We have purified to homogeneity a protein from bovine adrenal (fasciculata) cells that is capable of: 1) stimulating synthesis of pregnenolone (in a concentration-dependent fashion) by mitochondria when added with exogenous cholesterol; 2) increasing the concentration of cholesterol in outer and inner membrane when added to mitochondria with exogenous cholesterol; 3) increasing the rate of transport of cholesterol from outer membrane to inner membrane when the two membranes are incubated together in an aqueous buffer and subsequently separated by centrifugation; and 4) increasing the proportion of molecules of C27 side-chain cleavage P-450 that are bound to the substrate cholesterol. The protein is homogeneous according to two-dimensional polyacrylamide gels, shows a molecular weight of 8200 and is therefore referred to as 8.2 K. It is proposed that 8.2 K may be involved in the regulation of steroid synthesis in bovine fasciculata cells, possibly by stimulating entry of cholesterol into the outer membrane and by altering the intramitochondrial distribution of this substrate.

1. The effects of substrate concentration and enzyme source (human liver microsomes and recombinant cytochrome P450s, CYP) on the activation of 7-benzyloxyresorufin O-debenzylation and nifedipine oxidation were investigated. 2. 7-Benzyloxyresorufin O-debenzylase activity in human liver microsomes was inhibited by a monoclonal antibody against CYP2B6 and a polyclonal antibody against CYP3A2 by 53-69 and 19-44%, respectively, suggesting that CYP2B6 and CYP3A4 mainly catalyse 7-benzyloxyresorufin O-debenzylation in human liver microsomes. 3. 7-Benzyloxyresorufin O-debenzylase activity at 0.2-5 micro M substrate concentrations in human liver microsomes was increased by the addition of alpha-naphthoflavone, quinidine, testosterone and progesterone, and the V(max) of 7-benzyloxyresorufin O-debenzylation increased with increasing alpha-naphthoflavone concentrations, whereas the K(m) remained constant. Additionally, 7-benzyloxyresorufin O-debenzylation by recombinant CYP3A4 was increased by the addition of alpha-naphthoflavone, testosterone and progesterone but not by quinidine, whereas no chemicals tested could activate the O-debenzylation of 7-benzyloxyresorufin by CYP2B6. 4. The K(m) for nifedipine oxidation activity by CYP3A4 decreased by the addition of progesterone, whereas the V(max) remained constant. Quinidine and testosterone increased 7-benzyloxyresorufin O-debenzylase and nifedipine oxidase activities, respectively, in human liver microsomes, whereas activation was not observed in CYP3A4. 5. The results suggest that in vitro activation patterns are substrate dependent and that selection of the enzyme source can influence the activation phenomenon.

We investigated effects of variations in the cellular composition of G-CSF-mobilized peripheral blood progenitor cell (G-PBPC) allografts on clinical outcomes of allogeneic PBPC transplantation. We retrospectively analyzed transplanted doses of various immunocompetent cells from 27 HLA-identical sibling donors in relation to engraftment, incidence of graft-versus-host disease (GVHD), and survival. Significant variability was documented in both absolute numbers and relative proportions of CD34+, CD2+, CD3+, CD4(high)+, CD4+25+, CD8(high)+, CD19+, CD56+, and CD56+16+ cells contained in these allografts. Stepwise Cox regression analysis revealed that the CD56+ cell dose was significantly inversely correlated with the incidence of GVHD. Thus, there was a significantly higher incidence of grade II acute GVHD in patients receiving a lower CD56+16+ cell dose (hazard ratio (HR) 0.0090; 95% confidence interval (CI),<0.00001-3.38; P=0.031), a higher incidence of chronic GVHD in those receiving allografts with a lower CD56+16+ to CD34+ ratio (HR <0.00001; 95% CI <0.00001-0.0007; P=0.0035), and a higher incidence of extensive chronic GVHD in those receiving allografts with a lower CD56+ to CD34+ ratio (HR <0.00001; 95% CI <0.00001-0.053; P=0.0083). These results suggest that CD56+ cells in G-PBPC allografts from HLA-identical sibling donors may play an important role in preventing the development of GVHD.

In an attempt to elucidate the physiological relevance of the peripheral type of benzodiazepine receptor in adrenocortical mitochondria, we examined the effect of three different benzodiazepines (diazepam, Ro5-4864, and chlordiazepoxide) on the conversion of cholesterol to pregnenolone, the rate-limiting step in steroidogenesis, by using cholesterol-loaded mitochondria from bovine adrenal zona fasciculata. These benzodiazepines, except chlordiazepoxide, caused a dose-dependent stimulation of the cholesterol side chain cleavage in the mitochondria. The stimulatory effect of Ro5-4864 was approximately 10 times more potent than that of diazepam. No inhibitory effect of YM-684 (Ro15-1788), a potent antagonist to central-type benzodiazepine receptors, was observed in the stimulation induced by diazepam and Ro5-4864. Both external calcium ion and voltage-dependent calcium channel blocker, (+)-PN200-110, were without effect on the diazepam-induced steroidogenesis. By contrast, pretreatment of mitochondria with digitonin abolished the stimulatory effect of diazepam on the mitochondrial steroidogenesis. The present results indicate that the peripheral-type benzodiazepine receptor of adrenocortical mitochondria plays an essential role in regulating cholesterol side chain cleavage without any change of calcium channels.

The incidence of selective IgA deficiency (SIgAD) was determined in a healthy adult population of 222,597 Japanese volunteer blood donors. Of the blood donors screened, only 0.007% (1:14,840) were found to be IgA-deficient (less than 10 mg/dl) by means of the double diffusion method, while 0.005% (1:18,500) were less than 5 mg/dl, and 0.003% (1:31,800) were less than 1 mg/dl by means of the single radial immunodiffusion method. Statistical analysis of the results clearly showed that the incidence of SIgAD in Japanese blood donors is very much lower than that in blood donors of European ancestry. The Japanese population may occupy a unique position in the ethnical peculiarities. Anti-IgA antibodies were found in 3 (25.0%) of 12 IgA-deficient blood donors whose IgA levels were less than 5 mg/dl, a prevalence rate comparable to that in donors of European ancestry. Although it is difficult to develop a suitable file of IgA-deficient donors in Japan, the establishment of a Rare Donor Registry System on IgA deficiency is a matter of urgency.