Strain differences to effects of aging on concentrations of amino acids in cerebrospinal fluid between Sprague Dawley rat and Wistar Kyoto rat.

Age-related alterations and differences of weights and those of amino acid concentrations in the cerebrospinal fluid (CSF) were evaluated between Sprague Dawley (SD) rats and Wistar Kyoto (WKY) rats from eight to twenty weeks of age. The weights of SD rats were heavier than WKY rats at all ages. The age-related alterations of the CSF concentration of many amino acids within each strain were significant but showed no significant trend with age. Between the strains, the concentration differences of excitatory and inhibitory amino acids were not frequent although the concentrations of arginine, alanine and threonine were significantly higher in SD rats than in WKY rats. These results suggest that the different CSF concentrations of amino acids may relate to characteristics of rat strains.     

Function of RNA-binding protein Musashi-1 in stem cells

Musashi is an evolutionarily conserved family of RNA-binding proteins that is preferentially expressed in the nervous system. The first member of the Musashi family was identified in Drosophila. This protein plays an essential role in regulating the asymmetric cell division of ectodermal precursor cells known as sensory organ precursor cells through the translational regulation of target mRNA. In the CNS of Drosophila larvae, however, Musashi is expressed in proliferating neuroblasts and likely has a different function. Its probable mammalian homologue, Musashi-1, is a neural RNA-binding protein that is strongly expressed in fetal and adult neural stem cells (NSCs). Mammalian Musashi-1 augments Notch signaling through the translational repression of its target mRNA, m-Numb, thereby contributing to the self-renewal of NSCs. In addition to its functions in NSCs, the role of mammalian Musashi-1 protein in epithelial stem cells, including intestinal and mammary gland stem cells, is attracting increasing interest.     

Activation of big MAP kinase 1 (BMK1/ERK5) inhibits cardiac injury after myocardial ischemia and reperfusion

Big MAP kinase 1 (BMK1/ERK5) plays a critical role in pre-natal development of the cardiovascular system and post-natal eccentric hypertrophy of the heart. Of the two isoforms upstream of MAPK-kinase 5 (MEK5) known to exist, only the longer MEK5alpha isoform potently activates BMK1. We generated cardiac-specific constitutively active form of the MEK5alpha (CA-MEK5alpha transgenic (Tg) mice), and observed a 3 to 4-fold increase in endogenous BMK1 activation and hyperphosphorylation of connexin 43 in the ventricles of the Tg compared to wild-type mice. The CA-MEK5alpha-Tg-mice demonstrated a profoundly accelerated recovery of left ventricular developed pressure after ischemia/reperfusion. We propose a novel role for BMK1 in protecting the heart from ischemia/reperfusion-induced cardiac injury.     

Single gene deletions of mrpA to mrpG and mrpE point mutations affect activity of the Mrp Na+/H+ antiporter of alkaliphilic Bacillus and formation of hetero-oligomeric Mrp complexes

Mrp antiporters catalyze secondary Na(+)(Li(+))/H(+) antiport and/or K(+)/H(+) antiport that is physiologically important in diverse bacteria. An additional capacity for anion flux has been observed for a few systems. Mrp is unique among antiporters in that it requires all six or seven hydrophobic gene products (MrpA to MrpG) of the mrp operon for full antiporter activity, but MrpE has been reported to be dispensable. Here, the membrane complexes formed by Mrp proteins were examined using a cloned mrp operon from alkaliphilic Bacillus pseudofirmus OF4. The operon was engineered so that the seven Mrp proteins could be detected in single samples. Membrane extracts of an antiporter-deficient Escherichia coli strain expressing this construct were analyzed by blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mrp complexes of two sizes were identified containing all seven Mrp proteins. Studies of the single nonpolar mrp gene deletions in the construct showed that a subcomplex of MrpA, MrpB, MrpC, and MrpD was formed in the absence of MrpE, MrpF, or MrpG. By contrast, MrpE, MrpF, and MrpG were not observed in membranes lacking MrpA, MrpB, MrpC, or MrpD. Although MrpA and MrpD have been hypothesized to be the antiporter proteins, the MrpA-to-D complex was inactive. Every Mrp protein was required for an activity level near that of the wild-type Na(+)/H(+) antiporter, but a very low activity level was observed in the absence of MrpE. The introduction of an MrpE(P114G) mutation into the full Mrp complex led to antiport activity with a greatly increased apparent K(m) value for Na(+). The results suggested that interactions among the proteins of heterooligomeric Mrp complexes strongly impact antiporter properties.     

Cryoprotectant permeability of aquaporin-3 expressed in Xenopus oocytes

It has been shown that aquaporin-3, a water channel, is expressed in mouse embryos. This type of aquaporin transports not only water but also neutral solutes, including cell-permeating cryoprotectants. Therefore, the expression of this channel may have significant influence on the survival of cryopreserved embryos. However, permeability coefficients of aquaporin-3 to cryoprotectants have not been determined except for glycerol. In addition, permeability coefficients under concentration gradients are important for developing and improving cryopreservation protocols. In this study, we examined the permeability of aquaporin-3 to various cryoprotectants using Xenopus oocytes. The permeability of aquaporin-3 to cryoprotectants was measured by the volume change of aquaporin-3 cRNA-injected oocytes in modified Barth's solution containing either 10% glycerol, 8% ethylene glycol, 10% propylene glycol, 1.5 M acetamide, or 9.5% DMSO (1.51-1.83 Osm/kg) at 25 degrees C. Permeability coefficients of aquaporin-3 for ethylene glycol and propylene glycol were 33.50 and 31.45 x 10(-3) cm/min, respectively, which were as high as the value for glycerol (36.13 x 10(-3) cm/min). These values were much higher than those for water-injected control oocytes (0.04-0.11 x 10(-3) cm/min). On the other hand, the coefficients for acetamide and DMSO were not well determined because the volume data were poorly fitted by the two parameter model, possibly because of membrane damage. To avoid this, the permeability for these cryoprotectants was measured under a low concentration gradient by suspending oocytes in aqueous solutions containing low concentrations of acetamide or DMSO dissolved in water (0.20 Osm/kg). The coefficient for acetamide (24.60 x 10(-3) cm/min) was as high as the coefficients for glycerol, ethylene glycol, and propylene glycol, and was significantly higher than the value for control (6.50 x 10(-3) cm/min). The value for DMSO (6.33 x 10(-3) cm/min) was relatively low, although higher than the value for control (0.79 x 10(-3) cm/min). This is the first reported observation of DMSO transport by aquaporin-3.     

Localization of 9- and 13-oxo-octadecadienoic acids in tomato fruit

We previously reported that the two peroxisome proliferator-activated receptor-α agonists, 9- and 13-oxo-octadecadienoic acids (oxo-ODAs), were found in the tomato fruit. However, their localization remains unknown. Herein, we showed that oxo-ODAs localize primarily in the fruit peel and their amount increases after the homogenization of the tomato fruit.     

A distinct role of the queen in coordinated workload and soil distribution in eusocial naked mole-rats

We investigated how group members achieve collective decision-making, by considering individual intrinsic behavioural rules and behavioural mechanisms for maintaining social integration. Using a simulated burrow environment, we investigated the behavioural rules of coordinated workload for soil distribution in a eusocial mammal, the naked mole-rat (Heterocephalus glaber). We tested two predictions regarding a distinct role of the queen, a socially dominant individual in the caste system: the presence of a queen would increase the workload of other caste individuals, and the cues by a queen would affect the soil distribution. In experiment 1, we placed four individuals of various castes from the same colony into an experimental burrow. Workers exhibited the highest frequency of workload compared to other castes. The presence of a queen activated the workload by other individuals. Individuals showed a consistent workload in a particular direction so as to bias the soil distribution. These results suggest that individuals have a consensus on soil distribution and that the queen plays a distinct role. In experiment 2, we placed the odour of a queen in one of four cells and observed its effect on other individuals' workload and soil distribution. Relative to other cells, individuals frequently dug in the queen cell so the amount of soil in the queen cell decreased. These results suggest that queen odour is an important cue in coordinated workload and soil distribution in this species.     

Oscillatory membrane currents paradoxically induced via NO-activated pathways in detrusor cells

Oscillatory inward membrane currents (I(oscil-in)) reflecting intracellular Ca(2+) ([Ca(2+)](i)) activity in detrusor cells, are thought to play an important role in producing tonic bladder contractions during micturition. The present patch clamp study revealed a new activation mechanism: sodium nitroprusside (SNP), a nitric oxide (NO) donor induced I(oscil-in) in a subpopulation of detrusor cells. The inhibitory effect of niflumic acid on SNP-induced I(oscil-in) suggests that Ca(2+)-activated Cl(-) channels are responsible for this current. In addition, SNP-induced I(oscil-in) required the cooperation of Ca(2+) influx through SK&F96365-sensitive channels and intracellular Ca(2+) release channels sensitive to ryanodine but insensitive to xestospongin C (XeC). This is also true for muscarinic agonist (carbachol: CCh)-induced I(oscil-in). However, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylyl cyclase inhibitor, suppressed SNP-induced I(oscil-in) but not CCh-induced I(oscil-in). The results suggest that a subpopulation of detrusor cells employ the NO/cGMP cascade to potentiate bladder contraction. Mechanisms underlying NO-induced I(oscil-in) are likely to contribute not only to the physiology but also to the pathophysiology of the lower urinary tract.     

(3Z)-2-Acetylamino-3-octadecen-1-ol as a potent apoptotic agent against HL-60 cells

(2R,3Z)-, (2R,3E)-, (2S,3Z) and (2S,3E)-2-Acetylamino-3-octadecen-1-ol, and (2R)- and (2S)-2-acetylamino-octadecan-1-ol were prepared using the Wittig olefination of Garner's aldehyde (N-Boc-N,O-isopropylidene-L- or D-serinal) from L- or D-serine. The apoptotic activities of these saturated and unsaturated 2-acetylaminoalcohols were examined in human leukemia HL-60 cells using MTT assay. Among the newly synthesized compounds, the cis-isomers were the most potent. Despite their simple structures, (2R,3Z)- and (2S,3Z)-2-acetylamino-3-octadecen-1-ol showed high and comparable apoptotic activities compared with N-acetyl-D-erythro-sphingosine (D-e-C2-Cer, a well-known inducer of apoptosis). Their apoptotic activities were in the order D-e-C2-Cer approximately L-e-C2-Cer approximately (2R,3Z)- approximately (2S,3Z)->(2R,3E)- approximately (2S,3E)- approximately (2R)- approximately (2S)-derivative. Qualitative analysis of DNA fragmentation caused by these compounds was conducted using agarose gel electrophoresis, and typical DNA fragmentation was found in the cases of (2R,3Z)- and (2S,3Z)-isomers such as C2-Cer, but not trans and saturated isomers. The morphological features of the cells, the proteolytic processing of pro-caspase-3, and the cleavage of PARP as a result of exogenous treatment with (2R,3Z)- and (2S,3Z)-isomers indicated that cell death induced by these compounds was apoptosis. These observations suggest that these newly synthesized compounds, (3Z)-2-Acetylamino-3-octadecen-1-ol, have similar characteristics and apoptosis-inducing activities against HL-60 cells with C2-Cer.     

Effect of Protease Inhibitors on Early Events of Apoptosis

Proteolysis is an early event of apoptosis which appears to be associated with activation of the endonuclease which is responsible for internucleosomal DNA cleavage. The present study was designed to reveal the possible role of proteolysis in other early events, such as chromatin condensation, nuclear breakdown, and destabilization ofin situDNA double-stranded structure. Apoptosis of human leukemic HL-60 cells and rat thymocytes was induced by different agents, including DNA topoisomerase inhibitors, an RNA antimetabolite, and the glucocorticosteroid, prednisolone. DNA degradation was evaluated by pulsed field and conventional gel electrophoresis and by the presence ofin situDNA strand breaks. DNA stability was estimated by the measure of its sensitivityin situto denaturation. Chromatin condensation, nuclear breakdown, and other morphological changes were monitored by interference contrast and UV microscopy following cell staining with the DNA-specific fluorochrome 4′,6-diamidino-2-phenylindole. Several irreversible or reversible serine protease inhibitors prevented internucleosomal DNA degradation, nuclear breakdown, and destabilization of DNA double-stranded structure. The effective inhibitors, however, did not prevent the onset of chromatin condensation, nor the loss of the fine structural framework, nor the initial step of DNA cleavage generating DNA fragments of ≥50 kb in size. The data indicate that in both cell systems the activity of proteases sensitive to the inhibitors tested is needed for internucleosomal DNA cleavage to occur. The data also suggest that these proteases may be involved in dissolution of the nuclear envelope. Because nuclear matrix proteins and histones stabilize DNAin situ,and the decrease in DNA stability which occurs during apoptosis is precluded by the inhibitors, it is likely that serine proteases may degrade DNA stabilizing proteins. The activity of these proteases, however, appears needed neither for DNA cleavage to ≥50-kb fragments nor for the onset of chromatin condensation which is associated with dissolution of the structural framework of the nucleus.