Disruption of NAD~+ binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions

AIM:To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase(GAPDH),and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS:We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters(diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching(FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD+ cofactor binding.RESULTS:Using MALDI-TOF analysis,we identified novel phosphorylation sites within the NAD+ binding center of GAPDH at Y94,S98,and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH,we demonstrated accumulation of phospho-T99-GAPDH inthe nuclear fractions of A549,HCT116,and SW48 cancer cel s after cytotoxic stress. We performed site-mutagenesis,and estimated enzymatic properties,intranuclear distribution,and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD+ binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD+(Km = 741 ± 257 μmol/L in T99 I vs 57 ± 11.1 μmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD+ binding with GAPDH. FRAP(fluorescence recovery after photo bleaching) analysis showed that mutations in NAD+ binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION:Our results suggest an important functional role of phosphorylated amino acids in the NAD+ binding center in GAPDH interactions with its intranuclear partners.     

Protein kinase C impairs the coupling of the GTP-binding protein to LTB4 receptor in neutrophil.

In the present study, the mechanism of LTB4 receptor down regulation by protein kinase C (PKC) has been investigated using porcine neutrophil membranes. Pretreatment of intact porcine neutrophils with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 2 min prior to the preparation of plasma membrane, demonstrated a reduced binding sites (Bmax) for LTB4 without altering the receptor affinity (Kd). This effect of TPA on LTB4 receptor binding was found to be due to the activation of PKC as membrane treated with purified PKC (type III) produced the same effect. When membranes from neutrophils pretreated with TPA were exposed to non-hydrolyzable GTP analog, GTP-gamma S, or GMP-PNP, no further decrease in receptor Kd was observed, while the Bmax was reduced to the level observed in TPA treated samples. Treatment of isolated neutrophil membranes with purified PKC reduced the Bmax and blocked the effect of GTP analogs on the receptor affinity. These results suggest that, PKC interrupts the receptor binding to G-protein.     

Changes in brain cyclic AMP metabolism and acetylcholine and dopamine during narcotic dependence and withdrawal.

Effects of morphine administration were studied on cyclic AMP metabolism in several regions of rat brain. In the cortex, cerebellum and thalamus-hypothalamus, morphine dependence did not alter the activity of either adenylate cyclase or phosphodiesterase. However, during withdrawal from the opiate treatment, adenylate cyclase activity declined in all three regions studied. In contrast, the striatal cyclic AMP metabolism was enhanced during morphine treatment as reflected by elevated endogenous cyclic AMP and increased adenylate cyclase. Furthermore, narcotic dependence produced significant increases in acetylcholinesterase activity of rat striatum. Whereas morphine withdrawal reversed the changes in striatal acetylcholine levels and acetylcholinesterase activity, the enhanced striatal dopamine remained unaltered. Although the activity of striatal adenylate cyclase was significantly reduced when compared to the morphine-dependent rats, the drop in cyclic AMP levels was not significant. Methadone replacement did not affect the changes in striatal dopamine seen in morphine-withdrawn rats. Whereas dopamine stimulated equally well the striatal adenylate cyclase from control or morphine-dependent animals, it failed to stimulate the striatal enzyme from rats undergoing withdrawal. The crude synaptosomal fraction of the whole brain from morphine-dependent rats exhibited an increase in cyclic AMP which was accompanied by elevated adenylate cyclase and protein kinase activity. Naloxone administration suppressed this rise in cyclic AMP and reversed the morphine-stimulated increases in the activities of adenylate cyclase and protein kinase. Following the withdrawal of morphine treatment, alterations in cyclic AMP metabolism were similar to those noted in morphine-naloxone group. Furthermore, substitution of morphine with methadone antagonized the observed alterations in cyclic nucleotide metabolism during withdrawal.     

Sequential changes in hepatic polyamine, deoxyribonucleic acid, and cyclic adenosine 3',5'-monophosphate metabolism after subacute exposure to cadmium in rats.

Heavy metal treatment (2 X 1 mg/kg per day) for 3, 5, and 7 days resulted in progressive augmentation in the incorporation of [14C]thymidine into hepatic DNA. In contrast with the observed enhancement in DNA synthesis, cadmium exposure tended to produce a decrease in the activity of hepatic ornithine decarboxylase (EC 4.1.1.17) at 1, 3, or 5 days with the lowest (34% of control values) enzymic activity seen after 7 days. A similar reduction in the activity of S-adenosylmethionine decarboxylase (EC 4.1.1.50) was observed in livers of rats treated with cadmium for 1-7 days. Subacute exposure to cadmium significantly lowered the hepatic levels of spermidine and spermine whereas the endogenous concentrated of putrescine remained unaltered. In addition to the observed effects on the biosynthesis of polyamines and DNA, heavy metal treatment produced stimulation of the hepatic adenylate cyclase (EC 4.6.1.1)--cyclic AMP system. Significant increases in the activity of hepatic adenylate cyclase and endogenous cyclic AMP levels were detected as early as 1 day and the observed alterations persisted during the entire 1-week period of cadmium exposure. The depression in polyamine formation was accompanied by enhanced DNA biosynthesis as well as stimulation in the adenylate cyclase-cyclic AMP system of rat liver.     

Who Gets Prompt Access to Artemisinin-Based Combination Therapy? A Prospective Community-Based Study in Children from Rural Kilosa,Tanzania

Background

Effective and timely case management remains one of the fundamental pillars for control of malaria. Tanzania introduced artemisinin-combination therapy [ACT] for uncomplicated malaria; however, the policy change is challenged by limited availability of ACTs due to high cost. This study aimed to determine factors influencing prompt access to ACTs among febrile children in rural Kilosa, Tanzania.

Methods and Findings

In a community-based study, 1,235 randomly selected children under five were followed up weekly for six months, in 2008. Using a structured questionnaire, children''s caretakers were asked about the child''s febrile history in the last seven days, and treatment actions including timing, medicines used and source of care. Caretakers'' knowledge about malaria and socioeconomic and demographic data were also obtained. About half of followed-up children had at least one episode of fever. Less than half (44.8%) of febrile children were taken to government facilities. Almost one-third (37.6%; 95% CI 33.1–42.1) of febrile children had prompt access to ACT. Care-seeking from a government facility was the overriding factor, increasing the likelihood of prompt access to an ACT 18 times (OR 17.7; 95% CI 10.55–29.54; adjusted OR 16.9; 95% CI 10.06–28.28). Caretakers from the better-off household (3rd–5th quintiles) were more likely to seek care from government facilities (OR 3.66; 95% CI 2.56–5.24; adjusted OR 1.80; 95% CI 1.18–2.76). The majority of antimalarials accessed by the poor were ineffective [86.0%; 295/343], however, they paid more for them (median Tsh 500) compared to the better-offs (median Tsh 0).

Conclusions

Prompt access to ACT among febrile children was unacceptably low, due mainly to limited availability of subsidised ACT at the location where most caretakers sought care. There is urgent need to accelerate implementation of strategies that will ensure availability of ACT at an affordable price in remote rural areas, where the burden of malaria is highest.     

Malaria rapid testing by community health workers is effective and safe for targeting malaria treatment: randomised cross-over trial in Tanzania

Background

Early diagnosis and prompt, effective treatment of uncomplicated malaria is critical to prevent severe disease, death and malaria transmission. We assessed the impact of rapid malaria diagnostic tests (RDTs) by community health workers (CHWs) on provision of artemisinin-based combination therapy (ACT) and health outcome in fever patients.

Methodology/Principal Findings

Twenty-two CHWs from five villages in Kibaha District, a high-malaria transmission area in Coast Region, Tanzania, were trained to manage uncomplicated malaria using RDT aided diagnosis or clinical diagnosis (CD) only. Each CHW was randomly assigned to use either RDT or CD the first week and thereafter alternating weekly. Primary outcome was provision of ACT and main secondary outcomes were referral rates and health status by days 3 and 7. The CHWs enrolled 2930 fever patients during five months of whom 1988 (67.8%) presented within 24 hours of fever onset. ACT was provided to 775 of 1457 (53.2%) patients during RDT weeks and to 1422 of 1473 (96.5%) patients during CD weeks (Odds Ratio (OR) 0.039, 95% CI 0.029–0.053). The CHWs adhered to the RDT results in 1411 of 1457 (96.8%, 95% CI 95.8–97.6) patients. More patients were referred on inclusion day during RDT weeks (10.0%) compared to CD weeks (1.6%). Referral during days 1–7 and perceived non-recovery on days 3 and 7 were also more common after RDT aided diagnosis. However, no fatal or severe malaria occurred among 682 patients in the RDT group who were not treated with ACT, supporting the safety of withholding ACT to RDT negative patients.

Conclusions/Significance

RDTs in the hands of CHWs may safely improve early and well-targeted ACT treatment in malaria patients at community level in Africa.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00301015","term_id":"NCT00301015"}}NCT00301015     

Polyamine acetylation modulates polyamine metabolic flux, a prelude to broader metabolic consequences

Recent studies suggest that overexpression of the polyamine-acetylating enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) significantly increases metabolic flux through the polyamine pathway. The concept derives from the observation that SSAT-induced acetylation of polyamines gives rise to a compensatory increase in biosynthesis and presumably to increased flow through the pathway. Despite the strength of this deduction, the existence of heightened polyamine flux has not yet been experimentally demonstrated. Here, we use the artificial polyamine precursor 4-fluoro-ornithine to measure polyamine flux by tracking fluorine unit permeation of polyamine pools in human prostate carcinoma LNCaP cells. Conditional overexpression of SSAT was accompanied by a massive increase in intracellular and extracellular acetylated spermidine and by a 6-20-fold increase in biosynthetic enzyme activities. In the presence of 300 microM 4-fluoro-ornithine, SSAT overexpression led to the sequential appearance of fluorinated putrescine, spermidine, acetylated spermidine, and spermine. As fluorinated polyamines increased, endogenous polyamines decreased, so that the total polyamine pool size remained relatively constant. At 24 h, 56% of the spermine pool in the induced SSAT cells was fluorine-labeled compared with only 12% in uninduced cells. Thus, SSAT induction increased metabolic flux by approximately 5-fold. Flux could be interrupted by inhibition of polyamine biosynthesis but not by inhibition of polyamine oxidation. Overall, the findings are consistent with a paradigm whereby flux is initiated by SSAT acetylation of spermine and particularly spermidine followed by a marked increase in key biosynthetic enzymes. The latter sustains the flux cycle by providing a constant supply of polyamines for subsequent acetylation by SSAT. The broader metabolic implications of this futile metabolic cycling are discussed in detail.     

Aptamer-based diagnostic and therapeutic approaches in animals: Current potential and challenges

Fast and precise diagnosis of infectious and non-infectious animal diseases and their targeted treatments are of utmost importance for their clinical management. The existing biochemical, serological and molecular methods of disease diagnosis need improvement in their specificity, sensitivity and cost and, are generally not amenable for being used as points-of-care (POC) device. Further, with dramatic changes in environment and farm management practices, one should also arm ourselves and prepare for emerging and re-emerging animal diseases such as cancer, prion diseases, COVID-19, influenza etc. Aptamer – oligonucleotide or short peptides that can specifically bind to target molecules – have increasingly become popular in developing biosensors for sensitive detection of analytes, pathogens (bacteria, virus, fungus, prions), drug residues, toxins and, cancerous cells. They have also been proven successful in the cellular delivery of drugs and targeted therapy of infectious diseases and physiological disorders. However, the in vivo application of aptamer-mediated biosensing and therapy in animals has been limited. This paper reviews the existing reports on the application of aptamer-based biosensors and targeted therapy in animals. It also dissects the various modifications to aptamers that were found to be successful in in vivo application of the aptamers in diagnostics and therapeutics. Finally, it also highlights major challenges and future directions in the application of aptamers in the field of veterinary medicine.     

Proteomic analysis of oxidative stress-responsive proteins in human pneumocytes: insight into the regulation of DJ-1 expression

Oxidative injury is believed to play an important role in the pathogenesis of lung diseases such as emphysema and lung cancer. We examined the effects of a classic reactive oxygen species, H 2O 2, on the hydrogen peroxide response proteins (HPRP) in human pneumocytes using comparative two-dimensional gel electrophoresis (2DE) and peptide mass fingerprinting. Four HPRP-associated proteins (DJ-1, peroxiredoxins [Prxs] I and IV and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) were changed upon exposure to H 2O 2 (1 mM for 24 h). H 2O 2 exposure increased the acid (oxidized) form and decreased the basic (reduced) form of DJ-1 (pI 5.8 and 6.2, respectively), Prx I and IV and GAPDH. Mechanistic studies on DJ-1 indicated that the slow recovery of the reduced form was blocked by cyclohexamide, suggesting that the recovery was due to new protein synthesis. Total DJ-1 expression was decreased by increasing concentrations of H 2O 2. In contrast, a more complex mix of oxidants in the form of cigarette smoke extract (CSE) dose-dependently increased DJ-1 expression and produced a novel DJ-1 isoform (p I 5.6). Moreover, DJ-1 expression was higher in the lungs of chronic cigarette smokers compared with nonsmokers, a result which resembled the effects of CSE in cultured cells. These data indicate that in human pneumocytes, DJ-1 functions as an antioxidant but that no enzymatic system converts the oxidized to the reduced form. Up-regulation of DJ-1 by cigarette smoke may be a compensatory mechanism that protects the lung from oxidative stress-related injury.