In-vivo effects of nociceptin and its structural analogue [Orn9] nociceptin on the antioxidant status of rat blood and liver after carrageenan-induced paw inflammation

The production of reactive oxygen species (ROS) in cells is well balanced with their elimination by the antioxidant defence system. This balance is essential for maintenance of physiological conditions, and its disturbance (oxidative stress) has been suggested as a potential pathogenic mechanism in a variety of diseases, accompanied by inflammation. In this study, the in-vivo effects of nociceptin (N/OFQ(1–13)NH₂) and its structure analogue [Orn⁹]N/OFQ(1–13)NH₂ were studied on markers of oxidative stress in erythrocytes and liver of rats 4 hours after subplantar administration of carrageenan (CG) (1%, 100 µl) in the right hind paw. A considerable inflammatory oedema of the paw was observed. CG did not change blood haemoglobin content, hematocrit value, glutathione level and antioxidant enzyme activities in the erythrocytes, but there was an increase in lipid peroxidation. In liver, CG-induced imbalance was manifested by an increase in lipid peroxidation and a decrease in glutathione level. Both peptides (20 µg, i.p.), when administered alone, had no effect on all parameters tested. When either [Orn⁹]N/OFQ(1–13)NH₂ or N/OFQ(1–13)NH₂ was injected simultaneously with CG or 15 minutes before it, they did not affect the CG-induced changes in the antioxidant status of the erythrocytes and liver. Our results suggest that the peptides tested did not play a role in the free radical processes that accompany CG-induced paw inflammation.     

PRPS1基因功能缺失性突变导致X-连锁非综合征性DFN2型耳聋

本文报道了一个X连锁非综合征型语后聋的中国家系,该家系的致聋基因定位于X染色体一个遗传间距为5.41cM、物理距离为15.1 Mb的区域,与已知的DFN2位点重叠.对此家系及以前报道的3个国内外DFN2家系进行PRPS1基因突变筛查,鉴定出PRPS1基因的4种不同错义突变.通过对酶分子结构的分析,及来自于患者的红细胞和成纤维细胞的体外酶活性测定,证实这些突变可导致磷酸核楮焦磷酸(PRPP)合成酶1活性下降.通过原位杂交,我们证实Prvs1在小鼠的前庭和耳蜗毛细胞中皆有表达,并且在毛细胞中持续表达,出生后在螺旋神经节中仍有表达.PRPS1作为第二个X染色体上发现的非综合征型耳聋基因,是进行X连锁遗传性聋基因诊断很好的候选基因.     

Oral azathioprine leads to higher incorporation of 6-thioguanine in DNA of skin than liver: the protective role of the Keap1/Nrf2/ARE pathway

Azathioprine is a widely used anti-inflammatory, immunosuppressive, and anticancer agent. However, chronic treatment with this drug is associated with a profoundly increased risk (in certain cases by more than 100-fold) of developing squamous cell carcinoma of the skin. Incorporation of its ultimate metabolite, thio-dGTP, in DNA results in partial substitution of guanine with 6-thioguanine which, combined with exposure to UVA radiation, creates a source of synergistic mutagenic damage to DNA. We now report that oral treatment with azathioprine leads to a much greater incorporation of 6-thioguanine in DNA of mouse skin than liver. These higher levels of 6-thioguanine, together with the fact that the skin is constantly exposed to UV radiation from the sun, may be responsible, at least in part, for the increased susceptibility of this organ to tumor development. Genetic upregulation of the Keap1/Nrf2/ARE pathway, a major cellular regulator of the expression of a network of cytoprotective genes, reduces the incorporation of 6-thioguanine in DNA of both skin and liver following treatment with azathioprine. Similarly, pharmacologic activation of the pathway by the potent inducer sulforaphane results in lower 6-thioguanine incorporation in DNA and protects 6-thioguanine-treated cells against oxidative stress following exposure to UVA radiation. Protection is accompanied by increased levels of glutathione and induction of multidrug resistance-associated protein 4, an organic anion efflux pump that also exports nucleoside monophosphate analogues. Our findings suggest that activation of the Keap1/Nrf2/ARE pathway could reduce the risk for skin cancer in patients receiving long-term azathioprine therapy.     

A survey on patient educational needs in irritable bowel syndrome and attitudes toward participation in clinical research

BACKGROUND: The educational needs of patients with irritable bowel syndrome (IBS) are poorly understood and rarely studied. AIM: To determine the educational needs of IBS patients, regarding content, presentation format, and expectations from healthcare providers. METHODS: Fifteen functional GI clinic patients were asked open-ended questions to generate items for a questionnaire addressing the study aim. A total of 104 IBS patients received this questionnaire by mail (42 had declined to participate in a prior IBS study). To assess the frequency of endorsements and importance (on a scale of 1-3) of the items, an index was calculated (frequency of endorsements x mean rating per item, first priority scored 3, third priority scored 1). A higher index indicated greater endorsement based on frequency and rating of response. RESULTS: A total of 29 (28%) subjects (22 willing, 7 unwilling to participate previously in questionnaire research) completed the questionnaire (mean age, 42.6 years; SD, 14.2 years; 19 female, 10 male). The overall low response rate is likely related to the population studied; 40.4% of our study subjects have declined participation in prior research. The response rate of those who have previously agreed to participate was 36%. The typical response profile included: interest in learning disease management (index=1.4) and preference for information presented in person by an M.D. (2.4). Choice of presentation media included magazines (1.9), television (1.5), and Web sites (1.2). Doctors' qualities ranked high related to competency (0.8), allocation of sufficient time (0.7), and listening skills (0.4). Preferred incentives for research participation included a thank you note (0.4), summary of trial results (0.3), and monetary incentives (0.6). CONCLUSIONS: This qualitative study will provide pilot data for a national survey on the educational needs of IBS patients, for use in developing effective patient-centered, educational programs.     

Axonal neuropathy with optic atrophy is caused by mutations in mitofusin 2

OBJECTIVE: Charcot-Marie-Tooth (CMT) neuropathy with visual impairment due to optic atrophy has been designated as hereditary motor and sensory neuropathy type VI (HMSN VI). Reports of affected families have indicated autosomal dominant and recessive forms, but the genetic cause of this disease has remained elusive. METHODS: Here, we describe six HMSN VI families with a subacute onset of optic atrophy and subsequent slow recovery of visual acuity in 60% of the patients. Detailed clinical and genetic studies were performed. RESULTS: In each pedigree, we identified a unique mutation in the gene mitofusin 2 (MFN2). In three families, the MFN2 mutation occurred de novo; in two families the mutation was subsequently transmitted from father to son indicating autosomal dominant inheritance. INTERPRETATION: MFN2 is a mitochondrial membrane protein that was recently reported to cause axonal CMT type 2A. It is intriguing that MFN2 shows functional overlap with optic atrophy 1 (OPA1), the protein underlying the most common form of autosomal dominant optic atrophy, and mitochondrial encoded oxidative phosphorylation components as seen in Leber's hereditary optic atrophy. We conclude that autosomal dominant HMSN VI is caused by mutations in MFN2, emphasizing the important role of mitochondrial function for both optic atrophies and peripheral neuropathies.     

Eyeblink-related areas in human cerebellum as shown by fMRI

Classical eyeblink conditioning is used frequently to study the role of the cerebellum in associative learning. To understand the mechanisms involved in learning, the neural circuits that generate the eyeblink response should be identified. The goal of the present study was to examine cerebellar regions that are likely to control the human eyeblink response using event-related functional magnetic resonance imaging (fMRI). In 14 healthy volunteers eyeblinks were evoked by unilateral air-puff stimulation (total of 30 stimuli, inter-trial interval 27-44 sec). With eyes closed throughout the experiment, eyeblinks were recorded using a video-based system with infrared reflecting markers being attached to the upper eyelids. From each subject 500 scans were taken (TR = 2.2 sec, 22 slices per scan, slice thickness = 3 mm) using an echo planar imaging sequence (EPI). The statistical parametric maps of the experimental volume images were estimated with SPM99 specifying an appropriate event-related design matrix. Two main regions of significant activation were found in the ipsilateral posterior lobe of the cerebellar hemisphere. In the more anterior region the maxima of activation were located in hemispheral lobules VI and Crus I, and in the more posterior region in hemispheral lobules VIIb, Crus II and VIIIa (nomenclature according to Schmahmann et al. [2000]: MRI Atlas of the Human Cerebellum). Although less pronounced, activity was found also in corresponding areas of the contralateral cerebellar hemisphere. These eyeblink-related areas agree with trigeminal projection areas and blink reflex control areas shown in previous animal studies.     

Replication bypass of the acrolein-mediated deoxyguanine DNA-peptide cross-links by DNA polymerases of the DinB family

DNA-protein cross-links (adducts) are formed in cellular DNA under a variety of conditions, particularly following exposure to an alpha,beta-unsaturated aldehyde, acrolein. DNA-protein cross-links are subject to repair or damage-tolerance processes. These adducts serve as substrates for proteolytic degradation, yielding DNA-peptide lesions that have been shown to be actively repaired by the nucleotide excision repair complex. Alternatively, DNA-peptide cross-links can be subjected to replication bypass. We present new evidence about the capabilities of DNA polymerases to synthesize DNA past such cross-links. DNAs were constructed with site-specific cross-links, in which either a tetrapeptide or a dodecylpeptide was covalently attached at the N (2) position of guanine via an acrolein adduct, and replication bypass assays were carried out with members of the DinB family of polymerases, human polymerase (pol) kappa, Escherichia coli pol IV, and various E. coli polymerases that do not belong to the DinB family. Pol kappa was able to catalyze both the incorporation and the extension steps with an efficiency that was qualitatively indistinguishable from control (undamaged) substrates. Fidelity was comparable on all of these substrates, suggesting that pol kappa would have a role in the low mutation frequency associated with replication of these adducts in mammalian cells. When the E. coli orthologue of pol kappa, damage-inducible DNA polymerase, pol IV, was analyzed on the same substrates, pause sites were detected opposite and three nucleotides beyond the site of the lesion, with incorporation opposite the lesion being accurate. In contrast, neither E. coli replicative polymerase, pol III, nor E. coli damage-inducible polymerases, pol II and pol V, could efficiently incorporate a nucleotide opposite the DNA-peptide cross-links. Consistent with a role for pol IV in tolerance of these lesions, the replication efficiency of DNAs containing DNA-peptide cross-links was greatly reduced in pol IV-deficient cells. Collectively, these data indicate an important role for the DinB family of polymerases in tolerance mechanisms of N (2)-guanine-linked DNA-peptide cross-links.     

Consequences of p16 tumor suppressor gene inactivation in mycosis fungoides and Sézary syndrome and role of the bmi-1 and ras oncogenes in disease progression

In examining the expression of oncogenes and tumor suppressor genes in mycosis fungoides and Sézary syndrome, we found the cell cycle-regulating protein p16 to be absent in T cells. Immunohistochemical staining with p16-specific antibodies showed that the number of p16-expressing cells in cutaneous lesions decreases in late stages. The repression of p16 was not attributable to deletion or methylation of this gene; however, the Bmi-1 oncogene, a known suppressor of p16, was present in mycosis fungoides and Sézary syndrome cell lines and skin lesions. The absence of p16 correlated with the phosphorylation of the retinoblastoma protein on cyclin D/CDK4- or cyclin D/CDK6-specific sites. Ki-ras, which stimulates phosphorylation of retinoblastoma via cyclin-dependent kinases, was found in all tested cutaneous T-cell lymphoma samples; and its expression generally was stronger in advanced stages. Thus, cutaneous T-cell lymphoma cells show changes in oncogene and tumor suppressor gene expression that increase proliferation.