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Albumin, the most abundant protein in human serum, is applied to various diseases as a drug delivery carrier because of its superior blood retention, high biocompatibility, and a wide variety of drug binding abilities. Albumin is known to distribute widely in the blood and various interstitial fluids and organs. Different albumin receptors skillfully regulate the distribution characteristics of albumin in the body. Albumin receptors are a group of diverse proteins, such as FcRn, gp60, gp18, megalin, cubilin, SPARC, and CD36. Their tissue distributions in vivo are unique, with different albumin's recognition sites. Therefore, the distribution of albumin in vivo is ingeniously controlled by these multiple albumin receptors. Reevaluation of these albumin receptors opens up new possibilities for applying albumin as a drug delivery carrier. If the tissue distributions of albumin receptors were known and the albumin recognition site of the receptor was identified, organ-specific active targeting would be possible. In this review, we would like to scrutinize what is currently known and share information to develop next-generation albumin carriers that focus on interactions with albumin receptors.

Haemonchus contortus (H. contortus) has evolved sophisticated evasion mechanisms to ensure their survival, including generating excretion and secretion products (ESPs) to regulate the secretion of host cytokines. Interleukin 4 (IL4) is a classic T-helper cell type 2 (Th2)-type cytokine that plays an irreplaceable role against nematode infection. In this study, three proteins, glutathione S-transferase domain containing protein (HcGST), transthyretin domain containing protein (HcTTR) and calponin actin-binding domain containing protein (HcCab), were identified to bind to goat IL4 by co-immunoprecipitation (Co-IP) assays and yeast two-hybrid screening. Additionally, cell proliferation analysis showed that HcTTR blocked the IL4-induced proliferation of peripheral blood mononuclear cells in goats, while HcGST and HcCab did not. In addition, HcTTR could also downregulate the transcription of candidate genes in the IL4-induced JAK/STAT pathway. These results indicated that HcTTR is a novel antagonist against goat IL4 from HcESPs, and this information could improve our understanding of the relationship between host cytokines and parasite infections. Electronic supplementary material The online version of this article (10.1186/s13567-019-0661-z) contains supplementary material, which is available to authorized users.

Lipid-like nanoparticles (LNPs) have potential as non-viral delivery systems for mRNA therapies. However, repeated administrations of LNPs may lead to accumulation of delivery materials and associated toxicity. To address this challenge, we have developed biodegradable lipids which improve LNPs clearance and reduce toxicity. We modify the backbone structure of Dlin-MC3-DMA by introducing alkyne and ester groups into the lipid tails. We evaluate the performance of these lipids when co-formulated with other amine containing lipid-like materials. We demonstrate that these formulations synergistically facilitate robust mRNA delivery with improved tolerability after single and repeated administrations. We further identify albumin-associated macropinocytosis and endocytosis as an ApoE-independent LNP cellular uptake pathway in the liver. Separately, the inclusion of alkyne lipids significantly increases membrane fusion to enhance mRNA release, leading to synergistic improvement of mRNA delivery. We believe that the rational design of LNPs with multiple amine-lipids increases the material space for mRNA delivery., Lipid-like nanoparticles have applications as non-viral delivery systems for mRNA. Here, the authors develop biodegradable lipids with improved clearance and reduced toxicity.

Background/Aims: Liver progenitor cells (LPCs) were considered as a promising hepatocyte source of cell therapy for liver disease due to their self-renewal and differentiation capacities, while little is known about the mechanism of LPC differentiate into hepatocytes. This study aims to explore the effect of miR-382, a member of Dlk1-Dio3 microRNA cluster, during hepatic differentiation from LPCs. Methods: In this study, we used rat liver progenitor cell WB-F344 as LPC cell model and HGF as inducer to simulate the process of LPCs hepatic differentiation, then microRNAs were quantified by qPCR. Next, WB-F344 cell was transfected with miR-382 mimics, then hepatocyte cell trait was characterized by multiple experiments, including that periodic acid schiff staining and cellular uptake and excretion of indocyanine green to evaluate the hepatocellular function, qPCR and Western Blotting analysis to detect the hepatocyte-specific markers (ALB, Ttr, Apo E and AFP) and transmission electron microscopy to observe the hepatocellular morphology. Moreover, Luciferase reporter assay was used to determine whether Ezh2 is the direct target of miR-382. Results: We found that miR-382 increased gradually and was inversely correlated with the potential target, Ezh2, during WB-F344 hepatic differentiation. In addition, functional studies indicated that miR-382 increased the level of hepatocyte-specific genes. Conclusions: This study demonstrates that miR-382 may be a novel regulator of LPCs differentiation by targeting Ezh2.

Thyroid hormones (THs) are essential for the correct development of nearly every structure in the body from the very early stages of development, yet the embryonic thyroid gland is not functional at these stages. To clarify the roles of the egg yolk as a source of THs, the TH content in the yolk and the expression of TH regulator genes in the yolk sac membrane were evaluated throughout the 21-day incubation period of chicken embryos. The yolk TH content (22.3 ng triiodothyronine and 654.7 ng thyroxine per total yolk on day 4 of incubation) decreased almost linearly along with development. Real-time PCR revealed gene expression of transthyretin, a principal TH distributor in the chicken, and of a TH-inactivating iodothyronine deiodinase (DIO3), until the second week of incubation when the embryonic pituitary-thyroid axis is generally thought to start functioning. The TH-activating deiodinase (DIO2) and transmembrane transporter of thyroxine (SLCO1C1) genes were expressed in the last week of incubation, which coincided with a marked increase of circulating thyroxine and a reduction in the yolk sac weight. DIO1, which can remove iodine from inactive THs, was expressed throughout the incubation period. It is assumed that the chicken yolk sac inactivates THs contained abundantly in the yolk and supplies the hormones to the developing embryo in appropriate concentrations until the second week of incubation, while THs may be activated in the yolk sac membrane in the last week of incubation. Additionally, the yolk sac could serve as a source of iodine for the embryo. ispartof: The Journal of Reproduction and Development vol:63 issue:5 pages:463-472 ispartof: location:Japan status: published

Albumin is the most abundant protein in blood and acts as a molecular taxi for a plethora of small insoluble substances such as nutrients, hormones, metals and toxins. In addition, it binds a range of medical drugs. It has an unusually long serum half-life of almost 3 weeks, and although the structure and function of albumin has been studied for decades, a biological explanation for the long half-life has been lacking. Now, recent research has unravelled that albumin-binding cellular receptors play key roles in the homeostatic regulation of albumin. Here, we review our current understanding of albumin homeostasis with a particular focus on the impact of the cellular receptors, namely the neonatal Fc receptor (FcRn) and the cubilin–megalin complex, and we discuss their importance on uses of albumin in drug delivery.

Albumin re-uptake is a receptor-mediated pathway located in renal proximal tubuli. There is increasing evidence of glomerular protein handling by podocytes, but little is known about the mechanism behind this process. In this study, we found that human podocytes in vitro are committed to internalizing albumin through a receptor-mediated mechanism even after exposure to low doses of albumin. We show that these cells express cubilin, megalin, ClC-5, amnionless and Dab2, which are partners in the tubular machinery. Exposing human podocytes to albumin overload prompted an increase in CUBILIN, AMNIONLESS and CLCN5 gene expression. Inhibiting cubilin led to a reduction in albumin uptake, highlighting its importance in this mechanism. We demonstrated that human podocytes are committed to performing endocytosis via a receptor-mediated mechanism even in the presence of low doses of albumin. We also disclosed that protein overload first acts on the expression of the cubilin-amnionless (CUBAM) complex in these cells, then involves the ClC-5 channel, providing the first evidence for a possible role of the CUBAM complex in albumin endocytosis in human podocytes.

Transthyretin familial amyloid polyneuropathy is a rare, autosomal-dominant inherited multisystem disorder usually manifesting with a rapidly progressive, axonal, distally-symmetric polyneuropathy. The detection of nerve injury by nerve conduction studies is limited, due to preferential involvement of small-fibres in early stages. We investigated whether lower limb nerve-injury can be detected, localized and quantified in vivo by high-resolution magnetic resonance neurography. We prospectively included 20 patients (12 male and eight female patients, mean age 47.9 years, range 26–66) with confirmed mutation in the transthyretin gene: 13 with symptomatic polyneuropathy and seven asymptomatic gene carriers. A large age- and sex-matched cohort of healthy volunteers served as controls (20 male and 20 female, mean age 48.1 years, range 30–73). All patients received detailed neurological and electrophysiological examinations and were scored using the Neuropathy Impairment Score–Lower Limbs, Neuropathy Deficit and Neuropathy Symptom Score. Magnetic resonance neurography (3 T) was performed with large longitudinal coverage from proximal thigh to ankle-level and separately for each leg (140 axial slices/leg) by using axial T2-weighted (repetition time/ echo time = 5970/55 ms) and dual echo (repetition time 5210 ms, echo times 12 and 73 ms) turbo spin echo 2D sequences with spectral fat saturation. A 3D T2-weighted inversion-recovery sequence (repetition time/echo time 3000/202 ms) was acquired for imaging of the spinal nerves and lumbar plexus (50 axial slice reformations). Precise manual segmentation of the spinal/sciatic/ tibial/common peroneal nerves was performed on each slice. Histogram-based normalization of nerve–voxel signal intensities was performed using the age- and sex-matched control group as normative reference. Nerve-voxels were subsequently classified as lesion-voxels if a threshold of 41.2 (normalized signal-intensity) was exceeded. At distal thigh level, where a predominant nerve– lesion–voxel burden was observed, signal quantification was performed by calculating proton spin density and T2-relaxation time as microstructural markers of nerve tissue integrity. The total number of nerve–lesion voxels (cumulated from proximal-to-distal) was significantly higher in symptomatic patients (20 405 � 1586) versus asymptomatic gene carriers (12 294 � 3199; P= 0.036) and versus controls (6536 � 467; P5 0.0001). It was also higher in asymptomatic carriers compared to controls ( P= 0.043). The number of nerve–lesion voxels was significantly higher at thigh level compared to more distal levels (lower leg/ankle) of the lower extremities (f-value = 279.22, P5 0.0001). Further signal-quantification at this proximal site (thigh level) revealed a significant increase of proton-density (P5 0.0001) and T2-relaxation-time ( P= 0.0011) in symptomatic patients, whereas asymptomatic genecarriers presented with a significant increase of proton-density only. Lower limb nerve injury could be detected and quantified in vivo on microstructural level by magnetic resonance neurography in symptomatic familial amyloid polyneuropathy, and also in yet asymptomatic gene carriers, in whom imaging detection precedes clinical and electrophysiological manifestation. Although symptoms start and prevail distally, the focus of predominant nerve injury and injury progression was found proximally at thigh level with strong and unambiguous lesion-contrast. Imaging of proximal nerve lesions, which are difficult to detect by nerve conduction studies, may have future implications also for other distally-symmetric polyneuropathies.

To report the unique clinical and surgical characteristics encountered in eyes with vitreous amyloidosis. Systemic evaluation and visual outcome after vitrectomy are discussed. A novel mutation in the transthyretin gene (TTR) in Indian patients with familial amyloid polyneuropathy (FAP) is described.Retrospective, observational study.Ten eyes of 5 patients from 2 pedigrees with a diagnosis of vitreous amyloidosis.Detailed history, pedigree charting, systemic and ocular examination of 10 eyes (5 patients from 2 pedigrees) were carried out. Tests were performed to rule out vitreitis, retinal vasculitis, vitreous hemorrhage, and systemic amyloidosis. Genetic analysis to identify the mutation was performed in 1 patient. Vitreous biopsy, followed by 25-gauge pars plana vitrectomy, was performed in the same sitting in all cases. Samples were sent for Congo red staining and polarized microscopy. Patients were followed up on days 1, 7, and 28 and then every 2 months. Visual acuity assessment, intraocular pressure measurement, and fundus examination were performed each time.Mutations in TTR and postoperative visual acuity.Mean age at presentation was 32 years, with a 3:2 male-to-female distribution. Family history was positive in all patients. Nine eyes had pseudopodia lentis, whereas all 10 had glass wool-like vitreous. Glaucoma developed in 1 patient (2 eyes). Waxy paper-like vitreous with firm vitreous adhesions beyond major arcades and along retinal vessels was noted during surgery in all eyes. Congo red staining and apple green birefringence demonstrated vitreous amyloidosis. The mean preoperative best-corrected visual acuity (BCVA) was 1.39±0.64 logarithm of the minimum angle of resolution (logMAR), whereas the postoperative BCVA improved to 0.17±0.07 logMAR (P = 0.004). Gene sequencing revealed a phenylalanine→isoleucine mutation in the 33rd position of exon 2 of TTR in 1 patient of 1 pedigree, confirming the diagnosis of FAP. Two patients subsequently were found to have sensorimotor autonomic neuropathy, whereas 2 others had subclinical autonomic dysfunction.The clinical clues, management strategy, surgical characteristics, vitrectomy outcomes, and significance of systemic evaluation in vitreous amyloidosis are highlighted. A novel single mutation (Phe33Ile) in a case of FAP with vitreous amyloidosis from India is reported.

Transthyretin Familial Amyloid Polyneuropathy (TTR-FAP) is a rare disease with autosomal dominant transmission due to a point mutation of the TTR gene. By removing the main source of systemic mutant TTR, liver transplantation (LT) has become the reference therapy of this severe and fatal polyneuropathy of adult-onset, stopping disease progression in subgroup of patients. Recently, new therapeutic strategies have emerged, which intend to stabilize TTR or to silence the TTR gene. Amongst them, the TTR kinetic stabilizer tafamidis is the first drug approved in the EU.We shall review the natural history of TTR-FAP and the best indications for LT. Data on the efficacy, safety and tolerability of the TTR kinetic stabilizers, tafamidis and diflunisal, have been reviewed, from the pivotal Phase III clinical trials published in PubMed medical journals or presented at international meetings. We will review the ongoing phase III clinical trials of TTR gene silencing with RNAi therapeutics and ASO published in clinicaltrialgov.Due to the data on efficacy, tolerability, safety, tafamidis and diflunisal became the first line anti-amyloid treatment in stage 1 TTR-FAP. Both drugs slow progression of the disease. Only tafamidis got marketing authorization. We are waiting for results of the 2 phase III clinical trials of TTR gene silencing in varied stages of the disease.

A new type of surface receptor for serum albumin was detected in strains of Streptococcus pyogenes (group A). This receptor, called type e, was different from albumin receptors in other streptococcal species. Only mouse and human serum albumin was bound to the receptor. The albumin-binding capacity was high: 2 X 10(8) bacterial organisms bound 11 micrograms of mouse albumin. The receptor was stable even when treated at 100 degrees C for 5 min. Binding of albumin was not mediated by lipoteichoic acid (LTA) because of lack of correlation to surface LTA, restricted albumin reactivity, and positive binding in presence of 2% Tween 20. Presence of albumin receptor type e correlated to presence of M-protein as measured by growth in the bactericidal test. All 51 M-protein positive group A streptococcal strains tested could bind mouse albumin whereas only 3 out of 8 M-protein negative strains showed positive binding (P less than 0.001). The sensitivity to trypsin digestion suggests that the albumin receptor is of protein nature or mediated by a protein.

Recent studies of hepatitis B virus suggest that polymeric human serum albumin may facilitate the attachment of the virus via albumin receptors to hepatocytes during the infectious process. If this hypothesis is correct, hepatocytes should express binding sites for polymeric albumin. We employed the red blood cell adherence test using albumin-coated red blood cells as indicator cells on frozen sections of normal human livers to demonstrate these binding sites. Hepatocytes showed binding activity for both polymeric and monomeric albumin from different species. The receptor-ligand interaction was temperature and pH dependent, Ca++ independent and not altered by mercaptoethanol treatment. The binding activity was sensitive to neuraminidase and pronase, but resistant to trypsin, lipase and collagenase digestion. These findings suggest that human hepatocytes display species-non-specific albumin binding sites, which are glycoproteins.

Proteinuria is associated with progressive chronic kidney disease and poor cardiovascular outcomes. Exposure of proximal tubular epithelial cells to excess proteins leads to the development of proteinuric nephropathy with tubular atrophy, interstitial inflammation and scarring. Numerous signalling pathways are activated in proximal tubular epithelial cells under proteinuric conditions resulting in gene transcription, altered growth and the secretion of inflammatory and profibrotic mediators. Megalin, the proximal tubular scavenger receptor for filtered macromolecules, has intrinsic signalling functions and may also link albumin to growth factor receptor signalling via regulated intramembrane proteolysis. It now seems that endocytosis is not always a prerequisite for albumin-evoked alterations in proximal tubular cell phenotype. Recent evidence shows the presence of other potential receptors for proteins, such as the neonatal Fc receptor and CD36, in the proximal tubular epithelium.

Similar to many proteins trafficking through the secretory pathway, cellular prion protein (PrP) partly retrotranslocates from the endoplasmic reticulum to the cytosol through the endoplasmic reticulum-associated degradation (ERAD) pathway in an attempt to alleviate accumulation of cellular misfolded PrP. Surprisingly, familial PrP mutants fail to retrotranslocate and simultaneously block normal cellular PrP retrotranslocation. That impairments in retrotranslocation of misfolded proteins could lead to global disruptions in cellular homeostasis prompted further investigations into PrP mutant retrotranslocation defects. A gain- and loss-of-function approach identified human E3 ubiquitin ligase, Hrd1, as a critical regulator of PrP retrotranslocation in mammalian cells. Expression of familial human PrP mutants, V210I(129V) and M232R(129V), not only abolished PrP retrotranslocation, but also that of Hrd1-dependent ERAD substrates, transthyretin TTR(D18G) and α1-anti-trypsin A1AT(NHK). Mutant PrP expression decreased binding immunoglobulin protein (BiP) levels by 50% and attenuated ER stress-induced BiP by increasing BiP turnover 6-fold. Overexpression of BiP with PrP mutants rescued retrotranslocation of PrP, TTR(D18G) and A1AT(NHK). PrP mutants-induced cell death was also rescued by co-expression of BiP. These results show that PrP mutants highjack the Hrd1-dependent ERAD pathway, an action that would result in misfolded protein accumulation especially in terminally differentiated neurons. This could explain the age-dependent neuronal degeneration in familial prion diseases.

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant protein-folding disorder caused by over 100 distinct mutations in the transthyretin (TTR) gene. In ATTR, protein secreted from the liver aggregates and forms fibrils in target organs, chiefly the heart and peripheral nervous system, highlighting the need for a model capable of recapitulating the multisystem complexity of this clinically variable disease. Here, we describe detailed methodologies for the directed differentiation of protein folding disease-specific iPSCs into hepatocytes that produce mutant protein, and neural-lineage cells often targeted in disease. Methodologies are also described for the construction of multisystem models and drug screening using iPSCs.

The monophyletic group Caniformia in the order Carnivora currently comprises seven families whose relationships remain contentious. The phylogenetic positions of the two panda species within the Caniformia have also been evolutionary puzzles over the past decades, especially for Ailurus fulgens (the red panda). Here, new nuclear sequences from two introns of the beta-fibrinogen gene (beta-fibrinogen introns 4 and 7) and a complete mitochondrial (mt) gene (ND2) from 17 caniform representatives were explored for their utilities in resolving higher-level relationships in the Caniformia. In addition, two previously available nuclear (IRBP exon 1 and TTR intron 1) data sets were also combined and analyzed simultaneously with the newly obtained sequence data in this study. Combined analyses of four nuclear and one mt genes (4417 bp) recover a branching order in which almost all nodes were strongly supported. The present analyses provide evidence in favor of Ailurus fulgens as the closest taxon to the procyonid-mustelid (i.e., Musteloidea sensu stricto) clade, followed by pinnipeds (i.e., Otariidae and Phocidae), Ursidae (including Ailuropoda melanoleuca), and Canidae, the most basal lineage in the Caniformia. The potential utilities of different genes in the context of caniform phylogeny were also evaluated, with special attention to the previously unexplored beta-fibrinogen intron 4 and 7 genes.

Albumin is the most abundant plasmaprotein serving multiple functions as a carrier of metabolites, hormones, vitamins, and drugs, as an acid/base buffer, as antioxidant and by supporting the oncotic pressure and volume of the blood. The presence of albumin in urine is considered to be the result of the balance between glomerular filtration and tubular reabsorption. Albuminuria has been accepted as an independent risk factor and a marker for renal as well as cardiovascular disease, and during the past decade, evidence has suggested that albumin itself may cause progression of renal disease. Thus, the reduction of proteinuria and, in particular, albuminuria has become a target in itself to prevent deterioration of renal function. Studies have shown albumin and its ligands to induce expression of inflammatory and fibrogenic mediators, and it has been hypothesized that increased filtration of albumin causes excessive tubular reabsorption, resulting in inflammation and fibrosis, resulting in the loss of renal function. In addition, it is known that tubular dysfunction in itself may cause albuminuria owing to decreased reabsorption of filtered albumin, and, recently, it has been suggested that significant amounts of albumin fragments are excreted in the urine as a result of tubular degradation. Thus, although both tubular and glomerular dysfunction influences renal handling of albumin, it appears that tubular reabsorption plays a central role in mediating the effects of albumin on renal function. The present paper will review the mechanisms for tubular albumin uptake and the possible implications for the development of renal disease.

This review summarizes evidence on the effect of excess circulating glucose concentration and AGE-albumin on the aortic endothelial cells (ECs) phenotype, transport function, and expression of signalling molecules. The recent reports on the ECs dysfunction in diabetes are briefly reviewed, to provide a broader view on the link between ECs structural changes, functional alterations, and the underlying biochemical mechanisms. The original results emerging from streptozotocin-injected mice and human aortic endothelial cells grown in high (25 mM) glucose concentration are presented. Compared to physiological condition, in diabetes aortic ECs switch to a biosynthetic phenotype, present an increased number of caveolae, and enhance (by approximately 20%) transcytosis of AGE-albumin (AGE-Alb). In cultured ECs, 25 mM glucose induces approximately 2.6 fold increase in pSTAT-3 and pERK1 and approximately 1.8 fold increase in pERK2; further exposure to 5 microM AGE-Alb causes approximately 4.3 fold increase in pERK1/2 (vs. 5 mM glucose). Together, these data may explain the phenotypic change, enhanced permeability, and proliferation of aortic ECs in diabetic conditions.

ClC-5 is a chloride (Cl(-)) channel expressed in renal tubules and is critical for normal tubular function. Loss of function nonsense or missense mutations in ClC-5 are associated with Dent's disease, a condition in which patients present with low molecular weight (LMW) proteinuria (including albuminuria), hypercalciuria and nephrolithiasis. Several key studies in ClC-5 knockout mice have shown that the proteinuria results from defective tubular reabsorption of proteins. ClC-5 is typically regarded as an intracellular Cl(-) channel and thus the defect in this receptor-mediated uptake pathway was initially attributed to the failure of the early endosomes to acidify correctly. ClC-5 was postulated to play a key role in transporting the Cl(-) ions required to compensate for the movement of H(+) during endosomal acidification. However, more recent studies suggest additional roles for ClC-5 in the endocytosis of albumin. ClC-5 is now known to be expressed at low levels at the cell surface and appears to be a key component in the assembly of the macromolecular complex involved in protein endocytosis. Furthermore, mutations in ClC-5 affect the trafficking of v-H(+)-ATPase and result in decreased expression of the albumin receptor megalin/cubulin. Thus, the expression of ClC-5 at the cell surface as well as its presence in endosomes appears to be essential for normal protein uptake by the renal proximal tubule.

Long-chain fatty acids (FAs) are important substrates used by the heart to fulfil its energy requirements. Prior to mitochondrial oxidation, blood-borne FAs must pass through the cell membrane of the cardiac myocyte (sarcolemma). The mechanism underlying the sarcolemmal transport of FAs is incompletely understood. The aim of the present study was to estimate the trans-sarcolemmal FA uptake rate using a comprehensive computer model, in which the most relevant mechanisms proposed for cardiac FA uptake were incorporated. Our in silico findings show that diffusion of FA, present in its unbound form (uFA) in close proximity to the outer leaflet of the sarcolemma and serving as sole FA source, is insufficient to account for the physiological FA uptake rate. The inclusion of a hypothetical membrane-associated FA-TFPC (FA-transport-facilitating protein complex) in the model calculations substantially increased the FA uptake rate across the sarcolemma. The model requires that the biological properties of the FA-TFPC allow for increasing the rate of absorption of FA into the outer leaflet and the ‘flip-flop’ rate of FA from the outer to the inner leaflet of the sarcolemma. Experimental studies have identified various sarcolemma-associated proteins promoting cardiac FA uptake. It remains to be established whether these proteins possess the properties predicted by our model. Our findings also indicate that albumin receptors located on the outer leaflet of the sarcolemma facilitate the transfer of FA across the membrane to a significant extent. The outcomes of the computer simulations were verified with physiologically relevant FA uptake rates as assessed in the intact, beating heart in experimental studies.

We examined possible molecular mechanisms for the low-temperature arrest of T3-induced Rana catesbeiana metamorphosis. Scatchard plots revealed that the ratios of maximum binding capacity/dissociation constant for high-affinity sites of tadpole serum proteins for T3 at 20 and 28 C was 3.3-4.6 times less than that at 4 C, due to the decrease in maximum binding capacity values. Kinetic studies of T3 uptake into tadpole red blood cells demonstrated that the ratio of maximum uptake rate/Michaelis constant at 23 C was approximately 13 times greater than that at 4 C. The process of intracellular transport of T3 into the nucleus was not arrested at 4 C. The ratio of T3 incorporated into nuclei to that taken up into red blood cells was not significantly different at 4, 20, and 28 C, indicating the absence of temperature-sensitive sites in this process. T3 binding to the T3 receptors alpha and beta were not temperature sensitive at least at 4 and 20 C. Transcription of the tr genes, early primary T3 response genes, was activated by 10 nM T3 at 20 and 28 C but was barely detected at 4 C. These results indicate that the major molecular event causing the low-temperature arrest of amphibian metamorphosis occurs after T3 entry into the nucleus but before or during the transcriptional activation of the tr genes. Plasma proteins binding T3 and the cellular thyroid hormone uptake system on the plasma membrane may contribute to the slowing of the incorporation of T3 into nucleus at 4 C by decreasing the uptake velocity of T3.

To observe and compare the effects of Hanfangji Compound and IFN-gamma on expressions of transthyretin (TTR) , inter-alpha inhibitor H1 (ITIH1) and serpin peptidase inhibitor clade F member 2 (SERPINF2) of hepatic stellate cells (HSC-T6).Hanfangji Compound and IFN-gammaof different concentrations were used in hepatic stellate cell-T6 (HSC-T6) for 48 h. Flow cytometer was used to detect the effects of Hanfangji Compound and IFN-gamma on HSC proliferation. RT-PCR method was adopted to detect mRNA expressions of TFR, ITIH1 and SERPINF2. TTR, ITIH1 and SERPINF2 secretions were detected by ELISA. The protein localizations of TTR, ITIH1 and SERPINF2 were examined by immune fluorescence. The protein expression of TfR and ITIHI were determined by Western blot.After Hanfangji Compound and IFN-gamma were adopted in HSC-T6, compared with the control group, the cell proliferation was inhibited obviously (P0. 05) , protein expressions of TTR, ITIH1 and SERPINF2 and mRNA expression increased significantly, with certain correlation with concentrations of Hanfangji Compound. The 2. 5 g L-I Hanfangji Compound group was superior to the IFN-gamma group (P0. 05).Hanfangji Compound can inhibit HSC proliferation, upregulated TTR, ITIH1 and SERPINF2 proteins and mRNA expression, which may be one of mechanisms of anti-hepatic fibrosis of Hanfangji Compound.

Transthyretin (TTR) is involved in the transport of thyroxine (T4) and retinol-binding protein (RBP) in cerebrospinal fluid (CSF) and serum. TTR is secreted in the CSF by the epithelial cells of choroid plexus. The binding of [(125)I]TTR to cultured ependymoma cells which form the brain cerebrospinal barrier, was studied to determine whether these cells carry receptor(s) for TTR. TTR was bound by ependymoma cells in a time-dependent manner reaching equilibrium within 2 h. Scatchard analysis was consistent with a single class of high-affinity binding sites with a K(d) of approximately 18 nM. Saturable high-affinity binding of human TTR has previously been described in rat primary hepatocytes and human renal adenocarcinoma, neuroblastoma, hepatoma and astrocytoma cells, and also transformed lung cells. Endocytosis of fluorescent or biotinylated TTR was observed in ependymoma cells in cytoplasmic vesicles but TTR did not colocalize with clathrin in endocytic coated vesicles. Endocytosis of TTR was inhibited by high sucrose concentration (0.45 M). Finally, ligand blotting and chemical-linking experiments revealed the presence of a approximately 100 kDa putative TTR receptor on the ependymoma cell membrane. Receptor binding of TTR provides a potential mechanism for the delivery of T4 within the central nervous system.

Cholangiocarcinoma (CCA) is the second most common-primary liver cancer. The difficulties in diagnosis limit successful treatment of CCA. At present, histological investigation is the standard diagnosis for CCA. However, there are some poor-defined tumor tissues which cannot be definitively diagnosed by general histopathology. As molecular signatures can define molecular phenotypes related to diagnosis, prognosis, or treatment outcome, and CCA is the second most common cancer found after hepatocellularcarcinoma (HCC), the aim of this study was to develop a predictive model which differentiates CCA from HCC and normal liver tissues. An in-house PCR array containing 176 putative CCA marker genes was tested with the training set tissues of 20 CCA and 10 HCC cases. The molecular signature of CCA revealed the prominent expression of genes involved in cell adhesion and cell movement, whereas HCC showed elevated expression of genes related to cell proliferation/differentiation and metabolisms. A total of 69 genes differentially expressed in CCA and HCC were optimized statistically to formulate a diagnostic equation which distinguished CCA cases from HCC cases. Finally, a four-gene diagnostic equation (CLDN4, HOXB7, TMSB4 and TTR) was formulated and then successfully validated using real-time PCR in an independent testing set of 68 CCA samples and 77 non-CCA controls. Discrimination analysis showed that a combination of these genes could be used as a diagnostic marker for CCA with better diagnostic parameters with high sensitivity and specificity than using a single gene marker or the usual serum markers (CA19-9 and CEA). This new combination marker may help physicians to identify CCA in liver tissues when the histopathology is uncertain.

To evaluate the predictive and prognostic value of serum and plasma tumor markers, in comparison with clinical and biomedical parameters for response rate (RR), progression-free survival (PFS) and overall survival (OS) among patients with metastatic colorectal cancer (mCRC) treated with combination chemotherapy.One-hundred and six patients with mCRC from three centers, part of a multicenter study, received irinotecan with the Nordic bolus 5-fluorouracil (5-FU) and folinic acid schedule (FLIRI) or the de Gramont schedule (Lv5FU2-IRI). Blood samples for CEA, CA19-9, TPA, TIMP-1, SAA, transthyretin and CRP were taken at baseline and after two, four and eight weeks of treatment. Tumor marker levels at baseline and longitudinally were compared with responses evaluated (CT/MRI) after two and four months of treatment. The correlations to RR, PFS and OS were evaluated with regression analyses.A significant correlation to OS was seen for baseline levels of all markers. In multivariate analyses with clinical parameters, TPA, CRP, SAA and TIMP-1 provided independent information. The baseline values of CEA, TPA and TIMP-1 were also significantly correlated to PFS and TPA to RR. Changes during treatment, i.e. the slope gave with the exception of CA19-9 for OS less information about outcomes. The best correlation to response was seen for CEA, CA19-9 and TPA with AUC values of 0.78, 0.83 and 0.79, respectively, using a combined model based upon an interaction between the slope and the baseline value.Baseline tumor markers together with clinical parameters provide prognostic information about survival in patients with mCRC. The ability of the individual tumor markers to predict treatment response and PFS is limited. Changes in marker levels during the first two months of treatment are less informative of outcome.

Three types of bovine serum albumin (BSA) derivatives such as lactosylated BSA (LBSA), mannosylated BSA (Man-BSA), and cationized BSA (cBSA) were synthesized and their hepatic disposition characteristics in mice were evaluated by pharmacokinetic analysis. At lower doses (or = 1 mg/kg), LBSA and Man-BSA were very rapidly eliminated from the blood circulation due to uptake by parenchymal and nonparenchymal cells of the liver, respectively, via receptor-mediated endocytosis (Nishikawa et al., 1992; Nishida et al., 1991a, b). These uptake processes were nonlinear and the apparent hepatic uptake clearances (CLliver) were decreased at administered doses higher than 1 mg/kg, e.g. 10, 20, and 100 mg/kg. The liver accumulation of cBSA was also nonlinear, but its binding and/or uptake capacity in the liver was larger than those of LBSA and Man-BSA; i.e., CLliver decreased at doses higher than 20 mg/kg. In the next step, we modified these BSA derivatives by attaching polyethylene glycol (PEG), a modifier known to reduce the hepatic uptake and increase plasma retention, to achieve precise control of the in vivo disposition characteristics of BSA derivatives. By conjugation with PEG having a molecular weight of 10 kDa, the CLliver values of LBSA, Man-BSA, and cBSA were decreasing to one-seventh, one-fortyfifth, and one-onehundredthirtieth, respectively. However, liver accumulation of PEG modified LBSA and Man-BSA at 24 h after i.v. injection was not significantly different from unmodified BSA derivatives. These results suggest that it is possible to control the hepatic uptake of protein drugs by a combination of introduction of charge or sugar moieties and PEG conjugation.

SUMMARY: Members of the Streptococcus milleri group (SMG) that react with Lancefield group C antisera were shown to bind large amounts of albumin although there was no direct relation between these two properties as polyclonal antisera to Lancefield group C antigen did not prevent the binding of albumin. There was a specificity for albumin binding, with albumin from man, monkeys, cat, dog and mouse being bound to a greater degree than albumin from cow, horse, goat or rabbit. Gold-labelled albumin was shown to be located close to the surface of strains by transmission electron microscopy. A cell-surface protein of Mr 24000, which was liberated by Iysozyme treatment of cells, was shown to be the cell-surface receptor on Streptococcus intermedius C5. The receptor was physically dissimilar from protein G, an albumin- and IgG-binding protein of ‘large-colony’ Lancefield group C and G streptococci.

We investigated whether cardiomyocytes express specific albumin binding proteins (ABP) which may function in the dissociation of fatty acids from their non-covalent complexes with albumin. The experiments were performed on rat neonatal cardiomyocytes (freshly isolated and up to 3 days in culture) and on an enriched sarcolemmal fraction isolated from adult rabbit ventricular myocardium. Three types of experiments were conducted: (a) identification of ABP on electroblots of cardiomyocytes and sarcolemmal extracts reacted with [125I]-bovine serum albumin ([125I]Alb); (b) kinetic assays of [125I]Alb interaction with cardiomyocytes (at 37 degrees C), and with a sarcolemmal fraction (at 4 degrees C); (c) affinity isolation of ABP from solubilized radioiodinated sarcolemmal proteins interacted with an albumin-agarose matrix. The investigation showed that: first, two pairs of polypeptides (ABP of M(r) 18 and 31 kDa) in either cardiomyocytes or sarcolemmal fraction reacted on electroblots with [125I]Alb; second, the binding of the latter to cardiomyocytes was saturable and competed by unlabeled albumin: 50 microM albumin reduced by approximately 90% the binding of radiolabeled albumin. The sarcolemmal fraction bound [125I]Alb with a Kd of 3.66 x 10(-7) M. Thirdly, among the sarcolemmal proteins retained by the albumin-agarose matrix (18 and 31 kDa), the most prominent was the lower band (approximately 16 kDa) of the 18 kDa pair of ABP. The observations revealed that albumin interacts with relatively high affinity with specific binding sites on cardiomyocyte sarcolemma. This interaction may be a recognition step for subsequent fatty acid dissociation and translocation.

The pre-S2-coding region in the hepatitis B virus surface antigen M (P31; pre-S2 + S) protein gene was modified to identify a polymerized-albumin receptor (PAR) domain by deleting restriction fragments or performing site-directed mutagenesis. The modified M protein genes (M-P31x; x = d, e, f, h and i) were cloned into the yeast generalized-expression vector pGLD 906-1 and expressed in Saccharomyces cerevisiae under the control of yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter. The PAR activities of these gene products suggested that the PAR domain is located in the hydrophilic and highly conserved domain in the pre-S2 region (around Leu12 approximately Tyr21). Antibodies specific for a pre-S2 peptide (Phe8 approximately Pro34, subtype adr), which covers the PAR domain, were purified from sera of rabbits immunized with yeast-derived M protein particles having a natural PAR domain. Immune electron microscopy showed that the purified antibodies could aggregate HBV particles. Therefore, it was speculated that the PAR domain overlapped with the dominant virus-neutralizing and virus-protecting epitopes.

Nodular pulmonary amyloidosis, characterized by solitary or multiple parenchymal nodules, is primarily composed of amyloid immunoglobulin light chain protein. Pulmonary involvement by senile amyloidosis has been reported as an incidental finding with scattered or diffuse interstitial deposition of amyloid protein transthyretin mostly in patients with cardiac senile amyloidosis in a small number of autopsy cases. We report a unique case of pulmonary senile amyloidosis presenting with conglomerated nodular deposition of amyloid protein transthyretin as the main clinical manifestation. The patient was an 82-year-old man who presented with recurrent pleural effusions and nodular replacement of pulmonary parenchyma on a chest computed tomographic scan. He underwent a wedge resection of the lesion. Histologic examination revealed a massive deposition of Congo red-positive amyloid identified as amyloid protein transthyretin by both immunohistochemistry and mass spectrometry using formalin-fixed, paraffin-embedded tissues. Molecular testing did not show any mutation associated with familial amyloidosis in the TTR gene, further supporting the diagnosis of senile amyloidosis. To our knowledge, this is the first documented case of nodular senile amyloidosis of the lung that was confirmed with the current state-of-the-art methods.

There is currently a major debate on the mechanisms of albuminuria, and this review appraises recent studies in this area.The traditional view of albuminuria is that it is the result of damage to an essentially impermeable glomerular barrier. However, over the years, critical evidence for this traditional model has been shown to be flawed. An alternative explanation has evolved in which the glomerular filter governs albumin permeability by size selectivity alone. This means that the filter offers a significant barrier to albumin, but it is imperfect - the barrier leaks albumin. The virtue of this leakage is that it endows the filter an in-built anticlogging mechanism. The filtered albumin, if not rescued, would be excreted at nephrotic levels in the urine. There is evidence that proximal tubular cells participate in retrieving most of this filtered albumin to return it back to the blood supply intact. A small amount of the filtered albumin is not retrieved but directed toward lysosomal degradation, and the peptide products are exocytosed into the tubular lumen and excreted.In acquired and chemically induced kidney disease, albuminuria is the result of dysfunction in proximal tubular cell processing of albumin rather than alterations in glomerular permeability.

The degradation in Escherichia coli of the recombinant serum-albumin-binding receptor derived from streptococcal protein G was investigated using a dual-affinity fusion approach. The proteolytic degradation of the receptor was characterized when fused to human proinsulin and human secretin. Several cleavages occurred at sequences not normally regarded as proteolytically sensitive, such as the dipeptide sequences Ile-Gly, Val-Ser and Ser-Ala. Depending on the fusion partner, large differences in the degradation of the albumin-binding domain were observed. Thus, susceptibility to proteolysis of a recombinant protein can be affected by a neighbouring domain.

The albumin receptor expressed by bovine group G streptococci was extracted and affinity purified. The protein was characterized for species reactivity, and monospecific antibodies were prepared to the purified receptor. The bovine group G albumin receptor was compared functionally, antigenically, and for DNA homology with the albumin-binding protein expressed by human group G streptococci. In agreement with previous reports, the albumin-binding activity of human strains was mediated by a unique domain of the type III immunoglobulin G-Fc-binding molecule, protein G. The albumin receptor expressed by bovine group G strains was found to lack any immunoglobulin G-binding potential but displayed a wider profile of species albumin reactivity than protein G. Both albumin receptors could inhibit the binding of the other to immobilized human serum albumin, and each displayed similar binding properties. Antigenic comparison of the two albumin receptors demonstrated a low level of cross-reactivity; however comparison at the DNA level, using an oligonucleotide probe specific for the albumin-binding region of protein G, demonstrated that the two albumin receptors expressed by human and bovine group G streptococcal strains do not display significant homology.

We have previously shown that the uptake and transcytosis of albumin in astrocytes promote the synthesis of the neurotrophic factor oleic acid. Although the mechanism by which albumin induces oleic acid synthesis is well known, the mechanism of albumin uptake in astrocytes remains unknown. In this work, we found that astrocytes express megalin, an endocytic receptor for multiple ligands including albumin. In addition, when the activity of megalin is blocked by specific antibodies or by silencing megalin with specific siRNA, albumin binding and internalization is strongly reduced indicating that megalin is required for albumin binding and internalization in the astrocyte. Since the uptake of albumin in astrocytes aims at synthesizing the neurotrophic factor oleic acid, we tested the ability of megalin-silenced astrocytes to synthesize and release oleic acid in the presence of albumin. Our results showed that the amount of oleic acid found in the extracellular medium of megalin-silenced astrocytes was strongly reduced as compared with their controls. Together, the results of this work indicate that megalin is a receptor for albumin in astrocytes and is required for the synthesis of the neurotrophic factor oleic acid. Consequently, the possible involvement of albumin in the holoprosencephalic syndrome observed in megalin-deficient mice is suggested.

Cells that have evolved to produce large quantities of secreted proteins to serve the integrated functions of complex multicellular organisms are equipped to compensate for protein misfolding. Hepatocytes and plasma cells have well developed chaperone and proteasome systems to ensure that secreted proteins transit the cell efficiently. The number of neurodegenerative disorders associated with protein misfolding suggests that neurons are particularly sensitive to the pathogenic effects of aggregates of misfolded molecules because those systems are less well developed in this lineage. Aggregates of the amyloidogenic (Aβ 1–42 ) peptide play a major role in the pathogenesis of Alzheimer's disease (AD), although the precise mechanism is unclear. In genetic studies examining protein–protein interactions that could constitute native mechanisms of neuroprotection in vivo , overexpression of a WT human transthyretin (TTR) transgene was ameliorative in the APP23 transgenic murine model of human AD. Targeted silencing of the endogenous TTR gene accelerated the development of the neuropathologic phenotype. Intraneuronal TTR was seen in the brains of normal humans and mice and in AD patients and APP23 mice. The APP23 brains showed colocalization of extracellular TTR with Aβ in plaques. Using surface plasmon resonance we obtained in vitro evidence of direct protein–protein interaction between TTR and Aβ aggregates. These findings suggest that TTR is protective because of its capacity to bind toxic or pretoxic Aβ aggregates in both the intracellular and extracellular environment in a chaperone-like manner. The interaction may represent a unique normal host defense mechanism, enhancement of which could be therapeutically useful.

Transthyretin (TTR), a transport protein for T4 and retinol-binding protein, is the principal T4-binding protein of cerebrospinal fluid. Its function in regard to the delivery of its ligands and in other respects is unclear. The binding of [125I] TTR to cultured human astrocytoma cells was studied to determine whether these cells carry receptors for TTR. Scatchard analysis was consistent with a single class of binding sites with a Kd of 3 nM. No significant cross-reactivity with transferrin or serum albumin was observed. Internalization of TTR was temperature dependent and proportional to receptor occupancy. Dilutions of cerebrospinal fluid displaced [125I]TTR in proportion to their content of radioimmunoassayable TTR and in parallel with purified TTR. The uptake and internalization of TTR increased in the presence of high T4 or T3 concentrations. These results demonstrate that TTR binds to specific high affinity receptors on human astrocytoma cells. Receptor binding of TTR provides a potential mechanism for the delivery of its ligands within the central nervous system.

The mechanism of transfer of long chain fatty acids across the myocardial sarcolemmal membrane was investigated in isolated, calcium-resistant, rat cardiomyocytes. The initial rate of 14 C-palmitate uptake was determined at constant and increasing palmitate/albumin ratios. The latter condition led to a saturable dependence of uptake rate on palmitate concentration. At a constant palmitate/albumin ratio however, there was an almost constant rate of uptake even though the absolute concentration of palmitate increased. The enhanced metabolic rate resulting from electrically induced contractions of the myocytes decreased the apparent K m of uptake from 62 to 23 μ m . Thirty seconds after administration, there was no further increase in the [ 14 C]palmitate content of the myocytes. Moreover, from experiments using ghost membrane vesicles the concentration of palmitate in membranes increased almost linearly with increasing palmitate/albumin ratios. This concentration remained virtually constan if vesicles were pre-treated with diamide. Our results do not support the concept of an albumin receptor-mediated uptake but rather suggest that fatty acids are incorporated into cardiomyocytes by a simple diffusion process which is not rate-limiting. The rate of uptake is influenced both by the metabolic rate and by the concentration of fatty acids in the membranes. The rate-limiting step of fatty acid uptake is probably either the formation of acyl-CoA catalyzed by the membrane associated acyl-CoA synthetase, or the transfer of fatty acid carnitine esters across the mitochondrial matrix membrane.

The carboxylic acid 2-(4'-hydroxybenzeneazo)benzoic acid (HABA) binds to human and bovine albumin and this dye is a useful model ligand for the investigation of ligand-acceptor interactions

The binding characteristics of reduced hepatic membrane proteins for acetylated low-density lipoprotein (acetyl-LDL) and maleylated bovine serum albumin (Mal-BSA) have been examined. Two receptor activities were extracted from hepatic membranes in the presence of octyl beta-D-glucoside and beta-mercaptoethanol, and were separated by chromatography on Mal-BSA-Sepharose 4B. The receptors were revealed by ligand blotting. The active binding proteins had apparent molecular masses of 35 and 15 kDa in SDS/polyacrylamide gels. Equilibrium studies with protein-phosphatidylcholine complexes indicated that the reduced 35 kDa protein expresses two binding sites for Mal-BSA and one for acetyl-LDL, whereas the 15 kDa protein-phosphatidylcholine complex binds 131I-Mal-BSA and 131I-acetyl-LDL with a 4:1 stoichiometry. 131I-Mal-BSA binding was linear with both proteins, with a Kd of 4.8 nM at the 35 kDa protein and a Kd of 5.6 nM at the 15 kDa protein. The 35 kDa protein displayed saturable binding of 131I-acetyl-LDL with a Kd of 5 nM; the 15 kDa binding protein bound 131I-acetyl-LDL with a Kd of 2.3 nM. A 85 kDa protein was obtained by Mal-BSA-Sepharose chromatography when the hepatic membranes had been solubilized with Triton X-100 in presence of GSH/GSSG. This protein displayed saturable 131I-Mal-BSA binding with a Kd of 30 nM and 131I-acetyl-LDL binding with a Kd of 6.5 nM. The 131I-Mal-BSA binding capacity was four times higher than that of 131I-acetyl-LDL. Competition studies with the 35 kDa, 15 kDa and 85 kDa proteins binding Mal-BSA, acetyl-LDL, formylated albumin and polyanionic competitors provide evidence for the existence of more than one class of binding sites at the reduced binding proteins.

During early vertebrate evolution, a duplication event in the gene encoding 5-hydroxyisourate hydrolase (HIUase), a widely distributed enzyme of purine metabolism, gave rise to transthyretin (TTR), a thyroid hormone transporter. We report here on the crystal structure of zebra fish HIUase in two different crystal forms. Despite the phylogenetic distance, this structure compares well with those of newly characterized bacterial HIUases, especially with regard to catalytic regions, which are highly preserved. Comparison with TTR structure reveals a highly conserved scaffold, harbouring distinct functional sites located in the same regions of the two vertebrate proteins. Residues that are differentially conserved in HIUases compared to TTR map in putative catalytic regions occupying significant portions of the two halves of a central channel that transverses the whole TTR protein. The evolution of TTR has been accompanied by remarkable changes of the HIUase active sites that gave rise to a channel open at both ends, thus allowing free access to hormone molecules.

PURPOSE OF REVIEW: Clinical management of the amyloidoses has historically been the province of rheumatologists, because of the relation to long-standing inflammation in rheumatoid arthritis, ankylosing spondylitis, and juvenile chronic arthritis. Currently, nephrologists, hematologist-oncologists, neurologists, and transplant surgeons all have a diagnostic or therapeutic interest. Current advances, using the tools of physical biochemistry, cell biology, and genetics, have begun to impact the diagnosis and clinical management of these disorders and raise questions regarding our notions of protein conformation in vivo and how nonnatively folded proteins may produce disease. RECENT FINDINGS: It appears that all amyloidogenic precursors undergo some degree of misfolding that allows them to populate an immediate precursor pool from which they rapidly aggregate. Depending on the particular protein, a variety of mechanisms appear operative, some of which involve nonphysiologic proteolysis, defective physiologic proteolysis, mutations involving changes in thermodynamic or kinetic properties, and pathways that are yet to be defined. Whatever the particular process, the result is a tendency toward oligomeric aggregation followed by the assembly of higher order structures that become insoluble under physiologic conditions. Detailed analyses have been described for transthyretin (senile systemic amyloidosis and familial amyloid polyneuropathy), immunoglobulin light chains (light-chain amyloid), beta2 microglobulin (dialysis-related amyloid), and apolipoprotein A1, and are in process for others. SUMMARY Therapies have been proposed based on precursor stabilization (transthyretin), elimination of the synthesizing cell (light-chain amyloid), fibril disruption and immunization to induce host-mediated aggregate clearance (Alzheimer disease, light-chain amyloid, prions), and aggressive therapy of a primary inflammatory process (amyloid A). During the next decade, the value of these therapies, and others, suggested by studies on the basic properties of cells and proteins, will become clear.

Recently, we developed the method for measurement of the technetium-99m-diethylenetriamine-pentaacetic acid-galactosyl human serum albumin (Tc-GSA) receptor amount (R0) using a nonlinear 3-compartment model. We examined the usefulness of R0 for preoperative estimation of risk in hepatectomy.Sixty-three patients who underwent hepatectomy in our hospital were examined for R0. These patients consisted of 26 cases of normal liver, 16 cases of liver fibrosis, and 21 cases of cirrhosis. R0 was measured by the nonlinear 3-compartment model of ligand-receptor binding without blood sampling in Tc-GSA scintigraphy. The expected remnant liver R0 after hepatectomy was calculated from CT volumetry before hepatectomy.The preoperative mean R0 of liver was 15.8 +/- 3.8 mg for Z0, 13.8 +/- 3.9 mg for Z1, and 6.9 +/- 2.3 mg for Z2. R0 in Z2 was significantly lower than Z0 and Z1 (p0.0001). Every patient whose remnant liver R0 was over 5 mg tolerated hepatectomy without any postoperative complications. Among 63 cases, 5 patients developed postoperative complications (two liver failures, two postoperative jaundice and one hepatic coma), and remnant liver R0 of these patients were under 5 mg.From these results, it can be seen that R0 of remnant liver is a useful parameter to decide indication of hepatectomy and predict postoperative complications.

To investigate the relationship between interleukin-6 (IL-6) levels in serum and HBV replication and the role of IL-6 in liver damage in chronic hepatitis B patients.Detected IL-6 levels and polymerized human serum albumin receptor (PHSA-R) in serum of different type of HBV markers positive patients and of different degree of chronic hepatitis B patients.The results showed that the serum IL-6 levels were increased significantly in PHSA-R positive patients than in PHSA-R negative patients in chronic hepatitis B (P0.01). The serum IL-6 levels were correlated with the levels of PHSA-R (r=0.694, P0.01), and the degree of symptoms.This study suggested that the IL-6 levels in serum of hepatitis B patients correlated HBV replication and the degree of liver damage, serum IL-6 levels may be used as a value indicating of HBV replication, the degree of symptoms and the effect of treatment.

By screening phage display random peptide libraries with purified immunoglobulin E (IgE) from birch pollen-allergic patients, we previously defined peptides mimicking natural IgE epitopes (mimotopes) of the major birch pollen allergen Bet v 1. The present study aimed to define a monovalent carrier for the IgE mimotopes to induce protective antibodies directed to the IgE epitopes, suitable for mimotope-specific therapy. We expressed the selected mimotopes as fusion proteins together with streptococcal albumin binding protein (ABP). The fusion proteins were recognized specifically by anti-Bet v 1 human IgE, which demonstrated that the mimotopes fused to ABP resemble the natural IgE epitope. Bet v 1-specific IgG was induced by immunization of BALB/c mice with fusion proteins. These IgG antibodies could inhibit IgE binding to Bet v 1. Skin testing of Bet v 1 allergic mice showed that the ABP mimotope constructs did not elicit type I skin reactions, although they possess IgE binding structures. Our data suggest that IgE mimotopes are safe for epitope-specific immunotherapy of sensitized individuals, when presented in a monovalent form. Therefore, ABP-fused mimotopes are promising candidates for a new type of immunotherapy based on the precise induction of blocking antibodies.

The hepatitis B virus (HBV) envelope (env) protein is composed of three regions; the 108- or 119-residue pre-S1 region involved in the direct interaction with hepatocytes, the 55-residue pre-S2 region associated with the polymerized albumin-mediated interaction, and the major 226-residue S protein region. Thus, to improve the immunogenic potency of conventional HB vaccines, development of a new vaccine containing the entire pre-S1 region in addition to pre-S2 and S is desired. We previously reported the efficient production of the HBV env L (pre-S1 + pre-S2 + S) protein in the recombinant yeast cells [J Biol Chem 267 (1992) 1953]. In this study, the HBV env L protein produced as nano-particles in yeast has been purified and characterized. By equilibrium sedimentation, an average molecular weight of L particle was estimated to be approximately 6.4 x 10(6), indicating that about 110 molecules of L proteins are assembled into an L particle. By atomic force microscopy in a moist atmosphere, the L particles were observed as large spherical particles with a diameter of 50-500 nm. The L particles were stable on short-time heating at a high temperature and long-time storage at a low temperature but rather unstable on repeated freezing and thawing and treatment with dithiothreitol. When immunized in mice, L particles elicited efficiently and simultaneously the anti-S, anti-pre-S2, and anti-pre-S1 antibodies. The ED(50) values in mice for the anti-S and anti-pre-S2 antibodies were similar to those elicited by the M (pre-S2 + S) particles. Furthermore, the anti-pre-S1 rabbit antibodies were found to recognize various segments of the pre-S1 region, including the pre-S1 (21-47) segment. These results show the high ability of L particles to induce all antibodies against HBV env proteins, hence promising the future application of L particles for the next generation HB vaccine.

Transthyretin (TTR) is a plasma carrier of thyroxine and retinol-binding protein (RBP). Though the liver is the major site of TTR degradation, its cellular uptake is poorly understood. We explored TTR uptake using hepatomas and primary hepatocytes and showed internalization by a specific receptor. RBP complexed with TTR led to a 70% decrease of TTR internalization, whereas TTR bound to thyroxine led to a 20% increase. Different TTR mutants showed differences in uptake, suggesting receptor recognition dependent on the structure of TTR. Cross-linking studies using hepatomas and (125)I-TTR revealed a approximately 90-kDa complex corresponding to (125)I-TTR bound to its receptor. Given previous evidence that a fraction of TTR is associated with high-density lipoproteins (HDL) and that in the kidney, megalin, a member of the low-density lipoprotein receptor family (LDLr) internalizes TTR, we hypothesized that TTR and lipoproteins could share related degradation pathways. Using lipid-deficient serum in uptake assays, no significant changes were observed showing that TTR uptake is not lipoprotein-dependent or due to TTR-lipoprotein complexes. However, competition studies showed that lipoproteins inhibit TTR internalization. The scavenger receptor SR-BI, a HDL receptor, and known LDLr family hepatic receptors did not mediate TTR uptake as assessed using different cellular systems. Interestingly, the receptor-associated protein (RAP), a ligand for all members of the LDLr, was able to inhibit TTR internalization. Moreover, the approximately 90-kDa TTR-receptor complex obtained by cross-linking was sensitive to the presence of RAP. To confirm that RAP sensitivity observed in hepatomas did not represent a mechanism absent in normal cells, primary hepatocytes were tested, and similar results were obtained. The RAP-sensitive TTR internalization together with displacement of TTR uptake by lipoproteins, further suggests that a common pathway might exist between TTR and lipoprotein metabolism and that an as yet unidentified RAP-sensitive receptor mediates TTR uptake.

Based on the sequence of Hepatitis B Virus (type adr) (HBV) Pre S2 region, we synthesized a pair of PCR primers. After a test of specificity, the primers were employed to amplify the postulated transcripts of PHSAr gene in the liver samples of 3 normal human subjects. By RT-PCR and RNA-PCR, all amplification results were positive, while those from corresponding skeleton muscle controls were negative. It suggests that HBV Pre S2 region should be to some extent homologous to PHSAr gene exon(s). It also indicates that PHSAr gene is in expression at transcriptional level, but not in expression in tissues (e.g. skeleton muscle) un susceptible to HBV infection. Our data further confirm the role of PHSAr in HBV infection of hepatocytes, compared with those from membrane receptor researches.