Effects of organic zinc and phytase supplementation in a maize-soybean meal diet on the performance and tissue zinc content of broiler chicks.

1. The purpose of this study was to investigate the effects of Bioplex Zn (a chelated zinc proteinate) and phytase supplementation in a maize-soybean meal diet on the performance and tissue zinc (Zn) content of broiler chicks. Treatment structure consisted of a 2 x 6 factorial arrangement with two inclusions of phytase (0 or 500 PU/kg) and 6 of Bioplex Zn providing 0, 2, 4, 8, 16 and 32 mg Zn/kg diet. A total of 864 chicks were randomly assigned to each of 12 dietary treatments with 6 replicate cages of 12 chicks. 2. Dietary inclusion of phytase increased feed intake, weight gain, plasma Zn content, tibia Zn content, tibia and ash weight. 3. Dietary supplementation of Bioplex Zn linearly increased feed intake, weight gain, gain to feed ratio, plasma Zn concentration, liver Zn concentration, tibia Zn content, tibia and ash weight. 4. An interactive effect of phytase and Bioplex Zn on feed intake, weight gain, tibia Zn concentration and tibia ash weight was found. 5. One slope, straight broken-line analysis of weight gain regressed on the supplemental Zn level provided as Bioplex Zn indicated that 12 mg/kg supplemental Zn without phytase and 7.4 mg/kg supplemental Zn with phytase were required for the optimal weight gain of chicks.

Fidelity of mitotic double-strand-break repair in Saccharomyces cerevisiae: a role for SAE2/COM1.

Errors associated with the repair of DNA double-strand breaks (DSBs) include point mutations caused by misincorporation during repair DNA synthesis or novel junctions made by nonhomologous end joining (NHEJ). We previously demonstrated that DNA synthesis is approximately 100-fold more error prone when associated with DSB repair. Here we describe a genetic screen for mutants that affect the fidelity of DSB repair. The substrate consists of inverted repeats of the trp1 and CAN1 genes. Recombinational repair of a site-specific DSB within the repeat yields TRP1 recombinants. Errors in the repair process can be detected by the production of canavanine-resistant (can1) mutants among the TRP1 recombinants. In wild-type cells the recombinational repair process is efficient and fairly accurate. Errors resulting in can1 mutations occur in <1% of the TRP1 recombinants and most appear to be point mutations. We isolated several mutant strains with altered fidelity of recombination. Here we characterize one of these mutants that revealed an approximately 10-fold elevation in the frequency of can1 mutants among TRP1 recombinants. The gene was cloned by complementation of a coincident sporulation defect and proved to be an allele of SAE2/COM1. Physical analysis of the can1 mutants from sae2/com1 strains revealed that many were a novel class of chromosome rearrangement that could reflect break-induced replication (BIR) and NHEJ. Strains with either the mre11s-H125N or rad50s-K81I alleles had phenotypes in this assay that are similar to that of the sae2/com1Delta strain. Our data suggest that Sae2p/Com1p plays a role in ensuring that both ends of a DSB participate in a recombination event, thus avoiding BIR, possibly by regulating the nuclease activity of the Mre11p/Rad50p/Xrs2p complex.

Testing legal assumptions regarding the effects of dancer nudity and proximity to patron on erotic expression.

A field experiment was conducted in order to test the assumptions by the Supreme Court in Barnes v. Glen Theatre, Inc. (1991) and the Ninth Circuit Court of Appeals in Colacurcio v. City of Kent (1999) that government restrictions on dancer nudity and dancer-patron proximity do not affect the content of messages conveyed by erotic dancers. A field experiment was conducted in which dancer nudity (nude vs. partial clothing) and dancer-patron proximity (4 feet; 6 in.; 6 in. plus touch) were manipulated under controlled conditions in an adult night club. After male patrons viewed the dances, they completed questionnaires assessing affective states and reception of erotic, relational intimacy, and social messages. Contrary to the assumptions of the courts, the results showed that the content of messages conveyed by the dancers was significantly altered by restrictions placed on dancer nudity and dancer-patron proximity. These findings are interpreted in terms of social psychological responses to nudity and communication theories of nonverbal behavior. The legal implications of rejecting the assumptions made by the courts in light of the findings of this study are discussed. Finally, suggestions are made for future research.

The Saccharomyces cerevisiae RDN1 locus is sequestered from interchromosomal meiotic ectopic recombination in a SIR2-dependent manner.

Meiotic ectopic recombination occurs at similar frequencies among many sites in the yeast genome, suggesting that all loci are similarly accessible to homology searching. In contrast, we found that his3 sequences integrated in the RDN1 (rDNA) locus were unusually poor participants in meiotic recombination with his3 sequences at other sites. We show that the low rate of meiotic ectopic recombination resulted from the poor ability of RDN1::his3 to act as a donor sequence. SIR2 partially repressed interchromosomal meiotic ectopic recombination at RDN1, consistent with its role in regulating recombination, gene expression, and retrotransposition within RDN1. We propose that RDN1 is physically sequestered from meiotic homology searching mechanisms.

The Saccharomyces cerevisiae DNA recombination and repair functions of the RAD52 epistasis group inhibit Ty1 transposition.

RNA transcribed from the Saccharomyces cerevisiae retrotransposon Ty1 accumulates to a high level in mitotically growing haploid cells, yet transposition occurs at very low frequencies. The product of reverse transcription is a linear double-stranded DNA molecule that reenters the genome by either Ty1-integrase-mediated insertion or homologous recombination with one of the preexisting genomic Ty1 (or delta) elements. Here we examine the role of the cellular homologous recombination functions on Ty1 transposition. We find that transposition is elevated in cells mutated for genes in the RAD52 recombinational repair pathway, such as RAD50, RAD51, RAD52, RAD54, or RAD57, or in the DNA ligase I gene CDC9, but is not elevated in cells mutated in the DNA repair functions encoded by the RAD1, RAD2, or MSH2 genes. The increase in Ty1 transposition observed when genes in the RAD52 recombinational pathway are mutated is not associated with a significant increase in Ty1 RNA or proteins. However, unincorporated Ty1 cDNA levels are markedly elevated. These results suggest that members of the RAD52 recombinational repair pathway inhibit Ty1 post-translationally by influencing the fate of Ty1 cDNA.

Restoration of in vitro responses in platelets stored in plasma.

Conventional platelet storage in a blood bank is up to 5 days at room temperature in plasma. We investigated the optimal medium for assessing the quality of stored platelets by comparing in vitro test responses after resuspension in autologous plasma prepared from platelet-rich plasma after 5 days of storage at room temperature, autologous plasma stored cell-free for 5 days at room temperature, or autologous plasma stored cell-free for 5 days at -20 degrees C. Five-day-old platelets were prepared from aliquots of the same unit and resuspended in I of the 3 plasma preparations. The platelet-plasma mixtures were monitored for changes in pH, mean platelet volume, hypotonic shock response, P-selection expression, and aggregation. There were statistically significant differences between platelets resuspended in original plasma and platelets resuspended in either plasma stored cell-free at room temperature or frozen, with regard to hypotonic shock response, agonist-induced aggregation, and P-selectin expression. Plasma stored with platelets for 5 days yielded inferior platelet function test responses when compared with plasma stored cell-free at room temperature or frozen. Therefore, for direct comparison of platelet responses following novel storage methods, the resuspending plasma should be stored under the same conditions as the control platelet unit.

Peroxynitrite-induced tyrosine nitration and phosphorylation in human platelets.

Peroxynitrite (ONOO-) induces nitration of tyrosine residues and inhibits tyrosine phosphorylation in cell free systems. We investigated the effect of peroxynitrite on protein tyrosine nitration and phosphorylation in resting or thrombin-activated platelets. Peroxynitrite (150 microM) rapidly induced tyrosine nitration of 187, 164, 113, 89, and 61 kDa proteins in gel-filtered platelets which persisted up to 4.5 h. Repeated exposure of platelets to peroxynitrite produced increasing levels of nitration. Peroxynitrite also rapidly increased tyrosine phosphorylation of 120, 117, 95, 80-85, and 70 kDa platelet proteins, but this decreased by 5 min. The same pattern of tyrosine phosphorylation, but with higher intensity, was induced by thrombin in control platelets. Pretreatment of platelets with peroxynitrite decreased thrombin-induced tyrosine phosphorylation at 0.05 and 1 U/ml thrombin but not at 2 U/ml thrombin. Platelet activation responses such as P-selectin expression, serotonin secretion, and aggregation were also decreased by peroxynitrite treatment at low thrombin concentrations. Peroxynitrite exposure and tyrosine nitration decreased platelet sensitivity to thrombin but did not absolutely prevent tyrosine phosphorylation and other platelet responses.

Endogenous ADP prevents PGE1-induced tyrosine dephosphorylation of focal adhesion kinase in thrombin-activated platelets.

Prostaglandin E1(PGE1) inhibits tyrosine phosphorylation induced by low thrombin concentration (0.05 U/ml), but this is overcome by a high thrombin (2.0 U/ml) concentration. Thromboxane A2 and ADP are endogenous platelet agonists released during platelet activation which potentiate platelet responses. We investigated how these endogenous agonists influenced the effects of PGE1 on thrombin (2.0 U/ml)-induced tyrosine phosphorylation by removing released ADP with apyrase (2.0 U/ml) and by inhibiting thromboxane A2 synthesis with indomethacin (1 microM). Adding PGE1 (1 microM) before thrombin in apyrase/indomethacin(A/I)-treated platelets selectively prevented thrombin-induced tyrosine phosphorylation of a 117 kDa protein while other substrates were not affected. This selective effect was evident only in the presence of apyrase and was not dependent on indomethacin. Addition of PGE1 to A/I-treated platelets after thrombin also caused selective tyrosine dephosphorylation of the 117 kDa protein. Conditions which prevented thrombin-induced 117 kDa protein tyrosine phosphorylation also decreased fibrinogen binding to platelets. The 117 kDa protein was identified as the focal adhesion kinase (FAK) by immunoprecipitation with a monoclonal antibody to FAK and by absence of its tyrosine phosphorylation in the presence of RGDS peptide which inhibits fibrinogen binding and platelet aggregation. Thus, released endogenous ADP selectively prevents PGE1-mediated tyrosine dephosphorylation of platelet FAK most likely by stabilizing fibrinogen binding to platelets.

Thapsigargin-induced calcium influx in the absence of detectable tyrosine phosphorylation in human platelets.

Tyrosine phosphorylation is a potential mechanism for mediating store-operated calcium (SOC) influx in platelets and other nonexcitable cells. Thapsigargin induces calcium-dependent tyrosine phosphorylation and SOC influx in platelets. We prevented thapsigargin-induced tyrosine phosphorylation by buffering cytosolic calcium rise with the calcium chelator 1, 2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetomethoxyester (BAPTA-AM). Calcium influx, induced by thapsigargin and measured by 45Ca2+ accumulation, persisted in BAPTA-loaded platelets in the absence of tyrosine phosphorylation. This calcium influx was blocked by the SOC influx inhibitor SKF-96365. Tyrosine kinase inhibitors have been used to demonstrate a role for tyrosine phosphorylation in SOC influx. We compared the effects of four tyrosine kinase inhibitors genistein, methyl-2, 5-dihydroxycinnamate (erbstatin analog), tyrphostin A47, and lavendustin A, on thapsigargin-induced tyrosine phosphorylation in control platelets and on thapsigargin-induced SOC influx into BAPTA-loaded platelets in absence of tyrosine phosphorylation. Tyrphostin A47 prevented all measurable tyrosine phosphorylation in control platelets, but did not decrease calcium influx into BAPTA-loaded platelets. Genistein and the erbstatin analog were poor inhibitors of tyrosine phosphorylation but decreased SOC influx into BAPTA-loaded platelets to 55.8 +/- 3% and 51.9 +/- 7.5% of control, respectively. Lavendustin A did not decrease tyrosine phosphorylation or calcium influx. Thus, thapsigargin-induced SOC influx can occur without detectable tyrosine phosphorylation and the inhibition of SOC influx by tyrosine kinase inhibitors does not correlate with their ability to prevent tyrosine phosphorylation.

DNA synthesis errors associated with double-strand-break repair.

Repair of a site-specific double-strand DNA break (DSB) resulted in increased reversion frequency for a nearby allele. Site-specific DSBs were introduced into the genome of Saccharomyces cerevisiae by the endonuclease encoded by the HO gene. Expression of the HO gene from a galactose-inducible promoter allowed efficient DNA cleavage at a single site in large populations of cells. To determine whether the DNA synthesis associated with repair of DSBs has a higher error rate than that associated with genome duplication, HO-induced DSBs were generated 0.3 kb from revertible alleles of trp1. The reversion rate of the trp1 alleles was approximately 100-fold higher among cells that had experienced an HO cut than among uninduced cells. The reverted allele was found predominantly on the chromosome that experienced the DNA cleavage.

A ste12 allele having a differential effect on a versus alpha cells.

The transcriptional activator Ste12p is a key component of the yeast pheromone response pathway: phosphorylated as a consequence of signal transduction, it activates transcription of genes that promote mating and the subsequent fusion of the two cell types a and alpha. Activation by Ste12p requires three types of protein-protein interaction between DNA-binding activator proteins: (1) Ste12p by itself can induce non-cell-type-specific genes involved in mating; (2) cooperation of the transactivator Mcm1p with Ste12p induces a-specific genes; and (3) formation of a complex of the activator proteins Mcm1p and alpha 1 (a transcriptional activator of alpha-specific genes) with Ste12p is believed to induce alpha-specific genes. We isolated and characterized a partially functional ste12 allele (ste12-T50), that is defective only in the activation of alpha-specific genes. ste12-T50 was isolated as a second-site mutation conferring the a mating phenotype on mat alpha 2 mutant cells. In mat alpha 2 cells, where due to the lack of repressor, alpha 2, both sets of cell-type-specific genes are expressed, ste12-T50 apparently tips the balance in favor of a-specific gene expression. Thus, mat alpha 2 ste12-T50 cells mate like a cells. Additional ste12 mutants that confer the a mating phenotype on mat alpha 2 cells have also been isolated.

Isolation, identification and characterization of the FUN12 gene of Saccharomyces cerevisiae.

We have cloned and characterized the FUN12 gene which is found on chromosome 1 of Saccharomyces cerevisiae. The complete nucleotide (nt) sequence of the cDNA and the genomic clones shows that FUN12 is expressed as a 3.7-kb message and should encode a 97 kDa-protein. Immunoprecipitations using antipeptide antibodies showed that the cells contain a Fun12p of this size. The databases contain no nt sequences that are homologous to FUN12 and no protein homologous to Fun12p. Gene disruption experiments showed that FUN12 is an essential gene.

Mapping and sequencing of two yeast genes belonging to the ATP-binding cassette superfamily.

ATP-binding cassette (ABC) transporters share significant sequence identity within their ATP-binding domains. Degenerate oligonucleotides based on highly conserved portions of the ATP-binding domain genes were used to clone portions of two members of the ABC gene superfamily from Saccharomyces cerevisiae DNA. These genes were designated MDL1 and MDL2 (for multidrug resistance-like). Each MDL gene is predicted to encode a single set of transmembrane domains and a single ATP-binding domain, thus the MDL gene products are 'half-molecule' ABC proteins. The two genes were mapped to precise regions on chromosomes XII and XVI and show a considerable similarity to the mammalian P-glycoprotein/multidrug resistance (MDR) and peptide transporter (TAP) genes. Preliminary analysis of null mutants constructed by gene replacement has indicated that the MDL genes are not essential for viability of yeast. The sequences have been deposited in the GenBank data library under Accession Numbers L16958 (Locus YSCBCSA) and L16959 (Locus YSCBCSB).

Ends-in vs. ends-out recombination in yeast.

Integration of linearized plasmids into yeast chromosomes has been used as a model system for the study of recombination initiated by double-strand breaks. The linearized plasmid DNA recombines efficiently into sequences homologous to the ends of the DNA. This efficient recombination occurs both for the configuration in which the break is in a contiguous region of homology (herein called the ends-in configuration) and for "omega" insertions in which plasmid sequences interrupt a linear region of homology (herein called the ends-out configuration). The requirements for integration of these two configurations are expected to be different. We compared these two processes in a yeast strain containing an ends-in target and an ends-out target for the same cut plasmid. Recovery of ends-in events exceeds ends-out events by two- to threefold. Possible causes for the origin of this small bias are discussed. The lack of an extreme difference in frequency implies that cooperativity between the two ends does not contribute to the efficiency with which cut circular plasmids are integrated. This may also be true for the repair of chromosomal double-strand breaks.

Preoperative and postoperative nipple-areola sensibility in patients undergoing reduction mammaplasty.

Objective data on nipple and areola sensibility are scarce. For women with macromastia, there is little published information available indicating the incidence and intensity of postoperative nipple and areola sensibility. This prospective study was undertaken to evaluate nipple and areola sensibility in "small-breasted" control subjects as well as in patients with macromastia before and after reduction mammaplasty. Preoperative and postoperative Semmes-Weinstein pressure threshold testing was performed on 84 breasts in 43 patients and on 12 breasts of A or B cup size in the control group. The patients underwent reduction mammaplasty by the central parenchymal pedicle technique or the laterally based inferior pedicle technique. Nipple-areola sensibility was retained in 96 percent of breasts when the excision of breast tissue was less than 550 gm and 85 percent of breasts when the excision was greater than 550 gm. Overall, nipple-areola sensibility was retained in 90.5 percent of the 84 breasts tested. In those breasts in which nipple-areola sensibility was retained after surgery, there was no statistical difference in the preoperative and postoperative Semmes-Weinstein pressure threshold values. When pressure threshold values were compared in patients who had less than 550 gm of tissue resected, patients who had greater than 550 gm of tissue resected, and controls who had not undergone surgery, the trend of decreasing nipple-areola sensibility with increasing breast size was clearly seen.

Reduction mammaplasty improves symptoms of macromastia.

The condition macromastia has not been defined and characterized precisely by the medical community. Whether the patient with hypertrophic breasts is a candidate for or can be helped by reduction mammaplasty is unclear to both the medical and the lay community. A prospective study of 39 women undergoing reduction mammaplasty surgery was initiated to answer these questions. Patients rated the severity of their somatic pain symptoms and discomfort before reduction mammaplasty and again after complete recovery. The severity of their symptoms and complaints was numerically graded and analyzed. These data were compared with similar data obtained from 40 "small-breasted" women of similar age. Headache, neck pain, back pain, shoulder pain, and bra strap groove pain were present in 60 to 92 percent of patients, and 97 percent of patients had at least three of these pain symptoms preoperatively. All the patients had reduction of their pain symptomatology postoperative, and 25 percent of the study patients had total elimination of pain symptoms by reduction mammaplasty. The postoperative incidence and severity of pain symptoms and discomfort complaints were statistically equivalent to or less than the levels in the control group.

Alpha granule proteins in type I von Willebrand's disease.

Platelet von Willebrand factor (vWf) is located in the alpha granules. Individuals with type I von Willebrand's disease (vWd) with prolonged bleeding times are best discriminated from those who have normal bleeding times by the normal level of platelet vWf ristocetin cofactor activity (vWf activity) and, to a lesser extent, by their platelet vWf antigen content. We have studied the content of adhesive proteins and platelet factor-4 (PF-4), and beta-thromboglobulin (beta TG) in the platelet alpha granules of types I and III vWd patients to determine if other alterations in alpha granule contents of proteins occur in vWd. We found that type I vWd patients with prolonged or normal bleeding times could not be differentiated on the basis of their platelet levels of beta TG, PF-4, fibronectin, or fibrinogen. The levels of the alpha granule constituents in the type I vWd patient were similar to normal except for the platelet fibrinogen concentration. Patients with type I vWd, regardless of the level of platelet vWf activity of antigen, had increased levels of platelet fibrinogen. The patients with type III vWd who had undetectable levels of platelet and plasma vWf also had increased levels of platelet fibrinogen. In our study we could not attribute the variation in the platelet vWf activity and antigen in type I vWd to the size of the alpha granule pool as determined by the measurement of other alpha granule proteins. The mechanism(s) of increased platelet fibrinogen in these vWd patients is at present unknown.

Recombination initiated by double-strand breaks.

The HO endonuclease was used to introduce a site-specific double-strand break (DSB) in an interval designed to monitor mitotic recombination. The interval included the trp1 and his3 genes inserted into chromosome III of S. cerevisiae between the CRY1 and MAT loci. Mitotic recombination was monitored in a diploid carrying heteroalleles of trp1 and his3. The normal recognition sites for the HO endonuclease were mutated at the MAT alleles and a synthetic recognition site for HO endonuclease was placed between trp1 and his3 on one of the chromosomes. HO-induced cleavage resulted in efficient recombination in this interval. Most of the data can be explained by double-strand gap repair in which the cut chromosome acts as the recipient. However, analysis of some of the recombinants indicates that regions of heteroduplex were generated flanking the site of the cut, and that some recombinants were the result of the cut chromosome acting as the genetic donor.

Ty element-induced temperature-sensitive mutations of Saccharomyces cerevisiae.

Temperature-sensitive mutants of Saccharomyces cerevisiae were isolated by insertional mutagenesis using the HIS3 marked retrotransposon TyH3HIS3. In such mutants, the TyHIS3 insertions are expected to identify loci which encode genes essential for cell growth at high temperatures but dispensable at low temperatures. Five mutations were isolated and named hit for high temperature growth. The hit1-1 mutation was located on chromosome X and conferred the pet phenotype. Two hit2 mutations, hit2-1 and hit2-2, were located on chromosome III and caused the deletion of the PET18 locus which has been shown to encode a gene required for growth at high temperatures. The hit3-1 mutation was located on chromosome VI and affected the CDC26 gene. The hit4-1 mutation was located on chromosome XIII. These hit mutations were analyzed in an attempt to identify novel genes involved in the heat shock response. The hit1-1 mutation caused a defect in synthesis of a 74-kD heat shock protein. Western blot analysis revealed that the heat shock protein corresponded to the SSC1 protein, a member of the yeast hsp70 family. In the hit1-1 mutant, the TyHIS3 insertion caused a deletion of a 3-kb DNA segment between the delta 1 and delta 4 sequences near the SUP4 locus. The 1031-bp wild-type HIT1 DNA which contained an open reading frame encoding a protein of 164 amino acids and the AGG arginine tRNA gene complemented all hit1-1 mutant phenotypes, indicating that the mutant phenotypes were caused by the deletion of these genes. The pleiotropy of the HIT1 locus was analyzed by constructing a disruption mutation of each gene in vitro and transplacing it to the chromosome. This analysis revealed that the HIT1 gene essential for growth at high temperatures encodes the 164-amino acid protein. The arginine tRNA gene, named HSX1, is essential for growth on a nonfermentable carbon source at high temperatures and for synthesis of the SSC1 heat shock protein.

Disruption of a silencer domain by a retrotransposon.

A galactose-inducible Ty element carrying the HIS3 gene has been used as an insertional mutagen to generate alpha-factor resistant mutants. This collection of Ty-induced mutations includes insertions into the gene for the alpha-factor receptor (STE2), several nonspecific STE genes, and mutations that lead to the expression of the normally silent HML alpha locus. The hml alpha "on" mutations fall into two classes, those that disrupt trans-acting regulators involved in silencing HML alpha and a novel class of mutations that activate HML alpha by insertion at that locus. The hml alpha::Ty "on" mutations illustrate the unusual ability of these retrotransposons to activate genes by overcoming gene silencing mechanisms. The hml alpha::Ty "on" mutations include examples of multimeric Ty arrays. Single Ty and solo delta insertion derivatives of these Ty multimers restore the ability of the silencing mechanism to repress HML alpha.