Analysis of the intrinsic bend in the M13 origin of replication by atomic force microscopy

Atomic force microscopy (AFM) has been used to image a 471-bp bent DNA restriction fragment derived from the M13 origin of replication in plasmid LITMUS 28, and a 476-bp normal, unbent fragment from plasmid pUC19. The most probable angle of curvature of the 471-bp DNA fragment is 40-50 degrees, in reasonably good agreement with the bend angle determined by transient electric birefringence, 38 degrees +/- 7 degrees. The normal 476-bp DNA fragment exhibited a Gaussian distribution of bend angles centered at 0 degrees, indicating that this fragment does not contain an intrinsic bend. The persistence length, P, was estimated to be 60 +/- 8 and 62 +/- 8 nm for the 471- and 476-bp fragments, respectively, from the observed mean-square end-to-end distances in the AFM images. Since the P-values of the normal and bent fragments are close to each other, the overall flexibility of DNA fragments of this size is only marginally affected by the presence of a stable bend. The close agreement of AFM and transient electric birefringence results validates the suitability of both methods for characterizing DNA bending and flexibility.     

Validation of a reverse transcription nested polymerase chain reaction (RT-nPCR) to detect bovine viral diarrhea virus (BVDV) associated with in vitro-derived bovine embryos and co-cultured cells

Sensitive RT-nPCR assays can be used for the rapid detection of viruses. The objective of this research was to validate an RT-nPCR assay for detection of BVDV associated with various samples collected from an IVF system. In 12 research replicates, we maintained matured COCs as negative controls or exposed them to 1 of 4 noncytopathic strains (SD-1, NY-1, CD-87, or PA-131) of BVDV for 1 h immediately before IVF. After 4 d of IVC, we harvested groups of 5 nonfertile ova or degenerated embryos (NFD) and some associated cumulus cells and transferred developing embryos and the remaining cumulus cells into secondary IVC drops. On the seventh d of IVC, cumulus cells, groups of 5 washed NFD and groups of 5 developed, washed embryos were harvested. We also collected single developed embryos after washing, washing with trypsin, washing and cryopreservation in ethylene glycol, or washing with trypsin and cryopreservation in ethylene glycol. All washes were performed according to International Embryo Transfer Society standards. Developed embryos and NFD were sonicated prior to assay. All samples were assayed for BVDV using virus isolation and RT-nPCR. The virus isolation and RT-nPCR assays determined that all negative control samples were BVDV-free. Virus was detected in association with all exposed cumulus cells and groups of developed embryos using both virus isolation and RT-nPCR. Results from viral assays of other exposed samples indicate enhanced sensitivity of the RT-nPCR assay. The RT-nPCR assay used in this research exhibited acceptable sensitivity, specificity, predictive value and repeatability for rapid detection of BVDV associated with the various samples obtained from an IVF system.     

Structural characterization of the catalytic domain of the human 5-lipoxygenase enzyme

Leukotrienes are inflammatory mediators involved in several diseases. The enzyme 5-lipoxygenase initiates the synthesis of leukotrienes from arachidonic acid. Little structural information is available regarding 5-lipoxygenase. In this study, we found that the primary structure of the catalytic domain of human 5-lipoxygenase is similar to that of the rabbit 15-lipoxygenase. This similarity allowed the development of a theoretical model of the tertiary structure of the 5-lipoxygenase catalytic domain, using the resolved structure of rabbit 15-lipoxygenase as a template. This model was used in conjunction with primary and secondary structural information to investigate putative nucleotide binding sites, a MAPKAP kinase 2 phosphorylation site, and a Src homology 3 binding site on the 5-lipoxygenase protein, further. Results indicate that the putative nucleotide binding sites are spatially distinct, with one on the -barrel domain and the other(s) on the catalytic domain. The MAPKAP kinase 2 phosphorylation site involves a four amino acid insertion in mammalian 5-lipoxygenases that significantly alters molecular structure. This target for post-translational modification is both common and unique to 5-lipoxygenases. The Src homology 3 binding site, found in all lipoxygenases, appears to lack the characteristic left-handed type II helix structure of known Src homology 3 binding sites. These results, which highlight the unique nature of the MAPKAP kinase site, underscore the utility of structural information in the analysis of protein function. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00894-002-0076-y.Electronic Supplementary Material available.     

Structural and functional criteria reveal a new nuclear import sequence on the 5-lipoxygenase protein

Leukotrienes are lipid mediators with important roles in immunity. The enzyme 5-lipoxygenase initiates leukotriene synthesis; nuclear import of 5-lipoxygenase modulates leukotriene synthetic capacity. In this study, we used structural and functional criteria to identify potential nuclear import sequences. Specifically, we sought basic residues that 1) were common to different 5-lipoxygenases but not shared with other lipoxygenases, 2) were found on random coil/loop structures, and 3) could be replaced without eliminating catalytic activity. Application of these criteria to the putative bipartite nuclear import sequence of 5-lipoxygenase revealed that this region formed an alpha-helix rather than a random coil, that the critical residue arginine 651 serves a structural role, and that mutation of this residue eliminated catalytic activity. A previously unrecognized region corresponding to residues 518-530 on human 5-lipoxygenase was found to be unique to 5-lipoxygenase and on a random coil. This region alone was sufficient to drive import of green fluorescent protein to the same degree as complete 5-lipoxygenase. Replacement of basic residues in this region of the complete protein was capable of eliminating nuclear import without abolishing catalytic activity. Surprisingly, two subpopulations of cells expressing 5-lipoxygenase with this mutated region could be discerned: those with strongly impaired import and those with normal import. Taken together, these results show that the previously identified region with a bipartite motif is not a functional import sequence, whereas the newly identified basic region constitutes a true nuclear import sequence. Moreover, we suggest that another sequence that can mediate nuclear import of 5-lipoxygenase remains to be identified.     

Expression in E. coli and purification of Thermus thermophilus translation initiation factors IF1 and IF3

The initiation of protein translation in bacteria requires in addition to mRNA, fMet-tRNA, and ribosomal subunits three protein factors, the initiation factor 1 (IF1), initiation factor 2 (IF2), and initiation factor 3 (IF3). The genes coding for IF1 and IF3 from Thermus thermophilus have been identified and cloned into pET expression vector and were expressed as soluble proteins in Escherichia coli. IF1 was purified by a DEAE-cellulose chromatography, followed by heat denaturation, chromatography on Hydroxylapatit, and gel permeation chromatography using Sephacryl 200HR. For the purification of IF3, a heat denaturation step is followed by anion-exchange chromatography on Q-Sepharose FF and gel permeation chromatography on Sephacryl 200HR. Using these procedures we obtained chromatographically pure and biologically active preparations of both T. thermophilus IF1 and IF3.     

Leukotriene synthesis by epithelial cells

Leukotrienes (LTs) are intercellular signaling molecules that evoke a variety of responses. They are best known as potent promoters of inflammation. Normally, LTs are produced primarily by leukocytes. As a result, current models regarding the production of LTs in the context of disease focus on the leukocytes as the site of production. Structural cells, including epithelial cells, are typically relegated to supportive roles. It is recognized that epithelial cells normally contain all the components necessary for LT synthesis except the enzyme 5-lipoxygenase (5-LO). There is accumulating evidence that some populations of epithelial cells normally express low levels of 5-LO and can synthesize LTs autonomously. Moreover, certain factors, including bacterial and viral infection, can promote the expression of 5-LO in airway, gastrointestinal and skin epithelial cells. The appearance of active 5-LO enzyme in epithelial cells at these sites may contribute to diseases like cancer, colitis and psoriasis. This paper reviews the state of our knowledge regarding the expression of 5-LO in epithelial cells, the factors that modify that expression, and the implications regarding pathogenesis.     

Kupffer cell-initiated remote hepatic injury following bilateral hindlimb ischemia is complement dependent

Intravital fluorescence microscopy was applied to the livers of male Wistar rats to test the hypothesis that complement mobilization stimulates Kupffer cells and subsequently initiates hepatic injury after hindlimb ischemia/reperfusion (I/R). Following 3 h of limb reperfusion, hepatocellular viability (serum levels of alanine transaminase and cell death via propidium iodide labeling) decreased significantly from levels in sham-operated animals. Inhibition of complement mobilization with soluble complement receptor type 1 (20 mg/kg body wt) and interruption of Kupffer cell function with GdCl(3) (1 mg/100g body wt) resulted in significant hepatocellular protection. Although the effects of hindlimb I/R on hepatic microvascular perfusion were manifest as increased heterogeneity, both complement inhibition and suppression of Kupffer cell function resulted in marked improvements. No additional hepatocellular protection and microvascular improvements were provided by combining the interventions. Furthermore, inhibition of complement mobilization significantly depressed Kupffer cell phagocytosis by 42% following limb reperfusion. These results suggest that the stimulation of Kupffer cells via complement mobilization is necessary but is not the only factor contributing to the early pathogenesis of hepatic injury following hindlimb I/R.     

Mutational analysis of the C-terminus in ion transport peptide (ITP) expressed in Drosophila Kc1 cells

Ion transport peptide (ITP) stimulates Cl(-) transport (measured as short-circuit current, I(sc)) and fluid reabsorption in Schistocerca gregaria ilea. We report that Drosophila Kc1 cells transfected with preproITP cDNA secrete a peptide (KcITP(75)) that, while cleaved correctly at the N-terminus, had reduced (10-fold) stimulatory activity on ileal I(sc) compared to both native ITP (ScgITP) and synthetic ITP (synITP). We provide evidence that the reduced activity of KcITP(75) is due to incomplete processing of the C-terminal sequence LGKK (KcITP(75)) to L-amide. In support of this, in vitro amidation of glycine extended ITP (i.e., KcITP(73) ending in LG) but not KcITP(75) (ending in LGKK) significantly increased specific activity in the bioassay. Further evidence for C-terminus involvement includes complete loss of stimulation by truncated mutants (e.g., KcITP(71) which lacks LGKK) and a mutant in which alanine is substituted for the terminal glycine in KcITP(73). Moreover a natural homologue (KcITP-L, which differs only in the C-terminal sequence) expressed by Kc1 cells does not stimulate ileal I(sc). Rather KcITP-L acts as a weak ITP antagonist, as does the truncated mutant KcITP(71). KcITP(70) has no antagonistic effect. A short synthetic peptide fragment of the C-terminus (VEIL-amide) does not stimulate ileal I(sc), indicating that other regions of ITP are also essential to biological activity. Arch.