Analysis of the flanking regions of the human alpha-lactalbumin gene responsible for position-effect independent expression

Transgenic rats with the 130 kb bacterial artificial chromosome construct bLA, including the alpha-lactalbumin gene, had position-independent and copy number-dependent expression, which confirmed previous experiments using the 210 kb yeast artificial construct, yLALBA. To identify elements that confer a position effect, we compared the yLALBA and bLA sequences. yLALBA was chimeric. A common 32 kb region was identified and the total nucleotide sequence was determined. We previously analyzed transgenic rats using polymerase chain reaction to compare the integrity and expression of the transgenes. The -6 to +9 kb region is considered to be necessary for position-independent expression. Transgenic rats lacking the -3.4 to -0.85 kb region had a severe position effect. This 2.5 kb region contains two DNaseI hypersensitive sites at -1.0 and -2.8 kb. The 2.5 kb region is proposed to be a locus control region of the human alpha-lactalbumin gene.     

Distinctive solution conformation of phosphatase inhibitor CPI-17 substituted with aspartate at the phosphorylation-site threonine residue

We present solution NMR structures for wild-type and mutated forms of CPI-17, a phosphoinhibitor for protein phosphatase 1. Phosphorylation of Thr38 of CPI-17 produces a >1000-fold increase in inhibitory potency for myosin phosphatase. We compared the 1H-15N heteronuclear single quantum coherence spectroscopy (HSQC) chemical shifts of wild-type CPI-17, partially phosphorylated CPI-17 and CPI-17 with Thr38 replaced with Asp to introduce a negative charge. There was a switch in the protein conformation due to either Asp substitution or phosphorylation, so we determined the solution NMR structure of the CPI-17 T38D mutant as a model for the active (phospho-) conformation. The structures reveal a molecular switch in conformation that involves the rotation of two of the four helices in the four helix bundle. Despite this conformational switch, there was little increase in the inhibitory potency with T38D. We propose that for this inhibitor, a negative charge at residue 38 is sufficient to trigger an active conformation, but a phosphoryl group is required for full inhibitory potency against protein phosphatase-1.     

Categorical perception of orientation in monkeys

Structural and functional substrates of orientation processing in monkeys have been clarified. However, orientation perception in monkeys has not been fully studied. In this study, the cognitive mechanism that controls monkeys' perception of orientation was evaluated. After the monkeys were trained to discriminate between a cardinal and an oblique orientation (e.g., 0 degrees and 30 degrees), their perceptual mechanisms underlying orientation discrimination were tested by using six orientations, ranging from 0 degrees to 150 degrees, including ones used in the discrimination training. Generalization tests showed that the monkeys who were trained with cardinal orientations (e.g., 0 degrees) as positive stimuli generalized their responses to the other cardinal orientation (e.g., 90 degrees). Similarly, the monkeys who were trained with oblique orientations (e.g., 30 degrees) as positive stimuli generalized their responses to all other oblique orientations (e.g., 60 degrees, 120 degrees, and 150 degrees). These findings indicated that the monkeys abstracted the quality of the cardinal/oblique category from the physical features of orientation stimuli although they were not trained to do so. Such an abstraction also suggested a discrepancy between a continuously and orderly arranged cortical map and a discontinuously categorized perception of orientation. The present findings provide insight into the learning-correlated plasticity of cortical orientation preference.     

InsP3 receptor type 2 and oscillatory and monophasic Ca2+ transients in rat adrenal chromaffin cells

Muscarinic receptor stimulation induced oscillatory and monophasic Ca(2+) transients in rat adrenal chromaffin cells in the absence of external Ca(2+). As this Ca(2+) mobilization may be mediated by InsP(3), we first explored types of InsP(3) receptors and their intracellular distribution in chromaffin cells. The InsP(3) receptor type 1 was not immunodetected in precipitates of adrenal medulla homogenates and in dissociated adrenal chromaffin cells, whereas an anti-type 3 mAb recognized a faint band with about 250 kDa, but no significant immunoreaction was visible in chromaffin cells. The anti-type 2 mAb strongly detected a band with about 220 kDa and the immunoreaction was observed perinuclearly and at the cell periphery. These results indicate that InsP(3) receptor type 2 is predominant in chromaffin cells. The oscillatory and monophasic Ca(2+) transients were reproduced in simulation based on a three-state kinetic model (shut, open, and inactivated states). Ca(2+) ions were found experimentally and theoretically to turn over rapidly between stores and the cytosol during stimulation. The results suggest that InsP(3) receptor type 2 is responsible for both oscillatory and monophasic Ca(2+) transients and that change in mode of Ca(2+) responses may be accounted for by the kinetic property of the type 2 receptor.     

Disruption of a single copy of the p38alpha MAP kinase gene leads to cardioprotection against ischemia-reperfusion

The p38 mitogen-activated protein kinase (p38) is activated in the heart during ischemia-reperfusion. However, it is not clear whether the activation of p38 is the protective response or the kinase mediates the cellular damage by ischemia-reperfusion. We examined the role of p38alpha in ischemia-reperfusion injury by studying p38alpha(+/-) mice. The p38alpha protein level in the p38alpha(+/-) heart was 50+/-8.7% compared with that in the p38alpha(+/+) heart. Upon reperfusion following ischemia for 25min, p38alpha activity was transiently increased. The maximum level of p38 activity in p38alpha(+/-) was 60+/-10.5% compared with that in p38alpha(+/+). In the p38alpha(+/+) heart, 25min ischemia and 2h reperfusion resulted in necrotic injury (37.1+/-2.7% of the area at risk), whereas infarct size was drastically reduced to 7.2+/-0.7% in the p38alpha(+/-) heart. These suggested that p38alpha plays a pivotal role in the signal transduction pathway mediating myocardial cell death caused by ischemia-reperfusion.     

Induction of hepatic injury by hepatitis C virus-specific CD8+ murine cytotoxic T lymphocytes in transgenic mice expressing the viral structural genes

In the present study, we generated killer cells specific for hepatitis C virus (HCV) structural protein by re-stimulation of immune spleen cells from H-2(d) haplotype transgenic (Tg) mice, expressing the core, E1, E2, and NS2 genes of HCV regulated by the Cre/loxP switching system. The generated killer cells were conventional CD8(+)L(d) class-I MHC molecule-restricted cytotoxic T lymphocytes (CTLs) and specific for the HCV E1 structural protein. Because the CTLs could also kill hepatocytes from the Tg mice expressing HCV structural proteins in vitro, we attempted to transfer those CTLs intravenously into interferon regulatory factor-1 (IRF-1) negative, CD8-deficient Tg mice representing the HCV structural genes on hepatocytes to examine whether the inoculated CD8(+) CTLs can eliminate hepatocytes expressing the HCV genes in vivo. We observed an elevation of serum ALT level as well as damage of the liver tissue histologically. To our knowledge, this is the first demonstration to show that HCV-specific CD8(+) CTLs specifically attack hepatocytes expressing the HCV structural proteins both in vitro and in vivo.     

High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay

Background

Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP) that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA) of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods.

Results

A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA) fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA). Fluorescent signal output was measured in real time and as an end point.

Conclusions

Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample.     

RhoA-mediated Ca2+ sensitization in erectile function

A Rho-kinase inhibitor increases corpus cavernosum (CC) pressure in an in vivo rat model (Chitaley, K., Wingard, C. J., Webb, R. C., Branam, H., Stopper, V. S., Lewis, R. W., and Mills, T. M. (2001) Nat. Med. 7, 119-122) suggesting that Rho-mediated Ca(2+) sensitization of CC smooth muscle maintains the flaccid (contracted) state. We directly demonstrate Ca(2+) sensitization of permeabilized rabbit and human CC and identify a highly expressed molecular component of this pathway. Ca(2+) sensitization of force induced by endothelin or GTPgammaS was significantly greater in CC than in rabbit ileum smooth muscle and was accompanied by a 17-fold higher RhoA content. Pull-down assays with the RhoA binding domain of mDia showed the high RhoA content of CC to be available for activation by GTPgammaS. Ca(2+) sensitization induced by endothelin, phenylephrine, or GTPgammaS was completely relaxed by the Rho kinase inhibitor Y-27632. Human and rabbit CC both express the phosphatase inhibitor CPI-17, the myosin phosphatase regulatory (MYPT-1) and catalytic (PP1delta) subunits, and two isoforms of Rho kinase. We suggest that high expression of RhoA contributes, through RhoA-mediated Ca(2+) sensitization, to the flaccid state of CC that can be reversed by a water-soluble, orally active Rho kinase inhibitor suitable for therapy of erectile dysfunction.