A generalized conformational energy function of DNA derived from molecular dynamics simulations

Proteins recognize DNA sequences by two different mechanisms. The first is direct readout, in which recognition is mediated by direct interactions between the protein and the DNA bases. The second is indirect readout, which is caused by the dependence of conformation and the deformability of the DNA structure on the sequence. Various energy functions have been proposed to evaluate the contribution of indirect readout to the free-energy changes in complex formations. We developed a new generalized energy function to estimate the dependence of the deformability of DNA on the sequence. This function was derived from molecular dynamics simulations previously conducted on B-DNA dodecamers, each of which had one possible tetramer sequence embedded at its center. By taking the logarithm of the probability distribution function (PDF) for the base-step parameters of the central base-pair step of the tetramer, its ability to distinguish the native sequence from random ones was superior to that with the previous method that approximated the energy function in harmonic form. From a comparison of the energy profiles calculated with these two methods, we found that the harmonic approximation caused significant errors in the conformational energies of the tetramers that adopted multiple stable conformations.     

Absolute requirement for iron in the development of chemically induced uroporphyria in mice treated with 3-methylcholanthrene and 5-aminolevulinate

Accumulating evidence, including experiments using cytochrome P450 1a2 (Cyp1a2) gene knock-out mice (Cyp1a2(−/−)), indicates that the development of chemically induced porphyria requires the expression of CYP1A2. It has also been demonstrated that iron enhances and expedites the development of experimental uroporphyria, but that iron alone without CYP1A2 expression, as in Cyp1a2(−/−) mice, does not cause uroporphyria. The role of iron in the development of porphyria has not been elucidated. We examined the in vivo effect of iron deficiency on hepatic URO accumulation in experimental porphyria. Mice were fed diets containing low (iron-deficient diet (IDD), 8.5 mg iron/kg) or normal (normal diet (ND), 213.7 mg iron/kg) levels of iron. They were treated with 3-methylcholanthrene (MC), an archetypal inducer of CYP1A, and 5-aminolevulinate (ALA), precursors of porphyrin and heme. We found that uroporphyrin (URO) levels and uroporphyrinogen oxidation (UROX) activity were markedly increased in ND mice treated with MC and ALA, while the levels were not raised in IDD mice with the same treatments. CYP1A2 levels and methoxyresorufin O-demethylase (MROD) activities, the CYP1A2-mediated reaction, were markedly induced in the livers of both ND and IDD mice treated with MC and ALA. UROX activity, supposedly a CYP1A2-dependent activity, was not enhanced in iron-deficient mice in spite of the fact of induction of CYP1A2. We showed that a sufficient level of iron is essential for the development of porphyria and UROX activity.     

Palmitate Induces Insulin Resistance in H4IIEC3 Hepatocytes through Reactive Oxygen Species Produced by Mitochondria

Visceral adiposity in obesity causes excessive free fatty acid (FFA) flux into the liver via the portal vein and may cause fatty liver disease and hepatic insulin resistance. However, because animal models of insulin resistance induced by lipid infusion or a high fat diet are complex and may be accompanied by alterations not restricted to the liver, it is difficult to determine the contribution of FFAs to hepatic insulin resistance. Therefore, we treated H4IIEC3 cells, a rat hepatocyte cell line, with a monounsaturated fatty acid (oleate) and a saturated fatty acid (palmitate) to investigate the direct and initial effects of FFAs on hepatocytes. We show that palmitate, but not oleate, inhibited insulin-stimulated tyrosine phosphorylation of insulin receptor substrate 2 and serine phosphorylation of Akt, through c-Jun NH₂-terminal kinase (JNK) activation. Among the well established stimuli for JNK activation, reactive oxygen species (ROS) played a causal role in palmitate-induced JNK activation. In addition, etomoxir, an inhibitor of carnitine palmitoyltransferase-1, which is the rate-limiting enzyme in mitochondrial fatty acid β-oxidation, as well as inhibitors of the mitochondrial respiratory chain complex (thenoyltrifluoroacetone and carbonyl cyanide m-chlorophenylhydrazone) decreased palmitate-induced ROS production. Together, our findings in hepatocytes indicate that palmitate inhibited insulin signal transduction through JNK activation and that accelerated β-oxidation of palmitate caused excess electron flux in the mitochondrial respiratory chain, resulting in increased ROS generation. Thus, mitochondria-derived ROS induced by palmitate may be major contributors to JNK activation and cellular insulin resistance.Insulin is the major hormone that inhibits gluconeogenesis in the liver. Visceral adiposity in obesity causes hepatic steatosis and insulin resistance. In an insulin-resistant state, impaired insulin action allows enhancement of glucose production in the liver, resulting in systemic hyperglycemia (1) and contributing to the development of type 2 diabetes. In addition, we have demonstrated experimentally that insulin resistance accelerated the pathology of steatohepatitis in genetically obese diabetic OLETF rats (2). In contrast, lipid-induced oxidative stress caused steatohepatitis and hepatic insulin resistance in mice (3). In fact, steatosis of the liver is an independent predictor of insulin resistance in patients with nonalcoholic fatty liver disease (4).It remains unclear whether hepatic steatosis causally contributes to insulin resistance or whether it is merely a resulting pathology. Excessive dietary free fatty acid (FFA)² flux into the liver via the portal vein may cause fatty liver disease and hepatic insulin resistance. Indeed, elevated plasma FFA concentrations correlate with obesity and decreased target tissue insulin sensitivity (5).Experimentally, lipid infusion or a high fat diet that increases circulating FFA levels promotes insulin resistance in the liver. Candidate events linking FFA to insulin resistance in vivo are the up-regulation of SREBP-1c (6), inflammation caused by activation of c-Jun amino-terminal kinase (JNK) (7) or IKKβ (8), endoplasmic reticulum (ER) stress (9), ceramide (10, 11), and TRB3 (12).However, which event is the direct and initial target of FFA in the liver is unclear. Insulin resistance induced by lipid infusion or a high fat diet is complex and may be accompanied by alterations not restricted to the liver, making it difficult to determine the contribution of FFAs to hepatic insulin resistance. For example, hyperinsulinemia and hyperglycemia secondary to the initial event also may contribute to the development of diet-induced insulin resistance in vivo (6).To address the early event(s) triggering the development of high fat diet- or obesity-induced insulin resistance, we investigated the molecular mechanism(s) underlying the direct action of FFA on hepatocytes to cause insulin resistance in vitro, using the rat hepatocyte cell line H4IIEC3. We found that mitochondria-derived reactive oxygen species (ROS) were a cause of palmitate-induced insulin resistance in hepatocytes.     

HIV-1 Accessory Protein Vpu Internalizes Cell-surface BST-2/Tetherin through Transmembrane Interactions Leading to Lysosomes

Bone marrow stromal antigen 2 (BST-2, also known as tetherin) is a recently identified interferon-inducible host restriction factor that can block the production of enveloped viruses by trapping virus particles at the cell surface. This antiviral effect is counteracted by the human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein U (Vpu). Here we show that HIV-1 Vpu physically interacts with BST-2 through their mutual transmembrane domains and leads to the degradation of this host factor via a lysosomal, not proteasomal, pathway. The degradation is partially controlled by a cellular protein, β-transducin repeat-containing protein (βTrCP), which is known to be required for the Vpu-induced degradation of CD4. Importantly, targeting of BST-2 by Vpu occurs at the plasma membrane followed by the active internalization of this host protein by Vpu independently of constitutive endocytosis. Thus, the primary site of action of Vpu is the plasma membrane, where Vpu targets and internalizes cell-surface BST-2 through transmembrane interactions, leading to lysosomal degradation, partially in a βTrCP-dependent manner. Also, we propose the following configuration of BST-2 in tethering virions to the cell surface; each of the dimerized BST-2 molecules acts as a bridge between viral and cell membranes.     

Advances in molecular methods to alter chromosomes and genome in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A fundamental issue in biotechnology is how to breed useful strains of microorganisms for efficient production of valuable biomaterials. On-going and more recent developments in gene manipulation technologies and chromosomal and genomic modifications in particular have facilitated important contributions in this area. “Chromosome manipulation technology” as an outgrowth of “gene manipulation technology” may provide opportunities for creating novel strains of organisms with a variety of genomic constitutions. A simple and rapid chromosome splitting technology called “PCR-mediated chromosome splitting” (PCS) that we recently developed has made it possible to manipulate chromosomes and genomes on a large scale in an industrially important microorganism, Saccharomyces cerevisiae. This paper focuses on recent advances in molecular methods for altering chromosomes and genome in S. cerevisiae featuring chromosome splitting technology. These advances in introducing large-scale genomic modifications are expected to accelerate the breeding of novel strains for biotechnological purposes, and to reveal functions of presently uncharacterized chromosomal regions in S. cerevisiae and other organisms.     

Association analysis between canine behavioural traits and genetic polymorphisms in the Shiba Inu breed

The relationships between behavioural trait data and the genotype of 15 polymorphisms in eight neurotransmitter-related genes were analysed in 77 dogs of the Shiba Inu breed, an indigenous Japanese dog. The data were obtained from a 26-item questionnaire on the dog's behaviour, distributed to the dog's owners, through veterinary hospitals and the Shiba Inu breed magazine. A factor analysis of the questionnaire items extracted eight factors accounting for 66.8% of the variance. An association analysis between these factors and genetic polymorphisms indicated that the polymorphism of c.471T>C in the solute carrier family 1 ( neuronal/epithelial high-affinity glutamate transporter ) member 2 ( SLC1A2 ) gene was significantly associated with Factor 1, referred to as 'aggression to strangers'. This association remained stable in separate analyses of data from surveys obtained from the hospitals and those obtained from the magazine. The results suggest that the c.471T>C polymorphism is associated with some types of aggressive behaviour in the Shiba Inu. Further studies using other dog breeds are necessary to extend these findings to dogs in general.     

Crystal structure of the de-ubiquitinating enzyme UCH37 (human UCH-L5) catalytic domain

Ubiquitin C-terminal hydrolases (UCHs) are one of five sub-families of de-ubiquitinating enzymes (DUBs) that hydrolyze the C-terminal peptide bond of ubiquitin. UCH37 (also called UCH-L5) is the only UCH family protease that interacts with the 19S proteasome regulatory complex and disassembles Lys48-linked poly-ubiquitin from the distal end of the chain. The structures of three UCHs, UCH-L1, UCH-L3, and YUH1, have been determined by X-ray crystallography. However, little is known about their physiological substrates. These enzymes do not hydrolyze large adducts of ubiquitin such as proteins. To identify and characterize the hydrolytic specificities of their substrates, the crystal structure of the UCH37 catalytic domain (UCH-domain) was determined and compared with that of the other UCHs. The overall folding patterns are similar in these UCHs. However, helix-3 is collapsed in UCH37 and the pattern of electrostatic potential on the surface of the putative substrate-binding site (P′-site) is different. Helix-3 comprises an edge of the P′-site. As a result, the P′-site is wider than that in other UCHs. These differences indicate that UCH37 can interact with larger adducts such as ubiquitin.