Comparison of intravenous oxytocin and prostaglandin E2 for accelerating labour.

In a prospective study of 52 consecutive women who required acceleration of labour intravenous prostaglandin E2 (PGE2) was used as the oxytocic agent. These mothers were matched for age, parity, height, gestational age, initial cervical dilatation, and station and position of the fetal head with 52 women whose labours were accelerated with oxytocin; both drugs were equally effective. Acceleration to delivery intervals, second-stage durations, the number of assisted deliveries, and Apgar scores were similar regardless of the oxytocic used. Although PGE2 compares well with oxytocin, it offers no further advantages and is more expensive and less well tried than oxytocin.     

Molecular Basis for the Resistance of Human Mitochondrial 2-Cys Peroxiredoxin 3 to Hyperoxidation

Peroxiredoxins (Prxs) detoxify peroxides and modulate H₂O₂-mediated cell signaling in normal and numerous pathophysiological contexts. The typical 2-Cys subclass of Prxs (human Prx1–4) utilizes a Cys sulfenic acid (Cys-SOH) intermediate and disulfide bond formation across two subunits during catalysis. During oxidative stress, however, the Cys-SOH moiety can react with H₂O₂ to form Cys sulfinic acid (Cys-SO₂H), resulting in inactivation. The propensity to hyperoxidize varies greatly among human Prxs. Mitochondrial Prx3 is the most resistant to inactivation, but the molecular basis for this property is unknown. A panel of chimeras and Cys variants of Prx2 and Prx3 were treated with H₂O₂ and analyzed by rapid chemical quench and time-resolved electrospray ionization-TOF mass spectrometry. The latter utilized an on-line rapid-mixing setup to collect data on the low seconds time scale. These approaches enabled the first direct observation of the Cys-SOH intermediate and a putative Cys sulfenamide (Cys-SN) for Prx2 and Prx3 during catalysis. The substitution of C-terminal residues in Prx3, residues adjacent to the resolving Cys residue, resulted in a Prx2-like protein with increased sensitivity to hyperoxidation and decreased ability to form the intermolecular disulfide bond between subunits. The corresponding Prx2 chimera became more resistant to hyperoxidation. Taken together, the results of this study support that the kinetics of the Cys-SOH intermediate is key to determine the probability of hyperoxidation or disulfide formation. Given the oxidizing environment of the mitochondrion, it makes sense that Prx3 would favor disulfide bond formation as a protection mechanism against hyperoxidation and inactivation.     

The Memory Cytotoxic T-Lymphocyte (CTL) Response to Human Cytomegalovirus Infection Contains Individual Peptide-Specific CTL Clones That Have Undergone Extensive Expansion In Vivo

Human cytomegalovirus (HCMV)-specific CD8⁺ cytotoxic T lymphocytes (CTL) appear to play an important role in the control of virus replication and in protection against HCMV-related disease. We have previously reported high frequencies of memory CTL precursors (CTLp) specific to the HCMV tegument protein pp65 in the peripheral blood of healthy virus carriers. In some individuals, the CTL response to this protein is focused on only a single epitope, whereas in other virus carriers CTL recognized multiple epitopes which we identified by using synthetic peptides. We have analyzed the clonal composition of the memory CTL response to four of these pp65 epitopes by sequencing the T-cell receptors (TCR) of multiple independently derived epitope-specific CTL clones, which were derived by formal single-cell cloning or from clonal CTL microcultures. In all cases, we have observed a high degree of clonal focusing: the majority of CTL clones specific to a defined pp65 peptide from any one virus carrier use only one or two different TCRs at the level of the nucleotide sequence. Among virus carriers who have the same major histocompatibility complex (MHC) class I allele, we observed that CTL from different donors that recognize the same peptide-MHC complex often used the same Vβ segment, although other TCR gene segments and CDR3 length were not in general conserved. We have also examined the clonal composition of CTL specific to pp65 peptides in asymptomatic human immunodeficiency virus-infected individuals. We have observed a similarly focused peptide-specific CTL response. Thus, the large population of circulating HCMV peptide-specific memory CTLp in virus carriers in fact contains individual CTL clones that have undergone extensive clonal expansion in vivo.

CD8⁺ cytotoxic T lymphocytes (CTL) recognize virus-infected cells via the T-cell receptor (TCR), an αβ heterodimer that has specificity for the peptide antigen presented by major histocompatibility complex (MHC) class I molecules. During T-cell development in the thymus, the TCR β-chain is constructed by rearrangement of variable (V), diversity (D), and joining (J) gene segments, and the α-chain by rearrangement of V and J segments. Additional diversity is generated by imperfect joining of these segments, exonucleotide nibbling at the joins, and addition of non-germ line-encoded N-region nucleotides (25). The regions spanning the V-D-J and V-J joins constitute the hypervariable CDR3 regions which are thought to interact with the middle of the bound peptide and to account for approximately 50% of the TCR’s interaction with peptide (14, 15, 20). The α- and β-chain complementarity determining regions CDR1, which reside within the TCR V segments, are thought to interact with the N and C termini of a peptide that is bound to MHC. By contrast, Vα and Vβ CDR2s are thought to interact predominantly with the MHC itself (14, 15).Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that infects between 60 and 90% of individuals, depending on the population studied. After primary HCMV infection, the virus persists lifelong in a latent state in cells of the myeloid lineage and under the control of the immune system (5). HCMV reactivation can, however, cause serious disease in immunocompromised individuals, such as patients with advanced human immunodeficiency virus (HIV) infection (30) and patients who have undergone bone marrow transplantation (33). Evidence from animal models (32) and from studies of immunosuppressed humans (39) indicates that virus-specific CD8⁺ CTL have a role in protection against CMV disease.We previously studied in detail the HCMV-specific CTL response in healthy virus carriers. All seropositive donors had high frequencies of MHC-restricted HCMV-specific memory CTL precursors in peripheral blood and strongly recognized one of the viral tegument proteins, pp65. In some donors, the CTL response to this protein was highly focused, recognizing only a single epitope within pp65, whereas in others the CTL recognized multiple pp65 peptides (41 and unpublished data).The aim of this study was to examine the clonal composition of the memory CTL response to HCMV pp65 by determining how many different CTL clones are involved in the recognition of a given pp65 peptide. In order to do this, we analyzed the TCR α- and β-chain usage of multiple independently derived peptide-specific CTL clones from healthy virus carriers.Previous studies have examined the heterogeneity of the CTL response to other human virus infections within single subjects (2, 8, 11, 18, 19, 22, 38) or between different donors (2, 6, 8, 11, 23, 38). In the most extreme cases, a very high degree of TCR focusing has been seen: in a study of one HIV-positive individual’s CTL response to an HLA-B14-restricted HIV env peptide, the same TCR was used by 9 of 10 peptide-specific CTL clones, each derived at different time points over the course of 36 months (22). Similarly, multiple independent CTL clones specific to an HLA-B8-restricted Epstein-Barr virus (EBV) peptide derived from one virus carrier at one time point all used the same TCR (2). The CTL response to different human T-lymphotropic virus type 1 (HTLV-1) peptides has been observed to be oligoclonal within individual donors (38). However, in a variety of other human and mouse viral infections within a given individual, the repertoire of CTL specific for a given peptide has been highly heterogeneous (8, 11, 18, 19).The TCRs of CTL obtained from different donors that recognize the same peptide-MHC complex often show some conservation of gene segment usage, although they differ in hypervariable sequence. For example, Vβ segments and certain β-chain CDR3 motifs were conserved between TCR that recognized an HLA-A2-restricted influenza virus peptide in CTL clones derived from different donors (23); the same phenomenon has been seen for an HLA-B27 restricted influenza virus peptide (6) and an HLA-A11-restricted EBV peptide (8). A much higher degree of TCR conservation has also been seen; the same TCR α- and β-chain protein sequences were used by CTL clones from four of five unrelated donors that recognized an HLA-B8 restricted EBV peptide (2). In the case of HTLV-1, CTL from different donors that were specific to the same peptide used largely unrelated TCR (38).For all of the human viruses so far studied, the clonal composition of virus-specific CTL has only been examined for a very few viral peptide-MHC combinations, sometimes in only one donor or at only one time point. In this study, we have therefore examined multiple CTL clones specific to a total of four pp65 peptides, all restricted by three different HLA alleles. We have derived these clones from six healthy virus carriers at one to four time points up to 18 months apart. To identify CTL clonotypes for longitudinal studies and to determine whether HIV infection modifies the clonal composition of HCMV-specific CTL, we have also examined pp65-specific memory CTL in two asymptomatic HIV-infected subjects who are HCMV seropositive. For any given individual, whether HIV seropositive or seronegative, our results indicate that the memory CTL response to individual HCMV pp65 epitopes is highly focused and contains CTL clones that have undergone extensive expansion in vivo.     

A UK Isolate of Impatiens Necrotic Spot Virus From Glasshouse-grown Cineraria

A tospovirus was detected by ELISA in a Cineraria sample from a commercial protected crop in the UK. Its seroiogical properties indicated it to be Impatiens necrotic spot virus (INSV), results confirmed by RT-PCR tests and nucleotide sequence data on the nucleoprotein-encoding region of the genome. This is the first report of the presence of INSV in a protected crop grown in the UK.     

Comparative Analysis of Techniques to Purify Plasma Membrane Proteins

The aim of this project was to identify the best method for the enrichment of plasma membrane (PM) proteins for proteomics experiments. Following tryptic digestion and extended liquid chromatography-tandem mass spectrometry acquisitions, data were processed using MaxQuant and Gene Ontology (GO) terms used to determine protein subcellular localization. The following techniques were examined for the total number and percentage purity of PM proteins identified: (a) whole cell lysate (total number, 84–112; percentage purity, 9–13%); (b) crude membrane preparation (104–111; 17–20%); (c) biotinylation of surface proteins with N-hydroxysulfosuccinimydyl-S,S-biotin and streptavidin pulldown (78–115; 27–31%); (d) biotinylation of surface glycoproteins with biocytin hydrazide and streptavidin pulldown (41–54; 59–85%); or (e) biotinylation of surface glycoproteins with amino-oxy-biotin (which labels the sialylated fraction of PM glycoproteins) and streptavidin pulldown (120; 65%). A two- to threefold increase in the overall number of proteins identified was achieved by using stop and go extraction tip (StageTip)-based anion exchange (SAX) fractionation. Combining technique (e) with SAX fractionation increased the number of proteins identified to 281 (54%). Analysis of GO terms describing these proteins identified a large subset of proteins integral to the membrane with no subcellular assignment. These are likely to be of PM location and bring the total PM protein identifications to 364 (68%). This study suggests that selective biotinylation of the cell surface using amino-oxy-biotin in combination with SAX fractionation is a useful method for identification of sialylated PM proteins.