Disruption of the murine intestinal alkaline phosphatase gene Akp3 impairs lipid transcytosis and induces visceral fat accumulation and hepatic steatosis

Intestinal alkaline phosphatase (IAP) is involved in the process of fat absorption, a conclusion confirmed by an altered lipid transport and a faster body weight gain from 10 to 30 wk in both male and female mice with a homozygous null mutation of the IAP coding gene (Akp3(-/-) mice). This study was aimed to delineate morphologically and quantitatively the accelerated lipid absorption in male Akp3(-/-) mice. Feeding a corn oil bolus produced an earlier peak of triacylglycerol in serum (2 vs. 4 h for Akp3(-/-) and wild-type mice, respectively) and an approximately twofold increase in serum triacylglycerol concentration in Akp3(-/-) mice injected with a lipolysis inhibitor, Triton WR-1339. A corn oil load induced the threefold enlargement of the Golgi vacuoles in male wild-type mice but not in Akp3(-/-) mice, indicating that absorbed lipids rarely reached the Golgi complex and that the transcytosis of lipid droplets does not follow the normal pathway in male Akp3(-/-) mice. Force feeding an exaggerated fat intake by a 30% fat chow for 10 wk induced obesity in both male Akp3(-/-) and wild-type mice, and therefore no phenotypic difference was observed between the two. On the other hand, the forced high-fat chow induced an 18% greater body weight gain, hepatic steatosis, and visceral fat accumulation in female Akp3(-/-) mice but not in female wild-type controls. These results provide further evidence that IAP is involved in the regulation of the lipid absorption process and that its absence leads to progressive metabolic abnormalities in certain fat-forced conditions.     

Evolutionary graph theory: breaking the symmetry between interaction and replacement

We study evolutionary dynamics in a population whose structure is given by two graphs: the interaction graph determines who plays with whom in an evolutionary game; the replacement graph specifies the geometry of evolutionary competition and updating. First, we calculate the fixation probabilities of frequency dependent selection between two strategies or phenotypes. We consider three different update mechanisms: birth-death, death-birth and imitation. Then, as a particular example, we explore the evolution of cooperation. Suppose the interaction graph is a regular graph of degree h, the replacement graph is a regular graph of degree g and the overlap between the two graphs is a regular graph of degree l. We show that cooperation is favored by natural selection if b/c>hg/l. Here, b and c denote the benefit and cost of the altruistic act. This result holds for death-birth updating, weak-selection and large population size. Note that the optimum population structure for cooperators is given by maximum overlap between the interaction and the replacement graph (g=h=l), which means that the two graphs are identical. We also prove that a modified replicator equation can describe how the expected values of the frequencies of an arbitrary number of strategies change on replacement and interaction graphs: the two graphs induce a transformation of the payoff matrix.     

Exogenous expression of claudin-5 induces barrier properties in cultured rat brain capillary endothelial cells

Claudins are thought to be major components of tight junctions (TJs), and claudin-5 and -12 are localized at TJs of the blood-brain barrier (BBB). Claudin-5-deficient mice exhibit size-selective (<800 Da) opening of the BBB. The purpose of this study was to clarify the expression levels of claudin-5 and -12 in rat brain capillary endothelial cells, and to examine the ability of claudin-5 to form TJs in cultured rat brain capillary endothelial cells (TR-BBB). Expression of claudin-5 mRNA in rat brain capillary fraction was 751-fold greater than that of claudin-12. The level of claudin-5 mRNA in the rat brain capillary fraction (per total mRNA) was 35.6-fold greater than that in whole brain, while the level of claudin-12 mRNA was only 13.9% of that in whole brain, suggesting that expression of claudin-12 mRNA is not restricted to brain capillaries. Transfection of TR-BBB cells with the claudin-5 gene afforded TR-BBB/CLD5 cells, which showed no change in expression of claudin-12 or ZO-1, while the expressed claudin-5 was detected at the cell-cell boundaries. The permeability surface product of [(14)C]inulin at a TR-BBB/CLD5 cell monolayer was significantly smaller (P < 0.01) than that for the parental TR-BBB cells, and the values of the permeability coefficient (Pe) were 1.14 x 10(-3) and 11.6 x 10(-3) cm/min, respectively. These results indicate that claudin-5, but not claudin-12, is predominantly expressed in brain capillaries, and plays a key role in the appearance of barrier properties of brain capillary endothelial cells.     

Disassembly of actin filaments by botulinum C2 toxin and actin-filament-disrupting agents induces assembly of microtubules in human leukaemia cell lines

BACKGROUND INFORMATION: C(2) toxin produced by Clostridium botulinum types C and D ADP-ribosylates actin monomers and inactivates their polymerization activities. The disassembly of actin filaments by C(2) toxin induces a polarization of cultured human leukaemia cell lines. RESULTS: The polarization induced by C(2) toxin was temperature dependent and was prevented by nocodazole, a microtubule-disrupting agent, whereas it was promoted by paclitaxel, a microtubule-stabilizing agent. The fluorescence staining of polarized cells indicated an increase in microtubule assembly accompanying disassembly of actin filaments. Furthermore, several actin-filament-disrupting agents, other than C(2) toxin, also induced microtubule assembly and cell polarization, irrespective of their different mechanisms of action. The effects induced by some of the agents, which have lower binding affinities for actin, were reversible in response to the re-assembly of actin filaments. CONCLUSIONS: Thus the disassembly of actin filaments by C(2) toxin and actin-filament-disrupting agents induces assembly of microtubules followed by polarization of human leukaemia cell lines, indicating that the assembly/disassembly equilibrium of actin filaments influences the dynamics of microtubules, which control cell morphology and, in turn, diverse cellular processes.     

Comparative characterization of pulmonary surfactant aggregates and alkaline phosphatase isozymes in human lung carcinoma tissue

Alkaline phosphatase (AP) isozymes are surfactant-associated proteins (SPs). Since several different AP isozymes have been detected in the pneumocytes of lung cancer patients, we attempted to identify the relationship between pulmonary surfactant aggregate subtypes and AP isozymes. Pulmonary surfactant aggregates were isolated from carcinoma and non-carcinoma tissues of patients with non-small cell carcinoma of the lung. Upon analysis, ultraheavy, heavy, and light surfactant aggregates were detected in the non-carcinoma tissues, but no ultraheavy surfactant aggregates were found in the carcinoma tissues. Surfactant-associated protein A (SP-A) was detected as two bands (a 27-kDa band and a 54-kDa band) in the ultraheavy, heavy, and light surfactant aggregates found in the non-carcinoma tissues. Although both SP-A bands were detected in the heavy and light surfactant aggregates from adenocarcinoma tissues, the 54-kDa band was not detected in squamous cell carcinoma tissues. Liver AP (LAP) was detected in the heavy and light surfactant aggregates from both non-carcinoma and squamous carcinoma tissues, but not in heavy surfactant aggregates from adenocarcinoma tissues. A larger amount of bone type AP (BAP) was found in light surfactant aggregate fractions from squamous cell carcinomas than those from adenocarcinoma tissues or non-carcinoma tissues from patients with either type of cancer. LAP, BAP, and SP-A were identified immunohistochemically in type II pneumocytes from non-carcinoma tissues and adenocarcinoma cells, but no distinct SP-A staining was observed in squamous cell carcinoma tissues. The present study has thus revealed several differences in pulmonary surfactant aggregates and AP isozymes between adenocarcinoma tissue and squamous cell carcinoma tissue.     

Electrochemical study of the effect of nano-zinc oxide on microperoxidase and its application to more sensitive hydrogen peroxide biosensor preparation

Direct electron transfer reactions of microperoxidase were achieved with the help of semiconductive zinc oxide nanoparticles on a pyrolytic graphite electrode. The enzyme could also exhibit fine electrocatalytic activity towards the reduction of hydrogen peroxide. Thereby, a hydrogen peroxide biosensor was constructed based on the electrocatalysis of microperoxidase. Further studies revealed that after irradiating the microperoxidase/zinc oxide nanoparticles co-modified electrode with UV light for 4h, the catalytic ability of microperoxidase could be greatly promoted, which could be beneficial to developing more sensitive hydrogen peroxide biosensors. As comparison, it was found that the catalytic activity of the enzyme would be depressed if microperoxidase/agarose co-modified electrode was irradiated. We supposed it was the photovoltaic effect of the zinc oxide nanoparticles that improved the catalytic ability of microperoxidase.     

Purification, identification, characterization, and cDNA cloning of a high molecular weight extracellular superoxide dismutase of hamster that transiently increases in plasma during arousal from hibernation

We previously studied antioxidant profiles in the plasma of hibernating Syrian hamsters and found a transient increase of a superoxide radical-scavenging activity during the arousal phase. In this report, we purified and identified the high molecular weight superoxide dismutase (SOD)-like factor from the plasma of arousing hamsters. The cyanide-sensitive 240 kDa SOD-like factor showed a significant homology to mammalian extracellular SOD (EC-SOD) reported, although the molecular mass of EC-SOD was 135 kDa. The cDNA cloning revealed that the 240 kDa SOD-like factor was identical to the hamster ortholog of EC-SOD. It consisted of 245 amino acid residues including a signal sequence of 20 amino acid residues. Five cysteine residues that would participate in inner- and inter-subunit bonds were well conserved among species. Interestingly, there were four potential N-glycosylation sites in hamster EC-SOD, whereas there is only one site in other species. The amino acid sequence analysis indicated that three of the four sites were modified. These results suggest that the anomalistically high molecular weight of hamster EC-SOD is ascribed, at least in part, to the addition of extra sugar chains. Furthermore, results obtained here also propose the involvement of EC-SOD in the antioxidative defense of hibernating hamsters.     

Establishment and characterization of HEPFT, a cell line derived from hepatoid carcinoma of the fallopian tube, with special reference to α-fetoprotein, lectin affinity and histogenesis

A cell line designated "HEPFT" was established from a human fallopian tubal hepatoid carcinoma. This line grew well without interruption for 13 months and was subcultivated over 35 times. The cells were spherical and polygonal in shape and showed neoplastic and pleomorphic features such as a bizarre aggregation of chromatin granules, an irregular thickening membrane and multiple large nucleoli. The cells formed epithelial colonies with a jigsaw puzzle-like arrangement and multilayering without contact inhibition. The cells contained moderate to abundant amounts of eosinophilic cytoplasm and were immunohistochemically positive for alpha-fetoprotein. The cells proliferated rapidly, and the population doubling time was about 45 h. The chromosome number showed a wide distribution of aneuploidy. The modal chromosome number was stable in the hyper triploid range and many marker chromosomes were observed. The culture cells produced bile and a large amount of lentil lectin-reactive alpha-fetoprotein. The recently developed bacterial artificial chromosome array comparative genomic hybridization facilitated detailed analysis with high resolution and sensitivity. Different profiles of genomic copy-number abnormalities were demonstrated in various chromosomal regions in HEPFT cells.     

Major involvement of Na+‐dependent multivitamin transporter (SLC5A6/SMVT) in uptake of biotin and pantothenic acid by human brain capillary endothelial cells

The purpose of this study was to clarify the expression of Na⁺‐dependent multivitamin transporter (SLC5A6/SMVT) and its contribution to the supply of biotin and pantothenic acid to the human brain via the blood–brain barrier. DNA microarray and immunohistochemical analyses confirmed that SLC5A6 is expressed in microvessels of human brain. The absolute expression levels of SLC5A6 protein in isolated human and monkey brain microvessels were 1.19 and 0.597 fmol/μg protein, respectively, as determined by a quantitative targeted absolute proteomics technique. Using an antibody‐free method established by Kubo et al. (2015), we found that SLC5A6 was preferentially localized at the luminal membrane of brain capillary endothelium. Knock‐down analysis using SLC5A6 siRNA showed that SLC5A6 accounts for 88.7% and 98.6% of total [³H]biotin and [³H]pantothenic acid uptakes, respectively, by human cerebral microvascular endothelial cell line hCMEC/D3. SLC5A6‐mediated transport in hCMEC/D3 was markedly inhibited not only by biotin and pantothenic acid, but also by prostaglandin E2, lipoic acid, docosahexaenoic acid, indomethacin, ketoprofen, diclofenac, ibuprofen, phenylbutazone, and flurbiprofen. This study is the first to confirm expression of SLC5A6 in human brain microvessels and to provide evidence that SLC5A6 is a major contributor to luminal uptake of biotin and pantothenic acid at the human blood–brain barrier.