A thermostable laccase from Streptomyces lavendulae REN-7: purification, characterization, nucleotide sequence, and expression

We found a polyphenoloxidase (PPO) in the cell extract of Streptomyces lavendulae REN-7. About 0.8 mg of purified PPO was obtained from 200 g of the mycelia with a yield of 9.0%. REN-7-PPO showed broad substrate specificity toward various aromatic compounds. Moreover, this enzyme was capable of oxidation of syringaldazine, which is a specific substrate for laccase. Interestingly, REN-7-PPO retained its original activity after 20 min of incubation at even 70 degrees C. The gene encoding the PPO was cloned. Four copper-binding sites characteristics of laccases were contained in the deduced amino acid sequence. We constructed a high-level expression system of this gene in Escherichia coli. The properties of the recombinant enzyme were identical that of wild-type. In conclusion, this PPO is a thermostable laccase.     

Compositional changes of the xiphoid process and costal cartilage with aging

Because zinc attenuates endothelial cell dysfunction that proceeds atherosclerosis, depressed zinc status may be involved in the initiation of endothelial dysfunction. However, before recommending a zinc-enriched diet to reduce the risks for atherosclerosis, the effect of excess zinc on endothelial cell functions in normozincemic status should be known. Therefore, in this study, the effect of dietary zinc on normal endothelial cell functions in animals subjected to a diet containing 334±58 ppm zinc for 30 d was studied to see whether supplemented zinc has an effect on endothelial cells. Despite a slight increase in blood zinc, unaltered aortic and kidney zinc contents were associated with unchanged blood pressure in rats subjected to a zinc-enriched diet. Increased basal nitric oxide and prostacyclin were accompanied by a normal response to phenylephrine. Dietary zinc influenced neither endothelial-dependent nor endothelial-independent relaxations significantly. However, it elevated the share of M1-type cholinoceptor response as well as dilator prostaglandin release, which seems to be nitric oxide dependent. There was a strong correlation (r=0.826, p<0.05) between M1-type cholinoceptor response and prostacyclin release in zinc-treated rings. These results suggested that zinc ions increases M1-mediated prostacyclin release in normal endothelial cells without altering intracellular pathways.     

A novel murine model of oral candidiasis with local symptoms characteristic of oral thrush

A conventional and easy method to establish a murine oral candidiasis model, which has not only a stable yeast population in the oral cavity but also symptoms characteristic of oral thrush, was developed by using a sedative agent. Mice were immunosuppressed with prednisolone and were given tetracycline hydrochloride. They were orally infected with 10(6) viable cells of Candida albicans by means of a cotton swab and enough chlorpromazine chloride had been injected to keep them in a sedative state about for 3 hr after inoculation. From day 3 to day 7 post inoculation, 10(5)-10(6) colony forming units of Candida were recovered from the oral cavity of each mouse and whitish, curd-like patches were observed on most parts of tongue. Microscopically, germ tubes had appeared on the tongue surface. This model would be a useful experimental oral candidiasis for investigating the pathogenesis of C. albicans oral infection and the efficacy of various antifungal agents microbiologically and symptomatically.     

Transformation of yeast using calcium alginate microbeads with surface-immobilized chromosomal DNA

Yeast artificial chromosomes (YACs) are useful cloning vectors that have the capacity to carry large DNA inserts. The largest barrier to using such large DNA molecules in transformation experiments has been their physical instability in solution. We developed a new method of transforming yeast using chromosome-sized DNA. The method uses calcium alginate microbeads to immobilize high-density yeast chromosomal DNA. Chromosomal DNA immobilized on microbeads is physically stabilized compared with naked chromosomal DNA. The microbead-mediated transformation performed well, not only with respect to the transformation frequency with large DNA molecules (> 100 kb) but also in successful transformation using split chromosome DNA that exceeded 450 kb.     

Cholesterol is required for the polarized secretion of erythropoietin in Madin-Darby canine kidney cells

It has already been reported that stably expressed exogenous human wild-type EPO (wtEPO) is preferentially secreted to the apical side and one of the three N-linked carbohydrate chains critically acts as an apical sorting determinant in Madin-Darby canine kidney (MDCK) cells. It has been suggested that lipid rafts are involved in the apical sorting of membrane and secretory proteins. To investigate the involvement of lipid rafts in the apical sorting of wtEPO, we examined the effect of cholesterol depletion with methyl-beta-cyclodextrin on the secretion polarity of EPO and analyzed Triton X-100 insoluble cell extracts by sucrose density gradients centrifugation in MDCK cells. We found that wtEPO was shifted in non-polarized direction by cholesterol depletion. Most of the wtEPO was not detectable in the raft fractions by sucrose density gradients centrifugation analysis. These results indicate that apical secretion of EPO involves a cholesterol-dependent mechanism probably not involving lipid rafts.     

Improving the pyrophosphate-inosine phosphotransferase activity of Escherichia blattae acid phosphatase by sequential site-directed mutagenesis

Escherichia blattae acid phosphatase/phosphotransferase (EB-AP/PTase) exhibits C-5'-position selective pyrophosphate-nucleoside phosphotransferase activity in addition to its intrinsic phosphatase. Improvement of its phosphotransferase activity was investigated by sequential site-directed mutagenesis. By comparing the primary structures of higher 5'-inosinic acid (5'-IMP) productivity and lower 5'-IMP productivity acid phosphatase/phosphotransferase, candidate residues of substitution were selected. Then a total of 11 amino acid substitutions were made with sequential substitutions. As the number of substituted amino acid residues increased, the 5'-IMP productivity of the mutant enzyme increased, and the activity of the 11 mutant phosphotransferases of EB-AP/PTase reached the same level as that of Morganella morganii AP/PTase. This result shows that Leu63, Ala65, Glu66, Asn69, Ser71, Asp116, Thr135, and Glu136, whose relevance was not directly established by structural analysis alone, also plays an important role in the phosphotransferase activity of EB-AP/PTase.     

Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy

Autophagy is an intracellular process for vacuolar degradation of cytoplasmic components. Thus far, plant autophagy has been studied primarily using morphological analyses. A recent genome-wide search revealed significant conservation among autophagy genes (ATGs) in yeast and plants. It has not been proved, however, that Arabidopsis thaliana ATG genes are required for plant autophagy. To evaluate this requirement, we examined the ubiquitination-like Atg8 lipidation system, whose component genes are all found in the Arabidopsis genome. In Arabidopsis, all nine ATG8 genes and two ATG4 genes were expressed ubiquitously and were induced further by nitrogen starvation. To establish a system monitoring autophagy in whole plants, we generated transgenic Arabidopsis expressing each green fluorescent protein-ATG8 fusion (GFP-ATG8). In wild-type plants, GFP-ATG8s were observed as ring shapes in the cytoplasm and were delivered to vacuolar lumens under nitrogen-starved conditions. By contrast, in a T-DNA insertion double mutant of the ATG4s (atg4a4b-1), autophagosomes were not observed, and the GFP-ATG8s were not delivered to the vacuole under nitrogen-starved conditions. In addition, we detected autophagic bodies in the vacuoles of wild-type roots but not in those of atg4a4b-1 in the presence of concanamycin A, a V-ATPase inhibitor. Biochemical analyses also provided evidence that autophagy in higher plants requires ATG proteins. The phenotypic analysis of atg4a4b-1 indicated that plant autophagy contributes to the development of a root system under conditions of nutrient limitation.     

Hydrodynamics-based procedure involves transient hyperpermeability in the hepatic cellular membrane: implication of a nonspecific process in efficient intracellular gene delivery

BACKGROUND: The mechanisms underlying the efficient gene transfer by a large-volume and high-speed intravenous injection of naked plasmid DNA (pDNA), a so-called hydrodynamics-based procedure, remain unclear and require further investigation. In this report, we have investigated possible mechanisms for the intracellular transport of naked pDNA by this procedure. METHODS: Propidium iodide (PI), a fluorescent indicator for cell membrane integrity, and luciferase- or green fluorescent protein (GFP)-expressing pDNA were injected into mice by the hydrodynamics-based procedure. RESULTS: PI was efficiently taken up by hepatocytes which appeared to be viable following the hydrodynamics-based procedure. Pre-expressed GFP in the cytosol was rapidly eliminated from the hepatocytes by a large-volume injection of saline. The profiles of plasma ALT and AST showed a steady decline with the highest values observed immediately after the hydrodynamics-based procedure. These results suggest that the hydrodynamics-based procedure produces a transient increase in the permeability of the cell membrane. The cellular uptake process appeared nonspecific, since simultaneous injection of an excess of empty vector did not affect the transgene expression. Sequential injections of a large volume of pDNA-free saline followed by naked pDNA in a normal volume revealed that the increase in membrane permeability was transient, with a return to normal conditions within 30 min. Transgene expression was observed in hepatocyte cultures isolated 10 min after pDNA delivery and in the liver as early as 10 min after luciferase-expressing RNA delivery, indicating that pDNA delivered immediately by the hydrodynamics-based procedure has the potential to produce successful transgene expression. CONCLUSIONS: These findings suggest that the mechanism for the hydrodynamics-based gene transfer would involve in part the direct cytosolic delivery of pDNA through the cell membrane due to transiently increased permeability.     

Relationship between meniscal degeneration and element contents

The purpose of this study is to investigate the relationship between meniscal degeneration and element contents. The contents of elements (calcium, phosphorus, sulfur, and magnesium) in the menisci from 17 patients with osteoarthritis (OA) of the knee, 6 with rheumatoid arthritis (RA), and 2 who underwent the surgical operation for malignant tumors (control) were analyzed by inductively coupled plasma-atomic emission spectrometry, and the menisci were divided into four stages (Stage 0–3) of histological degeneration. The calcium contents of the menisci were 0.26±0.16 in Stage 0, 0.50±0.37 in Stage 1, and 0.69±0.66 in Stage 2, respectively (the values represent mg elements/g dry tissue). They increased with the progression of the stage. This tendency was found in the menisci with OA, but was not clear in those with RA. The calcium content in the control group was 0.17±0.09 mg/g. There was no significant relationship between the stage of degeneration and the contents of phosphorus, sulfur, or magnesium. The calcium content of the meniscus might indicate the degree of meniscal degeneration.