Immunohistochemical analysis of muscle cytochromec oxidase deficiency in children

Despite the demonstration of a clear biochemical defect, the genetic alterations causing childhood forms of cytochromec oxidase (COX) deficiency remain unknown. The double genetic origin (nuclear and mitochondrial DNA), and the complexity of COX enzyme structure and regulation, indicate the need for genetic iinvestigations of the molecular structure of individual COX subunits. In the present study a new monoclonal antibody, which reacts exclusively with heart-type human COX subunit VIIa (VIIa-H), and other monoclonal antibodies against human COX subunits, were used in the immunohistochemical analysis of skeletal muscle from children with different forms of mitochondrial myopathy with COX deficiency. By immunohistochemical investigation a normal reaction was seenn with antibodies to COX subunits IV, Va+Vb, and VIa+VIc in all four cases, and in two cases with antibodies to COX VIIa-H and VIIa+VIIb. In muscle from a fatal infantile case with cardiac and skeletal muscle involvement, no immunohistochemical reaction was seen with the monoclonal antibody against the tissue-specific subunit VIIa-H. In muscle from an 11-year-old boy with exclusive muscular symptoms and signs, immunohistological reactions were absent with COX subunit VIIa-H and COX subunits VIIa+VIIb, and slightly decreased with COX subunit II, thus demonstrating a different molecular mechanism in each case. It is concluded that the molecular basis of COX deficiency in childhood may vary greatly between patients.     

Evidence for lysosomal enzyme recognition by human fibroblasts via a phosphorylated carbohydrate moiety

Adsorptive endocytosis of five different lysosomal enzymes from various human and non-human sources was susceptible to inhibition by mannose and l-fucose, methyl α-d-mannoside, α-anomeric p-nitrophenyl glycosides of mannose and l-fucose, mannose 6-phosphate and fructose 1-phosphate. A few exceptions from this general scheme were observed for particular enzymes, particularly for β-glucuronidase from human urine. The inhibition of α-N-acetylglucosaminidase endocytosis by mannose, p-nitrophenyl α-d-mannoside and mannose 6-phosphate was shown to be competitive. The loss of endocytosis after alkaline phosphatase treatment of lysosomal enzymes supports the hypothesis that the phosphorylated sugars compete with a phosphorylated carbohydrate on the enzymes for binding to the cell-surface receptors [Kaplan, Achord & Sly (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2026–2030]. Endocytosis of `low-uptake' forms of α-N-acetylglucosaminidase and α-mannosidase was likewise susceptible to inhibition by sugar phosphates and by alkaline phosphatase treatment, suggesting that `low-uptake' forms are either contaminated with `high-uptake' forms or are internalized via the same route as `high-uptake' forms. The existence of an alternative route for adsorptive endocytosis of lysosomal enzymes is indicated by the unaffected adsorptive endocytosis of rat liver β-glucuronidase in the presence of phosphorylated sugars and after treatment with alkaline phosphatase.     

Stimulation of heparan sulphate synthesis in cultured rat hepatocytes by (+)-catechin.

The secretion of heparan sulphate by cultured rat hepatocytes was increased in the presence of (+)-catechin. The increase was due to a new species of heparan sulphate that lacked the carbohydrate-protein linkage between xylose and serine in normal heparan sulphate proteoglycan. The mean molecular weight of this heparan sulphate varied between 6300 and 9500, was not affected by treatment with alkali or Pronase and was 2-3-fold lower than that of chains released from heparan sulphate proteoglycan. After digestion with Pronase, only a minor fraction of chains contained serine, and after treatment with alkali and NaB3H4 reduction less than 5% of the chains exposed [3H]xylitol at the reducing terminals. These results suggested that (+)-catechin or metabolites of it acted as acceptors of heparan sulphate synthesis. In cultures treated wih cycloheximide, synthesis of heparan sulphate decreased to less than 5%. (+)-Catechin could restore the heparan sulphate synthesis to almost normal values. The (+)-catechin-induced heparan sulphate was secreted. Only a small fraction was incorporated into the plasma membrane or other cellular compartments. This may indicate that the protein core is essential for association of heparan sulphate with cellular compartments.     

Computation of the fraction of induced cells in enzyme induction systems

A theoretical model is developed for continuous multistage enzyme production systems, which consist of a growth fermentor used for growing microorganisms rapidly without enzyme production and a subsequent system of induction reactors in which enzymes induction and production occurs. The model allows the computation of the fraction of induced cells residing in the induction reactor for organisms exhibiting a lag phase in enzyme induction. For this model a general analytical solution was obtained for the cumulative internal residence time distribution of a series of n well-stirred vessels with a recycle. The theoretical results are compared in a preliminary way with experimentally measured cellulase productivities of continuous multistage cellulose fermentations with Trichoderma viride QM 9414.     

IgM and IgG but not cytokine secretion is restricted to the CD27+ B lymphocyte subset.

In a recent study we reported that CD27 is expressed on a subpopulation of human B lymphocytes and presented circumstantial phenotypic evidence that CD27 expression may be acquired late during B cell differentiation. Here we present functional data showing that, after in vitro stimulation, CD27+ but not CD27- B cells secrete large amounts of both IgM and IgG. Using double immunofluorescence staining of CD27 and IgD, three functionally different B cell subsets representing distinct stages of B cell differentiation can be isolated: 1) the CD27- IgD+ B cells, which do not secrete appreciable Ig; 2) the CD27+IgD+ B cells, which exclusively secrete IgM; and 3) the CD27+IgD- B cells, which comprise the IgG-producing cells. Furthermore, costimulation of CD27- B cells with low m.w. B cell growth factor, in the presence or in the absence of a CD40 mAb, does not induce these cells to become Ig-secreting cells. Although CD27- B cells hardly secrete Ig of any isotype in response to Staphylococcus aureus+IL-2, these cells proliferate vigorously and express the IL-2R alpha chain (CD25) under these stimulatory conditions. Furthermore, both CD27- and CD27+ B cells are capable of producing similar amounts of IL-6 and TNF-alpha. Taken together, these findings indicate that CD27 is a unique non-Ig surface marker discriminating naive from primed B lymphocytes. Furthermore, the capacity to proliferate and to secrete the B cell differentiation factors IL-6 and TNF-alpha already exists at an early B cell differentiation stage at which the cells lack CD27 expression and are not induced to produce Ig.     

Genetic variation in isozymes of wild boar (Sus scrofa ferus Linné)

Eleven isozyme systems were investigated in wild boar (Sus scrofa ferus L.) by means of horizontal starch gel-electrophoresis using liver and kidney extracts. Samples of 103 specimen that originated from three adjunct localities from Rhineland-Palatinate (Fed. Rep. Germany) were analysed. AAT, ACO, GDH, GPDH, LDH, ME, MPI, MR, PGDH, and PGI were invariant. Genetic polymorphism is described for PGM. The genetic polymorphism at Pgm2 is explained by a biallelic model. Observed genotypic structure did not differ significantly from Hardy-Wein-berg-proportions at this gene locus. The degree of heterozygosity is 58,3 % and Wright's Fixation Index F =–0.166 at Pgm2. The allelic structure at this gene locus differed to a great extent from that found in domestic swine, i. e. in domestic swine the faster migrating Pgm2 allozyme was only found in relative low frequencies compared to wild boar. The genetic polymorphisms were moderate in the wild boar population compared to electrophoretic data of other animal species.     

Use of activated clotting time for monitoring anticoagulation during cardiopulmonary bypass in infants and children with congenital heart disease

The use of a fixed dosage schedule was compared with the use of activated clotting time (ACT) for determining heparin and protamine dosages during and after cardiopulmonary bypass disease. Use of the ACT resulted in a statistically significant increase in heparin dosage and a statistically significant reduction of postoperative blood loss. With ACT use, chest tubes were retained for a shorter period of time, and the incidence of serious postoperative hemorrhage was reduced from 44% to 18%. These results confirm the superiority of the ACT method for monitoring intraoperative anticoagulation in pediatric patients with congenital heart disease.