Xanthine oxidase induced depolymerization of hyaluronic acid in the presence of ferritin

Oxygen free radicals generated by xanthine oxidase are able to depolymerize hyaluronic acid in the presence of ferritin-bound iron. This suggests that ferritin can catalyse the Haber-Weiss reaction, leading to the formation of highly damaging hydroxyl radicals.     

PPAR-gamma activation fails to provide myocardial protection in ischemia and reperfusion in pigs

Peroxisome proliferator-activated receptor (PPAR)-gamma modulates substrate metabolism and inflammatory responses. In experimental rats subjected to myocardial ischemia-reperfusion (I/R), thiazolidinedione PPAR-gamma activators reduce infarct size and preserve left ventricular function. Troglitazone is the only PPAR-gamma activator that has been shown to be protective in I/R in large animals. However, because troglitazone contains both alpha-tocopherol and thiazolidinedione moieties, whether PPAR-gamma activation per se is protective in myocardial I/R in large animals remains uncertain. To address this question, 56 pigs were treated orally for 8 wk with troglitazone (75 mg x kg(-1) x day(-1)), rosiglitazone (3 mg x kg(-1) x day(-1)), or alpha-tocopherol (73 mg x kg(-1) x day(-1), equimolar to troglitazone dose) or received no treatment. Pigs were then anesthetized and subjected to 90 min of low-flow regional myocardial ischemia and 90 min of reperfusion. Myocardial expression of PPAR-gamma, determined by ribonuclease protection assay, increased with troglitazone and rosiglitazone compared with no treatment. Rosiglitazone had no significant effect on myocardial contractile function (Frank-Starling relations), substrate uptake, or expression of proinflammatory cytokines during I/R compared with untreated pigs. In contrast, preservation of myocardial contractile function and lactate uptake were greater and cytokine expression was attenuated in pigs treated with troglitazone or alpha-tocopherol compared with untreated pigs. Multivariate analysis indicated that presence of an alpha-tocopherol, but not a thiazolidinedione, moiety in the test compound was significantly related to greater contractile function and lactate uptake and lower cytokine expression during I/R. We conclude that PPAR-gamma activation is not protective in a porcine model of myocardial I/R. Protective effects of troglitazone are attributable to its alpha-tocopherol moiety. These findings, in conjunction with prior rat studies, suggest interspecies differences in the response to PPAR-gamma activation in the heart.     

Vertebrate telomere repeat DNAs favor external loop propeller quadruplex structures in the presence of high concentrations of potassium

The circular dichroism, CD, spectra of the telomere repeats of vertebrates, d(TTAGGG), indicate that parallel type quadruplex structures or disordered single-stranded structures are formed in low salt. Anti-parallel quadruplex structures are favored in the presence of high concentrations, 140 mM, of sodium. External loop, also known as propeller, parallel type structures are favored in the presence of high concentrations, 100 mM, of potassium in the presence of either 5 or 140 mM sodium. The cation dependence of the CD spectra of the vertebrate telomere repeat DNAs is distinctly different from that of the telomere repeats of Tetrahymena and Oxytricha as well as that of the thrombin binding aptamer. These results indicate that the external loop structures may be present in vertebrate telomeres under the conditions of high potassium and low sodium concentration found in nuclei.     

The epidermal growth factor receptor juxtamembrane domain has multiple basolateral plasma membrane localization determinants,including a dominant signal with a polyproline core

The epidermal growth factor (EGF) receptor is located predominantly in the basolateral membrane of polarized epithelia, where it plays a pivotal role during organogenesis and tissue homeostasis. We have shown previously that a 22-amino acid sequence in the EGF receptor juxtamembrane domain contains autonomous sorting information necessary for basolateral localization using the Madin-Darby canine kidney epithelial cell model. The goal of this study was to determine the molecular basis of EGF receptor basolateral membrane expression using site-directed mutagenesis to modify specific residues in this region. We now show that this sequence has two different, functionally redundant basolateral sorting signals with distinct amino acid requirements: one dependent on residues (658)LL(659) conforming to well-characterized leucine-based sorting signals, and a second containing a polyproline core comprising residues Pro(667) and Pro(670) ((667)PXXP(670)). Our data also suggest that Arg(662) contributes to the function of the proline-based signal. (667)PXXP(670) was the dominant signal when both motifs were present and was more effective than (658)LL(659) at overriding strong apical sorting signals located in the same molecule. Site-directed mutations at Arg(662), Pro(667), and Pro(670) were also associated with increased apical expression of full-length EGF receptors, demonstrating for the first time that the juxtamembrane region is necessary for accurate polarized expression of the native molecule.     

Beta-secretase processing in the trans-Golgi network preferentially generates truncated amyloid species that accumulate in Alzheimer's disease brain

The amyloid beta (A beta) peptide that accumulates in Alzheimer's disease brain is derived from the proteolytic processing of the amyloid precursor protein by beta- and gamma-secretase activities. The beta-secretase enzyme beta-site amyloid precursor protein-cleaving enzyme (BACE) generates the N terminus of A beta by cleavage at either Asp(1) (beta-site) or Glu(11) (beta'-site), ultimately leading to the production of full-length A beta 1-40/42 or truncated A beta 11-40/42. The functional significance of this variable cleavage site specificity as well as the relative pathological impact of full-length versus N-terminally truncated A beta remains largely unknown. In our analysis of BACE reactivity in cell culture, we found that the preference of the protease for either beta- or beta'-cleavage was strongly dependent on intracellular localization. Within the endoplasmic reticulum, beta-site proteolysis predominated, whereas in the trans-Golgi network, beta'-cleavage was favored. Furthermore, the contrasting cleavage site specificities of BACE were not simply due to differences in organelle pH or the oligosaccharide composition of the glycoproteins involved. Examination of post-mortem brain specimens revealed significant levels of A beta 11-40/42 within insoluble amyloid pools. Taken together, these data support an important role for beta'-cleavage in the process of cerebral amyloid deposition and localize the processing event to the trans-Golgi network.     

Potential role for a novel AP180-related protein during endocytosis in MDCK cells

Clathrin assembly protein, AP180, was originally identified as a brain-specific protein localized to the presynaptic junction. AP180 acts to limit vesicle size and maintain a pool of releasable synaptic vesicles during rapid recycling. In this study, we show that polarized epithelial Madin-Darby canine kidney (MDCK) cells express two AP180-related proteins: the ubiquitously expressed 62-kDa clathrin assembly lymphoid myeloid leukemia (CALM, AP180-2) protein and a novel high-molecular-weight homolog that we have named AP180-3. Sequence analysis of AP180-3 expressed in MDCK cells shows high homology to AP180 from rat brain. AP180-3 contains conserved motifs found in brain-specific AP180, including the epsin NH₂-terminal homology (ENTH) domain, the binding site for the -subunit of AP-2, and DLL repeats. Our studies show that AP180-3 from MDCK cells forms complexes with AP-2 and clathrin and that membrane recruitment of these complexes is modulated by phosphorylation. We demonstrate by immunohistochemistry that AP180-3 is localized to cytoplasmic vesicles in MDCK cells and is also present in tubule epithelial cells from mouse kidney. We observed by immunodetection that a high-molecular-weight AP180-related protein is expressed in numerous cells in addition to MDCK cells. clathrin assembly lympoid myeloid leukemia; kidney epithelial cells; epsin NH₂-terminal homology domain; DLL repeats; clathrin; AP-2     

Variability in spore germination response by strains of proteolytic Clostridium botulinum types A,B and F

AIMS: The objective of the study was to evaluate the variability of germination response of 10 strains of proteolytic Clostridium botulinum. METHODS AND RESULTS: An automated turbidometric method was used to follow the fall in optical density. Spores of proteolytic Cl. botulinum germinated in response to l-alanine alone, with rate and extent of germination increased by addition of l-lactate or bicarbonate ions. Other hydrophobic amino acids also triggered germination of spores of proteolytic Cl. botulinum but not AGFK and inosine, germinants for Bacillus subtilis or B. cereus. CONCLUSIONS: Unlike spores of nonproteolytic Cl. botulinum, all proteolytic Cl. botulinum germinate in hydrophobic l-amino acids without l-lactate. However, a great variability of response to germinant is evidenced between the species. SIGNIFICANCE AND IMPACT OF THE STUDY: The selection of a model strain to study germination of Cl. botulinum spores should consider the variability in sensitivity to germinants shown in this work. In particular, the sequenced strain ATCC 3502 may not be the most appropriate model for germination studies.     

On the precision of the conditionally autoregressive prior in spatial models

Bayesian analyses of spatial data often use a conditionally autoregressive (CAR) prior, which can be written as the kernel of an improper density that depends on a precision parameter tau that is typically unknown. To include tau in the Bayesian analysis, the kernel must be multiplied by tau(k) for some k. This article rigorously derives k = (n - I)/2 for the L2 norm CAR prior (also called a Gaussian Markov random field model) and k = n - I for the L1 norm CAR prior, where n is the number of regions and I the number of "islands" (disconnected groups of regions) in the spatial map. Since I = 1 for a spatial structure defining a connected graph, this supports Knorr-Held's (2002, in Highly Structured Stochastic Systems, 260-264) suggestion that k = (n - 1)/2 in the L2 norm case, instead of the more common k = n/2. We illustrate the practical significance of our results using a periodontal example.     

A structural model for the membrane-bound form of the juxtamembrane domain of the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family involved in the regulation of cellular proliferation and differentiation. Its juxtamembrane domain (JX), the region located between the transmembrane and kinase domains, plays important roles in receptor trafficking. Two sorting signals, a PXXP motif and a 658LL659 motif, are responsible for basolateral sorting in polarized epithelial cells, and a 679LL680 motif targets the ligand-activated receptor for lysosomal degradation. To understand the regulation of these signals, we characterized the structural properties of recombinant JX domain in aqueous solution and in dodecylphosphocholine (DPC) detergent. JX is inherently unstructured in aqueous solution, albeit a nascent helix encompasses the lysosomal sorting signal. In DPC micelles, structures derived from NMR data showed three amphipathic, helical segments. A large, internally inconsistent group of long range nuclear Overhauser effects suggest a close proximity of the helices, and the presence of significant conformational averaging. Models were determined for the average JX conformation using restraints representing the translational restriction due to micelle-surface adsorption, and the helix orientations were determined from residual dipolar couplings. Two equivalent average structural models were obtained that differ only in the relative orientation between first and second helices. In these models, the 658LL659 and 679LL680 motifs are located in the first and second helices and face the micelle surface, whereas the PXXP motif is located in a flexible helix-connecting region. The data suggest that the activity of these signals may be regulated by their membrane association and restricted accessibility in the intact receptor.