Effects of protease activated receptor (PAR)2 blocking peptide on endothelin-1 levels in kidney tissues in endotoxemic rat mode

Aims

Septic shock, the severe form of sepsis, is associated with development of progressive damage in multiple organs. Kidney can be injured and its functions altered by activation of coagulation, vasoactive-peptide and inflammatory processes in sepsis. Endothelin (ET)-1, a potent vasoconstrictor, is implicated in the pathogenesis of sepsis and its complications. Protease-activated receptors (PARs) are shown to play an important role in the interplay between inflammation and coagulation. We examined the time-dependent alterations of ET-1 and inflammatory cytokine, such as tumor necrosis factor (TNF)-α in kidney tissue in lipopolysaccharide (LPS)-induced septic rat model and the effects of PAR2 blocking peptide on the LPS-induced elevations of renal ET-1 and TNF-α levels.

Main methods

Male Wistar rats at 8 weeks of age were administered with either saline solution or LPS at different time points (1, 3, 6 and 10 h). Additionally, we treated LPS-administered rats with PAR2 blocking peptide for 3 h to assess whether blockade of PAR2 has a regulatory role on the ET-1 level in septic kidney.

Key findings

An increase in ET-1 peptide level was observed in kidney tissue after LPS administration time-dependently. Levels of renal TNF-α peaked (around 12-fold) at 1 h of sepsis. Interestingly, PAR2 blocking peptide normalized the LPS-induced elevations of renal ET-1 and TNF-α levels.

Significance

The present study reveals a distinct chronological expression of ET-1 and TNF-α in LPS-administered renal tissues and that blockade of PAR2 may play a crucial role in treating renal injury, via normalization of inflammation, coagulation and vaso-active peptide.     

Synthetic inositol 1,3,4,5-tetrakisphosphate analogs and their effect on the binding to microsomal fraction of rat cerebellum.

Binding activity of [3H]inositol 1,3,4,5-tetrakisphosphate (InsP4) was characterized with rat cerebellar membranes. Two types of InsP4 analog with either the aminobenzoyl or the aminocyclohexanecarbonyl group on the 2nd position of InsP4 have been synthesized and their effects on the binding activity were also examined. [3H]InsP4 binding was gradually displaced by increasing amounts of unlabeled InsP4, with an IC50 of 60-170 nM, depending on the pH values. The binding was sharply increased at acidic pH and millimolar concentrations of Ca2+, this being in clear contrast with [3H]InsP3 binding noted in the same species of tissue. Heparin inhibited the binding, with an IC50 of 1.7, 3 or 20 micrograms/ml at pH 8.3, 7.2 or 5.0, respectively. Adenine nucleotide inhibited the binding more potently than did [3H]InsP3 binding. InsP4 analogs were as effective as InsP4 in displacing [3H]InsP4 from rat cerebellar membranes, thereby indicating that the 2nd hydroxyl group may not be involved in recognition of InsP4 by its binding sites.     

Refinement of the locus for X-linked recessive chondrodysplasia punctata

Although the locus for X-linked recessive chondrodysplasia punctata (CDPX1) has been mapped to the region between PABX and DXS31 (the critical region is about 3 Mb long), the precise location within the critical region has not been determined. In this paper, we describe a boy with a 46,Y,der(X)t(X;Y)(p22.3;q11)mat karyotype and review the genotype-phenotype correlations in three male patients with the combination of apparent lack of clinical features of CDPX1 and a partial deletion of the critical region. The results suggest that the region defined by the two BssHII sites at 3180 and 3570 kb from the Xp telomere may be the target region for the CDPX1 locus.     

Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo

Objective

Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs) are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1) whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2) whether aggregates of human MSCs promoted lubricin expression, and (3) whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats.

Methods

For in vitro analysis, human bone marrow (BM) MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages.

Results

In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4), which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and expressed lubricin in the superficial cartilage.

Conclusion

Cartilage derived from MSCs expressed lubricin protein both in vitro and in vivo. Aggregation promoted lubricin expression of MSCs in vitro and transplantation of aggregates of MSCs regenerated cartilage including the superficial zone in a rat osteochondral defect model. Our results indicate that aggregated MSCs could be clinically relevant for therapeutic approaches to articular cartilage regeneration with an appropriate superficial zone in the future.     

Microbiological degradation of bile acids. Ring a cleavage and 7α,12α-dehydroxylation of cholic acid by Arthrobacter simplex

The metabolism of cholic acid by Arthrobacter simplex was investigated. This organism effected both ring a cleavage and elimination of the hydroxyl groups at C-7 and C-12 and gave a new metabolite, (4R)-4-[4alpha-(2-carboxyethyl)-3aalpha-hexahydro-7abeta-methyl-5-oxoindan-1beta-yl]valeric acid, which was isolated and identified through its partial synthesis. A degradative pathway of cholic acid into this metabolite is tentatively proposed, and the possibility that the proposed pathway could be extended to the cholic acid degradation by other microorganisms besides A. simplex is discussed. The possibility that the observed reactions in vitro could occur during the metabolism of bile acids in vivo is considered.     

Sucrose concentratidns in the growth medium influence the membrane lipids of Streptococcus mutans

Streptococcus mutans was cultivated in media containing sucrose (10–40%, w/v) and the sucrose induced changes in chemical and physical properties of its membrane lipids were investigated. The degree of unsaturation in the fatty acids of both total lipid and glycolipid fractions decreased when the sucrose concentration was increased. An electron spin resonance spectroscopic study revealed the reduction of membrane lipid fluidity by adding sucrose to the growth medium. Liposomes prepared from membrane lipids of bacteria grown with sucrose showed less osmotic volume changes than those of bacteria grown without sucrose. These results suggest that modification of membrane lipid composition, fluidity and osmosis-resistance have an important role in the ability of Streptococcus mutans to grow in sucrose at high concentrations.     

Eicosapentaenoic acid prevents endothelin-1-induced cardiomyocyte hypertrophy in vitro through the suppression of TGF-beta 1 and phosphorylated JNK

The cardiovascular benefit of fish oil in humans and experimental animals has been reported. Endothelin (ET)-1 is a well-known cardiac hypertrophic factor. However, although many studies link a fish oil extract, eicosapentaenoic acid (EPA), to cardiac protection, the effects of EPA on cardiac hypertrophy and underlying mechanism(s) are unclear. The present study investigated whether EPA prevents ET-1-induced cardiomyocyte hypertrophy; the potential pathways likely to underlie such an effect were also investigated. Cardiomyocytes were isolated from neonatal rat heart, cultured for 3 days, and then treated for 24 h with vehicle only (control), treated with 0.1 nM ET-1 only, or pretreated with 10 microM EPA and then treated with 0.1 nM ET-1. The cells were harvested, and changes in cell surface area, protein synthesis, expression of a cytoskeletal (alpha-actinin) protein, and cell signaling were analyzed. ET-1 induced a 97% increase in cardiomyocyte surface area, a 72% increase in protein synthesis rate, and an increase in expression of alpha-actinin and signaling molecule [transforming growth factor-beta 1 (TGF-beta 1), c-Jun NH2-terminal kinase (JNK), and c-Jun]. Development of these ET-1-induced cellular changes was attenuated by EPA. Moreover, the hypertrophied cardiomyocytes showed a 1.5- and a 1.7-fold increase in mRNA expression of atrial and brain natriuretic peptides, the classical molecular markers of cardiac hypertrophy, respectively; these changes were also suppressed by EPA. Here we show that ET-1 induces cardiomyocyte hypertrophy and expression of hypertrophic markers, possibly mediated by JNK and TGF-beta 1 signaling pathways. These ET-1-induced effects were blocked by EPA, a major fish oil ingredient, suggesting that fish oil may have beneficial protective effects on cardiac hypertrophy.     

A Novel Bipartite Double-Stranded RNA Mycovirus from the White Root Rot Fungus Rosellinia necatrix: Molecular and Biological Characterization,Taxonomic Considerations,and Potential for Biological Control

White root rot, caused by the ascomycete Rosellinia necatrix, is a devastating disease worldwide, particularly in fruit trees in Japan. Here we report on the biological and molecular properties of a novel bipartite double-stranded RNA (dsRNA) virus encompassing dsRNA-1 (8,931 bp) and dsRNA-2 (7,180 bp), which was isolated from a field strain of R. necatrix, W779. Besides the strictly conserved 5′ (24 nt) and 3′ (8 nt) terminal sequences, both segments show high levels of sequence similarity in the long 5′ untranslated region of approximately 1.6 kbp. dsRNA-1 and -2 each possess two open reading frames (ORFs) named ORF1 to -4. Although the protein encoded by 3′-proximal ORF2 on dsRNA-1 shows sequence identities of 22 to 32% with RNA-dependent RNA polymerases from members of the families Totiviridae and Chrysoviridae, the remaining three virus-encoded proteins lack sequence similarities with any reported mycovirus proteins. Phylogenetic analysis showed that the W779 virus belongs to a separate clade distinct from those of other known mycoviruses. Purified virions ∼50 nm in diameter consisted of dsRNA-1 and -2 and a single major capsid protein of 135 kDa, which was shown by peptide mass fingerprinting to be encoded by dsRNA-1 ORF1. We developed a transfection protocol using purified virions to show that the virus was responsible for reduction of virulence and mycelial growth in several host strains. These combined results indicate that the W779 virus is a novel bipartite dsRNA virus with potential for biological control (virocontrol), named Rosellinia necatrix megabirnavirus 1 (RnMBV1), that possibly belongs to a new virus family.Viruses are found ubiquitously in major groups of filamentous fungi (40), and an increasing number of novel mycoviruses are being reported (3, 36). Mycoviruses with RNA genomes are now classified into 10 families, of which four accommodate double-stranded RNA (dsRNA) viruses and the remaining six comprise single-stranded RNA (ssRNA) viruses (23). While many ssRNA mycoviruses, like hypoviruses and endornaviruses, do not produce particles, dsRNA virus genomes, whether undivided (the family Totiviridae) or divided (11 or 12 segments for the family Reoviridae, 4 segments for the family Chrysoviridae, and 2 segments for the family Partitiviridae), are encapsidated in rigid particles. Most mycoviruses are considered to cause cryptic infections, while some cause phenotypic alterations that include hypovirulence and debilitation. However, the lack of artificial introduction methods for most mycoviruses has greatly hampered progress in exploring mycovirus-host interactions (23, 40). Thus, a virus etiology of altered fungal phenotypes was established only for a limited number of examples, including hypovirus-C. parasitica and mycoreovirus-C. parasitica.White root rot is one of the most devastating diseases of perennial crops worldwide, particularly highly valued fruits in Japan like apple, Japanese pear, and grapevine. The causal fungus, Rosellinia necatrix, is an ascomycete with a wide range of host plants of >197 species spanning 50 families (31) and is difficult to control by conventional methods, as is often the case for soilborne pathogens. Fungicide application, though it may be effective, is labor-intensive and raises environmental concerns, while cultural practices may not be effective. Successful biocontrol of chestnut blight disease in Europe with hypovirulent strains (25, 38) inspired a group of Japanese researchers to conduct an extensive search of a large collection of >1,000 field fungal isolates for mycoviruses that might serve as virocontrol agents. Virocontrol or virological control refers to one form of biological control utilizing viruses that infect organisms pathogenic to useful organisms (23). Approximately 20% of the collected isolates of R. necatrix were found to be dsRNA positive and presumed to be infected by mycoviruses (4, 29). Agarose gel profiles of dsRNAs suggested infections by members in the families Totiviridae, Partitiviridae, Reoviridae, and Chrysoviridae, as well as unassigned viruses (S. Kanematsu and A. Sasaki, unpublished results). Among those dsRNAs, the genomic segments of Mycoreovirus 3 (MyRV3) (55) and Rosellinia necatrix partitivirus 1 (RnPV1) (44) were well characterized. However, many other dsRNAs remain uncharacterized.Artificial virion introduction protocols, which are often unavailable for mycoviruses, have been developed for specific viruses infecting the white root rot fungus. Using a polyethylene glycol (PEG)-mediated method, as established for MyRV1 and MyRV2 infecting C. parasitaca (27, 28), RnPV1 and MyRV3 were shown to be infectious as particles (45, 46). Subsequently, the cause-effect relationship was established: MyRV3 was demonstrated to confer hypovirulence (attenuated virulence) on an isogenic strain and a few vegetatively incompatible virulent strains of R. necatrix (33, 45), while RnPV1 was shown to be associated with symptomless infection. Protoplast fusion is also available for introduction of partitiviruses and uncharacterized viruses into recipient fungal strains that are vegetatively incompatible with virus-containing ones (A. Sasaki, unpublished results). Furthermore, DNA transformation systems are available for foreign gene expression in R. necatrix (33, 42). These technical advances have made the R. necatrix-mycovirus systems attractive for studies of virus-host interactions and virocontrol (23, 37).R. necatrix strain W779 was isolated by Ikeda et al. (29, 30) from soil in Ibaraki Prefecture as a dsRNA-positive strain that had yet to be characterized. Here we describe the purification and biological and molecular properties of a novel virus isolated from W779. Particles ∼50 nm in diameter isolated from strain W779 consist of two dsRNA elements termed dsRNA-1 and -2 of approximately 9 and 7 kbp and a major protein of 135 kDa encoded by one of two open reading frames (ORFs) on dsRNA-1. Importantly, purified virus particles were shown to be infectious and confer hypovirulence on vegetatively incompatible fungal strains. The two dsRNA segments share the conserved terminal sequences at both ends, and both possess extremely long (>1.6 kb) 5′ untranslated regions (UTRs) similar to each other, two ORFs, and relatively short 3′ UTRs. The 3′-proximal ORF of dsRNA-1 encodes an RNA-dependent RNA polymerase (RdRp) showing low levels (22 to 32%) of sequence identity to those of members of the families Totiviridae and Chrysoviridae. A phylogenetic analysis with RdRp sequences revealed that the W779 virus is placed into a separate clade from the recognized virus families. These attributes indicate that dsRNA-1 and -2 represent the genome segments of a novel bipartite virus, designated Rosellinia necatrix megabirnavirus 1 (RnMBV1), with virolocontrol agent potential. We propose the establishment of a new family, Megabirnaviridae, to accommodate RnMBV1 as the type species.