Identification of citrullinated α-enolase as a candidate autoantigen in rheumatoid arthritis

Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis (RA), but little is understood about their citrullinated target antigens. We have detected a candidate citrullinated protein by immunoblotting lysates of monocytic and granulocytic HL-60 cells treated with peptidylarginine deiminase. In an initial screen of serum samples from four patients with RA and one control, a protein of molecular mass 47 kDa from monocytic HL-60s reacted with sera from the patients, but not with the serum from the control. Only the citrullinated form of the protein was recognised. The antigen was identified by tandem mass spectrometry as α-enolase, and the positions of nine citrulline residues in the sequence were determined. Serum samples from 52 patients with RA and 40 healthy controls were tested for presence of antibodies against citrullinated and non-citrullinated α-enolase by immunoblotting of the purified antigens. Twenty-four sera from patients with RA (46%) reacted with citrullinated α-enolase, of which seven (13%) also recognised the non-citrullinated protein. Six samples from the controls (15%) reacted with both forms. α-Enolase was detected in the RA joint, where it co-localised with citrullinated proteins. The presence of antibody together with expression of antigen within the joint implicates citrullinated α-enolase as a candidate autoantigen that could drive the chronic inflammatory response in RA.     

Proteins and Peptidases from Conidia and Mycelia of <Emphasis Type="Italic">Scedosporium apiospermum</Emphasis> Strain HLPB

The conidia–mycelia transformation is an essential step during the life cycle of the fungal human pathogens of the Pseudallescheria boydii complex. In the present study, we have analyzed the protein and peptidase profiles in two distinct morphological stages, conidia and mycelia, of Scedosporium apiospermum sensu stricto. Proteins synthesized by the mycelia, migrating at the ranges of 62–48 and 22–18 kDa, were not detected from the conidial extract. Conidia produced a single cellular peptidase of 28 kDa able to digest copolymerized albumin, while mycelia yielded 6 distinct peptidases ranging from 90 to 28 kDa. All proteolytic enzymes were active at acidic pH and fully inhibited by 1,10-phenanthroline, characterizing these activities as metallo-type peptidases. Quantitative peptidase assay, using soluble albumin, showed a high metallopeptidase production in mycelial cells in comparison with conidia. The regulated expression of proteins and peptidases in different morphological stages of S. apiospermum represents a potential target for isolation of stage-specific markers for biochemical and immunological analysis. Martha Machado Pereira and Bianca Alcantara Silva contributed equally to this work.     

Purification and characterization of κ-carrageenase from a novel γ-proteobacterium,<Emphasis Type="Italic">Pseudomonas elongata</Emphasis> (MTCC 5261) syn.<Emphasis Type="Italic">Microbulbifer elongatus</Emphasis> comb. Nov.

The phenotypic and carrageenolytic features of a novel halo tolerant marine bacterium, isolated from decayed red algal samples collected along the west coast of India were studied. This gram-negative strain was identified asPseudomonas elongala (MTCC 5261) syn.Microbulbifer elongalus comb. nov according to its morphological, physiological and molecular characterization. The extracellular κ-carrageenase was purified 106.54-fold by a combination of ammonium sulfate precipitation (40∼60%) and successive gel filtration chromatography. The purified protein fraction yielded significantly high activity of 426.19 units/mg protein and migrated as a single band on a sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of ∼128 kDa. For κ-carrageenase activity, optimum temperature was 40°C whereas two pH optimai.e. 5.6 and 7.7 were observed. For κ-carrageenan, the enzyme gave aK _m value of 6.66 mg/mL and aV _max value of 4 μmol/min/mg when the reaction was carried out at 40°C and pH 5.6. Isolated κ-carrageenase could successfully generate protoplasts ofKappaphycus alvarezii. This is the first report on the production of κ-carrageenase by this bacterium isolated from west coast of India. Molecular mass and various characteristics showed that the carrageenase fromP. elongata was much different from those previously reported.     

Jacalin Bound Plasma O-Glycoproteome and Reduced Sialylation of Alpha 2-HS Glycoprotein (A2HSG) in Rheumatoid Arthritis Patients

Glycosylation studies of plasma proteins can reveal information about the onset and progression of diseases, where in the glycan biosynthetic pathways are disturbed as in rheumatoid arthritis (RA). The present study was focused on analysis of O-linked glycoproteins of plasma in RA patients. Two dimensional gel electrophoresis of jacalin bound plasma of RA patients revealed a number of differentially expressed protein spots as compared to healthy controls. Eighteen protein spots were found to have statistically significant (p<0.05) difference in their expression level from four sets of gels and were identified by MALDI-TOF MS. Most of the identified proteins were predicted to be O-glycosylated proteins by Net–O-Gly 3.1 algorithm. Among these the alpha 2HS glycoprotein (A2HSG) was found to be down regulated whereas inter alpha trypsin inhibitor H4 (ITIH4) was up regulated and this was validated by Western blotting. The glycosylation studies showed the reduced N-linked sialylation of A2HSG in RA patients. Altered glycoprotein expression and functional as well as structural studies of glycans might help in the diagnosis of RA and understanding the disease pathogenesis.     

Primary biliary cirrhosis sera recognize not only gp210 but also proteins of the p62 complex bearing N-acetylglucosamine residues from rat liver nuclear envelope

We have recently observed reactivity of primary biliary cirrhosis (PBC) sera with several proteins bearing N-acetylglucosamine residues from rat liver nuclear envelopes. The aim of this study was to characterize the reactive antigens. Sera from 31 patients with PBC, 30 with rheumatoid arthritis (RA) and 30 with Sjögren's syndrome (SS) were examined. Rim-like immunofluorescence staining was observed in 15 of 31 (48%) sera from patients with PBC, in 1 of 30 with RA and in 1 of 30 with SS. Upon immunoblotting using preparations of whole rat liver nuclear envelopes and their Triton X 100-KCl extract as antigen souces, a 200 kDa protein band was observed in 9 of sera with PBC. Furthermore, upon immunoblotting using the wheat germ aggulutinin-bound fraction of rat liver envelope as antigen, 62, 60 and 54 kDa protein bands corresponding to components of the p62 complex in the nuclear pore complex (Kita et al. Biochem. 113, 377–382) were observed in 7, 5 and 6 samples respectively, of the 31 PBC sera. Our data suggest that PBC sera recognize not only the 210 kDa protein but also the p62 complex proteins.Abbreviations ANA antinuclear antibody - AMA anti-mitochondrial antibodies - IF immunofluorescence - LAP2 lamina-associated polypeptide 2 - LBR lamin B receptor - anti-NBP 60 anti-nuclear localization signal binding protein 60 - NE nuclear envelope - NPC nuclear pore complex - PBC primary biliary cirrhosis - RA rheumatoid arthritis - SLE systemic lupus erythematosus - SS Sjögren's syndrome - WGA wheat germ agglutinin     

Conventional protein kinase C isoenzymes undergo dephosphorylation in neutrophil-like HL-60 cells treated by chelerythrine or sanguinarine

The quaternary benzo[c]phenanthridine alkaloid chelerythrine is widely used as an inhibitor of protein kinase C (PKC). However, in biological systems chelerythrine interacts with an array of proteins. In this study, we examined the effects of chelerythrine and sanguinarine on conventional PKCs (cPKCs) and PKC upstream kinase, phosphoinositide-dependent protein kinase 1 (PDK1), under complete inhibition conditions of PKC-dependent oxidative burst. In neutrophil-like HL-60 cells, sanguinarine and chelerythrine inhibited N-formyl-Met-Leu-Phe, phorbol 12-myristate 13-acetate (PMA)-, and A23187-induced oxidative burst with IC₅₀ values not exceeding 4.6 μmol/L, but the inhibition of PMA-stimulated cPKC activity in intact cells required at least fivefold higher alkaloid concentrations. At concentrations below 10 μmol/L, sanguinarine and chelerythrine prevented phosphorylation of ∼80 kDa protein and sequestered ∼60 kDa phosphoprotein in cytosol. Moreover, neither sanguinarine nor chelerythrine impaired PMA-stimulated translocation of autophosphorylated PKCα/βII isoenzymes, but both alkaloids induced dephosphorylation of the turn motif in PKCα/βII. The dephosphorylation did not occur in unstimulated cells and it was not accompanied by PKC degradation. Furthermore, cell treatment with sanguinarine or chelerythrine resulted in phosphorylation of ∼70 kDa protein by PDK1. We conclude that PKC-dependent cellular events are affected by chelerythrine primarily by multiple protein interactions rather than by inhibition of PKC activity.     

Developmental study of thermotolerance and the heat shock response inLucilia cuprina (Weidemann)

The ability to withstand thermal stress in a laboratory population of the blowflyLucilia cuprina (measured as per cent adult survival following varying periods of exposure to elevated temperature up to a maximum of 48°C) was in the order pupa > larva > adult. Pre-exposure to a mild heat shock (37°C) induced tolerance to temperatures which were otherwise lethal. An analysis of heat shock-induced protein synthesis during development at similar elevated temperatures presented patterns corresponding to the above observations on thermotolerance. The induced level of synthesis of major heat shock proteins (viz., 79, 69, 28, 20 and 19 kDa) were greater in larval tissues than in most of the adult tissues except gonads. The response varied between young (2 days) and old (30 days) adults in a tissue-specific manner. In general, heat shock protein 69 kDa was most abundant in all the tissues studied. Control as well as heat shocked Malpighian tubules of adults uniquely showed two major [³⁵S]methionine labelled bands corresponding to approximately 62 and 64 kDa. Immunoblots showed the 62 kDa protein to cross react with an antibody againstHelioihis HSP60. Although the synthesis of the 62 kDa polypeptide was prominent only in Malpighian tubules of adult blowflies, nearly equal levels of this HSP60 family polypeptide were present in all tissues (control as well heat shocked) except the larval salivary glands.     

Effect of nutritional parameters on laccase production by the culinary and medicinal mushroom, Grifola frondosa

Summary Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100–500 (M Cu over the basal level (1.6 mM Cu) and peak levels observed at 300 mM Cu were ∼ ∼7-fold higher than in unsupplemented controls. Decreased laccase activity similar to levels detected in unsupplemented controls, as well as an adverse effect on fungal growth, occurred with further supplementation up to and including 0.9 mM Cu, but higher enzyme titres (2- to 16-fold compared with controls) were induced in cultures supplemented with 1–2 mM Cu²⁺. SDS-PAGE combined with activity staining revealed the presence of a single protein band (M _r ∼ ∼70 kDa) exhibiting laccase activity in control culture fluids, whereas an additional distinct second laccase protein band (M _r␣∼ ∼45 kDa) was observed in cultures supplemented with 1–2 mM Cu. Increased levels of extracellular laccase activity, and both laccase isozymes, were also detected in cultures of G. frondosa supplemented with ferulic, vanillic, veratric and 4-hydroxybenzoic acids, and 4-hydroxybenzaldehyde. The optimal temperature and pH values for laccase activity were 65 °C and pH 2.2 (using 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) {ABTS} as substrate), respectively, and the enzyme was relatively heat stable. In solid-state cultures of G. frondosa grown under conditions adopted for industrial-scale mushroom production, extracellular laccase levels increased during the substrate colonization phase, peaked when the substrate was fully colonized, and then decreased sharply during fruit body development.     

An Electrophoretic Analysis of the Seed Protein Body Proteins from Pinus Nigra

Protein bodies (PBs) of European black pine (Pinus nigra Arn.) were isolated from mature seeds. Extracted soluble matrix proteins and crystalloid proteins PBs proteins were investigated by SDS-PAGE electrophoresis in presence and absence of 2-mercaptoethanol. The proteins of molecular masses 16, 17, 18, 61 and 65 kDa were presented only in crystalloid protein samples. Only 15 kDa protein was present in soluble matrix proteins and not in crystalloid proteins. Another protein bands were present in both soluble matrix and crystalloid proteins. 20, 37, 38, 39 and 48 kDa proteins were strongly visible among crystalloid proteins. Bands of 23 and 32 kDa were more visible in soluble matrix protein samples. Different composition in crystalloid proteins was found in absence of 2-mercaptoethanol: no proteins with molecular mass 71 kDa and more proteins in soluble matrix. In case of crystalloid proteins we detected 7 protein bands in interval from 71 to 212 kDa.     

Identification of Autoantibodies against Transthyretin for the Screening and Diagnosis of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic, autoimmune, systemic and inflammatory rheumatic disease that leads to inflammation of the joints and surrounding tissues. Identification of novel protein(s) associated with severity of RA is a prerequisite for better understanding of pathogenesis of this disease that may also have potential to serve as novel biomarkers in the diagnosis of RA. Present study was undertaken to compare the amount of autoantigens and autoantibodies in the plasma of RA patients in comparison to healthy controls. Plasma samples were collected from the patients suffering from RA, Osteoarthritis (OA), Systemic lupus erythematosus (SLE) and healthy volunteers. The screening of plasma proteins were carried out using 2-dimensional gel electrophoresis followed by identification of differentially expressed protein by MALDI-TOF MS/MS. Among several differentially expressed proteins, transthyretin (TTR) has been identified as one of the protein that showed significantly up regulated expression in the plasma of RA patients. The results were further validated by Western blot analysis and ELISA. In comparison to OA synovium, an exclusive significantly high expression of TTR in RA has been validated through IHC, Western blotting and IEM studies. Most importantly, the increase in expression of TTR with the progression of severity of RA condition has been observed. The autoantibodies against TTR present in the RA plasma were identified using immunoprecipitation-Western methods. The significant production of autoantibodies was validated by ELISA and Western blot analysis using recombinant pure protein of TTR. Hence, these novel observations on increase in TTR expression with the increase in severity of RA conditions and significant production of autoantibodies against TTR clearly suggest that a systematic studies on the role TTR in the pathogenesis of RA is immediately required and TTR may be used as a serum diagnostic marker together with other biochemical parameters and clinical symptoms for RA screening and diagnosis.     

Molecular and phylogenetic characterization of Spodoptera litura granulovirus

Baculovirus, Spodoptera litura granulovirus (SlGV) was isolated from the infected S. litura larvae, and was characterized. The granule of SlGV was ovoidal shape with an approximate size of 240∼340 nm× 140∼180 nm. Each granule contained one single rod-shape virion with a mean size of 180∼200 nm×20∼40 nm. Restriction endonuclease fragment analysis estimated that the total genome size of SlGV is about 115 kb. Necleotide sequence analysis of the granulin gene showed that the gene encodes 249 amino acids with a predicted molecular mass of 29 kDa. When the phylogenic relationship was analyzed using the nucleotide sequence of the granulin gene, SlGV was most closely related to Trichoplusia ni granulovirus (TnGV) and Xestia c-nigrum granulovirus (XcGV) which belong to Type I granulovirus.     

Neutral endopeptidase neprilysin is copurified with Na,K-ATPase from rabbit outer medulla and hydrolyzes its α-subunit

Preparations of Na,K-ATPase from outer medulla of rabbit kidney purified in accordance with the method of P. L. Jorgensen were shown to contain as admixture a protease that moves with α-subunit (∼100 kDa) as a single protein band during one-dimensional SDS-PAGE. The electro-elution of proteins of this band from polyacrylamide gel results in the appearance of two protein fragments (∼67 and 55 kDa) that are stained with polyclonal antibodies against Na,K-ATPase α-subunit. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis showed that the neutral membrane-bound endopeptidase neprilysin is located in one protein band together with the Na,K-ATPase α-subunit. Addition of thiorphan, a specific inhibitor of neutral endopeptidase, eliminates proteolysis of the α-subunit. The data demonstrate that Na,K-ATPase α-subunit may be a natural target for neprilysin.     

Changes in protein spectra of transgenic plants carrying differentAgrobacterium tumefaciens C58 T-DNA genes

A series of binary vector plasmids derived from the T-DNA of theAgrobacterium tumefaciens strain C58, carrying the five plant morphoregulatory genes 1, 2, 4, 5 and 6b in different combinations, was used in the transformation ofNicotiana tabacum leaf discs. Protein patterns of the transgenic tobacco analysed through SDS-PAGE have shown changes in the polypeptides with M_r: ∼120, 60, 55, 43 and 27 kDa (for tobacco with transgene 4); ∼60, 55, 43, 26–25, 21, 18 kDa (for tobacco with transgenes 1, 2 and 5); ∼70, 60, 26, 25, 18 kDa (for tobacco with transgene 5); ∼60, 55, 48, 26, 18 kDa (for tobacco with transgenes 4, 5, 6b); ∼60, 55, 22 and 18 kDa (for tobacco with transgene 6b); ∼60, 55, 43, 26 and 18 kDa (for transgenes 5, 6b); ∼60, 55, 22, 18 and 16 kDa (for transgenes 4 and 6b). All types of transgenic plants showed quantitative changes in protein content. Mendelian segregation ratio to kanamycin resistance in the progeny of transgenic tobacco clones in the R1 generation was 3∶1 except in transgenic tobacco carrying transgenes 1, 2 and 5. Communicated by T. GICHNER     

Effect of crab shell size on bio-demineralization with lactic acid-producing bacterium, <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> subsp. <Emphasis Type="Italic">tolerans</Emphasis> KCTC-3074

Demineralization (DM) from crab shell (CS) waste was carried out using a lactic acid-producing bacterium, Lactobacillus paracasei subsp. tolerans KCTC-3074 for 7 days at 25, 30, and 35°C. DM rates were 89∼92% and slightly affected by temperature. DM was also performed for four particle-sized shell samples (0.84∼3.35, 3.35∼10, 10∼20, and 20∼35 mm) with 10% inoculum, 5% shell, and 10% glucose at 30°C and 180 rpm for 7 days. It was found out that the shell size had a slight effect on the rate of DM. Negative relationships were found between DM and residual dry weight (r² = 0.960), and between DM and pH (r² = 0.906). Conversely, positive relationships were found between DM and medium protein (r² = 0.696), and between DM and total titratable acidity (r² = 0.630).     

Expression and functional properties of antibodies to tissue inhibitors of metalloproteinases (TIMPs) in rheumatoid arthritis

Tissue inhibitors of matrix metalloproteinases (TIMPs) regulate the breakdown of extracellular matrix components and play an important role in tissue remodelling and growth, in both physiological and pathological conditions. We studied the autoimmune response to TIMPs in patients with rheumatoid arthritis (RA). Eighty-nine paired blood and synovial fluid samples from patients with RA were assessed for their reactivity with recombinant tissue inhibitors of metalloproteinases (TIMPs) 1 to 4 by an ELISA and were compared with blood from 62 healthy controls and 21 synovial fluid samples from patients with degenerative joint diseases. Presence of antibodies was established as the absorbance of the sample more than 2 standard deviations above the mean of the controls. In addition, immunoglobulin G (IgG) from blood samples of RA patients possessing TIMP antibodies was isolated on protein A–sepharose and tested for the in vitro ability to neutralize TIMP-2-dependent effects on metalloproteinase 9 (MMP9). Anti-TIMP antibodies were found in 56% of RA samples but in only 5% of the controls (P < 0.005). RA patients had high frequencies of antibodies against all TIMPs except TIMP-3. TIMP-2 antibodies were most frequently found (33%), being significantly more prevalent (P = 0.024) in patients with nonerosive than erosive RA. TIMP-1 antibodies were significantly more often found in synovial fluid samples than in the matched blood samples (P < 0.025). Importantly, the IgG fraction containing TIMP antibodies down-regulated the TIMP-2 inhibitory effect, thereby supporting MMP9 activity in vitro. In the present study, we show that RA patients frequently develop autoimmune response to TIMPs that may act as a functionally significant regulator of MMP activity and thereby of joint destruction.     

Typing of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> of fish and human diarrhoeal origin by outer membrane proteins and lipopolysaccharides

A study was undertaken to discriminate the strains of Aeromonas hydrophila isolated from fish and diarrhoeal samples by SDS-PAGE analysis of outer membrane proteins (OMPs) and lipopolysaccharides (LPSs). Common bands at 47 kDa positions for OMPs and at 31–38 kDa for LPSs were observed. No strain of A. hydrophila from clinical or fish samples was found identical in either OMPs or LPSs profile.     

Determination of oxidant stress in plasma of rheumatoid arthritis and primary osteoarthritis patients

Determination of oxidant stress in plasma of rheumatoid arthritis (RA) and primary osteoarthritis (POA) patients is important in understanding the pathogenesis of these diseases. In this study, we examined the relationship between oxidant stress and inflammation by measuring protein carbonyl content, thiol levels and plasma protein fractions as the oxidation markers and erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) tests as inflammation markers. Protein carbonyls content was higher in RA and POA patients, as compared to controls (p<0.0001), while the plasma thiol levels in both groups of patients were significantly lower than controls (p<0.0001). Increased levels of proteins under 40 kDa molecular mass were detected in the RA and POA patients compared to that of controls (p<0.0001) both in HPLC and SDS-PAGE analysis. Total protein concentration in plasma of RA patients was higher than the controls (p<0.001), while in POA patients was lower than that of controls (p<0.001). ESR and CRP levels were higher in both the patient groups than the normal group (p<0.001). These results suggested that alterations in the oxidant stress markers could be the cause of inflammation in these diseases. Thus, while working for RA/POA treatment strategies, consideration of the relationship between oxidant stress and inflammation would be worth evaluating.     

Identification of two putative adhesive polypeptides in Caulerpa prolifera rhizoids using an adhesion model system

The rhizoid section of the green alga Caulerpa prolifera (Cp) is active in attaching the developing plant to the substratum. A model system for the study of the adhesion of Cp rhizoids has been developed and identification of two putative adhesive polypeptides of Caulerpa (Vn-Cp) was revealed by immunodetection. A method for fast induction of new rhizoids was established using blade-base cutting followed by a few days of incubation. The new rhizoids were gently enclosed between two cover glasses and incubated until firm attachment developed. While analyzing protein extracts, two ∼60–70 kDa polypeptides (Vn-Cp I and Vn-Cp II) were identified by immunodetection with monoclonal antibodies to human vitronectin (Vn). The relative concentration values of the Vn-Cp proteins increased significantly in the ‘cell-wall’ fraction of the attached rhizoids during the incubation period. However, Vn-Cp proteins were not detected in non-attached rhizoids. Furthermore, the Vn-Cp proteins were also detectable on glass substratum subsequent to attached rhizoid removal. The induction and accumulation of Vn-Cp proteins on the ‘cell-wall’ of Caulerpa rhizoids and the firm attachment of the rhizoids to the glass substratum during the incubation period suggest that Vn-Cp proteins play a significant role in adhesion, which may be similar to the function of vitronectin in other adhesion systems. Furthermore, the high accumulation of Vn-Cp proteins on the glass substratum during attachment of new rhizoids suggests that the Vn-Cp proteins are secreted to the extracellular matrix and directly connect rhizoids to the glass substratum as an intermediate compound. These unique properties of Cp make it an excellent model system for the establishment of high amounts of adhesive material for future research. This revised version was published online in August 2006 with corrections to the Cover Date.     

Plasma Membrane Protein Clusters Appear in CFTR-Expressing Xenopus Laevis Oocytes after cAMP Stimulation

Membrane trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is supposed to be an important mechanism controlled by the intracellular messenger cAMP. This has been shown with fluorescence techniques, electron microscopy and membrane capacitance measurements. In order to visualize protein insertion we applied atomic force microscopy (AFM) to inside-out oriented plasma membrane patches of CFTR-expressing Xenopus laevis oocytes before and after cAMP-stimulation. In a first step, oocytes injected with CFTR-cRNA were voltage-clamped, verifying successful CFTR expression. Water-injected oocytes served as controls. Then, plasma membrane patches were excised, placed (inside out) on glass and scanned by AFM. Before cAMP-stimulation plasma membranes of both water-injected and CFTR-expressing oocytes contained about 200 proteins per μm². Molecular protein masses were estimated from molecular volumes measured by AFM. Before cAMP-stimulation, protein distribution showed a peak value of 11 nm protein height corresponding to 475 kDa. During cAMP-stimulation with 1 mm isobutylmethylxanthine (IBMX) plasma membrane protein density increased in water-injected oocytes to 700 proteins per μm² while the peak value shifted to 7 nm protein height corresponding to 95 kDa. In contrast, CFTR-expressing oocytes showed after cAMP-stimulation about 400 proteins per μm² while protein distribution exhibited two peak values, one peak at 10 nm protein height corresponding to 275 kDa and another one at 14 nm corresponding to 750 kDa. They could represent heteromeric protein clusters associated with CFTR. In conclusion, we visualized plasma membrane protein insertion upon cAMP-stimulation and quantified protein distribution with AFM at molecular level. We propose that CFTR causes clustering of plasma membrane proteins. Received: 11 September 2000/Revised: 13 December 2000