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.

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.

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.

Ecabet sodium alleviates neomycin-induced hair cell damage.

Ecabet sodium (ES) is currently applied to some clinical gastrointestinal disease primarily by the inhibition of the ROS production. In this study, the protective role of ES was evaluated against the neomycin-induced hair cell loss using zebrafish experimental animal model. Zebrafish larvae (5-7 dpf), were treated with each of the following concentrations of ES: 5, 10, 20, 40, and 80 µg/mL for 1 h, followed by 125 µM neomycin for 1h. The positive control group was established by 125 µM neomycin-only treatment (1h) and the negative control group with no additional chemicals was also established. Hair cells inside four neuromasts ( SO1, SO2, O1, OC1) were assessed using fluorescence microscopy (n = 10). Hair cell survival was calculated as the mean number of viable hair cells for each group. Apoptosis and mitochondrial damage were investigated using special staining (TUNEL and DASPEI assay, respectively), and compared among groups. Ultrastructural changes were evaluated using scanning electron microscopy. Pre-treatment group with ES increased the mean number of viable hair cells as a dose-dependent manner achieving almost same number of viable hair cells with 40 µM/ml ES treatment (12.98 ± 2.59 cells) comparing to that of the negative control group (14.15 ± 1.39 cells, p = 0.72) and significantly more number of viable hair cells than that of the positive control group (7.45 ± 0.91 cells, p < 0.01). The production of reactive oxygen species significantly increased by 183% with 125 µM neomycin treatment than the negative control group and significantly decreased down to 105% with the pre-treatment with 40 µM/ml ES (n = 40, p = 0.04). A significantly less number of TUNEL-positive cells (reflecting apoptosis, p < 0.01) and a significantly increased DASPEI reactivity (reflecting viable mitochondria, p < 0.01) were observed in 40 µM/ml ES pre-treatment group. Our data suggest that ES could protect against neomycin-induced hair cell loss possibly by reducing apoptosis, mitochondrial damages, and the ROS generation.

Protective effect of allicin against gentamicin-induced nephrotoxicity in rats.

In this study, the modulator effect of allicin on the oxidative nephrotoxicity of gentamicin in the kidneys of rats was investigated by determining indices of lipid peroxidation and the activities of antioxidant enzymes, as well as by histological analyses. Furthermore, the effect of allicin on gentamicin induced hypersensitivity of urinary bladder rings to ACh was estimated. Twenty-four male Wistar albino rats were randomly divided into three groups, control, gentamicin (100mg/kg, i.p.) and gentamicin+allicin (50mg/kg, orally). At the end of the study, all rats were sacrificed and then urine, blood samples and kidneys were taken. Gentamicin administration caused a severe nephrotoxicity as evidenced by an elevated kidney/body weight ratio, serum creatinine, blood urea nitrogen (BUN), serum lactate dehydrogenase (LDH) and proteinuria with a reduction in serum albumin and creatinine clearance as compared with control group. In addition, a significant increase in renal contents of malondialdehyde (MDA), myeloperoxidase (MPO), nitric oxide (NOx) and tumor necrosis factor-alpha (TNF-α) concomitantly with a significant decrease in renal reduced glutathione (GSH) and superoxide dismutase (SOD) activities was detected upon gentamicin injection. Exposure to gentamicin increased the sensitivity of isolated urinary bladder rings to ACh and induced acute renal tubular epithelial cells necrosis. Administration of allicin significantly decreased kidney/body weight ratio, serum creatinine, LDH, renal MDA, MPO, NOx and TNF-α while it significantly increased creatinine clearance, renal GSH content and renal SOD activity when compared to gentamicin-treated group. Additionally, allicin significantly reduced the responses of isolated bladder rings to ACh and ameliorated tissue morphology as evidenced by histological evaluation. Our study indicates that allicin exerted protection against structural and functional damage induced by gentamicin possibly due to its antioxidant, anti-inflammatory and immunomodulatory properties in addition to its ability to retaining nitric oxide level.

Structural basis for selective targeting of leishmanial ribosomes: aminoglycoside derivatives as promising therapeutics.

Leishmaniasis comprises an array of diseases caused by pathogenic species of Leishmania, resulting in a spectrum of mild to life-threatening pathologies. Currently available therapies for leishmaniasis include a limited selection of drugs. This coupled with the rather fast emergence of parasite resistance, presents a dire public health concern. Paromomycin (PAR), a broad-spectrum aminoglycoside antibiotic, has been shown in recent years to be highly efficient in treating visceral leishmaniasis (VL)-the life-threatening form of the disease. While much focus has been given to exploration of PAR activities in bacteria, its mechanism of action in Leishmania has received relatively little scrutiny and has yet to be fully deciphered. In the present study we present an X-ray structure of PAR bound to rRNA model mimicking its leishmanial binding target, the ribosomal A-site. We also evaluate PAR inhibitory actions on leishmanial growth and ribosome function, as well as effects on auditory sensory cells, by comparing several structurally related natural and synthetic aminoglycoside derivatives. The results provide insights into the structural elements important for aminoglycoside inhibitory activities and selectivity for leishmanial cytosolic ribosomes, highlighting a novel synthetic derivative, compound 3: , as a prospective therapeutic candidate for the treatment of VL.

E2F1 and E2F2 induction in response to DNA damage preserves genomic stability in neuronal cells.

E2F transcription factors regulate a wide range of biological processes, including the cellular response to DNA damage. In the present study, we examined whether E2F family members are transcriptionally induced following treatment with several genotoxic agents, and have a role on the cell DNA damage response. We show a novel mechanism, conserved among diverse species, in which E2F1 and E2F2, the latter specifically in neuronal cells, are transcriptionally induced after DNA damage. This upregulation leads to increased E2F1 and E2F2 protein levels as a consequence of de novo protein synthesis. Ectopic expression of these E2Fs in neuronal cells reduces the level of DNA damage following genotoxic treatment, while ablation of E2F1 and E2F2 leads to the accumulation of DNA lesions and increased apoptotic response. Cell viability and DNA repair capability in response to DNA damage induction are also reduced by the E2F1 and E2F2 deficiencies. Finally, E2F1 and E2F2 accumulate at sites of oxidative and UV-induced DNA damage, and interact with γH2AX DNA repair factor. As previously reported for E2F1, E2F2 promotes Rad51 foci formation, interacts with GCN5 acetyltransferase and induces histone acetylation following genotoxic insult. The results presented here unveil a new mechanism involving E2F1 and E2F2 in the maintenance of genomic stability in response to DNA damage in neuronal cells.

Green Tea Polyphenols Protects Cochlear Hair Cells from Ototoxicity by Inhibiting Notch Signalling.

Notch signalling pathway plays an essential role in the development of cochlea, which inhibits the proliferation of hair cells. Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol in green tea, which presents strong antioxidant activation and has been applied for anti-cancer and anti-inflammatory. In this study, we treated the cochlear explant cultures with EGCG and found that EGCG can protect cochlear hair cells from ototoxic drug gentamicin. We demonstrated that EGCG could down-regulate the expression of Notch signalling pathway target genes, such as Hes1, Hes5, Hey1 and Hey5. However, the Notch pathway ligands such as Delta1, Jag1 and Jag2 were not affected by EGCG. To further illustrate the mechanism of recover cochlear hair cells, we demonstrated that EGCG inhibited the activity of γ-secrectase to suppress Notch signalling pathway and promoted the proliferation and regeneration of hair cells in the damaged cochlea. Our results suggest for the first time the role of EGCG as an inhibitor of the Notch signalling pathway, and support its potential value in hearing-impaired recovery in clinical therapy.

Conjugation with polyamines enhances the antibacterial and anticancer activity of chloramphenicol.

Chloramphenicol (CAM) is a broad-spectrum antibiotic, limited to occasional only use in developed countries because of its potential toxicity. To explore the influence of polyamines on the uptake and activity of CAM into cells, a series of polyamine-CAM conjugates were synthesized. Both polyamine architecture and the position of CAM-scaffold substitution were crucial in augmenting the antibacterial and anticancer potency of the synthesized conjugates. Compounds 4 and 5, prepared by replacement of dichloro-acetyl group of CAM with succinic acid attached to N4 and N1 positions of N(8),N(8)-dibenzylspermidine, respectively, exhibited higher activity than CAM in inhibiting the puromycin reaction in a bacterial cell-free system. Kinetic and footprinting analysis revealed that whereas the CAM-scaffold preserved its role in competing with the binding of aminoacyl-tRNA 3'-terminus to ribosomal A-site, the polyamine-tail could interfere with the rotatory motion of aminoacyl-tRNA 3'-terminus toward the P-site. Compared to CAM, compounds 4 and 5 exhibited comparable or improved antibacterial activity, particularly against CAM-resistant strains. Compound 4 also possessed enhanced toxicity against human cancer cells, and lower toxicity against healthy human cells. Thus, the designed conjugates proved to be suitable tools in investigating the ribosomal catalytic center plasticity and some of them exhibited greater efficacy than CAM itself.

The fungal chimerolectin MOA inhibits protein and DNA synthesis in NIH/3T3 cells and may induce BAX-mediated apoptosis.

The Marasmius oreades mushroom agglutinin (MOA) is a blood group B-specific lectin carrying an active proteolytic domain. Its enzymatic activity has recently been shown to be critical for toxicity of MOA toward the fungivorous soil nematode Caenorhabditis elegans. Here we present evidence that MOA also induces cytotoxicity in a cellular model system (murine NIH/3T3 cells), by inhibiting protein synthesis, and that cytotoxicity correlates, at least in part, with proteolytic activity. A peptide-array screen identified the apoptosis mediator BAX as a potential proteolytic substrate and further suggests a variety of bacterial and fungal peptides as potential substrates. These findings are in line with the suggestion that MOA and related proteases may play a role for host defense.

Examination of calcium-binding protein expression in the inner ear of wild-type, heterozygous and homozygous pituitary adenylate cyclase-activating polypeptide (PACAP)-knockout mice in kanamycin-induced ototoxicity.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with diverse biological effects. It also occurs and exerts protective effects in sensory organs; however, little is known about its effects in the auditory system. Recently, we have shown that PACAP protects cochlear cells against oxidative-stress-induced apoptosis and homozygous PACAP-deficient animals show stronger expression of Ca(2+)-binding proteins in the hair cells of the inner ear, but there are no data about the consequences of the lack of endogenous PACAP in different ototoxic insults such as aminoglycoside-induced toxicity. In this study, we examined the effect of kanamycin treatment on Ca(2+)-binding protein expression in hair cells of wild-type, heterozygous and homozygous PACAP-deficient mice. We treated 5-day-old mice with kanamycin, and 2 days later, we examined the Ca(2+)-binding protein expression of the hair cells with immunohistochemistry. We found stronger expression of Ca(2+)-binding proteins in the hair cells of control heterozygous and homozygous PACAP-deficient mice compared with wild-type animals. Kanamycin induced a significant increase in Ca(2+)-binding protein expression in wild-type and heterozygous PACAP-deficient mice, but the baseline higher expression in homozygous PACAP-deficient mice did not show further changes after the treatment. Elevated endolymphatic Ca(2+) is deleterious for the cochlear function, against which the high concentration of Ca(2+)-buffers in hair cells may protect. Meanwhile, the increased immunoreactivity of Ca(2+)-binding proteins in the absence of PACAP provide further evidence for the important protective role of PACAP in ototoxicity, but further investigations are necessary to examine the exact role of endogenous PACAP in ototoxic insults.

Ribosome-inactivating proteins: potent poisons and molecular tools.

Ribosome-inactivating proteins (RIPs) were first isolated over a century ago and have been shown to be catalytic toxins that irreversibly inactivate protein synthesis. Elucidation of atomic structures and molecular mechanism has revealed these proteins to be a diverse group subdivided into two classes. RIPs have been shown to exhibit RNA N-glycosidase activity and depurinate the 28S rRNA of the eukaryotic 60S ribosomal subunit. In this review, we compare archetypal RIP family members with other potent toxins that abolish protein synthesis: the fungal ribotoxins which directly cleave the 28S rRNA and the newly discovered Burkholderia lethal factor 1 (BLF1). BLF1 presents additional challenges to the current classification system since, like the ribotoxins, it does not possess RNA N-glycosidase activity but does irreversibly inactivate ribosomes. We further discuss whether the RIP classification should be broadened to include toxins achieving irreversible ribosome inactivation with similar turnovers to RIPs, but through different enzymatic mechanisms.

Bax, Bcl2, and p53 differentially regulate neomycin- and gentamicin-induced hair cell death in the zebrafish lateral line.

Sensorineural hearing loss is a normal consequence of aging and results from a variety of extrinsic challenges such as excessive noise exposure and certain therapeutic drugs, including the aminoglycoside antibiotics. The proximal cause of hearing loss is often death of inner ear hair cells. The signaling pathways necessary for hair cell death are not fully understood and may be specific for each type of insult. In the lateral line, the closely related aminoglycoside antibiotics neomycin and gentamicin appear to kill hair cells by activating a partially overlapping suite of cell death pathways. The lateral line is a system of hair cell-containing sense organs found on the head and body of aquatic vertebrates. In the present study, we use a combination of pharmacologic and genetic manipulations to assess the contributions of p53, Bax, and Bcl2 in the death of zebrafish lateral line hair cells. Bax inhibition significantly protects hair cells from neomycin but not from gentamicin toxicity. Conversely, transgenic overexpression of Bcl2 attenuates hair cell death due to gentamicin but not neomycin, suggesting a complex interplay of pro-death and pro-survival proteins in drug-treated hair cells. p53 inhibition protects hair cells from damage due to either aminoglycoside, with more robust protection seen against gentamicin. Further experiments evaluating p53 suggest that inhibition of mitochondrial-specific p53 activity confers significant hair cell protection from either aminoglycoside. These results suggest a role for mitochondrial p53 activity in promoting hair cell death due to aminoglycosides, likely upstream of Bax and Bcl2.

A non-cytotoxic but ribonucleolytically specific ribotoxin variant: implication of tryptophan residues in the cytotoxicity of hirsutellin A.

Ribotoxins are a family of toxic proteins that exert a highly specific cleavage at the universally conserved sarcin/ricin loop (SRL) of the larger rRNA molecule. Before this ribonucleolytic action, passage through the cell membrane is a necessary step for ribotoxin internalization and the limiting factor for cytotoxicity. Although extensive knowledge of their ribonucleolytic activity and substrate recognition has been accumulated, little is known about the mechanisms of cell entry of ribotoxins. Hirsutellin A (HtA) is a recently described member of this family, which accommodates the main abilities of previously characterized ribotoxins into a shorter sequence, but exhibits some differences regarding membrane interaction properties. This work investigates the contribution of tryptophan (Trp) residues 71 and 78 to both endoribonucleolytic activity and cellular toxicity of this ribotoxin. Substitution mutants W71F and W78F, as well as the double mutant W71/78F, were obtained and assayed against isolated ribosomes, synthetic SRL, and human tumor cells. The results provide evidence that cell membrane passage and internalization, as well as substrate-specific recognition, require the participation of the region involving both Trp 71 and Trp 78. Additionally, the mutant W71/78F is the first non-cytotoxic but specific ribosome-cleaving ribotoxin mutant obtained to date.

Sustained Fos expression is observed in the developing brainstem auditory circuits of kanamycin-treated rats.

It has been demonstrated that kanamycin treatment during early developmental period induces partial cochlear destruction and enhanced glutamatergic transmission at the medial nucleus of the trapezoid body (MNTB) - the lateral superior olive (LSO) synapses in the superior olivary complex (SOC). As c-fos was expected to be expressed in the SOC by kanamycin-induced cochlear damage, the expression of c-fos protein (Fos) was investigated using immunohistochemistry in kanamycin-treated rat pups. In the control rat pups less than postnatal (P) day 9 in age, Fos-like immunoreactivity (Fos-IR) was transiently observed in the MNTB and LSO on P6, but disappeared on P9, which reflects a physiologic process. In contrast, in kanamycin-treated rats, Fos-IR was consistently observed through P9. Because a significant increase in terminal uridine deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick-end labeling (TUNEL) and glial fibrillary acidic protein (GFAP) IR was not demonstrated in the MNTB and LSO of kanamycin-treated rats, the increased Fos-IR does not appear to indicate an ongoing pathologic process, but may be related to the increased activity caused by the disturbance in excitatory and inhibitory balance between brainstem auditory circuits.