Wobble modification deficiency in mutant tRNAs in patients with mitochondrial diseases

Point mutations in mitochondrial (mt) tRNA genes are associated with a variety of human mitochondrial diseases. We have shown previously that mt tRNA(Leu(UUR)) with a MELAS A3243G mutation and mt tRNA(Lys) with a MERRF A8344G mutation derived from HeLa background cybrid cells are deficient in normal taurine-containing modifications [taum(5)(s(2))U; 5-taurinomethyl-(2-thio)uridine] at the anticodon wobble position in both cases. The wobble modification deficiency results in defective translation. We report here wobble modification deficiencies of mutant mt tRNAs from cybrid cells with different nuclear backgrounds, as well as from patient tissues. These findings demonstrate the generality of the wobble modification deficiency in mutant tRNAs in MELAS and MERRF.     

Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies

Exposure of antibodies to low pH is often unavoidable for purification and viral clearance. The conformation and stability of two humanized monoclonal antibodies (hIgG4-A and -B) directed against different antigens and a mouse monoclonal antibody (mIgG1) in 0.1M citrate at acidic pH were studied using circular dichroism (CD), differential scanning calorimetry (DSC), and sedimentation velocity. Near- and far-UV CD spectra showed that exposure of these antibodies to pH 2.7-3.9 induced only limited conformational changes, although the changes were greater at the lower pH. However, the acid conformation is far from unfolded or so-called molten globule structure. Incubation of hIgG4-A at pH 2.7 and 3.5 at 4 degrees C over the course of 24 h caused little change in the near-UV CD spectra, indicating that the acid conformation is stable. Sedimentation velocity showed that the hIgG4-A is largely monomeric at pH 2.7 and 3.5 as well as at pH 6.0. No time-dependent changes in sedimentation profile occurred upon incubation at these low pHs, consistent with the conformational stability observed by CD. The sedimentation coefficient of the monomer at pH 2.7 or 3.5 again suggested that no gross conformational changes occur at these pHs. DSC analysis of the antibodies showed thermal unfolding at pH 2.7-3.9 as well as at pH 6.0, but with decreased melting temperatures at the lower pH. These results are consistent with the view that the antibodies undergo limited conformational change, and that incubation at 4 degrees C at low pH results in no time-dependent conformational changes. Titration of hIgG4-A from pH 3.5 to 6.0 resulted in recovery of native monomeric proteins whose CD and DSC profiles resembled those of the original sample. However, titration from pH 2.7 resulted in lower recovery of monomeric antibody, indicating that the greater conformational changes observed at this pH cannot be fully reversed to the native structure by a simple pH titration.     

Identification of arylsulfonamides as Aquaporin 4 inhibitors

Carbonic anhydrase inhibitors AZA, EZA, and 4-acetamidobenzsulfonamide were found to inhibit human AQP4-M23 mediated water transport by 80%, 68%, and 23%, respectively, at 20 microM in an in vitro functional assay. AZA was found to have an IC50 against AQP4 of 0.9 microM. Phloretin was inactive under the same conditions.     

Sterol biosynthesis by a prokaryote: first in vitro identification of the genes encoding squalene epoxidase and lanosterol synthase from Methylococcus capsulatus

Sterol biosynthesis by prokaryotic organisms is very rare. Squalene epoxidase and lanosterol synthase are prerequisite to cyclic sterol biosynthesis. These two enzymes, from the methanotrophic bacterium Methylococcus capsulatus, were functionally expressed in Escherichia coli. Structural analyses of the enzymatic products indicated that the reactions proceeded in a complete regio- and stereospecific fashion to afford (3S)-2,3-oxidosqualene from squalene and lanosterol from (3S)-2,3-oxidosqualene, in full accordance with those of eukaryotes. However, our result obtained with the putative lanosterol synthase was inconsistent with a previous report that the prokaryote accepts both (3R)- and (3S)-2,3-oxidosqualenes to afford 3-epi-lanosterol and lanosterol, respectively. This is the first report demonstrating the existence of the genes encoding squalene epoxidase and lanosterol synthase in prokaryotes by establishing the enzyme activities. The evolutionary aspect of prokaryotic squalene epoxidase and lanosterol synthase is discussed.     

Past Japanese successes show the way to accomplish future goals

Many parasitic diseases have been eradicated in industrialized countries and well-proven tools and techniques exist to control them. However, the same diseases still cause incalculable ill health and suffering in the developing world. The difficulty remains how best to apply existing solutions where they are most needed. Within a period of 25 years following World War II, Japan eliminated many parasitic diseases and raised national health and living standards to world-leading levels. Gradually, the predominantly community-driven and intersectoral collaborative partnership systems (i.e. private sector, public sector, general public, etc.) and practices that worked in Japan are being extended to Asia and now Africa. These are backed by the provision of substantial human and financial resources from a nation whose population retains the reputation as being the healthiest and longest living in the world.     

Temporal and spatial variations of lipid droplets during adipocyte division and differentiation

By capturing time-lapse images of primary stromal-vascular cells (SVCs) derived from rat mesenteric adipose tissue, we revealed temporal and spatial variations of lipid droplets (LDs) in individual SVCs during adipocyte differentiation. Numerous small LDs (a few micrometers in diameter) appeared in the perinuclear region at an early stage of differentiation; subsequently, several LDs grew to more than 10 microm in diameter and occupied the cytoplasm. We have developed a method for the fluorescence staining of LDs in living adipocytes. Time-lapse observation of the stained cells at higher magnification showed that nascent LDs (several 100 nm in diameter) grew into small LDs while moving from lamellipodia to the perinuclear region. We also found that adipocytes are capable of division and that they evenly distribute the LDs between two daughter cells. Immunofluorescence observations of LD-associated proteins revealed that such cell divisions of SVCs occurred even after LDs were coated with perilipin, suggesting that the "final" cell division during adipocyte differentiation occurs considerably later than that characterized in 3T3-L1 cells. Our time-lapse observations have provided a detailed account of the morphological changes that SVCs undergo during adipocyte division and differentiation.     

Examination of protein sequence homologies: III. Ribosomal protein YS25 fromSaccharomyces cerevisiae and its counterparts fromSchizosaccharomyces pombe,rat liver,andEscherichia coli

Summary The sequences of the ribosomal proteins YS25, SP-S28, RL-S21, and Ec-S6, fromSaccharomyces cerevisiae, Schizosaccharomyces pombe, rat liver, andEscherichia coli, respectively, have been examined using a computer program that searches for homologous tertiary structures. Matrices of comparisons among the eukaryotic sequences show that they match each other sequentially without any internal gaps. The average values of the correlation coefficients obtained from the comparison matrices are higher for the first halves of the sequences than for the latter halves. This result suggests that the first halves of the sequences may represent a more important domain than the latter halves. The comparison matrices between the eukaryotic and bacterial sequences of ribosomal proteins, however, do not show sequentially arranged homology, though there are six well-matching segments arranged in different orders in the two types of sequences. This implies that the eukaryotic sequences of the ribosomal protein were reconstituted by two internal transpositions and six deletions of 4–12 residues each from the ancestral sequence during the divergence between bacterial and eukaryotic genes. These findings may give insight into structural and quantitative studies of evolutionary divergence between eukaryotes and prokaryotes.     

The Polymorphism of YWHAE,a Gene Encoding 14-3-3Epsilon,and Brain Morphology in Schizophrenia: A Voxel-Based Morphometric Study

Background

YWHAE is a possible susceptibility gene for schizophrenia that encodes 14-3-3epsilon, a Disrupted-in-Schizophrenia 1 (DISC1)-interacting molecule, but the effect of variation in its genotype on brain morphology remains largely unknown.

Methods

In this voxel-based morphometric magnetic resonance imaging study, we conducted whole-brain analyses regarding the effects of YWHAE single-nucleotide polymorphisms (SNPs) (rs28365859, rs11655548, and rs9393) and DISC1 SNP (rs821616) on gray matter volume in a Japanese sample of 72 schizophrenia patients and 86 healthy controls. On the basis of a previous animal study, we also examined the effect of rs28365859 genotype specifically on hippocampal volume.

Results

Whole-brain analyses showed no significant genotype effect of these SNPs on gray matter volume in all subjects, but we found significant genotype-by-diagnosis interaction for rs28365859 in the left insula and right putamen. The protective C allele carriers of rs28365859 had a significantly larger left insula than the G homozygotes only for schizophrenia patients, while the controls with G allele homozygosity had a significantly larger right putamen than the C allele carriers. The C allele carriers had a larger right hippocampus than the G allele homozygotes in schizophrenia patients, but not in healthy controls. No significant interaction was found between rs28365859 and DISC1 SNP on gray matter volume.

Conclusions

These different effects of the YWHAE (rs28365859) genotype on brain morphology in schizophrenia and healthy controls suggest that variation in its genotype might be, at least partly, related to the abnormal neurodevelopment, including in the limbic regions, reported in schizophrenia. Our results also suggest its specific role among YWHAE SNPs in the pathophysiology of schizophrenia.     

"Aqua-space", a new headspace method for isolation of natural floral aromas using humidified air as a carrier gas

A new method called "Aqua-space" was developed for the isolation of the natural fragrances of plants. Living flowers were enclosed in a space under simulated natural conditions, and humidified air was pumped into the space as a fragrance carrier. In a comparison among three isolation methods, i.e., Aqua-space, headspace, and solvent extraction, the Aqua-space method proved to be the most effective in retaining natural fragrances with abundant oxygenated components key to floral fragrances.     

C-Glucosidic ellagitannin oligomers from Melaleuca squarrosa Donn ex Sm., Myrtaceae

C-Glucosidic ellagitannin dimers were classified as types A-C according to a putative biogenetic oligomerization mode. They were characterized by different positions of the C-C bond between the phenolic acyl unit in one monomer and the benzylic C-1 of the open-chain glucose core in the other monomer. In recent years, four C-glucosidic tannins, melasquanins A-D (18-21), have been found in the leaves of Melaleuca squarrosa Donn ex Sm. (Myrtaceae). These are characterized as a dimer (melasquanin A) of a dimerization mode (type D), and trimers (melasquanins B-D) based on spectroscopic analysis including various two-dimensional nuclear magnetic resonance (2D NMR) experiments. Melasquanins B (19) and D (21) are C-glucosidic tannin trimers with a structure containing, non-repeating condensation modes, which was hitherto unknown.