CYCLIC AMP-DEPENDENT PROTEIN KINASE ACTIVITY AND SYNAPTOSOMAL PROTEIN PHOSPHORYLATION IN THE BRAINS OF AGED RATS

The activity of soluble protein kinase and phosphorylation of endogenous synaptosomal proteins were studied in vitro, in the hippocampus and cerebral cortex of rats 3, 12, or 24 months of age. No between-age differences in the activity of cyclic AMP-dependent or independent protein kinase were detected in either brain region. The degree of stimulation by cyclic AMP and the apparent K_a, for cyclic AMP were similar at all stages. Cyclic AMP stimulated the phosphorylation of synaptosomal proteins from the cerebral cortex, hippocampus, caudate nucleus, and cerebellum of rats at all ages. There were no significant differences across age in the extent of phosphorylation of any membrane proteins in any brain region. The number and staining density of synaptosornal proteins separated by polyacrylamide gel electrophoresis were also similar at all ages. These studies indicate that the cyclic AMP-dependent phosphorylation system in the rat brain does not change during advanced aging.     

Functional regulation of brain pyruvate dehydrogenase: Postnatal development, anesthesia and food-deprivation

The activity of brain pyruvate dehydrogenase complex (PDHC), is regulated by reversible phosphorylation of the alpha subunit of the E1 component (pyruvate dehydrogenase, EC 1.2.4.1) of PDHC. Using an in vitro back-titration assay, we have evaluated the postnatal development of E1 phosphorylation, as well as the effects of acute pentobarbital administration and food-deprivation on cerebral cortical E1 phosphorylation in synaptosomal and free mitochondrial compartments of the albino rat. Between birth and postnatal day 25, the back-titration phosphorylation increased ca 4-fold, with the largest increase occurring between days 15 and 20. The phosphorylation of E1 in the synaptosomal, but not free mitochondrial fraction, was decreased during pentobarbital anesthesia. Following 72 h of food-deprivation, E1 phosphorylation was decreased in both subcellular fractions.

The postnatal increase in E1 back-titration phosphorylation is consistent with and similar in magnitude to previously reported increases in the specific enzymatic activity of PDHC. These results also highlight the potential importance of localized subcellular alterations in mitochondrial metabolism and further validate the back-titration phosphorylation of E1 as a valuable tool for the study of central nervous system PDHC metabolism.     

Relative Validity of Three Food Frequency Questionnaires for Assessing Dietary Intakes of Guatemalan Schoolchildren

ObjectiveTo determine the relative validity of three food frequency questionnaires (FFQs) compared with results from 24-hour dietary recalls for measuring dietary intakes in Guatemalan schoolchildren.DesignA cross-sectional study of primary caregivers (mothers or grandmothers) of 6–11 year-old children. Caregivers completed one of three constructed FFQs to measure the child’s dietary consumption in the last week: FFQ1 did not incorporate portion sizes; FFQ2 provided portion sizes; and FFQ3 incorporated pictures of median portion sizes. During the same week, each caregiver also completed three 24-hour dietary recalls. Results from the FFQ were compared with corresponding results from the 24-hour dietary recalls.SettingSanta Catarina Pinula, peri-urban Guatemala City.SubjectsCaregivers (n = 145) of 6–11 year-old children: 46 completed FFQ1, 49 completed FFQ2, and 50 completed FFQ3.ResultsThe mean values for all nutrients obtained from the 24-hour dietary recall were lower than for those obtained from the FFQs, excluding folic acid in FFQ3, cholesterol and zinc in FFQ2, and cholesterol, folic acid, magnesium, potassium, sodium, and zinc in FFQ1. Energy-adjusted Pearson correlation coefficients ranged from 0.07 (protein) to 0.54 (cholesterol) for FFQ1 and from 0.05 to 0.74 for FFQ2 and FFQ3. Agreement by both methods (FFQ and 24-hour dietary recalls) of classifying children into the same or adjacent quartiles of energy-adjusted nutrient consumption ranged from 62.0% for cholesterol to 95.9% for vitamin B12 across all three FFQs.ConclusionsOur FFQs had moderate to good relative validity in measuring energy and nutrient intakes for 6–11 year-old Guatemalan children. More evidence is needed to evaluate their reproducibility and applicability in similar populations.     

Control of genotypic allelic inclusion through TCR surface expression

To gain insight into the molecular causes and functional consequences of allelic inclusion of TCR alpha-chains, we develop a computational model for thymocyte selection in which the signal that determines cell fate depends on surface expression. Analysis of receptor pairs on selected dual TCR cells reveals that allelic inclusion permits both autoreactive TCR and receptors not in the single TCR cell repertoire to be selected. However, in comparison with earlier theoretical studies, relatively few dual TCR cells display receptors with high avidity for thymic ligands because their alpha-chains compete aggressively for the beta-chain, which hinders rescue from clonal deletion. This feature of the model makes clear that allelic inclusion does not in itself compromise central tolerance. A specific experiment based on modulation of TCR surface expression levels is proposed to test the model.     

Porphyrin depth in lipid bilayers as determined by iodide and parallax fluorescence quenching methods and its effect on photosensitizing efficiency

Photosensitization by porphyrins and other tetrapyrrole chromophores is used in biology and medicine to kill cells. This light-triggered generation of singlet oxygen is used to eradicate cancer cells in a process dubbed "photodynamic therapy," or PDT. Most photosensitizers are of amphiphilic character and they partition into cellular lipid membranes. The photodamage that they inflict to the host cell is mainly localized in membrane proteins. This photosensitized damage must occur in competition with the rapid diffusion of singlet oxygen through the lipid phase and its escape into the aqueous phase. In this article we show that the extent of damage can be modulated by employing modified hemato- and protoporphyrins, which have alkyl spacers of varying lengths between the tetrapyrrole ring and the carboxylate groups that are anchored at the lipid/water interface. The chromophore part of the molecule, and the point of generation of singlet oxygen, is thus located at a deeper position in the bilayer. The photosensitization efficiency was measured with 9,10-dimethylanthracene, a fluorescent chemical target for singlet oxygen. The vertical insertion of the sensitizers was assessed by two fluorescence-quenching techniques: by iodide ions that come from the aqueous phase; and by spin-probe-labeled phospholipids, that are incorporated into the bilayer, using the parallax method. These methods also show that temperature has a small effect on the depth when the membrane is in the liquid phase. However, when the bilayer undergoes a phase transition to the solid gel phase, the porphyrins are extruded toward the water interface as the temperature is lowered. These results, together with a previous publication in this journal, represent a unique and precedental case where the vertical location of a small molecule in a membrane has an effect on its membranal activity.     

Associations between Serum C-reactive Protein and Serum Zinc, Ferritin, and Copper in Guatemalan School Children

Inflammation affects trace nutrient concentrations, but research on copper and particularly in children is limited. We assessed associations between serum C-reactive protein (CRP) and zinc, iron, copper, and other biomarkers (alkaline phosphatase, hemoglobin, and albumin), in 634 healthy 6- to 11-year-old Guatemalan schoolchildren. CRP was measured by a standardized, high-sensitive method. For significant associations with CRP, we stratified nutrient concentrations across categories of CRP and compared concentrations above and below several CRP cutoff points (0.5, 1, 3, 5, and 10 mg/L), and then adjusted values using correction factors (ratios of geometric means of the nutrients in the low and high groups). Prevalence of serum zinc (<65 μg/dL0, ferritin (<15 μg/L), and copper (<90 μg/dL) deficiency were 21%, 2.1%, and 23.8%, respectively. Median (25th and 75th percentiles) CRP was 0.56 (0.26 and 1.54) mg/L. CRP concentration was positively associated with ferritin and copper concentrations (r = 0.23 and 0.29, respectively; P < 0.0001) but not with zinc and other biomarkers (P > 0.05). Regardless of CRP cutoffs, high (> cutoff) vs. low (≤ cutoff) CRP levels had higher ferritin and copper concentrations and lower prevalence of copper deficiency of <90 μg/dL (P < 0.05). Adjustment for inflammation had the greatest influence on recalculated prevalence for the CRP 0.5 mg/L cutoff. The low ferritin prevalence hardly changed (from 2.1% to 2.5%) while the low copper prevalence changed appreciably (from 23.8% to 31.2%). In conclusion, CRP was positively associated with ferritin and copper but not with zinc concentrations. Adjustment for inflammation had little effect on low ferritin prevalence, low to begin with, and a large impact on low copper prevalence. High-sensitive CRP methods and the use of very low CRP cutoffs may be more accurate than traditional CRP methods in the adjustment of serum copper concentrations for inflammation in healthy school children.     

Annexin-1 mediates TNF-alpha-stimulated matrix metalloproteinase secretion from rheumatoid arthritis synovial fibroblasts

Annexins are intracellular molecules implicated in the down-regulation of inflammation. Recently, annexin-1 has also been identified as a secreted molecule, suggesting it may have more complex effects on inflammation than previously appreciated. We studied the role of annexin-1 in mediating MMP-1 secretion from rheumatoid arthritis (RA) synovial fibroblasts (SF) stimulated with TNF-alpha. TNF-alpha induced a biphasic secretion of annexin-1 from RA SF. Early (< or = 60 min), cycloheximide-independent secretion from preformed intracellular pools was followed by late (24 h) cycloheximide-inhibitable secretion requiring new protein synthesis. Exogenous annexin-1 N-terminal peptide Ac2-26 stimulated MMP-1 secretion in a dose- (EC(50) approximately 25 microM) and time- (8-24 h) dependent manner; full-length annexin-1 had a similar effect. Down-regulation of annexin-1 using small interfering RNA resulted in decreased secretion of both annexin-1 and MMP-1, confirming that annexin-1 mediates TNF-alpha-stimulated MMP-1 secretion. Erk, Jnk, and NF-kappaB have been implicated in MMP-1 secretion. Erk, Jnk, and NF-kappaB inhibitors had no effect on annexin-1 secretion stimulated by TNF-alpha but inhibited MMP-1 secretion in response to Ac2-26, indicating that these molecules signal downstream of annexin-1. Annexin-1 stimulation of MMP-1 secretion was inhibited by both a formyl peptide receptor antagonist and pertussis toxin, suggesting that secreted annexin-1 acts via formyl peptide family receptors, most likely FPLR-1. In contrast to its commonly appreciated anti-inflammatory roles, our data indicate that annexin-1 is secreted by RA SF in response to TNF-alpha and acts in an autacoid manner to engage FPRL-1, activate Erk, Jnk, and NF-kappaB, and stimulate MMP-1 secretion.     

EspM inhibits pedestal formation by enterohaemorrhagic Escherichia coli and enteropathogenic E. coli and disrupts the architecture of a polarized epithelial monolayer

Enterohaemorrhagic Escherichia coli and enteropathogenic E. coli are enteropathogens characterized by their ability to induce the host cell to form actin‐rich structures, termed pedestals. A type III secretion system, through which the pathogens deliver effector proteins into infected host cells, is essential for their virulence and pedestal formation. Enterohaemorrhagic E. coli encodes two similar effectors, EspM1 and EspM2, which activate the RhoA signalling pathway and induce the formation of stress fibres upon infection of host cells. We confirm these observations and in addition show that EspM inhibits the formation of actin pedestals. Moreover, we show that translocation of EspM into polarized epithelial cells induces dramatic changes in the tight junction localization and in the morphology and architecture of infected polarized monolayers. These changes are manifested by altered localization of the tight junctions and ‘bulging out’ morphology of the cells. Surprisingly, despite the dramatic changes in their architecture, the cells remain alive and the epithelial monolayer maintains a normal barrier function. Taken together, our results show that the EspM effectors inhibit pedestal formation and induce tight junction mislocalization as well as dramatic changes in the architecture of the polarized monolayer.     

PEG-ing down (and preventing?) the cause of pegloticase failure

Pegloticase is a powerful but underutilized weapon in the rheumatologist’s armamentarium. The drug’s immunogenicity leads to neutralizing antibody formation and rapid loss of efficacy in roughly one-half of all patients, which remains an impediment to broader use. New data, however, suggest that drug survival might improve with concomitant immunosuppressive agent (s), which merits further study. Efficacy appears to be unchanged when pegloticase is infused at 3-week (rather than 2-week) intervals. Stretching the time between infusions may also improve patient adherence and allow for earlier identification of transient responders.In the previous issue of Arthritis Research and Therapy, Hershfield and colleagues published a study putting forth a number of novel and potentially important findings regarding pegloticase, a powerful but underutilized weapon in the small but growing anti-hyperuricemic arsenal [1].The study examined the efficacy of pegloticase in a cohort of 30 patients with severe gout (93% tophaceous) utilizing an every 3-week infusion regimen, rather than the every 2-week schedule employed in previously published phase 3 trials. Despite the longer interval between infusions in the current study, the effectiveness of pegloticase is no worse (17/30 patients are persistent responders), and the pharmacokinetics of the drug suggest this should come as no surprise. The authors correctly note that a 3-week interval would be significantly more convenient for patients, and notably that such a regimen would also prove less costly to payors. Hershfield and colleagues’ dosing schedule would also help to identify transient responders earlier in the course of treatment – only four of 12 transient responders had uric acid >6 mg/dl 2 weeks after the first infusion (three of whom had previously been exposed to pegloticase in earlier phase 1 and 2 studies), whereas 11 of 12 transient responders had uric acid >6 mg/dl at 3 weeks (vs. only one of 17 persistent responders). For the reasons just elaborated upon, the paper demonstrates that dosing every 3 weeks may not just be as good as the current protocol, but may in some ways be superior.Hershfield and colleagues also upend the assumption that neutralizing antibodies to pegloticase are formed against uricase itself. Their paper clearly demonstrates that neutralizing antibodies develop in response to the polyethylene glycol (PEG) moiety of the drug, a finding that rebuffs a recent commentary which suggested antibodies to PEG are not pathogenic [2]. A septe and larger study (169 patients exposed to pegloticase) published in the previous issue Arthritis Research and Therapy by Lipsky and colleagues reaches the same conclusion regarding anti-pegloticase antibodies: anti-PEG antibodies are responsible for loss of efficacy rather than antibodies to the uricase enzyme itself, the latter of which rarely occur (positive more than once in only 11 subjects) and occur much later during the course of treatment, long after neutralizing anti-pegloticase antibodies have developed [3]. This larger study also demonstrates that an anti-pegloticase antibody titer >1:2,430 generally predicts loss of efficacy to the drug.Finally, and not least of all, Hershfield and colleagues’ smaller study included post-transplant patients (who were excluded from the phase 3 studies), a population particularly susceptible to developing gout. Of seven post-transplant subjects in the study, six proved to be persistent responders (86%) [1]. Although this is an admittedly small number of patients upon which to base any conclusion, it does raise the intriguing question of whether immunosuppression might lead to less of a mounted antibody response against pegloticase, and thus to more favorable outcomes. This is no small point; patients who are placed on pegloticase generally have severe, long-standing, and refractory gout, and should be given every chance to optimize their response to a potentially transformative therapy.While this signal is worth pursuing, some questions are immediately raised: how immunosuppressed must patients be to prevent neutralizing anti-PEG antibody formation, and with what should this be accomplished? Of the small subcohort in this trial, all patients were on cyclosporine or mycophenolate mofetil, and five of seven patients were on a combination of immunosuppressive agents. In choosing an immunosuppressant for the express purpose of preventing neutralizing antibodies, the risk of these drugs would very probably outweigh any proposed benefit (cyclosporine in particular might be the least desirable immunosuppressant for a patient with severe gout, because it both increases serum uric acid levels and decreases the glomerular filtration rate).If a trial was designed to investigate this line of query, a reasonable immunosuppressive agent of choice might be methotrexate. This drug has been shown to effectively prevent neutralizing antibodies from forming against monoclonal antibodies to anti-tumor necrosis factor [4,5]. Methotrexate’s inhibitory effect may not extend to preventing antibody formation against PEG, although methotrexate has also been shown to inhibit antibody formation against the polysaccharide 23-valent pneumococcal vaccine [6]. Methotrexate might also yield the unintended benefit of acting as an anti-inflammatory agent to suppress gouty attacks. However, because patients with severe gout have multiple comorbidities that place them at higher risk for medication side effects, methotrexate should not be considered in the clinical setting in the absence of data to support its use [7]. Nevertheless, methotrexate therapy is an avenue of inquiry that is clinically relevant and needs exploration to increase the likelihood that patients who begin this powerful drug can remain on it.