SH2A 基因对细胞信号转导的影响及其亚细胞定位

目的 研究Src同源域2(src homology 2，SH2)A基因在细胞信号转导中的作用并进行亚细胞定位。方法 通过RT—PCR方法扩增SH2A cDNA编码序列，构建真核重组表达载体pcDNA3．1-SH2A，利用脂质体转染肝癌Bel7402细胞、COS7细胞，检测蛋白酶C(protein kinaseC，PKC)、酪氨酸蛋白激酶(tyrosine protein kinase，TPK)、丝裂原激活蛋白激酶(mitogen activated protein kinase，MAPK)活性的改变；另构建pEGEP—SH2A，转染同前，荧光显微镜观察荧光定位。结果 测序结果显示真核重组表达载体pcDNA3．1-SH2A及pEGFP—SH2A中均含有SH2AcDNA编码序列；肝癌Bel7402细胞、COS7细胞转染pcDNA3．1-SH2A后，胞浆PKC的活性下降了40％左右，MAPK和TPK活性未见明显改变。荧光显微镜观察发现SH2A基因在细胞质中表达。结论 SH2A基因编码蛋白在PKC信号转导通路中起抑制作用；SH2A基因编码蛋白定位于细胞质。     

Molecular pathology of NEU1 gene in sialidosis

Lysosomal sialidase (EC 3.2.1.18) has a dual physiological function; it participates in intralysosomal catabolism of sialylated glycoconjugates and is involved in cellular immune response. Mutations in the sialidase gene NEU1, located on chromosome 6p21.3, result in autosomal recessive disorder, sialidosis, which is characterized by the progressive lysosomal storage of sialylated glycopeptides and oligosaccharides. Sialidosis type I is a milder, late-onset, normosomatic form of the disorder. Type I patients develop visual defects, myoclonus syndrome, cherry-red macular spots, ataxia, hyperreflexia, and seizures. The severe early-onset form, sialidosis type II, is also associated with dysostosis multiplex, Hurler-like phenotype, mental retardation, and hepatosplenomegaly. We summarize information on the 34 unique mutations determined so far in the sialidase gene, including four novel missense and one novel nonsense mutations found in two Czech and two French sialidosis patients. The analysis of sialidase mutations in sialidosis revealed considerable molecular heterogeneity, reflecting the diversity of clinical phenotypes that make molecular diagnosis difficult. The majority of sialidosis patients have had missense mutations, many of which have been expressed; their effects on activity, stability, intracellular localization, and supramolecular organization of sialidase were studied. A structural model of sialidase allowed us to localize mutations in the sialidase molecule and to predict their impact on the tertiary structure and biochemical properties of the enzyme.     

Study of CYP2D6 gene in children with autoimmune hepatitis and P450 IID6 autoantibodies.

Cytochrome P450 IID6 is an autoantigen recognized by the sera of children affected with a subtype of autoimmune hepatitis. It was hypothesized that a mutation in the CYP2D6 gene could explain the autoimmune response in these patients. To examine this question, genomic DNA from peripheral lymphocytes (n = 9) and liver (n = 1) of 10 patients with anti-LKM-1 antibody was analysed by Southern blot for genetic association studies between a particular CYP2D6 haplotype and autoimmune hepatitis. In addition, a region of CYP2D6, from the same genomic DNA, was amplified by polymerase chain reaction (PCR) and digested by BstNI, in a search for the most prevalent 29B mutation, described in subjects who do not express the P450 IID6. Total RNA and proteins, prepared from the liver of an anti-LKM-1+ patient, were analysed by Northern and Western (immunoblot) blots respectively. Our results do not reveal any major structural change in the DNA of this patient at the CYP2D6 locus that could explain their autoimmune response. Corroborating this observation, no changes were noted either in P450 IID6 mRNA size or in the corresponding protein. However, these data do not exclude the possibility of subtle changes in the protein due to point mutations in critical regions that might trigger an autoimmune response.     

A functionally active complement system is present in uterine secretion of the mouse prior to implantation.

Sephadex beads were placed carefully in the uterus on days 2 and 3 and left for 6 to 8 h to absorb uterine secretion. The beads were then removed with volatile silicon oil and mounted on small pieces of nitrocellulose paper. Immuno-staining of these bead blots showed they contained the complement components C1q, C3, C4, and C5. We demonstrated that complement component C3 in the uterine secretion could be activated and deposited on model immune complexes, and also that antibody-coated erythrocytes were lysed in utero, that is, a membrane attack complex was produced. Thus, the mouse uterine secretion at the preimplantation stage contains a functionally active complement system.     

Histidine-rich protein genes and their transcripts in Plasmodium falciparum and P. lophurae

The presence of histidine-rich protein (HRP) related genes and gene products in Plasmodium falciparum was demonstrated using a synthetic pentahistidine-encoding oligonucleotide and a cloned HRP cDNA probe prepared from the avian parasite P. lophurae. In Northern blotting experiments, two knobby clones of P. falciparum were found to contain a 3500 nucleotide RNA species that hybridized with the oligonucleotide and HRP cDNA probes. As this component had the expected size for an mRNA encoding an 80-90 kDa protein and was absent from two knobless clones of P. falciparum, we concluded that it represented a 'knob protein' mRNA. Using the restriction enzyme EcoRI, three identical cross-hydribizing HRP gene fragments were found in the DNA of both knobby and knobless clones of P. falciparum. These fragments differed in size from those present in P. lophurae. These results suggest that the absence of knob protein mRNA in knobless clones is not due to loss of the corresponding gene(s).     

The instability of membrane markers expressed by human monocytes and macrophages in culture

Surface markers were tested on freshly isolated human monocytes and following their in vitro maturation to macrophages. The markers tested were HLA-DR antigens, receptors for the Fc of IgG and complement as well as membrane markers defined by monoclonal antibodies. The results revealed a dynamic expression of some of the markers on monocytes which was influenced by several variables. The expression of the markers was modulated by the presence of different sera, by treatment with lymphokines and interferon and following the in vitro maturation of monocytes to macrophages. The most unstable marker was found to be the HLA-DR, which was modulated by all these variables. The 63D3 was affected by different sera and culture supernatant, as well as following the maturation of monocytes to macrophages, but not by lymphokines and interferon. One of the markers, the Mac 120, was found to be relatively stable and did not change significantly following the maturation of monocytes to macrophages. The Fc and complement receptors were also stable in their expression under these conditions, but were probably partially blocked in the presence of human serum. These results indicated that at least some of the heterogeneity related to the monocyte population was probably not due to the occurrence of stable subsets of cells, but rather to reversible changes in marker expression.     

Immunohistochemical analysis of centromere protein F expression in buccal and gingival squamous cell carcinoma

Centromere protein F (CENP-F) expression (localization and characteristics) in relation to tumor clinicopathological parameters was immunohistochemically examined and evaluated in 47 archival biopsy specimens of buccal and gingival squamous cell carcinomas (SCC). Centromere protein F expression was detected in 79% of the samples. An increase in the labeling index (LI) with WHO grading was obtained ( P  < 0.05). Correlations were obtained between the CENP-F LI and tumor size ( P  < 0.05). Immunoelectron microscopy showed CENP-F nuclear staining as punctate or fine dots. The present study shows that CENP-F expression and detection of a more specific cell subpopulation presents a theoretical advantage for the analysis of the precise cell cycle of G₂ to M cells, compared to Ki-67.     

Regulation of smooth muscle actin—myosin interaction and force by calponin

Smooth muscle contraction is regulated primarily by the reversible phosphorylation of myosin triggered by an increase in sarcoplasmic free Ca²⁺ concentration ([Ca²⁺]_i). Contraction can, however, be modulated by other signal transduction pathways, one of which involves the thin filament-associated protein calponin. The h1 (basic) isoform of calponin binds to actin with high affinity and is expressed specifically in smooth muscle at a molar ratio to actin of 1: 7. Calponin inhibits (i) the actin-activated MgATPase activity of smooth muscle myosin (the cross-bridge cycling rate) via its interaction with actin, (ii) the movement of actin filaments over immobilized myosin in the in vitro motility assay, and (iii) force development or shortening velocity in permeabilized smooth muscle strips and single cells. These inhibitory effects of calponin can be alleviated by protein kinase C (PKC)-catalysed phosphorylation and restored following dephosphorylation by a type 2A phosphatase. Three physiological roles of calponin can be considered based on its in vitro functional properties: (i) maintenance of relaxation at resting [Ca²⁺]_i, (ii) energy conservation during prolonged contractions, and (iii) Ca²⁺-independent contraction mediated by phosphorylation of calponin by PKCε, a Ca²⁺-independent isoenzyme of PKC.