Polychlorinated biphenyls and biotransformation enzymes in three species of sea turtles from the Baja California peninsula of Mexico.

Concentrations of polychlorinated biphenyls (PCBs) as well as the expression patterns of cytochrome P450 (CYP) enzymes and glutathione-S-transferase (GST) activities were measured in livers of loggerhead (Caretta caretta), green (Chelonia mydas), and olive ridley (Lepidocheyls olivacea) sea turtles from the Baja California peninsula of Mexico. The mean concentrations of total PCBs were 18.1, 10.5, and 15.2 ng/g wet weight (ww) respectively for the three species and PCB 153 was the dominant congener in all samples. Total PCB concentrations were dominated by penta- and hexa-chlorinated biphenyls. The mean estimated TEQs were 42.8, 22.9, and 10.4 pg/g (ww) for loggerhead, green, and olive ridley, respectively, and more than 70% was accounted for by non-ortho PCBs. Western blots revealed the presence of hepatic microsomal proteins that cross-reacted with anti-CYP2K1 and anti-CYP3A27 antibodies but not with anti-CYP1A antibody. There were no significant differences in GST activities between species. Grouping congeners based on structure-activity relationships for CYP isoenzymes suggested limited activity of CYP1A contribution to PCB biotransformation in sea turtles. These results suggest potential accumulation of PCBs that are CYP1A substrates and provide evidence for biotransformation capacity, which differs from known animal models, highlighting the need for further studies in reptiles, particularly those threatened with extinction.

Characterization of bovine coronavirus isolates/from eight different states in the USA.

Bovine coronavirus isolates from eight different states of the USA were compared for their antigenic properties and susceptibility to hygromycin B. Antigenic differences were observed among the isolates in a one-way hemagglutination-inhibition (HI) test using a polyclonal antiserum against the Mebus bovine coronavirus isolate. Differences were observed on isoelectric focusing among viral proteins with isoelectric points between 4.45-4.65. Most of the BCV isolates were susceptible to hygromycin B (0.5 mM) whereas a few hygromycin B resistant isolates were also found.

Relationship of breeding soundness evaluation score and its components with reproductive performance of beef bulls.

Angus, Polled Hereford and Santa Gertrudis bulls were subjected to a breeding soundness evaluation (BSE) just prior to being exposed to cows for 90 to 95 days in single-sire units under natural breeding conditions on pasture. Forty-eight of 55 bulls subjected to scrotal and semen evaluations passed the BSE and were considered acceptable for breeding. Of the bulls that passed BSE, 18 (six bulls from each breed) were used for breeding for each yr of the 2-yr study. Of the bulls used for breeding, breed of sire differences were significant for scrotal circumference and calving rate, while differences among sires within breed of sire were significant for secondary morphology, motility score and calving rate. The difference between means for bulls used vs bulls that failed BSE was significant for all eight traits. Correlations among various scrotal and semen evaluations were compared for bulls used vs bulls that failed BSE. Of the eight traits, only the motility score was significantly correlated with the calving rate. After the exclusion of bulls that failed to pass BSE, there remained differences (P<0.01) among bulls within breed of sire for calving rate. Thus, there is a need for an additional easy-to-use procedure that would more accurately predict the breeding performance of bulls.

Ovarian luteal activity and reproductive performance of heifers with norgestomet prototype implants.

A 122-day trial was conducted with grazing beef heifers (N=120; 14 to 17 months of age) to determine the effects of a silastic Norgestomet prototype implant on heifer weight gain and on the suppression of ovarian luteal activity. The retention rate of the Norgestomet implants and of silastic placebo implants, which were either left untreated, or were topically treated with oxytetracycline, was determined. Medication of the placebo implants with oxytetracycline increased (P < 0.01) implant retention rate at both Day 56 and Day 122. Heifers with Norgestomet implants had higher body weight gains during the last 66 days (P < 0.05), and during the entire 122-day trial (P < 0.07) than heifers with placebo implants. Serum progesterone concentrations on Day 111 or Day 112 indicated a decrease (P < 0.01) in percentage of heifers with ovarian luteal activity on the Norgestomet treatment compared with heifers receiving placebos (23.8% vs 87.7%, respectively). The Norgestomet implant has the potential for suppressing ovarian luteal activity while improving weight gain in beef heifers.

Serum alhpa 1-acid glycoprotein concentrations in healthy and tumor-bearing cats.

The purpose of this study was to evaluate alpha 1-acid glycoprotein (AGP) concentrations in tumor-bearing and healthy cats. The hypothesis of the present study was that AGP concentrations would be significantly increased in tumor-bearing cats. Serum from 51 healthy and 97 tumor-bearing, client-owned cats was harvested at the time of presentation and stored at -80 degrees C until assayed. Cats with measurable, histologically confirmed malignancies, and healthy cats of similar ages were included. Serum was assayed for AGP concentration by using a radial immunodiffusion method. AGP concentrations were significantly (P = .0051) higher in tumor-bearing (763 +/- 595 microg/mL; mean +/- SD) when compared to healthy cats (501 +/- 377 microg/mL; mean +/- SD). Of the tumor-bearing cats, 35 had carcinomas, 33 had sarcomas, and 26 had discrete, round cell tumors. AGP concentrations were 645 +/- 62 microg/mL, 660 +/- 540 microg/mL, and 967 +/- 860 microg/mL, respectively, and there were no significant differences among the groups.

Serum zinc, chromium, and iron concentrations in dogs with lymphoma and osteosarcoma.

We compared serum concentrations of zinc, chromium, and iron in dogs with cancer to those of normal dogs. Dogs with lymphoma (n = 50) and osteosarcoma (n = 52) were evaluated. Dogs with lymphoma had significantly lower (P = .0028) mean serum zinc concentrations (mean +/- SD; 1.0 +/- 0.3 mg/L) when compared to normal dogs (1.2 +/- 0.4 mg/L). Dogs with osteosarcoma also had lower mean serum zinc concentrations (1.1 +/- 0.4 mg/L), but this difference was not significant (P = .075). Serum chromium concentrations were significantly lower in dogs with lymphoma (2.6 +/- 2.6 microg/L, P = .0007) and osteosarcoma (2.4 +/- 3.1 microg/L, P = .0001) compared to normal dogs (4.7 +/- 2.8 microg/L). Serum iron concentrations and total iron-binding capacity were significantly lower in dogs with lymphoma (110.8 +/- 56.7 microg/dL, P < .0001, and 236.6 +/- 45.6 microg/dL, P < .0001, respectively) and osteosarcoma (99.6 +/- 49.3 microg/dL, P < .0001, and 245.0 +/- 43.8 microg/dL, P = .0011, respectively) when compared to normal dogs (175.1 +/- 56.7 microg/dL and 277.1 +/- 47.4 microg/dL). Mean ferritin concentration was significantly higher in dogs with lymphoma (1291.7 +/- 63.0 microg/L) than in normal dogs (805.8 +/- 291.1 microg/L, P < .0001) and dogs with osteosarcoma (826.5 +/- 309.2 microg/L, P < .0001). Further investigation is needed to explore the clinical significance of these mineral abnormalities in dogs with cancer.

Feedlot performance and pregnancy inhibition of heifers treated with depot-formulated melengestrol acetate.

In dose titration trials beef heifers received depot-formulated melengestrol acetate (DEPO-MGA) in single s.c. ear injections to determine effects on performance and pregnancy inhibition. In a 112-d feedlot trial, 105 heifers were assigned to light, medium and heavy weight blocks on five treatments: dietary MGA (.5 mg.hd-1.d-1), control (no MGA) or DEPO-MGA on d 1 at .5, 1.0 or 1.5 ml/hd (30, 60 or 90 mg MGA/hd, respectively). A high-energy cracked corn diet was fed to all heifers ad libitum. At d 56 and d 112, ADG and feed/gain were not affected (P greater than .05) by dietary MGA or DEPO-MGA treatments, although dietary MGA tended to increase 112-d ADG (1.67 vs 1.58 kg) compared with controls. In a pasture trial, 100 beef heifers were assigned by weight to five treatments: control (no MGA) or DEPO-MGA injected on d 1 at .5, 1.0, 1.5 or 2.0 ml/hd (30, 60, 90 or 120 mg MGA/hd, respectively). Heifers were pastured as a single herd and were exposed to fertile bulls from d 7 to d 181. Weights and pregnancy data were recorded at d 62, 90, 134, 181 and 225. Heifer ADG was not affected (P greater than .05) by DEPO-MGA treatment at any weighing date. By d 90, 90% of control heifers were pregnant, and 100% were pregnant by d 134. Pregnancy inhibition rates of 90% to 100% for each DEPO-MGA treatment at d 62, 90 and 134 were higher (P less than .01) than those in control heifers. Pregnancy was inhibited in 90% of heifers on 60-mg and 90-mg DEPO-MGA treatments through d 181, and in 95% of heifers on the 120 mg DEPO-MGA treatment through d 225.

Breeding performance of bulls assigned either to 40 cows per bull or 80 cows per two bulls during the breeding period.

The reproductive performance of Angus (A), Polled Hereford (PH) and Santa Gertrudis (SG) bulls was compared when exposed to 40 cows/bull vs 80 cows/two bulls during a 90- to 95-d breeding period on pasture. Cows were A, PH and SG straightbreds and crossbreds of these breeds. Each year, cows were allotted at random within breed composition and age of dam to breeding groups. A replicate consisted of two 40-cow single-sire units with bulls of two breeds and an 80-cow two-sire unit with two bulls of the same breeds and all four bulls of the same age. There were eight replicates of PH and SG bulls and five replicates of PH and A bulls. At breeding time, 20, 16, 12 and 4 bulls were 2-, 3-, 4- and 5-yr-olds, respectively. Reproductive performance of bulls was evaluated in terms of calving rate (CR) of cows exposed to them and number of days (NOD) from the beginning of the breeding period until calf birth. The 80-cow groups calved 3.7 d earlier (P less than .05) in the calving period and had a similar CR compared with the 40-cow groups. The PH-A replicates calved 3.5 d earlier (P less than .05) and had 7.3 percentage units higher (P less than .01) CR than PH-SG replicates. Results of this study indicated that 80-cow two-sire breeding groups had an advantage over the 40-cow single-sire groups in terms of calves born earlier in the calving period, with no reduction in CR.

Cow breeding and calf growth performance as affected by pregnancy status the previous year.

A 2-yr study was conducted to compare the subsequent cow breeding and calf performance of cows that were nonpregnant with cows that were pregnant at the time calves were weaned. Cows were Angus (A), Polled Hereford (PH), Santa Gertrudis (SG) straightbreds and crossbreds of these breeds. Nonpregnant cows (G1) were 4- to 9-yr-olds that had a calf the previous year and appeared to be physically sound with no detection (by rectal palpation) of an abnormal reproductive tract due to disease, abnormal growth or calving difficulties. Pregnant cows (G2) were of similar age and breed composition to G1 cows. The 93 G1 and the 193 G2 cows were assigned within age and breed composition to sire breeding groups on pasture in an approximate 1:2 ratio, respectively, per sire. There were six A, three PH and one SG sires. The year prior to G1 cows being nonpregnant, G1 cows calved 11 d later (P less than .01) than G2 cows. Subsequent to their being nonpregnant, G1 cows gained 27 kg more (P less than .001) weight during the breeding period, had 5.4 percentage units more (P less than .29) calves born, had calves 17 d earlier (P less than .001) in the calving period, had calves that gained at a similar rate to weaning and had calves that were 14 kg heavier (P less than .01) at weaning (due to their being 17 d older) compared with G2 cows and calves.

Breeding performance of beef bulls assigned to two-sire cow groups on pasture.

Angus, Polled Hereford and Santa Gertrudis bulls from ages 1 through 5 and 7 yr were assigned to 26 two-sire breeding groups. Each year, straightbred and crossbred cows of these breeds were allotted at random within breed composition, age of dam and calving date to breeding groups on pasture. Sires within each breeding group or pair were the same age at breeding and were two of the three breeds of sires. Neither calving rate nor the proportion of calves born by one vs the other sire in the two-sire breeding groups was affected by sire age among breeding groups. For a given breed, there was no uniformity among the sires in the proportion of calves they sired in their two-sire breeding groups. The proportion of calves born for the 26 sire pairs averaged .64 vs .36 (SE = 0.4 for either high or low value) for one vs the other sire in a sire pair with no indication that calving rate was affected by unequal proportions of calves by sires within sire pairs. Cows calved significantly earlier in the calving period (b = -.775 +/- .127) as calving rate increased among sire pairs. The number of days from the start of the breeding period to calf birth was affected by differences between sires in sire pairs for 8 of the 26 pairs, but there were no significant differences due to sire pair or breed of sire because of interaction between these two variables.

Subsequent reproduction and calf performance of nonpregnant cows compared with pregnant cows and replacement females.

This study was conducted with Angus, Polled Hereford and Santa Gertrudis straightbred and crossbred cows. The subsequent cow breeding and calf performance of cows that were nonpregnant (NP) were compared with cows that were pregnant (PG) at the time calves were weaned. All NP cows had a calf the year previous to their being nonpregnant. They were diagnosed as physically sound with no detection (by rectal palpation) of an abnormal reproductive tract. The NP and PG cows were aged 4 to 9 yr. Also, the NP cows were compared with replacement females exposed to calve first as 2- and 3-yr-olds (H2 and H3, respectively), and with cows exposed for second calving as 3-yr-olds (C2). Cows were assigned within breed composition and age to sire breeding groups on pasture. Subsequent calving and weaning rates were similar for NP, PG and H2 cows, similar for H3 and H2 cows and lowest (P less than .05) for C2 cows. Calves from NP and H3 cows were born earlier (P less than .05) in the calving period than calves from PG and H2 cows, whereas calves from C2 cows were born later (P less than .05) than those from NP, PG and H3 cows. Calving difficulty was similar for NP, PG and C2 and greatest (P less than .05) for H2 cows. Calf 205-d weights were highest (P less than .05) for NP, similar for PG and H3 and lowest (P less than .05) for C2 and H2 cows. Calf weaning weight per cow exposed for breeding from NP cows was 13.8, 32.3, 55.2 and 1.0 kg higher than from PG, H2, C2 and H3 cows, respectively. Causes for cows being nonpregnant were reported. Also, calving patterns during 6 consecutive calving periods (6 yr) were evaluated.

Effects of dietary cysteine on blood sulfur amino acid, glutathione, and malondialdehyde concentrations in cats.

OBJECTIVE: To determine effects of dietary cysteine on blood sulfur amino acids (SAA), reduced glutathione (GSH), oxidized glutathione (GSSG), and malondialdehyde (MDA) concentrations in cats. ANIMALS: 12 healthy adult cats. PROCEDURE: Cats were fed diets with a nominal (0.50 g/100 g dry matter [DM]), moderate (1.00 g/100 g DM), or high (1.50 g/100 g DM) cysteine content in a 3 X 3 Latin square design with blocks of 8 weeks' duration. Venous blood samples were collected after each diet had been fed for 4 and 8 weeks, and a CBC and serum biochemical analyses were performed; poikilocyte, reticulocyte, and Heinz body counts were determined; and MDA, GSH, GSSG, and SAA concentrations were measured. RESULTS: Blood cysteine and MDA concentrations were not significantly affected by dietary cysteine content. Blood methionine, homocysteine, and GSSG concentrations were significantly increased when cats consumed the high cysteine content diet but not when they consumed the moderate cysteine content diet, compared with concentrations obtained when cats consumed the nominal cysteine content diet. Blood GSH concentrations were significantly increased when cats consumed the moderate or high cysteine content diet. CONCLUSIONS: Increased dietary cysteine content promotes higher blood methionine, homocysteine, GSH, and GSSG concentrations in healthy cats. CLINICAL RELEVANCE: Supplemental dietary cysteine may be indicated to promote glutathione synthesis and ameliorate adverse effects of oxidative damage induced by disease or drugs.

Duration of effects of dietary fish oil supplementation on serum eicosapentaenoic acid and docosahexaenoic acid concentrations in dogs.

OBJECTIVE: To determine how long serum concentrations of omega-3 fatty acids remain elevated after cessation of dietary fish oil supplementation. ANIMALS: 12 healthy Beagles. PROCEDURE: Baseline serum concentrations of linoleic acid, linolenic acid, arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) were measured. Dogs were then fed a diet supplemented with soybean oil or fish oil for 8 weeks, and serum fatty acid concentrations were measured while dogs were fed the experimental diets and for 18 weeks after they were switched to a maintenance diet. RESULTS: For dogs fed the fish oil diet, serum EPA and DHA concentrations were significantly increased by week 1 and remained increased for 7 (DHA concentration) or 3 (EPA concentration) weeks after dietary fish oil supplementation was discontinued. CONCLUSIONS: In dogs, supplementation of the diet with fish oil may have effects for several weeks after dietary supplementation is discontinued. CLINICAL RELEVANCE: Studies of the effects of fish oil supplementation that use a crossover design should allow for an appropriate washout period.

Pyramiding and dissecting disease resistance QTL to barley stripe rust.

Quantitative resistance (QR) to disease is usually more durable than qualitative resistance, but its genetic basis is not well understood. We used the barley/barley stripe rust pathosystem as a model for the characterization of the QR phenotype and associated genomic regions. As an intermediate step in the preparation of near-isogenic lines representing individual QTL alleles and combinations of QTL alleles in a homogeneous genetic background, we developed a set of QTL introgression lines in a susceptible background. These intermediate barley near-isogenic (i-BISON) lines represent disease resistance QTL combined in one-, two-, and three-way combinations in a susceptible background. We measured four components of disease resistance on the i-BISON lines: latent period, infection efficiency, lesion size, and pustule density. The greatest differences between the target QTL introgressions and the susceptible controls were for the latter three traits. On average, however, the QTL introgressions also had longer latent periods than the susceptible parent (Baronesse). There were significant differences in the magnitudes of effects of different QTL alleles. The 4H QTL allele had the largest effect, followed by the alleles on 1H and 5H. Pyramiding multiple QTL alleles led to higher levels of resistance in terms of all components of QR except latent period.

Effect of population size on the estimation of QTL: a test using resistance to barley stripe rust.

The limited population sizes used in many quantitative trait locus (QTL) detection experiments can lead to underestimation of QTL number, overestimation of QTL effects, and failure to quantify QTL interactions. We used the barley/barley stripe rust pathosystem to evaluate the effect of population size on the estimation of QTL parameters. We generated a large (n = 409) population of doubled haploid lines derived from the cross of two inbred lines, BCD47 and Baronesse. This population was evaluated for barley stripe rust severity in the Toluca Valley, Mexico, and in Washington State, USA, under field conditions. BCD47 was the principal donor of resistance QTL alleles, but the susceptible parent also contributed some resistance alleles. The major QTL, located on the long arm of chromosome 4H, close to the Mlo gene, accounted for up to 34% of the phenotypic variance. Subpopulations of different sizes were generated using three methods-resampling, selective genotyping, and selective phenotyping-to evaluate the effect of population size on the estimation of QTL parameters. In all cases, the number of QTL detected increased with population size. QTL with large effects were detected even in small populations, but QTL with small effects were detected only by increasing population size. Selective genotyping and/or selective phenotyping approaches could be effective strategies for reducing the costs associated with conducting QTL analysis in large populations. The method of choice will depend on the relative costs of genotyping versus phenotyping.

Localization of the glucose phosphotransferase to a cytoplasmically accessible site on intracellular membranes.

UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor for this transfer in rat liver is a glycoprotein of 62 kDa. This acceptor was labeled in liver homogenates through incubation with the 35S-labeled phosphorothioate analogue of UDP-Glc, and its distribution following differential centrifugation was compared to that of the glycoproteins labeled by CMP-[3H]N-acetylneuraminic acid. Whereas 94% of the 3H-labeled macromolecules fractionated to the microsomal pellet, 85% of the 35S-labeled 62-kDa glycoprotein was found in the high-speed supernatant. The distribution of the Glc-phosphotransferase was also examined following differential centrifugation, and the bulk of the activity was found in the 100,000 x g pellet. In contrast to results obtained with the lumenal microsomal markers 4 beta-galactosyltransferase and mannose-6-phosphatase, however, optimal activity of the Glc-phosphotransferase was not dependent on the disruption of microsomal vesicles by detergent. In addition, Glc-phosphotransferase was degraded by exogenous proteases in the absence of detergent, whereas the lumenal markers were not. We conclude, therefore, that the 62-kDa acceptor glycoprotein is cytoplasmic and is glycosylated by the Glc-phosphotransferase at a site accessible to the cytoplasm. This may prove to be a model for the topography of glycosylation of other cytoplasmic glycoproteins as well.

Genetic instability in Drosophila melanogaster: P-element mutagenesis by gene conversion.

We report the molecular characterization of several P element-induced mutations and their revertants at the yellow (y) locus of Drosophila melanogaster. One of the mutants analyzed, y76d28, results from the insertion of a P element into the 5'-transcribed, untranslated portion of the y gene. Sequence analysis of several revertants of y76d28 shows that P excision occurs imprecisely. These events result in insertion of additional ATG codons in the y locus mRNA but are without phenotypic effect. In addition, we describe the molecular structure of P-associated mutations induced in a near wild-type revertant of y76d28 that carries an internally deleted 0.4-kilobase P element in the 5' noncoding region. Sequence analysis of two of these mutants demonstrates that they arose as a result of the integration of a larger P element at the exact location as in the parental stock without the 8-base-pair additional duplication associated with P insertions. The phenotype of these y alleles is dependent on the size and orientation of the integrated P element. We infer that P-element replacement in these mutants has occurred by a recombination/gene conversion mechanism.

An alpha-glucose-1-phosphate phosphodiesterase is present in rat liver cytosol.

UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha-Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor for this transfer in both mammalian cells and Paramecium is a cytoplasmic glycoprotein of 62-63 kDa. When cytoplasmic proteins from rat liver were fractionated by preparative isoelectric focusing following incubation of a liver homogenate with the 35S-labeled phosphorothioate analogue of UDP-Glc ([beta-35S]UDP-Glc), the acceptor was found to have a pI of about 6.0. This fraction, when not labeled prior to the focusing, became very heavily labeled when mixed with [beta-35S]. UDP-Glc and intact liver microsomes, a rich source of the Glc-phosphotransferase. In addition, it was observed that the isoelectric fractions of the cytosol having pI values of 2-3.2 contained a degradative activity, alpha-Glc-1-P phosphodiesterase, that was capable of removing alpha-Glc-1-P, monitored through radioactive labeling both in the sugar and the phosphate, as an intact unit from the 62-kDa acceptor. Identification of the product of this cleavage was substantiated by its partial transformation to UDP-Glc in the presence of UTP and UDP-Glc pyrophosphorylase. The alpha-Glc-1-P phosphodiesterase had a pH optimum of 7.5 and was not effectively inhibited by any of the potential biochemical inhibitors that were tested. Specificity for the Glc-alpha-1-P-6-Man diester was suggested by the diesterase's inability to degrade UDP-Glc or glucosylphosphoryldolichol. This enzyme may be important in the regulation of secretion since the alpha-Glc-1-P present on the 62-kDa phosphoglycoprotein appears to be removed and then rapidly replaced in response to secretagogue.

Resolution of phosphoglucomatase and the 62-kDA acceptor for the glucosylphosphotransferase.

The radioactive, photoactivatable labeling probe [beta-32P]5-azidouridine 5'-diphosphoglucose has recently been shown to label a 62-kDa protein in crude homogenates and in partially purified enzyme preparations without photoactivation. Here, we report that a portion of this radioactivity is due to labeling of phosphoglucomutase by contaminating levels of [32P]alpha Glc-1-P initially present at less than 1% of the total 32P. This conclusion is based in part on the ability of excess unlabeled alpha Glc-1-P and Glc-6-P, but not UDP-Glc, to block the labeling. In addition, the labeled protein in liver homogenates had a tryptic peptide pattern similar to that of authentic phosphoglucomutase. These findings, however, raised a second question. Assays for the UDP-Glc: glycoprotein glucosyl phosphotransferase (Glc phosphotransferase) have utilized [beta-32P]UDP-Glc and have resulted in the labeling of a small number of acceptors, including one of approximately 62 kDa. Despite the fact that these assays had routinely been performed in the presence of 1 mM alpha Glc-1-P, the coincidence in molecular weights led to these further studies. We conclude that the acceptor of approximately 62 kDa is distinct from phosphoglucomutase. This conclusion is based on differences in the time courses of incorporation, the specificity of blocking agents, the presence of covalently linked glucose, the products of acid hydrolysis and of beta-elimination, and isoelectric points.