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Diabetic cataracts are a common complication that can cause blindness among patients with diabetes mellitus. A novel nitro dihydrocapsaicin (NDHC), a capsaicin analog, was constructed to have a non-pungency effect. The objective of this research was to study the effect of NDHC on human lens epithelial (HLE) cells that lost function from hyperglycemia. HLE cells were pretreated with NDHC before an exposure to high glucose (HG) conditions. The results show that NDHC promoted a deacceleration of cellular senescence in HLE cells. This inhibition of cellular senescence was characterized by a delayed cell growth and lower production of reactive oxygen species (ROS) as well as decreased SA-β-galactosidase activity. Additionally, the expression of Sirt1 protein sharply increased, while the expression of p21 and phospho-p38 proteins decreased. These findings provide evidence that NDHC could exert a pharmacologically protective effect by inhibiting the senescence program of lens cells during diabetic cataracts.

Following T‐cell antigen receptor (TCR) engagement, rearrangement of the actin cytoskeleton supports intracellular signal transduction and T‐cell activation. The non‐catalytic region of the tyrosine kinase (Nck) molecule is an adapter protein implicated in TCR‐induced actin polymerization. Further, Nck is recruited to the CD3ε subunit of the TCR upon TCR triggering. Here we examine the role of actin polymerization in the recruitment of Nck to the TCR. To this end, Nck binding to CD3ε was quantified in Jurkat cells using the proximity ligation assay. We show that inhibition of actin polymerization using cytochalasin D delayed the recruitment of Nck1 to the TCR upon TCR triggering. Interestingly, CD3ε phosphorylation was also delayed. These findings suggest that actin polymerization promotes the recruitment of Nck to the TCR, enhancing downstream signaling, such as phosphorylation of CD3ε.

The T cell antigen receptor (TCR) is expressed on T cells, which orchestrate adaptive immune responses. It is composed of the ligand-binding clonotypic TCRαβ heterodimer and the non-covalently bound invariant signal-transducing CD3 complex. Among the CD3 subunits, the CD3ε cytoplasmic tail contains binding motifs for the Src family kinase, Lck, and the adaptor protein, Nck. Lck binds to a receptor kinase (RK) motif and Nck binds to a proline-rich sequence (PRS). Both motifs only become accessible upon ligand binding to the TCR and facilitate the recruitment of Lck and Nck independently of phosphorylation of the TCR. Mutations in each of these motifs cause defects in TCR signaling and T cell activation. Here, we investigated the role of Nck in proximal TCR signaling by silencing both Nck isoforms, Nck1 and Nck2. In the absence of Nck, TCR phosphorylation, ZAP70 recruitment, and ZAP70 phosphorylation was impaired. Mechanistically, this is explained by loss of Lck recruitment to the stimulated TCR in cells lacking Nck. Hence, our data uncover a previously unknown cooperative interaction between Lck and Nck to promote optimal TCR signaling.

Summary The T-cell receptor (TCR)–CD3 complex, expressed on T cells, determines the outcome of a T-cell response. It consists of the TCR-αβ heterodimer and the non-covalently associated signalling dimers of CD3eγ, CD3eδ and CD3ζζ. TCR-αβ binds specifically to a cognate peptide antigen bound to an MHC molecule, whereas the CD3 subunits transmit the signal into the cytosol to activate signalling events. Recruitment of proteins to specialized localizations is one mechanism to regulate activation and termination of signalling. In the last 25 years a large number of signalling molecules recruited to the TCR–CD3 complex upon antigen binding to TCR-αβ have been described. Here, we review knowledge about five of those interaction partners: Lck, ZAP-70, Nck, WASP and Numb. Some of these proteins have been targeted in the development of immunomodulatory drugs aiming to treat patients with autoimmune diseases and organ transplants.

Pharmaceutical treasure of herbs is well known and some of which have long been served as natural medicines or nourishments to enrich human health. It is conceivable that herbs offer unique habitats for endophytic microbes. Interestingly, the metabolic exchange between endophytes and their herbal hosts does not only allow the microbial subsistence but also enable them to form analog bioactive substances to those produced by their hosts. These challenge us to focus on searching for beneficial microbes from herbs and industrialize them as the producers of pharmaceutical products. In this work, we isolated 37 actinobacteria from rhizomes of fingerroot (Boesenbergia rotunda), ginger (Zingiber officinale), galangal (Alpinia galanga) and turmeric (Curcuma longa). These actinobacteria were classified preliminarily based on their morphology to the genus Streptomyces (86 %) and other unknown genera (14 %). To screen for anticancer activity, we found that 68% of all isolated actinobacteria produced L-asparaginase, which is a hydrolytic enzyme that acts as an inhibitor of leukemia. Streptomyces sp. ALP03 was among the active isolates exhibited the highest enzyme activity of L-asparaginase and showed a maximum 97.93 % similarity of its 16S rRNA gene sequence to Streptomyces spongiae Sp080513SC-24T. The cytotoxicity assays against diverse types of cancer cell lines will be carried out to assess the anticancer potential of isolate ALP03 and the others showing distinct L-asparaginase activity. With these findings, we conclude that herbal rhizomes have yet been a promising source for the discovery of useful microbes and their pharmaceutical products.

The fruit of Terminalia chebula Retz. (Combretaceae) has been used for several therapeutic purposes in Thai folk medicines. Currently, the ethanol extracts containing antioxidant compounds have shown the ability to promote collagen synthesis.This purpose of this work was to study the effects of the ethanol extract from T. chebula fruit on the inhibition of cutaneous photodamage.The viability of human skin fibroblasts after incubation with T. chebula at concentration 0.5-50 μg/mL for 24, 48 and 72 h was assessed by using sodium 3'-[(phenyl-amino)-carbonyl]-3,4,tetrazolium-bis(4-methoxy-6-notro)benzene-sulphonic acid hydrate (XTT). The levels of type I procollagen and matrix metalloproteinases (MMP)-1 and MMP-13 produced by UVB-irradiated fibroblasts were determined by ELISA. Skin thickness and collagen content caused by long-term UVB irradiation in male ICR mice were determined from haematoxylin and eosin stained tissue sections and spectrophotometric measurement of hydroxyproline.The extract (0.5-50 μg/mL) had no effect on cell viability or morphology of the human fibroblasts. In vitro studies showed that the T. chebula extract reduced the UVB-induced MMP-1 and MMP-13 expression, whereas an increased production of type I procollagen was observed. In a UVB-irradiated animal model, male ICR mice with hair shaved were chronically exposed to UVB which lead to epidermal thickness and loss of hydroxyproline. However, these effects were fully prevented by the topical application of the T. chebula ethanol extract.These data suggested that the T. chebula ethanol fruit extract is an efficacious pharmaceutical protectant of skin against photodamage.

An injectable hydrogel for chondrocyte delivery was developed by blending chitosan and starch derived from various sources with beta-glycerol phosphate (beta-GP) in the expectation that it would retain a liquid state at room temperature and gel at raised temperatures. Rheological investigation indicated that the system consisting of chitosan derived from crab shell and corn starch at 4:1 by weight ratio (1.53%, w/v of total polymers), and 6.0% (w/v) beta-GP (C/S/GP system) exhibited the sharpest sol-gel transition at 37+/-2 degrees C. The C/S/GP hydrogel was gradually degraded by 67% within 56 days in PBS containing 0.02 mg/ml lysozyme. The presence of starch in the system increased the water absorption of the hydrogel when compared to the system without starch. SEM observation revealed to the interior structure of the C/S/GP hydrogel having interconnected pore structure (average pore size 26.4 microm) whereas the pore size of the hydrogel without starch was 19.8 microm. The hydrogel also showed an ability to maintain chondrocyte phenotype as shown by cell morphology and expression of type II collagen mRNA and protein. In vivo study revealed that the gel was formed rapidly and localized at the injection site.

Ligand binding to the TCR causes a conformational change at the CD3 subunits to expose the CD3ε cytoplasmic proline-rich sequence (PRS). It was suggested that the PRS is important for TCR signaling and T cell activation. It has been shown that the purified, recombinant SH3.1 domain of the adaptor molecule noncatalytic region of tyrosine kinase (Nck) can bind to the exposed PRS of CD3ε, but the molecular mechanism of how full-length Nck binds to the TCR in cells has not been investigated so far. Using the in situ proximity ligation assay and copurifications, we show that the binding of Nck to the TCR requires partial phosphorylation of CD3ε, as it is based on two cooperating interactions. First, the SH3.1(Nck) domain has to bind to the nonphosphorylated and exposed PRS, that is, the first ITAM tyrosine has to be in the unphosphorylated state. Second, the SH2(Nck) domain has to bind to the second ITAM tyrosine in the phosphorylated state. Likewise, mutations of the SH3.1 and SH2 domains in Nck1 resulted in the loss of Nck1 binding to the TCR. Furthermore, expression of an SH3.1-mutated Nck impaired TCR signaling and T cell activation. Our data suggest that the exact pattern of CD3ε phosphorylation is critical for TCR functioning.

Background: The engagement of the T cell receptor (TCR)-CD3 complex induces the formation of multiple signalling complexes, which are required for actin cytoskeletal rearrangement. The Wiskott-Aldrich syndrome protein (WASp) is a key regulator of actin polymerization that is recruited to the TCR activation site. Since WASp is a binding partner of adaptor protein Nck, which is recruited directly to the TCR CD3e subunit upon TCR ligation, therefore we proposed that the direct recruitment of Nck to TCR-CD3 may also bring WASp directly to TCR-CD3. Objective: The aim of this present study was to assess the distribution of WASp, in relation to Nck, to the TCR-CD3e complex. Methods: Jurkat T cells were stimulated with anti-TCR antibody and then the cell lysates were immunoprecipitated with anti-CD3 antibody before immunoblotting with antibodies specific to WASp, Nck1, Nck2, SLP-76 and CD3e molecules. Results: WASp was recruited to SLP-76 and also directly to the TCR-CD3 complex upon TCR triggering. The inducible recruitment of WASp to the TCR-CD3 complex is partially dependent of tyrosine phosphorylation. Conclusions: The present findings provide an alternative mechanism of WASp recruitment to the site of TCR activation that may be involved in recruitment of Nck.

RNA interference (RNAi) is a potent gene delivery system for studying the regulation of gene expression in a wide variety of eukaryotic cells. In the present study, different RNAi approaches, namely, synthetic small interfering RNA (siRNA) and plasmid- and lentivirus-based short hairpin RNA (shRNA) were investigated to assess the down-regulation of Nck1 protein efficiency in the Jurkat T cell line. Jurkat T cells treated with these three different systems substantially and specifically reduced the expression of the Nck1 protein but not that of the Nck2 protein. Although the three systems showed a similar Nck1 knockdown efficiency, they led to different T cell activation outcomes. After stimulation, CD69 expression and IL-2 production were impaired in Nck1-siRNA and plasmid-based Nck1-shRNA transfected Jurkat cells. However, these T cell activation outcomes were increased in lentiviral vector based Nck1-shRNA transfected cells. These data suggest that the outcomes from transfection with the shRNA based lentiviral vector differ from those of siRNA and shRNA-based plasmids although they provide the same gene silencing efficiency. The verification of suitable RNAi strategies to silence target genes is therefore necessary before using it in experiments.

Background Signalling by the T cell antigen receptor (TCR) results in the activation of T lymphocytes. Nck1 and Nck2 are two highly related adaptor proteins downstream of the TCR that each contains three SH3 and one SH2 domains. Their individual functions and the roles of their SH3 domains in human T cells remain mostly unknown. Results Using specific shRNA we down-regulated the expression of Nck1 or Nck2 to approximately 10% each in Jurkat T cells. We found that down-regulation of Nck1 impaired TCR-induced phosphorylation of the kinases Erk and MEK, activation of the AP-1 and NFAT transcription factors and subsequently, IL-2 and CD69 expression. In sharp contrast, down-regulation of Nck2 hardly impacts these activation read-outs. Thus, in contrast to Nck2, Nck1 is a positive regulator for TCR-induced stimulation of the Erk pathway. Mutation of the third SH3 domain of Nck1 showed that this domain was required for this activity. Further, TCR-induced NFAT activity was reduced in both Nck1 and Nck2 knock-down cells, showing that both isoforms are involved in NFAT activation. Lastly, we show that neither Nck isoform is upstream of p38 phosphorylation or Ca2+influx. Conclusions In conclusion, Nck1 and Nck2 have non-redundant roles in human T cell activation in contrast to murine T cells.