Effect of elevated extracellular calcium on the proliferation of osteoblastic MC3T3-E1 cells:its direct and indirect effects via monocytes.

There has been evidence that elevated calcium concentration at the resorptive site of the bone directly regulates osteoclast function. In the present study, in order to clarify the role of elevated calcium concentration at the resorptive site in the regulation of osteoblast function, not only direct but also indirect effect via human monocytes of the increase in extracellular calcium (Cae) on the proliferation of osteoblastic MC3T3-E1 cells have been investigated in serum-free condition. The increase in Cae enormously stimulated osteoblast proliferation at the concentration of 3 to 20 mM. When human monocytes were cultured at the elevated Cae concentration, monocyte-conditioned medium-induced stimulation of osteoblast proliferation was significantly amplified. Present data demonstrate that elevated Cae has pronounced stimulatory effect on osteoblast proliferation not only directly but also indirectly via monocytes. Calcium released from bone matrix at the resorptive sites might be linked to the coupling of osteoclast and osteoblast functions.     

Beta-angonists modulate ACh-inhibition of a K current in intestinal smooth muscle cells.

ACh causes a long-lasting inhibition of STOCs via G proteins in intestinal smooth muscle cells. We examined the effects of isoproterenol (Iso) on the ACh-induced inhibition of STOCs in isolated ileal smooth muscle cells using the G omega-seal whole cell clamp technique. In control, ACh (1 microM) completely suppressed STOCs, which did not desensitize over a period lasting 20 minutes. When Iso (10 microM) was added to the bath in the presence of ACh, the ACh-induced inhibition of STOCs was gradually removed. This effect of Iso was prevented by propranolol (10 microM). Application of Db-cAMP (500 microM) mimicked the Iso effects. Intracellulary applied GTP-gamma S (100 microM) gradually suppressed STOCs in the absence of ACh, which could not be removed by either Iso or Db-cAMP. These results suggest that beta-adrenergic stimulation causes a removal of the muscarinic inhibition of STOCs via a cAMP-dependent process.     

Replication origin of the Escherichia coli K-12 chromosome: The size and structure of the minimum DNA segment carrying the information for autonomous replication

Summary A DNA fragment containing the replication origin of the Escherichia coli K-12 chromosome was inserted in two orientations at either the BamHI or SalI site of pBR322 DNA. All the resulting hybrid plasmids were found to replicate in both polA and polA ⁺ cells, whereas pBR322 replicates only in polA ⁺ cells. This characteristic provided a method for assaying the autonomously replicating ability (Ori function) of the E. coli origin.In order to define the minimum DNA region (ori) that determines Ori function, deletions of various sizes were introduced from either side of the ori-containing segment in the hybrid plasmids by in vitro techniques, and the correlation between the Ori phenotype and nucleotide sequence of the deletion derivatives was analyzed. It was found that the left end of ori is between positions 23 and 35, and the right end is either position 266 or 267 in our nucleotide coordinate (Sugimoto et al., 1979). Therefore, ori is present within a region of minimum 232 base pairs and maximum 245 base pairs in length. The Ori⁺ and Ori^- phenotypes were clearly resolved at both sides of these boundaries by the above assay procedure.To obtain information about the effect of mutations in the internal region of the defined ori stretch, short sequences were inserted or deleted in vitro in the vicinity of several restriction sites within ori on the hybrid plasmids. Most of these plasmids carrying modified sequences showed Ori^- phenotype, suggesting that most parts of the ori stretch play important roles in Ori function.     

Simplified determination of lorazepam and oxazepam in biological fluids by gas chromatography—mass spectrometry

Lorazepam and oxazepam in plasma and urine were measued by gas chromatography—mass spectrometry. Oxazepam was used as an internal standard in the assay of lorazepam and vice versa. After removal of interfering substances with n-hexane, the drugs were extracted with benzene and converted to N₁,O₃-bistrimethylsilyl derivatives. Glucuronide forms of the drugs were extracted after hydrolysis with β-glucuronidase. A common fragment ion at m/e 429 was used to monitor the two drugs. The sensitivity was 2 ng/ml for both drugs, which was sufficient to determine plasma and urine concentrations after therapeutic doses to humans.     

An unusual fluorescence spectrum of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor, a dimeric protein proteinase inhibitor isolated in crystalline form by Murae et al. in 1972, contains three tyrosine and one tryptophan residues per monomer unit and has unusual fluorescence properties. When excited at 280 nm, it shows a characteristic fluorescence spectrum having a peak at 307 nm and a shoulder near 340 nm, a feature which has been recognized only for a very few cases in proteins containing both tryosine and tryptophan residues. When excited at 295 nm, at which tryrosine scarcely absorbs, the inhibitor shows an emission spectrum with a peak at 340 nm characteristic of a tryptophan residue. The emission with a peak at 307 nm is considered to arise from the tryrosine residues. The tryptophan quantum yield of Streptomyces subtilisin inhibitor excited at 295 nm is very small, indicating that the tryptophan florescence is strongly quenched in the native state of the inhibitor. Below pH 4 the peak of the fluorescence spectrum of the inhibitor excited at 280 nm shifts toward 340-350 nm with a concomitant increase in the quantum yield. The structural change induced by low pH seems to release the tryptophan fluorescence from the quenching.     

Kinetic study of isomerization of ferricytochrome c at alkaline pH

Kinetic studies of the isomerization reaction of horse heart ferricytochrome c between pH 8.5 and pH 12.1 have been carried out by using stopped-flow and rapid scanning stopped-flow techniques. Below pH 10, our results were in good agreement with the scheme proposed earlier (Davis, L. A., Schejter, A. and Hess, G. P. (1974) J. Biol. Chem. 249, 2624–2632). Above pH 10, another faster first-order process was observed, which suggested the existence of a transient species in the isomerization reaction between the species with and without a 695 nm band. The probable scheme of the isomerization reaction is considered to be

where H denotes a proton, the colored forms are the species predominant at neutral pH with a 695 nm band and the noncolored forms are the species without a 695 nm band. The transient species has a small 695 nm absorbance which suggests that the sixth ligand is still Met-80, although the protein conformation might be different from that at neutral pH.     

Studies on metabolic role of 5′-methylthioadenosine in Ochromonas malhamensis and other microorganisms

Several compounds containing a thiomethyl group were found to replace vitamin B₁₂ in a protozoan, Ochromonas malhamensis. The order of the effectiveness was as follows: 5-methylthioadenosine > S-adenosylmethionine > 5-methylthioribose > L-methionine. A similar order was obtained with respect to the permeability of these compounds into the protozoan cells, except for S-adenosylmethionine. 5-Methylthioadenosine and 5-methylthioribose as well as l-methionine markedly increased the intracellular content of l-methionine. The level of S-adenosylmethionine was also increased by them, but to a lesser degree. The thiomethyl group of the compounds was established to be incorporated into S-adenosylmethionine. The metabolic fate of the thiomethyl group of 5-methylthioadenosine cannot be distinguished from that of l-methionine. A high activity of 5-methylthioadenosine nucleosidase was detected in the cell-free extracts of the protozoan. These results strongly suggest that 5-methylthioadenosine would be metabolized to l-methionine via 5-methylthioribose and then the l-methionine would be converted to S-adenosylmethionine. Like l-methionine and vitamin B₁₂, 5-methylthioadenosine and 5-methylthioribose may play an important role in maintenance of the C-1 pool in Ochromonas malhamensis.Neither 5-methylthioadenosine nor 5-methylthioribose replaced vitamin B₁₂ in some vitamin B₁₂-requiring bacteria. This result is consistent with the fact that neither compounds was significantly taken up by these bacteria.Abbreviations MTA 5-methylthioadenosine - AdoMet S-adenosylmethionine - MTR 5-methylthioribose - TCA trichloroacetic acid Paper II in the series. The first paper of the series has been published (Sugimoto and Fukui, 1974)     

Temperature-jump studies on benzeneboronic acid-serine protease interactions. Subtilisin and alpha-chymotrypsin.

The interactions of benzeneboronic acid (BBA) as a transition state analog with subtilisin (EC 3.4.21.4) and with alpha-chymotrypsin (EC 3.4.21.1) were investigated kinetically by the temperature-jump method using pH indicators. For both enzymes, the concentration dependence of the relaxation time was consistent with a two-step mechanism involving a fast bimolecular association followed by a slow, unimolecular process. The possibility of a trigonal-tetrahedral interconversion of BBA at the active site of the enzyme is discussed.     

The pH jump study of enzyme proteins. I. Liquefying alpha-amylase from Bacillus subtilis.

Rapid conformational changes due to pH jump were studied kinetically at 25 degrees mainly by the stopped-flow method using liquefying alpha-amylase from Bacillus subtilis [EC 3.2.1-.1, liquefying]. First, the conformational change due to a pH jump produced by mixing with alkali was monitored as a function of time at 245 nm through the ionization of phenolic hydroxyl groups of tyrosine residues which were originally buried and finally become exposed due to the pH jump. Three distinct phases of conformational change were clearly recognized by this method by varying the final pH values. Each phase involved the exposure of an essentially definite number of tyrosine residues, whose rate constant was crucially dependent on pH. Second, these phases of conformational change were subjected to examination in terms of the optical rotation change at 411 nm and the reversibility upon reverse pH jump with respect to conformational reconstitution, as observed through the protonation ofphenolic hydroxyl groups of ionized tyrosine residues and the enzyme activity. The first phase, which occurs above pH 12.5, involves no change in the optical rotation and is reversible as observed by the above two monitoring methods. In contrast, the other two phases, which are observed above pH 12.7, are accompanied by an optical rotation change and no appreciable reversibility was detected by these methods.