Uptake Of Serum Albumin Research Articles

Abstract 18522: Pyridoxamine (vitamin B6) Mitigation Of Structural Valve Degeneration Of Bioprosthetic Heart Valves

Introduction: Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde treated bovine pericardium (BP) have limited durability due to structural valve degeneration (SVD) resulting from both calcification and advanced glycation end product (AGE) deposition. Hypothesis: We investigated the hypothesis that PYR modification of BHV leaflets mitigates AGE-serum protein uptake, oxidation, and calcification mechanisms in model studies, both in vitro and in vivo. Methods: To model AGE accumulation in BHV, we used well-established in vitro and in vivo models. AGE and serum albumin (SA) uptake in BP were quantitated per immunohistochemistry (IHC) staining intensity. PYR uptake was quantified using a novel fluorescent assay. BP collagen was examined by second-harmonic generation (SHG) microscopy. Calcification studies investigated the activity of BP alkaline phosphatase (ALP), and BP calcium levels were quantitated using a colorimetric assay. In vivo studies used a juvenile rat subdermal BP implantation model with PYR oral administration or BP-PYR modification. Endpoints were calcification, SA, and AGE accumulation per IHC and ALP activity per enzyme histochemistry. Results: PYR modification of BP resulted in high-affinity binding of PYR with 2.0±0.2 μg/mg of PYR binding to BP that was stable for 28 days in physiological conditions. Modification had an inhibitory effect on SA uptake in vitro with 60±5% (p<0.001) reduction in IHC staining intensity; PYR modification of BP resulted in increased protection from oxidative damage in vitro with collagen structure preservation (by SHG) after ten days, while control tissues were degraded. After 28-day of in vivo subdermal BP implantation in rats, a significant 70±5% (p<0.001) decrease and 80±5% (p<0.001) decrease in SA IHC staining and AGE staining, respectively, as well as ALP activity was documented in BP-PYR. Quantitative analysis of calcium content demonstrated a reduction from 108±5 μg/mg in control to 77±5 μg/mg in BP-PYR explants (p<0.001). Conclusions: Our study demonstrated that PYR mitigates SVD mechanisms in vitro and in vivo, limiting both AGE-serum protein accumulation and calcification with associated inhibition of ALP.

Abstract 3067: Constitutive macropinocytosis in BRAFV600E thyroid cancer promotes response to a serum-albumin conjugate of monomethyl auristatin E

Abstract Background: The non-specific uptake of extracellular materials through macropinocytosis can fuel tumor growth and survival, and is especially studied in cancer-types that frequently harbor RAS or PTEN mutations. However, relatively little data describe macropinocytosis in follicular, papillary, and anaplastic thyroid cancers (FTC, PTC, and ATC, respectively) despite the abundance of prominent MAPK and PI3K pathway mutations in these malignancies. We hypothesized that thyroid cancers exhibit elevated macropinocytosis and efficiently accumulate chemotherapy covalently conjugated to serum albumin, a common macropinocytosis substrate. Methods: We assessed the impact of ectopic BRAFV600E expression or targeted kinase inhibition on macropinocytosis in vitro across a panel of thyroid cancer cell lines with varied BRAF mutation status. Cellular uptake of dextran and serum albumin was quantified across ≥ 246 individual cells using fluorescence microscopy in triplicate experiments. The in vivo uptake of serum albumin by both ATC cells and tumor infiltrating immune cells, including tumor-associated macrophages, was assessed across n = 4 replicates of an immunocompetent orthotopic model of BrafV600E p53-/- ATC in B6129SF1/J mice. The same mouse model was used to measure the effects of an albumin-drug conjugate based on the microtubule-destabilizing monomethyl auristatin E (MMAE) linked to serum albumin via a cathepsin-cleavable peptide (Alb-vc-MMAE) on tumor volume using n ≥ 10 tumors per group. Groups were compared by two-tailed Wilcoxon rank-sum tests. Results: FTC and ATC cells exhibited greater macropinocytosis than PTC and immortalized follicular thyroid cells. Fluorescence microscopy of thyroid cancer cells revealed punctate subcellular co-localization between dextran and serum albumin. Treatment with the Na+/H+ exchanger (NHE) inhibitor 5-(N-ethyl-N-isopropyl)amiloride (EIPA) reduced cellular uptake by roughly 50%, consistent with macropinocytosis (P < 0.01). Ectopic BRAFV600E enhanced macropinocytosis by 2-fold, while BRAF and MEK kinase inhibition decreased uptake by 50% (P < 0.01). In vivo, ATC tumors accumulated serum albumin at 8.5%ID/g (% injected dose per gram tissue). The albumin-drug-conjugate Alb-vc-MMAE, but not MMAE alone, blocked more than 90% of ATC tumor growth (P < 0.01). Conclusions: ATC exhibits high constitutive macropinocytosis driven by elevated oncogenic signaling, including via MAPK/ERK activity stimulated by BRAFV600E mutation. Macropinocytosis in ATC, therefore, may be a target for manipulating tumor metabolism and has implications for the delivery of some albumin-bound and other drugs that are affected by macropinocytic cell uptake. Citation Format: Huiyu Hu, Thomas S. Ng, Mikyung Kang, Ella Scott, Ran Li, Jeremy M. Quintana, Dylan Matvey, Venkata R. Vantaku, Ralph Weissleder, Sareh Parangi, Miles A. Miller. Constitutive macropinocytosis in BRAFV600E thyroid cancer promotes response to a serum-albumin conjugate of monomethyl auristatin E [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3067.

Abstract 13187: Inhibition of Advanced Glycation End Products Formation and Serum Protein Infiltration in Bioprosthetic Heart Valves: Investigations of Anti-Glycation Agents

Introduction: Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde pretreated bovine pericardium (BP), are the most commonly used heart valve replacements. However, BHV durability is limited by structural valve degeneration (SVD) resulting from both calcification and advanced glycation end product (AGE) deposition together with serum protein infiltration. This study investigated, for the first time, the application of anti-glycation agents as a way to mitigate AGE-related structural degeneration of BHV. Hypothesis: We investigated the hypothesis that anti-AGE agents (Aminoguanidine (AG), Pyridoxamine (PYR), and N-Acetylcysteine (NAC)) could mitigate BP glycation in model studies, both in vitro and in vivo. Methods: To model AGE accumulation in BHV, we used well-established accelerated in vitro glycation models using BP incubation with or without inhibitors. Incorporation of AGE in BP was monitored using radiolabeled glycation precursors. Changes in collagen structure were examined by two photon confocal microscopy and differential scanning calorimetry. In vivo studies used a juvenile rat subdermal BP implantation model with drug administration via drinking water. Endpoints were AGE formation, collagen structure, thermal denaturation temperature, serum albumin uptake, calcification, and serum glycation marker levels. Results: In vitro studies demonstrated that each of these agents significantly inhibited AGE formation in BP. However, after 28-day rat subdermal BP implantation in vivo, only PYR demonstrated significant inhibition of both AGE and serum albumin accumulation per immunostaining. BHV calcification was not mitigated by PYR, AG or NAC. Conclusions: This study is the first to demonstrate the use of AGE inhibitors in mitigating BHV glycation. PYR demonstrated significant efficacy both in vitro and in vivo, mitigating AGE formation and albumin infiltration in BP. Thus, this study provides a rationale for investigating PYR as a possible candidate for improving the durability and biocompatibility of clinically used BHV, providing the solution for this unmet clinical need.

Co-cultures of renal progenitors and endothelial cells on kidney decellularized matrices replicate the renal tubular environment in vitro.

Herein we propose creating a bilayer tubular kidney in-vitro model. It is hypothesized that membranes composed of decellularized porcine kidney extracellular matrix are valid substitutes of the tubular basement membrane by mimicking the physiological relevance of the in vivo environment and disease phenotypes. Extracellular matrix was obtained from decellularized porcine kidneys. After processing by lyophilization and milling, it was dissolved in an organic solvent and blended with poly(caprolactone). Porous membranes were obtained by electrospinning and seeded with human primary renal progenitor cells to evaluate phenotypic alterations. To create a bilayer model of the in vivo tubule, the same cells were differentiated into epithelial tubular cells and co-cultured with endothelial cells in opposite sites. Our results demonstrate increasing metabolic activity, proliferation and total protein content of renal progenitors over time. We confirmed the expression of several genes encoding epithelial transport proteins and we could also detect tubular-specific proteins by immunofluorescence stainings. Functional and transport assays were performed trough the bilayer by quantifying both human serum albumin uptake and inulin leakage. Furthermore, we validated the chemical modulation of nephrotoxicity on this epithelium-endothelium model by cisplatin exposure. The use of decellularized matrices in combination with primary renal cells was shown to be a valuable tool for modelling renal function and disease in vitro. We successfully validated our hypothesis by replicating the physiological conditions of an in vitro tubular bilayer model. The developed system may contribute significantly for the future investigation of advanced therapies for kidney diseases.

Clinical Significance of Preoperative Hepatocellular Carcinoma With High Lens culinaris Agglutinin-reactive Fraction of Alpha-Fetoprotein, But Low Alpha-Fetoprotein.

The aim of this study was to verify the significance of high Lens culinaris agglutinin-reactive fraction of α-fetoprotein (AFP-L3) in patients with hepatocellular carcinoma (HCC) with low AFP. There were 283 patients with low AFP who underwent initial hepatic resection with or without radiofrequency ablation for HCC. Patients were divided into two groups based on AFP-L3 values: >10%: high AFP-L3 (n=24); and ≤10%: low AFP-L3 (n=259). Overall survival (OS) and 2-year recurrence rates were compared, and independent prognostic factors were identified. The OS and 2-year recurrence rates of the high AFP-L3 group were significantly worse than those of the low AFP-L3 group. The independent prognostic factors for poor OS were des-gamma-carboxy prothrombin (DCP) of >40 mAU/ml, microvascular invasion, and invasive growth, and those for 2-year recurrence were 99mTc-galactosyl human serum albumin uptake ratio of <0.90, DCP of >40 mAU/ml, multiple tumors, microvascular invasion, and poor differentiation. DCP levels increased with AFP-L3, and cases with high DCP and AFP-L3 had worse prognoses and higher 2-year recurrence rates compared to those with elevation of only one of these. Patients with high AFP-L3 but low AFP have poor prognosis and high 2-year recurrence rates. DCP strongly reflects HCC malignancy in patients with low AFP.

Synergistic toxicity of zno nanoparticles and dimethoate in mice: Enhancing their biodistribution by synergistic binding of serum albumin and dimethoate to zno nanoparticles.

The extensive applications of ZnO nanoparticles (nano ZnO) and dimethoate (DM) have increased the risk of humans' co-exposure to nano ZnO and DM. Here, we report the synergistic effect of nano ZnO and DM on their biodistribution and subacute toxicity in mice. Nano ZnO and DM had a synergistic toxicity in mice. In contrast, bulk ZnO and DM did not cause an obvious synergistic toxicity in mice. Although nano ZnO was low toxic to mice, coexposure to nano ZnO and DM significantly enhanced DM-induced oxidative damage in the liver. Coadministration of nano ZnO with DM significantly increased Zn accumulation by 30.9±1.9% and DM accumulation by 45.6±2.2% in the liver, respectively. The increased accumulations of DM and Zn in the liver reduced its cholinesterase activity from 5.65±0.32 to 4.37±0.49U/mg protein and induced hepatic oxidative stress. Nano ZnO had 3-fold or 2.4-fold higher binding capability for serum albumin or DM, respectively, than bulk ZnO. In addition, serum albumin significantly increased the binding capability of nano ZnO for DM by approximately four times via the interaction of serum albumin and DM. The uptake of serum albumin- and DM-bound nano ZnO by the macrophages significantly increased DM accumulation in mice. Serum albumins play an important role in the synergistic toxicity of nano ZnO and DM. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1202-1212, 2017.

Neuronal uptake of serum albumin is associated with neuron damage during the development of epilepsy.

It is well established that brain blood barrier dysfunction following the onset of seizures may lead to serum albumin extravasation into the brain. However, the effect of albumin extravasation on the development of epilepsy is yet to be fully elucidated. Previous studies have predominantly focused on the effect of albumin absorption by astrocytes; however, the present study investigated the effects of neuronal uptake of albumin in vitro and in kainic acid-induced Sprague-Dawley rat models of temporal lobe epilepsy. In the present study, electroencephalogram recordings were conducted to record seizure onset, Nissl and Evans blue staining were used to detect neuronal damage and albumin extravasation, respectively, and double immunofluorescence was used to explore neuronal absorption of albumin. Cell counting was also conducted in vitro to determine whether albumin contributes to neuronal death. The results of the present study indicated that extravasated serum albumin was absorbed by neurons, and the neurons that had absorbed albumin died and were dissolved 28 days after seizure onset in vivo. Furthermore, significant neuronal death was detected after albumin absorption in vitro in a dose- and time-dependent manner. These results suggested that albumin may be absorbed by neurons following the onset of seizures. Furthermore, the results indicated that neuronal albumin uptake may be associated with neuronal damage and death in epileptic seizures. Therefore, attenuating albumin extravasation following epileptic seizures may reduce brain damage and slow the development of epilepsy.

In vivo biodistribution and synergistic toxicity of silica nanoparticles and cadmium chloride in mice

Silica nanoparticles (SiNPs) are now in daily use due to their low intrinsic toxicity. Cadmium is a ubiquitous environmental pollutant. In spite of real risk of humans’ co-exposure to SiNPs and cadmium, their synergistic toxicity is still unclear. Here, we report the synergistic effects of SiNPs and CdCl2 on their biodistribution and subacute toxicity in mice. The biodistributions, histopathological changes, serum biochemical parameters and oxidative stress responses were determined after intraperitoneal injection of SiNPs and/or CdCl2 to mice. SiNPs and CdCl2 have a positive synergistic toxicity in mice. Although SiNPs were low toxic to mice, co-exposure of SiNPs and CdCl2 significantly enhanced CdCl2-induced oxidative damage in the liver as indicated by the severe liver dysfunction and histopathological abnormalities. Co-exposure to SiNPs and CdCl2 markedly increased the cadmium accumulation in the liver, which induced significant hepatic oxidative stress. In vitro binding assays indicated that serum albumin and Cd2+ mutually enhanced the binding of each other to SiNPs via the interaction of serum albumin and Cd2+. The uptake of serum albumin- and Cd2+-bound SiNPs by the macrophages significantly increased cadmium accumulation in mice. These results demonstrate that serum albumins play an important role in the positive synergistic toxicity of SiNPs and CdCl2.

Abstract 3800: Nitric oxide acts a promoter of the enhanced permeation and retention effect in a xenograft pancreatic carcinoma model

Abstract Pancreatic cancer is the forth most common cause of cancer death worldwide and today there is no effective treatment. Gemcitabine and 5-fluorouracil have been approved for this indication, but merely show a poor response rate with a small benefit in overall survival. The use of albumin as a carrier for drugs is a potential therapeutic option for the treatment of this tumor indication, and we could recently show that INNO-206, an albumin-binding prodrug of doxorubicin under clinical evaluation, produces significant tumor growth inhibition and reduction of metastases in an orthotopic tumor model in mice (Graeser et al. Invest. New Drugs 2009, 28, 14-19). Since important benefits of drug-albumin conjugates can be attributed to the enhanced permeability and retention of the tumor vasculature (also known as the EPR effect), we were interested in how to further enhance this effect in pancreatic tumors which generally only exhibit a moderate vascularization. Maeda and co-workers have described three methods of EPR alteration: by the use of donors of nitric oxide such as nitroglycerin, by temporarily elevating the blood pressure with angiotensin II, and by treatment with a kininase II inhibitor such as enalapril in combination with angiotensin II. Thus, we set out to compare these methods in the MiaPaCa-2 xenograft model in nude mice. Instead of angiotensin II which has a very short half-life of a few seconds, we used the longer acting peptide endothelin I for inducing hypertension. The tumor-bearing mice were treated with nitroglycerin (topically, 1 mg per tumor), with endothelin I (3.7 µg/kg), and with enalapril (10 mg/kg) in combination with endothelin I (3.7 µg/kg) followed by an injection of the dye Evans Blue (10 mg/kg) which strongly binds to serum albumin thus forming a stable dye-albumin complex. Animals were sacrificed after 1 h, 6 h, 24 h, 48 h, and 168 h, respectively, and the tumors were dissected. The concentration of Evans Blue was determined after extraction of the tumor tissue with formamide. The lowest concentrations of Evans Blue correlating with respect to tumor uptake of serum albumin were found in the control group as well as the group treated with endothelin I. Higher levels of the dye were found in the mice treated with the combination enalapril/endothelin I whereas the highest concentrations were found after treatment with nitroglycerin. Generally, the concentrations of the dye increased with time and reached a maximum at the end of the experiment. These results indicate that vascular permeability for albumin can be enhanced most effectively by the NO donor nitroglycerin in mice bearing pancreas tumor xenografts. This seems to be a viable method for enhancing the EPR effect that is worthy of further investigation also with other nitric oxide releasing drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3800. doi:1538-7445.AM2012-3800

Age-Related Changes in Scavenger Receptor–Mediated Endocytosis in Rat Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) play an essential role in systemic waste clearance by effective endocytosis of blood-borne waste macromolecules. We aimed to study LSECs' scavenger function during aging, and whether age-related morphological changes (eg, defenestration) affect this function, in F344/BN F1 rats. Endocytosis of the scavenger receptor ligand formaldehyde-treated serum albumin was significantly reduced in LSECs from old rats. Ligand degradation, LSEC protein expression of the major scavenger receptors for formaldehyde-treated serum albumin endocytosis, stabilin-1 and stabilin-2, and their staining patterns along liver sinusoids, was similar at young and old age, suggesting that other parts of the endocytic machinery are affected by aging. Formaldehyde-treated serum albumin uptake per cell, and cell porosity evaluated by electron microscopy, was not correlated, indicating that LSEC defenestration is not linked to impaired endocytosis. We report a significantly reduced LSEC endocytic capacity at old age, which may be especially important in situations with increased circulatory waste loads.

Endocytosis of serum albumin in regenerating rat liver.

Lysosomes, isolated from rat liver after 70% partial hepatectomy (PHX), were found, by Western blotting, to contain a considerable amount of serum albumin. The level of intralysosomal serum albumin after PHX showed biphasic patterns: it increased immediately after PHX, peaked at 30 min, rapidly declined within a few hours, rose again with a peak at 15 hr, and gradually declined thereafter. At 15 hr after PHX, the content of lysosomal proteins in the liver increased to twice the level of unoperated control, and the electron-microscopic observation of the isolated lysosomes revealed numerous large membrane-delimited structures with ground substances of variable electron opacities. The increase in the intralysosomal serum albumin at 30 min and 15 hr was accompanied by changes in the buoyant densities of endosomes in Percoll density gradients. At both time points, the density profiles of endosomes isolated from hepatectomized rats shifted to the denser direction, suggesting that PHX activates fusion and/or maturation of endosomes. Formaldehyde-treated bovine serum albumin is known to be taken up by the liver by receptor-mediated endocytosis. The uptake of the modified heterologous albumin was shown to be activated as early as 30 min after PHX. Both the uptake of serum albumin into lysosomes and the shift of buoyant density profile of endosomes after PHX were inhibited by the administration of adrenergic receptor antagonists, particularly by the alpha r-antagonist prazosin. Further, the concentration of catecholamines in rat serum, particularly that of norepinephrine, was found to increase immediately after PHX, relative to that in serum from sham-operated rats. These results suggest that the elevation of serum norepinephrine levels after PHX activates endocytosis and facilitates delivery of endocytosed serum albumin to lysosomes, where albumin is digested to yield amino acids for possible use in protein synthesis during liver regeneration.

Inhibition of mouse embryonic, yolk sac, and limb-bud functions by the methyl isocyanate metabolite S-(N-methylcarbamoyl)glutathione.

We previously reported that S-(N-methylcarbamoyl)glutathione (SMG), a conjugate formed by the reversible reaction between methyl isocyanate and glutathione, inhibited the development of mouse embryos in culture. The present study was done to determine whether SMG produced embryotoxicity by inhibiting yolk sac functions. For this purpose we determined the effects of an embryotoxic concentration of SMG on mouse yolk sac uptake mechanisms and lysosomal proteolysis as well as on the incorporation of [3H]leucine in mouse embryonic and limb-bud proteins. After 5 h of culture, SMG inhibited the uptake of [14C]sucrose and 125I-labelled bovine serum albumin in isolated day 15 yolk sacs to 62 and 77% of control, respectively. Lysosomal proteolysis was not inhibited, as judged by the release of trichloroacetic acid soluble radioactivity into the culture media. Uptake and incorporation of [3H]leucine from free [3H]leucine or from [3H]leucine-labelled protein in SMG-treated day 9 embryos were inhibited, respectively, to 61 and 25% of the control uptake during a 16-h labelling period. SMG also inhibited the incorporation of free [3H]leucine into limb-bud proteins. SMG-induced inhibition of 125I-labelled bovine serum albumin uptake by yolk sacs was partially prevented by the thiol donors N-acetylcysteine and glutathione but not by acivicin (gamma-glutamyl transpeptidase inhibitor) and aminooxyacetic acid (cysteine conjugate beta-lyase inhibitor). These data suggest that SMG suppresses embryonic growth primarily by an inhibition of nutrient uptake by the yolk sac. We postulate that this inhibition is due to tissue carbamoylation by methyl isocyanate released from SMG.

Effect of endothelial integrity, transmural pressure, and time on the intimal-medial uptake of serum 125I-albumin and 125I-LDL in an in vitro porcine arterial organ-support system.

This report describes a new in vitro, metabolically supported, Sinclair Research Farm minipig aortic preparation in which the intimal-medial uptakes (M, mg.cm-2 of intimal surface) of porcine 125I-albumin and normocholesterolemic (nonoxidized) porcine 125I-low density lipoprotein (LDL) from a stirred, autogenous serum (containing a 125I-protein concentration of c0, mg.cm-3 at 37 degrees C and pH 7.4) were studied as functions of transmural pressure (0 < or = P < or = 150 mm Hg), time (30 < or = t < or = 120 minutes), and endothelial integrity. The following new observations were made: 1) The normalized transendothelial uptakes (M/c0, cm) of both albumin and LDL across normal intact aortic endothelial surfaces were insensitive to P. This indicated that these macromolecular solutes were not readily convected across the normal aortic endothelial surface despite increasing P. 2) However, the associated transendothelial M/c0 versus t relations for the normal intact surfaces were shown to increase monotonically with t in a manner consistent with a simple diffusive transport across a large surface barrier into the subjacent media, either with (Cases 2A and 2B) or without (Case 1) an associated transmural water convection. 3) The shapes of these temporal M/c0 curves of albumin and LDL were virtually the same; however, the magnitude of the albumin M/c0 curve was about sevenfold greater than that of LDL. 4) The M/c0 across the injured endothelial surface (Case 2C) not only increased monotonically with t but also increased significantly with P, indicating that in the absence of a normal endothelial surface, a very large convective component was added to the transport processes across the exposed aortic endothelial basement membrane and internal elastica. We conclude that: 1) the normal porcine aortic endothelial surface can provide a virtually complete barrier to the transendothelial convective transport of both albumin and LDL, 2) the diffusive barrier of the normal endothelial surface to LDL was sevenfold greater than that to albumin, 3) loss of the endothelial cell layer was associated with a threefold increase in the (P = 0) diffusive intimal-medial uptake of serum albumin in contrast to an eightfold increase in the pressurized (P = 150 mm Hg) combined diffusive-convective intimal-medial albumin uptake in the same vessel.

The role of albumin in developing rodent dental enamel: a possible explanation for white spot hypoplasia.

The uptake of serum albumin by maturation-stage rodent enamel and the resulting effects on the growth of enamel crystallites were investigated in vitro. Albumin uptake was demonstrated by means of gel electrophoresis and confirmed by Western blotting with use of monoclonal antibodies. Measurement of crystal size was carried out by direct TEM measurement of enamel crystallite outlines after incubations in metastable solutions of calcium phosphate. The ability of endogenous enamel enzymes to degrade albumin was investigated by substrate-specific zymography. The results showed that albumin could be taken up by maturation-stage enamel and produce inhibition of crystallite growth. There was no detectable proteolytic activity in the enamel against albumin substrate, which suggests that albumin entering enamel by extravasation in vivo may produce incomplete tissue maturation, resulting in a white, opaque appearance on eruption.

Subarachnoidal macrophages share a common epitope with resident non-cerebral macrophages and show receptor-mediated endocytosis of albumin-gold and IgG-gold complexes

Cerebral macrophages are supposed to exploit a pivotal role in scavenger functions of the central nervous system. We have examined the in vivo uptake of serum albumin and IgG conjugated with colloidal gold by subarachnoidal macrophages. These serum-borne proteins are endocytosed by receptor-mediated endocytosis by varying kinetics. Albumin-gold conjugates were found to be associated to a significant amount with coated pits and coated vesicles 1 min after superfusion of the cerebral surface. Within 25 min the major fraction of albumin-gold was transferred to the lysosomal compartment. IgG-conjugates revealed a less pronounced uptake. The uptake of albumin-gold could be competed by saturating of the 'receptor sites' with free albumin. It is suggested that the described receptor-mediated uptake of serum-borne proteins by subarachnoidal macrophages serves a cleansing function following blood-brain barrier disruption during acute or subacute inflammatory reactions.

The persistence of high uptake of serum albumin in the olfactory bulbs of mice throughout their adult lives

Brain to plasma concentration ratios of i.v. administered human serum albumin (HSA) in the olfactory bulb, frontal cortex and cerebellum were evaluated in DDD mice of different ages. We measured the brain uptake of serum albumin excluding intravascular content by using a double isotope technique and examined the time course of the brain uptake to evaluate the brain uptake at different time intervals. In young adult mice, the value was significantly higher in the olfactory bulb than in other brain regions 3–24 h after 125I-HSA injection. It was about 2.3 times higher in the olfactory bulb than in the cerebellum ( P<0.01). The high concentration ratios in the olfactory bulb were observed in all 4–22-month-old mice. Moreover, the ratio in the olfactory bulb 24 h after 125I-HSA injection was higher in 22-month-old mice than in younger animals. The high uptake of serum albumin in the olfactory bulb suggests that intravascular macromolecules can be transported into the olfactory bulb more easily than in other brain regions with tight endothelium, and the persistence of high uptake during adult life may be associated with age-related morphological changes in the olfactory bulb.

Synthetic glycopeptide substrates for receptor-mediated endocytosis by macrophages.

Mammalian macrophages contain a transport system that binds and internalizes glycoproteins with exposed mannose residues. This system and analogous systems on other types of cells require substrates to bear multiple nonreducing terminal residues of the appropriate sugar for effective uptake. Small multivalent synthetic glycopeptides with mannose residues covalently linked through a spacer arm to the alpha- and epsilon-amino groups of lysine, dilysine, and trilysine are competitive inhibitors of rat alveolar macrophage uptake of the neoglycoprotein mannosyl-bovine serum albumin with inhibition constants in the microM range. Various compounds could be covalently attached to the alpha-carboxyl group of these glycopeptides with substantial retention of inhibitory potency. This uptake system does not recognize galactose residues, and the galactosyl analog of an inhibitory mannosylpeptide did not inhibit uptake of mannosyl-bovine serum albumin. The trimannosyldilysine ligand is not only an inhibitor but also a substrate for specific uptake by macrophage, as shown with an 125I-labeled derivative. Macrophages bound 6.4 x 10(5) molecules per cell at 0 degrees C with a dissociation constant of 2 microM. At 21 degrees C the cells could internalize the labeled conjugate with an apparent Michaelis constant of 6 microM and a maximal velocity of 1.7 x 10(5) molecules per min per cel. The dissociation constant and Michaelis constant are similar to the inhibition constant of 9 microM determined at 21 degrees C for inhibition by this conjugate at mannosyl-bovine serum albumin uptake. These synthetic substrates may be useful in targeting pharmacologic agents to macrophages, and analogous compounds may target such agents to other types of cell.

Stimulation of triacylglycerol synthesis by intracellular serum albumin

A factor in the supernatant fraction of adipose tissue that stimulates the synthesis of triacylglycerols by microsomes has been identified as serum albumin. The stimulatory effect is directly proportional to the ratio of fatty acids bound to the albumin. Small amounts of serum albumin appear to be inside the adipocytes and albumin can be taken up by isolated adipocytes. The rate of uptake of fatty acids by the adipocytes is more than 1000 times the uptake of serum albumin. This difference provides counter-evidence for the proposal that serum albumin might function in the vesicular transport of fatty acids.

Conjugation of poly-L-lysine to albumin and horseradish peroxidase: a novel method of enhancing the cellular uptake of proteins.

The carbodiimide-catalyzed conjugation of a 6700 molecular weight fragment of poly-L-lysine to radiolabeled human serum albumin or to horseradish peroxidase enhances the membrane transport of each protein into cultured mouse fibroblasts approximately 11- and 200-fold, respectively. At least 50% of the peroxidase activity remained after conjugation. Trypsinization and carbamylation of the two conjugates demonstrates that the enhancement of their cellular uptake is related to their poly-L-lysine content. Simple addition to the medium of comparable amounts of free poly-L-lysine has no effect on the transport of either native protein. Addition of poly-L-ornithine (molecular weight 200,000) at 3-30 microgram/ml, a condition known to cause enhancement of 125I-labeled human serum albumin uptake by mouse sarcoma cells, has no visible effect on the cellular uptake of native horseradish peroxidase. The intracellular localization of the enzyme-poly-L-lysine conjugate can be demonstrated cytochemically by either light or transmission electron microscopy. A concentration of conjugate that increases the uptake more than 200-fold does not cause any detectable morphological change suggestive of cell toxicity. Furthermore, because poly-L-lysine is an excellent substrate for intracellular proteolytic enzymes, it can be expected to be broken down and reutilized in the cell.

Uptake and degradation of formaldehyde-treated 125I-labelled human serum albumin in rat liver cells in vivo and in vitro.

The uptake of formaldehyde-treated 125I-labelled human serum albumin in rat hepatocytes and nonparenchymal liver cells was measured in vivo and in vitro. Isolated liver cells were prepared by treating the perfused liver with collagenase. Purified hepatocytes and nonparenchymal cells were obtained by differential centrifugation. Human serum albumin was found to be taken up exclusively or almost exclusively by nonparenchymal cells in vitro and in vivo (after intravenous injection). The maximal rate of human serum albumin-uptake in vitro was comparable to that in vivo. Nonparenchymal cells degraded human serum albumin in vitro as indicated by release of trichloroacetic acid-soluble radioactivity. Degradation started about 20-30 min after addition of human serum albumin to cells and rate of degradation was proportional to rate of uptake. Human serum albumin-degradation could be studied without interference of concurrent uptake by separating cells that had been preincubated with human serum albumin from the medium and then reincubating them with human serum albumin-free medium. The lag phase before human serum albumin-degradation starts and the inhibitory effect of chloroquine on degradation indicate that human serum albumin is degraded in lysosomes. The data obtained show that enzymatically prepared nonparenchymal liver cells retain their endocytic activity in vitro. Denatured human serum albumin should be useful both as a marker for rat liver macrophages and for the study of intracellular proteolysis in these cells.