Identification and characterization of a structural protein of hepatitis B virus: a polymerase and surface fusion protein encoded by a spliced RNA

The hepatitis B virus (HBV) genome is known to contain four conserved and overlapped open reading frames (ORFs) encoding the viral core, polymerase (P), surface (S), and X proteins. Whether HBV encodes other proteins has long been a major interest in the field. Using (32)P-labeling of an introduced protein kinase A site attached to the N- or C-terminus of the HBV polymerase gene, a 43-kDa P-S fusion protein was detected in cell lysate, secreted virions, and 22-nm subviral particles. Immunobiochemical studies showed that the 43-kDa protein contains the epitopes of the N-terminus of polymerase and most parts of the surface proteins. This 43-kDa protein was shown to be a glycoprotein, similar to the surface protein. RT-PCR and sequence analyses identified a spliced mRNA which was derived from pregenomic RNA with a deletion of 454 nucleotides (nt) from nt 2447 to 2902. This splice event creates a P-S fusion ORF. This finding is consistent with the result obtained from an immunobiochemical study. Mutations at the splice donor or acceptor site on the HBV genome abrogated the production of the 43-kDa protein. These mutants had no effect on viral replication in transfected HuH-7 cells. However, this P-S fusion protein is able to substitute for the LS protein in virion maturation. On the basis of these results, we conclude that the 43-kDa protein is a polymerase-surface fusion protein encoded by a spliced RNA. Similar to the LS protein, the 43-kDa P-S fusion protein is a structural protein of HBV and might play a role in the HBV life cycle.     

The possible role of protein degradation in altered membrane structure and function

Possible changes in membrane lipid assemetry may result in altered function with aging. Membrane proteolysis is an additional factor which must be considered, both with respect to modulation of membrane function and also as a methodological problem in analyses of membrane dynamics.     

Cytochrome P450 1A1 and manganese superoxide dismutase genes polymorphisms in ankylosing spondylitis

OBJECTIVES: To investigate the associations of cytochrome p450 1A1 (CYP1A1) and manganese superoxide dismutase (MnSOD) genes polymorphisms with the susceptibility to AS in Taiwan. METHODS: The polymorphisms of CYP1A1 and MnSOD genes were determined in 70 patients with ankylosing spondylitis (AS) and 93 healthy controls by polymerase chain reaction (PCR)/restriction fragment length polymorphisms (RFLP) methods. RESULTS: The genotype frequency of CYP1A1 4887C/A was significantly lower in patients with AS than in controls. The phenotype frequency of CYP1A1 4887A also tended to be decreased in patients with AS. There were no significant differences in the genotype, allele, and phenotype frequencies of MnSOD gene polymorphisms between patients with AS and controls. CONCLUSION: CYP1A1 4887A may be a protective factor for the development of AS in Taiwan. However, MnSOD gene polymorphisms are not associated with the susceptibility to AS.     

Influence of the Length of the Lipooligosaccharide α Chain on Its Sialylation in Neisseria meningitidis

The sialylation of lipooligosaccharide (LOS) in Neisseria meningitidis plays a role in the resistance of the organism to killing by normal human serum. The length of the alpha chain extending out from the heptose I [Hep (I)] moiety of LOS influenced sialylation of N. meningitidis LOS in vitro and in vivo. The alpha chain required a terminal Gal and a trisaccharide or longer oligosaccharide to serve as an acceptor for sialylation. The disaccharide lactose (Galbeta1-4Glc) in the alpha chain of immunotype L8 LOS could not function as an acceptor for the sialyltransferase, probably due to steric hindrance imposed by the neighboring Hep (II) with phosphorylethanolamine and another group attached.     

Estimating the relative frequency of leukodystrophies and recommendations for carrier screening in the era of next‐generation sequencing

Leukodystrophies are a heterogeneous group of heritable disorders characterized by abnormal brain white matter signal on magnetic resonance imaging (MRI) and primary involvement of the cellular components of myelin. Previous estimates suggest the incidence of leukodystrophies as a whole to be 1 in 7,000 individuals, however the frequency of specific diagnoses relative to others has not been described. Next generation sequencing approaches offer the opportunity to redefine our understanding of the relative frequency of different leukodystrophies. We assessed the relative frequency of all 30 leukodystrophies (associated with 55 genes) in more than 49,000 exomes. We identified a relatively high frequency of disorders previously thought of as very rare, including Aicardi Goutières Syndrome, TUBB4A‐related leukodystrophy, Peroxisomal biogenesis disorders, POLR3‐related Leukodystrophy, Vanishing White Matter, and Pelizaeus‐Merzbacher Disease. Despite the relative frequency of these conditions, carrier‐screening laboratories regularly test only 20 of the 55 leukodystrophy‐related genes, and do not test at all, or test only one or a few, genes for some of the higher frequency disorders. Relative frequency of leukodystrophies previously considered very rare suggests these disorders may benefit from expanded carrier screening.     

Genome-wide profiling of oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a common malignancy, the incidence of which is particularly high in some Asian countries due to the geographically linked areca quid (AQ) chewing habit. In this study, array-based comparative genomic hybridization was used to screen microdissected OSCCs for genome-wide alterations. The highest frequencies of gene gain were detected for TP63, Serpine1, FGF4/FGF3, c-Myc and DMD. The highest frequencies of deletion were detected for Caspase8 and MTAP. Gained genes, classified by hierarchical clustering, were mainly on 17q21-tel; 20q; 11q13; 3q27-29 and the X chromosome. Among these, gains of EGFR at 7p, FGF4/FGF3, CCND1 and EMS1 at 11q13, and AIB1 at 20q were significantly associated with lymph node metastasis. The genomic profiles of FHIT and EXT1 in AQ-associated and non-AQ-associated OSCCs exhibited the most prominent differences. RT-PCR confirmed the significant increase of TP63 and Serpine1 mRNA expression in OSCC relative to non-malignant matched tissue. A significant increase in Serpine1 immunoreactivity was observed from non-malignant matched tissue to OSCC. However, there was no correlation between the frequent genomic loss of Caspase 8 and a significant decrease in Caspase8 expression. These data demonstrate that genomic profiling can be useful in analysing pathogenetic events involved in the genesis or progression of OSCC.     

The prevalence of IgE antibody reactivity against the alkaline serine protease major allergen of Penicillium chrysogenum increases with the age of asthmatic patients.

BACKGROUND: Penicillium species are prevalent airborne fungi. However, the prevalence of allergic sensitization to Penicillium antigens and the true impact of these ubiquitous fungi on atopic respiratory disorders remain to be determined. OBJECTIVE: The purpose of this study was to analyze the prevalence of immunoglobulin (Ig)E and IgG antibodies against Penicillium chrysogenum (Pen ch 13), the alkaline serine protease major allergen of P. chrysogenum, in asthmatic patients of different age groups. METHODS: Pen ch 13 was purified from a culture medium of P. chrysogenum. The reactivity of IgE and IgG antibodies to Pen ch 13 in the serum samples of 212 asthmatic patients was analyzed by immunoblotting methods. RESULTS: Sixty-nine (33%) of the 212 sera analyzed showed IgE and/or IgG immunoblot reactivity to Pen ch 13. Significant differences in the prevalence of IgE and/or IgG antibody reactivity to Pen ch 13 were found among eight different age groups of 212 asthmatic patients. The frequency of IgE-binding reactivity to Pen ch 13 increased significantly with the age of the patients. It was 7% for the group less than 10 years old and 42% for the group older than 70 years old. In addition, a significant difference between the prevalence of IgE (7%) and IgG (33%) antibodies against Pen ch 13 in the group aged 10 or less was also found. CONCLUSIONS: Our study demonstrates that IgE and IgG antibodies specific for Pen ch 13 were detected in approximately one-third of the 212 asthmatic patients analyzed. Our results suggest that allergic sensitization to Pen ch 13, and possibly to other airborne Penicillium species, is more common in older asthmatic patients.     

Comparison of different PCR approaches for characterization of Burkholderia (Pseudomonas) cepacia isolates.

In this study, we evaluated three PCR methods for epidemiological typing of Burkholderia (Pseudomonas) cepacia--PCR-ribotyping, arbitrarily primed PCR (AP-PCR) and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR)--and compared them with pulsed-field gel electrophoresis. The analysis was performed with 31 isolates of B. cepacia, comprising 23 epidemiologically unrelated isolates and 8 isolates collected from the same patient during two episodes of bacteremia. Pulsed-field gel electrophoresis, ERIC-PCR, and AP-PCR identified 23 distinct types among the 23 unrelated isolates, while PCR-ribotyping only identified 12 strain types, even after AluI digestion of the amplification products. Among the eight isolates collected from the same patient, all typing techniques revealed two clones of strains. The day-to-day reproducibilities of PCR-ribotyping and ERIC-PCR were good, while greater day-to-day variations were noted in the fingerprints obtained by AP-PCR. We conclude that all three PCR techniques are useful for rapid epidemiological typing of B. cepacia, but ERIC-PCR seems to be more reproducible and discriminative.     

Site-directed cytotoxic antibody against the C-terminal segment of the surface glycoprotein gp90 of avian reticuloendotheliosis virus

The major mature env-gene products of avian reticuloendotheliosis-associated virus (REV-A) are the surface glycoprotein (gp90) and the transmembrane protein (gp20). We have previously reported that gp90 was detected in the REV-A virus by Western blot analysis as well as in the REV-A-infected cells by radioimmunoprecipitation with antibodies raised in rabbits against the gp90 C-terminal tridecapeptide which was predicted from the nucleotide sequence (Wilhemsen et al., J. Virol., 52, 172, 1984). We have now shown that this antibody detected antigens on the REV-A-infected cells by fluorescence-activated cell sorter (FACS) analysis, and conferred specific cytotoxic effects on the infected cells in the presence of rabbit complement using the chromium release assay. These results clearly indicate that the C-terminal epitope of gp90 is situated on the surface of the REV-A-infected cells and accessible to site-directed antibodies which cause cytotoxicity by activating the complement system. The possible in vivo roles of this antibody are discussed.     

Thymoma and hypogammaglobulinemia (Good's syndrome): a case report.

Good's syndrome is extremely rare and refers to an acquired B and T cell immunodeficiency in thymoma patients. We report a 51-year-old female thymoma patient who presented with recurrent herpes zoster, pneumonia, diarrhea and opportunistic infections. She was found to have acquired hypogammaglobulinemia with absent B cells. Despite repeat intravenous immunoglobulin replacement and antibiotic therapy, she died of bacterial pneumonia-induced acute respiratory distress syndrome. Clinicians should look for evidence of immunologic dysfunction in thymoma patients presenting with recurrent infections.