Truncation of annexin A1 is a regulatory lever for linking epidermal growth factor signaling with cytosolic phospholipase A2 in normal and malignant squamous epithelial cells

Regulation of cell growth and apoptosis is one of the pleiotropic functions of annexin A1 (ANXA1). Although previous reports on the overexpression of ANXA1 in many human cancers and on growth suppression and/or induction of apoptosis by ANXA1 may indicate the tumor-suppressive nature of ANXA1, molecular mechanisms of the function of ANXA1 remain largely unknown. Here we provide evidence that ANXA1 mechanistically links the epidermal growth factor-triggered growth signal pathway with cytosolic phospholipase A(2) (cPLA(2)), an initiator enzyme of the arachidonic acid cascade, through interaction with S100A11 in normal human keratinocytes (NHK). Ca(2+)-dependent binding of S100A11 to ANXA1 facilitated the binding of the latter to cPLA(2), resulting in inhibition of cPLA(2) activity, which is essential for the growth of NHK. On exposure of NHK to epidermal growth factor, ANXA1 was cleaved solely at Trp(12), and this cleavage was executed by cathepsin D. In squamous cancer cells, this pathway was shown to be constitutively activated. The newly found mechanistic intersection may be a promising target for establishing new measures against human cancer and other cell growth disorders.     

Lower extremity muscle activity during different types and speeds of underwater movement

To compare the activity of lower extremity muscles during land walking (LW), water walking (WW), and deep-water running (DWR), 9 healthy young subjects were tested at self-selected low, moderate, and high intensities for 8 sec with two repetitions. Surface EMG electrodes were placed on the tibialis anterior (TA), soleus (SOL), medial gastrocnemius (GAS), rectus femoris (RF), and biceps femoris (BF). During DWR, the SOL and GAS activities were lower than LW and WW. The BF activities were higher during DWR than LW and WW. It was considered that the lower activity of SOL and GAS depended on water depth, and higher activity of BF occurred by greater flexion of the knee joint or extension of the hip joint during exercise.     

Regulation of cyclic longitudinal mechanical stretch on proliferation of human bone marrow mesenchymal stem cells

Mechanical stimulation is critical to both physiological and pathological states of living cells. Although a great deal of research has been done on biological and biochemical regulation of the behavior of bone marrow mesenchymal stem cells (MSCs), the influence of biomechanical factors on their behavior is still not fully documented. In this study, we investigated the modulation of mechanical stretch magnitude, frequency, and duration on the human marrow mesenchymal stem cells (hMSCs) proliferation by an in vitro model system using a mechanical stretch loading apparatus, and optimized the stretch regime for the proliferation of hMSCs. We applied 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrasodium bromide (MTT) assay to estimate the overall proliferative effects of the stretch on hMSCs. We found that fibronectin coating increased adhesion to silicone chamber surface, however, it did not show significant effect on proliferation of hMSCs. A frequency of 1 Hz was more effective in stimulating hMSCs proliferation. At 1 Hz, 5% strain for 15, 30, 60 min, the significant increase of hMSCs proliferation was observed. Proliferation was enhanced at 1 Hz, 10% strain for 15, 30 min, while decreased for 60 min. At 1 Hz, 15% strain, 15 min stretch resulted in the decrease of proliferation, and 30 min and 60 min stretch showed an increased proliferation. Long time (12 and 24 h) strain application blocked the proliferation. These results indicate that mechanical stretch plays an important role in hMSCs growth and proliferation; an appropriate mechanical stretch regime could be a novel approach to promoting proliferation of hMSCs in vitro.     

A functional polymorphism in the promoter/enhancer region of the<Emphasis Type="Italic"> FOXP3/Scurfin</Emphasis> gene associated with type 1 diabetes

FOXP3/Scurfin, a member of forkhead/winged-helix proteins, is involved in the regulation of T-cell activation, and essential for normal immune homeostasis. The FOXP3/Scurfin gene is located on chromosome Xp11.23, which includes one of the type 1 diabetes susceptible loci. Therefore, we investigated whether the human FOXP3/Scurfin gene might be a new candidate gene for type 1 diabetes. We first screened the human FOXP3/Scurfin gene for microsatellite and single nucleotide polymorphisms. Next, we performed an association study between the FOXP3/Scurfin gene and type 1 diabetes. Then, the evaluation of promoter/enhancer activity of the intron with (GT)(n) polymorphism was performed by dual luciferase reporter assay. We demonstrated two regions contained microsatellite polymorphisms; one was (GT)(n), located on intron zero and the other (TC)(n) on intron 5, which were under linkage-disequilibrium. The (GT)(15) allele showed a significantly higher frequency in patients with type 1 diabetes than in controls (43.1% vs 32.6%, P=0.0027). The genotype frequencies of (GT)(15)/(GT)(15) in female patients and of (GT)(15) in male patients tended to be higher than those in female ( P=0.064) and male ( P=0.061) controls, respectively. A significant difference in the enhancer activity between (GT)(15) and (GT)(16) dinucleotide repeats was detected. In conclusion, the FOXP3/Scurfin gene appears to confer a significant susceptibility to type 1 diabetes in the Japanese population.     

Development of bovine oocytes reconstructed with a nucleus from growing stage oocytes after fertilization in vitro

The developmental capacity of reconstructed bovine oocytes that contained nuclei from growing stage oocytes, 70-119 microm in diameter, was assessed after fertilization in vitro. Nuclei from growing stage oocytes of adult ovaries were transferred to enucleated, fully grown germinal vesicle (GV) stage oocytes. After culture in vitro, the reconstructed oocytes matured, forming the first polar body and MII plate. To supply the ability to form pronuclei, the resultant MII plate was transferred to enucleated MII oocytes, which were obtained by in vitro culture of cumulus-oocyte complexes. After fertilization in vitro, 11-15% of the reconstructed oocytes developed to morulae and blastocysts. To assess the ability to develop to term, a total of 27 late morulae and blastocysts were transferred to 19 recipient cows. Of the three cows that subsequently became pregnant, one recipient, who received two embryos derived from reconstructed oocytes with a nucleus from oocytes 100 to 109 microm in diameter, continued the pregnancy to Day 278 of gestation. This pregnancy, however, was unexpectedly a triplet pregnancy that included a set of identical twins and resulted in the premature birth of the calves, followed by death from lack of post-parturient treatment. These results show that bovine oocyte genomes are capable of supporting term development before the oocytes grow to their full size, which suggests that growing stage oocytes can be directly used as a source of maternal genomes.     

Properties of rice cooked with commercial water-soluble soybean polysaccharides extracted under weakly acidic conditions from soybean cotyledons

It has been found that commercial water-soluble soybean polysaccharides (SSPS) can make cooked rice and noodles non-sticky and prevent rice grains and noodles from adhering to each other. We studied in detail the phenomenon of rice cooked with SSPS. We assumed that the phenomenon resulted from the interaction between SSPS and starch during cooking, and studied the effects of SSPS on the gelatinizing behavior of rice starch by using a Rapid-Visco-Analyzer. The addition of SSPS reduced the viscosity of the gelatinized starch. This lower final viscosity of the rice starch was more distinct from than that of potato starch. These results imply that the properties of SSPS in forming a non-sticky condition might result from a decrease in the viscosity of the gelatinized starch.     

Different protofilament-dependence of the microtubule binding between MAP2 and MAP4

To see a molecular basis of the difference in the microtubule binding between MAP2 and MAP4, we compared the binding of them onto microtubule and Zinc-sheet in the presence of various concentrations of NaCl. The Zinc-sheet is the lateral association of protofilaments arranged in an antiparallel fashion with alternatively exposed opposite surfaces, so that binding requiring adjacent protofilaments is restricted. While the salt-dependence of the MAP2 desorption was not altered between these tubulin polymers, MAP4 dissociated from Zinc-sheet at lower concentrations of NaCl than from microtubule. These results suggest that single protofilament is sufficient for microtubule binding of MAP2 as observed by Al-Bassam et al. [J. Cell Biol. 157 (2002) 1187], but MAP4 appeared to interact with adjacent protofilaments during microtubule-binding. Weakened binding on Zinc-sheets was also observed in the projection domain-deletion mutants of MAP4, so that the difference in the protofilament-dependence would lie in the relatively conserved microtubule-binding domain.     

Involvement of Oct3/4 in the enhancement of neuronal differentiation of ES cells in neurogenesis-inducing cultures

Oct3/4 plays a critical role in maintaining embryonic stem cell pluripotency. Regulatable transgene-mediated sustained Oct3/4 expression in ES cells cultured in serum-free LIF-deficient medium caused accelerated differentiation to neuroectoderm-like cells that expressed Sox2, Otx1 and Emx2 and subsequently differentiated into neurons. Neurogenesis of ES cells is promoted by SDIA (stromal cell-derived inducing activity), which accumulates on the PA6 stromal cell surface. Oct3/4 expression in ES cells was maintained by SDIA whereas without it expression was promptly downregulated. Suppression of Oct3/4 abolished neuronal differentiation even after stimulation by SDIA. In contrast, sustained upregulated Oct3/4 expression enhanced SDIA-mediated neurogenesis of ES cells. Therefore, Oct3/4 appears to promote neuroectoderm formation and subsequent neuronal differentiation from ES cells.