mTOR Signaling pathway as a master regulator of memory CD8+ T-cells,Th17, and NK cells development and their functional properties

The mammalian target of rapamycin (mTOR) is a member of the evolutionary phosphatidylinositol kinase-related kinases (PIKKs). mTOR plays a pivotal role in the regulation of diverse aspects of cellular physiology such as body metabolism, cell growth, protein synthesis, cell size, autophagy, and cell differentiation. Immunologically, mTOR has a fundamental part in controlling and shaping diverse functions of innate and adaptive immune cells, in particular, T-cell subsets differentiation, survival, and metabolic reprogramming to ultimately regulate the fate of diverse immune cell types. Researchers report that rapamycin, a selective mTOR inhibitor, and immunosuppressive agent, has surprising immunostimulatory effects on inducing both quantitative and qualitative aspects of virus-specific memory CD8⁺ T-cells differentiation and homeostasis in a T-cell-intrinsic manner. The mTOR signaling pathway also plays a critical role in dictating the outcome of regulatory T cells (Treg), T helper 17 (Th17) cells, and natural killer (NK) cells proliferation and maturation, as well as the effector functions and cytotoxic properties of NK cells. Manipulation of mTOR activity is a critical therapeutic approach for pharmacological agents that seek to inhibit mTOR. This approach should enhance specific memory CD8 ⁺ T-cells responses and induce fully functional effector properties of NK cells to provoke their antitumor and antiviral activities.     

Comparative insight into expression of recombinant human VEGF111b,a newly identified anti-angiogenic isoform,in eukaryotic cell lines

VEGF-A is a critical growth factor in tumor growth and progression. Two families of VEGF-A isoforms are produced through alternative splicing including VEGFxxx pro-angiogenic and VEGFxxxb anti-angiogenic isoforms. VEGF111b is a new member of the VEGFxxxb family that is induced by mitomycin C and doesn't express in normal conditions. The potent anti-angiogenic properties of VEGF-111b and its remarkable resistance to proteolysis make it an interesting alternative candidate for therapeutic use in all types of cancers. Here, the recombinant VEGF-111b cDNA with insertion of intronic sequence was constructed by using a class IIs restriction enzyme-based method. The recombinant pBud-VEGF111b was transfected into CHO dhfr⁻ and HEK 293 cell lines which are currently the standard hosts for the production of candidate therapeutic proteins. Then, the VEGF-111b expression was evaluated in two cell lines using the Real-time PCR. The production of VEGF-111b protein was also investigated here by dot blotting. The VEGF expression was increased about 109 and 185-folds in transfected CHO-dhfr⁻ and HEK 293 cells, respectively, in comparison with the un-transfected cells. Dot blotting approach confirmed that both cell lines have successfully produced the VEGF-111b protein.     

Choline kinase beta is required for normal endochondral bone formation

Background

Choline kinase has three isoforms encoded by the genes Chka and Chkb. Inactivation of Chka in mice results in embryonic lethality, whereas Chkb^−/− mice display neonatal forelimb bone deformations.

Methods

To understand the mechanisms underlying the bone deformations, we compared the biology and biochemistry of bone formation from embryonic to young adult wild-type (WT) and Chkb^−/− mice.

Results

The deformations are specific to the radius and ulna during the late embryonic stage. The radius and ulna of Chkb^−/− mice display expanded hypertrophic zones, unorganized proliferative columns in their growth plates, and delayed formation of primary ossification centers. The differentiation of chondrocytes of Chkb^−/− mice was impaired, as was chondrocyte proliferation and expression of matrix metalloproteinases 9 and 13. In chondrocytes from Chkb^−/− mice, phosphatidylcholine was slightly lower than in WT mice whereas the amount of phosphocholine was decreased by approximately 75%. In addition, the radius and ulna from Chkb^−/− mice contained fewer osteoclasts along the cartilage/bone interface.

Conclusions

Chkb has a critical role in the normal embryogenic formation of the radius and ulna in mice.

General Significance

Our data indicate that choline kinase beta plays an important role in endochondral bone formation by modulating growth plate physiology.     

Phosphate concentration alters the effective bacterial quorum in the symbiosis of Medicago truncatula-Sinorhizobium meliloti

The symbiosis of Medicago truncatula-Sinorhizobium meliloti is affected by phosphate (P) deficiency in the environment. Quorum sensing (QS) is a regulatory pathway in S. meliloti that controls various functions of free-living and symbiotic bacteria in response to phosphate availability and regulation is mediated by a periplasmic protein PstS, and also bacterial density. The quorum sensing pathway of S. meliloti, involves three genes named sinI, sinR and expR and also some bacterial auto-inducers such as N-acyl homoserine lactones (AHLs). In the current study, the expression of the different genes of quorum sensing and pstS were evaluated under 0.1, 0.5 and 2 mM P. The qRT-PCR results showed an increased expression of pstS and also the quorum sensing genes sinI and sinR but not expR, following phosphate starvation. Indeed, the enhanced level of sinR induces the expression of sinI that is responsible for the N-acyl homoserine lactones (AHL) production in S. meliloti. The different response of expR may be due to its negative control on sinR expression. In the symbiosis of M. truncatula-S. meliloti, it was shown that the concentration of phosphate in the medium alters the effective inoculating bacterial quorum (density). By increasing the phosphate concentration in the medium from 0.1 to 0.5 and 2 mM, considering the optimal plant growth and pink nodule (nitrogen-fixing) formation, the effective inoculating bacterial densities were 10⁵, 10⁷ and 10⁹ CFU ml^?1, respectively. Therefore, low phosphate concentrations can compensate for a low bacterial density by inducing the quorum sensing pathway and establishing a symbiosis. Conversely, bacterial density plays the main role in the formation of symbiosis at high phosphate concentrations.     

Downregulation of Chloride Channel ClC-2 by Janus Kinase 3

Janus kinase-3 (JAK3) fosters proliferation and counteracts apoptosis of lymphocytes and tumor cells. The gain of function mutation ^A572VJAK3 has been discovered in acute megakaryoplastic leukemia. JAK3 is inactivated by replacement of lysine by alanine in the catalytic subunit (^K855AJAK3). Regulation of cell proliferation and apoptosis involves altered activity of Cl^? channels. The present study, thus, explored whether JAK3 modifies the function of the small conductance Cl^? channel ClC-2. To this end, ClC-2 was expressed in Xenopus oocytes with or without wild-type JAK3, ^A568VJAK3 or ^K851AJAK3, and the Cl^? channel activity determined by dual-electrode voltage clamp. Channel protein abundance in the cell membrane was determined utilizing chemiluminescence. As a result, expression of ClC-2 was followed by a marked increase of cell membrane conductance. The conductance was significantly decreased following coexpression of JAK3 or ^A568VJAK3, but not by coexpression of ^K851AJAK3. Exposure of the oocytes expressing ClC-2 together with ^A568VJAK3 to the JAK3 inhibitor WHI-P154 (4-[(3’-bromo-4’-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 22 μM) increased the conductance. Coexpression of ^A568VJAK3 decreased the ClC-2 protein abundance in the cell membrane of ClC-2 expressing oocytes. The decline of conductance in ClC-2 and ^A568VJAK3 coexpressing oocytes following inhibition of channel protein insertion by brefeldin A (5 μM) was similar in oocytes expressing ClC-2 with ^A568VJAK3 and oocytes expressing ClC-2 alone, indicating that ^A568VJAK3 might slow channel protein insertion into rather than accelerating channel protein retrieval from the cell membrane. In conclusion, JAK3 downregulates ClC-2 activity and thus counteracts Cl^? exit—an effect possibly influencing cell proliferation and apoptosis.     

X chromosome gene expression in human tissues: male and female comparisons

About 25% of X-linked genes may escape inactivation at least to some degree. However, in vitro results from somatic cell hybrids may not reflect what happens in vivo. Therefore, we analyzed the female/male (F/M) gene fold expression ratio for 299 X-linked and 7795 autosomal genes from 11 different tissues from an existing in vivo microarray database. On average 5.1 and 4.9% of genes showed higher expression in females compared with 7.4 and 7.9% in males, respectively, for X-linked and autosomal genes. A trend was found for F/M gene fold ratios greater than 1.5 for several X-linked genes indicating overexpression in females among multiple tissues. Nine X-linked genes showed overexpression in females in at least 3 of the 11 studied tissues. Of the 9 genes, 6 were located on the short arm and 3 on the long arm of the X chromosome. Six of the 9 genes have previously been reported to escape X inactivation. However, in general, no consistent pattern was seen for the expression of X-linked genes between in vitro and in vivo systems. This study indicates that factors other than the X-inactivation process may impact on the expression of X-linked genes resulting in an overall similar gender expression for both X-linked and autosomal genes.     

Synthesis, characterization and catalytic oxidation of cyclohexane using a novel host (zeolite-Y)/guest (binuclear transition metal complexes) nanocomposite materials

Transition metal (M = Mn(II), Co(II), Ni(II) and Cu(II)) complexes with octahydro-Schiff base (H₄-N₄O₄) = 2,7,13,18-tetramethyl-3,6,14,17-tetraazatricyclo-[17.3.1.1]-tetracosa-1(23),2,6,8(24),9,11,13,17,19,21-decaene-9,11,20,22-tetraol; H₄([H]₈-N₄O₄) = 2,7,13,,18-tetramethyl-3,6,14,17-tetraazatricyclo-[17.13.1.1.]-tetracosa-1(23),8(24),9,11,19,21-hexane-9,11,20,22-tetraol) have been encapsulated in nanopores of zeolite-Y; [M([H]₈-N₄O₄)]@NaY; with Flexible Ligand Method (FLM) for the first time. The new Host-Guest Nanocomposite Materials (HGNM) was characterized by several techniques: chemical analysis, spectroscopic methods (DRS, FT-IR and UV/Vis), BET technique, conductometric and magnetic measurements. The catalytic activities for oxidation of cyclohexane with HGNM complexes are reported. Zeolite encapsulated octahydro-Schiff base copper(II) complex; [Cu([H]₈-N₄O₄)]@NaY; was found to be more active than the corresponding cobalt(II), manganese(II) and nickel(II) complexes for cyclohexane oxidation. The catalytic properties of the complexes are influenced by their geometry and by the steric environment of the active sites. HGNM are stable enough to be reused and are suitable to be utilized as partial oxidation catalysts. The encapsulated catalysts systems; [M([H]₈-N₄O₄)]@NaY; were more active than the corresponding neat complexes; [M([H]₈-N₄O₄))].     

Butyrylcholinesterase K variant and the APOE-ε4 allele work in synergy to increase the risk of coronary artery disease especially in diabetic patients

We have previously shown that butyrylcholinesterase-K (BCHE-K, G1615A/Ala539Thr) variant increases the risk of coronary artery disease (CAD). In addition, we have found that the presence of APOE-ε4 allele augments the risk of CAD in patients with type II diabetes mellitus (T2DM/CAD). Here we explored the concomitant presences of two alleles of the BCHE-K and APOE-ε4 in increasing the risk of CAD or diabetes in T2DM patients with or without CAD and CAD patients without T2DM. This case–control study comprised 631 subjects undergoing their first coronary angiography. They were matched and randomly assigned into four groups: type II diabetic patients with no sign of CAD (T2DM), type II diabetic patients with CAD/ND (T2DM/CAD), CAD patients with no sign of diabetes (CAD/ND), and healthy individuals (NCAD/ND). BCHE-K variant and APOE genotypes were detected by PCR-RFLP and serum lipid level was measured enzymatically. We found that BCHE-K and APOE-ε4 allele act synergistically to increase the risk of CAD in both T2DM, non-diabetic and total CAD (TCAD = T2DM/CAD + CAD/ND) individuals. The level of synergy 1.5 and 1.2 fold are higher in CAD patients (OR = 4.5; P = 0.011) with T2DM than the non-diabetic CAD patients (OR = 3.07; P = 0.024) and TCAD patients (OR = 3.74; P = 0.018), respectively. The CAD subjects with and without T2DM and TCAD patients carrying both APOE-ε4 allele and BCHE-K had significantly lower plasma HDL-C (P values = 0.008, 0.047, and 0.036, respectively) and higher plasma LDL-C (P values = 0.025, 0.048, and 0.04, respectively), than that of the control carriers both APOE-ε4 and BCHE-K. We have found that BCHE-K and APOE-ε4 allele not only act synergistically to increase the risk of CAD, particularly in T2DM subjects in population from western Iran, who have high levels of LDL-C and low levels of HDL-C, suggesting that a specific therapeutic intervention should be considered for these particular groups of patients.