Crystal Structure of Transglutaminase 2 with GTP Complex and Amino Acid Sequence Evidence of Evolution of GTP Binding Site

Transglutaminase2 (TG2) is a multi-functional protein involved in various cellular processes, including apoptosis, differentiation, wound healing, and angiogenesis. The malfunction of TG2 causes many human disease including inflammatory disease, celiac disease, neurodegenerative diseases, tissue fibrosis, and cancers. Protein cross-linking activity, which is representative of TG2, is activated by calcium ions and suppressed by GTP. Here, we elucidated the structure of TG2 in complex with its endogenous inhibitor, GTP. Our structure showed why GTP is the optimal nucleotide for interacting with and inhibiting TG2. In addition, sequence comparison provided information describing the evolutionary scenario of GTP usage for controlling the activity of TG2.     

Morphogenetic and neuronal characterization of human neuroblastoma multicellular spheroids cultured under undifferentiated and all-trans-retinoic acid-differentiated conditions

In this study, we aimed to compare the morphogenetic and neuronal characteristics between monolayer cells and spheroids. For this purpose, we established spheroid formation by growing SH-SY5Y cells on the hydrophobic surfaces of thermally-collapsed elastin-like polypeptide. After 4 days of culture, the relative proliferation of the cells within spheroids was approximately 92% of the values for monolayer cultures. As measured by quantitative assays for mRNA and protein expressions, the production of synaptophysin and neuronspecific enolase (NSE) as well as the contents of cell adhesion molecules (CAMs) and extracellular matrix (ECM) proteins are much higher in spheroids than in monolayer cells. Under the all-trans-retinoic acid (RA)-induced differentiation condition, spheroids extended neurites and further up-regulated the expression of synaptophysin, NSE, CAMs, and ECM proteins. Our data indicate that RA-differentiated SH-SY5Y neurospheroids are functionally matured neuronal architectures. [BMB Reports 2013; 46(5): 276-281]     

The Optimal Cut-Off Value of Neutrophil-to-Lymphocyte Ratio for Predicting Prognosis in Adult Patients with Henoch–Sch?nlein Purpura

BackgroundThe development of gastrointestinal (GI) bleeding and end-stage renal disease (ESRD) can be a concern in the management of Henoch–Schönlein purpura (HSP). We aimed to evaluate whether the neutrophil-to-lymphocyte ratio (NLR) is associated with the prognosis of adult patients with HSP.MethodsClinical data including the NLR of adult patients with HSP were retrospectively analyzed. Patients were classified into three groups as follows: (a) simple recovery, (b) wax & wane without GI bleeding, and (c) development of GI bleeding. The optimal cut-off value was determined using a receiver operating characteristics curve and the Youden index.ResultsA total of 66 adult patients were enrolled. The NLR was higher in the GI bleeding group than in the simple recovery or wax & wane group (simple recovery vs. wax & wane vs. GI bleeding; median [IQR], 2.32 [1.61–3.11] vs. 3.18 [2.16–3.71] vs. 7.52 [4.91–10.23], P<0.001). For the purpose of predicting simple recovery, the optimal cut-off value of NLR was 3.18, and the sensitivity and specificity were 74.1% and 75.0%, respectively. For predicting development of GI bleeding, the optimal cut-off value was 3.90 and the sensitivity and specificity were 87.5% and 88.6%, respectively.ConclusionsThe NLR is useful for predicting development of GI bleeding as well as simple recovery without symptom relapse. Two different cut-off values of NLR, 3.18 for predicting an easy recovery without symptom relapse and 3.90 for predicting GI bleeding can be used in adult patients with HSP.     

Xenoreactivity of human clonal mesenchymal stem cells in a major histocompatibility complex-matched allogeneic graft-versus-host disease mouse model

Effects of mesenchymal stem cells (MSCs) on graft-versus-host disease (GVHD) have been actively investigated since the discovery of the immunomodulation property of MSCs about a decade ago. Human clonal MSCs (hcMSCs) were isolated from human bone marrow aspirate according to our newly established isolation protocol called subfractionation culturing method, and were evaluated for their efficacy on GVHD treatment, using a mouse MHC-matched B6 → BALB.B GVHD model system. Although the hcMSCs can suppress the allogeneic proliferation of human peripheral blood mononuclear cells in in vitro, the administration of the hcMSCs failed to reduce the GVHD-related mortality of the murine recipients. One of the reasons might be that murine cytokines such as IFN-γ and TNF-α cannot activate the hcMSCs. Based on these results, we suggest that xenogeneic MSCs may not be used for the treatment of GVHD.     

Crowdsourced identification of multi-target kinase inhibitors for RET- and TAU- based disease: The Multi-Targeting Drug DREAM Challenge

A continuing challenge in modern medicine is the identification of safer and more efficacious drugs. Precision therapeutics, which have one molecular target, have been long promised to be safer and more effective than traditional therapies. This approach has proven to be challenging for multiple reasons including lack of efficacy, rapidly acquired drug resistance, and narrow patient eligibility criteria. An alternative approach is the development of drugs that address the overall disease network by targeting multiple biological targets (‘polypharmacology’). Rational development of these molecules will require improved methods for predicting single chemical structures that target multiple drug targets. To address this need, we developed the Multi-Targeting Drug DREAM Challenge, in which we challenged participants to predict single chemical entities that target pro-targets but avoid anti-targets for two unrelated diseases: RET-based tumors and a common form of inherited Tauopathy. Here, we report the results of this DREAM Challenge and the development of two neural network-based machine learning approaches that were applied to the challenge of rational polypharmacology. Together, these platforms provide a potentially useful first step towards developing lead therapeutic compounds that address disease complexity through rational polypharmacology.     

Superoxide Dismutase Activity and Lipid Peroxidation in the Liver of Guinea Pig Infected with Leptospira interrogans

Superoxide dismutase (SOD) activity and the degree of lipid peroxidation were studied over a two week period in guinea pigs infected with Leptospira interrogans derived from wild mice. The total SOD activity in infected host liver increased by four-fold two days after infection; this was followed by a 20% decrease resulting in levels comparable to normal, uninfected liver. During the period of decreasing SOD activity after day two, the levels of TBA-reactive material (TBARS) are increased by three-fold in infected guinea pig, liver, compared to uninfected liver. The results indicate that SOD attenuates intracellular superoxide-mediated toxic effects in guinea pigs infected with L. interrogans. In addition, electron microscopy structure demonstrates correlated pathogenic shrinkage of mitochondrial and Kupffer cell structures.     

Bax inhibitor-1 enhances survival and neuronal differentiation of embryonic stem cells via differential regulation of mitogen-activated protein kinases activities

Bax inhibitor-1 (BI-1), a member of the BI-1 family of integral membrane proteins, was originally identified as an inhibitor of stress-induced cell death in mammalian cells. Previous studies have shown that the withdrawal of leukemia inhibitory factor (LIF) results in differentiation of the majority of mouse embryonic stem (mES) cells into various cell lineages, while some ES cells die within 3days. Thus, to investigate the function of BI-1 in ES cell survival and neuronal differentiation, we generated mES cell lines that overexpress BI-1 or a carboxy-terminal BI-1ΔC mutant. Overexpression of BI-1 in mES cells significantly increased cell viability and resistance to apoptosis induced by LIF withdrawal, while the control vector or BI-1ΔC-overexpressing mES cells had no effect. Moreover, overexpression of BI-1 produced significant inhibition of the p38 mitogen-activated protein kinases (MAPK) pathway in response to LIF withdrawal, while activity of the extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK) MAPK pathway was increased. Interestingly, we found that BI-1-overexpressing cells showed higher expression levels of neuroectodermal markers (Otx1, Lmx1b, En1, Pax2, Wnt1, Sox1, and Nestin) and greater neuronal differentiation efficiency than control or BI-1ΔC-overexpressing mES cells did. Considering these findings, our results indicated that BI-1-modulated MAPK activity plays a key role in protecting mES cells from LIF-withdrawal-induced apoptosis and in promoting their differentiation toward neuronal lineages.     

Investigating the effects of positive charge and hydrophobicity on the cell selectivity, mechanism of action and anti-inflammatory activity of a Trp-rich antimicrobial peptide indolicidin

To investigate the effects of positive charge and hydrophobicity on the cell selectivity, mechanism of action and anti-inflammatory activity of a Trp-rich antimicrobial peptide indolicidin (IN), a series of IN analogs with Trp→Lys substitution were synthesized. All IN analogs displayed an approximately 7- to 18-fold higher cell selectivity, compared with IN. IN, IN-1 and IN-2 depolarized (50−90%) the cytoplasmic membrane potential of Staphylococcus aureus close to minimal inhibitory concentration (5–10 μg mL⁻¹). However, other IN analogs (IN-3 and IN-4) displayed very low ability in membrane depolarization even at 40 μg mL⁻¹. Confocal laser-scanning microscopy revealed that IN-3 and IN-4 penetrated the Escherichia coli cell membrane, whereas IN, IN-1 and IN-2 did not enter the cell membrane. In the gel retardation assay, IN-3 and IN-4 bound more strongly to DNA compared with IN, IN-1 and IN-2. These findings suggest that the mechanism of antimicrobial action of IN-3 and IN-4 may be involved in the inhibition of intracellular functions via interference with DNA/RNA synthesis. Unlike IN, all IN analogs did not inhibit nitric oxide production or inducible nitric oxide synthase mRNA expression in lipopolysaccharide-stimulated mouse macrophage RAW264.7 cells, indicating that the hydrophobicity of IN is more important for anti-inflammatory activity in lipopolysaccharide-treated macrophage cells than the positive charge.