Suppression of hepatitis B viral gene expression by protein-tyrosine phosphatase PTPN3

Summary Protein-tyrosine phosphatase PTPN3 is a membrane-associated non-receptor protein-tyrosine phosphatase. PTPN3 contains a N-terminal FERM domain, a middle PDZ domain, and a C-terminal phosphatase domain. Upon co-expression of PTPN3, the level of human hepatitis B viral (HBV) RNAs, 3.5 kb, 2.4/2.1 kb, and 0.7 kb transcribed from a replicating HBV expression plasmid is significantly reduced in human hepatoma HuH-7 cells. When the expression of endogenous PTPN3 protein is diminished by specific small interfering RNA, the expression of HBV genes is enhanced, indicating that the endogenous PTPN3 indeed plays a suppressive role on HBV gene expression. PTPN3 can interact with HBV core protein. The interaction is mediated via the PDZ domain of PTPN3 and the carboxyl-terminal last four amino acids of core. Either deletion of PDZ domain of PTPN3 or substitution of PDZ ligand in core has no effect on PTPN3-mediated suppression. These results clearly show that the interaction of PTPN3 with core is not required for PTPN3 suppressive effect. Mutation of ³⁵⁹serine and ⁸³⁵serine of 14-3-3β binding sites to alanine, which slightly reduces the interaction with 14-3-3β, does not influence the PTPN3 effect. In contrast, mutation of the invariant ⁸⁴²cysteine residue in phosphatase domain to serine, which makes the phosphatase activity inactive, does not change its subcellular localization and interaction with core or 14-3-3β, but completely abolishes PTPN3-mediated suppression. Furthermore, deletion of FERM domain does not affect the phosphatase activity or interaction with 14-3-3β, but changes the subcellular localization from cytoskeleton-membrane interface to cytoplasm and nucleus, abolishes binding to core, and diminishes the PTPN3 effect on HBV gene expression. Taken together, these results demonstrate that the phosphatase activity and FERM domain of PTPN3 are essential for its suppression of HBV gene expression. En-Chi Hsu, Yen-Cheng Lin have equal contributions to this work.     

Photosynthetic characteristics and productivity of spring rape plants as related to crop density

Optimal density of spring rape (Brassica napus L.) crop stand was determined by plant photosynthetic characteristics at the beginning of flowering. As crop density increased from 100 to 350 plants/m², leaf surface index (LSI) of the crop was found to increase by 18.2–80.2%, and LSI decreased by 38.8–67.3% as compared with the sparsest crop (50–100 plants/m²). LSI depended on the rate of incident PAR reaching 0.5 and 0.25 heights of the crop stand and to the soil surface. When crop density increased from 100 to 350 plants/m², the photosynthetic potential (PP) of the crop increased 1.8 times as compared with the sparsest crop. PP of the densest rape crop stand was 3 times lower than in the sparsest crop. When the crop density increased from 100 to 250 plants/m², the daily increment in biomass calculated per leaf surface unit increased by 27.0% as compared with the sparsest crop and depended on LSI. When leaf area decreased, the daily increment in biomass calculated per leaf surface unit declined; in the densest stand, this characteristic was by 58.3% lower than in the sparsest crop. Rape productivity at the flowering stage depended on the crop density, LSI of plants, rate of PAR reaching 0.5 and 0.25 heights of the crop stand and to the soil surface, PP, and the daily increment in biomass calculated per leaf surface unit. Crop productivity at the flowering stage and the rape seed yield were associated by a significant parabolic relationship. When crop density increased from 100 to 350 plants/m², seed yield per plant considerably decreased (by 33.1–78.5%) as compared with the sparsest crop. The greatest influence on seed yield per plant was exerted by LSI and the daily increment in biomass calculated per leaf surface unit. When crop density increased to 250–300 plants/m², the seed yield considerably rose (by 28.6–58.8%) as compared with the sparsest crop; when this index reached 300–350 plants/m², the seed yield decreased because plant growth was suppressed, with the productivity reduced. The results thus obtained suggest that the photometric characteristics of spring rape were at optimum at crop density of 100–250 plants/m². The agroclimatic conditions of Lithuania ensure potential for rapid accumulation of total biomass and high seed yield.     

Expression, purification, and refolding of recombinant collagen alpha1(XI) amino terminal domain splice variants

The amino terminal domain of collagen type XI alpha1 chain is a noncollagenous structure that is essential for the regulation of fibrillogenesis in developing cartilage. The amino terminal domain is alternatively spliced at the mRNA level, resulting in proteins expressed as splice variants. These splice variants, or isoforms, have unique distribution in growing tissues, alluding to distinct roles in development. We report here a rapid and straightforward method for expression, purification and in vitro folding of recombinant collagen XI isoforms alpha1(XI) NTD[p7] and alpha1(XI) NTD[p6b+7]. The recombinant isoforms were expressed in Escherichia coli as bacterial inclusion bodies. Unfolded carboxy terminal polyhistidine tagged proteins were purified via nickel affinity chromatography and refolded with specific protocols optimized for each isoform. Purity was assessed by SDS-PAGE and correct secondary structure by a comparison of circular dichroism data with that obtained for Npp. Protein expression and purification of the recombinant collagen XI splice variants will allow further studies to elucidate the structure and molecular interactions with components of the extracellular matrix. This research will clarify the mechanism of collagen XI mediated regulation of collagen fibrillogenesis.     

Forms of LonB protease from Archaeoglobus fulgidus devoid of the transmembrane domain: The contribution of the quaternary structure to the regulation of enzyme proteolytic activity

Deletion of the transmembrane domain (TM-domain) of Archaeoglobus fulgidus LonB protease (Archaeoglobus fulgidus (AfLon)) was shown to result in uncontrollable activation of the enzyme proteolytic site and in vivo autolysis yielding a stable and functionally inactive fragment consisting of both α-helical and proteolytic domains (αP). The ΔTM-AfLon-S509A enzyme form, obtained by site-directed mutagenesis of the catalytic Ser residue, is capable of recombination with the αP fragment. The mixed oligomers were shown to be proteolytically active, which indicates a crucial role of subunit interactions in the activation of the AfLon proteolytic site. The thermophilic nature of AfLon protease was found to be due to the special features of the enzyme activity regulation, the structure of ATPase domain, and the quaternary structure.     

Role of Drosophila gene dunc-115 in nervous system

Axonal guidance signals are transduced through growth cone surface receptors to the interior leading to changes of actin dynamics and actin binding proteins, which are critical in determining the outcome of actin cytoskeleton reorganization. We report here the characterization of the Drosophila actin binding protein abLIM/Unc-115 homolog Dunc-115 and its role in the nervous system. Three Dunc-115 isoforms are identified as Dunc-115L, M and S, respectively. While Dunc-115L is a canonical homolog of Unc-115 with four LIM domains and one villin headpiece domain, Dunc-115M and S are novel isoforms without counterparts in other species. Our molecular modeling shows Dunc-115L is likely to bind to actin. Mutant analysis reveals that Dunc-115 is involved in axonal projection in both the visual and central nervous system.     

TGFBIp/betaig-h3 protein: a versatile matrix molecule induced by TGF-beta

TGFBIp/βig-h3 protein is an extracellular matrix molecule initially cloned from human adenocarcinoma cells treated with TGF-β. Its precise function remains obscure but a number of studies have demonstrated it to be an intriguingly versatile molecule role in a wide range of physiological and pathological conditions. To date, the most extensively studied and reported action of TGFBIp/βig-h3 protein is in corneal dystrophy and several excellent reviews are available on this. Work from various laboratories on this molecule has compiled a tremendous amount of information over the past decade and a half. Here we review the current understanding on TGFBIp/βig-h3 protein and its functions in morphogenesis, extracellular matrix interactions, adhesion/migration, corneal dystrophy, tumorigenesis, angiogenesis, nephropathies, osteogenesis, wound healing and inflammation.     

Overexpression of phospholipase C-gamma1 inhibits NGF-induced neuronal differentiation by proliferative activity of SH3 domain

Since the biological role of phospholipase C (PLC) gamma1 in neuronal differentiation still barely understood, here, we report that overexpression of PLC gamma1 inhibits neurite outgrowth and prolonged proliferation ability of PLC gamma1 contribute to the alteration of cell cycle regulatory proteins, subsequently exiting from cell growth arrest. Deletion of the SH3 or the entire SH223 domains, but not deletion of the N-SH2 or both the N-SH2 and C-SH2 domains expressing cells abolishes the differentiation-inhibitory effects of PLC gamma1, displaying depression of PCNA and elevation of cyclin D1. Moreover, these cells declined CDK1 and CDK2 expression and increased p21WAF-1, accompanying with G2/M accumulation. Some antiproliferative reagents are able to restore neurite outgrowth in PLC gamma1 cells, showing G2/M arrest. Our findings suggest that the proliferation activity of PLC gamma1 via its SH3 domain may be coupled with the flight from growth arrest by NGF, thereby inhibiting neuronal differentiation.     

Parameters controlling the gene-targeting frequency at the Sphingomonas species rrn site and expression of the methyl parathion hydrolase gene

AIMS: To investigate the key parameters controlling the exogenous methyl parathion hydrolase (MPH) gene mpd-targeting frequency at the ribosomal RNA operon (rrn) site of Sphingomonas species which has a wide range of biotechnological applications. METHODS AND RESULTS: Targeting vectors with different homology lengths and recipient target DNA with different homology identities were used to investigate the parameters controlling the targeting frequency at the Sphingomonas species rrn site. Targeting frequency decreased with the reduction of homology length, and the minimal size for normal homologous recombination was >100 bp. Homologous recombination could succeed even if there were 3-4% mismatches; however, targeting frequency decreased with increasing sequence divergence. The Red recombination system could increase the targeting frequency to some extent. Targeting of the mpd gene to the rrn site did not affect cell viability and resulted in an increase of MPH-specific activity in recombinants. CONCLUSIONS: Targeting frequency was affected by homology length, identity and the Red recombination system. The rrn site is a good target site for the expression of exogenous genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is useful as a foundation for a better understanding of recombination events involving homologous sequences and for the improved manipulation of Sphingomonas genes in biotechnological applications.     

Subcellular localization determines the delicate balance between the anti- and pro-apoptotic activity of Livin

Livin is a member of the Inhibitor of Apoptosis Protein family which inhibits apoptosis induced by a variety of stimuli. We previously identified Livin and demonstrated that following apoptotic stimuli, Livin is cleaved by effector caspases to produce a truncated form with paradoxical pro-apoptotic activity. In the present study, we reveal that while full-length Livin shows diffuse cytoplasmic localization, truncated Livin (tLivin) is found in a peri-nuclear distribution with marked localization to the Golgi apparatus. Using mutation analysis, we identified two domains that are crucial for the pro-apoptotic activity of tLivin: the N-terminal region of tLivin which is exposed by cleavage, and the RING domain. We demonstrate that, of the N-terminal sequence, only the first N-terminal glycine residue dictates the peri-nuclear distribution of tLivin. However, while the perinuclear localization of tLivin is essential, it is not sufficient for tLivin to exert its pro-apoptotic function. Once tLivin is properly localized, an intact RING domain enables its pro-apoptotic function. Electronic Supplementary Material Supplementary material is available in the online version of this article at .     

Record error and range contraction, real and imagined, in the restricted shrub Banksia hookeriana in south-western Australia

Banksia hookeriana Meissn. (Proteaceae) is a fire‐killed shrub endemic to the northern sandplains of south‐western Australia that could be described as endangered based on its small geographical range (< 5000 km²) and area of occupancy (～500 km²). Impacts on the species’ geographical range by land clearance for farming and mining, and by altered fire regime, were investigated using three lines of evidence: records of herbarium collections, a comprehensive field survey of extant populations, and air photo and satellite images revealing the recent history of land clearance and fires. These show that the species’ range has contracted by up to 40% in area and 26% latitudinally through the loss of outlier and range limit populations since 1960. In addition, 22% of remaining native shrubland on the Eneabba sandplain has been lost over this period through clearing for farming and mining, representing further habitat loss for B. hookeriana. Detailed investigation of B. hookeriana herbarium collections (n = 46) revealed important errors that artificially affected the geographical range of the species and emphasized the importance of close examination of all data captured from collection records. Recorded locations occurred hundreds of kilometres outside the current geographical range of the species in areas with different climate and substrate. Incorrect species identification of herbarium specimens further extended the apparent geographical range of the species. On the other hand, credible records indicated the loss of the species from localities at the limits of its range. Overall, a disconcertingly high proportion of records contained errors that may be difficult to identify without close examination of the original collections and detailed ground‐truthing. Were these records to be used to model climate envelopes, identify potential habitat where the species might occur, or might migrate to either as pests or under climate change scenarios, or to analyse evolutionary or ecological theory (for example) — as is now becoming commonplace — large errors may ensue.