Short communication: A splice site mutation in CENPU is associated with recessive embryonic lethality in Holstein cattle.

A genome scan for homozygous haplotype deficiency coupled with whole-genome sequence data analysis is a very effective method to identify embryonic lethal mutations in cattle. Among other factors, the power of the approach depends on the availability of a greater amount of genotyping and sequencing data. In the present study, we analyzed the largest known panel of Illumina BovineSNP50 (Illumina Inc., San Diego, CA) genotypes, comprising 401,896 Holstein animals, and we report the mapping of a new embryonic lethal haplotype on chromosome 27, called HH7. We fine mapped the locus in a 2.0-Mb interval using an identical-by-descent approach and analyzed genome sequence data from 4 carrier and 143 noncarrier Holstein bulls to identify the causative mutation. We detected a strong candidate variant in the gene encoding centromere protein U (CENPU), a centromere component essential for proper chromosome segregation during mitosis. The mutant allele is a deletion of 4 nucleotides located at position +3 to +6 bp after the splicing donor site of exon 11. Cross-species nucleotide alignment revealed that the nucleotide at position +3 is entirely conserved among vertebrates, suggesting that it plays an important role in the regulation of CENPU splicing. For verification, we genotyped the candidate variant in 232,775 Holstein individuals and did not observe any homozygotes, whereas 16 were expected (Poisson P-value = 1.1 × 10-7; allele frequency = 0.8%). In addition, genotyping of 250,602 animals from 19 additional breeds revealed that the mutant allele is restricted to animals of Holstein descent. Finally, we estimated the effect of the candidate variant on 2 fertility traits in at-risk mating (i.e., between carrier bulls and daughters of carrier bulls) versus non-risk mating. In agreement with a recessive lethal inheritance pattern, we observed a marked reduction in both conception rate and 56-d nonreturn rate in heifers and cows. The effect on 56-d nonreturn rate suggests that a substantial proportion of homozygous mutants die before 35 d after insemination, which is consistent with the early embryonic death previously reported in CENPU-/- mouse embryos. In conclusion, we demonstrate that with more than 400,000 genotypes, we can map very rare recessive lethal mutations segregating at a frequency below 1% in the population. We recommend performing new analyses regularly as data are accumulating.

A missense mutation (p.Tyr452Cys) in the CAD gene compromises reproductive success in French Normande cattle.

We scanned the genome of 77,815 Normande cattle with different Illumina SNP chips (Illumina Inc., San Diego, CA) to map recessive embryonic lethal mutations using homozygous haplotype deficiency. We detected 2 novel haplotypes on chromosomes 11 and 24 but did not confirm 6 previously reported haplotypes. The one on chromosome 11 showed a marked reduction in conception rates and moderate decrease in nonreturn rate in at-risk versus control mating, supporting late embryonic mortality. After fine mapping and analyzing whole-genome sequences, we prioritized a missense mutation in CAD (g.72399397T>C; p.Tyr452Cys)-a gene encoding a protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) essential for de novo pyrimidine biosynthesis-as a candidate causal variant. This transition mutation replaces a tyrosine residue, which is perfectly conserved among living organisms, with a cysteine residue in the carbamoyl-phosphate synthetase 2 domain of the protein. A single animal was confirmed to be homozygous for the mutation based on Sanger sequencing. However, large-scale genotyping of the candidate variant with the Illumina EuroG10k BeadChip revealed an absence of live homozygotes in a panel of 33,323 Normande animals and an absence of carriers in 348,593 animals from 19 other cattle breeds. These results support recessive embryonic lethality with nearly complete penetrance, as was previously reported in CAD mutants in several eukaryote species. The only homozygous cow had extremely poor udder conformation, suggesting a potential role of CAD in udder development, but no effect was detected when comparing daughter yield deviations of 250 heterozygous bulls with that of 2,912 homozygotes for the ancestral allele. Together, our results showed the importance of large-scale screening for homozygous haplotype deficiency with hundreds of thousands of animals, validating results with an independent data set, and considering unexpected live homozygotes, to avoid both false-positive and false-negative discoveries. These discoveries will be used primarily in mating decisions to avoid at-risk mating. In addition, we recommend including CAD in the breeding objectives of Normande cattle.

Use of meta-analyses and joint analyses to select variants in whole genome sequences for genomic evaluation: An application in milk production of French dairy cattle breeds.

As a result of the 1000 Bull Genome Project, it has become possible to impute millions of variants, with many of these potentially causative for traits of interest, for thousands of animals that have been genotyped with medium-density chips. This enormous source of data opens up very interesting possibilities for the inclusion of these variants in genomic evaluations. However, for computational reasons, it is not possible to include all variants in genomic evaluation procedures. One potential approach could be to select the most relevant variants based on the results of genome-wide association studies (GWAS); however, the identification of causative mutations is still difficult with this method, partly because of weak imputation accuracy for rare variants. To address this problem, this study assesses the ability of different approaches based on multi-breed GWAS (joint and meta-analyses) to identify single-nucleotide polymorphisms (SNP) for use in genomic evaluation in the 3 main French dairy cattle breeds. A total of 6,262 Holstein bulls, 2,434 Montbéliarde bulls, and 2,175 Normande bulls with daughter yield deviations for 5 milk production traits were imputed for 27 million variants. Within-breed and joint (including all 3 breeds) GWAS were performed and 3 models of meta-analysis were tested: fixed effect, random effect, and Z-score. Comparison of the results of within- and multi-breed GWAS showed that most of the quantitative trait loci identified using within-breed approaches were also found with multi-breed methods. However, the most significant variants identified in each region differed depending on the method used. To determine which approach highlighted the most predictive SNP for each trait, we used a marker-assisted best unbiased linear prediction model to evaluate lists of SNP generated by the different GWAS methods; each list contained between 25 and 2,000 candidate variants per trait, which were identified using a single within- or multi-breed GWAS approach. Among all the multi-breed methods tested in this study, variant selection based on meta-analysis (fixed effect) resulted in the most-accurate genomic evaluation (+1 to +3 points compared with other multi-breed approaches). However, the accuracies of genomic evaluation were always better when variants were selected using the results of within-breed GWAS. As has generally been found in studies of quantitative trait loci, these results suggest that part of the genetic variance of milk production traits is breed specific in Holstein, Montbéliarde, and Normande cattle.

Inbreeding and crossbreeding parameters for production and fertility traits in Holstein, Montbéliarde, and Normande cows.

Breed differences and nonadditive genetic effects for milk production traits, somatic cell score (SCS), conception rate (CR), and days to first service (DFS) were estimated for Holstein × Montbéliarde and Holstein × Normande crossbreds, using an animal model adapted from the French genetic evaluation and extended to across-breed analysis. Inbreeding and breed differences were estimated from all purebred recorded cows. Only records from 1,137 herds with Holstein × Montbéliarde crossbred cows and from 1,033 herds with Holstein × Normande crossbred cows were used to estimate crossbreeding parameters. In these herds, crossbred cows represented about 13% of the total number of recorded animals compared with <1% when all herds were considered. Compared with the Montbéliarde and Normande breeds, the Holstein breed was genetically superior for production [+951kg and +2,444kg for 305-d mature-equivalent (305ME) milk, +40kg and +102kg for 305ME fat, +17kg and +54kg for 305ME protein, respectively] and inferior for fertility traits (-12 and -9% for CR, respectively). Inbreeding depression caused loss of yield for production traits (from -32 to -41kg of 305ME milk, -1.4 to -1.7kg of 305ME fat, and -1.1 to -1.3kg of 305ME protein per inbreeding percentage), a small increase in SCS (+0.001 to 0.006) and DFS (+0.12d), and a decrease in CR (-0.27 to -0.44%). Favorable heterosis effects were found for all traits (+494 to 524kg of 305ME milk, +21 to 22kg of 305ME fat, +15 to 16kg of 305ME protein, -0.05 to -0.04 SCS, +2 to 3% for CR, and -3 to 6d of DFS), to such a point that F1 crossbreds could compete with Holstein cows for milk production while having a better fertility. However, recombination losses suggested that some F1 heterosis was lost for backcross cows.

Genetic tools to improve reproduction traits in dairy cattle.

Fertility is a major concern in the dairy cattle industry and has been the subject of numerous studies over the past 20 years. Surprisingly, most of these studies focused on rough female phenotypes and, despite their important role in reproductive success, male- and embryo-related traits have been poorly investigated. In recent years, the rapid and important evolution of technologies in genetic research has led to the development of genomic selection. The generalisation of this method in combination with the achievements of the AI industry have led to the constitution of large databases of genotyping and sequencing data, as well as refined phenotypes and pedigree records. These resources offer unprecedented opportunities in terms of fundamental and applied research. Here we present five such examples with a focus on reproduction-related traits: (1) detection of quantitative trait loci (QTL) for male fertility and semen quality traits; (2) detection of QTL for refined phenotypes associated with female fertility; (3) identification of recessive embryonic lethal mutations by depletion of homozygous haplotypes; (4) identification of recessive embryonic lethal mutations by mining whole-genome sequencing data; and (5) the contribution of high-density single nucleotide polymorphism chips, whole-genome sequencing and imputation to increasing the power of QTL detection methods and to the identification of causal variants.

Consequences of the inscription of local breeds in protected designation of origin cow cheese specifications for the genetic management of the herds.

In the mountainous areas of Europe with a humid climate, dairy cattle production is a major agricultural activity, and the milk is often processed into cheese according to protected designation of origin (PDO) specifications. We analyzed the extent to which PDO specifications and/or a mountain environment influence the spatial distribution of estimated breeding values (EBVs) of cows and the herd-year effects (HYEs) for milk yield (kg/lactation) and protein and fat contents (g/kg), as well as lactation ranks and calving months. The study focused on the northern French Alps. A total of 37 023 lactations, recorded in 2006, in 1153 herds were analyzed. The cows belonged to the Montbéliarde (21 516 lactations), Abondance (10 346 lactations) and Tarentaise (5161 lactations) breeds. The two factors of variation considered were the status of the commune where the farm was located in relation to PDO (three categories: area with no PDO, area with a PDO with no milk yield limit, area with a PDO with a milk yield limit) and 'mountain' environment (four categories based on the European regulation: plain, piedmont, mountain and high mountain). In the Abondance breed, the average lactation rank increased with an increase in production constraints due to the PDO or to a mountain environment. In the Abondance and Tarentaise breeds, grouping of calving in winter was most marked in the 'PDO with a milk yield limit' and 'high-mountain' categories. In the Tarentaise breed, no significant effect on any trait and any variable was found in the 'PDO' or 'mountain' categories. In the other two breeds, the average EBV for milk yield decreased with an increase in the constraints due to PDO, with differences of 226 and 93 kg between extreme values in the Abondance and Montbéliarde breeds, respectively. The average HYE for milk yield was higher in the Abondance breed in the 'PDO with no milk yield limit' category than in the other categories (+740 and +1110 kg, respectively); HYE was not affected by the 'PDO' factor in the Montbéliarde breed or by the 'mountain' factor in either breed. Concerning the protein and fat contents, the effect of the 'PDO' and 'mountain' factors depended on the trait, the variable and the breed. The proportion of individual decisions (the farmer makes the decision) v. collective decisions (breed management) concerning herd dynamics in the face of existing constraints is discussed.

Environmental effects on lactation curves included in a test-day model genetic evaluation.

A large number of environmental factors affect the daily milk production of a cow. Lactation curves included in the French test-day model are modelled as a function of days in milk with semi-parametric curves (splines). The proper modelling of environmental effects in the test-day analysis was investigated using test-day records collected from the first three lactations of French Montbéliarde cows from 1988 to 2005. Four lactation-curve effects describing calving month, length of dry period, age at calving and gestation defined within parity-class were fitted. The shape of lactation curves did not depend on year of calving, which can be modelled as a constant over the whole lactation. To reduce computational requirements and time, data were pre-adjusted in a first step for fixed effects with no year interaction, and then used for genetic evaluation. Correlations for each lactation between 305-day estimates of genetic and permanent environment effects computed using pre-adjustment factors obtained at a 4-year interval were virtually one. The use of a two-step procedure had a very limited impact on the estimates of genetic and permanent environment effects. The minimum correlations with values estimated with a one-step procedure were 0.9984 and 0.9974, respectively. The knowledge of systematic environmental effects affecting the cow daily yield through lactation curves offers interesting perspectives to predict future daily milk production.

Structural and functional lesions in brush border of human polarized intestinal Caco-2/TC7 cells infected by members of the Afa/Dr diffusely adhering family of Escherichia coli.

Diffusely adhering Escherichia coli (DAEC) strains expressing F1845 fimbrial adhesin or Dr hemagglutinin belonging to the Afa/Dr family of adhesins infect cultured polarized human intestinal cells through recognition of the brush border-associated decay-accelerating factor (DAF; CD55) as a receptor. The wild-type Afa/Dr DAEC strain C1845 has been shown to induce brush border lesions by an adhesin-dependent mechanism triggering apical F-actin rearrangements. In the present study, we undertook to further characterize cell injuries following the interaction of wild-type Afa/Dr DAEC strains C1845 and IH11128 expressing fimbrial F1845 adhesin and Dr hemagglutinin, respectively, with polarized, fully differentiated Caco-2/TC7 cells. In both cases, bacterium-cell interaction was followed by rearrangement of the major brush border-associated cytoskeletal proteins F-actin, villin, and fimbrin, proteins which play a pivotal role in brush border assembly. In contrast, distribution of G-actin, actin-depolymerizing factor, and tubulin was not modified. Using draE mutants, we found that a mutant in which cysteine replaces aspartic acid at position 54 conserved binding capacity but failed to induce F-actin disassembly. Accompanying the cytoskeleton injuries, we found that the distribution of brush border-associated functional proteins sucrase-isomaltase (SI), dipeptidylpeptidase IV (DPPIV), glucose transporter SGLT1, and fructose transporter GLUT5 was dramatically altered. In parallel, SI and DPPIV enzyme activity decreased.

Postmitotic differentiation of colon carcinoma caco-2 cells does not prevent reentry in the cell cycle and tumorigenicity.

Our purpose was to analyze whether postmitotic Caco-2 colon cancer cells, although they express most of the differentiation characteristics of terminally differentiated intestinal epithelial cells, still maintain, unlike normal cells, a proliferation potential. Experiments were performed with clone TC7 of the Caco-2 cell line. Dividing TC7 cells are undifferentiated and express detectable levels of thymidylate synthase (TS) and cytochrome P450 1A1 (CYP1A1) mRNAs. When reaching confluence TS and CYP1A1 are downregulated, mitosis is no longer detectable, and differentiation takes place, as demonstrated by appearance and increasing levels of differentiation-associated marker mRNAs (e.g., sucrase-isomaltase (SI), dipeptidylpeptidase-IV (DPP-IV) or GLUT5), increasing activities of sucrase and DPP-IV, and increasing expression, on immunofluorescence analysis, of SI on the surface of the cell layer. Trypsinization and seeding of late postconfluent cells (day 30) expressing complete differentiation results within 24 h in upregulation of TS and CYP1A1, a concomitant and dramatic disappearance of differentiation marker mRNAs associated with a decrease in sucrase and DPP-IV activities, and delayed resumption of cell division. This is followed, after the cells have reached confluence again, by downregulation of TS and CYP1A1 and resumption of cell differentiation. The ability of differentiated cells to dedifferentiate was further confirmed by wounding the cell layer of late postconfluent differentiated cultures: within 24 h following the wound, cells migrate from the wound edge and dedifferentiate, as demonstrated by transmission electron microscopy and disappearance of SI from the cell surface of migrating cells. Late postconfluent differentiated cells were tumorigenic in nude mice. These results raise the question of the validity of the concept of differentiation therapy when applied to colon cancer cells.

Afa/Dr diffusely adhering Escherichia coli C1845 infection promotes selective injuries in the junctional domain of polarized human intestinal Caco-2/TC7 cells.

The Afa/Dr diffusely adhering Escherichia coli (DAEC) C1845 strain harboring the F1845 fimbrial adhesin interacts with the brush border-associated CD55 molecule and promotes elongation of brush border microvilli resulting from rearrangement of the F-actin network. This phenomenon involves the activation of a cascade of signaling coupled to the glycosylphosphatidylinositol-anchored receptor of the F1845 adhesin. We provide evidence that infection of the polarized human intestinal cell line Caco-2/TC7 by strain C1845 is followed by an increase in the paracellular permeability for [(3)H]mannitol without a decrease of the transepithelial resistance of the monolayers. Alterations in the distribution of tight-junction (TJ)-associated occludin and ZO-1 protein are observed, whereas the distribution of the zonula adherens-associated E-cadherin is not affected. Using the recombinant E. coli strains HB101(pSSS1) and -(pSSS1C) expressing the F1845 fimbrial adhesin, we demonstrate that the adhesin-CD55 interaction is not sufficient for the induction of structural and functional TJ lesions. Moreover, using the actin filament-stabilizing agent Jasplakinolide, we demonstrate that the C1845-induced functional alterations in TJs are independent of the C1845-induced apical cytoskeleton rearrangements. The results indicated that pathogenic factor(s) other than F1845 adhesin may be operant in Afa/Dr DAEC C1845.

Listeriolysin O-induced stimulation of mucin exocytosis in polarized intestinal mucin-secreting cells: evidence for toxin recognition of membrane-associated lipids and subsequent toxin internalization through caveolae.

Lysteriolysin O (LLO) induces a microtubule-dependent activation of mucin exocytosis in the human mucin-secreting HT29-MTX. Cholesterol inhibits the LLO-induced mucin exocytosis, whereas the oxidized form of cholesterol had no inhibitory effect. LLO-induced mucin exocytosis inhibited by cholesterol can be restored by enzymatic treatment with cholesterol oxidase. Inhibition of cholesterol synthesis in HT29-MTX cells results in a decrease in the LLO-induced mucin exocytosis. Other lipids such as gangliosides are able to inhibit the LLO-induced mucin exocytosis, suggesting that the binding of the toxin occurs at a multiplicity of membrane-associated lipids acting as receptors. Incubation of the toxin with lipids such as cholesterol or gangliosides does not decrease binding of LLO to target membranes. The present work also provides evidence that the LLO-induced mucin exocytosis develops independently of the pore-forming activity of the toxin. Finally, we demonstrated that the toxin associates with detergent-insoluble glycolipid microdomains (DIGs) containing VIP/21 caveolin, allowing internalization of the toxin and subsequent activation of the mucin exocytosis.

Selecting agent hygromycin B alters expression of glucose-regulated genes in transfected Caco-2 cells.

Incorporation into plasmids of genes conferring resistance to aminoglycoside antibiotics such as hygromycin B is currently utilized for selection in experiments involving gene transfer in eukaryotic cells. Using a subclone of Caco-2 cells stably transfected with an episomal plasmid containing the hygromycin resistance gene, we observed that transformed cells subcultured in the presence of hygromycin B exhibit, compared with the same cells subcultured in antibiotic-free medium, a sixfold increase in the rates of glucose consumption and lactic acid production and dramatic changes, at mRNA and protein level, of the expressions of sucrase-isomaltase and hexose transporter GLUT-2, which are downregulated, contrasting with an upregulation of hexose transporter GLUT-1. This occurs without significant modifications of the differentiation status of the cells, as demonstrated by the normal expression of villin, ZO-1, dipeptidyl peptidase IV, or Na(+)-K(+)-ATPase. The plasmid copy number is, however, the same, whether or not the cells are cultured in the presence of hygromycin B. These results draw attention to the need to consider antibiotic-dependent alterations of metabolism and gene expression in transfection experiments.

GalNAc-alpha-O-benzyl inhibits NeuAcalpha2-3 glycosylation and blocks the intracellular transport of apical glycoproteins and mucus in differentiated HT-29 cells.

Exposure for 24 h of mucus-secreting HT-29 cells to the sugar analogue GalNAc-alpha-O-benzyl results in inhibition of Galbeta1-3GalNAc:alpha2,3-sialyltransferase, reduced mucin sialylation, and inhibition of their secretion (Huet, G., I. Kim, C. de Bolos, J.M. Loguidice, O. Moreau, B. Hémon, C. Richet, P. Delannoy, F.X. Real., and P. Degand. 1995. J. Cell Sci. 108:1275-1285). To determine the effects of prolonged inhibition of sialylation, differentiated HT-29 populations were grown under permanent exposure to GalNAc-alpha-O-benzyl. This results in not only inhibition of mucus secretion, but also in a dramatic swelling of the cells and the accumulation in intracytoplasmic vesicles of brush border-associated glycoproteins like dipeptidylpeptidase-IV, the mucin-like glycoprotein MUC1, and carcinoembryonic antigen which are no longer expressed at the apical membrane. The block occurs beyond the cis-Golgi as substantiated by endoglycosidase treatment and biosynthesis analysis. In contrast, the polarized expression of the basolateral glycoprotein GP 120 is not modified. Underlying these effects we found that (a) like in mucins, NeuAcalpha2-3Gal-R is expressed in the terminal position of the oligosaccharide species associated with the apical, but not the basolateral glycoproteins of the cells, and (b) treatment with GalNAc-alpha-O-benzyl results in an impairment of their sialylation. These effects are reversible upon removal of the drug. It is suggested that alpha2-3 sialylation is involved in apical targeting of brush border membrane glycoproteins and mucus secretion in HT-29 cells.

Resistance to high concentrations of methotrexate and 5-fluorouracil of differentiated HT-29 colon-cancer cells is restricted to cells of enterocytic phenotype.

Adaptation of HT-29 cells to increasing concentrations of methotrexate (MTX) results in the selection of differentiated populations which show sequential dose-dependent changes of their differentiated phenotype with, at the highest concentrations (0.1 and 1 mM), a shift of differentiation from a mucus-secreting to an enterocytic phenotype coinciding with an amplification of the DHFR gene. We show here that DHFR gene amplification itself does not play a role in the shift of differentiation. An alternative explanation is the presence, within the mucus-secreting population, of an undetectable minor population of cells committed to enterocytic differentiation and able to develop resistance to higher concentrations of MTX. This was confirmed by cloning the population of cells resistant to 10 microM MTX. Out of 19 isolated clones, 17 were found to be mucus-secreting and 2 enterocytic. We tested 9 of these clones for their ability to develop resistance to 0.1 mM MTX: only 1 of enterocytic phenotype, was found to develop resistance to this higher concentration and to amplify the DHFR gene. The ability of enterocytic cells to develop resistance to elevated MTX concentration through amplification of the DHFR gene was demonstrated in another enterocytic HT-29 population selected by glucose deprivation. Enterocytic cells resistant to 10 microM MTX were also found, unlike mucus-secreting cells, to be readily adaptable to 5-fluorouracil, this occurring without amplification of the thymidylate synthase gene. Together these results highlight a previously uncharacterized relationship between commitment to enterocytic differentiation of colon-cancer cells and their ability to develop resistance to MTX and 5-fluorouracil.

Resistance of the human colon carcinoma cell line HCT-8 to methotrexate results in selection of cells with features of enterocytic differentiation.

Results obtained previously with the human colon carcinoma cell line HT-29 have shown that the ability of the cells to develop resistance against methotrexate (MTX) or 5-fluorouracil is restricted to cells committed to differentiate. With the aim of investigating whether this observation is cell type-specific or more general, we have extended our studies to another colon cell line, HCT-8. We have compared HCT-8 parental cells and the MTX-resistant subline HCT8-MTX using transmission electron microscopy and immuno-fluorescence detection of markers of cell polarity and differentiation. Post-confluent parental HCT-8 cells appeared highly heterogeneous and occurred in clusters of piled-up cells in which the majority were unpolarized and undifferentiated, with a minority exhibiting features of enterocyte-like cells. In contrast, HCT8-MTX cells formed domes and appeared as a monolayer of polarized cells with tight junctions and a discrete apical brush border which expressed villin, dipeptidylpeptidase-IV, CEA and the epithelial mucin MUC1. Together, our results suggest that, as in HT-29 cells, induction of resistance to MTX of HCT-8 cells results in the selection of differentiated cell types.

Regulation of sucrase-isomaltase and hexose transporters in Caco-2 cells: a role for cytochrome P-4501A1?

Involvement of cytochrome P-4501A1 (CYP1A1) in the regulation of sucrase-isomaltase and hexose transporters was analyzed in low (TC7)- and high (PF11)-glucose-consuming Caco-2 clones. CYP1A1 mRNA is elevated in exponentially growing cells concomitantly with high rates of glucose consumption and high levels of GLUT-1 and GLUT-3 mRNA. After confluency, CYP1A1 is not detectable in TC7 cells; this is associated with a decreased glucose consumption, a downregulation of GLUT-1 and GLUT-3, and an upregulation of sucrase-isomaltase, SGLT-1, GLUT-2, and GLUT-5. In PF11 cells CYP1A1 mRNA remains elevated, along with high glucose consumption, high levels of GLUT-1 and GLUT-3, and minimal expression of sucrase-isomaltase, SGLT-1, GLUT-2, and GLUT-5. Exposure of TC7 cells to inducers of CYP1A1 results in high levels of CYP1A1 mRNA, a 10-fold increase of glucose consumption after confluency, an upregulation of GLUT-1 and GLUT-3, and a downregulation of sucrase-isomaltase, GLUT-2, and, to a lesser extent, SGLT-1 and GLUT-5. These results suggest that activation of CYP1A1, whether spontaneous or drug induced, is involved in the variations of glucose utilization and in the associated modifications of expression of sucrase-isomaltase and hexose transporters.

Two stages of enteropathogenic Escherichia coli intestinal pathogenicity are up and down-regulated by the epithelial cell differentiation.

Pathogens and eucaryotic cells are active partners during the process of pathogenicity. To gain access to enterocytes and to cross the epithelial membrane, many enterovirulent microorganisms interact with the brush border membrane-associated components as receptors. Recent reports provide evidence that intestinal cell differentiation plays a role in microbial pathogenesis. Human enteropathogenic Escherichia coli (EPEC) develop their pathogenicity upon infecting enterocytes. To determine if intestinal epithelial cell differentiation influences EPEC pathogenicity, we examined the infection of human intestinal epithelial cells by JPN 15 (pMAR7) [EAF+ eae+] EPEC strain as a function of the cell differentiation. The human embryonic intestinal INT407 cells, the human colonic T84 cells, the human undifferentiated HT-29 cells (HT-29 Std) and two enterocytic cell lines, HT-29 glc-/+ and Caco-2 cells, were used as cellular models. Cells were infected apically with the EPEC strain and the cell-association and cell-entry were examined by quantitative determination using metabolically radiolabeled bacteria, as well as by light, scanning and transmission electron microscopy. [EAF+ eae+] EPEC bacteria efficiently colonized the cultured human intestinal cells. Diffuse bacterial adhesion occurred to undifferentiated HT-29 Std and INT407 cells, whereas characteristic EPEC cell clusters were observed on fully differentiated enterocytic HT-29 glc-/+ cells and on colonic crypt T84 cells. As shown using the Caco-2 cell line, which spontaneously differentiates in culture, the formation of EPEC clusters increased as a function of the epithelial cell differentiation. In contrast, efficient cell-entry of [EAF+ eae+] EPEC bacteria occurred in recently differentiated Caco-2 cells and decreased when the cells were fully differentiated.(ABSTRACT TRUNCATED AT 250 WORDS)

Three methods to validate the estimation of genetic trend for dairy cattle.

Three methods are proposed to validate the estimation of genetic trend for dairy cattle. With the first method, official evaluations, which are generally derived from a repeatability animal model applied to lactation records from several parities, are compared with evaluations based only on first parity to determine whether estimates of genetic trend are similar. With the second method, daughter yield deviations are analyzed within sire by calving year to determine whether these deviations remain stable over time. With the third method, variations of successive official evaluations are analyzed by regression to determine any systematic trend associated with information from additional daughters. Access to raw data is needed for the first two methods, but only published evaluations are needed for the third method. The three methods were applied to French Holstein evaluations calculated with 1990 and 1993 animal models and validated estimates of genetic trend based on information from the 1993 animal model.

Expression of cytochrome P-450 3A in HT29-MTX cells and Caco-2 clone TC7.

HT-29 sublines and Caco-2 clones were analyzed for the expression of cytochrome P-450 3A. The enzyme was found to be expressed in differentiated HT-29 cells selected by resistance to methotrexate and in one of seven Caco-2 clones, TC7. Its expression parallels the differentiation process, with highest levels being observed at late confluency. P-450 3A mRNA and protein patterns, as well as subcellular distribution, are intermediate between those observed in human adult intestine and fetal liver.

Presence and differential expression of SGLT1, GLUT1, GLUT2, GLUT3 and GLUT5 hexose-transporter mRNAs in Caco-2 cell clones in relation to cell growth and glucose consumption.

Seven clones from the Caco-2 cell line, three isolated from passage 29 (PD7, PD10, PF11) and four from passage 198 (TB10, TC7, TF3, TG6), all of them selected on the basis of differences in the levels of expression of sucrase-isomaltase and rates of glucose consumption, were analysed for the expression of hexose-transporter mRNAs (SGLT1, GLUT1-GLUT5) in relation to the phases of cell growth and the associated variations of the rates of glucose consumption. All clones showed a similar pattern of evolution of the rates of glucose consumption, which decreased from the exponential to the late-stationary phase, but differed, in a 1-40-fold range, in the values observed at late postconfluency. According to these values, clones could be divided into high- (PD10, PF11) and low-glucose-consuming cells (PD7, TB10, TC7, TF3 and TG6). GLUT1 and GLUT3 mRNAs were expressed in all clones and showed a similar pattern of evolution: their level decreased, from the exponential to the stationary phase, in close correlation with the decrease in rates of glucose consumption, with only high-glucose-consuming clones maintaining high levels in the stationary phase. In contrast, SGLT1, GLUT2 and GLUT5 mRNAs were only expressed, like sucrase-isomaltase mRNA, in the low-glucose-consuming clones, and their level increased from the exponential to the stationary phase, in parallel with the differentiation of the cells. GLUT4 was undetectable in all the clones. Glucose deprivation generally resulted in a discrete decrease in the levels of all transporter mRNAs in all clones, one exception being GLUT2, which in the high-glucose-consuming clones is only detectable when the cells are grown in low glucose. These clones should be ideal tools with which to study in vitro, at the single-cell level, how these transporters concur to the utilization and transport of hexoses and how their exclusive or co-ordinated expression is regulated.