日本血吸虫亲免素基因的扩增及序列分析

[目的]研究日本血吸虫亲免素基因编码序列的结构及进行初步的功能预测.[方法]用5'端和3'端锚定PCR从尾蚴文库中扩增,以获取日本血吸虫中国大陆株亲免素基因完整的开放阅读框(open readingframe,ORF);用生物信息学技术确认其是否为亲免素编码基因并进行结构功能的初步分析和预测.[结果]用5'端和3'端锚定PCR从尾蚴文库中获得了亲免素基因5'端和3'端所缺序列,得到的亲免素基因cDNA的总序列长为1 438 bp,其ORF长1 296 bp,编码431个氨基酸.利用生物信息学技术鉴定其为日本血吸虫亲免素基因的完整cDNA序列.并用RT-PCR从日本血吸虫尾蚴mRNA扩增出亲免素基因的ORF.[结论]成功获得了日本血吸虫亲免素编码基因的全长cDNA,为进一步的功能研究打下了基础.     

From Drosophila to Humans: Reflections on the Roles of the Prolyl Isomerases and Chaperones,Cyclophilins, in Cell Function and Disease

Abstract: Despite remarkable advances in human genetics and other genetic model systems, the fruit fly, Drosophila melanogaster, remains a powerful experimental tool to probe with ease the inner workings of a myriad of biological and pathological processes, even when evolutionary forces impart apparent divergences to some of such processes. The understanding of such evolutionary differences provides mechanistic insights into genotype-phenotype correlations underpinning biological processes across metazoans. The pioneering work developed by the William Pak laboratory for the past four decades, and the work of others, epitomize the notion of how the Drosophila system breaks new fertile ground or complements research fields of high scientific and medical relevance. Among the three major genetic complementation groups produced by the Pak's laboratory and impairing distinct facets of photoreceptor neuronal function, the nina group (ninaA, …., ninaJ) selectively affects the biogenesis of G protein–coupled receptors (GPCRs), mediating the photoconversion and transduction of light stimuli. Among the nina genes identified, ninaA arguably assumes heightened significance for several reasons. First, it presents unique physiological selectivity toward the biogenesis of a subset of GPCRs, a standalone biological manifestation yet to be discerned for most mammalian homologues of NinaA. Second, NinaA belongs to a family of proteins, immunophilins, which are the primary targets for immunosuppressive drugs at the therapeutic forefront of a multitude of medical conditions. Third, NinaA closest homologue, cyclophilin B (CyPB/PPIB), is an immunophilin whose loss-of-function was found recently to cause osteogenesis imperfecta in the human. This report highlights advances made by studies on some members of immunophilins, the cyclophilins. Finally, it reexamines critically data and dogmas derived from past and recent genetic, structural, biological, and pathological studies on NinaA and few other cyclophilins that support some of such paradigms to be less than definite and advance our understanding of the roles of cyclophilins in cell function, disease, and therapeutic interventions.     

亲免素结合类免疫抑制剂作用的分子机理

随着环孢素、FK5 0 6及雷帕霉素等生物制剂类免疫抑制剂在器官移植时的免疫抑制及治疗自身免疫性疾病的成功应用 ,对其生物作用机理有了一定认识。发现了生物体内存在其天然配基亲免素 ,并通过形成药物 亲免素复合物发挥免疫抑制作用 ,故又称为亲免素结合类免疫抑制剂。近年来研究证实 ,药物 亲免素复合物分别作用于细胞信号传递体系中的钙调磷酸酶与雷帕霉素靶蛋白 ,从而影响免疫细胞的蛋白质生物合成 ,细胞的增殖和生存 ,达到免疫抑制的作用。     

FK506-binding protein localizations in human penile innervation

OBJECTIVE

To determine if FK506‐binding proteins (FKBPs) are localized to the autonomic nerve supply of the human penis because FK506 (an immunosuppressant drug) has been linked to enhanced nerve regeneration after nerve injury and neurodegenerative diseases by binding to FKBPs, a select group of immunophilins.

MATERIALS AND METHODS

Human lower genitourinary tract specimens were obtained and embedded in paraffin wax. The tissue was sectioned (10 µm) and processed for immunohistochemistry using antibodies for FKBPs 12, 38, 52, 65, 135 and neuronal nitric oxide synthase (nNOS). To confirm specificity of the antibody, we processed some tissue in the absence of primary antibody, with mouse or rabbit IgG, and with a blocking peptide for FKBP12.

RESULTS

In the pelvis, immunoreactivity for all the FKBPs and nNOS was localized to the periprostatic ganglia although FKBP12 was the only FKBP localized to nerve bundles in this location. In penile tissue, immunoreactivity for all five FKBPs and nNOS was localized to nerves, although immunoreactivity for FKBP38 was minimal. The FKBPs were also evident in epithelial, endothelial and smooth muscle cells of the prostate and penis. Negative controls did not produce staining.

CONCLUSIONS

Identification and localization of immunophilins to nerves coursing in prostate and penile tissue suggest a likely molecular basis to apply immunophilin ligand therapy to protect or regenerate cavernosal nerves. Our findings support the hypothesis that immunosuppressant drugs such as FK506, working via specific receptor mechanisms, are potentially useful to sustain erectile function in men after radical prostatectomy.     

Neuroimmunophilin ligands: evaluation of their therapeutic potential for the treatment of neurological disorders

Neuroimmunophilin ligands are a class of compounds that hold great promise for the treatment of nerve injuries and neurological disease. In contrast to neurotrophins (e.g., nerve growth factor), these compounds readily cross the blood-brain barrier, being orally effective in a variety of animal models of ischaemia, traumatic nerve injury and human neurodegenerative disorders. A further distinction is that neuroimmunophilin ligands act via unique receptors that are unrelated to the classical neurotrophic receptors (e.g., trk), making it unlikely that clinical trials will encounter the same difficulties found with the neurotrophins. Another advantage is that two neuroimmunophilin ligands (cyclosporin A and FK-506) have already been used in humans (as immunosuppressant drugs). Whereas both cyclosporin A and FK-506 demonstrate neuroprotective actions, only FK-506 and its derivatives have been clearly shown to exhibit significant neuroregenerative activity. Accordingly, the neuroprotective and neuroregenerative properties seem to arise via different mechanisms. Furthermore, the neuroregenerative property does not involve calcineurin inhibition (essential for immunosuppression). This is important since most of the limiting side effects produced by these drugs arise via calcineurin inhibition. A major breakthrough for the development of this class of compounds for the treatment of human neurological disorders was the ability to separate the neuroregenerative property of FK-506 from its immunosuppressant action via the development of non-immunosuppressant (non-calcineurin inhibiting) derivatives. Further studies revealed that different receptor subtypes, or FK-506-binding proteins (FKBPs), mediate immunosuppression and nerve regeneration (FKBP-12 and FKBP-52, respectively, the latter being a component of steroid receptor complexes). Thus, steroid receptor chaperone proteins represent novel targets for future drug development of novel classes of compounds for the treatment of a variety of human neurological disorders, including traumatic injury (e.g., peripheral nerve and spinal cord), chemical exposure (e.g., vinca alkaloids, Taxol?) and neurodegenerative disease (e.g., diabetic neuropathy and Parkinson’s disease).     

Increases in [3H]FK-506 and [3H]L-NG-Nitro-Arginine Binding in the Rat Brain After Nigrostriatal Dopaminergic Denervation

Receptor autoradiographic technique was studied to investigate sequential changes in FK-506 binding proteins, nitric oxide synthase and dopamine uptake sites in the brain 1 week to 8 weeks after unilateral 6-hydroxydopamine injection of the medial forebrain bundle in rats. [³H]FK-506, [³H]L-N^G-nitro-arginine and [³H]mazindol were used to label FK-506 binding proteins (immunophilin), nitric oxide synthase and dopamine uptake sites, respectively. [³H]FK-506 binding showed about 13-25% increase in the ipsilateral striatum from 2 to 8 weeks after degeneration of nigrostriatal pathway. However, no significant change in [³H]FK-506 binding was observed in the ipsilateral substantia nigra during the postlesion periods. In the contralateral side, [³H]FK-506 binding also showed about 13-25% increase in the striatum from 2 to 8 weeks postlesion. The substantia nigra showed a 21% increase in [³H]FK-506 binding only 2 weeks after the lesioning. On the other hand, [³H]L-N^G-nitro-arginine binding showed about 21-31% increase in the parietal cortex and striatum 1 week or 2 weeks postlesion. In the contralateral side, a 21% increase in [³H]L-N^G-nitro-arginine binding was found in the dorsolateral striatum only 1 week postlesion. In contrast, degeneration of nigrostriatal pathway caused a conspicuous loss of [³H]mazindol binding in the ipsilateral striatum (87-96%), substantia nigra (36-73%) and ventral tegmental area (91-100%) during the postlesion periods. In the contralateral side, no significant changes in [³H]mazindol binding were observed in these areas upto 8 weeks after the postlesion. The present study demonstrates that unilateral injection of 6-hydroxydopamine into the medial forebrain bundle of rats can cause a significant increase in [3H]FK-506 and [³H]L-N^G-nitro-arginine bindings in the brains. In contrast, a marked reduction in [³H]mazindol binding is observed in the brains after the lesioning, indicating severe damage to nigrostriatal dopaminergic pathway. These results suggest that immunophilin and nitric oxide synthase may play some role in the pathogenesis of neurodegenerative disorders such as Parkinson's disease.     

Emerging neuromodulatory molecules for the treatment of neurogenic erectile dysfunction caused by cavernous nerve injury

Advances in the neurobiology of growth factors, neural development, and prevention of cell death have resulted in a heightened clinical interest for the development of protective and regenerative neuromodulatory strategies for the cavernous nerves （CNs）, as therapies for prostate cancer and other pelvic malignancies often result in neuronal damage and debilitating loss of sexual function. Nitric oxide released from the axonal end plates of these nerves within the corpora cavernosa causes relaxation of smooth muscle, initiating the haemodynamic changes of penile erection as well as contributing to maintained tumescence; the loss of CN function is primarily responsible for the development of erectile dysfunction （ED） after pelvic surgery and serves as the primary target for potential neuroprotective or regenerative strategies. Evidence from pre-clinical studies for select neuromodulatory approaches is reviewed, including neurotrophins, glial cell line-derived neurotrophic factors （GDNF）, bone morphogenic proteins, immunophilin ligands, erythropoetin （EPO）, and stem cells.     

Diabetic neuropathy: therapies on the horizon

Objectives This is a review of emerging interventions from the recent preclinical and clinical literature that demonstrate the potential for effectiveness in the therapy of diabetic neuropathy (DN). DN is the most common complication of diabetes mellitus and up to 50% of patients with type 1 and type 2 forms have some or other form of neuropathy. The pathology of DN is characterized by progressive nerve fibre loss that gives rise to positive and negative clinical signs and symptoms such as pain, paraesthesiae and loss of sensation. Key findings There are very few drugs available to directly treat DN. Those that are clinically indicated provide symptomatic relief but do not repair or reverse underlying nerve damage. However, some agents are in clinical development that may support adult neurons and direct reparative processes after injury stages. Several disease modifying drugs such as aldose reductase inhibitors and protein kinase C inhibitors are in phase III development. Agents on the horizon include neurotrophic factors, growth factors, gene therapy, immunotherapy, poly(ADP‐ribose) polymerase inhibitors and non‐immunosuppressive immunophilin ligands. Summary Progress has been made toward understanding the biochemical mechanisms leading to diabetic neuropathy, and as a result, new treatment modalities are being explored. The pathogenesis, types and approaches for treating DN together with the newer therapeutic interventions on the horizon are discussed.     

Non-FK506-binding protein-12 neuroimmunophilin ligands increase neurite elongation and accelerate nerve regeneration

Neurotrophic activity of neuroimmunophilin ligands (FK506 and its nonimmunosuppressant derivatives) has been assumed to be mediated by the FK506-binding protein-12 (FKBP-12). We recently showed that activity is retained in hippocampal neurons from FKBP-12 knockout mice, indicating that binding to FKBP-12 is not necessary. Here we show that three nonimmunosuppressant FK506 derivatives (V-13,450, V-13,629, and V-13,670) that do not bind FKBP-12 (>12.5 mM affinity) are equipotent to FKBP-12 ligands (FK506, V-10,367, and V-13,449) for increasing neurite elongation in SH-SY5Y cells. One non-FKBP-12 ligand (V-13,670) is also shown to accelerate functional recovery and nerve regeneration in the rat sciatic nerve crush model. Surprisingly, it exhibited an unusual dose-response effect upon oral administration, showing a novel bimodal dose-response for behavioral functional recovery and myelination, but not for axonal size, suggesting both Schwann cell and neuronal targets. Orally active non-FKBP-12 neuroimmunophilin ligands may be useful for the treatment of human neurological disorders without any potential side effects resulting from FKBP-12 binding.