A27L protein mediates vaccinia virus interaction with cell surface heparan sulfate

Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.     

The value of immediate cytologic evaluation for needle aspiration lung biopsy

RATIONALE AND OBJECTIVES: The authors evaluate the role of immediate cytologic evaluation (ICE) with fine-needle aspiration biopsy (FNAB) for lung lesions at highest risk for pneumothorax. METHODS: A prospective randomized study was conducted of 80 patients with lung lesions surrounded by aerated parenchyma undergoing FNAB with and without ICE (47 and 33 patients, respectively). An analysis of needle passes, procedure time, complications, specimen adequacy, diagnostic yield, and accuracy of procedure was made. RESULTS: There was an increased number of needle passes with ICE (> or = three passes: 23% [11 biopsies] versus 3% [1 biopsy]; P = 0.01). Fluoroscopic procedures took longer with ICE (median time: 15 versus 9 minutes; P = 0.002) with no difference in complication rates. Specimen adequacy was similar (74% and 64%) and the procedure was diagnostic in 79% (37 biopsies) with ICE and in 70% (33 biopsies) without ICE. There were no significant differences in the sensitivity, specificity, or accuracy of the biopsy. CONCLUSIONS: Immediate cytologic evaluation improved results marginally with increased procedure time and needle passes. Immediate cytologic evaluation may be most useful for lesions at lowest risk of complications to assure that a second procedure is not required.     

DNA binding of hypothalamic nuclear proteins on estrogen response element and preproenkephalin promoter: modification by estrogen

Preproenkephalin (PPE) gene expression is specifically induced by estrogen in hypothalamus of ovariectomized (OVX) females, better than in male rats. To study estrogen actions on gene regulation, we have presently characterized protein-DNA interactions by use of a consensus estrogen response element (ERE) and a putative ERE from PPE gene, with nuclear extracts from hypothalamus. By use of the electrophoretic mobility shift assay (EMSA), ERE binding activity was detected in nuclear extracts from neuronal tissues including hypothalamus, hippocampus, striatum, cerebellum and frontal cortex, and non-neuronal tissues such as pituitary and uterus, but not lung of OVX female rats with a consensus ERE, as well as a 129-bp PCR fragment from PPE promoter and a hairpin oligonucleotide that contains a putative ERE of the rat PPE gene. The ERE binding was eliminated by the addition of specific ERE-containing oligonucleotide, but not control oligonucleotides. Protein and DNA associated and dissociated very rapidly. By use of supershift assay, interactions of estrogen receptor with ERE were demonstrated in hypothalamic nuclear extracts. The initial levels of specific ERE binding in the hypothalamic nuclear extracts were comparable between castrated male and OVX female rats. However, estrogen treatment, either estradiol or estradiol benzoate, produced a rapid and tissue-specific induction of a slow mobility complex of ERE binding in hypothalamic nuclear extracts from females, better than in male rats, presumably from other associated factors, or a conformational change or other posttranslational modifications. This estrogen-induced slow mobility complex of ERE binding in hypothalamus was not observed after treatment with progesterone or tamoxifen. These results suggest that specific ERE binding is present in rat hypothalamic nuclear proteins, which may contribute to the upregulation of PPE gene expression by estrogen, and that the sexually differentiated action of estrogen may be related to an estrogen-induced conformational change, but not to the initial level of ERE-binding activity.     

Brain acetylhydrolase that inactivates platelet-activating factor is a G-protein-like trimer

The platelet-activating factor PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent lipid first messenger active in general cell activation, fertilization, inflammatory and allergic reactions, asthma, HIV pathogenesis, carcinogenesis, and apoptosis. There is substantial evidence that PAF is involved in intracellular signalling, but the pathways are poorly understood. Inactivation of PAF is carried out by specific intra- and extracellular acetylhydrolases (PAF-AHs), a subfamily of phospholipases A2 that remove the sn-2 acetyl group. Mammalian brain contains at least three intracellular isoforms, of which PAF-AH(Ib) is the best characterized. This isoform contains a heterodimer of two homologous catalytic subunits alpha1 and alpha2, each of relative molecular mass 26K, and a non-catalytic 45K beta-subunit, a homologue of the beta-subunit of trimeric G proteins. We now report the crystal structure of the bovine alpha1 subunit of PAF-AH(Ib) at 1.7 A resolution in complex with a reaction product, acetate. The tertiary fold of this protein is closely reminiscent of that found in p21(ras) and other GTPases. The active site is made up of a trypsin-like triad of Ser 47, His 195 and Asp 192. Thus, the intact PAF-AH(Ib) molecule is an unusual G-protein-like (alpha1/alpha2)beta trimer.