An 80-kilodalton protein that binds to the pre-S1 domain of hepatitis B virus

It has been suggested that hepatitis B virus (HBV) binds to a receptor on the plasma membrane of human hepatocytes via the pre-S1 domain of the large envelope protein as an initial step in HBV infection. However, the nature of the receptor remains controversial. In an attempt to identify a cell surface receptor for HBV, purified recombinant fusion protein of the pre-S1 domain of HBV with glutathione S-transferase (GST), expressed in Escherichia coli, was used as a ligand. The surface of human hepatocytes or HepG2 cells was biotinylated, and the cell lysate (precleared lysate) which did not bind to GST and glutathione-Sepharose beads was used as a source of receptor molecules. The precleared lysate of the biotinylated cells was incubated with the GST-pre-S1 fusion protein, and the bound proteins were visualized by Western blotting and enhanced chemiluminescence. An approximately 80-kDa protein (p80) was shown to bind specifically to the pre-S1 domain of the fusion protein. The receptor binding assay using serially or internally deleted segments of pre-S1 showed that amino acid residues 12 to 20 and 82 to 90 are essential for the binding of pre-S1 to p80. p80 also bound specifically to the pre-S1 of native HBV particles. Analysis of the tissue and species specificity of p80 expression in several available human primary cultures and cell lines of different tissue origin showed that p80 expression is not restricted to human hepatocytes. Taken together the results suggest that p80 may be a component of the viral entry machinery.     

Suppression of ceramide-induced cell death by hepatitis C virus core protein

The hepatitis C virus (HCV) core protein is believed to be one of viral proteins that are capable of preventing virus-infected cell death upon various stimuli. But, the effect of the HCV core protein on apoptosis that is induced by various stimuli is contradictory. We examined the possibility that the HCV core protein affects the ceramide-induced cell death in cells expressing the HCV core protein through the sphingomyelin pathway. Cell death that is induced by C(2)-ceramide and bacterial sphingomyelinase was analyzed in 293 cells that constitutively expressed the HCV core protein and compared with 293 cells that were stably transfected only with the expression vector. The HCV core protein inhibited the cell death that was induced by these reagents. The protective effects of the HCV core protein on ceramide-induced cell death were reflected by the reduced expression of p21(WAF1/Cip1/Sid1) and the sustained expression of the Bcl-2 protein in the HCV core-expressing cells with respect to the vector-transfected cells. These results suggest that the HCV core protein in 293 cells plays a role in the modulation of the apoptotic response that is induced by ceramide. Also, the ability of the HCV core protein to suppress apoptosis might have important implications in understanding the pathogenesis of the HCV infection.     

Agrodrench: a novel and effective agroinoculation method for virus-induced gene silencing in roots and diverse Solanaceous species

Summary Virus-induced gene silencing (VIGS) is an extremely powerful tool for plant functional genomics. We used Tobacco rattle virus (TRV)-derived VIGS vectors expressed from binary vectors within Agrobacterium to induce RNA silencing in plants. Leaf infiltration is the most common method of agroinoculation used for VIGS but this method has limitations as it is laborious for large-scale screening and some plants are difficult to infiltrate. Here we have developed a novel and simple method of agroinoculation, called 'agrodrench', where soil adjacent to the plant root is drenched with an Agrobacterium suspension carrying the TRV-derived VIGS vectors. By agrodrench we successfully silenced the expression of phytoene desaturase (PDS), a 20S proteasome subunit (PB7) or Mg-protoporphyrin chelatase (Chl H) encoding genes in Nicotiana benthamiana and in economically important crops such as tomato, pepper, tobacco, potato, and Petunia, all belonging to the Solanaceae family. An important aspect of agrodrench is that it can be used for VIGS in very young seedlings, something not possible by the leaf infiltration method, which usually requires multiple fully expanded leaves for infiltration. We also demonstrated that VIGS functioned to silence target genes in plant roots. The agrodrench method of agroinoculation was more efficient than the leaf infiltration method for VIGS in roots. Agrodrench will facilitate rapid large-scale functional analysis of cDNA libraries and can also be applied to plants that are not currently amenable to VIGS technology by conventional inoculation methods.     

Transgenic Expression of the Formin Protein Fhod3 Selectively in the Embryonic Heart: Role of Actin-Binding Activity of Fhod3 and Its Sarcomeric Localization during Myofibrillogenesis

Fhod3 is a cardiac member of the formin family proteins that play pivotal roles in actin filament assembly in various cellular contexts. The targeted deletion of mouse Fhod3 gene leads to defects in cardiogenesis, particularly during myofibrillogenesis, followed by lethality at embryonic day (E) 11.5. However, it remains largely unknown how Fhod3 functions during myofibrillogenesis. In this study, to assess the mechanism whereby Fhod3 regulates myofibrillogenesis during embryonic cardiogenesis, we generated transgenic mice expressing Fhod3 selectively in embryonic cardiomyocytes under the control of the β-myosin heavy chain (MHC) promoter. Mice expressing wild-type Fhod3 in embryonic cardiomyocytes survive to adulthood and are fertile, whereas those expressing Fhod3 (I1127A) defective in binding to actin die by E11.5 with cardiac defects. This cardiac phenotype of the Fhod3 mutant embryos is almost identical to that observed in Fhod3 null embryos, suggesting that the actin-binding activity of Fhod3 is crucial for embryonic cardiogenesis. On the other hand, the β-MHC promoter-driven expression of wild-type Fhod3 sufficiently rescues cardiac defects of Fhod3-null embryos, indicating that the Fhod3 protein expressed in a transgenic manner can function properly to achieve myofibril maturation in embryonic cardiomyocytes. Using the transgenic mice, we further examined detailed localization of Fhod3 during myofibrillogenesis in situ and found that Fhod3 localizes to the specific central region of nascent sarcomeres prior to massive rearrangement of actin filaments and remains there throughout myofibrillogenesis. Taken together, the present findings suggest that, during embryonic cardiogenesis, Fhod3 functions as the essential reorganizer of actin filaments at the central region of maturating sarcomeres via the actin-binding activity of the FH2 domain.     

Antioxidant Activity and α-Glucosidase Inhibitory Activities of the Polycondensate of Catechin with Glyoxylic Acid

In order to investigate polymeric flavonoids, the polycondensate of catechin with glyoxylic acid (PCG) was prepared and its chemically antioxidant, cellular antioxidant (CAA) and α-glucosidase inhibitory activities were evaluated. The DPPH and ABTS radical scavenging activities and antiproliferative effect of PCG were lower than those of catechin, while PCG had higher CAA activity than catechin. In addition, PCG had very high α-glucosidase inhibitory activities (IC₅₀ value, 2.59 μg/mL) in comparison to catechin (IC₅₀ value, 239.27 μg/mL). Inhibition kinetics suggested that both PCG and catechin demonstrated a mixture of noncompetitive and anticompetitive inhibition. The enhanced CAA and α-glucosidase inhibitor activities of PCG could be due to catechin polymerization enhancing the binding capacity to the cellular membrane and enzymes.     

Pericyte-derived extracellular vesicle-mimetic nanovesicles ameliorate erectile dysfunction via lipocalin 2 in diabetic mice

Diabetes mellitus is one of the main causes of erectile dysfunction (ED). Men with diabetic ED do not respond well to oral phosphodiesterase-5 inhibitors owing to neurovascular dysfunction. Pericyte-derived extracellular vesicle-mimetic nanovesicles (PC-NVs) are known to promote nerve regeneration in a mouse model of cavernous nerve injury. Here, we report that administration of PC-NVs effectively promoted penile angiogenesis and neural regeneration under diabetic conditions, thereby improving erectile function. Specifically, PC-NVs induced endothelial proliferation and migration and reduced cell apoptosis under diabetic conditions. In addition, PC-NVs induced neural regeneration in STZ-induced diabetic mice in dorsal root ganglion and major pelvic ganglion explants in vivo and ex vivo under high-glucose conditions. We found that lipocalin 2 (Lcn2) is a new target of PC-NVs in this process, demonstrating that PC-NVs exert their angiogenic and nerve-regeneration effects by activating MAP kinase and PI3K/Akt and suppressing P53 signaling pathway in an Lcn2-dependent manner. Our findings provide new conclusive evidence that PC-NVs can promote neurovascular regeneration and recovery of erectile function under diabetic conditions via an Lcn2-dependent mechanism. Thus, local administration of PC-NVs may be a promising treatment strategy for the treatment of diabetic ED.     

Separation and partial characterization of three distinct intracellular GLUT4 compartments in rat adipocytes. Subcellular fractionation without homogenization

Insulin recruits GLUT4 from an intracellular location to the plasma membrane in rat adipocytes. The process involves multiple intracellular compartments and multiple protein functions, details of which are largely unknown partly due to our inability to separate individual GLUT4 compartments. Here, by hypotonic lysis, differential centrifugation, and glycerol density gradient sedimentation, we separated intracellular GLUT4 compartments in rat adipocytes into three fractions: plasma membrane-containing fraction T and plasma membrane-free fractions H and L. The GLUT4 contents in fractions T, H, and L were approximately 25, 56, and 18% of total GLUT4, respectively, in basal adipocytes and 55, 42, and 3-4% in insulin-stimulated adipocytes. The plasma membrane GLUT4 contents estimated separately further revealed that intracellular GLUT4 in fraction T amounts to approximately 20% in both basal and insulin-stimulated adipocytes. Organelle-specific marker and membrane traffic-related protein distribution data suggested that intracellular GLUT4 in fraction T represents sorting endosomes, whereas GLUT4 in fractions H and L represents storage endosomes and exocytic vesicles, respectively. The subcellular fractionation without homogenization described here should be useful in identifying the role of the individual GLUT4 compartments and the associated proteins in insulin-induced GLUT4 recruitment in rat adipocytes.     

Hyponatremia Predicts New-Onset Cardiovascular Events in Peritoneal Dialysis Patients

Background and Aim

Cardiovascular (CV) disease is the leading cause of morbidity and mortality in patients on peritoneal dialysis (PD). Hyponatremia was recently shown to be a modifiable factor that is strongly associated with increased mortality in PD patients. However, the clinical impact of hyponatremia on CV outcomes in these patients is unclear.

Methods

To determine whether a low serum sodium level predicts the development of CV disease, we carried out a prospective observational study of 441 incident patients who started PD between January 2000 and December 2005. Time-averaged serum sodium (TA-Na) levels were determined to investigate the ability of hyponatremia to predict newly developed CV events in these patients.

Results

During a mean follow-up of 43.2 months, 106 (24.0%) patients developed new CV events. The cumulative incidence of new-onset CV events after the initiation of PD was significantly higher in patients with TA-Na levels ≤ 138 mEq/L than in those with a TA-Na > 138 mEq/L. After adjustment for multiple potentially confounding covariates, an increase in TA-Na level was found to be associated with a significantly lower risk of CV events (subdistribution hazard ratio per 1 mEq/L increase, 0.90; 95% confidence interval, 0.83–0.96; p = 0.003). Patients with a TA-Na ≤ 138 mEq/L had a 2.31-fold higher risk of suffering a CV event.

Conclusions

These results provide evidence of a clear association between low serum sodium and new-onset CV events after dialysis initiation in PD patients. Whether the correction of hyponatremia for this indication provides additional protection for the development of CV disease in these patients remains to be addressed in interventional studies.     

SESN2/sestrin2 suppresses sepsis by inducing mitophagy and inhibiting NLRP3 activation in macrophages

Proper regulation of mitophagy for mitochondrial homeostasis is important in various inflammatory diseases. However, the precise mechanisms by which mitophagy is activated to regulate inflammatory responses remain largely unknown. The NLRP3 (NLR family, pyrin domain containing 3) inflammasome serves as a platform that triggers the activation of CASP1 (caspase 1) and secretion of proinflammatory cytokines. Here, we demonstrate that SESN2 (sestrin 2), known as stress-inducible protein, suppresses prolonged NLRP3 inflammasome activation by clearance of damaged mitochondria through inducing mitophagy in macrophages. SESN2 plays a dual role in inducing mitophagy in response to inflammasome activation. First, SESN2 induces “mitochondrial priming” by marking mitochondria for recognition by the autophagic machinery. For mitochondrial preparing, SESN2 facilitates the perinuclear-clustering of mitochondria by mediating aggregation of SQSTM1 (sequestosome 1) and its binding to lysine 63 (Lys63)-linked ubiquitins on the mitochondrial surface. Second, SESN2 activates the specific autophagic machinery for degradation of primed mitochondria via an increase of ULK1 (unc-51 like kinase 1) protein levels. Moreover, increased SESN2 expression by extended LPS (lipopolysaccharide) stimulation is mediated by NOS2 (nitric oxide synthase 2, inducible)-mediated NO (nitric oxide) in macrophages. Thus, Sesn2-deficient mice displayed defective mitophagy, which resulted in hyperactivation of inflammasomes and increased mortality in 2 different sepsis models. Our findings define a unique regulatory mechanism of mitophagy activation for immunological homeostasis that protects the host from sepsis.