The role of cysteine proteinase and carboxypeptidase in the breakdown of storage proteins in buckwheat seeds

Two proteolytic enzymes, a cysteine proteinase and a carboxypeptidase, responsible for breakdown of the main storage protein, 13S globulin, were purified from buckwheat seedlings (Fagopyrum esculentum Moench) by (NH₄)₂SO₄ fractionation, gel-filtration on Sephadex G-150, ionexchange chromatography on DEAE-Toyopearl 650 M and chromatofocusing. The cysteine proteinase was purified 74-fold. It has a pH optimum of 5.5, a pI of 4.5 and an apparent molecular mass (M_r) of 71000. The carboxypeptidase was purified 128-fold. It has a pH optimum of 5.3, a pI of 5.8 and a M_r of 78500. Cysteine proteinase hydrolyzed the modified 13S globulin only if the reaction products were eliminated from the incubation mixture by dialysis. Storage protein degradation by the proteinase increased in the presence of carboxypeptidase. We suggest that the two enzymes complete the digestion of 13S globulin after its preliminary hydrolysis by the earlier described enzyme, metalloproteinase, present in dry buckwheat seeds.Abbreviations BSA bovine serum albumin - DEAE diethylaminoethyl - M_r apparent molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

In vivo apoprotein catabolism of high density lipoproteins in copper-deficient, hypercholesterolemic rats

High density lipoprotein (HDL) apoprotein catabolism was examined in male Sprague-Dawley rats deficient in dietary copper. Twenty-four rats were randomly divided into two groups: copper-adequate (control, 5 mg of copper/kg diet) and copper-deficient (0.6 mg of copper/kg diet). After 5 weeks, animals were administered a tracer dose of iodinated HDL protein previously isolated from donor rats that were subjected to the same dietary treatments as the test animals. Copper-deficient rats exhibited a 54% increase in plasma volume and a 26% increase in HDL protein concentration above controls. Consequently, the intravascular pool of total HDL protein was increased 2-fold. The fractional catabolic rate of total HDL protein was similar between groups. However, because of the increased intravascular HDL pool in copper-deficient animals, the absolute catabolic rate was greater (640 +/- 49 micrograms/hr vs 316 +/- 12 micrograms/hr in controls). Tissue uptake of total HDL protein in copper-deficient rats tended to be greater in the kidneys, spleen, and testes compared with controls; the heart exhibited a significant 2.3-fold increase. In contrast, the catabolic rate of HDL protein in the liver and adrenal gland were not different between treatment groups. That an obligatory increase in HDL protein uptake was not observed in the liver and adrenal gland (organs which are sensitive to and can further metabolize cholesterol) suggests that these organs may be regulated, possibly contributing to the observed hypercholesterolemia in this model. These data imply that total HDL apoprotein catabolism is increased in response to the increased intravascular pool of HDL in copper-deficient rats.     

54Mn absorption and excretion in rats fed soy protein and casein diets

Rats were fed diets containing either soy protein or casein and different levels of manganese, methionine, phytic acid, or arginine for 7 days and then fed test meals labeled with 2 microCi of 54Mn after an overnight fast. Retention of 54Mn in each rat was measured every other day for 21 days using a whole-body counter. Liver manganese was higher (P less than 0.0001) in soy protein-fed rats (8.8 micrograms/g) than in casein-fed rats (5.2 micrograms/g); manganese superoxide dismutase activity also was higher in soy protein-fed rats than in casein-fed rats (P less than 0.01). There was a significant interaction between manganese and protein which affected manganese absorption and biologic half-life of 54Mn. In a second experiment, rats fed soy protein-test meals retained more 54Mn (P less than 0.001) than casein-fed rats. Liver manganese (8.3 micrograms/g) in the soy protein group was also higher than that (5.7 micrograms/g) in the casein group (P less than 0.0001), but manganese superoxide dismutase activity was unaffected by protein. Supplementation with methionine increased 54Mn retention from both soy and casein diets (P less than 0.06); activity of manganese superoxide dismutase increased (P less than 0.05) but liver manganese did not change. The addition of arginine to casein diets had little effect on manganese bioavailability. Phytic acid affected neither manganese absorption nor biologic half-life in two experiments, but it depressed liver manganese in one experiment. These results suggest that neither arginine nor phytic acid was the component in soy protein which made manganese more available from soy protein diets than casein diets.     

Brefeldin A inhibits oligosaccharide processing of glycoproteins in mouse hypothyroid pituitary tissue at several subcellular sites

We have studied the effects of brefeldin A (BFA) and monensin on the processing of the oligosaccharides of thyrotropin (TSH), free alpha-subunits, and cellular glycoproteins of mouse pituitary tissue to clarify the subcellular sites of action of BFA. BFA was previously shown to inhibit the translocation of glycoproteins from the rough endoplasmic reticulum to the Golgi apparatus but action at other sites was possible. Pituitaries from hypothyroid mice were incubated with [35S]methionine, [3H]mannose, [3H]galactose, [3H]fucose, N-[3H]acetylmannosamine, or [35S]sulfate for 2 hr in the absence or presence of 5 micrograms of BFA/ml or 2 microM monensin. TSH and free alpha-subunits were immunoprecipitated from tissue lysates and analyzed by sodium dodecyl sulfate-gel electrophoresis. The tryptic glycopeptides of TSH were separated using high-performance liquid chromatography. Total glycoproteins in cell lysates were precipitated using trichloroacetic acid. Labeled oligosaccharides were released from the tryptic glycopeptides of TSH and cellular glycoproteins by endoglycosidase H and they were analyzed by paper chromatography. Compared with control incubations, BFA caused the intracellular accumulation of glycoproteins having less than expected amounts of Man9GlcNAc2 units, but with excess Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, and Man5GlcNAc2 units. There was a lesser accumulation of glucose-containing oligosaccharides, especially Glc1Man9GlcNAc2. Monensin also caused the accumulation of certain high mannose species, but the pattern differed from that seen for BFA, since Man9GlcNAc2 units were preserved and there was less excess of Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, and Man5GlcNAc2 units. BFA did not block the initial attachment of oligosaccharides at any of the three Asn-glycosylation sites of TSH, but caused the accumulation of Man5-8GlcNAc2 units at each site. Both monensin and BFA inhibited fucosylation, sulfation, and sialylation more markedly than mannose incorporation. Thus, in addition to its previously described action of inhibiting rough endoplasmic reticulum to Golgi transport, BFA appears to partially inhibit the glucose-trimming enzymes as well as some Golgi enzymes.     

Isolation and characterization of streptomycin-resistant mutants in Nicotiana plumbaginifolia

Summary Streptomycin-resistant colonies were isolated from protoplast cultures of haploid Nicotiana plumbaginifolia based on their ability to green in medium containing 1 mg/ml streptomycin sulfate. The frequency of resistant colonies was 0.9×10^–5 in nonmutagenized culture, and increased ten-fold following treatment of culture with 10 g/ml N-methyl-N-nitro-N-nitrosoguanidine. Of a total of 52 resistant clones isolated, 2 gave rise to haploid, 15 to diploid, and 3 to tetraploid plants upon transfer of calli to differentiation medium. Leaf-segment and protoplast assays showed that all diploid regenerates were resistant to streptomycin but sensitive to chloramphenicol, kanamycin, lincomycin, neomycin, and spectinomycin. Plants in most diploid clones were fertile and able to set seeds when self-fertilized and crossed reciprocally to wild-type plants. Inheritance of streptomycin resistance was studied in the diploid clones and, without exception, the resistance was transmitted maternally. Comparative studies of the ultrastructure of organelles and protein synthesis in isolated chloroplasts between wild-type and resistant clones in the presence of streptomycin suggest that streptomycin resistance is controlled by chloroplasts.     

Some physicochemical properties of rice mitochondrial DNA

Summary Certain physicochemical properties of rice mitochondrial DNA (mtDNA) were determined. Certain low-molecular-weight mtDNA bands were found in addition to the major mtDNA band. Rice mtDNA appeared in the electron microscope as a collection of linear molecules with heterogeneous length in the range of 1–156 kb. The major distribution area was 60–105 kb. A small fraction (less than 5%) of rice mtDNA was found in the form of a circular molecule. Some molecules had the appearance of being supercoiled. Replication fork structures were found in both circular and linear mtDNA molecules. In one rice species, Jin Nante, 15 different circular molecules were found. Rice mtDNA was digested with different restriction enzymes. The total molecular weight of rice mtDNA was calculated to be about 300 kb according to the data of restriction enzyme digestion and electron microscopy.     

Anatomical features associated with preaxial duplication (pd): a recessive mutation in the rat

Preaxial polydactyly of the fore- and hindlimbs was found in Wistar-derived rats in 1978. Genetic analysis indicated that the polydactyly was due to the effects of an autosomal recessive gene (gene symbol; pd). Polydactylous homozygous rats had two or three pollices (six or seven digits) in the forelimbs and one to three preaxial extra digits (six to eight digits) in the hindlimbs. Skeletal examination revealed the presence of the extra carpal, metacarpal, and phalangeal bones that seemed to be complete or incomplete duplication of the navicular, greater multangular, first metacarpal, and phalanges of digit I in the forelimbs. In the hindlimbs, extra tarsal, metatarsal, and phalangeal bones were also observed preaxially. These extra elements seemed to be mirror-image duplications of the talus, navicular, second cuneiform, third cuneiform, cuboid, and metatarsals and phalanges of digits II-V with the absence of the first cuneiform, tibiale, first metatarsal, and phalanges of digit I. In addition, morphological changes were observed in the humerus, radius, and ulna in the forelimbs and femur, tibia, and fibula in the hindlimbs. Especially in the radius and tibia, thickening and bifurcation were found, indicating incomplete duplication of these bones. Based on these findings, the limb anomaly was classified as preaxial carpometacarpal/tarsometatarsal-type polydactyly with incomplete duplication of the radius and tibia. The mutant rats had other associated anomalies such as accessory spleens and cryptorchism. The males are sterile, whereas the females breed normally.     

Changes in cerebral level of monoamines by Japanese encephalitis virus infection

The changes in monoamine levels of different brain regions following Japanese encephalitis virus (JEV) intraperitoneal inoculation were examined in experimentally JEV-infected mice. In addition, virus distribution was studied using infectivity assay and immuno-histochemistry of viral antigen. 1) The level of monoamines in brain tissues was not affected by 48 hours after viral inoculation, but marked effects were elicited at 96 hours after the inoculation. The cerebral concentration of 5-hydroxyindole-3-acetic acid (5 HIAA) was increased, while that of dopamine (DA) showed a decrease. Especially these alteration were observed in the cerebral cortex, but not in the cerebellum. 2) The viral growth in the brain was observed at 48 hours after the inoculation. The growth in the cerebellum, however, was found to be lower than those in other cerebral regions. 3) The viral antigen was detected in the cerebral cortex, hippocampus, mesencephalon and diencephalon in addition to the substantia nigra and striatum. From these results, it is presumed that clinical manifestation of JEV infection may involve the changes in the metabolism of neurotransmitter, especially those of DA and serotonin in the brain.