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Intra-amniotic inflammation (IAI) may cause preterm birth with poor neonatal out-come. To identify novel biomarkers for IAI, we analyzed amniotic and cervical fluid samples from 27 patients with signs of threatening preterm birth with or without IAI by surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF−MS). Seventeen proteins were significantly overexpressed in amniotic fluid from IAI cases and more often in women with preterm labor than those with rupture of membranes. Five of these were identified as human neutrophil protein 1−3, calgranulin A and B. Keywords: proteomics • surface-enhanced laser desorption ionization time-of-flight mass spectrometry • SELDI-TOF−MS • preterm birth • intra-amniotic inflammation • preterm labor • preterm prelabor rupture of membranes

The insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene determines the concentration of ACE in serum and local tissues. The role of this polymorphism in progressive chronic renal disease is still not fully clear.

Epstein‐Barr virus (EBV)‐negative, Burkitt‐like lymphoma‐derived cells were transformed with a transducing vector (pSV2‐gpt) containing the Escherichia coli gene coding for xanthine‐guanine phosphoribosyltransferase (XGPRT) and with a derivative of PSV2‐gpt that carries the genes for the EBV‐associated small RNAs on the EcoRI J fragment of B95‐8 EBV DNA inserted at the unique EcoRI site (pJ‐gpt). Cells transformed with PSV2‐gpt and pJ‐gpt express the E. coli gpt gene to approximately the same extent, judged by determinations of the XGPRT activity of cell extracts. Blot hybridisation experiments with restriction endonuclease‐cleaved DNA from the transformants have revealed the presence of vector DNA sequences in the cells, at least some of which are most probably integrated into high mol. wt. chromosomal DNA. Northern blot hybridisation analysis of cytoplasmic RNA from pJ‐gpt‐transformed cells revealed the presence of an EcoRI J DNA complementary RNA species of the same size as the EBV DNA‐encoded small RNAs found in EBV‐transformed cells.

Cytoplasmic RNA prepared from several human cell lines and tissues was hybridised to DNA from Epstein‐Barr virus, human adenovirus types 2, 3 and 12 and human papovaviruses BK and JC. RNA from all the cells, regardless of whether or not they were virally infected, hybridised to specific regions of the Epstein‐Barr virus or adenovirus genomes but not to papovavirus DNA. The cellular cross‐hybridising species appear to be repetitive sequences which are conserved in higher eukaryotes. Mismatch estimations indicate a high degree of homology between the viral and host sequences. Detailed analysis of selected regions of viral DNA failed to reveal any primary‐structural peculiarities.

Large Epstein-Barr virus (EBV) DNA restriction fragments corresponding to regions transcribed in transformed, proliferating cells were cloned in a cosmid derivative of the dominant-acting selection vector pSV2-gpt. Recombinant vectors carrying the EcoRI A fragment of EBV DNA were modified in the region corresponding to the deletion of the virion DNA in the non-transforming viral substrain P3HR-1, to create a series of recombinants lacking parts of this region. The recombinant vectors were introduced into 3T3 mouse fibroblasts under selective conditions, and resistant clones shown to contain EBV DNA sequences were analyzed for the expression of EBV-related antigens detectable by direct, indirect, and anticomplement immunofluorescence techniques. Cells that contained the BamHI K fragment expressed the EBV-determined nuclear antigen (EBNA) as expected. Cells transfected with recombinant vectors containing the BamHI W, Y, and H fragment part of the EcoRI A fragment also express a nuclear antigen detectable with certain anti-EBNA-positive human sera in anticomplement immunofluorescence tests. The BamHI WYH-induced EBNA polypeptide is similar in size to the EBNA2 polypeptide in Raji cells, as shown by gel electrophoresis and immunoblotting. The antigen is not detected in cells transfected with EcoRI A-derived vectors in which the BamHI H fragment has been deleted or in cells transformed with vectors carrying the BamHI H fragment alone. Direct and indirect immunofluorescence did not reveal the presence of antigens associated with productive infection in any of the EBV DNA-transfected fibroblast clones.

Recombinant vectors carrying DNA fragments from the BamHI E region of the B95-8 Epstein-Barr virus (EBV) genome were transfected into COS-1 cells, and the transient expression of EBV-encoded nuclear antigens (EBNAs) was analyzed by using polyvalent human antisera and rabbit antibodies to synthetic peptides. Vector DNA containing two rightward open reading frames in the BamHI E fragment, BERF2a and BERF2b, induced the expression of a nuclear antigen identical serologically and with respect to size to the larger of the two polypeptides previously designated as EBNA4 in B95-8 cells. An antigen corresponding to the smaller polypeptide was induced in cells transfected with constructs that contained two neighboring reading frames, BERF3 and BERF4. This antigen also reacted with a rabbit antiserum to the synthetic peptide 203, deduced from BERF4. Thus, the findings show that the two components of the EBNA4 doublet in B95-8 cells are encoded by separate genes. The antigen encoded by BERF2a and/or BERF2b has been designated as EBNA4 and the antigen encoded by BERF3 and/or BERF4 has been designated as EBNA6. Polyvalent human antisera detected EBNA4 and EBNA6 in 9 of 11 lymphoid cell lines carrying independent EBV isolates. In the remaining two lines, either EBNA4 or EBNA6 was not detectable.

Cytoplasmic RNA prepared from five lymphoid cell lines and a Burkitt lymphoma biopsy was radioactively labeled in vitro and hybridized to cloned EcoRI restriction endonuclease fragments of B95-8 Epstein-Barr virus DNA. The results confirmed that the most abundant cytoplasmic RNA species in such cells is specified by a small region of the genome defined by the EcoRI J fragment. Detailed mapping experiments precisely localized these transcripts within the sequence of the rightmost one-third of the EcoRI J fragment. DNA sequencing suggested that this region of the Epstein-Barr virus genome is unable to code for protein. The major early transcripts consisted of two non-polyadenylated RNA species, each about 170 nucleotides in length. They were both transcribed off the same strand of the DNA and showed significant sequence homology with each other. The coding sequences of the two small RNAs contained potential intragenic control regions for RNA polymerase III.

The intracellular Epstein-Barr virus (EBV) DNA present in virus-transformed cells was partly purified from 23 cell lines or biopsies of Burkitt lymphoma, nasopharyngeal carcinoma, infectious mononucleosis, or healthy carrier origin. Such DNA was cleaved in fragments (A-K) of molecular weights between 1 x 10(6) and 30 x 10(6) with restriction enzyme EcoRI, and these fragments were analyzed by standard methods involving agarose gel electrophoresis, transfer to nitrocellulose filters, and hybridization with radioactive EBV DNA or complementary RNA. Sequence variability among different EBV DNA isolates was largely confined to the A, C, and I fragments. These results are discussed in relation to the linkage map of the EcoRI fragments of EBV DNA. The EcoRI cleavage pattern of intracellular viral DNA of an EBV-like virus from baboon cells, Herpesvirus papio, was entirely different from that of human EBV isolates.

RNA was extracted from the Burkitt lymphoma-derived cell line Raji and from Burkitt lymphoma tumor biopsies, isotope labeled in vitro by iodination with 125I, and hybridized to electrophoretically separated restriction endonuclease fragments of Epstein-Barr virus DNA on nitrocellulose membranes. The results indicated that only certain parts of the Epstein-Barr virus genome are represented as polyribosomal RNA in Raji cells, with a pronounced dominance of RNA sequences complementary to a 2.0 x 10(6)-dalton segment of Epstein-Barr virus DNA located close to the left end of the viral genome. A map of virus-specific polyribosomal RNA sequences was constructed, which indicated that a minimum of three regions of the Epstein-Barr virus genome are expressed in Raji cells. Total-cell RNA preparations from five Burkitt lymphoma biopsies contained RNA sequences homologous to the same regions of Epstein-Barr virus DNA as polyribosomal RNA from Raji cells, albeit at different relative proportions.

Differentiation of muscle cells is characterized morphologically by the acquisition of contractile filaments and characteristic shape changes, and on the molecular level by induction of the expression of several genes, including those for the muscle-specific alpha-actin isoforms. IFN-gamma is an inhibitor of proliferation for several cells, including vascular smooth muscle, and is also an inducer of differentiated properties for several hematopoietic cells. We have therefore investigated whether IFN-gamma affects the expression of alpha-smooth muscle actin in cultured arterial smooth muscle cells. Cells exposed to IFN-gamma show a reduction of alpha-smooth muscle actin-containing stress fibers, as detected by immunofluorescence. The effect was observed in all phases of the cell cycle, and was caused by a reduction of the synthesis of alpha-smooth muscle actin protein as revealed by two-dimensional electrophoretic analysis of actin isoforms. RNA hybridization using a cRNA probe that hybridizes to all actin mRNAs showed that IFN-gamma-treated cells have a reduced content of the 1.7-kb mRNA that codes for alpha-smooth muscle actin, and to a lesser extent, also of the 2.1-kb mRNA encoding the beta and gamma-cytoplasmic actins. The reduction of alpha-smooth muscle actin mRNA was confirmed using an alpha-smooth muscle actin-specific cRNA probe. The reduction of alpha-smooth muscle actin mRNA occurs within 12 h, and is dependent on protein synthesis, since cycloheximide treatment reversed the effect. The inhibition of this mRNA species was dose dependent, and detectable by RNA hybridization at a dose of 50 U/ml IFN-gamma. These results suggest that the differentiation of arterial smooth muscle cells is not necessarily coupled to an inhibition of cellular proliferation. Instead, IFN-gamma may regulate the expression of several genes that control both proliferation and expression of differentiation markers.

Five distinct Epstein-Barr virus (EBV)-determined nuclear antigens (EBNA-1 to EBNA-5) were recently identified. Antibody responses to these antigens could conceivably differ, and thus prove of serodiagnostic value, in EBV-associated disease processes. As a first step, murine or human cell lines transfected with appropriate EBV DNA fragments and stably expressing either EBNA-1 or EBNA-2 were used to determine the frequency and time of emergence of antibodies to these two antigens in the course of acute and chronic infectious mononucleosis (IM) and to assess their titers in so-called chronic active EBV infections. Following IM, antibodies to EBNA-2 arose first and, after reaching peak titers, declined again in time to lower persistent or even nondetectable levels. Antibodies to EBNA-1 emerged several weeks or months after anti-EBNA-2 and gradually attained the titers at which they persisted indefinitely. The ratios between the anti-EBNA-1 and anti-EBNA-2 titers therefore were generally well below 1.0 during the first 6-12 months after IM and turned to well above 1.0 during the second year. In clear cases of chronic IM, the inversion of this ratio was delayed or prevented. In the less well-defined chronic EBV infections, low ratios were observed in only some of the patients. Because many of these illnesses were not ushered in by a proven IM and often showed EBV-specific antibody profiles within the normally expected range, a causal role of the virus in these cases remains doubtful.