MARCH8 Targets Cytoplasmic Lysine Residues of Various Viral Envelope Glycoproteins.

The host transmembrane protein MARCH8 is a RING finger E3 ubiquitin ligase that downregulates various host transmembrane proteins, such as MHC-II. We have recently reported that MARCH8 expression in virus-producing cells impairs viral infectivity by reducing virion incorporation of not only HIV-1 envelope glycoprotein but also vesicular stomatitis virus G-glycoprotein through two different pathways. However, the MARCH8 inhibition spectrum remains largely unknown. Here, we show the antiviral spectrum of MARCH8 using viruses pseudotyped with a variety of viral envelope glycoproteins. Infection experiments revealed that viral envelope glycoproteins derived from the rhabdovirus, arenavirus, coronavirus, and togavirus (alphavirus) families were sensitive to MARCH8-mediated inhibition. Lysine mutations at the cytoplasmic tails of rabies virus-G, lymphocytic choriomeningitis virus glycoproteins, SARS-CoV and SARS-CoV-2 spike proteins, and Chikungunya virus and Ross River virus E2 proteins conferred resistance to MARCH8. Immunofluorescence showed impaired downregulation of the mutants of these viral envelope glycoproteins by MARCH8, followed by lysosomal degradation, suggesting that MARCH8-mediated ubiquitination leads to intracellular degradation of these envelopes. Indeed, rabies virus-G and Chikungunya virus E2 proteins proved to be clearly ubiquitinated. We conclude that MARCH8 has inhibitory activity on a variety of viral envelope glycoproteins whose cytoplasmic lysine residues are targeted by this antiviral factor. IMPORTANCE A member of the MARCH E3 ubiquitin ligase family, MARCH8, downregulates many different kinds of host transmembrane proteins, resulting in the regulation of cellular homeostasis. On the other hands, MARCH8 acts as an antiviral factor when it binds to and downregulates HIV-1 envelope glycoprotein and vesicular stomatitis virus G-glycoprotein that are viral transmembrane proteins. This study reveals that, as in the case of cellular membrane proteins, MARCH8 shows broad-spectrum inhibition against various viral envelope glycoproteins by recognizing their cytoplasmic lysine residues, resulting in lysosomal degradation.

Mapping protein carboxymethylation sites provides insights into their role in proteostasis and cell proliferation.

Posttranslational mechanisms play a key role in modifying the abundance and function of cellular proteins. Among these, modification by advanced glycation end products has been shown to accumulate during aging and age-associated diseases but specific protein targets and functional consequences remain largely unexplored. Here, we devise a proteomic strategy to identify sites of carboxymethyllysine modification, one of the most abundant advanced glycation end products. We identify over 1000 sites of protein carboxymethylation in mouse and primary human cells treated with the glycating agent glyoxal. By using quantitative proteomics, we find that protein glycation triggers a proteotoxic response and indirectly affects the protein degradation machinery. In primary endothelial cells, we show that glyoxal induces cell cycle perturbation and that carboxymethyllysine modification reduces acetylation of tubulins and impairs microtubule dynamics. Our data demonstrate the relevance of carboxymethyllysine modification for cellular function and pinpoint specific protein networks that might become compromised during aging.

Allyl isothiocyanate regulates lysine acetylation and methylation marks in an experimental model of malignant melanoma.

OBJECTIVE(S): Isothiocyanates (ITCs) are biologically active plant secondary metabolites capable of mediating various biological effects including modulation of the epigenome. Our aim was to characterize the effect of allyl isothiocyanate (AITC) on lysine acetylation and methylation marks as a potential epigenetic-induced anti-melanoma strategy. METHODS: Our malignant melanoma model consisted of (1) human (A375) and murine (B16-F10) malignant melanoma as well as of human; (2) brain (VMM1) and lymph node (Hs 294T) metastatic melanoma; (3) non-melanoma epidermoid carcinoma (A431) and (4) immortalized keratinocyte (HaCaT) cells subjected to AITC. Cell viability, histone deacetylases (HDACs) and acetyltransferases (HATs) activities were evaluated by the Alamar blue, Epigenase HDAC Activity/Inhibition and EpiQuik HAT Activity/Inhibition assay kits, respectively, while their expression levels together with those of lysine acetylation and methylation marks by western immunoblotting. Finally, apoptotic gene expression was assessed by an RT-PCR-based gene expression profiling methodology. RESULTS: AITC reduces cell viability, decreases HDACs and HATs activities and causes changes in protein expression levels of various HDACs, HATs, and histone methyl transferases (HMTs) all of which have a profound effect on specific lysine acetylation and methylation marks. Moreover, AITC regulates the expression of a number of genes participating in various apoptotic cascades thus indicating its involvement in apoptotic induction. CONCLUSIONS: AITC exerts a potent epigenetic effect suggesting its potential involvement as a promising epigenetic-induced bioactive for the treatment of malignant melanoma.

Insulin action on protein synthesis and its association with eIF5A expression and hypusination.

The hormone insulin plays a central role in the metabolism of carbohydrates, lipids, and proteins. In relation to protein metabolism, insulin stimulates amino acid uptake and activates protein synthesis in responsive cells by modulation of signal transduction pathways, such as associated to Akt/PkB, mTOR, S6Ks, 4E-BP1, and several translation initiation/elongation factors. In this context, there is no information on direct cellular treatment with insulin and effects on eukaryotic translation initiation factor 5A (eIF5A) regulation. The eIF5A protein contains an exclusive amino acid residue denominated hypusine, which is essential for its activity and synthesized by posttranslational modification of a specific lysine residue using spermidine as substrate. The eIF5A protein is involved in cellular proliferation and differentiation processes, as observed for satellite cells derived from rat muscles, revealing that eIF5A has an important role in muscle regeneration. The aim of this study was to determine whether eIF5A expression and hypusination are influenced by direct treatment of insulin on L6 myoblast cells. We observed that insulin increased the content of eIF5A transcripts. This effect occurred in cells treated or depleted of fetal bovine serum, revealing a positive insulin effect independent of other serum components. In addition, it was observed that hypusination follows the maintenance of eIF5A protein content in the serum depleted cells and treated with insulin. These results demonstrate that eIF5A is modulated by insulin, contributing the protein synthesis machinery control, as observed by puromycin incorporation in nascent proteins.

Pyridoxamine: A novel treatment for schizophrenia with enhanced carbonyl stress.

AIM: The aim of this clinical trial was to obtain proof of concept for high-dose pyridoxamine as a novel treatment for schizophrenia with enhanced carbonyl stress. METHODS: Ten Japanese schizophrenia patients with high plasma pentosidine, which is a representative biomarker of enhanced carbonyl stress, were recruited in a 24-week, open trial in which high-dose pyridoxamine (ranging from 1200 to 2400 mg/day) was administered using a conventional antipsychotic regimen. Main outcomes were the total change in Positive and Negative Syndrome Scale score and the Brief Psychiatric Rating Scale score from baseline to end of treatment at week 24 (or at withdrawal). RESULTS: Decreased plasma pentosidine levels were observed in eight patients. Two patients showed marked improvement in their psychological symptoms. A patient who harbors a frameshift mutation in the Glyoxalase 1 gene also showed considerable reduction in psychosis accompanied with a moderate decrease in plasma pentosidine levels. A reduction of greater than 20% in the assessment scale of drug-induced Parkinsonism occurred in four patients. Although there was no severe suicide-related ideation or behavior, Wernicke's encephalopathy-like adverse drug reactions occurred in two patients and were completely suppressed by thiamine supplementation. CONCLUSION: High-dose pyridoxamine add-on treatment was, in part, effective for a subpopulation of schizophrenia patients with enhanced carbonyl stress. Further randomized, placebo-controlled trials with careful monitoring will be required to validate the efficacy of high-dose pyridoxamine for these patients.

Evaluación piloto de la reducción de niveles séricos de productos de glicación avanzada (AGEs) sobre la electrofisiología de la visión, en pacientes con diabetes mellitus tipo 2 / Pilot evaluation of the serum levels reduction of advanced glication products (AGEs) on the vision electrophysiology in type 2 diabetic patients

BACKGROUND: Diabetic vascular complications are associated with elevated concentrations of advanced glycation end-products (AGEs). These substances can be originated endogenously by hyperglycaemia and oxidative stress, but also by dietary intake. There is indirect evidence suggesting that these complications can be prevented by lowering AGEs levels by dietary or pharmacological interventions, however its clinical benefits are still not clear enough because this would require long periods of treatment. Specific neuro-ophthalmologic tests like Multifocal Electroretinogram (MFERG) and visual evoked potentials (VEP) can detect retinal and myelinic nerve early changes, and thus could represent good methods to study the results of certain interventions in shorter lapses. The aim of this preliminary study was to evaluate the effects of a pharmacological intervention designed to lower AGEs levels, on these variables. PATIENTS AND METHODS: We included 7 patients with type 2 diabetes (DM2), with more than 5 and less than 10 years of disease, without clinically evident micro and macrovascular disease, without renal failure, hypothyroidism nor vitamin B12 deficiency, whose AGEs dietary intake was moderately elevated or high (according to dietary recalls). Upon admission, a clinical evaluation, urine and blood samples were obtained for routine labs, plus ultrasensitive C Reactive Protein (usCRP) as an inflammatory marker, and carboxymethyl-lysine (CML) as representative of AGEs. Then a complete ophthalmologic evaluation was performed, including fundus, MFERG and VEP. After the initial evaluation, placebo capsules were prescribed (12 daily capsules, 4 with each main meal) during 3 months, repeating the same initial evaluation at completion of this period. Then the active treatment followed, with capsules containing cholestyramine (4 capsules containing 500 mg each, totaling 6 g per day). Patients were cited each month, to register adverse events and repeating the same evaluation after this second 3 months period. RESULTS: The sample was composed of 2 male patients, mean age was 55.1 ± 3.8 years, and diabetes was managed with metformin plus other oral agents or o insulin (4 cases). In addition, 4 patients received lipid lowering and 4 antihypertensive drugs. Metabolic control and lipid levels were variable (ranges of HbA1c 6.2-8.4%, LDL cholesterol 45-141 mg/dL, triglycerides 70-220 mg/dL). AGEs levels represented by CML were highly variable (median 31.7, range min-max 3.4-58.9 ug/uL). Basal usCRP was also variable (median 405.9, range min-max 265.6-490.7 mg/L). The treatment was well tolerated, except for mild constipation associated with cholestiramine intake. No significant changes in electroretinography or evoked potentials were observed when comparing the initial placebo period with cholestyramine treatment. A significant increase in triglyceride levels and decrease of vitamin D levels after cholestyramine treatment was observed. No changes were detected in serum concentrations of CML, usCRP or glycemic control, after treatment. The latter variables were not correlated with neurophthalmologic studies. DISCUSSION: In this preliminary study we did not observe changes in MFERG nor VEP after 6 g/day cholestyramine treatment, which did not induce lowering of CML levels. This could be attributed to the many limitations of a pilot study, such as a small sample size, short duration of treatment, reduced doses. However this design allowed to evaluate the patients´ tolerance to the drug and rule out adverse effects, in order to plan further studies using the necessary doses to obtain lowering of AGEs

A proteasome inhibitor produced by Burkholderia pseudomallei modulates intracellular growth.

The NRPS/PKS cluster encodes the enzymes necessary for glidobactin synthesis it is partially conserved in various members of the Burkholderia genus including B. pseudomallei. In this study we have shown that the insertional inactivation or deletion of glbC in this cluster in B. pseudomallei could reduce the ability of the bacterium to survive or grow in murine macrophages or in human neutrophils. Exogenously added proteasome inhibitors were able to chemically complement the mutation. The insertional inactivation or deletion of glbC increased virulence in an acute model of infection in Balb/c or C57BL/6 mice but virulence in a chronic model of infection was similar to that of the wild type. Our findings contrast with the previous finding that inactivation of the glb gene cluster in B. pseudomallei strain 1026b resulted in marked attenuation, and provides evidence of differential roles for some genes in virulence of different strains of B. pseudomallei.

A Proteomic Approach to Analyze the Aspirin-mediated Lysine Acetylome.

Aspirin, or acetylsalicylic acid is widely used to control pain, inflammation and fever. Important to this function is its ability to irreversibly acetylate cyclooxygenases at active site serines. Aspirin has the potential to acetylate other amino acid side-chains, leading to the possibility that aspirin-mediated lysine acetylation could explain some of its as-yet unexplained drug actions or side-effects. Using isotopically labeled aspirin-d3, in combination with acetylated lysine purification and LC-MS/MS, we identified over 12000 sites of lysine acetylation from cultured human cells. Although aspirin amplifies endogenous acetylation signals at the majority of detectable endogenous sites, cells tolerate aspirin mediated acetylation very well unless cellular deacetylases are inhibited. Although most endogenous acetylations are amplified by orders of magnitude, lysine acetylation site occupancies remain very low even after high doses of aspirin. This work shows that while aspirin has enormous potential to alter protein function, in the majority of cases aspirin-mediated acetylations do not accumulate to levels likely to elicit biological effects. These findings are consistent with an emerging model for cellular acetylation whereby stoichiometry correlates with biological relevance, and deacetylases act to minimize the biological consequences of nonspecific chemical acetylations.

Ptena, but not Ptenb, reduces regeneration after spinal cord injury in adult zebrafish.

Based on the observation that the tumor suppressor gene PTEN (phosphatase and tensin homolog) reduces regeneration after spinal cord injury (SCI) as evidenced in the PTEN knockout mouse, we have investigated the function of Ptena and Ptenb, the two zebrafish homologs of mammalian PTEN, in adult zebrafish after spinal cord injury with the aim to assess the contribution of the two zebrafish genes to functional recovery in an animal species that spontaneously recovers from central nervous system injury. The inhibition of Ptena expression by antisense morpholino (MO) application improved spinal cord regeneration through 4 to 5weeks after injury. Retrograde tracing showed regrowth of axons from neurons of the regeneration-competent nucleus of the medial longitudinal fascicle in the brainstem in the Ptena MO-treated fish. Ptenb MO-treated fish recovered as well as control MO-treated fish at 4 and 5weeks after SCI, with their locomotion being similar to that of sham-injured and non-injured fish. The mRNA levels of Ptena were upregulated after SCI at the early stage after injury (12h and 6days) caudal to the lesion site, compared to the non-injured control, while the levels of Ptenb were upregulated only at 12h after injury. In situ hybridization experiments were in agreement with the qPCR measurements. At the protein level, Ptena was found to be expressed in spinal motoneurons and immature neurons. These results indicate that Ptena, but not Ptenb, inhibits regeneration in zebrafish, thus sharing this feature with PTEN in mammals. The fact that zebrafish regenerate better than mammals despite the inhibitory presence of Ptena is likely due to regeneration-conducive molecules that tip the balance from inhibition to enhancement. Interestingly, although Ptena and Ptenb have been shown to be functionally redundant in promoting the development of the fish larval central nervous system, they are not functionally redundant in the adult, suggesting that regeneration in fish is not predominantly due to the overall recapitulation of development.

Acute insulin resistance mediated by advanced glycation endproducts in severely burned rats.

OBJECTIVE: Hyperglycemia often occurs in severe burns; however, the underlying mechanisms and importance of managing postburn hyperglycemia are not well recognized. This study was designed to investigate the dynamic changes of postburn hyperglycemia and the underlying mechanisms and to evaluate whether early glycemic control is beneficial in severe burns. DESIGN: Prospective, randomized experimental study. SETTING: Animal research laboratory. SUBJECTS: Sprague-Dawley rats. INTERVENTIONS: Anesthetized rats were subjected to a full-thickness burn injury comprising 40% of the total body surface area and were randomized to receive vehicle, insulin, and a soluble form of receptor for advanced glycation endproducts treatments. An in vitro study was performed on cultured H9C2 cells subjected to vehicle or carboxymethyllysine treatment. MEASUREMENTS AND MAIN RESULTS: We found that blood glucose change presented a distinct pattern with two occurrences of hyperglycemia at 0.5- and 3-hour postburn, respectively. Acute insulin resistance evidenced by impaired insulin signaling and glucose uptake occurred at 3-hour postburn, which was associated with the second hyperglycemia and positively correlated with mortality. Mechanistically, we found that serum carboxymethyllysine, a dominant species of advanced glycation endproducts, increased within 1-hour postburn, preceding the occurrence of insulin resistance. More importantly, treatment of animals with soluble form of receptor for advanced glycation endproducts, blockade of advanced glycation endproducts signaling, alleviated severe burn-induced insulin resistance. In addition, early hyperglycemic control with insulin not only reduced serum carboxymethyllysine but also blunted postburn insulin resistance and reduced mortality. CONCLUSIONS: These findings suggest that severe burn-induced insulin resistance is partly at least mediated by serum advanced glycation endproducts and positively correlated with mortality. Early glycemic control with insulin or inhibition of advanced glycation endproducts with soluble form of receptor for advanced glycation endproducts ameliorates postburn insulin resistance.

A biochemical correlate of dimorphism in a zygomycete Benjaminiella poitrasii: characterization of purified NAD-dependent glutamate dehydrogenase, a target for antifungal agents.

The fungal organisms, especially pathogens, change their vegetative (Y, unicellular yeast and H, hypha) morphology reversibly for survival and proliferation in the host environment. NAD-dependent glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2) from a non-pathogenic dimorphic zygomycete Benjaminiella poitrasii was previously reported to be an important biochemical correlate of the transition process. The enzyme was purified to homogeneity and characterized. It is a 371 kDa native molecular weight protein made up of four identical subunits. Kinetic studies showed that unlike other NAD-GDHs, it may act as an anabolic enzyme and has more affinity towards 2-oxoglutarate than L-glutamate. Chemical modifications revealed the involvement of single histidine and lysine residues in the catalytic activity of the enzyme. The phosphorylation and dephosphorylation study showed that the NAD-GDH is present in active phosphorylated form in hyphal cells of B. poitrasii. Two of the 1,2,3 triazole linked ß-lactam-bile acid conjugates synthesized in the laboratory (B18, B20) were found to be potent inhibitors of purified NAD-GDH which also significantly affected Y-H transition in B. poitrasii. Furthermore, the compound B20 inhibited germ tube formation during Y-H transition in Candida albicans strains and Yarrowia lipolytica. The possible use of NAD-GDH as a target for antifungal agents is discussed.

Is a positive nasal lysine-aspirin challenge test associated with a more severe phenotype of chronic rhinosinusitis and asthma?

BACKGROUND: Current guidelines recommend a greater use of aspirin challenge testing in the diagnosis of aspirin-intolerant rhinosinusitis and asthma, a disorder with high burden of illness and resistance to treatment. The indications for these tests and their clinical significance remain unclear. This study was designed to characterize the phenotype of patients with chronic rhinosinusitis with nasal polyposis (CRSwNP) with or without asthma undergoing a nasal lysine-aspirin (L-ASA) challenge to evaluate which factors strongly predict a positive test. METHODS: Seventy-five patients with CRSwNP underwent nasal challenge with 16 mg (total) of L-ASA after 30 minutes of acclimatization and diluent challenge. A positive challenge was defined as a 25% drop in total nasal volume measured by acoustic rhinometry. RESULTS: Twenty-three (31%) participants gave a history of aspirin intolerance and 38 (51%) had a positive nasal L-ASA challenge. Upper airway measures (CT scan score, olfaction, polyp grading, peak nasal inspiratory flow, nasal symptoms, etc.) and lower airway measures (methacholine provocative concentration required to produce a 20% drop in forced expiratory volume in 1 second, effective special airway resistance, and spirometry) were not significantly worse in patients with a positive aspirin challenge. Test sensitivity was 48%, specificity was 52%, positive predictive value was 29%, and negative predictive value was 68%. A regression analysis identified forced expiratory flow at 25-75% (FEF(25-75)), history of aspirin intolerance, and duration of rhinosinusitis as significant predictors of a positive aspirin challenge. CONCLUSION: A positive response to nasal L-ASA challenge is not associated with a more severe phenotype of CRSwNP with or without asthma. A history of aspirin intolerance, duration of rhinosinusitis, and FEF(25-75) predict a greater response to aspirin.

Initiation of epileptiform activity in a rat model of periventricular nodular heterotopia.

PURPOSE: Periventricular nodular heterotopia (PNH) is, in humans, often associated with difficult-to-control epilepsy. However, there is considerable controversy about the role of the PNH in seizure generation and spread. To study this issue, we have used a rat model in which injection of methylazoxymethanol (MAM) into pregnant rat dams produces offspring with nodular heterotopia-like brain abnormalities. METHODS: Electrophysiologic methods were used to examine the activity of the MAM-induced PNH relative to activity in the neighboring hippocampus and overlying neocortex. Recordings were obtained simultaneously from these three structures in slice preparations from MAM-exposed rats and in intact animals. Bath application or systemic injection of bicuculline was used to induce epileptiform activity. KEY FINDINGS: In the in vitro slice, epileptiform discharge was generally initiated in hippocampus. In some cases, independent PNH discharge occurred, but the PNH never "led" discharges in hippocampus or neocortex. Intracellular recordings from PNH neurons confirmed that these cells received synaptic drive from both hippocampus and neocortex, and sent axonal projections to these structures-consistent with anatomic observations of biocytin-injected PNH cells. In intact animal preparations, bicuculline injection resulted in epileptiform discharge in all experiments, with a period of ictal-like electrographic activity typically initiated within 2-3 min after drug injection. In almost all animals, the onset of ictus was seen synchronously across PNH, hippocampal, and neocortical electrodes; in a few cases, the PNH electrode (histologically confirmed) did not participate, but in no case was activity initiated in the PNH electrode. Interictal discharge was also synchronized across all three electrodes; again, the PNH never "led" the other two electrodes, and typically followed (onset several milliseconds after hippocampal/neocortical discharge onset). SIGNIFICANCE: These results do not support the hypothesis that the PNH lesion is the primary epileptogenic site, since it does not initiate or lead epileptiform activity that subsequently propagates to other brain regions.

Muscarinic receptor occupancy and cognitive impairment: a PET study with [11C](+)3-MPB and scopolamine in conscious monkeys.

The muscarinic cholinergic receptor (mAChR) antagonist scopolamine was used to induce transient cognitive impairment in monkeys trained in a delayed matching to sample task. The temporal relationship between the occupancy level of central mAChRs and cognitive impairment was determined. Three conscious monkeys (Macaca mulatta) were subjected to positron emission tomography (PET) scans with the mAChR radioligand N-[(11)C]methyl-3-piperidyl benzilate ([(11)C](+)3-MPB). The scan sequence was pre-, 2, 6, 24, and 48 h post-intramuscular administration of scopolamine in doses of 0.01 and 0.03 mg/kg. Occupancy levels of mAChR were maximal 2 h post-scopolamine in cortical regions innervated primarily by the basal forebrain, thalamus, and brainstem, showing that mAChR occupancy levels were 43-59 and 65-89% in doses of 0.01 and 0.03 mg/kg, respectively. In addition, dose-dependent impairment of working memory performance was measured 2 h after scopolamine. A positive correlation between the mAChR occupancy and cognitive impairment 2 and 6 h post-scopolamine was the greatest in the brainstem (P<0.00001). Although cognitive impairment was not observed 24 h post-scopolamine, sustained mAChR occupancy (11-24%) was found with both doses in the basal forebrain and thalamus, but not in the brainstem. These results indicate that a significant degree of mAChRs occupancy is needed to produce cognitive impairment by scopolamine. Furthermore, the importance of the brainstem cholinergic system in working memory in monkey is described.

Low-dose valproic acid enhances radiosensitivity of prostate cancer through acetylated p53-dependent modulation of mitochondrial membrane potential and apoptosis.

Histone deacetylase inhibitors (HDI) have shown promise as candidate radiosensitizers for many types of cancers, including prostate cancer. However, the mechanisms of action are not well understood. In this study, we show in prostate cancer cells that valproic acid (VPA) at low concentrations has minimal cytotoxic effects yet can significantly increase radiation-induced apoptosis. VPA seems to stabilize a specific acetyl modification (lysine 120) of the p53 tumor suppressor protein, resulting in an increase in its proapoptotic function at the mitochondrial membrane. These effects of VPA are independent of any action of the p53 protein as a transcription factor in the nucleus, since these effects were also observed in native and engineered prostate cancer cells containing mutant forms of p53 protein having no transcription factor activity. Transcription levels of p53-related or Bcl-2 family member proapoptotic proteins were not affected by VPA exposure. The results of this study suggest that, in addition to nuclear-based pathways previously reported, HDIs may also result in radiosensitization at lower concentrations via a specific p53 acetylation and its mitochondrial-based pathway(s).

Characterization of spontaneous recurrent epileptiform discharges in hippocampal-entorhinal cortical slices prepared from chronic epileptic animals.

Epilepsy, a common neurological disorder, is characterized by the occurrence of spontaneous recurrent epileptiform discharges (SREDs). Acquired epilepsy is associated with long-term neuronal plasticity changes in the hippocampus resulting in the expression of spontaneous recurrent seizures. The purpose of this study is to evaluate and characterize endogenous epileptiform activity in hippocampal-entorhinal cortical (HEC) slices from epileptic animals. This study employed HEC slices isolated from a large series of control and epileptic animals to evaluate and compare the presence, degree and localization of endogenous SREDs using extracellular and whole cell current clamp recordings. Animals were made epileptic using the pilocarpine model of epilepsy. Extracellular field potentials were recorded simultaneously from areas CA1, CA3, dentate gyrus, and entorhinal cortex and whole cell current clamp recordings were obtained from CA3 neurons. All regions from epileptic HEC slices (n=53) expressed SREDs, with an average frequency of 1.3Hz. In contrast, control slices (n=24) did not manifest any SREDs. Epileptic HEC slices demonstrated slow and fast firing patterns of SREDs. Whole cell current clamp recordings from epileptic HEC slices showed that CA3 neurons exhibited paroxysmal depolarizing shifts associated with these SREDs. To our knowledge this is the first significant demonstration of endogenous SREDs in a large series of HEC slices from epileptic animals in comparison to controls. Epileptiform discharges were found to propagate around hippocampal circuits. HEC slices from epileptic animals that manifest SREDs provide a novel model to study in vitro seizure activity in tissue prepared from epileptic animals.

Supplementation of green tea catechins in dentifrices suppresses gingival oxidative stress and periodontal inflammation.

OBJECTIVE: this study examined the effects of a dentifrice containing green tea catechins on gingival oxidative stress and periodontal inflammation using a rat model. DESIGN: twenty-four male Wister rats were randomly divided into four groups. The first group (Control group) received no treatment for 8 weeks. Periodontal inflammation was induced in the second group for 8 weeks. Periodontal inflammation was induced in the last two groups for 8 weeks and dentifrices with or without green tea catechins were topically applied to the gingival sulcus daily for 4 weeks prior to the end of the experimental period. RESULTS: rats that had experimental periodontal inflammation showed apical migration of the junctional epithelium, alveolar bone loss and inflammatory cell infiltration in the connective tissue subjacent to the junctional epithelium at 8 weeks, whilst the control group showed no pathologic changes. Topical application of a green tea catechin-containing dentifrice reduced inflammatory cell infiltration in the periodontal lesions to a greater degree than the control dentifrice at 8 weeks. The gingiva in which green tea catechin-containing dentifrice was applied also showed a lower level of expression of hexanoyl-lysine (a marker of lipid peroxidation), nitrotyrosine (a marker of oxidative protein damage), and tumour necrosis factor-α (an indicator of pro-inflammatory cytokines) at 8 weeks compared to gingiva in which the control dentifrice was applied. CONCLUSIONS: adding green tea catechins to a dentifrice may contribute to prevention of periodontal inflammation by decreasing gingival oxidative stress and expression of pro-inflammatory cytokines.

Reaction of human albumin with aspirin in vitro: mass spectrometric identification of acetylated lysines 199, 402, 519, and 545.

The aspirin esterase activity of human plasma is due to butyrylcholinesterase and albumin. Our goal was to identify the amino acid residues involved in the aspirin esterase activity of albumin. Fatty acid-free human albumin and human plasma were treated with aspirin for 5 min-24 h. Acetylated residues were identified by LC/MS/MS and MALDI-TOF/TOF mass spectrometry of tryptic peptides. Treatment with 0.3 mM aspirin resulted in acetylation of Lys-199, Lys-402, Lys-519, and Lys-545. Treatment with 20 mM aspirin resulted in acetylation of 26 lysines. There was no acetylation of Tyr-411, under any conditions. Acetylated lysine was stable for at least 21 days at pH 7.4, 37 degrees C. Albumin acetylated by aspirin had reduced esterase activity with beta-naphthyl acetate as shown on gels stained for esterase activity. It was concluded that the aspirin esterase activity of albumin is a pseudo-esterase activity in which aspirin stably acetylates lysines and releases salicylate.

Crystallization of a pentapeptide-repeat protein by reductive cyclic pentylation of free amines with glutaraldehyde.

The pentapeptide-repeat protein EfsQnr from Enterococcus faecalis protects DNA gyrase from inhibition by fluoroquinolones. EfsQnr was cloned and purified to homogeneity, but failed to produce diffraction-quality crystals in initial crystallization screens. Treatment of EfsQnr with glutaraldehyde and the strong reducing agent borane-dimethylamine resulted in a derivatized protein which produced crystals that diffracted to 1.6 A resolution; their structure was subsequently determined by single-wavelength anomalous dispersion. Analysis of the derivatized protein using Fourier transform ion cyclotron resonance mass spectrometry indicated a mass increase of 68 Da per free amino group. Electron-density maps about a limited number of structurally ordered lysines indicated that the modification was a cyclic pentylation of free amines, producing piperidine groups.

Role of maltol in advanced glycation end products and free radicals: in-vitro and in-vivo studies.

Inhibitors of advanced glycation end products (AGEs) have potential as preventive agents against diabetic complications. In-vitro AGE inhibitory activity, transition metal chelating, and free radical scavenging activity tests have been used to screen for and identify effective AGE inhibitors. In an ongoing project to elucidate AGE inhibiting active components of heat-processed ginseng, maltol was selected for more detailed investigation. Although there are several lines of evidence concerning the antioxidant activity of maltol, the in-vitro and in-vivo inhibitory effects of maltol on AGE generation have not been evaluated. In the present study, the in-vitro AGE inhibitory effects and free radical scavenging activity of maltol were investigated. In addition, the in-vivo therapeutic potential of maltol against diabetic renal damage was tested using streptozotocin (STZ)-diabetic rats. Maltol showed a stronger AGE inhibitory effect than aminoguanidine, a well known AGE inhibitor. In addition, the hydroxyl radical scavenging activity of maltol on electron spin resonance (ESR) spectrometry was slightly stronger than that of aminoguanidine. Therefore, maltol was found to have stronger in-vitro AGE inhibiting activity compared with aminoguanidine. The administration of 50 mgkg(-1) per day of maltol suppressed the elevated serum levels of glycosylated protein, renal fluorescent AGEs, carboxymethyllysine, receptors for AGEs, and nuclear factor-kappaB p65 in diabetic control rats. These beneficial effects of maltol against STZ-diabetic renal damage were thought to result from its free radical scavenging and AGE inhibitory effects.