Gender Differences in Elements of Human Anterior Commissure and Olfactory Bulb and Tract

To examine whether there were gender differences in the various brain regions, the authors investigated the gender differences in seven element contents of the anterior commissure, mammillary body, and olfactory bulb and tract by direct chemical analysis. After ordinary dissection at Nara Medical University was finished, the anterior commissures, mammillary bodies, and olfactory bulbs and tracts were resected from the cerebra cut at median line. The brain samples were treated with 99.5% ethanol three times to remove lipids. After ashing with nitric acid and perchloric acid, the seven element contents Ca, P, S, Mg, Zn, Fe, and Na were determined by inductively coupled plasma–atomic emission spectrometry. It was found that the Zn content was significantly higher in the anterior commissures of men than in those of women. In the olfactory bulbs and tracts, it was found that the Ca, P, and Zn contents were significantly higher in men than in women. In contrast, no significant difference was found between the mammillary bodies of men and women regarding the seven element contents.     

Formation of a complex of 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin with G-quadruplex DNA

A water-soluble cationic porphyrin, 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin (TmPyP4), has been studied extensively because of its unique physicochemical properties that lead to interactions with nucleic acids, as well as its therapeutic application. Formation of a complex between TmPyP4 and parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d(TTAGGG), has been characterized in an effort to elucidate the mode of molecular recognition between TmPyP4 and the DNA. The study demonstrated that TmPyP4 intercalates into the A3pG4 step of [d(TTAGGG)]4 with an association constant of 6.2 x 10(6) M(-1) and a stoichiometric ratio of 1:1. The binding of TmPyP4 to the A3pG4 step of [d(TTAGGG)]4 was found to be stabilized by the pi-pi stacking interaction of the porphyrin ring of TmPyP4 with the G4 quartet as well as the A3 bases of the G-quadruplex DNA. These findings provide novel insights for the design of porphyrin derivatives that bind to DNA with high affinity and specificity.     

Rapid sexing of water buffalo (Bubalus bubalis) embryos using loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The objective of this study was to identify a Y chromosome-specific sequence in water buffalo and to establish an efficient procedure for embryo sexing by LAMP. The homologues of a Y chromosome-specific sequence, bovine repeat Y-associated.2, in swamp and river buffalo were cloned, and designated swamp buffalo repeat Y-associated.2 and river buffalo repeat Y-associated.2, respectively. Sexing by LAMP was performed using primers for swamp buffalo repeat Y-associated.2. A 12S rRNA was also amplified by LAMP as a control reaction in both male and female. The minimal amount of the template DNA required for LAMP appeared to be 0.1-10 pg. The sensitivity was further examined using swamp buffalo fibroblasts as templates. When fibroblasts were lysed with NaOH, the minimal cell number required for detection of both male-specific and male-female common DNA appeared to be two cells, whereas correct determination of sex could not be achieved using fibroblasts lysed by heat denaturation. Embryo sexing was also performed using blastomeres from interspecies nuclear transfer embryos. The sex determined by LAMP for blastomeres corresponded with the sex of nuclear donor cells in analyses using four or five blastomeres as templates. The LAMP reaction required only about 45 min, and the total time for embryo sexing, including DNA extraction, was about 1 h. In conclusion, the present procedure without thermal cycling and electrophoresis was reliable and applicable for water buffalo embryos.     

ATP autocrine/paracrine signaling induces calcium oscillations and NFAT activation in human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the potential to differentiate into several types of cells. Calcium ions (Ca(2+)) play an important role in the differentiation and proliferation of hMSCs. We have demonstrated that spontaneous [Ca(2+)](i) oscillations occur without agonist stimulation in hMSCs. However, the precise mechanism of its generation remains unclear. In this study, we investigated the mechanism and role of spontaneous [Ca(2+)](i) oscillations in hMSCs and found that IP(3)-induced Ca(2+) release is essential for spontaneous [Ca(2+)](i) oscillations. We also found that an ATP autocrine/paracrine signaling pathway is involved in the oscillations. In this pathway, an ATP is secreted via a hemi-gap-junction channel; it stimulates the P(2)Y(1) receptors, resulting in the activation of PLC-beta to produce IP(3). We were able to pharmacologically block this pathway, and thereby to completely halt the [Ca(2+)](i) oscillations. Furthermore, we found that [Ca(2+)](i) oscillations were associated with NFAT translocation into the nucleus in undifferentiated hMSCs. Once the ATP autocrine/paracrine signaling pathway was blocked, it was not possible to detect the nuclear translocation of NFAT, indicating that the activation of NFAT is closely linked to [Ca(2+)](i) oscillations. As the hMSCs differentiated to adipocytes, the [Ca(2+)](i) oscillations disappeared and the translocation of NFAT ceased. These results provide new insight into the molecular and physiological mechanism of [Ca(2+)](i) oscillations in undifferentiated hMSCs.     

Trace elements in human tendons and ligaments

Tendons and ligaments are key structures in promoting joint movement and maintaining joint stability. Although numerous reviews have detailed their structure, molecular composition, and biomechanical properties, far less attention has been paid to their content of trace elements. Tendons and ligaments are generally rich in calcium, sulfur, and phosphorus, although there are intriguing differences between one tendon/ligament and another. Furthermore, there can be significant regional variations that correlate with the presence or absence of fibrocartilage in the “wraparound” regions of tendons or ligaments, where they change direction and press against bone. Here, their sulfate and calcium contents are particularly high. This is undoubtedly associated with the high levels of proteoglycans that are found in these cartilaginous tissues and the occasional presence of sesamoid bones within them.     

Age-dependent increase of magnesium in the cerebral arteries of Thai

To elucidate compositional changes of the cerebral arteries with aging, the authors investigated age-related changes of elements in the cerebral arteries by inductively coupled plasma-atomic emission spectrometry (ICP-AES). After an ordinary dissection by medical students at Chiang Mai University was finished, the anterior, middle, and posterior cerebral arteries and the anterior and posterior communicating arteries were resected from the subjects. The subjects consisted of six men and seven women, ranging in age from 34 to 86 yr. The element content was determined by ICP-AES. It was found that the Mg content increased progressively with aging in all of the anterior, middle, and posterior cerebral arteries, but the Ca and P content did not increase significantly with aging in the anterior, middle, and posterior cerebral arteries, with the one exception being that the P content increased significantly in the posterior cerebral arteries with aging. The average content of Ca was higher in the order of the anterior communicating, anterior cerebral, posterior communicating, posterior cerebral, and middle cerebral arteries.     

Roles of cAMP in regulating microtubule sliding and flagellar bending in demembranated hamster spermatozoa

To understand the mechanism regulating spermatozoa motility, it is important to investigate the mechanism regulating the conversion of microtubule sliding into flagellar bending. Therefore, we analyzed microtubule sliding and its conversion into flagellar bending using a demembranated spermatozoa model in which microtubule sliding and flagellar bending could be analyzed separately by treating the demembranated spermatozoa with and without dithiothreitol, respectively. Using this model, we examined the roles of cAMP and its target molecules in regulating flagellar bending and microtubule sliding. Although flagellar bending did not occur in the absence of cAMP, microtubule extrusion occurred without it, suggesting that cAMP is necessary for the conversion of microtubule sliding into flagellar bending, but not for microtubule sliding itself. The target of cAMP for regulating flagellar bending was not cAMP-dependent protein kinase (PKA), since flagellar bending was still observed in the spermatozoa treated with a PKA-specific inhibitor. Alternatively, the Epac/Rap pathway may be the target. Epac2 and Rap2 were detected in hamster spermatozoa using immunoblotting. Since Rap2 is a GTPase, we investigated the flagellar bending of demembranated spermatozoa treated with GTPgammaS. The treatment markedly increased the beat frequency and bending rate. These results suggest that cAMP activates the Epac/Rap pathway to regulate the conversion of microtubule sliding into flagellar bending.     

Genetic networks responsive to sodium butyrate in colonic epithelial cells

We performed microarray and computational gene network analyses to identify the detailed mechanisms by which sodium butyrate (SB) induces cell growth arrest and the differentiation of mouse colonic epithelial MCE301 cells. Two thousand six hundred four differentially expressed probe sets were identified in the cells treated with 2mM SB and were classified into four groups. Of these, the gradually increased group and the gradually and remarkably decreased group contained the genetic networks for cellular development and cell cycles or canonical pathways for fatty acid biosynthesis and pyrimidine metabolism, respectively. The present results provide a basis for understanding the detailed molecular mechanisms of action of SB in colonic epithelial cells.     

Intravenous administration of mannosylated cationic liposome/NFkappaB decoy complexes effectively prevent LPS-induced cytokine production in a murine liver failure model

The purpose of this study was to inhibit endotoxin induced cytokines production and liver injury by liver non-parenchymal cell (NPC) selective delivery of nuclear factor kappaB (NFkappaB) decoy using mannosylated cationic liposomes (Man-liposomes). In this study, we examined the distribution, inhibitory effect on cytokines production and ALT/AST of intravenously injected Man-liposome/NFkappaB decoy complex. Man-liposome/[(32)P] NFkappaB decoy complexes mostly accumulated in the liver, preferentially in NPC. In a murine lipopolysaccharide-induced liver failure model, the production of tumor necrosis factor-alpha (TNFalpha), IFNgamma, IL1-beta, ALT and AST were effectively reduced by Man-liposome complexes. However, cationic or galactosylated cationic liposome complexes could not inhibit TNFalpha production.     

Molecular Epidemiology of Echoviruses 11 and 13, Based on an Environmental Surveillance Conducted in Toyama Prefecture, 2002-2003

Nineteen echovirus 11 (E11) and 12 E13 isolates were isolated from three rivers in Toyama Prefecture, Japan, during an environmental surveillance conducted from April 2002 to March 2003. The nucleotide sequences of E13 isolates were closely related to those from patients with aseptic meningitis, with less than 1.3% divergence in the VP1 region of the viral capsid gene, and belonged to the same clade responsible for a worldwide outbreak that started in 2000. In contrast, E11 isolates were clustered into three genomic groups and were not closely related to echovirus strains isolated from patients. These results suggest that the combination of both virus isolation from environmental sources and phylogenetic analysis could be complementary assessment approaches to trace prevalent and minor circulating enteroviruses in the human population.