Trace element status and inflammation parameters during chronic indomethacin treatment in adjuvant arthritic rats

Oral indomethacin administration (2 mg/kg/d) was investigated in rats with adjuvant arthritis up to a period of 5 wk. Baseline low serum zinc levels in arthritic rats increased rapidly from the first week of indomethacin treatment (started 1 or 2 wk after disease induction), whereas baseline high serum copper decreased after 1–2 wk. After 3–4 wk of treatment, serum zinc levels returned to control values, but serum copper was somewhat higher in arthritic animals having received indomethacin 2 wk after disease induction than in controls. Clinical indices of inflammation simultaneously improved to reach control values at the end of the trial. Biological indicators of inflammation also improved, but did not reach control levels. Serum zinc correlated negatively with plasma fibrinogen (r=?0.69,p<0.005) and serum copper correlated positively with serum ceruloplasmin (r=0.92,p<0.0005) both in indomethacin-treated and untreated arthritic rats. Contrary to long-term glucocorticoid administration that was previously reported to maintain or aggravate hypozincemia, indomethacin treatment normalized perturbed zinc and copper status in arthritic animals.     

Single alpha-domain constructs of the Na+/Ca2+ exchanger, NCLX, oligomerize to form a functional exchanger

Spliced isoforms of the Na+/Ca2+ exchanger, NCLX, truncated at the alpha-repeat region have been identified. The activity and functional organization of such proteins are, however, poorly understood. In the present work, we have studied Na+/Ca2+ exchange mediated by single alpha-repeat constructs (alpha1 and alpha2) of NCLX. Sodium-dependent calcium transport was fluorescently detected in both the reversal and forward modes; calcium-dependent outward currents were also recorded using a whole cell patch configuration in HEK293 cells heterologously expressing either the alpha1 or alpha2 single-domain proteins. In contrast, calcium transport and reversal currents were not detected when cells were transfected with a vector or with an alpha2 mutant (alpha2-S273T). Thus, our data indicate that the single alpha-domain constructs mediate electrogenic Na+/Ca2+ exchange. The alpha1 domain, but not the alpha2, exhibited partial sensitivity to the NCX inhibitor, KB-R7943, while Li+-dependent Ca2+ efflux was detected in cells expressing either the alpha1 or alpha2 construct. The functional organization of the single alpha-domain constructs was assessed using a dominant-negative approach. Coexpression of the alpha1 or alpha2 constructs with the nonfunctional alpha2-S273T mutant had a synergistic inhibitory effect on Na+/Ca2+ transport. Dose-dependence analysis of the inhibition of alpha2 construct activity by the alpha2-S273T mutant indicated that the functional unit is either a dimer or a trimer. Immunoprecipitation analysis indicated that the alpha2 construct indeed interacts with the alpha2-S273T mutant. Taken together, our data indicate that although single alpha1 or alpha2 domain constructs are independently capable of Na+/Ca2+ exchange, oligomerization is required for their activity. Such organization may give rise to transport activity with distinct kinetic parameters and physiological roles.     

Mechanism of Cl- selection by a glutamate-gated chloride (GluCl) receptor revealed through mutations in the selectivity filter

To learn about the mechanism of ion charge selectivity by invertebrate glutamate-gated chloride (GluCl) channels, we swapped segments between the GluClbeta receptor of Caenorhabditis elegans and the vertebrate cationic alpha7-acetylcholine receptor and monitored anionic/cationic permeability ratios. Complete conversion of the ion charge selectivity in a set of receptor microchimeras indicates that the selectivity filter of the GluClbeta receptor is created by a sequence connecting the first with the second transmembrane segments. A single substitution of a negatively charged residue within this sequence converted the selectivity of the GluClbeta receptor's pore from anionic to cationic. Unexpectedly, elimination of the charge of each basic residue of the selectivity filter, one at a time or concomitantly, moderately reduced the P(Cl)/P(Na) ratios, but the GluClbeta receptor's mutants retained high capacity to select Cl(-) over Na(+). These results indicate that, unlike the proposed case of anionic Gly- and gamma-aminobutyric acid-gated ion channels, positively charged residues do not play the key role in the selection of ionic charge by the GluClbeta receptor. Taken together with measurements of the effective open pore diameter and with structural modeling, the study presented here collectively indicates that in the most constricted part of the open GluClbeta receptor's channel, Cl(-) interacts with backbone amides, where it undergoes partial dehydration necessary for traversing the pore.     

Tyrosine phosphatases epsilon and alpha perform specific and overlapping functions in regulation of voltage-gated potassium channels in Schwann cells

Tyrosine phosphatases (PTPs) epsilon and alpha are closely related and share several molecular functions, such as regulation of Src family kinases and voltage-gated potassium (Kv) channels. Functional interrelationships between PTPepsilon and PTPalpha and the mechanisms by which they regulate K+ channels and Src were analyzed in vivo in mice lacking either or both PTPs. Lack of either PTP increases Kv channel activity and phosphorylation in Schwann cells, indicating these PTPs inhibit Kv current amplitude in vivo. Open probability and unitary conductance of Kv channels are unchanged, suggesting an effect on channel number or organization. PTPalpha inhibits Kv channels more strongly than PTPepsilon; this correlates with constitutive association of PTPalpha with Kv2.1, driven by membranal localization of PTPalpha. PTPalpha, but not PTPepsilon, activates Src in sciatic nerve extracts, suggesting Src deregulation is not responsible exclusively for the observed phenotypes and highlighting an unexpected difference between both PTPs. Developmentally, sciatic nerve myelination is reduced transiently in mice lacking either PTP and more so in mice lacking both PTPs, suggesting both PTPs support myelination but are not fully redundant. We conclude that PTPepsilon and PTPalpha differ significantly in their regulation of Kv channels and Src in the system examined and that similarity between PTPs does not necessarily result in full functional redundancy in vivo.     

Translocation of active heparanase to cell surface regulates degradation of extracellular matrix heparan sulfate upon transmigration of mature monocyte-derived dendritic cells

After Ag capture and exposure to danger stimuli, maturing dendritic cells (DCs) migrate to regional lymph nodes, where antigenic peptides are presented to T lymphocytes. To migrate from peripheral tissue such as the epidermis to regional lymph nodes, Ag-bearing epidermal Langerhans cells must move through an extracellular matrix (ECM) of various compositions. The nature of their capacity to transmigrate via ECM is not well understood, although MIP-3beta and CCR7 play critical roles. We were interested in verifying whether heparanase, a heparan sulfate-degrading endo-beta-d-glucuronidase that participates in ECM degradation and remodeling, is expressed and functional in monocyte-derived DCs. Using immunohistochemistry, confocal microscopy, RT-PCR, Western blot analysis, assays for heparanase activity, and Matrigel transmigration, we show that heparanase is expressed in both nuclei and cytoplasm of immature DCs, and that gene expression and synthesis take place mainly in monocytes and early immature DCs. We also found that both nuclear and cytoplasm fractions show heparanase activity, and upon LPS-induced maturation, heparanase translocates to the cell surface and degrades ECM heparan sulfate. Matrigel transmigration assays showed a MIP-3beta-comparable role for heparanase. Because heparan sulfate glycosaminoglycans play a key role in the self-assembly, insolubility, and barrier properties of the ECM, the results of this study suggest that heparanase is a key enzyme in DC transmigration through the ECM.     

Chromatin Remodelers Act Globally, Sequence Positions Nucleosomes Locally

The precise placement of nucleosomes has large regulatory effects on gene expression. Recent work suggests that nucleosome placement is regulated in part by the affinity of the underlying DNA sequence for the histone octamer. Nucleosome locations are also regulated by several different ATP-dependent chromatin remodeling enzymes. This raises the question of whether DNA sequence influences the activity of chromatin remodeling enzymes. DNA sequence could most simply regulate nucleosome remodeling through its effect on nucleosome stability. In such a model, unstable nucleosomes would be remodeled faster than stable nucleosomes. It is also possible that certain DNA elements could regulate remodeling by inhibiting the interaction of nucleosomes with the remodeling enzyme. A third possibility is that DNA sequence could regulate the outcome of remodeling by influencing how reaction intermediates collapse into a particular set of stable nucleosomal positions. Here we dissect the contribution from these potential mechanisms to the activities of yeast RSC and human ACF, which are representative members of two major classes of remodeling complexes. We find that varying the histone-DNA affinity over 3 orders of magnitude has negligible effects on the rates of nucleosome remodeling and ATP hydrolysis by these two enzymes. This suggests that the rate-limiting step for nucleosome remodeling may not involve the disruption of histone-DNA contacts. We further find that a specific curved DNA element previously hypothesized to inhibit ACF activity does not inhibit substrate binding or remodeling by ACF. The element, however, does influence the distribution of nucleosome positions generated by ACF. Our data support a model in which remodeling enzymes move nucleosomes to new locations by a general sequence-independent mechanism. However, consequent to the rate-limiting remodeling step, the local DNA sequence promotes a collapse of remodeling intermediates into highly resolved positions that are dictated by thermodynamic differences between adjacent positions.     

Aβ40 oligomers identified as a potential biomarker for the diagnosis of Alzheimer's disease

Alzheimer's Disease (AD) is the most prevalent form of dementia worldwide, yet the development of therapeutics has been hampered by the absence of suitable biomarkers to diagnose the disease in its early stages prior to the formation of amyloid plaques and the occurrence of irreversible neuronal damage. Since oligomeric Aβ species have been implicated in the pathophysiology of AD, we reasoned that they may correlate with the onset of disease. As such, we have developed a novel misfolded protein assay for the detection of soluble oligomers composed of Aβ x-40 and x-42 peptide (hereafter Aβ40 and Aβ42) from cerebrospinal fluid (CSF). Preliminary validation of this assay with 36 clinical samples demonstrated the presence of aggregated Aβ40 in the CSF of AD patients. Together with measurements of total Aβ42, diagnostic sensitivity and specificity greater than 95% and 90%, respectively, were achieved. Although larger sample populations will be needed to confirm this diagnostic sensitivity, our studies demonstrate a sensitive method of detecting circulating Aβ40 oligomers from AD CSF and suggest that these oligomers could be a powerful new biomarker for the early detection of AD.