Replacement of C305 in Heart/Muscle-Type Isozyme of Human Carnitine Palmitoyltransferase I with Aspartic Acid and Other Amino Acids

Liver- and heart/muscle-type isozymes of human carnitine palmitoyltransferase I (L- and M-CPTI, respectively) show a certain similarity in their amino acid sequences, and mutation studies on the conserved amino acids between these two isozymes often show essentially the same effects on their enzymatic properties. Earlier mutation studies on C305 in human M-CPTI and its counterpart residue, C304, in human L-CPTI showed distinct effects of the mutations, especially in the aspect of enzyme stability; however, simple comparison of these effects on the conserved Cys residue between L- and M-CPTI was difficult, because these studies were carried out using different expression systems and distinct amino acids as replacements. In the present study, we carried out mutation studies on the C305 in human M-CPTI using COS cells for the expression system. Our results showed that C305 was replaceable with aspartic acid but that substitution with other amino acids caused both loss of function and reduced expression.     

Phylogeny of Local Sheep Breeds in East Asia, Focusing on the Bayanbulak Sheep in China and the Sipsu Sheep in Bhutan

The phylogenetic positions of the Bayanbulak sheep in China and the Sipsu sheep in Bhutan in the northern Asian sheep group were determined on the basis of allele frequency data for five informative and polymorphic loci of blood protein and nonproteins, such as transferrin (TF), arylesterase (ES), hemoglobin-β (HB-β), X-protein (XP), and potassium transport (KE), using different electrophoretic and ion-densitometric techniques. Based on Nei’s genetic distance, clustering analysis by the UPGMA method showed that the Bayanbulak sheep is clustered in the northern Asian sheep group. Furthermore, the Bayanbulak sheep belongs to a subgroup containing the Khalkhas and Hu sheep of the Mongolian sheep group, which is distinguished from another subgroup of the small-tailed Han, Tan, Tong, and Wadi sheep. The Bayanbulak sheep was closest to the Hu sheep, despite a morphological difference in the fat deposits. In addition to these findings, the Sipsu sheep was verified to belong to the Baruwal sheep.     

Surface-induced phase separation of a sphingomyelin/cholesterol/ganglioside GM1-planar bilayer on mica surfaces and microdomain molecular conformation that accelerates Aβ oligomerization

Ganglioside GM1 mediates the amyloid beta (Aβ) aggregation that is the hallmark of Alzheimer's disease (AD). To investigate how ganglioside-containing lipid bilayers interact with Aβ, we examined the interaction between Aβ40 and supported planar lipid bilayers (SPBs) on mica and SiO₂ substrates by using atomic force microscopy, fluorescence microscopy, and molecular dynamics computer simulations. These SPBs contained several compositions of sphingomyelin, cholesterol, and GM1 and were treated at physiological salt concentrations. Surprisingly high-speed Aβ aggregation of fibril formations occurred at all GM1 concentrations examined on the mica surface, but on the SiO₂ surface, only globular agglomerates formed and they formed slowly. At a GM1 concentration of 20 mol%, unique triangular regions formed on the mica surface and the rapidly formed Aβ aggregations were observed only outside these regions. We have found that some unique surface-induced phase separations are induced by the GM1 clustering effects and the strong interactions between the GM1 head group and the water layer adsorbed in the ditrigonal cavities on the mica surface. The speed of Aβ40 aggregation and the shape of the agglomerates depend on the molecular conformation of GM1, which varies depending on the substrate materials. We identified the conformation that significantly accelerates Aβ40 aggregation, and we think that the detailed knowledge about the GM1 molecular conformation obtained in this work will be useful to those investigating Aβ-GM1 interactions.     

Crystal structure of cyclic Lys48-linked tetraubiquitin

Lys48-linked polyubiquitin chains serve as a signal for protein degradation by 26S proteasomes through its Ile44 hydrophobic patches interactions. The individual ubiquitin units of each chain are conjugated through an isopeptide bond between Lys48 and the C-terminal Gly76 of the preceding units. The conformation of Lys48-linked tetraubiquitin has been shown to change dynamically depending on solution pH. Here we enzymatically synthesized a wild-type Lys48-linked tetraubiquitin for structural study. In the synthesis, cyclic and non-cyclic species were obtained as major and minor fractions, respectively. This enabled us to solve the crystal structure of tetraubiquitin exclusively with native Lys48-linkages at 1.85 Å resolution in low pH 4.6. The crystallographic data clearly showed that the C-terminus of the first ubiquitin is conjugated to the Lys48 residue of the fourth ubiquitin. The overall structure is quite similar to the closed form of engineered tetraubiquitin at near-neutral pH 6.7, previously reported, in which the Ile44 hydrophobic patches face each other. The structure of the second and the third ubiquitin units [Ub(2)-Ub(3)] connected through a native isopeptide bond is significantly different from the conformations of the corresponding linkage of the engineered tetraubiquitins, whereas the structures of Ub(1)-Ub(2) and Ub(3)-Ub(4) isopeptide bonds are almost identical to those of the previously reported structures. From these observations, we suggest that the flexible nature of the isopeptide linkage thus observed contributes to the structural arrangements of ubiquitin chains exemplified by the pH-dependent closed-to-open conformational transition of tetraubiquitin.     

The Met268Pro mutation of mouse TRPA1 changes the effect of caffeine from activation to suppression

The transient receptor potential A1 channel (TRPA1) is activated by various compounds, including isothiocyanates, menthol, and cinnamaldehyde. The sensitivities of the rodent and human isoforms of TRPA1 to menthol and the cysteine-attacking compound CMP1 differ, and the molecular determinants for these differences have been identified in the 5th transmembrane region (TM5) for menthol and TM6 for CMP1. We recently reported that caffeine activates mouse TRPA1 (mTRPA1) but suppresses human TRPA1 (hTRPA1). Here we aimed to identify the molecular determinant that is responsible for species-specific differences in the response to caffeine by analyzing the functional properties of various chimeras expressed in Xenopus oocytes. We initially found that the region between amino acids 231 and 287, in the distal N-terminal cytoplasmic region of mTRPA1, is critical. In a mutagenesis study of this region, we subsequently observed that introduction of a Met268Pro point mutation into mTRPA1 changed the effect of caffeine from activation to suppression. Because the region including Met-268 is different from other reported ligand-binding sites and from the EF-hand motif, these results suggest that the caffeine response is mediated by a unique mechanism, and confirm the importance of the distal N-terminal region for regulation of TRPA1 channel activity.     

Keratinocytes in culture accumulate phagocytosed melanosomes in the perinuclear area

There are many techniques for evaluating melanosome transfer to keratinocytes but the spectrophotometric quantification of melanosomes incorporated by keratinocyte phagocytosis has not been previously reported. Here we describe a new method that allows the spectrophotometric visualization of melanosome uptake by normal human keratinocytes in culture. Fontana-Masson staining of keratinocytes incubated with isolated melanosomes showed the accumulation of incorporated melanosomes in the perinuclear areas of keratinocytes within 48 h. Electron microscopic observations of melanosomes ingested by keratinocytes revealed that many phagosomes containing clusters of melanosomes or their fragments were localized in the perinuclear area. A known inhibitor of keratinocyte phagocytosis which inhibits protease-activated receptor-2, i.e., soybean trypsin inhibitor, decreased melanosome uptake by keratinocytes in a dose-dependent manner. These data suggest that our method is a useful model to quantitate keratinocyte phagocytosis of melanosomes visually in vitro.     

Paleoceanographic evolution of North Pacific surface water off Japan during the past 150,000 years

Hydrographic variability in the Mixed Water Region of the Northwest Pacific Ocean at latitudes 35°–40°N, between the Kuroshio Extension and Oyashio Front, causes complex upwelling, leading to large primary productivity and thus great fishery resources. We reconstructed the periodicity of the variability in North Pacific Intermediate Water upwelling and surface ocean hydrography based on the high-resolution analysis of diatom assemblages in seven cores, representing the last 150,000 years. We derived annual sea surface temperatures (SSTs) through a diatom-based proxy (Td′). The Td′-derived annual SSTs (°C) are controlled by orbital forcing, and show a reversed saw-tooth in southern cores, in contrast to a normal saw-tooth pattern in the northern cores. Oceanic diatom abundances along the northern margin of the Mixed Water Region are twice times as high as beneath the axis of the Kuroshio Extension, and fluctuated in a revised saw-tooth pattern with higher overall abundances interglacials. After the last deglaciation, annual SSTs declined markedly during Heinrich and Bond events in the northern North Atlantic, when ice-rafted detritus transported by icebergs was abundant. Wavelet analyses of the record of oceanic diatom abundances show significant variability at 2.0-kyr, 2 to 5.6-kyr and 3.2 to 9.6-kyr periods. Wavelet analyses of the annual SST records show significant periodicity at 1.4 to 2.6-kyr, 3.3 to 4.0-kyr, 7.2 to 12.8-kyr cycles.     

Genetic diversity of gamma-hexachlorocyclohexane-degrading sphingomonads isolated from a single experimental field

Aim:  To determine the effect of the surface roughness of denture acrylic on the attachment of Streptococcus oralis .
Methods and Results:  Roughened denture acrylic samples were assessed for bacterial attachment, over time, using microscopy. The area of the image covered by bacteria was calculated and converted into a percentage of the total area sampled. The results showed an increasing bacterial coverage with time of incubation and increasing roughness. Differences were seen between heat cured acrylic and cold cured acrylic.
Conclusion:  This study successfully demonstrated a system for the assessment of the amount of attached bacteria on denture acrylic varying roughness. The system was able to discern the difference in surface area coverage by attached bacteria over a roughness range relevant to brushing dentures with dentifrices.
Significance and Impact of Study:  This study provides strong support for the scratches caused by brushing dentures with dentifrice encouraging bacterial attachment. This is likely to have a significant effect on efficacy of denture cleaning, general hygiene and biofilm re-formation between cleaning regimens and may indicate that alternative low abrasive cleaners, such as antimicrobial denture-cleaning tablets, offer a more appropriate regimen.     

Curved EFC/F-BAR-domain dimers are joined end to end into a filament for membrane invagination in endocytosis

Pombe Cdc15 homology (PCH) proteins play an important role in a variety of actin-based processes, including clathrin-mediated endocytosis (CME). The defining feature of the PCH proteins is an evolutionarily conserved EFC/F-BAR domain for membrane association and tubulation. In the present study, we solved the crystal structures of the EFC domains of human FBP17 and CIP4. The structures revealed a gently curved helical-bundle dimer of approximately 220 A in length, which forms filaments through end-to-end interactions in the crystals. The curved EFC dimer fits a tubular membrane with an approximately 600 A diameter. We subsequently proposed a model in which the curved EFC filament drives tubulation. In fact, striation of tubular membranes was observed by phase-contrast cryo-transmission electron microscopy, and mutations that impaired filament formation also impaired membrane tubulation and cell membrane invagination. Furthermore, FBP17 is recruited to clathrin-coated pits in the late stage of CME, indicating its physiological role.