N-ethylmaleimide inhibits Ca2+ influx induced by collagen or arachidonate on rabbit platelets

N-Ethylmaleimide dose dependently inhibited platelet aggregation induced by collagen or arachidonate but did not inhibit the aggregation by thrombin or ionophore A23187 within the concentrations tested. [3H]Arachidonate release from membrane phospholipids of the collagen-stimulated platelets was inhibited by N-ethylmaleimide in parallel with the inhibition of aggregation, but not in response to A23187. N-Ethylmaleimide prevented 45Ca2+ influx into platelet cells from outer medium induced by collagen, and also inhibited the increase in the concentration of cytoplasmic free Ca2+, which probably results from Ca2+ influx, as monitored by quin2 fluorescence, under stimulation with arachidonate. The concentration of N-ethylmaleimide giving a complete inhibition of Ca2+ influx was consistent with that required to inhibit collagen- or arachidonate-induced aggregation. Prostaglandin metabolism from arachidonate to thromboxane A2 was not disturbed by N-ethylmaleimide, while phosphatidate formation induced by arachidonate was slightly inhibited by it at concentrations at which aggregation was completely inhibited. These data suggest that N-ethylmaleimide preferentially suppresses increase in cytoplasmic free Ca2+ which is linked to thromboxane A2-receptor occupation in collagen- or arachidonate-stimulated platelets, probably due to blockage of Ca2+ influx through Ca2+-channel protein, thereby inhibiting aggregation induced by these agonists.     

A case of von Recklinghausen's disease associated with pheochromocytoma and papillary carcinoma of the thyroid gland

A 58-year-old woman was admitted to our hospital complaining of headache, dizziness and intermittent elevation of blood pressure. Multiple café-au-lait spots and neurofibromas had appeared on the back and the limbs since the age of 30 years. At the age of 54 years she underwent total thyroidectomy because of papillary carcinoma of the thyroid gland. On admission, the levels of plasma norepinephrine and epinephrine, urinary norepinephrine and normetanephrine were all within the normal range. However, urinary excretion of metanephrine was markedly increased to 1.49 +/- 0.45 (Mean +/- SD) mg/day and that of epinephrine was also slightly increased. The computed tomographic scans of the abdomen and the scintigraphy with 131I-metaiodobenzylguanidine revealed a tumor mass in the region of the right adrenal gland. The tumor was histologically confirmed to be pheochromocytoma at the operation. In her family history, her mother and one of her two sisters had von Recklinghausen's disease and another sister suffered from follicular carcinoma of the thyroid gland. As far as we know, this paper is the first report of a patient with von Recklinghausen's disease associated with both pheochromocytoma and non-medullary carcinoma of the thyroid gland, and her family.     

Cholecystokinin-stimulated pepsinogen secretion and cholecystokinin receptors on gastric chief cells in guinea pigs

We investigated cholecystokinin (CCK) receptors on isolated gastric chief cells from guinea pig. CCK stimulated pepsinogen secretion from chief cells at the same efficacy as that induced by carbamylcholine. Binding of 125I-labeled CCK-33 (125I-CCK) to chief cells was temperature-dependent, and was saturable and reversible at 37 degrees C. Hofstee plots of the ability of CCK-8 to inhibit binding of 125I-CCK showed a linear regression line, suggesting that CCK receptors possessed one binding site. The dissociation constant of the binding site was calculated to be 3.8 x 10(-10) M. The dose-response curve of CCK for pepsinogen secretion was superimposed on that for the binding to its receptors. These results indicated that gastric chief cells from the guinea pig possess CCK receptors that relate closely to the action of CCK involved in pepsinogen secretion.     

The Relationship between Dipeptidase Activity Variation and Larval Viability in Drosophila melanogaster

The enzyme dipeptidase-A (DIP-A) in Drosophila melanogaster is coded by a second chromosome locus that is polymorphic for three allozymes in natural populations. DIP-A appears to be the only enzyme in D. melanogaster capable of hydrolyzing the dipeptide glycyl-L-isoleucine, since flies homozygous for null alleles at this locus have no detectable glycyl-L-isoleucine-ase activity. DIP-A activity occurs in many tissues and throughout development, but is particularly high in the larval midgut, suggesting an important role in protein digestion. These observations suggested an experimental design for investigating the adaptive significance of genetic variation in DIP-A activity. Fitness components of DIP-A variants could be estimated and compared under two environmental conditions (defined diets under axenic conditions). In the restrictive environment, the essential amino acid L-isoleucine is provided only in the form of glycyl-L-isoleucine, whereas in the permissive environment, L-isoleucine is provided in free form. We predicted that DIP-A activity would be essential in the restrictive, but not in the permissive environment. The results reported here clearly contradict this prediction. Two stocks homozygous for DIP-A null alleles from different geographic locations are each viable on the restrictive diet. Furthermore, relative viability experiments in which null allele larvae compete with larvae having DIP-A activity provide no evidence for even a partial reduction in egg to adult survival on the restrictive diet. Apparently, the null allele larvae have some alternative mechanism for obtaining L-isoleucine from the dipeptide, even though no glycyl-L-isoleucine-ase activity can be detected in vitro. These results, along with the viability of null alleles for many other enzymes, support the idea that eukaryotes have an intricate network of alternative biochemical pathways through which the same necessary function may be achieved. Such "buffering capacity" makes it very difficult to analyze the effects of enzyme variants on fitness components.     

Homoeosis in Drosophila: The Lethal Syndrome of the Regulator of bithorax (or trithorax) Locus and Its Interaction with Other Homoeotic Loci

Regulator of bithorax (Rg-bx)^- [or trithorax (trx)^-] lethal zygotes show anterior transformations of various cuticular features of the larval thorax and abdomen. The Rg-bx^- lethal syndrome depends on the dosage of the bithorax gene complex (BX-C), and lack of Rg-bx⁺ function is antagonistic to posterior transformations displayed by Polycomb ( Pc)^- embryos. Significantly, when the BX-C is deleted, the Rg-bx^- embryos disclose homoeosis of mesothoracic to prothoracic cuticular structures. This homoeotic transformation is due to a reduction in Antennapedia (Antp)⁺ gene activity and is consequently dependent on the dosage of the Antennapedia gene complex (ANT-C), suggesting that the Rg-bx⁺ activity is necessary for proper expression of the Antp⁺ gene. However, the functional relationship between the Rg-bx and Sex combs reduced (Scr) loci in embryogenesis is still to be established.     

Effect of roasting on ochratoxin A level in green coffee beans inoculated with Aspergillus ochraceus

The heat stability of ochratoxin A in green coffee beans inoculated with Aspergillus ochraceus was studied. Heat treatment (roasting) at 200 °C for 10 or 20 min reduced the levels of ochratoxin A by only 0–12% in the dried whole beans. Almost all of the ochratoxin A was infused into the coffee decoction when the roasted samples were ground and extracted with boiling water. Therefore, the reduction of ochratoxin A concentration of contaminated coffee beans by roasting under these conditions is ineffective.     

Photosynthetic Characteristics of Photoautotrophically Cultured Cells of Tobacco

The photosynthetic characteristics of photoautotrophically culturedcells of tobacco (Nicotiana tabacum cv. Samsun NN) as well asthose of photomixotrophically cultured cells and green leaveswere investigated. Analyses revealed that on a fresh weightbasis cultured tobacco cells had lower chlorophyll contentsthan cells of green leaves. The chlorophyll content per chloro-plast,however, was almost the same in both types of cell, and thechloroplast number per cell accounted for only small differencesin the cellular chlorophyll content. This indicates that thelarger cell volume of cultured cells is the main factor in thedifference in the chlorophyll content of these cells. Photosynthetic activity measurements also showed differencesin the chloroplasts of cultured and leaf cells. The maximumactivities of photosystem I and the Hill reaction for the culturedcells were about half those for leaf cells on a per unit chlorophyllbasis. Moreover, photo-autotrophic cells had relatively constantphotosystem I and Hill reaction activities during growth; whereas,on a fresh weight basis these activities in leaf cells reflecteddevelopmental changes in the chlorophyll content. Lithium dodecyl sulfate-polyacrylamide gel electrophoresis showedqualitatively similar thylakoid polypeptide compositions forcultured and leaf cells at all stages of growth even thoughthere were quantitative decreases in the contents of severalpolypeptides in the cultured green cells (especially in photomixotrophiccells) in comparison to the polypeptide contents of tobaccoleaves. We speculate that the lower photosynthetic activityof the cultured cells may be caused by this reduction in thecontents of certain thylakoid polypeptides. (Received November 14, 1988; Accepted June 19, 1989)     

Polymorphism of T-cell receptor genes among laboratory and wild mice: Diverse origins of laboratory mice

Southern blots of genomic DNA from 23 strains of laboratory mice and 19 individual wild mice were examined for restriction fragment length polymorphisms in their loci encoding the T-cell receptors (Tcr): the constant regions of the α, β, and γ chains (C _α,C _β, andC _γ) and a variable region family of the β chain (V _β8). Only a few polymorphisms were observed for each locus in the laboratory mice after using three restriction enzymes,Bam HI,Eco RI, andHind III. All the laboratory mice examined fall into one of two types for theC _α,C _β andV _β8 loci and one of three types for theC _γ. These types are found in some of the wild mice studied, indicating that they were already present in the founder mice of laboratory mouse strains. In contrast, theTcr genes are highly polymorphic among wild mice. Analysis of the polymorphisms in these loci suggests that laboratory mice have inherited their genes not only fromMus musculus domesticus, but also from other subspecies, and much more than previously believed from Asian subspecies.     

Conformational change of bovine serum albumin by heat treatment

The thermal denaturation of bovine serum albumin (BSA) was studied at pH 2.8 and 7.0 in the range of 2–65°C. The relative proportions of -helix, -structure, and disordered structure in the protein conformation were determined as a function of temperature, by the curve-fitting method of circular dichroism spectra. With the rise of temperature at pH 7.0, the proportion of -helix decreased above 30°C and those of -structure and disordered structure increased in the same temperature range. The structural change was reversible in the temperature range below 45°C. However, the structural change was partially reversible upon cooling to room temperature subsequent to heating at 65°C. On the other hand, the structural change of BSA at pH 2.3 was completely reversible in the temperature range of 2–65°C, probably because the interactions between domains and between subdomains might disappear due to the acid expansion. The secondary structure of disulfide bridges-cleaved BSA remained unchanged during the heat treatment up to 65°C at pH 2.8 and 7.0.