Oviductal extracellular vesicles interact with the spermatozoon's head and mid-piece and improves its motility and fertilizing ability in the domestic cat.

Fertilization and early embryo development are regulated by a unique maternal-gamete/embryo cross-talk within the oviduct. Recent studies have shown that extracellular vesicles (EVs) within the oviduct play important roles in mediating this developmental process. Here, we examined the influence of oviductal EVs on sperm function in the domestic cat. We demonstrated that (1) EVs are enriched in proteins related to energy metabolism, membrane modification, and reproductive function; (2) EVs bound and fused with the membranes of the acrosome and mid piece; and (3) incubating sperm with EVs improved motility, fertilizing capacity of cat spermatozoa and prevented acrosomal exocytosis in vitro. These findings indicated that oviductal EVs mediate sperm function and fertilization in the cat and provides new insights to improve sperm cryopreservation and in vitro fertilization in the domestic and wild felids and human.

Estimating human inbreeding coefficients: comparison of genealogical and marker heterozygosity approaches.

We have used genealogies and genomic polymorphisms to estimate individual inbreeding coefficients (F) in 50 subjects with an expected range (based on recent genealogies) of F from 0.0 to 0.0625. The estimates were based on two approaches, using genotypes respectively from 410 microsatellite markers (410-STR panel) and from 10,000 SNPs (10K-SNP panel). The latter was performed in a sub-sample of 15 individuals. We concluded that for both marker panels measures of inbreeding based on the excess of homozygosity over Hardy-Weinberg expectation were not closely correlated with 4-5 generation genealogical F-values. For the 10K-SNP panel we found two alternative measures which correlated more closely with F, based respectively on standard errors and on paired homozygosity of nearby SNPs over distances of 2-4 cM. We propose an empirical method for estimating standard errors and hence individual F-values, based on the variation between individual autosomes. This method could provide useful estimates of average F-values for groups of individuals in population-based studies of the effects of inbreeding/homozygosity on quantitative traits.

Extent of linkage disequilibrium in a Sardinian sub-isolate: sampling and methodological considerations.

The extent of linkage disequilibrium (LD) is an important factor when designing experiments for mapping disease or trait loci using LD mapping methods. It depends on the population history and hence is a characteristic of each population. Here, we have assessed the extent of LD in a sub-isolate of the general Sardinian population (775 members of one village) using 22 polymorphic markers on chromosome 19. We found high levels of disequilibrium that extended to 8 cM, when based on D', and 11 cM when based on the significance level of the allelic association. The fact that conclusions based on both methods are similar suggests that the estimates are quite robust. We have also shown, through a simple resampling technique, that small sample sizes can overestimate both the mean value of D' and its variance up to a factor of about 2 and 16, respectively, when the number of diplotypes (the pair of haplotypes that compose the genotype) decreased from 186 to 26. We evaluated the effect on D' of the depth of the pedigree available when using phased founders, and compared the estimates with those obtained when using unphased founders, and also the effect of grouping alleles on the value of D' and the significance level. Owing to the high sampling variance of LD, we recommend the use of at least 200 unrelated individuals when characterizing the extent of LD.

Power of linkage disequilibrium mapping to detect a quantitative trait locus (QTL) in selected samples of unrelated individuals.

We considered a strategy to map quantitative trait loci (QTLs) using linkage disequilibrium (LD) when the QTL and marker locus were multiallelic. The strategy involved phenotyping a large number of unrelated individuals and genotyping only selected individuals from the two tails of the trait distribution. Power to detect trait-marker association was assessed as a function of the number of QTL and marker alleles. Two patterns of LD were used to study their influence on power. When the frequency of the QTL allele with the largest effect and that of the marker allele linked in coupling were equal, power was maximum. In this case, increasing the number of QTL alleles reduced the power. The maximum difference in power between the two LD patterns studied was approximately 30%. For low QTL heritabilities (h2QTL<0.1) and single trait studies we recommend selecting around 5% of the upper and lower tails of the trait distribution.

2-Methoxyestradiol induces p53-associated apoptosis of colorectal cancer cells.

Menopausal estrogen replacement therapy is thought to be responsible for the recent decline in colorectal cancer (CRC) incidence among women. In the C57BL/6J-Min/+ mouse, an animal model of CRC, 17beta-estradiol (E(2)) prevents tumor formation in ovariectomized females. We examined human CRC intestinal cell lines to determine whether particular E(2) metabolites produced anti-tumor effects. Treatment of CRC cells with 2-methoxyestradiol (2-MeOE(2)) increased expression of p53 and p21(WAF1/CIP1) proteins and induced apoptosis, but did not produce changes in expression of estrogen receptor (ER)alpha or ERbeta. The finding that 2-MeOE(2) induces p53-mediated colon cell apoptosis in vitro supports a role for 2-MeOE(2) as an endogenous mediator of intestinal tumor suppression.

Search for ethnic, geographic, and other factors in the epidemiology of Down syndrome in South America: analysis of data from the ECLAMC project, 1967-1997.

We have analyzed data on 3,157 cases of Down syndrome (DS) from nine South American countries in consecutive series of hospital live births over a 30-year period, with particular emphasis on possible ethnic or geographic variations in maternal age-adjusted incidence. The data constitute the largest series of DS cases assembled to date from an area lacking advanced health care systems. Absolute incidence rates were estimated from total hospital live births; relative rates were estimated from matched case-control data using conditional logistic regression. Maternal age-adjusted rates were closely similar to those reported elsewhere, and showed little or no dependency on other factors investigated, including paternal age, birth order, ancestral origin, country of birth, maternal educational level, maternal ABO and Rhesus blood groups, interval to and outcome of mother's previous pregnancy, and parental consanguinity. The absence of an effect of high birth order was particularly notable because of the relatively large number of grand multipara resulting from high fertility in this population. The study adds to a body of evidence suggesting that maternal age-adjusted DS rates vary little across human populations, and are therefore unlikely to be greatly influenced by genetic or environmental factors that differ between them. An unusual finding was of a markedly lower sex ratio (98 males per 100 females) than has been reported in other DS samples.

Progressive changes in adherens junction structure during intestinal adenoma formation in Apc mutant mice.

The C57BL/6J-Min/+ (Min/+) mouse bears a mutant Apc gene and therefore is an important in vivo model of intestinal tumorigenesis. Min/+ mice develop adenomas that exhibit loss of the wild-type Apc allele (Apc(Min/-)). Previously, we found that histologically normal enterocytes bearing a truncated Apc protein (Apc(Min/+)) migrated more slowly in vivo than enterocytes with either wild-type Apc (Apc(+/+)) or with heterozygous loss of Apc protein (Apc(1638N)). To study this phenotype further, we determined the effect of the Apc(Min) mutation upon cell-cell adhesion by examining the components of the adherens junction (AJ). We observed a reduced association between E-cadherin and beta-catenin in Apc(Min/+) enterocytes. Subcellular fractionation of proteins from Apc(+/+), Apc(Min/+), and Apc(Min/-) intestinal tissues revealed a cytoplasmic localization of intact E-cadherin only in Apc(Min/+), suggesting E-cadherin internalization in these enterocytes. beta-Catenin tyrosine phosphorylation was also increased in Apc(Min/+) enterocytes, consistent with its dissociation from E-cadherin. Furthermore, Apc(Min/+) enterocytes showed a decreased association between beta-catenin and receptor protein-tyrosine phosphatase beta/zeta (RPTPbeta/zeta), and Apc(Min/-) cells demonstrated an association between beta-catenin and receptor protein-tyrosine phosphatase gamma. In contrast to the Apc(Min/+) enterocytes, Apc(Min/-) adenomas displayed increased expression and association of E-cadherin, beta-catenin, and alpha-catenin relative to Apc(+/+) controls. These data show that Apc plays a role in regulating adherens junction structure and function in the intestine. In addition, discovery of these effects in initiated but histologically normal tissue (Apc(Min/+)) defines a pre-adenoma stage of tumorigenesis in the intestinal mucosa.

Reciprocal expression of ERalpha and ERbeta is associated with estrogen-mediated modulation of intestinal tumorigenesis.

Menopausal hormone replacement therapy has been widely used to alleviate the symptoms of menopause and to decrease the detrimental effects of ovarian hormone loss on bone density and cardiovascular health. Multiple studies of colorectal cancer epidemiology also support a role for hormone replacement therapy in prevention of colorectal cancer. We studied the effect of ovariectomy and estrogen replacement on tumor formation in C57BL/6J-Min/+ (Min/+) mice, animals that bear a germline mutation in murine Apc. These mice develop multiple intestinal tumors that show loss of wild-type Apc protein. After ovariectomy, intestinal adenomas in Min/+ mice increased by 77% (P = 0.0004). Ovariectomized Min/+ mice that were treated with a replacement dose of 17beta-estradiol had the same number of tumors as Min/+ mice that were neither castrated nor treated with estrogen replacement (P = 0.85). Examination of estrogen receptor (ER) levels in intestinal tissue by immunoblot showed changes in relative expression levels of ERalpha and ERbeta, with highest ERalpha and lowest ERbeta expression in the normal-appearing intestine of Min/+ mice, and lowest ERalpha and highest ERbeta expression in the enterocytes of animals that received 17beta-estradiol. These results suggest that endogenous estrogens protect against Apc-associated tumor formation and that tumor prevention by 17beta-estradiol is associated with an increase in ERbeta and a decrease in ERalpha expression in the target tissue.

(+)-Catechin inhibits intestinal tumor formation and suppresses focal adhesion kinase activation in the min/+ mouse.

Colorectal cancer is sensitive to dietary influences. Epidemiological data linking high intake of fruits and vegetables to decreased cancer risk have prompted the search for specific plant constituents implicated in tumor prevention. This task is difficult because of the complex chemical composition of plant foods and the multifactorial nature of carcinogenesis. Researchers are aided in this effort by the C57BL/6J-Min/+ (Min/+) mouse, an animal bearing a germline defect in Apc that is similar to the initiating genetic event in the majority of human colorectal cancers. In this study, we treated Min/+ mice with (+)-catechin, a phenolic antioxidant abundant in certain fruits. Administration of (+)-catechin in an AIN-76A diet at doses of 0.1 and 1% decreased the intestinal tumor number by 75 and 71%, respectively. Mechanistic studies linked this effect to (+)-catechin-induced changes in integrin-mediated intestinal cell-survival signaling, including structural alteration of the actin cytoskeleton and decreased focal adhesion kinase (FAK) tyrosine phosphorylation. Immunoblot analysis of small intestine scrapings from Min/+ mice and Apc+/+ wild-type C57BL/6J littermates together with excised Min/+ adenomas showed increased expression of phosphorylated FAK in the macroscopically normal enterocytes of untreated Min/+ mice and adenomas. Confirming the relevance of this signaling pathway, treatment of Min/+ mice with (+)-catechin reduced the expression of phosphorylated FAK to a level similar to the wild-type littermate controls. Thus, the natural abundance and favorable bioavailability of (+)-catechin make it a promising addition to the list of potential colorectal cancer chemopreventive agents.

apolipoprotein-E dependent role for the FAS receptor in early onset Alzheimer's disease: finding of a positive association for a polymorphism in the TNFRSF6 gene.

The TNFRSF6 gene encodes FAS, a cell-surface receptor involved in apoptosis initiation. Elevated levels of FAS have been reported in the brains of Alzheimer's disease (AD) patients. We have tested a G/A polymorphism at position -670 in the TNFRSF6 gene for association with non-familial, early onset Alzheimer's disease (EOAD) by using dynamic allele-specific hybridization. In an initial set of Scottish EOAD cases (n=78) and controls (n=152), we found that, for individuals carrying one or two APOE4 alleles, the homozygous GG-genotype was enriched in the patients (26.7% versus 10.9% in controls). A second study was conducted on an independent set of Scottish individuals (87 EOAD, 358 controls). In this material, the TNFRSF6 GG-genotype frequency was elevated in patients regardless of APOE4 status (28.7% versus 15.1%) and was even more enriched in APOE4 carriers (35.9% versus 15.3%). A combination of the two sample sets (165 cases, 510 controls) gave a significant disease association for the TNFRSF6 GG-genotype that was irrespective of APOE4 (P=0.0020) and that was almost completely attributable to the enrichment present within the set of APOE4 carriers (P=0.0016). This represents an odds ratio of 8.71 for GG-homozygotes carrying at least one APOE4 allele compared with other TNFRSF6 genotypes in APOE4 non-carriers. The TNFRSF6 variation was further explored in Scottish late-onset Alzheimer's disease (n=159) but no associations were found. These results imply that TNFRSF6, in interaction with APOE4, is a genetic risk factor for sporadic EOAD. Hence, the AD risk contributed by APOE4 could be mechanistically related to a pathway in common with FAS-mediated apoptosis.

Population carrier frequency of hMSH2 and hMLH1 mutations.

Knowledge of population carrier frequency for DNA mismatch repair (MMR) gene mutations would contribute to understanding the burden of cancer due to genetic susceptibility, but robust prevalence estimates are lacking. To estimate carrier frequency, we genotyped a cohort of relatives of mutation carriers and determined their colorectal cancer prevalence. Systematic Finnish and US data were combined with Scottish genotype and cancer prevalence data in a Bayesian calculation. The estimated carrier prevalence in the population aged 15-74 years is 1:3139 (95% Cl = 1:1247-1:7626) and these carriers are at high risk of colorectal and other cancers.

Pitfalls in homozygosity mapping.

There is much interest in use of identity-by-descent (IBD) methods to map genes, both in Mendelian and in complex disorders. Homozygosity mapping provides a rapid means of mapping autosomal recessive genes in consanguineous families by identifying chromosomal regions that show homozygous IBD segments in pooled samples. In this report, we point out some potential pitfalls that arose during the course of homozygosity mapping of the enhanced S-cone syndrome gene, resulting from (1) unexpected allelic heterogeneity, so that the region containing the disease locus was missed as a result of pooling; (2) identification of a homozygous IBD region unrelated to the disease locus; and (3) the potential for inflation of LOD scores as a result of underestimation of the extent of inbreeding, which Broman and Weber suggest may be quite common.

Plant phenolics decrease intestinal tumors in an animal model of familial adenomatous polyposis.

Epidemiological studies consistently indicate that consumption of fruits and vegetables lowers cancer risk in humans and suggest that certain dietary constituents may be effective in preventing colon cancer. Plant-derived phenolic compounds manifest many beneficial effects and can potentially inhibit several stages of carcinogenesis in vivo. In this study, we investigated the efficacy of several plant-derived phenolics, including caffeic acid phenethyl ester (CAPE), curcumin, quercetin and rutin, for the prevention of tumors in C57BL/6J-Min/+ (Min/+) mice. These animals bear a germline mutation in the Apc gene and spontaneously develop numerous intestinal adenomas by 15 weeks of age. At a dietary level of 0.15%, CAPE decreased tumor formation in Min/+ mice by 63%. Curcumin induced a similar tumor inhibition. Quercetin and rutin, however, both failed to alter tumor formation at dietary levels of 2%. Examination of intestinal tissue from the treated animals showed that tumor prevention by CAPE and curcumin was associated with increased enterocyte apoptosis and proliferation. CAPE and curcumin also decreased expression of the oncoprotein beta-catenin in the enterocytes of the Min/+ mouse, an observation previously associated with an antitumor effect. These data place the plant phenolics CAPE and curcumin among a growing list of anti-inflammatory agents that suppress Apc-associated intestinal carcinogenesis.

Colon cancer chemopreventive drugs modulate integrin-mediated signaling pathways.

Epidemiological studies of colorectal cancer incidence suggest that the development of this disease can be modulated by dietary factors. Among the micronutrients showing significant efficacy in tumor prevention are polyphenolic antioxidants found in fruits and vegetables. Epidemiological studies also indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) decrease the incidence of colorectal cancer. Integrin-mediated cell-matrix contact provides critical signaling that regulates cellular proliferation, migration, and apoptosis. A signaling mediator for this system is focal adhesion kinase (FAK). Thus far, FAK has not been identified as a target for the inhibitory action of any chemopreventive drug in vivo or in vitro. However, the loss of integrin-mediated cell-matrix contact can induce apoptosis (anoikis), and effective chemopreventive agents typically increase the rate of enterocyte apoptosis. Therefore, we asked whether the NSAID, sulindac sulfide, and the phenolic antioxidant, caffeic acid phenethyl ester (CAPE), affected FAK expression or tyrosine phosphorylation in human colon carcinoma cells. We show that subapoptotic doses of both sulindac sulfide and CAPE caused a rearrangement of the actin cytoskeleton and consequently the loss of focal adhesion plaques. These drugs also reduced the tyrosine phosphorylation of FAK and an associated factor, p130Cas. Steady-state levels of these proteins, together with other relevant signaling molecules, remained unchanged after treatments. Finally, we show that both CAPE and sulindac reduced cell invasion, a functional assay for the inhibition of signaling downstream of FAK. These data strongly suggest that chemopreventive drugs can regulate FAK activity. In conclusion, these novel studies add modulation of integrin-mediated signaling to the spectrum of activity of NSAIDs and plant phenolics.

Population choice in mapping genes for complex diseases.

The difficulty of identifying susceptibility genes for common diseases has polarized geneticists' views on what disease models are appropriate and how best to proceed once high-density genome maps become available. Different disease models have different implications for using linkage or linkage-disequilibrium-based approaches for mapping complex disease genes. We argue that the choice of study population is a critical factor when designing a study, and that genetically simplified isolates are more useful than diverse continental populations under most assumptions.

International variation in reported livebirth prevalence rates of Down syndrome, adjusted for maternal age.

Reported livebirth prevalence of Down syndrome (DS) may be affected by the maternal age distribution of the population, completeness of ascertainment, accuracy of diagnosis, extent of selective prenatal termination of affected pregnancies, and as yet unidentified genetic and environmental factors. To search for evidence of the latter, we reviewed all published reports in which it was possible to adjust both for effects of maternal age and for selective termination (where relevant). We constructed indices that allowed direct comparisons of prevalence rates after standardising for maternal age. Reference rates were derived from studies previously identified as having near complete ascertainment. An index value significantly different from 1 may result from random fluctuations, as well as from variations in the factors listed above. We found 49 population groups for which an index could be calculated. Methodological descriptions suggested that low values could often be attributed to under-ascertainment. A possible exception concerned African-American groups, though even among these most acceptable studies were compatible with an index value of 1. As we have reported elsewhere, there was also a suggestive increase in rates among US residents of Mexican or Central American origin. Nevertheless, our results suggest that "real" variation between population groups reported to date probably amounts to no more than +/-25%. However, reliable data in many human populations are lacking including, surprisingly, some jurisdictions with relatively advanced health care systems. We suggest that future reports of DS livebirth prevalence should routinely present data that allow calculation of an index standardised for maternal age and adjusted for elective prenatal terminations.

Inhibitory effects of caffeic acid phenethyl ester on the activity and expression of cyclooxygenase-2 in human oral epithelial cells and in a rat model of inflammation.

We investigated the mechanisms by which caffeic acid phenethyl ester (CAPE), a phenolic antioxidant, inhibited the stimulation of prostaglandin (PG) synthesis in cultured human oral epithelial cells and in an animal model of acute inflammation. Treatment of cells with CAPE (2.5 microg/ml) suppressed phorbol ester (12-O-tetradecanoylphorbol-13-acetate; TPA) and calcium ionophore (A23187)-mediated induction of PGE2 synthesis. This relatively low concentration of CAPE did not affect amounts of cyclooxygenase (COX) enzymes. CAPE nonselectively inhibited the activities of baculovirus-expressed hCOX-1 and hCOX-2 enzymes. TPA- and A23187-stimulated release of arachidonic acid from membrane phospholipids was also suppressed by CAPE (4-8 microg/ml). Higher concentrations of CAPE (10-20 microg/ml) suppressed the induction of COX-2 mRNA and protein mediated by TPA. Transient transfections using human COX-2 promoter deletion constructs were performed; the effects of TPA and CAPE were localized to a 124-bp region of the COX-2 promoter. In the rat carrageenan air pouch model of inflammation, CAPE (10-100 mg/kg) caused dose-dependent suppression of PG synthesis. Amounts of COX-2 in the pouch were markedly suppressed by 100 mg/kg CAPE but were unaffected by indomethacin. These data are important for understanding the anticancer and anti-inflammatory properties of CAPE.