Localization of blood coagulation factors and fibrinolysis factors within lymphoid germinal centers in human lymph nodes.

The presence of nineteen blood coagulation factors and fibrinolysis factors was immunohistochemically evaluated in human lymph node germinal centers (GCs). Twelve of these factors were detected within lymphoid GCs. The predominant pattern was dendritic with occasional crescent-shaped, ring-shaped or 'moth-eaten' appearance. Immunostains of factor VIII-related antigen, factor I, protein C, tetranectin, antithrombin III, type 2-plasminogen activator inhibitor, and alpha 2-plasmin inhibitor were almost entirely absent from GCs, although they reacted in vascular wall and lumen, respectively. The immunostaining to high molecular weight kininogen, kallikrein, factors XII, X, V, II, XIIIa, XIIIs, plasminogen, tissue-plasminogen activator, and type 1-plasminogen activator inhibitor more frequently revealed a positive dendritic pattern. Immuno-electron microscopy demonstrated factor X and factor XIIIa attached to the cell surfaces of lymphocytes, macrophages, and follicular dendritic cells (FDCs); and in the intercellular space within GCs, especially attached to the labyrinthine-like structure of FDCs. No reaction products were observed in the perinuclear cisternae and rough endoplasmic reticulum in either lymphocytes or FDCs. Our data demonstrate that human lymphoid GCs really contain some of the proteins related to the blood coagulation and fibrinolysis cascades.     

Sleep‐related behaviors in zoo‐housed giraffes (Giraffa camelopardalis reticulata): Basic characteristics and effects of season and parturition

Despite increasing interest in the behavior of zoo animals, studies of nocturnal behavior of zoo animals are limited. In this study, we investigated the relationship between parturition, season, and the sleep‐related behaviors in captive reticulated giraffes to better understand the nocturnal life in giraffes. The subjects were two adult reticulated giraffes living in Kyoto City Zoo, Japan. Observations were made via an infrared camera that was mounted in the indoor enclosure between June 2007 and August 2009. We analyzed video clips that were recorded between 16:30 and 09:00 the next morning, over a total of 199 days. Sleep‐related behaviors were classified into two categories based on the posture of the giraffes; recumbent posture and paradoxical sleep. We also recorded the laterality of recumbent posture, which was coded based on the direction of the legs against the torso (right or left). Seasonal differences in sleep behaviors between summer and winter were observed in both individuals. They tended to start to lie down earlier in the winter than in the summer. Parturition also affected the behaviors as both individuals decreased the behaviors before and after the parturition of the female. Additionally, the female lay on her left side less frequently than her right when resuming a recumbent posture in the pre‐parturition period, while such laterality was not observed in the baseline and post‐parturition period. These results suggested that season and parturition are important factors for determining the sleep‐related behaviors in giraffes. Further studies are needed to understand how these changes in sleep affect other welfare parameters.     

Evaluation of the antimicrobial activity of methylglyoxal bis(guanylhydrazone) analogues, the inhibitors for polyamine biosynthetic pathway

Metabolic and antiproliferative effects of methylglyoxal bis(butylamidinohydrazone) (MGBB) and methylglyoxal bis(cyclopentylamidinohydrazone) (MGBCP), inhibitors for polyamine biosynthetic pathway, on Escherichia coli, Shigella sonnei, Aeromonas sobria, Aeromonas hydrophila and Vibrio cholerae were investigated. MGBB at the concentration of 100 mumol/l depleted intracellular putrescine and spermidine concentrations of E. coli to 25 and 20% of the controls, respectively, while MGBCP depressed their concentrations to 38 and 24%, respectively. In these polyamine-depleted E. coli cells the syntheses of RNA, DNA and protein decreased to 13, 54 and 29% of the control, respectively, with MGBB and to 23, 71 and 55%, respectively, with MGBCP. The minimum inhibitory concentrations (MIC) of MGBB for the growth of A. sobria, E. coli, A. hydrophila, V. cholerae and Sh. sonnei were estimated to be 50, 160, 240, 285 and 320 mumol/l, respectively, whereas those of MGBCP were slightly higher for respective bacteria.     

On the validity of the spectrophotometric determination of oxygen saturation of hemoglobin. The wavelength dependence of observed oxygen equilibrium parameter values

Spectral changes of oxyhemoglobin induced by such anions as 2,3-diphosphoglycerate, inositol hexaphosphate, and Cl- may affect the validity of the spectrophotometric determination of oxygen saturation of hemoglobin. Therefore, the anion-induced difference spectra were extensively measured under a variety of conditions and accurate oxygen equilibrium curves were determined under representative conditions with detection at different wavelengths selected from peaks, troughs, and zero difference points of the difference spectra in the visible and Soret regions. Oxygen equilibrium parameters including the four Adair constants (i.e., equilibrium constants for four steps of oxygenation) estimated from the equilibrium curves did not show any dependence on wavelength within the limits of experimental error. These results indicate that anion-induced spectral changes do not invalidate the spectrophotometric determination of oxygen saturation and confirm the validity of the previous conclusions drawn in our series of studies on the effects of anions, pH and temperature on oxygen equilibrium parameters.     

Mitochondrial phospholipid hydroperoxide glutathione peroxidase suppresses apoptosis mediated by a mitochondrial death pathway.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a key enzyme in the protection of biomembranes exposed to oxidative stress. We investigated the role of mitochondrial PHGPx in apoptosis using RBL2H3 cells that overexpressed mitochondrial PHGPx (M15 cells), cells that overexpressed non-mitochondrial PHGPx (L9 cells), and control cells (S1 cells). The morphological changes and fragmentation of DNA associated with apoptosis occurred within 15 h in S1 and L9 cells upon exposure of cells to 2-deoxyglucose (2DG). The release of cytochrome c from mitochondria was observed in S1 cells after 4 h and was followed by the activation of caspase-3 within 6 h. Overexpression of mitochondrial PHGPx prevented the release of cytochrome c, the activation of caspase-3, and apoptosis, but non-mitochondrial PHGPx lacked the ability to prevent the induction of apoptosis by 2DG. An ability to protect cells from 2DG-induced apoptosis was abolished when the PHGPx activity of M15 cells was inhibited by diethylmalate, indicating that the resistance of M15 cells to apoptosis was indeed due to the overexpression of PHGPx in the mitochondria. The expression of members of the Bcl-2 family of proteins, such as Bcl-2, Bcl-xL, Bax, and Bad, was unchanged by the overexpression of PHGPx in cells. The levels of hydroperoxides, including hydrogen and lipid peroxide, in mitochondria isolated from S1 and L9 cells were significantly increased after the exposure to 2DG for 2 h, while the level of hydroperoxide in mitochondria isolated from M15 cells was lower than that in S1 and L9 cells. M15 cells were also resistant to apoptosis induced by etoposide, staurosporine, UV irradiation, cycloheximide, and actinomycin D, but not to apoptosis induced by Fas-specific antibodies, which induces apoptosis via a pathway distinct from the pathway initiated by 2DG. Our results suggest that hydroperoxide, produced in mitochondria, is a major factor in apoptosis and that mitochondrial PHGPx might play a critical role as an anti-apoptotic agent in mitochondrial death pathways.     

Direct suppression of TCR-mediated activation of extracellular signal-regulated kinase by leukocyte protein tyrosine phosphatase, a tyrosine-specific phosphatase.

Leukocyte protein tyrosine phosphatase (LC-PTP)/hemopoietic PTP is a human cytoplasmic PTP that is predominantly expressed in the hemopoietic cells. Recently, it was reported that hemopoietic PTP inhibited TCR-mediated signal transduction. However, the precise mechanism of the inhibition was not identified. Here we report that extracellular signal-regulated kinase (ERK) is the direct target of LC-PTP. LC-PTP dephosphorylated ERK2 in vitro. Expression of wild-type LC-PTP in 293T cells suppressed the phosphorylation of ERK2 by a mutant MEK1, which was constitutively active regardless of upstream activation signals. No suppression of the phosphorylation was observed by LC-PTPCS, a catalytically inactive mutant. In Jurkat cells, LC-PTP suppressed the ERK and p38 mitogen-activated protein kinase cascades. LC-PTP and LC-PTPCS made complexes with ERK1, ERK2, and p38alpha, but not with the gain-of-function sevenmaker ERK2 mutant (D321N). A small deletion (aa 1-46) in the N-terminal portion of LC-PTP or Arg to Ala substitutions at aa 41 and 42 resulted in the loss of ERK binding activity. These LC-PTP mutants revealed little inhibition of the ERK cascade activated by TCR cross-linking. On the other hand, the wild-type LC-PTP did not suppress the phosphorylation of sevenmaker ERK2 mutant. Thus, the complex formation of LC-PTP with ERK is the essential mechanism for the suppression. Taken collectively, these results indicate that LC-PTP suppresses mitogen-activated protein kinase directly in vivo.