Is diabetic hypercoagulability an acquired annexinopathy? Glycation of annexin II as a putative mechanism for impaired fibrinolysis in diabetic patients.

Diabetics die mainly from thrombotic complications and there is clear evidence that diabetes is a hypercoagulable state. Epidemiological and prospective intervention data link hyperglycemia to vascular complications and glycation of proteins is one favored molecular basis to explain this fact. Cell surface receptors may support fibrinolytic surveillance in both intravascular and extravascular locations by stimulating plasmin generation and by protecting plasmin from its inhibitors. The existing experimental evidence suggests that annexin II in its tetrameric form is the main physiological receptor for plasminogen on the extracellular surface of endothelial cells. We have recently shown that annexin II is an extremely vulnerable target for glycation, quickly responding to restoration of normoglycemia. We hypothesize that glycation of endothelial membrane annexin II impairs the appropriate formation of the plasminogen/tissue plasminogen activator/annexin II complex, disrupting a key regulatory mechanism in fibrinolytic vigilance. This would in turn produce decreased fibrinolytic activity and indirectly promote a thrombophilic state in diabetic patients. We base our hypothesis on our observation and on evidence for the mechanism of action of two major independent risk factors for CV events: lipoprotein (a) and hyperhomocysteinemia. Binding of plasminogen to annexin II is inhibited by Lp (a) and binding of tissue plasminogen activator to annexin II is blocked by homocysteine. If our hypothesis is correct, one of the components of the increased thrombogenicity seen in diabetic patients might then be an acquired annexinopathy.

Morpho-functional studies of the blood-brain barrier in streptozotocin-induced diabetic rats.

AIMS/HYPOTHESIS: We undertook the characterization of the capillary bed of the rat frontal cortex and their permeability properties in short-term and long-term diabetic rats. METHODS: Diabetes was induced by strepozotocin injection. Rats were maintained hyperglycaemic without insulin treatment during 4 to 5 months (short-term) and 8 to 13 months (long-term). Rats from an additional short-term hyperglycaemic group received an injection of exogenous dinitrophenylated albumin 15 min before being killed. Tissues were processed for electron microscopy and quantitative immunocytochemistry. Endogenous and dinitrophenylated exogenous albumin were revealed with high resolution over the capillary wall using specific antibodies and the protein A-gold complex. Morphometrical analyses were carried out. RESULTS: Albumin is transported across endothelial cells by plasmalemmal vesicles or caveolae and larger vacuolar structures. This transport increased in diabetic rats by an increment in the number of vesicles. Albumin distribution across the capillary basement membrane showed that the restrictive properties of the basement membrane present in normoglycaemic rats are altered in the diabetic condition, as was its thickness. Similar alterations of the basement membrane structure and function were encountered in old normoglycaemic rats but to a lesser extent. CONCLUSION/INTERPRETATION: The results indicate that diabetes seems to accelerate the ageing process of the vascular wall and that the central nervous system capillary bed is also a target for diabetic microangiopathy.

Vascular immunotargeting to endothelial surface in a specific macrodomain in alveolar capillaries.

A novel 85 kD glycoprotein (gp85) is a marker of the avesicular zone, a thin part of pulmonary endothelial cells separating alveolar and vascular compartments and lacking vesicles. This report presents the first evaluation whether mAb 30B3, a monoclonal antibody to gp85, can be used for targeting of drugs to the surface of lung endothelium. 125I-mAb 30B3 accumulated in isolated perfused lungs (IPL) (22.8 +/- 1.1 versus 0.5 +/- 0.1 %ID/g for 125I-IgG) and accumulated preferentially in the lungs after intravenous or intraarterial injection (10.9 +/- 0.7 and 11.0 +/- 1.5 versus 0.9 +/- 0.2 %ID/g for 125I-IgG). 125I-mAb 30B3 uptake in IPL was rapid (T1/2 15 min), saturable (Bmax appr. 10(5) molecules/cell), specific (inhibited by nonlabeled mAb 30B3) and temperature independent (26.3 +/- 2.1 versus 22.8 +/- 1.1 %ID/g at 6 degrees C versus 37 degrees C). Biotinylated mAb 30B3 permitted subsequent accumulation of perfused avidin derivative in IPL. Because these data indicated that mAb 30B3 binds to an accessible, poorly internalizable antigen in the lung, we conjugated mAb 30B3 with a plasminogen activator, 125I-tPA. After intravenous injection in rats, lung-to-blood ratio was 8.4 +/- 0.9 for mAb 30B3/125I-tPA versus 0.4 +/- 0.1 for IgG/125I-tPA, indicating that mAb 30B3 may deliver drugs, which was supposed to exert therapeutic action in the vascular lumen (e.g., antithrombotic proteins), to the surface of pulmonary endothelium.

Actin and annexins I and II are among the main endothelial plasmalemma-associated proteins forming early glucose adducts in experimental diabetes.

An immunochemical and biochemical study was performed to reveal which of the endothelial plasma membrane proteins become glycated during the early phases of diabetes. The blood front of the lung microvascular endothelial plasmalemma was purified by the cationic colloidal silica method from normal and diabetic (streptozotocin-induced) rats and comparatively analyzed by two-dimensional electrophoresis. No major qualitative differences in the general spectrum of endothelial plasmalemmal proteins were recorded between normoglycemic and hyperglycemic animals. By probing with anti-glucitollysine antibodies, we found that at 1 month after the onset of diabetes, several endothelial membrane polypeptides contained glucose covalently linked to their lysyl residues. Ten days of insulin treatment restored euglycemia in the diabetic animals and completely abolished the membrane nonenzymatic glycosylation. All the glycated polypeptides of the endothelial plasma membrane belong to the peripheral type and are associated with its cytoplasmic face (cell cortex). They were solubilized by buffers of high pH and were not detected in the lung cytosolic fraction (100,000 g). By microsequencing, the major proteins labeled by the anti-glucitollysine have been identified as being actin, annexin I, annexin II, the p34 subunit of the Arp2/3 complex, and the Ras suppressor protein-1. Conversely, the intrinsic endothelial membrane proteins do not seem to be affected by hyperglycemia. This defines the internal face of the endothelial plasma membrane, particularly the cortical cytoskeleton, as a preferential target for nonenzymatic glycosylation in diabetes, with possible consequences on the fluidity of the endothelial plasmalemma and impairment of the endothelial mechanotransducing ability.

Alterations of the rat mesentery vasculature in experimental diabetes.

The alteration induced by diabetes on vascular permeability to serum albumin was investigated in the mesentery of streptozotocin-induced hyperglycemic rats. Double-tagged ((125)I and dinitrophenol-haptenated) heterologous albumin was intravenously administered in normal and hyperglycemic animals, and the extravasation of the tracer was evaluated by radioactivity measurements and by morphometry at the ultrastructural level using quantitative protein A-colloidal gold immunocytochemistry. The results demonstrate that diabetes induces a significant increase in the permeability of the mesentery vessels to albumin. This increase is due to a more efficient transport of macromolecules by endothelial plasmalemmal vesicles and not to leakier interendothelial junctions. Passage across the endothelial basement membranes did not appear to be restricted in either the control or diabetic condition. However, in diabetes, the mesothelial basement membrane appeared to become modified and to restrain the passage of albumin toward the peritoneal cavity. After 3 months of diabetes, the rats presented a net increase in the average diameter of the blood vessels localized in the mesentery arcada (macrovascular hyperplasy) and a notable angiogenesis, manifested at the level of the microvasculature in the mesenteric windows.

A novel, 85 kDa endothelial antigen differentiates plasma membrane macrodomains in lung alveolar capillaries.

A monoclonal antibody raised against purified rat lung endothelial plasma membranes was found to recognize an apparently novel, 85 kD, integral endothelial plasma membrane glycoprotein. By immunofluorescence and electron microscope immunocytochemistry, this endothelial antigen was detected at the luminal and tissue fronts of all rat endothelia, except those of discontinuous type (liver and spleen sinusoids). In lung alveolar capillaries the antigen appeared to be uniquely associated with the very attenuated endothelial processes forming the blood-air barrier, and virtually absent on the surface of the rest of the cell, where the nucleus and the organelles are located. No other cells, except fibroblasts, appeared to be labeled by this monoclonal antibody.

Isolation, cloning, and localization of rat PV-1, a novel endothelial caveolar protein.

By using an immunoisolation procedure (Stan, R.-V., W.G. Roberts, K. Ihida, D. Predescu, L. Saucan, L. Ghitescu, and G.E. Palade. 1997. Mol. Biol. Cell. 8:595-605) developed in our laboratory, we have isolated a caveolar subfraction from rat lung endothelium and we have partially characterized the proteins of this subfraction which include an apparently caveolae-specific glycoprotein we propose to call PV-1 (formerly known as gp68). The isolation and partial sequencing of PV-1, combined with the cloning of the full length PV-1 cDNA led to the following conclusions: (a) PV-1 is a novel single span type II integral membrane protein (438 amino acids long) which forms homodimers in situ; (b) the transmembrane domain of PV-1 is near the NH2 terminus defining a short cytoplasmic endodomain and a large COOH-terminal ectodomain exposed to the blood plasma; (c) PV-1 is N-glycosylated and its glycan antennae bear terminal nonreducing galactosyl residues in alpha1-3 linkage. PV-1 is expressed mostly in the lung but both the messenger RNA and the protein can be detected at lower levels also in kidney, spleen, liver, heart, muscle, and brain. No signal could be detected in testis and two lower molecular weight forms were detected in brain. Immunocytochemical studies carried out by immunodiffusion on rat lung with an anti-PV-1 polyclonal antibody directed against a COOH-terminal epitope reveal a specific localization of PV-1 to the stomatal diaphragms of rat lung endothelial caveolae and confirm the extracellular orientation of the PV-1 COOH terminus.

P-glycoprotein is strongly expressed in the luminal membranes of the endothelium of blood vessels in the brain.

Luminal membranes of the vascular endothelium were isolated from brain, heart and lungs by modification of their density. The presence of P-glycoprotein (P-gp) was detected by Western blotting in luminal membranes from the endothelium of the three tissues. Strong enrichment in brain capillary luminal membranes, compared with brain capillaries (17-fold) and whole membranes (400-500-fold), indicates that P-gp is mainly located on the luminal side of the brain endothelium. Western blotting was also performed with antibodies directed against GLUT1, glial fibrillary acidic protein, adaptin, IP3R-3, integrins alphav and collagen IV as controls to determine whether the preparations were contaminated by other membranes. Strong enrichment of GLUT1 in brain capillary luminal membranes (9.9-fold) showed that the preparation consisted mainly of endothelial cell plasma membranes. Poor enrichment of glial fibrillary acidic protein (1.4-fold) and adaptin (2.4-fold) and a decreased level of IP3R-3, integrins alphav and collagen IV excludes the possibility of major contamination by astrocytes or internal and anti-luminal membranes. High levels of P-gp in the luminal membranes of brain capillary endothelial cells suggests that it may play an important role in limiting the access of anti-cancer drugs to the brain.

Immunoisolation and partial characterization of endothelial plasmalemmal vesicles (caveolae).

Plasmalemmal vesicles (PVs) or caveolae are plasma membrane invaginations and associated vesicles of regular size and shape found in most mammalian cell types. They are particularly numerous in the continuous endothelium of certain microvascular beds (e.g., heart, lung, and muscles) in which they have been identified as transcytotic vesicular carriers. Their chemistry and function have been extensively studied in the last years by various means, including several attempts to isolate them by cell fractionation from different cell types. The methods so far used rely on nonspecific physical parameters of the caveolae and their membrane (e.g., size-specific gravity and solubility in detergents) which do not rule out contamination from other membrane sources, especially the plasmalemma proper. We report here a different method for the isolation of PVs from plasmalemmal fragments obtained by a silica-coating procedure from the rat lung vasculature. The method includes sonication and flotation of a mixed vesicle fraction, as the first step, followed by specific immunoisolation of PVs on anticaveolin-coated magnetic microspheres, as the second step. The mixed vesicle fraction, is thereby resolved into a bound subfraction (B), which consists primarily of PVs or caveolae, and a nonbound subfraction (NB) enriched in vesicles derived from the plasmalemma proper. The results so far obtained indicate that some specific endothelial membrane proteins (e.g., thrombomodulin, functional thrombin receptor) are distributed about evenly between the B and NB subfractions, whereas others are restricted to the NB subfraction (e.g., angiotensin converting enzyme, podocalyxin). Glycoproteins distribute unevenly between the two subfractions and antigens involved in signal transduction [e.g., annexin II, protein kinase C alpha, the G alpha subunits of heterotrimeric G proteins (alpha s, alpha q, alpha i2, alpha i3), small GTP-binding proteins, endothelial nitric oxide synthase, and nonreceptor protein kinase c-src] are concentrated in the NB (plasmalemma proper-enriched) subfraction rather than in the caveolae of the B subfraction. Additional work should show whether discrepancies between our findings and those already recorded in the literature represent inadequate fractionation techniques or are accounted for by chemical differentiation of caveolae from one cell type to another.

Antibodies specific to the plasma membrane of rat lung microvascular endothelium.

The highly purified, luminal domain of rat lung endothelial plasma membranes was used as an immunogen to obtain monoclonal antibodies to the endothelial cell surface. The procedure was highly efficient, yielding antibodies which recognize three seemingly novel endothelial integral membrane glycoproteins of molecular weights of 170, 114, and 95 kDa, respectively. By immunofluorescence, two of these antigens (170 and 95 kDa) appeared to be uniquely expressed by the lung microvascular endothelium. The third one, the 114-kDa polypeptide, was detected in the continuous endothelium of the lung, but also in the fenestrated endothelia of pancreas, intestinal villi, and kidney peritubular capillaries. Partition in Triton X-100-soluble and -insoluble plasmalemmal components suggests that two of these novel endothelial antigens (170 and 114 kDa) are specific for the plasma membrane proper only, while that of 95 kDa is present both in the caveolae and on the rest of the cell surface.

Endocytotic functions of ameloblasts and odontoblasts: immunocytochemical and tracer studies on the uptake of plasma proteins.

BACKGROUND: Biochemical, (immuno)cytochemical, and radioautographic data accumulated over several years have lead to the view that ameloblasts carry out both secretory and degradative functions throughout amelogenesis. Whereas it has been assumed that maturation stage ameloblasts endocytose aged enamel proteins from the enamel layer, the origin of the newly formed ones detected in the endosomal/lysosomal compartment of ameloblasts from all stages remains to be elucidated. One possible source is from secretory products released ectopically along basolateral surfaces. METHODS: To test this hypothesis, we have investigated, using colloidal gold immunocytochemistry, whether plasma proteins (albumin and alpha 2HS-glycoprotein) found in the interstitial fluid are endocytosed by rat incisor ameloblasts and other cells from hard and soft tissues. Rat albumin, tagged with dinitrophenol, was injected intravenously to trace the movement of this protein. RESULTS: Plasma proteins were immunodetected along the baso-lateral surfaces and in multivesicular bodies of ameloblasts where enamel proteins were also found. By 2 hours following intravenous administration of dinitrophenylated albumin, the tracer had left the blood and diffused into the enamel organ and between odontoblasts and osteoblasts. The tracer was also found in multivesicular bodies of all cells examined. CONCLUSIONS: The uptake of albumin by many different cell types suggests that this process is not restricted to ameloblasts and likely occurs in a nonselective manner. Hence, baso-lateral uptake in ameloblasts may play a role not only in the continuous removal of plasma proteins leaking from the blood, but also of enamel proteins 'dumped' laterally between these cells. Likewise, odontoblasts may use the same mechanism to internalize some of the plasma proteins and any enamel protein that diffuse toward them.

Immunocytochemical investigation of the in vivo endocytosis by renal tubular epithelial cells.

The internalization and degradation of glomerular filtered serum proteins by the proximal tubular epithelium has been extensively studied by microperfusion methods. By using a cationic probe that easily traverses the glomerular wall into the urinary space, we have performed a morpho-cytochemical and quantitative study of the in vivo endocytotic activity of the proximal tubular epithelial cell. Bovine serum albumin (BSA) was tagged with dinitrophenol (DNP) and cationized to pI over 8. It was introduced into the circulation of normal mice for 5, 10, and 30 minutes and the distribution of the labeling was determined by protein A-gold immunocytochemistry, using specific antiDNP antibodies on tissue sections of routinely aldehyde-fixed, osmiumpostfixed, and Epon-embedded kidneys. Cationic BSA-DNP was detected at the endothelial and epithelial sides of the glomerular basement membrane, and over capillary and tubular basement membranes. In the proximal tubular epithelial cell, labeling was present over microvilli as well as over endosomal and lysosomal compartments, with labeling intensities varying from one compartment to the other. Morphometric evaluations of the labeling demonstrated a progressive incorporation of the probe from microvilli and endocytic compartments at 5 minutes to endocytic and lysosomal compartments at 10 and then 30 minutes. When considering labeling densities, no significant differences were found on microvilli and basolateral membranes between times of circulation; however, the labeling density over endosomal and lysosomal compartments was very intense at 10 minutes compared with 5 minutes, decreasing at 30 minutes. Results from this study validate the cationic albumin tagged with DNP as a tool in the study of the quantitative aspects of protein endocytosis at the ultrastructural level, in the kidney tubular epithelium.

Glomerular handling of circulating glycated albumin in the normal mouse kidney.

In the present study, we have evaluated the glomerular handling of circulating glycated albumin in the normal mouse kidney by quantitative immunocytochemistry. Bovine serum albumin (BSA) was glycated in vitro and dinitrophenylated. Glycated and nonglycated probes were introduced into the circulation of anesthetized mice and traced by postembedding immunogold cytochemistry after 10 and 30 min of circulation. Endogenous albumin, as well as dinitrophenylated native BSA (DNP-BSA) and glycated albumins (DNP-gBSA), were localized within the capillary lumen, glomerular and peritubular basement membranes, and the mesangial matrix. Morphometric evaluation of the labeling over the glomerular basement membrane (GBM) revealed a peak of labeling in the endothelial side for either endogenous albumin or DNP-BSA. In contrast, the labeling distribution for DNP-gBSA showed a shift toward the epithelial side, suggesting a further penetration of the glycated probe into the GBM. When coinjected with gBSA, DNP-BSA was found to display a labeling distribution similar to that displayed by DNP-gBSA. These results indicate that the glycated tracer penetrates the normal glomerular wall deeper than the nonglycated one. Moreover, glycated albumin increases the infiltration of the nonglycated tracer through the normal glomerular wall. Circulating glycated serum proteins thus appear to play an important role in the onset of the glomerular dysfunction and proteinuria, which take place in long-term hyperglycemic states.

Hapten-tagged plasma proteins as immunocytochemical probes for the study of vascular permeability.

Bovine serum albumin and transferrin were covalently coupled with fluorescein isothiocyanate and digoxigenin, respectively, and intravenously co-injected in equal amounts in mouse. The derivation of the two proteins induces minor alterations of their physicochemical properties as well as of their physiological functions. The two tracers were revealed within vascular and extravascular compartments of diaphragm by quantitative postembedding immunocytochemistry, using antibodies against each of the haptens in conjunction with the protein AG-gold complexes. The influence of different fixatives and embedding protocols on the immunodetectability of the hapten-tagged proteins was assessed. Both resist reasonably well to osmication and embedding in Epon. None of the haptens reacted with the heterologous antibody. At 30 minutes after injection, the tracers were detected in blood plasma, interstitium, and endothelial plasmalemmal vesicles. The presence of both proteins within the interendothelial clefts was inconspicuous. The ratios between the labeling densities found over endothelium, interstitial space, and vascular lumen were similar for both tracers. This suggests that the endothelium of mouse diaphragm capillaries might exhibit comparable permeabilities towards serum albumin and transferrin which are similar in size and charge. The study shows that hapten-tagged polypeptides are close to the corresponding native macromolecules, and represent interesting tools for the morphological study of dynamic processes such as transcytosis.

Immunocytochemical study of glomerular permeability to anionic, neutral and cationic albumins.

The renal handling of albumin of various isoelectric points (pI) was studied in mice by high resolution quantitative immunocytochemistry. Bovine serum albumin (BSA) was tagged with dinitrophenol (DNP) and cationized to pI 6.5 to 7.5 and to pI over 8.5. These tracers, including the anionic BSA-DNP (pI 4.8) were injected in the iliac vein of mice and the post-embedding protein A-gold technique was used with antibodies against DNP to localize the different tracers in renal tissue. Morphometric analysis of the labeling over the glomerular wall has demonstrated variations in its distribution according to the pI of the markers. Anionic and neutral BSA molecules were detected on the endothelial side of the basement membrane, while labeling for cationic BSA was present mainly in the laminae rarae interna and externa known to display fixed polyanionic sites. Neutral and cationic BSA-DNP reached the urinary space and were detected within the endocytotic apparatus of the proximal tubule epithelium. Neutralization of the anionic sites of the basement membrane with cationic BSA resulted in an increase in the permeability towards anionic BSA-DNP. In addition to the demonstration of glomerular permeability properties towards various probes, the present study has demonstrated that dinitrophenylated albumin represents an excellent versatile tool for the quantitative morphological investigation of glomerular permeability.

Transendothelial transport of serum albumin: a quantitative immunocytochemical study.

The steady-state distribution of endogenous albumin in mouse diaphragm was determined by quantitative postembedding protein A-gold immunocytochemistry using a specific anti-mouse albumin antibody. Labeling density was recorded over vascular lumen, endothelium, junctions, and subendothelial space. At equilibrium, the volume density of interstitial albumin was 18% of that in circulation. Despite this large difference in albumin concentration between capillary lumen and interstitium, plasmalemmal vesicles labeling was uniformly distributed across the endothelial profile. 68% of the junctions displayed labeling for albumin, which was however low and confined to the luminal and abluminal sides. The scarce labeling of the endothelial cell surface did not confirm the fiber matrix theory. The kinetics of albumin transcytosis was evaluated by injecting radioiodinated and DNP-tagged BSA. At 3, 10, 30, and 60 min, and 3, 5, and 24 h circulation time, blood radioactivity was measured and diaphragms were fixed and embedded. Anti-DNP antibodies were used to map the tracer in aforementioned compartments. A linear relationship between blood radioactivity and vascular labeling density was found, with a detection sensitivity approaching 1 gold particle per DNP-BSA molecule. Tracer presence over endothelial vesicles reached rapidly (10 min) a saturation value; initially localized near the luminal front, it evolved towards a uniform distribution across endothelium during the first hour. An hour was also needed to reach the saturation limit within the subendothelial space. Labeling of the junctions increased slowly, out of phase with the inferred transendothelial albumin fluxes. This suggests that they play little, if any, role in albumin transcytosis, which rather seems to proceed through the vesicular way.

Distribution of endogenous apoprotein B-containing lipoproteins in normal and injured aortas of normocholesterolemic rabbits.

The distribution of endogenous apolipoprotein B (apo B) was studied in both normal and balloon catheter-injured aortas of standard fed rabbits. Using light and electron microscopy, the distribution within entire aortic walls and individual tissue compartments was investigated by immunocytochemistry using an antibody raised against rabbit apo B. The concentration of apo B across the vessel wall dropped sharply from the luminal front towards the media of the normal aortas. The strong superficial reaction was mainly due to a heavy, yet specific, labelling of endothelial cells. Significant concentrations of apo B were also detected within the innermost regions of the extracellular space. The characteristics associated with the labelling of the intimal layer suggested an intense uptake and transcellular transport of apo B by endothelial cells. In contradistinction, normal smooth muscle cells did not appear to be labelled. In the previously injured aortas, the same features of strong superficial apo B labelling were present in the areas covered by regenerated endothelial cells, but not in those persistently deendothelialized. The smooth muscle cells of these regions appeared to show a low uptake of apo B. The increased concentrations of apo B in deeper interstitial areas of injured aortas, indicated the contribution of the extracellular matrix to apo B accumulation. This was especially prominent in the advanced lesions, selectively developed within neointima covered by regenerated endothelium. A rather uniform labelling pattern accompanying small lipid particle deposits, suggested a direct extracellular accumulation of circulating lipoproteins. Intensely labelled foam cells and irregularly distributed apo B within areas of cellular necrosis were detected as well. Injury-mediated responses of the cellular and extracellular aortic components can trigger the development of lipoprotein accumulations characteristic of atherosclerosis within aortas of normocholesterolemic animals.

Protein AG-gold complex: an alternative probe in immunocytochemistry.

The purpose of this study was to evaluate the use of protein AG tagged with colloidal gold as a reliable immunocytochemical reagent. Protein AG is a recombinant of 47.3 KD molecular weight and pI = 4.3, which displays immunoglobulin Fc binding sites for both staphylococcal protein A and streptococcal protein G. It adsorbs to 10-nm colloidal gold particles with a lower affinity than does protein A, and is saturable. A maximal number of 12 protein AG molecules could be accommodated on the gold particle surface. Protein AG-gold conjugates yielded positive signals in post-embedding immunocytochemical assays when used as a secondary reagent in conjunction with several species and classes of polyclonal (rabbit, goat, sheep, guinea pig) and mouse monoclonal immunoglobulins (IgG1, IgG2, and IgG3). In addition, protein AG-gold was found to be a useful reagent in immunoblot analysis because of its ability to bind and identify nitrocellulose-immobilized IgGs (rabbit, mouse, goat, sheep, rat, and cow). Its spectrum of specificity towards various types of antibodies combines those of the parental protein A and protein G molecules. The protein AG-gold complex therefore appears to be a highly versatile and convenient alternative probe for immunochemical and immunocytochemical studies.

Immunolabeling efficiency of protein A-gold complexes.

A systematic study of the adsorption of protein A on colloidal gold particles varying in size from 5-16 nm was performed at different protein concentrations. The number of protein A molecules bound per colloidal particle was evaluated and the Scatchard analysis of the adsorption parameters was applied for each size of the colloid. The binding of protein A to the colloidal gold surface exhibited the same affinity pattern for all of the particle sizes. At low concentrations of stabilizing protein, adsorption took place with high affinity (Kd 1.96-3.3 nM) and the maximum number of protein A molecules attached with this affinity correlated well with the surface of the particle. At higher concentrations of protein A, adsorption exhibited a significantly lower affinity (Kd 530-800 nM), and no saturation was recorded. Competition by albumin did not reveal a preferential removal of the "low-affinity" bound protein A molecules, contradicting the model of successive shells of stabilizing protein around the colloidal particle. The immunolabeling efficiency of conjugates having the same size of gold nucleus but carrying different numbers of protein A molecules was comparatively investigated by quantitative post-embedding immunocytochemistry. Protein A-gold formed with 5-10-nm colloids gave the highest intensity of labeling when carrying the maximum number of protein A molecules that could be adsorbed with high affinity. Overloading as well as underloading these complexes resulted in a significant decrease of their immunoreactivity. The most efficient conjugates were obtained when stabilization was performed with 6 micrograms protein A/ml gold sol of 5 and 10 nm particle diameter, and 15 micrograms protein/ml of 15-nm colloid.

Fatty acids binding to albumin increases its uptake and transcytosis by the lung capillary endothelium.

To determine whether uptake and transcytosis of albumin (A) in continuous capillary endothelia are modified when this protein carries fatty acids, the transport of albumin-oleic acid and albumin-palmitic acid complexes was compared with that of defatted albumin. The probes, either radioiodinated or tagged with 5-nm gold particles (Au), or both, were perfused in situ or injected in vivo; after 3 or 30 min lung fragments were radioassayed or examined by electron microscopy. Both in situ and in vivo, the uptake of fatty acid-carrying albumin (A-FA) was consistently 2 to 3 times higher than that of defatted A. Electron microscopy revealed that A-FA complexes tagged with gold were taken up and transported across the endothelium by plasmalemmal vesicles. Morphometric analysis showed that as compared with A-Au, at 3 min the density of (A-FA)Au bound to plasmalemmal vesicles was 2 to 3 times higher, and the extent of transcytosis was increased. Uptake of the iodinated albumin was more effectively competed by A-FA complexes than by defatted A, suggesting a higher affinity of the former for the albumin binding sites of the endothelium. The results indicate that when carrying fatty acids, albumin is taken up specifically and with high affinity by the capillary endothelium, a process that may play a role in the transport of fatty acids from the plasma to the cells where they are metabolized.