The squamous cell carcinoma cell line OM-1 retains both p75-dependent stratified epithelial progenitor potential and cancer stem cell properties

The low-affinity nerve growth factor receptor p75 is a stratified epithelial stem/progenitor marker of human epithelia. We found OM-1, a human squamous cell carcinoma (SCC) cell line, showed distinct cells with p75 cluster, especially located at the center of a growing colony in a monolayer culture. A cell with p75 cluster was surrounded by cytokeratin 14- and cytokeratin 13-expressing cells that settled at the outer margin of the colony. OM-1 cells were also capable of forming tumor spheres in a cell suspension culture, an ability which was attenuated by the inhibition of p75-signaling. Intriguingly, we also found a p75-negative cell population from a growing culture of OM-1 that re-committed to become p75-clustering cells. These results indicated the possibility that SCC with epithelial multi-layering capacity can exploit the p75-dependent stratified epithelial progenitor property for the cancer stemness.     

Biomass density of wild and domestic herbivores and carrying capacity on a working ranch in Laikipia District,Kenya

This study of biomass densities of wild animals and domestic stock was carried out on the Lolldaiga Hills ranch in the Laikipia District, Kenya, between 1989 and 1995, as part of the study of the ecology of leopards and their impact on the working ranch. The ranch is set amongst the Lolldaiga Mountains, north-west of Mount Kenya, and lies in ecological Zone IV. The vegetation in the area was classified as a scattered tree grassland. The weighted mean annual rainfall was 554 mm. The ranch carried 1543 kg km^?2 of wild animals and 6512 kg km^?2 of cattle and sheep. The ranch has produced beef, milk and wool since its foundation over 70 years ago, with a steadily maintained output and without any apparent degradation of the land. The milk production of normally over 450,000 kg per year was reduced, or ceased, in periods of drought and may represent a useful ‘safety valve’. The feasibility of multiple land use and conservation of wildlife are discussed.     

Herpes simplex virus type 2 US3 blocks apoptosis induced by sorbitol treatment

Previously, we established HEp2 cell lines which express the US3 protein kinase of herpes simplex virus type 2 upon induction with IPTG. Using these cells, we examined whether expression of US3 is sufficient to protect cells from apoptotic cell death induced by sorbitol. Cells expressing US3 showed significantly reduced nuclear fragmentation in the degree that DNA fragmentation and caspase-3 activation were suppressed. It is known that stressors such as osmotic shock and UV irradiation induce the activation of the JNK (c-Jun N-terminal kinase), which can lead to apoptotic cell death. Expression of US3 resulted in the suppression of sorbitol-induced phosphorylation of JNK and MKK4/SEK1, suggesting that the suppression of apoptotic cell death was due to the attenuation of JNK activity.     

Selective reaction to a flipping cytidine of the duplex DNA mediated by triple helix formation

A new nucleoside derivative (2) with a butyl spacer between the sugar part and the 2-amino-6-vinylpurine motif has been synthesized. The triplex-forming oligodeoxynucleotide incorporating 2 has achieved strand- and cytidine-selective cross-linking reaction to the G-C target site mediated by triple helix formation. It has been suggested that 2 reacts with a flipping cytidine at the target site.     

Robo4 Plays a Role in Bone Marrow Homing and Mobilization,but Is Not Essential in the Long-Term Repopulating Capacity of Hematopoietic Stem Cells

Roundabout (Robo) family proteins are immunoglobulin-type surface receptors critical for cellular migration and pathway finding of neuronal axons. We have previously shown that Robo4 was specifically expressed in hematopoietic stem and progenitor cells and its high expression correlated with long-term repopulating (LTR) capacity. To reveal the physiological role of Robo4 in hematopoiesis, we examined the effects of Robo4 disruption on the function of hematopoietic stem cells (HSCs) and progenitors. In Robo4-deficient mice, basic hematological parameters including complete blood cell count and differentiation profile were not affected. In contrast to the previous report, HSC/hematopoietic progenitor (HPC) frequencies in the bone marrow (BM) were perfectly normal in Robo4^−/− mice. Moreover, Robo4^−/− HSCs were equally competitive as wild-type HSCs in transplantation assays and had normal long-term repopulating (LTR) capacity. Of note, the initial engraftment at 4-weeks after transplantation was slightly impaired by Robo4 ablation, suggesting a marginal defect in BM homing of Robo4^−/− HSCs. In fact, homing efficiencies of HSCs/HPCs to the BM was significantly impaired in Robo4-deficient mice. On the other hand, granulocyte-colony stimulating factor-induced peripheral mobilization of HSCs was also impaired by Robo4 disruption. Lastly, marrow recovery from myelosuppressive stress was equally efficient in WT- and Robo4-mutant mice. These results clearly indicate that Robo4 plays a role in HSC trafficking such as BM homing and peripheral mobilization, but is not essential in the LTR and self-renewal capacity of HSCs.     

A Resealed-Cell System for Analyzing Pathogenic Intracellular Events: Perturbation of Endocytic Pathways under Diabetic Conditions

Cell-based assay systems that can serve as cellular models of aberrant function in pathogenic organs would be novel and useful tools for screening drugs and clarifying the molecular mechanisms of various diseases. We constructed model cells that replicated the conditions in diabetic hepatocytes by using the cell resealing technique, which enables the exchange of cytosol. The plasma membrane of HeLa cells was permeabilized with the streptococcal toxin streptolysin O, and cytosol that had been prepared from wild-type or db/db diabetic mice was introduced into the resulting semi-intact cells. By resealing the plasma membrane by exposure to Ca(2+), we created WT or Db model cells, in which the cytosolic conditions replicated those of healthy or diabetic liver. Interestingly, phosphorylation of p38 MAPK was promoted, whereas the level of endosomal phosphatidylinositol-3-phosphate was decreased, in Db cells. We investigated several endocytic pathways in WT and Db cells, and found that retrograde endosome-to-Golgi transport was delayed in a p38 MAPK-dependent manner in Db cells. Furthermore, the degradation pathway of the EGF receptor from endosomes to lysosomes was enhanced in Db cells, and this did not depend on the activation of p38 MAPK. The disease model cell system should become a powerful tool for the detection of aberrant processes in cells under pathogenic conditions and for therapeutic applications.     

Dual contradictory roles of cAMP signaling pathways in hydroxyl radical production in the rat striatum

Studies have suggested that cAMP signaling pathways may be associated with the production of reactive oxygen species. In this study, we examined how modifications in cAMP signaling affected the production of hydroxyl radicals in rat striatum using microdialysis to measure extracellular 2,3-dihydroxybenzoic acid (2,3-DHBA), which is a hydroxyl radical adduct of salicylate. Up to 50 nmol of the cell-permeative cAMP mimetic 8-bromo-cAMP (8-Br-cAMP) increased 2,3-DHBA in a dose-dependent manner (there was no additional increase in 2,3-DHBA at 100 nmol). Another cAMP mimetic, dibutyryl cAMP (db-cAMP), caused a nonsignificant increase in 2,3-DHBA at 50 nmol and a significant decrease at 100 nmol. Up to 20 nmol of forskolin, which is a direct activator of adenylyl cyclase, increased 2,3-DHBA, similar to the effect of 8-Br-cAMP; however, forskolin resulted in a much greater increase in 2,3-DHBA. A potent inhibitor of protein kinase A (PKA), H89 (500 μM), potentiated the 8-Br-cAMP- and forskolin-induced increases in 2,3-DHBA and antagonized the inhibitory effect of 100 nmol of db-cAMP. Interestingly, the administration of 100 nmol of 8-bromo-cGMP alone or in combination with H89 had no significant effect on 2,3-DHBA levels. Doses of 100 nmol of a preferential PKA activator (6-phenyl-cAMP) or a preferential PKA inhibitor (8-bromoadenosine-3',5'-cyclic monophosphorothionate, Rp-isomer; Rp-8-Br-cAMPS), which also inhibits the cAMP-mediated activation of Epac (the exchange protein directly activated by cAMP), suppressed or enhanced, respectively, the formation of 2,3-DHBA. Up to 100 nmol of 8-(4-chlorophenylthio)-2'-O-methyladenosine-cAMP, which is a selective activator of Epac, dose-dependently stimulated the formation of 2,3-DHBA. These findings suggest that cAMP signaling plays contradictory roles (stimulation and inhibition) in the production of hydroxyl radicals in rat striatum by differential actions of Epac and PKA. These roles might contribute to the production of hydroxyl radicals concomitant with cAMP in carbon monoxide poisoning, because the formation of 2,3-DHBA was potentiated by the PKA inhibitor H89 and suppressed by Rp-8-Br-cAMPS, which inhibits PKA and Epac.     

Specific interaction of the envelope glycoproteins E1 and E2 with liver heparan sulfate involved in the tissue tropismatic infection by hepatitis C virus

The first step in the process of infections by the hepatitis C virus (HCV) is attachment to the host cell, which is assumed to be mediated by interaction of the envelope glycoproteins E1 and E2 with cell surface glycosaminoglycans. In this study, a variety of glycosaminoglycans, heparan sulfate (HS) from various bovine tissues as well as chondroitin sulfate (CS)/dermatan sulfate from bovine liver, were used to examine the direct interaction with recombinant E1 and E2 proteins. Intriguingly, among HS preparations from various bovine tissues, only liver HS strongly bound to both E1 and E2. Since HS from liver, which is the target tissue of HCV, contains highly sulfated structures compared to HS from other tissues, the present results suggest that HS-proteoglycan on the liver cell surface appears to be one of the molecules that define the liver-specific tissue tropism of HCV infection. The interaction assay with chemically modified heparin derivatives provided evidence that the binding of the viral proteins to heparin/HS is not only mediated by simple ionic interactions, but that the 6-O-sulfation and N-sulfation are important. Heparin oligosaccharides equal to or larger than 10-mer were required to inhibit the binding. Notably, a highly sulfated CS-E preparation from squid cartilage also strongly interacted with both viral proteins and inhibited the entry of pseudotype HCV into the target cells, suggesting that the highly sulfated CS-E might be useful as an anti-HCV drug.     

Two conformational states of Ras GTPase exhibit differential GTP-binding kinetics

Previous ³¹P NMR studies revealed that small GTPases H-Ras and K-Ras in complex with GTP assume two interconverting conformational states, state 1 and state 2. While state 2 corresponds to an active conformation, little is known about the function of state 1, an inactive conformation incapable of effector binding. To address the biochemical properties of state 1, we measured the ³¹P NMR spectra of five Ras family small GTPases; H-Ras, M-Ras, Rap1A, Rap2A and RalA, and find that they exhibit distinctive state 2/state 1 populations with the ratios ranging from 0.072 for M-Ras to 16 for Rap2A. Further, we show that GTPases with higher populations of state 1 exhibit higher dissociation and association rate constants for GTP. These results imply that GTP loading to the nucleotide-free small GTPases preferentially yields state 1, which is subsequently converted to state 2, rendering the GTP-bound form functional.