A disintegrin and metalloproteinase 17 (ADAM17) mediates epidermal growth factor receptor transactivation by angiotensin II on hepatic stellate cells

Aims

Epidermal growth factor receptor (EGFR) transactivation induced by angiotensin II (Ang II) participates in the progression of various diseases. A disintegrin and metalloproteinase 17 (ADAM17) is thought to promote renal fibrosis, cardiac hypertrophy with fibrosis and atherosclerosis by activation of the EGFR through secretion of EGFR ligands. The purpose of this study was to investigate whether Ang II-induced EGFR transactivation occurs on hepatic stellate cells (HSCs) and whether the reaction is mediated via ADAM17.

Main methods

Ang II-induced EGFR transactivation and cellular proliferation of the human HSC line LI90 were investigated using Western blotting and ATP assay, respectively. Ang II-induced secretion of mature amphiregulin into the cell culture medium was evaluated by enzyme-linked immunosorbent assay (ELISA).

Key findings

An inhibitor of ADAM17, TAPI-1, as well as antagonists of EGFR and angiotensin II type-1 receptor (AT1), attenuated Ang II-induced EGFR transactivation and proliferation of LI90 cells. Furthermore, silencing of ADAM17 inhibited Ang II-induced secretion of mature amphiregulin in addition to EGFR transactivation.

Significance

These results indicate that ADAM17 mediates Ang II-induced EGFR transactivation on HSCs, and that this process may participate in the progression of liver fibrosis.     

Comparison of pollination characteristics between the insular shrub Clerodendrum izuinsulare and its widespread congener C. trichotomum

Impact of pollinator shift on differentiation of floral morphology has attracted the interest of naturalists for many years. A comparative investigation was conducted for determining the pollination characteristics, including pollinator assemblage, floral morphology, flowering phenology, and self‐compatibility, of two closely related Clerodendrum species—insular C. izuinsulare and widespread C. trichotomum. Japanese black swallowtail butterflies were the predominant flower visitors in mainland Japan, whereas diurnal hawk moths were predominantly found on the Izu Islands, a chain of oceanic islands located off the southeastern coast of the main Japanese island of Honshu in the west Pacific Ocean. The corolla tube of C. izuinsulare was longer than that of C. trichotomum, whereas the filaments and petals of C. izuinsulare were shorter than those of C. trichotomum. The flowering season of C. izuinsulare was later than that of C. trichotomum. The self‐compatibility of C. izuinsulare was higher than that of C. trichotomum. These differences might be associated with the low density of Japanese black swallowtail butterflies and dominance of diurnal hawk moths on the Izu Islands.     

Molecular cloning,characterization and functional assessment of the myosin light polypeptide chain 2 (mylz2) promoter of farmed carp,Labeo rohita

We cloned the 5′-flanking region (1.2 kb) of a muscle-specific gene, encoding myosin light chain 2 polypeptide (mylz2) of a farmed carp, Labeo rohita (rohu). Sequence analysis using TRANSFAC-database search identified the consensus cis acting regulatory elements of TATA-box and E (CANNTG)-box, including the monocyte enhancer factor 2 motif, implying that it is likely to be a functional promoter. The proximal promoter (~620 bp) was highly homologous with that of Danio rerio (zebrafish) as compared to Channa striatus (snakehead murrel) counterparts and showed less identity with Sparus auratus (gilthead sea bream), Xenopus laevis (African clawed frog) and Rattus norvegicus (Norway rat). Direct muscular (skeletal) injection of the construct containing the mylz2 promoter (0.6 kb) fused to a green fluorescent protein (GFP) reporter gene showed efficient expression in L. rohita, validating its functional activity. Further, the functional activity was confirmed by the observation that this promoter drove GFP expression in the skeletal muscle of transgenic rohu. The promoter may have potential applications for value-addition in ornamental fishes and studying gene regulatory functions.     

Spinocerebellar Ataxia Type 31 Is Associated with “Inserted” Penta-Nucleotide Repeats Containing (TGGAA)n

Spinocerebellar ataxia type 31 (SCA31) is an adult-onset autosomal-dominant neurodegenerative disorder showing progressive cerebellar ataxia mainly affecting Purkinje cells. The SCA31 critical region was tracked down to a 900 kb interval in chromosome 16q22.1, where the disease shows a strong founder effect. By performing comprehensive Southern blot analysis and BAC- and fosmid-based sequencing, we isolated two genetic changes segregating with SCA31. One was a single-nucleotide change in an intron of the thymidine kinase 2 gene (TK2). However, this did not appear to affect splicing or expression patterns. The other was an insertion, from 2.5–3.8 kb long, consisting of complex penta-nucleotide repeats including a long (TGGAA)_n stretch. In controls, shorter (1.5–2.0 kb) insertions lacking (TGGAA)_n were found only rarely. The SCA31 repeat insertion''s length inversely correlated with patient age of onset, and an expansion was documented in a single family showing anticipation. The repeat insertion was located in introns of TK2 and BEAN (brain expressed, associated with Nedd4) expressed in the brain and formed RNA foci in the nuclei of patients'' Purkinje cells. An electrophoretic mobility-shift assay showed that essential splicing factors, serine/arginine-rich splicing factors SFRS1 and SFRS9, bind to (UGGAA)_n in vitro. Because (TGGAA)_n is a characteristic sequence of paracentromeric heterochromatin, we speculate that the insertion might have originated from heterochromatin. SCA31 is important because it exemplifies human diseases associated with “inserted” microsatellite repeats that can expand through transmission. Our finding suggests that the ectopic microsatellite repeat, when transcribed, might cause a disease involving the essential splicing factors.     

Multiunit Floating Drug Delivery System of Rosiglitazone Maleate: Development, Characterization, Statistical Optimization of Drug Release and In Vivo Evaluation

A multiunit floating drug delivery system of rosiglitazone maleate has been developed by encapsulating the drug into Eudragit® RS100 through nonaqueous emulsification/solvent evaporation method. The in vitro performances of microspheres were evaluated by yield (%), particle size analysis, drug entrapment efficiency, in vitro floating behavior, surface topography, drug–polymer compatibility, crystallinity of the drug in the microspheres, and drug release studies. In vitro release was optimized by a {3, 3} simplex lattice mixture design to achieve predetermined target release. The in vivo performance of the optimized formulation was evaluated in streptozotocin-induced diabetic rats. The results showed that floating microspheres could be successfully prepared with good yields (69–75%), high entrapment (78-97%), narrow size distribution, and desired target release with the help of statistical design of experiments from very small number of formulations. In vivo evaluation in albino rats suggested that floating microspheres of rosiglitazone could be a promising approach for better glycemic control.     

Glycosylation at 158N of the Hemagglutinin Protein and Receptor Binding Specificity Synergistically Affect the Antigenicity and Immunogenicity of a Live Attenuated H5N1 A/Vietnam/1203/2004 Vaccine Virus in Ferrets

A live attenuated influenza A/Vietnam/1203/2004 (H5N1) vaccine virus (VN04 ca) has receptor binding specificity to α2,3-linked sialosides (α2,3SAL), and a single dose induces a minimal serum antibody response in mice and ferrets. In contrast, A/Hong Kong/213/2003 (H5N1) vaccine virus (HK03 ca) binds to both α2,6SAL and α2,3SAL and generates a stronger serum antibody response in animals. Among the 9 amino acids that differed between the two H5 HA1 proteins, several HK03-specific residues enabled the VN04 ca virus to bind to both α2,3SAL and α2,6SAL receptors, but only the removal of the 158N glycosylation, together with an S227N change, resulted in more-efficient viral replication in the upper respiratory tract of ferrets and an increased serum antibody response. However, the antibody response was HK03 strain specific and did not significantly cross-neutralize VN04 virus. A second approach was taken to adapt the H5N1 VN04 ca virus in MDCK cells to select HA variants with larger plaque morphology. Although a number of large-plaque-size HA variants with amino acid changes in the HA receptor binding region were identified, none of these mutations affected virus receptor binding preference and immunogenicity. In addition, the known receptor binding site changes, Q226L and G228S, were introduced into the HA protein of the VN04 ca virus. Only in conjunction with the removal of the 158N glycosylation did the virus replicate efficiently in the upper respiratory tract of ferrets and became more immunogenic, yet the response was also HK03 specific. Thus, the mask of the antigenic epitopes by 158N glycosylation at the HA globular head and its α2,3SAL binding preference of VN04 ca virus affect virus antigenicity and replication in the host, resulting in a lower antibody response.Influenza A viruses have the potential to cause pandemics of various severities. The emergence of new influenza virus strains to which the general population has low or no immunity, such as the 2009 swine-origin influenza A H1N1 viruses, will continue to challenge public health authorities and the scientific community to develop quick and efficient mitigation responses (18). Highly pathogenic avian influenza A (HPAI) H5N1 viruses pose a serious pandemic threat due to their virulence and high mortality in humans, and their increasingly expanding host reservoir and significant ongoing evolution could enhance their human-to-human transmissibility (8). Currently, the case fatality rate of HPAI H5N1 viruses in humans is estimated to be approximately 60% (30).Although HPAI H5N1 viruses are now endemic in several countries (2), direct transmission of influenza viruses from avian species to humans remains a relatively rare event. The hemagglutinin (HA) protein''s affinity for cell surface sialic acid-containing molecules is one of the determinants of influenza A virus host range restriction. Human and avian influenza virus isolates differ in their recognition of host cell receptors; human strains mainly bind α2,3-linked sialosides (α2,6SAL), whereas the avian strains have a high affinity to α2,3SAL (15, 32). The influenza pandemics of the last century have been suggested to result from switching of HA receptor-binding specificity from α2,3SAL to α2,6SAL receptors (6, 26, 31).The receptor-binding specificity of the HA protein can be influenced by several critical residues. For influenza H3 subtype viruses, substitutions of Q226L and G228S could completely reverse receptor-binding specificity from α2,3SAL to α2,6SAL (4, 21). For the H1 subtype viruses, the E190D and D225G residues switch virus receptor binding specificity from α2,3SAL to α2,6SAL for the 1918 pandemic H1N1 viruses (6, 25). However, based on glycan microarray analysis, the 190E and 225D residues cannot alter the HA binding preference from α2,3SAL to α2,6SAL for H5N1 viruses (26).Vaccination is considered a preferred approach to prevent influenza-related illness in the community. A pandemic influenza vaccine should stimulate protective immunity in the target population using the smallest amount of antigen possible, thus enabling availability of maximal vaccine doses. The inactivated H5N1 VN04 vaccines have been found to be poorly immunogenic in humans, and adjuvants are needed to enhance vaccine immunogenicity (13). Live attenuated influenza vaccines (LAIV) have several desirable attributes: the stimulation of a durable mucosal and systemic immunity, broad efficacy against homologous and drifted strains, and efficient production (17).Several H5N1 LAIV vaccines possessing a modified HA and neuraminidase (NA) of an H5N1 virus and the six internal protein gene segments (PB1, PB2, PA, NP, M, and NS) of the A/Ann Arbor/6/60 (H2N2) cold-adapted (AA ca) master donor virus were previously generated and evaluated for their immunogenicity and efficacy in mice and ferrets (29). A single dose of A/Vietnam/1203/2004 (VN04 ca) LAIV elicited very low levels of serum neutralizing antibodies against homologous and heterologous wild-type (wt) H5N1 viruses 4 weeks after administration to mice and ferrets. In contrast, a single dose of A/Hong Kong/213/2003 (H5N1) (HK03 ca) LAIV was more immunogenic (29). A specific amino acid residue at position 227 in the HK03 HA has been reported to be responsible for the greater immunogenicity of HK03 (9). VN04 and HK03 also differ in their receptor binding specificities. The VN04 HA mainly recognizes α2,3SAL, while the HK03 HA recognizes both α2,3SAL and α2,6SAL (7, 14, 22, 36). Sequence alignment of the two H5 HA proteins revealed nine amino acid differences in their HA1 region (9). The current analysis evaluates the impact of these amino acid differences on H5N1 VN ca vaccine strain replication and immunogenicity. In addition, adaptive mutations selected from MDCK passage of the H5N1 VN04 ca virus and introduction of known receptor binding sites were evaluated for their effect on antigenicity and immunogenicity of the H5N1 VN04 ca virus.     

Synovial fluid leukocyte apoptosis is inhibited in patients with very early rheumatoid arthritis

Synovial leukocyte apoptosis is inhibited in established rheumatoid arthritis (RA). In contrast, high levels of leukocyte apoptosis are seen in self-limiting crystal arthritis. The phase in the development of RA at which the inhibition of leukocyte apoptosis is first apparent, and the relationship between leukocyte apoptosis in early RA and other early arthritides, has not been defined. We measured synovial fluid leukocyte apoptosis in very early arthritis and related this to clinical outcome. Synovial fluid was obtained at presentation from 81 patients with synovitis of < or = 3 months duration. The percentages of apoptotic neutrophils and lymphocytes were assessed on cytospin preparations. Patients were assigned to diagnostic groups after 18 months follow-up. The relationship between leukocyte apoptosis and patient outcome was assessed. Patients with early RA had significantly lower levels of neutrophil apoptosis than patients who developed non-RA persistent arthritis and those with a resolving disease course. Similarly, lymphocyte apoptosis was absent in patients with early RA whereas it was seen in patients with other early arthritides. The inhibition of synovial fluid leukocyte apoptosis in the earliest clinically apparent phase of RA distinguishes this from other early arthritides. The mechanisms for this inhibition may relate to the high levels of anti-apoptotic cytokines found in the early rheumatoid joint (e.g. IL-2, IL-4, IL-15 GMCSF, GCSF). It is likely that this process contributes to an accumulation of leukocytes in the early rheumatoid lesion and is involved in the development of the microenvironment required for persistent RA.     

Lack of digalactosyldiacylglycerol increases the sensitivity of Synechocystis sp. PCC 6803 to high light stress

The physiological role of digalactosyldiacylglycerol (DGDG) in photosynthesis was examined using a dgdA mutant of Synechocystis sp. PCC 6803 that is defective in the biosynthesis of DGDG. The dgdA mutant cells showed normal growth under low light (LL) conditions. However, their growth was retarded under high light (HL) conditions and under Ca²⁺- and/or Cl⁻-limited conditions compared to wild-type cells. The retardation in growth of the mutant cells was recovered by exogenous supply of DGDG in the growth medium. The dgdA mutant showed increased sensitivity to photoinhibition. Although both photodamage and repair processes of photosynthesis were affected, the repair process was more severely affected than the photodamage process, suggesting that DGDG plays an important role in the photosynthetic repair cycle.