Clinical significance of serum bone Gla protein and urinary gamma-Gla as biochemical markers in primary hyperparathyroidism

The serum bone Gla-protein (BGP) and urinary gamma-carboxyglutamic acid (gamma-Gla) levels were determined in patients with primary hyperparathyroidism (PHP). The mean serum BGP and urinary gamma-Gla levels were 18.6 +/- 2.34 ng/ml and 65.5 +/- 4.62 nmoles/mgCr, respectively, for the 11 patients with the skeletal type of PHP, 5.13 +/- 0.85 ng/ml and 45.2 +/- 1.33 nmoles/mgCr for the 4 with the chemical type, and 7.91 +/- 2.43 ng/ml and 43.2 +/- 3.47 nmoles/mgCr for the 5 with the renal type. Thus, patients with skeletal-type PHP had significantly higher serum BGP and urinary gamma-Gla levels than those with the other type of PHP. Serum BGP levels had significant positive correlations with serum Ca (r = 0.64, P less than 0.005), serum A1-p (r = 0.77, P less than 0.001) and serum PTH (r = 0.45, P less than 0.005). Urinary gamma-Gla levels also had significant positive correlations with serum Ca (r = 0.50, P less than 0.05), serum A1-p (r = 0.67, P less than 0.005), serum 1,25(OH)2D (r = 0.62, P less than 0.02), and serum BGP (r = 0.72, P less than 0.001). Mineral content in the left radius had significant negative correlations with serum BGP levels (r = -0.73, P less than 0.001) and urinary gamma-Gla levels (r = -0.59, P less than 0.01). As these data show, serum BGP and urinary gamma-Gla levels clearly reflect the abnormal bone metabolism and can therefore be useful biochemical markers in PHP.     

Suppressive effect of human natural killer cells on Epstein-Barr virus-induced immunoglobulin synthesis

The suppressive effect of human natural killer (NK) cells on Epstein-Barr virus (EBV)-induced immunoglobulin (Ig) synthesis by autologous B cells was investigated. By Percoll discontinuous density gradient centrifugation, low-density fractions enriched for NK cells were isolated from human peripheral blood lymphocytes. These NK-enriched fractions were added to purified autologous B cells in the presence of EBV, were cultivated for 8 days, and were examined for their suppressive effect on Ig synthesis by an enzyme-linked immunosorbent assay. The fractions markedly suppressed both IgM and IgG synthesis induced by EBV. It was possible to reduce the suppressive effect of NK-enriched cells by complement-dependent lysis of NK cells and Leu-11, but not by OKT3 monoclonal antibody, indicating that NK cells may be responsible for the suppression of Ig synthesis. Upon close examination of interferon (IFN) activity, it was revealed that the co-cultures of NK-enriched cells and EBV-infected B cells generated production of IFN-alpha, which might be produced by NK cells in response to EBV-stimulated B cells. Addition of anti-IFN-alpha but not anti-IFN-gamma serum almost completely abrogated the suppressive effect of NK-enriched cells on Ig synthesis, indicating that IFN-alpha produced are required for the NK cell-mediated suppression of Ig synthesis. However, addition of IFN-alpha into purified B cells showed no direct suppressive effect on EBV-induced Ig synthesis by B cells in the absence of NK cells. Nevertheless, NK cells when previously incubated with IFN-alpha and added to B cells showed a suppressor activity on Ig synthesis to a level higher than that of untreated NK controls. These results strongly suggest the possibility that NK cells display an interaction with EBV-infected B cells and produce IFN-alpha, which in turn activates NK cells. These activated NK cells suppress the Ig synthesis by B cells, which undergo transformation induced by EBV.     

The pyrogallol related compounds reduce UV-induced mutations in Escherichia coli B/r WP2

Plant components with bio-antimutagenic activity were screened on UVC (254 nm)-induced mutagenesis using E. coli B/r WP2. The components with a pyrogallol moiety including gallic acid, (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG) reduced the mutation induction, but other components such as caffeic acid, chlorogenic acid and quercetin did not. The above compounds with a pyrogallol moiety were also effective on UVAB (295-400 nm)-induced mutagenesis, while they showed little effect on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutagenesis. As this bio-antimutagenic effect was not seen in the DNA excision-repair-deficient strains WP2s and ZA159, the activity by the above plant components might be based on the promotion of the excision-repair system in E. coli B/r WP2.     

Improved method for the isolation and preliminary characterization of human DAF (decay-accelerating factor)

DAF (decay-accelerating factor) is one of the integral membrane proteins of erythrocytes, and is considered to play an important role in the regulation of complement activation. The purification of DAF has been impeded by the difficulty in removing glycophorin. We devised an effective method for removing glycophorin. Through the limited trypsinization of stromata prior to the extraction of DAF, glycophorin was readily digested so that the DAF could be purified free of glycophorin by DEAE-Sephacel and Bio-Gel A 0.5 m chromatographies. On SDS-PAGE, DAF from trypsinized stromata showed the same mobility as that from native stromata: its molecular weight was estimated to be about 70 kDa. Amino acid analysis of DAF showed high contents of serine and glutamic acid. The amino-terminal sequence of DAF prepared by the present method, determined for the 29 residues, did not show significant homology with that of glycophorin.     

GDPmannose dolicholphosphate mannosyltransferase of chicken liver mitochondria

Chicken liver mitochondria contain enzymes for the dolichol cycle. GDPmannose dolicholphosphate mannosyltransferase has been solubilized with Emulgen 909 and purified. The purified enzyme was not homogeneous, but highly specific for GDPmannose and dolichyl phosphate. The enzyme activity was stimulated by MgCl2 (3 mM optimum) and exhibited a pH optimum at around 7.2. Bisubstrate kinetic analysis indicated that the enzyme follows a sequential mechanism. The Km values for GDPmannose and dolichyl phosphate were 0.43 and 14.3 microM, respectively. The purified enzyme was labile and lost its activity on storage at 0 degree C overnight or incubation at 30 degrees C or higher temperature. Inactivation could be prevented by the addition of heat-denatured mitochondrial extract. Further investigation revealed that phospholipids and dolichyl phosphate are responsible for the stabilization. Single addition of either phospholipid or dolichyl phosphate showed little activity, but the combination of these lipids enhanced the stabilizing activity greatly. Eight naturally occurring phospholipids were tested and found to be effective in combination with dolichyl phosphate. Among these, sphingomyelin was the most effective. Dolichol could partially substitute dolichyl phosphate but worked at higher concentrations.     

Specific binding sites for atrial natriuretic peptide (ANP) in cultured mesenchymal nonmyocardial cells from rat heart

Specific binding sites for atrial natriuretic peptide (ANP) were studied in cultured mesenchymal nonmyocardial cells (NMC) from rat heart. Binding study using 125I-labeled synthetic rat (r) ANP revealed the presence of a single class of high-affinity binding sites for rANP in cultured NMCs derived from both atria and ventricles; the apparent dissociation constant (Kd) was approximately 0.2 - 0.3 nM and the number of maximal binding sites was approximately 190,000 - 300,000 sites/cell. rANP significantly stimulated intracellular cGMP formation of cardiac NMCs in a dose-dependent manner (1.6 X 10(-8) M - 3.2 X 10(-7) M). rANP had no effect on synthesis of prostaglandin I2 by cultured cardiac NMCs. The physiological significance of ANP action on cardiac tissue remains to be determined.     

Defective endocytosis of low-density lipoprotein in monensin-resistant mutants of the mouse Balb/3T3 cell line

Two monensin-resistant clones show similar low-density lipoprotein binding activity but less internalization or degradation of low-density lipoprotein than the parental Balb/3T3 or other resistant clone. Sterol synthesis from radioactive acetate in the resistant mutant, MO-5, is inhibited by more than 70% of control in the presence of tenfold higher amounts of low-density lipoprotein than the dose that inhibits the parental Balb/3T3 to similar level. 3-Hydroxy-3-methylglutaryl coenzyme A reductase activity of Balb/3T3 and MO-5 is inhibited by 48% and 27% of control, respectively, in the presence of 10 micrograms/ml of low-density lipoprotein. Colloidal silica gradient centrifugation shows that transport of low-density lipoprotein from the surface membrane to the lysosome is much slower in MO-5 cells than in Balb/3T3 cells. Down regulation of low-density lipoprotein receptors on the cell surface in Balb/3T3 is observed by exposing the cells to 5-15 micrograms/ml low-density lipoprotein, whereas only slight if any down regulation is observed when MO-5 cells are treated with low-density lipoprotein. The altered endocytosis of low-density lipoprotein behaves as a dominant trait in hybrids of MO-5 and THO2-2, a derivative of Balb/3T3 resistant to both ouabain and 6-thioguanine.     

Methyl cinnamate derivatives enhance UV-induced mutagenesis due to the inhibition of DNA excision repair in Escherichia coli B/r

UV-induced mutagenesis in Escherichia coli B/r WP2 was enhanced by certain derivatives of methyl cinnamate which themselves were not mutagenic. Methyl ferulate, methyl isoferulate and methyl sinapate showed this effect markedly. Such an enhancement effect was absent with the derivatives of cinnamic acid and ethyl cinnamate and was not observed in Escherichia coli WP2s uvrA. Methyl sinapate also enhanced 4NQO-induced mutation and suppressed liquid-holding recovery in the above repair-proficient strain. The presence of methyl sinapate in plating agar medium decreased the survival of UV-irradiated cells of a recombination-repair-deficient strain, CM571 recA. However, the effect was not observed with those of WP2s uvrA. In an in vitro experiment in which the removal rate of thymine dimers was measured, methyl sinapate clearly inhibited this repair event. From these results, we conclude that methyl sinapate inhibits DNA excision repair, thus enhancing UV mutagenicity.     

The standardization of the candidate national standard anti-D blood-typing serum with the Groupamatic MG50 system

The utility of the Groupamatic MG50 system for quantitative expression of agglutination in terms of continuous response was studied. By use of the characteristics of the design of this system, in which the change in voltage reflects the degree of agglutination, a linear regression relationship between the log ratio of light flux obtained by transformation of the voltage and the log dilution factor of the serum was demonstrated. The availability of the parallel line assay method to the standardization of the blood grouping antisera was also described.     

Isolation and characterization of rabbit H of the alternative complement pathway

Rabbit factor H, a control protein of the alternative complement pathway, was isolated from rabbit serum by polyethylene glycol precipitation, DEAE-Sephacel chromatography, and gel chromatography on Sephadex G200. The protein migrated as a single-chain polypeptide with a molecular weight of 160,000 on sodium dodecyl sulfate-gel electrophoresis with Laemmli's buffer system, but hardly migrated into the gel with Fairbanks' buffer system. Physical and chemical properties of rabbit H were similar to those of human H, except that fragments produced by limited tryptic digestion from rabbit H had different molecular sizes from those produced from human H. Significant species-specificity was observed in the functional activity of factor H; activation of the alternative complement pathway was inhibited more efficiently with homologous H than with heterologous H. In contrast, factor H inhibited the hemolysis of homologous erythrocytes less than that of heterologous erythrocytes.