Choline transporter‐like protein 4 (CTL4) links to non‐neuronal acetylcholine synthesis

Synthesis of acetylcholine (ACh) by non‐neuronal cells is now well established and plays diverse physiologic roles. In neurons, the Na⁺‐dependent, high affinity choline transporter (CHT1) is absolutely required for ACh synthesis. In contrast, some non‐neuronal cells synthesize ACh in the absence of CHT1 indicating a fundamental difference in ACh synthesis compared to neurons. The aim of this study was to identify choline transporters, other than CHT1, that play a role in non‐neuronal ACh synthesis. ACh synthesis was studied in lung and colon cancer cell lines focusing on the choline transporter‐like proteins, a five gene family choline‐transporter like protein (CTL)1–5. Supporting a role for CTLs in choline transport in lung cancer cells, choline transport was Na⁺‐independent and CTL1–5 were expressed in all cells examined. CTL1, 2, and 5 were expressed at highest levels and knockdown of CTL1, 2, and 5 decreased choline transport in H82 lung cancer cells. Knockdowns of CTL1, 2, 3, and 5 had no effect on ACh synthesis in H82 cells. In contrast, knockdown of CTL4 significantly decreased ACh secretion by both lung and colon cancer cells. Conversely, increasing expression of CTL4 increased ACh secretion. These results indicate that CTL4 mediates ACh synthesis in non‐neuronal cell lines and presents a mechanism to target non‐neuronal ACh synthesis without affecting neuronal ACh synthesis.     

Melatonin attenuates the effects of sub-acute administration of lead on kidneys in rats without altering the lead-induced reduction in nitric oxide

Exposure to lead induces oxidative stress and renal damage. Although most forms of oxidative stress are characterized by simultaneous elevation of nitrogen and oxidative species, lead-induced oxidative stress is unusual in that it is associated with a reduction in nitric oxide (NO) levels in the kidney. The role of NO in kidney injury is controversial; some studies suggest that it is associated with renal injury, whereas others show that it exerts protective effects. Concentration-dependent effects have also been proposed, linking low levels with vasodilatation and high levels with toxicity. The aim of this study was to evaluate the effects of melatonin co-exposure on the lead-induced reduction in renal NO levels. We found that sub-acute intraperitoneal administration of 10 mg/kg/day of lead for 15 days induced toxic levels of lead in the blood and caused renal toxicity (pathological and functional). Under our experimental conditions, lead induced an increase in lipid peroxidation and a decrease in NO. Melatonin co-treatment decreased lead-induced oxidative stress (peroxidation level) and toxic effects on kidneys without altering the lead-induced reduction in renal NO. These results suggest that, in our experimental model, the reduction in renal NO levels by lead exposure is not the only responsible factor for lead-induced kidney damage.     

Plasma Proteome Signature of Sepsis: a Functionally Connected Protein Network

Sepsis is an extreme host response to infection that leads to loss of organ function and cardiovascular integrity. Mortality from sepsis is on the rise. Despite more than three decades of research and clinical trials, specific diagnostic and therapeutic strategies for sepsis are still absent. The use of LFQ‐ and TMT‐based quantitative proteomics is reported here to study the plasma proteome in five mouse models of sepsis. A knowledge‐based interpretation of the data reveals a protein network with extensive connectivity through documented functional or physical interactions. The individual proteins in the network all have a documented role in sepsis and are known to be extracellular. The changes in protein abundance observed in the mouse models of sepsis have for the most part the same directionality (increased or decreased abundance) as reported in the literature for human sepsis. This network has been named the Plasma Proteome Signature of Sepsis (PPSS). The PPSS is a quantifiable molecular readout that can supplant the current symptom‐based approach used to diagnose sepsis. This type of molecular interpretation of sepsis, its progression, and its response to therapeutic intervention are an important step in advancing our understanding of sepsis, and for discovering and evaluating new therapeutic strategies.     

Ca2+ signal summation and NFATc1 nuclear translocation in sympathetic ganglion neurons during repetitive action potentials

NFATc-mediated gene expression constitutes a critical step during neuronal development and synaptic plasticity. Although considerable information is available regarding the activation and functionality of specific NFATc isoforms, in neurons little is known about how sensitive NFAT nuclear translocation is to specific patterns of electrical activity. Here we used high-speed fluo-4 confocal imaging to monitor action potential (AP)-induced cytosolic Ca2+ transients in rat sympathetic neurons. We have recorded phasic and repetitive AP patterns, and corresponding Ca2+ transients initiated by either long (100-800 ms) current-clamp pulses, or single brief (2 ms) electrical field stimulation. We address the functional consequences of these AP and Ca2+ transient patterns, by using an adenoviral construct to express NFATc1-CFP and evaluate NFATc1-CFP nuclear translocation in response to specific patterns of electrical activity. Ten Hertz trains stimulation induced nuclear translocation of NFATc1, whereas 1 Hz trains did not. However, 1 Hz train stimulation did result in NFATc1 translocation in the presence of 2 mM Ba2+, which inhibits M-currents and promotes repetitive firing and the accompanying small (approximately 0.6 DeltaF/F0) repetitive and summating Ca2+ transients. Our results demonstrate that M-current inhibition-mediated spike frequency facilitation enhances cytosolic Ca2+ signals and NFATc1 nuclear translocation during trains of low frequency electrical stimulation.     

Multiple insulin degrading enzyme variants alter in vitro reporter gene expression

The insulin degrading enzyme (IDE) variant, v311 (rs6583817), is associated with increased post-mortem cerebellar IDE mRNA, decreased plasma β-amyloid (Aβ), decreased risk for Alzheimer''s disease (AD) and increased reporter gene expression, suggesting that it is a functional variant driving increased IDE expression. To identify other functional IDE variants, we have tested v685, rs11187061 (associated with decreased cerebellar IDE mRNA) and variants on H6, the haplotype tagged by v311 (v10; rs4646958, v315; rs7895832, v687; rs17107734 and v154; rs4646957), for altered in vitro reporter gene expression. The reporter gene expression levels associated with the second most common haplotype (H2) successfully replicated the post-mortem findings in hepatocytoma (0.89 fold-change, p = 0.04) but not neuroblastoma cells. Successful in vitro replication was achieved for H6 in neuroblastoma cells when the sequence was cloned 5′ to the promoter (1.18 fold-change, p = 0.006) and 3′ to the reporter gene (1.29 fold change, p = 0.003), an effect contributed to by four variants (v10, v315, v154 and v311). Since IDE mediates Aβ degradation, variants that regulate IDE expression could represent good therapeutic targets for AD.     

A novel protease from Entamoeba histolytica homologous to members of the family S28 of serine proteases

Serine proteases are one of the biologically most important and widely distributed enzyme families. A protease capable of degrading the substrate Suc-AAF-AMC was isolated from axenically grown trophozoites of Entamoeba histolytica. The enzyme was purified by ion-exchange chromatography and electroelution, and appeared on 2D-PAGE as a spot of 60 kDa and pI of 4.65. Data obtained from zymogram suggest the active protease is present either as homodimer (130 kDa) or homotetramer (250 kDa). The optimal temperature of the enzyme was 37 degrees C, and it exhibited activity over a broad pH range. The protease was strongly inhibited by TPCK and chelating agents. The enzymatic activity was restored upon addition of calcium. BLAST analysis with the sequence of internal peptides of the protein revealed two open reading frames within the genome of E. histolytica, homologous to members of the family S28, clan SC of serine proteases.     

Quantitative proteomic analysis of drug-induced changes in mycobacteria

A new approach for qualitative and quantitative proteomic analysis using capillary liquid chromatography and mass spectrometry to study the protein expression response in mycobacteria following isoniazid treatment is discussed. In keeping with known effects on the fatty acid synthase II pathway, proteins encoded by the kas operon (AcpM, KasA, KasB, Accd6) were significantly overexpressed, as were those involved in iron metabolism and cell division suggesting a complex interplay of metabolic events leading to cell death.     

High incidence of deafness from three frequent connexin 26 mutations in an isolated community

In a small village founded by few ancestors, three mutations in GJB2, the gene for connexin 26, are responsible for the high prevalence of deafness. A total of 15% of healthy individuals from a random sample were carriers of either 35Gdel (7.8%), W77R (2.4%), or V37I (4.8%). The three mutations appeared in the village approximately 100-150 years ago. The question of why three distinct mutations of similar age are observed at high frequency within a genetic isolate is discussed.