Genomic organization and mutational analysis of HERG, a gene responsible for familial long QT syndrome

Familial long QT syndrome (LQTS) is characterized by prolonged ventricular repolarization. Clinical symptoms include recurrent syncopal attacks, and sudden death may occur as a result of ventricular tachyarrhythmias. Three genes responsible for this syndrome (KVLQT1, HERG, and SCN5A) have been identified so far, and mutations have been reported on the basis of partially characterized genomic organization. To optimize the search for HERG mutations, we have determined the genomic structure of HERG and investigated mutations in LQTS families. Human genomic clones containing the HERG gene were isolated from a human genomic library by using reverse-transcribed polymerase chain reaction (RT-PCR) products from this gene as probes. We determined exon/intron boundaries and flanking intronic sequences by using primers synthesized on the basis of the HERG cDNA sequence available in the DNA database. HERG was shown to consist of 15 exons spanning approximately 19 kb on chromosome 7q35. Subsequently, we synthesized oligonucleotide primers to cover the entire coding region and searched for mutations in 36 Japanese LQTS families. When genomic DNA from each proband was examined by the PCR/single-strand conformation polymorphism technique followed by direct DNA sequencing, five novel mutations were detected. Each mutation was present in affected relatives of the respective proband. This work should increase the efficiency of screening mutations associated with HERG. Received: 4 November 1997 / Accepted: 5 January 1998     

Effect of methyl substitution in a ligand on the selectivity and binding affinity for a nucleobase: A case study with isoxanthopterin and its derivatives

Isoxanthopterin (IX) has two edges with hydrogen bond-forming sites suitable for binding to thymine (T) and cytosine (C). The binding affinity of IX for T or C is stronger than for adenine (A) and guanine (G), whereas the base selectivity of IX for T over C (and vice versa) is moderate. In order to improve both the binding affinity and base selectivity for T over C or C over T, a methyl group is introduced respectively at the N-3 or N-8 position of IX. This leads to the known ligands 3-methyl isoxanthopterin (3-MIX) and 8-methyl isoxanthopterin (8-MIX), and the binding affinity for C or T is expected to be tuned and improved by methyl substitution. Indeed, 3-MIX selectively binds to T more strongly than IX with a binding constant of 1.5 × 10⁶ M^?1 and it loses its binding affinity for C. In contrast, 8-MIX selectively binds to C over T with a binding constant of 1.0 × 10⁶ M^?1 and the binding affinity is greatly improved compared to the parent ligand IX. The thermodynamics of the ligand–nucleotide interaction is analyzed by isothermal calorimetric titrations, and the results show that the interaction follows a 1:1 stoichiometry and is enthalpy-driven. The introduction of methyl groups at both N-3 and N-8 positions results in an increase in enthalpy of the ligand–nucleotide interaction, which leads to the improved binding affinity.     

Novel polymer biomaterials and interfaces inspired from cell membrane functions

Background

Materials with excellent biocompatibility on interfaces between artificial system and biological system are needed to develop any equipments and devices in bioscience, bioengineering and medicinal science. Suppression of unfavorable biological response on the interface is most important for understanding real functions of biomolecules on the surface. So, we should design and prepare such biomaterials.

Scoop of review

One of the best ways to design the biomaterials is generated from mimicking a cell membrane structure. It is composed of a phospholipid bilayered membrane and embedded proteins and polysaccharides. The surface of the cell membrane-like structure is constructed artificially by molecular integration of phospholipid polymer as platform and conjugated biomolecules. Here, it is introduced as the effectiveness of biointerface with highly biological functions observed on artificial cell membrane structure.

Major conclusions

Reduction of nonspecific protein adsorption is essential for suppression of unfavorable bioresponse and achievement of versatile biomedical applications. Simultaneously, bioconjugation of biomolecules on the phospholipid polymer platform is crucial for a high-performance interface.

General significance

The biointerfaces with both biocompatibility and biofunctionality based on biomolecules must be installed on advanced devices, which are applied in the fields of nanobioscience and nanomedicine.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.     

Recovery of saltwater crocodiles following unregulated hunting in tidal rivers of the Northern Territory,Australia

Saltwater crocodiles (Crocodylus porosus) in the Northern Territory of Australia were protected in 1971, after a severe population decline resulting from 26 yr of intense commercial hunting. By that time wild saltwater crocodiles were rarely sighted anywhere and they were commercially extinct in areas where they had once been abundant. Standardized monitoring by spotlight surveys started in 1975 and provided relative density indices over time (1975–2009) as a unique record of the post-protection recovery of a wild crocodilian population. We examined the survey data for populations at 12 major tidal rivers, individually and as a single subpopulation. The pattern of recovery in the subpopulation in both abundance and biomass was approximated by logistic curves, predicting 5.26 non-hatchling crocodiles weighing 387.64 kg sighted per kilometer of river in 2010. We predicted potential carrying capacity as 5.58 non-hatchling crocodiles (5.73% higher than 2010) weighing 519.0 kg (25.31% higher than 2010). Individual rivers showed largely different abundance and biomass among rivers. The statistical model that best described the recovery in individual rivers was not always logistic. However, where it was logistic, expected carrying capacity of different rivers showed considerable variation in abundance and biomass. The variation indicates different habitat quality among the rivers. Recovery occurred despite various consumptive uses, particularly a widespread egg-harvest program, which has been an integral part of the incentive-driven conservation program for saltwater crocodiles in the Northern Territory since 1983. We suggest that the saltwater crocodile population of the Northern Territory is achieving full recovery from uncontrolled hunting in 1945–1971. Although saltwater crocodiles are considered an important natural resource, their increase in number, size, and distribution is posing management issues for public safety. Continuation of human–crocodile conflict management through public education and strategic removal of problem crocodiles will be essential. © 2011 The Wildlife Society.     

Accumulation of 8-oxo-deoxyguanosine in cardiovascular tissues with the development of hypertension

Accumulation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in DNA is associated with mutagenesis and cell death. Little attention has been given to the biological significance of 8-oxo-dG accumulation in cardiovascular tissues during the different stage of hypertension and its prevention. We thus investigated the levels and localization of both 8-oxo-dG accumulation and expression of MTH1, which hydrolyzes 8-oxo-dGTP to prevent its incorporation into DNA, in the thoracic aorta prepared from stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched Wister-Kyoto rats (WKY), aged 5-32 weeks. HPLC-MS/MS analysis revealed that the levels of nuclear 8-oxo-dG in the aorta increased significantly in SHRSP, but not WKY, with aging. Immunohistochemical study revealed that both TUNEL reactivity and 8-oxo-dG immunoreactivity were increased in smooth muscle cells (SMC) and endothelial cells (EC) of the aorta with aging, and they exhibited similar distributions in serial sections. The number of 8-oxo-dG and TUNEL positive cells in EC, but not in SMC, was significantly higher in SHRSP than WKY at 32 weeks of age. In contrast, the expression levels of Mth1mRNA and MTH1 protein in the aorta were similarly decreased both in SHRSP and WKY with aging. However, the number of MTH1 expressing EC was remarkably increased in the older SHRSP compared to the younger ones or age-matched WKY. Hypertension significantly increased not only 8-oxo-dG accumulation but also the expression of MTH1 in EC of the aorta during aging. While accumulation of 8-oxo-dG in SMC of the aorta was slightly increased, the expression of MTH1 protein in SMC was rather decreased by hypertension. We thus suggest that MTH1 may protect EC in the aorta from the oxidative damage increased by hypertension.     

Structural studies of [2',6'-dimethyl-L-tyrosine1]endomorphin-2 analogues: enhanced activity and cis orientation of the Dmt-Pro amide bond

Analogues of endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-NH(2)) (1) were designed to examine the importance of each residue on mu-opioid receptor interaction. Replacement of Tyr(1) by 2',6'-dimethyl-L-tyrosine (Dmt) (9-12) exerted profound effects: [Dmt(1)]EM-2 (9) elevated mu-opioid affinity 4.6-fold (K(i mu=0.15 nM) yet selectivity fell 330-fold as delta-affinity rose (K(i)delta=28.2 nM). This simultaneous increased mu- and delta-receptor bioactivities resulted in dual agonism (IC(50)=0.07 and 1.87 nM, respectively). While substitution of Phe(4) by a phenethyl group (4) decreased mu affinity (K(i)mu=13.3 nM), the same derivative containing Dmt (12) was comparable to EM-2 but also acquired weak delta antagonism (pA(2)=7.05). 1H NMR spectroscopy revealed a trans configuration (1:2 to 1:3, cis/trans) in the Tyr-Pro amide bond, but a cis configuration (5:3 to 13:7, cis/trans) with Dmt-Pro analogues.     

Chromatographic and mass spectrometric analysis of glutathione in biological samples

Biological thiol compounds are classified into high-molecular-mass protein thiols and low-molecular-mass free thiols. Endogenous low-molecular-mass thiol compounds, namely, reduced glutathione (GSH) and its corresponding disulfide, glutathione disulfide (GSSG), are very important molecules that participate in a variety of physiological and pathological processes. GSH plays an essential role in protecting cells from oxidative and nitrosative stress and GSSG can be converted into the reduced form by action of glutathione reductase. Measurement of GSH and GSSG is a useful indicator of oxidative stress and disease risk. Many publications have reported successful determination of GSH and GSSG in biological samples. In this article, we review newly developed techniques, such as liquid chromatography coupled with mass spectrometry and tandem mass spectrometry, for identifying GSH bound to proteins, or for localizing GSH in bound or free forms at specific sites in organs and in cellular locations.     

The RNA ligands for mouse proline-rich RNA-binding protein (mouse Prrp) contain two consensus sequences in separate loop structure

Mouse proline-rich RNA-binding protein (mPrrp) is a mouse ortholog of Xenopus Prrp, which binds to a vegetal localization element (VLE) in the 3′-untranslated region (3′-UTR) of Vg1 mRNA and is expected to be involved in the transport and/or localization of Vg1 mRNA to the vegetal cortex of oocytes. In mouse testis, mPrrp protein is abundantly expressed in the nuclei of pachytene spermatocytes and round spermatids, and shifts to the cytoplasm in elongating spermatids. To gain an insight into the function of mPrrp in male germ cells, we performed in vitro RNA selection (SELEX) to determine the RNA ligand sequence of mPrrp. This analysis revealed that many of the selected clones contained both of two conserved elements, AAAUAG and GU_1–3AG. RNA-binding study on deletion mutants and secondary structure analyses of the selected RNA revealed that a two-loop structure containing the conserved elements is required for high-affinity binding to mPrrp. Furthermore, we found that the target mRNAs of Xenopus Prrp contain intact AAAUAG and GU_1–3AG sequences in the 3′-UTR, suggesting that these binding sequences are shared by Prrps of Xenopus and mouse.     

Small RNA class transition from siRNA/piRNA to miRNA during pre-implantation mouse development

Recent studies showed that small interfering RNAs (siRNAs) and Piwi-interacting RNA (piRNA) in mammalian germ cells play important roles in retrotransposon silencing and gametogenesis. However, subsequent contribution of those small RNAs to early mammalian development remains poorly understood. We investigated the expression profiles of small RNAs in mouse metaphase II oocytes, 8–16-cell stage embryos, blastocysts and the pluripotent inner cell mass (ICM) using high-throughput pyrosequencing. Here, we show that during pre-implantation development a major small RNA class changes from retrotransposon-derived small RNAs containing siRNAs and piRNAs to zygotically synthesized microRNAs (miRNAs). Some siRNAs and piRNAs are transiently upregulated and directed against specific retrotransposon classes. We also identified miRNAs expression profiles characteristic of the ICM and trophectoderm (TE) cells. Taken together, our current study reveals a major reprogramming of functional small RNAs during early mouse development from oocyte to blastocyst.