[Polysaccharide from Phellinus igniarius alleviates oxidative stress and hepatic fibrosis in Schistosoma japonicum-infected mice].

OBJECTIVE: To evaluate the role of polysaccharide from Phellinus igniarius (PPI) in the improvement of oxidative stress, hepatic granuloma and hepatic fibrosis in Schistosoma japonicum-iniected in mice. METHODS: The mouse model of schistosomiasis was established by S. japonicum cercariae infection via the abdomen. Balb/c mice were randomly assigned into 5 groups, including the healthy control group (Group A), infection control group (Group B), PPI treatment group (Group C), praziquantel treatment group (Group D) and PPI-praziquantel combination group (Group E), of 10 mice in each group. Each mouse in groups B, C, D and E was infected with (30 ± 2) S. japonicum cercariae. Then, mice in groups D and E were given praziquantel by gavage at a dose of 500 mg/kg for successive two days on day 42 post-infection, while mice in groups C and E were given PPI by gavage at a dose of 400 mg/kg for successive 30 days on day 42 post-infection. Histopathological changes of hepatic tissues were observed using hematoxylin-eosin (HE) staining, and serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), laminin (LN) were determined, while the activities of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), glutathione reductase (GSH-R) and glutathione (GSH) were detected in Mouse liver homogenates. The expression of transforming growth factor-beta (TGF-ß) and alpha-smooth muscle actin (α-SMA) was quantified in hepatic tissues using immunohistochemistry, and the Nrf2 and Gsta4 gene expression was quantified using quantitative real-time PCR (qPCR) assay. RESULTS: Untreated mice presented typical pathological changes of schistosomal hepatic disorders, while PPI treatment effectively alleviated hepatic egg granulomas and collagen deposition. S. japonicum infection resulted in aggravation of hepatic lipid peroxidation, induction of oxidative stress, elevated serum MDA level and a reduction in the activity of GSH and antioxidant enzymes activities in mice. As compared to infected but untreated mice, PPI treatment suppressed hepatic lipid peroxidation, increased the GSH activity and restored the activity of antioxidant enzymes. In addition, PPI treatment inhibited the TGF-ß signaling pathway and up-regulated the Nrf2 and Gsta4 gene expression. CONCLUSIONS: PPI plays a critical role in the treatment of schistosomiasis-induced hepatic fibrosis. It may improve oxidative stress damages through up-regulating Nrf2 and Gsta4 gene expression, thereby suppressing the development of hepatic egg granulomas and hepatic fibrosis.

The B-Raf(V600E) inhibitor dabrafenib selectively inhibits RIP3 and alleviates acetaminophen-induced liver injury.

Receptor-interacting protein (RIP)3 is a critical regulator of necroptosis and has been demonstrated to be associated with various diseases, suggesting that its inhibitors are promising in the clinic. However, there have been few RIP3 inhibitors reported as yet. B-Raf(V600E) inhibitors are an important anticancer drug class for metastatic melanoma therapy. In this study, we found that 6 B-Raf inhibitors could inhibit RIP3 enzymatic activity in vitro. Among them, dabrafenib showed the most potent inhibition on RIP3, which was achieved by its ATP-competitive binding to the enzyme. Dabrafenib displayed highly selective inhibition on RIP3 over RIP1, RIP2 and RIP5. Moreover, only dabrafenib rescued cells from RIP3-mediated necroptosis induced by the necroptosis-induced combinations, that is, tumor necrosis factor (TNF)α, TNF-related apoptosis-inducing ligand or Fas ligand plus Smac mimetic and the caspase inhibitor z-VAD. Dabrafenib decreased the RIP3-mediated Ser358 phosphorylation of mixed lineage kinase domain-like protein (MLKL) and disrupted the interaction between RIP3 and MLKL. Notably, RIP3 inhibition of dabrafenib appeared to be independent of its B-Raf inhibition. Dabrafenib was further revealed to prevent acetaminophen-induced necrosis in normal human hepatocytes, which is considered to be mediated by RIP3. In acetaminophen-overdosed mouse models, dabrafenib was found to apparently ease the acetaminophen-caused liver damage. The results indicate that the anticancer B-Raf(V600E) inhibitor dabrafenib is a RIP3 inhibitor, which could serve as a sharp tool for probing the RIP3 biology and as a potential preventive or therapeutic agent for RIP3-involved necroptosis-related diseases such as acetaminophen-induced liver damage.

Tanshinone-1 induces tumor cell killing, enhanced by inhibition of secondary activation of signaling networks.

Tumor multidrug resistance (MDR) can result from overexpression of drug transporters and deregulation of cellular signaling transduction. New agents and strategies are required for overcoming MDR. Here, we report that tanshinone-1, a bioactive ingredient in traditional Chinese medicine, directly killed MDR tumor cells and their corresponding parental cells, which was potentiated by inhibition of secondary activation of signaling networks. Tanshinone-1 was slightly more potent at inducing cytotoxicity and apoptosis in MDR cells than in corresponding parental cells. Tanshinone-1-induced MDR cell killing was independent of the function and expression of drug transporters but was partially correlated with the phosphatase-dependent reduction of phospho-705-Stat3, which secondarily activated p38-, AKT-, and ERK-involved signaling networks. Cotreatments with p38, AKT, and ERK inhibitors potentiated the anti-MDR effects of tanshinone-1. Our study presents a model for MDR cell killing using a compound of natural origin. This model could lead to new therapeutic strategies for targeting signaling network(s) in MDR cancers as well as new strategies for multitarget design.

Structures and characterizations of bundles of collapsed double-walled carbon nanotubes.

The performance of carbon nanotube fibers (CNTFs) significantly depends on the packing styles of carbon nanotube (CNT) bundles. Revealing the structures and characterizations of CNT bundles is contributive to understanding the structures, properties and even the formation of CNTFs during chemical vapor deposition (CVD) processing. In this paper, bundles consisting of collapsed double-walled carbon nanotubes (CDWNT) in continuous CNTFs fabricated from CVD processing were characterized and analyzed by transmission electronic microscopy (TEM) and x-ray diffraction (XRD). TEM observations show that the continuous CNTFs are composed of CDWNT-bundle units. CDWNT-bundle units of 10-20 nm in thickness contain near numbers of collapsed tubes. The degree of collapse of the CDWNTs varies with their location in the bundle and their own diameter. CDWNT-bundle units pack side by side or face to face, assembling into super-bundles with diameters of 200-300 nm. XRD patterns show that three novel and strong peaks appear at 10°-15°, 21.3° and 23.7°, respectively, corresponding to CDWNT two side pores (10°-15°) and CDWNT layers (21.3° and 23.7°), which indicates the collapsed tube structures in CNTFs are common characterizations. Finally, a collapse mechanism is discussed from the observation and analysis.

Regulation of Ca(2+)-entry in pancreatic α-cell line by transient receptor potential melastatin 4 plays a vital role in glucagon release.

Elevation in the intracellular Ca(2+) concentration stimulates glucagon secretion from pancreatic α-cells. The Transient Receptor Potential Melastatin 4 channel (TRPM4) is critical for Ca(2+) signaling. However, its role in glucagon secreting α-cells has not been investigated. We identified TRPM4 gene expression and protein in the αTC1-6 cell line using RT-PCR and immunocytochemistry. Furthermore, we performed a detailed biophysical characterization of the channel using the patch-clamp technique to confirm that currents typical for TRPM4 were present in αTC1-6 cells. To investigate TRPM4 function, we generated a stable knockdown clone using shRNA and a lentiviral vector. Inhibition of TRPM4 significantly reduced the responses to different agonists during Ca(2+) imaging analysis with Fura-2AM. The reduction in the magnitude of Ca(2+) signals resulted in decreased glucagon secretion. These results suggested that depolarization by TRPM4 may play an important role in controlling glucagon secretion from α-cells and perhaps glucose homeostasis.

A bovine herpesvirus type 1 mutant virus with truncated glycoprotein E cytoplasmic tail has defective anterograde neuronal transport in rabbit dorsal root ganglia primary neuronal cultures in a microfluidic chamber system.

Bovine herpesvirus type 1 (BHV-1) is an important component of the bovine respiratory disease complex (BRDC) in cattle. Following primary intranasal and ocular infection of cattle, BHV-1 establishes lifelong latent infection in trigeminal ganglia (TG). Upon reactivation from latency, the virus is transported from neuronal cell bodies in the TG to projected nerve endings in nose and cornea of latently infected cattle where the virus shedding occurs. This property of BHV-1 plays a significant role in the pathogenesis of BRDC and maintenance of BHV-1 in the cattle population. Recently, we have reported that a glycoprotein E (gE) cytoplasmic tail-truncated BHV-1 (BHV-1 gEAm453) did not reactivate from latency and was not shed in the nasal and ocular secretions of calves and rabbits. Here we describe the methods to establish rabbit primary dorsal root ganglia (DRG) neuron cultures in a microfluidic chamber system and to characterize in vitro anterograde and retrograde axonal transport properties of BHV-1 gE-deleted and BHV-1 cytoplasmic tail-truncated gEAm453 mutant viruses relative to BHV-1 gEAm453-rescued/wild-type viruses. The results clearly demonstrated that whereas the BHV-1 gE-deleted, BHV-1 gEAm453, and BHV-1 gEAm453-rescued/wild-type viruses were transported equally efficiently in the retrograde direction, only the BHV-1 gEAm453-rescued/wild-type virus was transported anterogradely. Therefore, we have concluded that sequences within the BHV-1 gE cytoplasmic tail are essential for anterograde axonal transport and that primary rabbit DRG neuronal cultures in the microfluidic chambers are suitable for BHV-1 neuronal transport studies.

Rapid diagnosis in early stage renal tuberculosis by real-time polymerase chain reaction on renal biopsy specimens.

OBJECTIVE: To determine the early rapid diagnosis of renal tuberculosis (RTB) by real-time polymerase chain reaction (PCR) on renal biopsy specimens. METHODS: Ninety patients were selected for this study. The patients were divided into the following three groups: RTB, non-RTB (N-RTB) and clinically suspected RTB (CS-RTB). The renal biopsy specimens of these patients were used for Mycobacterium tuberculosis DNA detection by real-time PCR, using 35 and 40 as cycle threshold (C(T)) cut-off values. Morning urine samples were collected for M. tuberculosis culture. RESULTS: In the RTB group, 25 C(T)35 and 28 C(T)40 patients were PCR-positive, seven of whom were urine M. tuberculosis culture-positive. In the N-RTB group, four C(T)35 and 13 C(T)40 patients were PCR-positive, none of whom were urine M. tuberculosis culture-positive. In the CS-RTB group, nine C(T)35 and 14 C(T)40 patients were PCR-positive, two of whom were urine M. tuberculosis culture-positive during 12 months of follow-up. The sensitivity and specificity of real-time PCR (C(T)40) were respectively 93.3% and 56.7%. The sensitivity and specificity of real-time PCR (C(T)35) were respectively 83.3% and 86.7%. The sensitivity and specificity of the urine M. tuberculosis culture were respectively 23.3% and 100%. CONCLUSIONS: The detection of M. tuberculosis DNA in renal biopsy tissue by real-time PCR is highly sensitive. Real-time PCR can increase diagnostic accuracy and provide valuable information regarding the early diagnosis of RTB.

TRPM4 impacts on Ca2+ signals during agonist-induced insulin secretion in pancreatic beta-cells.

TRPM4 is a Ca(2+)-activated non-selective cation (CAN) channel that functions in cell depolarization, which is important for Ca(2+) influx and insulin secretion in pancreatic beta-cells. We investigated TRPM4 expression and function in the beta-cell lines HIT-T15 (hamster), RINm5F (rat), beta-TC3 (mouse), MIN-6 (mouse) and the alpha-cell line INR1G9 (hamster). By RT-PCR, we identified TRPM4 transcripts in alpha- and beta-cells. Patch-clamp recordings with increasing Ca(2+) concentrations resulted in a dose-dependent activation of TRPM4 with the greatest depolarizing currents recorded from hamster-derived cells. Further, Ca(2+) imaging experiments revealed that inhibition of TRPM4 by a dominant-negative effect significantly decreased the magnitude of the Ca(2+) signals generated by agonist stimulation compared to control cells. The decrease in the [Ca(2+)](i) resulted in reduced insulin secretion. Our data suggest that depolarizing currents generated by TRPM4 are an important component in the control of intracellular Ca(2+) signals necessary for insulin secretion and perhaps glucagon from alpha-cells.

Thymocytes express the golli products of the myelin basic protein gene and levels of expression are stage dependent.

The golli products of the myelin basic protein gene have been shown to be expressed in mouse thymus and brain. The full repertoire of thymic cell types expressing golli products has not yet been determined, although immunoreactivity has been found in some macrophages. We have analyzed the cellular expression of golli mRNAs and proteins in the thymus. The results showed that MTS5(+) cortical/MTS10(+) medullary epithelial cells and NLDC145(+) dendritic cells did not express golli, while some macrophages did exhibit strong immunoreactivity. GOLLI: mRNAs were not detected in macrophages by in situ hybridization. Thymocytes expressed significant levels of golli mRNAs and proteins by in situ hybridization and immunohistochemistry. Interestingly, golli immunoreactivity varied with thymocyte stage of differentiation. For example, CD4(-)CD8(-) (double-negative) thymocytes expressed relatively high levels of golli. Upon further differentiation into CD4(-)CD8(-) (double-positive) thymocytes, golli protein expression declined dramatically. When thymocytes developed into CD8(-) or CD4(+) (single-positive) thymocytes, golli protein expression increased again, but it never achieved the levels found in double-negative thymocytes. Thus, the altered levels of expression of golli proteins in developing thymocytes correlated with the transitions from double-negative to double-positive and double-positive to single-positive stages. The lack of significant golli expression in thymic stromal cells may offer an alternative explanation for the mechanism of inefficient negative selection of those autoreactive thymocytes with specificity for myelin basic proteins.

Preliminary study on HA coating percutaneously implanted in bone.

A comparative investigation on the possibility of hydroxyapatite (HA) coating and pure Ti column to form biological sealing with skin tissue was completed in this study. HA coating and pure Ti column were percutaneously implanted in the tibia of rabbits. Compared with titanium (Ti) implant, HA coating forms epithelial sealing with skin tissue at 6 weeks postoperatively, while the Ti implant may loosen from the implanted site and be lost. The Ti column loosing rate at this time was 50%. However, once the Ti implant becomes fixed with the bone tissue, it can form epithelial sealing with skin tissue just like the HA coating, at 8 weeks postoperatively. At 8 weeks postoperatively, the epithelial sealing is not destroyed in spite of the fact that the HA coating is biodegraded. Our results show that the HA coating can become fixed with the bone faster than the Ti, which is beneficial for epithelial sealing formation. The main role of HA coating for epithelial sealing is beneficial for sealing at the initial period after it is implanted.

[Morphological evidence for thymocytes apoptosis outside the thymus].

In vitro thymus explants culture designed in this paper can mimic the thymic microenvironment as it were in vivo. Theoretically, thymus explants are cut off free from blood stream. So if some developing or developed thymocytes had the inclination to migrate into the periphery, they would only be accumulated in the blood vessels within thymus explants. After 3-day's culture, under transmission electron microscope we observed the migrating thymocytes accumulated in the blood vessels of C57BL/6 mice thymus explants, and these thymocytes were occurring apoptosis at different stage. To our knowledge, this findings offers the first morphological evidence that thymocytes do not necessarily die inside the thymus in situ, and that having acquired the death signals thymocytes can migrate into the blood stream and die quickly outside the thymus. But this is not to say that we deny the intrathymic death hypothesis. On the contrary, we found the number of thymocytes occurring in situ apoptosis on the surfaces of stromal cells is far more than that of migrating into the blood vessels. So, our proposal is that there are two sites for thymocytes apoptosis, some die inside the thymus and the others die outside the thymus.