Purification and Storage of Ribulose 1,5-bisphosphate Carboxylase from Rice Leaves

Ribulose 1,5-bisphosphate (RuBP) carboxylase was purified fromrice leaves. By using a buffer containing 12.5% (v/v) glycerolthroughout purification, the enzyme was protected from coldlability and was obtained at a high yield (5.5 mg/g fresh wt).The purified enzyme exhibited different rates of CO₂/Mg²⁺-activationby temperature pretreatment/storage. The purified enzyme was stable for at least one year in phosphatebuffer containing 12.5% (v/v) glycerol at 4°C or 50% (v/v)glycerol at –20°C. (Received March 1, 1983; Accepted June 27, 1983)     

Regulation of the pho regulon in Escherichia coli K-12. Genetic and physiological regulation of the positive regulatory gene phoB

phoB is a positive regulatory gene for phoA, which codes for alkaline phosphatase, as well as for other genes belonging to the phosphate (pho) regulon whose expression is inducible by phosphate limitation in Escherichia coli. A hybrid plasmid that contains a phoB-lacZ fused gene was constructed in vitro. This plasmid enabled us to study phoB gene expression by measuring the beta-galactosidase level in the cells. The plasmid was introduced into various regulatory mutants related to the phosphate regulon, and phoB gene expression in these strains was studied under limited and excess phosphate conditions. It was found that the regulation of phoB expression was very similar to that of phoA expression. Expression of both genes was induced by phosphate starvation. Both genes were constitutively expressed in phoR, phoS, phoT and phoU mutants and were not expressed in a phoR-phoM double mutant. The implications of these findings for the regulatory mechanism of the pho regulon are discussed.     

A new method for oligo(ADP-ribose) fractionation according to chain length

A new method was developed to separate mono- and oligo-(ADP-ribose) with chain lengths below 11 ADP-ribose units by size difference of one ADP-ribose residue. The separation was performed on a DEAE-cellulose column by elution with a NaCl gradient (0–0.3 $\text{M}$) in the presence of 7 $\text{M}$ urea at pH 7.6. Using this method, the chain length distribution of oligo(ADP-ribose) molecules attached to histones by incubation of isolated nuclei with radioactive NAD was determined. The average chain length estimated from this distribution coincided exactly with the value obtained by the phosphodiesterase digestion method, suggesting that the oligomers were synthesized directly on histones and not elongated from pre-existing ADP-ribose.     

Intestinal microflora and bile acids. Effect of bile acids on the distribution of microflora and bile acid in the digestive tract of the rat

The population levels of intestinal microflora and bile acid composition in the digestive tract were examined in rats fed bile acids to determine the relationships between gastrointestinal microflora and the host. The population level of Bacteroides was increased in the ceca of rats fed cholic acid or deoxycholic acid. In the ileum, the concentration of conjugated bile acid in rats fed cholesterol, cholic acid, hyodeoxycholic acid or lithocholic acid was higher than that in control rats, and was very low in ceca and feces of all the rats. The concentration of total free bile acid was much higher in the ceca than in the ilea of rats fed hyodeoxycholic acid or lithocholic acid. Cholic acid and deoxycholic acid were found in the ilea, ceca and feces of the cholic acid-fed rats. In the deoxycholic acid-fed rats, cholic acid was localized in the ileum. 7-Ketodeoxycholic acid was also found in the ceca of the cholic acid-fed rats. 12-Ketolithocholic acid was found in the feces of rats fed cholic acid or deoxycholic acid. 3-Ketocholanic acid was found in some samples from the lithocholic acid-fed rats. Therefore, some kinds of bile acids influence the population levels of gastrointestinal microflora and bile acid composition in the intestine.     

The occurrence of C4 species in the genusRhynchospora and its significance in kranz anatomy of the cyperaceae

Rhynchospora rubra was found to have a low CO₂ compensation point, high δ¹³C value, Kranz leaf anatomy, starch present in the bundle sheath cells and narrow interveinal distance. These observations suggest thatR. rubra is a C₄ plant. A further anatomical survey revealed seven otherRhynchospora species presumably having the C₄ photosynthetic pathway. In the family Cypraceae C₄ plants therefore occur in the tribe Rhynchosporeae as well as in the Scirpeae and Cypereae. The C₄ species ofRhynchospora have a normal Kranz type of leaf anatomy, although the C₄ species ofCyperus andFimbristylis presently known have an abnormal one in which the mestome sheath without chloroplasts is interposed between the Kranz tissue and the rest of the chlorenchyma. Thus inRhynchospora the Kranz tissue is in direct contact with the rest of the chlorenchyma, and it is suggested that the Kranz tissue may be homologous with the mestome sheath.     

Specific induction of indoleamine 2,3-dioxygenase by bacterial lipopolysaccharide in the mouse lung

Indoleamine 2,3-dioxygenase activity in the supernatant fractions (30,000g, 30 min) from various tissues of mice increased almost linearly after a single intraperitoneal administration of bacterial lipopolysaccharide (5 to 20 μg/mouse). The most prominent effect was observed in the lung, where both specific and total enzyme activities increased 40 to 80-fold during the first 24 h. Significant (10- to 20-fold) stimulation was also observed in the seminal vesicle, coagulating gland, colon, and caecum, and severalfold in the trachea, stomach, heart, small intestine, and spleen. Lipid A fraction, the biologically active unit in the lipopolysaccharide complex, was as active as the lipopolysaccharide preparations from either Escherichia coli or Salmonella S and R mutant strains, whereas the polysaccharide fraction was inactive under identical experimental conditions. When mice were pretreated with a series of daily injections of bacterial lipopolysaccharide, enzyme induction was no longer evident, indicating that tolerance to this agent had developed and that enzyme induction was caused by lipopolysaccharide but not by possible contaminants in the preparations. The enzyme activities from normal and lipopolysaccharide-treated mice were exclusively found in the soluble fractions of mouse lung homogenates. Other enzyme activities in the lung such as lysosomal (acid phosphatase), microsomal (prostaglandin cyclooxygenase), mitochondrial (monoamine oxidase and superoxide dismutase), and soluble enzyme activities (lipooxygenase and superoxide dismutase) were not significantly altered by this treatment. This increase in the enzyme activity with the lipopolysaccharide treatment was abolished with a simultaneous administration of cycloheximide or actinomycin D, and an immunological analysis with antibody for mouse enzyme (rabbit IgG) demonstrated that the observed increment of the enzyme activity was essentially due to an increase in the enzyme protein.     

Effects of 2,4-substituents of deuteroheme upon peroxidase functions. II. Reactions of peroxidases and metmyoglobin with azide, cyanide and fluoride

Artificial horseradish peroxidases and metmyoglobins were reconstituted from their apoproteins and the following unnatural hemes: mesoheme, deuteroheme, hematoheme, chlorocruoroheme, and diacetyldeuteroheme. The electron-withdrawing effects of the 2,4-substituents upon affinities of these hemoproteins for azide, cyanide, and fluoride were investigated. Peroxidase preparations used were acidic (A₁ + A₂) and neutral (B + C) enzymes.The electron withdrawal of the substituents increased the strength of ligaud binding. Plotting the logaritms of dissociation constants for the hemoprotein-ligand complexes against pK₃, a measure of relative basicities for metal-free porphyrins, the Hammett relationship was found to be applicable to this case except for a few. The affinities of deuterohemoproteins for ligands were definitely higher than those expected from the above relationship.The constant ρ, slope of the Hammett equation, varied with the ligand. For any particular ligand, the two peroxidase preparations gave the same ρ value, but it was different from the value for metmyoglobin.     

The ultrastructure of organelles appearing in spermatids and nutritive cells of Cipangopaludina malleata

Summary The ultrastructure of organelles appearing in the early typical and atypical spermatids, and the nutritive cells of Cipangopaludina malleata has been examined by a Siemens' electron microscope Elmiskop I.Mitochondria appearing in the early typical spermatid have doughnut-like profiles in which the internal ridges appear as triple-layered membranes arranged radially and extending into the interior of the organelle without reaching the other side. Each membrane 40–60 Å in width, separated by a clear interspace 60–90 Å wide, is characterized by a porous structure 20–30 Å in diameter which suggests a filtration apparatus for enzymes.Walls of the flattened saccules consisting the Golgi apparatus are calculated 35–60 Å thick, in which an electron-lucent, porous structure about 30 Å wide has been revealed.The smooth-surfaced endoplasmic reticulum is bordered by a triple-layered membrane consisting of two opaque layers with a less opaque interspace 20–30 Å wide. The outer membrane ca. 15 Å wide presents a more linear appearance than the dotted arrangement of the inner membrane 20–25 Å thick.The plasma membrane is composed of a triple-layered structure where two dense lines 15 Å wide are separated by a layer 20–30 Å thick of less density.The electron micrographs for the present studies were taken with the Siemens electron microscope, model Elmiskop I, at the Anatomical Institute of Kiel University, Germany. The one of the authors, G. Yasuzumi is deeply grateful to Prof. Dr. W. Bargmann and Dr. A. Knoop for the privilege of using this instrument and other equipments in the Laboratory.     

Spermatogenesis in Animals as Revealed by Electron Microscopy : VIII. Relation between the Nutritive Cells and the Developing Spermatids in a Pond Snail, Cipangopaludina malleata Reeve

This paper deals with spermatogenesis in Cipangopaludina malleata Reeve, with special regard to the relation between the nutritive cells and the developing spermatids. The nutritive cell gives rise to numerous, slender or broad, elongate pseudopodia which extend from its surface toward the seminiferous lumen. They are characteristically provided with rows of circular, oval, and elongate profiles identical in form and position with the profiles of the endoplasmic reticulum. As the elongate pseudopodia increase in number, they become more slender and more closely packed until they coalesce into a continuous sheet circumferentially disposed around the nucleus and the full length of the middle piece of the typical spermatid. Thus the mantle of the typical spermatozoon of the pond snail is formed by a thin fold of the cytoplasm of the nutritive cells. This wrapping appears to contain 16 to 18 elements of the smooth surfaced endoplasmic reticulum, which run parallel and helically (50 to 100 mµ apart). It is suggested that these constitute a conductor system for nutritional supply from the nutritive cells to the developing typical spermatids. The mantle is assumed to be a transient structure which disappears when the sperms are detached. The atypical spermatids develop while lodged in deep indentations of the surface of the nutritive cells.