AVP_(4-8)增加海马组织内NGF mRNA和蛋白含量

本文采用了Ｎｏｒｔｈｅｒｎ杂交和ＥＬＩＳＡ法，观察了神经肽ＡＶＰ４－８对大鼠海马组织中神经生长因子（ＮＧＦ）ｍＲＮＡ和蛋白水平的影响，结果显示：给大鼠皮下注射ＡＶＰ４－８１２ｈ后，海马组织中ＮＧＦｍＲＮＡ的水平明显高于生理盐水对照组。而给大鼠皮下注射精氨酸加压素（ＡＶＰ）和催产素（ＯＴ）对ＮＧＦｍＲＮＡ的水平均无显著影响。另外，用ＡＶＰ４－８孵育离体大鼠海马组织切片，能使ＮＧＦ蛋白表达量升高，且在４．５ｈ左右达到高峰。     

Immunohistochemical presence of 5 α-reductase rat type I -containing cells in the rat brain

We showed immunohistochemically the localization of 5 α-reductase-containing cells in the rat brain, using a rabbit antibody generated against 5α-reductase rat type 1. The antibody was produced by injecting the synthetic peptide corresponding to the amino acids 38–53 of 5α-reductase rat type 1, conjugated to keyhole limpet hemocyanin with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Western blot analysis revealed that this antiserum recognized the protein with a molecular weight of 29 000 Da. The inununoreactive cells were distributed throughout the brain and they were preferentially located in the white matter rather than in the grey matter. These cells were mostly small and round and had a few fine processes. The immunoreaction was confined to the cytoplasm and processes. These findings indicate that 5α-reductase rat type 1-containing cells are widely distributed in the rat brain and are located preferentially in the white matter rather than in the grey matter.     

Application of DNA filter hybridization and PCR to distinguish between human and non-human tissues of poor quality

Summary The present report describes a novel approach for the identification of human or non-human specimens after long-term storage in a badly preserved state. The application of the PCR-technique (polymerase chain reaction) using human-specific primers as well as Southern blot (filter) hybridization of the sample DNA to a primate-specific DNA probe enabled us to extend the positive identification beyond the limits of conventional methods such as serological or morphological examinations.     

Ca2+/CaM-sensitive adenylyl cyclase activity is decreased in the Alzheimer's brain: Possible relation to type I adenylyl cyclase

Summary Immunoreactivities of four subtypes of adenylyl cyclase (AC) (types I, II, IV and V/VI), and basal, forskolin- and Mn²⁺-stimulated AC activities with or without calcium and calmodulin (Ca²⁺/CaM) were estimated in parietal cortex membranes from cases with dementia of the Alzheimer type (DAT) and age-matched controls. Immunoreactivities of AC-I and AC-II were significantly decreased, but those of AC-IV and AC-V/VI did not change in DAT brains. There was a significant correlation of AC-I immunoreactivity with Ca²⁺/CaM-sensitive AC activity, but not with the Ca²⁺/CaM-insensitive activity. Ca²⁺/CaM-sensitive AC activity was significantly lower in DAT than in the control, indicating that impairment of Ca²⁺/CaM-sensitive AC-I is clearly involved in the pathophysiology of DAT.     

Expression of pendrin in benign and malignant human thyroid tissues

The Pendred syndrome gene (PDS) encodes a transmembrane protein, pendrin, which is expressed in follicular thyroid cells and participates in the apical iodide transport. Pendrin expression has been studied in various thyroid neoplasms by means of immunohistochemistry (IHC), Western blot and RT-quantitative real-time PCR. The expression was related to the functional activity of the thyroid tissue. Follicular cells of normal, nodular goitre and Graves' disease tissues express pendrin at the apical pole of the thyrocytes. In follicular adenomas, pendrin was detected in cell membranes and cytoplasm simultaneously in 10 out of 15 cases. Pendrin protein was detected in 73.3 and 76.7% of the follicular (FTC) and papillary (PTC) thyroid carcinomas, respectively, where pendrin was solely localised inside the cytoplasm. An extensive intracellular immunostaining of pendrin was observed in six out of 11 (54.5%) of positive FTCs and 19 out of 23 (82%) of PTCs. Focal reactivity was detected in one follicular- and three papillary carcinomas, whereas pendrin protein was absent in three of 15 FTC and four of 30 PTC; mRNA of pendrin was detected in 92.4% of thyroid tumours. The relative mRNA expression of pendrin was lower in cancers than in normal thyroid tissues (P<0.001). The pendrin protein level was found to parallel its mRNA expression, which was not, however, related to the tumour size and tumour stage. In conclusion, pendrin is expressed in the majority of differentiated thyroid tumours with high individual variability but its targeting to the apical cell membrane is affected.     

Expression of KIT, EGFR, HER-2 and tyrosine phosphorylation in undifferentiated thyroid carcinoma: implication for a new therapeutic approach

The KIT, epidermal growth factor receptor (EGFR) and HER-2 oncoproteins have tyrosine kinase activity and are molecular targets in human cancer therapy. To clarify the significance of KIT, EGFR, and HER-2 in undifferentiated thyroid carcinoma (UTC), the expression of these receptors and tyrosine phosphorylation was examined immunohistochemically in resected cases of UTC and papillary thyroid carcinoma (PTC). KIT, EGFR, and HER-2 were also examined at the protein and mRNA levels in five UTC cell lines. KIT expression (1+), EGFR overexpression (2+/3+), HER-2 expression (1+), and tyrosine phosphorylation were detected immunohistochemically in 40%, 70%, 10%, and 50% of the 10 UTC. In 20 PTC, KIT, EGFR, and HER-2 were not detected, but tyrosine phosphorylation was detected in 25% of cases. In the five UTC cell lines, KIT expression (1+), EGFR overexpression (3+), HER-2 expression (1+), and tyrosine phosphorylation were detected immunocytochemically in 60%, 100%, 20%, and 40%, respectively. Western blot analysis did not detect KIT expression, but did detect EGFR and HER-2 expression in all five cell lines. Real-time polymerase chain reaction detected KIT mRNA in two of the cell lines (40%), EGFR in five (100%), and HER-2 in three (60%). The present findings suggest that EGFR overexpression was involved in the proliferation and development of UTC and was frequently accompanied by tyrosine phosphorylation. Expression of KIT and HER-2 appeared to be weak but significant, suggesting a possible role in the development of UTC. Molecular therapies targeting KIT, EGFR, HER-2, and/or tyrosine phosphorylation might be indicated for UTC.     

Levothyroxin restores hypothyroidism-induced impairment of LTP of hippocampal CA1: electrophysiological and molecular studies

Hypothyroidism impairs synaptic plasticity as well as learning and memory. Clinical reports are conflicting about the ability of thyroid hormone replacement therapy to fully restore the hypothyroidism-induced learning and memory impairment. Recently, we have shown that hypothyroidism impairs LTP and cognition in adult rats. We have studied the effect of thyroxin replacement therapy on hypothyroidism-induced LTP impairment using electrophysiological and molecular approaches. Recording from CA1 region of the hippocampus in anesthetized adult rat indicated that 6 weeks of thyroxin replacement therapy (20 microg/kg/day) fully restored LTP impaired by hypothyroidism. Western blotting showed reduction in phosphorylated (P)-CAMKII, total-CaMKII, neurogranin, and calmodulin basal levels in the CA1 region of the hippocampus of hypothyroid rats. The levels of these molecules were normalized by thyroxin replacement therapy. The hypothyroid-induced elevation of basal calcineurin levels and activity was also normalized by thyroxin treatment. However, thyroxin replacement therapy did not restore hypothyroidism-induced reduction in PKCgamma basal protein levels. Additionally, real-time PCR, showed a reduction in basal neurogranin mRNA level that was normalized by thyroxin replacement therapy. In the sham (control) rats, induction of LTP by high-frequency stimulation increases P-CaMKII, and total CaMKII levels as well as CaMKII phosphotransferase activity. However, in hypothyroid rats, the same stimulation protocol induced an increase only in total-CaMKII. Thyroxin treatment normalized the levels and activity of these molecules. The results demonstrated that thyroxin therapy normalized the electrophysiological and molecular effects of hypothyroidism on the CA1 region and emphasized the critical role P-CaMKII plays in hypothyroidism-induced LTP impairment.     

Proteomics approaches to identify tumor antigen directed autoantibodies as cancer biomarkers

The identification of autoantibodies to tumor cell proteins by proteomics approaches has great potential impact on cancer biomarker discovery. The humoral immune response represents a form of biological amplification of signals that are otherwise weak due to very low concentrations of antigen, especially in the early stages of cancers. In addition, proteomics can detect immunoreactivity directed against protein post-translational modifications. Two-dimensional gel based Western blots, protein antigen microarrays, and multiplex ELISA reactions have been applied by our group to antigen based biomarker detection and validation. The latter two are based on liquid-phase separations that are suitable for automation. This work has resulted in the identification of numerous cancer biomarker candidates. Large clinical studies are currently planned to establish their value in early cancer diagnosis.     

2-Chlorodeoxyadenosine alone and in combination with cyclophosphamide and mitoxantrone induce apoptosis in B chronic lymphocytic leukemia cells in vivo

The purpose of the study was to determine some apoptotic events in mononuclear cells obtained from peripheral blood of patients with B-cell chronic lymphocytic leukemia (B-CLL) during and after therapy with 2-chlorodeoxyadenosine (2-CdA; C), and the combination of 2-CdA with cyclophosphamide (CC), or 2-CdA with mitoxantrone and cyclophosphamide (CMC). Western blot technique was performed to estimate expression/proteolytic degradation of generally accepted apoptotic markers, i.e., Bcl-2 protein, lamin B, PARP-1, and caspase-3 in leukemic cells isolated from blood samples of patients before treatment and subjected to drug(s) administration. The decrease of antiapoptotic protein Bcl-2 expression and proteolytic cleavage of nuclear proteins--lamin B and PARP-1 were observed in leukemic cells of patients treated according to the above therapy protocols, however, each to a different level among the studied groups. The obtained results indicated also that procaspase-3 was cleaved and activated in leukemic cells of three drug(s) treated groups. However, the cleavage of procaspase-3 and the generation of fragments with mol. mass of 17/20 kDa occurred especially effectively among patients treated according to CMC regimen. The changes in expression/proteolytic degradation of the above selected apoptotic markers, are accompanied by the appearance of apoptotic morphology in leukemic cells originated from blood of patients treated with the above drug(s) in comparison to untreated ones.