On the possible presence of a beta 2-microglobulin-like protein in extracts of livers from normal chickens and chickens with erythroblastosis--V. Studies on an immunological relationship of a small molecular weight liver protein (SMWP) to serum albumin

An extract of the livers of normal chickens (N) and chickens (Eb) infected with avian erythroblastosis virus (EbV) contained a small molecular weight protein (SMWP, mol. wt 11,000). When the extract was not dehaeminized, SMWP in agarose electrophoresis was shown to have peroxidase activity probably due to the presence of haem. When this non-dehaeminized extract was chromatographed on Con-A Sepharose neither SMWP nor an antigen (EbAg) present in Eb livers were retained. The association of EbAg with SMWP is still unexplained. Immunoelectrophoresis shows a reaction of identity between chicken SMWP and serum albumin. Chicken SMWP is thus not a beta 2-microglobulin. The finding of an immunological relationship between SMWP and albumin confirms the biochemical homology of SMWP with serum albumin in terms of amino acid residues. It has been suggested that SMWP may be a precursor or fragment of albumin but the possibility of its being a distinct entity, a microalbumin, should not be discounted.     

On the possible presence of beta 2-microglobulin-like protein in extracts of livers from normal chickens and chickens with erythroblastosis--II. Amino acid analysis of the protein

1. Amino acid analyses are presented for a small molecular weight (mol. wt 11,000) protein (SMWP) obtained from the livers of normal chickens and chickens infected with erythroblastosis virus. It is resistant to acid denaturation and was isolated following acidification of liver homogenates and removal of haem and lipids. 2. Close homology exists between SMWP of fowl and human liver, and a protein of the same size obtained from chicken serum, (similar in size to human beta 2-microglobulin). 3. Human beta 2-microglobulin, virus core proteins and chicken prealbumin show a lower order relationship. 4. The amino acid residues of SMWP from healthy and erythroblastosis infected chicken livers are identical despite immunologic differences.     

On the possible presence of a beta 2-microglobulin-like protein in extracts of livers from normal chickens and chickens with erythroblastosis-IV. Homology of small molecular weight (mol.wt 11,000) protein with serum albumin based on the content of amino acid residues

A small molecular weight (mol.wt 11,000) protein (SMWP) was obtained from the livers of normal chickens and chickens infected with erythroblastosis virus. SMWP, which had been shown to have no biochemical homology with beta 2-microglobulin, is homologous with chicken serum albumin. SMWP shows a similar order of homology with another small molecular weight protein isolated by others from chicken plasma. Like chicken albumin, SMWP is more closely homologous with bovine albumin than with human albumin.     

On the possible presence of a beta 2-microglobulin-like protein in extracts of livers from normal chickens and chickens with erythroblastosis-III. Immunological relationship to serum albumin of a small molecular weight human liver protein

Human liver contains a small molecular weight protein (SMWP) previously shown to be biochemically homologous with a chicken liver protein in terms of its amino acid residues. This human protein, which reacts immunologically as a human serum protein, has been tested for its reactions against a battery of antisera that react specifically with many of the human serum proteins. Material prepared from four human livers gave strong reactions in double gel immunodiffusion with an antiserum against human albumin. One of the liver preparations reacted weakly with antiserum against human ferritin; this ferritin is assumed to be a contaminant. Because of the biochemical homology of human liver SMWP with chicken liver SMWP the latter would be expected to react immunologically as serum albumin.     

Genetic control of liver esterase forms in chickens

Six regions of esterase activity designated I to VI were resolved from liver extracts of chickens by horizontal starch gel electrophoresis. These esterases were further characterized on the basis of their substrate affinities and differential responses to various inhibitors.
Genetic variation was found in esterases of region VI which appeared to be ali-esterase. Four phenotypes, A, B, AB and O, were observed. These phenotypes were shown to be controlled by one autosomal locus, designated Es-3 , with alleles Es-3 ^A, Es-3 ^B and Es-3 ^O. This locus is not closely linked to the blood group loci A and B , serum alkaline phosphatase ( Ap ), liver acid phosphatase ( Acp-2 ) and serum esterase ( Es-1 ) loci.     

Hepatic lipogenesis in genetically lean and fat chickens. In vitro studies

1. Acetyl-coenzyme A carboxylase, malic enzyme, glucose 6-phosphate dehydrogenase and delta 9-desaturase activity was measured in liver extracts from 5- to 11-week-old genetically lean or fat chickens. 2. A significant difference between the two lines of chickens was shown as concerns desaturating activity only, which was 45% higher in the fat animals than in the lean ones. 3. This result is consistent with the hypothesis of a higher rate of lipoprotein processing and secretion in the liver of the fat line chickens.     

Production of recombinant human erythropoietin/Fc fusion protein by genetically manipulated chickens

We previously reported the production of human erythropoietin (hEpo) using genetically manipulated (GM) chickens. The recombinant hEpo was produced in the serum and egg white of the GM chickens, and the oligosaccharide chain structures of the serum-derived hEpo were more favorable than those of the egg white-derived hEpo. In the present study, a retroviral vector encoding an expression cassette for a fusion protein of hEpo and the Fc region of human immunoglobulin G (hEpo/Fc) was injected into developing chicken embryos, with the aim of recovering the serum-derived hEpo from egg yolk through the yolk accumulation mechanism of maternal antibodies. The GM chickens that hatched stably produced the hEpo/Fc fusion protein not only in their serum and egg white, but also in the egg yolk as expected. Lectin blot analyses revealed that significant amounts of the oligosaccharide chains of hEpo/Fc produced in the serum and eggs of GM chickens terminated with galactose, and that the oligosaccharide chains of the serum- and yolk-derived hEpo/Fc incorporated sialic acid residues. Moreover, biological activity assessment using Epo-dependent cells revealed that the yolk-derived hEpo/Fc exhibited a comparable performance to the serum- and CHO-derived hEpo/Fc. These results indicate that transport of Fc fusion proteins from the blood circulation to the yolk in chickens represents an effective strategy for the production of pharmaceutical glycoproteins using transgenic chicken bioreactors.     

Effect of bacterial protein meal on protein and energy metabolism in growing chickens

This experiment investigates the effect of increasing the dietary content of bacterial protein meal (BPM) on the protein and energy metabolism, and carcass chemical composition of growing chickens. Seventy-two Ross male chickens were allocated to four diets, each in three replicates with 0% (D0), 2% (D2), 4% (D4), and 6% BPM (D6), BPM providing up to 20% of total dietary N. Five balance experiments were conducted when the chickens were 3-7, 10-14, 17-21, 23-27, and 30-34 days old. During the same periods, 22-h respiration experiments (indirect calorimetry) were performed with groups of 6 chickens (period 1), 5 chickens (period 2), and one chicken (periods 3-5). After each balance period, one chicken in each cage was killed and the carcass weight was recorded. Chemical analyses were performed on the carcasses from periods 1, 3, and 5. Weight gain, feed intake, and feed conversion rate were found to be similar for all diets. Chickens on D0 retained 1.59 g N x kg(-0.75) x d(-1), significantly more than chickens on D2, D4, and D6, which retained 1.44 g, 1.52 g, and 1.50 g N x kg(-0.75) x d(-1), respectively. This was probably caused by the higher nitrogen content of DO. Neither the HE (p = 0.92) nor the retention of energy (p = 0.88) were affected by diet. Carcass composition was similar between diets, in line with the values for protein and energy retention found in the balance and respiration experiments. It was concluded that the overall protein and energy metabolism as well as carcass composition were not influenced by a dietary content of up to 6% BPM corresponding to 20% of dietary N.     

伴性矮小型鸡GH、GHR和IGF-1基因的表达变化

采用荧光实时定量PCR的方法, 从转录水平上分析了伴性矮小型鸡和普通鸡肝脏中GH、GHR和IGF-1基因的表达变化趋势。结果表明：伴性矮小型鸡和普通鸡肝脏组织中GH的mRNA表达量没有明显差异, 而GHR在矮小鸡中的表达量明显比普通鸡的高3倍多, 但IGF-1基因在矮小鸡肝脏中的表达量却远远低于普通鸡, 差异达到2个数量级。这表明, 伴性矮小型鸡GHR外显子10 和3′非翻译区的长片断缺失并没有降低GHR基因的表达, 相反有所增高, 这一过程中可能存在相应的功能代偿机制。与此同时, 在伴性矮小型鸡肝脏中几乎观察不到IGF-1基因的表达, 证明正是由于GHR基因的缺陷影响了GH生理效应的发挥。实验结果印证了伴性矮小表型与GH和GHR的转录水平无关, 而可能是GHR编码产物异常阻碍了GH-GHR-IGF信号通路, 导致IGF-1表达受阻, 不能发挥正常的生理功能。     

Identification of a butanol-extractable human placenta-specific antigen with alkaline phosphatase activity.

N-Butanol extracts of whole-term placenta from different individuals were prepared, and used as immunogens to raise heterologous hyperimmune sera in rabbits. Upon immunoelectrophoresis the anti-placenta antisera could recognize at least six antigenic components in the placental extract even after they had been completely absorbed with pooled male serum proteins. However, the antisera so absorbed, designated (-PMS) antisera, could still react strongly with several normal adult tissue extracts including kidney. Systematic and quantitative absorptions of the (-PMS) antisera were thus further carried out with individual butanol extracts of normal adult liver, lung, intestine, stomach, kidney, bone, pancreas, spleen, heart, cerebrum, cerebellum, breast, and packed red cells, as well as a composite extract containing equal amounts of each of the 13 adult tissue extracts. Of the six antigenic components in the placental extracts reacting with the (-PMS) antisera the only one which retained its reactivity with the antisera throughout exhaustive absorptions was associated with alkaline phosphatase activity. This immunologic and enzymologic identity was confirmed with homogeneous placental alkaline phosphatase. Extracts from each of three placentae injected into three pairs of rabbits all produced an identical antibody reaction with the unique determinant(s) of placental alkaline phosphatase. The same identity of precipitin reaction was also found with extracts of 14 other placentae against each of these antisera. It thus firmly establishes that placental alkaline phosphatase is a characteristic placenta-specific fetal protein.     

Inhibition of the glycogen phosphorylase system during ochratoxicosis in chickens.

Graded doses of ochratoxin A incorporated into the diet (0, 0.5, 1.0, 2.0, 4.0, and 8.0 micrograms/g) of broiler chickens significantly (P < 0.05) inhibited activity of protein kinase, the initiator enzyme of the glycogen phosphorylase system, in the livers at all dose levels. Only the highest dose, 8.0 micrograms/g, significantly reduced the total activity of phosphorylase kinase, which is activated by protein kinase. The total activity of phosphorylase, which is activated by phosphorylase kinase, was unaltered by ochratoxin A at any level. Additon of ochratoxin A to liver extracts control birds inhibited protein kinase but not phosphorylase kinase. When added to extracts of livers from control birds, cyclic adenosine 3',5'-monophosphate stimulated protein kinase but not phosphorylase kinase. The cyclic adenosine 3',5'-monophosphate had no effect when added to extracts from birds fed ochratoxin A. These results suggest that ochratoxin A affects primarily the cyclic adenosine 3',5'-monophosphate-dependent protein kinase which initiates the enzymatic cascade leading to glycogenolysis. Furthermore, these results conform an earlier assignment on morphological criteria of the glycogenosis of ochratoxicosis as a type X glycogen storage disease.     

Molecular characterization and expression of the cholesteryl ester transfer protein gene in chickens

The cDNA for cholesteryl ester transfer protein (CETP), a protein that catalyzes cholesteryl ester transfer between very low density and high density lipoproteins in plasma, was isolated from chicken liver. When the recombinant protein was overexpressed in HEK293 cells, cholesteryl ester transfer activity was observed in media and cell lysates. By Northern blot analysis, chicken CETP mRNA expression was detected in liver, brain, heart, and spleen. Changes in chicken CETP mRNA expression and plasma CETP activity with nutritional state were examined and found to increase following dietary supplementation with cholesterol in a similar way as in humans. Both the hepatic CETP mRNA levels and plasma CETP activity were significantly lower in mature (i.e egg-laying) hens than in immature female chickens, but were unaffected by age in male animals. Similar changes to those observed in female chickens were observed upon estradiol administration of males. The present study is the first to report the molecular characterization of an avian CETP, and the impairments of CETP gene and activity, which might be regulated by estrogen, play an important role in egg production in laying hens, demonstrating species-specific differences in the lipid metabolism of avian and mammalian species.     

gamma-Glutamyltranspeptidase and dipeptidyl peptidase IV activity in the serum of normal and hepatoma-bearing chickens and in the plasma membranes from liver and hepatoma Mc-29.

Investigations on the activity of gamma-glutamyltranspeptidase (GGT) and dipeptidyl peptidase IV (DPP IV) in the serum of healthy chickens and those bearing hepatoma Mc-29, and in liver and hepatoma plasma membranes were carried out. There was no difference in the serum enzyme activities of control and tumor-bearing chickens but the activity of GGT was twice higher and that of DPP IV 20 times lower in hepatoma plasma membranes than in chicken liver plasma membranes. Using thin-layer analytical isoelectric focusing in agarose gels it was established that the pI range of GGT from host serum and hepatoma plasma membranes was shifted to more acidic values. This could be interpreted as a specific feature for this enzyme considered as a tumor marker.     

Effect of zearalenone on female White Leghorn chickens.

Acute toxic effects of purified zearalenone were studied in growing female White Leghorn chickens. In the first experiment, zearalenone in gelatin capsules was administered to 10 chickens (zearalenone-treated chickens [ZC]) in a single oral dose of 15.0 g/kg. Another 10 control chickens (CC) received empty gelatin capsules. All chickens survived the 10-day experiment and did not show any noticeable gross or histopathological lesions. There were no differences between CC and ZC in weight gain, oviduct, comb and liver weights, hematological parameters, and serum cholesterol. ZC had significantly less (P less than 0.05) serum calcium but significantly greater (P less than 0.01) serum phosphorus than CC. In the second experiment, zearalenone was administered orally or intramuscularly (pectoral muscle) at levels of 0, 50, 200, 400, and 800 mg/kg for 7 consecutive days. The oviduct weight increased with increasing toxin levels in both orally (OZC) and intramuscularly (IZC) administered groups: there were more pronounced effects in the IZC. The liver weight increased and comb weight decreased in IZC. The relative estrogenic biopotency of zearalenone in IZC, using estradiol dipropionate as a standard, was 1.37%. The results of this experiment demonstrate that chickens are highly tolerant to zearalenone and that the estrogenic effects of the toxin are greater when it is administered in multiple doses than in a single dose and in IZC than in OZC.     

A cellular protein is immunologically crossreactive with and functionally homologous to the Fujinami sarcoma virus transforming protein

We obtained a regressing-tumor antiserum specific for the unique sequence of the transforming protein P140 of Fujinami sarcoma virus by injecting Fischer rats with syngeneic embryo cells transformed with Fujinami sarcoma virus. This serum is capable of immunoprecipitating a protein of 98,000 daltons from cell extracts of normal, uninfected chicken bone marrow cells. This normal cellular protein (NCP98) was shown to be structurally related to P140, sharing the majority of ³⁵S-methionine-labeled tryptic peptides with the viral gene product P140. NCP98 is a phosphoprotein in vivo, with an associated in vitro protein kinase activity, capable of phosphorylating specifically at tyrosine residues of NCP98 itself and a-casein, an externally added substrate. This kinase activity is biochemically indistinguishable from the kinase activity associated with P140 by all criteria tested. Moreover, in vitro-phosphorylated NCP98 and P140 shared the same phosphopeptides. The expression of NCP98 is tissue-specific. It is readily detectable in bone marrow cells and detectable to a lesser extent in liver and lung cells from 6–18 day old chickens.     

Identification of a breast tumor-associated orosomucoid by concanavalin A affinity chromatography.

Con A-Sepharose affinity chromatography was utilized to examine the glycoproteins in phosphosaline extracts of normal and breast tumor tissues and breast patient sera. In extracts of normal breast tissue, normal sera and patient sera, all glycoproteins were eluted from the Con A-Sepharose with a linear gradient of 0.0-0.5 M alpha-methylmannose. Using breast tumor extracts, a glycoprotein peak which could not be eluted as with normal tissue extracts was observed. This tightly-binding peak could be eluted from the Con A-Sepharose with acetate buffer containing 1.0 M KCl. Polyacrylamide electrophoresis of this tightly-binding glycoprotein peak revealed one major glycoprotein and four minor glycoproteins. The major glycoprotein obtained from electrophoresis represented about 60% of the Con A-Sepharose tightly-binding protein and reacted with antiserum to human orosomucoid (alpha 1-acid glycoprotein). All glycoproteins isolated from tumor tissue extracts appeared to represent normal serum constituents as they were retained on an immunoadsorbent containing antibodies to normal serum proteins. The possible significance of the isolated tumor-associated orosomucoid is discussed.     

Absence of cross-reacting material in isolated propionyl CoA carboxylase deficiency: nature of residual carboxylating activity.

Fibroblast extracts and fetal liver homogenates from patients with propionic acidemia due to inherited deficiency of propionyl CoA carboxylase (PCC) were analyzed for the presence of immunologically cross-reactive PCC protein. Using several rabbit antisera raised against homogeneous human liver PCC, homogeneous pig heart PCC, or the individual non-identical subunits of the human liver enzyme, we found no detectable cross-reacting material by direct or competitive immunotitration in several cell lines from patients in either major complementation group (pcc A; pcc C) with isolated PCC deficiency. In contrast, cells of a patient from the bio complementation group contained normal amounts of immunoreactive PCC. Further analysis of the pcc A and pcc C mutants revealed that their residual propionyl CoA carboxylating activity varied greatly depending on the concentration of extract or homogenate protein used in the PCC assay. When propionyl CoA carboxylation was assayed at high protein concentration in a fetal liver homogenate from a pcc C patient, the apparent PCC activity was comparable to that found in normal human fetal liver. Significantly, the specific activity in the mutant, but not in the control, extract declined steeply as protein concentration was lowered, and this loss could not be prevented by adding PCC substrates, bovine serum albumin, glycerol, or 2-mercaptoethanol. Moreover, detailed analyses of immunotitration curves of control fibroblasts extracts showed that fresh extracts contained an amount of nonimmunotitratable carboxylating activity corresponding to the residual activity present in fresh extracts of mutant cell lines. We conclude that the residual propionyl CoA carboxylating activity found in isolated PCC deficiency represents another carboxylase that can utilize propionyl CoA as a substrate rather than a mutant form of PCC with markedly different immunochemical and physicochemical properties.     

Incorporation of intraportal ammonia-N into blood and tissue nitrogenous compounds in chickens fed low and high protein diets

1. 15N-Percentage of the amide of glutamine in total blood non-protein-15N was 42 and 48% in chickens fed 5 and 20% protein diets, respectively, when 15N-ammonia was intraportally-infused for 6 hr. 2. The infused ammonia-15N also appeared in the amide of free glutamine in the liver and kidney in large amounts at both levels of protein intake. 3. The 15N incorporated into glutamine-amide in the blood, liver and kidney and non-protein-15N in plasma were greater in chickens fed the low protein diet than in those fed the high protein diet (P less than 0.05 except kidney of P less than 0.01). 4. About 60% of the amide-N of the glutamine which was increased during 6 hr infusion of ammonia was derived from infused ammonia-N and the remainder from endogenous nitrogen, irrespective of protein intake. 5. These results suggest that glutamine is the most important intermediate in detoxication of intraportal ammonia in chickens.     

Characterization of a novel protein that specifically binds to DNA modified by N-acetoxy-acetylaminofluorene and cis-diamminedichloroplatinum

Proteins recognizing DNA damaged by the chemical carcinogen N-acetoxy-acetylaminofluorene (AAAF) were analyzed in nuclear extracts from rat tissues, using a 36 bp oligonucleotide as a substrate and electrophoretic mobility shift and Southwestern blot assays. One major damage-recognizing protein was detected, whose amount was estimated as at least 10(5) copies per cell. Levels of this protein were similar in extracts from brain, kidney and liver, but much lower in extracts from testis. The affinity of the detected protein for DNA damaged by AAAF was about 70-fold higher than for undamaged DNA. DNA damaged by cis-diamminedichloroplatinum (cis-DDP), benzo(a)pyrene diolepoxide (BPDE) or UV-radiation also bound this protein with an increased affinity, the former more strongly and the latter two more weakly as compared to AAAF-damaged DNA. The detected AAAF/DDP-damaged-DNA-binding (AAAF/DDP-DDB) protein had a molecular mass of about 25 kDa and was distinct from histone H1 or HMGB proteins, which are known to have a high affinity for cis-DDP-damaged DNA. The level of this damage-recognizing protein was not affected in rats treated with the carcinogen 2-acetylaminofluorene. The activity of an AAAF/DDP-DDB protein could also be detected in extracts from mouse liver cells but not from the Hep2G human hepatocellular carcinoma.     

Oxidation state of tissue thiol groups and content of protein carbonyl groups in chickens with inherited muscular dystrophy.

Indirect evidence suggests that oxidative stress may play a role in the pathogenesis of inherited muscular dystrophy, but the significance and precise extent of this contribution is poorly understood. Compared with normal muscle, significantly higher contents of glutathione, glutathione disulphide, protein-glutathione mixed disulphides and protein carbonyl groups, and significantly lower contents of free protein thiol groups, were found in pectoralis major muscle of genetically dystrophic chickens (the muscle affected by this disease) at 4 weeks of age. Other tissues did not show such marked disease-related differences. Interestingly, the protein pool in normal, but not dystrophic, pectoralis major muscle was relatively less oxidized in relation to the glutathione pool as compared with other tissues studied. The mechanisms by which this unique relationship between the thiol pools is maintained remain unknown. Although the physiological consequences of the increased content of protein carbonyl groups and the altered thiol pools in dystrophic muscle are not clear, the changes evident at such a young age are consistent with the occurrence of oxidative stress and may reflect significant damage to cellular proteins in this disease.