Icing on the Cake: Imidazole-Anchored Strategy To Enhance the Proton Conductivity of Two Isostructural Ce(IV)/Hf(IV) Metal-Organic Frameworks.

In the field of proton conduction, the acquisition of crystalline metal-organic frameworks (MOFs) with high stability and ultrahigh proton conductivity has been of great research value and is worth continuous exploration. Here, we greenly synthesized a three-dimensional porous MOF (MOF-801-Ce) by using [(NH4)2Ce(NO3)6 and fumaric acid as starting materials and solvothermally synthesized Hf-UiO-66-NO2 by using HfCl4 and 2-nitroterephthalic acid as starting materials. A series of measurements have shown that both MOFs exhibit good water stability, acid-base stability, and thermal stability and demonstrate outstanding proton conductivity. At 100 °C and 98% relative humidity (RH), the proton conductivities (σ) could be 2.59 × 10-3 S·cm-1 for MOF-801-Ce and 0.89 × 10-3 S·cm-1 for Hf-UiO-66-NO2. To pursue higher proton conductivity, we further adopted the evaporation approach to encapsulate imidazole molecules in the pores of the two compounds, achieving the imidazole-encapsulated MOFs, Im@MOF-801-Ce and Im@Hf-UiO-66-NO2. As expected, their σ values were significantly boosted by almost an order of magnitude up to 10-2 S·cm-1. Finally, their proton-conductive mechanisms were explored in light of the structural information, gas adsorption/desorption, and other tests. The outstanding structural stability of these MOFs and their durability of the proton conduction capability manifested that they have great promise in electrochemical fields.

Comparative Studies on the Proton Conductivities of Hafnium-Based Metal-Organic Frameworks and Related Chitosan or Nafion Composite Membranes.

Hafnium (Hf)-based UiO-66 series metal-organic frameworks (MOFs) have been widely studied on gas storage, gas separation, reduction reaction, and other aspects since they were first prepared in 2012, but there are few studies on proton conductivity. In this work, one Hf-based MOF, Hf-UiO-66-fum showing UiO-66 structure, also known as MOF-801-Hf, was synthesized at room temperature using cheap fumaric acid as the bridging ligand, and then imidazole units were successfully introduced into MOF-801-Hf to obatin a doped product, Im@MOF-801-Hf. Note that both MOF-801-Hf and Im@MOF-801-Hf demonstrate excellent thermal, water, and acid-base stabilities. Expectedly, the maximum proton conductivity (σ) of Im@MOF-801-Hf (1.46 × 10-2 S·cm-1) is nearly 4 times greater than that of MOF-801-Hf (3.98 × 10-3 S·cm-1) under 100 °C and 98% relative humidity (RH). To explore their possible practical application value, we doped them into chitosan (CS) or Nafion membranes as fillers, namely, CS/MOF-801-Hf-X, CS/Im@MOF-801-Hf-Y, and Nafion/MOF-801-Hf-Z (X, Y, and Z are the doping percentages of MOF in the membrane, respectively). Intriguingly, it was found that CS/MOF-801-Hf-6 and CS/Im@MOF-801-Hf-4 indicated the highest σ values of 1.73 × 10-2 and 2.14 × 10-2 S·cm-1, respectively, under 100 °C and 98% RH and Nafion/MOF-801-Hf-9 also revealed a high σ value of 4.87 × 10-2 S·cm-1 under 80 °C and 98% RH, which showed varying degrees of enhancement compared to the original MOFs or pure CS and Nafion membranes. Our study illustrates that these Hf-based MOFs and related composite membranes offer great potential in electrochemical fields.

S-type Anion Channels SLAC1 and SLAH3 Function as Essential Negative Regulators of Inward K+ Channels and Stomatal Opening in Arabidopsis.

Drought stress induces stomatal closure and inhibits stomatal opening simultaneously. However, the underlying molecular mechanism is still largely unknown. Here we show that S-type anion channels SLAC1 and SLAH3 mainly inhibit inward K+ (K+in) channel KAT1 by protein-protein interaction, and consequently prevent stomatal opening in Arabidopsis. Voltage-clamp results demonstrated that SLAC1 inhibited KAT1 dramatically, but did not inhibit KAT2. SLAH3 inhibited KAT1 to a weaker degree relative to SLAC1. Both the N terminus and the C terminuses of SLAC1 inhibited KAT1, but the inhibition by the N terminus was stronger. The C terminus was essential for the inhibition of KAT1 by SLAC1. Furthermore, drought stress strongly up-regulated the expression of SLAC1 and SLAH3 in Arabidopsis guard cells, and the over-expression of wild type and truncated SLAC1 dramatically impaired K+in currents of guard cells and light-induced stomatal opening. Additionally, the inhibition of KAT1 by SLAC1 and KC1 only partially overlapped, suggesting that SLAC1 and KC1 inhibited K+in channels using different molecular mechanisms. Taken together, we discovered a novel regulatory mechanism for stomatal movement, in which singling pathways for stomatal closure and opening are directly coupled together by protein-protein interaction between SLAC1/SLAH3 and KAT1 in Arabidopsis.

The S-Type Anion Channel ZmSLAC1 Plays Essential Roles in Stomatal Closure by Mediating Nitrate Efflux in Maize.

Diverse stimuli induce stomatal closure by triggering the efflux of osmotic anions, which is mainly mediated by the main anion channel SLAC1 in plants, and the anion permeability and selectivity of SLAC1 channels from several plant species have been reported to be variable. However, the genetic identity as well as the anion permeability and selectivity of the main S-type anion channel ZmSLAC1 in maize are still unknown. In this study, we identified GRMZM2G106921 as the gene encoding ZmSLAC1 in maize, and the maize mutants zmslac1-1 and zmslac1-2 harboring a mutator (Mu) transposon in ZmSLAC1 exhibited strong insensitive phenotypes of stomatal closure in response to diverse stimuli. We further found that ZmSLAC1 functions as a nitrate-selective anion channel without obvious permeability to chloride, sulfate and malate, clearly different from SLAC1 channels of Arabidopsis thaliana, Brassica rapa ssp. chinensis and Solanum lycopersicum L. Further experimental data show that the expression of ZmSLAC1 successfully rescued the stomatal movement phenotypes of the Arabidopsis double mutant atslac1-3atslah3-2 by mainly restoring nitrate-carried anion channel currents of guard cells. Together, these findings demonstrate that ZmSLAC1 is involved in stomatal closure mainly by mediating the efflux of nitrate in maize.

A protein kinase, calcineurin B-like protein-interacting protein Kinase9, interacts with calcium sensor calcineurin B-like Protein3 and regulates potassium homeostasis under low-potassium stress in Arabidopsis.

Potassium (K⁺) is an essential macronutrient for plant growth and development. Previous studies have demonstrated that Calcineurin B-Like Protein1 (CBL1) or CBL9 and CBL-Interacting Protein Kinase23 (CIPK23) regulate K⁺ uptake in Arabidopsis (Arabidopsis thaliana) roots by modulating K⁺ channel Arabidopsis K⁺ Transporter1. In this study, we show that the protein kinase CIPK9 interacts with the calcium sensor CBL3 and plays crucial roles in K⁺ homeostasis under low-K⁺ stress in Arabidopsis. Arabidopsis wild-type plants showed leaf chlorotic symptoms when grown for 10 d on low-K⁺ (100 µM) medium. Here, we show that plants lacking CIPK9 displayed a tolerant phenotype to low-K⁺ stress, which still maintained green leaves when the wild-type plants showed typical K⁺-deficient symptoms. Overexpressing lines of CIPK9 resulted in a low-K⁺-sensitive phenotype compared with wild-type plants. Furthermore, CBL2 and CBL3 were identified as upstream regulators of CIPK9. Both CBL2- and CBL3-overexpressing lines displayed similar low-K⁺-sensitive phenotypes and K⁺ contents to CIPK9-overexpressing lines. However, only cbl3 mutant plants, but not cbl2 mutant plants, showed the low-K⁺-tolerant phenotype similar to cipk9 mutants. Taken together, these results demonstrate that CIPK9 and CBL3 work together and function in K⁺ homeostasis under low-K⁺ stress in Arabidopsis.

Identification of cyclic GMP-activated nonselective Ca2+-permeable cation channels and associated CNGC5 and CNGC6 genes in Arabidopsis guard cells.

Cytosolic Ca(2+) in guard cells plays an important role in stomatal movement responses to environmental stimuli. These cytosolic Ca(2+) increases result from Ca(2+) influx through Ca(2+)-permeable channels in the plasma membrane and Ca(2+) release from intracellular organelles in guard cells. However, the genes encoding defined plasma membrane Ca(2+)-permeable channel activity remain unknown in guard cells and, with some exceptions, largely unknown in higher plant cells. Here, we report the identification of two Arabidopsis (Arabidopsis thaliana) cation channel genes, CNGC5 and CNGC6, that are highly expressed in guard cells. Cytosolic application of cyclic GMP (cGMP) and extracellularly applied membrane-permeable 8-Bromoguanosine 3',5'-cyclic monophosphate-cGMP both activated hyperpolarization-induced inward-conducting currents in wild-type guard cells using Mg(2+) as the main charge carrier. The cGMP-activated currents were strongly blocked by lanthanum and gadolinium and also conducted Ba(2+), Ca(2+), and Na(+) ions. cngc5 cngc6 double mutant guard cells exhibited dramatically impaired cGMP-activated currents. In contrast, mutations in CNGC1, CNGC2, and CNGC20 did not disrupt these cGMP-activated currents. The yellow fluorescent protein-CNGC5 and yellow fluorescent protein-CNGC6 proteins localize in the cell periphery. Cyclic AMP activated modest inward currents in both wild-type and cngc5cngc6 mutant guard cells. Moreover, cngc5 cngc6 double mutant guard cells exhibited functional abscisic acid (ABA)-activated hyperpolarization-dependent Ca(2+)-permeable cation channel currents, intact ABA-induced stomatal closing responses, and whole-plant stomatal conductance responses to darkness and changes in CO2 concentration. Furthermore, cGMP-activated currents remained intact in the growth controlled by abscisic acid2 and abscisic acid insensitive1 mutants. This research demonstrates that the CNGC5 and CNGC6 genes encode unique cGMP-activated nonselective Ca(2+)-permeable cation channels in the plasma membrane of Arabidopsis guard cells.

Infection-responsive long-term antibacterial bone plates for open fracture therapy.

The infections in open fracture induce high morbidity worldwide. Thus, developing efficient anti-infective orthopedic devices is of great significance. In this work, we designed a kind of infection-responsive long-term antibacterial bone plates. Through a facile and flexible volatilization method, a multi-aldehyde polysaccharide derivative, oxidized sodium alginate, was crosslinked with multi-amino compounds, gentamycin and gelatin, to fabricate a uniform coating on Ti bone plates via Schiff base reaction, which was followed by a secondary crosslinking process by glutaraldehyde. The double-crosslinked coating was stable under normal condition, and could responsively release gentamycin by the triggering of the acidic microenvironment caused by bacterial metabolism, owning to the pH-responsiveness of imine structure. The thickness of the coating was ranging from 22.0 µm to 63.6 µm. The coated bone plates (Ti-GOGs) showed infection-triggered antibacterial properties (>99%) and high biocompatibility. After being soaked for five months, it still possessed efficient antibacterial ability, showing its sustainable antibacterial performance. The in vivo anti-infection ability was demonstrated by an animal model of infection after fracture fixation (IAFF). At the early stage of IAFF, Ti-GOGs could inhibit the bacterial infection (>99%). Subsequently, Ti-GOGs could promote recovery of fracture of IAFF. This work provides a convenient and universal strategy for fabrication of various antibacterial orthopedic devices, which is promising to prevent and treat IAFF.

Effect of trehalose on PC12 cells overexpressing wild-type or A53T mutant α-synuclein.

Accumulation of α-synuclein (α-Syn) is a common pathology for both familiar and sporadic Parkinson's disease (PD), enhancing its clearance might be a promising strategy for treating PD. To assess the potential of trehalose in this regard, we investigated its effect on the PC12 cells overexpressing wild type (WT) or A53T mutant α-Syn and the implicated pathway it might mediated. We observed that trehalose promoted the clearance of A53T α-Syn but not WT α-Syn in PC12 cells, and confirmed the increased LC3 and Lysotracker RED positive autolysosomes by using lysotracker and LC3 staining, the enhanced expression of LC3-II in Western blot, and more autophagosomes under Transmission Electron Microscope in a dose dependent manner after the trehalose treatment. The activation of autophagy can be alleviated by applying macroautophagy inhibitor 3-methyladenine (3-MA). In addition, degradation of A53T and WT α-Syn was blocked after Ubiquitin Proteasome System (UPS) inhibitor (MG132) was applied in those PC12 cells overexpressing A53T or WT α-Syn, suggesting that A53T α-Syn could be degraded by both UPS and macroautophagy. But the effect of trehalose on A53T α-Syn is mainly mediated through the macroautophagy pathway, which is not a dominant way for WT α-Syn clearance. Further in vivo research will be needed to verify the effectiveness of trehalose in treating PD.

Proton Conductive Lanthanide-Based Metal-Organic Frameworks: Synthesis Strategies, Structural Features, and Recent Progress.

In the fields of proton exchange membrane fuel cells as well as impedance recognition, molecular sieve, and biochemistry, the development of proton conductive materials is essential. The design and preparation of the next generation of proton conductive materials-crystalline metal-organic framework (MOF) materials with high proton conductivity and excellent water stability-are facing great challenges. Due to the large radius and high positive charge of lanthanides, they often interact with organic ligands to exhibit high coordination numbers and flexible coordination configurations, resulting in the higher stability of lanthanide-based MOFs (Ln-MOFs) than their transition metal analogues, especially regarding water stability. Therefore, Ln-MOFs have attracted considerable attention. This review offers a view of the latest progress of proton conductive Ln-MOFs, including synthesis strategy, structural characteristics, and advantages, proton conductivity, proton conductive mechanism, and applications. More importantly, by discussing structure-property relationships, we searched for and analyzed design techniques and directions of development of Ln-MOFs in the future. The latest progress of synthesis strategy, structural characteristics, proton conductive properties and mechanism and applications on Ln-MOFs. Ln-MOFS Lanthanide-based MOFs, MOF metal-organic framework, PEMFC proton exchange membrane fuel cells.

Diffuse uterine leiomyomatosis: A case report and review of literature.

BACKGROUND: Diffuse uterine leiomyomatosis (DUL) is a benign uterine smooth muscle neoplasm with unknown etiology. Since DUL is rarely reported, knowledge regarding it is limited. The rate of early diagnosis is low, and DUL is often misdiagnosed as common multiple uterine leiomyomas before surgery. CASE SUMMARY: A 27-year-old patient with no sexual activity presented to the Emergency Department of our hospital complaining of heavy vaginal bleeding. She had a history of uterine fibroids and menorrhagia. Pelvic examination showed a regularly enlarged uterus, similar in size to that associated with a 4-mo pregnancy. Pelvic magnetic resonance imaging (MRI) revealed numerous multiple uterine fibroids, and a transabdominal myomectomy (TM) was performed. Intraoperative exploration revealed that the myometrium was full of myoma nodules of variable sizes. Over 50 leiomyomas were removed. The pathology report confirmed leiomyoma. The patient was discharged and received a gonadotropin-releasing hormone analog (3.75 mg) for 6 mo. Ten months after surgery, the patient presented to the hospital again for abnormal uterine bleeding. MRI showed an irregular mass with a diameter of 5.2 cm without sharp demarcation in the uterine cavity. Submucosal leiomyoma was considered first, and the patient underwent a hysteroscopic myomectomy plus hymen repair. Intraoperative exploration showed that there were several leiomyomatosis masses in the cavity. Postoperative pathological examination confirmed submucosal leiomyoma and necrotic and generative tissue. Although the menstrual cycle was still irregular, the patient did not have symptoms of menorrhagia for a period of 28 mo after the second surgery. CONCLUSION: Individuals with DUL are easily misdiagnosed due to the lack of specific manifestations of this disease. MRI is helpful for early identification and preoperative evaluation. There is currently no unified method of diagnosis. For women who want to preserve fertility, conservative surgery should be made an option. When TM is chosen, a modified new myomectomy should be considered to avoid the drawbacks of traditional TM.

Clinical and pathological features in 15 Chinese patients with calpainopathy.

BACKGROUND: Calpainopathy is comprised of a group of myopathies caused by deficiency in calcium-activated, neutral protease (calpain-3). In this study we identify calpainopathy in a cohort of Chinese patients with unclassified myopathy and analyze its clinical and pathological features. METHODS: Sixty-six muscle biopsies were selected for combined Western blotting of dysferlin and calpain-3 after immunohistochemical staining. Clinical and pathological parameters of 15 confirmed calpainopathy cases were determined. RESULTS: The diagnosis of calpainopathy in 15 Chinese patients was confirmed by Western blot analysis. Fourteen subjects had progressive proximal muscle weakness; 1 presented with bilateral distal muscle atrophy of the lower extremities. Scapular winging was observed in 12 patients (80%), and joint contractures were found in 10 others (66.7%). Histopathological studies showed a high prevalence of lobulated fibers (66.7%). CONCLUSIONS: Chinese patients with calpainopathy share some common clinical and pathological features with the reported characteristics of non-Chinese patients.

The bradykinin B2 receptor mediates hypoxia/reoxygenation induced neuronal cell apoptosis through the ERK1/2 pathway.

The bradykinin B2 receptor (B2R) mediates many physiological processes such as hypotension, inflammation and blood-vessel permeability. Hypoxia/reoxygenation (H/R) induces neuronal cell apoptosis. It was found that B2R expression was enhanced in primary cultured cortical neurons after H/R treatment. Addition of bradykinin (BK) alleviated the neuronal damage from H/R. This protective effect of BK was inhibited by the B2R antagonist, HOE140, and the ERK1/2 antagonist, PD98059. The phosphorylation of ERK1/2 was increased under H/R, and the addition of BK enhanced this effect. These results indicate that B2R plays an important role in protecting neurons from damage induced by H/R and this effect may function through the ERK1/2 pathway.

Localization of alpha-synuclein to mitochondria within midbrain of mice.

The interrelationship between alpha-synuclein (alpha-syn) and mitochondria is not clearly understood. Owing to the lack of the signal peptide and its predominant localization in the cytosol, alpha-syn is generally considered to affect mitochondrial function through some secondary effects. Contrary to this assumption, here, we show that a portion of alpha-syn is present in the membrane of mitochondria in normal dopaminergic neurons. The same profile is also found in other alpha-syn-positive neurons. Thus, binding to the membrane of mitochondria is the physiological nature of alpha-syn and might also contribute to the pathological role of this protein in the mitochondrial dysfunction in Parkinson's disease.

[Effect of dizocilpine on P-glycoprotein expression in hippocampus in limbic seizure: experiment with rats].

OBJECTIVE: To observe the effect of dizocilpine (MK801), a noncompetitive antagonist of N-methyl-D-aspartic acid (NMDA) receptor, on P-glycoprotein (P-gp) expression after limbic seizure, and to explore whether NMDA receptor play a role in the regulation of P-gp expression during limbic seizure. METHODS: 120 Wistar rats were randomly divided into 2 equal sets. 50 rats in Set 1 underwent intraperitoneal injection of lithium chloride, scopolamine, and pilocarpine so as to cause status epilepticus (SE) for 90 min. Then diazepam was given to terminate the SE. The rats were killed 0, 3, 6, 14, and 72 h after the SE respectively. The hippocampus was isolated. Realtime fluorescent quantitative RT-PCR (qRT-PCR) was used to detect the expression of multidrug resistance gene 1a (mdr1a) and mdr1b. Immunohistochemistry was used to detect the P-gp. The rats were used as controls. Another 60 rats (Set 2) were randomly divided into 3 equal groups: control group, given with normal saline (NS) only, SE group, given with NS 20 min before administration of pilocarpine, and MK801 group, given with MK801 20 min before administration of pilocarpine. The 3 groups in Set 2 were further divided into 2 equal subgroups of 10 rats to be killed 6 or 24 h after SE. RESULTS: The mdr1a expression in hippocampus within 72 h after seizure was much higher at each time point: the level of mdr1a expression instantly after the seizure was terminated was [5.6 (2.9) x 10(5) mRNA copies/40 ng total RNA], significantly higher than that of the controls [2.4 (1.1) x 10(5) mRNA copies/40 ng total RNA, P < 0.05], increased to the level of [7.6 (6.3) x 10(5), P < 0.01] 3 h after, and kept at such level till 72 h after. The msr1b expression transiently increased 2.2 and 2.4 times that of the controls respectively 3 h and 6 h after the seizure was terminated [(3.3 +/- 0.4) x 10(4), and (3.6 +/- 1.0) x 10(4), both P < 0.01)]. The expression level of mdr1a 6 h after the seizure was terminated of the MK801 group was (4.3 +/- 0.8) x 10(5) and the expression level of mdr1b 6 h after the seizure was terminated of the MK801 group was (2.0 +/- 0.7) x 10(4), both significantly lower than those of the SE group (both P < 0.01). The P-gp expression level 24 h after the seizure was terminated of the MK801 group was 26.6 +/- 5.0 pieces of microvessels/400 times field, significantly lower than that of the SE group (39.0 +/- 4.1, P < 0.01). CONCLUSION: MK801 down-regulates the overexpression of P-gp after seizure, which indicates that NMDA receptor may be involved in the regulation of P-gp expression during seizure. Therefore, it is possible to prevent the overexpression of P-gp after seizure by inhibiting NMDA receptor's overactivation effectively.

Effects of ischemic preconditioning on blood-brain barrier permeability and MMP-9 expression of ischemic brain.

The study was designed to investigate the effects of ischemic preconditioning (IP) on permeability of blood-brain barrier (BBB) and expression of matrix metalloproteinase-9 (MMP-9) in subsequent ischemic hemisphere. Rats were divided into four groups, one group was used as control, and the other three groups were given three different pretreatments: the first group received a saline injection into the right internal carotid artery (SI), the second group underwent both left and right carotid arteries occlusion (BCAO), and the third group was treated with BCAO and SI simultaneously (BS). After 24 hours of pretreatments, the focal cerebral ischemia was induced by inserting a thread into the right middle cerebral artery causing occlusion (MCAO). Brain water content, BBB permeability and MMP-9 expression of ischemic hemisphere brains were measured at 24 and 48 hours after MCAO. After 24 and 48 hours MCAO, averages for brain water content were 82.92 and 83.12% in BS group, 85.19 and 85.73% in SI group and 86.06 and 85.88% in BCAO group. Evans blue content of ischemic hemispheres were 14.01 and 11.74 microg/mm(3) at 24 and 48 hours after MCAO in BS group, which were lower than the other two groups, 16.22, 15.01 and 16.61, 15.58 microg/mm(3), respectively (p<0.01). The expression levels of MMP-9 in ischemic hemisphere in BS were lower than that in other two groups (p<0.01). Therefore, ischemic preconditioning could ameliorate brain edema and BBB disruption caused by subsequent cerebral ischemia. Ischemic preconditioning could decrease MMP-9 protein and mRNA expression, which may be an important mechanism of cerebral ischemic tolerance.

[Neurodegenerative conformational disease and its molecular bases].

In recent years, the term "conformational disease" has been used to describe a range of disorders which are linked to misfolding and aberrant structural change in proteins. The molecular bases underlying the pathogenesis of neurodegenerative diseases are gradually being disclosed, and almost all of the diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease and prion disease are increasingly being realized to have common molecular mechanisms including the accumulation of misfolded or aggregation-prone proteins, thus, they can be termed "neurodegenerative conformational disease". There is now increased understanding of the molecular pathways involved in protein misfolding and aggregation and cellular toxicity in neurodegenerative conformational diseases. These are leading to approaches toward rational therapeutics.

The effects of the total saponin of Dipsacus asperoides on the damage of cultured neurons induced by beta-amyloid protein 25-35.

According to the pathogenesis of Alzheimer's disease (AD), beta-amyloid protein (A beta) was directly toxic to neurons, leading to neurodegeneration. Total saponin of Dipsacus asperoides (tSDA) is one of the main ingredients of Dipsacus asperoide, a traditional Chinese medicine. To explore the effects of tSDA on neuronal damage induced by A beta in vitro, biochemical analysis combining primary cultured neurons were adopted. Neurons were treated with 35 mmol/L A beta for 24 h, and tSDA at concentrations of 1-300 mg/L were added to A beta-treated cultures 24 h in advance, A beta for 24 h, the survival rate of neurons decreased closely by 50%. Lactate dehydrogenase release and the Malondialdehyde (MDA) level increased substantially. However, if neurons were pretreated with tSDA, the survival rate of neurons was higher than A beta-treated alone. Lactate dehydrogenase release and the MDA level decreased distinctly. Results demonstrated that tSDA possessed a neuroprotective action and that tSDA protected neurons against the toxicity of A beta, most likely by relieving oxidative stress or inhibiting the process of A beta, inducing free radical generation.

Expression and subcellular localization of P9-ZFD protein in patients with myasthenia gravis.

OBJECTIVE: To express and purify the protein coded by the TRAF-type zinc finger domain of myasthenia gravis (MG)-related gene P9 (P9-ZFD) and to prepare P9-ZFD antiserum for detecting expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of patient with MG. METHODS: The cDNA encoding P9-ZFD was amplified by RT-PCR. The cloned P9-ZFD cDNA was ligated into pET24a, and the P9-ZFD recombinant protein was induced via E. coli. BL21 (DE3) and purified by histidine affinity chromatography. P9-ZFD antiserum was prepared and its titer and specificity were determined by ELISA and Western blot. Expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of MG and control were studied. RESULTS: The molecular weight of purified P9-ZFD protein was about 30 kD. Its purity was more than 95%. Antiserum specific for P9-ZFD was excellent. P9-ZFD protein is fully confined to the cytoplasm membrane of skeletal muscle cell of MG, obvious immunostaining was absent in the A, I, and Z bands of cytoplasm and no immunoreactivity was observed in the skeletal muscle cell of control. CONCLUSION: P9-ZFD protein is expressed as a cytoplasm membrane-bound protein and has obvious distribution difference in the skeletal muscle cells of patient with MG and normal control.

[New mutations of the 12th exon of CCM1 gene in Chinese patients with intracranial cavernous angiomas].

OBJECTIVE: To study the effect of CCM1 gene mutations in Chinese patients with intracranial cavernous angiomas(ICCA). METHODS: Twenty-one ICCA patients confirmed by pathology after operations in hospital from June 2002 to Feb.2003 and 15 healthy individuals as contrast were recruited. The peripheral venous blood samples of all the individuals were collected, and then DNA was extracted from the blood samples followed by amplification of exon 12 and some of its intron sequence using PCR. After purification, the PCR products were directly sequenced by ABI PRISM377 sequencing instrument. RESULTS: Three mutations of CCM1 gene were found in 5 patients and reported firstly. There existed a missense mutation of 1172C-->T in exon 12 in 5 patients, which led the No.391 amino acid of KRIT1 protein, serine, to phenyalanine. There existed a missense mutation of 1160A-->C in one patient, which led the No.387 amino acid, glutamine, to proline. Another mutation was an intronic mutation of IVS12-4C-->T in 4 patients. In contrast no mutations were found. CONCLUSION: The authors firstly report that mutations of CCM1 gene in exon 12 also exist in Chinese ICCA patients and those mutations are related with the occurring of ICCA.

[Expression and tissue and species specificity of P25 protein in patients with myasthenia gravis].

OBJECTIVES: To confirm the specific decrease of P25 protein, a novel myasthenia gravis (MG) associated protein, in myasthenia gravis (MG) patients and to study the tissue and species specificity of its expression. METHODS: Samples of great pectoral muscle were taken from 28 MG patients, 17 males and 11 females, 10 of which being complicated with thymoma and 18 with hyperplasia of thymus, during thymectomy, and 24 wounded persons as normal controls, 14 males and 10 females, during thoracic surgery. Ten samples of skeletal muscle were taken from 10 patients with other muscular disorders (OMD) during skeletal muscle biopsy. Six samples of normal skeletal muscle, smooth muscle, brain, lung, kidney, and skin were taken from persons who died of accident. Two samples of normal cardiac muscle and thymus gland were taken from a patient undergoing cardiac valve replacement. Eight samples of animal skeletal muscle were taken from the thighs of pig, cattle, dog, rabbit, rat, mouse, chicken, and frog. Immunohistochemistry was used to examine the expression of P25 protein in the samples from the MG patients and the normal controls. Western blotting was used to detect the expression of P25 in the samples from MG patients, normal controls, OMD patients, 8 samples of different human tissues, and skeletal muscle samples from 8 different animals. RESULTS: Immunohistochemistry showed that the staining intensity of P25 protein in skeletal muscle of MG patients was much lower than that of the normal controls. Western blotting showed that the relative density value of P25 protein in MG patients was 1.04 +/- 0.18, significantly lower than those in the normal controls and OM patients (1.27 +/- 0.21 and 1.21 +/- 0.15 respectively. P < 0.01 and P < 0.05). No statistically significant difference in expression of P25 protein was found among different clinical and pathological types of MG patients. Among the 8 human tissues P25 protein was expressed only in skeletal muscle and among the 8 animal samples of skeletal muscle P25 protein expression was found only in swine skeletal muscle. CONCLUSION: The expression of P25 protein is significantly decreased in skeletal muscle of MG patients. The expression of P25 protein is tissue- and species specific.