A high-frequency null mutant of an odorant-binding protein gene, Obp57e, in Drosophila melanogaster

We have found a null mutant of an odorant-binding protein, Obp57e, in Drosophila melanogaster. This frameshift mutation, which is a 10-bp deletion in the coding region, is at a high frequency in the Kyoto population and is also present in Taiwan and Africa. We have sequenced a 1.5-kb region including the tandemly duplicated gene, Obp57d, from 16 inbred lines sampled in Kyoto, Japan. The analyses showed a peak of nucleotide diversity and strong linkage disequilibrium around this mutation. This pattern suggests an elevated mutation rate or an influence of balancing selection in this region. The level of nucleotide divergence between D. melanogaster and D. simulans does not support the former possibility. Thus, this presence/absence polymorphism may be due to balancing selection, which takes advantage of the relatively weak functional constraint in members of a large gene family. In addition, the Obp57d gene region showed an excess of high-frequency-derived mutants that is consistent with a pattern predicted under positive natural selection.     

De-N-acetyllactotriaosylceramide as a novel cationic glycosphingolipid of bovine brain white matter: isolation and characterization

A novel cationic lipid was separated from bovine brain white matter by a series of chromatographies on carboxymethyl-Sephadex and silica gel in chloroform and methanol. Its structure was identified unambiguously as de-N-acetyllactotriaosylceramide (deNAcLc(3)Cer) by mass spectrometry and (1)H NMR. The natural occurrence of this glycolipid in white matter extract was detected by immunostaining of thin-layer chromatography with monoclonal antibody 5F5, which is directed to deNAcLc(3)Cer and recognizes the terminal beta-glucosaminyl (GlcNH(2)) residue, having a free NH(2) group. A de-N-acetylase capable of hydrolyzing the N-acetyl group of Lc(3)Cer was detected in bovine brain extract using N-[(14)C]acetyl-labeled Lc(3)Cer as a substrate. The biogenesis and possible functional significance of deNAcLc(3)Cer are discussed.     

A new cancer diagnostic system based on a CDK profiling technology

A series of molecular pathological investigations of the molecules that stimulate the cyclin dependent kinases (CDK1, 2, 4, and 6) have led to enormous accumulation of knowledge of the clinical significance of these molecules for cancer diagnosis. However, the molecules have yet to be applied to clinical cancer diagnosis, as there is no available technology for application of the knowledge in a clinical setting. We hypothesized that the direct measurement of CDK activities and expressions (CDK profiling) might produce clinically relevant values for the diagnosis. This study investigated the clinical relevance of CDK profiling in gastrointestinal carcinoma tissues by using originally developed expression and activity analysis methods. We have established novel methods and an apparatus for analyzing the expression and activities of the CDK molecules in lysate of tumor tissue in a clinical setting, and examined 30 surgically dissected gastrointestinal carcinomas and corresponding normal mucosal specimens. We demonstrate here that remarkably elevated CDK2 activity is evident in more than 70% of carcinoma tissues. Moreover, a G1-CDK activity profiling accurately mirrored the differences in proliferation between tumor and normal colonic tissues. Our results suggest that CDK profiling is a potent molecular-clinical approach to complement the conventional pathological diagnosis, and to further assist in the individualized medications.     

Cytochrome P450 2E polymorphism in feline liver

Only one isoform of cytochrome P450 (CYP) 2E subfamily was known in human and various animals. Three cDNAs corresponding to CYP 2E subfamily members (CYP2E-a, CYP2E-b and CYP2E-c) were obtained from feline liver. These cDNAs each had a 1488-bp nucleotide coding region encoding a predicted amino acid sequence of 495 residues. Eleven amino acid substitutions were observed between CYP2E-a and CYP2E-b, but only one substitution between CYP2E-b and CYP2E-c. The CO difference spectrums about 450 nm wave length and similar values of Vmax and Km of 6-hydoxygenase activity toward chlorzoxazone were observed in all three isoforms expressed in AH22 yeast cells. By PCR-RFLP, mRNA of the CYP2E-a was found to be expressed in liver, mononuclear cells, kidney, lung, stomach, intestine and pancreas, whereas CYP2E-b and CYP2E-c were expressed mainly in the liver and mononuclear cells. Expression of CYP2E-a was observed in the livers of all felines tested, but CYP2E-b and CYP2E-c were not expressed in all cats. The sequences of two different introns between exons I and II and between exons VII and VIII were obtained in genomic DNA from the feline liver. Based on these results, we conclude that cats have two highly similar CYP2E genes.     

Measurement and characterization of C-3 epimerization activity toward vitamin D3

Recently, epimerization of the hydroxyl group at C-3 has been identified as a unique metabolic pathway of vitamin D compounds. We measured C-3 epimerization activity in subcellular fractions prepared from cultured cells and investigated the basic properties of the enzyme responsible for the epimerization. C-3 epimerization activity was detected using a NADPH-generating system containing glucose-6-phosphate, NADP, glucose-6-phosphate dehydrogenase, and Mg(2+). The highest level of activity was observed in a microsomal fraction prepared from rat osteoblastic UMR-106 cells but activity was also observed in microsomal fractions prepared from MG-63, Caco-2, Hep G2, and HUH-7 cells. In terms of maximum velocity (V(max)) and the Michaelis constant (K(m)), 25-hydroxyvitamin D(3) [25(OH)D(3)] exhibited the highest specificity for the epimerization at C-3 among 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], 25(OH)D(3), 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)], and 22-oxacalcitriol (OCT). The epimerization activity was not inhibited by various cytochrome P450 inhibitors and antiserum against NADPH cytochrome P450 reductase. Neither CYP24, CYP27A1, CYP27B1 nor 3(alpha-->beta)hydroxysteroid epimerase (HSE) catalyzed the epimerization in vitro. Based on these results, the enzyme(s) responsible for the epimerization of vitamin D(3) at C-3 are thought to be located in microsomes and different from cytochrome P450 and HSE.     

Direct determination of a membrane-peptide interface using the nuclear magnetic resonance cross-saturation method

Membrane-peptide interactions are involved in many crucial biological and pharmacological activities. To clarify the interaction mode of membrane-peptide complexes, it is important to analyze both the dynamic properties and the contact residues of the membrane-bound peptide. In this study, we investigated the dynamic properties of a peptide bound to a lipid bilayer, using relaxation and amide-water exchange analyses, and directly determined the membrane-peptide interface, using the cross-saturation method. For the models of a lipid bilayer and a peptide, isotropic bicelles and mastoparan were used, respectively. The results indicate that mastoparan had a heterogeneous distribution of motion over various timescales and interacted with the lipid bilayer by using its hydrophobic side; the molecule was located within the lipid bilayer rather than on the surface, as thought previously. This study shows that the cross-saturation method is useful for determining the interface of not only protein-protein but also membrane-peptide complexes.     

Insertion/deletion polymorphism in clusterin gene influences serum lipid levels and carotid intima-media thickness in hypertensive Japanese females

Clusterin has been implicated in lipid metabolism and atherogenesis, however, the influence of genetic variation has not been examined in Japanese. In this study, we identified 11 single nucleotide polymorphisms (SNPs) of clusterin gene by direct sequencing. Among them, one promoter SNP (-4453T>G), one missense SNP (4183G>A), and 2 common SNPs (5608T>C and 6316delT) were genotyped in 525 asymptomatic hypertensives not treated with lipid lowering agents. -4453T>G, 4183G>A, and 5608T>C showed no correlation with the clinical characteristics, however, in the 6316delT, an insertion (I)/deletion (D) polymorphism, D/D subjects had significantly higher levels of total cholesterol and low-density lipoprotein (LDL)-cholesterol than I/I subjects in females but not in males. Female subjects with the D allele (D/D+I/D) had greater intima-media thickness of the carotid artery than I/I subjects. In a multiple logistic regression analysis, the D allele of 6316delT was detected as an independent predictor for the plaque prevalence. In conclusion, the clusterin gene polymorphism may contribute to the serum lipid levels and the progression of carotid atherosclerosis in hypertensive Japanese females.     

Two mouse cofilin isoforms, muscle-type (MCF) and non-muscle type (NMCF), interact with F-actin with different efficiencies

Two cofilin isoforms, a muscle-type (MCF) and a non-muscle-type (NMCF), are co-expressed in developing mammalian skeletal and cardiac muscles. To clarify how they are involved in the actin filament dynamics during myofibrillogenesis, we examined their localization in muscle tissues and cultured muscle cells using immunocytochemical methods, and their interaction with F-actin in vitro. NMCF was mostly detected in a diffuse pattern in the cytoplasm but MCF was partly localized to the striated structures in myofibrils. The location of chicken cofilin, a homologue of MCF, in the I-bands of myofibrils was determined by an immunocytochemical method. It is suggested that MCF could be associated with actin filaments in muscle cells more efficiently than NMCF. Using purified recombinant MCF and NMCF, their interaction with F-actin was examined in vitro by a cosedimentation assay method. We observed that MCF was precipitated with F-actin more effectively than NMCF. When MCF and NMCF were simultaneously incubated with F-actin, MCF was preferentially associated with F-actin. MCF and NMCF inhibited the interaction of F-actin with tropomyosin, but the former suppressed the actin-tropomyosin interaction more strongly than the latter. These results suggest that MCF interacts with F-actin with higher affinity than NMCF, and although both of them are involved in the regulation of actin assembly in developing myotubes, the two proteins may play somewhat different roles.     

Green fluorescent protein causes mitochondria to aggregate in the presence of the Bcl-2 family proteins

Green fluorescent protein (GFP) has been widely used in a variety of experiments in cell biology. When cells were co-transfected with the GFP gene and the bcl-2 family genes bcl-2, bcl-x(L), and bax, mitochondria appeared to aggregate at the periphery of the nucleus specifically where GFP was expressed. Little aggregation was seen in the presence of other members of the GFP family, EGFP (enhanced GFP), ECFP (enhanced cyan variant), and EYFP (enhanced yellow-green variant). GFP but not EGFP seemed to promote cell death induced by pro-apoptotic Bax. Thus, GFP specifically promotes the aggregation of mitochondria when co-expressed with a member of the Bcl-2 family in association with apoptosis.