Two thimet oligopeptidase-like Pz peptidases produced by a collagen-degrading thermophile, Geobacillus collagenovorans MO-1

A collagen-degrading thermophile, Geobacillus collagenovorans MO-1, was found to produce two metallopeptidases that hydrolyze the synthetic substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz-PLGPR), containing the collagen-specific sequence -Gly-Pro-X-. The peptidases, named Pz peptidases A and B, were purified to homogeneity and confirmed to hydrolyze collagen-derived oligopeptides but not collagen itself, indicating that Pz peptidases A and B contribute to collagen degradation in collaboration with a collagenolytic protease in G. collagenovorans MO-1. There were many similarities between Pz peptidases A and B in their catalytic properties; however, they had different molecular masses and shared no antigenic groups against the respective antibodies. Their primary structures clarified from the cloned genes showed lower identity (22%). From homology analysis for proteolytic enzymes in the database, the two Pz peptidases belong to the M3B family. In addition, Pz peptidases A and B shared high identities of over 70% with unassigned peptidases and oligopeptidase F-like peptidases of the M3B family, respectively. Those homologue proteins are putative in the genome database but form two distinct segments, including Pz peptidases A and B, in the phylogenic tree. Mammalian thimet oligopeptidases, which were previously thought to participate in collagen degradation and share catalytic identities with Pz peptidases, were found to have lower identities in the overall primary sequence with Pz peptidases A and B but a significant resemblance in the vicinity of the catalytic site.     

Inhibition by adiponectin of IL-8 production by human macrophages upon coculturing with late apoptotic cells

Adiponectin, an adipocyte-derived hormone, reportedly suppresses the production of TNF-alpha and IL-6 by LPS-stimulated human or porcine macrophages, and the phagocytosis of microbeads by human macrophages. In this study, we used a high molecular weight form of adiponectin purified from human plasma to examine its effects on the phagocytosis of late apoptotic cells by human macrophages and the subsequent IL-8 production. Adiponectin suppressed both the phagocytosis of apoptotic cells and the IL-8 production. In contrast, adiponectin augmented both the phagocytosis of apoptotic cells and the IL-8 production in the presence of LPS. These results suggest that adiponectin is not an anti-inflammatory hormone but rather a dual modulator of innate responses.     

Selective delivery of rifampicin incorporated into poly(DL-lactic-co-glycolic) acid microspheres after phagocytotic uptake by alveolar macrophages, and the killing effect against intracellular Mycobacterium bovis Calmette-Guérin

Macrophages and their phagocytotic abilities play a dominant role for defense against infected organisms. However, Mycobacterium tuberculosis can survive in the phagosomes of macrophages. In this study, the effective delivery of a drug and the killing effect of tubercle bacilli within macrophages were investigated utilizing the phagocytotic uptake of rifampicin (RFP) that had been incorporated into poly(DL-lactic-co-glycolic) acid (PLGA) microspheres. The microspheres were composed of PLGA that had a monomer ratio (lactic acid/glycolic acid) of either 50/50 or 75/25. They had molecular weights from 5000 to 20,000, and diameters of 1.5, 3.5, 6.2 and 8.9 microm. The most significant factor for phagocytotic activity of macrophages was the diameter of the microspheres. By contrast, molecular weight and monomer ratio of PLGA did not influence phagocytosis. The amount of RFP delivered into cells was also investigated. RFP-PLGA microspheres composed of PLGA with a molecular weight of 20,000 and monomer ratio of 75/25 showed the highest amount of delivery (4 microg/1 x 10(6) cells). Fourteen days after infection, the survival rate of treated intracellular bacilli was 1% when compared with untreated cells. There was almost no killing effect of free RFP (4 or 15 microg/ml) on intracellular bacilli. In vivo efficacy of RFP-PLGA was also examined in rats infected with M. tuberculosis Kurono. Intratracheal administration of RFP-PLGA microspheres was shown to be superior to free RFP for killing of intracellular bacilli and preventing granuloma formation in some lobes. These results suggest that phagocytotic activity could be part of a new drug delivery system that selectively targeted macrophages.     

A case of intramuscular cysticercosis diagnosed definitively by mitochondrial DNA analysis of extremely calcified cysts

A case of obsolete intramuscular cysticercosis diagnosed definitively by mitochondrial DNA analysis of extremely calcified cysts was reported. X-ray and computed tomography findings highly suggested cysticercosis due to Taenia solium; however, no direct evidence of cysticercosis was obtained through serological or histopathological examinations. Mitochondrial DNA analysis of a histopathological specimen confirmed the causative agent to be the Asian genotype of T. solium.     

Two-dimensional HPLC on-line analysis of phosphopeptides using titania and monolithic columns

Conventional and comprehensive two-dimensional (2D) HPLC systems using the combination of titania and monolithic columns were established for the on-line analysis of phosphopeptides. Compared with immobilized metal affinity chromatography of a general method for the analysis of phosphopeptides, the use of titania columns in the analysis permits the specific isolation of phosphopeptides in a higher yield. Using the current 2D HPLC systems, phosphopeptides were specifically isolated from nonphosphorylated peptides by the first-dimension titania column, followed by the high-speed separation of the phosphopeptides by the second-dimension monolithic column. Proteolytic digests of beta-casein were analyzed within 30 min using the comprehensive 2D HPLC system; all phosphopeptides from beta-casein could be efficiently isolated and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The comprehensive 2D HPLC system coupled with mass spectrometry will be useful for high-throughput and on-line phosphoproteome analyses.     

Conferring cadmium resistance to mature tobacco plants through metal-adsorbing particles of tomato mosaic virus vector

Tomato mosaic virus vectors were designed that produced, by a translational readthrough, a fusion protein consisting of coat protein and metal-binding peptide, as a result of which particles were expected to present the metal-binding peptides on their surface. When inoculated in plants, they were expected to replicate and form a metal-adsorbing artificial sink in the cytoplasm, so as to reduce metal toxicity. Vectors were constructed harbouring sequences encoding various lengths of polyhistidine as a metal-binding peptide. One of the vectors, TLRT6His, which contains a 6 x histidine sequence, moved systemically in tobacco plants, and its particles were shown to retain cadmium ions by an in vitro assay. When a toxic amount of cadmium was applied, the toxic effect was much reduced in TLRT6His-inoculated tobacco plants, probably as a result of cadmium adsorption by TLRT6His particles in the cytosol. This shows the possible use of an artificial sink for metal tolerance and the advantage of employing a plant viral vector for phytoremediation.     

A comparison of the cellular actions of polaprezinc (zinc-L-carnosine) and ZnCl2

AimsZinc supplementation has been proven to be beneficial for the prevention of some health problems. Many zinc supplements are used for medical and nutritional purposes. However, it is difficult to distinguish between them in terms of their cellular actions. We compared the cellular actions of polaprezinc (zinc-l-carnosine) with those of ZnCl₂ in order to determine whether polaprezinc has greater zinc-related actions than ZnCl₂.Main methodsCellular actions of polaprezinc and ZnCl₂ were estimated by flow-cytometric techniques with appropriate fluorescent probes in rat thymocytes.Key findingsBoth agents had almost equal stimulatory effects on the intracellular Zn^2 + level and cellular level of nonprotein thiol in a similar concentration-dependent manner. However, the increase in cell lethality caused by ZnCl₂ under severe oxidative stress was significantly greater than that caused by polaprezinc.SignificanceThere are various zinc supplements, for example, zinc gluconate, zinc picolinate, and zinc methionine. However, the differences in their cellular actions have not been elucidated to date. Such studies could distinguish between zinc supplements.     

Construction of intraspecific linkage maps, detection of a chromosome inversion, and mapping of QTL for constitutive root aerenchyma formation in the teosinte Zea nicaraguensis

The teosinte Zea nicaraguensis, a wild relative of maize, possesses a flooding tolerance-related trait: the formation of constitutive root aerenchyma under drained (non-flooded) soil conditions. A previous study suggested that the degree of constitutive aerenchyma formation varies within Z. nicaraguensis. The objectives of this study were to construct linkage maps, to determine the marker order in a region of chromosome 4 in which recombination between maize and Z. nicaraguensis is suppressed, and to identify quantitative trait loci (QTL) controlling constitutive root aerenchyma formation in two segregating populations of Z. nicaraguensis. A total of 236 simple sequence repeat (SSR) markers were screened for polymorphism in an S1 population of Z. nicaraguensis. Seventy-one polymorphic SSR markers were assigned to 10 chromosomes, and a linkage map was constructed covering 793.5 cM. In the S1 map, a paracentric inversion was detected on the long arm of chromosome 4; this rearrangement was confirmed in an S1 linkage map of a different Z. nicaraguensis accession. Composite interval mapping analysis in 96 S1 plants revealed QTL for aerenchyma formation on chromosomes 1 (bins 1.06–1.07) and 7 (bin 7.01), explaining 17 and 12% of the total phenotypic variance, respectively. The QTL on chromosome 1 was verified by using 156 S2 plants. Near-isogenic lines exhibiting the presence or absence of the aerenchyma QTL have been developed that should be useful for genetic and physiological analyses of root aerenchyma formation.