Isolation and characterization of a collagen fibril-associated dermatan sulphate proteoglycan from bovine lung

Dermatan sulphate proteoglycans have been extracted from bovine lung with 2.0 M CaCl2 and isolated using CsCl density gradient centrifugation, DEAE ion-exchange chromatography, gel chromatography and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Ultrastructurally these proteoglycans are specifically associated with collagen fibrils. Dermatan sulphate (Mr 15.10(3)-35.10(3), with a strong prevalence for the higher Mr) is link via an O-glycosidic bond to a protein core, which is rich in Asx, Glx and Leu. Of the total uronic acid, 91% is iduronic acid. A part of the glucuronic acid residues is located near the protein core and a large cluster of disaccharides is devoid of glucuronic acid residues. An inhibition enzyme immunoassay has been developed to quantitate the proteoglycan. A model for the interaction between dermatan sulphate proteoglycans and collagen fibrils is proposed.     

Stages of B-lymphocyte differentiation in acute lymphoblastic leukemia

Blasts phenotype was determined in 61 children with the acute lymphoblastic leukemia. Non-T-cell acute lymphoblastic leukemia was diagnosed in 51 children. Stages of blasts differentiation were determined with the aid of monoclonal antibodies set using alkaline phosphatase-anti-alkaline phosphatase technique. Blasts in 50 patients belonged to B subpopulation confirmed by the presence of panB CD19 and CD22 antigens. Common antigen was seen in 76.5% of the examined patients with non-T-cell acute lymphoblastic leukemia. Cases of non-T-cell acute lymphoblastic leukemia were divided into 8 subgroups depending on the antigens of B-cells differentiation. An identification of pre-B subgroups of the acute lymphoblastic leukemia indicates heterogenicity of the acute lymphoblastic leukemias in childhood and enables their classification into groups corresponding to the early stages of lymphoblasts maturation.     

DNA nucleotide sequence analysis of the immediate-early gene of pseudorabies virus.

The complete DNA sequence coding for the immediate-early protein (IE180) of pseudorabies virus was determined. The coding region of IE180 is 4380 nucleotides for 1460 amino acid residues. G+C content of the non-coding portion of the IE gene is 70.3% while the G+C content of the coding portion is considerably higher at 80.1%. Correspondingly, codons consisting mainly of Gs and Cs are favoured. Clusters of amino acid homologies are observed among IE180 of pseudorabies virus, ICP4 of herpes simplex virus type-1 and IE140 of varicella-zoster virus, and are organized similarly in all three polypeptides. Functions exhibited by IE180 are assigned, tentatively, to structural domains of the molecule by analogy to the HSV-1 ICP4 polypeptide.     

Vitamin K-dependent carboxylase: influence of the "propeptide" region on enzyme activity

The liver microsomal vitamin K-dependent carboxylase catalyzes the post-translational conversion of specific glutamyl to gamma-carboxyglutamyl (Gla) residues in precursor forms of a limited number of proteins. These proteins contain an amino-terminal extension (propeptide) that is presumed to serve as an enzyme recognition site to assure their normal processing. The free, noncovalently bound propeptide has also been shown to stimulate the in vitro activity of this enzyme. This peptide has now been shown to lower the app Km of a low-molecular-weight Glu site substrate while having no influence on the app Km of the other substrates, vitamin KH2, O2, and CO2/HCO3-. Propeptide addition was shown to have no influence on the ratio of the two products of the enzyme, Gla and vitamin K-2,3-epoxide. Stimulation of carboxylase activity by the propeptide from human factor X was observed in a number of rat tissues and in the liver of a number of different species. Stability of the enzyme in crude microsomal preparations was greatly enhanced by the presence of propeptide. These observations are consistent with the hypothesis that this region of the protein substrates for the carboxylase not only serves an enzyme recognition or docking function but also modulates the activity of the enzyme by altering the affinity for one of its substrates.     

Augmentation of inorganic pyrophosphate elaboration in cartilage by serum factors

The disordered production of inorganic pyrophosphate (PPi) by articular cartilage is thought to have an important role in the pathogenesis of calcium pyrophosphate dihydrate deposition disease and perhaps osteoarthritis. We have previously shown that fetal calf serum added to the culture media of porcine articular cartilage explants increases the elaboration of PPi into the ambient media. We have examined this PPi stimulatory activity by studying the effects of adult human serum (HS), serum derived from adult human plasma (HP), and an acid-alcohol extract of human platelets (PE) on PPi production in cartilage organ culture. Ten percent HS produces a 1.4-fold increase in PPi production after 48 h of culture, while cartilage incubated in media containing 10% HP produces no more PPi than that incubated in media alone. PE stimulates a mean 2-fold increase in PPi production at 48 h in the presence of low concentrations of HP, and has no effect alone. It does not appear to up-regulate the activity of the ectoenzyme nucleoside triphosphate pyrophosphohydrolase (NTPPPH), nor does it promote the release of enzyme substrate into the extracellular space. Cartilage exposed to 0.5% HP and PE has 1.51 +/- 0.36 units of NTPPPH activity whereas cartilage exposed to 0.5% HP alone has 1.52 +/- 0.41 units of enzyme activity. PE does not increase the release of [14C]adenine-labeled compounds into the media. Approximately 13% of soluble 14C counts was found in the media of chondrocytes treated with PE while 18% of counts was released in the presence of HP alone. We have demonstrated a factor or factors present in FCS, HS, and an acid-ethanol extract of human platelets which represent(s) the first known physiologic modulators of PPi production in articular cartilage and may increase PPi production without affecting NTPPPH activity.     

NMR studies of differences in the conformations and dynamics of ligand complexes formed with mutant dihydrofolate reductases

Two mutants of Lactobacillus casei dihydrofolate reductase, Trp 21----Leu and Asp 26----Glu, have been prepared by using site-directed mutagenesis methods, and their ligand binding and structural properties have been compared with those of the wild-type enzyme. 1H, 13C, and 31P NMR studies have been carried out to characterize the structural changes in the complexes of the mutant and wild-type enzymes. Replacement of the conserved Trp 21 by a Leu residue causes a decrease in activity of the enzyme and reduces the NADPH binding constant by a factor of 400. The binding of substrates and substrate analogues is only slightly affected. 1H NMR studies of the Trp 21----Leu enzyme complexes have confirmed the original resonance assignments for Trp 21. In complexes formed with methotrexate and the mutant enzyme, the results indicate some small changes in conformation occurring as much as 14 A away from the site of substitution. For the enzyme-NADPH complexes, the chemical shifts of nuclei in the bound coenzyme indicate that the nicotinamide ring binds differently in complexes with the mutant and the wild-type enzyme. There are complexes where the wild-type enzyme has been shown to exist in solution as a mixture of conformations, and studies on the corresponding complexes with the Trp 21----Leu mutant indicate that the delicately poised equilibria can be perturbed. For example, in the case of the ternary complex formed between enzyme, trimethoprim, and NADP+, two almost equally populated conformations (forms I and II) are seen with the wild-type enzyme but only form II (the one in which the nicotinamide ring of the coenzyme is extended away from the enzyme structure and into the solvent) is observed for the mutant enzyme complex. It appears that the Trp 21----Leu substitution has a major effect on the binding of the nicotinamide ring of the coenzyme. For the Asp 26----Glu enzyme there is a change in the bound conformation of the substrate folate. Further indications that some conformational adjustments are required to allow the carboxylate of Glu 26 to bind effectively to the N1 proton of inhibitors such as methotrexate and trimethoprim come from the observation of a change in the dynamics of the bound trimethoprim molecule as seen from the increased rate of the flipping of the 13C-labeled benzyl ring and the increased rate of the N1-H bond breaking.     

MHC class II sequences of an HLA-DR2 narcoleptic

Narcolepsy has a 98% association with the DR2-Dw2/DQw1 haplotype. To establish if a disease-specific allele is present in narcolepsy, a cDNA library was made from a B-cell line from a DR2,4/DQw1,3 narcoleptic. Clones encoding the two expressed DR2 chains, along with DQw1 and chains, were isolated and completely sequenced. The coding regions of these four genes were similar to published nucleotide and protein sequences from corresponding healthy controls, with some minor exceptions. The 3 untranslated region of one of the DR2 genes in the narcoleptic was extended by 42 bp. Complete sequences were not available for DQw1.2 or from healthy individuals, but first domain nucleotide sequences showed only a single nonproductive difference in DQ. Partial protein sequences of both DQ and from published data were identical. Although the effects of minor differences cannot be ruled out completely, it is concluded that there are probably no narcolepsy-specific DR or DQ / sequences, and that the alleles found in narcolepsy are representative of those found in the healthy population.     

Morphological differentiation of immobilizedClaviceps paspali mycelium during semi-continuous cultivation

Summary The morphological development ofClaviceps paspali immobilized in Ca-alginate gel was examined. During consecutive reincubations, the immobilized mycelia differentiated into swollen, arthrosporoid-like cells, which never appeared during fermentation of free mycelium. Such differentiation could be connected with the improved, prologed vitality and metabolic activity of the immobilized cells.