Preparative purification of the rat mast cell chymase. Characterization and Interaction with granule components

The rat mast cell granule chymotrypsinlike enzyme was purified to homogeneity from 1 M NaCl solubilized membrane and granule-rich fractions of concentrated rat peritoneal mast cells by a preparative technique utilizing chromatography on Dowex 1, filtration on Sephadex G-75, and affinity chromatography with D-tryptophan methyl ester. Acid disk gel electrophoresis of the purified chymase disclosed a single stained band with activity being eluted from a replicate sliced gel in the same region. SDS-polyacrylamide gel electrophoresis of purified protein gave a single stained band that did not change in position with reduction and alkylation. Mast cell chymase is thus a cationic protein of 25,000 mol wt composed of a single polypeptide chain. The apparent K(m) of the chymase for BTEE was 1.5 x 10(-3) M and the V(max) was 67.8 μmol/min per mg. The enzyme was inhibited by TPCK and not by TLCK. The chymase complexed with native macromolecular rat mast cell heparin in molar ratios of 12:1 and 16:1, and complete heparin uptake occurred at a 40:1 ratio of chymase to heparin. Chymase activity was partially masked by combination with heparin in the isolated granule or experimental chymase-heparin complex, and soluble purified chymase was inhibited by concentrations of 5-HT comparable to those present in mast cells. It is therefore possible that the active site of chymase in the mast cell granule is largely masked by the combined effects of macromolecular heparin and 5-HT.     

Anti-VH antibodies interfere with antigen binding by human T lymphocytes.

The effect of antisera against a VH fragment have been investigated in several T cell proliferative assay systems. Anti-VH antisera raised in sheep, rabbits and chicken induced profound inhibition of PPD stimulated lymphoproliferation. Likewise were both mixed lymphocyte reaction (MLR) and autologous mixed lymphocyte reaction (AMLR) severely hampered while stimulation induced by mitogens was only minimally affected. Specificity testing indicates that the inhibiting antibodies in these experiments are not directed against native immunoglobulin determinants but rather against determinants specific for the VH fragment. These results thus support the notion that T cells express VH antigens and that these antigens are part of or closely associated with the antigen receptor on human T lymphocytes.     

Dose-response study of probiotic bacteria Bifidobacterium animalis subsp lactis BB-12 and Lactobacillus paracasei subsp paracasei CRL-341 in healthy young adults

OBJECTIVE: This study was performed to investigate the dose-response effects of supplementation with Bifidobacterium animalis subsp lactis (BB-12) and Lactobacillus paracasei subsp paracasei (CRL-431) on blood lipids, recovery from feces and bowel habits. Changes of the fecal microflora was analyzed in the 10(10) CFU/day probiotic and placebo group. DESIGN: The study was designed as a randomized, placebo-controlled, double-blinded, parallel dose-response study. SUBJECTS: Healthy young adults (18-40 years) were recruited by advertising in local newspapers. Of the 75 persons enrolled, 71 (46 women, 25 men, mean age 25.6 years (range 18-40 years)) completed the study. INTERVENTION: The volunteers were randomly assigned into five groups receiving either placebo or a mixture of the two probiotics in the concentration of 10(8), 10(9), 10(10) or 10(11) CFU/day in 2 weeks run-in period, 3 weeks intervention and 2 weeks wash-out. Diary reporting bowel habits and well being (abdominal bloating, flatulence and headache) was kept for all 7 weeks and blood lipids, fecal recovery of BB-12 and CRL-431, as well as fecal microflora was tested before, immediately and 2 weeks after intervention. RESULTS: The fecal recovery of BB-12 increased significantly (P < 0.001) with increasing dose. In the group receiving 10(11) CFU/day BB-12 was recovered from 13 out of 15 volunteers. CRL-431 was not recovered in any of the fecal samples. Supplementation with probiotics did not change the fecal bacterial composition. A significant linear increase in fecal consistency (looser stool) with increasing probiotic dose (P = 0.018) was observed. No overall dose-response effect was found on the blood lipids. High doses of probiotics were well tolerated. CONCLUSION: A dose-related recovery of BB-12 from feces was observed.     

Iron supplementation increases small intestine permeability in primary schoolchildren in Lusaka, Zambia

Elevated intestinal permeability, measured as an increased lactulose:mannitol (L:M) ratio, indicates injury of the small intestinal mucosa. As part of a randomized iron and multi-micronutrient (without iron) supplementation trial (Nchito et al., 2004), we determined intestinal permeability in a subgroup of schoolchildren at 10 months' follow-up to assess the effect of the interventions. Among 153 children (mean age 10.2 years and 53.6% girls) iron supplementation resulted in a higher L:M ratio compared with placebo (0.29 vs. 0.21, P=0.025). There was no effect of multi-micronutrient supplementation, and no interaction between the interventions. The finding could be one of the mechanisms explaining the negative effects of medicinal iron supplementation on morbidity found in some other studies.     

Alteration of the conformation of human IgG subclasses by reduction of the hinge S-S bonds

Human IgG changed molecular size upon mild reduction and alkylation as shown by HPLC gel filtration. IgG1, IgG2 and IgG4 proteins increased in molecular size while IgG3 proteins were decreased in molecular size by this treatment. Several proteins within each subclass covering different light chain types and Gm types were tested all showing the same effect. A plausible explanation was related to the hinge and to the CH2 region since Fab fragments experienced unchanged molecular size irrespective of IgG subclass while Fc (of IgG1, IgG2, IgG3, containing only two aa of the 62 aa long hinge of IgG3 and IgG4) increased in size and Fch (which contains most of the 62 aa long hinge region of IgG3) decreased in size upon reduction and alkylation. It is postulated that reduction of the hinge S-S bonds permit the IgG and Fc molecules to open up in the CH2 region due to the lack of trans-interaction here, resulting in a larger molecular size. For IgG3 and Fch (from IgG3) molecules there was an opposite and even greater effect on the open polyproline like structure of the gamma 3 hinge which depends on intact S-S bonds (there are 11 bonds here). Reduction of these S-S bonds apparently breaks down this open hinge structure resulting in a net decrease in molecular size of IgG3 and Fch molecules.