The ORNATE India Project: United Kingdom-India Research Collaboration to tackle visual impairment due to diabetic retinopathy.

INTRODUCTION: The ORNATE India project is funded by the UK Research and Innovation (UKRI) through the Global Challenges Research Fund. The aim is to build research capacity and capability in India and the UK to tackle global burden of diabetes-related visual impairment. As there are over 77 million people with diabetes in India, it is challenging to screen every person with diabetes annually for sight-threatening diabetic retinopathy (DR). Therefore, alternate safe approaches need to be developed so that those at-risk of visual impairment due to DR is identified promptly and treated. METHODS: The project team utilised diverse global health strategies and research methods to co-design work packages to build research capacity and capability to ensure effective, affordable and efficient DR services are made available for the population. The strategies and methods employed included health system strengthening; implementation science; establishing care pathways; co-designing collaborative studies on affordable technologies, developing quality standards and guidelines to decrease variations in care; economic analysis; risk modelling and stratification. Five integrated work packages have been developed to deal with all aspects of DR care. These included implementation of a DR screening programme in the public health system in a district in Kerala, evaluating regional prevalence of diabetes and DR and assessing ideal tests for holistic screening for diabetes and its complications in 20 areas in India, utilising artificial intelligence on retinal images to facilitate DR screening, exploring biomarker and biosensor research to detect people at risk of diabetes complications, estimating cost of blindness in India and risk modelling to develop risk-based screening models for diabetes and its complications. A large collaborative network will be formed to propagate research, promote shared learning and bilateral exchanges between high- and middle-income countries to tackle diabetes-related blindness.

Impact of parvovirus B19 infection on paediatric patients with haematological and/or oncological disorders.

To determine the frequency and the impact of parvovirus B19 (B19V) infection and its influence on the course of haematological and/or oncological diseases in paediatric patients, consecutive serum and bone marrow samples from 110 were analyzed for markers of acute, past and persistent B19V-infection using qPCR, ELISA and WesternLine. Twenty-seven out of 110 (24.5%) children suffered from non-malignant diseases (anaemia, pancytopenia, autoimmune disorders); 68/110 (61.8%) patients had developed leukaemia, malignant lymphoma or solid malignant tumours; 15/110 patients (13.6%) presented with other symptoms. At admission, B19V-specific IgM and IgG indicating acute or previous B19V-infection were observed in 5 (4.5%) and 48 patients (43.6%), respectively. B19V-DNA (10(3) -10(9) geq/mL) was detectable in serum and/or bone marrow of 22 patients (20.0%). These suffered from leukaemia (5), non-Hodgkin lymphoma (2), solid tumours (6), autoimmune (4) and haematological (4) disease and fever (1). During clinical observation four further leukaemia patients developed viraemia and persistent B19V-infection was observed in 13/22 DNA-positive patients. Treatment of B19V-DNA-positive cancer patients was associated with more supportive therapy involving erythrocyte and thrombocyte transfusion and/or antibiotic therapy. Acute B19V-infection has been frequently observed in paediatric patients with haematological and/or oncological disease. In patients with non-malignant diseases anaemia or autoimmune disorders were diagnosed in association with B19V-infection. Furthermore, a significant number of cancer patients displayed markers for acute, recent or persistent B19V-infection. This association may be strengthened by frequent treatment with blood products combined with therapeutic immune suppression. In B19V-infected cancer patients supportive therapy was more complex.

Transcriptomic "portraits" of canine mammary cancer cell lines with various phenotypes.

In light of the high incidence of mammary cancer in dogs and completion of the canine genome sequencing, the new possibilities of gene profiling by using DNA microarrays give hope to veterinary oncology. The cell lines isolated from mammary tumors are a valuable tool in developing and testing new pathway-specific cancer therapeutics. Differential cytometric analysis of 6 canine mammary cancer cell lines was performed. We divided cell lines into 3 groups based on their phenotype: 2 lines with high proliferative potential, 2 lines with high antiapoptotic potential, and 2 lines with high metastatic potential. DNA microarray analysis revealed common genes for cell lines of each group. We found that genes encoding the receptors for growth hormone and ghrelin are related to high proliferation rate, while ABR (active BCR-related) and TMD1 (TM2 domain containing 1) genes are related to a high antiapoptotic potential of the cancer cells. Metastatic properties of mammary cancer cells seem to be associated with elevated expression of PGP (P glycoprotein), SEMA3B (semaphorin 3B), and STIM1 (stromal interaction molecule 1).

Transcriptomic signature of cell lines isolated from canine mammary adenocarcinoma metastases to lungs.

Metastasis is a final step in the progression of mammary gland cancer, usually leading to death. Potentially, a molecular signature of metastasis can be defined via comparison of primary tumors with their metastases. Currently, there is no data in the literature regarding the molecular portrait of metastases in dogs and only few reports regarding human cancer. This is the first report describing the transcriptomic signature of canine cancer metastatic cells. Two adenocarcinoma cell lines isolated from the canine mammary gland (CMT-W1 and CMT-W2) were compared with cell lines isolated from their lung metastases (CMT-W1M and CMT-W2M) with regards to the following cytometric parameters: cell cycle, ploidy, Bcl-2 expression, susceptibility to induced apoptosis, and transcriptomic profile. Cytometric analyses revealed significant differences in cell cycle and antiapoptotic potential between the examined cells. Using oligonucleotide microarrays, we found 104 up-regulated genes in the metastatic cell line CMT-W1M and 21 up-regulated genes in the primary CMT-W1 cell line. We also found 83 up-regulated genes in the CMT-W2M cell line and only 21 up-regulated genes in the CMT-W2 cell line. Among the up-regulated genes in both metastatic cell lines, we found 15 common genes. These differently expressed genes are involved mainly in signal transduction, cell structure and motility, nucleic acid metabolism, developmental processing, and apoptosis (GHSR, RASSF1, ARF1GAP, WDR74, SMOC2, SFRP4, DIAPH1, FSCN1, ALX4, SNX15, PLD2, WNT7B, POU6F2, NKG7, and POLR2F). Seven of them are involved in a cellular pathway dependent on ghrelin via growth hormone secretagogue receptor (GHSR). Our results suggest that this pathway may be essential for mammary cancer cells to have a metastatic potential.

The influence of xenon on regulation of the autonomic nervous system in patients at high risk of perioperative cardiac complications.

BACKGROUND: As xenon anaesthesia (XE) does not produce haemodynamic depression its use may be of benefit in patients at high risk of intraoperative haemodynamic instability and perioperative cardiac complications. XE (n=22) was compared with total i.v. anaesthesia (TIVA, n=22) for differences in autonomic regulation, peri- and postoperative performance. METHODS: Patients undergoing abdominal aortic surgery were studied at five events: T1: baseline awake; T2: anaesthesia induction; T3: before aortic cross-clamping; T4: after aortic cross-clamping; T5: after aortic declamping. T3-T5: end-tidal xenon concentration 60 (5)%. Intraoperative analysis: heart rate, heart rate variability, blood pressure and cardiac output. Postoperative analysis: 24 h Holter ECG, intensive care unit and hospital stay, and patient's outcome after 6 months. RESULTS: XE in contrast to TIVA increased parasympathetic and decreased sympathetic activity. Median low to high frequency decreased significantly in the XE group after start of XE (P<0.05) and remained significantly lower during all events after start of XE as compared with TIVA (P=0.0001). After start of XE heart rate of these patients was significantly lower as compared with TIVA (P=0.04). Cardiac output increased significantly in TIVA after aortic declamping (P<0.05). Outcome parameters did not differ significantly between groups. CONCLUSIONS: XE patients demonstrated lower sympathetic and higher parasympathetic activity as compared with TIVA patients. This was reflected by significant differences in haemodynamics but did not correlate with a better postoperative outcome. Thus, it remains controversial whether XE provides benefits in high risk patients.

Comparison of xenon-based anaesthesia compared with total intravenous anaesthesia in high risk surgical patients.

Xenon, a noble gas with anaesthetic and analgesic properties, has gained renewed interest due to its favourable physical properties which allow a rapid emergence from anaesthesia. However, high costs limit its use to a subset of patients who may benefit from xenon, thereby offsetting its costs. To date, there are only limited data available on the performance of xenon in high risk patients. We studied 39 patients with ASA physical status III undergoing aortic surgery. The patients were randomly assigned to either a xenon (Xe, n = 20) or a TIVA (T, n = 19) group. Global cardiac performance and myocardial contractility were assessed using transoesophageal echocardiography, and myocardial cell damage with troponin T and CK-MB. Echocardiographic measurements were made prior to xenon administration, following xenon administration, and after clamping of the abdominal aorta, after declamping and at corresponding time points in the TIVA group. Laboratory values were determined repeatedly for up to 72 h. Data were analysed using two-way anova factoring for time and anaesthetic agent or with ancova comparing linear regression lines. No significant differences were found in global myocardial performance, myocardial contractility or laboratory values at any time during the study period. Mean (SEM) duration of stay on the ICU (xenon: 38 +/- 46 vs. TIVA 25 +/- 15 h) or in hospital (xenon: 14 +/- 12 vs. TIVA 10 +/- 6 days) did not differ significantly between the groups. Although xenon has previously been shown to exert superior haemodynamic stability, we were unable to demonstrate an advantage of xenon-based anaesthesia compared to TIVA in high risk surgical patients.

Blood-brain barrier-specific properties of a human adult brain endothelial cell line.

Establishment of a human model of the blood-brain barrier has proven to be a difficult goal. To accomplish this, normal human brain endothelial cells were transduced by lentiviral vectors incorporating human telomerase or SV40 T antigen. Among the many stable immortalized clones obtained by sequential limiting dilution cloning of the transduced cells, one was selected for expression of normal endothelial markers, including CD31, VE cadherin, and von Willebrand factor. This cell line, termed hCMEC/D3, showed a stable normal karyotype, maintained contact-inhibited monolayers in tissue culture, exhibited robust proliferation in response to endothelial growth factors, and formed capillary tubes in matrix but no colonies in soft agar. hCMEC/D3 cells expressed telomerase and grew indefinitely without phenotypic dedifferentiation. These cells expressed chemokine receptors, up-regulated adhesion molecules in response to inflammatory cytokines, and demonstrated blood-brain barrier characteristics, including tight junctional proteins and the capacity to actively exclude drugs. hCMEC/D3 are excellent candidates for studies of blood-brain barrier function, the responses of brain endothelium to inflammatory and infectious stimuli, and the interaction of brain endothelium with lymphocytes or tumor cells. Thus, hCMEC/D3 represents the first stable, fully characterized, well-differentiated human brain endothelial cell line and should serve as a widely usable research tool.

Vimentin dephosphorylation by protein phosphatase 2A is modulated by the targeting subunit B55.

The intermediate filament protein vimentin is a major phosphoprotein in mammalian fibroblasts, and reversible phosphorylation plays a key role in its dynamic rearrangement. Selective inhibition of type 2A but not type 1 protein phosphatases led to hyperphosphorylation and concomitant disassembly of vimentin, characterized by a collapse into bundles around the nucleus. We have analyzed the potential role of one of the major protein phosphatase 2A (PP2A) regulatory subunits, B55, in vimentin dephosphorylation. In mammalian fibroblasts, B55 protein was distributed ubiquitously throughout the cytoplasm with a fraction associated to vimentin. Specific depletion of B55 in living cells by antisense B55 RNA was accompanied by disassembly and increased phosphorylation of vimentin, as when type 2A phosphatases were inhibited using okadaic acid. The presence of B55 was a prerequisite for PP2A to efficiently dephosphorylate vimentin in vitro or to induce filament reassembly in situ. Both biochemical fractionation and immunofluorescence analysis of detergent-extracted cells revealed that fractions of PP2Ac, PR65, and B55 were tightly associated with vimentin. Furthermore, vimentin-associated PP2A catalytic subunit was displaced in B55-depleted cells. Taken together these data show that, in mammalian fibroblasts, the intermediate filament protein vimentin is dephosphorylated by PP2A, an event targeted by B55.

The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs.

The PR65/A subunit of protein phosphatase 2A serves as a scaffolding molecule to coordinate the assembly of the catalytic subunit and a variable regulatory B subunit, generating functionally diverse heterotrimers. Mutations of the beta isoform of PR65 are associated with lung and colon tumors. The crystal structure of the PR65/Aalpha subunit, at 2.3 A resolution, reveals the conformation of its 15 tandemly repeated HEAT sequences, degenerate motifs of approximately 39 amino acids present in a variety of proteins, including huntingtin and importin beta. Individual motifs are composed of a pair of antiparallel alpha helices that assemble in a mainly linear, repetitive fashion to form an elongated molecule characterized by a double layer of alpha helices. Left-handed rotations at three interrepeat interfaces generate a novel left-hand superhelical conformation. The protein interaction interface is formed from the intrarepeat turns that are aligned to form a continuous ridge.

Phosphorylation of an N-terminal motif enhances DNA-binding activity of the human SRY protein.

Of the several strategies that eukaryotes have evolved to modulate transcription factor activity, phosphorylation is regarded as one of the major mechanisms in signal-dependent transcriptional control. To conclusively demonstrate that the human sex-determining gene SRY is affected by such a post-translational control mechanism, we have analyzed its phosphorylation status in living cells. In the present study, we show that the cyclic AMP-dependent protein kinase (PKA) phosphorylates the human SRY protein in vitro as well as in vivo on serine residues located in the N-terminal part of the protein. This phosphorylation event was shown to positively regulate SRY DNA-binding activity and to enhance the ability of SRY to inhibit a basal promoter activity located downstream of an SRY DNA-binding site concatamer. Together these results strongly support the hypothesis that human SRY is a natural substrate for PKA in vivo and that this phosphorylation significantly modulates its major activity, DNA-binding, thereby possibly altering its biological function.

Modulation of the enzymatic properties of protein phosphatase 2A catalytic subunit by the recombinant 65-kDa regulatory subunit PR65alpha.

All protein phosphatase 2A (PP2A) holoenzymes contain a 36-kDa catalytic subunit (PP2Ac) and a regulatory subunit of 65 kDa (PR65). We have studied the interaction between PP2Ac and PR65 in an in vitro system, using PP2Ac isolated from rabbit skeletal muscle and recombinant PR65alpha expressed in bacteria or insect cells. Bacterially expressed PR65alpha exhibited identical biochemical properties to the protein expressed and isolated from the baculoviral expression system. The association of recombinant PR65 with PP2Ac was very tight (K(D)app = 85 pM) and led to a suppression of PP2A activity, which was maximal (70-80%) when phosphoproteins were used as substrates. When less-structured or smaller substrates (such as phosphopeptides) were used, this inhibition was only 30%. PR65 stimulated PP2Ac activity when the assays were performed in the presence of polycations. This indicates that the PR65 not only serves the previously predicted structural role as a molecular scaffold, but also allosterically modulates the enzymatic properties of PP2Ac. Furthermore, we identified a site of interaction between PP2Ac and PR65alpha by disruption of a stretch of basic amino acids by introduction of a glutamate at position 416. This produced an almost 100-fold reduced affinity for PP2Ac and indicated that this basic motif is an important determinant for the interaction of PR65 and PP2Ac.

Differential inhibition and posttranslational modification of protein phosphatase 1 and 2A in MCF7 cells treated with calyculin-A, okadaic acid, and tautomycin.

Calyculin-A (CA), okadaic acid (OA), and tautomycin (TAU) are potent inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A) and are widely used on cells in culture. Despite their well characterized selectivity in vitro, their exact intracellular effects on PP1 and PP2A cannot be directly deduced from their extracellular concentration because their cell permeation properties are not known. Here we demonstrate that, due to the tight binding of the inhibitors to PP1 and/or PP2A, their cell penetration could be monitored by measuring PP1 and PP2A activities in cell-free extracts. Treatment of MCF7 cells with 10 nM CA for 2 h simultaneously inhibited PP1 and PP2A activities by more than 50%. A concentration of 1 microM OA was required to obtain a similar time course of PP2A inhibition in MCF7 cells to that observed with 10 nM CA, whereas PP1 activity was unaffected. PP1 was predominantly inhibited in MCF7 cells treated with TAU but even at 10 microM TAU PP1 inhibition was much slower than that observed with 10 nM CA. Furthermore, binding of inhibitors to PP2Ac and/or PP1c in MCF7 cells led to differential posttranslational modifications of the carboxyl termini of the proteins as demonstrated by Western blotting. OA and CA, in contrast to TAU, induced demethylation of the carboxyl-terminal Leu309 residue of PP2Ac. On the other hand, CA and TAU, in contrast to OA, elicited a marked decrease in immunoreactivity of the carboxyl terminus of the alpha-isoform of PP1c, probably reflecting proteolysis of the protein. These results suggest that in MCF7 cells OA selectively inhibits PP2A and TAU predominantly affects PP1, a conclusion supported by their differential effects on cytokeratins in this cell line.

Negative modulation of membrane localization of the Raf-1 protein kinase by hyperphosphorylation.

The serine/threonine-specific protein kinase Raf-1 plays a key role in mitogenic signal transduction by coupling Ras to the mitogen-activated protein (MAP) kinase cascade. Ras-mediated translocation to the plasma membrane represents a crucial step in the process of serum-stimulated Raf-1 kinase activation. The exact role of the multisite phosphorylation in Raf regulation, however, is not clear. We have previously reported that the mobility shift-associated hyperphosphorylation of Raf correlates with a reduction of serum-stimulated Raf kinase activity (Wartmann, M., and Davis, R. J. (1994) J. Biol. Chem. 269, 6695-6701). Here we show that incubation of serum-starved CHO cells with D609, a purported inhibitor of phosphatidylcholine-specific phospholipase C, also results in a mobility shift of Raf-1 that is due to hyperphosphorylation on sites identical to those observed following mitogen stimulation. Subcellular fractionation analyses revealed that D609-induced mobility shift-associated hyperphosphorylation was paralleled by a decreased membrane association of Raf-1. Similar results were obtained in an in vitro reconstitution system. Furthermore, PD98059, a specific inhibitor of activation of the MAP kinase kinase MEK, prevented D609-induced Raf hyperphosphorylation and restored the amount of membrane-bound Raf to control levels. Taken together, these data suggest that mobility shift-associated hyperphosphorylation of Raf-1, by virtue of reducing the amount of plasma membrane-bound Raf-1, represents a negative feedback mechanism contributing to the desensitization of the MAP kinase signaling cascade.

Human cytomegalovirus carries serine/threonine protein phosphatases PP1 and a host-cell derived PP2A.

Human cytomegalovirus (CMV), a herpesvirus, is an important cause of morbidity and mortality in immunocompromised patients. When studying hyper-immediate-early events after contact between CMV virions and the cell membrane, we observed a hypophosphorylation of cellular proteins within 10 min. This can be explained in part by our finding that purified CMV contains serine/threonine protein phosphatase activities. Biochemical analyses indicate that this protein phosphatase activity has all characteristics of type 1 and 2A protein phosphatases (PP1 and PP2A). Specifically, PP1 accounts for approximately 30% and PP2A accounts for the remaining 70% of the phosphorylase phosphatase activity found. CMV produced in astrocytoma cells stably expressing an amino-terminally tagged PP2A catalytic subunit contained tagged enzyme, thus demonstrating the cellular origin of CMV-associated PP2A. PP2A is specifically found inside the virus, associated with the nucleocapsid fraction. Western blot (immunoblot) analysis of purified virus revealed the presence of the catalytic subunits of PP2A and PP1. Furthermore, the catalytic subunit of PP2A appears to be complexed to the regulatory subunits PR65 and PR55, which is also the most abundant configuration of this enzyme found in the host cells. Incubation of virus with okadaic acid before contact of CMV with cells prevented hypophosphorylation of cellular proteins, thus demonstrating the role of CMV-associated phosphatases in this phenomenon. CMV can thus transport an active enzyme from one cell to another.

Deregulation of translational control of the 65-kDa regulatory subunit (PR65 alpha) of protein phosphatase 2A leads to multinucleated cells.

Efficient translation of the mRNA encoding the 65-kDa regulatory subunit (PR65 alpha) of protein phosphatase 2A (PP2A) is prevented by an out of frame upstream AUG and a stable stem-loop structure (delta G = -55.9 kcal/mol) in the 5'-untranslated region (5'-UTR). Deletion of the 5'-UTR allows efficient translation of the PR65 alpha message in vitro and overexpression in COS-1 cells. Insertion of the 5'-UTR into the beta-galactosidase leader sequence dramatically inhibits translation of the beta-galactosidase message in vitro and in vivo, confirming that this sequence functions as a potent translation regulatory sequence. Cells transfected or microinjected with a PR65 alpha expression vector lacking the 5'-UTR, express high levels of PR65 alpha, accumulating in both nucleus and cytoplasm. PR65 alpha overexpressing rat embryo fibroblasts (REF-52 cells) become multinucleated. These data and previous results (Mayer-Jaekel, R. E., Ohkura, H., Gomes, R., Sunkel, C. E., Baumgartner, S., Hemmings, B. A., and Glover, D. M. (1993) Cell 72, 621-633) suggest that PP2A participates in the regulation of both mitosis and cytokinesis.

Differential methylation and altered conformation of cytoplasmic and nuclear forms of protein phosphatase 2A during cell cycle progression.

Protein phosphatase 2A (PP2A) appears to be involved in the regulation of many cellular processes. Control mechanisms that lead to the activation (and deactivation) of the various holoenzymes to initiate appropriate dephosphorylation events remain obscure. The core components of all PP2A holoenzymes are the catalytic (PP2Ac) and 63-65-kD regulatory (PR65) subunits. Monospecific and affinity-purified antibodies against both PP2Ac and PR65 show that these proteins are ubiquitously localized in the cytoplasm and the nucleus in nontransformed fibroblasts. As determined by quantitative immunofluorescence the core subunits of PP2A are twofold more concentrated in the nucleus than in the cytoplasm. Detailed analysis of synchronized cells reveals striking changes in the nuclear to cytoplasmic ratio of PP2Ac-specific immunoreactivity albeit the total amounts of neither PP2Ac nor PR65 in each compartment alters significantly during the cell cycle. Our results imply that differential methylation of PP2Ac occurs at the G0/G1 and G1/S boundaries. Specifically a demethylated form of PP2Ac is found in the cytoplasm of G1 cells, and in the nucleus of S and G2 cells. In addition nuclear PP2A holoenzymes appear to undergo conformational changes at the G0/G1 and G1/S boundaries. During mitosis PP2A is lost from the nuclear compartment, and unlike protein phosphatase 1 shows no specific association with the condensed chromatin.

The catalytic subunit of protein phosphatase 2A is carboxyl-methylated in vivo.

We have used polyclonal antibodies against an internal peptide (residues 169 to 182; Ab169/182) and a peptide corresponding to the carboxyl terminus (residues 299 to 309; Ab299/309) to look for in vivo modifications of protein phosphatase 2A catalytic (PP2Ac) subunit. Treatment of extracts from human breast cancer (MCF7) cells with either alkali or ethanol increased immunoreactivity of PP2Ac subunit severalfold on Western blots with Ab299/309, but did not apparently change molecular weight or isoelectric point of the protein. In contrast, immunoreactivity with Ab169/182 was unchanged by these treatments. Subsequently, we demonstrated that the increase in PP2Ac subunit recognition by Ab299/309 coincides with the demethylation of this protein at the carboxyl-terminal leucine (Leu309). Methylation of PP2Ac subunit, in vitro, increases its activity toward both phosphorylase a and a phosphopeptide. The carboxyl-terminal sequence (TPDYFL) of PP2Ac subunit is completely conserved between mammals, yeast, fruit fly, and plants which suggests that regulation of this enzyme activity by carboxyl-terminal methylation has been conserved during evolution.

Intramolecular electron transfer rate between active-site copper and topa quinone in pea seedling amine oxidase.

The equilibrium between the two substrate-reduced forms of pea seedling amine oxidase, one containing Cu(II) and reduced 3-(2,4,5-trihydroxyphenyl)-L-alanine (topa) cofactor and one containing Cu(I) and topa semi-quinone, was investigated by visible spectroscopy as a function of temperature. To determine the rate of interconversion between the two species, temperature jump relaxation studies were performed on the substrate-reduced enzyme near room temperature. The yellow radical species was found to approach its equilibrium concentration with a maximum rate constant of 43,000 +/- 3,000 s-1. This rapid equilibration is attributed to intramolecular electron transfer between copper and topa. The data indicate that the Cu(I)/topaSQ species is a kinetically competent intermediate in the reaction of amine oxidases with substrates. Furthermore, the extremely rapid electron transfer rate (kET congruent to 20,000 s-1) suggests that the topa cofactor is in close proximity to the copper atom.

Analysis of subunit isoforms in protein phosphatase 2A holoenzymes from rabbit and Xenopus.

A dimeric and two trimeric forms of protein phosphatase 2A (PP2A) were purified from rabbit and Xenopus tissues and analyzed using antisera specific for the catalytic and regulatory subunits. The dimeric holoenzyme consists of a complex between a 36-kDa catalytic subunit associated with a approximately 65-kDa regulatory subunit. The two trimeric holoenzymes consist of the catalytic subunit complexed with 65- and 55-kDa subunits, or 65- and 72-kDa subunits. Antisera were raised against synthetic peptides specific for the alpha- and beta-isoforms of the 65-kDa (PR65 alpha/beta) and 55-kDa (PR55 alpha/beta) subunits identified by molecular cloning. Anti-peptide antisera to the 36-kDa catalytic subunit of PP2A were prepared against two selected regions: one specific for the alpha-isoform and one to a peptide common to both the alpha- and beta-isoforms. Immunochemical analysis of all three mammalian holoenzymes showed that the catalytic, 55- and 65-kDa subunits are both predominantly of the alpha-isoform, which is consistent with the peptide sequence data. The 65-kDa subunit of PP2A holoenzymes isolated from Xenopus skeletal muscle reacted with both anti-alpha and anti-beta PR65-specific antisera whereas the PP2A holoenzymes isolated from Xenopus oocytes reacted preferentially with the beta-specific antisera, indicating developmental changes in the expression of the 65-kDa subunit isoform. Taken together, these results show that the "core" subunits of the PP2A holoenzymes consist of the catalytic complexed with the 65-kDa subunit and that the association of the third subunit does not appear to be influenced by the isoform of these two core subunits.