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.     

Identification of three protein disulfide isomerase members from Haemaphysalis longicornis tick

Three genes encoding putative protein disulfide isomerase (PDI) were isolated from the Haemaphysalis longicornis EST database and designed as HlPDI-1, HlPDI-2, and HlPDI-3. All three PDI genes contain two typical PDI active sites CXXC and encode putative 435, 499, and 488 amino acids, respectively. The recombinant proteins expressed in Escherichia coli all show PDI activities, and the activities were inhibited by a PDI-specific inhibitor, zinc bacitracin. Western blot analysis and real-time PCR revealed that three HlPDIs were present in all the developmental stages of the tick as well as in the midgut, salivary glands, ovary, hemolymph, and fatbody of adult female ticks, but the three genes were expressed at the highest level in the egg stage. HlPDI-1 is expressed primarily in the ovary and secondarily in the salivary glands. HlPDI-2 and HlPDI-3 are expressed primarily in the salivary gland, suggesting that the PDI genes are important for tick biology, especially for egg development, and that they play distinct roles in different tissues. Blood feeding induced significantly increased expression of HlPDI-1 and HlPDI-3 in both partially fed nymphs and adults. Babesia gibsoni-infected larval ticks expressed HlPDI-1 and HlPDI-3 2.0 and 4.0 times higher than uninfected normal larval ticks, respectively. The results indicate that HlPDI-1 and HlPDI-3 might be involved in tick blood feeding and Babesia parasite infection in ticks.     

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.     

Influence of avilamycin administration and its subsequent withdrawal on emergence and disappearance of antimicrobial resistance in enterococci in the intestine of broiler chickens

AIMS: To investigate the influence of avilamycin (AVM) administration and its subsequent withdrawal on the emergence and disappearance of AVM-resistant enterococci in the intestine of broiler chickens. METHODS AND RESULTS: Five chicks each of C, L and H groups were given the basal diet, the basal diet supplemented with 5 g AVM/ton and the basal diet supplemented with 50 g AVM/ton, respectively. The AVM-resistant Enterococcus faecalis population did not emerge during 30 days of the AVM administration period, whereas the AVM-resistant Enterococcus faecium with a minimum inhibitory concentration of >512 microg ml(-1) in the faeces of chicks of the L and H groups emerged on 3 and 1 days after the AVM administration, respectively. Thereafter, the AVM-resistant Ent. faecium population density in both L and H groups maintained high levels during the AVM administration period. Twenty days after the AVM withdrawal, the AVM-resistant Ent. faecium population disappeared from the intestines of both four of five chicks of L group and three of five chicks of H group. The AVM-resistant Ent. faecium population density in one chick from each of the groups, L and H, did not change before and after the AVM removal. CONCLUSIONS: The AVM-resistant Ent. faecium emerged during the AVM administration, and disappeared from the intestine of most chicks after the AVM withdrawal. However, the AVM-resistant Ent. faecium persisted in some chicks 20 days after AVM withdrawal. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that introducing an AVM withdrawal period could minimize the risk of AVM-resistant Ent. faecium becoming carcass contaminants, and that prudent antibiotic use alone is not sufficient to stem emergence of the AVM-resistant Ent. faecium.     

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.