Biomedical subjects

M Sapp

Publications and source records attributed to M Sapp.

48 records · Page 3Linked to original sources

PubMed1995

Binding and internalization of human papillomavirus type 33 virus-like particles by eukaryotic cells.

Infection of cells by human papillomaviruses (HPVs) associated with malignant genital lesions has not been studied because of the lack of an in vitro system and the unavailability of virions. We have now used virus-like particles (VLPs) of HPV type 33 to analyze the initial events in the interaction of the HPV capsid with cell lines. Binding of VLPs to HeLa cells was observed in biochemical assays and by immunofluorescence. VLP binding was inhibited by antisera raised against VLPs but not by monoclonal antibodies recognizing either L1 or L2 epitopes accessible on VLPs. Under saturating conditions, approximately 2 x 10(4) VLPs were bound per cell, with a dissociation constant of about 100 pM. VLPs composed of L1 alone bound as well as VLPs composed of both capsid proteins, indicating that L2 is not required for initial binding. VLPs dissociated into capsomers did not bind, demonstrating that intercapsomer contacts are required. Neither capsomers nor simian virus 40 virions competed with VLP binding. Uptake of VLPs by small and smooth endocytic vesicles was demonstrated by immunoelectron microscopy. Cellular binding of VLPs was sensitive to trypsin but not to sialidase, N-glycosidase, or octyl-beta-D-glycopyranoside treatment, suggesting that a cell surface protein is involved in the VLP binding. Cell lines originating from a variety of tissues and organisms as distantly related as insects and humans bound VLPs with similar efficiency and specificity. Therefore, the putative receptor mediating VLP attachment should be highly conserved and cannot be responsible for the species and tissue specificity of HPVs.

Animals↗

PubMed1994

Assembly of the major and the minor capsid protein of human papillomavirus type 33 into virus-like particles and tubular structures in insect cells.

Native virions of human papillomaviruses (HPV) can be isolated from genital lesions only in very limited amounts. Recent studies have shown that virus-like particles can be obtained by expression of the capsid proteins using vaccinia virus recombinants or the baculovirus system. We now present the first detailed characterization of virus-like particles of a human papillomavirus associated with malignant genital lesions, HPV-33, produced in high yield using the baculovirus expression system. Assembly of the major capsid protein L1 alone or together with the minor capsid protein L2 has been obtained. Both spherical virus-like particles of 50-60 nm diameter and tubular structures of either 25-30 nm or 50-60 nm diameter and variable length were extracted from nuclei of the infected insect cells. However, predominantly 50- to 60-nm spherical particles were found in the cell culture medium in long-term infections. The particles have icosahedral symmetry and a density of 1.29 g/cm3 in cesium chloride corresponding to empty papillomavirus capsids. Immunoelectron microscopy confirmed the presence of L1 and L2 in the virus-like particles. The L2 protein seemed to be modified and was shown to be tightly associated with L1 using density gradient and sedimentation analysis. Approximately 50% of the L1 molecules are cross-linked by intermolecular disulfide bonds. This is the first example for the production of HPV-like particles containing both the major and the minor capsid protein using the baculovirus expression system.

Animals↗

PubMed1994

Analysis of type-restricted and cross-reactive epitopes on virus-like particles of human papillomavirus type 33 and in infected tissues using monoclonal antibodies to the major capsid protein.

A panel of six monoclonal antibodies recognizing at least three different antigenic regions has been raised against the L1 major capsid protein of human papillomavirus type 33 (HPV-33), which is associated with cervical carcinoma. The antigenic sites defined by these antibodies have been mapped and classified as type-restricted or broadly cross-reactive using bacterially expressed L1 fusion proteins of a variety of HPV types. Conformational and linear epitopes have been distinguished using native and denatured virus-like particles. HPV infection of genital lesions has been analysed using both monoclonal antibodies and DNA amplification by PCR. The antibodies obtained should be useful to probe the structure of HPV capsids and to develop a general assay for the detection and classification of productive HPV infections.

Amino Acid Sequence↗

PubMed1993

Development of type-specific and cross-reactive serological probes for the minor capsid protein of human papillomavirus type 33.

Human papillomavirus type 33 (HPV33) is associated with malignant tumors of the cervix. In an attempt to develop immunological probes for HPV33 infections, antisera against various bacterial fusion proteins carrying sequences of the minor capsid protein encoded by L2 were raised in animals. Antigenic determinants on the HPV33 L2 protein were identified by using truncated fusion proteins and were classified as type specific or cross-reactive with respect to HPV1, -8, -11, -16, and -18. Cross-reactive epitopes map to amino acids 98 to 107 or to amino acids 102 to 112 and 107 to 117, respectively, depending on the fusion protein used for immunization. Antibodies directed toward these epitopes detect L2 proteins of HPV11, -16, and -18, but not of HPV1 and -8, in Western immunoblots and enzyme-linked immunosorbent assays. HPV33 L2 amino acids 82 to 94 and 117 to 130 induce type-specific antibodies, with the major response directed to amino acids 117 to 130. By using a synthetic peptide corresponding to L2 amino acids 117 to 130, high-titered, type-specific antisera were obtained. These antisera should be useful as immunological probes for HPV33 infection.

Amino Acid Sequence↗

PubMed1992

Human papillomavirus type 42: new sequences, conserved genome organization.

We report the nucleotide sequence and genome organization of the human papillomavirus type 42. HPV42 DNA was isolated from vulvar papillomas. It has been detected in benign forms of proliferative lesions only. The genome of HPV42 is 7917 bp long and shows the open reading frame pattern conserved in all HPVs sequenced so far. HPV42 has no high degree of sequence homology to any of the known HPVs. It shows characteristics previously found either exclusively in HPVs associated with invasive carcinomas or exclusively in nongenital HPVs. Therefore it cannot be readily ascribed to any of the established subgroups of human papillomaviruses.

Base Sequence↗

PubMed1990

Molecular structures of two human DNA topoisomerase I retrosequences.

We have isolated recombinant lambda-phage clones that contain sequences complementary to the 3' half of the cDNA encoding human topoisomerase I (hTOP1). These lambda clones belong to three distinct classes: class-I clones contain sequences from the active gene located on human chromosome 20. Class-II and class-III clones contain sequences corresponding to the cDNA encoding hTOP1 from nucleotide (nt) 2208 to 3434 and from nt 1639 to 3434, respectively. These sequences exhibit the characteristic features of retroposons or retrosequences. They are most likely derived from truncated mRNA transcripts of the active gene. We propose to designate the truncated hTOP1 sequence located on chromosome 1 as the pseudogene 1 (psi 1-hTOP1) and the sequence on chromosome 22 as the pseudogene 2 (psi 2-hTOP1). Pseudogene psi 1-hTOP1 has two unique properties: it is flanked by upstream sequences which display promoter activity in transient expression assays, and it contains an open reading frame which could code for the 211 C-terminal amino acids of hTOP1. Pseudogene psi 2-hTOP1 is located within an AluI repetitive element and is flanked on one side by a (CA)21 stretch.

Base Sequence↗

PubMed1990

Purification and mechanism of action of a nucleosome assembly factor from Xenopus oocytes.

Chromatin with nucleosomes spaced at 180-base pair intervals can be formed in vitro on circular DNA molecules using a Xenopus oocyte S-150 extract, but the ability to form a periodic chromatin structure is lost upon fractionation of this extract. To identify factors other than the known ones involved in chromatin assembly, we have first depleted the extract by incubating it in batch with charged resins, and we have subsequently reconstituted it with purified fractions. Studies performed with the fractionated components indicate that formation of periodically spaced nucleosomes on the relaxed, closed circular DNA proceeds in two steps and does not require DNA topoisomerases. In a first step, histones H3/H4 are transferred from the endogenous H3/H4-N1 complex to the DNA, forming a nascent chromatin structure. This structure can then be rapidly complemented in a subsequent and independent step with a stoichiometric amount of histone H2A/H2B dimers. Under these experimental conditions, excess histone H2A/H2B dimers inhibit DNA supercoiling and nucleosome formation. We describe the purification of a factor from the Xenopus oocyte S-150 which permits DNA supercoiling and nucleosome formation under conditions of excess histone H2A/H2B. The activity purifies as a complex of five nonacidic polypeptides with apparent molecular masses ranging between 56 and 62 kDa. This factor prevents the binding of excess histone H2A/H2B to the DNA, and it can also remove excess histone H2A/H2B already bound to the DNA, thus ensuring that stoichiometric amounts of all four nucleosomal histones associate with the DNA.

Animals↗

PubMed1989

Characterization of a 48-kDa nucleic-acid-binding fragment of nucleolin.

Nucleolin (C23 or 100 kDa) is an abundant single-stranded-nucleic-acid-binding nucleolar protein proposed to be involved in the early stages of ribosome assembly. A stable 48-kDa fragment of the protein was produced either by proteolytic activity present in nucleolar extracts or by added trypsin. The hydrodynamic and DNA-binding properties of the 48-kDa fragment were compared with the parent molecule. Protein sequencing indicated that the fragment begins at residue 282; amino acid composition of the fragment including 10-12 methylated arginine residues suggested that the fragment contains the entire COOH-terminal two-thirds of the protein. The 48-kDa fragment was more globular than nucleolin, as indicated by a lower frictional coefficient (1.3 vs. 2.0 for nucleolin) and a similar sedimentation coefficient (4.1-4.3S) in spite of the reduction in molecular mass. Although the 48-kDa fragment retained single-stranded-DNA-binding activity, the binding capacity and the ability to reassociate DNA were about fivefold and sixfold lower, respectively, than nucleolin. Similarly, tenfold higher concentrations of the 48-kDa fragment were required to form nucleoprotein aggregates. These results suggest that nucleolin contains a globular COOH-terminal domain for nucleic-acid binding and a NH2-terminal region which is involved in protein-protein interactions and modulating nucleic-acid-binding activity.

Amino Acid Sequence↗

PubMed1989

Histone H1 represses transcription from minichromosomes assembled in vitro.

We have previously shown that transcription from a Xenopus 5S rRNA gene assembled into chromatin in vitro can be repressed in the absence of histone H1 at high nucleosome densities (one nucleosome per 160 base pairs of DNA) (A. Shimamura, D. Tremethick, and A. Worcel, Mol. Cell. Biol. 8:4257-4269, 1988). We report here that transcriptional repression may also be achieved at lower nucleosome densities (one nucleosome per 215 base pairs of DNA) when histone H1 is present. Removal of histone H1 from the minichromosomes with Biorex under conditions in which no nucleosome disruption was observed led to transcriptional activation. Transcriptional repression could be restored by adding histone H1 back to the H1-depleted minichromosomes. The levels of histone H1 that repressed the H1-depleted minichromosomes failed to repress transcription from free DNA templates present in trans. The assembly of transcription complexes onto the H1-depleted minichromosomes protected the 5S RNA gene from inactivation by histone H1.

Animals↗

PubMed1986

DNA binding properties of a 110 kDa nucleolar protein.

A single strand specific DNA binding protein was purified to homogeneity from calf thymus nucleoprotein. The monomeric protein is elongated in shape and has a molecular mass of 110 kDa. Since immunocytochemistry revealed that the protein is predominantly located in the nucleolus we refer to it as the 110 kDa nucleolar protein. The protein binds not only to single stranded DNA but also to single stranded RNA, including homopolymeric synthetic RNA. We have used the single stranded DNA binding properties of the 110 kDa protein in model studies to investigate its effects on the configuration of nucleic acid. Our results are: only 50-55 protein molecules are sufficient to saturate all binding sites on the 6408 nucleotides of phage fd DNA; protein binding cause a compaction of single stranded DNA; large nucleoprotein aggregates are formed in the presence of divalent cations; this is due to protein-protein interactions which occur at moderately high concentrations of magnesium-, calcium or manganese ions; the protein induces the reassociation of complementary nucleic acid sequences. We speculate that the 110 kDa protein performs similar reactions in vivo and may have a function related to the processing and packaging of preribosomal RNA.

Animals↗

PubMed1985

A newly detected class of mammalian single strand-specific DNA-binding proteins. Effects on DNA polymerase alpha-catalyzed DNA synthesis.

Using a rapid purification scheme, we have isolated from calf thymus cells two single strand-specific DNA-binding proteins, with apparent molecular masses of 48 and 61 kDa. These proteins remained undetected in earlier studies on DNA-binding proteins from calf thymus because they are extremely sensitive to proteolytic breakdown occurring during purification. The proteins are immunologically and biochemically related. They also share a number of functional properties. Both proteins bind noncooperatively to single-stranded DNA and almost totally ignore double-stranded DNA. Both proteins stimulate DNA synthesis catalyzed by mammalian DNA polymerase alpha in the presence of an excess of "activated" DNA as primer-template. The stimulation factor was between 2 and 100 depending on the protein/DNA ratio. We have analyzed this effect in more detail with specifically primed single-stranded phage fd DNA as template and concluded that the proteins block nonproductive polymerase-binding sites on single-stranded DNA sequences, thereby increasing the probability for an association of polymerase with 3'-OH primer ends.

Animals↗

PubMedDate not supplied

Comparison of development of serum antibodies to HPV16 and HPV33 and acquisition of cervical HPV DNA among sexually experienced and virginal young girls. A longitudinal cohort study.

OBJECTIVES: To study the importance of sexual activity and early coitus debut on the risk for acquiring infection with human papillomavirus (HPV) type 16 or 33. STUDY DESIGN: Ninety-eight healthy adolescent girls were followed up with consecutive interviews and donations of serum and cervical brush samples during 2 years. RESULTS: Fourteen percent of sexually experienced girls had serum immunoglobulin G to HPV16 and/or HPV33 capsids, and 14% also had cervical HPV16 or HPV33 DNA. Seropositivity for HPV correlated with detection of cervical HPV DNA. None of the 36 girls without coital experience was seropositive or harbored cervical HPV DNA. Seropositivity for HPV was correlated strongly with the number of sexual partners: Odds ratio for > 1 sexual partner was 16.3 (P < 0.001), and for early coitus debut (younger than 17 years of age), it was 14.3 (P < 0.002). CONCLUSIONS: Both HPV serology and HPV DNA testing indicated that the number of sexual partners and earliness of coitus debut determined the risk for acquiring HPV infection and that nonsexually transmitted infections are rare or nonexistent among adolescent girls.

Adolescent↗