The LightTyper: high-throughput genotyping using fluorescent melting curve analysis.

Instrumentation, chemistry, and software for high-throughput genotyping using fluorescent melting curves are described. The LightTyper system provides post-amplification genotyping within 10 min using samples in 96- or 384-well microplate formats. The system is homogenous because all reagents are added at the beginning of the reaction and there is no sample manipulation between amplification and genotyping. High-resolution melting curves are achieved by slow and steady heating. As samples are heated, panels of blue light-emitting diodes excite the probes, and fluorescence emission is acquired with a cooled charge-coupled device camera. A variety of probe chemistries are compatible for genotyping on the LightTyper, including dsDNA dyes, single-labeled probes, and fluorescence resonance energy transfer systems. Genotyping is performed automatically, and each sample is given a score reflecting the similarity of the genotype to the standards provided. Standard genotypes can be selected from within the run or imported from other files. Samples and genotypes can be grouped to allow multiple-allele detection on one or many samples. The utility of the LightTyper is illustrated by genotyping samples for the Factor V Leiden mutation and for mutations in the CFTR gene.

Performance of two strains of laying hens fed ground and whole barley with and without access to insoluble grit.

The live performance from 19 to 43 wk of age of two strains of commercial White Leghorn hens fed two levels of whole barley (0 or 60%) and insoluble grit (0 or 4 g/bird per wk) was compared. The 0 and 60% whole barley diets differed only in feed form and were formulated to the same nutrient specifications. No dilution of nutrients or ingredients occurred. The 0% whole barley diet was fed in mash form. The 60% whole barley diet was fed as whole grain and mash concentrate blended into a complete diet and fed in the same feed trough. Feeding whole barley reduced egg production, feed efficiency, and egg specific gravity and increased feed intake, egg weight, and body weight gain. Access to insoluble grit had no effect on any of the production variables measured. The two strains of hens responded similarly to whole barley but differed in feed intake, feed efficiency, egg weight, egg specific gravity, and body weight gain. Feeding whole barley combined with a mash concentrate depressed hen performance compared to birds fed a similar diet in mash form. Strain of hen and access to insoluble grit did not alter the response to feeding whole barley.

Feeding broiler chickens wheat and barley diets containing whole, ground and pelleted grain.

The effect of grain form (whole, mash, or pelleted) on the live performance of broiler chickens was determined. In the first trial, six regimens compared the feeding of whole wheat: 1) 0% of diet for the whole trial (0 to 48 d); 2) 5% at 6 d, 20% at 13 d, 35% at 27 d; 3) 5% at 6 d, 35% at 13 d, 50% at 27 d; 4) 5% at 0 d, 20% at 6 d, 35% at 13 d, 50% at 27 d; 5) 5% at 6 d, 50% at 13 d, 65% at 27 d; and 6) 5% at 0 d, 20% at 6 d, 50% at 13 d, 65% at 27d. Each feeding regimen was replicated with steam-pelleted and mash supplements. None of the feed was diluted. The second trial was similar, except that whole barley was fed instead of whole wheat. Feeding mash supplements slowed growth at all ages and lowered mortality caused by sudden death syndrome and ascites plus right heart failure. Cumulative feed:gain was increased by feeding mash supplements in Trial 1. Total weight gain was unaffected by feeding whole wheat but was decreased by most levels of whole barley. Whole-grain diets increased cumulative feed:gain. Feeding whole wheat decreased skeletal problems. Whole-grain diets increased gizzard size but did not alter carcass yield. Feeding whole-grain and mash supplements caused at least a temporary loss in growth rate and feed efficiency but in some cases improved bird health.

Influence of whole barley and grit on live performance and health of turkey toms.

Live performance to 96 d was compared for 1,584 turkey toms reared on diets containing four levels of whole barley and two levels of insoluble grit (0 or 9 g/bird per wk). Nutrient specifications for all diets were similar. The six dietary treatments were 1) 0% whole barley plus grit, 2) Treatment 1 minus grit, 3) grit plus 5% whole barley at 0 d increasing to 35% by 19 d, 4) grit plus 5% whole barley at 0 d increasing to 50% by 19 d, 5) Treatment 4 minus grit, and 6) grit plus 5% whole barley at 19 d increasing to 50% by 40 d. The concentrate blended with the whole barley was fed as crumbles or pellets. Nutrients were not diluted. Compared to the control treatments, feeding 35% or more whole barley temporarily reduced weight gain and increased feed:gain prior to 68 d. Cumulative weight gain was reduced in Treatments 3 and 5 compared to treatments in which no barley was fed. Cumulative feed:gain was increased in Treatment 5 compared to Treatments 2 and 3. Total mortality and leg and skeletal problems were reduced in treatments where whole grain slowed early growth rate. Feeding grit had no effect on the live performance of birds fed similar levels of whole barley. In treatments in which whole barley was introduced at 0 d, gizzard pH was decreased at 18 d, and gizzard weight was increased at 32 d. Jejunal viscosity was unaffected by dietary treatment.

A study of electrocardiographic patterns in a population of commercial broiler chickens.

A study was completed to characterize the electrocardiographic (ECG) patterns of male broiler chickens. Data were collected from 300 commercial broilers. ECG readings were collected from all birds between 12 and 15 days of age and then twice more at 10-day intervals. The measurements. included heart rate and rhythm, QRS complex duration, amplitude and mean electrical axis (MEA), incidence of ascites and incidence of sudden death syndrome (SDS). Eight birds died from SDS and 4 birds died from ascites. Twelve birds were condemned for ascites at the processing plant. The overall population heart rate declined with age. Birds that died of SDS had a higher heart rate, whereas those that developed ascites had a lower heart rate than the remainder of the population. The normal MEA was found to be between 0 degree and 180 degrees. On average 30% of birds showed left or right QRS axis deviation, and this pattern was observed in 14 of the 16 birds that developed ascites. Several types of cardiac arrhythmias were observed, the most common being premature ventricular contractions (PVC). The incidence of PVC increased with age, ranging from 1% at 12-15 days of age to 8.9% at 32-35 days of age. QRS axis deviation was present in 5 SDS birds. It is concluded that some 30% of the broiler flock tested was at risk of developing heart failure or heart-related disease.

Activity of the rat osteocalcin basal promoter in osteoblastic cells is dependent upon homeodomain and CP1 binding motifs.

A detailed analysis of the transcriptional machinery responsible for osteoblast-specific gene expression should provide tools useful for understanding osteoblast commitment and differentiation. We have defined three cis-elements important for basal activity of the rat osteocalcin (OC) promoter, located at about -200 to -180, -170 to -138, and -121 to -64 relative to the transcription initiation site. A motif (TCTGATTGTGT) present in the region between -200 and -170 that binds a multisubunit CP1/NFY/CBF-like CAAT factor complex contributes significantly to high level basal activity and presumably functions as the CAAT box for the rat OC promoter. We show that the region -121 to 32 is sufficient to confer osteoblastic cell type specificity in transient transfection assays of cultured cell lines using luciferase as a reporter. The basal promoter is active in rodent osteoblastic cell lines, but not in rodent fibroblastic or muscle cell lines. Although the rat OC box (-100 to -74) contains a CAAT motif, we could not detect CP1-like CAAT factor binding to this region. In fact, we demonstrate that a Msx-1 (Hox 7.1) homeodomain binding motif (ACTAATTG; bottom strand) in the 3'-end of the rat OC box is necessary for high level activity of the rat OC basal promoter in osteoblastic cells. A nuclear factor that recognizes this motif appears to be present in osteoblastic ROS 17/2.8 cells, which produce OC, but not in fibroblastic ROS 25/1 cells, which fail to express OC. This ROS 17/2.8 nuclear factor also recognizes the A/T-rich DNA cognates of the homeodomain-containing POU family of transcription factors. Taken together, these data suggest that a ubiquitous CP1-like CAAT factor and a cell type-restricted homeodomain containing (Msx or POU family) transcription factor interact with the proximal rat OC promoter to direct appropriate basal OC transcription in osteoblastic cells.

Inhibition of HIV-1 reverse transcriptase by pyridinone derivatives. Potency, binding characteristics, and effect of template sequence.

The inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by pyridinone compounds has been investigated using as templates synthetic RNA with sequences based on the HIV-1 genome sequence. In reactions catalyzed by the enzyme that incorporated more than one nucleotide per primer, inhibition by a representative pyridinone inhibitor, 3-[2-(1,3-benzoxazol-2-yl)ethyl]-5-ethyl-6-methyl-pyridin-2(1H)one (L-696,229), was noncompetitive against deoxynucleotide triphosphate. For reactions that incorporated one deoxynucleotide per primer, IC50 values ranged from 20 to 200 nM, depending on the position of incorporation of the incoming deoxynucleotide base on the template. Inhibition of synthesis on a set of four templates differing only at the template base complementary to the incoming nucleotide had similar IC50 values. These results demonstrate that inhibitory potency is dependent on the primary structure of the template and that inhibitory potency is largely independent of the identity of the incoming nucleotide base. The inhibition of HIV-1 reverse transcription by L-696,229 also displayed slow-binding characteristics. The slow-binding aspect was exploited to gauge the interaction between inhibitor and enzyme. By titrating the reduction in the extent of the burst of synthesis observed in a reaction incorporating dideoxythymidine monophosphate into poly(rA)-oligo(dT)18, the apparent equilibrium constant for dissociation of the reverse transcriptase-L-696,229 complex was estimated to be 400 nM.

Body composition of the broiler-breeder pullet.

Broiler breeder pullets were reared on a standard breeder-grower diet in accordance with breeder recommendations to 10 wk of age, with samples of the birds being selected for carcass analysis at 2, 3, 4, 5, 6, 7, 8 and 10 wk of age. At 10 wk of age, 264 pullets were assigned to one of three diets formulated to contain 15% CP and to provide 2,550, 2,800 or 3,080 kcal of ME per kg. The birds on the 2,800-kcal diet were fed to attain the body weight recommended by the breeder; the birds on the other diets received the same feed allotment. At 14, 16, 18, 20, 22, and 24 wk of age, 12 birds per treatment were slaughtered for carcass analysis. All of the birds were light-stimulated at 20 wk of age. From 2 to 10 wk of age, carcass analysis of the feed-restricted pullets revealed a relatively constant protein content, 20 to 21%, accompanied by a declining proportion of fat. Feeding a high-energy diet from 10 to 24 wk of age increased body weight and the absolute and proportional content of fat. The percentage of body protein was largely unaffected by the diet, but there was a consistent effect on fat content (P less than .05). The body fat and protein contents were highly correlated with body weight.

High-level expression in Escherichia coli of a chemically synthesized gene for [Leu-28]echistatin.

A gene (Ecs) encoding a platelet aggregation inhibitor, echistatin (Ecs), has been chemically synthesized. Met at position 28 of the native protein was replaced by Leu in the recombinant Ecs. To express this synthetic gene in Escherichia coli, an expression vector, pJC264, was constructed by inserting portions of the E. coli cheB and cheY gene complex into the plasmid pUC13. High-level expression of the synthetic [Leu-28]Ecs was achieved by its fusion with the E. coli cheY gene in the expression vector. Recombinant [Leu-28]Ecs was liberated from the fusion protein by CNBr cleavage at the Met inserted between the CheY protein and [Leu-28]Ecs. The recombinant [Leu-28]Ecs was purified to homogeneity by reverse-phase high-performance liquid chromatography. The refolded [Leu-28]Ecs was identical to native Ecs in inhibiting platelet aggregation, suggesting that Met at position 28 is not essential for the biological activity of this platelet aggregation inhibitor.

Growth of broiler breeder pullets with skip-a-day versus daily feeding.

One hundred and sixty-eight broiler breeder pullets were fed skip-a-day or half the same feed allotment on a daily basis from 5 to 20 wk of age. When weighed on the same day, daily fed birds were significantly larger than skip-a-day-fed birds, but this difference appears to be partly due to feed retained in the digestive tract. Body composition and uniformity were unaffected by feeding regimen.

Water usage of broiler breeders.

Water usage of daily fed and skip-a-day-fed broiler pullets housed in cages was measured in two trials. Daily fed birds in both trials had free access to water. In Trial 1, skip-a-day-fed birds were restricted to 4 h water every day or only on feed days. In Trial 2, the skip-a-day-fed birds were water restricted 4 h either every day, only on feed days, or had free access to water. Skip-a-day-fed birds tended to drink more water on feed days than on off-feed days but amounts were significantly different only in Trial 2. Water intake of these skip-a-day-fed birds on off-feed days was less than that of comparable birds fed on a daily basis with free access to water. Conversely, skip-a-day-fed birds drank more water on feed days than did daily fed birds (P less than .05) when both groups were given free access to water. Overall mean daily water consumption and spillage were unaffected by feeding or water restriction regimens. The main influence of skip-a-day feeding and water restriction appears to be on the pattern of water consumption and not on overall water usage. Daily feeding increased body weight gain relative to that of birds fed skip-a-day, although water restriction did not influence growth. Metabolizable energy derived from a feed was unaffected by feeding or water restriction regimens.

Hearing aid use with minimal high-frequency hearing loss.

This study evaluated the use of hearing aids by patients with hearing threshold levels of 20 dB or less at 500 and 1000 Hz and 35 dB or less at 2000 Hz. Ninety-eight patients completed a 30-day trial with amplification. Six months later, patients were interviewed by telephone and questioned on hearing aid use and perceived unaided and aided difficulty in various listening environments. Results of the study demonstrated that patients with minimal high-frequency hearing loss can benefit from the use of hearing aids. Ninety-two percent of the patients elected to purchase the hearing aids and 85% considered the aids a worthwhile investment after 6 months of use. Patients showed a mean improvement from moderate unaided to slight aided difficulty at work and in general social situations. The only variable that predicted success with hearing aids was degree of unaided difficulty at work. Patients who perceived less unaided difficulty at work were less likely to obtain benefit from the use of the hearing aids.

Cloning and expression of human leukocyte 5-lipoxygenase.

In conclusion, we have cloned a full-length cDNA for human leukocyte 5-LO from differentiating HL-60 cells. The complete amino acid sequence of 5-LO has been determined from the nucleotide sequence of the cDNA. Some interesting features of the sequence include potential lipid and Ca2+ binding sites and sequence homologies with other lipoxygenases. Human osteosarcoma cells transfected with the 5-LO cDNA expressed 5-LO and LTA4 synthase activities that were indistinguishable from those of the human leukocyte enzyme confirming that the cloned cDNA was the correct gene.

Chemical synthesis and enzymatic activity of a 99-residue peptide with a sequence proposed for the human immunodeficiency virus protease.

Retroviral proteins, including those from the human immunodeficiency virus (HIV), are synthesized as polyprotein precursors that require proteolytic cleavage to yield the mature viral proteins. A 99-residue polypeptide, encoded by the 5' end of the pol gene, has been proposed as the processing protease of HIV. The chemical synthesis of the 99-residue peptide was carried out by the solid-phase method, and the isolated product was found to exhibit specific proteolytic activity upon folding under reducing conditions. Upon size-exclusion chromatography, enzymatic activity was eluted at a point consistent with a dimeric molecular size. Specificity was demonstrated by the cleavage of the natural substrate HIV gag p55 into gag p24 and gag p17, as well as cleavage of small peptide substrates representing processing sites of HIV fusion proteins. The proteolytic action of the synthetic product could be inhibited by pepstatin, an aspartic protease inhibitor.

Human cytotoxic lymphocyte tryptase. Its purification from granules and the characterization of inhibitor and substrate specificity.

A trypsin-like enzyme (tryptase) has been purified to homogeneity from the granules of a human cytolytic lymphocyte (CTL) line, Q31, by a three-step procedure. By including 0.3% (v/v) Triton X-100 and 1 mg/ml heparin in purification buffers, near total yields of tryptase activity were obtained during the purification. The enzyme, referred to as Q31 tryptase, migrated in polyacrylamide gels with sodium dodecyl sulfate at a position corresponding to 28 kDa with and to 45 kDa without 2-mercaptoethanol. It had an amino-terminal sequence identical to a previously reported human CTL tryptase at 20 of 22 positions identified. It hydrolyzed N alpha-carbobenzyloxy-L-lysyl-thiobenzyl ester (BLT), and this BLT esterase activity was most efficient at slightly alkaline pH and was relatively more active near neutral pH than mouse CTL tryptase. Human alpha 1-protease inhibitor, human antithrombin III, phenylmethanesulfonyl fluoride, and p-aminobenzamidine inhibited the Q31 tryptase. The inhibition by human antithrombin III was rapid enough to be of physiological significance. A survey of oligopeptide p-nitroanilides found that the best substrate for human Q31 tryptase is H-D-(epsilon-carbobenzyloxy)Lys-L-Pro-L-Arg-p-nitroanilide. The Q31 tryptase appears to have broad specificity for amino acid residues at P2 and P3, i.e. at 2 and 3 residues amino-terminal to the scissile bond.

Amino acid sequence of the N-terminus and selected tryptic peptides of the active subunit of human plasma carboxypeptidase N: comparison with other carboxypeptidases.

Human plasma carboxypeptidase N was purified to homogeneity and its active and inactive subunits were separated. By introducing a novel technique, both forms of the active subunit (Mr = 55,000 and Mr = 48,000) were isolated. N-terminal sequencing of the active subunit of human carboxypeptidase N revealed significant homology with the N-terminal sequence of bovine carboxypeptidase H (43% identity) and to a lesser extent with carboxypeptidase A (29% identity) or carboxypeptidase B (18% identity). The active subunit of carboxypeptidase N was hydrolyzed with trypsin and 4 of the tryptic peptides were isolated by HPLC and sequenced. The sequences of the four peptides were homologous (39-64% identity) with regions of carboxypeptidase H corresponding to the middle (residues 148-175) and C-terminal portion (residues 321-408). These regions had essentially no homology with carboxypeptidase A or B. These data indicate that carboxypeptidase H and the active subunit of carboxypeptidase N may have diverged from a common ancestral gene.

Cloning of the cDNA for human 5-lipoxygenase.

The enzyme 5-lipoxygenase (5-LO) catalyzes the first two reactions in the pathway leading to the formation of leukotrienes from arachidonic acid. Leukotrienes are potent arachidonic acid metabolites possessing biological activities that suggest a role in the pathophysiology of allergic and inflammatory diseases. To obtain structural information about 5-LO for use in developing anti-inflammatory chemotherapeutic agents, the enzyme was purified from human polymorphonuclear leukocytes and the amino acid sequences were determined for several cyanogen bromide-derived peptides. A cDNA clone encoding a 674-amino acid protein containing all of the derived peptide sequences was isolated from a dimethyl sulfoxide differentiated HL60 cell cDNA library. The mRNA encoding 5-LO was detected only in differentiated HL60 cells and not in the undifferentiated cell line, indicating that the expression of 5-LO in this cell line is transcriptionally regulated. In addition, the human protein displays some amino acid sequence homology with several lipases and significant homology with the partial sequences of soybean and reticulocyte lipoxygenases. Thus, 5-LO appears to be a member of a larger family of related enzymes.

Occurrence of beta-hydroxylated asparagine residues in non-vitamin K-dependent proteins containing epidermal growth factor-like domains.

Vitamin K-dependent bovine protein S has been shown to contain a posttranslationally hydroxylated asparagine within a conserved sequence in three of its epidermal growth factor (EGF)-like domains. In a review of amino acid sequences deduced from cDNA data, we have observed that a conserved sequence containing a potential asparagine hydroxylation site exists within EGF-like domains of a variety of functionally diverse proteins. We have studied a number of these and report the presence of erythro-beta-hydroxyasparagine (e-beta Hyn) in three non-vitamin K-dependent proteins: the plasma complement proteins C1r and C1s (where overbar indicates activated form) and the urinary protein uromodulin. For each protein, e-beta Hyn was identified in enzyme digests following the initial observation of erythro-beta-hydroxyaspartic acid (e-beta Hya) in acid hydrolysates of the proteins. e beta Hya and e-beta Hyn residues are detected by a postcolumn derivatization cation-exchange HPLC method herein described. HPLC isolation of the presumptive e-beta Hyn residue from enzyme digests of intact C1r allowed confirmation of its structure by GC/MS. Based upon available cDNA sequence data and observation of e-beta Hya in acid hydrolysates, we suggest other proteins in which e-beta Hyn may occur.

Isolation of multiple biologically and chemically diverse species of epidermal growth factor.

We have analyzed several lots of epidermal growth factor (EGF) purified from murine submaxillary glands including "receptor grade" EGF from Collaborative Research and EGF from Boehringer Mannheim Biochemicals. New England Nuclear uses "receptor grade" EGF to produce 125I-labeled EGF. Though these reagents are reported to be homogeneous, we found them to be a mixture of six species. A method was developed to separate this mixture into its component parts. The individual components were chemically characterized and tested for biological potency. N-terminal sequence analysis of the unfractionated EGF-mixture reveals three different sequences starting with residues 1, 2, or 3 of the mature peptide. Each component exhibited different degrees of mitogenic and EGF receptor binding activity indicating that the N-terminal region contributes to the biological response. The species representing the complete EGF peptide is the most active species in all biological assays. A rapid method for purification of homogeneous complete EGF from commercial EGF preparations is described.