Persimmon leaf extract inhibits the ATM activity during DNA damage response induced by Doxorubicin in A549 lung adenocarcinoma cells

Persimmon leaf (PL) has been commonly recognized for its wide variety of health benefits. A previous study has reported that persimmon leaf extract (PLE) contained flavonols with the 2″-galloly moiety (PLEg). Galloylated homologues generically show stronger activity in their biological function, so enhanced functions can be expected for PLEg. We investigated in this present study the effect of PLEg on the cellular DNA damage checkpoint signaling to sensitize cancer chemotherapy. Treatment with PLE and PLEg significantly increased the cytotoxicity of doxorubicin (DOX) in A549 adenocarcinoma cells. PLE and PLEg reduced the phosphorylation of checkpoint proteins such as structural maintenance of chromosomes 1 (SMC1), checkpoint kinase 1 (Chk1), and p53 in DOX-treated cells. Moreover, PLE decreased the phosphorylation of ATM (ataxia telangiectasia mutated) in a dose-dependent manner. PLE, and especially PLEg, abrogated the G2/M checkpoint during DOX-induced DNA damage. These results suggest that PLEg specifically inhibited ATM-dependent checkpoint activation by DOX, and that PLEg might be a useful sensitizer in cancer chemotherapy.     

Double-knockout of putative endo-β-N-acetylglucosaminidase (ENGase) genes in Arabidopsis thaliana: loss of ENGase activity induced accumulation of high-mannose type free N-glycans bearing N,N'-acetylchitobiosyl unit

Endo-β-N-acetylglucosaminidase (ENGase) is involved in the production of high-mannose type free N-glycans during plant development and fruit maturation. In a previous study (K. Nakamura et al. Biosci. Biotechnol. Biochem., 73, 461-464 (2009)), we identified the tomato ENGase gene and found that gene expression remained relatively constant. In the present study, we constructed an Arabidopsis thaliana mutant in which the expression of two putative ENGase genes was suppressed. The mutant showed no ENGase activity, but produced high-mannose type free N-glycans carrying the N,N'-acetylchitobiosyl unit that is produced by peptide:N-glycanase, indicating that both these genes encode Arabidopsis ENGase.     

Design and synthesis of C35-fluorinated solamins and their growth inhibitory activities against human cancer cell lines

The convergent synthesis of C35-fluorinated analogues of solamin, a mono-THF Annonaceous acetogenin, has been achieved by the Sonogashira coupling of the THF ring fragment and the fluorinated γ-lactone fragment. It was revealed that the number of fluorine atoms on the γ-lactone moiety affects the growth inhibitory activities against human cancer cell lines.     

Identification of 4-quinolone derivatives as inhibitors of reactive oxygen species production from human umbilical vein endothelial cells

Oxidative stress is widely recognized as being associated with a number of disorders, including metabolic dysfunction and atherosclerosis. A series of substituted 4-quinolone derivatives were prepared and evaluated as inhibitors of reactive oxygen species (ROS) production from human umbilical vein endothelial cells (HUVECs). One compound in particular, 2-({[4-(3-hydroxy-3-methylbutoxy)pyridin-2-yl]oxy}methyl)-3-methylquinolin-4(1H)-one (25b), inhibited ROS production from HUVECs with an IC(50) of 140 nM. This compound also exhibited low CYP2D6 inhibitory activity, high aqueous solubility, and good in vitro metabolic stability. An in vivo pharmacokinetic study of this compound in SD rats revealed high oral bioavailability and a long plasma half-life.     

Calpain-7 binds to CHMP1B at its second α-helical region and forms a ternary complex with IST1

Some intracellular proteins involved in the endosomal sorting complex required for transport (ESCRT) system have microtubule interacting and transport (MIT) domains and bind to ESCRT-III protein family members named charged multivesicular body proteins (CHMPs) at their C-terminal regions containing MIT-interacting motifs (MIMs). While two types of MIMs (MIM1 and MIM2) have been reported, CHMP1B has MIM1 and IST1 has both MIM1 and MIM2. Previously, we demonstrated that CHMP1B and IST1 directly interacted with a tandem repeat of MIT domains of calpain-7 (CL7MIT) and that autolytic activity of calpain-7 was enhanced by IST1 in vitro but not by overexpression of IST1 in HEK293T cells. In this study, we detected enhancement of autolysis of mGFP-fused calpain-7 by coexpression with CHMP1B and observed further activation by additional coexpression of IST1 in HEK293T cells. We found that CL7MIT interacted with the second α-helical region of CHMP1B but not with the canonical C-terminal region containing MIM1 in vitro. Co-immunoprecipitation assays demonstrated that the interaction between CL7MIT and CHMP1B and between CL7MIT and IST1 became stronger when IST1 or CHMP1B was additionally coexpressed, suggesting formation of ternary complex of calpain-7, IST1 and CHMP1B. Moreover, subcellular fractionation analyses revealed increase of calpain-7 in membrane/organelle fractions by concomitant overexpression of these ESCRT-III family member proteins.     

Molecular cloning and characterization of a beta-L-Arabinobiosidase in Bifidobacterium longum that belongs to a novel glycoside hydrolase family

Extensin is a glycoprotein that is rich in hydroxyprolines linked to β-L-arabinofuranosides. In this study, we cloned a hypBA2 gene that encodes a novel β-L-arabinobiosidase from Bifidobacterium longum JCM 1217. This enzyme does not have any sequence similarity with other glycoside hydrolase families but has 38-98% identity to hypothetical proteins in Bifidobacterium and Xanthomonas strains. The recombinant enzyme liberated L-arabinofuranose (Araf)-β1,2-Araf disaccharide from carrot extensin, potato lectin, and Araf-β1,2-Araf-β1,2-Araf-β-Hyp (Ara(3)-Hyp) but not Araf-α1,3-Araf-β1,2-Araf-β1,2-Araf-β-Hyp (Ara(4)-Hyp) or Araf-β1,2-Araf-β-Hyp (Ara(2)-Hyp), which indicated that it was specific for unmodified Ara(3)-Hyp substrate. The enzyme also transglycosylated 1-alkanols with retention of the anomeric configuration. This is the first report of an enzyme that hydrolyzes Hyp-linked β-L-arabinofuranosides, which defines a new family of glycoside hydrolases, glycoside hydrolase family 121.     

Paired immunoglobulin-like receptor B knockout does not enhance axonal regeneration or locomotor recovery after spinal cord injury

Myelin components that inhibit axonal regeneration are believed to contribute significantly to the lack of axonal regeneration noted in the adult central nervous system. Three proteins found in myelin, Nogo, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, inhibit neurite outgrowth in vitro. All of these proteins interact with the same receptors, namely, the Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PIR-B). As per previous reports, corticospinal tract (CST) regeneration is not enhanced in NgR-knock-out mice after spinal cord injury. Therefore, we assessed CST regeneration in PIR-B-knock-out mice. We found that hindlimb motor function, as assessed using the Basso mouse scale, footprint test, inclined plane test, and beam walking test, did not differ between the PIR-B-knock-out and wild-type mice after dorsal hemisection of the spinal cord. Further, tracing of the CST fibers after injury did not reveal enhanced axonal regeneration or sprouting in the CST of the PIR-B-knock-out mice. Systemic administration of NEP1-40, a NgR antagonist, to PIR-B knock-out mice did not enhance the regenerative response. These results indicate that PIR-B knock-out is not sufficient to induce extensive axonal regeneration after spinal cord injury.     

Functional activation of Src family kinase yes protein is essential for the enhanced malignant properties of human melanoma cells expressing ganglioside GD3

The possible roles of Src family kinases in the enhanced malignant properties of melanomas related to GD3 expression were analyzed. Among Src family kinases only Yes, not Fyn or Src, was functionally involved in the increased cell proliferation and invasion of GD3-expressing transfectant cells (GD3+). Yes was located upstream of p130Cas and paxillin and at an equivalent level to focal adhesion kinase. Yes underwent autophosphorylation even before serum treatment and showed stronger kinase activity in GD3+ cells than in GD3- cells following serum treatment. Coimmunoprecipitation experiments revealed that Yes bound to focal adhesion kinase or p130Cas more strongly in GD3+ cells than in GD3- cells. As a possible mechanism for the enhancing effects of GD3 on cellular phenotypes, it was shown that majority of Yes was localized in glycolipid-enriched microdomain/rafts in GD3+ cells even before serum treatment, whereas it was scarcely detected in glycolipid-enriched microdomain/rafts in GD3- cells. An in vitro kinase assay of Yes revealed that coexistence of GD3 with Yes in membranous environments enhances the kinase activity of GD3- cell-derived Yes toward enolase, p125, and Yes itself. Knockdown of GD3 synthase resulted in the alleviation of tumor phenotypes and reduced activation levels of Yes. Taken together, these results suggest a role of GD3 in the regulation of Src family kinases.     

Spectral tuning in sensory rhodopsin I from Salinibacter ruber

Organisms utilize light as energy sources and as signals. Rhodopsins, which have seven transmembrane α-helices with retinal covalently linked to a conserved Lys residue, are found in various organisms as distant in evolution as bacteria, archaea, and eukarya. One of the most notable properties of rhodopsin molecules is the large variation in their absorption spectrum. Sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII) function as photosensors and have similar properties (retinal composition, photocycle, structure, and function) except for their λ(max) (SRI, ～560 nm; SRII, ～500 nm). An expression system utilizing Escherichia coli and the high protein stability of a newly found SRI-like protein, SrSRI, enables studies of mutant proteins. To determine the residue contributing to the spectral shift from SRI to SRII, we constructed various SRI mutants, in which individual residues were substituted with the corresponding residues of SRII. Three such mutants of SrSRI showed a large spectral blue-shift (>14 nm) without a large alteration of their retinal composition. Two of them, A136Y and A200T, are newly discovered color tuning residues. In the triple mutant, the λ(max) was 525 nm. The inverse mutation of SRII (F134H/Y139A/T204A) generated a spectral-shifted SRII toward longer wavelengths, although the effect is smaller than in the case of SRI, which is probably due to the lack of anion binding in the SRII mutant. Thus, half of the spectral shift from SRI to SRII could be explained by only those three residues taking into account the effect of Cl(-) binding.     

Activation of TLR2 enhances tight junction barrier in epidermal keratinocytes

The epidermis has developed physical and immunological barriers that prevent infiltration of deleterious chemicals and pathogens. As a first step to understanding the relationship between these barriers, we investigated whether TLR2 activation functionally alters tight junctions (TJs) in cultured human keratinocytes. Stimulation with peptidoglycan, a ligand for TLR2, elevated the TJ-associated barrier in the space of 3 h. The increase in TJ-associated barrier function due to peptidoglycan stimulation was suppressed by the knockdown of TLR adaptor MyD88 or the pretreatment with TLR2-neutralizing Ab, indicating that TLR2 activation enhanced TJ-associated barrier. One and 3 h after peptidoglycan stimulation, expression levels of the TJ proteins occludin, claudin-1, claudin-4, and ZO-1 were unchanged. However, immunoprecipitation studies demonstrated that the association of phospho-atypical protein kinase Cζ/ι, crucial for TJ biogenesis, with occludin was increased. Significantly, inhibition of atypical protein kinase Cζ/ι activity completely blocked the immediate elevation of the TJ-associated barrier. Finally, peptidoglycan was applied to the stratum corneum surface of a human skin equivalent, and the TJ barrier was evaluated. In the space of 3 h after the stimulation, the amount of intercellular tracer in the stratum corneum incubated from the dermal side was reduced, indicating that the TJ barrier is strengthened via TLR2 activation. Taken together, our findings indicated that infiltration of pathogens into the epidermis immediately enhanced TJ function via TLR2 signaling. Furthermore, the dynamically controlled TJs in skin are considered fundamental in preventing further invasion of pathogens and maintaining cutaneous barrier homeostasis.