Release of galactosyl oligosaccharides by endo-β-N-acetylglucosaminidase D

Endo-β-N-acetylglucosaminidase D from Diplococcus pneumoniae released galactosyl oligosaccharides from IgG glycopeptides treated with β-N-acetylglucosaminidase. The structure of the major oligosaccharide was proposed to be as follows.

Since α-mannosidase digestion of the β-N-acetylglucosaminidase-treated glycopeptides made them again resistant to the endoglycosidase, we concluded that an unsubstituted α-mannosyl residue was required for the enzymic action.     

Phenotype of a Temperature-Sensitive, Respiration-Deficient (cyt) Mutant of Yeast

A temperature-sensitive respiration-deficient mutant of yeast lacks hemoproteins and accumulates coproporphyrin III when cultivated at elevated temperatures. Cells grown at 20 C respired normally and contained cytochromes a, b, and c. Cells grown at 35 C showed respiration-deficient mutant characters; they did not respire, lacked cytochromes, and accumulated coproporphyrin III. Addition of protoporphyrin IX or protohemin IX to the culture medium restored the respiratory activity of this mutant during growth at 35 C. The activities of various enzymes, including succinate-2,6-dichlorophenol indophenol (DCPIP), reduced nicotinamide adenine dinucleotide (NADH(2))-DCPIP, succinate-cytochrome c, and NADH(2)-cytochrome c oxidoreductase, and cytochrome oxidase, and the cytochrome c content of cells cultured in various conditions were determined. Changes in the number and structure of mitochondria were associated with changes in respiratory activity.     

T296→M,a common mutation causing mild hemophilia B in the Amish and others: founder effect,variability in factor IX activity assays,and rapid carrier detection

Summary By direct genomic sequencing, we have delineated the causative mutation in 64 families of European descent with hemophilia B. Six (9%) had a CT transition at base 31008, which substitutes methionine for threonine 296 (T²⁹⁶M) in the catalytic domain of factor IX. Five of the patients had the same haplotype (frequency of 16% in the northern European population). These individuals are of Amish/German descent and they are likely to share a common ancestor. The sixth patient had a different haplotype, which indicates that his mutation had an independent origin. The data highlight the importance of clinical criteria for the classification of hemophilia B. All six patients had clinically mild disease and their factor IX coagulant activities were in the range of 3%–6% when tested simultaneously in one laboratory, yet the factor IX activities provided with patient records varied 40-fold. Due to the high frequency of this mutation, we have utilized the technique of polymerase chain reaction amplification of specific alleles (PASA) to perform rapid and inexpensive carrier diagnoses in the families with this mutation. This is of particular importance for the Amish since the mutation should account for much of, if not all, the mild hemophilia B that is commonly found in this population.     

Sequence selective binding of ditrisarubicin B to DNA: comparison with daunomycin

DNase I footprinting has been used to examine the sequence selective binding of ditrisarubicin B, novel anthracycline antibiotic, to DNA. At 37°C no footprinting pattern is observed, the drug protects all sites from enzymic cleavage with equal efficiency. At 4°C a footprinting pattern is induced with low drug concentrations which is different from that produced by daunomycin. The best binding sites contain the dinucleotide step GpT (ApC) and are located in regions of alternating purines and pyrimidines.     

Characterization of the pTZ2162 encoding multidrug efflux gene qacB from Staphylococcus aureus

The plasmid-borne multidrug efflux gene qacB is widely distributed in methicillin-resistant Staphylococcus aureus (MRSA). We analyzed the complete nucleotide sequence of the plasmid pTZ2162 (35.4 kb) encoding qacB. The plasmid pTZ2162 contains 47 ORFs and four copies of IS257 (designated IS257A to D). The 24.7-kb region of pTZ2162, which excluding the region flanked by IS257A and IS257D, is 99.9% identical to pN315 carried by MRSA N315. However, the repA-like region of pTZ2162 was divided into two ORFs, ORF46 and ORF47. Functional analysis with the pUC19-based vector pTZN03 showed that both ORF46 and ORF47 were essential for the replication of pTZ2162 and ORF1 is required for the stable maintenance of pTZ2162 in S. aureus. When pTZ2162 was searched for evidence of mobile elements, an 8-bp duplicated sequence (GATAAAGA) was existed at the left boundary of IS257A and the right boundary of IS257D. Therefore, the 10.7-kb region between IS257A and IS257D in pTZ2162 has the potential to act as a transposon. In addition to qacB, the pTZ2162 transposon-like element contains a novel fosfomycin resistance determinant fosD and an aminoglycoside resistance determinant aacA-aphD. This transposon-like element appears to have translocated into the beta-lactamase gene blaZ. Our data suggest that qacB is transferred between MRSA as a multiple antibiotic resistance transposon.     

Purification and Characterization of Streptomyces Sialidases

Some strains of Streptomyces produce sialidases. Two sialidases were purified over 1,000-fold from a culture filtrate of two Streptomyces species. They had the same properties in molecular weight, behavior to ions and other reagents, and substrate specificity. They showed very small differences in kinetic properties, pH optima, and heat stability. These Streptomyces sialidases differed markedly from Clostridium perfringens sialidase in molecular weight, p-chloromercuribenzoate sensitivity, and substrate specificity. Approximate molecular weights of the sialidases from Streptomyces and C. perfringens were 32,000 and 57,000, respectively. p-Chloromercuribenzoate (10(-3) M) caused complete inhibition of C. perfringens sialidase but not of Streptomyces sialidases.     

Loss of plastidic lysophosphatidic acid acyltransferase causes embryo-lethality in Arabidopsis

Phosphatidic acid is a key intermediate for chloroplast membrane lipid biosynthesis. De novo phosphatidic acid biosynthesis in plants occurs in two steps: first the acylation of the sn-1 position of glycerol-3-phosphate giving rise to lysophosphatidic acid; second, the acylation of the sn-2 position of lysophosphatidic acid to form phosphatidic acid. The second step is catalyzed by a lysophosphatidic acid acyltransferase (LPAAT). Here we describe the identification of the ATS2 gene of Arabidopsis encoding the plastidic isoform of this enzyme. Introduction of the ATS2 cDNA into E. coli JC 201, which is temperature-sensitive and carries a mutation in its LPAAT gene plsC, restored this mutant to nearly wild type growth at high temperature. A green-fluorescent protein fusion with ATS2 localized to the chloroplast. Disruption of the ATS2 gene of Arabidopsis by T-DNA insertion caused embryo lethality. The development of the embryos was arrested at the globular stage concomitant with a transient increase in ATS2 gene expression. Apparently, plastidic LPAAT is essential for embryo development in Arabidopsis during the transition from the globular to the heart stage when chloroplasts begin to form.     

Proteins involved in biophoton emission and flooding-stress responses in soybean under light and dark conditions

To know the molecular systems basically flooding conditions in soybean, biophoton emission measurements and proteomic analyses were carried out for flooding-stressed roots under light and dark conditions. Photon emission was analyzed using a photon counter. Gel-free quantitative proteomics were performed to identify significant changes proteins using the nano LC–MS along with SIEVE software. Biophoton emissions were significantly increased in both light and dark conditions after flooding stress, but gradually decreased with continued flooding exposure compared to the control plants. Among the 120 significantly identified proteins in the roots of soybean plants, 73 and 19 proteins were decreased and increased in the light condition, respectively, and 4 and 24 proteins were increased and decreased, respectively, in the dark condition. The proteins were mainly functionally grouped into cell organization, protein degradation/synthesis, and glycolysis. The highly abundant lactate/malate dehydrogenase proteins were decreased in flooding-stressed roots exposed to light, whereas the lysine ketoglutarate reductase/saccharopine dehydrogenase bifunctional enzyme was increased in both light and dark conditions. Notably, however, specific enzyme assays revealed that the activities of these enzymes and biophoton emission were sharply increased after 3 days of flooding stress. This finding suggests that the source of biophoton emission in roots might involve the chemical excitation of electron or proton through enzymatic or non-enzymatic oxidation and reduction reactions. Moreover, the lysine ketoglutarate reductase/saccharopine dehydrogenase bifunctional enzyme may play important roles in responses in flooding stress of soybean under the light condition and as a contributing factor to biophoton emission.