Development and application of a novel recombinant Aleuria aurantia lectin with enhanced core fucose binding for identification of glycoprotein biomarkers of hepatocellular carcinoma.

The Aleuria aurantia lectin (AAL) derived from orange peel fungus contains five fucose-binding sites that recognizes fucose bound in α-1,2, α-1,3, α-1,4, and α-1,6 linkages to N-acetylglucosamine and galactose. Recently, we have created several recombinant AAL (rAAL) proteins that had altered binding affinity to fucose linkages. In this report, we further characterize the binding specificity of one of the mutated lectins, N224Q lectin. This lectin was characterized by lectin Western blotting, surface plasmon resonance, and glycan microarray and shown to have increased binding to fucosylated glycan. Subsequently, we used this lectin to identify secreted fucosylated glycoproteins from a fetal hepatic cell line. Proteomic analysis revealed several glycoproteins secreted by the fetal cell line that were bound by N224Q lectin. These findings were confirmed by subsequent proteomic analysis of human serum from control patients or patients with hepatocellular carcinoma. These represent candidate oncofetal markers for liver cancer.

Glucosidase inhibition enhances presentation of de-N-glycosylated hepatitis B virus epitopes by major histocompatibility complex class I in vitro and in woodchucks.

UNLABELLED: In this report, the possibility of pharmacologically altering the hepatitis B virus (HBV) epitopes presented by major histocompatibility complex class I on infected cells is demonstrated. The HBV middle envelope glycoprotein (MHBs) maturation appears to require calnexin-mediated folding. This interaction is dependent on glucosidases in the endoplasmic reticulum. Prevention of HBV envelope protein maturation in cultured cells through use of glucosidase inhibitors, such as 6-O-butanoyl castanospermine and N-nonyl deoxynorjirimycin, resulted in MHBs degradation by proteasomes. The de-N-glycosylation associated with polypeptide degradation was predicted to result in conversion of asparagine residues into aspartic acid residues. This prediction was confirmed by showing that peptides corresponding to the N-glycosylation sequons of MHBs, but with aspartic acid replacing asparagine, (1) can prime human cytotoxic T lymphocytes that recognize HBV-producing cells and (2) that the presentation of these envelope motifs by major histocompatibility complex class I is enhanced by incubation with glucosidase inhibitors. Moreover, although peripheral blood mononuclear cells isolated from woodchucks chronically infected with woodchuck hepatitis virus and vaccinated with woodchuck hepatitis virus surface antigen could be induced to recognize the natural MHBs asparagine-containing peptides, only cells isolated from animals treated with glucosidase inhibitor recognized the aspartic acid-containing peptides. CONCLUSION: These data suggest that pharmacological intervention with glucosidase inhibitors can alter the MHBs epitopes presented. This editing of the amino acid sequence of the polypeptide results in a new epitope, or "editope", with possible medical significance.

Role of chromosome 1q copy number variation in hepatocellular carcinoma.

Chromosome 1q often has been observed to be amplified in hepatocellular carcinoma. This review summarizes literature reports of multiple genes that have been proposed as possible 1q amplification drivers. These largely fall within 1q21-1q23. In addition, publicly available copy number alteration data from The Cancer Genome Atlas project were used to identify additional candidate genes involved in carcinogenesis. The most frequent location for gene amplification was 1q22, consistent with the results of the literature search. The genes TPM3 and NUF2 were found to be candidates whose amplification and/or mRNA up-regulation was most highly associated with poorer hepatocellular carcinoma outcomes.

A Decade of Disproportionality: A State-Level Analysis of African American Students Enrolled in the Primary Disability Category of Speech or Language Impairment.

Purpose This study aimed to determine if African American students were disproportionately represented between the years of 2004 and 2014 in the primary disability category of Speech or Language Impairment (S/LI) under the 2004 reauthorized Individuals with Disabilities Education Improvement Act. Method S/LI enrollment data from the Office of Special Education Programs and general enrollment data from the National Center for Education Statistics were analyzed to compare the risk of primary S/LI category enrollment of African American students to that of all other students. Risk ratios with 99% confidence intervals were calculated for each state across the 10 years studied. Results An average of 75% of states disproportionately represented African American students in the S/LI category each year; on average, 62% underrepresented African American students, and 14% overrepresented them. A post hoc analysis of the relationship between African American student representation and population densities revealed that states with high African American population densities almost exclusively underrepresented African American students and states with low densities tended toward a proportionate representation. Conclusions African American students were largely underrepresented in the category of S/LI in the years studied. These findings, alongside historic and chronic overrepresentation in other categories of special education, are discussed in the context of the fragmented harm theory ( Payne, 1984 ; Voulgarides, 2018 ; Voulgarides, Zwerger, & Noguera, 2013 ) and the disability rights and critical race theory ( Annamma, Connor, & Ferri, 2013 ). Supplemental Material https://doi.org/10.23641/asha.7967024.

Expression of genes that control core fucosylation in hepatocellular carcinoma: Systematic review.

BACKGROUND: Changes in N-linked glycosylation have been observed in the circulation of individuals with hepatocellular carcinoma. In particular, an elevation in the level of core fucosylation has been observed. However, the mechanisms through which core fucose is increased are not well understood. We hypothesized that a review of the literature and related bioinformatic review regarding six genes known to be involved in the attachment of core fucosylation, the synthesis of the fucosylation substrate guanosine diphosphate (GDP)-fucose, or the transport of the substrate into the Golgi might offer mechanistic insight into the regulation of core fucose levels. AIM: To survey the literature to capture the involvement of genes regulating core N-linked fucosylation in hepatocellular carcinoma. METHODS: The PubMed biomedical literature database was searched for the association of hepatocellular carcinoma and each of the core fucose-related genes and their protein products. We also queried The Cancer Genome Atlas Liver hepatocellular carcinoma (LIHC) dataset for genetic, epigenetic and gene expression changes for the set of six genes using the tools at cBioportal. RESULTS: A total of 27 citations involving one or more of the core fucosylation-related genes (FPGT, FUK, FUT8, GMDS, SLC35C1, TSTA3) and hepatocellular carcinoma were identified. The same set of gene symbols was used to query the 371 patients with liver cancer in the LIHC dataset to identify the frequency of mRNA over or under expression, as well as non-synonymous mutations, copy number variation and methylation level. Although all six genes trended to more samples displaying over expression relative to under-expression, it was noted that a number of tumor samples had undergone amplification of the genes of the de novo synthesis pathway, GMDS (27 samples) and TSTA3 (78 samples). In contrast, the other four genes had undergone amplification in 2 or fewer samples. CONCLUSION: Amplification of genes involved in the de novo pathway for generation of GDP-fucose, GMDS and TSTA3, likely contributes to the elevated core fucose observed in hepatocellular carcinoma.

N-linked glycosylation of the liver cancer biomarker GP73.

The association between elevated circulating levels of GP73 (and fucosylated GP73 in particular) and hepatocellular carcinoma suggests that a thorough analysis of the extent of GP73 glycosylation is warranted. Detailed analysis of the glycosylation patterns of such low abundance proteins are hampered by technical difficulties. Using conventional lectin affinity chromatography, we have established that three quarters of the GP73 secreted from a cell line derived from HCC is fucosylated. Using mass spectrometry, we have established that at least two of three potential sites of N-linked glycosylation are occupied on most molecules of GP73 secreted from cultured hepatoma cells. Furthermore, the oligosaccharides added to recombinant GP73 resemble those present in the bulk of secreted protein, mostly bi-antennary with core fucose, with a smaller fraction of tri- and tetra-antennary structures. The frequency of fucosylation observed on the recombinant protein agrees well with the pattern of lectin binding of the endogenous secreted protein. Finally, we have developed a method to interrogate the glycans added to either the near full length protein or at a particular sequon, providing proof of concept that a small peptide embedded in a heterologous context can preserve both fucosylation and a high level of branching of oligosaccharides added.

Antiviral profiles of novel iminocyclitol compounds against bovine viral diarrhea virus, West Nile virus, dengue virus and hepatitis B virus.

The antiviral activity of iminocyclitol compounds with a deoxynojirimycin (DNJ) head group and either a straight chain alkyl or alkylcycloalkyl group attached to the nitrogen atom have been tested in vitro against multiple-enveloped viruses. Several of these analogues were superior to previously reported DNJ compounds. Iminocyclitols that inhibit the glycan-processing enzyme endoplasmic-reticular glucosidase have been shown to inhibit the morphogenesis of viruses that bud from the endoplasmic reticulum (ER) at non-cytotoxic concentrations. Bovine viral diarrhoea virus (BVDV) has been used as a surrogate system for study of the hepatitis C virus, which belong to the virus family (Flaviviridae) as West Nile virus (WNV) and dengue virus (DV). N-Nonyl-DNJ (NNDNJ) was previously reported to have micromolar antiviral activity against BVDV, but a limiting toxicity profile. N-Butylcyclohexyl-DNJ (SP169) was shown to be as potent as NNDNJ in assays against BVDV and less toxic. However, it was inactive against hepatitis B virus (HBV). The present study reports efforts to improve the performance profiles of these compounds. Introduction of an oxygen atom into the N-alkyl side chain of DNJ, either as an ether or a hydroxyl functionality, reduced toxicity but sacrificed potency. Introduction of a hydroxyl group at the tertiary carbon junction of the cycloalkyl and linear alkyl group, as in N-pentyl-(1-hydroxycyclohexyl)-DNJ (OSL-9511), led to a structure that was as well tolerated as DNJ (CC50>500 microM), but retained micromolar antiviral activity against all ER morphogenesis budding viruses tested: BVDV, WNV, DV and HBV. The implication of this modification to the development of broad-spectrum antiviral agents is discussed.

Intrinsic hepatocyte dedifferentiation is accompanied by upregulation of mesenchymal markers, protein sialylation and core alpha 1,6 linked fucosylation.

Alterations in N-linked glycosylation have long been associated with cancer but for the most part, the reasons why have remained poorly understood. Here we show that increased core fucosylation is associated with de-differentiation of primary hepatocytes and with the appearance of markers indicative of a transition of cells from an epithelial to a mesenchymal state. This increase in core fucosylation was associated with increased levels of two enzymes involved in α-1,6 linked fucosylation, GDP-mannose 4, 6-dehydratase (Gmds) and to a lesser extent fucosyltransferase 8 (Fut8). In addition, the activation of cancer-associated cellular signaling pathways in primary rat hepatocytes can increase core fucosylation and induce additional glycoform alterations on hepatocyte proteins. Specifically, we show that increased levels of protein sialylation and α-1,6-linked core fucosylation are observed following activation of the ß-catenin pathway. Activation of the Akt signaling pathway or induction of hypoxia also results in increased levels of fucosylation and sialylation. We believe that this knowledge will help in the better understanding of the genetic factors associated with altered glycosylation and may allow for the development of more clinically relevant biomarkers.

Assays for glucosidase inhibitors with potential antiviral activities: secreted alkaline phosphatase as a surrogate marker.

As secretion of the middle (MHBs) glycoprotein of hepatitis B virus is highly dependent upon the action of the host oligosaccharide processing enzymes glucosidase I and II, drugs that inhibit this enzyme have been proposed as potential antiviral agents. To facilitate the identification of new, more effective inhibitors of MHBs secretion, an assay has been developed based on the expression of this glycoprotein alone by transfection of Huh7 hepatoma cells. The data clearly demonstrate that both mono- and di-glycosylated forms of MHBs are produced in this system and both forms are equally dependent upon glucosidase processing for secretion. In addition, inclusion of a co-transfected reporter construct that encodes secreted alkaline phosphatase (SEAP) to permit normalization of transfection revealed that the SEAP gene product was itself sensitive to glucosidase inhibition. This sensitivity also was observed in HepG2 human hepatoma cells. Thus, measuring SEAP secretion may be another method for evaluating glucosidase inhibition. In addition, this finding has important implications for the use of a SEAP reporter in screens of potential antiviral agents.

Increased levels of tetra-antennary N-linked glycan but not core fucosylation are associated with hepatocellular carcinoma tissue.

BACKGROUND: Alterations in glycosylation have long been associated with the development of cancer. In the case of primary hepatocellular carcinoma (HCC), one alteration that has often been associated is increased amounts of fucose attached to the N-glycans of serum proteins secreted by the liver. METHODS: In an effort to determine the origin of this increased fucosylation, we have conducted N-linked glycan analysis of HCC tissue, the surrounding nontumor tissue, and compared this to tissue from a nondiseased adult liver. RESULTS: Surprisingly, no difference in the level of fucosylation was observed from the three donor groups, suggesting that the increased levels of fucosylation observed in serum of those with HCC is not the result of increased synthesis of fucosylated proteins in the cancer tissue. On the other hand, increased levels of a tetra-antennary glycan were observed in the HCC tissue as compared with the surrounding tissue or to the nondiseased livers. CONCLUSIONS: This represents, to our knowledge, one of the first reports associating increased levels of branching with the development of HCC. IMPACT: The identification of increased levels of tetra-antennary glycan on liver tumor tissue, as opposed to adjacent or nondiseased tissue may lead to improved detection of HCC.

Interleukin-6 and oncostatin M are elevated in liver disease in conjunction with candidate hepatocellular carcinoma biomarker GP73.

The Golgi phosphoprotein GP73 is elevated in the circulation of individuals with a diagnosis of hepatocellular carcinoma. Its usefulness as a biomarker of HCC is questioned, since it has also been reported to be elevated in the circulation of people with liver cirrhosis. Regulation of GP73 by inflammatory cytokines is therefore of interest. The interleukin-6 (IL-6) family cytokines were tested for effects on GP73 mRNA and/or protein levels in human hepatoblastoma HepG2 cells. Levels of GP73 mRNA and protein were up-regulated in HepG2 cells following treatment with either proinflammatory cytokine IL-6 or the related cytokine oncostatin M (OSM). Induction required the shared receptor subunit gp130, and correlated with increased tyrosine phosphorylation of STAT3. Maximal cytokine-mediated induction was not observed in the presence of protein synthesis inhibitor cycloheximide, suggesting additional regulatory factors play an important role. ELISA measurement of GP73 and IL-6 levels in the sera of patients with pre-malignant liver disease revealed a significant correlation between circulating levels of the two proteins. Similarly, a sensitive ELISA assay was developed to measure circulating OSM. OSM levels were elevated 6-7 fold in sera from patients with either cirrhosis or HCC relative to controls without liver disease. Although there was an association between levels of GP73 and OSM in serum from people with liver cirrhosis, there was not a statistically significant correlation in HCC, suggesting that the role of the cytokines in determining circulating levels may be complex. To our knowledge, this is the first report of OSM elevation being associated with liver disease.

Linkage specific fucosylation of alpha-1-antitrypsin in liver cirrhosis and cancer patients: implications for a biomarker of hepatocellular carcinoma.

BACKGROUND: We previously reported increased levels of protein-linked fucosylation with the development of liver cancer and identified many of the proteins containing the altered glycan structures. One such protein is alpha-1-antitrypsin (A1AT). To advance these studies, we performed N-linked glycan analysis on the five major isoforms of A1AT and completed a comprehensive study of the glycosylation of A1AT found in healthy controls, patients with hepatitis C- (HCV) induced liver cirrhosis, and in patients infected with HCV with a diagnosis of hepatocellular carcinoma (HCC). METHODOLOGY/PRINCIPAL FINDINGS: Patients with liver cirrhosis and liver cancer had increased levels of triantennary glycan-containing outer arm (alpha-1,3) fucosylation. Increases in core (alpha-1,6) fucosylation were observed only on A1AT from patients with cancer. We performed a lectin fluorophore-linked immunosorbent assay using Aleuria Aurantia lectin (AAL), specific for core and outer arm fucosylation in over 400 patients with liver disease. AAL-reactive A1AT was able to detect HCC with a sensitivity of 70% and a specificity of 86%, which was greater than that observed with the current marker of HCC, alpha-fetoprotein. Glycosylation analysis of the false positives was performed; results indicated that these patients had increases in outer arm fucosylation but not in core fucosylation, suggesting that core fucosylation is cancer specific. CONCLUSIONS/SIGNIFICANCE: This report details the stepwise change in the glycosylation of A1AT with the progression from liver cirrhosis to cancer and identifies core fucosylation on A1AT as an HCC specific modification.

Inhibition of cellular alpha-glucosidases results in increased presentation of hepatitis B virus glycoprotein-derived peptides by MHC class I.

Inhibitors of alpha glucosidases prevent the trimming of oligosaccharides on certain nascent glycoproteins, including the hepatitis B virus MHBs envelope glycoprotein. MHBs proteins with untrimmed oligosaccharides do not interact with calnexin, increasing protein misfolding and subsequent degradation by proteasomes. As peptides loaded onto newly synthesized MHC class I complexes are predominantly derived from proteasomes, the possibility that glucosidase inhibition could increase presentation by MHC class I was determined. Using either a model epitope, or a natural MHBs epitope, it was demonstrated that glucosidase inhibitors enhanced presentation by MHC class I and promoted activation of antigen-specific CTLs, suggesting a pharmacologic approach to immune modulation.

Detection of mutated K-ras DNA in urine, plasma, and serum of patients with colorectal carcinoma or adenomatous polyps.

Our previous studies demonstrated that urine contains DNA derived from the circulation and that this DNA originated, in part, from organ sites and tumors distal to the urinary tract. To explore the potential use of DNA from urine as compared to other body fluids as a source for circulating DNA for cancer detection, the DNA concentration and the frequency of detection of mutated Kristin-ras (K-ras) DNA in serum, plasma, and urine were examined. The concentration of DNA in the urine was similar to that in the serum, but the DNA concentration in plasma was significantly lower than in either urine or serum (P < 0.05). When DNA derived from 10 muL of body fluid was used in each mutation assay, the detection frequency of mutated K-ras DNA was comparable among serum, plasma, and urine. However, when DNA derived from 200 muL of body fluid was used, the incidence of detecting mutated K-ras DNA in urine was significant higher (95%) than in either serum (35%) or plasma (40%) (P < 0.0005), suggesting that inhibitory factors in serum/plasma may be more limiting than in urine. The use and practicality of urine as a source of circulating DNA for cancer detection are discussed.

Overexpression of SR proteins and splice variants modulates chondrogenesis.

Fibronectin alternative exon EIIIA is largely included in undifferentiated mesenchymal cells of the developing limb bud, whereas the exon is excluded in differentiated chondrocytes. Inclusion of exon EIIIA in chondrocytic cells is increased by overexpression of SRp40, and, to a lesser extent, SRp75, but not SRp55. RT-PCR analysis using real-time PCR revealed that the levels of the mRNAs for these three proteins did not vary significantly in chick chondrocytes versus mesenchymal cells of the developing limb bud. However, a variant spliced form of SRp40, termed, SRp40LF, is detected preferentially in chondrocytes and in chondrifying mesenchymal cells. Forced overexpression of SRp40 or SRp75, but not SRp55, enhanced chondrogenic differentiation of chick limb mesenchymal cells in a high-density micromass assay. Overexpression of SRp40LF, which produces a truncated form of SRp40, also was strongly pro-chondrogenic. In a HeLa cell-based assay, SRp40LF fails to substitute for SRp40 in mediating an increase in exon EIIIA inclusion, suggesting that the latter event is not essential for the pro-chondrogenic effect. These results demonstrate the ability of these highly conserved splicing factors to modulate chondrogenesis and are consistent with earlier results that implicated exon EIIIA-containing isoforms of fibronectin in formation of chondrogenic condensations.

The role of the downstream signal sequences in the maturation of the HBV middle surface glycoprotein: development of a novel therapeutic vaccine candidate.

The signal sequences that mediate entry of the hepatitis B virus (HBV) envelope proteins into the endoplasmic reticulum (ER) are located within the S domain at positions 11-32 and at positions 80-98 (from the start of the S domain). In addition, hydrophobic patches at positions 160-184 and 189-210 of the S domain may also be involved in entry into the ER. The role of each of these domains in the entry of the HBV M glycoprotein into the ER was studied by deletion mutations of each of the signal sequences. Glycosylation of proteins was used as a marker of entry into the ER. Our results indicate that association with the ER could not be prevented by the deletion of either individual or combinations of the HBV signal sequences. M protein lacking signal sequence I was able to enter the ER and had limited secretion. In contrast, M protein lacking signal sequence II could not be secreted but still entered the ER. M protein lacking signal sequences I and II, while still associated with the ER, was rapidly degraded by the cytosolic proteasome. The potential use of such a vector as a CTL vaccine was tested through an in vitro antigen presentation assay. In this assay, a DNA vaccine candidate lacking signal sequences I and II lead to a >6-fold increase in CTL activation, as compared to the vector expressing wild type M protein. These results suggest that increased degradation of the HBV envelope proteins can lead to enhanced antigen presentation.

Herpes simplex virus type 1 ICP4 deletion mutant virus d120 infection failed to induce apoptosis in nerve growth factor-differentiated PC12 cells.

It has been suggested that terminally differentiated neuronal cells and mitotic cells respond differently in many aspects to herpes simplex virus type 1 (HSV-1) infection. The ICP4-deleted, Us3-defective, HSV-1 mutant strain d120 induces classical apoptosis in a variety of mitotic cell lines. Its behavior in postmitotic cells is not known. Here the authors report that mutant d120 virus failed to induce apoptosis in neuronal-like, nerve growth factor (NGF)-differentiated PC12 cells. More strikingly, rather than inducing apoptosis, d120 infection prolonged the life of nondividing NGF-differentiated PC12 cells in the culture flask. The virus genome had a half-life of 30 days. Unlike in other cells, such as Vero, neither wild-type nor d120 infection of NGF-differentiated PC12 cells induced the nuclear factor (NF)-kappa B p65 pathway, which has been associated with virus-induced apoptosis. Thus, the authors demonstrate, for the first time, that a potent apoptosis inducer mutant d120 failed to induce apoptosis in neuronal-like NGF-differentiated PC12 cells, unlike a number of other cell lines studied. The possible mechanisms involved in the failure of d120 to induce apoptosis in neuronal-like NGF-differentiated PC12 cells are discussed.

Transforming growth factor-beta1 regulates fibronectin isoform expression and splicing factor SRp40 expression during ATDC5 chondrogenic maturation.

Fibronectin (FN) isoform expression is altered during chondrocyte commitment and maturation, with cartilage favoring expression of FN isoforms that includes the type II repeat extra domain B (EDB) but excludes extra domain A (EDA). We and others have hypothesized that the regulated splicing of FN mRNAs is necessary for the progression of chondrogenesis. To test this, we treated the pre-chondrogenic cell line ATDC5 with transforming growth factor-beta1, which has been shown to modulate expression of the EDA and EDB exons, as well as the late markers of chondrocyte maturation; it also slightly accelerates the early acquisition of a sulfated proteoglycan matrix without affecting cell proliferation. When chondrocytes are treated with TGF-beta1, the EDA exon is preferentially excluded at all times whereas the EDB exon is relatively depleted at early times. This regulated alternative splicing of FN correlates with the regulation of alternative splicing of SRp40, a splicing factor facilitating inclusion of the EDA exon. To determine if overexpression of the SRp40 isoforms altered FN and FN EDA organization, cDNAs encoding these isoforms were overexpressed in ATDC5 cells. Overexpression of the long-form of SRp40 yielded an FN organization similar to TGF-beta1 treatment; whereas overexpression of the short form of SRp40 (which facilitates EDA inclusion) increased formation of long-thick FN fibrils. Therefore, we conclude that the effects of TGF-beta1 on FN splicing during chondrogenesis may be largely dependent on its effect on SRp40 isoform expression.

A substituted tetrahydro-tetrazolo-pyrimidine is a specific and novel inhibitor of hepatitis B virus surface antigen secretion.

The high levels of hepatitis B virus (HBV) surface antigen (HBsAg)-bearing subviral particles in the serum of chronically infected individuals are thought to play a role in suppressing the HBV-specific immune response. Current therapeutics are not directed at reducing this viral antigenemia; thus, our group has focused on identifying inhibitors of HBsAg secretion. By using the HBV-expressing cell line HepG2.2.15, high-throughput screening of an 80,288-compound synthetic small-molecule library identified HBF-0259, an aromatically substituted tetrahydro-tetrazolo-(1, 5-a)-pyrimidine. Following resynthesis, HBF-0259 had a 50% effective concentration of approximately 1.5 microM in a secondary, HBV-expressing cell line, with a concentration that exhibited 50% cytotoxicity of >50 microM. The equilibrium concentration of HBF-0259 in aqueous solution at physiological pH was 15 to 16 microM; the selective index was thus >9. As intended by our screening paradigm, HBF-0259 is a selective, potent inhibitor of secretion of both subviral and DNA-containing viral particles, while the secretion of alpha-1-acid glycoprotein and alpha-1-antitrypsin was unaffected. The HBV e antigen, which is not a constituent of HBV particles, was also unaffected, suggesting that the secretion of particles bearing HBV structural glycoproteins is targeted directly. Inhibitory activity was also confirmed by transfection of HBsAg, indicating that the action of the compound is independent of those of other viral proteins. HBF-0259 had no effect on HBV DNA synthesis, demonstrating that inhibition is independent of viral genomic replication. Finally, HBF-0259 had little or no effect on the cell-to-cell spread of two unrelated viruses, suggesting that it is a specific inhibitor of secretion of HBsAg. Possible mechanisms of action and the implications for its development are discussed.

Transforming growth factor-beta1 (TGF-beta1) regulates ATDC5 chondrogenic differentiation and fibronectin isoform expression.

Regulated splicing of fibronectin (FN) occurs during the mesenchymal to chondrocyte transition and ultimately results in the relative enrichment of an extra domain B (EDB) exon-containing FN isoform with the suggestion that FN isoforms may play a functional role in chondrogenesis. Promotion of chondrogenesis can also be achieved by treatment with transforming growth factor-beta (TGF-beta), which also regulates FN isoform expression. We have examined the effects of TGF-beta treatment on the assumption of the chondrogenic phenotype in the teratoma-derived cell line ATDC5 and tested whether these effects on chondrogenesis are paralleled by appropriate changes in FN isoform expression. ATDC5 cells were maintained in a pre-chondrogenic state and, in this state, treated with 10 ng/ml TGF-beta. The cells started to elaborate a matrix rich in sulfated proteoglycans, such that within the first 12 days of culture, TGF-beta1 treatment appeared to slightly accelerate early acquisition of an Alcian blue-stained matrix, and caused a dose- and time-dependent decrease in collagen type I expression; changes in collagen type II expression were variable. At later times, cells treated with TGF-beta became indistinguishable from those of the controls. Interestingly, TGF-beta treatment caused a significant dose- and time-dependent decrease in the proportion of FN containing the extra domain A (EDA) and the EDB exons. These data suggest that TGF-beta induces the early stages of chondrogenic maturation in this pre-chondrogenic line and that TGF-beta treatment increases expression of FN isoforms that lack the EDA and EDB exons.