Sub-systems for optical frequency measurements: application to the 282-nm (199)Hg(+) transition and the 657-nm Ca line.

We are developing laser frequency measurement technologies that should allow us to construct an optical frequency synthesis system capable of measuring optical frequencies with a precision limited by the atomic frequency standards. The system will be used to interconnect and compare new advanced optical-frequency references (such as Ca, Hg(+ ), and others) and eventually to connect these references to the Cs primary frequency standard. The approach we are taking is to subdivide optical frequency intervals into smaller and smaller pieces until we are able to use standard electronic-frequency-measurement technology to measure the smallest interval.

Sub-Doppler Heterodyne Frequency Measurements on OCS Near 2900 cm-1 Using a CO Overtone Sideband Spectrometer.

We present sub-Doppler heterodyne frequency measurements on 10 rovibrational transitions of carbonyl sulfide (OCS) between 2894 and 2910 cm-1. The measurements were made using a CO overtone laser which had limited tuneability through the generation of microwave sidebands in a CdTe crystal. With this technique the laser frequencies were shifted to the desired OCS transition frequencies. The transition frequencies could be measured with uncertainties less than 30 kHz (Deltanu/nu = 3 x 10(-10)) by frequency offset-locking the CO overtone laser to combination frequencies of two saturation-stabilized CO2 laser standards. The measured transition frequencies of OCS were combined with previous sub-Doppler, Fourier transform, and microwave measurements to recalculate improved calibration tables for the 10(0)1-00(0)0, 11(1e)1-01(1e)0, and 11(1f)1-01(1f)0 bands. These tables are suitable for the calibration of infrared spectrometers in the 87 THz region (near 2900 cm-1). Copyright 1998 Academic Press.

Tunable carbon monoxide overtone laser sideband system for precision spectroscopy from 2.6 to 4.1 microm.

We report a source of tunable laser radiation for high-precision molecular spectroscopy in the 2.6- 4.1-microm spectral region. Laser light from a CO overtone laser is mixed with microwaves, generating tunable sidebands of ~1 mW of power. We achieve very high absolute frequency accuracy by frequency-offset locking the CO laser to a CO(2) laser secondary frequency standard. The uncertainty of the laser frequency is less than 30 kHz (Dnu/nu=3x10(-10)) , and the laser linewidth is of the order of 100 kHz. This tunable and ultrastable laser system is suitable for very accurate molecular spectroscopy and metrology in a most interesting wavelength region. We demonstrate an application of the system to saturated-absorption spectroscopy of a rovibrational transition of carbonyl sulfide.

Identification of latent membrane protein 2A (LMP2A) domains essential for the LMP2A dominant-negative effect on B-lymphocyte surface immunoglobulin signal transduction.

Epstein-Barr virus (EBV) recombinants which carry three different deletion mutations in the LMP2A cytoplasmic amino-terminal domain were constructed. The presence of each mutation, LMP2A delta 21-36, LMP2A delta 21-64, and LMP2A delta 21-85, in EBV-infected transformed lymphoblastoid cell lines was confirmed by PCR analysis and Southern blot hybridization. Confirmation of mutant LMP2A protein expression was by immunofluorescence and immunoblotting with a newly identified rat monoclonal antibody that recognizes each of the LMP2A deletion mutations. Lymphoblastoid cell lines infected with recombinant EBV DNAs containing the mutations were analyzed for loss of LMP2A's dominant-negative effect on surface immunoglobulin signal transduction by monitoring induction of tyrosine phosphorylation, calcium mobilization, and activation of lytic replication following surface immunoglobulin cross-linking. Domains of LMP2A important for induction of tyrosine phosphorylation, calcium mobilization, and activation of lytic replication were identified.

Inducible nitric oxide synthase in cattle. Differential cytokine regulation of nitric oxide synthase in bovine and murine macrophages.

We assessed bovine bone marrow-derived macrophages and monocyte-derived macrophages for expression of inducible nitric oxide synthase (iNOS) activity. Both cell types expressed iNOS activity upon stimulation with Salmonella dublin (S. dublin) or with LPS. A 372-bp fragment of the bovine iNOS mRNA could be amplified by reverse transcription-PCR from mRNA of stimulated macrophages. Cloning and sequencing of the fragment revealed a high degree of homology to human hepatocyte, rat vascular smooth muscle cell, and mouse macrophage iNOS both at the nucleotide (87 to 92%) and amino acid levels (94 to 97%). iNOS mRNA was expressed maximally 6 h after stimulation with S. dublin, whereas maximal nitrite accumulation in supernatants was measured at 24 to 48 h. Significant differences with regard to cytokine regulation of iNOS were observed between murine and bovine macrophages cultured under identical conditions. The most striking difference was the inability of homologous IFN-gamma to induce iNOS both at the level of nitrite production and of mRNA expression in bovine macrophages. TNF-alpha, IL-2, and IL-1 alone or together with IFN-gamma neither induced iNOS nor primed bovine macrophages for enhanced iNOS expression or activity upon stimulation with S. dublin. RhuIL-4, but not rhuTGF-beta, down-regulated S. dublin-induced iNOS activity and mRNA expression in bovine macrophages. Thus, an enzyme with a high degree of homology to rodent iNOS is inducible by stimulation of bovine macrophages with bacteria, but induction and regulation by cytokines occur under more restricted conditions than in rodent macrophages.

Noncytopathic strains of bovine viral diarrhea virus prime bovine bone marrow-derived macrophages for enhanced generation of nitric oxide.

Bovine bone marrow-derived macrophages (BBMM) were infected in vitro with a cytopathic (cp) and a noncytopathic (ncp) biotype of bovine viral diarrhea virus (BVDV). The virus strains used, TGAN (ncp) and TGAC (cp), originate from one animal and are antigenically closely related. Both TGAC and TGAN infected a subset of BBMM. Only cp BVDV induced a cytopathic effect. Infection of BBMM resulted in the modulation of certain macrophage functions. Only ncp strains of BVDV primed BBMM for enhanced reactive nitrogen production in response to Salmonella dublin. In contrast, infection with both biotypes did not influence bacteria-induced procoagulant activity and both biotypes equally reduced PMA-induced superoxide production. This suggests that the two biotypes differentially and selectively affect certain macrophage functions related to host defense. For the first time, a BVDV biotype-associated difference has been related to a biochemical parameter of the host cell.

Characterization of the antibody response to the latent infection terminal proteins of Epstein-Barr virus in patients with nasopharyngeal carcinoma.

Human sera were tested for antibodies against the Epstein-Barr virus (EBV) latent infection terminal proteins (TPs). Anti-TP IgG and IgA antibodies were detected by an indirect immunofluorescence assay of insect cells expressing a recombinant TP1. Out of 301 human sera of patients with EBV-related and EBV-unrelated disorders, only sera from patients with nasopharyngeal carcinoma (NPC) (32/83; 38%) showed anti-TP antibodies. Studies on serial sera from German and Hong Kong NPC patients revealed a decline of anti-TP antibodies during tumour therapy, and none of these antibodies were identified in patients with early tumour stages or in remission. Comparative studies of TP1-specific polyclonal rabbit antisera and human TP-positive sera showed clear differences in the TP epitopes recognized by each. Human antisera contained antibodies only to native epitopes in exons 2 to 7 of TP1 whereas rabbit antisera reacted only with epitopes located in the first exon and, additionally, exhibited EBV strain specificities.

Down-regulation of integrated Epstein-Barr virus nuclear antigen 1 and 2 genes in a Burkitt lymphoma cell line after somatic cell fusion with autologous EBV-immortalized lymphoblastoid cells.

Epstein Barr virus (EBV) latent gene expression was analyzed in somatic cell hybrids between an EBV-positive Burkitt lymphoma (BL) cell line (BL 60) and an autologous EBV-immortalized lymphoblastoid cell line (LCL, IARC 277). The EBV genomes carried by the parental cell lines differ in sequence and in their physical state. The BL 60 EBV genome is integrated into the host cell genome whereas the LCL IARC 277 carries exclusively episomal EBV molecules. The hybrid cells contain both EBV genomes and display the differentiation phenotype of the parental LCL with regard to growth characteristics and cell-surface antigen expression in vitro and in vivo. While the EBNA 1 and EBNA 2 gene expression of the LCL-derived EBV is maintained in these hybrid cells, the BL-60-derived EBNA 1 and EBNA 2 genes are transcriptionally down-regulated. Mapping of the genomic region surrounding the latent Cp promoter of the BL-60-derived EBV revealed an extensive deletion upstream of the Cp promoter including the enhancer element in the ori P region, the origin of latent viral replication (ori P), the coding sequences for the EBV latent membrane protein (LMP) and the EBV terminal protein (TP), and suggested that one viral-cellular junction sequence is located near the Cp promoter. Integration of EBV into the host cell genome together with the extensive deletion might be causally related with the altered latent gene expression pattern after introduction of a lymphoblastoid host-cell background by somatic cell fusion. Down-regulation of the BL-60-derived EBNA genes could be due to loss of regulatory sequences in the BL-derived EBV necessary for EBNA 1 and EBNA 2 transcription in the lymphoblastoid hybrid cells, but not in the parental BL cells.

Identification of Epstein-Barr virus terminal protein 1 (TP1) in extracts of four lymphoid cell lines, expression in insect cells, and detection of antibodies in human sera.

The terminal proteins TP1 and TP2 are putative products of Epstein-Barr virus (EBV) genes expressed during the latent cycle of the virus. They are predicted to code for 53- and 40-kilodalton integral membrane proteins. We used the baculovirus Autographa californica nuclear polyhedrosis virus as an expression vector to produce TP1 in large amounts in insect cells. The DNA sequences used to express TP1 originated from a TP1 cDNA derived from an M-ABA/CBL1 cDNA library. Rabbit antisera raised against procaryotic TP1 fusion proteins recognized a monomer and a dimer of the recombinant TP1 protein in the infected insect cells. Immunofluorescence studies of living insect cells showed that the recombinant protein is located in the plasma membrane. The insect cells infected with the recombinant baculovirus producing TP1 provided a test system to screen human antisera for TP1 antibodies. A total of 168 human EBV-positive and EBV-negative antisera were studied. TP1 antibodies were detected only in sera from nasopharyngeal carcinoma patients (16 out of 42). Rabbit antiserum raised against the recombinant TP1 protein expressed in the baculovirus system specifically recognized a protein of about 54 kilodaltons in the lymphoblastoid cell lines M-ABA and M-ABA/CBL1 and in the Burkitt's lymphoma cell lines BL18 and BL72. This protein could be located in the total membrane fraction of M-ABA cells and is upregulated by treating the cells with 12-O-tetradecanoylphorbol-13-acetate.