Chromatoid bodies: aggresome-like characteristics and degradation sites for organelles of spermiogenic cells.

We investigated the localization of several markers for lysosomes and aggresomes in the chromatoid bodies (CBs) by immunoelectron microscopy. We found so-called aggresomal markers such as Hsp70 and ubiquitin in the core of the CBs and vimentin and proteasome subunit around the CBs. Ubiquitin-conjugating enzyme (E2) was also found in the CBs. In tubulovesicular structures surrounding the CBs, lysosomal markers were detected but an endoplasmic reticulum retention signal (KDEL) was not. Moreover, proteins located in each subcellular compartment, including the cytosol, mitochondria, and nucleus, were detected in the CBs. Signals for cytochrome oxidase I (COXI) coded on mitochondrial DNA were also found in the CBs. Quantitative analysis of labeling density showed that all proteins examined were concentrated in the CBs to some extent. These results show that the CBs have some aggresomal features, suggesting that they are not a synthetic site as proposed previously but a degradation site where unnecessary DNA, RNA, and proteins are digested.     

Development of an ON/OFF switchable fluorescent probe targeting His tag fused proteins in living cells

The fluorescent labeling of target proteins is useful for analyzing their functions and localization in cells, and several fluorescent probes have been developed. However, the fusion of tags such as green fluorescent protein (GFP) to target proteins occasionally affects their functions and/or localization in living cells. Therefore, an imaging method that uses short peptide tags such as hexa-histidine (the His tag) has been attracting increasing attention. Few studies have investigated ON/OFF switchable fluorescent probes for intracellular His-tagged proteins. We herein developed a novel ON/OFF switchable probe for imaging targeted intracellular proteins fused with a CH6 tag, which is composed of one cysteine residue and six histidine residues.     

Migratory history of the spotted flagtail, <Emphasis Type="Italic">Kuhlia marginata</Emphasis>

The spotted flagtail, Kuhlia marginata (Kuhliidae) is commonly found in streams throughout the Ryukyu Archipelago, including Okinawa Island, Japan. Although it has been suggested that they spawn at sea, little is known about their migratory history. The aim of the study was to clarify their migration history based on otolith microchemistry analysis, size composition and the gonadal development of fish collected from the Genka River on Okinawa Island. All strontium/calcium (Sr/Ca) ratios of otoliths remained high around their cores, and then dropped rapidly at some distance from the core. The estimated standard length corresponding to decreases correlated with the size of new recruits collected in the river (ca. 20 mm in standard length: SL). Multiple increases, the sign for migrations into the high saline area, were recognized intermittently around the otolith margin. As the estimated SL at these increases in the Sr/Ca ratio occurred above the minimum maturation size (female: 95.5 mm SL; male: 83.5 mm SL), such multiple increases in Sr/Ca ratios were caused by spawning migration. Therefore, we conclude that the catadromous pattern of K. marginata is as follows; this species grows in the sea during the early life stage until ca. 20 mm SL and then grow in the freshwater area until maturation, before they migrate again to the sea for spawning. Sr/Ca ratio profiles also suggested that a large number of males did not return to the freshwater area after spawning, whereas females might spawn several times during their lives around the inshore area.     

Molecular characterization of alanine racemase from Bifidobacterium bifidum

Bifidobacterium bifidum is a useful probiotic agent exhibiting health-promoting properties, and its peptidoglycans have the potential for applications in the fields of food science and medicine. We investigated the bifidobacterial alanine racemase, which is essential in the synthesis of -alanine as an essential component of the peptidoglycans. Alanine racemase was purified to homogeneity from a crude extract of B. bifidum NBRC 14252. It consisted of two identical subunits with a molecular mass of 50 kDa. The enzyme required pyridoxal 5′-phosphate (PLP) as a coenzyme. The activity was lost in the presence of a thiol-modifying agent. The enzyme almost exclusively catalyzed the alanine racemization; other amino acids tested, except for serine, were inactive as substrates. The kinetic parameters of the enzyme suggested that the B. bifidum alanine racemase possesses comparatively low affinities for both the coenzyme (9.1 μM for PLP) and substrates (44.3 mM for -alanine; 74.3 mM for -alanine). The alr gene encoding the alanine racemase was cloned and sequenced. The alr gene complemented the -alanine auxotrophy of Escherichia coli MB2795, and an abundant amount of the enzyme was produced in cells of the E. coli MB2795 clone. The enzymologic and kinetic properties of the purified recombinant enzyme were almost the same as those of the alanine racemase from B. bifidum NBRC 14252.     

Photosystem II Complex in Vivo Is a Monomer

Photosystem II (PS II) complexes are membrane protein complexes that are composed of >20 distinct subunit proteins. Similar to many other membrane protein complexes, two PS II complexes are believed to form a homo-dimer whose molecular mass is ∼650 kDa. Contrary to this well known concept, we propose that the functional form of PS II in vivo is a monomer, based on the following observations. Deprivation of lipids caused the conversion of PS II from a monomeric form to a dimeric form. Only a monomeric PS II was detected in solubilized cyanobacterial and red algal thylakoids using blue-native polyacrylamide gel electrophoresis. Furthermore, energy transfer between PS II units, which was observed in the purified dimeric PS II, was not detected in vivo. Our proposal will lead to a re-evaluation of many crystallographic models of membrane protein complexes in terms of their oligomerization status.Photosystem II (PS II)3 complexes convert solar energy to biological redox energy. Through this reaction process, water molecules are oxidized and molecular oxygen is released as a byproduct (reviewed in Ref. 1), which is the only source of molecular oxygen upon which all aerobic organisms on earth rely. PS II core complexes are membrane protein complexes that are composed of >20 distinct subunit proteins and many functional cofactors, including chlorophylls (Chls), carotenoids, plastoquinone, and metal ions (2–5). Similar to many other membrane protein complexes (6–10), two PS II core complexes are believed to associate together to form a homo-dimer with a molecular mass of ∼650 kDa, as shown by crystallographic models (2, 3, 5).The PS II complex turns over dynamically, although it is quite an integrated complex; our current understanding is that the PS II complex that is damaged by high light is disintegrated into a monomeric form and is further dissociated to replace a degraded D1 protein with a de novo synthesized D1 (reviewed in Refs. 11 and 12). After the replacement, the PS II complex is integrated into a functional form as a dimer. It is supposed that PS II subunit proteins such as PsbI (13) or PsbTc (14) participate in the formation of the PS II dimer.Crystallographic models of PS II have enabled the determination of the accurate molecular architecture of PS II complexes, all of which are in a dimeric form. The most recent crystallographic model of the PS II dimer at 3.0-Å resolution revealed the presence of six detergent molecules located at the interface of the two monomers (5). Small structural fluctuations during the purification process might allow the invasion of those detergent molecules. However, it is also probable that the PS II complexes exist in the form of a monomer in vivo and the two distinct monomers become a dimer during the purification step incorporating detergents between their interfaces. This idea led us to investigate the actual form of PS II in vivo. Contrary to the above well known dimeric model of a functional PS II core complex, here we show that the PS II core complex functions and exists in a monomeric form in vivo.     

The NAD World: A New Systemic Regulatory Network for Metabolism and Aging—Sirt1, Systemic NAD Biosynthesis,and Their Importance

For the past several years, it has been demonstrated that the NAD-dependent protein deacetylase Sirt1 and nicotinamide phosphoribosyltransferase (Nampt)-mediated systemic NAD biosynthesis together play a critical role in the regulation of metabolism and possibly aging in mammals. Based on our recent studies on these two critical components, we have developed a hypothesis of a novel systemic regulatory network, named “NAD World”, for mammalian aging. Conceptually, in the NAD World, systemic NAD biosynthesis mediated by intra- and extracellular Nampt functions as a driver that keeps up the pace of metabolism in multiple tissues/organs, and the NAD-dependent deacetylase Sirt1 serves as a universal mediator that executes metabolic effects in a tissue-dependent manner in response to changes in systemic NAD biosynthesis. This new concept of the NAD World provides important insights into a systemic regulatory mechanism that fundamentally connects metabolism and aging and also conveys the ideas of functional hierarchy and frailty for the regulation of metabolic robustness and aging in mammals.