Detergent fractionation with subsequent subtractive suppression hybridization as a tool for identifying genes coding for plasma membrane proteins

Abstract:  The identification of tumor-specific proteins located at the plasma membrane is hampered by numerous methodological pitfalls many of which are associated with the post-translational modification of such proteins. Here, we present a new combination of detergent fractionation of cells and of subtractive suppression hybridization (SSH) to gain overexpressed genes coding for membrane-associated or secreted proteins. Fractionation of subcellular components by digitonin allowed sequestering mRNA of the rough Endoplasmatic reticulum and thereby increasing the percentage of sequences coding for membrane-bound proteins. Fractionated mRNAs from the cutaneous T-cell lymphoma (CTCL) cell line HuT78 and from normal peripheral blood monocytes were used for SSH leading to the enrichment of sequences overexpressed in the tumor cells. We identified some 21 overexpressed genes, among them are GPR137B, FAM62A, NOMO1, HSP90, SLIT1, IBP2, CLIF, IRAK and ARC. mRNA expression was tested for selected genes in CTCL cell lines, skin specimens and peripheral blood samples from CTCL patients and healthy donors. Several of the detected sequences are clearly related to cancer, but have not yet been associated with CTCL. qPCR confirmed an enrichment of these mRNAs in the rough endoplasmic reticulum fraction. RT-PCR confirmed the expression of these genes in skin specimens and peripheral blood of CTCL patients. Western blotting verified protein expression of HSP90 and IBP2 in HuT78. GPR137B could be detected by immunohistology in HuT78 and in keratinocytes of dysplastic epidermis, but also in sweat glands of healthy skin. In summary, we developed a new technique, which allows identifying overexpressed genes coding preferentially for membrane-associated proteins.     

Proton magnetic resonance spectroscopy in childhood brainstem lesions

Background Diagnosis of brainstem lesions in children based on magnetic resonance imaging alone is a challenging problem. Magnetic resonance spectroscopy (MRS) is a noninvasive technique for spatial characterization of biochemical markers in tissues and gives information regarding cell membrane proliferation, neuronal damage, and energy metabolism. Methods We measured the concentrations of biochemical markers in five children with brainstem lesions and evaluated their potential diagnostic significance. Images and spectra were acquired on a 1.5-T imager. The concentrations of N-acetylaspartate, tetramethylamines (e.g., choline), creatine, phosphocreatine, lactate, and lipids were measured within lesions located at the brainstem using Point-resolved spectroscopy sequences. Results Diagnosis based on localized proton spectroscopy included brainstem glioma, brainstem encephalitis, demyelination, dysmyelination secondary to neurofibromatosis type 1 (NF 1), and possible infection or radiation necrosis. In all but one patient, diagnosis was confirmed by biopsy or by clinical follow-up. Conclusions This small sample of patients suggests that MRS is important in the differential diagnosis between proliferative and nonproliferative lesions in patients without neurofibromatosis. Unfortunately, in cases of NF 1, MRS can have a rather misdiagnosis role.     

Central Sensitization Theory of Migraine: Clinical Implications

The clinical science of migraine headache continues to evolve. Theories of the pathophysiology of migraine have progressed from the early vascular basis of migraine to more complex current theories that emphasize the centrality of neuronal dysfunction. The most recently articulated theory of migraine is the central sensitization hypothesis, which proposes that altered processing of sensory input in the brainstem, principally the trigeminal nucleus caudalis, could account for many of the temporal and symptomatic features of migraine, as well as its poor response to triptan therapy when such treatment is initiated hours after the onset of pain. Both preclinical and clinical data support the central sensitization theory. A critical clinical implication of this theory is that drugs that are capable of either aborting or arresting the process of central sensitization, most prominently dihydroergotamine, may have a unique role in the treatment of migraine. An additional, and highly practical, implication is based upon the finding that cutaneous allodynia—pain arising from innocuous stimulation of the skin, as in hair brushing or the application of cosmetics—is an easily identifiable marker of central sensitization. Thus, the presence or absence of cutaneous allodynia can be integrated into the routine clinical assessment of migraine and utilized as a determinant of treatment. Future basic and clinical research on central sensitization is likely to be of ongoing importance to the field.     

Thrombotic occlusion of the common carotid artery (CCA) in acute ischemic stroke treated with intravenous tissue plasminogen activator (TPA)

Although common carotid artery (CCA) occlusions are rare, acute clinical presentations vary from mild to devastating strokes primarily due to tandem occlusions in the intracranial arteries. Three patients with acute CCA occlusions were treated with systemic tissue plasminogen activator (TPA). Blood pressures were kept at the upper limits allowed with TPA therapy with fluid balance and the 'head-down' position. Recanalization occurred in intracranial vessels only. Marked early neurological improvement occurred in two of three patients. CCA occlusions should not be considered contra-indication to systemic thrombolysis.     

Lower limb blocks for children

Femoral and lateral cutaneous nerve of the thigh blocks have been performed in a group of 50 children; the method has not previously been described in paediatric practice. The technique was judged to have been successful in 48 (96%) of the children. There were no early or late complications. It is concluded that these blocks are easy to perform, even in small children and infants, and that they can produce reliable postoperative analgesia for a variety of orthopaedic and plastic procedures.     

Cutaneous leishmaniasis: Clinical report of two cases and review of the recent literature

With 1–1.5 million cases reported every year cutaneous leishmaniasis represents an increasing health problem. The course of cutaneous leishmaniasis varies from a single self‐healing ulcer to a persistent ulcer or progressive mucosal disease with nasopharyngeal destruction. An enormous array of topical and systemic treatment modalities has been endorsed. The response to treatment depends on the species of parasite as well as the host's immunological and genetic status. Species‐specific treatment guidelines based on evidence from controlled studies are highly desirable. We present two cases of cutaneous leishmaniasis, one in a child and one during pregnancy, reviewing various diagnostic and therapeutic measures with special attention to problems in young and pregnant patients.     

结节性皮肤狼疮黏蛋白病

报告1例盘状红斑狼疮并发结节性皮肤狼疮黏蛋白病。患者男，45岁因而颈部红斑、皮肤萎缩20余年．背部、双上肢斑块、结节半年余就诊实验室检查抗核抗体（ANA）（＋），滴度1：320．抗ds—DNA抗体、抗Sm抗体均（-），背部皮损组织病理检查提示真皮中上部大量黏蛋白沉积。     

冠心病危险因素与冠状动脉病变程度的相关性研究

目的:探讨冠心病危险因素与冠状动脉病变程度的关系.方法:选取150例冠心病患者行冠状动脉造影术及各项化验检查,冠状动脉病变程度以Gensini积分值表示,对冠心病危险因素和冠状动脉病变程度进行统计分析.结果:经多因素线性回归证实:非高密度脂蛋白胆固醇为冠状动脉病变程度的独立危险因素(P=0.030),而性别、年龄、空腹血糖、糖尿病、高血压、吸烟、家族史及甘油三酯、低密度脂蛋白胆固醇均与冠状动脉病变程度无明确相关.结论:非高密度脂蛋白胆固醇对冠状动脉病变严重程度具有重要的影响.     

尖牙的生理性与病理性迁徙的临床分析

目的：探讨阻生尖牙与迁徙尖牙的联系、生理性迁徙或病理性迁徙与阻生尖牙的关系及迁徙尖牙的治疗方法。方法：对历年的X线片资料进行分析并复习有关文献。结果：阻生尖牙迁徙者有12例（上颌2例、下颌10例），其中生理性迁徙5例，病理性迁徙7例。结论：部分尖牙阻生可能为迁徙发生的早期阶段，尖牙迁徙包括生理性迁徙和病理性迁徙，尖牙生理性迁徙和病理性迁徙在发病原因、临床表现和治疗方法上都明显不同。     

新型经皮渗透促进剂的研究进展

介绍国内外近年来报道的新型经皮渗透促进剂的研究与开发进展,重点介绍若干具代表性的新合成的化合物以及新发现的天然产物,包括内酰胺类、糖苷类、氨基酸衍生物、聚合物、挥发油和酶类等,为合成和发现更加安全、高效的促渗剂提供线索.