Immunolabeling of carbonic anhydrase isoenzyme C and glial fibrillary acidic protein in paraffin-embedded tissue sections of human brain and retina

The specificities of carbonic anhydrase isoenzyme C (CA C) and glial fibrillary acidic (GFA) protein as immunocytochemical markers for different glial cell populations in human brain and retina were studied using indirect immunofluorescence and peroxidase-antiperoxidase complex methods. With antibodies against CA C, only those cerebral cells that were morphologically oligodendrocytes and Müller cells of the retina showed positive immunostaining reaction, whereas antibodies against GFA protein selectively labeled cerebral astrocytes and a part of the glial cells and fibers in the inner layers of the retina. In double labeling, when both glial cell markers were successively localized in the same cerebral tissue sections, GFA protein immunofluorescence was never found in the immunoperoxidase-stained CA C-positive cells, which further supports the oligodendrocyte-specificity of CA C in human brain.     

Immunofluorescence study of fimbrial phase variation in Escherichia coli KS71

An immunofluorescence assay was developed to study fimbrial phase variation in a pyelonephritogenic Escherichia coli strain, KS71. By using fluorochrome-labeled antibodies specific for either P, type-1C, or type-1 fimbriae of strain KS71, it was shown that in a broth culture of strain KS71 the fimbrial types mostly occurred on different cells. Only 9% of the cells carried more than one fimbrial type. The KS71 cell population was fractionated into subpopulations expressing only one of the fimbrial types or lacking fimbriae. Immunofluorescence assay of the subpopulations revealed a rapid phase variation in fimbrial synthesis. Kinetic analyses of a nonfimbriated cell population suggested that a change from one fimbrial phase to another was not totally random.     

Escherichia coli fimbriae recognizing sialyl galactosides

Fimbriae recognizing sialyl galactosides (S fimbriae) were purified from an Escherichia coli strain. The S fimbriae were morphologically identical to type 1 and P fimbriae of E. coli and showed a hemagglutination that was abolished when erythrocytes were treated with neuraminidase. Hemagglutination by the purified fimbriae was inhibited by orosomucoid but not by its desialylated derivative. Of the oligosaccharides tested, sialyl-(alpha 2-3)-lactose and sialyl-(alpha 2-3)-N-acetyllactosamine had the strongest inhibitory activities. It was concluded that S fimbriae have the strongest affinity for (alpha 2-3)-linked sialyl galactosides. In the enzyme-linked immunosorbent assay, the hyperimmune serum to the S fimbriae reacted strongly with the homologous antigen but not with type 1, P, or nonhemagglutinating KS71C fimbriae of E. coli. Analogously, the hyperimmune sera to the other E. coli fimbriae did not react with the purified S fimbriae. The immunoprecipitation assay showed that S fimbriae on different E. coli serotypes shared immunological cross-reactivity.     

Type 3 fimbriae of Klebsiella sp.: molecular characterization and role in bacterial adhesion to plant roots.

Type 3 fimbriae of Klebsiella were purified and characterized. The fimbriae were 4 to 5 nm in diameter and 0.5 to 2 microns long. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the fimbrillin had an apparent molecular weight of 23,500, and it differed from enterobacterial type 1 fimbrillins in its amino acid composition. Hydrophobic amino acids comprised 33.6% of all amino acids in the fimbrillin, which lacked cystine, phenylalanine, and arginine. Serologically, the type 3 fimbriae were also distinct from the type 1 fimbriae. Purified type 3 fimbriae agglutinated tannin-treated human blood group O erythrocytes; this confirms the role of type 3 fimbriae as hemagglutinins. Purified 125I-labeled type 3 fimbriae bound to the roots of Poa pratensis, and this binding could be inhibited by Fab fragments to the purified fimbriae. Anti-type 3 fimbriae Fab fragments also inhibited bacterial adhesion to plant roots. These results demonstrate that type 3 fimbriae mediate adhesion of klebsiellas to plant roots. Eight nitrogen-fixing strains of Klebsiella also produced type 3 fimbriae when grown under anaerobic nitrogen fixation conditions. It is proposed that type 3 fimbriae are involved in the establishment of the plant-bacterium association concerning nitrogen-fixing Klebsiella strains.     

Induction of aryl hydrocarbon hydroxylase in human fetal liver cell and fibroblast cultures by polycyclic hydrocarbons

Cells originating from the human fetal liver and grown as a primary monolayer culture for 4 to 11 days contain an enzyme system that metabolizes benzo(α) pyrene. The basal level of the enzyme varied about three-fold. The activity was increased from 1.4- to 5.1-fold by the exposure of cells for 24 hours to benz(α) anthracene, the magnitude of increase depending on the amount of inducer, on the individual cell batch studied and on the stage of cell growth. Also 3-methylcholanthrene, but not benzo(α)pyrene, induced the enzyme activity in fetal liver cell cultures at concentrations used. Fibroblast cultures derived from the human fetal lung or skin exhibited less benzo(α)pyrene metabolism and the inducibility of the enzyme activity was less marked than in hepatic cell cultures.     

Role of clozapine in the occurrence of chromosomal abnormalities in human bone-marrow cells in vivo and in cultured lymphocytes in vitro

Summary Bone-marrow chromosomes were examined from 38 mentally and physically retarded and two psychiatric patients who were being treated with a variety of neuropharmacologic drugs. Twenty of these patients used clozapine (Leponex^®). The clastogenic effects of clozapine in vitro were studied in the lymphocyte cultures of three patients-one free of hematologic disease and two who 6 months earlier had had agranulocytosis attributed to the use of clozapine. The mean frequency of cytogenetic abnormalities in the bonemarrow cells of patients who used clozapine was significantly increased (P> 0.05). The two patients who had had agranulocytosis had a greater frequency of cytogenetic abnormalities in their cultured lymphocytes in vivo and in vitro than the patient free of hematologic disease. A clone with a 13/14 chromosome translocation was detected in one of the patients. As all patients received a number of drugs during the in vivo and in vitro studies no definite conclusions could be drawn regarding the role played by clozapine in the occurrence of chromosomal abnormalities.     

Organ scaling in the raccoon dog Nyctereutes procyonoides gray 1834, as monitored by influences of internal and external factors

1. Animals fed a high energy ration had bigger body weight, and bigger heart, brain and genitals then animals fed a normal diet, but they had substantially smaller liver, kidneys, adrenals and thyroid glands than the otherwise smaller animals. Restricted feeding did not necessarily produce smaller organ sizes than normal. 2. The yearly variation in organ sizes was astonishingly large whereas the sex differences were rather rare. 3. For organs like liver, kidneys and thyroid glands the conclusion from the results was independent of the method of expressing the organ mass. The organ sizes seemed to be influenced by many coexisting factors like yearly differences, sex and age of animals, feed and farm.     

PKCepsilon-mediated phosphorylation of vimentin controls integrin recycling and motility

PKCepsilon controls the transport of endocytosed beta1-integrins to the plasma membrane regulating directional cell motility. Vimentin, an intermediate filament protein upregulated upon epithelial cell transformation, is shown here to be a proximal PKCepsilon target within the recycling integrin compartment. On inhibition of PKC and vimentin phosphorylation, integrins become trapped in vesicles and directional cell motility towards matrix is severely attenuated. In vitro reconstitution assays showed that PKCepsilon dissociates from integrin containing endocytic vesicles in a selectively phosphorylated vimentin containing complex. Mutagenesis of PKC (controlled) sites on vimentin and ectopic expression of the variant leads to the accumulation of intracellular PKCepsilon/integrin positive vesicles. Finally, introduction of ectopic wild-type vimentin is shown to promote cell motility in a PKCepsilon-dependent manner; alanine substitutions in PKC (controlled) sites on vimentin abolishes the ability of vimentin to induce cell migration, whereas the substitution of these sites with acidic residues enables vimentin to rescue motility of PKCepsilon null cells. Our results indicate that PKC-mediated phosphorylation of vimentin is a key process in integrin traffic through the cell.     

Characterisation of a secondary alcohol dehydrogenase from Xanthomonas campestris DSM 3586

The chromosomal locus NP_636946 of Xanthomonas campestris DSM 3586 (ATCC 33913) which was earlier presumed to encode a quinoprotein glucose dehydrogenase has been cloned, expressed in Escherichia coli and the recombinant enzyme has been characterised. It was found to have no glucose dehydrogenase activity but to be active on many different polyols and diols, aliphatic alcohols, certain aldonic acids and amino-sugars. The product of d-gluconic acid oxidation was 5-keto-d-gluconic acid. The enzyme differs from polyol/gluconate dehydrogenases found in Gluconobacter by its single-chain architecture, different substrate specificity and much higher (20- to 30-fold) expression level in E.coli.     

Reconstitution of a minimal mtDNA replisome in vitro

We here reconstitute a minimal mammalian mitochondrial DNA (mtDNA) replisome in vitro. The mtDNA polymerase (POLgamma) cannot use double-stranded DNA (dsDNA) as template for DNA synthesis. Similarly, the TWINKLE DNA helicase is unable to unwind longer stretches of dsDNA. In combination, POLgamma and TWINKLE form a processive replication machinery, which can use dsDNA as template to synthesize single-stranded DNA (ssDNA) molecules of about 2 kb. The addition of the mitochondrial ssDNA-binding protein stimulates the reaction further, generating DNA products of about 16 kb, the size of the mammalian mtDNA molecule. The observed DNA synthesis rate is 180 base pairs (bp)/min, corresponding closely to the previously calculated value of 270 bp/min for in vivo DNA replication. Our findings provide the first biochemical evidence that TWINKLE is the helicase at the mitochondrial DNA replication fork. Furthermore, mutations in TWINKLE and POLgamma cause autosomal dominant progressive external ophthalmoplegia (adPEO), a disorder associated with deletions in mitochondrial DNA. The functional interactions between TWINKLE and POLgamma thus explain why mutations in these two proteins cause an identical syndrome.