Lysosomal Proteolysis Research Articles

Vitamin B6Deficiency Accelerates Metabolic Turnover of Cystathionase in Rat Liver

Although most of cystathionase was found to exist as an inactive apoenzyme in the liver of vitamin B6-deficient rats, the concentrations of the immunoreactive enzyme protein were virtually the same for control and vitamin B6-deficient livers. Under vitamin B6deficiency, however, the rate of synthesis of cystathionase, measured by incorporation of labeled amino acid into the immunoprecipitated enzyme, was increased severalfold due to an increased level of cystathionase mRNA. Western blot analysis of lysosomal proteins showed that the amount of cystathionase in the lysosomes from the liver of vitamin B6-deficient rats was also increased severalfold. This observation suggests that lysosomes specifically recognize the apocystathionase for sequestration in preference to the holoenzyme. The present study provides the molecular basis for dual roles of vitamin B6in controlling the metabolic turnover of cystathionase; it regulates synthesis of the enzyme by modulating the expression of cystathionase gene, and it regulates degradation of the enzyme by different susceptibilities of apo- and holoenzymes to lysosomal proteolysis.

Lysosomal metabolism of proteins.

The lysosome is a major site of mammalian protein degradation and is calculated to be capable of degrading as much protein as the digestive tract (Barrett and Kirschke, 1981). Detailed characteristics are known for several of the enzymes involved in lysosomal proteolysis, although recent data suggest that the current catalog of enzymes involved in protein degradation is by no means complete. Although the enzymes are the necessary tools for hydrolysis, they are only effective in the right environment. In this chapter the relationship between the different enzymes and their environment is discussed, including mechanisms by which lysosomal proteolysis is controlled. Delivery of substrates to the lysosome, the pH in the lysosome, and removal of products from the lysosome are discussed in depth elsewhere in this volume (Chapters 3, 4, 5, 10, and 11), so discussion of these topics will be restricted to how they relate specifically to protein turnover. This chapter focuses on how the proteases function to hydrolyze proteins in the lysosome (for information on the pathological roles and the chemistry of the lysosomal proteases, see Lee and Marzella, 1994, and Mason and Wilcox, 1993).

Assessment of the in vivo hepatic lysosomal processing of horseradish peroxidase

The lysosomal processing of horseradish peroxidase (HRP) was assessed in this study, i.e., its lysosomal proteolysis and the biliary output of its possible lysosomal metabolites by rat liver in vivo. HRP was covalently linked to [14C]sucrose to provide a label that remains trapped within lysosomes after proteolysis. The [14C]sucrose-labelled HRP was injected into the portal vein of rat, and after 30 min about 34% of the injected radiolabel was present in the liver. Subcellular fractionation by differential centrifugation and further purification of lysosomes in a Percoll gradient showed that radiolabel was concentrated in lysosomes and indicated that about 91% of the total proteolysis of HRP in liver could be attributed to these organelles. The in vivo lysosomal degradation rate of HRP at 30 min was about 40%/h, decreasing over time. The lysosomal inhibitors chloroquine and leupeptin suppressed proteolysis of HRP by about 30 and 60%, respectively. Analysis of the 14C excreted in bile by trichloroacetic acid precipitation and by SDS-polyacrylamide gel electrophoresis showed a minor fraction, which was intact HRP (40 kDa), and a major fraction, which was associated with material smaller than 3 kDa. The biliary output of these low molecular mass products, in contrast to that of intact HRP, did not gradually decline with time and represented about 3% of the corresponding amounts in liver. Chloroquine and leupeptin specifically decreased their biliary excretion (about 60%), giving additional support to their lysosomal origin. In addition, the overall hepatic processing of [14C]sucrose-labelled HRP did not differ from that of the native HRP measured by enzyme assay, indicating no significant alteration caused by the labelling procedure.

Selective proteolysis: 70-kDa heat-shock protein and ubiquitin-dependent mechanisms?

The proteolysis of the vast majority of cytoplasmic proteins, once inside the lysosome compartment, would be expected to occur rapidly; therefore, the rate-limiting step in the lysosomal degradation of cytoplasmic proteins must be their initial sequestration. Indeed, the initial sequestration serves to remove cytoplasmic proteins from their functional site in the cell. Bulk sequestration of cytoplasm (macro-autophagy), described elsewhere in this volume (Chapter 4), is considered essentially nonselective. Microautophagy has been described by a number of investigators over the years (see Chapter 4) and may provide a mechanism for the selective sequestration of cytoplasmic proteins into the lysosome system. However, controversy surrounds the whole topic of selective lysosomal proteolysis, with some investigators probably of the view that the lysosome system is entirely nonselective. However, recent work from the laboratory of Dice (Dice, 1990) describes a selective mechanism of sequestration of cytosolic proteins into lysosomes for degradation in a process regulated by serum and involving a member of the 70-kDa heat-shock protein family.

Activation of a selective pathway of lysosomal proteolysis in rat liver by prolonged starvation.

Lysosomal uptake and degradation of polypeptides such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribonuclease A (RNase A), and RNase S-peptide (residues 1-20 of RNase A) are progressively activated in rat liver by starvation before isolation of lysosomes. This pathway of proteolysis is selective, since it is stimulated by the heat shock cognate protein of 73 kDa (HSC73) and ATP-MgCl2, and lysosomal uptake of RNase A could be competed by GAPDH but not by ovalbumin. A portion of intracellular HSC73 is associated with certain lysosomes, and the amount of lysosomal HSC73 increases by 5- to 10-fold during prolonged starvation. The lysosome-associated HSC73 is primarily within the lysosomal lumen. Double immunogold labeling of lysosomes incubated in vitro with RNase A detects this protein substrate as well as HSC73 within lysosomes. More than two-thirds of the labeled lysosomes contain both RNase A and HSC73. The possible physiological significance of the activation of this selective pathway of lysosomal proteolysis in long-term starvation is discussed.

A lysosomal targeting signal in the cytoplasmic tail of the beta chain directs HLA-DM to MHC class II compartments.

In human B cells, class II molecules of the major histocompatibility complex (MHC-II) accumulate in an endosomal/lysosomal compartment, the MIIC, in which they may encounter and bind peptides. An additional molecule required for MHC-II peptide binding, HLA-DM (DM), has also been localized to the MIIC. Neither the relationship of the MIIC to the endosomal system nor the mechanisms by which DM localizes to the MIIC are understood. To address these issues, DM localization was analyzed in cells that do or do not express MHC-II. DM alpha beta heterodimers were localized in transfected MHC-II-negative HeLa and NRK cells, in the absence of the MHC-II-associated invariant chain, to a prelysosomal/lysosomal compartment by immunofluorescence microscopy. To identify a potential targeting determinant, we analyzed the localization of a chimeric protein, T-T-Mb, in which the cytoplasmic tail of murine DM beta (Mb) was appended to the lumenal and transmembrane domains of a cell surface protein, Tac. Like intact DM, T-T-Mb was localized to a lysosomal compartment in HeLa and NRK cells, as judged by immunofluorescence and immunoelectron microscopy. T-T-Mb was rapidly degraded in this compartment by a process that was blocked by inhibitors of lysosomal proteolysis. The DM beta cytoplasmic tail also mediated internalization of anti-Tac antibody from the cell surface and delivery to lysosomes. Deletion from the DM beta cytoplasmic tail of the tyrosine-based motif, YTPL, resulted in cell surface expression of T-T-Mb and a loss of both degradation and internalization; alanine scanning mutagenesis showed that the Y and L residues were critical for these functions. Similarly, mutation of the same Y residue within full-length DM beta resulted in cell surface expression of DM alpha beta heterodimers. Lastly, T-T-Mb was localized by immunoelectron microscopy to the MIIC in a human B lymphoblastoid cell line. Our results suggest that a motif, YTPL, in the cytoplasmic tail of the beta chain of DM is sufficient for targeting either to lysosomes or to the MIIC.

Some Growth Factors Stimulate Cultured Adult Rabbit Ventricular Myocyte Hypertrophy in the Absence of Mechanical Loading

Cultured adult rabbit cardiac myocytes treated with recombinant growth factors display enhanced rates of protein accumulation (ie, growth) in response to insulin and insulin-like growth factors (IGFs), but epidermal growth factor, acidic or basic fibroblast growth factor, and platelet-derived growth factor failed to increase contractile protein synthesis or growth of the heart cells. Insulin and IGF-1 increased growth rates by stimulating anabolic while simultaneously inhibiting catabolic pathways, whereas IGF-2 elevated growth modestly by apparently inhibiting lysosomal proteolysis. Neutralizing antibodies directed against either IGF-1 or IGF-2 or IGF binding protein 3 blocked protein accumulation. A monoclonal antibody directed against the IGF-1 receptor also inhibited changes in protein turnover provoked by recombinant human IGF-1 but not IGF-2. Of the other growth factors tested, only transforming growth factor-beta 1 increased the fractional rate of myosin heavy chain (MHC) synthesis, with beta-MHC synthesis being elevated and alpha-MHC synthesis being suppressed. However, the other growth factors were able to modestly stimulate the rate of DNA synthesis in this preparation. Bromodeoxyuridine labeling revealed that these growth factors increased DNA synthesis in myocytes and nonmyocytes alike, but the heart cells displayed neither karyokinesis or cytokinesis. In contrast, cocultures of cardiac myocytes and nonmyocytes and nonmyocyte-conditioned culture medium failed to enhance the rate of cardiac MHC synthesis or its accumulation, implying that quiescent heart cells do not respond to "conditioning" by cardiac nonmyocytes. These findings demonstrated that insulin and the IGFs promote passively loaded cultured adult rabbit heart cells to hypertrophy but suggest that other growth factors tested may be limited in this regard.

Mice deficient for the lysosomal proteinase cathepsin D exhibit progressive atrophy of the intestinal mucosa and profound destruction of lymphoid cells.

Mice deficient for the major lysosomal aspartic proteinase cathepsin D, generated by gene targeting, develop normally during the first 2 weeks, stop thriving in the third week and die in a state of anorexia at day 26 +/- 1. An atrophy of the ileal mucosa first observed in the third week progresses towards widespread intestinal necroses accompanied by thromboemboli. Thymus and spleen undergo massive destruction with fulminant loss of T and B cells. Lysosomal bulk proteolysis is maintained. These results suggest, that vital functions of cathepsin D are exerted by limited proteolysis of proteins regulating cell growth and/or tissue homeostasis, while its contribution to bulk proteolysis in lysosomes appears to be non-critical.

Radiolabeled metabolites of proteins play a critical role in radioactivity elimination from the liver

We have recently reported that the behavior of radiolabeled metabolites in the liver appears to be responsible for the hepatic radioactivity levels after administration of protein radiopharmaceuticals. To better understand the role played by radiolabeled metabolites in hepatic radioactivity levels, two benzyl-EDTA derivatives rendering different radiolabeled metabolites, 1-(4-isothiocyanatobenzyl) ethylenediaminetetraacetic acid (SCN-Bz-EDTA) and 1-[ p-(5-maleimidopentyl)aminobenzyl] ethylenediaminetetraacetic acid (ECMS-Bz-EDTA), were selected as bifunctional chelating agents (BCAs), and 111In labeling of galactosyl-neoglycoalbumin (NGA) and mannosyl-neoglycoalbumin (NMA) was performed. Biodistribution of radioactivity in mice and subcellular distribution of radioactivity in hepatocytes were then compared. After accumulation in hepatic parenchymal cells, NGA-EMCS-Bz-EDTA- 111 In rendered a faster elimination rate of radioactivity from the liver than NGA-SCN-Bz-EDTA- 111 In. Although each 111In-NMA exhibited a delayed elimination rate of radioactivity from the liver compared to the 111In-NGA counterpart, NMA-EMCS-Bz-EDTA- 111In showed faster elimination rate of radioactivity than NMA-SCN-Bz-EDTA- 111In. Analyses of radioactivity excreted in feces and urine and remaining in the liver indicated that both BCAs rendered mono-amino acid adducts as the major radiolabeled metabolites (cysteine-EMCS-Bz-EDTA- 111In and lysine-SCN-Bz-EDTA- 111In), which were generated in both cell types of the liver within 1 h postinjection. Subcellular distribution of radioactivity indicated that the radioactivity was copurified with lysosomes. These results demonstrate that although in vivo stability of radiometal chelates is essential, the biological properties of the radiolabeled metabolites generated after lysosomal proteolysis in hepatocytes play a critical role in radioactivity elimination from the liver.

Chloroquine does not exert insulin-like actions on human forearm muscle metabolism

Insulin's anabolic action on skeletal muscle and whole body protein is attributable to its action to slow tissue proteolysis. The antimalarial chloroquine inhibits lysosomal proteolysis and is reported to improve glycemia in poorly controlled diabetic patients. We infused chloroquine into the brachial artery of seven healthy postabsorptive volunteers over 3 h during a steady-state infusion of L-[ring-2,6-3H]phenylalanine (Phe) to study its effect on muscle glucose and protein turnover. Compared with basal, chloroquine increased forearm blood flow (P < 0.01) but did not change glucose uptake or lactate release. Neither Phe released from muscle by proteolysis (78 +/- 15 vs. 94 +/- 16 nmol Phe.min-1.100 ml-1) nor Phe balance (-37 +/- 7 vs. -50 +/- 6 nmol.min-1.100 ml-1) was reduced from basal. We conclude that in postabsorptive human skeletal muscle: 1) lysosomal proteolysis does not make a major contribution to proteolysis; and 2) chloroquine does not cause an acute increase in glucose uptake. These findings suggest that the inhibition of postabsorptive muscle protein degradation provoked by physiological increases in plasma insulin is likely mediated by a nonlysosomal proteolytic pathway.

The Structure of the Bovine Cathepsin B Gene

Cathepsin B is a cysteine proteinase which plays an important role in lysosomal proteolysis. We report here the characterization of a 15‐kbp genomic clone of the bovine cathepsin B gene. Bovine preprocathep‐sin B is encoded by nine exons from translational initiation to stop codon. The last exon is sandwiched between Alu‐like short interspersed nuclear elements. Alternate use of polyadenylation sites generates three transcripts encoding bovine cathepsin B. In all tissues tested, 3′ end cleavage was found to occur in equal proportion at the first and second polyadenylation site, producing transcripts of 2.6‐kb. Polyadenylation at the third polyadenylation site generates a 3.2‐kb mRNA, only expressed at low levels and in a developmental and tissue‐specific manner. Due to micro‐heterogeneities between genomic and cDNA clones, sequence polymorphism was investigated in the trailer region. DNA sequencing of PCR‐amplified genomic fragments revealed that, in contrast to the protein‐encoding region, genetic variability exists in the 3′ untranslated region. Polymorphism in the trailer was confirmed in cathepsin B mRNA by ribo‐nuclease‐protection assays. We finally emphasize that while exon‐exon boundaries in mature cathepsin B are well conserved from nematodes to mammals, exons also tend to correspond to discrete units of cathepsin B structure.

The Structure of the Bovine Cathepsin B Gene. Genetic variability in the 3' untranslated region

Cathepsin B is a cysteine proteinase which plays an important role in lysosomal proteolysis. We report here the characterization of a 15-kbp genomic clone of the bovine cathepsin B gene. Bovine preprocathepsin B is encoded by nine exons from translational initiation to stop codon. The last exon is sandwiched between Alu-like short interspersed nuclear elements. Alternate use of polyadenylation sites generates three transcripts encoding bovine cathepsin B. In all tissues tested, 3' end cleavage was found to occur in equal proportion at the first and second polyadenylation site, producing transcripts of 2.6-kb. Polyadenylation at the third polyadenylation site generates a 3.2-kb mRNA, only expressed at low levels and in a developmental and tissue-specific manner. Due to micro-heterogeneities between genomic and cDNA clones, sequence polymorphism was investigated in the trailer region. DNA sequencing of PCR-amplified genomic fragments revealed that, in contrast to the protein-encoding region, genetic variability exists in the 3' untranslated region. Polymorphism in the trailer was confirmed in cathepsin B mRNA by ribonuclease-protection assays. We finally emphasize that while exon-exon boundaries in mature cathepsin B are well conserved from nematodes to mammals, exons also tend to correspond to discrete units of cathepsin B structure.

Delivery of nascent MHC class II-invariant chain complexes to lysosomal compartments and proteolysis of invariant chain by cysteine proteases precedes peptide binding in B-lymphoblastoid cells.

The intracellular trafficking, proteolysis, and dissociation of invariant chain (li) associated with nascent class II molecules was examined in B-lymphoblastoid cells. Metabolic labeling and Percoll gradient centrifugation was used to assess the kinetics of delivery and processing of class II-li complexes within the endocytic pathway. Catabolism of class II-li complexes rapidly followed their delivery from post-Golgi compartments to dense lysosome-like compartments distinct from early and late endosomes. Direct peptide binding assays revealed that class II molecules associated with even small N-terminal fragments of li failed to bind peptide. Cysteine protease inhibitors alone blocked li proteolysis/dissociation and accumulation of class II-li biosynthetic intermediates within lysosome-containing compartments. Active-site labeling of cysteine proteases in B cells was used to identify cysteine proteases capable of mediating li proteolysis within endosomal compartments. Our results indicate rapid, possibly direct, transport of nascent class II-li complexes from the Golgi/trans-Golgi network to dense lysosomal compartments wherein cysteine protease(s), likely including cathepsin B, mediate complete removal of li. Inhibition of cysteine protease activity results in the accumulation of incompletely processed class II-li complexes, which lack peptide binding ability, within lysosomal compartments.

Lipofuscin fluorophore inhibits lysosomal protein degradation and may cause early stages of macular degeneration.

One of the autofluorescent compounds that accumulates within the lipofuscin granules of the human retinal pigment epithelium (RPE) has now been identified as a quaternary nitrogen-containing cationic amphiphile (the bis-retinoid pyridinium salt, A2-E). Experimental evidence suggests that it may be responsible for lipofuscinogenesis in the RPE through its ability to inhibit lysosomal proteolysis. Furthermore, it may be involved in the events that trigger the changes leading to age-related macular degeneration (AMD), the leading cause of untreatable blindness in the elderly. It is suggested that if similar weakly basic nitrogenous compounds or cationic amphiphiles arise in reactions between amines and aldehydes in other tissues, a "self-assembling lysosomotropic amine" mechanism may provide an alternative explanation for lipofuscinogenesis those cell types as well.

タンパク質代謝の栄養制御

Regulatory mechanisms of hepatic protein metabolism and gene expression mediated by amino acids and vitamin B6 were investigated. 1) Rats were nourished by total parenteral nutrition, and the effects of variations in the amino acid supply on the rates of hepatic protein synthesis and degradation were investigated. Amino acids decreased the rate of degradation whilst the rate of synthesis was relatively unaffected. Protein degradation, therefore, predominates the regulation of liver protein mass. 2) Studies on cystathionase and aspartate aminotransferase (AspAT) in the liver of vitamin B6-deficient rats showed that their rates of synthesis and degradation were both increased. The enhanced degradation was caused by different susceptibilities of apo- and holo-enzymes to lysosomal proteolysis. 3) Pyridoxal phosphate (PLP) was found to modulate AspAT gene expression by preventing the glucocorticoid receptor from binding to glucocorticoid-responsive elements. PLP also modulated albumin gene expression by inactivat ng the DNA-binding activity of tissue-specific transcriptional factors such as HNF-1 and C/EBP. 4) Amino acid vinfusion increased the albumin mRNA level and decreased the PLP concentration. The decrease in PLP concentration would prevent the inactivation of tissue-specific transcription factors and enhance albumin gene expression. The present study sheds new light on the physiological role of PLP as a mediator of gene regulation in protein/amino acid nutrition.

Sepsis stimulates nonlysosomal, energy-dependent proteolysis and increases ubiquitin mRNA levels in rat skeletal muscle.

We tested the role of different intracellular proteolytic pathways in sepsis-induced muscle proteolysis. Sepsis was induced in rats by cecal ligation and puncture; controls were sham operated. Total and myofibrillar proteolysis was determined in incubated extensor digitorum longus muscles as release of tyrosine and 3-methylhistidine, respectively. Lysosomal proteolysis was assessed by using the lysosomotropic agents NH4Cl, chloroquine, leupeptin, and methylamine. Ca(2+)-dependent proteolysis was determined in the absence or presence of Ca2+ or by blocking the Ca(2+)-dependent proteases calpain I and II. Energy-dependent proteolysis was determined in muscles depleted of ATP by 2-deoxyglucose and 2.4-dinitrophenol. Muscle ubiquitin mRNA and the concentrations of free and conjugated ubiquitin were determined by Northern and Western blots, respectively, to assess the role of the ATP-ubiquitin-dependent proteolytic pathway. Total and myofibrillar protein breakdown was increased during sepsis by 50 and 440%, respectively. Lysosomal and Ca(2+)-dependent proteolysis was similar in control and septic rats. In contrast, energy-dependent total and myofibrillar protein breakdown was increased by 172% and more than fourfold, respectively, in septic muscle. Ubiquitin mRNA was increased severalfold in septic muscle. The results suggest that the increase in muscle proteolysis during sepsis is due to an increase in nonlysosomal energy-dependent protein breakdown, which may involve the ubiquitin system.

Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules

Reagents that inhibit the ubiquitin-proteasome proteolytic pathway in cells have not been available. Peptide aldehydes that inhibit major peptidase activities of the 20S and 26S proteasomes are shown to reduce the degradation of protein and ubiquitinated protein substrates by 26S particles. Unlike inhibitors of lysosomal proteolysis, these compounds inhibit the degradation of not only abnormal and short-lived polypeptides but also long-lived proteins in intact cells. We used these agents to test the importance of the proteasome in antigen presentation. When ovalbumin is introduced into the cytosol of lymphoblasts, these inhibitors block the presentation on MHC class I molecules of an ovalbumin-derived peptide by preventing its proteolytic generation. By preventing peptide production from cell proteins, these inhibitors block the assembly of class I molecules. Therefore, the proteasome catalyzes the degradation of the vast majority of cell proteins and generates most peptides presented on MHC class I molecules.

Maleimidoethyl 3-(tri-n-butylstannyl)hippurate: a useful radioiodination reagent for protein radiopharmaceuticals to enhance target selective radioactivity localization.

In pursuit of radiolabeled monoclonal antibodies (mAbs) with rapid urinary excretion of radioactivity from nontarget tissues, radioiodinated mAbs releasing a m-iodohippuric acid from the mAbs in nontarget tissues were designed. A novel reagent, maleimidoethyl 3-(tri-n-butylstannyl)hippurate (MIH), was synthesized by reacting N-(hydroxyethyl)maleimide with N-Boc-glycine before coupling with N-succinimidyl 3-(tri-n-butylstannyl)benzoate (ATE). MIH possessed a maleimide group for mAb conjugation and a butylstannyl moiety for high-yield and site-specific radioiodination, and the two functional groups were linked via an ester bond to release m-iodohippuric acid. To investigate the fate of radiolabels after lysosomal proteolysis, hepatic parenchymal cells were used as a model nontarget tissue and 131I-labeled MIH was conjugated with galactosyl-neoglycoalbumin (NGA). Further conjugation of [131I]MIH with a mAb against osteogenic sarcoma (OST7) after reduction of its disulfide bonds was followed up. In murine biodistribution studies, [131I]MIH-NGA exhibited rapid accumulation in the liver followed by radioactivity elimination from the liver at a rate that was identical to and faster than those of 131I-labeled NGA via direct iodination ([131I]NGA) and [131I]ATE-labeled NGA, respectively. While [131I]NGA indicated high radioactivity levels in the murine neck, stomach, and blood, such increases in the radioactivity count were not detectable by the administration of either [131I]MIH-NGA or [131I]ATE-NGA. At 6 h postinjection of [131I]MIH-NGA, 80% of the injected radioactivity was recovered in the urine. Analyses of urine samples indicated that m-iodohippuric acid was the sole radiolabeled metabolite. In biodistribution studies using [131I]-MIH-OST7 and [131I]ATE-OST7, while both 131I-labeled OST7s registered almost identical radioactivity levels in the blood up to 6 h postinjection, the former demonstrated a lower radioactivity level than [131I]ATE-OST7 in nontarget tissues throughout the experiment. Such chemical and biological characteristics of MIH would enable high target/nontarget ratios in diagnostic and therapeutic nuclear medicine using mAbs and other polypeptides.

Transendothelial transport of modified low-density lipoproteins

Human umbilical vein endothelial cell monolayers were grown as monolayers on porous filters and their transcellular transport and degradation of 125I-labelled native and modified forms of LDL, supplied to either the intimal or the luminal face, were measured. Intact native, acetylated and oxidized LDL were all transported in both directions across the cell monolayers by receptor-independent mechanisms, and all forms of LDL were transported at similar rates. However, the mass of intact LDL transported from the intimal to the luminal face of the monolayer was always four-fold more than that transported in the opposite direction under similar conditions. In addition to LDL transport, endothelial cell monolayers also degraded native and modified forms of LDL by predominantly receptor-dependent routes, in that these could be inhibited ( > 70%) by the addition of a 20-fold excess of the same form of (but unlabelled) LDL. The measured amounts of lipoprotein degraded were the same whether supplied to the intimal or the luminal face. Incubation of endothelial cells with oxidized LDL led to intracellular accumulation of a pool of macromolecular apo B which was apparently resistant to lysosomal proteolysis.

Amino acid requirements in the early post-implantation rat conceptus

Amino acid analysis has been performed on hydrolysates of embryos/fetuses, visceral yolk sacs and ectoplacental cones/placentae from early post-implantation rat conceptuses. The increments in each amino acid between 10.5 and 11.5 days, between 11.5 and 12.5 days, and between 12.5 and 13.5 days, are expressed as percentages of the total amino acid increment. These three profiles are very similar to each other and also strongly resemble the amino acid composition of hydrolysates of rat serum. The results are discussed in the context of the hypothesis that a transudate of plasma is responsible for the amino acid nutrition of the embryo at this stage of gestation, and that inhibition of this pathway can lead to the induction of congenital defects. The results suggest that an inhibition of pinocytosis or lysosomal proteolysis would affect the supply of all protein-derived amino acids to approximately the same extent: there is no indication that the supply of any particular amino acid would be particularly vulnerable.