Angiotensin-converting enzyme inhibitors attenuated advanced glycation end products-induced renal tubular hypertrophy via enhancing nitric oxide signaling

Advanced glycation end products (AGE) and angiotensin II were closely correlated with the progression of diabetic nephopathy (DN). Nitric oxide (NO) is a protective mediator of renal tubular hypertrophy in DN. Here, we examined the molecular mechanisms of angiotensin-converting enzyme inhibitor (ACEI) and NO signaling responsible for diminishing AGE-induced renal tubular hypertrophy. In human renal proximal tubular cells, AGE decreased NO production, inducible NOS activity, guanosine 3′,5′-cyclic monophosphate (cGMP) synthesis, and cGMP-dependent protein kinase (PKG) activation. All theses effects of AGE were reversed by treatment with ACEIs (captopril and enalapril), the NO donor S-nitroso-N-acetylpenicillamine (SNAP), and the PKG activator 8-para-chlorophenylthio-cGMPs (8-pCPT-cGMPs). In addition, AGE-enhanced activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) were clearly reduced by captopril, enalapril, SNAP, and 8-pCPT-cGMPs. The abilities of ACEIs and NO/PKG activation to inhibit AGE-induced hypertrophic growth were verified by the observation that captopril, enalapril, SNAP, and 8-pCPT-cGMPs decreased protein levels of fibronectin, p21 ^Waf1/Cip1, and receptor for AGE. The results of the present study suggest that ACEIs significantly reduced AGE-increased ERK/JNK/p38 MAPK activation and renal tubular hypertrophy partly through enhancement of the NO/PKG pathway.     

CDKN1B Val 109 Gly variant is not related to risk of prostate cancer

Association between CDKN1B gene Val 109 Gly polymorphism and prostate cancer (PCa) susceptibility has been investigated in several studies but with inconsistent conclusions. We adopted odds ratios (ORs) and 95% confidence intervals (CIs) to assess the correlation between CDKN1B Val 109 Gly variant and PCa susceptibility. Moreover, we used in-silico tools to evaluate the relationship of CDKN1B expression and overall survival (OS) or disease free survival (DFS) time in PCa patients. The overall results demonstrated no association of the CDKN1B variant on PCa risk [allelic contrast (OR = 0.78, 95% CI = 0.45 − 1.35, P_{heterogeneity} = 0.038); GV vs VV (OR = 0.83, 95% CI = 0.56 − 1.25, P_{heterogeneity} = 0.253); GG vs VV (OR = 0.48, 95% CI = 0.23 − 1.01, P_{heterogeneity} = 0.161); GG+GV vs VV (OR = 0.75, 95% CI = 0.52 −1.08, P_{heterogeneity} = 0.132) and GG vs GV+VV (OR = 0.63, 95% CI = 0.25 − 1.11, P_{heterogeneity} = 0.152)]. In subgroup analysis by ethnicity and source of control, we also identified similar results. In-silico results showed that expression of CDKN1B was decreased in PCa tissue, especially in less advanced PCa (Gleason score = 6 or 7). No significant difference of OS or DFS time was indicated between the low and high expression of CDKN1B. Our present study showed evidence that CDKN1B Val 109 Gly variant is not related to PCa risk. Future studies with large sample size are needed to confirm this correlation in more details.     

Reciprocal regulatory interaction between human herpesvirus 8 and human immunodeficiency virus type 1

Human herpesvirus 8 (HHV8) is the primary viral etiologic agent in Kaposi's sarcoma (KS). However, individuals dually infected with both HHV8 and human immunodeficiency virus type 1 (HIV-1) show an enhanced prevalence of KS when compared with those singularly infected with HHV8. Host immune suppression conferred by HIV infection cannot wholly explain this increased presentation of KS. To better understand how HHV8 and HIV-1 might interact directly in the pathogenesis of KS, we queried for potential regulatory interactions between the two viruses. Here, we report that HHV8 and HIV-1 reciprocally up-regulate the gene expression of each other. We found that the KIE2 immediate-early gene product of HHV8 interacted synergistically with Tat in activating expression from the HIV-1 long terminal repeat. On the other hand, HIV-1 encoded Tat and Vpr proteins increased intracellular HHV8-specific expression. These results provide molecular insights correlating coinfection with HHV8 and HIV-1 with an unusually high incidence of KS.     

Structure-activity relationships in flexible protein domains: regulation of rho GTPases by RhoGDI and D4 GDI

The guanine dissociation inhibitors RhoGDI and D4GDI inhibit guanosine 5'-diphosphate dissociation from Rho GTPases, keeping these small GTPases in an inactive state. The GDIs are made up of two domains: a flexible N-terminal domain of about 70 amino acid residues and a folded 134-residue C-terminal domain. Here, we characterize the conformation of the N-terminal regions of both RhoGDI and D4GDI using a series of NMR experiments which include (15)N relaxation and amide solvent accessibility measurements. In each protein, two regions with tendencies to form helices are identified: residues 36 to 58 and 9 to 20 in RhoGDI, and residues 36 to 57 and 20 to 25 in D4GDI. To examine the functional roles of the N-terminal domain of RhoGDI, in vitro and in vivo functional assays have been carried out with N-terminally truncated proteins. These studies show that the first 30 amino acid residues are not required for inhibition of GDP dissociation but appear to be important for GTP hydrolysis, whilst removal of the first 41 residues completely abolish the ability of RhoGDI to inhibit GDP dissociation. The combination of structural and functional studies allows us to explain why RhoGDI and D4GDI are able to interact in similar ways with the guanosine 5'-diphosphate-bound GTPase, but differ in their ability to regulate GTP-bound forms; these functional differences are attributed to the conformational differences of the N-terminal domains of the guanosine 5'-diphosphate dissociation inhibitors. Therefore, the two transient helices, appear to be associated with different biological effects of RhoGDI, providing a clear example of structure-activity relationships in a flexible protein domain.     

Mechanisms for GroEL/GroES-mediated folding of a large 86-kDa fusion polypeptide in vitro

Our understanding of mechanisms for GroEL/GroES-assisted protein folding to date has been derived mostly from studies with small proteins. Little is known concerning the interaction of these chaperonins with large multidomain polypeptides during folding. In the present study, we investigated chaperonin-dependent folding of a large 86-kDa fusion polypeptide, in which the mature maltose-binding protein (MBP) sequence was linked to the N terminus of the alpha subunit of the decarboxylase (E1) component of the human mitochondrial branched-chain alpha-ketoacid dehydrogenase complex. The fusion polypeptide, MBP-alpha, when co-expressed with the beta subunit of E1, produced a chimeric protein MBP-E1 with an (MBP-alpha)2beta2 structure, similar to the alpha2 beta2 structure in native E1. Reactivation of MBP-E1 denatured in 8 M urea was absolutely dependent on GroEL/GroES and Mg2+-ATP, and exhibited strikingly slow kinetics with a rate constant of 376 M-1 s-1, analogous to denatured untagged E1. Chaperonin-mediated refolding of the MBP-alpha fusion polypeptide showed that the folding of the MBP moiety was about 7-fold faster than that of the alpha moiety on the same chain with rate constants of 1.9 x 10(-3) s-1 and 2.95 x 10(-4) s-1, respectively. This explained the occurrence of an MBP-alpha. GroEL binary complex that was isolated with amylose resin from the refolding mixture and transformed Escherichia coli lysates. The data support the thesis that distinct functional sequences in a large polypeptide exhibit different folding characteristics on the same GroEL scaffold. Moreover, we show that when the alpha.GroEL complex (molar ratio 1:1) was incubated with GroES, the latter was capable of capping either the very ring that harbored the 48-kDa (His)6-alpha polypeptide (in cis) or the opposite unoccupied cavity (in trans). In contrast, the MBP-alpha.GroEL (1:1) complex was capped by GroES exclusively in the trans configuration. These findings suggest that the productive folding of a large multidomain polypeptide can only occur in the GroEL cavity that is not sequestered by GroES.     

Dynamic analysis of T-lymphocyte function in relation to hepatopathologic changes and effect of interleukin-12 treatment in mice infected with Schistosoma japonicum

We analyzed the dynamics of splenic T-lymphocyte function in relation to hepatopathologic changes in C3H/Hc mice, experimentally infected with Schistosoma japonicum. Vigorous granuloma formation was observed at 7 wk postinfection. At 10 wk postinfection, granuloma formation entered into the down-modulation stage, as represented by the diminished granuloma size. The Th2 response was activated when eggs appeared in the liver, whereas Th1 responses were depressed and the proliferation of T lymphocytes was decreased. The level of IgG antibodies to the worm and egg antigens rose continually after infection. Interleukin-12 treatment of infected mice inhibited Th2 responses and T-cell proliferation, decreased granuloma formation and fibrosis, but had no effect on the fecundity of the worms. These data suggest that egg deposition is the major factor driving Th2 responses, depressing Th1 cytokine expression as well as T-cell proliferation in S. japonicum-infected mice.     

The initiative role of XPC protein in cisplatin DNA damaging treatment-mediated cell cycle regulation

XPC is an important DNA damage recognition protein involved in DNA nucleotide excision repair. We have studied the role of the XPC protein in cisplatin treatment-mediated cell cycle regulation. Through the comparison of microarray data obtained from human normal fibroblasts and two individual XPC-defective cell lines, 486 genes were identified as XPC-responsive genes in the cisplatin treatment (with a minimal 1.5-fold change) and 297 of these genes were further mapped to biological pathways and gene ontologies. The cell cycle and cell proliferation-related genes were the most affected genes by the XPC defect in the cisplatin treatment. Many other cellular function genes were also affected by the XPC defect in the treatment. Western blot hybridization results revealed that the XPC defect reduced the p53 responses to the cisplatin treatment. The ability to activate caspase-3 was also attenuated in the XPC cells with the treatment. These results suggest that the XPC protein plays a critical role in initiating the cisplatin DNA damaging treatment-mediated signal transduction process, resulting in activation of the p53 pathway and cell cycle arrest that allow DNA repair and apoptosis to take place. These results reveal an important role of the XPC protein in the cancer prevention.     

The super-cooling agent icilin reveals a mechanism of coincidence detection by a temperature-sensitive TRP channel

TRPM8, a member of the transient receptor potential family of ion channels, depolarizes somatosensory neurons in response to cold. TRPM8 is also activated by the cooling agents menthol and icilin. When exposed to menthol or cold, TRPM8 behaves like many ligand-gated channels, exhibiting rapid activation followed by moderate Ca(2+)-dependent adaptation. In contrast, icilin activates TRPM8 with extremely variable latency followed by extensive desensitization, provided that calcium is present. Here, we show that, to achieve full efficacy, icilin requires simultaneous elevation of cytosolic Ca2+, either via permeation through TRPM8 channels or by release from intracellular stores. Thus, two stimuli must be paired to elicit full channel activation, illustrating the potential for coincidence detection by TRP channels. Determinants of icilin sensitivity map to a region of TRPM8 that corresponds to the capsaicin binding site on the noxious heat receptor TRPV1, suggesting a conserved molecular logic for gating of these thermosensitive channels by chemical agonists.     

Four DEF-like MADS box genes displayed distinct floral morphogenetic roles in Phalaenopsis orchid

The complex flower organization of orchids offers an opportunity to discover new variant genes and different levels of complexity in the morphogenesis of flowers. In this study, four B-class Phalaenopsis DEF-like MADS-box genes were identified and characterized, including PeMADS2, PeMADS3, PeMADS4 and PeMADS5. Differential expression profiles of these genes were detected in the floral organs of P. equestris, suggesting distinctive roles in the floral morphogenesis of orchids. Furthermore, expressions of these genes were varied to different extents in the peloric mutants with lip-like petals. Expression of PeMADS4 was in lips and columns of wild type, and it extended to the lip-like petals in the peloric mutant. Expression of PeMADS5 was mainly in petals and to a lesser extent in columns in the wild type, whereas it was completely eliminated in the peloric mutant. Disruption of the PeMADS5 promoter region of the peloric mutant was detected at nucleotide +312 relative to the upstream of translational start codon, suggesting that a DNA rearrangement has occurred in the peloric mutant. Genomic structure analysis of the PeMADS5 showed that the exon length was conserved in exons 1-6, similar to DEF-like genes of other plants. Collectively, this is the first report that four DEF-like MADS genes were identified in a single monocotyledonous species and that they may play distinctive morphogenetic roles in the floral development of an orchid.