The soluble carnitine palmitoyltransferase from bovine liver. A comparison with the enzymes from peroxisomes and from the mitochondrial inner membrane.

The properties of two carnitine acyltransferases (CPT) purified from bovine liver are compared to confirm that they are different proteins. The soluble CPT and the inner CPT from mitochondria differ in subunit Mr, native Mr, pI and reactivity with thiol reagents. All eight free thiol groups in soluble CPT react with 5,5'-dithiobis-(2-nitrobenzoate) in the absence of any unfolding reagent, and activity is gradually lost. The inner CPT activity is completely stable in the presence of 5,5'-dithiobis-(2-nitrobenzoate), and only one thiol group per molecule of subunit is modified in the native enzyme. Antisera to each enzyme inhibit that enzyme, but do not cross-react. CPT activity in subcellular fractions can now be identified by titration with these antibodies. The soluble CPT from bovine liver is probably peroxisomal in origin, but, although antigenically similar, it differs from the peroxisomal carnitine octanoyltransferase found in rat and mouse liver in its specificity for the longer-chain acyl-CoA substrates.     

Identification, purification, and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules

A novel Mr 28,000 integral membrane protein ("28kDa") was identified in human erythrocytes and found entirely associated with the Triton X-100 insoluble membrane skeletons. Antibodies to 28kDa reacted strongly on immunoblots with 28kDa and a diffuse region of Mr 35,000-60,000 ("HMW-28kDa"). Selective proteolytic digestions of membranes demonstrated that HMW-28kDa has an extracellular domain, and both 28kDa and HMW-28kDa have intracellular domains. 28kDa and HMW-28kDa were purified to homogeneity. Quantitative immunoblots indicate that each erythrocyte contains 120,000-160,000 copies of 28kDa. Two-dimensional iodopeptide maps of 28kDa and HMW-28kDa were nearly identical; peptide-N-glycosidase digestion of purified HMW-28kDa demonstrated that it is the N-glycosylated form of 28kDa. When concentrated, 28kDa formed a series of larger oligomers which were stable in sodium dodecyl sulfate. Of several nonerythroid tissues studied with anti-28kDa immunoblots, only kidney displayed immunoreactive 28kDa. Purified rat kidney 28kDa was nearly identical to rat erythrocyte 28kDa when compared by two-dimensional iodopeptide mapping. Immunohistochemical staining of human kidney with anti-28kDa demonstrated prominent staining over the apical brush borders of proximal convoluted tubules. A novel integral membrane protein has been purified from erythrocyte and kidney membranes. This new protein may play a role in linkage of the membrane skeleton to the lipid bilayer.     

Isolation and characterization of different forms of thioredoxins from the green alga Acetabularia mediterranea: identification of an NADP/thioredoxin system in the extrachloroplastic fraction.

A procedure has been developed for the simultaneous purification to apparent homogeneity of chloroplast thioredoxins f and m, and nonchloroplast thioredoxin h, from the green alga Acetabularia mediterranea. In the chloroplast fraction, three thioredoxins were isolated: one f type thioredoxin (Mr 13.4 kDa) and two m type thioredoxin forms (Mr of 12.9 and 13.8 kDa). A Western blot analysis of crude and purified chloroplast thioredoxin preparations revealed that Acetabularia thioredoxin m was immunologically related to its higher-plant counterparts whereas thioredoxin f was not. In the nonchloroplast fraction, a single form of thioredoxin h (Mr 13.4 kDa) and its associated enzyme NADP-thioredoxin reductase (NTR) were evidenced. Acetabularia NTR was partially purified and shown to be an holoenzyme composed of two 33.0-kDa subunits as is the case for other plant and bacterial NTRs. Similarity was confirmed by immunological tests: the algal enzyme was recognized by antibodies to spinach and Escherichia coli NTRs. Acetabularia thioredoxin h seemed to be more distant from higher-plant type h thioredoxins as recognition by antibodies to thioredoxin h from spinach and wheat was weak. The algal thioredoxin h was also slightly active with spinach and E. coli NTRs. These results suggest that in green algae as in the green tissues of higher plants the NADP and chloroplast thioredoxin systems are present simultaneously, and might play an important regulatory role in their respective cellular compartments.     

Purification and reconstitution of phosphatidylinositol 4-kinase from human erythrocytes.

A membrane-bound phosphatidylinositol 4-kinase (PtdIns kinase) has been purified to apparent homogeneity from human erythrocytes. Enzyme activity was solubilized from urea-KCl-stripped, inside-out membrane vesicles by 3% Triton X-100. Purification to apparent homogeneity was accomplished by cation-exchange chromatography on phosphocellulose, followed by heparin-acrylamide chromatography. This resulted in a nearly 3900-fold purification of PtdIns kinase activity to a specific activity of 44 nmol min-1 mg-1. The purified enzyme has an Mr of 59,000 on silver-stained SDS-PAGE; however, many preparations also contain 54 kDa and 50 kDa proteins which are related to the 59 kDa protein and have PtdIns kinase activity. Kinetic analysis of the PtdIns kinase indicate apparent Km values of 40 and 35 microM for phosphatidylinositol and ATP, respectively. The purified enzyme has been reconstituted into phospholipid liposomes and shown to phosphorylate phosphatidylinositol.     

Identification of a protein with properties of tenascin in the matrix of embryonal cartilage tissue and in a culture of human embryonal fibroblasts]

A protein with properties of tenascine has been discovered in extracellular matrix of human embryonic cartilage tissue. Its Mr, as determined by electrophoresis in PAAG with SDS, is 205 and 195 kDa. Immunoblotting with monoclonal antibodies raised against tenascine, revealed 2 precipitation bands, which permitted to identify the discovered protein as tenascine. This is the first report of detection of tenascine in rib cartilage matrix of human embryos. A protein identified as tenascine, has been also detected in a culture of human embryonic fibroblasts.     

The small molecular mass ubiquinone-binding protein (QPc-9.5 kDa) in mitochondrial ubiquinol-cytochrome c reductase: isolation, ubiquinone-binding domain, and immunoinhibition

The small molecular mass ubiquinone-binding protein (QPc-9.5 kDa) was purified to homogeneity from 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinol+ ++- labeled bovine heart mitochondrial ubiquinol-cytochrome c reductase. The N-terminal amino acid sequence of the isolated protein is Gly-Arg-Gln-Phe-Gly-His-Leu-Thr-Arg-Val-Arg-His-, which is identical with that of a Mr = 9500 protein in the reductase [Borchart et al. (1986) FEBS Lett. 200, 81-86]. A ubiquinone-binding peptide was prepared from [3H]azidoubiquinol-labeled QPc-9.5 kDa protein by trypsin digestion followed by HPLC separation. The partial N-terminal amino acid sequence of this peptide, Val-Ala-Pro-Pro-Phe-Val-Ala-Phe-Tyr-Leu-, corresponds to amino acid residues 48-57 in the reported Mr = 9500 protein. According to the proposed structural model for the Mr = 9500 protein, the azido-Q-labeled peptide is located in the membrane on the matrix side. These results confirm our previous assessment that the Mr = 13,400 subunit is not the small molecular weight Q-binding protein. Purified antibodies against QPc-9.5 kDa have a high titer with isolated QPc-9.5 kDa protein and complexes that contain it. Although antibodies against QPc-9.5 kDa do not inhibit intact succinate- and ubiquinol-cytochrome c reductases, a decrease of 85% and 20% in restoration of succinate- and ubiquinol-cytochrome c reductases, respectively, is observed when delipidated succinate- or ubiquinol-cytochrome reductases are incubated with antibodies prior to reconstitution with ubiquinone and phospholipid, indicating that epitopes at the catalytic site of QPc-9.5 kDa are buried in the phospholipid environment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and partial purification of the insulin-responsive glucose transporter from 3T3-L1 adipocytes

The glucose transporter in 3T3-L1 adipocytes has been identified as a polypeptide of average Mr 51000 by means of its reaction with antibodies raised against the purified human erythrocyte glucose transporter and by photolabeling with [3H]cytochalasin B. The finding that the antibodies immunoprecipitated the photolabeled polypeptide demonstrated that both methods detected the same polypeptide. The 3T3-L1 adipocyte glucose transporter has been partially purified. The main steps in the purification procedure were the preparation of salt-washed cellular membranes, Triton X-100 solubilization, and immunoaffinity chromatography on affinity-purified antibodies against the human erythrocyte transporter. A simple method of affinity purification of these antibodies, which consists of adsorption from serum onto protein-depleted erythrocyte membranes and release with acid, and an assay for the 3T3-L1 adipocyte transporter polypeptide, which employs immunoblotting, have been developed.     

Isolation and characterization of methylmalonyl-CoA mutase from human placenta

Methylmalonyl-CoA mutase, one of two known cobalamin-dependent enzymes present in mammalian tissues, has been isolated from 2.5 kg of human placenta utilizing affinity chromatography on 5'-deoxyadenosylcobalamin-Sepharose as the major purification step. The enzyme gives a single band on polyacrylamide disc gel electrophoresis. The Mr of the enzyme is 145,000 and it has two subunits of Mr = 72,000. Amino acid analysis reveals major differences from other human cobalamin-binding proteins. Based on x-ray fluorescence, the enzyme has 2 mol of cobalamin bound/mol of enzyme. In contrast to purified cobalamin transport proteins, most of the cobalamin bound to the enzyme is not released by boiling at low pH in the presence of KCN, or dialysis against 7.5 M guanidine containing 0.2 M dithiothreitol, or both, suggesting the possibility that cobalamin may be covalently attached to the purified enzyme. Both precipitating antibodies and antibodies that inhibit enzyme activity have been raised in a chicken.     

Protein X, a proteolysis product of human pancreatic juice. Immunological relationship with trypsinogen 1

Protein X (PX) previously isolated from human pancreatic juice is an inactive protein of 14 kDa which has been shown to be a degradation product liberated by proteolysis of 19 kDa precursors. Polyclonal antibodies against P19 and PX were prepared in rabbits by injection of the two proteins purified by SDS polyacrylamide gel electrophoresis. These antibodies reacted with a form of trypsin 1 (DFP-trypsin 1) which was shown to be partly proteolysed. Immunological studies were performed with pancreatic juice proteins and partially purified trypsinogen 1 using antibodies directed against PX, P19 and trypsin 1. The results of immunoprecipitation and immunoadsorbent chromatography show that these different antisera recognized a protein of 25 kDa. Immunoblotting has permitted to characterize this protein as a trypsinogen 1-like molecule which would be a form of inert protein generated by uncontrolled trypsinogen activation.     

Monoclonal antibodies to the Mr 74,000 cholesteryl ester transfer protein neutralize all of the cholesteryl ester and triglyceride transfer activities in human plasma

A cholesteryl ester transfer protein (CETP) of apparent Mr 74,000 has recently been purified from human plasma. Three monoclonal neutralizing antibodies to the CETP were obtained by immunizing mice with purified CETP. The antibodies, each recognizing a similar epitope on CETP, caused parallel and complete immunotitration of plasma cholesteryl ester and triglyceride transfer activities but only partial inhibition of phospholipid transfer activity. Monoclonal immunoaffinity chromatography of plasma or its fractions showed complete removal of cholesteryl ester and triglyceride transfer activities but incomplete removal of phospholipid transfer activity. Sodium dodecyl sulfate gel electrophoresis and immunoblotting of the immunoaffinity-retained fractions showed that only the Mr 74,000 protein was immunoreactive. The results suggest that the previously characterized CETP accounts for all of the cholesteryl ester and triglyceride transfer activity in human plasma but only part of the phospholipid transfer activity.     

Cholesteryl ester transfer protein is secreted by Hep G2 cells and contains asparagine-linked carbohydrate and sialic acid

A cholesteryl ester transfer protein (CETP) of apparent Mr 74,000 has recently been purified from human plasma. Cholesteryl ester transfer activity was found to accumulate in the medium of cultured Hep G2 cells. The transfer activity was removed by immunoprecipitation with specific antibodies to the plasma CETP. Sodium dodecyl sulfate gel electrophoresis of immunoprecipitates prepared from the medium of cells pulsed with [35S]methionine revealed a broad specific band of protein of Mr 72,000 to 76,000; by contrast, immunoprecipitates of cellular homogenates showed a sharp specific band of Mr 58,000. The Mr 72,000 to 76,000 band disappears, concomitant with the appearance of lower Mr products, upon neuraminidase or glycopeptidase F treatment of medium immunoprecipitates or of purified CETP. The results indicate that liver cells have the capacity to synthesize and secrete CETP. The CETP peptide acquires asparagine-linked carbohydrate and sialic acid during intracellular processing.     

Cardiac alpha-crystallin. I. Isolation and identification

A water soluble protein, a major component of the cytosolic fraction of rat heart cells, was purified using either reverse phase HPLC or antibodies affinity chromatography procedures and characterized. The protein has an apparent Mr of 24 k, as judged by SDS-gel electrophoresis. Under non-denaturing conditions, however, the protein occurs as a homomultimer (Mr between 400 and 650 k) of the monomeric 24 kDa species and could be selectively enriched by fractionation of the cytosolic fraction on 10 to 40% sucrose gradients. Polyclonal antibodies, raised against the denatured 24 kDa protein, were used to investigate its tissue distribution. Besides the heart, where it is very abundant, the 24 kDa protein is expressed also in other red muscles and in kidneys, but was not detectable in stomach, thymus, liver, and brain. The amino acid composition of the protein and the partial amino acid sequence of various proteolytic fragments was determined. A search for homologies of the primary structure of known proteins has shown that the 24 kDa protein is strikingly similar, if not identical to alpha-B-crystallin. In fact, the two proteins were found to be indistinguishable also by immunological criteria. This study demonstrates that the lens protein alpha B-crystallin is a major cytosolic component of heart cells.     

Inter-tissue and inter-species characteristics of the mitochondrial carnitine palmitoyltransferase enzyme system

Properties of the carnitine palmitoyltransferase (EC 2.3.1.21) (CPT) enzyme system were compared in isolated mitochondria from a range of tissues in rodents, monkey, and man. Common features were as follows: (a) while membrane-bound, CPT I, but not CPT II, was inhibited reversibly by malonyl-coenzyme A (CoA) and irreversibly by CoA esters of certain oxirane carboxylic acids; (b) the detergent, Tween-20, readily solubilized CPT II in active form while leaving CPT I membrane associated and catalytically functional; (c) octyl glucoside and Triton X-100 released active CPT II but caused essentially complete loss of CPT I activity. Use of [3H]tetradecylglycidyl-CoA, a covalent ligand for CPT I, yielded estimates of the enzyme's monomeric molecular size: approximately 86 kDa in non-hepatic tissues and approximately 90-94 kDa in liver, depending upon species. A polyclonal antibody to purified rat liver CPT II recognized a single protein in each tissue; its apparent molecular mass was approximately 70 kDa in all rat tissues and approximately 68 kDa in all mouse tissues as well as monkey and human liver. On Northern blot analysis a rat liver CPT II cDNA probe detected a single approximately 2.5-kilobase mRNA in all rat and mouse tissues examined. The following points are emphasized. First, CPT I and II are different proteins. Second, within a species CPT II, but not CPT I, is probably conserved across tissue lines. Third, slight variations in size of both enzymes were found in different species, although, at least in the case of CPT II, significant amino acid identity exists among the various isoforms. Fourth, CPT I, unlike CPT II, requires membrane integrity for catalytic function. Finally, the strategic use of detergents provides a simple means of discriminating between the two enzyme activities.     

Specific identification of tissue kallikrein in exocrine tissues and in cell-free translation products with monoclonal antibodies.

A panel of six mouse monoclonal antibodies (IgG1) has been prepared against purified rat urinary kallikrein (EC 3.4.21.35) and characterized. In radioimmunoassay, the antibody titres of ascitic fluid giving 50% binding to 125I-kallikrein range from 1:2 X 10(3) to 1:1 X 10(6). Antibodies from four of the clones show no cross-reactivity with human urinary kallikrein, rat urinary esterase A or tonin. However, antibodies from a fifth clone cross-react with tonin and, from a sixth, with both urinary esterase A and tonin. Three of the kallikrein affinity-purified monoclonal antibodies inhibited, whereas one of the antibodies stimulated, kallikrein activity. Tissue kallikrein from rat submandibular-gland and pancreatic extracts and urine were labelled with [14C]di-isopropyl phosphofluoridate, immunoprecipitated with each of the six monoclonal antibodies and identified to be 38 kDa proteins, similar in size to purified rat urinary kallikrein. Western-blot analysis shows that 125I-labelled kallikrein monoclonal antibodies (V4D11) bind directly to a 38 kDa protein in submandibular-gland and pancreatic extracts and urine. Cell-free translation products of submandibular-gland polyadenylylated[poly(A)+]mRNA were immunoprecipitated with affinity-purified sheep anti-kallikrein antibodies and three monoclonal antibodies (V4D11, V4G6 and V1C3). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these immunoprecipitates revealed that two kallikrein precursors with Mr values of 37 000 and 35 000 are encoded by submandibular-gland mRNA. The third monoclonal antibody, V1C3, which binds to active kallikrein, did not recognize either precursor form. Collectively, the data show that these monoclonal antibodies comprise a set of powerful and specific reagents for studies of tissue kallikreins.     

Affinity purification and refined structural characterization of terminal deoxynucleotidyltransferase.

A total of 56 stable murine hybridoma monoclones that produce homogeneous antibodies against human or calf terminal deoxynucleotidyltransferase have been established. All of the antibodies exhibited specific binding to various Mr forms of terminal transferase and eight possessed neutralizing activity. Results are presented that permitted characterization of ten of these antibodies with respect to their immunoglobulin class, their recognition of calf or human terminal-transferase Mr species by immunoblotting techniques and their recognition of distinct antigenic sites. Terminal transferase was purified in a single step by using an immunoaffinity column constructed with a monoclonal antibody exhibiting a high binding affinity for the enzyme. Single monoclonal antibodies were also used to bind selectively to terminal-transferase antigen in tissue slices and individual cells.     

NADP-malic enzyme and Hsp70: co-purification of both proteins and modification of NADP-malic enzyme properties by association with Hsp70

Different preparations of antibodies against 62 kDa NADP-malic enzyme (NADP-ME) from purified maize leaves cross-react with a 72 kDa protein from diverse tissues in many species. A 72 kDa protein, suggested to be a non-photosynthetic NADP-ME, has been purified from several plant species. However, to date, a cDNA coding for this putative 72 kDa NADP-ME has not been isolated. The screening of maize and tobacco leaf expression libraries using antibodies against purified 62 kDa NADP-ME allowed the identification of a heat shock protein (Hsp70). In addition, tandem mass spectrometry (MS/MS) studies indicate that along with NADP-ME, a 72 kDa protein, identified as an Hsp70 and reacting with the antibodies, is also purified from maize roots. On the other hand, the screening of a maize root cDNA library revealed the existence of a cDNA that encodes a mature 66 kDa NADP-ME. These results suggest that the 72 kDa protein is not actually an NADP-ME but in fact an Hsp70, at least in maize and tobacco. Probably, NADP-ME-Hsp70 association, taking place at least when preparing crude extracts, can lead to a co-purification of the proteins and can thus explain the cross-reaction of the antibodies. In the present work, we analyse and discuss a probable interaction of NADP-ME with Hsp70.     

Identification, immunoaffinity purification and partial characterization of a human decidua-associated protein

A crude extract of pooled early-pregnancy decidual tissue was enriched for soluble decidual proteins by exhaustive affinity absorption with antibodies to human serum proteins immobilized on Eupergit C. The partly purified extract was used to prepare monoclonal antibodies. A monoclonal antibody was obtained recognizing an antigen present in extract of decidual tissue and not in extract of proliferative endometrium. The monoclonal antibody was used for immunoaffinity purification of the decidua-associated protein. By SDS-PAGE analysis, under reducing conditions it yielded 2 bands at apparent molecular weights of 55,000 and 25,000. Under non-reducing conditions a single protein band at apparent molecular weight of 200,000 was observed. The Mr 200,000 protein was named hDP200 and the Mr 55,000 protein was named hDP55. It is suggested that hDP55 is a subunit of the hDP200. The hDP200 did not react with polyclonal antibodies specific for PP12 and PP14. PP14 has been shown to be immunologically indistinguishable from PEP and alpha 2-PEG. Our data therefore suggest that hDP200 is a novel human decidua-associated protein.     

Purification and characterization of M3 protein expressed on the surface of group A streptococcal type 3 strain C203

Abstract Monoclonal antibodies (mAbs) have been produced by immunizing BALB/C mice with whole M⁺ bacteria in incomplete Freund adjuvant and the resulting mAbs for M3 protein have been selected by an indirect immuno-fluorescent technique using formaldehyde-fixed M⁺ and M⁻ bacteria. Four mAbs reacted with a 65 kDa protein in an extract obtained from the cell wall of M⁺ bacteria after treatment with N -acetyl muramidase and lysozyme. The purified 65 kDa protein neutralized the phagocytic activity of rabbit anti-M3 antibody. The N-terminal amino acid sequence of the 65 kDa protein was identical with that of protein generated by the M3 gene which has been previously cloned and sequenced. The evidence indicates that the 65 kDa protein is M3 protein. The M3 protein bound not only human fibrinogen but also human serum albumin (HSA). When the M3 protein was purified by gel-filtration and ion-exchange chromatography in the absence of phenylmethyl sulfonyl fluoride (PMSF), four fragments (35 kDa, 32 kDa, 30 kDa, and 25 kDa) in addition to the intact molecule appeared. N-terminal amino acid sequence analysis showed that 35 kDa and 25 kDa fragments were ANAAD and DARSV, respectively, being identical at positions 1–5 and 198–202 to the M3 gene derived protein. Therefore, the 35 kDa and 25 kDa fragments, which were presumed to be cleavage products, may be derived from the C-terminal part and N-terminal part of the intact molecule, respectively. When the effect of purified M3 protein in the bactericidal activity of normal human blood in the presence of M⁻ bacteria was investigated, the M3 protein was responsible for the organism's resistance to attack by phagocytic cells.     

A general method for rapid purification of soluble versions of glycosylphosphatidylinositol-anchored proteins expressed in insect cells: an application for human tissue-nonspecific alkaline phosphatase.

A soluble form of tissue-nonspecific alkaline phosphatase was purified to apparent homogeneity from the culture media of Sf9 cells which had been infected with recombinant baculoviruses encoding human tissue-nonspecific alkaline phosphatase (TNSALP). To facilitate purification, an oligonucleotide consisting of 6 tandem codons for histidine and a stop codon was engineered into the TNSALP cDNA. The molecular mass of the enzyme purified through a nickel-chelate column was estimated to be 54 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. That of the native enzyme was 90 kDa as estimated by gel filtration, indicating that the purified soluble TNSALP is dimeric. The enzyme was used for production of antibodies specific for human TNSALP. Immunoblotting analysis showed a single 80-kDa band in the cell homogenate prepared from Saos-2 (human osteosarcoma) cells. However, upon digestion with peptide: N-glycosidase F, the 80-kDa TNSALP of human origin and the soluble enzyme of insect origin migrated to the same position on SDS-polyacrylamide gel, indicating that the size difference between the two enzymes is ascribed to N-linked oligosaccharides. The antibodies prepared against the purified TNSALP were found to be useful also for immunoprecipitation and immunofluorescence studies.     

Monoclonal antibodies to the glucose transporter from human erythrocytes. Identification of the transporter as a Mr = 55,000 protein

Using the preparation of purified glucose transporter from human erythrocytes as antigen, we have prepared and characterized six monoclonal antibodies. Three of these antibodies have been shown to be to the glucose transporter by several criteria: they immunoprecipitate the transport activity, the cytochalasin B binding activity, and 75% of the protein from the solubilized purified preparation. The remaining three antibodies were shown to recognize the same polypeptide by a Western blot procedure. All of the antibodies reacted with the deglycosylated transporter and are thus against peptide determinants; most bound to the cytoplasmic domain of the transporter. The antibodies exhibited a range of effects on cytochalasin B binding, from slight enhancement to modest inhibition to strong inhibition; for this reason they must bind to at least three different epitopes. Western blot analysis of erythrocyte membranes prepared in the presence of protease inhibitors showed that all six antibodies bound to a polypeptide of average Mr = 55,000. Moreover, by immunological assay this polypeptide accounted for 5.3% of the membranes protein, a value similar to that given by cytochalasin B binding. Thus, the proposal that the native transporter is a Mr = 100,000 polypeptide is highly unlikely. The antibodies also react with the glucose transporter in other human cell types, but not with that in rodent or avian cells.