Effects of Phosphate in Medium on Protein Secretion in a Protein-Producing Bacterium, Bacillus brevis 47

Bacillus brevis 47 secreted up to 1 mg of protein per ml in a chemically defined medium, depending on phosphate concentration. The composition of exoproteins was altered quantitatively by the concentration of external phosphate. Morphologically, B. brevis 47 showed a distinct three-layered cell wall structure and shed the outer two layers during growth.     

Two new subspecies of Bacillus thuringiensis isolated in Japan: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) and Bacillus thuringiensis subsp. tochigiensis (serotype 19)

Bacteriological and serological characteristics of three Bacillus thuringiensis isolates obtained in Japan were investigated. They formed typical rhomboidal parasporal inclusions but flagellar (H) antigens of these isolates were different from those of the known 17 H serotypes of B. thuringiensis. The three isolates were divided into two new serotypes (serotypes 18 and 19). The serotype 18 isolate (3–71) produced thermostable exotoxin and the inclusions of this isolate were toxic to larvae of the silkworm, Bombyx mori, but nontoxic to larvae of the mosquito, Aedes aegypti. The other isolate (119-72) belonging to serotype 18 produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. However, other bacteriological properties of the isolate 119-72 were similar to those of the isolate 3–71. The serotype 19 isolate (117-72) produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. Acid production from saccharose and the production of brownish purple pigment were observed in the two serotype 18 isolates, while neither of them was observed in the serotype 19 isolate. In other 29 biochemical properties tested, there was no difference among the three isolates. Based on these characteristics, the following two subspecies names are proposed: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) for the type strain 3–71 and Bacillus thuringiensis subsp. tochigiensis (serotype 19) for the type strain 117-72.     

Studies on the constituents of the seeds of Cassia obtusifolia: The structures of two new lactones,isotoralactone and cassialactone

Torosachrysone and two new naphthalenic lactones, isotoralactone and cassialactone, were isolated from the seeds of Cassia obtusifolia. Their structures were established as 9,10-dihydroxy-7-methoxy-3-methylene-¹H-naphtho(2,3-c)dihydropyrone-1-one and 8-methoxy-4-methyl-1-oxo-4,10,11-trihydroxy-naphtho(2,3-c)oxepin, respectively.     

Soluble and bound forms of intracellular acid carboxypeptidase inAspergillus saitoi

The enzymological, physical, and immunological properties of soluble and bound forms of intracellular acid carboxypeptidase isolated from fresh mycelia ofAspergillus saitoi are reported. In the broken mycelia, about 60% of the total activity was found in the 2,000×g precipitate, with most of the remainder in the 100,000×g supernantant. The highly purified enzymes, I_a and I_b, from the 100,000×g supernatant were found to be homogeneous by such criteria as disc gel electrophoresis at pH 9.4 The bound enzyme, II, was solubilized from the 2,000×g precipitate by self-digestion at pH 6.4 and was highly purified by chromotography. The two forms of intracellular enzymes, the soluble enzymes (I_a and I_b) from the 100,00×g supernatant and the solubilized enzyme (II) from the 2,000×g precipitate, were closely related to, but not completely identical with, the extracellular acid carboxypeptidase.     

Sugar hydrolases of the infant rat intestine and their arrangement of the brush border membrane.

Lactase and maltase, the predominant sugar hydrolases associated with the intestinal brush bordermembrane of the suckling rat, were purified essentially free of the other to near homogeneity (lactase at specific activity 23, maltase at specific activity 58), and their specific physiocochemical properties determined. Antisera prepared to each showed by immunodiffusion a single common precipitin line with pure enzyme and solubilized proteins of the brush border membrane. Brush border membranes were purified 26--35-fold from infant rat intestine. Membranes prepared from 10-day-old rats contained 32% protein, 43% lipid and 25% carbohydrate with lactase and maltase estimated to comprise in excess of 10% and 2%, respectively, of the membrane protein. Immunotitration curves of lactase and maltase showed equivalent antibody binding by the membrane-bound and free enzyme forms. Furthermore, antibody binding to one enzyme did not affect the immunotitration curve or the extractability (by papain or Triton X-100) of the other membrane-bound enzyme. It was concluded that the lactase and maltase molecules are attached singly on the external membrane surface in a spatially independent manner with their antigenic sites as freely available to antibody binding as exhibited by their papain-solubilized counterparts.     

Nucleic acid of flacherie viruses of the silkworm, Bombyx mori

It was reported previously that two spherical flacherie viruses of silkworm, FVS I and FVS II, had been isolated from flacherie silkworm larvae and the nucleic acid of FVS II was RNA as suggested by the experiments of incorporation of [³H]-uracil. In this paper, it has been confirmed by biochemical methods that the nucleic acid of FVS I and FVS II is RNA. FVS I and FVS II were labeled with ³²P in flacherie silkworms, and the viruses were analyzed by sucrose density gradient centrifugation. When the ³²P-labeled compound in the viruses was treated with 0.5 n KOH, the acid-insoluble ³²P-labeled compound changed to acid-soluble compounds. It was determined by paper chromatography and ion-exchange column chromatography that the alkali-decomposed compounds included four ribonucleotides. Therefore, the viral nucleic acid of FVS I and FVS II was determined to be RNA. The correlations between FVS I and FVS II particles were discussed, and it was suggested that FVS I and FVS II might be closely related or were the same viral species.     

Adenylate kinase of human erythrocyte. Isolation and properties of the predominant inherited form.

Adenylate kinase exists in the human erythrocyte in a number of molecular forms with two allels at a single polymorphic locus coding for most of the enzyme forms. The predominant enzyme form, AK a, was purified to constant specific activity in excess of 3000 and appeared homogeneous by chromatography, electrophoresis, and ultracentrifugation. Sedimentation velocity and partial specific volume measurments of AK a yielded values of s20,w = 2.1 S and 0.722 cm-3per g. The molecular weight of the native enzyme was estimated to be 22,500 by sedimentation equilibrium and gel filtration analyses. The molecular weight of the denatured enzyme did not differ, indicating an absence of subunit structure in confirmation of genetic evidence of a single locus coding for the enzyme. The isolated enzyme demonstrated remarkable stability to denaturants (heat, guanidine HCl, urea) in the presence of appropriate stabilizing agents and could not be distinguished from rabbit muscle enzyme on this basis (as well as by a number of other kinetic and physicochemical parameters). The erythrocyte adenylate kinases have a common molecular size but differ in their charge properties. They demonstrate anomalous electrophoretic behavior, migrating anionic to hemoglobin in starch gel, yet exhibit isoelectric points considerably alkaline to hemoglobin (e.g. AK a, pI = 9.0) by isoelectric focusing.     

Radioimmunoassay for 11-deoxycortisol using iodine-labeled tracer.

A simple and sensitive radioimmunoassay for 11-deoxycortisol was developed. The antiserum produced in rabbits by immunizing with a complex of 11-deoxycortisol-3-oxime and bovine serum albumin (BSA) has little cross-reactivity with other endogenous steroids. The immunoassay procedure requires only one-step ethanol denaturation of binding proteins in plasma and extraction by an organic solvent can be omitted. Furthermore, use of 125I-labeled tracer significantly simplify the counting procedure. The method is sensitive enough to detect 1 microng/100 ml of 11-deoxycortisol. Plasma 11-deoxycortisol levels measured by this method after the administration of a single dose of metyrapone ranged from 5.0 to 19.2 microng/100 ml, whereas they were 0 to 4.0 microng/100 ml in hypopituitary patients. It is concluded that this simple method is useful for the routine assay of plasma 11-deoxycortisol as a parameter of the metyrapone tests.     

Pulse-labeled, small closed circular DNA in cultured mouse and human cells

Mouse (erythroleukemia, TSA8, and FM3A) cells and human (HeLa and HL-60) cells were pulse-labeled with [3H]thymidine and covalently closed circular DNA in the extrachromosomal fraction was analyzed by fluorography following polyacrylamide gel electrophoresis. Two discrete bands for mouse and at least one, different, band for human cells emerged in the position to which small circular DNA (less than 1 kb) migrate, suggesting there to be species-specific, preferentially labeled, small circular DNA in mammalian cells. The incorporation of [3H]thymidine into the DNA was inhibited by cycloheximide but unaffected by aphidicolin. Restriction enzyme (AluI) digestion of the DNA fraction from MEL cells produced approximately 120-, 100-, and 50-bp labeled DNA fragments. The origin of the pulse-labeled DNAs is discussed.     

The isolation and culture of lily pollen protoplasts

Methods for the enzymatic isolation of lily protoplasts and their successful culture are described. When pre-anthesis binucleate pollen (immature pollen grains) was treated in enzyme solution containing macerozyme and cellulase, up to 80% lost their exine and gave rise to intact protoplasts within 1 h. These pollen protoplasts were uniform in size and densely cytoplasmic with two prominent generative and vegetative nuclei. The isolated pollen protoplasts regenerated a cell wall within 1 day of culture and produced a structure resembling a pollen tube after 10–12 days of culture. During this culture period, dividing generative nuclei or 2 sperm nuclei were observed in many protoplasts with regenerated cell walls.