High sodium chloride affects BMP-7 and 1α-hydroxylase levels through NCC and CLC-5 in NRK-52E cells.

A diet high in sodium chloride (NaCl) can affect renal function damage and increase urinary calcium excretion, leading to bone loss. in renal tubules, Na-Cl co-transporter (NCC) and chloride channel 5 (CLC-5) are involved in regulating urinary calcium excretion. In addition, some cytokines, such as Bone morphogenetic protein 7 (BMP-7) and 1α-hydroxylase, are synthesized by renal tubules, which target on bone and play important roles on bone metabolism. However, the specific mechanisms between NaCl and these ion channels or cytokines still need investigations from many aspects. This study, in culture normal rat renal tubular epithelial NRK-52E cells, showed that high concentrations of NaCl significantly inhibited the cell viability and increased the cell apoptosis. High concentration of NaCl reduce bone mineral density (BMD), as demonstrated by the significantly increased mRNA and protein levels of NCC and osteopontin (OPN), but decreased the levels of CLC-5, BMP-7, and 1α-hydroxylase. In addition, we found that ovariectomized (OVX) rats on a high-salt diet for 12 weeks had altered levels of these indices in the renal cortices. Moreover, the BMD in fourth and fifth lumbar vertebra (LV4 and 5) and femurs were significantly decreased and bone microstructure was destroyed of these rats. We also demonstrated that high concentration of NaCl enhanced the inhibition of these cytokines which is beneficial to increase BMD, induced by modulating ion channels NCC and CLC-5. In conclusion, our results indicate that high concentration of NaCl reduce BMD by regulating ion channels NCC and CLC-5.

Neovascular glaucoma regulation by arylsulfonyl indoline-benzamide (ASIB) through targeting NF-kB signalling pathway.

The present study investigated the effect of arylsulfonyl indoline-benzamide (ASIB) on neovascular glaucoma in the mice model in vivo. In the mice model of glaucoma, ASIB treatment significantly (P < 0.05) increased PDGF-B-positive cell count in the corneal tissues. ASIB treatment at 5, 10, 15 and 20 mg/kg doses raised the level of PDGF-B mRNA in the mice cornea by 2.3-, 3.8-, 5.4- and 5.5-fold, respectively. Pre-treatment of the glaucoma mice with ASIB leads to inhibition of TNF-α and IL-6 production. In the glaucoma mice, treatment with ASIB leads to a marked decrease in the level of NOD2 mRNA and protein. ASIB treatment caused a significant decrease in the glaucoma-induced up-regulation of NF-κB p65 activation. The phosphorylation of NF-κB p65 was almost completely inhibited in the glaucoma mice on treatment with 15 mg/kg dose of ASIB. ASIB exhibited inhibitory effect on glaucoma-induced inflammatory cytokine and oxidative factor damage in the mice. It caused up-regulation of PDGF expression and down-regulated NF-κB activation. Therefore, ASIB can be of therapeutic significance for neovascular glaucoma treatment. However, more studies need to be performed to fully understand the molecular mechanism of ASIB in glaucoma treatment.

Lead exposure inhibits osteoblastic differentiation and inactivates the canonical Wnt signal and recovery by icaritin in MC3T3-E1 subclone 14 cells.

Exposure to lead (Pb) poses a threat to human bone health, including changes in bone mineral composition and the inhibition of skeletal growth and bone maturation. However, little is known about how Pb directly affects osteoblasts. In this work, we found that sub-toxic Pb concentrations suppressed bone nodule formation and inhibited differentiation in MC3T3-E1 subclone 14 cells, as shown by decreased expression levels of the differentiation markers alkaline phosphatase (ALP), type 1 collagen (COL1), osteocalcin (OC), and runt-related transcription factor 2 (RUNX2). Moreover, Pb inactivated the canonical Wnt pathway by regulating key components, such as Wnt3a, Dkk-1, pGSK3ß, and ß-catenin. Icaritin (ICT), a hydrolytic product of icariin from the genus Epimedium, attenuates the inhibitory effect of Pb on osteoblastic differentiation, as well as activate the canonical Wnt signal pathway. Taken together, the results suggest ICT as a potential bone protectant that may be used to prevent bone damage caused by Pb and can activate the canonical Wnt signal pathway.

Influence of polymorphisms in VEGF, ACE, TNF and GST genes on the susceptibility to retinopathy of prematurity among Chinese infants.

AIM: To investigate common polymorphisms in VEGF, ACE, TNF and GST genes with retinopathy of prematurity (ROP) risk among Chinese infants. METHODS: Nine polymorphisms in the above genes were genotyped on 724 advanced cases of ROP and 878 prematurely-born infants of low birth weight who were without any ophthalmologic disease. The frequencies of the polymorphisms were compared between cases and controls to identify the association present, if any. RESULTS: Of the nine polymorphisms, only two showed significant associations: ACE insertion deletion (ID) polymorphism (P=0.031) and TNF -308G/A polymorphism (P<0.001). The former was associated with a reduced ROP risk [ID genotype, adjusted OR (aOR): 0.603, 95%CI: 0.427-0.893, P=0.034; DD genotype, aOR: 0.468, 95%CI: 0.229-0.626, P=0.002], while the latter showed an increased risk (GA genotype, aOR: 1.956, 95%CI: 1.396-2.465, P<0.001; AA genotype, aOR: 2.809, 95%CI: 1.802-4.484, P<0.001). The association was also noted at the allele level (ACE D allele aOR: 0.698, 95%CI: 0.294-0.883, P<0.001; TNF -308A allele aOR: 1.776, 95%CI: 1.446-2.561, P<0.001). CONCLUSION: The ACE ID polymorphism can protect against ROP development while the TNF -308G/A can increase the risk of the disease among Chinese infants.

Protective effect of Pterocarpus marsupium bark extracts against cataract through the inhibition of aldose reductase activity in streptozotocin-induced diabetic male albino rats.

The present study was aimed to investigate the protective effect of Pterocarpus marsupium bark extracts against cataract in streptozotocin-induced diabetic male albino rats. Aldose reductase is a key enzyme in the intracellular polyol pathway, which plays a major role in the development of diabetic cataract. Rats were divided into five groups as normal control, diabetic control, and diabetic control treated with different concentrations of Pterocarpus marsupium bark extracts. Presence of major constituents in Pterocarpus marsupium bark extract was performed by qualitative analysis. Body weight changes, blood glucose, blood insulin, and reduced glutathione (GSH) and aldose reductase mRNA and protein expression were determined. Rat body weight gain was noted following treatment with bark extracts. The blood glucose was reduced up to 36% following treatment with bark extracts. The blood insulin and tissue GSH contents were substantially increased more than 100% in diabetic rats following treatment with extracts. Aldose reductase activity was reduced up to 79.3% in diabetic rats following treatment with extracts. Vmax, Km, and Ki of aldose reductase were reduced in the lens tissue homogenate compared to the diabetic control. Aldose reductase mRNA and protein expression were reduced more than 50% following treatment with extracts. Treatment with Pterocarpus marsupium bark was able to normalize these levels. Taking all these data together, it is concluded that the use of Pterocarpus marsupium bark extracts could be the potential therapeutic approach for the reduction of aldose reductase against diabetic cataract.

The processing of human rhomboid intramembrane serine protease RHBDL2 is required for its proteolytic activity.

RHBDL2, a human homolog of the rhomboids, belongs to a unique class of serine intramembrane proteases; little is known about its function and regulation. Here, we show that RHBDL2 is produced as a proenzyme and that the processing of RHBDL2 is required for its cellular protease activity. The processing of RHBDL2 was shown by both Western blot and immunofluorescence analysis. We have demonstrated that a highly conserved Arg residue on loop 1 of RHBDL2 plays a critical role in the activation of the proenzyme. The activation of RHBDL2 is catalyzed by a protease that is sensitive to a class of sulfonamide compounds. Furthermore, endogenous RHBDL2 exists as the processed form and treatment of cells with a sulfonamide inhibitor led to an accumulation of the full length of RHBDL2. Therefore, this study has demonstrated that RHBDL2 activity is regulated by proenzyme activation, revealed a role for the conserved WR residues in loop 1 in RHBDL2 activity, and provided critical insights into the regulation and function of this human rhomboid protease.

Soluble oligomers of the intramembrane serine protease YqgP are catalytically active in the absence of detergents.

Rhomboid, a polytopic membrane serine protease, represents a unique class of proteases that cleave substrates within the transmembrane domain. Elucidating the mechanism of this extraordinary catalysis comes with inherent challenges related to membrane-associated peptide hydrolysis. Here we established a system that allows expression and isolation of YqgP, a rhomboid homologue from Bacillus subtilis, as a soluble protein. Intriguingly, soluble YqgP is able to specifically cleave a peptide substrate that contains the transmembrane domain of Spitz. Mutation of the catalytic dyad abolished protease activity, and substitution of another highly conserved residue, Asn241, with Ala or Asp significantly reduced the catalytic efficiency of YqgP. We have identified the cleavage site that resides in the middle of the transmembrane domain of Spitz. Replacement of two residues that contribute to the scissile bond by Ala did not eliminate cleavage, but rather led to additional or alternative cleavages. Moreover, we have demonstrated that soluble YqgP exists as oligomers that are required for catalytic activity. These results suggest that soluble oligomers of maltose binding protein-YqgP complexes form micellelike structures that are able to retain the active conformation of the protease for catalysis. Therefore, this work not only provides a unique system for elucidating the reaction mechanism of rhomboid but also will facilitate the characterization of other intramembrane proteases as well as non-protease membrane proteins.

Rabies virus nucleoprotein is phosphorylated by cellular casein kinase II.

It has been reported that phosphorylation of rabies virus N plays an important role in the process of viral transcription and replication. Rabies virus N is phosphorylated when expressed alone, indicating that cellular kinase phosphorylates rabies virus N. To identify what cellular kinase phosphorylates rabies virus N, the N was expressed in Escherichia coli and purified by metal affinity chromatography. The recombinant N was phosphorylated by BHK cellular extracts and by purified CK-II. In addition, the phosphorylation of the recombinant N in vitro can be blocked by a CK-II inhibitor, heparin. Furthermore, N phosphorylation in the virus-infected cells can be inhibited by a CK-II specific inhibitor, 5,6-dichloro-beta-D-ribofuranosyl benzimidazole. However, PKC did not phosphorylate the recombinant N in vitro; nor did staurosporine, a PKC and other kinase inhibitor, prevent rabies virus N from phosphorylation. Thus, our data demonstrate that cellular CK-II phosphorylates rabies virus N.