Purification and characterization of endo-xylanases from Aspergillus niger. II. An enzyme of pl 4.5

A homogeneous endo-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) was obtained from a crude Aspergillus niger pentosanase by chromatography with Ultrogel AcA 54, SP-Sephadex C-25 at pH 4.5, DEAE-Sephadex A-25 at pH 5.4, Sephadex G-50, and SP-Sephadex C-25 with a gradient from pH 2.8 to pH 4.6. It was much more active on soluble than on insoluble xylan, yielding large amounts of unreacted xylan and a mixture of oligosaccharides with chain lengths from two to six. No xylose or L-arabinose was produced. There was high activity on a xylopentaose through xylononaose mixture, but not on xylobiose, xylotriose, or xylotetraose. The enzyme had slight activity on untreated cellulose, carboxymethylcellulose, and pectin. Molecular weight was ca. 1.4 x 10(4), with an isoelectric point of 4.5 and an amino acid profile high in acidic but low in sulfur-containing residues. In a 25-min assay at pH 4.7, this endo-xylanase was most active at 45 degrees C, with an activation energy from 5 to 35 degrees C of 33.3 kJ/mol. The optimum pH for activity was 4.9. Decay in buffer was first order, with an activation energy at pH 4.7 from 48 to 53 degrees C of 460 kJ/mol. Optimum pH for stability was about 5.6, where the half-life at 48 degrees C in buffer was ca. 40 h.     

Inactivation of bradykinin and kallidin by cathepsin G and mast cell chymase

Human neutrophil cathepsin G and human skin chymase can inactivate bradykinin by cleavage at the carboxy terminal phenylalanyl-arginyl peptide bond of this polypeptide. The mast cell enzyme is far more effective than cathepsin G, the rates of hydrolysis being comparable to that found for angiotensin I to angiotensin II conversion (C.F. Reilly, D. Tewksbury, N. Schechter, and J. Travis, J. Biological Chemistry 257:8619-8622). This ability to both inactivate bradykinin and accelerate the production of angiotensin II may be of significance in the development of biochemical events associated with inflammation.     

The expression of human glycerol-3-phosphate dehydrogenase in human/rodent somatic-cell hybrids

Our previous studies using rodent/human somatic-cell hybrids suggested that the expression of human mitochondrial glycerol-3-phosphate dehydrogenase (GPDM) is dependent on the presence of human mitochondria. This has now been tested directly by analysis of GPDM activity in a series of nine hybrid-cell lines, four segregating human chromosomes and five losing rodent chromosomes (reverse segregants). The chromosome composition of the hybrids was deduced from analysis of biochemical markers and examination of G- and G11-banded metaphase spreads and the mitochondrial content was determined by Southern blot analysis, using cloned mouse and human mtDNA sequences as probes. We found that the mtDNA species present in these hybrids correlated exactly with the pattern of chromosome segregation such that the conventional hybrids contained rodent mtDNA and the reverse segregants human mtDNA. However, the pattern of GPDM expression was not directly correlated with the species of chromosomes or mitochondria present: all the hybrids showed strong rodem GPDM activity and two from each class of hybrid also showed human GPDM activity but the other hybrids were negative for human GPDM. We conclude that rodent GPDM readily integrates into human mitochondria, that the expression of rodent GPDM is not dependent on the presence of rodent mitochondria, and that GPDM is not coded by mtDNA. Human GPDM either is not capable of being inserted into the rodent mitochondrial membrane or is regulated in some way in the hybrid cells by an unidentified rodent factor.     

Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: prohead restoration for DNA-gp3 packaging and assembly.

The DNA-protein complex DNA-gp3 of phi 29 is efficiently packaged into purified proheads with the aid of plasmid-derived gp16. The filled heads can be assembled to phage by addition of an extract providing the products for neck-tail assembly (Bjornsti et al., J. Virol. 50:766-772, 1984). However, purified proheads lost their competence to package DNA-gp3 upon storage for 2 months at 4 degrees C. Competence was restored by complementation with extracts of certain mutant-infected cells, and these experiments demonstrated that late proteins were not involved; restoration obtained with 4-8-14--infected cells was indistinguishable from that obtained with 7-8-14--infected cells. 2-8-14- and 3-8-14- extracts restored about one-third of the capacity to package exogenous DNA-gp3. A 1-8-14- extracts restored activity to package 20.6% of the DNA-gp3 added, but phage were not produced.     

Seasonal Fluctuations of Globodera tabacum solanacearum as Estimated by Two Soil Extraction Techniques

Two soil extraction methods were compared to determine their efficiency in recovering cysts and juveniles of a tobacco cyst nematode, Globodera tabacum solanacearum. The methods were equally efficient when extracting nematodes from soil samples seeded in the laboratory; however, there was a significant extraction method × month interaction when the methods were used to estimate field soil populations over 2 years. The centrifugal sugar flotation method recovered greater numbers of cysts when densities were near 400 cysts/100 cm³ soil and greater numbers of juveniles in all samples. The sugar flotation method recovered greater numbers of cysts during months when densities were less than 400 cysts/100 cm³ soil. Numbers of cysts and juveniles were lowest in June and July following land tillage in May. A soil freeze in January 1982 may have been responsible for unusually high numbers of recovered cysts in February and March 1982, a pattern that did not occur in 1983.     

Application of cross-linked carboxymethyl cellulose degradation by beta-glucosidase and vaginal microbes to toxic shock syndrome

Eleven bacterial and two yeast strains, four of which were previously identified as having activity on a lightly cross-linked carboxymethyl cellulose (CLD-2) found in one type of superabsorbent tampon, were grown on a variety of substrates, most containing cellulosics. None produced detectable amounts of cellulases, but all elaborated beta-glucosidase. None of these 13 strains nor 3 commercially obtained beta-glucosidase preparations could hydrolyze CLD-2, although a commercial cellulase and two other bacterial preparations known to produce cellulases could. Based on these results, it appears that previous work suggesting that the degradation of CLD-2 by vaginal microbes and beta-glucosidase is implicated in the production by Staphylococcus aureus of toxin causing toxic shock syndrome must be reevaluated.     

Regeneration of cells from protoplasts ofClostridium acetobutylicum B643

Summary Conditions that allow regeneration of cells fromClostridium acetobutylicum strain B643 protoplasts were studied. Protoplast formation and stabilization in minimal media with 50 mM CaCl₂, 50 mM MgCl₂ and 0.3 M sucrose were crucial to subsequent regeneration on soft yeast extract agar containing 25 mM CaCl₂ and 25 mM MgCl₂. A regeneration frequency of 8–25% was consistently obtained.     

Locus determining the human sperm-specific lactate dehydrogenase,LDHC, is syntenic with LDHC

From the data presented in this report, the human LDHC gene locus is assigned to chromosome 11. Three genes determine lactate dehydrogenase (LDH) in man. LDHA and LDHB are expressed in most somatic tissues, while expression of LDHC is confined to the germinal epithelium of the testes. A human LDHC cDNA clone was used as a probe to analyze genomic DNA from rodent/human somatic cell hybrids. The pattern of bands with LDHC hybridization is easily distinguished from the pattern detected by LDHA hybridization, and the LDHC probe is specific for testis mRNA. The structural gene LDHA has been previously assigned to human chromosome 11, while LDHB maps to chromosome 12. Studies of pigeon LDH have shown tight linkage between LDHB and LDHC leading to the expectation that these genes would be syntenic in man. However, the data presented in this paper show conclusively that LDHC is syntenic with LDHA on human chromosome 11. The terminology for LDH genes LDHA, LDHB, and LDHC is equivalent to Ldhl, Ldh2, and Ldh3, respectively.