Degradations of human immunoglobulins and hemoglobin by a 60 kDa cysteine proteinase of Trichomonas vaginalis

The present study was undertaken to investigate the role of cysteine proteinase of Trichomonas vaginalis in escaping from host defense mechanism. A cysteine proteinase of T. vaginalis was purified by affinity chromatography and gel filtration. Optimum pH for the purified proteinase activity was 6.0. The proteinase was inhibited by cysteine and serine proteinase inhibitors such as E-64, NEM, IAA, leupeptin, TPCK and TLCK, and also by Hg²⁺, but not affected by serine-, metallo-, and aspartic proteinase inhibitors such as PMSF, EDTA and pepstatin A. However, it was activated by the cysteine proteinase activator, DTT. The molecular weight of a purified proteinase was 62 kDa on gel filtration and 60 kDa on SDS-PAGE. Interestingly, the purified proteinase was able to degrade serum IgA, secretory IgA, and serum IgG in time- and dose-dependent manners. In addition, the enzyme also degraded hemoglobin in a dose-dependent manner. These results suggest that the acidic cysteine proteinase of T. vaginalis may play a dual role for parasite survival in conferring escape from host humoral defense by degradation of immunoglobulins, and in supplying nutrients to parasites by degradation of hemoglobin.     

In vitro modeling of liver fibrosis with 3D co-culture system using a novel human hepatic stellate cell line

Hepatic stellate cells (HSCs) play an important role in liver fibrosis; however, owing to the heterogeneity and limited supply of primary HSCs, the development of in vitro liver fibrosis models has been impeded. In this study, we established and characterized a novel human HSC line (LSC-1), and applied it to various types of three-dimensional (3D) co-culture systems with differentiated HepaRG cells. Furthermore, we compared LSC-1 with a commercially available HSC line on conventional monolayer culture. LSC-1 exhibited an overall upregulation of the expression of fibrogenic genes along with increased levels of matrix and adhesion proteins, suggesting a myofibroblast-like or transdifferentiated state. However, activated states reverted to a quiescent-like phenotype when cultured in different 3D culture formats with a relatively soft microenvironment. Additionally, LSC-1 exerted an overall positive effect on co-cultured differentiated HepaRG, which significantly increased hepatic functionality upon long-term cultivation compared with that achieved with other HSC line. In 3D spheroid culture, LSC-1 exhibited enhanced responsiveness to transforming growth factor beta 1 exposure that is caused by a different matrix-related protein expression mechanism. Therefore, the LSC-1 line developed in this study provides a reliable candidate model that can be used to address unmet needs, such as development of antifibrotic therapies.     

Independent prediction of naphthalene transport and biodegradation in soil with a mathematical model.

Experiments were performed to test the ability of a mathematical model to predict naphthalene transport and biodegradation. Pseudomonas putida G7, a model bacterial strain capable of degrading naphthalene, was added to a column packed with the soil that had been pre-equilibrated with naphthalene. Model prediction for transport and degradation were based on predetermined parameters that described naphthalene desorption kinetics and the utilization of naphthalene by the test bacterium. However, initial prediction for naphthalene biodegradation was high, and the formation of cell aggregates is advanced as a plausible explanation. Access of substrate to cells in the interior of an aggregate would be restricted. When the numerical simulation was conducted with a factor to account for cell aggregation, it successfully described the experimental data. Thus, with a single adjustable parameter (an average effectiveness factor), the model predicted macroscopic responses of naphthalene in soil-columns where naphthalene was subject to transport and biodegradation.     

Structural characterization of the membrane-associated regulatory subunit of type I cAMP-dependent protein kinase by mass spectrometry: identification of Ser81 as the in vivo phosphorylation site of RIalpha.

The mechanism by which the type Ialpha regulatory subunit (RIalpha) of cAMP-dependent protein kinase is localized to cell membranes is unknown. To determine if structural modification of RIalpha is important for membrane association, both beef skeletal muscle cytosolic RI and beef heart membrane-associated RI were characterized by electrospray ionization mass spectrometry. Total sequence coverage was 98% for both the membrane-associated and cytosolic forms of RI after digestion with AspN protease or trypsin. Sequence data indicated that membrane-associated and cytosolic forms of RI were the same RIalpha gene product. A single RIalpha phosphorylation site was identified at Ser81 located near the autoinhibitory domain of both membrane-associated and cytosolic RIalpha. Because both R subunit preparations were 30-40% phosphorylated, this post-translational modification could not be responsible for the membrane compartmentation of the majority of RIalpha. Mass spectrometry also indicated that membrane-associated RIalpha had a higher extent of disulfide bond formation in the amino-terminal dimerization domain. No other structural differences between cytosolic and membrane-associated RIalpha were detected. Consistent with these data, masses of the intact proteins were identical by LCQ mass spectrometry. Lack of detectable structural differences between membrane-associated and cytosolic RIalpha strongly suggests an interaction between RIalpha and anchoring proteins or membrane lipids as more likely mechanisms for explaining RIalpha membrane association in the heart.     

Development of a virus-induced gene silencing (VIGS) system for <Emphasis Type="Italic">Spinacia oleracea</Emphasis> L.

Virus-induced gene silencing (VIGS) is known as a rapid and efficient system for studying functions of interesting genes in plants. Tobacco rattle virus (TRV) is widely applied for the gene silencing of many plants. Although spinach is a TRV-susceptible plant, a TRV-based VIGS system has not yet been developed for spinach. In this study, we established a TRV-based VIGS system for spinach. To evaluate the functionality of the TRV-based VIGS system, the phytoene desaturase gene (SoPDS) was first isolated from spinach as a marker gene. Then, the VIGS vector pTRV2 was combined with the partial fragment of SoPDS gene in sense or antisense orientation. Using the Agrobacterium infiltration method, we introduced the pTRV2-SoPDS clone to silence the SoPDS gene in spinach. SoPDS was efficiently silenced, and consequently, greater than 90% of newly emerging leaves exhibited severe chlorosis symptoms in the treated plants. Levels of chlorosis symptoms were similar in both plants infected with pTRV2 vectors harboring sense (SoPDS_S) or antisense (SoPDS_A) gene fragments. Quantitative analysis of SoPDS gene expression by qRT-PCR revealed that gene expression was reduced by greater than 90% in both SoPDS_S and SoPDS_A VIGS plants. Chlorosis on leaves was prolonged up to 4~5 wk after Agrobacterium infiltration. The TRV-based VIGS system was effective in silencing the SoPDS gene in spinach, suggesting that it can be a useful reverse genetics tool for the functional study of spinach genes.     

Growth Phase and pH Influence Peptide Signaling for Competence Development in Streptococcus mutans

The development of competence by the dental caries pathogen Streptococcus mutans is mediated primarily through the alternative sigma factor ComX (SigX), which is under the control of multiple regulatory systems and activates the expression of genes involved in DNA uptake and recombination. Here we report that the induction of competence and competence gene expression by XIP (sigX-inducing peptide) and CSP (competence-stimulating peptide) is dependent on the growth phase and that environmental pH has a potent effect on the responses to XIP. A dramatic decline in comX and comS expression was observed in mid- and late-exponential-phase cells. XIP-mediated competence development and responses to XIP were optimal around a neutral pH, although mid-exponential-phase cells remained refractory to XIP treatment, and acidified late-exponential-phase cultures were resistant to killing by high concentrations of XIP. Changes in the expression of the genes for the oligopeptide permease (opp), which appears to be responsible for the internalization of XIP, could not entirely account for the behaviors observed. Interestingly, comS and comX expression was highly induced in response to endogenously overproduced XIP or ComS in mid-exponential-phase cells. In contrast to the effects of pH on XIP, competence induction and responses to CSP in complex medium were not affected by pH, although a decreased response to CSP in cells that had exited early-exponential phase was observed. Collectively, these results indicate that competence development may be highly sensitive to microenvironments within oral biofilms and that XIP and CSP signaling in biofilms could be spatially and temporally heterogeneous.     

Investigation of Oncogenic Cooperation in Simple Liver-Specific Transgenic Mouse Models Using Noninvasive In Vivo Imaging

Liver cancer is a complex multistep process requiring genetic alterations in multiple proto-oncogenes and tumor suppressor genes. Although hundreds of genes are known to play roles in hepatocarcinogenesis, oncogenic collaboration among these genes is still largely unknown. Here, we report a simple methodology by which oncogenic cooperation between cancer-related genes can be efficiently investigated in the liver. We developed various non-germline transgenic mouse models using hydrodynamics-based transfection which express HrasG12V, SmoM2, and a short-hairpin RNA down-regulating p53 (shp53) individually or in combination in the liver. In this transgenic system, firefly luciferase was co-expressed with the oncogenes as a reporter, allowing tumor growth in the liver to be monitored over time without an invasive procedure. Very strong bioluminescence imaging (BLI) signals were observed at 4 weeks post-hydrodynamic injection (PHI) in mice co-expressing HrasG12V and shp53, while only background signals were detected in other double or single transgenic groups until 30 weeks PHI. Consistent with the BLI data, tumors were observed in the HrasG12V plus shp53 group at 4 weeks PHI, while other transgenic groups failed to exhibit a hyperplastic nodule at 30 weeks PHI. In the HrasG12V plus shp53 transgenic group, BLI signals were well-correlated with actual tumor growth in the liver, confirming the versatility of BLI-based monitoring of tumor growth in this organ. The methodology described here is expected to accelerate and facilitate in vivo studies of the hepatocarcinogenic potential of cancer-related genes by means of oncogenic cooperation.