Rax-CreERT2 Knock-In Mice: A Tool for Selective and Conditional Gene Deletion in Progenitor Cells and Radial Glia of the Retina and Hypothalamus

To study gene function in neural progenitors and radial glia of the retina and hypothalamus, we developed a Rax-CreER^T2 mouse line in which a tamoxifen-inducible Cre recombinase is inserted into the endogenous Rax locus. By crossing Rax-CreER^T2 with the Cre-dependent Ai9 reporter line, we demonstrate that tamoxifen-induced Cre activity recapitulates the endogenous Rax mRNA expression pattern. During embryonic development, Cre recombinase activity in Rax-CreER^T2 is confined to retinal and hypothalamic progenitor cells, as well as progenitor cells of the posterior pituitary. At postnatal time points, selective Cre recombinase activity is seen in radial glial-like cell types in these organs – specifically Müller glia and tanycytes – as well as pituicytes. We anticipate that this line will prove useful for cell lineage analysis and investigation of gene function in the developing and mature retina, hypothalamus and pituitary.     

On the Theory of Speciation Induced by Transposable Elements

A simple methematical model describes the invasion of panmictic, sexually reproducing populations by a newly introduced transposon. The model places important constraints on the properties that transposons must have to successfully invade a population and describes the kinetics with which such an invasion will occur. Invasibility conditions serve as a basis for new, detailed scenarios whereby transposon-mediated depression in fitness produces reproductive isolation of populations. These scenarios, in turn, lead to several speculations concerning the role of transposons in evolution.     

Human hair growth enhancement in vitro by green tea epigallocatechin-3-gallate (EGCG)

Green tea is a popular worldwide beverage, and its potential beneficial effects such as anti-cancer and anti-oxidant properties are believed to be mediated by epigallocatechin-3-gallate (EGCG), a major constituent of polyphenols. Recently, it was reported that EGCG might be useful in the prevention or treatment of androgenetic alopecia by selectively inhibiting 5alpha-reductase activity. However, no report has been issued to date on the effect of EGCG on human hair growth. This study was undertaken to measure the effect of EGCG on hair growth in vitro and to investigate its effect on human dermal papilla cells (DPCs) in vivo and in vitro. EGCG promoted hair growth in hair follicles ex vivo culture and the proliferation of cultured DPCs. The growth stimulation of DPCs by EGCG in vitro may be mediated through the upregulations of phosphorylated Erk and Akt and by an increase in the ratio of Bcl-2/Bax ratio. Similar results were also obtained in in vivo dermal papillae of human scalps. Thus, we suggest that EGCG stimulates human hair growth through these dual proliferative and anti-apoptotic effects on DPCs.     

Molecular mapping of the C locus for presence of pungency in Capsicum.

Pungency owing to the presence of capsaicinoids is a unique character of pepper (Capsicum spp.). Capsaicinoids are produced in the placenta and it has long been known that a single dominant gene, C, is required for pungent genotypes to produce capsaicinoids. We mapped C to pepper chromosome 2 in a cross between a pungent Capsicum frutescens wild accession and a non-pungent Capsicum annuum bell pepper. This position confirmed results from earlier studies. The RFLP marker TG 205 cosegregated with C and two additional RFLP markers were also located within 1 cM. The recessive allele at the C locus is used in breeding programs around the world focused on very diverse germplasm, hence any of these tightly linked markers may be of value as potential sources of useful markers for marker-assisted selection. To demonstrate this point, we developed a PCR-based CAPS (cleaved amplified polymorphic sequence) marker linked to C using the sequence of the Capsicum fibrillin gene located 0.4 cM from C. The use of molecular markers for high-throughput screening for the c allele in pepper breeding programs is discussed.     

Construction of malate-sensing <Emphasis Type="Italic">Escherichia coli</Emphasis> by introduction of a novel chimeric two-component system

In an attempt to develop a high-throughput screening system for screening microorganisms which produce high amounts of malate, a MalKZ chimeric HK-based biosensor was constructed. Considering the sequence similarity among Escherichia coli (E. coli) MalK with Bacillus subtilis MalK and E. coli DcuS, the putative sensor domain of MalK was fused with the catalytic domain of EnvZ. The chimeric MalK/EnvZ TCS induced the ompC promoter through the cognate response regulator, OmpR, in response to extracellular malate. Real-time quantitative PCR and GFP fluorescence studies showed increased ompC gene expression and GFP fluorescence as malate concentration increased. By using this strategy, various chimeric TCS-based bacteria biosensors can be constructed, which may be used for the development of biochemical-producing recombinant microorganisms.     

Orexinergic Neurotransmission in Temperature Responses to Methamphetamine and Stress: Mathematical Modeling as a Data Assimilation Approach

Experimental Data

Orexinergic neurotransmission is involved in mediating temperature responses to methamphetamine (Meth). In experiments in rats, SB-334867 (SB), an antagonist of orexin receptors (OX1R), at a dose of 10 mg/kg decreases late temperature responses (t>60 min) to an intermediate dose of Meth (5 mg/kg). A higher dose of SB (30 mg/kg) attenuates temperature responses to low dose (1 mg/kg) of Meth and to stress. In contrast, it significantly exaggerates early responses (t<60 min) to intermediate and high doses (5 and 10 mg/kg) of Meth. As pretreatment with SB also inhibits temperature response to the stress of injection, traditional statistical analysis of temperature responses is difficult.

Mathematical Modeling

We have developed a mathematical model that explains the complexity of temperature responses to Meth as the interplay between excitatory and inhibitory nodes. We have extended the developed model to include the stress of manipulations and the effects of SB. Stress is synergistic with Meth on the action on excitatory node. Orexin receptors mediate an activation of on both excitatory and inhibitory nodes by low doses of Meth, but not on the node activated by high doses (HD). Exaggeration of early responses to high doses of Meth involves disinhibition: low dose of SB decreases tonic inhibition of HD and lowers the activation threshold, while the higher dose suppresses the inhibitory component. Using a modeling approach to data assimilation appears efficient in separating individual components of complex response with statistical analysis unachievable by traditional data processing methods.     

Distal NF-kB binding motif functions as an enhancer for nontypeable H. influenzae-induced DEFB4 regulation in epithelial cells

Among the antimicrobial molecules produced by epithelial cells, DEFB4 is inducible in response to proinflammatory signals such as cytokines and bacterial molecules. Nontypeable Haemophilus influenzae (NTHi) is an important human pathogen that exacerbates chronic obstructive pulmonary disease in adult and causes otitis media and sinusitis in children. Previously, we have demonstrated that DEFB4 effectively kills NTHi and is induced by NTHi via TLR2 signaling. The 5′-flanking region of DEFB4 contains several NF-κB binding motifs, but their NTHi-specific activity remains unclear. In this study, we aimed to elucidate molecular mechanism involved in DEFB4 regulation, focusing on the role of the distal NF-κB binding motif of DEFB4 responding to NTHi. Here, we show that the human middle ear epithelial cells up-regulate DEFB4 expression in response to NTHi via NF-κB activation mediated by IκKα/β−IκBα signaling. Deletion of the distal NF-κB binding motif led to a significant reduction in NTHi-induced DEFB4 up-regulation. A heterologous construct containing the distal NF-κB binding motif was found to increase the promoter activity in response to NTHi, indicating a NTHi-responding enhancer activity of the distal NF-κB binding motif. Furthermore, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that the p65 domain of NF-κB binds to the distal NF-κB binding motif in response to NTHi. Taken together, our results suggest that NTHi-induced binding of p65 NF-κB to the distal NF-κB binding motif of DEFB4 enhances NTHi-induced DEFB4 regulation in epithelial cells.     

Anticancer activity of TTAC-0001, a fully human anti-vascular endothelial growth factor receptor 2 (VEGFR-2/KDR) monoclonal antibody,is associated with inhibition of tumor angiogenesis

Vascular endothelial growth factor (VEGF) and its receptors are considered the primary cause of tumor-induced angiogenesis. Specifically, VEGFR-2/kinase insert domain receptor (KDR) is part of the major signaling pathway that plays a significant role in tumor angiogenesis, which is associated with the development of various types of tumor and metastasis. In particular, KDR is involved in tumor angiogenesis as well as cancer cell growth and survival. In this study, we evaluated the therapeutic potential of TTAC-0001, a fully human antibody against VEGFR-2/KDR. To assess the efficacy of the antibody and pharmacokinetic (PK) relationship in vivo, we tested the potency of TTAC-0001 in glioblastoma and colorectal cancer xenograft models. Antitumor activity of TTAC-0001 in preclinical models correlated with tumor growth arrest, induction of tumor cell apoptosis, and inhibition of angiogenesis. We also evaluated the combination effect of TTAC-0001 with a chemotherapeutic agent in xenograft models. We were able to determine the relationship between PK and the efficacy of TTAC-0001 through in vivo single-dose PK study. Taken together, our data suggest that targeting VEGFR-2 with TTAC-0001 could be a promising approach for cancer treatment.     

An extremely alkaline mannanase from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CS428 hydrolyzes galactomannan producing series of mannooligosaccharides

An alkaline-thermostable mannanase from Streptomyces sp. CS428 was produced, purified, and biochemically characterized. The extracellular mannanase (Mn428) was purified to homogeneity with 12.4 fold, specific activity of 2406.7 U/mg, and final recovery of 37.6 %. The purified β-mannanase was found to be a monomeric protein with a molecular mass of approximately 35 kDa as analyzed by SDS-PAGE and zymography. The first N-terminal amino acid sequences of mannanase enzyme were HIRNGNHQLPTG. The optimal temperature and pH for enzyme were 60 °C and 12.5, respectively. The mannanase activities were significantly affected by the presence of metal ions, modulators, and detergents. K_m and V_max values of Mn428 were 1.01 ± 3.4 mg/mL and 5029 ± 85 µmol/min mg, respectively when different concentrations (0.6–10 mg/mL) of locust bean gum galactomannan were used as substrate. The substrate specificity of enzyme showed its highest specificity towards galactomannan which was further hydrolyzed to produce mannose, mannobiose, mannotriose, and a series of mannooligosaccharides. Mannooligosaccharides can be further converted to ethanol production, thus the purified β-mannanase isolated from Streptomyces sp. CS428 was found to be attractive for biotechnological applications.