Role of T helper 17 cells and T regulatory cells in alopecia areata: comparison of lesion and serum cytokine between controls and patients.

BACKGROUND: Alopecia areata (AA) is an organ-specific autoimmune disease with T-cell-mediated attack of hair follicle autoantigens. As T helper 17 (Th17) cells and T regulatory (Treg) cells are crucially involved in the pathogenesis, the role of Th17 and Treg cytokines has not been studied yet. OBJECTIVE: To determine whether AA is associated with alterations in lesional and serum Th17 and Treg cytokines and studied whether they were associated with clinical type. METHODS: Scalp skin samples from 45 patients and eight normal controls were obtained for PCR specific for IFN-γ, TNF-α, TGF-ß, IL-1, IL-2, IL-4, IL-10, IL-12A, IL-13, IL-17, IL-22 and IL-23. Serum cytokines were measured from 55 patients and 15 normal controls using ELISA. RESULTS: Lesional IL-17 and IL-22 were significantly increased in patient group. Moreover, positive correlations were shown between lesional IL-17, IL-22 and disease severity. Serum IL-1, IL-17, TNF-α and TGF-ß were significantly increased, and positive correlation was shown between serum IL-17 and disease severity. CONCLUSION: These results showed significantly high Th17 cytokines in both lesion and serum in AA patients, which may highlight a functional role of these cytokines in the pathogenesis of AA.

Identification of new kinase clusters required for neurite outgrowth and retraction by a loss-of-function RNA interference screen.

Disruption of synaptic integrity, loss of connectivity and axodendritic degeneration are early and essential components of neurodegeneration. Although neuronal cell death mechanisms have been thoroughly investigated, less is known about the signals involved in axodendritic damage and the processes involved in regeneration. Here we conducted a genome-wide RNA interference-based forward genetic screen, using small interfering RNA targeting all human kinases, and identified clusters of kinases families essential for growth cone collapse, neurite retraction and neurite outgrowth. Of 59 kinases identified as positive regulators of neurite outgrowth, almost 50% were in the tyrosine kinase/tyrosine kinase-like (TK/TKL) receptor subgroups, underlining the importance of extracellular ligands in this process. Neurite outgrowth was inhibited by 66 other kinases, none of which were TK/TKL members, whereas 79 kinases inhibited lysophosphatidic acid-induced neurite retraction. Twenty kinases were involved in both inhibitory processes suggesting shared mechanisms. Within this group of 20 kinases, some (ULK1, PDK1, MAP4K4) have been implicated previously in axonal events, but others (MAST2, FASTK, CKM and DGUOK) have not. For a subset of kinases, the effect on neurite outgrowth was validated in rat primary cerebellar cultures. The ability to affect regeneration was further tested in a model of axodendritic lesion using primary rat midbrain cultures. Finally, we demonstrated that haploinsufficiency of two members of the AGC kinase subgroup, ROCK1 and PKN1, was able to suppress retinal degeneration in Drosophila model of class III Autosomal Dominant Retinitis Pigmentosa.

Formulation of chelating agent with surfactant in cloud point extraction of methylphenol in water.

Cloud point extraction (CPE) is a separation and preconcentration of non-ionic surfactant from one liquid phase to another. In this study, Sylgard 309 and three different types of additives for CPE, namely CPE-Sylgard, CPE-Sylgard-BMIMBr and CPE-Sylgard-GLDA, are investigated to extract methylphenol from water samples. The methylphenols are well separated by reversed-phase high-performance liquid chromatography (HPLC) with isocratic elution of acetonitrile : water; 60 : 40 (v/v) and detection at 260 nm. The optimized parameters for the effect of salt, surfactant, temperature, time of extraction, pH, interference study and the performance of different additives on methylphenol extraction are investigated. CPE-Sylgard-GLDA is chosen because it gives us a high peak and good peak area compared with CPE-Sylgard and CPE-Sylgard-BMIMBr. The recovery extractions of CPE-Sylgard-GLDA are obtained in the range of 80-99% as the percentage of relative standard deviation (RSD) is less than 10. The LOD and LOQ are 0.05 ppm and 0.18 ppm, respectively. The method developed for CPE-Sylgard-GLDA coupled with HPLC is feasible for the determination of methylphenol because it is simple, effective, cheap, and produces a high percentage of recovery.

Parp mutations protect against mitochondrial dysfunction and neurodegeneration in a PARKIN model of Parkinson's disease.

The co-enzyme nicotinamide adenine dinucleotide (NAD(+)) is an essential co-factor for cellular energy generation in mitochondria as well as for DNA repair mechanisms in the cell nucleus involving NAD(+)-consuming poly (ADP-ribose) polymerases (PARPs). Mitochondrial function is compromised in animal models of Parkinson's disease (PD) associated with PARKIN mutations. Here, we uncovered alterations in NAD(+) salvage metabolism in Drosophila parkin mutants. We show that a dietary supplementation with the NAD(+) precursor nicotinamide rescues mitochondrial function and is neuroprotective. Further, by mutating Parp in parkin mutants, we show that this increases levels of NAD(+) and its salvage metabolites. This also rescues mitochondrial function and suppresses dopaminergic neurodegeneration. We conclude that strategies to enhance NAD(+) levels by administration of dietary precursors or the inhibition of NAD(+)-dependent enzymes, such as PARP, that compete with mitochondria for NAD(+) could be used to delay neuronal death associated with mitochondrial dysfunction.

Mitofusin-mediated ER stress triggers neurodegeneration in pink1/parkin models of Parkinson's disease.

Mutations in PINK1 and PARKIN cause early-onset Parkinson's disease (PD), thought to be due to mitochondrial toxicity. Here, we show that in Drosophila pink1 and parkin mutants, defective mitochondria also give rise to endoplasmic reticulum (ER) stress signalling, specifically to the activation of the protein kinase R-like endoplasmic reticulum kinase (PERK) branch of the unfolded protein response (UPR). We show that enhanced ER stress signalling in pink1 and parkin mutants is mediated by mitofusin bridges, which occur between defective mitochondria and the ER. Reducing mitofusin contacts with the ER is neuroprotective, through suppression of PERK signalling, while mitochondrial dysfunction remains unchanged. Further, both genetic inhibition of dPerk-dependent ER stress signalling and pharmacological inhibition using the PERK inhibitor GSK2606414 were neuroprotective in both pink1 and parkin mutants. We conclude that activation of ER stress by defective mitochondria is neurotoxic in pink1 and parkin flies and that the reduction of this signalling is neuroprotective, independently of defective mitochondria. A video abstract for this article is available online in the supplementary information.

Suppression of the development of hypertension by the inhibitor of inducible nitric oxide synthase.

Our previous study demonstrated that the aortic inducible nitric oxide synthase (iNOS) expression and the plasma nitrite level in spontaneously hypertensive rats (SHR) were greater than that in age-matched Wistar-Kyoto rats (WKY). We subsequently hypothesized that the over-expression of iNOS might play an important role in the pathogenesis of hypertension in SHR. In the present study, pyrrolidinedithiocarbamate (PDTC, 10 mg kg(-1) day(-1), p.o., antioxidant and nuclear factor-kappa B inhibitor) and aminoguanidine (15 mg kg(-1) day(-1), p.o., selective inhibitor of iNOS) was used to treat SHR and WKY from age of 5 weeks through 16 weeks. We found that PDTC and aminoguanidine significantly suppressed the development of hypertension and improved the diminished vascular responses to acetylcholine in SHR but not in WKY. Likewise, the increase of iNOS expression, nitrotyrosine immunostaining, nitric oxide production and superoxide anion formation in adult SHR were also significantly suppressed by chronic treatment with PDTC and aminoguanidine. In conclusion, this study demonstrated that both PDTC and aminoguanidine significantly attenuated the development of hypertension in SHR. The results suggest that PDTC suppresses iNOS expression due to its anti-oxidant and/or nuclear factor-kappa B inhibitory properties. However, the effect of aminoguanidine was predominantly mediated by inhibition of iNOS activity, thereby reducing peroxynitrite formation. We propose that the development of a more specific and potent inhibitor of iNOS might be beneficial in preventing pathological conditions such as the essential hypertension.

Effect of Hoe 694, a novel Na(+)-H+ exchange inhibitor, on intracellular pH regulation in the guinea-pig ventricular myocyte.

1. Hoe 694 (3-methylsulphonyl-4-piperidinobenzoyl, guanidine hydrochloride) is a Na+/H+ exchange (NHE) inhibitor exhibiting cardioprotective properties during ischaemia and reperfusion in animal hearts. We have (i) tested the selectivity of Hoe 694 for NHE over other pHi-regulating mechanisms in the myocardium, and (ii) tested if the functionally important NHE isoform contributing to intracellular pH regulation in heart is NHE-1, as suggested from molecular biology studies of this protein. 2. pHi was recorded by fluorescence microscopy with carboxy SNARF-1, AM-loaded into single ventricular myocytes of guinea-pig. 3. In nominally HCO3-free media, recovery of pHi from an intracellular acid load is mediated by NHE, and was inhibited by Hoe 694, amiloride (an NHE inhibitor) or dimethyl amiloride (DMA, a high affinity NHE inhibitor) with potency values of 2.05, 87.3 and 1.96 microM respectively, giving the potency series: Hoe 694 congruent to DMA > > amiloride. This potency series, and the potency values (corrected for drug competition with extracellular Na+) match those determined previously for cloned NHE-1 expressed in mutant fibroblasts. In the absence of extracellular Na+ (to inhibit NHE), Hoe 694 had no effect on pHi. 4. In 5% CO2/HCO3(-)-buffered solution containing DMA, pHi recovery from acidosis is mediated by Na(+)-HCO3- symport and was unaffected by Hoe 694. The drug also had no effect on pHi recovery from an alkali-load, a process largely mediated by Cl(-)-HCO3- exchange. Finally, the fall of pHi upon adding extracellular Na-lactate is assisted by H(+)-lactate symport, and this too was unaffected by Hoe 694. 5. We conclude (i) Hoe 694 has no detectable inhibitory potency for pH-regulating carriers in heart other than NHE. (ii) native NHE functioning during pHi-regulation in the cardiomyocyte is the NHE-1 isoform. These data strengthen the case for NHE-1 being the receptor for mediating the cardioprotective effects of Hoe 694.

Ionic mechanisms responsible for the antiarrhythmic action of dehydroevodiamine in guinea-pig isolated cardiomyocytes.

1. Dehydroevodiamine alkaloid (DeHE), an active ingredient of a Chinese herbal medicine Wu-Chu-Yu (Evodiae frutus), has been shown to decrease aterial blood pressure in experimental animals and prolong action potential duration in cardiac cells. The aim of the present study was to explore the ionic basis of its possible antiarrhythmic effects. 2. Guinea-pig atrial and ventricular myocytes were isolated enzymatically and the ionic currents were recorded under whole-cell patch-clamp with single suction pipettes. 3. DeHE at a concentration of 0.1 microM inhibited reversibly the time-dependent outward K current (delayed rectifier, Ik) and the Na-dependent inward current (INa). 4. In low-K (1 mM) and high-Ca (9 mM) solution, DeHE also depressed the delayed afterdepolarizations (DAD) and the transient inward current (Iti) induced by 2 microM strophanthidin. On the other hand, DeHE occasionally induced early afterdepolarizations and slow response action potentials at a depolarized level. 5. At higher concentrations (1 microM and above), the L-type Ca current (ICa,L) was moderately inhibited. 6. The present findings indicate that DeHE may depress triggered arrhythmias in Ca-overloaded guinea-pig cardiac myocytes through its inhibitory actions on INa, Iti and, to a smaller extent, ICa. DeHE may also exert class III antiarrhythmic effect through a reduction of outward K currents (Ik) across the sarcolemma.

Depressant Effects of Prostacyclin in Human Atrial Fibers and Cardiomyocytes.

The aim of this study was to characterize the electropharmacological effects of prostacyclin (PGI(2)) in human atrial fibers and cardiomyocytes. Atrial tissues obtained from the hearts of 28 patients undergoing corrective cardiac surgery were used. Transmembrane action potentials were recorded using a conventional microelectrode technique, and twitch force by a transducer. Effects of PGI(2) (1 nM-10 &mgr;M) on action potential characteristics and contraction of atrial fibers were evaluated in normal [K](o) (4 mM) and high [K](o) (27 mM) in the absence and presence of cardiotonic agents. In addition, atrial and ventricular myocytes were isolated enzymatically from atrial tissues and hearts of 4 patients undergoing cardiac transplant. The effects of PGI(2) on Na- and Ca-dependent inward currents (I(Na) and I(Ca)) of cardiomyocytes were tested. In 9 human atrial fibers showing fast-response action potentials (mean dV/dt(max) = 101 +/- 15 Vs(-1)) in 4 mM [K](o), PGI(2) did not influence dV/dt(max) of phase 0 depolarization even at 1 &mgr;M. However, at a concentration as low as 10 nM, PGI(2) depressed spontaneous rhythms or slow-response action potentials in high-K-depolarized fibers. PGI(2) also depressed delayed afterdepolarizations and aftercontractions induced by cardiotonic agents. In isolated cardiomyocytes, PGI(2) reduced I(Ca) but not I(Na). The present findings show that, in human atrial fibers and cardiomyocytes, PGI(2) induces greater depressant effects on the slow-response action potential, I(Ca) and triggered activity than on the fast-response action potential. It is suggested that PGI(2) may act through a selective reduction of transmembrane Ca influx. Copyright 1994 S. Karger AG, Basel

Inhibitory effect of endothelin-1 on the isoproterenol-induced chloride current in human cardiac myocytes.

It is still controversial whether the cAMP-activated Cl(-) current (I(Cl,cAMP)) is expressed in human cardiomyocytes. The whole-cell configuration of the voltage-clamp technique was used to examine in detail the I(Cl,cAMP) in single human atrial and ventricular myocytes. Human cardiomyocytes were enzymatically isolated from atrial or ventricular specimens obtained from open-heart surgery or cardiac transplantation, respectively. Isoproterenol (1 microM) or forskolin (10 microM) was used to activate the cAMP second-messenger system. The isoproterenol- or forskolin-induced Cl(-) current was elicited in 12 of 54 atrial myocytes but was completely absent from ventricular myocytes. The isoproterenol-induced Cl(-) current in atrial myocytes was time-independent and had a reversal potential close to zero. Endothelin-1 (30 nM) inhibited the isoproterenol-induced Cl(-) current by 75+/-6% (n=4). This inhibitory effect of endothelin-1 was attenuated by pretreating atrial myocytes with the endothelin ET(A) receptor antagonist, BQ485, but not with the ET(B) receptor antagonist, BQ-788. The results provide evidence that the I(Cl,cAMP) exists in human atria, but not ventricle, and is inhibited by endothelin-1.

Cl--dependent and Cl--independent Na+/ HCO3- acid extrusion in cultured rat cerebellar astrocytes.

It is still uncertain whether the Na+-dependent Cl--HCO3- exchanger (NCBE) is expressed in mammalian astrocytes. Using fluorescent indicators to monitor the intracellular pH (pHi) and intracellular Na+ or Cl- levels, the NCBE in cultured rat cerebellar astrocytes was examined in detail. In nominally bicarbonate-free (Hepes-buffered) medium, a marked pHi recovery from internal acid load was seen which could be blocked completely by 30 microM HOE 694, a specific Na+-H+ exchanger isoform 1(NHE-1) inhibitor, at a pHi above 6.9. These conditions were therefore used to block NHE activity in CO2/HCO3-buffered media when the NCBE was being studied at pHi above 6.9. After internal acid loading in completely Cl--free bicarbonate-buffered medium (containing HOE 694), the rates of pHi recovery and transient Na+ influx were considerably slowed, and the Cl--dependent acid extrusion was both Na+- and 4,4-diisothiocyano-stilbene-disulphonic acid (DIDS)-sensitive. Moreover, a HCO3-dependent Cl- efflux during internal acid injection was seen. These results suggest that the NCBE is present in astrocytes. Following repetitive internal acid loading by addition of 5% CO2 to internal Cl- depleted cells, a similar rate of pHi recovery was consistently seen, suggesting Cl--independent pHi regulation also occurred in astrocytes. Moreover, this pHi recovery was completely blocked in the absence of sodium or on addition of DIDS, confirming that the Na+-HCO3 cotransporter (NBC) is present. Thus, the present study provides evidence that both the NCBE and NBC play important roles in acid extrusion in cultured mammalian astrocytes.

Drosophila Trap1 protects against mitochondrial dysfunction in a PINK1/parkin model of Parkinson's disease.

Mitochondrial dysfunction caused by protein aggregation has been shown to have an important role in neurological diseases, such as Parkinson's disease (PD). Mitochondria have evolved at least two levels of defence mechanisms that ensure their integrity and the viability of their host cell. First, molecular quality control, through the upregulation of mitochondrial chaperones and proteases, guarantees the clearance of damaged proteins. Second, organellar quality control ensures the clearance of defective mitochondria through their selective autophagy. Studies in Drosophila have highlighted mitochondrial dysfunction linked with the loss of the PTEN-induced putative kinase 1 (PINK1) as a mechanism of PD pathogenesis. The mitochondrial chaperone TNF receptor-associated protein 1 (TRAP1) was recently reported to be a cellular substrate for the PINK1 kinase. Here, we characterise Drosophila Trap1 null mutants and describe the genetic analysis of Trap1 function with Pink1 and parkin. We show that loss of Trap1 results in a decrease in mitochondrial function and increased sensitivity to stress, and that its upregulation in neurons of Pink1 mutant rescues mitochondrial impairment. Additionally, the expression of Trap1 was able to partially rescue mitochondrial impairment in parkin mutant flies; and conversely, expression of parkin rescued mitochondrial impairment in Trap1 mutants. We conclude that Trap1 works downstream of Pink1 and in parallel with parkin in Drosophila, and that enhancing its function may ameliorate mitochondrial dysfunction and rescue neurodegeneration in PD.

Drosophila ref(2)P is required for the parkin-mediated suppression of mitochondrial dysfunction in pink1 mutants.

Autophagy is a critical regulator of organellar homeostasis, particularly of mitochondria. Upon the loss of membrane potential, dysfunctional mitochondria are selectively removed by autophagy through recruitment of the E3 ligase Parkin by the PTEN-induced kinase 1 (PINK1) and subsequent ubiquitination of mitochondrial membrane proteins. Mammalian sequestrome-1 (p62/SQSTM1) is an autophagy adaptor, which has been proposed to shuttle ubiquitinated cargo for autophagic degradation downstream of Parkin. Here, we show that loss of ref(2)P, the Drosophila orthologue of mammalian P62, results in abnormalities, including mitochondrial defects and an accumulation of mitochondrial DNA with heteroplasmic mutations, correlated with locomotor defects. Furthermore, we show that expression of Ref(2)P is able to ameliorate the defects caused by loss of Pink1 and that this depends on the presence of functional Parkin. Finally, we show that both the PB1 and UBA domains of Ref(2)P are crucial for mitochondrial clustering. We conclude that Ref(2)P is a crucial downstream effector of a pathway involving Pink1 and Parkin and is responsible for the maintenance of a viable pool of cellular mitochondria by promoting their aggregation and autophagic clearance.

Genetic analysis of mitochondrial protein misfolding in Drosophila melanogaster.

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.

Drosophila happyhour modulates JNK-dependent apoptosis.

Mitogen-activated protein kinase kinase kinase kinase-3 (MAP4K3) is a Ste20 kinase family member that modulates multiple signal transduction pathways. We recently identified MAP4K3 as proapoptotic kinase using an RNA interference screening approach. In mammalian cells, MAP4K3 enhances the mitochondrial apoptosis pathway through the post-transcriptional modulation of selected proapoptotic Bcl-2 homology domain 3-only proteins. Recent data suggest that MAP4K3 mutations contribute to pancreatic cancer, which highlights the importance of studying the in vivo function of this kinase. To determine whether the cell death function is conserved in vivo and which downstream signalling pathways are involved, we generated transgenic flies expressing happyhour (hppy), the Drosophila MAP4K3 orthologue. Here, we show that the overexpression of hppy promotes caspase-dependent apoptosis and that the hypothetical kinase domain is essential for inducing cell death. In addition, we show that hppy expression triggers the activation of both the c-Jun N-terminal kinase (JNK) and target of rapamycin (TOR) signalling pathways; however, only JNK signalling is required for apoptosis. Together, our results show that hppy has a JNK-dependent proapoptotic function in Drosophila, which reinforces the hypothesis that MAP4K3 might act as tumour suppressor by regulating apoptosis in higher eukaryotes.

Reality basis for teaching psychomotor skills in a tertiary nursing curriculum.

This paper, written whilst the authors were working at Curtin University, Western Australia, describes a process of determining the psychomotor skills to be taught in an undergraduate nursing programme. It outlines how consultation with clinical agencies enhanced the planning of the skills component within the new nursing curriculum and details the outcomes in terms of faculty development and curriculum design.

Characterization of a zebrafish (Danio rerio) desmin cDNA: an early molecular marker of myogenesis.

Desmin is a muscle-specific protein and a constitutive subunit of the intermediate filaments (IF) in skeletal, cardiac and smooth muscles. It is an early marker of skeletal muscle myogenesis. We have characterized a clone of desmin cDNA from an embryonic zebrafish (Danio rerio) cDNA library. The full-length cDNA comprised 1798 nucleotides, encoding a protein of 473 amino acids. The predicted amino acid sequence of the zebrafish desmin shares a high degree of similarity to other vertebrate desmins, but also contains a sequence at the carboxyl terminal of the tail domain that is unique to the zebrafish. It carries many features which are distinctive of IF subunit proteins. These include the T/SSYRRXF/Y motif in the head domain, and the intermediate filament signature consensus, [I/V]-X-[T/A/C/I]-Y-[R/K/H]-X-[L/M]-L-[D/E], located in the carboxyl terminus of the central helical rod. Unlike other 3' UTR sequences, the 3' UTR of the zebrafish cDNA sequence has two CAYUG elements flanking a single polyadenylation site. The temporal and spatial expression patterns of desmin mRNA during early zebrafish development were studied. The onset of desmin expression occurred at the 1-3 somite stage (11 hpf). It increased throughout somitogenesis, with maximum expression at the Prim-6 stage (25 hpf), and decreasing expression towards the protruding-mouth stage (72 hpf). Desmin mRNA was initially localised exclusively to the somites, but was subsequently also detected in other musculature in the developing heart and fins. The onset of expression and the spatial localization of desmin mRNA in the zebrafish coincides with that reported for MyoD and myogenin.

Downregulation of phospholipase C delta3 by cAMP and calcium.

Four different isoforms of mammalian phospholipase C delta (PLCdelta) have been described. PLCdelta1, the best-understood isoform, is activated by an atypical GTP-binding protein. It has been suggested that it is a calcium signal amplifier. However, very less is known about other subtypes, including PLCdelta3. Therefore, in the present study, we examined the expression of PLCdelta3 in different human tissues. Moreover, the cellular underlying regulation for PLCdelta3 was studied in different cell lines. Our study showed that the mRNA and protein levels differed significantly among human tissues. The human PLCdelta3 gene was composed of 15 exons and 1 putative cAMP response element in the 5'-end promoter region. PLCdelta3 mRNA expression was downregulated by cAMP and calcium in both the human normal embryonic lung tissue diploid WI38 cell line and the glioblastoma/astrocytoma U373 cell line. However, mRNA expression showed no impact by PKC activators or inhibitors. This study shows the human PLCdelta3 expression pattern and is the first report that PLCdelta3 gene expression is downregulation by cAMP and calcium.