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We describe the structural and variable temperature magnetic susceptibility properties of an unusual homoleptic bimetallic iron(iii) thiocyanate tetraanion. This work represents the first structurally characterized bis(μ-1,3-thiocyanato) dimer of iron(iii). A weak antiferromagnetic exchange interaction is observed between the two iron(iii) ions, which is supported by broken symmetry density functional theory (DFT) calculations. [ABSTRACT FROM AUTHOR]

High levels of BCL-2 family proteins are implicated in a failed/ineffective apoptotic programme, often resulting in diseases, including cancer. Owing to their potential as drug targets in cancer therapy, several inhibitors of BCL-2 family proteins have been developed. These primarily target specific members of the BCL-2 family, particularly BCL-2 and BCL-XL but are ineffective against MCL-1. Major efforts have been invested in developing inhibitors of MCL-1, which is commonly amplified in human tumours and associated with tumour relapse and chemoresistance. In this report, the specificity of several BCL-2 family inhibitors (ABT-263, UCB-1350883, apogossypol and BH3I-1) was investigated and compared with putative MCL-1 inhibitors designed to exhibit improved or selective binding affinities for MCL-1 (TW-37, BI97C1, BI97C10, BI112D1, compounds 6 and 7, and MCL-1 inhibitor molecule (MIM-1)). ABT-263, BI97C1, BI112D1, MIM-1 and TW-37 exhibited specificity in inducing apoptosis in a Bax/Bak- and caspase-9-dependent manner, whereas the other agents showed no killing activity, or little or no specificity. Of these inhibitors, only ABT-263 and UCB-1350883 induced apoptosis in a BCL-2- or BCL-XL-dependent system. In cells that depend on MCL-1 for survival, ABT-263 and TW-37 induced extensive apoptosis, suggesting that at high concentrations these inhibitors have the propensity to inhibit MCL-1 in a cellular context. TW-37 induced apoptosis, assessed by chromatin condensation, caspase processing and phosphatidylserine externalisation, in a BAK-dependent manner and in cells that require MCL-1 for survival. TW-37-mediated apoptosis was also partly dependent on NOXA, suggesting that derivatives of TW-37, if engineered to exhibit better selectivity and efficacy at low nanomolar concentrations, may provide useful lead compounds for further synthetic programmes. Expanded medicinal chemistry iteration, as performed for the ABT series, may likewise improve the potency and specificity of the evaluated MCL-1 inhibitors. [ABSTRACT FROM AUTHOR]

Canonical endoplasmic reticulum (ER) stress, which occurs in many physiological and disease processes, results in activation of the unfolded protein response (UPR). We now describe a new, evolutionarily conserved cellular stress response characterised by a striking, but reversible, reorganisation of ER membranes that occurs independently of the UPR, resulting in impaired ER transport and function. This reorganisation is characterised by a dramatic redistribution and clustering of ER membrane proteins. ER membrane aggregation is regulated, in part, by anti-apoptotic BCL-2 family members, particularly MCL-1. Using connectivity mapping, we report the widespread occurrence of this stress response by identifying several structurally diverse chemicals from different pharmacological classes, including antihistamines, antimalarials and antipsychotics, which induce ER membrane reorganisation. Furthermore, we demonstrate the potential of ER membrane aggregation to result in pathological consequences, such as the long-QT syndrome, a cardiac arrhythmic abnormality, arising because of a novel trafficking defect of the human ether-a-go-go-related channel protein from the ER to the plasma membrane. Thus, ER membrane reorganisation is a feature of a new cellular stress pathway, clearly distinct from the UPR, with important consequences affecting the normal functioning of the ER. [ABSTRACT FROM AUTHOR]

Protein misfolding has a key role in several neurological disorders including Parkinson's disease. Although a clear mechanism for such proteinopathic diseases is well established when aggregated proteins accumulate in the cytosol, cell nucleus, endoplasmic reticulum and extracellular space, little is known about the role of protein aggregation in the mitochondria. Here we show that mutations in both human and fly PINK1 result in higher levels of misfolded components of respiratory complexes and increase in markers of the mitochondrial unfolded protein response. Through the development of a genetic model of mitochondrial protein misfolding employing Drosophila melanogaster, we show that the in vivo accumulation of an unfolded protein in mitochondria results in the activation of AMP-activated protein kinase-dependent autophagy and phenocopies of pink1 and parkin mutants. Parkin expression acts to clear mitochondria with enhanced levels of misfolded proteins by promoting their autophagic degradation in vivo, and refractory to Sigma P (ref(2)P), the Drosophila orthologue of mammalian p62, is a critical downstream effector of this quality control pathway. We show that in flies, a pathway involving pink1, parkin and ref(2)P has a role in the maintenance of a viable pool of cellular mitochondria by promoting organellar quality control. [ABSTRACT FROM AUTHOR]

Cell death requires coordinated intracellular signalling before disassembly of cell architecture by degradative enzymes. Although the death signalling cascades that involve the mitochondria, the ER and the plasma membrane have been extensively characterized, only a handful of studies have examined the functional and structural alterations of the nuclear pore complex (NPC) during neuronal death. Here, we show that during excitotoxic neuronal degeneration calpains redistributed across the nuclear envelope and mediated the degradation of NPC components causing altered permeability of the nuclear membrane. In primary dissociated neurons, simultaneous recording of cytosolic [Ca2+] and localization of fluorescent proteins showed that the onset of Ca2+ overload signalled a progressive increase in the diffusion of small reporter molecules across the nuclear envelope. Later, calpain-mediated changes in nuclear pore permeability allowed accumulation of large proteins in the nucleus. Further, in a model of excitotoxic neuronal degeneration in Caenorhabditis elegans, we found similar nuclear changes and redistribution of fluorescent probes across the nuclear membrane in dying neurons. Our findings strongly suggest that increased leakiness of the nuclear barrier affects nucleocytoplasmic transport, alters the localization of proteins across the nuclear envelope and it is likely to be involved in Ca2+-dependent cell death, including ischemic neuronal demise. [ABSTRACT FROM AUTHOR]

Several inhibitors of BCL2 proteins have been identified that induce apoptosis in a variety of tumor cells, indicating their potential in cancer therapy. We investigated the specificity of six putative BCL2 inhibitors (obatoclax, gossypol, apogossypol, EM20-25, chelerythrine and ABT-737). Using cells deficient either for Bax/Bak or caspase-9, we found that only ABT-737 specifically targeted BCL2 proteins and induced apoptosis by activation of caspase-9, as only ABT-737 induced apoptosis was completely inhibited in cells deficient for Bax/Bak or caspase-9. Our data show that only ABT-737 is a specific BCL2 inhibitor and all other compounds investigated were not specific for BCL2 proteins. Furthermore, investigations of the effects of these compounds in primary chronic lymphocytic leukemic cells showed that all compounds induced certain biochemical hallmarks of apoptosis, such as release of cytochrome c and caspase cleavage. However, they all caused strikingly different ultrastructural changes. ABT-737 induced all the characteristic ultrastructural changes of apoptosis together with early rupture of the outer mitochondrial membrane, whereas obatoclax, chlelerythrine and gossypol induced pronounced mitochondrial swelling with formation of phospholipid inclusions. Therefore, we conclude that biochemical measurements used earlier to define apoptosis like mitochondrial release of cytochrome c and caspase cleavage, are insufficient to distinguish between classic apoptosis and other forms of cell death.Cell Death and Differentiation (2009) 16, 1030–1039; doi:10.1038/cdd.2009.48; published online 24 April 2009 [ABSTRACT FROM AUTHOR]

Despite tremendous advances over the last 15 years in understanding fundamental mechanisms of apoptosis, this has failed to translate into improved cancer therapy for patients. However, there may now be light at the end of this long tunnel. Antiapoptotic Bcl-2 family members may be divided into two subclasses, one comprising Bcl-2, Bcl-XL and Bcl-w and the other Mcl-1 and Bcl2A1. Neutralization of both subclasses is required for apoptosis induction. Solution of the structure of antiapoptotic Bcl-2 family proteins has led to the design of novel small molecule inhibitors. Although many such molecules have been synthesized, rigorous verification of their specificity has often been lacking. Further studies have revealed that many putative Bcl-2 inhibitors are not specific and have other cellular targets, resulting in non-mechanism based toxicity. Two notable exceptions are ABT-737 and a related orally active derivative, ABT-263, which bind with high affinity to Bcl-2, Bcl-XL and Bcl-w and may prove to be useful tools for mechanistic studies. ABT-263 is in early clinical trials in lymphoid malignancies, small-cell lung cancer and chronic lymphocytic leukemia, and some patients have shown promising results. In in vitro studies, primary cells from patients with various B-cell malignancies are exquisitely sensitive to ABT-737, exhibiting novel morphological features of apoptosis including marked outer mitochondrial membrane rupture.Cell Death and Differentiation (2009) 16, 360–367; doi:10.1038/cdd.2008.137; published online 19 September 2008 [ABSTRACT FROM AUTHOR]

Primary chronic lymphocytic leukemia (CLL) cells are exquisitely sensitive to ABT-737, a small molecule BCL2-antagonist, which induces many of the classical biochemical and ultrastructural features of apoptosis, including BAX/BAK oligomerization, cytochrome c release, caspase activation and chromatin condensation. Surprisingly, ABT-737 also induces mitochondrial inner membrane permeabilization (MIMP) resulting in mitochondrial matrix swelling and rupture of the outer mitochondrial membrane (OMM), so permitting the rapid efflux of cytochrome c from mitochondrial cristae and facilitating rapid caspase activation and apoptosis. BAX and BAK appear to be involved in the OMM discontinuities as they localize to the OMM break points. Notably, ABT-737 induced mitochondrial matrix swelling and OMM discontinuities in other primary B-cell malignancies, including mantle cell, follicular and marginal zone lymphoma cells but not in several cell lines studied. Thus, we describe a new paradigm of apoptosis in primary B-cell malignancies, whereby targeting of BCL2 results in all the classical features of apoptosis together with OMM rupture independent of caspase activation. This mechanism may be far more prevalent than hitherto recognized due to the failure of most methods, used to measure apoptosis, to recognize such a mechanism.Cell Death and Differentiation (2008) 15, 820–830; doi:10.1038/cdd.2008.25; published online 29 February 2008 [ABSTRACT FROM AUTHOR]

Epidermal development requires the transcription factor p63, as p63−/− mice are born dead, without skin. The gene expresses two proteins, one with an amino-terminal transactivation domain (TAp63) and one without (ΔNp63), although their relative contribution to epidermal development is unknown. To address this issue, we reintroduced TAp63α and/or ΔNp63α under the K5 promoter into p63−/− mice by in vivo genetic complementation. Whereas p63−/− and p63−/−;TA mice showed extremely rare patches of poorly differentiated keratinocytes, p63−/−;ΔN mice showed significant epidermal basal layer formation. Double TAp63α/ΔNp63α complementation showed greater patches of differentiated skin; at the ultrastructural level, there was clear reformation of a distinct basal membrane and hemidesmosomes. At the molecular level, ΔNp63 regulated expression of genes characteristic of the basal layer (K14), interacting (by Chip, luc assay) with the third p53 consensus site. Conversely, TAp63 transcribed the upper layer's genes (Ets-1, K1, transglutaminases, involucrin). Therefore, the two p63 isoforms appear to play distinct cooperative roles in epidermal formation.Cell Death and Differentiation (2006) 13, 1037–1047. doi:10.1038/sj.cdd.4401926; published online 7 April 2006 [ABSTRACT FROM AUTHOR]

We present the case of a woman who developed respiratory failure in the postoperative period secondary to previously unsuspected motor neurone disease. This case highlights the difficulty in detecting subtle neuromuscular weakness during anaesthetic pre-assessment. [ABSTRACT FROM AUTHOR]

Chromium-based catalysts are used for the synthesis of polyethylene, but little is known about the hazard and biomonitoring possibilities of this type of chromium for workers who may be occupationally exposed to such compounds. Therefore, the bioavailability and toxicokinetics of chromium were studied in male Wistar rats after a single intratracheal instillation (2 ml/kg body weight) of various doses (1, 5, or 25 mg/kg body weight) of the catalyst (approximately 1% chromium bound to an amorphous silica matrix), either before (CAT-Cr[III]) or after (CATCr[VI]) heat treatment. The results were compared with those of equivalent amounts of two chromium salts (CrCl[sub 3] and K[sub 2]Cr[sub 2]O[sub 7]). Each dose group was composed of three rats. The concentration of chromium was determined by atomic absorption spectrometry in urine (collected daily for 7 d) and in plasma, erythrocytes, lung, and liver tissue obtained 2 d (only highest concentration) and 7 d after dosing. On d 2, a significant increase in lung weight was found in the animals treated with the highest dose of the hexavalent Cr products. On d 7, on the basis of body weights, lung weights, and lung histology, there was no overt toxicity, except after the highest dose of CAT-Cr(VI). The elimination of all forms of chromium was apparently monoexponential, with calculated half-life elimination times in urine of 4-11 h for Cr(III) (CAT-Cr[III] and CrCI[sub 3]) and 8-21 h for Cr(VI) (CAT-Cr[VI] and K[sub 2]Cr[sub 2]O[sub 7]). On d 2, the erythrocytes Cr concentrations were significantly higher for the hexavalent Cr products than for the trivalent Cr products. After 7 d, the erythrocytes Cr concentrations were significantly increased above control values (3 µg/L) only in rats treated with the 2 highest doses of Cr(VI) compounds (12 and 64 µg/L for K[sub 2]Cr[sub 2]O[sub 7], and 14 and 79 µg/L for CAT-Cr[VI]). The present study shows that intratracheally instilled Cr(VI) and Cr(III) have... [ABSTRACT FROM AUTHOR]

Proteasome inhibitors, including lactacystin and MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal), potently induce apoptosis in leukaemic B cells from patients with B cell chronic lymphocytic leukaemia (B-CLL). This pro-apoptotic effect occurs in cells from patients at all stages of the disease, including those resistant to conventional chemotherapy, suggesting that proteasome inhibitors may be useful for treatment of B-CLL. Following initial inhibition of proteasomal activity, these agents induce mitochondrial cytochrome c release and caspase-dependent apoptosis, involving cleavage/activation of caspases -2, -3, -7, -8 and -9. Pre-treatment with the cell permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe)fluoromethyl ketone (Z-VAD.fmk), did not prevent the release of cytochrome c or partial processing of caspase-9 but prevented activation of effector caspases and the induction of apoptosis. These results suggest that the release of cytochrome c is caspase independent and that caspase-9 is the initiator caspase in proteasome inhibitor-induced apoptosis of B-CLL cells. Activation of B-CLL lysates with dATP results in the formation of an approximately 700 kDa caspase-activating apoptosome complex containing Apaf-1. We describe for the first time the formation of a similar approximately 700 kDa caspase-activating apoptosome complex in B-CLL cells induced to undergo apoptosis by proteasome inhibitors. [ABSTRACT FROM AUTHOR]

Repeated oral administration of chemopreventive retinoids such as isotretinoin over extended periods of time is associated with intolerable systemic toxicity. Here isotretinoin was formulated as a powder aerosol, and its delivery to the lungs of rats was studied with the aim to explore the possibility of minimizing adverse effects associated with its oral administration. Rats received isotretinoin orally (0.5, 1 or 10 mg kg[SUP-1]) or by inhalation (theoretical dose ∼1 or ∼10 mg kg[SUP-1]) in a nose-only inhalation chamber. Isotretinoin was quantitated by high-pressure liquid chromatography in plasma and lung tissue. The ratios of mean area of concentration-vs-time curve (AUC) values in the lungs over mean AUCs in the plasma for isotretinoin following single or repeated aerosol exposure surpassed those determined for the oral route by factors of between two (single low-dose) and five (single high-dose). Similarly, the equivalent ratios for the maximal peak concentrations in lungs and plasma obtained after aerosol exposure consistently exceeded those seen after oral administration, suggesting that lungs were exposed to higher isotretinoin concentrations after aerosol inhalation than after oral administration of similar doses. Repeated high doses of isotretinoin by inhalation resulted in moderate loss of body weight, but microscopic investigation of ten tissues including lung and oesophagus did not detect any significant aerosol-induced damage. The results suggest that administration of isotretinoin via powder aerosol inhalation is probably superior to its application via the oral route in terms of achieving efficacious drug concentrations in the lungs. [ABSTRACT FROM AUTHOR]

Some Aspects of the Toxicology of Trimethyl and Triethyl Phosphorothioates. ALDRIDGE, W. N., DINSDALE, D., NEMERY, B., AND VERSCHOYLE, R. D. (1985). 5, S47–S60. The pharmacokinetics (disposal curves) of trimethyl and triethyl phosphorothioates have been determined. The concentrations to which the lung has been exposed at the LD50 dose of different chemical structures have been compared with the dose administered to the animal; the variation of LD50 of different chemical structures is little reduced. The kinetics of the reaction of -trimethyl phosphorodithioate or -triethyl phosphorothioate with plasma cholinesterase and carboxylesterase and brain acetylcholinesterase have been determined. The relation between inhibition and circulating concentrations have been examined. Changes in Clara cells reported by others seem to be physiological rather than pathological. -Trimethyl phosphorodithioate is metabolised by rat lung and liver slices and microsomes. From these studies and the effect of various pretreatments of the rats on toxicity to the lung, it is probable that the proximal toxin is produced in the lung by oxidative attack on the alkylthio moeity of the compounds. [ABSTRACT FROM PUBLISHER]

1. Selective induction and inhibition experiments have been used to identify the cytochrome P450 (CYP) isoforms responsible for butylated hydroxytoluene (BHT) bioactivation in mouse lung. 2. Pre-treatment of BALB c mice with O , O , O -trimethylphosphorothioate (OOOMeP(S)), which prevented all the signs of toxicity observed following BHT treatment,inhibitedthe pulmonary activity of pentoxyresorufin O -dealkylase (PROD) and coumarin hydroxylase but not 4-nitrophenol hydroxylase. 3. Pulmonary coumarin hydroxylase activity was greater inDBA thanin BALB c mice but the severity of BHT-induced lung injury was similar. 4. Pre-treatment with pyrazole, which exacerbated BHT-induced lung injury, did not affect pulmonary coumarin hydroxylase or 4-nitrophenol hydroxylase activity but increased that of PROD. 5. Pre-treatment with OOOMeP(S) prevented the lethargy and weight-loss associated with naphthalene poisoning but not the pulmonary injury.Pre-treatment with pyrazole did not exacerbate naphthalene-induced injury. 6. Members of both CYP2F and 2B sub-families have been shown to exhibit PROD activity and 2F2 activates naphthalene in mouse lung. The current studies, however, indicate that 2F2 is unlikely to be a significant component of PROD activity in mouse lung. 2F2, like coumarin hydroxylase (2A5) and 4-nitrophenol hydroxylase (2E1), is not responsible for the pulmonary activation of BHT, which is largely attributable to an isoform of 2B, probably 2B10. [ABSTRACT FROM AUTHOR]

Background: The polyamines (putrescine, spermidine, and spermine) are involved in cellular growth, proliferation, and differentiation. In the lungs of various species, polyamines are accumulated by an active uptake system which also mediates the uptake of cystamine and paraquat. In the rat lung putrescine uptake has been shown to be cell-specific, occurring predominantly in the alveolar epithelium. The aim of this study was to characterise the uptake of putrescine in human lung.Methods: Lung tissue was obtained from 31 patients undergoing surgery for lung cancer. Slices (0.7 mm thick) from non-tumour containing lung parenchyma were incubated for 15-60 minutes in Krebs-Ringer phosphate buffer with various concentrations of putrescine (2.5 to 80 mumol/l) containing 0.1 microCi [1,4-14C]-putrescine. Uptake was assessed from tissue radioactivity. For autoradiographic imaging, slices were incubated for 30 minutes with 2.5 mumol/l putrescine containing 2.5 mCi [1,4n-3H]-putrescine.Results: The accumulation of [14C]-putrescine into slices was time-dependent and energy-dependent, and obeyed saturation kinetics, with mean calculated values for Vmax (maximal rate of uptake) of 414 nmol/g/hour and for Km (medium concentration at which the rate of uptake is half Vmax) of 7.2 mumol/l, with a large interindividual variation. Competitive inhibition was observed on incubation with cystamine, which appears to have a high affinity for the uptake system since its calculated Ki (concentration of inhibitor at which the Km is doubled) was 3.2 mumol/l. Ultrastructural autoradiography showed labelling over both type I and type II cells of the alveolar epithelium, but not over the endothelium or any cells of the interstitium. Alveolar macrophages were also devoid of label.Conclusions: These results show that the human lung possesses an active uptake system for putrescine, and probably also cystamine, which is located in both cell types of the alveolar epithelium. These findings may be used to develop tests for the assessment of the alveolar epithelium. [ABSTRACT FROM AUTHOR]