Kinetic studies on CO binding to reconstituted myoglobins with four synthetic hemes; structural control in ligand binding to myoglobin.

Examination was made of CO binding reactions to four kinds of modified sperm whale myoglobin (Mb), whose heme was reconstituted by iron complexes of synthetic porphyrins such as porphine (Por), meso-tetramethylporphyrin (TMeP), meso-tetraethylporphyrin (TEtP) and meso-tetra(n-propyl)porphyrin (TnPrP), using flash photolysis and stopped-flow methods. The CO association rate was found to be 5- to 20-times and dissociation rate 10- to 36-times accelerated by replacement with synthetic hemes. These features could be explained based on characteristic structures of modified Mbs indicated by X-ray crystallography. The side chain of Arg-45 protruded from the heme vicinity into the solvent region and heme was tilted by interactions of meso-alkyl side chains with surrounding peptides, resulting in the formation of widely opened channels and pockets for ligand passage. These structural features indicate the CO ligand to more easily enter or exit from heme pockets of reconstituted myoglobins, compared to native Mb.     

ClpX protein of Escherichia coli activates bacteriophage Mu transposase in the strand transfer complex for initiation of Mu DNA synthesis.

During transposition bacteriophage Mu transposase (MuA) catalyzes the transfer of a DNA strand at each Mu end to target DNA and then remains tightly bound to the Mu ends. Initiation of Mu DNA replication on the resulting strand transfer complex (STC1) requires specific host replication proteins and host factors from two partially purified enzyme fractions designated Mu replication factors alpha and beta (MRFalpha and beta). Escherichia coli ClpX protein, a molecular chaperone, is a component required for MRFalpha activity, which removes MuA from DNA for the establishment of a Mu replication fork. ClpX protein alters the conformation of DNA-bound MuA and converts STC1 to a less stable form (STC2). One or more additional components of MRFalpha (MRFalpha2) displace MuA from STC2 to form a nucleoprotein complex (STC3), that requires the specific replication proteins and MRFbeta for Mu DNA synthesis. MuA present in STC2 is essential for its conversion to STC3. If MuA is removed from STC2, Mu DNA synthesis no longer requires MRFalpha2, MRFbeta and the specific replication proteins. These results indicate that ClpX protein activates MuA in STC1 so that it can recruit crucial host factors needed to initiate Mu DNA synthesis by specific replication enzymes.     

Structure and Chromosomal Assignment of the Human S1-5 Gene (FBNL) That Is Highly Homologous to Fibrillin

The human S1-5 gene (fibrillin-like; FBNL) was originally isolated from a subtractively enriched cDNA library established from a subject with Werner syndrome (WS). We isolated genomic clones containing the entire S1-5 gene and determined its genomic structure including the exon–intron organization. The gene spanned approximately 18 kb of genomic DNA and consisted of 12 exons. Its expression was abundant in all tissues examined except brain and peripheral leukocytes, where it was undetectable. In addition, we have mapped S1-5 by fluorescencein situhybridization to chromosome 2p16, a position that excludes it as a candidate for WS. Our data should facilitate an understanding of the function and regulation of S1-5 in human tissues.     

Inhibitory effect of oxygen accumulation on the growth of Spirulina platensis

Summary The growth of Spirulina platensis was studied in a light-limited culture under various dissolved oxygen (DO) concentrations. At high DO concentration, e.g. at 1.25 mM DO, the growth rate was decreased up to 36 % compared with that of 0.063 mM DO. The retarded growth rate at high DO concentrations seemed to be coupled with the degeneration of photosynthetic activity in terms of O₂ evolution. Under higher DO concentrations, superoxide dismutase and ascorbate peroxidase activities tended to increase, while the contents of photosynthetic pigment, such phycocyanin, carotenoid and chlorophyll-a decreased distinctly.     

In vivo and in vitro studies on the appearance of LHRH neurons in the hypothalamus of perinatal rats

Summary Ontogenetic development of LHRH-containing neurons was studied by fluorescence and enzyme immunohistochemistry in rats. In in vitro studies, the tissues of the septal-chiasmatic and mediobasal hypothalamic areas of fetal rats on day 16.5 or 18.5 of gestation were trypsinized separately for dissociation of the neural cells, and cultured for several days. Immunopositive reaction against LHRH was first detected in nerve cells derived from both areas of the hypothalamus of the fetuses on days 16.5 and 18.5 of gestation, after 8 and 6 days culture, respectively. The cells were small, and seemed to be bipolar in morphology indicating an axon and arborized dendrites. Immunopositive material occurred in the cell soma as well as in the cellular processes. In in vivo studies, immunopositive material, possibly deposited in nerve fibers, appeared first in OVLT and simultaneously in the external layer of the median eminence of fetuses on day 20.5 of gestation. The immunoreactive fibers increased in number in both parts with development, especially after birth in the median eminence. No immunopositive material was detected within any neural cell bodies nor in the cytoplasm of any ependymal cells.This work was financed by the Ministry of Education, Japan. No. 257008. We would like to thank Dr. Katsuhiko Saito (Department of Surgery, Tokushima University) for his kind advice on the preparation of the antibody used for the immunofluorescence study.     

Effects of polyamines on nucleosidediphosphate kinase activity.

Effects of naturally occurring polyamines were tested on the activity of bovine liver nucleosidediphosphate kinase with ATP as phosphoryl donor and eight nucleosidediphosphates as phosphoryl acceptors. The enzyme was either stimulated, inhibited, or unaffected depending upon the nucleosidediphosphate substrate and the polyamine, indicating that a general cation effect is not a sole mechanism of polyamine action. This selectivity and specificity of the effects with respect to both the polyamines and the nucleosidediphosphates leads us to speculate that an action of polyamines on nucleosidediphosphate kinase may play a significant role in the regulation of specific nucleosidetriphosphate synthesis $\text{in vivo}$.     

Cloning of a Phosphate-Regulated Hemolysin Gene (Phospholipase C) from Pseudomonas aeruginosa

Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa is a phosphate (P_i)-regulated extracellular protein which may be a significant virulence factor of this organism. The gene for this hemolytic enzyme was cloned on a 4.1-megadalton (Mdal) fragment from a BamHI digest of P. aeruginosa PAO1 genomic DNA and was inserted into the BamHI sites of the multicopy Escherichia coli(pBR322) and P. aeruginosa(pMW79) vectors. The E. coli and P. aeruginosa recombinant plasmids were designated pGV26 and pVB81, respectively. A restriction map of the 4.1-Mdal fragment from pGV26 was constructed, using double and single digestions with BamHI and EcoRI and several different restriction enzymes. Based on information from this map, a 2.4-Mdal BamHI/BglII fragment containing the gene for phospholipase C was subcloned to pBR322. The hybrid plasmids pGV26 and pVB81 direct the synthesis of enzymatically active phospholipase C, which is also hemolytic. The plasmid-directed synthesis of phospholipase C in E. coli or P. aeruginosa is not repressible by P_i as is the chromosomally directed synthesis in P. aeruginosa. Data are presented which suggest that the synthesis of phospholipase C from pGV26 and pVB81 is directed from the tetracycline resistance gene promoter. The level of enzyme activity produced by E. coli(pGV26) is slightly higher than the levels produced by P. aeruginosa(pMW79) under repressed conditions. In contrast, the levels produced by P. aeruginosa(pVB81) are at least 600-fold higher than the levels produced by P. aeruginosa(pMW79) under repressed conditions and approximately 20-fold higher than those produced by P. aeruginosa(pMW79) under derepressed conditions. The majority (85%) of the enzyme produced by E. coli(pGV26) remained cell associated, whereas >95% of the enzyme produced by P. aeruginosa(pVB81) was extracellular. Analysis of extracellular proteins from cultures of P. aeruginosa(pMW79) and P. aeruginosa(pVB81) by high-performance liquid chromotography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the phospholipase C gene was cloned intact, and it is likely that several additional genes were cloned on the 4.1-Mdal fragment of DNA. It was also found that some of these genes encode proteins which are the same molecular weight as some previously described P_i-repressible proteins of P. aeruginosa. The existence of a P_i regulon of P. aeruginosa is proposed. It is likely that one of these genes also regulates the level of pyocyanin production by P. aeruginosa and that one or more play a role in transport or binding of P_i. The availability of the hybrid plasmids described herein will be useful in further studies on the role of this hemolysin in the virulence of P. aeruginosa and in the study of the genetics and physiology of P_i-regulated proteins.     

Single cell protein production from mandarin orange peel

Summary As the hydrolysis of mandarin orange peel with macerating enzyme (40°C, 24 h) produced 0.59 g g^–1 reducing sugar per dry peel compared to 0.36 by acid-hydrolysis (15 min at 120°C with 0.8 N H₂SO₄), the production of single cell protein (SCP) from orange peel was studied mostly using enzymatically hydrolyzed orange peel.When the enzymatically hydrolyzed peel media were used, the utilization efficiency of reducing sugars (%) and the growth yield from reducing sugars (g g^–1) were: 63 and 0.51 for Saccharomyces cerevisiae; 56 and 0.48 for Candida utilis; 74 and 0.69 for Debaryomyces hansenii and 64 and 0.70 for Rhodotorula glutinis. SCP production from orange peel by D. hansenii and R. glutinis were further studied. Batch cultures for 24 h at 30°C using 100 g dried orange peel produced 45 g of dried cultivated peel (protein content, 33%) with D. hansenii and 34 g (protein content, 50%) with R. glutinis, and 38 g (protein content, 44%) with a mixture of both yeasts.