Induction of epithelial cell apoptosis in the uterus by a mouse uterine ischemia-reperfusion model: possible involvement of tumor necrosis factor-alpha

Menstruation in primates is preceded by a period of intense vasoconstriction, with resultant ischemia-reperfusion. Although apoptosis is involved in endometrial breakdown, the relationship between ischemia-reperfusion and apoptosis in the female genital tract has not been determined. To investigate the relationship between ischemia-reperfusion and apoptosis in the uterus, we analyzed a uterine ischemia-reperfusion model using BDF1 and C57BL/6 mice. Ischemia was induced by clamping the uterine horn and uterine artery for 5 to 30 min, followed by 6, 12, 24, or 48 h of reperfusion (n = 4 for each group). The number of TUNEL-positive endometrial cells increased with the duration of ischemia and reached a maximum at 24 h of reperfusion, but then tended to decrease at 48 h. Transmission electron micrographs of endometrial cells revealed a typical nuclear condensation, confirming the occurrence of apoptosis. The mRNA expression level of the proinflammatory cytokine tumor necrosis factor-alpha (TNFalpha) in the uterus increased after reperfusion. Ischemia-reperfusion-induced endometrial apoptosis was markedly decreased in TNF-R p55-deficient mice, confirming the essential role of TNFalpha in the induction of apoptosis by ischemia-reperfusion (n = 4). Our results suggest that ischemia-reperfusion and subsequent TNFalpha expression may be critical factors in inducing endometrial cell apoptosis. Our mouse model could be suitable for investigating ischemia-related uterine injury in humans, particularly in menstruation.     

The neurofibromatosis type 1 gene product neurofibromin enhances cell motility by regulating actin filament dynamics via the Rho-ROCK-LIMK2-cofilin pathway

Neurofibromin is a neurofibromatosis type 1 (NF1) tumor suppressor gene product with a domain that acts as a GTPase-activating protein and functions, in part, as a negative regulator of Ras. Loss of neurofibromin expression in NF1 patients is associated with elevated Ras activity and increased cell proliferation, predisposing to a variety of tumors of the peripheral and central nervous systems. We show here, using the small interfering RNA (siRNA) technique, that neurofibromin dynamically regulates actin cytoskeletal reorganization, followed by enhanced cell motility and gross cell aggregation in Matrigel matrix. NF1 siRNA induces characteristic morphological changes, such as excessive actin stress fiber formation, with elevated negative phosphorylation levels of cofilin, which regulates actin cytoskeletal reorganization by depolymerizing and severing actin filaments. We found that the elevated phosphorylation of cofilin in neurofibromin-depleted cells is promoted by activation of a Rho-ROCK-LIMK2 pathway, which requires Ras activation but is not transduced through three major Ras-mediated downstream pathways via Raf, phosphatidylinositol 3-kinase, and RalGEF. In addition, the exogenous expression of the NF1-GTPase-activating protein-related domain suppressed the NF1 siRNA-induced phenotypes. Neurofibromin was demonstrated to play a significant role in the machinery regulating cell proliferation and in actin cytoskeletal reorganization, which affects cell motility and adhesion. These findings may explain, in part, the mechanism of multiple neurofibroma formation in NF1 patients.     

Decline in the HIV-1 isolation rate in Japan: a 12-year observation

Since 1988, we have isolated HIV-1 from 614 HIV-1-infected persons (total sample=2,785) in Japan. During the past 12 years, we have found a decline in the HIV-1 isolation rate in Japan, with two identifiable turning points, 1991-1992 and 1996-1997. The two turning points correspond to shifts in anti-HIV-1 therapy. These findings suggest that HIV-1 in Japan is currently biologically well controlled, probably due to anti-HIV-1 therapy. On the other hand, this decline is inconsistent with the recent increase of genetic drug-resistant HIV-1 in Japan. Further studies are needed to clarify mechanisms that might explain the discrepancy.     

Epidermal sphingomyelins are precursors for selected stratum corneum ceramides

Epidermal ceramides (Cer) comprise a heterogeneous family of seven species, including two unique omega-hydroxylated Cer, that are key components of the stratum corneum (SC) intercellular lamellar membranes responsible for the epidermal permeability barrier. Although both glucosylceramide (GlcCer) and the phospho-sphingolipid sphingomyelin (SM) are potential precursors of SC Cer, based on reported chemical structures of epidermal GlcCer and SC Cer, it is assumed that all major subfractions of SC Cer are generated from lamellar body-derived GlcCer. Yet, we and others have shown that SM-derived Cer are required for normal barrier homeostasis. Moreover, two pools of SM, one from plasma membrane, the other from lamellar body-derived contents, are potentially available for Cer production. To clarify the role of SM as a potential precursor of bulk or specific SC Cer, we compared Cer moieties in epidermal SM, Cer generated from epidermal SM by sphingomyelinase treatment, Cer within SC, and Cer that persist in Gaucher SC, where GlcCer cannot generate Cer due to an absence of beta-glucocerebrosidase. Using gas chromatography-mass spectrometry, fast atom bombardment-mass spectrometry, and nuclear magnetic resonance for Cer characterization, epidermal SM comprise three major subfractions with distinctive amide-linked (N-acyl) fatty acid (FA) compositions: that is, either long-chain FA (SM-1; C(22;-26)), short-chain FA (SM-2; primarily C(16)), and short-chain alpha-hydroxy FA (SM-3; C(16;-18)). In contrast, only trace quantities of omega-hydroxy FA were present. For each SM subfraction, the sphingoid base was either sphingosine or sphinganine, but phytosphingosine was not detected. Comparison of these SM with corresponding sphingomyelinase-generated epidermal Cer and SC Cer revealed that the Cer moieties of SM-1 and SM-3 are equivalent to Cer 2 (NS) and Cer 5 (AS), respectively. Moreover, both Cer 2 and Cer 5 occurred in Gaucher SC, whereas other Cer subfractions did not occur.These results indicate that two epidermal SM, that is, SM-1 and SM-3, are important precursors of two corresponding Cer in mammalian SC, that is, Cer 2 and Cer 5, but other Cer species, including the omega-hydroxy Cer species, do not derive from SM.     

14-3-3 protein family members have a regulatory role in retinoic acid-mediated induction of cytokeratins in F9 cells

We have found that the expression of five 14-3-3 protein isoforms is induced during the retinoic acid (RA)-mediated differentiation of mouse embryonal carcinoma F9 cells. The induced expression of the 14-3-3 proteins is presumed to have a role in enhancing the mitogen-activated protein kinase (MAPK) activity during RA-mediated F9 cell differentiation, because using genetically engineered budding yeast we showed that these isoforms enhanced the signaling in the MAPK cascade mainly through the interaction with Raf-1. Then we assessed the role of increased MAPK activity in F9 cell differentiation by interfering with signaling in the MAPK cascade in F9 cells. The exogenous expression of dominant-negative MEK1 efficiently abrogated RA-mediated induction of the cytokeratins EndoA and EndoC in the F9 cells. These results suggest that the 14-3-3 proteins play a role in the efficient induction of the cytokeratins during F9 cell differentiation through their signal enhancing activity in the MAPK cascade.     

Inhibition of influenza virus replication in cultured cells by RNA-cleaving DNA enzyme

Influenza virus replication has been effectively inhibited by antisense phosphothioate oligonucleotides targeting the AUG initiation codon of PB2 mRNA. We designed RNA-cleaving DNA enzymes from 10-23 catalytic motif to target PB2-AUG initiation codon and measured their RNA-cleaving activity in vitro. Although the RNA-cleaving activity was not optimal under physiological conditions, DNA enzymes inhibited viral replication in cultured cells more effectively than antisense phosphothioate oligonucleotides. Our data indicated that DNA enzymes could be useful for the control of viral infection.     

D-Serine inhibits serine palmitoyltransferase, the enzyme catalyzing the initial step of sphingolipid biosynthesis

Serine palmitoyltransferase (SPT), responsible for the initial step of sphingolipid biosynthesis, catalyzes condensation of palmitoyl coenzyme A and L-serine to produce 3-ketodihydrosphingosine (KDS). For determination of the stereochemical specificity of the amino acid substrate, a competition analysis of the production of [(3)H]KDS from L-[(3)H]serine was performed using purified SPT. D-Serine inhibited [(3)H]KDS production as effectively as non-radioactive L-serine, whereas neither D-alanine nor D-threonine showed any significant effect. Incubation of purified SPT with [palmitoyl 1-(14)C]palmitoyl coenzyme A and D-serine did not produce [(14)C]KDS, while the control incubation with L-serine did. These results suggest that D-serine competes with L-serine for the amino acid recognition site of SPT, but that D-serine is not utilized by this enzyme to produce KDS.     

Zyxin, a regulator of actin filament assembly, targets the mitotic apparatus by interacting with h-warts/LATS1 tumor suppressor

The mitotic apparatus plays a pivotal role in dividing cells to ensure each daughter cell receives a full set of chromosomes and complement of cytoplasm during mitosis. A human homologue of the Drosophila warts tumor suppressor, h-warts/LATS1, is an evolutionarily conserved serine/threonine kinase and a dynamic component of the mitotic apparatus. We have identified an interaction of h-warts/LATS1 with zyxin, a regulator of actin filament assembly. Zyxin is a component of focal adhesion, however, during mitosis a fraction of cytoplasmic-dispersed zyxin becomes associated with h-warts/LATS1 on the mitotic apparatus. We found that zyxin is phosphorylated specifically during mitosis, most likely by Cdc2 kinase, and that the phosphorylation regulates association with h-warts/LATS1. Furthermore, microinjection of truncated h-warts/LATS1 protein, including the zyxin-binding portion, interfered with localization of zyxin to mitotic apparatus, and the duration of mitosis of these injected cells was significantly longer than that of control cells. These findings suggest that h-warts/LATS1 and zyxin play a crucial role in controlling mitosis progression by forming a regulatory complex on mitotic apparatus.     

Analysis of capsid formation of human polyomavirus JC (Tokyo-1 strain) by a eukaryotic expression system: splicing of late RNAs, translation and nuclear transport of major capsid protein VP1, and capsid assembly

Human polyomavirus JC (JCV) can encode the three capsid proteins VP1, VP2, and VP3, downstream of the agnoprotein in the late region. JCV virions are identified in the nucleus of infected cells. In this study, we have elucidated unique features of JCV capsid formation by using a eukaryotic expression system. Structures of JCV polycistronic late RNAs (M1 to M4 and possibly M5 and M6) generated by alternative splicing were determined. VP1 would be synthesized from M2 RNA, and VP2 and VP3 would be synthesized from M1 RNA. The presence of the open reading frame of the agnoprotein or the leader sequence (nucleotides 275 to 409) can decrease the expression level of VP1. VP1 was efficiently transported to the nucleus in the presence of VP2 and VP3 but distributed both in the cytoplasm and in the nucleus in their absence. Mutation analysis indicated that inefficiency in nuclear transport of VP1 is due to the unique structure in the N-terminal sequence, KRKGERK. Within the nucleus, VP1 was localized discretely and identified as speckles in the presence of VP2 and VP3 but distributed diffusely in their absence. These results suggest that VP1 was efficiently transported to the nucleus and localized in the discrete subnuclear regions, possibly with VP2 and VP3. By electron microscopy, recombinant virus particles were identified in the nucleus, and their intranuclear distribution was consistent with distribution of speckles. This system provides a useful model with which to understand JCV capsid formation and the structures and functions of the JCV capsid proteins.     

Dipole estimation of alpha EEG during alcohol ingestion in males genotypes for ALDH2

Using a dipole tracing method based on the two-dipole model, the purpose of the present study was to investigate alcohol-induced changes in the alpha band of electroencephalogram (EEG) and its equivalent current dipoles (ECDs) in 12 healthy male volunteers, who were genetically typed for mitochondrial aldehyde dehydrogenase-2 (ALDH2). The alpha power and the mean interval dipolarity, which represents the goodness of fit of alpha EEG with the two-dipole model, increased at 30 min after 0.75 ml/kg of alcohol ingestion, when breath alcohol concentration showed its peak. However, the location of ECDs and distribution of alpha EEG did not change after alcohol ingestion. These findings indicate that alcohol enhances alpha EEG but does not change the location of its electrical sources. Interestingly, the time course of alcohol-induced EEG changes differed significantly according to the aversive flushing reaction after its intake. From 60 to 120 min, the non-flushing group which had homozygous ALDH2* 1 (active type) displayed significant increase not only in the alpha power but also in the interval dipolarity compared to the baseline, whereas the flushing group with heterozygous ALDH2*1/2*2 (inactive type) did not exhibit this significant increase. The difference in the time course was discussed from the viewpoint of the protective effect of ALDH2*2 allele against the risk for alcoholism. These results suggest that the dipole tracing method could provide an alternative neurophysiological marker for the risk for alcoholism.