Calumin, a Ca²⁺-binding protein on the endoplasmic reticulum, alters the ion permeability of Ca²⁺ release-activated Ca²⁺ (CRAC) channels

Store-operated channels (SOC) are Ca(2+)-permeable channels that are activated by IP(3)-receptor-mediated Ca(2+) depletion of the endoplasmic reticulum (ER). Recent studies identify a membrane pore subunits, Orai1 and a Ca(2+) sensor on ER, STIM1 as components of Ca(2+) release-activated Ca(2+) (CRAC) channels, which are well-characterized SOCs. On the other hand, proteins that act as modulators of SOC activity remain to be identified. Calumin is a Ca(2+)-binding protein that resides on the ER and functional experiments using calumin-null mice demonstrate that it is involved in SOC function, although its role is unknown. This study used electrophysiological analysis to explore whether calumin modulates CRAC channel activity. CRAC channel currents were absent in HEK293 cells co-expressing calumin with the CRAC channel components, Orai1 or STIM1. Meanwhile, HEK cells that co-expressed calumin with CRAC channels exhibited larger currents with slower inactivation than cells expressing CRAC channels alone. The current-voltage relationship showed an inwardly rectifying current, but a negative shift in the reversal potential of greater than 60mV was observed in HEK cells co-expressing calumin with CRAC channels. In addition, the permeability coefficient ratio of Ca(2+) over monovalent cations was much lower than that of cells expressing CRAC channels alone. Replacement of Na(+) with N-methyl-d-glucamine(+) in the external solution noticeably diminished the CRAC current in HEK cells co-expressing calumin and CRAC channels. In a Cs(+)-based external solution, CRAC current was not observed in either cell-type. In addition, Ca(2+) imaging analysis revealed that co-transfection of calumin reduced extracellular Ca(2+) influx via CRAC channels. Further, calumin was shown to be directly associated with CRAC channels. These results reveal a novel mechanism for the regulation of CRAC channels by calumin.     

Down-regulation of MutS homolog 3 by hypoxia in human colorectal cancer

Down-regulation of hMSH3 is associated with elevated microsatellite alterations at selected tetranucleotide repeats and low levels of microsatellite instability in colorectal cancer (CRC). However, the mechanism that down-regulates hMSH3 in CRC is not known. In this study, a significant association between over-expression of glucose transporter 1, a marker for hypoxia, and down-regulation of hMSH3 in CRC tissues was observed. Therefore, we examined the effect of hypoxia on the expression of hMSH3 in human cell lines. When cells with wild type p53 (wt-p53) were exposed to hypoxia, rapid down-regulation of both hMSH2 and hMSH3 occurred. In contrast, when null or mutated p53 (null/mut-p53) cells were exposed to hypoxia, only hMSH3 was down-regulated, and at slower rate than wt-p53 cells. Using a reporter assay, we found that disruption of the two putative hypoxia response elements (HREs) located within the promoter region of the hMSH3 abrogated the suppressive effect of hypoxia on reporter activity regardless of p53 status. In an EMSA, two different forms of HIF-1α complexes that specifically bind to these HREs were detected. A larger complex containing HIF-1α predominantly bound to the HREs in hypoxic null/mut-p53 cells whereas a smaller complex predominated in wt-p53 cells. Finally, HIF-1α knockdown by siRNA significantly inhibited down-regulation of hMSH3 by hypoxia in both wt-p53 and mut-p53 cells. Taken together, our results suggest that the binding of HIF-1α complexes to HRE sites is necessary for down-regulation of hMSH3 in both wt-p53 and mut-p53 cells.     

Embryological ontogeny of aromatase gene expression in Chrysemys picta and Apalone mutica turtles: comparative patterns within and across temperature-dependent and genotypic sex-determining mechanisms

Although the role of aromatase in many estrogen-dependent reproductive and metabolic functions is well documented in vertebrates, its involvement in the ovarian development of species exhibiting temperature-dependent sex determination (TSD) is incompletely understood. This is partly due to the conflicting temporal and spatial pattern of aromatase expression and activity across taxa. To help resolve this ongoing debate, we compared for the first time the embryological ontogeny of aromatase expression in turtles possessing genotypic sex determination (GSD) (Apalone mutica) and TSD (Chrysemys picta) incubated under identical conditions. As anticipated, we found no significant thermal differences in aromatase expression at any stage examined (prior to until the end of the thermosensitive period) in A. mutica. Surprisingly, the same was true for C. picta. When placed in a phylogenetic context, our results suggest that aromatase expression is evolutionarily plastic with respect to sex determination in reptiles, and that differences between reptilian TSD and GSD are not aromatase-driven. Further research across TSD and GSD species is warranted to fully decipher the evolution of functional differences among sex-determining mechanisms.     

The gene MACCHI-BOU 4/ENHANCER OF PINOID encodes a NPH3-like protein and reveals similarities between organogenesis and phototropism at the molecular level

Intercellular transport of the phytohormone auxin is a significant factor for plant organogenesis. To investigate molecular mechanisms by which auxin controls organogenesis, we analyzed the macchi-bou 4 (mab4) mutant identified as an enhancer of pinoid (pid). Although mab4 and pid single mutants displayed relatively mild cotyledon phenotypes, pid mab4 double mutants completely lacked cotyledons. We found that MAB4 was identical to ENHANCER OF PINOID (ENP), which has been suggested to control PIN1 polarity in cotyledon primordia. MAB4/ENP encodes a novel protein, which belongs to the NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) family thought to function as a signal transducer in phototropism and control lateral translocation of auxin. MAB4/ENP mRNA was detected in the protodermal cell layer of the embryo and the meristem L1 layer at the site of organ initiation. In the mab4 embryo, the abundance of PIN1:GFP was severely decreased at the plasma membrane in the protodermal cell layer. In addition, subcellular localization analyses indicated that MAB4/ENP resides on a subpopulation of endosomes as well as on unidentified intracellular compartments. These results indicate that MAB4/ENP is involved in polar auxin transport in organogenesis.     

Determination of microsatellite repeats in the human thyroid peroxidase (TPOX) gene using an automated gene analysis system with nanoscale engineered biomagnetite

The number of repeat in the microsatellite region (AATG)(5-14) of the human thyroid peroxidase gene (TOPX) was determined using an automated DNA analysis system with nano-scale engineered biomagnetite. Thermal melting curve analysis of DNA duplexes on biomagnetite indicated that shorter repeat sequences (less than 9 repeats) were easily discriminated. However, it was difficult to determine the number of repeats at more than nine. In order to improve the selectivity of this method for the longer repeats, a "double probe hybridization assay" was performed in which an intermediate probe was used to replace a target repeat sequence having more than 9 repeats with a shorter sequence possessing less than 9 repeats. Thermal probe melting curve analyses and Tm determination confirmed that the target with 10 repeats was converted to 5 repeats, 11 repeats converted to 4 and 12 to 3, respectively. Furthermore, rapid determination of repeat numbers was possible by measuring fluorescence intensities obtained by probe dissociation at 56 and 66 degrees C, and 40, 60 and 80 degrees C for signal normalization.