S-Nitrosylation of Tissue Transglutaminase in Modulating Glycolysis, Oxidative Stress, and Inflammatory Responses in Normal and Indoxyl-Sulfate-Induced Endothelial Cells.

Circulating uremic toxin indoxyl sulfate (IS), endothelial cell (EC) dysfunction, and decreased nitric oxide (NO) bioavailability are found in chronic kidney disease patients. NO nitrosylates/denitrosylates a specific protein's cysteine residue(s), forming S-nitrosothios (SNOs), and the decreased NO bioavailability could interfere with NO-mediated signaling events. We were interested in investigating the underlying mechanism(s) of the reduced NO and how it would regulate the S-nitrosylation of tissue transglutaminase (TG2) and its substrates on glycolytic, redox and inflammatory responses in normal and IS-induced EC injury. TG2, a therapeutic target for fibrosis, has a Ca2+-dependent transamidase (TGase) that is modulated by S-nitrosylation. We found IS increased oxidative stress, reduced NADPH and GSH levels, and uncoupled eNOS to generate NO. Immunoblot analysis demonstrated the upregulation of an angiotensin-converting enzyme (ACE) and significant downregulation of the beneficial ACE2 isoform that could contribute to oxidative stress in IS-induced injury. An in situ TGase assay demonstrated IS-activated TG2/TGase aminylated eNOS, NFkB, IkBα, PKM2, G6PD, GAPDH, and fibronectin (FN), leading to caspases activation. Except for FN, TGase substrates were all differentially S-nitrosylated either with or without IS but were denitrosylated in the presence of a specific, irreversible TG2/TGase inhibitor ZDON, suggesting ZDON-bound TG2 was not effectively transnitrosylating to TG2/TGase substrates. The data suggest novel roles of TG2 in the aminylation of its substrates and could also potentially function as a Cys-to-Cys S-nitrosylase to exert NO's bioactivity to its substrates and modulate glycolysis, redox, and inflammation in normal and IS-induced EC injury.

Prediction of large-for-gestational-age infant by fetal growth charts and hemoglobin A1c level in pregnancy complicated by pregestational diabetes.

OBJECTIVES: To compare the ability of three fetal growth charts (Fetal Medicine Foundation (FMF), Hadlock and National Institutes of Child Health and Human Development (NICHD) race/ethnicity-specific) to predict large-for-gestational age (LGA) at birth in pregnant individuals with pregestational diabetes, and to determine whether inclusion of hemoglobin A1c (HbA1c) level improves the predictive performance of the growth charts. METHODS: This was a retrospective analysis of individuals with Type-1 or Type-2 diabetes with a singleton pregnancy that resulted in a non-anomalous live birth. Fetal biometry was performed between 28 + 0 and 36 + 6 weeks of gestation. The primary exposure was suspected LGA, defined as estimated fetal weight ≥ 90th percentile using the Hadlock (Formula C), FMF and NICHD growth charts. The primary outcome was LGA at birth, defined as birth weight ≥ 90th percentile, using 2017 USA natality reference data. The performance of the three growth charts to predict LGA at birth, alone and in combination with HbA1c as a continuous measure, was assessed using the area under the receiver-operating-characteristics curve (AUC), sensitivity, specificity, positive predictive value and negative predictive value. RESULTS: Of 358 assessed pregnant individuals with pregestational diabetes (34% with Type 1 and 66% with Type 2), 147 (41%) had a LGA infant at birth. Suspected LGA was identified in 123 (34.4%) by the Hadlock, 152 (42.5%) by the FMF and 152 (42.5%) by the NICHD growth chart. The FMF growth chart had the highest sensitivity (77% vs 69% (NICHD) vs 63% (Hadlock)) and the Hadlock growth chart had the highest specificity (86% vs 76% (NICHD) and 82% (FMF)) for predicting LGA at birth. The FMF growth chart had a significantly higher AUC (0.79 (95% CI, 0.74-0.84)) for LGA at birth compared with the NICHD (AUC, 0.72 (95% CI, 0.68-0.77); P < 0.001) and Hadlock (AUC, 0.75 (95% CI, 0.70-0.79); P < 0.01) growth charts. Prediction of LGA improved for all three growth charts with the inclusion of HbA1c measurement in comparison to each growth chart alone (P < 0.001 for all); the FMF growth chart remained more predictive of LGA at birth (AUC, 0.85 (95% CI, 0.81-0.90)) compared with the NICHD (AUC, 0.79 (95% CI, 0.73-0.84)) and Hadlock (AUC, 0.81 (95% CI, 0.76-0.86)) growth charts. CONCLUSIONS: The FMF fetal growth chart had the best predictive performance for LGA at birth in comparison with the Hadlock and NICHD race/ethnicity-specific growth charts in pregnant individuals with pregestational diabetes. Inclusion of HbA1c improved further the prediction of LGA for all three charts. © 2022 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

HLA-DQ genotype and biochemical characterization of anti-transglutaminase 2 antibodies in patients with type 1 diabetes mellitus in Taiwan.

Human Leukocyte Antigen (HLA)-DQ2 and HLA-DQ8 are genetic risk factors for Type 1 Diabetes Mellitus (T1DM) and Celiac disease (CD) in Caucasians, but their association with Taiwanese Han population is unknown. We screened 532 Taiwanese T1DM patients for CD biomarkers including anti-tissue transglutaminase (TGM2), anti-gliadin and anti-neoepitope antibodies (Abs), sequencing DQB1 genotypes, and characterized the TGM2 Abs. We report that 3.76% of Taiwanese patients had TGM2-Abs and all had no CD's symptoms. In contrast to Caucasian's CD patients, DQ2/DQ8 only constituted ~4/5 of TGM2-Abs positive patients, while the other ~1/5 patients belonged to different HLA genotypes. Either anti-gliadin or anti-neoepitope Abs coexisted with ~3/4 of TGM2-Abs positive patients that were likely due to gluten-ingestion, while the cause of TGM2-Abs production for other ~1/4 of patients was unknown. Purified anti-TGM2 IgA (TGA) and anti-TGM2 IgG (TGG) could bind on endothelial cells surface, recognized native better than denatured forms of TGM2, and TGA inhibited TGM2's transamidation activity by up to 80% but TGG had no effects. Epitope mapping of all TGM2-Abs positive sera demonstrated that TGM2-Abs had heterogeneity in specificities. This is the first study on the differences between Taiwanese Han group and Caucasian in HLA genotypes and properties of TGM2-Abs.

An evaluation of fetal heart rate characteristics associated with neonatal encephalopathy: a case-control study.

OBJECTIVE: We sought to identify fetal heart rate (FHR) characteristics that are associated with neonatal encephalopathy (NE). DESIGN: Retrospective case-control study. SETTING: A single medical centre in Shanghai, China, 2006-2015. SAMPLE: Women delivering a singleton, non-anomalous infant at ≥36 weeks' gestation diagnosed with NE (cases, n = 109) were compared with a group of women with unaffected infants (controls, n = 233). METHODS: Two physicians blinded to the outcome independently reviewed FHR tracings during the last 30 minutes of tracing prior to delivery. FHR characteristics were compared in the two groups and multivariable logistic regression was used to adjust for confounding. MAIN OUTCOME MEASURES: Adjusted odds ratio (aOR) and 95% confidence interval (CI) for the presence of specific FHR categories and characteristics. RESULTS: Category II FHR tracings were observed in 89% of women prior to delivery and were not independently associated with NE. Notably, a category III FHR was observed in 17.4% of women in the NE group compared with 0.9% of women in the control group (aOR 44.99, 95% CI 7.23-279.97). Bradycardia, minimal/absent variability, late decelerations and prolonged decelerations were independently associated with NE, whereas accelerations were protective. Similar findings were found when the cases were limited to NE with arterial cord pH <7.1 and in a subgroup analysis of women with category II tracings. CONCLUSIONS: Category III tracings, while infrequent, are not uncommon prior to delivery among fetuses who develop NE. In contrast, most FHR tracings are category II prior to delivery; however, individual FHR characteristics within this category are associated with NE. FUNDING: This research was supported by the Interdisciplinary Programme of Shanghai Jiao Tong University. TWEETABLE ABSTRACT: Category III tracings are not uncommon prior to delivery among fetuses who develop neonatal encephalopathy.

Results of Fabry Disease Screening in Male Pre-End Stage Renal Disease Patients with Unknown Etiology Found Through the Platform of a Chronic Kidney Disease Education Program in a Northern Taiwan Medical Center.

BACKGROUND/AIMS: Fabry disease (FD), a rare x-lined genetic disorder is a cause of renal deterioration. The phenotype of FD is highly variable and nonspecific, and correct diagnosis has always been delayed. We aimed to explore the prevalence and clinical presentation of FD in this high-risk male population in a Northern Taiwan medical center. METHODS: This is the first study to survey the incidence of FD in this high-risk population through the platform of a chronic kidney disease (CKD) education program in Asia. A total of 1,012 male patients with unknown CKD causes were screened using an assay of alpha-galactosidase A activity (α-Gal A) by dried blood spots (DBS). A final GLA gene analysis was also done for those with low enzyme activity. RESULTS: We identified two new patients with classic FD and four patients with late-onset FD. One novel GLA mutation with c.413 G>A was found in one classic FD patient (index 5). The prevalence of FD is about 0.59 % (6 in 1,012) in the high-risk population group with CKD. The clinical symptoms of FD patients are nonspecific except in those with various degrees of renal failure. Those patients' correct diagnosis was delayed, taking years and even decades. Three patients received enzyme replacement therapy and one started regular hemodialysis due to persistent renal function deterioration. Another two patients were found from family screening through a new index. In addition, a false negative result occurred in one patient who was proved to have FD by his kidney pathology as determined by this screening. CONCLUSION: FD is not such as rare a disease and its prevalence is greater in this high-risk male population. Clinicians need to be aware that FD should be included in the differential diagnosis in CKD with unknown etiology.

Role of tissue transglutaminase-2 (TG2)-mediated aminylation in biological processes.

Post-translational modification (PTM) is an important mechanism in modulating a protein's structure and can lead to substantial diversity in biological function. Compared to other forms of PTMs such as phosphorylation, acetylation and glycosylation, the physiological significance of aminylation is limited. Aminylation refers to the covalent incorporation of biogenic/polyamines into target protein by calcium-dependent transglutaminases (TGs). The development of novel and more sensitive techniques has led to more proteins identified as tissue transglutaminase (TG2) substrates and potential targets for aminylation. Many of these substrate proteins play a role in cell signaling, cytoskeleton organization, muscle contraction, and inflammation. TG2 is well studied and widely expressed in a variety of tissues and will be the primary focus of this review on recent advance in transglutaminase-mediated aminylation.

Histaminylation of fibrinogen by tissue transglutaminase-2 (TGM-2): potential role in modulating inflammation.

Plasma fibrinogen plays an important role in hemostasis and inflammation. Fibrinogen is converted to fibrin to impede blood loss and serves as the provisional matrix that aids wound healing. Fibrinogen also binds to cytokine activated endothelial cells and promotes the binding and migration of leukocytes into tissues during inflammation. Tissue transglutaminase (TGM-2) released from injured cells could cross-link fibrinogen to form multivalent complexes that could promote adhesion of platelets and vascular cells to endothelium. Histamine released by mast cells is a potent biogenic amine that promotes inflammation. The covalent attachment of histamine to proteins (histaminylation) by TGM-2 could modify local inflammatory reactions. We investigated TGM-2 crosslinking of several biogenic amines (serotonin, histamine, dopamine and noradrenaline) to fibrinogen. We identified histaminylation of fibrinogen by TGM-2 as a preferred reaction in solid and solution phase transglutaminase assays. Histamine caused a concentration-dependent inhibition of fibrinogen cross-linking by TGM-2. Fibrinogen that was not TGM-2 crosslinked bound to unactivated endothelial cells with low affinity. However, the binding was increased by sevenfold when fibrinogen was cross-linked by TGM-2. Histaminylation of fibrinogen also inhibited TGM-2 crosslinking of fibrinogen and the binding to un-activated HUVEC cells by 75­90 %. In summary, the histaminylation of fibrinogen by TGM-2 could play a role in modifying inflammation by sequestering free histamine and by inhibiting TGM-2 crosslinking of fibrinogen.

Evaluating the cost-effectiveness of prenatal surgery for myelomeningocele: a decision analysis.

OBJECTIVE: To determine whether prenatal myelomeningocele repair is a cost-effective strategy compared to postnatal repair. METHODS: Decision-analysis modeling was used to calculate the cumulative costs, effects and incremental cost-effectiveness ratio of prenatal myelomeningocele repair compared with postnatal repair in singleton gestations with a normal karyotype that were identified with myelomeningocele between T1 and S1. The model accounted for costs and quality-adjusted life years (QALYs) in three populations: (1) myelomeningocele patients; (2) mothers carrying myelomeningocele patients; and (3) possible future siblings of these patients. Sensitivity analysis was performed using one-way, two-way and Monte Carlo simulations. RESULTS: Prenatal myelomeningocele repair saves $ 2 066 778 per 100 cases repaired. Additionally, prenatal surgery results in 98 QALYs gained per 100 repairs with 42 fewer neonates requiring shunts and 21 fewer neonates requiring long-term medical care per 100 repairs. However, these benefits are coupled to 26 additional cases of uterine rupture or dehiscence and one additional case of neurologic deficits in future offspring per 100 repairs. Results were robust in sensitivity analysis. CONCLUSION: Prenatal myelomeningocele repair is cost effective and frequently cost saving compared with postnatal myelomeningocele repair despite the increased likelihood of maternal and future pregnancy complications associated with prenatal surgery.

Universal cervical-length screening to prevent preterm birth: a cost-effectiveness analysis.

OBJECTIVE: To determine whether routine measurement of second-trimester transvaginal cervical length by ultrasound in low-risk singleton pregnancies is a cost-effective strategy. METHODS: We developed a decision analysis model to compare the cost-effectiveness of two strategies for identifying pregnancies at risk for preterm birth: (1) no routine cervical length screening and (2) a single routine transvaginal cervical length measurement at 18-24 weeks' gestation. In our model, women identified as being at increased risk (cervical length < 1.5 cm) for preterm birth would be offered daily vaginal progesterone supplementation. We assumed that vaginal progesterone reduces preterm birth at < 34 weeks' gestation by 45%. We also assumed that a decreased cervical length could result in additional costs (ultrasound scans, inpatient admission) without significantly improved neonatal outcomes. The main outcome measure was incremental cost-effectiveness ratio. RESULTS: Our model predicts that routine cervical-length screening is a dominant strategy when compared to routine care. For every 100,000 women screened, $12,119,947 can be potentially saved (in 2010 US dollars) and 423.9 quality-adjusted life-years could be gained. Additionally, we estimate that 22 cases of neonatal death or long-term neurologic deficits could be prevented per 100,000 women screened. Screening remained cost-effective but was no longer the dominant strategy when cervical-length ultrasound measurement costs exceeded $187 or when vaginal progesterone reduced delivery risk at < 34 weeks by less than 20%. CONCLUSION: In low-risk pregnancies, universal transvaginal cervical length ultrasound screening appears to be a cost-effective strategy under a wide range of clinical circumstances (varied preterm birth rates, predictive values of a shortened cervix and costs).

Identification of chemical inhibitors to human tissue transglutaminase by screening existing drug libraries.

Human tissue transglutaminase (TGM2) is a calcium-dependent crosslinking enzyme involved in the posttranslational modification of intra- and extracellular proteins and implicated in several neurodegenerative diseases. To find specific inhibitors to TGM2, two structurally diverse chemical libraries (LOPAC and Prestwick) were screened. We found that ZM39923, a Janus kinase inhibitor, and its metabolite ZM449829 were the most potent inhibitors with IC(50) of 10 and 5 nM, respectively. In addition, two other inhibitors, including tyrphostin 47 and vitamin K(3), were found to have an IC(50) in the micromolar range. These agents used in part a thiol-dependent mechanism to inhibit TGM2, consistent with the activation of TGM2 by reduction of an intramolecular disulfide bond. These inhibitors were tested in a polyglutamine-expressing Drosophila model of neurodegeneration and found to improve survival. The TGM2 inhibitors we discovered may serve as valuable lead compounds for the development of orally active TGM2 inhibitors to treat human diseases.

Correction: The role of indoxyl sulfate in renal anemia in patients with chronic kidney disease.

[This corrects the article DOI: 10.18632/oncotarget.18789.].

Identification of two GTP-independent alternatively spliced forms of tissue transglutaminase in human leukocytes, vascular smooth muscle, and endothelial cells.

Tissue transglutaminase (tTG) is a multifunctional enzyme with transglutaminase crosslinking (TGase), GTP binding, and hydrolysis activities that play a role in many different disorders. We identified, characterized, and investigated the function and stability of two alternatively spliced forms of tTG using biochemical, cellular, and molecular biological approaches. Using a human aortic vascular smooth muscle cells (VSMC) cDNA library, we identified two cDNAs encoding C-terminal truncated forms, tTG(V1) and tTG(V2). tTG(V1,2) mRNAs were synthesized by a rare splicing event using alternate splice sites within exons 12 and 13 of the tTG gene, respectively. Quantitative PCR and immunoblotting demonstrated that there was unique expression and localization of tTG(V1,2) compared with tTG in human umbilical vein endothelial cells (HUVECs), VSMC, and leukocytes. The loss of C-terminal 52 amino acid residues (AAs) in tTG(V1,2) altered GTP binding, enhanced GTP hydrolysis, rendered the variants insensitive to GTP inhibition, and resulted in <10% residual Ca(+2)-dependent TGase activity. Transfection in HEK293 demonstrated a 28- and 5-fold reduction in the expression of tTG(V1) and tTG(V2), respectively, demonstrating that the C-terminal GTP-binding domain is important in stabilizing and promoting the half-life of tTG. The altered affinity for GTP allowed tTG(V1,2) to exhibit enhanced TGase activity when there is a transient increase in Ca(+2) levels. The abundance of tTG(V1,2) and its distinct intracellular expression patterns in human vascular cells and leukocytes indicate these isoforms likely have unique physiological functions.

Effect of ischemia-reperfusion on the incidence of acute cellular rejection and timing of histologic hepatitis C virus recurrence after liver transplantation.

BACKGROUND: Because of a critical shortage of deceased donor (DD) livers, more extended criteria allografts are being utilized; these allografts are at increased risk for ischemia-reperfusion injury (IRI). We assessed whether, in a large cohort of patients transplanted for hepatitis C virus (HCV) either via a DD or live donor (LD), there was a relationship between the degree of IRI and the frequency and timing of acute cellular rejection (ACR) and histologic HCV recurrence. METHODS: During an 8-year study, patients were separated into four groups based on peak alanine aminotransferase (ALT) levels and three groups based on severity of IRI on postreperfusion liver biopsy. RESULTS: The mean follow-up time of 433 DD and 44 LD recipients was 1212 days. We noted a strong correlation in DD between peak ALT and the histologic degree of IRI (P = .01). There was no difference in the incidence or grade of ACR among the four groups. There was no correlation between the severity of IRI and the incidence or time to histologic recurrence of HCV. CONCLUSIONS: The magnitude of peak ALT correlated with the severity of IRI on postreperfusion liver biopsy. Among this large HCV cohort, there was no correlation between the severity of IRI and the incidence or timing of histologic HCV recurrence or incidence of ACR.

Physicochemical and immunohistological studies of a sulfobromophthalein- and bilirubin-binding protein from rat liver plasma membranes.

Affinity chromatography over bilirubinagarose and sulfobromophthalein (BSP)-agarose was used to isolate two proteins, with high affinities for bilirubin and BSP, respectively, from Triton X-100-solubilized rat liver plasma membranes. The protein eluted from either affinity column migrated as a single band of approximately 55,000 D on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and either protein cochromatographed with both [14C]bilirubin and [35S]BSP on Sephadex G-75. On gradient gels without reduction or SDS, or on Sephadex G-150, the native BSP-binding protein had an estimated molecular mass of approximately 100,000 D. After incubation with SDS, an additional Sephadex G-150 peak of molecular mass of 56,000 D was observed. Both, the 100,000- and 56,000-D G-150 peaks cochromatographed with [35S]BSP. The native protein had an isoelectric point of 3.5, stained with periodic acid-Schiff but not Sudan black, and contained 4 mol of sialic acid per mol of protein. A rabbit antibody to the BSP-binding protein gave a line of identity with both the BSP- and bilirubin-binding antigens, and inhibited the binding of [14C]bilirubin and [35S]BSP, but not [14C]oleate or [14C]taurocholate, to rat liver plasma membranes. Immunohistochemical studies revealed the presence of the antigen on all surface domains of rat hepatocytes, but not on other cell populations from normal rat liver. It was not found in other organs. These data are compatible with the hypothesis that a specific liver cell plasma membrane protein mediates the hepatocytic sequestration of bilirubin and BSP.

Tissue transglutaminase (TG2) and mitochondrial function and dysfunction.

Mitochondria are the cell's power plant to satisfy the energy demands. However, dysfunctional mitochondria can cause overproduction of reactive oxygen species (ROS), oxidative stress, and alteration of calcium homeostasis, which are the hallmarks of mitochondrial diseases. Under prolong oxidative stress, repeated cytosolic calcium elevations even only transiently, can lead to activation of some enzymes. One calcium-activated enzyme with demonstrated pathophysiological important in mitochondrial disease is tissue transglutaminase (TG2). TG2 is known as a post-translational modification (PTM) enzyme that is induced by oxidative stress. Compared to other types of PTMs, the physiological significance of TG2 mediated PTM is just beginning to be understood. Once activated, TG2 can modulate transcription, inactivate metabolic enzymes, and cause aggregation of critical proteins. Recent data indicate that TG2's activity not only can modulate the assembly of respiratory chain complexes but can also modulate the transcription of critical genes including PGC-1alpha and cytochrome C that are important for function and biogenesis of mitochondria. Here, we summarize dysfunctional mitochondria in diseases such as in neurodegenerative disorders can modulate TG2's activity and function.  TG2 is also important for normal function of mitochondria.

Endothelial cell-surface tissue transglutaminase inhibits neutrophil adhesion by binding and releasing nitric oxide.

Nitric oxide (NO) produced by endothelial cells in response to cytokines displays anti-inflammatory activity by preventing the adherence, migration and activation of neutrophils. The molecular mechanism by which NO operates at the blood-endothelium interface to exert anti-inflammatory properties is largely unknown. Here we show that on endothelial surfaces, NO is associated with the sulfhydryl-rich protein tissue transglutaminase (TG2), thereby endowing the membrane surfaces with anti-inflammatory properties. We find that tumor necrosis factor-α-stimulated neutrophil adherence is opposed by TG2 molecules that are bound to the endothelial surface. Alkylation of cysteine residues in TG2 or inhibition of endothelial NO synthesis renders the surface-bound TG2 inactive, whereas specific, high affinity binding of S-nitrosylated TG2 (SNO-TG2) to endothelial surfaces restores the anti-inflammatory properties of the endothelium, and reconstitutes the activity of endothelial-derived NO. We also show that SNO-TG2 is present in healthy tissues and that it forms on the membranes of shear-activated endothelial cells. Thus, the anti-inflammatory mechanism that prevents neutrophils from adhering to endothelial cells is identified with TG2 S-nitrosylation at the endothelial cell-blood interface.

The role of indoxyl sulfate in renal anemia in patients with chronic kidney disease.

Renal anemia is a common complication in patients with advanced chronic kidney disease. In vitro studies have shown that indoxyl sulfate decreases erythropoietin production. Whether this effect is seen in vivo remains unclear. Our goal was to explore the role of indoxyl sulfate in renal anemia. We found serum indoxyl sulfate levels are significantly and negatively associated with erythropoietin levels in human. A multiple stepwise linear regression analyses after adjustment for other independent parameters revealed that free indoxyl sulfate, and total indoxyl sulfate were significantly associated with erythropoietin levels. In animal studies, erythropoietin gene and protein expression were markedly inhibited in rats with chronic kidney disease; however, this effect was significantly reversed by lowering serum indoxyl sulfate with AST-120. Indoxyl sulfate may also inhibit erythropoietin expression in animal models with chronic kidney disease. These findings further support the role of indoxyl sulfate in the development of renal anemia.

Tumorigenicity in nude mice of a human hepatoma cell line containing hepatitis B virus DNA.

The human hepatocellular carcinoma cell line PLC/PRF/5, which synthesizes and secretes hepatitis B surface antigen, was grown under optimal conditions in tissue culture, using Eagle's minimal essential medium supplemented with 10% fetal bovine serum and 10(-11) M triiodothyronine on collagen rafts. Injection s.c. of the PLC/PRF/5 cell line into athymic BALB/c nude mice resulted in the growth of a well-circumscribed, moderately differentiated hepatocellular carcinoma. The intervals until tumor appearance and tumor "take" rates were dependent on inoculum dose. Four to 5 x 10(6) cells induced tumor growth in 29% of 14 injected mice within 29 to 40 days, while 7 to 13 X 10(6) cells induced tumors in all 15 mice within 10 to 12 days after inoculation. Hepatitis B surface antigen was detected in the nude mouse serum and tumor tissue, and its concentration roughly correlated with tumor weight. A low level of antibody against hepatitis B surface antigen was detected in five tumor-bearing animals, as well as in one mouse which did not produce a tumor. Hepatitis B core antigen and its antibody and hepatitis B e antigen and its antibody were not detected in 26 mice, using immunohistochemical and radioimmunoassay methods. alpha-Fetoprotein, carcinoembryonic antigen, and alpha-antitrypsin were detected in nude mice tumors, using the peroxidase-antiperoxidase technique. Finally, hepatitis B virus DNA, identified in the nude mouse tumor by molecular hybridization techniques, was compared to PLC/PRF/5 cell line hepatitis B virus DNA.

The kinetics of sulfobromophthalein uptake by rat liver sinusoidal vesicles.

The kinetics of bromo[35S]sulfophthalein (35S-BSP) binding by and uptake across the hepatocyte sinusoidal membrane were investigated using isolated rat liver sinusoidal membrane vesicles containing K+ as the principal internal inorganic cation. Uptake of 35S-BSP into vesicles was found to be temperature dependent, with maximum uptake between 35 and 40 degrees C; only binding occurred at or below 15 degrees C. Uptake at 37 degrees C was saturable and resolvable by Eadee-Hofstee analysis into two components: one with high affinity (Km = 53.1 microM) but low capacity, and the second of low affinity (Km = 1150 microM) but high capacity. By pre- or post-incubation, respectively, with unlabelled BSP, trans-stimulation and counter transport of 35S-BSP could also be demonstrated in these vesicles. Uptake was inhibited competitively using 5 microM Rose bengal and 10 microM indocyanine green, and non-competitively using 10 microM DIDS. Taurocholate did not inhibit uptake, and actually enhanced transport at concentrations greater than or equal to 250 microM. Imposition of inwardly directed inorganic ion gradients resulted in the enhancement of 35S-BSP transport when chloride ions were part of this gradient, irrespective of the cation employed whereas there was no apparent cation effect. However, substitution of 10 mM Na+ for 10 mM K+ as the internal cation resulted in a significant increase in uptake in the presence of external K+ as compared to Na+ gradients. This effect was not observed when 10 mM Tris+ was employed as the internal cation. The kinetics of 35S-BSP uptake by isolated sinusoidal membrane vesicles are indicative of facilitated transport. While the observed inorganic ion effects suggest a possible electrogenic component, the driving forces for hepatic BSP uptake remain uncertain. Isolated sinusoidal membrane vesicles provide a useful technique for studying hepatic uptake processes independent of circulatory or subsequent cellular phenomena.