An NDPase links ADAM protease glycosylation with organ morphogenesis in C. elegans

In the nematode Caenorhabditis elegans, the gonad acquires two U-shaped arms through the directed migration of its distal tip cells (DTCs), which are located at the tip of the growing gonad arms. A member of the ADAM (a disintegrin and metalloprotease) family, MIG-17, regulates directional migration of DTCs: MIG-17 is synthesized and secreted from the muscle cells of the body wall, and diffuses to the gonad where it is required for DTC migration. The mig-23 mutation causes defective migration of DTCs and interacts genetically with mig-17. Here, we report that mig-23 encodes a membrane-bound nucleoside diphosphatase (NDPase) required for glycosylation and proper localization of MIG-17. Our findings indicate that an NDPase affects organ morphogenesis through glycosylation of the MIG-17 ADAM protease.     

Properties of chlorogenic acid quinone: relationship between browning and the formation of hydrogen peroxide from a quinone solution

Chlorogenic acid is the major polyphenol in foods derived from plants and is a good substrate for polyphenol oxidase. Chlorogenic acid quinone (CQA-Q), which is an oxidative product of chlorogenic acid by polyphenol oxidase, is an important intermediate compound in enzymatic browning. CQA-Q was prepared, and its properties and the relationship with browning were examined. The quinone solution was yellow or orange, and its molecular absorption coefficient was estimated to be 1.7 x 10(3) for 325 nm and 9.7 x 10(2) for 400 nm in an acidic aqueous solution. Chlorogenic acid and H2O2 were spontaneously generated in the CQA-Q solution as the yellowish color of the solution gradually faded. A pale colored polymer was the major product in the reaction solution. Amino acids such as lysine and arginine added to CQA-Q solution did not repress the fading of the yellowish color of the solution. We concluded from these results that CQA-Q itself and a mixture of CQA-Q and amino acids did not form intensive brown pigments in the acidic aqueous solution. H2O2 spontaneously formed in the CQA-Q solution, and other polyphenols might have played an important role in the formation of the brown color by enzymatic browning.     

Design of a highly potent anti-hypertensive peptide based on ovokinin(2-7)

Ovokinin(2-7) (RADHPF), an orally active antihypertensive peptide derived from ovalbumin, lowers blood pressure in SHRs at a dose of 10 mg/kg. Attempts were made to potentiate its anti-hypertensive activity by replacing the amino acid residues in [Pro2, Phe3]-ovokinin(2-7), which was previously reported to have 33-fold stronger activity than ovokinin(2-7). The anti-hypertensive activity of [Pro2, Phe3]-ovokinin(2-7) was improved by replacement of the C-terminal Phe residue with Trp. Then, the best amino acid residues at other positions for the anti-hypertensive effect were selected. RPLKPW, the most potent derivative obtained, showed significant anti-hypertensive activities at a dose of 0.1 mg/kg after oral administration in spontaneously hypertensive rats (SHRs). Thus, RPLKPW showed 100-fold more potent anti-hypertensive activity than ovokinin(2-7).     

Changes in activity coefficient gamma(w) of water and the foaming capacity of protein during hydrolysis

The changes in the interaction between food proteins and water and in their surface functional property during enzymatic hydrolysis were investigated. Ovalbumin, a soy protein isolate (SPI), and casein were hydrolyzed with trypsin, and the degree of hydrolysis, water activity a(w), and foaming capacity of each hydrolysate were measured. Ovalbumin showed the minimum value for a(w), and the values for SPI and casein progressively decreased during hydrolysis. Therefore, the activity coefficient of water, gamma(w) (=a(w)/x(w), where x(w) is the mole fraction of water) was obtained to remove the influence of mole change and to examine the interaction of protein hydrolysates with water. In order to calculate x(w) in a sample during protein hydrolysis, a method for roughly estimating the number of moles of the protein hydrolysate in a solution was developed. The strategy was to modify the TNBS (2,4,6-trinitrobenzenesulfonic acid) method and to combine this method with the modified Ellman method and the determination of lysine by an amino acid analyzer. During enzymatic hydrolysis, each protein sample showed a minimum gamma(w) value and maximum foaming capacity.     

Apoptosis induction by lectin isolated from the mushroom Boletopsis leucomelas in U937 cells

A 15-kDa lectin was isolated from the edible mushroom Kurokawa by affinity chromatography using N,N'-diacetylchitobiose-Sepharose 4B. The results of microsequencing analysis indicated that the lectin has a partial amino acid sequence similar to the mushroom lectin, Agaricus bisporus agglutinin (ABA). We found that the Kurokawa lectin inhibited proliferation of human monoblastic leukemia U937 cells dose-dependently. Several lines of evidence indicated that this inhibition was due to its apoptosis induction. We observed that the lectin induced apoptotic bodies formation, chromatin condensation, and DNA ladder formation, features of apoptosis. The DNA ladder formation was inhibited by a general inhibitor of caspases, which are known to play essential roles in apoptosis. In contrast, ABA did not have cell growth-inhibiting or apoptosis-inducing activities. Thus, the Kurokawa lectin is the first mushroom lectin with apoptosis-inducing activity.     

Stretch-activated cation channels in skeletal muscle myotubes from sarcoglycan-deficient hamsters

Deficiency of -sarcoglycan(-SG), a component of the dystrophin-glycoprotein complex, causescardiomyopathy and skeletal muscle dystrophy in Bio14.6 hamsters. Usingcultured myotubes prepared from skeletal muscle of normal and Bio14.6hamsters (J2N-k strain), we investigated the possibility that the-SG deficiency may lead to alterations in ionic conductances, whichmay ultimately lead to myocyte damage. In cell-attached patches (withBa²⁺ as the charge carrier), an ~20-pS channel wasobserved in both control and Bio14.6 myotubes. This channel is alsopermeable to K⁺ and Na⁺ but not toCl. Channel activity was increased by pressure-inducedstretch and was reduced by GdCl₃ (>5 µM). The basal openprobability of this channel was fourfold higher in Bio14.6 myotubes,with longer open and shorter closed times. This was mimicked bydepolymerization of the actin cytoskeleton. In intact Bio14.6 myotubes,the unidirectional basal Ca²⁺ influx was enhanced comparedwith control. This Ca²⁺ influx was sensitive toGdCl₃, signifying that stretch-activated cation channelsmay have been responsible for Ca²⁺ influx in Bio14.6hamster myotubes. These results suggest a possible mechanism by whichcell damage might occur in this animal model of muscular dystrophy.

     

Ascorbate-induced high-affinity binding of copper to cytosolic proteins

Copper chaperones are necessary for intracellular trafficking of copper to target proteins. This is probably because the milieu inside the cell has a large capacity for sequestering this metal. By fluorometry using a fluorescent Cu(II) chelator and by centrifugal ultrafiltration, we have studied copper binding of the whole cytosolic proteins from mouse brain and liver, and found that their binding capacity and affinity for copper were markedly increased by ascorbate. Brain cytosolic protein bound, with high affinity, 63 nmol of copper/mg, more than half of which was redox-inactive, as indicated by its inability to catalyze oxidation of ascorbate. Most of the bound copper was in the Cu(I) state, coordinating to thiol groups of protein. Cytosolic protein competed for copper more strongly than GSH when compared at their relative concentrations in tissues. The results taken together suggest that protein thiols of cytosol can strongly sequester copper.     

Weight Change and Risk of Developing Type 2 Diabetes

Objective: To examine the association between weight change and risk of type 2 diabetes and whether initial weight modifies the association. Research Methods and Procedures: This is a prospective cohort study of 20, 187 alumni from Harvard University and the University of Pennsylvania. At baseline in 1962 or 1966, men (mean age, 45.9 years) reported their weight, height, and other risk factors. They also had had their weight and height measured at university entry (mean age, 18.5 years). Participants were followed from baseline to 1998 for type 2 diabetes. Results: During follow‐up, 1223 men developed type 2 diabetes. Weight gain significantly increased the risk of this disease. The multivariate relative risks associated with BMI change from university entry to baseline of <?0.5, ±0.5, >0.5 to 1.0, >1.0 to 1.5, >1.5 to 2.0, >2.0 to 3.0, and >3.0 kg/m² per decade were 0.88, 1.00 (referent), 1.29, 2.09, 2.69, 4.67, and 7.02, respectively (p for trend < 0.0001). Even among men with a low initial BMI < 21 kg/m², weight gain significantly increased risk; the corresponding relative risks were (no cases), 1.00 (referent), 1.00, 1.93, 2.47, 4.82, and 7.68, respectively (p for trend < 0.0001). Discussion: A low initial BMI does not ameliorate the increase in risk of type 2 diabetes with weight gain. Avoidance of weight gain, even among lean individuals, is important to reduce the risk of this disease.