Optimized protocol for detection of native, full-length HIV-1 envelope on the surface of transfected cells.

AIMS: Designing therapeutics against the HIV envelope glycoprotein (Env) is only as accurate as the structure of the Env they are targeting. Conserving the structure of the Env trimer is crucial for proper experimental assessment of antibody binding and neutralization. However, Env is notably difficult to express by transfection of a recombinant Env plasmid. To increase surface expression, researchers commonly utilize c-tail mutants of the gp41 transmembrane glycoprotein of HIV-1, but mutations and deletions in this region can impact the overall conformation and stability of the Env trimer. Multiple studies have shown that while tail mutants have higher Env surface expression, they are easier to neutralize and have altered trimer conformations compared with wild-type Env found in vivo on infected cells. To assess and characterize native cell surface Env structures, we sought a protocol that could reliably detect wild-type Env surface expression by flow cytometry. METHODS AND RESULTS: By avoiding fetal bovine serum-based buffers, significantly increasing the amounts of transfected plasmid and Env-specific antibody and by selecting a bright, biotin + streptavidin-PE detection system, we were able to increase the surface expression of transfected Env protein. CONCLUSION: This protocol will allow for more precise assessment of antibody binding, epitope exposure, and Env structure, all of which will contribute to designing more effective vaccines and immunotherapeutics.

A process for energy-efficient high-solids fed-batch enzymatic liquefaction of cellulosic biomass.

The enzymatic hydrolysis of cellulosic biomass is a key step in the biochemical production of fuels and chemicals. Economically feasible large-scale implementation of the process requires operation at high solids loadings, i.e., biomass concentrations >15% (w/w). At increasing solids loadings, however, biomass forms a high viscosity slurry that becomes increasingly challenging to mix and severely mass transfer limited, which limits further addition of solids. To overcome these limitations, we developed a fed-batch process controlled by the yield stress and its changes during liquefaction of the reaction mixture. The process control relies on an in-line, non-invasive magnetic resonance imaging (MRI) rheometer to monitor real-time evolution of yield stress during liquefaction. Additionally, we demonstrate that timing of enzyme addition relative to biomass addition influences process efficiency, and the upper limit of solids loading is ultimately limited by end-product inhibition as soluble glucose and cellobiose accumulate in the liquid phase.

Investigation of country bias in international genetic evaluations using full-brother information.

International Bull Evaluation Service evaluations from May 2005 were examined for country bias by comparing Holstein full-brother families. Countries with > or =25 bulls in multicountry full-brother families were included. The model fit evaluations of US estimated breeding values (EBV) by absorbing full-brother family and producing solutions for country of brothers. For yield and somatic cell score, 24,611 and 22,802 bulls, respectively, were included in the analysis. The study was repeated fitting evaluations on the scales of 9 countries other than the United States. On all countries' scales, bulls from Australia, Germany, Great Britain, and Japan had greater EBV for milk yield than did their full brothers from the United States; Italian bulls had lower EBV. Bulls from Australia, Great Britain, and South Africa had an advantage in EBV for fat yield. For EBV for protein yield, bulls from Germany, Great Britain, Japan, and South Africa had an advantage, whereas bulls from the Netherlands were disadvantaged. For somatic cell score, US bulls were advantaged compared with bulls from South Africa. Significance and rankings of apparent biases were similar across country scales of the international evaluations. Causes of those differences are unknown; differences in incorporation of parental data in national and International Bull Evaluation Service evaluations are a possibility.

Consistency of maturity rate for milk yield across countries and generations.

Differences among bulls in maturity rate of their daughters for milk yield were investigated. Milk records for US Holsteins with first-parity calving dates between 1960 and 1998 were used to calculate 3 evaluations for bulls based on daughter records from parity 1, parities 1 and 2, and parities 1, 2, and 3. The 3 evaluations were used to estimate parity-specific evaluations for parities 2 and 3. Maturity rate of Holstein bull daughters in Canada and The Netherlands was compared with that for daughters of the same bulls in the United States by using official November 2004 Canadian and August 2005 Dutch parity-specific evaluations. For bulls with > or = 500 first-parity daughters, correlations among parity-specific evaluations within country and birth year of bull were 0.88 between parities 1 and 2, 0.84 between parities 1 and 3, and 0.96 between parities 2 and 3 for the United States; 0.90, 0.86, and 0.97, respectively, for Canada; and 0.92, 0.89, and 0.98, respectively, for The Netherlands. Correlations between Canada and the United States for within-country differences between evaluations for parities 1 and 2 were 0.72 for bulls with > or = 50 first-parity daughters and 0.89 for bulls with > or = 500 first-parity daughters; corresponding correlations between The Netherlands and the United States were 0.66 and 0.82. Correlations between countries for differences between evaluations for parities 1 and 3 were slightly less, and corresponding correlations between evaluations for parities 2 and 3 were still lower. To establish whether differences between parity-specific evaluations were genetic, comparisons were made across a generation. Coefficients for regression of son on sire within country and birth years of sire and son for parity-specific evaluations and differences between parity-specific evaluations ranged from 0.35 to 0.53, with standard errors of < or = 0.04. Differences in maturity rate of bull daughters were quite consistent across country, and those differences were transmitted to the sons' daughters. Modeling to account for maturity differences should increase the accuracy of US evaluations and reduce fluctuation between evaluations, especially for bulls with daughters that deviate substantially from the population mean for maturity rate for milk yield.

Major advances in genetic evaluation techniques.

The past quarter-century in genetic evaluation of dairy cattle has been marked by evolution in methodology and computer capacity, expansion in the array of evaluated traits, and globalization. Animal models replaced sire and sire-maternal grandsire models and, more recently, application of Bayesian theory has become standard. Individual test-day observations have been used more effectively in estimation of lactation yield or directly as input to evaluation models. Computer speed and storage are less limiting in choosing procedures. The increased capabilities have supported evaluation of additional traits that affect the net profitability of dairy cows. The importance of traits other than yield has increased, in a few cases due to an antagonistic relationship with yield. National evaluations combined internationally provide evaluations for bulls from all participating countries on each of the national scales, facilitating choices from among many more bulls. Selection within countries has increased inbreeding and the use of similar genetics across countries reduces the previously available outcross population. Concern about inbreeding has prompted changes in evaluation methodology and mating practices, and has promoted interest in crossbreeding. In just the past decade, distribution of genetic evaluations has gone from mailed paper or computer tapes for a limited audience to publicly accessible, request-driven distribution via the Internet. Among the distributed information is a choice of economic indices that combine an increasing array of traits into numbers reflecting breeding goals under different milk-pricing conditions. Considerable progress in genomics and the mapping of the bovine genome have identified markers for some deleterious recessive genes, but broader benefits of marker-assisted selection are still in the future. A possible exception is the proprietary use of DNA testing by semen producers to select among potential progeny test bulls. The collection and analysis of industry-wide data to evaluate genetic merit will continue to be the most important tool for genetic progress into the foreseeable future.

Impact of estimated genetic correlations on international evaluations to predict milk traits.

The Interbull procedure for combining dairy bull evaluations uses estimated genetic correlations between countries. It is important to know whether the resulting difficulties from differences in ranking in each country are justified by improved accuracy relative to a system assuming unity correlations. Data submitted for the May 2001 yield and somatic cell score (SCS) Interbull evaluations were processed once with the usual estimated genetic correlations (E01) and again assuming these correlations to be essentially unity (0.995; U01). The 2 sets of resulting evaluations were compared with August 2004 national evaluations (N04) for bulls not having local evaluations used in the 2001 evaluations. Thus, the examination was of Interbull evaluations from foreign data in predicting national evaluations. Countries in the study for yield were Australia, Canada, France, Germany, Great Britain, Ireland, Italy, The Netherlands, New Zealand, and the United States. Countries included for SCS were Canada, France, Germany, Great Britain, The Netherlands, and the United States. For most countries' evaluations, standard deviations of differences between E01 or U01 and N04 were smaller for E01 by about 5 to 7% and correlations between E01 and N04 were higher by 0.01 or the same as for U01 and N04. Although use of estimated correlations tended to improve prediction, the advantage was small. A previous study had concluded no difference in accuracy for yield but did not include Australia and New Zealand, countries with the lowest correlations with other countries. Excluding bulls from those countries produced results for the other 8 countries more like the previous study, but still favoring E01 slightly. Those 2 countries were not in the SCS data. Estimated genetic correlations improved the prediction of future national evaluations slightly in most countries but more substantially for the evaluations and bulls of Australia and New Zealand.

Impact of maturity rate of daughters on genetic ranking of Holstein bulls.

If genetic evaluations are calculated with a single-trait repeatability model, evaluation changes may be attributed in part to bulls that have daughters that deviate considerably from the typical response to aging. Differences in maturity rate of bull daughters were examined to determine whether they influence change in bull evaluations. Standardized milk records for Holsteins that first calved between 1960 and 1998 were used to calculate 12 tailored predicted transmitting abilities (PTA) for each bull. Predicted transmitting abilities were tailored from combinations of 4 annual cut-off dates and 3 parities. Date screening selected cows first calving before January of 1996, 1997, 1998, or 1999. Parity screening selected milk records from the first 1, 2, or 3 parities. Therefore, 4 evaluations (PTA1) included only first-parity records available for daughters and contemporaries prior to the respective years designated. Four more evaluations (PTA(1,2)) included the records from the first 2 parities for cows first calving prior to those same year cutoffs; likewise, the last 4 evaluations (PTA(1,2,3)) included records from the first 3 parities. Stability of bull evaluations (standard deviations of differences as well as correlations between bull evaluations) across time was compared. Bulls born after 1984 with > or =500 daughters were of interest because of the high precision of evaluations and recent activity. Tailored PTA of those bulls had more uniformity across years in mean records per daughter than did official USDA PTA. Standard deviation of differences in PTA1, PTA(1,2), and PTA(1,2,3) for milk between evaluation years 1996 and 1997 were 28, 28, and 27 kg compared with 63 kg for official evaluations; similarly, between 1996 and 1999, SD were 36, 32, and 32 kg compared with 80 kg. Results suggested that a modification to the current evaluation model to account for maturity rate should reduce fluctuations in individual bull PTA across time and may improve accuracy of evaluations.

Accuracy and stability of national and international somatic cell score evaluations.

Somatic cell score (SCS) evaluations have been published in the United States since 1994 and international evaluations have been available through Interbull since May 2001. The accumulated data provides an opportunity to investigate the accuracy and stability of SCS evaluations. United States domestic evaluations from January 1995 through August 2004, for 21,500 Holstein bulls were considered, over time and sequentially within bull, for changes to the November 2004 evaluation. On average, predicted transmitting ability (PTA) SCS increased (worsened) by 0.002 from earlier evaluations to November 2004. Although bias was small, PTA changes were more than expected based on change in reliability. When looked at sequentially, bulls' earlier evaluations were generally lower (i.e., merit was overestimated) relative to November 2004. Differences were small, and PTA SCS increased steadily with the addition of second-crop daughters. All 524,081 evaluations were considered pairwise providing over 8,000,000 pairs of bulls' evaluations for analysis of PTA differences relative to change in reliability. Agreement of observed and expected SD improved for larger changes in reliability. The November 2004 US and Interbull PTA were matched with US and Interbull PTA from May 2001 (US04, IB04, US01, and IB01, respectively) for 14,652 Holstein bulls. For bulls having only US daughters in IB01, correlations were similar for US01 and IB01 with US04, and IB01 with IB04. Corresponding regressions were all nearly 1.00. For bulls also having nonUS daughters in IB01, correlations with yield deviations calculated for later daughters (used as source of independent data) were higher (0.747 vs. 0.714) for IB01 than for US01. For bulls with added US daughters, correlation with US04 was also higher for IB01 than US01, showing that inclusion of foreign data improved predictive value of SCS evaluations.

Effectiveness of national and regional sire evaluations in predicting future-daughter milk yield.

National and regional bull evaluations were compared for ability to predict standardized milk yield of future daughters. Correlations between evaluations and first-, second-, and third-parity yields of future daughters were calculated within herd-year-month group. Mean correlations with predicted yield of future daughters across the United States were higher for national (0.109, 0.111, and 0.082 for first, second, and third parities, respectively) than for Northeast (0.098, 0.085, and 0.061) Holstein evaluations; corresponding correlations for future Northeast daughters were similar. Bull evaluations based on the first 5 parities of daughters that first calved through 1991 from either California, North Central, Northeast, or Southeast regions as well as from the entire United States were compared with standardized milk yields of daughters that calved later. Correlations with first-, second-, and third-parity yields of future daughters were higher (from 0.001 to 0.011) for national than for regional evaluations. National evaluations were better predictors of future-daughter yield, especially for California and the Southeast. Evaluations based on only first parity were slightly better than those based on the first 5 parities in predicting first-parity yield for 3 of 4 regions but were far less useful in predicting second-or third-parity yield regardless of region. Regional evaluations included fewer bulls because of limited numbers of daughters in each region. The top 100 bulls for genetic merit for milk yield based on regional rankings were inferior to the top 100 bulls based on national ranking by 25 to 173 kg. Reliance on regional rather than national evaluations would reduce current US genetic gains.

Stability of genetic evaluations for active artificial insemination bulls.

Genetic evaluations for milk, fat, and protein from 1995 through August 2003 for 17,987 Holstein bulls in active artificial insemination (AI) service were examined for changes to the November 2003 evaluation. Evaluations for active AI bulls at each of 31 evaluation dates showed mean declines to November 2003. No evidence was seen of a worsening situation over time. Bulls' early evaluations with active AI status showed much larger declines, but this overevaluation diminished and essentially disappeared after 3 yr. The bulls with first active AI evaluations since 1995 were the primary focus of the study. The influx of second-crop daughters did not appear to cause a decline in evaluations for these bulls, attesting to the successful modification to the genetic evaluation system by expanding the genetic variance of short records. Mean declines and the variation of those differences were generally similar by bull sampling organization. A change from active to inactive AI status was generally concurrent with a decline in predicted transmitting ability (PTA). Bulls coded as having standard AI sampling declined less than bulls coded as having other sampling, but the differences were much less than in previous reports. Larger increases in reliability were generally associated with greater declines in PTA, and the magnitude of these changes decreased over time (increasing evaluation number). Change in reliability underpredicted the variance of change in PTA, indicating that other important factors contribute or that the assumptions for the calculation of the expected change in PTA are not met. Declines in estimated merit over time are not sufficient to alter present genetic selection programs, but reasons for the declines continue to elude explanation.

Accuracy of foreign dairy bull evaluations in predicting United States evaluations for yield.

Combining foreign daughter data with domestic information in dairy bull genetic evaluations has been shown to improve prediction of future domestic evaluations for US bulls. This study focused on the accuracy of Interbull evaluations of bulls with only foreign daughters, in predicting the latest domestic evaluations (based on US daughters). August 2003 USDA evaluations based only on US daughters were matched with the most recent February or August Interbull evaluations without US daughters. A minimum reliability of at least 80% for yield and 70% for somatic cell score (SCS) was required in both evaluations. This provided pairs of evaluations based on different daughters (foreign or US) for 286 bulls (60 bulls for SCS). Mean Interbull reliabilities on the US scale were 88% for yield and 84% for SCS, and the mean US reliability for the current evaluations was 91% for yield and 80% for SCS. Correlations between the Interbull and domestic evaluations were 0.90, 0.87, 0.90, and 0.87 for milk, fat, protein, and SCS respectively. Expected correlations were 0.89 for yield and 0.82 for SCS. Mean differences between the Interbull and current domestic evaluations were near zero. These foreign bulls had graduated from progeny test programs (selected for positive Mendelian sampling) before being marketed in the United States. Thus, parent average was a substantial underestimate of merit. The small average differences between evaluations from foreign and US daughters and high correlations indicate that Interbull evaluations based solely on foreign daughters are useful predictors of the US evaluations for yield and SCS, providing accuracy in agreement with reliabilities and much better estimates than the alternative, parent averages.

Performance of Holstein clones in the United States.

Phenotypic and genetic performance of US Holstein clones from embryo splitting (ETS) and nuclear transfer (ETN) was documented for yield and fitness traits. Holstein Association USA has registered 2319 ETS and 215 ETN clones. The number of male clones has decreased. Animals selected for cloning were slightly superior genetically to population mean for yield traits. For females, mean superiority of pedigree merit of ETS clones was 186 kg for milk, 9 kg for fat, and 7 kg for protein compared with the population for the same birth year; for ETN clones, superiority to the population was 165, 10, and 8 kg. Advantage in pedigree merit for male clones generally was slightly greater. The small pedigree advantage for female clones of <1 standard deviation above breed mean indicates that selection of animals to clone was not based primarily on yield. Yield deviations were lower for ETS clones than for their full siblings, which indicates a possible impact of the technology on performance. Yields were lower for ETN clones than for their noncloned full siblings, but differences were small and based on small numbers of clones. Milk composition for cloned cows was not different from that for the population. Estimated genetic merit based on daughter yield was more similar for male clone pairs with apparent identical genotype than for clone pairs from the same biotechnology but nonidentical as confirmed by blood typing. For ETS clones with confirmed identical genotypes, identical genetic merit should be assigned.

Progeny testing and selection intensity for Holstein bulls in different countries.

International Bull Evaluation Service (Interbull) Holstein evaluations from February 1995 through February 2003 were used to determine characteristics of progeny testing for Holstein bulls in Australia, Canada, Denmark, France, Germany, Italy, New Zealand, Sweden, The Netherlands, and the United States. The decision to graduate a bull from progeny test (PT) was assumed to have been made based on the second Interbull evaluation, and graduation was defined as the addition of 200 daughters in the period 2.5 to 4.5 yr later. Mean bull age at PT decision varied across countries by 12 mo. Mean numbers of herds and daughters ranged from 39 to 111 and 54 to 144, respectively. Countries with higher requirements for official evaluations generally had more herds and daughters but older bulls at PT decision. Mean estimated breeding values for yield traits of sires of tested bulls were most similar across countries for fat, differing by only 6.4 kg. The four countries highest for sire protein differed only by 1 kg; however, the range was 12 kg. Percentages of bulls graduated ranged from 4.4 to 14.7 across countries. Selection intensities (standardized selection differentials) tended to be about 1.0 for yield traits. Selection intensities for somatic cell score were generally unfavorable, reflecting selection for negatively correlated yield traits. Reflecting variation in national breeding goals, selection intensities for stature were positive for most countries and highly negative for New Zealand. Selection intensity for fore udder was generally the lowest among the traits examined. All but one country showed positive selection for udder support. These statistics permit comparison of the components of PT programs across country, illustrating possible opportunities for improvement.

Timeliness and effectiveness of progeny testing through artificial insemination.

Progeny-test (PT) programs of US artificial-insemination (AI) organizations were examined to determine timeliness of sampling, PT daughter distribution, rate of return of PT bulls to widespread service, and genetic merit of PT bulls compared with AI-proven and natural-service (NS) bulls. Bull age at semen release and at birth and calving of PT daughters was documented by breed (Ayrshire, Brown Swiss, Guernsey, Holstein, Jersey, and Milking Shorthorn) for bulls that entered AI service since 1960. Mean Holstein bull age at semen release (16 mo) changed little over time, but standard deviations (SD) decreased from 4.0 mo during the 1960s to 2.4 mo during the 1990s. Most Holstein bulls (80%) had semen released by 18 mo. Mean age of Holstein bulls at birth and calving of PT daughters during the 1990s was 29 and 56 mo, respectively (a decline of 4 mo from the 1960s); SD decreased from 6 to 3 mo. Bulls of other breeds usually were older at birth and calving of PT daughters, and SD were larger. Mean Holstein bull age when 80% of PT daughters had been born declined from 36 mo during the 1960s to 31 mo during the early 1990s; for other breeds, bulls showed the same trend but at older ages. Mean Holstein bull age when 80% of PT daughters had calved declined from 65 mo during the 1960s to 59 mo during the 1990s; for other breeds, bulls were older. Percentage of herds with PT daughters has increased over time. For Holsteins, herds with five or more usable first-parity records that had PT daughters with usable records increased from 15% during 1965 to 61% during 1998; percentage of herds with from 1 to 19% PT records increased from 11 to 38%, and percentage of herds with >50% PT daughters increased from 1 to 5%. Percentage of Holstein PT bulls returned to AI service declined to about 12% for bulls with PT entry around 1990; for other breeds, 12 to 23% of most recent PT bulls were returned to service. Percentage of milking daughters that had records usable for genetic evaluation that were sired by PT bulls increased steadily from 10 to 18%, whereas percentage of daughters with usable records that were sired by NS bulls declined from 14 to 7%. Milk yield of daughters of AI-proven bulls was 107 to 200 kg greater than for daughters of PT bulls and 366 to 444 kg greater than for daughters of NS bulls for all years. More extensive and rapid sampling and increased selection intensity of PT programs have led to more rapid genetic progress. More extensive use of AI could increase US producer income by millions of dollars annually.

International dairy bull evaluations expressed on national, subglobal, and global scales.

Genetic evaluations on a global scale were calculated for Holstein bulls using the May 2001 International Bull Evaluation Service (Interbull) evaluations expressed on each of 27 national scales. National scale data were weighted by the country's proportion of total daughters from all bulls (population size) to represent market share. Correlations between Interbull evaluations on national scales and evaluations on a global scale ranged from 0.961 to 0.998 (mean of 0.988). Number of top 100 bulls for protein yield that were in common between national and global scales ranged from 54 to 94 and was related significantly to mean genetic correlation between a country and the other 26 countries. Weighting of evaluations on national scales by population size, inverse of population size weight, or equal weight produced practically the same group of top bulls and correlations among the three global scales were 0.999. Thus, the method for combining Interbull evaluations expressed on national scales had only minor impact and was much less important than use of all data. Subglobal scales were established by a clustering technique that gave two to five groups. For grazing countries or other atypical systems, a subglobal scale may provide better guidance, although a scale representing three grazing countries did not provide the expected improvement over a global scale in the relationship with the three country scales. If conditions in non-participating countries are generally represented by participating countries, most needs are met by a global scale.

Mapping the unique activation function 3 in the progesterone B-receptor upstream segment. Two LXXLL motifs and a tryptophan residue are required for activity.

Progesterone receptors (PR) contain three activation functions (AFs) that together define the extent to which they regulate transcription. AF1 and AF2 are common to the two isoforms of PR, PR-A and PR-B, whereas AF3 lies within the N-terminal 164 amino acids unique to PR-B, termed the "B-upstream segment" (BUS). To define the BUS regions that contribute to AF3 function, we generated a series of deletion and amino acid substitution mutants and tested them in three backgrounds as follows: BUS alone fused to the PR DNA binding domain (BUS-DBD), the entire PR-B N terminus linked to its DBD (NT-B), and full-length PR-B. Analyses of these mutants identified two regions in BUS whose loss reduces AF3 activity by more than 90%. These are associated with amino acids 54-90 (R1) and 120-154 (R2). R1 contains a consensus (55)LXXLL(59) motif (L1) identical to ones found in nuclear receptor co-activators. R2 is adjacent to a second nuclear receptor box (L2) at (115)LXXLL(119) and contains a conserved tryptophan (Trp-140). Their mutation completely disrupts AF3 activity in a promoter and cell type-independent manner. Critical mutations elicited similar effects on all three B-receptor backgrounds. This underscores the probability that these mutations alter a process linking BUS structure to the function of full-length PR-B in a fundamental way.

Overview of progeny-test programs of artificial-insemination organizations in the United States.

Characteristics of progeny-test (PT) programs of artificial insemination (AI) organizations in the United States were examined for changes since 1960. Mean number of bulls that were progeny tested annually by major AI organizations during the mid 1990s was 11 for Ayrshires, 24 for Brown Swiss, 21 for Guernseys, 1261 for Holsteins, 112 for Jerseys, and 3 for Milking Shorthorns. Mean parent age at progeny-test (PT) bull birth decreased except for Milking Shorthorns; mean age of maternal grandsire at bull birth decreased for Holsteins and Jerseys but increased for other breeds. For Holsteins, mean ancestor ages at PT bull birth were 85 mo for sires, 47 mo for dams, and 136 mo for maternal grandsires during the mid 1990s. Percentage of PT bulls that resulted from embryo transfer increased to 78% for Brown Swiss and 80% for Holsteins by 1999. Inbreeding in PT bulls increased over time and ranged from 3.8% for Brown Swiss to 6.4% for Jerseys (5.6% for Holsteins) during the mid 1990s. Mean numbers of daughters and herds per PT bull generally declined except for Holsteins, which increased during the early 1990s to 61 daughters and 44 herds. Mean number of states in which PT daughters are located increased; for Holstein PT bulls during 1994, 22% of daughters were in California, 13% in Wisconsin, 12% in New York, and 10% in Pennsylvania and Minnesota. Percentage of first-lactation cows that were PT daughters increased and ranged from 6% for Milking Shorthorns to 22% for Ayrshires (14% for Holsteins) during 1998. Percentage of PT daughters that were registered declined and was 19% for Holsteins and around 80% for other breeds.

Impact of paternity errors in cow identification on genetic evaluations and international comparisons.

The impact of paternity identification errors on US genetic evaluations and international comparisons of Holstein dairy bulls for milk, fat, and protein yields was investigated. Sire identification was replaced for 11% of Holstein cows that were sired by AI bulls and had records in the US database for national genetic evaluations; US evaluations were computed based on those modified pedigrees and compared with official national evaluations. Estimated breeding values from the data with introduced paternity errors were biased, especially for later generations. Estimated genetic trends decreased by 11 to 15%. Estimates of standard deviations of sire transmitting ability also decreased by 8 to 9%. International multitrait across-country comparisons of bulls were computed based on national evaluations from the United States, Canada, New Zealand, and The Netherlands. Estimates of genetic correlations between the United States and other countries decreased by 0.04 to 0.06 when US evaluations were based on modified pedigree. The resulting bias toward selection of domestic bulls and the inability to identify truly superior animals that are available internationally could decrease potential selection differentials by 0.07 to 0.09 standard deviation units on the US scale, which corresponds to sire breeding values of approximately 50 kg for milk, 3 kg for fat, and 1.7 kg for protein. Losses for the other countries were lower and ranged from 0.02 to 0.05 standard deviation units, because a correlation of less than unity with the United States decreased the impact of US cow paternity errors on the scales of other countries. Although paternity verification is desirable and technically feasible, commercial implementation would require low testing costs.

Retrospective analysis of the accuracy of conversion equations and multiple-trait, across-country evaluations of Holstein bulls used internationally.

In 1995, the multiple-trait across country genetic evaluation procedure replaced regression-based conversion equations as the preferred method for international genetic comparisons of dairy bulls. In the present study, February 1999 estimated breeding values of 632 foreign Holstein bulls that were used in Canada, Germany, Italy, The Netherlands, Sweden, and the US were compared with January 1995 predictions from home country data only. January 1995 predicted breeding values for each importing country were calculated using three methods: the multiple-trait, across-country evaluation procedure; conversion equations based on the multiple-trait, across-country evaluations; and conversion equations based on the Wilmink method. Mean correlations between 1999 estimated breeding values in the importing countries and 1995 predictions from international data were from 0.76 to 0.81 for all methods. The multiple-trait, across-country evaluation procedure is expected to lead to selection of different bulls, because bulls were allowed to be ranked differently in each country, but no significant increase in accuracy of selection was observed. The lack of improvement in accuracy of prediction was most likely due to limitations in data structure. International genetic comparisons are largely driven by data from a relatively small number of evaluated bulls with exported semen. Data from siblings and more distant relatives provide only weak, indirect genetic links between countries, and inclusion of such data seems to provide a minimal improvement in accuracy. Limitations in data structure might be alleviated by methods that define environments by climate or management factors rather than country borders.

Tamoxifen resistant breast cancer: coregulators determine the direction of transcription by antagonist-occupied steroid receptors.

Pharmacological antagonists of steroid receptor action had been thought to exert their effects by a passive mechanism driven principally by the ability of the antagonist to compete with agonist for the ligand binding site. However, recent analyses of antagonist-occupied receptor function suggest a more complex picture. Antagonists can be subdivided into two groups, type I, or pure antagonists, and type II, or mixed antagonists that can have variable transcriptional activity based upon differential dimerization and DNA binding properties. This led us to propose that receptor antagonism may not simply be a passive competition for the ligand binding site, but may, in some cases, involve active recruitment of corepressor or coactivator proteins to produce a mixed transcriptional phenotype. We used a yeast two-hybrid screen to identify proteins that interact specifically with antagonist-occupied receptors. Two proteins have been characterized: L7/SPA, a ribosome-associated protein that is localized in both the cytoplasm and nucleus, but with no known extranucleolar nuclear function; and hN-CoR, the human homolog of the mouse thyroid receptor corepressor mN-CoR. In in vivo transcription assays we show that L7/SPA enhances the partial agonist activity of type II mixed antagonists, and that N-CoR and the related corepressor, SMRT, suppresses it. The coregulators do not affect agonists or pure antagonists. Moreover, the net agonist activity seen with mixed antagonists is a function of the ratio of coactivator to corepressor. Based upon these results, we proposed that in breast tumors the inappropriate agonist activity seen with therapeutic antagonists such as tamoxifen is responsible for the hormone-resistant state. To confirm this, we are quantitating coactivator/corepressor ratios in breast tumor cells lines and clinical breast cancers. Results should provide new insights into the mechanisms underlying the progression of breast cancer to hormone resistance, and may suggest strategies for delaying or reversing this process.