Genetic variation in catechol-O-methyltransferase modifies effects of clonidine treatment in chronic fatigue syndrome.

Clonidine, an α2-adrenergic receptor agonist, decreases circulating norepinephrine and epinephrine, attenuating sympathetic activity. Although catechol-O-methyltransferase (COMT) metabolizes catecholamines, main effectors of sympathetic function, COMT genetic variation effects on clonidine treatment are unknown. Chronic fatigue syndrome (CFS) is hypothesized to result in part from dysregulated sympathetic function. A candidate gene analysis of COMT rs4680 effects on clinical outcomes in the Norwegian Study of Chronic Fatigue Syndrome in Adolescents: Pathophysiology and Intervention Trial (NorCAPITAL), a randomized double-blinded clonidine versus placebo trial, was conducted (N=104). Patients homozygous for rs4680 high-activity allele randomized to clonidine took 2500 fewer steps compared with placebo (Pinteraction=0.04). There were no differences between clonidine and placebo among patients with COMT low-activity alleles. Similar gene-drug interactions were observed for sleep (Pinteraction=0.003) and quality of life (Pinteraction=0.018). Detrimental effects of clonidine in the subset of CFS patients homozygous for COMT high-activity allele warrant investigation of potential clonidine-COMT interaction effects in other conditions.

Effect of increased body mass index and anaesthetic duration on recovery of protective airway reflexes after sevoflurane vs desflurane.

BACKGROUND: Increased BMI may increase the body's capacity to store potent inhaled anaesthetics, more so with more soluble agents. Accordingly, we asked whether increased BMI and longer anaesthesia prolonged airway reflex recovery. METHODS: We measured time from anaesthetic discontinuation until first response to command (T1); from response to command until ability to swallow (T2); and from anaesthetic discontinuation to recovery of ability to swallow (T3) in 120 patients within three BMI ranges (18-24, 25-29, and >or=30 kg m(-2)). All received sevoflurane or desflurane, delivered via an LMA. RESULTS: T1 and T3 after sevoflurane exceeded T1 and T3 after desflurane: 6.6 (sd 4.2) vs 4.0 (1.9) min (P<0.001), and 14.1 (sd 8.3) vs 6.1 (2.0) min (P<0.0001). T3 correlated more strongly with BMI after sevoflurane (28 s per kg m(-2), P=0.02) than desflurane (7 s per kg m(-2), P=0.03). Regarding T2, patients receiving sevoflurane with BMI >or=30 kg m(-2) were less often able to swallow 2 min after response to command than were those with BMI 18-24 or 25-29 kg m(-2) (3/20 vs 10/20 or 9/20, P<0.05). Each sevoflurane MAC-hour delayed T3 by 4.5 min (268 s) (R=0.46, P<0.001) whereas each desflurane MAC-hour delayed T3 by 0.2 min (16 s) (R=0.10, P=0.44). CONCLUSIONS: Prolonged sevoflurane administration and greater BMI delay airway reflex recovery. The contribution of BMI to this delay is more pronounced after sevoflurane than desflurane.

A herpesvirus saimiri-based gene therapy vector with potential for use in cancer immunotherapy.

The herpesvirus saimiri (HVS) genome has the capacity to incorporate large amounts of heterologous DNA and can be maintained episomally in many different human cell types. To evaluate the efficacy of HVS-mediated gene transfer into human hemopoietic cells, we investigated the ability of an HVS-based construct, carrying the enhanced green fluorescent protein (EGFP) and neomycin resistance genes, to transduce a variety of human hemopoietic cell lines and primary CD34+ cells. As measured by flow cytometry, the numbers of EGFP+ cells at 2 days postinfection differed between various cell types ranging, from 1.3% for KG1 cells to 56.8% for THP-1 cells. In addition, the expression of EGFP in Jurkat cells was retained at >95% per round of cell division over a period of 6 weeks (comparable with Epstein-Barr virus-derived gene therapy systems). Although the virus was not specifically disabled, no lytic viral mRNAs could be detected in transduced Jurkat cells, and infectious virus could not be detected by sensitive virus recovery assay. We also describe a simple centrifugation method that increases the efficiency of transduction by >100% in some cases and may be generally applicable to other herpesvirus-based vectors for ex vivo gene delivery. Using this technique, we were able to demonstrate a tropism for CD34+/CD14+ cells, transducing 30% of the population. These cells are known to give rise to dendritic cells (the most potent of the antigen-presenting cells), suggesting that the vector could be used to deliver DNA sequences encoding tumor antigens for cancer immunotherapy.

Specific oncolytic activity of herpesvirus saimiri in pancreatic cancer cells.

The potential use of oncolytic viruses in the treatment of cancer has been investigated for some time. A variety of agents have been studied, including some which appear to be selectively replication-competent in cancer cell lines. In this study, we have investigated the ability of herpesvirus saimiri to specifically lyse selected human cancer cell lines. Upon infection with a replication-competent virus carrying the EGFP reporter gene and a neomycin resistance marker, the pancreatic cancer lines MIAPACA and PANC-1 exhibited definite cytopathic effects. In contrast, the colonic carcinoma cell lines SW480 and HCT116 were phenotypically unaltered. In addition, stable cell lines could not be generated from PANC-1 infected cultures, in marked contrast to cultures of cells from other human tissues. Virus recovery assays demonstrated that all of the cell lines produced a small amount of virus post-infection, but that virus replication was minimal after 1 week in culture. In addition, treatment with acyclovir inhibited virus replication but paradoxically increased cytopathic effect. These data suggest that herpesvirus saimiri may have potential as an oncolytic agent for the treatment of pancreatic cancer.

Analysis of gene expression in a human cell line stably transduced with herpesvirus saimiri.

Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus; it has significant homology to the human gammaherpesviruses Kaposi's sarcoma-associated virus and Epstein-Barr virus and the murine gammaherpesvirus murine herpesvirus 68. HVS causes a persistent asymptomatic infection in its natural host, the squirrel monkey. Both subgroups A and C possess the ability to immortalize common marmoset T lymphocytes to interleukin-2-independent proliferation. However, only subgroup C is capable of transforming human, rabbit, and rhesus monkey lymphocytes in vitro. In addition, HVS can stably transduce a variety of human cell lines where the virus persists as a nonintegrating circular episome. In this study, we have developed a system in which the HVS DNA is stably maintained as a nonintegrated circular episome in the human lung carcinoma cell line A549. Virus production can be reactivated using chemical inducing agents, including tetradecanoyl phorbol acetate and n-butyrate, suggesting that the infection in human A549 cells is latent. To analyze virus gene expression in these stably transduced cells, Northern blot analysis was performed using a series of probes produced from restriction fragments spanning the entire coding region of the HVS genome. This demonstrated that an adjacent set of genes containing open reading frames (ORFs) 71 to 73 are expressed in this stably transduced cell line. Moreover, these genes are transcribed as a polycistronic mRNA species produced from a common promoter upstream of ORF 73. This model may serve as a useful tool in the further analysis of the role of ORFs 71 to 73 in gamma-2 herpesvirus latency.

The activation domain of herpesvirus saimiri R protein interacts with the TATA-binding protein.

The herpesvirus saimiri open reading frame (ORF) 50 produces two transcripts. The first is spliced, contains a single intron, and is detected at early times during the productive cycle, whereas the second is expressed later and is produced from a promoter within the second exon. Analysis of their gene products has shown that they function as sequence specific transactivators. In this report, we demonstrate that the carboxy terminus of ORF 50b contains an activation domain which is essential for transactivation. This domain contains positionally conserved hydrophobic residues found in a number of activation domains, including the herpes simplex virus VP16 and the Epstein-Barr virus R proteins. Mutational analysis of this domain demonstrates that these conserved hydrophobic residues are essential for ORF 50 transactivation capability. Furthermore, this domain is required for the interaction between the ORF 50 proteins and the basal transcription factor TATA-binding protein.

The open reading frame 57 gene product of herpesvirus saimiri shuttles between the nucleus and cytoplasm and is involved in viral RNA nuclear export.

The herpesvirus saimiri open reading frame (ORF) 57 is homologous to genes identified in all classes of herpesviruses. It has previously been shown to regulate gene expression through a posttranscriptional mechanism. We demonstrate in this report that the expression of the ORF 57 protein leads to the cytoplasmic accumulation of glycoprotein B and capsid mRNAs. We also demonstrate that ORF 57 has the ability to specifically bind viral RNA transcripts. Utilizing an interspecies heterokaryon assay, we show that ORF 57 has the ability to shuttle between the nucleus and the cytoplasm. Furthermore, we show that ORF 57 contains a relatively leucine-rich sequence which shares some homology with nuclear export signals (NES) found in a number of proteins with the ability to shuttle between the nucleus and the cytoplasm. Moreover, we demonstrate that the ORF 57 NES enables the nuclear export of a heterologous protein and that mutation of the conserved leucine residues contained within the ORF 57 NES signal abrogates the ability of the ORF 57 protein to shuttle between the nucleus and cytoplasm. These results suggest that ORF 57 is involved in mediating the nuclear export of viral transcripts.

The gene product encoded by ORF 57 of herpesvirus saimiri regulates the redistribution of the splicing factor SC-35.

The herpesvirus saimiri (HVS) gene product encoded by ORF 57 shares limited C-terminal similarity with herpes simplex virus 1 ICP27, a protein that has been demonstrated to be involved in the inhibition of host-cell splicing and is responsible for the redistribution of components of the spliceosome. It has previously been shown that ORF 57 can either activate or repress viral gene expression by a post-transcriptional mechanism. Furthermore, repression of gene expression by ORF 57 is dependent on the presence of an intron within the target gene coding region. In this report, it is shown that HVS infection results in the redistribution of the SC-35 splicing factor in the infected cell nucleus. Furthermore, the redistributed SC-35 colocalized with the ORF 57 protein product and expression of the protein alone was sufficient to cause the redistribution of the spliceosome components. These results suggest that the mechanism by which ORF 57 down-regulates expression of intron-containing genes involves the redistribution of the spliceosome complex.

Transcriptional regulation of MHC class I gene expression in rat oligodendrocytes.

MHC class I molecules are normally expressed at very low levels in the brain and their up-regulation in response to cytokines and viral infections has been associated with a number of neurological disorders. Here we demonstrate that the down-regulation of surface class I molecules in differentiated primary rat oligodendrocytes was accompanied by reduced steady-state levels of class I heavy-chain mRNA. Transient expression assays were performed in oligodendrocytes and fibroblasts, using a mouse H-2Kb class I promoter chloramphenicol acetyltransferase plasmid termed pH2KCAT (which contained 5'-flanking sequences from -2033 to +5 bp of the H-2Kb gene relative to the transcriptional start site at +1 bp). These assays showed that H-2Kb promoter activity was reduced in oligodendrocytes but not in class I-expressing fibroblasts. H-2Kb promoter activity was up-regulated in oligodendrocytes co-transfected with a plasmid expression vector encoding the transcriptional activator tax of human T-cell leukaemia virus type I, showing that down-regulation of promoter activity was reversible. Deletion mutant analysis of the H-2Kb promoter revealed the presence of negative regulatory elements that were functional in oligodendrocytes at -1.61 to -1.07 kb and -242 to -190 bp. Deletion of sequences in pH2KCAT encompassing the downstream element totally abolished promoter activity in both oligodendrocytes and fibroblasts, whereas a deletion within the upstream negative regulatory element increased promoter activity specifically in oligodendrocytes. The upstream negative regulatory element also down-regulated a linked heterologous herpes simplex virus thymidine kinase promoter in oligodendrocytes, but not in fibroblasts. Gel retardation assays using overlapping DNA probes that spanned the entire -1.61 to -1.07 kb region revealed the presence of a number of DNA-binding activities that were present in oligodendrocyte, but not in fibroblast nuclear extracts.

The open reading frame (ORF) 50a gene product regulates ORF 57 gene expression in herpesvirus saimiri.

We have previously demonstrated that open reading frame (ORF) 50 and ORF 57 encode transcriptional regulating genes in herpesvirus saimiri. ORF 50, a homolog of Epstein-Barr virus R protein, is a sequence-specific transactivator, whereas ORF 57 acts posttranscriptionally. In this report, we demonstrate that the ORF 57 gene is regulated by the ORF 50a gene product. We show that the ORF 57 gene is expressed at basal levels early in the virus replication cycle and that thereafter it is transactivated by the ORF 50a gene product, due to an increase in RNA levels. As it has been shown that the ORF 57 gene product downregulates ORF 50a due to the presence of its intron, these combined observations identify a feedback mechanism modulating gene expression in herpesvirus saimiri, whereby ORF 50a transcription is downregulated by the ORF 57 gene product, a gene which it specifically transactivates. Furthermore, we propose that the intron-containing ORF 57 gene downregulates itself by the same mechanism as that for ORF 50a, as both genes are downregulated at similar times during the replication cycle.

Carbocyclic frenolicin analogues: novel anticoccidial agents.

Carbocyclic analogues of the antibacterial natural product frenolicin B have been synthesised. These analogues were active against parasitic protozoa of the genus Eimeria and represent a new series of anticoccidial agents. The synthesis of simplified analogues helped to define a possible pharmacophore for frenolicin.

Human CD100, a novel leukocyte semaphorin that promotes B-cell aggregation and differentiation.

Herein we describe the molecular characterization of the human leukocyte activation antigen CD100 and identify it as the first semaphorin, to our knowledge, in the immune system. Semaphorins have recently been described as neuronal chemorepellants that direct pioneering neurons during nervous system development. In this study we demonstrate that CD100 induces B cells to aggregate and improves their viability in vitro. We show that CD100 modifies CD40-CD40L B-cell signaling by augmenting B-cell aggregation and survival and down-regulating CD23 expression. Thus, these results suggest that semaphorins as exemplified by CD100 also play a functional role in the immune system.

Complementation of the ionizing radiation sensitivity, DNA end binding, and V(D)J recombination defects of double-strand break repair mutants by the p86 Ku autoantigen.

Two ionizing radiation-sensitive (IRs) and DNA double-strand break (DSB) mutants, sxi-3 and sxi-2, were shown to be severely deficient in a DNA end binding activity, similar to a previously described activity of the Ku autoantigen, correlating with the xrs (XRCC5) mutations. Cell fusions with xrs-6, another IRs, DSB repair-deficient cell line, defined these sxi mutants in the XRCC5 group. sxi-3 cells have low expression levels of the p86Ku mRNA. Introduction of the Ku p86 gene, but not the p70 Ku gene, complemented the IRs, DNA end binding, and variable (diversity) joining [V(D)J] recombination signal and coding junction deficiencies of sxi-3. Thus, the p86 Ku gene product is essential for DSB repair and V(D)J recombination.

Complementation of V(D)J recombination defect and X-ray sensitivity of scid mouse cells by human chromosome 8.

Cells derived from mice homozygous for the severe combined immune deficiency (scid) mutation exhibit hypersensitivity to ionizing radiation, and defects in DNA double-strand break repair and V(D)J recombination. Using the technique of microcell-mediated chromosome transfer, we have introduced a number of dominantly marked human chromosomes into scid cells to localize the human homolog of the murine scid gene. Analysis of human-scid hybrid clones revealed that the presence of human chromosome 8 partially restored accurate V(D)J recombination and radioresistance to scid cells. Subsequent loss of the human chromosome 8 from human-scid hybrid clones rendered these cells sensitive to gamma-radiation and impaired their ability to catalyse V(D)J recombination. Introduction of chromosomes 2, 14, 16 and 19 that encode other repair genes did not result in the correction of these two scid defects. These observations demonstrate that the human homolog of the mouse scid gene resides on human chromosome 8.

Characterization of ligand binding to a carbohydrate-recognition domain of the macrophage mannose receptor.

The extracellular portion of the macrophage mannose receptor, an endocytic receptor involved in clearance of glycoconjugates, contains eight domains related to the Ca(2+)-dependent carbohydrate-recognition domains (CRDs) of other C-type animal lectins. The characteristics of ligand binding to an expressed form of one of these CRDs (CRD-4) have been investigated. The expressed domain was found to be a monomer in solution. Results of a solid phase binding assay and a protease resistance assay show that CRD-4 of the mannose receptor undergoes a conformational rearrangement upon binding of Ca2+, correlating with its ability to bind sugar. CRD-4 requires two Ca2+ for sugar binding, even though sequence comparisons with other C-type CRDs suggested that it might bind only one Ca2+. The results are consistent with a ternary complex being formed between CRD-4, sugar, and Ca2+ as is seen in the crystal structure of the CRD of rat mannose-binding protein in complex with an oligosaccharide. The stability of Ca2+ binding is shown to be pH-dependent, a result that is pertinent to release of ligand by the receptor in the endosome. However, CRD-4 retains sugar binding activity at a lower pH than does the whole receptor, suggesting that the conformational change in this CRD alone may not be sufficient to allow release of ligand in the endosomes.