Purification and properties of an extracellular β-glucosidase from the cellulolytic thermophile Clostridium stercorarium

Summary Clostridium stercorarium cultures grown on cellobiose contain both an extracellular and a cell-bound -glucosidase activity. A substantial portion of the cell-bound enzyme could be extracted by osmotic shock, suggesting a periplasmic localization. The -glucosidase present in culture supernatants was purified to homogeneity. It was found to be identical in all aspects tested with the cell-bound -glucosidase. The enzyme exists as a monomer with an apparent molecular weight of 85.000 (SDS-PAGE) and a pI of 4.8. It shows optimal activity as pH 5.5 and 65° C. Thiol groups are essential for enzyme activity. In the presence of reducing agents and divalent cations the half-life of the purified enzyme was more than 5 h at 60°C. The enzyme hydrolyses at different rates a wide range of substrates including aryl--glucosides, cellobiose, and disordered cellulose. K _m values were determined as 0.8 mM for p-nitrophenyl--glucoside (PNPG) and 33 mM for cellobiose. The cellular localization and the substrate specificity pattern are consistent with a dual role of the C. stercorarium -glucosidase in cellulose saccharification: (1) Cleavage of cellobiose formed by exoglucanase and (2) degradation of cellodextrins produced by endoglucanase action.     

Cardiovascular and Pulmonary Effects of a New Sedative/Analgesic (Medetomidine) as a Preanaesthetic Drug in the Dog

Cardiovascular and pulmonary effects of a new sedative/analgesic (medetomidine) as a preanaesthetic drug in the dog. A study was carried out to investigate the possible usefulness of medetomidine (Farmos Group, Turku, Finland) for premedication prior to general anaesthesia with thiopental sodium and halothane. The main emphasis was laid on the circulatory and respiratory effects of medetomidine. Dogs treated with xylazine (2 mg/kg) or placebo (physiological saline solution) served as controls. Medetomidine caused a decrease in blood pressure, heart rate and respiratory rate at all dose levels tested. These decreases were essentially dose -dependent, but there were great individual variations. It is concluded that the drug can be useful for premedication at the lowest dose level tested (10 μ/kg). The sedative effect, however, is so strong that an even lower dose might be sufficient for the present purpose.     

Glycosphingolipid expression in spontaneously aborted fetuses and placenta from blood group p women. Evidence for placenta being the primary target for anti-Tja-antibodies

A 12-week-old fetus and one 17-week-old fetus + placenta were obtained after spontaneous abortions from two women of blood group p. The 17-week-old fetus was dissected into intestine, liver, brain and residual tissue. Nonacid glycosphingolipid fractions were prepared from the tissues. Glycolipid characterization was carried out using thin layer chromatography immunostained with monoclonal antibodies and bacteria and by¹H NMR spectroscopy and mass spectrometry. In the placental fraction substantial amounts of globotetraosylceramide (P-antigen) and globotriaosylceramide (P^k-antigen) were identified. In contrast, the fetuses contained only trace amounts of these structures, as revealed by immunostaining. These results indicate that the primary target for the antibodies of the anti-Tj^a serum is the placenta tissue, resulting in termination of the pregnancy.     

Cytogenetic and molecular characterization of a newly established neuroblastoma cell line LS

Summary A new human neuroblastoma cell line (LS) that originated from an abdominal tumor of a 16-month-old girl is presented; it was classified, according to Evans, as being stage III. Morphological (dense-core particles) and biochemical characteristics (dopamine--hydroxylase, acetylcholinesterase, neuron-specific-enolase) confirmed the diagnosis. In addition to a slightly variable modal chromosome number of 48 or 49 (because of marker-chromosomes and autosomal trisomies), cytogenetic analysis revealed two constantly appearing chromosomes with homogeneously stained regions (HSR's). The karyo-type remained constant over 50 passages in vitro [49,XX, –12,+der5, + 17,+mar1,+mar2]. Double minutes were a rare phenomenon and appeared only in a few metaphases. In situ hybridization showed that some of the HSR's consisted of amplified N-myc copies. The distribution of the N-myc copies according to in situ hybridization signals along the HSR's was compared with the data of Southern and Northern blotting analyses.     

Genotype-specific soluble auxin-binding in etiolated pea epicotyls

Using different independent procedures for assaying soluble auxin-binding in etiolated pea epicotyls, wo could prove the reliability of the (XH₄)₂SO₄-pelleting assay both for crude cytosols as well as for specific protein fractions obtained after chromatofocusing. Three distinct genotypes (two parent lines, one tall recombinant) investigated so far exhibit characteristic differences with respect to soluble auxin-binding kinetics in their cytosols.     

ESCHERICHIA COLI Rho Factor Is Involved in Lysis of Bacteriophage T4-Infected Cells

A Rid (Rho interaction deficient) phenotype of bacteriophage T4 mutants was defined by cold-sensitive restriction (lack of plaque formation) on rho+ hosts carrying additional polar mutations in unrelated genes, coupled to suppression (plaque formation) in otherwise isogenic strains carrying either a polarity-suppressing rho or a multicopy plasmid expressing the rho+ allele. This suggests that the restriction may be due to lower levels of Rho than what is available to T4 in the suppressing strains.--Rid394 X 4 was isolated upon hydroxylamine mutagenesis and mapped in the t gene; other t mutants (and mot, as well as dda dexA double mutants) also showed a Rid phenotype. In liquid culture in strains that restricted plaque formation Rid394 X 4 showed strong lysis inhibition (a known t- phenotype) but no prolonged phage production (another well-known t- phenotype). This implies that when Rho is limiting the t mutant shuts off phage production at the normal time. Lysis inhibition was partially relieved, and phage production prolonged to varying extents depending on growth conditions in strains that allowed plaque formation. No significant effect on early gene expression were found. Apparently, both mutant (polarity-suppressing) and wild-type Rho can function in prolonging phage production and partially relieving lysis inhibition of Rid394 X 4 when present at a sufficiently high level, and Rho may play other role(s) in T4 development than in early gene regulation.     

Organization of the ribosomal ribonucleic acid genes in various wild-type strains and wild-collected strains of Neurospora

Summary The organization of the ribosomal DNA (rDNA) repcat unit in the standard wild-type strain of Neurospora crassa, 74-OR23-1A, and in 30 other wild-type strains and wild-collected strains of N. crassa, N. tetrasperma, N. sitophila, N. intermedia, and N. discreta isolated from nature, was investigated by restriction enzyme digestion of genomic DNA, and probing of the Southern-blotted DNA fragments with specific cloned pieces of the rDNA unit from 74-OR23-1A. The size of the rDNA unit in 74-OR23-1A was shown to be 9.20 kilobase pairs (kb) from blotting data, and the average for all strains was 9.11+0.21 kb; standard error=0.038; coefficient of variation (C.V.)=2.34%. These data indicate that the rDNA repeat unit size has been highly conserved among the Neurospora strains investigated. However, while all strains have a conserved HindIII site near the 5 end of the 25 S rDNA coding sequence, a polymorphism in the number and/or position of HindIII sites in the nontranscribed spacer region was found between strains. The 74-OR23-1A strain has two HindIII sites in the spacer, while others have from 0 to at least 3. This restriction site polymorphism is strain-specific and not species-specific. It was confirmed for some strains by restriction analysis of clones containing most of the rDNA repeat unit. The current restriction map of the 74-OR23-1A rDNA repeat unit is presented.