Changes in Expression of Aquaporin-4 and Aquaporin-9 in Optic Nerve after Crushing in Rats

The purpose of this study was to determine the temporal and spatial changes in the expression of AQP4 and AQP9 in the optic nerve after it is crushed. The left optic nerves of rats were either crushed (crushed group) or sham operated (sham group), and they were excised before, and at 1, 2, 4, 7, and 14 days later. Four optic nerves were pooled for each time point in both groups. The expression of AQP4 and AQP9 was determined by western blot analyses. Immunohistochemistry was used to determine the spatial expression of AQP4, AQP9, and GFAP in the optic nerve. Optic nerve edema was determined by measuring the water content in the optic nerve. The barrier function of the optic nerve vessels was determined by the extravasated Evans blue dye on days 7 and 14. The results showed that the expression of AQP4 was increased on day 1 but the level was significantly lower than that in the sham group on days 4 and 7 (P<0.05). In contrast, the expression of AQP9 gradually increased, and the level was significantly higher than that in the sham group on days 7 and 14 (P<0.05, Tukey-Kramer). The down-regulation of AQP4 was associated with crush-induced optic nerve edema, and the water content of the nerve was significantly increased by 4.3% in the crushed optic nerve from that of the untouched fellow nerve on day 7. The expression of AQP4 and GFAP was reduced at the crushed site where AQP4-negative and AQP9-positive astrocytes were present. The barrier function was impaired at the crushed site on days 7 and 14, restrictedly where AQP4-negative and AQP9-positive astrocytes were present. The presence of AQP9-positive astrocytes at the crushed site may counteract the metabolic damage but this change did not fully compensate for the barrier function defect.     

Traditional but Not HIV-Related Factors Are Associated with Nonalcoholic Fatty Liver Disease in Asian Patients with HIV-1 Infection

Background

The prevalence and factors associated with nonalcoholic fatty liver disease (NAFLD) are largely unknown in HIV-1 monoinfected patients.

Methods

The present study elucidated the prevalence and factors associated with NAFLD among Asian patients with HIV-1 infection who underwent abdominal ultrasonography between January 2004 and March 2013. Diagnosis of NAFLD was based on the liver to kidney contrast and diffusion in hepatic echogenicity. Uni- and multi-variate logistic regression analyses were applied to estimate factors associated with NAFLD.

Results

435 Asian patients free of chronic hepatitis B or C virus infection and without excessive alcohol intake were analyzed. NAFLD was diagnosed in 135 (31%) patients. Obesity (BMI >30 kg/m²) was evident in 18 (4.1%) patients, and BMI was >25 kg/m² in 103 (24%). Multivariate analysis identified higher BMI (per 1 kg/m² increment, adjusted OR = 1.198; 95% CI, 1.112–1.290; p<0.001), dyslipidemia (adjusted OR = 2.045; 95% CI, 1.183–3.538; p = 0.010), and higher ALT to AST ratio (per 1 increment, adjusted OR = 3.557; 95% CI, 2.129–5.941; p<0.001) as significant factors associated with NAFLD. No HIV-specific variables, including treatment with dideoxynucleoside analogues (didanosine, stavudine, and zalcitabine) and cumulative duration of antiretroviral therapy (ART), were associated with NAFLD.

Conclusions

The incidence of NALFD among Asian patients with HIV-1 infection is similar to that in Western countries. NAFLD was associated with high BMI, dyslipidemia, and high ALT/AST ratio, but not with HIV-related factors. The results highlight the importance of early recognition and management of NAFLD and traditional factors associated with NAFLD for Asian patients with HIV-1 infection.     

Involvement of TNF-α Converting Enzyme in the Development of Psoriasis-Like Lesions in a Mouse Model

TNF-α plays a crucial role in psoriasis; therefore, TNF inhibition has become a gold standard for the treatment of psoriasis. TNF-α is processed from a membrane-bound form by TNF-α converting enzyme (TACE) to soluble form, which exerts a number of biological activities. EGF receptor (EGFR) ligands, including heparin-binding EGF-like growth factor (HB-EGF), amphiregulin and transforming growth factor (TGF)-α are also TACE substrates and are psoriasis-associated growth factors. Vascular endothelial growth factor (VEGF), one of the downstream molecules of EGFR and TNF signaling, plays a key role in angiogenesis for developing psoriasis. In the present study, to assess the possible role of TACE in the pathogenesis of psoriasis, we investigated the involvement of TACE in TPA-induced psoriasis-like lesions in K5.Stat3C mice, which represent a mouse model of psoriasis. In this mouse model, TNF-α, amphiregulin, HB-EGF and TGF-α were significantly up-regulated in the skin lesions, similar to human psoriasis. Treatment of K5.Stat3C mice with TNF-α or EGFR inhibitors attenuated the skin lesions, suggesting the roles of TACE substrates in psoriasis. Furthermore, the skin lesions of K5.Stat3C mice showed down-regulation of tissue inhibitor of metalloproteinase-3, an endogenous inhibitor of TACE, and an increase in soluble TNF-α. A TACE inhibitor abrogated EGFR ligand-dependent keratinocyte proliferation and VEGF production in vitro, suggesting that TACE was involved in both epidermal hyperplasia and angiogenesis during psoriasis development. These results strongly suggest that TACE contributes to the development of psoriatic lesions through releasing two kinds of psoriasis mediators, TNF-α and EGFR ligands. Therefore, TACE could be a potential therapeutic target for the treatment of psoriasis.     

Rearranging the domain order of a diabody-based IgG-like bispecific antibody enhances its antitumor activity and improves its degradation resistance and pharmacokinetics

One approach to creating more beneficial therapeutic antibodies is to develop bispecific antibodies (bsAbs), particularly IgG-like formats with tetravalency, which may provide several advantages such as multivalent binding to each target antigen. Although the effects of configuration and antibody-fragment type on the function of IgG-like bsAbs have been studied, there have been only a few detailed studies of the influence of the variable fragment domain order. Here, we prepared four types of hEx3-scDb-Fc, IgG-like bsAbs, built from a single-chain hEx3-Db (humanized bispecific diabody [bsDb] that targets epidermal growth factor receptor and CD3), to investigate the influence of domain order and fusion manner on the function of a bsDb with an Fc fusion format. Higher cytotoxicities were observed with hEx3-scDb-Fcs with a variable light domain (VL)–variable heavy domain (VH) order (hEx3-scDb-Fc-LHs) compared with a VH–VL order, indicating that differences in the Fc fusion manner do not affect bsDb activity. In addition, flow cytometry suggested that the higher cytotoxicities of hEx3-scDb-Fc-LH may be attributable to structural superiority in cross-linking. Interestingly, enhanced degradation resistance and prolonged in vivo half-life were also observed with hEx3-scDb-Fc-LH. hEx3-scDb-Fc-LH and its IgG2 variant exhibited intense in vivo antitumor effects, suggesting that Fc-mediated effector functions are dispensable for effective anti-tumor activities, which may cause fewer side effects. Our results show that merely rearranging the domain order of IgG-like bsAbs can enhance not only their antitumor activity, but also their degradation resistance and in vivo half-life, and that hEx3-scDb-Fc-LHs are potent candidates for next-generation therapeutic antibodies.     

Isothermal amplification of DNA using quadruplex primers with fluorescent pteridine base analogue 3‐methyl isoxanthopterin

We previously developed a method, known as quadruplex priming amplification (QPA), which greatly simplifies DNA amplification and quantification assays. QPA employs specific primers based on GGGTGGGTGGGTGGG (G3T) sequence, which upon polymerase elongation spontaneously dissociates from the target and folds into a stable quadruplex. Fluorescent nucleotide analogs, when incorporated into these primers, emit light upon quadruplex formation and permit simple, specific, and sensitive quantification without the attachment of probe molecules. Here, we studied optical [fluorescence and circular dichroism (CD)] and thermodynamic properties of the G3T sequence and variants incorporating 3‐methylisoxanthopterin (3MI), a highly fluorescent nucleotide analog suitable for QPA. CD studies demonstrate that the incorporation of 3MI does not change the overall tertiary structure of G3T; however, thermal unfolding experiments revealed that it significantly destabilizes the quadruplex. Enzymatic studies revealed that Taq and Bst are practically unable to incorporate any nucleotides opposite to template 3MI. Based on this knowledge, we designed QPA assays with truncated targets that demonstrate efficient amplification around 55°C. Overall, these studies suggest that 3MI‐based QPA is a useful assay for DNA amplification and detection. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 583–590, 2014.     

Quadruplex priming amplification combined with nicking enzyme for diagnostics

Quadruplex priming amplification (QPA) is a straightforward assay that allows isothermal amplification of DNA and possesses an intrinsic real-time detection mechanism. QPA can be employed as a diagnostic tool for both linear and exponential signal amplification. The linear QPA, which is less prone to background activity characteristics of exponential systems, suffers from low sensitivity. To increase the sensitivity, here we introduce specific probe molecules that are designed for combined activities of Bst 2.0 polymerase and Nt.BstNBI nicking enzyme. The current assay, which is suitable for single-tube isothermal signal amplification, has increased sensitivity of plain linear QPA by three orders of magnitude to levels of low femtomolar concentration of target molecules.     

Microbial fluorescence sensing for human neurotensin receptor type 1 using Gα-engineered yeast cells

Neurotensin receptor type-1 (NTSR1) is a member of the G-protein-coupled receptor (GPCR) family. The natural ligand of NTSR1 is neurotensin (NT), a neuromodulator of the central nervous system. Because NT is also involved in many oncogenic actions, the signaling mediator NTSR1 is a significant molecular target in medicinal and therapeutic fields. In the current study, we constructed a fluorescence-based microbial yeast biosensor that can monitor the activation of human NTSR1 signaling responding to its agonist. To increase the sensitivity of the biosensor, a yeast strain with the green fluorescent protein (GFP) reporter gene was genetically engineered to enhance binding with human NTSR1 expressed on the membrane. Following previous reports, the 5 carboxy-terminal amino acid residues of the guanine nucleotide binding protein α-subunit (Gα) in yeast Gpa1p were substituted with the equivalent human Gα_q sequences (Gpa1/Gα_q transplant). After optimizing the assay conditions, the Gα-engineered yeast demonstrated significantly improved sensing for NTSR1 signaling. Because detection using a GFP fluorescence reporter considerably simplifies the measurement procedure, this microbial fluorescence sensor holds promise for use in the diagnosis of NTSR1-associated diseases and the development of agonists.     

Alkylation of a human telomere sequence by heterotrimeric chlorambucil PI polyamide conjugates

We designed and synthesized human telomere alkylating N-methylpyrrole-N-methylimidazole (PI) polyamide conjugates (1–6). The C-type conjugates 1–3 possessed a chlorambucil moiety at the C terminus, whereas the N-type conjugates 4–6 had one of these moieties at the N terminus. The DNA alkylating activity of these conjugates was evaluated by high-resolution denaturing polyacrylamide gel electrophoresis using a 220 bp DNA fragment containing the human telomere repeat sequence 5′-(GGGTTA)₄-3′/5′-(TAACCC)₄-3′. C-type conjugates are designed to alkylate the G-rich-strand-containing 5′-GGGTTA-3′ and N-type conjugates were designed to alkylate the complementary C-rich strand-containing 5′-TAACCC-3′ sequence. The difference between conjugates 1–3 and 4–6 lies in the linker region between the polyamide moiety and chlorambucil. Conjugates 1 and 4 efficiently alkylated the 5′-GGTTAGGGTTA-3′ and 5′-CCCTAACCCTAA-3′ sequences, respectively, by recognizing 11 bp in the presence of distamycin A (Dist), in a heterotrimeric manner: one long alkylating polyamide conjugate (1–6) and two short partners (Dist).     

Efficient trafficking of ceramide from the endoplasmic reticulum to the Golgi apparatus requires a VAMP-associated protein-interacting FFAT motif of CERT

Ceramide is synthesized at the endoplasmic reticulum (ER) and transported to the Golgi apparatus by CERT for its conversion to sphingomyelin in mammalian cells. CERT has a pleck-strin homology (PH) domain for Golgi targeting and a START domain catalyzing the intermembrane transfer of ceramide. The region between the two domains contains a short peptide motif designated FFAT, which is supposed to interact with the ER-resident proteins VAP-A and VAP-B. Both VAPs were actually co-immunoprecipitated with CERT, and the CERT/VAP interaction was abolished by mutations in the FFAT motif. These mutations did not affect the Golgi targeting activity of CERT. Whereas mutations of neither the FFAT motif nor the PH domain inhibited the ceramide transfer activity of CERT in a cell-free system, they impaired the ER-to-Golgi transport of ceramide in intact and in semi-intact cells at near endogenous expression levels. By contrast, when overexpressed, both the FFAT motif and the PH domain mutants of CERT substantially supported the transport of ceramide from the ER to the site where sphingomyelin is produced. These results suggest that the Golgi-targeting PH domain and ER-interacting FFAT motif of CERT spatially restrict the random ceramide transfer activity of the START domain in cells.     

Dmbx1, a novel evolutionarily conserved paired-like homeobox gene expressed in the brain of mouse embryos.

To identify novel homeobox genes expressed during mouse embryogenesis, we searched the databases and found a novel mouse paired-like homeobox gene, Dmbx1(diencephalon/mesencephalon-expressed brain homeobox gene 1), that is also conserved in zebrafish and human. Linkage analysis mapped mouse Dmbx1 to the mid-portion of chromosome 4 that is the homologous gene cluster region of human chromosome 1, where human DMBX1 is located. Both mouse and human Dmbx1/DMBX1 have four coding exons and their gene structures are conserved. Whole-mount in situ hybridization revealed that Dmbx1 expression is detected in 7.5-9.5 dpc mouse embryos. At 7.5 and 8.5 dpc, Dmbx1 is expressed in a sub-region of the anterior head folds. At 9.5 dpc, expression is observed in the caudal diencephalon as well as in the mesencephalon and is restricted to the neuroepithelium. Expression in adult tissues was detected in brain, stomach, and testis. Dmbx1 provides a unique marker of the developing anterior nervous system and should provide a useful molecular resource to elucidate the mechanisms that pattern the vertebrate brain.