Molecular cloning and expression ofThiobacillus ferrooxidans chromosomal ribulose bisphosphate carboxylase genes inEscherichia coli

The genes coding ford-ribulose-1,5-bisphosphate carboxylase (RuBPCase) from an iron-oxidizing bacterium,Thiobacillus ferrooxidans, were cloned into anEscherichia coli plasmid, pUC18. The recombinant plasmid, termed pTR11, contained a 4.0-kb PstI fragment including the entire coding regions for both large and small subunits of RuBPCase.Escherichia coli carrying pTR11 did not show any CO₂-fixing activity. However, a derivative plasmid with an appropriate deletion, which was placed under the control of atac promoter, conferred ribulose bisphosphate-dependent CO₂-fixing activity on the host cell. Analysis of gel-filtration chromatography of the RuBPCase synthesized inE. coli revealed that it had a hexadecameric form like the native enzyme ofT. ferrooxidans.     

Selective phagocytosis of gram-positive bacteria and interleukin 1-like factor production by a subpopulation of large granular lymphocytes

There has been a consensus that a large granular lymphocyte (LGL) population with natural killer (NK) function is nonadherent and nonphagocytic. However, a significant proportion of the nonadherent cells purified by the two-step depletion of adherent cells with a plastic surface and nylon wool columns engulfed Sta. aureus into their cytoplasm. These cells were morphologically identified as LGL in light and electron microscopies. Two-color immunofluorescence tests, furthermore, demonstrated that Leu-11+ LGL, Leu-11+7-, and Leu-11+7+, but not Leu-11-7+, phagocytosed Sta. aureus. Among the particles tested here, only Gram(+) bacteria were preferentially phagocytosed, whereas Gram(-) bacteria, other large-sized microbes (e.g., baker's yeast and Candida albicans), latex, silica, and carbonyl iron were not. LGL exhibited a substantial level of bactericidal activity against Sta. aureus, although the level was one third of that mediated by monocytes. When Gram(+) bacteria were incubated with nonadherent cells for 18 hr, significant amounts of interleukin 1 (IL 1)-like factors (or IL 1 itself) as well as interferon were detected in the supernatants. On the other hand, this incubation did not induce interleukin 2 (IL 2). The IL 1-like factor producer cells were demonstrated to be the low-density lymphocytes on Percoll separation and to have the Leu-11+ phenotype. The phagocytosis was suggested to be an important stimulus in producing IL 1-like factors from LGL. Thus, the treatment of cells with cytochalasin B, a microfilament disrupting agent, completely abrogated both phagocytosis and IL 1-like factor production. Some cell wall components of Gram(+) bacteria might be important to a recognition process of the phagocytosis, since the protoplasts of Sta. aureus, when prepared by the treatment of bacteria with lysostaphin, were no longer phagocytosed by LGL. The present results therefore identify an additional unique characteristic similar to, but not identical with, the myelomonocytic nature of Leu-11+ LGL.     

A New Myelin-Deficient Mutant Hamster: Biochemical and Morphological Studies

Biochemical and morphological studies were done on a new trembling mutant hamster CBB. The yield of myelin from the mutant was 30 and 40% of the control at 46 and 140 days of age, respectively, but myelin composition and 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNPase) activity were normal. Morphologically, about 18% of the axons were myelinated in the mutant optic nerve at 46 days of age, in which the myelinated fibers were those with larger diameters (more than 0.6 micron), while the control had a peak at 0.4 micron in diameter. The ultrastructure and thickness of compact myelin lamellae in the mutant were normal. Myelination and the structure of peripheral nerve myelin appeared normal. The results indicate that the essential defect is the delay and arrest of myelination in the CNS, which is probably caused by either a decreased rate of synthesis of myelin components in oligodendrocytes or a defect in the oligodendrocyte-axon recognition in smaller axons.     

Correlation between X-chromosome inactivation and cell differentiation in female preimplantation mouse embryos

By means of a cytological method involving BrdU incorporation and acridine orange fluorescence staining in combination with embryo manipulation, we studied X-chromosome activity in female preimplantation mouse embryos with special reference to the correlation between X-chromosome inactivation and cell differentiation. There was no sign of asynchronous replication between the two X chromosomes from the one-cell to intermediate blastocyst stage. The allocyclic X chromosome, first detected in late blastocysts, was paternal in origin, mostly replicating early in the S phase and limited to the trophectoderm. Subsequent X-chromosome inactivation occurring in the primary endoderm was also characterized by the involvement of the paternal X and early replication. Both X chromosomes continued to replicate synchronously in the embryonic ectoderm or epiblast at this stage. It was evident that overt cell differentiation preceded the appearance of the asynchronously replicating X chromosome in the trophectoderm and primary endoderm. This finding seems to support the view that cell differentiation is an important correlate of X-chromosome inactivation.     

Intra uterine survival and growth of Wistar and Wistar x Brown Norway rat embryos

Embryos of the Wistar strain and its F(1) cross (Wistar females mated with Brown Norway males) of rats were transferred nonsurgically to 48 Wistar, 17 F(1) cross and 20 Wistar-Imamichi recipients. The two types of embryos were transferred together to each recipient to compare the viability of the embryos. Pregnancy rate was 78.8% (67 85 ). The survival rate of fetuses to term was 11.5% (20 174 ) and 25.1% (42 168 ) for the Wistar and F(1) embryos, respectively. Placental weight differed significantly (P<0.05) between embryo types and among recipient types while fetus weight differed (P<0.01) only among recipient types, with a significant interaction between recipient and embryo types (P<0.01). It was concluded that the F(1) embryos (Wistar x Brown Norway) were twice as viable as Wistar embryos under the conditions provided.     

A versatile bacterial enzyme: l-methionine γ-lyase

The properties and application of l-methionine γ-lyase [methioninase, l-methionine methanethiol-lyase (deaminating), EC 4.4.1.11], a pyridoxal 5′-phosphate enzyme, purified from Pseudomonas putida and Aeromonas sp. are presented. The enzyme has multicatalytic functions: it catalyses α,γ-elimination and γ-replacement reactions of l-methionine and its analogues (e.g. ethionine, homocysteine, O-acetylhomoserine and selenomethionine), α,β-elimination and β-replacement reactions of l-cysteine and its analogues (e.g. S-methylcysteine, O-acetylserine and Se-methylselenocysteine), deamination and γ-addition of vinylglycine, and deuterium labelling at the α and β positions of l-methionine and other straight-chain l-amino acids. These reactions are applicable to the synthesis of various optically active sulphur and selenium amino acids, preparation of deuterium or tritium labelled l-amino acids, and determination of sulphur amino acids. In addition, the enzyme shows potent anti-neoplastic activity.     

Actions of amino-beta-carbolines on induction of sister-chromatid exchanges

Synthetic 3-aminoharman and 3-aminonorharman (amino-beta-carbolines) caused slight but definite induction of sister-chromatid exchanges (SCEs) in human lymphoblastoid cells NL3 and Chinese hamster cells CHO-K1. These amino-beta-carbolines are ranked between 2-amino-alpha-carboline and 2-amino-6-methyl-9a-aza-delta-carboline (Glu-P-2) and much lower than 3-amino-gamma-carbolines (Trp-P-1 and 2) in inductive activity. 1-Amino-beta-carboline, harman and norharman had very weak, if any, SCE-inducer activity. Norharman had a synergistic effect with aromatic amines such as Trp-P-2 and aniline on SCE induction, while 3-aminoharman suppressed SCE induction by more potent inducers such as Trp-P-2 and benzo[a]pyrene.     

Reversal of X-inactivation in female mouse somatic cells hybridized with murine teratocarcinoma stem cells in vitro

A series of near-diploid embryonal carcinoma-like hybrid cells were obtained from polyethylene glycol mediated cell fusion between murine embryonal carcinoma cells (PSA-6TG1 or OTF9-63) having one X chromosome and thymocytes or bone marrow cells from female mice carrying Cattanach's or Searle's translocation. Prior to fusion with EC cells the somatic cells are presumed to contain only one active X chromosome. Following hybrid formation, the chronology of X chromosome replication and the expression of X-linked gene Pgk-1 indicated that all X chromosomes contributed by both parents were active in these hybrids. Experiments were performed to rule out the possibility that the hybrids were formed by fusion of EC cells with rare somatic cells in which both X chromosomes were active. Taken together the data indicate that within four days of fusion there is reactivation of the entire inactive X chromosome.     

Measurement of turbulence intensity in the center of the canine ascending aorta with a hot-film anemometer

The blood flow velocity near the central axis of the canine ascending aorta was measured with a hot-film anemometer. The cardiac output and the heart rate were controlled at will by means of an extracorporeal circulation and by atrial pacing. The turbulent component of the blood flow velocity was calculated using an ensemble average technique. Ensemble average turbulent intensity was also calculated to show the time course of turbulence in the aorta. The ratio of the mean turbulence intensity to the time mean sectional average velocity in the aorta was constant in most animals regardless of the changes in fluid mechanical parameters. The correlation between the frequency parameter and the relative mean turbulence intensity was weakly positive. The power spectrum of the turbulence was also calculated.