Corynebacterium thomssenii sp. nov., a Corynebacterium with N-acetyl-beta-glucosaminidase activity from human clinical specimens.

A strain of a previously undescribed non-lipophilic coryneform bacterium was isolated from pleural fluids of a patient with chronic renal failure, stroke and pneumonia. Slow fermentative acid production from glucose, maltose and sucrose, and strong N-acetyl-beta-glucosaminidase activity were the most characteristic features of the bacterium. Chemotaxonomic characterization unambiguously indicated that the organism belonged to the genus Corynebacterium. The results of comparative 16S rRNA gene sequence analysis revealed that the isolate represented a new species within the genus, for which the name Corynebacterium thomssenii sp. nov. is proposed. The type strain is DSM 44276.

Human papillomavirus in tissue of bladder and bladder carcinoma specimens. A preliminary study.

OBJECTIVE: To evaluate the significance of HPV type 6b, 11, 16 and 18 together with type-specific antibodies in the serum of bladder carcinoma. METHODS: The prevalence of HPV type 6b, 11, 16 and 18 in bladder tumor, normal bladder and urethra together with type-specific antibodies in serum was investigated in 23 patients with bladder cancer and 9 patients with chronic cystitis. HPV DNA analysis was done by polymerase chain reaction (PCR). Open reading frames of HPV were expressed in Escherichia coli as beta-galactosidase fusion proteins. RESULTS: HPV 6b was demonstrated in the tumor tissue of 6 patients (19%), and in the nonmalignant specimens of 6 further patients (19%). HPV 16/18 was only found in the urethral swabs of 2 patients (6%). Anti-HPV antibodies were positive in 7 patients (22%). There was no association between the demonstration of HPV 6b and the occurrence of bladder tumor in this study. CONCLUSION: Though, in this study, HPV was not associated with bladder cancer, further investigation is necessary to elucidate the role of HPV 6b in bladder tissue possibly by a semiquantitative PCR in tissue samples and of anti-HPV antibodies in serum.

Diagnostic significance of antibodies to hepatitis B virus polymerase in acutely and chronically HBV-infected individuals.

The prevalence and time course of the occurrence of antibodies to the hepatitis B virus polymerase (anti-HBpol) were investigated in acutely and in chronically HBV-infected individuals by using recombinant HBpol protein for Western blot analysis. One group consisted of 19 patients who were acutely infected and recovered completely. Five of these patients (26%, 69 serum samples examined) exhibited anti-HBpol. Among those anti-HBpol positive patients, recovery from the disease was combined with a complete loss of this antibody. In contrast, in a second group of 15 individuals who developed chronic hepatitis B, 13 (87%, 102 serum samples examined) had anti-HBpol during the acute phase of the disease. The difference between the anti-HBpol prevalence rates of the two patient groups is statistically significant (Exact Fisher test, P < .002), implying that the occurrence of anti-HBpol may be indicative of a potential chronic course of hepatitis B. Remarkably, anti-HBpol was found in one case of a clinically suspected hepatitis B in which no other serological HBV parameters were found. This serum sample was positive in HBV PCR, supporting a possible diagnostic value of anti-HBpol.

Characterization of the endogenous protein kinase activity of the hepatitis B virus.

During the assembly of the nucleocapsid of the hepatitis B virus a protein kinase, probably of cellular origin, is encapsidated. This enzyme phosphorylates serine residue(s) localized within the lumen of the particle. By using purified, liver-derived core particles, we characterized the protein kinase activity in the presence of different ions and inhibitors. Controls were performed with cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) and recombinant core particles. We showed that the endogenous protein kinase of the core particles was not inhibited by H89, a specific inhibitor of PKA. Staurosporine, a selective inhibitor of PKC inhibited the endogenous kinase activity only within the first minutes of the reaction. In contrast, quercetine, a selective inhibitor of the protein kinase M (PKM) did not inhibit during the first minutes but inhibited efficiently during later phases of incubation. PKM represents an enzymatically active proteolytic fragment of PKC. These results suggest that PKC is encapsidated into human core particles and is converted to PKM during the in vitro reaction. This conclusion implies the association of a protease activity localized with the HBV nucleocapsid inside liver-derived core particles.

Association of hepatitis C virus in human sera with beta-lipoprotein.

Hepatitis C virus (HCV)-RNA in sera of patients with viral hepatitis C is supposed to be included, at least partially, into HCV particles. We found that the density of HCV-RNA-carrying material was variable, as determined by sucrose gradient density centrifugation (1.03-1.20 g/cm3). In some of the sera examined HCV-RNA was restricted to low densities between 1.03 and 1.08 g/cm3. In other sera additional densities of HCV-RNA were found distributed over the whole gradient with peaks at 1.12 and 1.17 and at 1.19-1.20 g/cm3. HCV-RNA banding at low densities could be completely co-precipitated with anti-beta lipoprotein, whereas HCV-RNA fractions of higher densities were only partially precipitated or not at all. In 8 of 20 sera directly examined, HCV-RNA could be completely and in 9 sera only partially co-precipitated by anti-beta lipoprotein. In 3 sera no significant precipitation could be observed.

Occurrence of antibodies to L1, L2, E4 and E7 gene products of human papillomavirus types 6b, 16 and 18 among cervical cancer patients and controls.

Sera from 118 women of 33 to over 90 years of age, with or without a history of cervical squamous-cell carcinoma, were examined for the presence of antibodies to HPV-6b, HPV-16 and HPV-18, L1, L2, E4, and E7 gene products by the use of bacterially derived beta-Gal fusion proteins and Western-blot analysis. Among the cervical cancer patients, 29/46 (63.0%) were positive for antibodies to E4 and/or E7 of HPV-16 and/or E7 of HPV-18. In contrast, only 2 of 31 (6.5%) non-genital cancer patients and 4 of 41 (9.8%) healthy individuals were antibody-positive for HPV-16 E4 or E7, while antibodies to the homologous proteins of HPV-18 could not be detected. Prevalence rates of antibodies to the HPV-16/18 late proteins were 25/46 (54.3%) in the cervical carcinoma group, 13/31 (41.9%) among women with non-genital cancer types, and 18/41 (43.9%) among normal, healthy individuals. Antibodies to HPV-6b late gene products ranged between 6.5% and 12.2% in the different patient groups. Antibodies to HPV-6b E4 and E7 were detected only once. By studying an additional control group of 207 women with a different age distribution, age-dependence of antibodies to HPV gene products could be ruled out. Whereas antibodies to late proteins may indicate that, regardless of clinical stage, HPV infections are wide-spread among the female population, the striking difference between the prevalence rates of antibodies to early proteins of HPV-16 and HPV-18 among cervical cancer patients and controls (p less than 0.001) supports the idea of the involvement of these virus types in carcinogenesis of the cervix.

Antibodies to human papillomavirus type-16 in human sera as revealed by the use of prokaryotically expressed viral gene products.

Open reading frames of human papillomaviruses were expressed in Escherichia coli as beta-galactosidase fusion proteins. These bacterially derived papillomaviral gene products were used to examine sera from 67 women (63 healthy subjects, 4 patients with genital carcinoma) for antibodies to papillomavirus type-16 antigens (E1, E2, E4, E5, E6, E7, L1, L2) and the L2 proteins of HPV-6b and HPV-18 by Western-blot analysis. The serologic data were compared with cytological findings classified according to Papanicolaou and with nucleic acid hybridization data from cervical smears of the same individuals. Twenty-three of the normal individuals showed antibodies exclusively directed against L2 gene products; whereas in the sera from the four genital cancer patients, antibodies to the early gene products E4 and/or E7 could be detected. In one case these antibodies were found to be combined with antibodies to L2 of HPV-16 and -18 and in another case with those to E1 and E2 of HPV-16. In none of the sera examined could antibodies to L1, E5 or E6 be identified. Three of the antibody positive normal women were found to be also positive for HPV-16/18 DNA, while all of the 40 seronegative women were HPV-16/18 DNA negative. These data indicate that serology may be a valuable means to study the epidemiology of genital human papillomavirus infection.

Identification of a binding site in the hepatitis B virus RNA pregenome for the viral Pol gene product.

The hepatitis B virus, although containing a DNA genome, replicates by reverse transcription of an RNA pregenome. The viral Pol gene encodes the reverse transcriptase which catalyzes viral DNA synthesis. To study the interaction of this protein with HBV RNA, the entire Pol gene product was expressed except its eight amino-terminal codons in Escherichia coli as fusion protein with beta-galactosidase. In the absence of competing nucleic acids full-length expression products were able to nonspecifically bind in vitro synthesized HBV RNAs of different polarity and length. However, if competed with an excess of unspecific RNA, only those HBV RNAs were bound which contained besides the direct repeats 1 and 2 nucleotide sequences downstream of direct repeat 1. The corresponding binding site was found to be located within the adjacent 134 nucleotides downstream of DR1. We conclude from our data that this region which is in part homologous to the U5 region of retroviral genomes may be important for the binding of the HBV Pol gene product to the viral pregenome.

Occurrence of human papillomavirus DNA types 16 and 18 (HPV-16/18) in cervical smears as compared to cytological findings.

Cervical smears from 2336 women were examined for the presence of HPV-16/18 by dot-blot hybridization using 32P-labeled HPV-16/18 DNA under high stringency conditions. The hybridization data were compared with cytological findings classified according to Papanicolaou. The ages of the patients ranged from under 20 to over 70 years. Ninety-eight (4.4%) of the 2237 cytologically normal cervical samples (Pap I and II) were HPV-16/18 positive. Thirteen out of 32 (40.6%) samples showing signs of mild and moderate dysplasia (Pap IIID) were found to be HPV-16/18 positive. In 5 out of 7 (71.4%) samples from women with severe dysplasia or carcinoma in situ (Pap IV) and in 9 out of 25 (32.1%) samples from patients with invasive cervical carcinoma (Pap V) HPV-16/18 DNA was detected. Thirty-two smears were from women with severe unspecific cervical inflammation (Pap III). Two (6.2%) out of them were HPV-16/18 positive. Normal smears showed an apparent age-dependent pattern of HPV-16/18 positivity with a peak prevalence of 10.6% among women younger than 20 years old. The majority of premalignant lesions was detected among women younger than 40 years old; whereas all invasive lesions were from women older than 39 years. Compared to the HPV-16/18 prevalence rate in normal smears, abnormal smears harbored HPV-16/18 DNA approximately 9 times more frequently. This finding supports the hypothesis that HPV-16/18 may be involved in the development of cervical cancer.

Expression of an antigenic polypeptide of the human parvovirus B19.

The DNA fragment of the human parvovirus B19, with 715 nucleotides between nucleotide positions 3141-3856 was expressed in Escherichia coli as a beta-galactosidase fusion protein. The plasmid vector pSS20d used for this purpose permits cleavage of the viral gene product from the beta-galactosidase moiety by collagenase. After purification by p-aminophenyl-beta-D-thiogalactoside-sepharose and superose, a soluble protein with a molecular mass of 28 kDa was isolated. It represents a common part of the viral capsid proteins VP1 and VP2. This bacterially derived parvoviral gene product can be used for detection of anti-B19 antibodies in human sera.

Precore sequence of hepatitis B virus inducing e antigen and membrane association of the viral core protein.

Hepatitis B virus (HBV) DNA contains a precore (pre-c) sequence of 29 codons with unknown function upstream of its gene for the major core protein. Its significance was studied by expression of core proteins with and without pre-c in Escherichia coli. Core protein without pre-c, P22c, assembled spontaneously to core particles and formed core antigen. It had the same size and antigenicity as core particles from infected liver. Core protein with pre-c, P25e, instead formed membrane-associated e antigen (HBeAg). The data suggest that pre-c functions as a signal peptide for the attachment of core protein P25e to cellular membranes. This hypothesis can explain the not yet understood relation between viremia and HbeAg and the protective role of anti-HBe antibody.

Mitochondrial L-rRNA from Aspergillus nidulans: potential secondary structure and evolution.

The alignment of gene sequences coding for A. nidulans mitochondrial L-rRNA and E. coli 23S rRNA indicates a strong conservation of primary and potential secondary structure of both rRNA molecules, except that homologies to the 5'-terminal 5.8S-like region and the 3'-terminal 4.5S-like region of bacterial rRNA are not detectable on mtDNA. The structural organization of the A. nidulans mt L-rRNA gene corresponds to that of yeast omega + strains: both genes are interrupted by a large intron sequence (1678 and 1143 bp, respectively) and by another smaller insert (91 and 66 bp) at homologous positions within domain V. An evolutionary tree derived from conserved L-rRNA gene sequences of yeast nuclei, E. coli, maize chloroplasts and six mitochondrial species exhibits a common root of organelle and bacterial sequences separating early from the nuclear branch.

Nucleotide sequence of Aspergillus nidulans mitochondrial genes coding for ATPase subunit 6, cytochrome oxidase subunit 3, seven unidentified proteins, four tRNAs and L-rRNA.

The complete nucleotide sequence of a 14 kb segment of A. nidulans mtDNA reveals a rather compact organization of genes transcribed from the same strand and coding for two functionally known proteins, seven unidentified polypeptides (URFs), 24 tRNAs and two rRNAs. One of the URFs is located in the intron of the L-rRNA gene and codes for a basic protein of 410 residues. The other URFs are in spacer regions and code for hydrophobic proteins. URFa is homologous to human URF4, and URFb produces a polypeptide of 48 residues resembling the human URF6L product (hydrophobic N-terminus, basic C-terminus). The ATPase subunit 6 genes from mitochondria and E. coli appear to share a common ancestor. The codon frequencies of identified genes and URFs are similar, and codons ending with G or C are rarely used. The structures of tRNAs specific for arginine, asparagine, tyrosine and histidine are deduced from gene sequences.

Nucleotide sequence of the Aspergillus nidulans mitochondrial gene coding for the small ribosomal subunit RNA: homology to E. coli 16S rRNA.

The complete primary structure of the 1437 bp gene coding for mitochondrial 15S rRNA and its flanking regions was determined by Maxam-Gilbert sequencing of cloned HindIII fragment H3 of A. nidulans mtDNA. The gene product reveals significant homology (59%) to E. coli 16S rRNA, and the potential secondary structures of both rRNA molecules are very similar, except that the hairpin structures 7, 8 and 30 of the Brimacombe 16S rRNA model are deleted, and that two sequences of 8 and 31 nucleotides are inserted in the mitochondrial species.