Idiosyncratic reactions and metabolism of sulfur-containing drugs.

INTRODUCTION: Idiosyncratic drug reactions (IDRs) that involve the formation of toxic metabolites followed by covalent binding to cellular proteins often go undiscovered until after post-marketing. The goal of this article is to review the current status of IDRs, potential mechanisms and the challenges associated with predicting drug toxicity. AREAS COVERED: The authors review the metabolic pathways of five select classes of sulfur-containing drugs (captopril, troglitazone, tienilic acid, zileuton, methimazole and sudoxicam) suggesting that bioactivation plays a crucial role in the occurrence of IDRs. The reader will gain further awareness that the sulfur atom can propagate as the bioactivation site for the formation of reactive and conceivably toxic metabolites. As such, it is the body's capacity to detoxify these drug products that may determine whether IDRs occur. EXPERT OPINION: Incomplete understanding of mechanisms culminating in IDR occurrence represents a monumental impediment toward their abrogation. Moreover, current technology utilized to predict their manifestation (including structure-toxicity relationships) is not infallible and thus, development of novel tools and strategies is indispensible. In an attempt to streamline clinical development and drug approval processes, consortiums have been instated under the US FDA Critical Path Initiative. Collectively, these parameters along with the availability of validated biomarkers and new/updated regulatory guidance could positively influence the outcome of drug toxicity profiles and direct future drug development.

TMEM237 is mutated in individuals with a Joubert syndrome related disorder and expands the role of the TMEM family at the ciliary transition zone.

Joubert syndrome related disorders (JSRDs) have broad but variable phenotypic overlap with other ciliopathies. The molecular etiology of this overlap is unclear but probably arises from disrupting common functional module components within primary cilia. To identify additional module elements associated with JSRDs, we performed homozygosity mapping followed by next-generation sequencing (NGS) and uncovered mutations in TMEM237 (previously known as ALS2CR4). We show that loss of the mammalian TMEM237, which localizes to the ciliary transition zone (TZ), results in defective ciliogenesis and deregulation of Wnt signaling. Furthermore, disruption of Danio rerio (zebrafish) tmem237 expression produces gastrulation defects consistent with ciliary dysfunction, and Caenorhabditis elegans jbts-14 genetically interacts with nphp-4, encoding another TZ protein, to control basal body-TZ anchoring to the membrane and ciliogenesis. Both mammalian and C. elegans TMEM237/JBTS-14 require RPGRIP1L/MKS5 for proper TZ localization, and we demonstrate additional functional interactions between C. elegans JBTS-14 and MKS-2/TMEM216, MKSR-1/B9D1, and MKSR-2/B9D2. Collectively, our findings integrate TMEM237/JBTS-14 in a complex interaction network of TZ-associated proteins and reveal a growing contribution of a TZ functional module to the spectrum of ciliopathy phenotypes.

Deciphering the structure and function of Als2cr4 in the mouse retina.

PURPOSE: The role of Als2cr4 (amyotrophic lateral sclerosis 2 [juvenile] chromosome region, candidate 4; also known as hypothetical protein FLJ33282) in the mouse retina was determined by characterizing the molecular structure, cellular interacting partners, and potential biochemical functions. Previous in situ hybridization and gene expression profiles show that the mRNAs encoding Als2cr4 are abundant in the eye, hippocampus, cerebellum, and olfactory bulb. METHODS: From predicted antigenic epitopes of Als2cr4, two novel antibodies were developed to examine protein expression and morphologic localization in retinas from light-adapted and dark-adapted mice by immunohistochemistry, immunoblot analysis, and immunoelectron microscopy, and then immunoprecipitation was performed to identify interacting proteins by mass spectroscopy. RESULTS: Peptide antibodies with Als2cr4 antigenic epitopes from either the amino- or carboxyl terminus were characterized with Als2cr4 recombinant proteins and peptide competition assays. Als2cr4 is a 45-kDa insoluble protein, highly enriched in retina, and localizes to photoreceptor outer segments, ciliary complex, and horizontal cells in the outer plexiform layer. Immunoelectron microscopy for Als2cr4 verified its expression in the discs of photoreceptor outer segments. Immunoprecipitation and mass spectroscopy identified eight potential interacting partners: vimentin, actin, myosin Va, myosin VI, myosin X, myosin XIV, kinesin 1, Als2cr4, and lamin B-1. CONCLUSIONS: Als2cr4 is a novel protein, with a probable tetraspanin-like membrane structure, that is localized in photoreceptors and in the postsynaptic outer plexiform layer and that interacts with cytoskeletal proteins. Als2cr4 may be involved in membrane transport between the photoreceptor inner and outer segments and may be a key component in maintaining the structural integrity of the outer segment.

Mouse cones require an arrestin for normal inactivation of phototransduction.

Arrestins are proteins that arrest the activity of G protein-coupled receptors (GPCRs). While it is well established that normal inactivation of photoexcited rhodopsin, the GPCR of rod phototransduction, requires arrestin (Arr1), it has been controversial whether the same requirement holds for cone opsin inactivation. Mouse cone photoreceptors express two distinct visual arrestins: Arr1 and Arr4. By means of recordings from cones of mice with one or both arrestins knocked out, this investigation establishes that a visual arrestin is required for normal cone inactivation. Arrestin-independent inactivation is 70-fold more rapid in cones than in rods, however. Dual arrestin expression in cones could be a holdover from ancient genome duplication events that led to multiple isoforms of arrestin, allowing evolutionary specialization of one form while the other maintains the basic function.

Antisense and short hairpin RNA (shRNA) constructs targeting PIN (Protein Inhibitor of NOS) ameliorate aging-related erectile dysfunction in the rat.

INTRODUCTION: Over-expression of penile neuronal nitric oxide synthase (PnNOS) from a plasmid ameliorates aging-related erectile dysfunction (ED), whereas over-expression of the protein inhibitor of NOS (PIN), that binds to nNOS, increases ED. AIM: To improve this form of gene therapy for ED by comparing the electrical field response of short hairpin RNA (shRNA) for PIN with that of antisense PIN RNA. MAIN OUTCOME MEASURE: Both shRNA and antisense RNA gene therapy vectors increased intracavernosal pressure in aged rats. METHODS: PIN small interfering RNA (siRNA), and plasmid constructs for cytomegalovirus promoter plasmid vector (pCMV-PIN), pCMV-PIN antisense RNA, pSilencer2.1-U6-PIN-shRNA; and pSilencer2.1-U6-randomer-shRNA were prepared and validated by transfection into HEK293 cells, determining the effects on PIN expression by Western blot. Plasmid constructs were then injected, followed by electroporation, into the penile corpora cavernosa of aged (20-month-old) Fisher 344 rats and, 1 month later, the erectile response was measured by intracavernosal pressure increase following electrical field stimulation (EFS) of the cavernosal nerve. PIN was estimated in penile tissue by Western blot and real-time reverse transcriptase-polymerase chain reaction. Cyclic guanosine monophosphate (cGMP) measurements were conducted by competitive enzyme immunoassay (EIA). Immunohistofluorescence detected PIN in corporal tissue sections. RESULTS: In cell culture, PIN siRNA and plasmid-expressed pU6-PIN-shRNA effectively reduced PIN expression from pCMV-PIN. pSilencer2.1-U6-PIN-shRNA corrected the impaired erectile response to EFS in aged rats and raised it above the value for young rats, more efficiently than pCMV-PIN antisense RNA. PIN mRNA expression in the penis was decreased by >70% by the shRNA but remained unaffected by the antisense RNA, whereas PIN protein expression was reduced in both cases, particularly in the dorsal nerve. PIN antisense increased cGMP concentration in treated tissue by twofold. CONCLUSION: pSilencer2.1-U6-PIN-shRNA gene therapy was more effective than the antisense PIN mRNA in ameliorating ED in the aged rat, thereby suggesting that PIN is indeed a physiological inhibitor of nNOS and nitrergic neurotransmission in the penis.

Myostatin short interfering hairpin RNA gene transfer increases skeletal muscle mass.

BACKGROUND: Myostatin negatively regulates skeletal muscle growth. Myostatin knockout mice exhibit muscle hypertrophy and decreased interstitial fibrosis. We investigated whether a plasmid expressing a short hairpin interfering RNA (shRNA) against myostatin and transduced using electroporation would increase local skeletal muscle mass. METHODS: Short interfering RNAs (siRNAs) targeting myostatin were co-transfected with a myostatin-expressing plasmid into HEK293 cells and identified for myostatin silencing by Western blot. Corresponding shRNAs were cloned into plasmid shRNA expression vectors. Myostatin or a randomer negative control shRNA plasmid was injected and electroporated into the tibialis anterior or its contralateral muscle, respectively, of nine rats that were sacrificed after 2 weeks. Six other rats received a beta-galactosidase reporter plasmid and were sacrificed at 1, 2, and 4 weeks. Uptake of plasmid was examined by beta-galactosidase expression, whereas myostatin expression was determined by real-time polymerase chain reaction (PCR) and Western blotting. Muscle fiber size was determined by histochemistry. Satellite cell proliferation was determined by PAX7 immunohistochemistry. Myosin heavy chain type II (MHCII) expression was determined by Western blot. RESULTS: beta-Galactosidase reporter plasmid was expressed at 1 and 2 weeks but diminished by 4 weeks in tibialis anterior skeletal muscle. Myostatin shRNA reduced myostatin mRNA and protein expression by 27 and 48%, respectively. Tibialis anterior weight, fiber size, and MHCII increased by 10, 34, and 38%, respectively. Satellite cell number was increased by over 2-fold. CONCLUSIONS: This is the first demonstration that myostatin shRNA gene transfer is a potential strategy to increase muscle mass.

Rho GTPases regulate rhabdom morphology in octopus photoreceptors.

In the cephalopod retina, light/dark adaptation is accompanied by a decrease/increase in rhabdom size and redistribution of rhodopsin and retinochrome. Rearrangements in the actin cytoskeleton probably govern changes in rhabdom size by regulating the degradation/formation of rhabdomere microvilli. Photopigment movements may be directed by microtubules present in the outer segment core cytoplasm. We believe that rhodopsin activation by light stimulates Rho and Rac signaling pathways, affecting these cytoskeletal systems and their possible functions in controlling rhabdom morphology and protein movements. In this study, we localized cytoskeletal and signaling proteins in octopus photoreceptors to determine their concurrence between the lighting conditions. We used toxin B from Clostridium difficile to inhibit the activity of Rho/Rac and observed its effect on the location of signaling proteins and actin and tubulin. In both lighting conditions, we found Rho in specific sets of juxtaposed rhabdomeres in embryonic and adult retinas. In the light, Rho and actin were localized along the length of the rhabdomere, but, in the dark, both proteins were absent from a space beneath the inner limiting membrane. Rac colocalized with tubulin in the outer segment core cytoplasm and, like Rho, the two proteins were also absent beneath the inner limiting membrane in the dark. The distribution of actin and Rho was affected by toxin B and, in dark-adapted retinas, actin and Rho distribution was similar to that observed in the light. Our results suggest that the Rho/Rac GTPases are candidates for the regulation of rhabdomere size and protein movements in light-dark-adapted octopus photoreceptors.

Peyronie's disease associated with increase in plasminogen activator inhibitor in fibrotic plaque.

OBJECTIVES: To investigate whether tissue expression of plasminogen activator inhibitor type 1 (PAI-1) is increased in the fibrotic plaque of human Peyronie's disease (PD). Increased tissue levels of PAI-1, an inhibitor of both fibrinolysis and collagenolysis, have been found in a variety of fibrotic conditions. Recently, it was reported that PAI-1 expression was also increased in the fibrotic plaque of an animal model of PD induced by the injection of fibrin into the tunica albuginea (TA) of the penis. METHODS: Tissue (n = 10/group) and cells (n = 4/group) obtained from the penile TA plaque of patients with PD or from normal TA were subjected to RNA extraction and real-time reverse transcriptase-polymerase chain reaction. Tissues were also analyzed by immunohistochemistry (n = 8/group) for the detection of PAI-1 expression at the transcription and protein levels. RESULTS: A significant 3.5-fold to 16-fold increase was found in both PAI-1 mRNA and protein levels in the human PD plaque and the respective fibroblast cultures compared with the normal non-PD TA. CONCLUSIONS: The observed increase in PAI-1 in the human PD plaque agrees with what has been observed in the rat and suggests that PAI-1 may be a key pro-fibrotic factor in the development of human PD.