Anti-tumor and anti-invasive effects of diverse delta-alkyllactones: dependence on molecular side-chain length, action period and intracellular uptake

New delta-alkyllactones (DALs) with diverse side-chain lengths (184-254 Da), which are structurally different from the widespread, naturally occurring delta-lactones of higher molecular weight (348-439 Da), such as camptothecin and sultriecin, were chemically synthesized and analyzed for their carcinostatic activity. Of the DALs with 11, 12, 13, 14, or 16 carbon atoms, delta-hexadecalactone (DH16:0) was the most carcinostatic when administered to Ehrlich ascites tumor (EAT) cells at 37 degrees C for 20 h, and measured by the mitochondrial dehydrogenase-based WST-1 assay. Prolongation of the administration period to 72 h enhanced the carcinostatic activity more markedly for DH16:0 than for other DALs. The carcinostatic activity of DALs was unexpectedly augmented by increasing the number of carbon atoms, in contrast to the conventional view that carcinostatic activity is attenuated by the addition of carbon atoms to fatty acids. Intracellular accumulation of DH16:0, as analyzed by gas chromatography, was detected (1.5 Pg/cell), whereas other DALs studied were rarely found. The results indicate a close relationship between carcinostatic activity and intracellular accumulation. Invasion of human fibrosarcoma HT-1080 cells through the reconstituted basement membrane was inhibited by several DALs, even at doses as low as 5-10% of those necessary for carcinostatic activity, suggesting an invasive mechanism different from carcinostasis. The invasion-inhibitory activity was intensified by increasing the number of carbon atoms, in a manner similar to that for the carcinostatic activity. The lifespan of EAT-cell-transplanted mice was markedly prolonged with DH16:0, presumably due to excellent distribution throughout the body and tumor cells. Thus DH16:0 may be a potent anticancer agent, in term of its carcinostatic, anti-invasive, and lifespan-prolonging activities.     

The Himalayas as a directional barrier to gene flow

High-resolution Y-chromosome haplogroup analyses coupled with Y-short tandem repeat (STR) haplotypes were used to (1) investigate the genetic affinities of three populations from Nepal--including Newar, Tamang, and people from cosmopolitan Kathmandu (referred to as "Kathmandu" subsequently)--as well as a collection from Tibet and (2) evaluate whether the Himalayan mountain range represents a geographic barrier for gene flow between the Tibetan plateau and the South Asian subcontinent. The results suggest that the Tibetans and Nepalese are in part descendants of Tibeto-Burman-speaking groups originating from Northeast Asia. All four populations are represented predominantly by haplogroup O3a5-M134-derived chromosomes, whose Y-STR-based age (+/-SE) was estimated at 8.1+/-2.9 thousand years ago (KYA), more recent than its Southeast Asian counterpart. The most pronounced difference between the two regions is reflected in the opposing high-frequency distributions of haplogroups D in Tibet and R in Nepal. With the exception of Tamang, both Newar and Kathmandu exhibit considerable similarities to the Indian Y-haplogroup distribution, particularly in their haplogroup R and H composition. These results indicate gene flow from the Indian subcontinent and, in the case of haplogroup R, from Eurasia as well, a conclusion that is also supported by the admixture analysis. In contrast, whereas haplogroup D is completely absent in Nepal, it accounts for 50.6% of the Tibetan Y-chromosome gene pool. Coalescent analyses suggest that the expansion of haplogroup D derivatives--namely, D1-M15 and D3-P47 in Tibet--involved two different demographic events (5.1+/-1.8 and 11.3+/-3.7 KYA, respectively) that are more recent than those of D2-M55 representatives common in Japan. Low frequencies, relative to Nepal, of haplogroup J and R lineages in Tibet are also consistent with restricted gene flow from the subcontinent. Yet the presence of haplogroup O3a5-M134 representatives in Nepal indicates that the Himalayas have been permeable to dispersals from the east. These genetic patterns suggest that this cordillera has been a biased bidirectional barrier.     

Differential regulation of cholera toxin-inhibited Na-H exchange isoforms by butyrate in rat ileum

Electroneutral Na absorption occurs in the intestine via sodium-hydrogen exchanger (NHE) isoforms NHE2 and NHE3. Bicarbonate and butyrate both stimulate electroneutral Na absorption through NHE. Bicarbonate- but not butyrate-dependent Na absorption is inhibited by cholera toxin (CT). Long-term exposure to butyrate also influences expression of apical membrane proteins in epithelial cells. These studies investigated the effects of short- and long-term in vivo exposure to butyrate on apical membrane NHE and mRNA, protein expression, and activity in rat ileal epithelium that had been exposed to CT. Ileal loops were exposed to CT in vivo for 5 h and apical membrane vesicles were isolated. 22Na uptake was measured by using the inhibitor HOE694 to identify NHE2 and NHE3 activity, and Western blot analyses were performed. CT reduced total NHE activity by 70% in apical membrane vesicles with inhibition of both NHE2 and NHE3. Reduced NHE3 activity and protein expression remained low following removal of CT but increased to control values following incubation of the ileal loop with butyrate for 2 h. In parallel there was a 40% decrease in CT-induced increase in cAMP content. In contrast, NHE2 activity partially increased following removal of CT and was further increased to control levels by butyrate. NHE2 protein expression did not parallel its activity. Neither NHE2 nor NHE3 mRNA content were affected by CT or butyrate. These results indicate that CT has varying effects on the two apical NHE isoforms, inhibiting NHE2 activity without altering its protein expression and reducing both NHE3 activity and protein expression. Butyrate restores both CT-inhibited NHE2 and NHE3 activities to normal levels but via different mechanisms.     

Noradrenergic afferents and receptors in the medial preoptic area: neuroanatomical and neurochemical links between the regulation of sleep and body temperature

Several studies have shown the importance of the medial preoptic area in the regulation of sleep-wakefulness and of body temperature. The medial preoptic area has a rich noradrenergic innervation, coming mostly from the lateral tegmental noradrenergic system. The accumulating evidences show that the noradrenergic afferents to the medial preoptic area are involved in the induction of sleep. This hypnogenic mechanism operates through the postsynaptic alpha1 and alpha2-adrenergic receptors. Noradrenergic afferents are also involved in the thermoregulatory mechanisms, and the activation of these fibers brings about a fall in body temperature. Though the body temperature changes are brought about by the same receptor subtypes as those involved in hypnogenesis, observations suggest the possibility of separate sets of noradrenergic afferents in the medial preoptic area for sleep regulation and thermoregulation. In this review, we present the compelling evidences, which showed that the noradrenergic afferents of the medial preoptic area bring about a fall in body temperature and other thermoregulatory behavioral alterations associated with sleep.     

Structure and function of extracellular phospholipase A1 belonging to the pancreatic lipase gene family

Phospholipase A1 (PLA1) is an enzyme that hydrolyzes phospholipids and produces 2-acyl-lysophospholipids and fatty acids and is conserved in a wide range of organisms. Mammals have several enzymes that exhibit PLA1 activity in vitro. The extracellular PLA1s include phosphatidylserine (PS)-specific PLA1 (PS-PLA1), membrane-associated phosphatidic acid (PA)-selective PLA1s (mPA-PLA1alpha and mPA-PLA1beta), hepatic lipase (HL), endothelial lipase (EL) and pancreatic lipase-related protein 2 (PLRP2), all of which belong to the pancreatic lipase gene family. The former three PLA1s differ from other members in their substrate specificities, structural features and gene organizations, and form a subfamily in the pancreatic lipase gene family. PS-PLA1, mPA-PLA1alpha and mPA-PLA1beta exhibit only PLA1 activity, while HL, EL and PLRP2 show triacylglycerol-hydrolyzing activity in addition to PLA1 activity. The tertiary structures of lipases have two surface loops, the lid and the beta9 loop. The lid and the beta9 loop cover the active site in its closed conformation. An alignment of amino acid sequences of the pancreatic lipase gene family members revealed two molecular characteristics of PLA1s in the two surface loops. First, lipase members exhibiting PLA1 activity (PS-PLA1, mPA-PLA1alpha and mPA-PLA1beta, EL, guinea pig PLRP2 and PLA1 from hornet venom (DolmI)) have short lids. Second, PS-PLA1, mPA-PLA1alpha, mPA-PLA1beta and DolmI, which exhibit only PLA(1) activity, have short beta9 loops. Thus, the two surface loops appear to be involved in the ligand recognition. PS-PLA1 and mPA-PLA1s specifically hydrolyze PS and PA, respectively, producing their corresponding lysophospholipids. Lysophosphatidylserine and lysophosphatidic acid have been defined as lipid mediators with multiple biological functions. Thus, these PLA1s have a role in the production of these lysophospholipid mediators.     

A computational docking study for prediction of binding mode of diospyrin and derivatives: Inhibitors of human and leishmanial DNA topoisomerase-I

A computational approach was utilized to study the relative binding modes of diospyrin (bisnaphthoquinonoid) with the crystal structure of human DNA-TopoI and the recently reported Leishmania donavani DNA-TopoI. Additionally, the binding site interactions of amino derivatives of diospyrin with human TopoI were studied extensively. Based on the docking results, binding modes of diospyrin with the human and leishmanial TopoI catalytic core were predicted. The parallel use of two efficient and predictive docking programs, GOLD and Ligandfit, allowed mutual validation of the predicted binding poses. A reasonably good correlation coefficient between the calculated docking scores and the experimentally determined cytotoxicity helped in validating the docking method. Furthermore, a structure-based pharmacophore model was developed for L. donavani DNA-TopoI inhibition which helped in elucidating the topological and spatial requirements of the ligand-receptor interactions. This study provides an understanding of the structural basis of ligand binding to the topoisomerase receptor, which may be used for the structure-based design of potent and novel ligands for anticancer and antileishmanial therapy. To our knowledge, this is the first report of a binding mode exploration study for diospyrin and its derivatives as inhibitors of the leishmanial and human TopoI enzymes.     

Mode of infection of Hokkaido virus (Genus Hantavirus) among grey red-backed voles, Myodes rufocanus, in Hokkaido, Japan

Hokkaido virus (HOKV) is a member of the genus Hantavirus, in the family Bunyaviridae. To investigate HOKV infection in the host Myodes rufocanus, the grey red-backed vole, 199 animals were captured at Tobetsu (October 2004 and July 2005) and Nakagawa (October 2004) in Hokkaido, Japan, for detection of antibody, antigen, and viral RNA. In the surveys in Tobetsu (2004) and Nakagawa (2004), seropositive animals were detected at a frequency of 6.0% (5/84) and 10.4% (5/48), respectively. No seropositive animals were detected in Tobetsu in 2005. Seroprevalence in males in Tobetsu and Nakagawa in 2004 was 25% (1/4) and 45.5% (5/11), respectively, which was higher than in females, at 5.0% (4/80) and 0% (0/37), respectively (P<0.01). These results suggest that male animals play an important role in the maintenance of HOKV in M. rufocanus. Two females were seronegative but viral RNA-positive, indicating that these animals had acute infections before antibody was produced. Another five infected animals in Nakagawa were all male and had high levels of antibodies and viral RNA, suggesting that they had persistent infections. Viral RNA copies in organs of infected animals in Nakagawa were quantified by real-time polymerase chain reaction. Two acutely infected animals had > or = 10 times the number of RNA copies in their lungs compared to those of persistently infected animals. In most cases, lungs or spleen had the highest RNA copy number, regardless of infection status.     

Evidence for cancer-associated expression of NADPH oxidase 1 (Nox1)-based oxidase system in the human stomach

Helicobacter pylori infection has been suggested to stimulate expression of the NADPH oxidase 1 (Nox1)-based oxidase system in guinea pig gastric epithelium, whereas Nox1 mRNA expression has not yet been documented in the human stomach. PCR of human stomach cDNA libraries showed that Nox1 and Nox organizer 1 (NOXO1) messages were absent from normal stomachs, while they were specifically coexpressed in intestinal- and diffuse-type adenocarcinomas including signet-ring cell carcinoma. Immunohistochemistry showed that Nox1 and NOXO1 proteins were absent from chronic atrophic gastritis (15 cases), adenomas (4 cases), or surrounding tissues of adenocarcinomas (45 cases). In contrast, Nox1 and its partner proteins were expressed in intestinal-type adenocarcinomas (19/21 cases), diffuse-type adenocarcinomas (15/15 cases), and signet-ring cell carcinomas (9/9 cases). Confocal microscopy revealed that Nox1, NOXO1, Nox activator 1, and p22^phox were predominantly associated with Golgi apparatus in these cancer cells, while diffuse-type adenocarcinomas also contained cancer cells having Nox1 and its partner proteins in their nuclei. Nox1-expressing cancer cells exhibited both gastric and intestinal phenotypes, as assessed by expression of mucin core polypeptides. Thus, the Nox1-base oxidase may be a potential marker of neoplastic transformation and play an important role in oxygen radical- and inflammation-dependent carcinogenesis in the human stomach.     

Effects of dipeptides having a C-terminal lysine on the cholesterol 7alpha-hydroxylase mRNA level in HepG2 cells

Inducing expression of the cholesterol-catabolizing enzyme cholesterol 7alpha-hydroxylase (CYP7A1) in the liver can be an effective strategy in preventing hypercholesterolemia and atherosclerosis. We used HepG2 cells to investigate the effects of 1 mM dipeptides having a C-terminal lysine group on the CYP7A1 mRNA level. We found that the dipeptides Asp-Lys, Glu-Lys, and Trp-Lys significantly increased the CYP7A1 mRNA level.     

Relevance of anti-reactive oxygen species activity to anti-inflammatory activity of components of Eviprostat, a phytotherapeutic agent for benign prostatic hyperplasia

Inflammation is a common finding in benign prostatic hyperplasia (BPH). The phytotherapeutic agent eviprostat is a popular treatment for BPH in Japan and Germany. This agent consists of five components; four are extracted from Chimaphila umbellata, Populus tremula, Pulsatilla pratensis and Equisetum arvense (coded as EVI-1, EVI-2, EVI-3 and EVI-4, respectively) and the fifth is germ oil from Triticum aestivum (coded as EVI-5). In this study, the effects of each component on the reactive oxygen species (ROS), superoxide anion (O2-) and hydroxyl radical (OH*) generated in cell-free systems and human neutrophils, and on carrageenin-induced paw edema in rats were investigated. EVI-1, EVI-2 and EVI-4 suppressed the O2- levels in the xanthine/xanthine oxidase system, and EVI-1, EVI-2, EVI-3 and EVI-4 abolished the OH* produced in a Fenton-type reaction system, so that EVI-1, EVI-2 and EVI-4 possessed inhibitory action with respect to both O2- and OH*. EVI-1, EVI-2 and EVI-4 also reduced ROS levels in phorbol myristate acetate-stimulated neutrophils. The paw swelling was inhibited by a mixture of EVI-1, EVI-2, EVI-3, EVI-4 and EVI-5 (a mixture which is equivalent to eviprostat) or by a mixture of EVI-1, EVI-2 and EVI-4, even though each component alone did not significantly inhibit the swelling. These findings suggest that the suppression of ROS by EVI-1, EVI-2 and EVI-4 may partly contribute to the anti-inflammatory action of eviprostat, and this action may be implicated in its therapeutic effect on BPH.