Structural and dynamic studies of two antigenic loops from haemagglutinin: A relaxation matrix approach

Summary We have investigated the dynamics and structural behaviour of two antigenic peptides using ¹H NMR. The two cyclic peptides mimic the antigenic site A of influenza haemagglutinin protein; they only differ in the way they were cyclized and in the size of their respective linkers. Homonuclear relaxation parameters extracted from a complete NOE matrix were interpreted in terms of local dynamics. A set of distance constraints was deduced from these parameters which allowed 3D models to be constructed using distance geometry. NOE back-calculation was used to check the validity of the final models. Strong variations of internal motion amplitude have been found in both peptides along their backbone. Motions with high amplitudes have been localized in the Gly-Pro-Gly sequence which forms a -turn in both structures.Abbreviations DSS 3-(trimethylsilyl)-1-propanesulfonic acid - D-loop aspartic acid loop - ELISA enzyme-linked immunoabsorbent assay - f.i.d free induction decay - HOHAHA homonuclear Hartmann-Hahn spectroscopy - HPLC high pressure liquid chromatography - K-loop lysine loop - NMR nuclear magnetic resonance - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - r.m.s.d. root-mean-square deviation of atomic positions     

Uterine epithelial cells specifically induce interferon-stimulated genes in response to polyinosinic-polycytidylic acid independently of estradiol

Interferon β (IFNβ) is an antiviral cytokine secreted in response to pathogenic exposure that creates a restrictive intracellular environment through the action of downstream interferon-stimulated genes (ISG). The objective of this study was to examine the expression of IFNβ and ISG in both human uterine epithelial cells (UEC) and the ECC-1 uterine epithelial cell line and determine if expression changes with TLR stimulation and hormone exposure. Stimulation of primary uterine epithelial cells and ECC-1 cells with the TLR3 agonist poly (I:C) induced the mRNA expression of IFNβ, MxA, OAS2 and PKR. Other TLR agonists including imiquimod and CpG had no effect on either IFNβ or ISG expression. In contrast to ECC-1 cell responses which were slower, maximal IFNβ upregulation in UEC occurred 3 hours post-stimulation and preceded the ISG response which peaked approximately 12 hours after poly (I:C) exposure. Unexpectedly, estradiol, either alone or prior to treatment with poly (I:C), had no effect on IFNβ or ISG expression. Blockade of the IFN receptor abrogated the upregulation of MxA, OAS2 and PKR. Furthermore, neutralizing antibodies against IFNβ partially inhibited the upregulation of all three ISG. Estradiol, directly and in the presence of poly (I:C) had no effect on IFNβ and ISG expression. These results indicate that uterine epithelial cells are important sentinels of the innate immune system and demonstrate that uterine epithelial cells are capable of mounting a rapid IFN-mediated antiviral response that is independent of estradiol and is therefore potentially sustained throughout the menstrual cycle to aid in the defense of the uterus against potential pathogens.     

High-affinity folate binding in human mammary gland

High-affinity³H-folate binding in Triton X-100 solubilized human mammary gland tissue displayed characteristics, e.g. apparent positive cooperativity and increasing affinity with decreasing concentration of folate binding protein, shown to be typical of specific folate binding. Radioligand dissociation was slow at pH 7.4. A major fraction of the bound radioligand dissociated rapidly at pH 3.5, while a residual binding of 20% persisted even after prolonged dialysis at pH 3.5. Gel chromatography revealed two major folate binding proteins (M_r100 kDa and 25 kDa). However, only one single band was detectable on SDS-PAGE immunoblotting. The highest folate binding activity per g protein was associated with the upper triglyceride-containing layer of the 1000 g supernatant of the homogenate. The folate binding protein extracted from this layer had a low cross-reactivity (<5%) with rabbit antibodies against 25 kDa human milk folate binding protein. The folate binding protein in the 1000 g pellet and the aqueous phase of the 1000 g supernatant was present at a low concentration and had a cross-reactivity of 100%.     

Shelf-life extension program (SLEP) as a significant contributor to Strategic National Stockpile Maintenance: the Israeli experience with ciprofloxacin

In the past decade, the 2001 anthrax incident in the U.S. and the 2003 SARS epidemic have highlighted the biological threat to civilian populations. The risk posed by the natural or manmade spread of biological agents among the population dictates a need for better national preparedness. One key component of this preparation is the establishment of a Strategic National Stockpile (SNS) of pharmaceuticals that would provide appropriate medical countermeasures in case of an outbreak. However, to reduce the expense of such a stockpile and to make it worthwhile, there is also a need for a shelf-life extension program (SLEP) through which pharmaceuticals could be extended beyond manufacturer-ascribed shelf life, as long as they meet regulation standards. In this article, we review the Israeli experience with the national ciprofloxacin stockpile procurement and shelf-life extension program.