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Candidate Topical Microbicides Bind Herpes Simplex Virus Glycoprotein B and Prevent Viral Entry and Cell-to-Cell Spread. Natalia Cheshenko, 2004.Topical microbicides designed to prevent acquisition of sexually transmitted infections are urgently needed . Nonoxynol-9, the only commercially available spermicide, damages epithelium and may enhance human immunodeficiency virus transmission . The observation that herpes simplex virus (HSV) and human immunodeficiency virus bind heparan sulfate provided the rationale for the development of sulfated or sulfonated polymers as topical agents . Although several of the polymers have advanced to clinical trials, the spectrum and mechanism of anti-HSV activity and the effects on soluble mediators of inflammation have not been evaluated . The present studies address these gaps . The results indicate that PRO 2000, polystyrene sulfonate, cellulose sulfate, and polymethylenehydroquinone sulfonate inhibit HSV infection 10,000-fold and are active against clinical isolates, including an acyclovir-resistant variant . The compounds formed stable complexes with glycoprotein B and inhibit viral binding, entry, and cell-to-cell spread . The effects may be long lasting due to the high affinity and stability of the sulfated compound-virus complex, as evidenced by surface plasmon resonance studies . The candidate microbicides retained their antiviral activities in the presence of cervical secretions and over a broad pH range . There was little reduction in cell viability following repeated exposure of human endocervical cells to these compounds, although a reduction in secretory leukocyte protease inhibitor levels was observed . These studies support further development and rigorous evaluation of these candidate microbicides . ilvIH Operon Expression in Escherichia coli Requires Lrp Binding to Two Distinct Regions of DNA. Samina Jafri, 2002.The leucine-responsive regulatory protein Lrp regulates the expression of a number of operons in Escherichia coli, including the ilvIH operon . Earlier in vitro experiments showed purified Lrp binding to two regions of DNA proximal to the ilvIH promoter, an upstream region (-260 to -190) and a downstream region (-150 to -40) . The effect of mutations in these regions on ilvIH promoter expression in vivo led to the proposal that activation of transcription required Lrp binding to downstream sites 3, 4, 5, and 6 . Binding of Lrp to upstream sites 1 and 2 seemed to enhance promoter expression but was not absolutely required (Q . Wang and J . M . Calvo, J . Mol . Biol . 229:306-318, 1993) . Here we present data that require a reevaluation of the above conclusion . Constructs having either a deletion of DNA or a 100-bp substitution of DNA upstream of position -160 showed no ilvIH promoter activity in vivo . These results unambiguously establish that DNA at or upstream of position -160 is required for ilvIH promoter expression . Together with previous results, we conclude that Lrp bound at downstream sites is necessary but not sufficient for promoter activation . In addition, insertion of 4, 6, 8, or 10 bp between the upstream and downstream regions also resulted in a very strong reduction of in vivo promoter expression, even though the binding of Lrp in vitro was not greatly affected by these mutations . Closer inspection showed that the affinity of Lrp for the upstream region of all of these constructs was about the same but that Lrp bound to the downstream region of the wild-type construct with a higher degree of cooperativity than in the case of the others . These mutations may have reduced promoter activity in vivo by eliminating a binding site for some transcription factor other than Lrp . Alternatively, the small-addition mutations may have affected the geometry of these complexes, preventing either an interaction between Lrps bound at upstream and downstream sites (which might be necessary for promoter expression) or preventing the positioning of Lrp bound at upstream sites for productive interaction with the promoter .
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