Analysis of integrated human papillomavirus type 16 DNA in cervical cancers: amplification of viral sequences together with cellular flanking sequences.

We have isolated four clones of integrated human papillomavirus type 16 (HPV-16) DNA from four different primary cervical cancer specimens. All clones were found to be monomeric or dimeric forms of HPV-16 DNA with cellular flanking sequences at both ends. Analysis of the viral sequences in these clones showed that E6/E7 open reading frames and the long control region were conserved and that no region specific for the integration was detected. Analysis of the cellular flanking sequences revealed no significant homology with any known human DNA sequences, except Alu sequences, and no homology among the clones, indicating no cellular sequence specific for the integration. By probing with single-copy cellular flanking sequences from the clones, it was demonstrated that the integrated HPV-16 DNAs, with different sizes in the same specimens, shared the same cellular flanking sequences at the ends. Furthermore, it was shown that the viral sequences together with cellular flanking sequences were amplified. The possible process of viral integration into cell chromosomes in cervical cancer is discussed.     

Effect of onion (Allium cepa) root exudates on the hyphal growth of Gigaspora margarita

 The effect of root exudates from onions differing in P status on spore germination and hyphal growth of arbuscular mycorrhizal fungi was investigated. Onion (Allium cepa) was grown in solution culture at different phosphorus concentrations (0, 0.1, 1.0, 8.0 and 24.0 mg P l^–1) and root exudates were collected. When spores of the arbuscular mycorrhizal fungus, Gigaspora margarita were incubated with these root exudates, spore germination was only slightly affected but hyphal growth was greatly affected, particularly with exudates from P-deficient plants. This suggests that the P nutrition of host plants influences the composition of root exudates and thereby the hyphal growth of arbuscular mycorrhizal fungi. Accepted: 25 June 1995     

Endogenous expression of angiogenesis-related factors in response to muscle injury

Gene therapy has developed a new strategy to treat a variety of ischemic diseases using angiogenic growth factors. However, the endogenous expression pattern of angiogenesis-related factors in response to muscle injury is not fully characterized. In the present study, we investigated the expression of angiogenesis-related factors, vascular endothelial growth factor, angiopoietin-1, -2, monocyte chemoattractant protein-1, and their receptors during muscle regeneration. Mice underwent freeze injury, and then the gastrocnemius muscles were isolated 1, 3, 5, 7, 10, 14, and 28 days after surgery. Generally, changes in gene expression were most dramatic during the early stage of muscle regeneration, and were attenuated as angiogenesis progressively developed and then returned to steady-state levels. VEGF mRNA began to increase from day 3 and peaked at day 5 after muscle injury. VEGF receptors, Flt-1, KDR/Flk-1, and neuropilin-1 mRNAs were increased from 3- to 9-fold at day 3 after muscle injury. At the same time, angiopoietin-1 and angiopoietin-2 mRNA were increased by 3- and 15-fold respectively, concomitantly with an increase in their receptors and Tie-2 mRNA. Finally, MCP-1 and CC-chemokine receptor 2 mRNAs were sharply up-regulated by 1600- and 100-fold, respectively, at day 3 after muscle injury. These results suggest that the molecular events implicated in angiogenesis occur at an early stage of muscle regeneration.     

Zinc transport via ZNT5-6 and ZNT7 is critical for cell surface glycosylphosphatidylinositol-anchored protein expression

Glycosylphosphatidylinositol (GPI)-anchored proteins play crucial roles in various enzyme activities, cell signaling and adhesion, and immune responses. While the molecular mechanism underlying GPI-anchored protein biosynthesis has been well studied, the role of zinc transport in this process has not yet been elucidated. Zn transporter (ZNT) proteins mobilize cytosolic zinc to the extracellular space and to intracellular compartments. Here, we report that the early secretory pathway ZNTs (ZNT5–ZNT6 heterodimers [ZNT5-6] and ZNT7–ZNT7 homodimers [ZNT7]), which supply zinc to the lumen of the early secretory pathway compartments are essential for GPI-anchored protein expression on the cell surface. We show, using overexpression and gene disruption/re-expression strategies in cultured human cells, that loss of ZNT5-6 and ZNT7 zinc transport functions results in significant reduction in GPI-anchored protein levels similar to that in mutant cells lacking phosphatidylinositol glycan anchor biosynthesis (PIG) genes. Furthermore, medaka fish with disrupted Znt5 and Znt7 genes show touch-insensitive phenotypes similar to zebrafish Pig mutants. These findings provide a previously unappreciated insight into the regulation of GPI-anchored protein expression and protein quality control in the early secretory pathway.     

Palmitate-induced Down-regulation of Sortilin and Impaired GLUT4 Trafficking in C2C12 Myotubes

Elevated saturated FFAs including palmitate (C16:0) are a primary trigger for peripheral insulin resistance characterized by impaired glucose uptake/disposal in skeletal muscle, resulting from impaired GLUT4 translocation in response to insulin. We herein demonstrate that palmitate induces down-regulation of sortilin, a sorting receptor implicated in the formation of insulin-responsive GLUT4 vesicles, via mechanisms involving PKCθ and TNF-α-converting enzyme, but not p38, JNK, or mitochondrial reactive oxygen species generation, leading to impaired GLUT4 trafficking in C2C12 myotubes. Intriguingly, unsaturated FFAs such as palmitoleate (C16:1) and oleate (C18:1) had no such detrimental effects, appearing instead to effectively reverse palmitate-induced impairment of insulin-responsive GLUT4 recycling along with restoration of sortilin abundance by preventing aberrant PKCθ activation. On the other hand, shRNA-mediated reduction of sortilin in intact C2C12 myotubes inhibited insulin-induced GLUT4 recycling without dampening Akt phosphorylation. We found that the peroxisome proliferator-activated receptor γ agonist troglitazone prevented the palmitate-induced sortilin reduction and also ameliorated insulin-responsive GLUT4 recycling without altering the palmitate-evoked insults on signaling cascades; neither highly phosphorylated PKCθ states nor impaired insulin-responsive Akt phosphorylation was affected. Taken together, our data provide novel insights into the pathogenesis of PKCθ-dependent insulin resistance with respect to insulin-responsive GLUT4 translocation, which could occur not only through defects of insulin signaling but also via a reduction of sortilin, which directly controls trafficking/sorting of GLUT4 in skeletal muscle cells. In addition, our data suggest the insulin-sensitizing action of peroxisome proliferator-activated receptor γ agonists to be at least partially mediated through the restoration of proper GLUT4 trafficking/sorting events governed by sortilin.     

Simple micropatterning method for enhancing fusion efficiency and responsiveness to electrical stimulation of C2C12 myotubes

Cultured myotubes induced in vitro from myoblast cell lines have been widely used to investigate muscle functional properties and disease‐related biological phenotypes. Until now, several cell patterning techniques have been applied to regulate in vitro myotube structures. However, these previous studies required specific geometry patterns or soft materials for inducing efficient myotube formation. Thus, more simple and easy handling method will be promising. In this study, we aimed to provide a method to form C2C12 myotubes with regulated sizes and orientations in simple line patterns. We used a poly(dimethylsiloxane) (PDMS) stamp and a 2‐methacryloyloxyethyl phosphorylcholine (MPC) polymer solution to fabricate line patterns for myotube formation onto a culture dish. We confirmed that C2C12 myotubes of well‐defined size and orientation were reproducibly formed. In particular, myotubes formed in the micropatterned lines showed the increased fusion efficiency. Then, functional dynamics in the micropatterned myotubes were detected and analyzed using a calcium imaging method. We confirmed micropatterning in line patterns enhanced the responsiveness of myotubes to external electrical stimulations. These results indicate that micropatterning myoblasts with the MPC polymer is a simple and effective method to form functional myotube networks. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:220–225, 2015     

Prenatal diagnosis of Hurler's syndrome—Biochemical studies on the affected fetus

Summary A prenatal diagnosis of Hurler's syndrome was made in a pregnancy at risk in a family with two affected children. The fetus was diagnosed as having Hurler's syndrome on the basis of a deficiency of -L-iduronidase in the cultured amniotic cells. The glycosaminoglycans (GAG) content in the supernatant of the amniotic fluid was increased about 1.5 fold compared with that in the control, and increases of heparan sulfate and dermatan sulfate were observed on electrophoresis.The diagnosis could be confirmed by the deficiencies of -L-iduronidase in the liver and brain from the affected fetus. GAG content in the liver from the affected fetus was increased approximately 10 fold as compared with that in the control fetal liver, and most of the GAG were degraded. The GAG content was observed to be increased two fold in the brain, and dermatan sulfate, which was not detected in normal fetal brain, was identified. -galactosidase activities in the affected liver and brain were decreased to 30tt50% of the control, and an altered hexosaminidase A was also observed in the liver.     

Phthalocyanine-dyed fibers adsorb allergenic proteins

Summary. Phthalocyanine (Pc)-dyed fiber is reported to reduce atopic symptoms in some patients when they use underwear made of the fiber. We investigated the adsorption of allergens on Pc-fiber. Pc-fiber trapped house dust/pollen/food allergens with varied molecular weight and pI. The adsorbed allergens were released in the presence of mild detergent. Pc-fiber did not change the molecular weight or disulfide bonding of the allergens. These observations imply that Pc-fiber is applicable as an “allergen trap” for a wide variety of products.     

Stem diameter changes before bud opening in <Emphasis Type="Italic">Zelkova serrata</Emphasis> saplings

It is well known that stems of woody plants shrink and swell diurnally. These fluctuations of stem diameter are induced mainly by the changes of water contents in plants, which are caused by the combination of leaf transpiration and root absorption of water. This implies that dormant-like deciduous broadleaved trees in a leafless state should show no or less changes in stem diameter. However, some physiological activities in woody plants are also known to precede their winter bud opening. Whether and how diameter changes occur in deciduous tree stems during winter was investigated using Zelkova serrata saplings in a leafless state. Measurements of stem diameter changes were done for more than 4 months continuously. The saplings showed distinct diameter changes with periodicities from diurnal to a few weeks, and these changes were initiated 2 months before winter bud opening. These results indicate that some physiological and/or developmental activities occur in the stem of deciduous trees before winter bud opening, and do not correspond to changes in water relations as a result of leaf transpiration. These internal activities cause fluctuations in stem diameter prior to winter bud opening in deciduous trees. Electronic Publication     

The early snail acquires the learning. Comparison of scores for conditioned taste aversion between morning and afternoon

The pond snail Lymnaea stagnalis acquires conditioned taste aversion (CTA) and maintains its memory for more than a month. Snails in our laboratory were cultured at 20 degrees C on a 12:12 light-dark cycle (light from 7 am to 7 pm). To examine the hours during which snails acquire CTA effectively, we trained some snails in the morning and others in the afternoon, and then compared their scores. CTA developed in both cases, but scores were significantly better in the morning than in the afternoon. To elucidate the cause of this difference in scores, we observed the voluntary activity of snails and found the circadian rhythm reflected in the snails' free-movement distances; distances at the circadian time 0-12 (daytime) were significantly longer than those at the circadian time 12-24 (nighttime). This rhythm was kept up for at least 3 days, even in constant darkness. In conclusion, L. stagnalis should be trained in the morning to acquire associative learning, possibly because of its greater propensity to roam about at that time as opposed to the afternoon.