the reaction period, the factors that affected reaction stability were examined. The hydroxylation activity of the cells incubated in buffer was significantly stabilized by the inclusion of an inducer such as methyl ethyl ketone. It is suggested that a regulatory mechanism is involved in controlling the activity. This study resulted in the development of a convenient method to stabilize the hydroxylation activity, involving the addition of an inducer, such as acetone, to the reaction system. This method permitted the hydroxylation reaction to continue for more than 67 h.

Efficient callus initiation from leaf of mangrove plant,Bruguiera sexangula in amino acid medium: Effect of NaCl on callus initiation.

Experimental conditions for efficient callus initiation from mangrove plants were investigated. As a source explant, leaf ofBruguiera sexangula was used. Mangrove plant is one of the most famous woody plants which can grow at the salty area. The initiated callus can be a suitable material for the investigation of salt tolerant mechanisms of mangrove plants.Leaf pieces cultured in an Amino Acid medium supplemented with 2 µM 2,4-dichlorophenoxyacetic acid and 2 µMN-(2-chloro-4-pyridyl)-N'-phenylurea at 30 C developed calluses. Microscopic observation suggested that the callus was initiated from the tissue in the vascular bundles in the leaf.We also examined the effect of NaCl on callus initiation and short-term culture of the calluses on the leaves. Callus initiation rate decreased with increasing NaCl concentration higher than 100 mM in the culture media. The medium containing 100 mM NaCl produced the largest callus on the leaf, compared with higher or lower concentrations of NaCl.

An anti-HIV peptide, T22, forms a highly active complex with Zn(II).

T22 ([Tyr5,12, Lys7]-polyphemusin II) has been shown to have strong anti-human immunodeficiency virus (HIV) activity, comparable to that of 3'-azide-2', 3'-dideoxythymidine (AZT). T22 takes an antiparallel beta-sheet structure maintained by two disulfide bridges and contains two antiparallel repeats of Cys-Tyr-Arg-Lys-Cys. As reported herein, fully reduced T22 was found by HPLC and ion spray mass spectrometric analyses to form a complex in a molar ratio of 1:1 with Zn(II) ion at neutral pH in aqueous solution. Complexation of Zn(II) ion to this peptide appears to result in tetracoordinate bonding to sulfur atoms of four Cys residues. We also found that the anti-HIV activity of the T22-Zn(II) complex was fourfold stronger than that of T22.

Vacuolar function in the phosphate homeostasis of the yeast Saccharomyces cerevisiae.

We studied physiological roles of the yeast vacuole in the phosphate metabolism using 31P-in vivo nuclear magnetic resonance (NMR) spectroscopy. Under phosphate starvation wild-type yeast cells continued to grow for two to three generations, implying that wild-type cells contain large phosphate pool to sustain the growth. During the first four hours under the phosphate starved condition, the cytosolic phosphate level was maintained almost constant, while the vacuolar pool of phosphate decreased significantly. 31P-NMR spectroscopy on the intact cells and perchloric acid (PCA) extracts showed that drastic decrease of polyphosphate took place during this phase. In contrast, delta slp1 cells, which were defective in the vacuolar compartment, thus lacked polyphosphate, ceased their growth immediately when they faced to phosphate starvation. Taken together, we conclude that vacuolar polyphosphate provides an active pool for phosphate and is mobilized to cytosol during phosphate starvation and sustained cell growth for a couple rounds of cell cycle.

Comparative effects of insulin-like growth factor II (IGF-II) and IGF-II mutants specific for IGF-II/CIM6-P or IGF-I receptors on in vitro hematopoiesis.

This report presents the results of studies investigating the effect of insulin-like growth factor II (IGF-II) on the proliferation and differentiation of CD34+ bone marrow cells in serum-substituted liquid cultures. Bone marrow cells were enriched for CD34+ cells and then placed in liquid cultures supplemented with either interleukin 3 (IL-3) or IL-3 and c-kit ligand with and without the addition of IGF-II. When CD34+ cells were incubated with IL-3, cellularity increased throughout four weeks of culture. Cellularity was twofold greater when cultures also contained IGF-II. IGF-II also promoted an increase in cellularity in cultures with IL-3 and c-kit ligand. In combination with IL-3 or IL-3 and c-kit ligand, IGF-II promoted an earlier differentiation of granulocytes, as well as an increase in the number of megakaryocyte lineage cells. There were approximately two-fold more colony-forming units for granulocytes and macrophages (CFU-GM) and burst-forming units for erythroid cells (BFU-E) in cultures containing both IL-3 and IGF-II than in cultures with IL-3 alone. These results demonstrate that in cytokine-supplemented media, physiological concentrations of IGF-II augmented both the proliferation and differentiation of CD34+ bone marrow cells while maintaining a greater number of progenitor cells. To identify the receptors through which IGF-II enhances in vitro hematopoiesis, IGF-II was substituted with one of the mutant forms of IGF-II that selectively interacts with either IGF-II/CIM6-P receptors or with IGF-I and insulin receptors. The results with the mutant forms of IGF-II demonstrate that IGF-II augments in vitro hematopoiesis primarily through its interaction with IGF-I and possibly insulin receptors, rather than IGF-II/CIM6-P receptors.

N-terminal deletion mutants of insulin-like growth factor-II (IGF-II) show Thr7 and Leu8 important for binding to insulin and IGF-I receptors and Leu8 critical for all IGF-II functions.

To define the role of the N-terminal region of insulin-like growth factor-II (IGF-II) in its binding to insulin and IGF receptors, deletion mutants des-(1-5)-, des-(1-7)-, and des-(1-8)-recombinant (r) IGF-II, and the Gly8 for Leu substitution mutant of rIGF-II were prepared by site-directed mutagenesis, expressed in Escherichia coli, and purified. The binding affinity and mitogenic activity of these rIGF-II mutants as well as commercially available des-(1-6)-rIGF-II were analyzed. While the relative affinity of des-(1-5)- and des-(1-6)-rIGF-II for purified human insulin and IGF-I receptors remained at > or = 50% levels of that of rIGF-II, the affinity of des-(1-7)-rIGF-II decreased to approximately 10% and approximately 3%, respectively, of that of rIGF-II. When the octapeptide including Leu8 was removed prior to the Cys9-Cys47 intrachain bond, the relative affinity of this deletion mutant, des-(1-8)-rIGF-II, for these receptors dramatically decreased to < 1% of that of rIGF-II. Substituting Gly8 for Leu in rIGF-II decreased the affinity of this mutant for the IGF-I and insulin receptors to about the same extent. These results suggest that the side chains of Thr7 and Leu8 may play an important role in retaining all of the IGF-II functions. Decreases in the relative affinity for binding of the mutants to these receptors paralleled the decreases in their mitogenic potency for cultured Balb/c 3T3 cells. Although the relative affinity of des-(1-8)- or [Gly8]rIGF-II for rat IGF-II/CIM6-P (cation-independent mannose 6-phosphate) receptors was also < 1% of that of rIGF-II, the relative affinities of des-(1-5)-, des-(1-6)-, and des-(1-7)-rIGF-II for these receptors was significantly greater than that of rIGF-II. These results clearly demonstrate that Thr7 and Leu8 are important for binding to insulin and IGF-I receptors and Leu8 is critical for expression of all IGF-II functions.

Lack of Control in Inorganic Phosphate Uptake by Catharanthus roseus (L.) G. Don Cells (Cytoplasmic Inorganic Phosphate Homeostasis Depends on the Tonoplast Inorganic Phosphate Transport System?).

Inorganic phosphate (Pi) uptake by Catharanthus roseus (L.) G. Don cells was studied in relation to its apparent uncontrolled uptake using 31P-nuclear magnetic resonance spectroscopy. Kinetics of Pi uptake by the cells indicated that apparent Km and Vm were about 7 [mu]M and 20 [mu]mol g-1 fresh weight h-1, respectively. Pi uptake in Murashige-Skoog medium under different Pi concentrations and different initial cell densities followed basically the same kinetics. When supplied with abundant Pi, cells absorbed Pi at a constant rate (Vm) for the first hours and accumulated it in the vacuole. As the endogenous pool expanded, the rate of Pi uptake gradually decreased to nil. Maximum Pi accumulation was 100 to 120 [mu]mol g-1 fresh weight if cell swelling during Pi uptake (about 2-fold in cell volume) was not considered. Results indicated that (a) the rate of Pi uptake by Catharanthus cells was independent of initial cell density and was constant over a wide range of Pi concentrations (2 mM to about 10 [mu]M) unless the cells were preloaded with excess Pi, and (b) there was no apparent feedback control over the Pi uptake process in the plasma membrane to avoid Pi toxicity. The importance of the tonoplast Pi transport system in cytoplasmic Pi homeostasis is discussed.

Solution structure of human insulin-like growth factor II; recognition sites for receptors and binding proteins.

The three-dimensional structure of human insulin-like growth factor II was determined at high resolution in aqueous solution by NMR and simulated annealing based calculations. The structure is quite similar to those of insulin and insulin-like growth factor I, which consists of an alpha-helix followed by a turn and a strand in the B-region and two antiparallel alpha-helices in the A-region. However, the regions of Ala1-Glu6, Pro31-Arg40 and Thr62-Glu67 are not well-defined for lack of distance constraints, possibly due to motional flexibility. Based on the resultant structure and the results of structure-activity relationships, we propose the interaction sites of insulin-like growth factor II with the type 2 insulin-like growth factor receptor and the insulin-like growth factor binding proteins. These sites partially overlap with each other at the opposite side of the putative binding surface to the insulin receptor and the type 1 insulin-like growth factor receptor. We also discuss the interaction modes of insulin-like growth factor II with the insulin receptor and the type 1 insulin-like growth factor receptor.

Stimulation of glucose uptake by insulin-like growth factor II in human muscle is not mediated by the insulin-like growth factor II/mannose 6-phosphate receptor.

Although the growth-promoting effects of insulin-like growth factor II (IGF-II) have been intensively studied, the acute actions of this hormone on glucose metabolism have been less well evaluated, especially in skeletal muscle of humans. We and other groups have shown that IGFs reduce glycaemic levels in humans and stimulate glucose uptake in rat muscle. The purpose of the present study was to evaluate the effect of IGF-II on glucose transport in muscle of normal and obese patients with and without non-insulin-dependent diabetes mellitus (NIDDM), as well as to identify the receptor responsible for this action. 2-Deoxyglucose transport was determined in vitro using a muscle-fibre strip preparation. IGF-II were investigated in biopsy material of rectus abdominus muscle taken from lean and obese patients and obese patients with NIDDM at the time of surgery. In the lean group, IGF-II (100 nM) stimulated glucose transport 2.1-fold, which was slightly less than stimulation by insulin (2.8-fold) at the same concentration. Binding of IGF-II was approx. 25% of that of insulin at 1 nM concentrations of both hormones. Obesity with or without NIDDM significantly reduced IGF-II-stimulated glucose uptake compared with the lean group. In order to explore which receptor mediated the IGF-II effect, we compared glucose uptake induced by IGF-II and two IGF-II analogues: [Leu27]IGF-II, with high affinity for the IGF-II/Man 6-P receptor but markedly reduced affinity for the IGF-I and insulin receptors, and [Arg54,Arg55]IGF-II was similar to that of IGF-II, whereas [Leu27]IGF-II had a very diminished effect. Results show that IGF-II is capable of stimulating muscle glucose uptake in lean but not in obese subjects and this effect seems not to be mediated via an IGF-II/Man 6-P receptor.