Estimation of stature in children from second metacarpal measurements.

On the basis of 552 boys and 542 girls aged 6 to 20 years, this study examines the estimation of stature from dimensions and maturity of second metacarpals by means of linear regression equations. A combination of length and width measurements provided a more accurate estimation than each measurement individually. When taken alone, length produced a more accurate estimation than width. Sex and age factors are useful for the estimation of stature, though these variables are often unknown in the isolated bone. The samples are divided into immature and mature groups (according to skeletal maturity). Regardless of sex, stature could be estimated from the metacarpal length and width with a standard error of 4.19 cm by means of a multiple linear equation in the immature group. The mature group should be considered with adults for this purpose. Thus, taking into account their skeletal maturity, living stature could be practically estimated from the second metacarpal with significant degrees of accuracy in children.     

Estimation of age at death from second metacarpals.

This study examined the estimation of age at death from the second metacarpal in 227 individuals aged 30-98 years. Variables ascertained from each bone were: cortical thickness and microdensitometric cortical bone density measured on radiographs of the bone and total osteon count and density recorded on microradiographs of the complete cross section at its midshaft. Based on the latter two variables, two age groups were formed; a middle age group representing those individuals aged 30-65 years, and an older group aged 65+. Stepwise regression analysis of the four variables produced a series of regression equations for age estimation for the middle, old and combined age groups for each sex and sexes combined. Sex-specific equations provided better results than nonspecific ones, especially in females. Total osteon density and combined cortical thickness were found to be the most useful estimators in the middle and the old age group, respectively. The standard error of estimate was 6.71 and 6.90 years in each age group for the sexes combined. In the combined age group, age could be estimated accurately from total osteon count, cortical thickness and MD cortical bone density with the standard error of estimate of 11.10 years. The relative error of estimate ranged within +/- 30% in almost all individuals aged above 60 years.     

Production of S-lactoylglutathione by organic-solvent-extracted glyoxalase I from Hansenula mrakii

Summary Glyoxalase I was extracted from Hansenula mrakii IFO 0895 by incubating the cells with buffer solution containing 50% acetone (enzyme activity 35 units/g cells) or 50% ethyl acetate (enzyme activity 28 units/g cells) at 30°C for 10 h. Glyoxalase II was also extracted from the cells, although the activity of the enzyme was lost during incubation with organic solvents, especially at higher temperature (30°C). By using the organic-solvent-extracted fraction of H. mrakii, enzymatic production of S-lactoylglutathione was studied, and approximately 82 mmol/l (30 g/l) of S-lactoylglutathione was produced from 120 mmol/l glutathione. Offprint requests to: A. Kimura     

A sensitive nonisotopic method for the determination of intracellular azidothymidine 5'-mono-, 5'-di-, and 5'-triphosphate.

In order to analyze the efficacy of azidothymidine (AZT), it is important to know intracellular concentrations of AZT metabolites. However, it has been impossible to measure intracellular AZT 5'-monophosphate (AZT-MP), AZT 5'-diphosphate (AZT-DP), and AZT 5'-triphosphate (AZT-TP) without using isotopes. In the present study, we developed a new method to measure intracellular AZT metabolites without radiolabeled compounds. The method employed was a high-performance liquid chromatography (HPLC) system programmed for column switching technique, in which two columns were used: column 1 (TSK-G2000-SW, 300 x 7.5 mm) to preseparate AZT metabolites from major cell components, and column 2 (YMC-A-312-ODS, 150 x 6 mm) to determine the metabolites. The limit of detectability of this system was 3.3 pmol/injection. When MT-4 cells were incubated with various concentrations of AZT, intracellular concentrations of AZT-MP increased in parallel with extracellular AZT. Those of AZT-DP and AZT-TP, however, reached plateaus at 5 and 2 microM of AZT, respectively. In MT-4 and Molt-4 cells incubated with 5 microM AZT, concentrations of AZT-MP increased time dependently, while the AZT-DP/AZT-MP ratios decreased with time. These data suggest that high dose of AZT may not necessarily increase intracellular concentration of AZT-TP. The concentrations of AZT metabolites in peripheral blood mononuclear cells in a patient with AIDS and an asymptomatic carrier were measured; the concentrations were comparable to those in cultured cells. Quantitative analysis of intracellular AZT metabolites without the use of isotopes will increase safety and convenience of measurement, and take an effective step in studying pharmacokinetics of AZT in clinical materials.     

Oxidative modification of glycated low density lipoprotein in the presence of iron

This is the first observation for contributing to the glycation of low density lipoprotein (LDL) to oxidative modification of its own lipids and protein. Human plasma LDL was glycated by incubation with glucose (G-LDL). Glucose incorporated into apoprotein B was approximately 10 mol/mol of apoprotein (2.8% modification of lysine residues) and 84% of G-LDL was adsorbed on phenylboronate affinity column. G-LDL incubated with Fe3+ for 4 h caused a significantly higher level of lipid peroxidation than U-LDL (untreated with glucose), and a higher molecular weight protein was observed in apoprotein B on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), increasing with incubation period. Corresponding to change on SDS-PAGE, G-LDL exposed to Fe3+ moved faster than G-LDL per se or U-LDL to anode on agarose gel electrophoresis. The higher the Fe3+ concentration, the more lipid peroxidation was caused. Alpha-tocopherol or probucol suppressed the lipid peroxidation of G-LDL exposed to Fe3+.     

Synthesis of bombyxin-IV,an insulin superfamily peptide from the silkworm,Bombyx mori,by stepwise and selective formation of three disulfide Bridges

We report the synthesis of bombyxin-IV, a disulfide-linked, heterodimeric, insulin superfamily peptide from the silkworm,Bombyx mori. The two chains (A- and B-chains) were synthesized separately by the solid-phase method using fluoren-9-ylmethoxycarbonyl (Fmoc) group as a protecting group for α-amino group. Three disulfide bonds were bridged step by step (A6–A11, A20–B22, and A7–B10) in a good yield. Synthetic bombyxin-IV was identical with natural one with regard to the retention time on a reversed-phase column and the molecular weight measured by mass spectrometry. Circular dichroism (CD) spectrum of the synthetic bombyxin-IV was very similar to that of the natural one. The specific activity of synthetic bombyxin-IV is equal to that of natural one (0.1 ng/Samia unit). These results suggest that the synthetic bombyxin-IV has the tertiary structure identical with the natural peptide. Our method developed for synthesis of bombyxin-IV would be generally applicable to the synthesis of insulin-like heterodimeric peptides.     

Affinity labelling of endothelin receptor and characterization of solubilized endothelin-endothelin-receptor complex

Chick cardiac membranes were affinity labelled by cross-linking to membrane-bound 125I-endothelin-1 with disuccinimidyl tartarate. SDS/PAGE and autoradiographic analysis of the 125I-endothelin-1-labelled material in the presence or absence of 2-mercaptoethanol revealed one major labelled band, corresponding to a molecular mass of 53 kDa, whose appearance was dose-dependently inhibited by the addition of unlabelled endothelin-1 (1-100 nM). Subtracting the molecular mass of 125I-endothelin-1 and disuccinimidyl tartarate, the binding protein appeared to have a molecular mass of 50 kDa. To investigate further the molecular properties of endothelin receptor, the 125I-endothelin-1-endothelin-receptor complex was solubilized from chick cardiac membranes using the detergent digitonin. Sucrose gradient sedimentation of the solubilized complex indicated a sedimentation coefficient of 13 S, whereas the complex of (+)-[3H]PN200-110, a dihydropyridine derivative, and dihydropyridine-sensitive Ca2+ channels sedimented at 22 S. A monoclonal antibody raised against dihydropyridine-sensitive Ca2+ channels from the chick brain did not immunoprecipitate the 125I-endothelin-1-endothelin-receptor complex. These data suggest that endothelin receptor is clearly distinct from dihydropyridine-sensitive Ca2+ channels and endothelin has its own specific 50-kDa receptor.