Functional structures of the recA protein found by chimera analysis.

We developed a novel genetic method for finding functional regions of a protein by the analysis of chimeras formed between homologous proteins. Sets of chimeric genes were made by intramolecular homologous recombination in a linearized plasmid DNA carrying both recA genes of Escherichia coli and Pseudomonas aeruginosa. A recBCsbcA strain of E. coli was used for isolation of plasmids carrying recombinants between these genes. Examination of properties of E. coli strains deleting the recA gene and carrying a plasmid with a chimeric gene shows that chimera formation at certain positions inactivates a RecA function. Frequently, all chimeras with a junction in a certain region of the protein inactivate a function. Rather than a direct effect of the presence of the junction at a particular position, mismatching of the regions both sides of the junction that are derived from the different species is responsible for the inactivation. For a chimeric protein to be functional, certain pairs of sequences in different regions of the protein must derive from the same parent. Four pairs of such sequences were found: two are involved in activities for genetic recombination and for resistance to ultraviolet light irradiation and the others in formation of active oligomers. Regions defined by these sequences are located in the looped regions of the protein. A pair of regions may co-operate to form a functional folded structure.     

Roles of Inorganic Nitrogen in Gametophytic Growth and in Initiation and Development of Sporophytic Shoots of Equisetum arvense

The formation of sporophytic shoots, which is induced by treatmentwith benzylaminopurine of gametophyte tissue of Equisetum arvense,can be divided into initiation and developmental phases. Thenitrogen in MS medium was suitable for two phases as well as gametophytic growth, buta reduction in the concentration of available nitrogen was neededfor the development of shoots. ions alone were effective for gametophytic growth and the initiationof sporophytic shoots, but both and ions was required for the developmental phase. (Received February 18, 1992; Accepted April 14, 1992)     

Synthetic sialyl glycolipids (sialo-cholesterol and sialo-diglyceride) induce granulocytic differentiation of human myelogenous leukemia cell line HL-60

When HL-60 cells were cultivated with synthetic sialyl glycolipids, sialo-cholesterol and sialo-diglyceride, the cells were found to be differentiated into mature granulocytes on morphological and functional criteria. The differentiation of cells was accompanied by inhibition of cell proliferation. The differentiation-inducing activity of sialo-cholesterol was greater than that of sialo-diglyceride on a molar basis, and the alpha-anomer of each compound was more potent than the beta-anomer, suggesting that the stereospecific structure of the compounds is important for the differentiation-inducing activity.     

A novel TIMP-insensitive type IV collagen-degrading metalloproteinase from murine metastatic sarcoma cells

A novel type IV collagen-degrading metalloproteinase was purified from the conditioned media of a murine metastatic sarcoma cell line. The molecular weight of the purified enzyme was determined to be 100 kDa by SDS-PAGE, while 700 kDa by gel filtration suggesting that the enzyme has a multimer structure. This enzyme degrades type IV collagen, but neither type I collagen nor casein. The failure of trypsin treatment to enhance the enzyme activity suggested that the purified enzyme did not require activation. Although the enzyme seems to be classified as a matrix metalloproteinase, it was inhibited by neither tissue inhibitor of metalloproteinases (TIMP) nor TIMP-2 and thus represents a novel type IV collagen-degrading metalloproteinase.     

“Active” conformation of an inactive semi-synthetic ribonuclease-S

We have studied the integrity of folded structure of a fully active semi-synthetic ribonuclease-S which lacks amino acid residues 16 through 20, and an inactive one with the same residues deleted and 4-fluoro-l-histidine substituted for active site histidine 12. Using “Y” form crystals, we obtained X-ray structural data to a resolution of 2·6 Å and, incorporating phase information calculated from refined ribonuclease-S coordinates, prepared several types of electron density maps. These showed that the overall backbone structure and active site configuration of both analogues do not differ noticeably from those of the native protein. Structural homology extends to the catalytically relevant side-chain at position 12; 4-F-His² assumes the same position as does His in active ribonuclease-S. This supports the view that the 4-F-Hisl2 analogue is inactive due to a change in histidine 12 imidazole basicity, rather than to any significant conformational distortion within the active site.     

Effects of Dietary Amino Acids on Brain Amino Acids and Transmitter Amines in Rats with a Portacaval Shunt

The etiologic relationship between disturbances in metabolism of amino acids and amines and hepatic coma was investigated by examining the effects of diets containing various mixtures of amino acids on brain amine metabolism in rats with a portacaval shunt, using a method for simultaneous analysis of amino acids and amines. Rats with a portacaval shunt were fed on four different amino acid compositions with increased amounts of various amino acids suspected to be etiologically related to hepatic coma, such as methionine, phenylalanine, tyrosine, and tryptophan. The animals were killed 4 weeks after operation. During the experimental period, these animals did not become comatose, but exhibited various behavioral abnormalities. Marked increase in the plasma and brain levels of the augmented amino acids, especially methionine and tyrosine, were observed in rats with a portacaval shunt. Brain noradrenaline, dopamine, and serotonin levels were significantly decreased when the brain tyrosine level was increased. These results indicate that in rats with a portacaval shunt the dietary levels of amino acids greatly influence the brain levels of both amino acids and transmitter amines.     

Hydrocephalus in suckling rats infected intracerebrally with mouse hepatitis virus, MHV-A59

After intracerebral inoculation of mouse hepatitis virus, MHV-A59 strain, into 3- to 5-day-old Wistar rats, some survivors at 14 days postinoculation (p.i.) were found to lack the cerebral cortex and to have an accumulation of considerable amount of cerebrospinal fluid. The virus titer in the brain increased exponentially after inoculation, reaching a maximum 4 to 6 days p.i. when immunofluorescence revealed virus-specific antigen within neurons in the cerebral cortex. A small amount of infectious virus was also detectable 14 days p.i. when the cerebral anomaly was evident. This brain malformation causing hydrocephalus was due to cerebral damage by viral infection.     

Synthesis of 2,2,2-trichloroethyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside,and its reaction with glycosyl halides.

2,2,2-trichloroethyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside (9) was synthesized in 6 steps from the readily available 1,3,4,6-tetra-O-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranose in 25% overall yield by employing the stannyl method for the regioselective activation of hydroxyl groups. Dibenzyl ether 9 was then glycosylated with appropriate glycosyl donors to afford lactosamine and chitobiose derivatives in good yield.     

Nuclear magnetic resonance studies of hemoproteins. VIII. Proton NMR studies of the binding of various nitrogenous ligands to ferric cytochrome c.

The structure of the heme environment of horse heart ferric cytochrome c was examined in the presence of various nitrogenous bases at several temperatures with the aid of hyperfine shifted proton NMR spectra at 220 MHz. The resonance positions and line widths of the signals for the peripheral methyl groups of the heme exhibited distinctive features of its low-spin state characteristic of each external ligand. In the imidazole complex of ferric cytochrome c, remarkable line sharpening of the heme-linked proton signals was encountered on raising the temperature. This may be related to the apoprotein perturbation on the binding of external ligand to the heme iron. These spectral peculiarities were discussed in relation to the electronic structure of the heme, the basicity of the external ligand and the van der Waals contact interaction between heme side chains and apoprotein.