ABSTRACT
BACKGROUND/AIMS: In approximately 5% of chronic liver disease cases, no aetiology can be identified. We selected sera from 50 patients with chronic hepatitis of unknown aetiology who were enrolled in this follow-up study whose aim is to gain insight into the possible role of viruses and to define potential clinical outcomes. METHODS: Patients' sera were screened with highly sensitive polymerase chain reaction assays for hepatitis B (HBV), C, D, and G viruses and TT virus. Sera were also retested for antibodies against the core antigen of HBV. RESULTS: Surprisingly, HBV DNA was detected in both serum and liver in 15/50 (30%) patients. Immunostaining for HBV antigens on biopsies from patients positive for HBV DNA showed HBcAg and/or HBsAg expression at low levels in 9/15 samples. Eleven of the fifteen patients were anti-HBc positive. With one exception, all patients carried HBV genomes at low levels (10(4) copies/ml or less). Histological signs of chronic liver disease were observed in all patients. CONCLUSION: Unrecognised HBV infections may account for a high proportion of chronic hepatitis cases of unknown aetiology. Improved HBV detection tests, which appear mandatory for the diagnosis and management of non-A non-E hepatitis as well as for improved safety of transfusions and transplantations are needed.
Subject(s)
Hepatitis B/epidemiology , Hepatitis, Chronic , Adult , Antigens, Viral/analysis , Base Sequence/genetics , DNA, Viral/blood , DNA, Viral/genetics , DNA, Viral/metabolism , Female , Genome, Viral , Hepatitis B virus/genetics , Hepatitis B virus/immunology , Hepatitis, Chronic/immunology , Hepatitis, Chronic/metabolism , Hepatitis, Chronic/pathology , Humans , Incidence , Liver/immunology , Liver/metabolism , Liver/pathology , Male , Middle Aged , Molecular Sequence Data , Viral LoadABSTRACT
Human sperm is characterized by a high heterogeneity of its basic nuclear protein complement of pro-protamines, protamines and histones. This heterogeneity is increased by the persistence of phosphorylated protamines in mature spermatozoa. Alkaline phosphatase treatment of whole protein indicated that protamines HP1 and HP2 were phosphorylated to various degrees. Presence of non-phosphorylated and phosphorylated protamines HP1 and HP2 was further demonstrated by electrospray mass spectrometry. Phosphorylation sites of mono- and di-phosphorylated protamine HP1 were identified by automatic Edman degradation of the protein after phosphoserine derivatization to S-ethylcysteine. In both phosphorylated forms, Ser-10 was found phosphorylated; in the di-phosphorylated form, Ser-8 was identified as the second site of phosphorylation. In protamine HP2, the unique site of phosphorylation (Ser-14) was located after limited acid hydrolysis of enzymic peptides and thin-layer electrophoresis.
Subject(s)
Protamines/metabolism , Spermatozoa/metabolism , Alkaline Phosphatase , Amino Acid Sequence , Binding Sites , Chymotrypsin , Humans , Male , Mass Spectrometry/methods , Metalloendopeptidases , Molecular Sequence Data , Peptide Fragments/chemistry , Phosphorylation , Protamines/chemistry , Protamines/isolation & purificationABSTRACT
Three monoclonal antibodies against human protamines were obtained by immunization with total human basic nuclear proteins or purified protamine HP3. The specificity of antibodies was assessed by enzyme-linked immunosorbent assay (ELISA) and Western blot. They recognized three distinct epitopes: One was specific for the protamine P1 family, another was specific for the protamine P2 family and the third was common to both families. All were specific for the human species. Antibodies were used to detect protamines in germ cells by indirect immunofluorescence and by immunoelectron microscopy. Protamines appeared in spermtid nuclei at steps 4-5 of spermiogenesis, i.e., during the chromatin condensation process, and were not accumulated in the cytoplasm before entering the nucleus.
Subject(s)
Antibodies, Monoclonal , Protamines/immunology , Protamines/metabolism , Spermatozoa/metabolism , Animals , Antibodies, Monoclonal/biosynthesis , Antibody Specificity , Cell Nucleus/metabolism , Epitopes , Fluorescent Antibody Technique , Humans , Immunohistochemistry , Male , Mice , Mice, Inbred BALB C , Microscopy, Immunoelectron , Spermatozoa/ultrastructureABSTRACT
Human intermediate basic protein 2 (HPI2) is a low-molecular-mass basic protein present in small amounts in human sperm nuclei. The amino acid composition of the protein, its N-terminal amino acid sequence and peptide maps obtained after digestion with endoproteinases Lys-C and Glu-C, reveal that HPI2 is structurally related to human protamine species P2 (HP2), which is rich in Arg, His and Cys residues. Compared to HP2, which is one of the two major sperm protamines, HPI2 has an N-terminal extension of 24 residues which includes six acidic residues and does not possess any Arg residues. The amino acid sequence of HPI2 (81 residues) is identical to the sequence of the C-terminal region of another minor sperm nuclear protein, human intermediate basic protein 1 (HPI1, 101 residues), which was sequenced previously [Martinage, A., Arkhis, A., Alimi, E., Sautière, P. & Chevaillier, P. (1990) Eur. J. Biochem. 191, 449-451]. Due to this structural similarity, HPI2 must be considered as an intermediate in the maturation of proprotamine HPI1 limited proteolysis.
Subject(s)
Cell Nucleus/chemistry , Nuclear Proteins/chemistry , Protamines/chemistry , Spermatozoa/ultrastructure , Amino Acid Sequence , Humans , Male , Metalloendopeptidases/metabolism , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Mapping , Sequence Homology, Amino Acid , Serine Endopeptidases/metabolism , Spermatozoa/chemistryABSTRACT
Protamine HP4 is a minor protein which was purified from human sperm nuclei. It was characterized by its amino acid composition, peptide mapping after digestion with highly specific endoproteinases and finally by its amino acid sequence. Protamine HP4 contains high amounts of arginine, cysteine and histidine. The primary structure of the protein was established by sequence analysis of intact protamine and of its fragments. HP4 is a P2-type protamine of 58 residues (Mr 7783) structurally related to human protamines HP2 and HP3 from which it only differs by an amino-terminal extension of one and four residues, respectively. These three protamines exhibit a close structural relationship with mouse protamine mP2. The heterogeneity of protamines in human sperm nuclei is discussed.
Subject(s)
Nuclear Proteins/chemistry , Protamines/chemistry , Spermatozoa/chemistry , Amino Acid Sequence , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Humans , Male , Molecular Sequence Data , Nuclear Proteins/isolation & purification , Peptide Mapping , Protamines/analysis , Protamines/isolation & purificationABSTRACT
The largest intermediate basic protein HPI1 (101 residues) from human sperm chromatin was isolated and characterized. The amino acid composition and sequence analysis of the protein and of tryptic peptides together with peptide mapping of endoproteinases Lys-C and Glu-C hydrolysates showed that the C-terminal region (residues 45-101) of HPI1 is identical to protamine HP2. These structural data strongly suggest that protein HPI1 is a precursor of human sperm protamines HP2 and HP3 (57 and 54 residues, respectively) as well as of two other intermediate basic proteins HPS1 and HPS2 (69 and 66 residues, respectively) sequenced previously.
Subject(s)
Nuclear Proteins/analysis , Protamines/isolation & purification , Protein Precursors/isolation & purification , Spermatozoa/analysis , Amino Acid Sequence , Humans , Male , Molecular Sequence Data , Peptide Mapping , TrypsinABSTRACT
Basic proteins isolated from human sperm nuclei are highly heterogeneous. Three groups of nuclear basic proteins have been characterized: somatic-type as well as testis-specific histones, protamines and basic proteins with an electrophoretic mobility which is intermediate between that of histones and that of protamines. Human protamines can be separated into 2 protein families with different amino acid composition and amino-acid sequence. Protamines HP1 differ in their degree of phosphorylation. Protamines HP2, 3 and 4 differ by their amino-terminal sequence. Intermediate basic proteins (HPI1, HPI2, HPS1, HPS2) share a common C-terminal sequence of 54 residues identical to the amino-acid sequence of protamine HP3; only their N-terminal regions are different. Taking into account these structural homologies, the intermediate basic protein HPI1 appears as a precursor of protamines HP2 and HP3.
Subject(s)
Protamines/analysis , Protein Precursors/analysis , Spermatozoa/analysis , Amino Acid Sequence , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Humans , Male , Molecular Sequence Data , PhosphorylationABSTRACT
Two intermediate nuclear basic proteins HPS1 and HPS2 were isolated from human sperm. They were characterized by their electrophoretic mobility in acid-urea gels, their amino acid composition, and their peptide maps after digestion by endoproteinase Lys-C and by endoproteinase Glu-C. Their amino-terminal amino acid sequences have also been determined. The structural data thus obtained suggest that HPS1 and HPS2 are precursors of human protamines HP2 and HP3.