Evolution of the neuropeptide Y receptor family: gene and chromosome duplications deduced from the cloning and mapping of the five receptor subtype genes in pig

Neuropeptide Y (NPY) receptors mediate a variety of physiological responses including feeding and vasoconstriction. To investigate the evolutionary events that have generated this receptor family, we have sequenced and determined the chromosomal localizations of all five presently known mammalian NPY receptor subtype genes in the domestic pig, Sus scrofa (SSC). The orthologs of the Y(1) and Y(2) subtypes display high amino acid sequence identities between pig, human, and mouse (92%-94%), whereas the Y(4), Y(5), and y(6) subtypes display lower identities (76%-87%). The lower identity of Y(5) is due to high sequence divergence in the large third intracellular loop. The NPY1R, NPY2R, and NPY5R receptor genes were localized to SSC8, the NPY4R to SSC14, and NPY6R to SSC2. Our comparisons strongly suggest that the tight cluster of NPY1R, NPY2R, and NPY5R on human chromosome 4 (HSA4) represents the ancestral configuration, whereas the porcine cluster has been split by two inversions on SSC8. These 3 genes, along with adjacent genes from 14 other gene families, form a cluster on HSA4 with extensive similarities to a cluster on HSA5, where NPY6R and >13 other paralogs reside, as well as another large cluster on HSA10 that includes NPY4R. Thus, these gene families have expanded through large-scale duplications. The sequence comparisons show that the NPY receptor triplet NPY1R-NPY2R-NPY5R existed before these large-scale duplications.     

T- and B-Cell Immune Responses of Patients Who Had Undergone Colectomies to Oral Administration of Salmonella enterica Serovar Typhi Ty21a Vaccine

The capacity of an oral live attenuated Salmonella enterica serovar Typhi Ty21a vaccine to induce immune responses in patients who had undergone colectomies because of ulcerative colitis was evaluated, and these responses were compared with those of healthy volunteers. Purified CD4⁺ and CD8⁺ T cells from peripheral blood were stimulated in vitro by using the heat-killed Ty21a vaccine strain, and the proliferation and gamma interferon (IFN-γ) production were measured before and 7 or 8 days after vaccination. Salmonella-specific immunoglobulin A (IgA) and IgG antibody responses in serum along with IgA antibody responses in ileostomy fluids from the patients who had undergone colectomies were also evaluated. Three doses of vaccine given 2 days apart failed to induce proliferative T-cell responses in all the six patients who had undergone colectomies, and increases in IFN-γ production were found only among the CD8⁺ cells from three of the patients. In contrast, both proliferative responses and increased IFN-γ production were observed among CD4⁺ and CD8⁺ T cells from 3 and 6 of 10 healthy volunteers, respectively. Salmonella-specific IgA and/or IgG antibody responses in serum were observed for five (56%) of nine patients who had undergone colectomies and in 15 (88%) of 17 healthy volunteers. In ileostomy fluids, significant anti-Salmonella IgA antibody titer increases were detected in six (67%) of nine patients who had undergone colectomies. The impaired T- and B-cell immune responses found after vaccination in the circulation of patients who have undergone colectomies may be explained by a diminished colonization of the Ty21a vaccine strain due to the lack of a terminal ileum and colon.     

Chromosomal integrity maintained in five human embryonic stem cell lines after prolonged in vitro culture

There have been recent reports of human embryonic stem cell (hESC) lines developing chromosomal aberrations after long-term culture, indicating an unstable genomic status due to the in vitro milieu. This raises concern, since it would limit their use in therapeutics. In this study the chromosomal status of five well-characterized hESC lines, SA002, SA002.5, AS034.1.1, SA121 and SA461, was monitored during long-term in vitro culture. The criteria of defined hESCs were met by all of the five hESC lines (four diploid and one trisomic for chromosome 13). The genomes were screened for chromosomal aberrations and rearrangements using comparative genomic hybridization (CGH), interphase fluorescence in situ hybridization (FISH) and traditional karyotyping on several occasions while in culture. The genomic integrity was shown to be maintained after repeated freeze-thaw procedures and continuous culture in vitro for up to 22 months (148 passages). We discuss the most common de novo chromosomal aberrations reported in hESCs, as well as their possible origin.     

A 3 year retrospective review of intrauterine insemination, using cryopreserved donor spermatozoa and cycle monitoring by urinary or serum luteinizing hormone measurements

Insemination with donor spermatozoa is an integral part of infertility treatment. For the last 3 years in our unit, intrauterine insemination with donor spermatozoa (IUID) has been used in preference to vaginal insemination. In this retrospective study, patients were offered an initial course of five single intrauterine inseminations with cryopreserved donor spermatozoa and treatment was then reviewed. A total of 389 patients received 1465 inseminations. In all, 1119 cycles were monitored using luteinizing hormone serum analyses and 346 cycles using the urine home test kits. The clinical pregnancy rate per insemination for the cycles monitored by the serum assay was 18.0% (202/1119) compared with the urine cycles (13.7%, 46/346) (P <05). The pregnancy loss rate was not significantly different (14.4%, 29/202 and 21.7%, 10/46) (serum and urine cycles respectively). The viable clinical pregnancy rate was significantly higher (P <03) for the serum cycles than for the cycles using the urinary monitoring (15.5%, 173/1119 and 10.4%, 36/346 respectively). The cycles monitored by serum assay had a significantly higher cumulative viable clinical pregnancy rate (P <0001) of 70.2% after nine inseminations compared with the urine monitored cycles of 54.8%. The majority of patients opted for the serum cycles, with a minority self-selecting the urine cycles mainly for travelling convenience. The explanation for the significant differences between the viable clinical pregnancy rates per insemination and the cumulative viable clinical pregnancy rates may be due to the sensitivity of the urine home test kit or the patients' interpretation of the result.