Purification of Ribulose 5-phosphate Kinase and Minor Polypeptides of Pyrenoid from the Green Alga Bryopsis maxima

Ribulose 5-phosphate (Ru5P) kinase (ATP:D-ribulose 5-phosphate1-phosphotrans- ferase; EC 2.7.1.19 [EC] ), an enzyme in the reductivepentose phosphate cycle, was purified from the green alga Bryopsismaxima and its activity and peptide composition were studied.The specific activity of purified Ru5P kinase was 20 µmoleRuBP formed (mg protein)^–1 min^–1 corresponding toa 490-fold purification from the supernatant of chloroplasts.The K_m values of Ru5P kinase for ATP and Ru5P were 69 µMand 330 µM, respectively. The molecular size of Ru5P kinase was estimated as 90 kDa bygel filtration and that of its polypeptide as 41 kDa by SDS-polyacrylamidegel electrophoresis. A small portion of the Ru5P kinase wasfound in a large molecular state (500 kDa) which was consideredto be an inactive form of the enzyme. Ru5P kinase activity has been reported in the pyrenoid of Eremosphaeraviridis as well as ribulose 1,5-bisphosphate carboxylase-oxygenase(RuBisCO) and ribose 5-phosphate isomerase activity (Holdsworth1971). In Bryopsis maxima, among the pyrenoid polypeptides otherthan that of RuBisCO, we found a polypeptide of 42 kDa, similarto that of Ru5P kinase in molecular size and ratio to RuBisCO.A peptide map of the 42 kDa pyrenoid polypeptide, however, showedthat it differed from that of Ru5P kinase. In conclusion, Ru5Pkinase may be not involved in the pyrenoid of this alga. (Received January 19, 1985; Accepted May 15, 1985)     

The heterogeneity of human fibroblasts as determined from the effects of hydrocortisone on cell growth and specific dexamethasone binding

To elucidate the heterogeneity of human fibroblasts from lung and skin, the effects of hydrocortisone on cell proliferation and the specific dexamethasone binding to cells were studied. Hydrocortisone at physiological concentrations stimulated the proliferation in three strains of human fetal lung fibroblasts and inhibited it in two strains. There are two kinds of fibroblasts in the human fetal lung in addition to the human skin fibroblasts reported previously. Dexamethasone-binding experiments showed that human fibroblasts may be classified into two groups with respect to the dissociation constant (Kd) of the binding reaction. The heterogeneity of human fibroblasts shown by Kd could not be correlated to classification on the basis of the effects of hydrocortisone on cell proliferation. The differences in Kd for the binding reactions suggest differences in donor tissues from which human fibroblasts are derived.     

A Study on Erythrocyte Membrane Plasmalogen in Myotonic Dystrophy

Phospholipid classes that included plasmalogens of erythrocyte membranes in seven myotonic dystrophy (MyD) patients and seven normal controls were analyzed by HPLC. No significant difference in phospholipid classes was found between patients with MyD and normal controls, but there was a visible difference in peak profiles of compounds of the phosphatidylethanolamine class. In the study of plasmalogens, we used two preparation methods: exposure to HCl and deacylation with mild alkaline. The area ratio of the plasmalogen form to the diacyl form in the phosphatidylethanolamine class of MyD erythrocyte membranes was significantly lower than that of normal controls. Fatty acid analyses showed that fatty acids of both phosphatidylethanolamine subclasses have less unsaturation in MyD.     

The Escherichia coli cytochrome b556 gene, cybA, is assignable as sdhC in the succinate dehydrogenase gene cluster

Abstract The cytochrome b556 -deficient mutant Escherichia coli K12 strain TK3D11 [7] could not grow with succinate as the sole carbon source, but could grow well on dl -lactate. This finding suggested that cytochrome b556 is primarily responsible for oxidative metabolism and utilization of succinate. 24 Amino acid residues at the amino-terminal of purified cytochrome b556 were determined. This sequence coincided completely with amino acid residues 4 to 27, predicted from the DNA sequence of the sdhC gene, one of the unassigned open reading frames of the sdh gene cluster recently reported by Wood et al. [16]. Based on these and other results, we concluded that cybA , the gene for cytochrome b556 , is assignable as sdhC .     

The morphology and anatomy ofHydrastis (Ranunculales): Systematic reevaluation of the genus

Evidence from morphology and anatomy (including embryology), as well as from palynology, chemistry and cytology, indicates thatHydrastis is quite divergent from Ranunculaceae (in which the genus has been most often included) as well as from both Glaucidiaceae and Berberidaceae. Distinctive features ofHydrastis, which demarcate it from Ranunculaceae but which are sometimes shared by Berberidaceae, are: the unique mode of origin of the vascular supply to stamens and carpels; the micropyle being formed by both integuments; the xylem not V-shaped in cross section; scalariform vessel perforations present; haploid chromosome number 13; pollen tectum consisting of a compound layer of striae; leaf mesophyll not differentiated; the unique course of stem medullary bundles; D-galactose present. Its distinctive higher haploid chromosome number, as well as its many less-specialized character states (in floral structure, leaf anatomy, and xylem and vessel morphology), suggest thatHydrastis is a relictual primitive group which diverged early from a common ancestral stock of Ranunculaceae, Berberidaceae and probably of Circaeasteraceae; at least some of the features shared byHydrastis and one or another of the families concerned seem to be a heritage from their common ancestor. We propose a reestablishment of a monotypic family, Hydrastidaceae.     

Spontaneous Mutations Modifying the Activity of Alcohol Dehydrogenase (Adh) in DROSOPHILA MELANOGASTER

In a marked-inversion balanced lethal system of the second chromosome of Drosophila melanogaster, mutations were accumulated under minimum pressure of natural selection in 1000 individual lines that originated essentially from two individuals. After about 300 generations, the specific activities of alcohol dehydrogenase of 69 randomly selected individual lines were measured with replications using four replicated vials (on 2 days—two replications per day) by observing the reduction of NAD⁺ to NADH at 340 nm. Total soluble protein as the basis of standardization of enzyme activity was measured by the Lowry method for each vial. A control experiment was made immediately after the establishment of 20 individual lines from a single genotype. A significant increase in genetic variance was observed among the mutation-accumulating lines but was not detected in the control experiment. The statistical analysis of the data on the basis of the one-band/one-gene hypothesis suggests that many mutations controlling the activity of alcohol dehydrogenase occurred in regions different from the alcohol dehydrogenase locus itself, mainly in the noncoding DNA. Furthermore, it is suggested that transposon-like elements are related to the induction of these changes in alcohol dehydrogenase specific activities. Additional experimental evidence supporting this conclusion is also given.     

Distribution of Leu 7 (HNK-1) antigen in human digestive organs: an immunohistochemical study with monoclonal antibody

Summary Leu 7 immunoreactivity was demonstrated with the indirect peroxidase-labelled antibody method on frozen and paraffin-embedded tissue sections of human digestive organs. Anti-Leu 7 monoclonal antibody, which allegedly detects mononuclear cells with natural killer or killer activity, recognized lymphoid cells among intestinal epithelial cells and in the germinal centres of solitary lymphoid follicles of small and large intestine, and a few in gallbladder, liver and the lamina propria of the intestine. In addition, peripheral nerve fibres, endocrine cells in the gut and pancreas and carcinoid and islet cell tumours were also positively stained. At the ultrastructural level, Leu 7 antigen was localized on the plasma membrane of granulated lymphoid cells in the gut mucosa and on the secretory granules of intestinal endocrine cells. In normal pancreas, Leu 7 immunoreactivity was demonstrated in most cells containing pancreatic polypeptide and in many cells containing somatostatin or glicentin. Insulin-containing cells, however, lacked Leu 7 immunoreactant. These findings were obtained in both frozen sections and paraffin-embedded sections. The possible cross-reactivities of monoclonal antibodies are discussed as they raise an important caveat in immunohistochemical studies using these antibodies.     

Differences in Ca2+ mobilization induced by alpha-adrenergic agonist and phosphatidic acid in cultured hepatocytes

In an attempt to elucidate the relationship between phosphatidylinositol breakdown and alpha-adrenergic responses, effects of phosphatidic acid and phosphatidylinositol related metabolites on Ca²⁺ mobilization and glucose output in cultured hepatocytes were examined. Norepinephrine induced the net ⁴⁵Ca²⁺ efflux from preloaded cells and stimulated glucose output via alpha-adrenergic receptor stimulation, whereas phosphatidic acid caused ⁴⁵Ca²⁺ uptake to cells and did not stimulate glucose output. Myo-inositol-monophosphate, diglyceride and arachidonic acid, which are released by phosphatidylinositol breakdown, had no effect on ⁴⁵Ca²⁺ efflux and glucose output in cells. These results suggest that phosphatidic acid and phosphatidylinositol related metabolites can not mimic the alpha-adrenergic actions in cultured hepatocytes.     

Chromosomal location of the Escherichia coli cytochrome b556 gene, cybA

Summary The amounts of cytochrome b556 in the cytoplasmic membranes of several Escherichia coli K12 strains having F-prime factors and a lambda transducing phage were determined. The amount was amplified about two-fold in strains having F100-12 and F152, but not in strains having F100-11, F8 and psu ⁺ 2glnS ⁺. The strain TK3D11, which lacks the kdp-gltA region (deletion D-01) of the E. coli chromosome, did not synthesize cytochrome b556 at all. From these results, the gene cybA encoding cytochrome b556 was located in the kdp-gltA region.In the cytochrome b556-deficient mutant, a novel b type cytochrome, cytochrome b561 which is a product of the gene cybB, was identified. It seems to function as a physiological electron transferring cytochrome in place of cytochrome b556 in this mutant.Abbeviations HPLC high performance liquid chromatography - EDTA ethylenediamine tetraacetic acid - SDS sodium dodecyl sulfate - NADH reduced form of nicotinamide adenine dinucleotide