Progression of Human Renal Cell Carcinoma via Inhibition of RhoA-ROCK Axis by PARG1

Renal cell carcinoma (RCC) is the most lethal urological malignancy with high risk of recurrence; thus, new prognostic biomarkers are needed. In this study, a new RCC antigen, PTPL1 associated RhoGAP1 (PARG1), was identified by using serological identification of recombinant cDNA expression cloning with sera from RCC patients. PARG1 protein was found to be differentially expressed in RCC cells among patients. High PARG1 expression is significantly correlated with various clinicopathological factors relating to cancer cell proliferation and invasion, including G3 percentage (P = .0046), Ki-67 score (p expression is also correlated with high recurrence of N0M0 patients (P = .0084) and poor prognosis in RCC patients (P = .0345). Multivariate analysis has revealed that high PARG1 expression is an independent factor for recurrence (P = .0149) of N0M0 RCC patients. In in vitro studies, depletion of PARG1by siRNA in human RCC cell lines inhibited their proliferation through inducing G1 cell cycle arrest via upregulation of p53 and subsequent p21^Cip1/Waf1, which are mediated by increased RhoA-ROCK activities. Similarly, PARG1 depletion cells inhibited invasion ability via increasing RhoA-ROCK activities in the RCC cell lines. Conversely, overexpression of PARG1 on human embryonic kidney cell line HEK293T promotes its cell proliferation and invasion. These results indicate that PARG1 plays crucial roles in progression of human RCC in increasing cell proliferation and invasion ability via inhibition of the RhoA-ROCK axis, and PARG1 is a poor prognostic marker, particularly for high recurrence of N0M0 RCC patients.     

The broad range di- and tri-nucleotide exchanger SLC35B1 displays asymmetrical affinities for ATP transport across the ER membrane

In eukaryotic cells, uptake of cytosolic ATP into the endoplasmic reticulum (ER) lumen is critical for the proper functioning of chaperone proteins. The human transport protein SLC35B1 was recently postulated to mediate ATP/ADP exchange in the ER; however, the underlying molecular mechanisms mediating ATP uptake are not completely understood. Here, we extensively characterized the transport kinetics of human SLC35B1 expressed in yeast that was purified and reconstituted into liposomes. Using [α³²P]ATP uptake assays, we tested the nucleotide concentration dependence of ATP/ADP exchange activity on both sides of the membrane. We found that the apparent affinities of SLC35B1 for ATP/ADP on the internal face were approximately 13 times higher than those on the external side. Because SLC35B1-containing liposomes were preferentially inside-out oriented, these results suggest a low-affinity external site and a high-affinity internal site in the ER. Three different experimental approaches indicated that ATP/ADP exchange by SLC35B1 was not strict, and that other di- and tri-nucleotides could act as suitable counter-substrates for ATP, although mononucleotides and nucleotide sugars were not transported. Finally, bioinformatic analysis and site-directed mutagenesis identified that conserved residues K117 and K120 from transmembrane helix 4 and K277 from transmembrane helix 9 play critical roles in transport. The fact that SLC35B1 can promote ATP transport in exchange for ADP or UDP suggest a more direct coupling between ATP import requirements and the need for eliminating ADP and UDP, which are generated as side products of reactions taking place in the ER-lumen.     

Temporal Expression of Mouse Glial Fibrillary Acidic Protein mRNA Studied by a Rapid In Situ Hybridization Procedure

A rapid and sensitive in situ hybridization technique is described for the detection of mRNA sequences in 6-8-micron cryostat sections. The method incorporates the use of alpha-thio-35S-labelled nucleoside triphosphates for the generation of high-specific-activity DNA probes and a high-stringency washing procedure that virtually eliminates background without unduly compromising histological integrity. Whereas signal resolution is less than that observed using 3H probes, 35S-labelled probes are well-suited for experiments where resolution at the cellular level is required. The method has been applied to a study of the developmental regulation of glial fibrillary acidic protein (GFAP) mRNA expression in developing mouse brain. GFAP-specific sequences are first detectable after the second postnatal day, and thereafter rise to a level that is maintained throughout development and into adulthood. The distribution of GFAP-encoding sequences broadly reflects the known distribution of astrocytes, but the levels of mRNA within these cells vary by a surprisingly large amount depending on their location. For example, in adult animals, the astrocytes of the glial limitans contain an abundance of GFAP-specific mRNA that is higher than corresponding levels in astrocytes in the cerebellar white matter, whereas these cells in turn contain considerably more GFAP-specific mRNA than astrocytes in the gray matter of the cerebrum. Unexpectedly, parallel RNA blot transfer experiments show the existence of some GFAP-encoding mRNA size heterogeneity that is restricted to the first postnatal week.     

Ebola virus replication is regulated by the phosphorylation of viral protein VP35

Ebola virus (EBOV) is a zoonotic pathogen, the infection often results in severe, potentially fatal, systematic disease in human and nonhuman primates. VP35, an essential viral RNA-dependent RNA polymerase cofactor, is indispensable for Ebola viral replication and host innate immune escape. In this study, VP35 was demonstrated to be phosphorylated at Serine/Threonine by immunoblotting, and the major phosphorylation sites was S187, S205, T206, S208 and S317 as revealed by LC-MS/MS. By an EBOV minigenomic system, EBOV minigenome replication was shown to be significantly inhibited by the phosphorylation-defective mutant, VP35 S187A, but was potentiated by the phosphorylation mimic mutant VP35 S187D. Together, our findings demonstrate that EBOV VP35 is phosphorylated on multiple residues in host cells, especially on S187, which may contribute to efficient viral genomic replication and viral proliferation.     

S100β Protein Expression: Gender- and Age-Related Daily Changes

S100β is a soluble protein released by glial cells mainly under the activation of the 5-HT1A receptor. It has been reported as a neuro-trophic and -tropic factor that promotes neurite maturation and outgrowth during development. This protein also plays a role in axonal stability and the plasticity underlying long-term potentiation in adult brains. The ability of S100β to rapidly regulate neuronal morphology raises the interesting point of whether there are daily rhythm or gender differences in S100β level in the brain. To answer this question, the S100β expression in adult female and male rats, as well as in adult female CD-21 and S100β −/− female mice, were investigated. Scintillation counting and morphometric analysis of the immunoreactivity of S100β, showed rhythmic daily expression. The female and male rats showed opposite cycles. Females presented the highest value at the beginning of the rest phase (5:00 h), while in males the maximum value appeared in the beginning of the motor activity period (21:00 h). These results confirm previous S100β evaluations in human serum and cerebrospinal fluid reporting the protein’s function as a biomarker for brain damage (Gazzolo et al. in Clin Chem 49:967–970, 2003; Clin Chim Acta 330:131–133, 2003; Pediatr Res 58:1170–1174, 2005), similar behavior was also observed for GFAP in relation to Alzheimer Disease (Fukuyama et al. in Eur Neurol 46:35–38, 2001). The data should be taken into account when considering S100β as a biomarker of health condition. In addition, the results raise questions on which structure or condition imposes these rhythms as well as on the physiological meaning of the observed gender differences.     

Study on Calceolarioside A Anti-Aβ25–35-Induced Damage in SH-SY5Y cells by Atomic Force Microscopy

Calceolarioside A is a phenylethyl glycoside compound, originally isolated from the bark of Fraxinus mandshurica Rupr. In this work, the protective effect of Calceolarioside A on beta Amyloid protein induced toxicity in SH-SY5Y cells was studied. DPPH experiment, MTT assay, SEM, Atomic force microscopy and Colony formation were used to study the activity of Calceolarioside A. The results in this article show that the survival rate of the cells with Calceolarioside A (20–40 mg/mL) was significantly higher than that of the model cells without Calceolarioside A. Calceolarioside A could protect SH-SY5Y cells by improving some parameters in cells, such as the cell height, Young's modulus, adhesion and branch. In summary, Calceolarioside A can reduce Aβ_25–35-induced damage in SH-SY5Y cells. It can be a potential medicine to treatment with AD.     

Fibrotic enzymes modulate wound‐induced skin tumorigenesis

Fibroblasts are a major component of the microenvironment of most solid tumours. Recent research elucidated a large heterogeneity and plasticity of activated fibroblasts, indicating that their role in cancer initiation, growth and metastasis is complex and context‐dependent. Here, we performed genome‐wide expression analysis comparing fibroblasts in normal, inflammatory and tumour‐associated skin. Cancer‐associated fibroblasts (CAFs) exhibit a fibrotic gene signature in wound‐induced tumours, demonstrating persistent extracellular matrix (ECM) remodelling within these tumours. A top upregulated gene in mouse CAFs encodes for PRSS35, a protease capable of collagen remodelling. In human skin, we observed PRSS35 expression uniquely in the stroma of high‐grade squamous cell carcinomas. Ablation of PRSS35 in mouse models of wound‐ or chemically‐induced tumorigenesis resulted in aberrant collagen composition in the ECM and increased tumour incidence. Our results indicate that fibrotic enzymes expressed by CAFs can regulate squamous tumour initiation by remodelling the ECM.     

“Trace Metabolites”–Effect of High Concentrations on Leishmania tarentolae Promastigotes*

SYNOPSIS. Relatively high concentrations (10–100 μM) of chromate, selenate, and vanadate ions accelerated growth of Leishmania tarentolae in a chemically defined medium containing taurine as the sole sulfur source. These ions also enhanced the incorporation of [³⁵S]taurine label in whole cell, 10% trichloroacetic acid insoluble, and 10% trichloroacetic acid soluble cell fractions.     

Choline Uptake by the Neuroblastoma X Glioma Hybrid, NG108-15

The neuroblastoma X glioma hybrid clone NG108-15 is able to release acetylcholine upon depolarization and form cholinergic neuromuscular synapses in culture. Normal functioning of cholinergic synapses is thought to be dependent on the ability of a neuron to take up extracellular choline, since neurons are unable to synthesize choline de novo. For these two reasons it became important to characterize the choline uptake system of NG108-15 cells. The uptake system appears to bear little if any resemblance to the Na⁺-dependent high-affinity choline uptake system normally associated with cholinergic neurons. Although the cells appear to possess both high- and low- affinity choline uptake systems, neither system is dependent on Na⁺ and uptake actually is increased about 60% by the substitution of sucrose for NaCl. Acetylcholine synthesis also is not dependent on Na⁺, since sucrose, substituted for NaCl, also stimulates acetylcholine synthesis. Changes in the concentrations of the other ions in the uptake medium have little effect on uptake, with the exception that elevated Ca²⁺ or Mg²⁺ reverses the stimulation of choline uptake produced by substitution of sucrose for NaCl. Choline uptake is inhibited by hemicholinium-3, but only at high concentrations of the drug (IC₅₀= 30–80 μm ). The metabolic poisons cyanide and iodoacetate inhibit uptake by only 30-40%. Growth of the cells in N⁶,O^2′ dibutyryladenosine-3′,5′-cyclic monoposphate, which promotes functional and morphological differentiation of the cells, decreased slightly the total amount of choline taken up but had no additional effect on the uptake system. Thus, it appears that NG108-15 cells are capable of forming functional cholinergic synapses with muscle cells even though the neuroblastoma does not possess the high-affinity choline uptake system normally associated with cholinergic neurons.