Down-regulation of natural resistance-associated macrophage protein-1 (Nramp1) is associated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/1-methyl-4-phenylpyridinium (MPP+ )-induced α-synuclein accumulation and neurotoxicity.

AIMS: The accumulation of α-synuclein is a hallmark in the pathogenesis of Parkinson's disease (PD). Natural resistance-associated macrophage protein-1 (Nramp1) was previously shown to contribute to the degradation of extracellular α-synuclein in microglia under conditions of iron overload. This study was aimed at investigating the role of Nramp1 in α-synuclein pathology in the neurone under 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/1-methyl-4-phenylpyridinium (MPP+ ) treatment. METHODS: The expression of Nramp1 and pathological features (including iron and α-synuclein accumulation) were examined in the dopaminergic neurones of humans (with and without PD) and of mice [with and without receiving chronic MPTP intoxication]. The effects of Nramp1 expression on low-dose MPP+ -induced α-synuclein expression and neurotoxicity were determined in human dopaminergic neuroblastoma SH-SY5Y cells. RESULTS: Similar to the findings in the substantia nigra of human PD, lower expression of Nramp1 but higher levels of iron and α-synuclein were identified in the dopaminergic neurones of mice receiving chronic MPTP intoxication, compared to controls. In parallel to the loss of dopaminergic neurones, the numbers of glial fibrillary acidic protein- and ionized calcium-binding adapter molecule-1-positive cells were significantly increased in the substantia nigra of MPTP-treated mice. Likewise, in human neuroblastoma SH-SY5Y cells exposed to low-dose MPP+ , Nramp1 expression and cathepsin D activity were decreased, along with an increase in α-synuclein protein expression and aggregation. Overexpression of functional Nramp1 restored cathepsin D activity and attenuated α-synuclein up-regulation and neuronal cell death caused by MPP+ treatment. CONCLUSIONS: These data suggest that the neuronal expression of Nramp1 is important for protecting against the development of MPTP/MPP+ -induced α-synuclein pathology and neurotoxicity.

Leukemia inhibitory factor (LIF) potentiates antinociception activity and inhibits tolerance induction of opioids.

BACKGROUND: The efficacy of opioids typically decreases after long-term use owing to the development of tolerance. Glial activation and the upregulation of proinflammatory cytokines are related to the induction of tolerance. We investigated the effect of leukemia inhibitory factor (LIF) on morphine analgesia and tolerance. METHODS: LIF concentrations in rat spinal cords were measured by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) after morphine administration. LIF distribution was examined using confocal microscopy. To evaluate the effects of LIF on morphine analgesia and tolerance, LIF was intrathecally administered 30 min before morphine injection. The analgesic effect of morphine was evaluated by measuring tail-flick latency. Human LIF concentrations from the cerebrospinal fluid (CSF) of opioid tolerant patients were also determined by specific ELISA. RESULTS: Chronic morphine administration upregulated LIF concentrations in rat spinal cords. Intrathecal injection of LIF potentiated the analgesic action of morphine. Patch clamp recording of spinal cord slices showed that LIF enhanced DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin)-induced outward potassium current. The development of tolerance was markedly suppressed by exogenous LIF, whereas neutralizing the endogenously released LIF with anti-LIF antibodies accelerated the tolerance induction. Moreover, LIF concentrations in the CSF of opioid-tolerant patients were higher than those in the opioid-naive controls. CONCLUSIONS: Intrathecal administration of LIF potentiated morphine antinociceptive activity and attenuated the development of morphine tolerance. Upregulation of endogenously released LIF by long-term use of opioids might counterbalance the tolerance induction effects of other proinflammatory cytokines. LIF might be a novel drug candidate for inhibiting opioid tolerance induction.

The association between miR-499a polymorphism and oral squamous cell carcinoma progression.

OBJECTIVE: To investigate the association of miR-499a genetic polymorphism with the risk of oral leukoplakia, oral submucous fibrosis (OSF), oral squamous cell carcinoma (OSCC), and clinicopathological outcomes of OSCC. METHODS: The genotyping of miR-499a T>C (rs3746444) using TagMan assay was conducted in two case-control studies of 1549 subjects. miR-499a-5p and miR-499a-3p were assayed using stem-loop RT-PCR for 63 paired OSCC and adjacent normal tissues. RESULTS: T/C+C/C genotypes [adjusted odds ratio (AOR) 1.84, P = 0.032] and C allelic type (AOR 1.91, P = 0.007) at miR-499a T>C were associated with an increased risk of BQ-related OSF as compared to those with T/T genotype or T allelic type, respectively. Conversely, T/C+C/C genotypes and C allelic type decreased the risk of OSCC, especially for non-BQ-related OSCC (for genotype: AOR 0.49, P = 0.010; for allelic type: AOR 0.50, P = 0.007). Additionally, downregulation of miR-499a-5p was found in OSCC tissues (P = 0.001) and correlated with the TT genotype (P = 0.001). CONCLUSION: The T/C+C/C genotypes of MiR-499a may contribute to an increased risk of BQ-related OSF, but a decreased risk of OSCC. miR-499a T>C influences the expression levels of miR-499a-5p during the tumorigenesis of OSCC.

Clinical presentations and skin denervation in amyloid neuropathy due to transthyretin Ala97Ser.

OBJECTIVE: Familial amyloid polyneuropathy (FAP) due to amyloidogenic transthyretin (TTR) is often associated with impairment of thermonociceptive functions. This study investigated skin innervation and its clinical significance in genetically defined FAP due to a hot-spot Ala97Ser TTR mutation (Ala97Ser). METHODS: Skin biopsies were performed on the distal leg of patients with Ala97Ser, and intraepidermal nerve fiber (IENF) densities were quantified. RESULTS: There were 19 unrelated patients with Ala97Ser manifesting a late-onset (59.47 +/- 5.70 years) generalized neuropathy with disabling motor, sensory, and autonomic symptoms. Against a background of a slowly progressive course, 7 patients (36.8%) exhibited additional rapid declines in neurologic deficits, which were associated with elevation of the protein content in the CSF (p < 0.001). The IENF density was markedly reduced in Ala97Ser patients compared to age- and gender-matched controls (0.99 +/- 1.11 vs 8.31 +/- 2.87 fibers/mm, p < 0.001). Skin denervation was present in all patients and was lower in patients with a higher disability grade (0.17 +/- 0.26 vs 1.37 +/- 1.16 fibers/mm, p = 0.003). Albuminocytologic dissociation in the CSF was observed in 14 patients (73.7%), and the IENF density was negatively correlated with the CSF protein concentration (p = 0.015). CONCLUSIONS: Skin denervation was common in Ala97Ser, and degeneration of cutaneous nerve terminals was correlated with the severity of clinical phenotypes and the level of CSF protein.

Gabapentin activates ROMK1 channels by a protein kinase A (PKA)-dependent mechanism.

BACKGROUND AND PURPOSE: Gabapentin is an effective anticonvulsant. The major physiological function of renal outer medullary potassium (ROMK1) channels is to maintain the resting membrane potential (RMP). We investigated the effect of gabapentin on ROMK1 channels and the mechanism involved. EXPERIMENTAL APPROACH: Xenopus oocytes were injected with mRNA coding for wild-type or mutant ROMK1 channels and giant inside-out patch-clamp recordings were performed. KEY RESULTS: Gabapentin increased the activity of ROMK1 channels, concentration-dependently and enhanced the activity of wild-type and an intracellular pH (pH(i))-gating residue mutant (K80M) channels over a range of pH(i). Gabapentin also increased activity of channels mutated at phosphatidylinositol 4,5-bisphosphate (PIP(2))-binding sites (R188Q, R217A and K218A). However, gabapentin failed to enhance channel activity in the presence of protein kinase A (PKA) inhibitors and did not activate phosphorylation site mutants (S44A, S219A or S313A), mutants that mimicked the negative charge carried by a phosphate group bound to a serine (S44D, S219D or S313D), or a mutated channel with a positive charge (S219R). These findings show that gabapentin activates ROMK1 channels independently of the pH(i) and not via a PIP(2)-dependent pathway. The effects of gabapentin on ROMK1 channels may be due to a PKA-mediated phosphorylation-induced conformational change, but not to charge-charge interactions. CONCLUSIONS AND IMPLICATIONS: ROMK1 channels are the main channels responsible for maintaining the RMP during cellular excitation. Gabapentin increased the activity of ROMK1 channels by a PKA-dependent mechanism, reducing neuronal excitability, and this may play an important role in its antiepileptic effect.

Structural influence of hanatoxin binding on the carboxyl terminus of S3 segment in voltage-gated K(+)-channel Kv2.1.

The voltage-sensing domains of voltage-gated potassium channels Kv2.1 (drk1) contain four transmembrane segments in each subunit, termed S1 to S4. While S4 is known as the voltage sensor, the carboxyl terminus of S3 (S3C) bears a gradually broader interest concerning the site for gating modifier toxins like hanatoxin and thus the secondary structure arrangement as well as its surrounding environment. To further examine the putative three-dimensional (3-D) structure of S3C and to illustrate the residues required for hanatoxin binding (which may, in turn, show the influence on the S4 in terms of changes in channel gating), molecular simulations and dockings were performed. These were based on the solution structure of hanatoxin and the structural information from lysine-scanning results for S3C fragment. Our data suggest that several basic and acidic residues of hanatoxin are electrostatically and stereochemically mapped onto their partner residues on S3C helix, whereas some aromatic or hydrophobic residues located on the same helical fragment interact with the hydrophobic patch of the toxin upon binding. Therefore, a slight distortion of the S3C helix, in a direction toward the N-terminus of S4, may exist. Such conformational change of S3C upon toxin binding is presented as a possible explanation for the observed shift in hanatoxin binding-induced gating.

Risk factors associated with infantile spasms: a hospital-based case-control study in Taiwan.

We investigated the risk factors associated with infantile spasms (IS) by a hospital-based case-control study in Taiwan. Twenty-five patients with IS were recruited from one medical center (National Taiwan University Hospital) between 1990 and 1997. Based on a close-structured questionnaire, standardized interviews were carried out to obtain information on risk factors associated with IS. Two comparison groups are used, including a total of 106 subjects in the Disease Control group, and 139 subjects in the Normal Control group. Unconditional logistic regression is used to calculate odds ratios (OR) and 95% confidence interval (CI). Univariate analysis revealed gestational age, congenital cerebral anomalies, tuberous sclerosis (TS), asphyxia, febrile seizure, and developmental delay (before onset of spasm) were at increased risk of IS. After adjustment of multiple risk factors through unconditional logistic regression, significant risk factors for IS include congenital cerebral anomalies, TS, asphyxia, postterm, and developmental delay were highly associated with IS. The risk factors of IS may closely relate to underlying neurological abnormalities. Our results are consistent with the previous findings.

Modulation of protein kinase A activation by fibronectin matrix proteins at developing neuromuscular synapses in Xenopus laevis cell cultures.

Extracellular matrix proteins, such as fibronectin, laminin, and collagen, have been implicated in a wide variety of cellular properties, which include cell adhesion, migration, differentiation, and proliferation. In this study, we investigated the modulation of protein kinase A (PKA) activity by matrix proteins at developing motoneurons. The cultures of spinal neurons and myotomal cells were prepared from 1-day-old Xenopus laevis embryos. Spontaneous synaptic currents (SSC) were recorded from innervated myocytes of natural synapses by whole-cell voltage-clamped recordings (V(h) = -60 to approximately -65 mV). Bath application of agents, which directly or indirectly activate PKA, such as forskolin (20 microM), dibutyryl cAMP (DBcAMP) (1 mM), isoproterenol (10 microM), or albuterol (10 microM), significantly increased SSC frequency in cultures grown on fibronectin (FN)-coated substratum, but not on laminin- or collagen-coated glasses. The evoked synaptic currents increased in response to forskolin in neurons grown on FN substratum. Triflavin, an Arg-Gly-Asp-dependent disintegrin, inhibited potentiating action of isoproterenol in neurons grown on FN substratum, suggesting that integrin is involved in the potentiation of the PKA pathway in the regulation of acetylcholine (ACh) release. There is collaboration of neurotrophic factors and the FN matrix in regulating synaptic transmission in response to DBcAMP. Chronic treatment with neurotrophic factors, such as ciliary neurotrophic factor (150 ng/ml), glial cell line-derived neurotrophic factor (30 ng/ml), or neurotrophin-3 (50 ng/ml), enhanced the SSC-increasing action of DBcAMP in neurons grown on FN-coated glasses. These results suggest that the FN matrix potentiates synaptic transmission in response to PKA activation. Neurotrophic factors may collaborate with FN to regulate spontaneous ACh secretion at developing motoneurons, which may play an important role in the maturation of embryonic neuromuscular synapses.

Collaboration of fibronectin matrix and neurotrophin in regulating spontaneous transmitter release at developing neuromuscular synapses in Xenopus cell cultures.

Integrins mediate cell-extracellular matrix connection and are particularly important during neuronal development. We here investigated the regulation of fibronectin (FN) matrix and neurotrophins on the embryonic synaptic transmission. Spontaneous synaptic currents (SSCs) were recorded from innervated myocytes of 1-day-old Xenopus cultures by whole-cell recordings. The SSC increasing action of alpha,beta-methylene adenosine triphosphate was enhanced in neurons grown on FN substratum, which was further potentiated by chronic treatment with brain-derived neurotrophic factor (BDNF). The SSC increasing action of thapsigargin, carbonyl cyanide m-chlorophenylhydrazone and N-methyl-D-aspartate was also markedly potentiated in neurons grown on FN-coated glass coverslips and chronically treated with BDNF. FN matrix or BDNF alone only exerts slight potentiation on the SSC increasing action of these three drugs. Our results suggest that FN matrix can collaborate with neurotrophin in regulating synaptic transmission at developing motoneurons, which may play an important role in the maturation of embryonic neuromuscular junction.

Regulation of ROMK1 channel by protein kinase A via a phosphatidylinositol 4,5-bisphosphate-dependent mechanism.

ROMK inward-rectifier K+ channels control renal K+ secretion. The activity of ROMK is regulated by protein kinase A (PKA), but the molecular mechanism for regulation is unknown. Having found that direct interaction with membrane phosphatidylinositol 4, 5-bisphosphate (PIP2) is essential for channel activation, we investigate here the role of PIP2 in regulation of ROMK1 by PKA. By using adenosine-5'-[gamma-thio]triphosphate) (ATP[gammaS]) as the substrate, we found that PKA does not directly activate ROMK1 channels in membranes that are devoid of PIP2. Rather, phosphorylation by PKA + ATP[gammaS] lowers the concentration of PIP2 necessary for activation of the channels. In solution-binding assays, anti-PIP2 antibodies bind PIP2 and prevent PIP2-channel interaction. In inside-out membrane patches, antibodies inhibit the activity of the channels. PKA treatment then decreases the sensitivity of ROMK1 for inhibition by the antibodies, indicating an enhanced interaction between PIP2 and the phosphorylated channels. Conversely, mutation of the PKA phosphorylation sites in ROMK1 decreases PIP2 interaction with the channels. Thus, PKA activates ROMK1 channels by enhancing PIP2-channel interaction.

Environmental risk factors and Parkinson's disease: a case-control study in Taiwan.

To explore environmental risk factors for Parkinson's disease (PD) in Taiwan, we investigated 120 patients with PD and 240 hospital control subjects matched with patients on age (+/-2 years) and sex. Based on a structured open-ended questionnaire, we carried out standardized interviews to obtain history of exposure to environmental factors, including place of residence, source of drinking water, and environmental and occupational exposures to various agricultural chemicals. In the univariate analysis, the history of living in a rural environment, farming, use of herbicides/pesticides, and use of paraquat were associated with an increased PD risk in a dose-response relationship. After adjustment for multiple risk factors through conditional logistic regression, the biological gradient between PD and previous uses of herbicides/pesticides and paraquat remained significant. The PD risk was greater among subjects who had used paraquat and other herbicides/pesticides than those who had used herbicides/pesticides other than paraquat. There were no significant differences in occupational exposures to chemicals, heavy metals, and minerals between PD patients and matched control subjects. The duration of drinking well water and alcohol consumption was not significantly associated with PD. There was an inverse relationship between cigarette smoking and PD. Environmental factors, especially exposures to paraquat and herbicides/pesticides, may play important roles in the development of PD in Taiwan.

Churg-Strauss syndrome presented as multiple intracerebral hemorrhage.

Intracerebral hemorrhage is an uncommon sequel of Churg-Strauss syndrome. We describe a 27 y old Taiwanese male patient who was clinicopathologically diagnosed as Churg-Strauss syndrome. The patient experienced a sudden onset of blurring of vision and slowness of motion and speech. Magnetic resonance imaging of the brain revealed lobar hemorrhage on right parieto-occipital and left parietal areas. The cause of cerebral hemorrhage was probably due to poorly controlled high blood pressure and vasculitis. He received pulse therapy of methylprednisolone and cyclophosphamide followed by oral prednisolone. His neurological symptoms responded well to such a regimen. Cerebral hemorrhage is a major cause of morbidity and death in patients with Churg-Strauss syndrome. Uncontrolled high blood pressure may cause cerebral hemorrhage. Careful monitor of blood pressure is critical for the management of Churg-Strauss syndrome patients.

Elevation of cytosolic calcium of rat cardiac myocytes in phosphate depletion.

Phosphate depletion is associated with a rise in cytosolic calcium ([Ca2+]i) of cells and such a derangement is responsible in major part for organ dysfunction in phosphate depletion (PD). Cardiac function is impaired in PD, and it is possible that PD is also associated with rise in [Ca2+]i of cardiac myocytes. The present study examined the effect of PD on [Ca2+]i of cardiac myocytes and explored the mechanisms that may lead to the rise in their [Ca2+]i. The [Ca2+]i of cardiac myocytes began to rise and ATP content began to fall at the third week of PD. After six weeks of PD, the values of [Ca2+]i were significantly higher (P < 0.01) and those of ATP content were significantly lower (P < 0.01) than in control (PW) rats. The Vmax of Ca2(+)-ATPase and Na+,K(+)-ATPase as well as the Na(+)-Ca2+ exchange were significantly lower (P < 0.01) in PD than in PW animals. The data of the present study are consistent with the notion that the rise in [Ca2+]i of cardiac myocytes of PD rats is due to a decrease in calcium efflux out of them.

Allergic granulomatosis and angiitis (Churg-Strauss syndrome) presenting as prominent neurologic lesions and optic neuritis.

We describe a Chinese male patient with clinicopathologically definite Churg-Strauss syndrome. He had a clinical pattern of mononeuropathy multiplex and unusual optic neuritis, the latter probably being due to vasculitis related demyelination of the optic nerve. Magnetic resonance imaging of the brain revealed many clinically silent central nervous system lesions. He initially received steroid therapy, but an early cessation of treatment resulted in acute exacerbation. Later on, he was treated with combined prednisolone-cyclophosphamide therapy for 1 year. His ophthalmological and neurological symptoms responded well to such a regimen and disappeared within 6 months.

Interactions of anticholinesterases with Achatina fulica acetylcholine responses and electrogenic sodium pump.

The dose-dependent effects of the anticholinesterases, neostigmine and mycotoxin territrem-B, were determined on: (i) Cl(-)-responses of voltage clamped Achatina fulica neurons to microperfused acetylcholine; (ii) the 4 K(+)-induced outward currents evoked by an electrogenic sodium pump in the same neuron; and (iii) acetylcholinesterase activity of Achatina fulica ganglionic homogenates. Both compounds at low doses potentiated the peak acetylcholine responses. However, they had different effects at higher (> 1 microM) doses in that neostigmine now antagonized acetylcholine responses, while territrem-B still produced a maximal potentiation. At all doses neostigmine produced a dose-dependent inhibition of acetylcholinesterase activity. The cholinolytic effect of high doses of neostigmine was associated with the inhibition of 4 K(+)-induced current in the same neuron, while territrem-B neither altered the K(+)-induced current nor antagonized acetylcholine responses. The cholinolytic effect of neostigmine was completely antagonized by the inhibition of electrogenic sodium pump by ouabain or by perfusion with K(+)-free solution. These results suggest that neostigmine at high concentrations inhibits the electrogenic sodium pump and that the cholinolytic effect of high doses of neostigmine is secondary to this action. Territrem-B, on the other hand, had no effect on the electrogenic sodium pump and had no effect on the neuronal membrane properties other than to inhibit acetylcholinesterase. Thus, territrem-B may be a useful tool for studying the interaction between acetylcholinesterase and acetylcholine receptors.

The association of aldosterone-producing adrenal adenoma in a patient with autosomal dominant polycystic kidney disease.

We report a young female with autosomal dominant polycystic kidney disease associated with right adrenal adenoma. Refractory hypertension and hypokalemia were the findings that led to this diagnosis. The diagnostic approach included plasma potassium and aldosterone levels, plasma renin activity, captopril test, saline infusion test, NP-59 scintigraphy, computed tomography, and magnetic resonance imaging. Adrenalectomy completely normalized hypertension and hypokalemia in this patient.