Kirkiin: A New Toxic Type 2 Ribosome-Inactivating Protein from the Caudex of Adenia kirkii.

Ribosome-inactivating proteins (RIPs) are plant toxins that irreversibly damage ribosomes and other substrates, thus causing cell death. RIPs are classified in type 1 RIPs, single-chain enzymatic proteins, and type 2 RIPs, consisting of active A chains, similar to type 1 RIPs, linked to lectin B chains, which enable the rapid internalization of the toxin into the cell. For this reason, many type 2 RIPs are very cytotoxic, ricin, volkensin and stenodactylin being the most toxic ones. From the caudex of Adenia kirkii (Mast.) Engl., a new type 2 RIP, named kirkiin, was purified by affinity chromatography on acid-treated Sepharose CL-6B and gel filtration. The lectin, with molecular weight of about 58 kDa, agglutinated erythrocytes and inhibited protein synthesis in a cell-free system at very low concentrations. Moreover, kirkiin was able to depurinate mammalian and yeast ribosomes, but it showed little or no activity on other nucleotide substrates. In neuroblastoma cells, kirkiin inhibited protein synthesis and induced apoptosis at doses in the pM range. The biological characteristics of kirkiin make this protein a potential candidate for several experimental pharmacological applications both alone for local treatments and as component of immunoconjugates for systemic targeting in neurodegenerative studies and cancer therapy.

Structure and Function of the Bacterial Protein Toxin Phenomycin.

Phenomycin is a bacterial mini-protein of 89 amino acids discovered more than 50 years ago with toxicity in the nanomolar regime toward mammalian cells. The protein inhibits the function of the eukaryotic ribosome in cell-free systems and appears to target translation initiation. Several fundamental questions concerning the cellular activity of phenomycin, however, have remained unanswered. In this paper, we have used morphological profiling to show that direct inhibition of translation underlies the toxicity of phenomycin in cells. We have performed studies of the cellular uptake mechanism of phenomycin, showing that endosomal escape is the toxicity-limiting step, and we have solved a solution phase high-resolution structure of the protein using NMR spectroscopy. Through bioinformatic as well as functional comparisons between phenomycin and two homologs, we have identified a peptide segment, which constitutes one of two loops in the structure that is critical for the toxicity of phenomycin.

Nicotine Self-administration Is Not Increased in the Methylazoxymethanol Acetate Rodent Model of Schizophrenia.

INTRODUCTION: Patients with schizophrenia (SCZ) smoke at a rate of 4-5 times higher than the general population, contributing to negative health consequences in this group. One possible explanation for this increased smoking is that individuals with SCZ find nicotine (NIC) more reinforcing. However, data supporting this possibility are limited. METHODS: The present experiments examined self-administration of NIC, alone or in combination with other reinforcers, across a range of doses in the methylazoxymethanol acetate (MAM) rodent model of SCZ. RESULTS: MAM and control animals did not differ in NIC self-administration across a range of doses and schedules of reinforcement, in both standard 1-hour self-administration sessions and 23-hour extended access sessions. However, MAM animals responded less for sucrose or reinforcing visual stimuli alone or when paired with NIC. CONCLUSIONS: To the extent that MAM-treated rats are a valid model of SCZ, these results suggest that increased NIC reinforcement does not account for increased smoking in SCZ patients. IMPLICATIONS: This study is the first to utilize nicotine self-administration, the gold standard for studying nicotine reinforcement, in the methylazoxymethanol acetate model of schizophrenia, which is arguably the most comprehensive animal model of the disease currently available. Our assessment found no evidence of increased nicotine reinforcement in methylazoxymethanol acetate animals, suggesting that increased reinforcement may not perpetuate increased smoking in schizophrenia patients.

Prolonged inhibition and incomplete recovery of mitochondrial function in oxazolidinone-treated megakaryoblastic cell lines.

Thrombocytopenia is commonly seen in patients receiving linezolid for >14 days. Linezolid is a reversible inhibitor of mitochondrial function in various cell types. This study investigated the inhibitory effects of linezolid and tedizolid, and their potential recovery on (i) CYTox I expression (subunit I of cytochrome c-oxidase; encoded by the mitochondrial genome), (ii) cytochrome c-oxidase activity and (iii) mitochondrial respiration (Seahorse bioanalysis) in two megakaryocytic cell lines [UT-7 WT (human acute megakaryoblastic leukaemia cells) and UT-7 MPL (transduced to express the thrombopoietin receptor)]. Cells were exposed to linezolid (0.5-25 mg/L) or tedizolid (0.1-5 mg/L) for up to 5 days and recovery followed after drug removal. Both oxazolidinones caused concentration- and time-dependent inhibition of CYTox I expression, cytochrome c-oxidase activity and mitochondrial spare capacity. On electron microscopy, mitochondria appeared dilated with a loss of cristae. Globally, tedizolid exerted stronger effects than linezolid. While CYTox I expression recovered completely after 6 days of drug washout, only partial (linezolid) or no (tedizolid) recovery of cytochrome c-oxidase activity, and no rescue of mitochondrial spare capacity (after 3 days) was observed. Thus, and in contrast to previous studies using a variety of cell lines unrelated to megakaryocytic lineages, the inhibitory effects exerted by oxazolidinones on the mitochondrial function of megakaryoblastic cells appear to be particularly protracted. Given the dynamics of platelet production and destruction, these results may explain why oxazolidinone-induced thrombocytopenia is one of the most common side effects in patients exposed to these antibiotics.

Protein synthesis inhibitors induce both memory impairment and its recovery.

The involvement of protein synthesis in the mechanisms of conditioned food aversion memory impairment and recovery in grape snails was studied. It was found that protein synthesis inhibitor (cycloheximide) injections before a reminder by the conditioned stimulus (CS) caused amnesia development. Three days after amnesia induction, injections of cycloheximide or another protein synthesis inhibitor, anisomycin, combined with a reminder by four CSs resulted in memory retrieval, which was saved for 24 h. Cycloheximide injections and the administration of one CS as a reminder to an amnestic animals caused the memory expression only in response to this CS, while it was absent the next day. The isolated administration of a reminder or inhibitor injections without a reminder was not effective. It is suggested that amnesia is an active process and that one of its mechanisms may be a protein-dependent amnesia reactivation caused by a reminder. The administration of protein synthesis inhibitors led to impairment of amnesia reactivation and to recovery of the state formed before amnesia induction and thus to the recovery of conditioned food aversion memory.

Fruticulosin: A novel type 2 ribosome-inactivating protein from Abrus fruticulosus seeds that exhibits toxic and antileishmanial activity.

Plant ribosome-inactivating proteins (RIPs) are a family of toxins that inhibit protein synthesis. In this study, we have isolated a novel type 2 ribosome-inactivating protein (RIP) present in seeds of the Abrus fruticulosus, named of fruticulosin. Fruticulosin, shows characteristics common to other type 2 RIPs, as specificity by galactosides (d-galactose, N-acetyl-d-galactosamine, and d-lactose), mass of approximately 60 kDa and presence of the of disulfide bonds. The N-terminal amino acid sequence (26 residues) of A-chain fruticulosin, determined by Edman degradation, revealed high similarity of the A-chain with those of other type 2 RIPs. The secondary structure of fruticulosin was analysed by circular dichroism, which showed that fruticulosin contains α-helices (22.3%), ß-sheets (43.5%), and random coils and corners (34.2%). Furthermore, fruticulosin showed high toxicity in Artemia sp. (3.12 µg/mL), inhibited in vitro protein synthesis by a cell-free system and showed RNA N-glycosidase activity. Fruticulosin presented biological activities such as agglutination and antileishmanial activity on promastigote forms of Leishmania major.

Genetic disruption of fractalkine signaling leads to enhanced loss of cochlear afferents following ototoxic or acoustic injury.

Cochlear hair cells are vulnerable to a variety of insults like acoustic trauma and ototoxic drugs. Such injury can also lead to degeneration of spiral ganglion neurons (SGNs), but this occurs over a period of months to years. Neuronal survival is necessary for the proper function of cochlear prosthetics, therefore, it is of great interest to understand the mechanisms that regulate neuronal survival in deaf ears. We have recently demonstrated that selective hair cell ablation is sufficient to attract leukocytes into the spiral ganglion, and that fractalkine signaling plays a role in macrophage recruitment and in the survival of auditory neurons. Fractalkine (CX3 CL1), a chemokine that regulates adhesion and migration of leukocytes is expressed by SGNs and signals to leukocytes via its receptor CX3 CR1. The present study has extended the previous findings to more clinically relevant conditions of sensorineural hearing loss by examining the role of fractalkine signaling after aminoglycoside ototoxicity or acoustic trauma. Both aminoglycoside treatment and acoustic overstimulation led to the loss of hair cells as well as prolonged increase in the numbers of cochlear leukocytes. Lack of CX3 CR1 did not affect macrophage recruitment after injury, but resulted in increased loss of SGNs and enhanced expression of the inflammatory cytokine interleukin-1ß, when compared to mice with intact CX3 CR1. These data indicate that the dysregulation of macrophage response caused by the absence of CX3 CR1 may contribute to inflammation-mediated neuronal loss in the deafened ear, suggesting a key role for inflammation in the long-term survival of target-deprived afferent neurons.

Coordinated Plasticity of Synapses and Astrocytes Underlies Practice-Driven Functional Vicariation in Peri-Infarct Motor Cortex.

Motor rehabilitative training after stroke can improve motor function and promote topographical reorganization of remaining motor cortical movement representations, but this reorganization follows behavioral improvements. A more detailed understanding of the neural bases of rehabilitation efficacy is needed to inform therapeutic efforts to improve it. Using a rat model of upper extremity impairments after ischemic stroke, we examined effects of motor rehabilitative training at the ultrastructural level in peri-infarct motor cortex. Extensive training in a skilled reaching task promoted improved performance and recovery of more normal movements. This was linked with greater axodendritic synapse density and ultrastructural characteristics of enhanced synaptic efficacy that were coordinated with changes in perisynaptic astrocytic processes in the border region between head and forelimb areas of peri-infarct motor cortex. Disrupting synapses and motor maps by infusions of anisomycin (ANI) into anatomically reorganized motor, but not posterior parietal, cortex eliminated behavioral gains from rehabilitative training. In contrast, ANI infusion in the equivalent cortical region of intact animals had no effect on reaching skills. These results suggest that rehabilitative training efficacy for improving manual skills is mediated by synaptic plasticity in a region of motor cortex that, before lesions, is not essential for manual skills, but becomes so as a result of the training. These findings support that experience-driven synaptic structural reorganization underlies functional vicariation in residual motor cortex after motor cortical infarcts.SIGNIFICANCE STATEMENT Stroke is a leading cause of long-term disability. Motor rehabilitation, the main treatment for physical disability, is of variable efficacy. A better understanding of neural mechanisms underlying effective motor rehabilitation would inform strategies for improving it. Here, we reveal synaptic underpinnings of effective motor rehabilitation. Rehabilitative training improved manual skill in the paretic forelimb and induced the formation of special synapse subtypes in coordination with structural changes in astrocytes, a glial cell that influences neural communication. These changes were found in a region that is nonessential for manual skill in intact animals, but came to mediate this skill due to training after stroke. Therefore, motor rehabilitation efficacy depends on synaptic changes that enable remaining brain regions to assume new functions.

Targeting plasminogen activator inhibitor-1 in tetracycline-induced pleural injury in rabbits.

Elevated active plasminogen activator inhibitor-1 (PAI-1) has an adverse effect on the outcomes of intrapleural fibrinolytic therapy (IPFT) in tetracycline-induced pleural injury in rabbits. To enhance IPFT with prourokinase (scuPA), two mechanistically distinct approaches to targeting PAI-1 were tested: slowing its reaction with urokinase (uPA) and monoclonal antibody (mAb)-mediated PAI-1 inactivation. Removing positively charged residues at the "PAI-1 docking site" (179RHRGGS184→179AAAAAA184) of uPA results in a 60-fold decrease in the rate of inhibition by PAI-1. Mutant prourokinase (0.0625-0.5 mg/kg; n = 12) showed efficacy comparable to wild-type scuPA and did not change IPFT outcomes ( P > 0.05). Notably, the rate of PAI-1-independent intrapleural inactivation of mutant uPA was 2 times higher ( P < 0.05) than that of the wild-type enzyme. Trapping PAI-1 in a "molecular sandwich"-type complex with catalytically inactive two-chain urokinase with Ser195Ala substitution (S195A-tcuPA; 0.1 and 0.5 mg/kg) did not improve the efficacy of IPFT with scuPA (0.0625-0.5 mg/kg; n = 11). IPFT failed in the presence of MA-56A7C10 (0.5 mg/kg; n = 2), which forms a stable intrapleural molecular sandwich complex, allowing active PAI-1 to accumulate by blocking its transition to a latent form. In contrast, inactivation of PAI-1 by accelerating the active-to-latent transition mediated by mAb MA-33B8 (0.5 mg/kg; n = 2) improved the efficacy of IPFT with scuPA (0.25 mg/kg). Thus, under conditions of slow (4-8 h) fibrinolysis in tetracycline-induced pleural injury in rabbits, only the inactivation of PAI-1, but not a decrease in the rate of its reaction with uPA, enhances IPFT. Therefore the rate of fibrinolysis, which varies in different pathologic states, could affect the selection of PAI-1 inhibitors to enhance fibrinolytic therapy.

Influence of Mitochondrial Genetics on the Mitochondrial Toxicity of Linezolid in Blood Cells and Skin Nerve Fibers.

The antibiotic linezolid is a ribosomal inhibitor with excellent efficacy. Although the administration period has been reduced to 28 days, side effects, usually of hematologic or neuropathic origin, are still reported due to secondary inhibition of mitochondrial protein synthesis. Susceptibility to linezolid toxicity remains unknown. Therefore, the objective of this study was to gain an understanding of clinical heterogeneity in response to identical linezolid exposures through exhaustive examination of the molecular basis of tissue-dependent mitotoxicity, consequent cell dysfunction, and the association of mitochondrial genetics with adverse effects of linezolid administered for the recommended period. Peripheral blood mononuclear cells (PBMC) and skin nerve fibers from 19 and 6 patients, respectively, were evaluated before and after a 28-day linezolid treatment in order to assess toxic effects on mitochondria and cells. Mitochondrial DNA haplotypes and single nucleotide polymorphisms (SNPs) in ribosomal sequences where linezolid binds to mitochondrial ribosomes were also analyzed to investigate their genetic contributions. We found that linezolid reduced mitochondrial protein levels, complex IV activity, and mitochondrial mass in PBMC and was associated with a trend toward an increase in the rate of apoptosis. In skin tissue, mitochondrial mass increased within nerve fibers, accompanied by subclinical axonal swelling. Mitochondrial haplogroup U, mutations in 12S rRNA, and the m.2706A→G, m.3197T→C, and m.3010G→A polymorphisms in 16S rRNA showed a trend toward an association with increased mitochondrial and clinical adverse effects. We conclude that even when linezolid is administered for a shorter time than formerly, adverse effects are reported by 63% of patients. Linezolid exerts tissue-dependent mitotoxicity that is responsible for downstream cellular consequences (blood cell death and nerve fiber swelling), leading to adverse hematologic and peripheral nervous side effects. Multicentric studies should confirm genetic susceptibility in larger cohorts.

Metal-Based Combinations That Target Protein Synthesis by Fungi.

A wide range of fungicides (or antifungals) are used in agriculture and medicine, with activities against a spectrum of fungal pathogens. Unfortunately, the evolution of fungicide resistance has become a major issue. Therefore, there is an urgent need for new antifungal treatments. Certain metals have been used for decades as efficient fungicides in agriculture. However, concerns over metal toxicity have escalated over this time. Recent studies have revealed that metals like copper and chromate can impair functions required for the fidelity of protein synthesis in fungi. This occurs through different mechanisms, based on targeting of iron-sulphur cluster integrity or competition for uptake with amino acid precursors. Moreover, chromate at least acts synergistically with other agents known to target translation fidelity, like aminoglycoside antibiotics, causing dramatic and selective growth inhibition of several fungal pathogens of humans and plants. As such synergy allows the application of decreased amounts of metals for effective inhibition, it lessens concerns about nonspecific toxicity and opens new possibilities for metal applications in combinatorial fungicides targeting protein synthesis.

Exploring hazards of acute exposure of Acephate in Drosophila melanogaster and search for l-ascorbic acid mediated defense in it.

This study reveals protective role of l-ascorbic acid (25, 50 and 100µg/mL) against toxic impacts of acute sub-lethal exposure of Acephate (5µg/mL) in a non-target organism Drosophila melanogaster. Organismal effect was evident from increased impairment in climbing activities (9 folds) of treated individuals who also manifested altered ocular architecture. These anomalies were reduced with l-ascorbic acid (l-AA) supplementation. Acephate induced apoptotic lesions in eye imaginal discs and gut confirmed tissue damage that also reduced with l-AA co-treatment. Reduction in viability of fat body cells (∼41%), neural cells (∼42%) and hemocytes (3 folds) indicates cytotoxic and immunotoxic potential of Acephate, which were significantly mitigated with l-AA co-administration. The sub-cellular toxic impacts of Acephate treatment became obvious from enhancement in activities of antioxidant enzymes (CAT by ∼1.63 folds, SOD by ∼1.32 folds), detoxifying enzymes (Cyp450 by ∼1.99 folds and GST by ∼1.34 folds), 2.1 times boost in HSP 70 expression, and inhibition of cholinesterase activity (by ∼0.66 folds). DNA breaks evident through comet assay confirmed Acephate triggered genotoxicity which could also be prevented through co-administration of. L-AA Furthermore, the study proposes the use of Drosophila as a model to screen chemicals for their protective potential against pesticide toxicity.

Structural changes in the inner ear over time studied in the experimentally deafened guinea pig.

Today a cochlear implant (CI) may significantly restore auditory function, even for people with a profound hearing loss. Because the efficacy of a CI is believed to depend mainly on the remaining population of spiral ganglion neurons (SGNs), it is important to understand the timeline of the degenerative process of the auditory neurons following deafness. Guinea pigs were transtympanically deafened with neomycin, verified by recording auditory brainstem responses (ABRs), and then sacrificed at different time points. Loss of SGNs as well as changes in cell body and nuclear volume were estimated. To study the effect of delayed treatment, a group of animals that had been deaf for 12 weeks was implanted with a stimulus electrode mimicking a CI, after which they received a 4-week treatment with glial cell-derived neurotrophic factor (GDNF). The electrical responsiveness of the SGNs was measured by recording electrically evoked ABRs. There was a rapid degeneration during the first 7 weeks, shown as a significant reduction of the SGN population. The degenerative process then slowed, and there was no difference in the amount of remaining neurons between weeks 7 and 18. © 2016 The Authors Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

Angiotensin converting-enzyme inhibition restores glomerular glycosaminoglycans in rat puromycin nephrosis.

BACKGROUND: Aberrant glomerular polyanionic charge of glycosaminoglycans (GAGs) and sialic acid expression has been observed in proteinuric human and experimental glomerular diseases. Angiotensin-converting enzyme inhibitors (ACEI) lower proteinuria and amend renal function deterioration via hemodynamic mechanisms. We tested the hypothesis that ACEI modulate proteinuria additionally by modifying glomerular GAGs. METHODS: In this study, we explored the effects of the ACEI enalapril on proteinuria and GAG synthesis in puromycin aminonucleoside (PAN)-treated rats. We employed cationic colloidal gold (CCG) localization in glomerular basement membranes (GBM) to identify GAGs by electron microscopy and determined sialic acid residues by immunohistochemical staining with lectins. To clarify ACEI effects on GAG production in vitro, we studied de novo GAG synthesis into newly synthesized proteoglycans in podocytes and mesangial cells using 35S incorporation. Cells were incubated with or without PAN, and with increasing doses of the ACEI enalaprilat. RESULTS: PAN rats developed severe proteinuria that was significantly improved by enalapril treatment. In non-treated PAN rats GBM GAGs were reduced, whereas in the enalapril-treated group GBM GAGs were significantly increased to control levels. Enalapril did not affect glomerular sialic acid. Furthermore, in cultured podocytes and mesangial cells PAN decreased de novo GAG synthesis, an effect which was significantly ameliorated by enalaprilat treatment. CONCLUSION: Treatment with ACEI improves permselectivity properties of the glomerular capillary wall by maintaining its GAG content. This finding provides an additional new mechanism, whereby ACEI exert anti-proteinuric effects.

Quantitative profiling of the in vivo enzymatic activity of ricin reveals disparate depurination of different pulmonary cell types.

The plant-derived toxins ricin and abrin, operate by site-specific depurination of ribosomes, which in turn leads to protein synthesis arrest. The clinical manifestation following pulmonary exposure to these toxins is that of a severe lung inflammation and respiratory insufficiency. Deciphering the pathways mediating between the catalytic activity and the developing lung inflammation, requires a quantitative appreciation of the catalytic activity of the toxins, in-vivo. In the present study, we monitored truncated cDNA molecules which are formed by reverse transcription when a depurinated 28S rRNA serves as template. We found that maximal depurination after intranasal exposure of mice to 2LD50 ricin was reached 48h, where nearly 40% of the ribosomes have been depurinated and that depurination can be halted by post-exposure administration of anti-ricin antibodies. We next demonstrated that the effect of ricin intoxication on different cell types populating the lungs differs greatly, and that outstandingly high levels of damage (80% depurination), were observed in particular for pulmonary epithelial cells. Finally, we found that the magnitude of depurination induced by the related plant-derived toxin abrin, was significantly lower in comparison to ricin, and can be attributed mostly to reduced depurination of pulmonary epithelial cells by abrin. This study provides for the first time vital information regarding the scope and timing of the catalytic performance of ricin and abrin in the lungs of intact animals.

Detection of Burkholderia pseudomallei toxin-mediated inhibition of protein synthesis using a Caenorhabditis elegans ugt-29 biosensor.

Toxins are believed to play a crucial role in Burkholderia pseudomallei pathogenicity, however to date, only a few have been identified. The discovery of additional toxic molecules is limited by the lack of a sensitive indicator of B. pseudomallei toxicity. Previously, from a whole genome transcriptome analysis of B. pseudomallei-infected Caenorhabditis elegans, we noted significant overexpression of a number of worm genes encoding detoxification enzymes, indicating the host's attempt to clear bacterial toxic molecules. One of these genes, ugt-29, a family member of UDP-glucuronosyltransferases, was the most robustly induced phase II detoxification gene. In this study, we show that strong induction of ugt-29 is restricted to infections by the most virulent species among the pathogens tested. We also noted that ugt-29 is activated upon disruption of host protein synthesis. Hence, we propose that UGT-29 could be a promising biosensor to detect B. pseudomallei toxins that compromise host protein synthesis. The identification of bactobolin, a polyketide-peptide hybrid molecule, as a toxic molecule of B. pseudomallei further verifies the utilization of this surveillance system to search for bacterial toxins. Hence, a ugt-29 based reporter should be useful in screening for other molecules that inhibit host protein synthesis.

c-Jun N-Terminal Phosphorylation: Biomarker for Cellular Stress Rather than Cell Death in the Injured Cochlea.

Prevention of auditory hair cell death offers therapeutic potential to rescue hearing. Pharmacological blockade of JNK/c-Jun signaling attenuates injury-induced hair cell loss, but with unsolved mechanisms. We have characterized the c-Jun stress response in the mouse cochlea challenged with acoustic overstimulation and ototoxins, by studying the dynamics of c-Jun N-terminal phosphorylation. It occurred acutely in glial-like supporting cells, inner hair cells, and the cells of the cochlear ion trafficking route, and was rapidly downregulated after exposures. Notably, death-prone outer hair cells lacked c-Jun phosphorylation. As phosphorylation was triggered also by nontraumatic noise levels and none of the cells showing this activation were lost, c-Jun phosphorylation is a biomarker for cochlear stress rather than an indicator of a death-prone fate of hair cells. Preconditioning with a mild noise exposure before a stronger traumatizing noise exposure attenuated the cochlear c-Jun stress response, suggesting that the known protective effect of sound preconditioning on hearing is linked to suppression of c-Jun activation. Finally, mice with mutations in the c-Jun N-terminal phosphoacceptor sites showed partial, but significant, hair cell protection. These data identify the c-Jun stress response as a paracrine mechanism that mediates outer hair cell death.

Inhibitions of mTORC1 and 4EBP-1 are key events orchestrated by Rottlerin in SK-Mel-28 cell killing.

Earlier studies demonstrated that Rottlerin exerts a time- and dose-dependent antiproliferative effect on SK-Mel-28 melanoma cells during 24 h of treatment, but cytotoxicity due to cell death began only after a 48 h exposure. In the current study, in order to identify the type of cell death in this cell line, which is notoriously refractory to most anticancer therapies, and to clarify the underlying mechanisms of this delayed outcome, we searched for apoptotic, necrotic/necroptotic and autophagic traits in Rottlerin-exposed cells. Although SK-Mel-28 cells are both apoptosis and autophagy competent, Western blotting analysis, caspase activity assay, nuclear imaging and the effects of autophagy, apoptosis and necroptosis inhibitors, indicated that Rottlerin cytotoxicity was due to none of the aforementioned death mechanisms. Nevertheless, in growth arrested cells, the death did occur after a prolonged treatment and most likely ensued from the observed blockage of protein synthesis that reached levels expected to be incompatible with cell survival. From a mechanistic point of view, we ascribed this effect to the documented inhibition of mTORC1 activity; mTORC1 inhibition on the one hand led to a not deadly, rather protective autophagic response but, on the other hand caused a near complete arrest of protein synthesis. Interestingly, no cytotoxicity was found towards normal skin fibroblasts, which only resulted mildly growth arrested by the drug.

Neomycin damage and regeneration of hair cells in both mechanoreceptor and electroreceptor lateral line organs of the larval Siberian sturgeon (Acipenser baerii).

The lateral line found in some amphibians and fishes has two distinctive classes of sensory organs: mechanoreceptors (neuromasts) and electroreceptors (ampullary organs). Hair cells in neuromasts can be damaged by aminoglycoside antibiotics and they will regenerate rapidly afterward. Aminoglycoside sensitivity and the capacity for regeneration have not been investigated in ampullary organs. We treated Siberian sturgeon (Acipenser baerii) larvae with neomycin and observed loss and regeneration of sensory hair cells in both organs by labeling with DASPEI and scanning electron microscopy (SEM). The numbers of sensory hair cells in both organs were reduced to the lowest levels at 6 hours posttreatment (hpt). New sensory hair cells began to appear at 12 hpt and were regenerated completely in 7 days. To reveal the possible mechanism for ampullary hair cell regeneration, we analyzed cell proliferation and the expression of neural placodal gene eya1 during regeneration. Both cell proliferation and eya1 expression were concentrated in peripheral mantle cells and both increased to the highest level at 12 hpt, which is consistent with the time course for regeneration of the ampullary hair cells. Furthermore, we used Texas Red-conjugated gentamicin in an uptake assay following pretreatment with a cation channel blocker (amiloride) and found that entry of the antibiotic was suppressed in both organs. Together, our results indicate that ampullary hair cells in Siberian sturgeon larvae can be damaged by neomycin exposure and they can regenerate rapidly. We suggest that the mechanisms for aminoglycoside uptake and hair cell regeneration are conserved for mechanoreceptors and electroreceptors. J. Comp. Neurol. 524:1443-1456, 2016. © 2015 Wiley Periodicals, Inc.

Evaluation of Canine Pancreas-Specific Lipase Activity, Lipase Activity, and Trypsin-Like Immunoreactivity in an Experimental Model of Acute Kidney Injury in Dogs.

BACKGROUND: Diagnosis of pancreatitis in dogs is complicated by extrapancreatic disorders that can alter the results of laboratory tests. Extrapancreatic disorders can also affect the diagnosis of exocrine pancreatic insufficiency (EPI). The effects of acute kidney injury (AKI) on pancreas-specific lipase activity (Spec cPL(®) Test), serum lipase activity and trypsin-like immunoreactivity (TLI) in dogs have not been evaluated. HYPOTHESIS/OBJECTIVES: Serum Spec cPL, lipase activity, and TLI concentrations will increase secondary to decreased kidney function. ANIMALS: Five purpose-bred dogs. METHODS: Experimental prospective study. Gentamicin was used to induce AKI in 5 purpose-bred dogs. Serum samples were collected for measurement of creatinine, Spec cPL, lipase activity and TLI over 60 days, during both induction of, and recovery from, AKI. RESULTS: All dogs developed and recovered from AKI. Six of 52 (12%) serum Spec cPL concentrations were increased (2 in the equivocal zone and 4 consistent with pancreatitis) in 2 of 5 (40%) dogs. Two of 51 (4%) serum lipase activity values were increased in 2 of 5 dogs. Serum TLI was increased above the reference range in 17 of 50 (34%) samples in 3 of 5 dogs. For all biomarkers, there was no consistent correlation with increases in serum creatinine concentration. CONCLUSIONS AND CLINICAL IMPORTANCE: Decreased renal excretion during experimental AKI did not cause consistent and correlated increases in serum Spec cPL, lipase activity, or TLI in this cohort of dogs.