An Archaeal Homolog of Proteasome Assembly Factor Functions as a Proteasome Activator

Assembly of the eukaryotic 20S proteasome is an ordered process involving several proteins operating as proteasome assembly factors including PAC1-PAC2 but archaeal 20S proteasome subunits can spontaneously assemble into an active cylindrical architecture. Recent bioinformatic analysis identified archaeal PAC1-PAC2 homologs PbaA and PbaB. However, it remains unclear whether such assembly factor-like proteins play an indispensable role in orchestration of proteasome subunits in archaea. We revealed that PbaB forms a homotetramer and exerts a dual function as an ATP-independent proteasome activator and a molecular chaperone through its tentacle-like C-terminal segments. Our findings provide insights into molecular evolution relationships between proteasome activators and assembly factors.     

A Lactobacillus mutant capable of accumulating long-chain polyphosphates that enhance intestinal barrier function

Inorganic polyphosphate (polyP) was previously identified as a probiotic-derived substance that enhances intestinal barrier function. PolyP-accumulating bacteria are expected to have beneficial effects on the human gastrointestinal tract. In this study, we selected Lactobacillus paracasei JCM 1163 as a strain with the potential to accumulate polyP, because among the probiotic bacteria stored in our laboratory, it had the largest amount of polyP. The chain length of polyP accumulated in L. paracasei JCM 1163 was approximately 700 phosphate (Pi) residues. L. paracasei JCM 1163 accumulated polyP when Pi was added to Pi-starved cells. We further improved the ability of L. paracasei JCM 1163 to accumulate polyP by nitrosoguanidine mutagenesis. The mutant accumulated polyP at a level of 1500 nmol/mg protein—approximately 190 times that of the wild-type strain. PolyP extracted from the L. paracasei JCM 1163 significantly suppressed the oxidant-induced intestinal permeability in mouse small intestine. In conclusion, we have succeeded in breeding the polyP-accumulating Lactobacillus mutant that is expected to enhance intestinal barrier function.     

Novel dual-reporter transgenic rodents enable cell tracking in animal models of stem cell transplantation

In the present study, we have established a novel transgenic mouse and transgenic rats with dual reporters of EGFP and ELuc. In these transgenic (Tg) rodents, both GFP fluorescent and luciferase luminescent signals were ubiquitously detected in the heart, liver, kidney and testis, while only the GFP signal was detected in the brain. This expression system is based on a P2A linked EGFP/ELuc protein allowing both signals to be generated simultaneously. Microscopy experiments, FCM, and luciferase assays showed strong expression in freshly isolated ADSCs from Tg rodents upon transplantation of Tg rat-derived ADSCs into wild-type-mice. The ELuc transgene signal was observed and traced in vivo, and EGFP positive cells could be recovered from ELuc positive tissues in engraftment sites of wild-type mice for multiple analysis. These dual reporter Tg rodents are a useful reconstituted model system of regenerative medicine and are a valuable tool to study stem cells.     

A new membrane-bound cytochrome c works as an electron donor to the photosynthetic reaction center complex in the purple bacterium, Rhodovulum sulfidophilum

A new type of membrane-bound cytochrome c was found in a marine purple photosynthetic bacterium, Rhodovulum sulfidophilum. This cytochrome c was significantly accumulated in cells growing under anaerobic photosynthetic conditions and showed an apparent molecular mass of approximately 100 kDa when purified and analyzed by SDS-PAGE. The midpoint potential of this cytochrome c was 369 mV. Flash-induced kinetic measurements showed that this new cytochrome c can work as an electron donor to the photosynthetic reaction center. The gene coding for this cytochrome c was cloned and analyzed. The deduced molecular mass was nearly equal to 50 kDa. Its C-terminal heme-containing region showed the highest sequence identity to the water-soluble cytochrome c(2), although its predicted secondary structure resembles that of cytochrome c(y). Phylogenetic analyses suggested that this new cytochrome c has evolved from cytochrome c(2). We, thus, propose its designation as cytochrome c(2m). Mutants lacking this cytochrome or cytochrome c(2) showed the same growth rate as the wild type. However, a double mutant lacking both cytochrome c(2) and c(2m) showed no growth under photosynthetic conditions. It was concluded that either the membrane-bound cytochrome c(2m) or the water-soluble cytochrome c(2) work as a physiological electron carrier in the photosynthetic electron transfer pathway of Rvu. sulfidophilum.     

Essential roles of lipoyl domains in the activated function and control of pyruvate dehydrogenase kinases and phosphatase isoform 1.

Four pyruvate dehydrogenase kinase and two pyruvate dehydrogenase phosphatase isoforms function in adjusting the activation state of the pyruvate dehydrogenase complex (PDC) through determining the fraction of active (nonphosphorylated) pyruvate dehydrogenase component. Necessary adaptations of PDC activity with varying metabolic requirements in different tissues and cell types are met by the selective expression and pronounced variation in the inherent functional properties and effector sensitivities of these regulatory enzymes. This review emphasizes how the foremost changes in the kinase and phosphatase activities issue from the dynamic, effector-modified interactions of these regulatory enzymes with the flexibly held outer domains of the core-forming dihydrolipoyl acetyl transferase component.     

Stroke and glide of wing-propelled divers: deep diving seabirds adjust surge frequency to buoyancy change with depth

In order to increase locomotor efficiency, breath-holding divers are expected to adjust their forward thrusts in relation to changes of buoyancy with depth. Wing propulsion during deep diving by Brünnich's guillemots (Uria lomvia) was measured in the wild by high-speed (32 Hz) sampling of surge (tail-to-head) and heave (ventral-to-dorsal) accelerations with bird-borne data loggers. At the start of descent, the birds produced frequent surges (3.2 Hz) during both the upstroke and the downstroke against buoyancy to attain a mean speed of 1.2-1.8 m s(-1) that was close to the expected optimal swim speed. As they descended deeper, the birds decreased the frequency of surges to 2.4 Hz, relaying only on the downstroke. During their ascent, they stopped stroking at 18 m depth, after which the swim speed increased to 2.3 m s(-1), possibly because of increasing buoyancy as air volumes expanded. This smooth change of surge frequency was achieved while maintaining a constant stroke duration (0.4-0.5 s), presumably allowing efficient muscle contraction.     

Sonic hedgehog is involved in osteoblast differentiation by cooperating with BMP-2

The roles of Sonic hedgehog (Shh) and Bone morphogenetic protein-2 (Bmp-2) in osteoblast differentiation were investigated using in vitro cell systems. Recombinant amino-terminal portion of SHH (rSHH-N) dose dependently stimulated ALP activity in C3H10T1/2 and MC3T3-E1 cells. rSHH-N induced expression of Osteocalcin mRNA in C3H10T1/2 cells. A soluble form of the receptor for type IA BMP receptor antagonized rSHH-N-induced ALP activity in C3H10T1/2 and MC3T3-E1 cells, indicating that BMPs are involved in SHH-induced osteoblast differentiation. Simultaneous supplement with rSHH-N and BMP-2 synergistically induced ALP activity and expression of Osteocalcin mRNA in C3H10T1/2 cells. Pretreatment with rSHH-N for 6 h enhanced the response to BMP-2 by increasing ALP activity in C3H10T1/2 and MC3T3-E1 cells. Stimulatory effects of rSHH-N and additive effects with rSHH-N and BMP-2 on ALP activity were also observed in mouse primary osteoblastic cells. Transplantation of BMP-2 (1 microg) into muscle of mice induced formation of ectopic bone, whereas transplantation of r-SHH-N (1-5 microg) failed to generate it. These results indicate that Shh plays important roles in osteoblast differentiation by cooperating with BMP.     

Abiotic Formation of Valine Peptides Under Conditions of High Temperature and High Pressure

We investigated the oligomerization of solid valine and the stabilities of valine and valine peptides under conditions of high temperature (150–200 °C) and high pressure (50–150 MPa). Experiments were performed under non-aqueous condition in order to promote dehydration reaction. After prolonged exposure of monomeric valine to elevated temperatures and pressures, the products were analyzed by liquid chromatography mass spectrometry comparing their retention times and masses. We identified linear peptides that ranged in size from dimer to hexamer, as well as a cyclic dimer. Previous studies that attempted abiotic oligomerization of valine in the absence of a catalyst have never reported valine peptides larger than a dimer. Increased reaction temperature increased the dissociative decomposition of valine and valine peptides to products such as glycine, β-alanine, ammonia, and amines by processes such as deamination, decarboxylation, and cracking. The amount of residual valine and peptide yields was greater at higher pressures at a given temperature, pressure, and reaction time. This suggests that dissociative decomposition of valine and valine peptides is reduced by pressure. Our findings are relevant to the investigation of diagenetic processes in prebiotic marine sediments where similar pressures occur under water-poor conditions. These findings also suggest that amino acids, such as valine, could have been polymerized to peptides in deep prebiotic marine sediments within a few hundred million years.     

Docosahexaenoic acid induces ERK1/2 activation and neuritogenesis via intracellular reactive oxygen species production in human neuroblastoma SH-SY5Y cells

Docosahexaenoic acid (22: 6n-3; DHA) is a long chain polyunsaturated fatty acid that exists highly enriched in fish oil, and it is one of the low molecular weight food chemicals which can pass a blood brain barrier. A preliminary survey of several fatty acids for expression of growth-associated protein-43 (GAP-43), a marker of axonal growth, identified DHA as one of the most potent inducers. The human neuroblastoma SH-SY5Y cells exposed to DHA showed significant and dose-dependent increases in the percentage of cells with longer neurites. To elucidate signaling mechanisms involved in DHA-enhanced basal neuritogenesis, we examined the role of extracellular signal-regulated kinase (ERK)1/2 and intracellular reactive oxygen species (ROS) production using SH-SY5Y cells. From immunoblotting experiments, we observed that DHA induced the ROS production, protein tyrosine phosphatase inhibition, mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) phosphorylation, and sequentially ERK1/2 phosphorylation, the last of which was significantly reduced by MEK inhibitor U0126. Both antioxidants and MEK inhibitor affected DHA-induced GAP-43 expression, whereas the specific PI3K inhibitor LY294002 did not. We found that total protein tyrosine phosphatase activity was also downregulated by DHA treatment, which was counteracted by antioxidant pretreatment. These results suggest that the ROS-dependent ERK pathway, rather than PI3K, plays an important role during DHA-enhanced neurite outgrowth.