Studies on a glycopeptide from ovalbumin

1. The structure of the carbohydrate component of the glycopeptide isolated from the proteolytic digest of ovalbumin has been investigated by chemical and enzymic methods. 2. The results are consistent with the presence of a single carbohydrate prosthetic group, linked through its reducing end group to the peptide chain. 3. Further, all the 2-amino-2-deoxy-d-glucose units appear to be in the N-acylated form, the phenolic hydroxyl group of tyrosine is free and the ω-carboxyl group of aspartic acid is substituted. 4. The carbohydrate component has a branched-chain structure, the two non-reducing ends being terminated by a d-mannopyranosyl and a 2-acetamido-2-deoxy-d-glucopyranosyl residue respectively. 5. The terminal d-mannopyranosyl unit is probably linked through at least one other d-mannopyranosyl residue to the remainder of the carbohydrate.     

Mitigation under Section 404 of the Clean Water Act: where it comes from, what it means

The requirement to mitigate impacts to wetlands and streams is a frequently misunderstood policy with a long and complicated history. We narrate the history of mitigation since the inception of the Clean Water Act Section 404 permit program in 1972, through struggles between the US Environmental Protection Agency and the US Army Corps of Engineers, through the emerging importance of wetland conservation on the American political landscape, and through the rise of market-based approaches to environmental policy. Mitigation, as it is understood today, was not initially foreseen as a component of the Section 404 permitting program, but was adapted from 1978 regulations issued by the Council on Environmental Quality as a way of replacing the functions of filled wetlands where permit denials were unlikely. EPA and the Corps agreed in 1990 to define mitigation as the three steps of avoidance, minimization, and compensation, principles which must be applied to permit decisions in the form of the environmental criteria in EPA’s 404(b)(1) Guidelines. Through the 1980s and 1990s, the compensation component of mitigation has become nearly the sole focus of mitigation policy development, and has been the subject of numerous guidance documents and memoranda since 1990. Avoidance and minimization have received far less policy attention, and this lack of policy development may represent a missed opportunity to implement effective wetland conservation.

CaMK activation during exercise is required for histone hyperacetylation and MEF2A binding at the MEF2 site on the Glut4 gene

The role of CaMK II in regulating GLUT4 expression in response to intermittent exercise was investigated. Wistar rats completed 5 x 17-min bouts of swimming after receiving 5 mg/kg KN93 (a CaMK II inhibitor), KN92 (an analog of KN93 that does not inhibit CaMK II), or an equivalent volume of vehicle. Triceps muscles that were harvested at 0, 6, or 18 h postexercise were assayed for 1) CaMK II phosphorylation by Western blot, 2) acetylation of histone H3 at the Glut4 MEF2 site by chromatin immunoprecipitation (ChIP) assay, 3) bound MEF2A at the Glut4 MEF2 cis-element by ChIP, and 4) GLUT4 expression by RT-PCR and Western blot. Compared with controls, exercise caused a twofold increase in CaMK II phosphorylation. Immunohistochemical stains indicated increased CaMK II phosphorylation in nuclear and perinuclear regions of the muscle fiber. Acetylation of histone H3 in the region surrounding the MEF2 binding site on the Glut4 gene and the amount of MEF2A that bind to the site increased approximately twofold postexercise. GLUT4 mRNA and protein increased approximately 2.2- and 1.8-fold, respectively, after exercise. The exercise-induced increases in CaMK II phosphorylation, histone H3 acetylation, MEF2A binding, and GLUT4 expression were attenuated or abolished when KN93 was administered to rats prior to exercise. KN92 did not affect the increases in pCaMK II and GLUT4. These data support the hypothesis that CaMK II activation by exercise increases GLUT4 expression via increased accessibility of MEF2A to its cis-element on the gene.     

HIF-VEGF pathways are critical for chronic otitis media in Junbo and Jeff mouse mutants

Otitis media with effusion (OME) is the commonest cause of hearing loss in children, yet the underlying genetic pathways and mechanisms involved are incompletely understood. Ventilation of the middle ear with tympanostomy tubes is the commonest surgical procedure in children and the best treatment for chronic OME, but the mechanism by which they work remains uncertain. As hypoxia is a common feature of inflamed microenvironments, moderation of hypoxia may be a significant contributory mechanism. We have investigated the occurrence of hypoxia and hypoxia-inducible factor (HIF) mediated responses in Junbo and Jeff mouse mutant models, which develop spontaneous chronic otitis media. We found that Jeff and Junbo mice labeled in vivo with pimonidazole showed cellular hypoxia in inflammatory cells in the bulla lumen, and in Junbo the middle ear mucosa was also hypoxic. The bulla fluid inflammatory cell numbers were greater and the upregulation of inflammatory gene networks were more pronounced in Junbo than Jeff. Hif-1α gene expression was elevated in bulla fluid inflammatory cells, and there was upregulation of its target genes including Vegfa in Junbo and Jeff. We therefore investigated the effects in Junbo of small-molecule inhibitors of VEGFR signaling (PTK787, SU-11248, and BAY 43-9006) and destabilizing HIF by inhibiting its chaperone HSP90 with 17-DMAG. We found that both classes of inhibitor significantly reduced hearing loss and the occurrence of bulla fluid and that VEGFR inhibitors moderated angiogenesis and lymphangiogenesis in the inflamed middle ear mucosa. The effectiveness of HSP90 and VEGFR signaling inhibitors in suppressing OM in the Junbo model implicates HIF-mediated VEGF as playing a pivotal role in OM pathogenesis. Our analysis of the Junbo and Jeff mutants highlights the role of hypoxia and HIF-mediated pathways, and we conclude that targeting molecules in HIF-VEGF signaling pathways has therapeutic potential in the treatment of chronic OM.     

The synthesis of 2,1′-anhydro-,2,1′:3,6-dianhydro-, and 2,1′:3,6: 3′,6′-trianhydro-sucrose

1′-O-Mesyl-6,6′-di-O-tritylsucrose and the corresponding 1′-O-tosyl derivative were prepared from 6,6′-di-O-tritylsucrose by selective sulphonylation. Both sulphonates underwent intramolecular cyclisation reactions, to give 2,1′-anhydrosucrose in high yields rather than the isomeric 1′,4′-anhydride. Sequential benzoylation, detritylation, and mesylation of the 2,1′-anhydride afforded 2,1′-anhydro-6,6′-di-O-mesylsucrose tetrabenzoate which, in the presence of base, gave 2,1′:3,6:3′,6′-trianhydrosucrose that was not identical with the product previously claimed to have this structure. Several derivatives of 2,1′-anhydrosucrose were prepared possessing different functional groups at either the 6,6′- or 4,6′-positions. Dimolar mesitylene-sulphonylation of 3,3′,4′6′-tetra-O-acetylsucrose gave the 6,1′-disulphonate, which, in the presence of alkali, gave 2,1′:3,6-dianhydrosucrose, which was transformed into the 2,1′:3,6:3′,6′-trianhydride by sequential bromination at C-6′ (carbon tetrabromide-triphenylphosphine) and base-catalysed cyclisation. Treatment of 3,3′,4′,6′-tetra-O-benzoylsucrose with sulphuryl chloride furnished the 4,6,1′-trichloro derivative, which, on alkaline hydrolysis, was converted into 2,1′:3,6-dianhydro-4-chloro-4-deoxy-galacto-sucrose.     

Substrate-enzyme interactions and catalytic mechanism in phospholipase C: a molecular modeling study using the GRID program.

Based on the high-resolution X-ray crystallographic structure of phospholipase C from Bacillus cereus, the orientation of the phosphatidylcholine substrate in the active site of the enzyme is proposed. The proposal is based on extensive calculations using the GRID program and molecular mechanics geometry relaxations. The substrate model has been constructed by successively placing phosphate, choline and diacylglycerol moieties in the positions indicated from GRID calculations. On the basis of the resulting orientation of a complete phosphatidylcholine molecule, we propose a mechanism for the hydrolysis of the substrate.     

Circular pancreatic trypsin inhibitor. A novel substrate for studies on intracellular proteolysis

Several recent studies indicate that substrates for ubiquitin-dependent proteolysis must possess unblocked alpha-amino termini. To examine further the importance of free amino groups for proteolytic susceptibility we selected pancreatic trypsin inhibitor (PTI) as a test substrate. PTI can be circularized to form cPTI, a molecule that lacks alpha-amino groups in the absence of an endoproteolytic cleavage. We compared the breakdown of radioiodinated PTI and cPTI in rabbit reticulocyte lysate and found that cPTI was not stabilized relative to PTI. In addition, proteolysis of PTI or cPTI was not inhibited upon conversion of their lysine residues to homoarginine. However, neither degradation of PTI nor cPTI required ATP, and ubiquitin conjugation to either molecule was minor relative to known substrates of the ubiquitin pathway. Thus, PTI and cPTI are cleaved by an ATP-independent endoprotease(s) that does not require the substrate to be ubiquitinated. Such an activity was identified in low salt fractions obtained upon DEAE chromatography of reticulocyte lysate. The ubiquitin/ATP-dependent protease and another large multisubunit protease, both of which elute from DEAE-Fractogel at higher salt concentrations, do not degrade PTI or cPTI. Although monomeric PTI was rapidly degraded in reticulocyte lysate, cross-linked PTI molecules were stable both in reticulocyte lysate and following introduction into cultured cells using red blood cell-mediated microinjection. Thus, increased rates of turnover do not necessarily correlate with greater molecular mass of the substrate.