Association of beta-glucosidase with intact cells of Thermoactinomyces.

The location of the B-glucosidase activity in a whole culture broth of the thermophilic organism Thermoactinomyces has been studied. Little beta-glucosidase activity was found in the culture filtrate, while the culture solids contained the major part of the activity of the whole culture broth. The activity does not appear to be adsorbed to the culture solids; rather there is evidence that it is an intracellular soluble enzyme(s). The pH and temperature optima for a crude beta-glucosidase preparation were determined to be pH 6.5 and 50--55 degrees C. Enzyme activity studies indicate that the same enzyme(s) accounts for the beta-glucosidase and the cellobiase activities. The validity of using the filter paper activity of culture filtrates from Thermoactinomyces to predict the total saccharification of cellulosic materials to glucose is discussed.     

Interleukin 1 preferentially stimulates the production of tissue-type plasminogen activator by human articular chondrocytes

Interleukin 1, derived from human placenta, stimulates plasminogen activator activity in human articular chondrocytes. The stimulation of plasminogen activator activity can be abolished by preincubation of placental interleukin 1 with an antiserum to homogeneous 22K factor, a species of interleukin 1 beta, indicating that the stimulation of plasminogen activator activity is due to interleukin 1 and not contaminating factors. Chondrocytes produce three species of plasminogen activator, with apparent Mr approximately 50,000, 65,000 and 100,000 as determined after sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis with gels containing casein and plasminogen. Both placental interleukin 1 and 22K factor enhance the production of the species of Mr approximately 65,000 and 100,000. Comparison of the mobility of the plasminogen activator species on SDS-polyacrylamide gel electrophoresis with human urokinase (u-PA) and human melanoma tissue-type plasminogen activator (t-PA) and studies with antibodies to these enzymes indicate that the Mr approximately 50,000 species is a u-PA and the Mr approximately 65,000 a t-PA. The Mr approximately 100,000 species is possibly an enzyme-inhibitor complex. Interleukin 1 therefore appears to enhance the production of t-PA and a putative enzyme-inhibitor complex. Abolition of plasminogen activator activity in the fibrin plate assay with antibodies to t-PA and u-PA also confirms enhanced t-PA production on interleukin 1 stimulation, though there is also evidence for increased cell-associated production of u-PA.     

Modulation of human chondrocyte metabolism by recombinant human interferon gamma: in-vitro effects on basal and IL-1-stimulated proteinase production, cartilage degradation and DNA synthesis

Human articular chondrocytes in monolayer culture and fragments of human articular cartilage were treated with recombinant human interferon gamma (IFN-gamma) both alone and in combination with interleukin 1 (IL-1). IFN-gamma alone inhibits metalloproteinase production, as measured in the caseinase assay, and decreases glycosaminoglycan release from cartilage fragments in culture. The synthesis of DNA, as measured by [3H]thymidine incorporation, is stimulated by IFN-gamma. Similar effects are seen in the presence of IL-1. Thus, IFN-gamma opposes the stimulatory effect of IL-1 on caseinase production and decreases IL-1-stimulated cartilage degradation, as measured by glycosaminoglycan release. In contrast, IFN-gamma has no effect on IL-1-stimulated prostaglandin production, and acts synergistically with IL-1 to cause a large stimulation of DNA synthesis. These results show that IFN-gamma has a number of effects on articular chondrocytes in-vitro and suggest a possible role for IFN-gamma in limiting cartilage degradation in inflammatory joint conditions.     

Reaction of anti-HLA-B monoclonal antibodies with envelope proteins of Shigella species. Evidence for molecular mimicry in the spondyloarthropathies

Immunologic cross-reactivity between enteric bacteria and the HLA-B27 protein may play a role in the etiology of Reiter's syndrome and reactive arthritis. The reactivity of two anti-B27 mAb (B27M1 and B27M2) with envelope proteins of Shigella flexneri isolated from Reiter's syndrome patients was studied by Western blot analysis. Proteins with an apparent Mr of approximately 36 and 23 kDa reacted with both mAb in ascites. mAb against related HLA class I Ag B7 and B40 did not react with the 23 kDa protein. Relatively high concentrations of antibody were required for reactivity, suggesting a low affinity interaction. Additional evidence for cross-reactive epitopes was obtained by ELISA against whole envelope and by using unsolubilized envelope to inhibit binding of M1 and M2 to B27-positive cell lines, as measured by quantitative flow microfluorimetry. The presence of cross-reactive proteins was not related to the presence of the intact virulence-associated plasmid or the invasive phenotype. Two Shigella sonnei isolates not implicated as causative agents of Reiter's syndrome or reactive arthritis showed a similar pattern of cross-reactivity. These results indicate that cross-reactive epitopes may be present on "arthritogenic" bacteria, but their presence is not a unique feature of such strains and is not the sole factor in induction of arthritis in B27-positive individuals.     

Linkage to Xq28 in a family with nonspecific X-linked mental retardation

Linkage analysis was performed in a family with nonspecific X-linked mental retardation (MRX). Affected individuals had no clinical characteristics other than mental retardation. Linkage was detected to the marker loci DXS477, DXS465, DXS52, DXS15 and F8C with maximum lod scores of 1.70, 1.32, 2.52, 1.70, and 1.09, respectively ( = 0.0). The results strongly indicate that the gene for mental retardation in the family studied maps close to DXS52.     

Some Acridine-Resistant Mutations of Bacteriophage T4d

Three new 9-aminoacridine (9AA) resistant mutations of bacteriophage T4D have been isolated and characterized. Two of the mutations, rs and rc, have identical patterns of acridine resistance, but they map on opposite sides of the rII region. In addition, rs has an effect on the plaque morphology of r mutations, whereas rc does not. The third mutation, ama, maps very close to rs but exhibits a different pattern of resistance to 9AA. None of the three is resistant to acridines by virtue of reduced permeability. Taken together with other mutations that have been previously characterized, these new mutations permit us to set the minimum number of acridine-sensitive processes in T4 development at four.     

Location of the SPO2 Attachment Site and the Bryamycin Resistance Marker on the Bacillus subtilis Chromosome

The attachment site on the Bacillus subtilis chromosome for the lysogenic bacteriophage SPO2 has been mapped by PBS1-mediated transduction and was found to be between spc-2 and lin-2, showing 90% linkage to the former and 30% linkage to the latter marker. In the course of these studies the bry-2 marker was mapped between the cysA14 and str-1 loci.     

ENZYME LOCALIZATION DURING MELANOGENESIS

The DOPA-reaction was used to identify tyrosinase in the nucleus and cytoplasm of the neural crest melanoblast of Taricha torosa, the California newt. In this urodele there is a nuclear DOPA-positive response during the normal embryonic development from the late blastula stage to the nucleus of the early melanocyte. During the gastrula stages, all nuclei of this newt are DOPA-positive. This positive nuclear response fades away after the formation of the neural crest, save in the melanoblasts. The only cells that give a positive DOPA marking in the cytoplasm are the melanoblasts. This cytoplasmic reaction appears while the melanoblast nucleus still gives a DOPA-positive reaction. Tyrosinase activity, as marked by unlabeled DOPA, has ceased in the fully mature melanocyte. The red nuclei, seen in some of the animals in the maturing melanocyte and adjacent tissues, may be in the hallachrome stage of melanin formation. There is a diffuse distribution of DOPA reactivity in the resting nucleus, and an adherence of the DOPA-marking in the region of the dividing chromosomes in the mitosis of DOPA-positive nuclei of the melanoblast. These observations suggest that tyrosinase may be among the chromosomally bound enzymes of the chromatin space.     

Micro Radiometric Method for Study of Acid-insoluble Materials in Monolayer Cell Cultures

A simple radiometric procedure for study of acid-insoluble products synthesized in monolayer cell cultures is described. Cell cultures were produced directly on the bottom surface of scintillation vials or on glass cover slips (8 X 30 mm). The cells were labeled and extracted; the radioactivity was determined while the cells remained affixed to the glass surface upon which they were grown. This procedure enabled rapid investigations of certain biosynthetic processes to be carried out by using many individual cell cultures. The method was applied to an investigation of (3)H-thymidine incorporation induced by vaccinia virus in a 5-bromodeoxyuridine-resistant cell line. (14)C-labeling was evaluated as an alternate procedure for cell quantitation.     

Vaccinia Neutralization Test Using a Radioisotope Assay for Virus-induced Enzyme Activity

Vaccinia virus induction of a metabolic activity in host cell cultures forms the basis of a new assay for neutralizing antibodies. A direct relationship between the amount of vaccinia virus infecting cell cultures and the induced incorporation of tritium-labeled thymidine into the acid-insoluble fraction of the cells provided an indicator system. A liquid scintillation spectrometer was used to determine radioactivity associated with cell materials, and it provided a method for partial automation of an immunological procedure. Reproducibility of the method was satisfactory, and agreement with conventional vaccinia serum neutralization tests was demonstrated.