Preparation of specific antisera to 15alpha-hydroxyestrogens.

The synthesis of haptens of 15alpha-hydroxyestrone, 15alpha-hydroxyestradiol, and 15alpha-hydroxyestriol (estetrol) was undertaken, to obtain specific antisera required for enzyme immunoassay. 3-(1-Carboxypropyl) ethers of these 15alpha-hydroxyestrogens were prepared and conjugated with bovine serum albumin and horseradish peroxidase. The specificity of antisera elicited against bovine serum albumin conjugates was checked by the enzyme immunoassay by using horseradish peroxidase-labeled antigen, and proved to be satisfactory in terms of cross-reactivities to related compounds.     

Circular dichroism and structure of the complex of acridine orange with poly(L-glutamic acid)

Circular dichroism and absorption spectra have been measured on solutions of acridine orange and poly(L -glutamic acid) mixed at two molar ratios of carboxyl group-to-dye, P/D 25 and 0.8, and at different pH's. Characteristic circular dichroism is induced at the absorption bands of acridine orange when the P/D is 25 and the solution is acidic. Another type of circular dichroism is manifest at neutral and alkaline pH when P/D is 0.8. For the induction of the former type of circular dichroism, a helical array of acridine-orange dimers bound to the α-helix is postulated, in which the dye molecular planes are almost perpendicular to the helical axis. Assuming the helical geometry and optical parameters, combined with the observed magnitude of transition electric moment, the rotatory strength of the complex is calculated to the zeroth order approximation, and the observed circular dichroism spectra have been reproduced.     

Functional Role for Piezo1 in Stretch-evoked Ca2+ Influx and ATP Release in Urothelial Cell Cultures

The urothelium is a sensory structure that contributes to mechanosensation in the urinary bladder. Here, we provide evidence for a critical role for the Piezo1 channel, a newly identified mechanosensory molecule, in the mouse bladder urothelium. We performed a systematic analysis of the molecular and functional expression of Piezo1 channels in the urothelium. Immunofluorescence examination demonstrated abundant expression of Piezo1 in the mouse and human urothelium. Urothelial cells isolated from mice exhibited a Piezo1-dependent increase in cytosolic Ca²⁺ concentrations in response to mechanical stretch stimuli, leading to potent ATP release; this response was suppressed in Piezo1-knockdown cells. In addition, Piezo1 and TRPV4 distinguished different intensities of mechanical stimulus. Moreover, GsMTx4, an inhibitor of stretch-activated channels, attenuated the Ca²⁺ influx into urothelial cells and decreased ATP release from them upon stretch stimulation. These results suggest that Piezo1 senses extension of the bladder urothelium, leading to production of an ATP signal. Thus, inhibition of Piezo1 might provide a promising means of treating bladder dysfunction.     

Histo-Blood Group Antigen-Like Substances of Human Enteric Bacteria as Specific Adsorbents for Human Noroviruses

Histo-blood group antigens (HBGAs) have been suggested to be receptors or coreceptors for human noroviruses (HuNoVs) expressed on the intestinal epithelium. We isolated an enteric bacterium strain (SENG-6), closely related to Enterobacter cloacae, bearing HBGA-like substances from a fecal sample of a healthy individual by using a biopanning technique with anti-HBGA antibodies. The binding capacities of four genotypes of norovirus-like particles (NoVLPs) to Enterobacter sp. SENG-6 cells were confirmed by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy demonstrated that NoVLPs bound mainly to extracellular polymeric substances (EPS) of Enterobacter sp. SENG-6, where the HBGA-like substances were localized. EPS that contained HBGA-like substances extracted from Enterobacter sp. SENG-6 was shown by enzyme-linked immunosorbent assay (ELISA) to be capable of binding to NoVLPs of a GI.1 wild-type strain (8fIIa) and a GII.6 strain that can recognize A antigen but not to an NoVLP GI.1 mutant strain (W375A) that loses the ability to bind to A antigen. Enzymatic cleavage of terminal N-acetyl-galactosamine residues in the bacterial EPS weakened bacterial EPS binding to the GI.1 wild-type strain (8fIIa). These results indicate that A-like substances in the bacterial EPS play a key role in binding to NoVLPs. Since the specific binding of HuNoVs to HBGA-positive enteric bacteria is likely to affect the transmission and infection processes of HuNoVs in their hosts and in the environment, further studies of human enteric bacteria and their binding capacity to HuNoVs will provide a new scientific platform for understanding interactions between two types of microbes that were previously regarded as biologically unrelated.     

Novel insight into the agonistic mechanism of alefacept in vivo: differentially expressed genes may serve as biomarkers of response in psoriasis patients

Alefacept is an LFA3-Ig fusion protein that binds to CD2 and is thought to inhibit T cell activation by antagonism of CD2 signaling or by lysis of CD2(+) cells. Alefacept is potential future therapeutic for organ transplant recipients or graft-vs-host disease and is an approved therapeutic for psoriasis vulgaris, which is a T cell-mediated inflammatory disease. However, alefacept improves psoriasis in only approximately 50% of patients treated for 12 wk. We studied the immunologic effects of alefacept in a group of psoriasis patients during treatment. We found that T cells, especially CD8(+) T cells, were rapidly decreased in the peripheral circulation. Decreases in circulating T cells were not associated with induced apoptosis. Unexpectedly, in addition to suppression of inflammatory genes, we found a marked induction of mRNAs for STAT1, IL-8, and monokine induced by IFN-gamma during the first day of treatment in PBMC. We confirmed the agonistic effects of alefacept in PBMC in vitro, which were similar to CD3/CD28 ligation on T cells. These data establish that alefacept activates gene expression programs in leukocytes and suggest that its therapeutic action may be as a mixed agonist/antagonist. Furthermore, responding patients to alefacept treatment show unique patterns of gene modulation. Whereas alefacept down-regulated TCRs CD3D and CD2 in responders, nonresponders reveal a higher expression of T cell activation genes such as CD69 in pretreatment PBMC. These finding suggest a potential basis for categorizing responders vs nonresponders at an early time point in treatment or before treatment of a broad range of proinflammatory diseases. This study 1) establishes alefacept as a novel CD2 agonist molecule for induction of leukocyte activation genes (prior work proposed its mechanism as a CD2 antagonist) and 2) that differential activation of genes may categorize clinical responders to this agent, critical for cost-effective use of this drug.     

NRFL-1, the C. elegans NHERF Orthologue, Interacts with Amino Acid Transporter 6 (AAT-6) for Age-Dependent Maintenance of AAT-6 on the Membrane

The NHERF (Na(+)/H(+) exchanger regulatory factor) family has been proposed to play a key role in regulating transmembrane protein localization and retention at the plasma membrane. Due to the high homology between the family members, potential functional compensations have been a concern in sorting out the function of individual NHERF numbers. Here, we studied C. elegans NRFL-1 (C01F6.6) (nherf-like protein 1), the sole C. elegans orthologue of the NHERF family, which makes worm a model with low genetic redundancy of NHERF homologues. Integrating bioinformatic knowledge of C. elegans proteins into yeast two-hybrid scheme, we identified NRFL-1 as an interactor of AAT-6, a member of the C. elegans AAT (amino acid transporter) family. A combination of GST pull-down assay, localization study, and co-immunoprecipitation confirmed the binding and characterized the PDZ interaction. AAT-6 localizes to the luminal membrane even in the absence of NRFL-1 when the worm is up to four-day old. A fluorescence recovery after photobleaching (FRAP) analysis suggested that NRFL-1 immobilizes AAT-6 at the luminal membrane. When the nrfl-1 deficient worm is six-day or older, in contrast, the membranous localization of AAT-6 is not observed, whereas AAT-6 tightly localizes to the membrane in worms with NRFL-1. Sorting out the in vivo functions of the C. elegans NHERF protein, we found that NRFL-1, a PDZ-interactor of AAT-6, is responsible for the immobilization and the age-dependent maintenance of AAT-6 on the intestinal luminal membrane.     

Delimitation of a QTL region controlling cold tolerance at booting stage of a cultivar, ‘Lijiangxintuanheigu’, in rice, <Emphasis Type="Italic">Oryza sativa</Emphasis> L.

Low temperature at the booting stage of rice causes male sterility resulting in severe yield loss. Cold tolerance has long been an important objective in rice breeding. We identified a quantitative trait locus (QTL) for cold tolerance on the long arm of chromosome 3 from the cold-tolerant breeding line ‘Ukei 840’ by using F₂ and BC₁F₂ populations from crosses between ‘Ukei 840’ and ‘Hitomebore’. The cold tolerance of ‘Ukei 840’ is derived from the Chinese cultivar ‘Lijiangxintuanheigu’. The effect of this QTL on cold tolerance was confirmed by developing ‘Hitomebore’ chromosome segment substitution lines having ‘Lijiangxintuanheigu’ alleles on chromosome 3. By producing recombinants in chromosome 3, the QTL region for cold tolerance was delimited to the region of about 1.2-Mb region between RM3719 and RM7000. All lines heterozygous for the QTL showed seed fertilities as low as that of ‘Hitomebore’, suggesting that the ‘Lijiangxintuanheigu’ allele for cold tolerance in the QTL region is recessive. Determination of a 1.2-Mb nucleotide sequence of ‘Ukei 840’ and comparison with the published genomic sequence of ‘Nipponbare’ showed 254 SNPs, of which 11 were in coding regions of genes, seven in five genes being non-synonymous. SNPs were detected in the 5-kb upstream regions of 89 genes, but no differences of gene expression levels were detected between alleles of these genes. Although further delimitation is required to identify the gene responsible for cold tolerance of ‘Lijiangxintuanheigu’, SNP markers developed here will be useful for marker-assisted selection in a breeding program using ‘Lijiangxintuanheigu’ as a donor of cold tolerance.