assessment-dendritic-arborization-dentate-gyrus-hippocampal-region

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

The major high molecular weight complex of aminoacyl-tRNA synthetases is purified about 1000-fold with 30% yield from rat liver. The synthetase complex sediments at 24 S with a molecular weight of 900,000 +/- 75,000 and contains aminoacylation activities for lysine, arginine, isoleucine, leucine, methionine, glutamine, glutamate, and proline. The 24 S synthetase complex dissociates into 21 S, 18 S, 13 S, 12 S, and 10 S complexes with specific enzymatic activities. Dissociation of the 24 S complex into active free synthetases is achieved by hydrophobic interaction chromatography. The disassembly of the synthetase complex is consistent with the structural model of a heterotypic multienzyme complex and suggests that the complex formation is due to the specific intermolecular interactions among the synthetases.

Disassembly and gross structure of particulate aminoacyl-tRNA synthetases from rat liver. Isolation and the structural relationship of synthetase complexes.

Maximum in the stability of the amorphous state in the system water--glycerol--ethanol.

We describe a strategy and reagents for study of protein-protein interactions in mammalian cells, termed the karyoplasmic interaction selection strategy (KISS). With this strategy, specific protein-protein interactions are identified by reconstitution of the functional activity of the yeast transcriptional activator GAL4 and the resultant transcription of a GAL4-regulated reporter gene. Reconstitution of GAL4 function results from specific interaction between two chimeric proteins: one contains the DNA-binding domain of GAL4; the other contains a transcriptional activation domain. Transcription of the reporter gene occurs if the two chimeric proteins can form a complex that reconstitutes the DNA-binding and transcriptional activation functions of GAL4. Using the KISS system, we demonstrate specific interactions for sequences from three different pairs of proteins that complex in the cytoplasm. In addition, we demonstrate that reporter genes encoding cell surface or drug-resistance markers can be specifically activated as a result of protein-protein interactions. With these selectable markers, the KISS system can be used to screen specialized cDNA libraries to identify novel protein interactions.

Karyoplasmic interaction selection strategy: a general strategy to detect protein-protein interactions in mammalian cells.

Arginyl- and lysyl-tRNA synthetases copurify throughout a six-step chromatographic procedure resulting in a purification of 605- and 559-fold, respectively. The purified enzymes were estimated to be 98% pure with a stoichiometry of 1:1 from acrylamide gel electrophoresis under denaturing conditions. On the basis of a native molecular weight of 285000 calculated from s20,w, Rs, and V and subunit molecular weights of 73000 and 65000 obtained by sodium dodecyl sulfate gel electrophoresis, the synthetases appear to exist as a tetramer. The tetrameric structure was also supported by cross-linking studies. These results are consistent with an alpha 2 beta 2 structure, but an alpha beta structure has not been ruled out.

Physical and biochemical characterization of a purified arginyl-tRNA synthetase-lysyl-tRNA-synthetase complex from rat liver.

Characterization of a homogeneous arginyl- and lysyl-tRNA synthetase complex isolated from rat liver. Arginyl- and lysyl-tRNA synthetases contain carbohydrates.

The major aminoacyl-tRNA synthetase complex (the 24 S complex) isolated from rat liver, which contains lysyl-, leucyl-, and arginyl-tRNA synthetase activities, is dissociated into fully active free aminoacyl-tRNA synthetases by column chromatography on diaminooctyl-Sepharose. During the hydrophobic interaction chromatography, more than a quantitative yield of the lysyl-tRNA synthetase activity is obtained. The free lysyl-tRNA synthetase, dissociated from the synthetase complex, is purified about 2,000-fold with a 13% yield by conventional column chromatography. Lysyl-tRNA synthetase is also purified from the 24 S synthetase complex by affinity column chromatography on lysyl-diaminohexyl-Sepharose. Free lysyl-tRNA synthetase as dissociated from the synthetase complex, is evidently a dimeric enzyme with a subunit molecular weight of 66,000 +/- 3,000, as determined by gel electrophoresis, sucrose gradient centrifugation and gel filtration.

Purification and characterization of lysyl-tRNA synthetase after dissociation of the particulate aminoacyl-tRNA synthetases from rat liver.

The presence of circulating factors in the plasma of shocked dogs which would be capable of causing lung lesions when infused into normotensive dogs was demonstrated by light and electron microscopy. Light microscopy only identified significant differences in atelectasis, vascular congestion, and interstitial edema in the shock and shock-plasma recipient animals. On electron microscopy, interstitial edema and disruption of collagen bundles were consistently found in shocked dogs and in dogs that had received shocked plasma. There was a consistent increase in pulmonary interstitial sodium in both shock and shock-plasma recipient groups. It is concluded that a plasma factor present in the serum of shocked dogs may be transferred to other dogs and cause histopathologic changes seen in the lungs of shocked animals.

A humoral factor in the plasma of dogs with hemorrhagic hypotension causing histopathologic changes when infused into normals.

The maturation of megakaryocytes in vivo requires polyploidization or repeated duplication of DNA without cytokinesis. As DNA replication and cytokinesis are tightly regulated in somatic cells by cyclins and cyclin-dependent kinases, we sought to determine the pattern of cyclin gene expression in cells that undergo megakaryocytic differentiation and polyploidization. The Dami megakaryocytic cell line differentiates and increases ploidy in response to phorbol 12-myristate 13-acetate (PMA) stimulation in vitro. We used Northern blotting to analyze mRNA levels of cyclins A, B, C, D1, and E in PMA-induced Dami cells and found that cyclin D1 mRNA levels increased dramatically (18-fold). Similar increases in cyclin D1 mRNA were obtained for other cell lines (HEL and K562) with megakaryocytic properties, but not in HeLa cells. The increase in cyclin D1 was confirmed by Western immunoblotting of PMA-treated Dami cells. This finding suggested that cyclin D1 might participate in megakaryocyte differentiation by promoting endomitosis and/or inhibiting cell division. To address these possibilities, we constructed two stable Zn+2-inducible, cyclin D1-overexpressing Dami cell lines. Cyclin D1 expression alone was not sufficient to induce polyploidy, but in conjunction with PMA-induced differentiation, polyploidization was slightly enhanced. However, unlike other cell systems, cyclin D1 overexpression caused cessation of cell growth. Although the mechanism by which cyclin D1 may affect megakaryocyte differentiation is not clear, these data demonstrate that cyclin D1 is upregulated in differentiating megakaryocytic cells and may contribute to differentiation by arresting cell proliferation.

Overexpression of cyclin D1 in the Dami megakaryocytic cell line causes growth arrest.

TaqI polymorphism of the human MXI1 gene.

The MYC onco-protein is a transcription factor that regulates cell proliferation, metabolism, protein synthesis, mitochondrial function and stem cell renewal. A region on chromosome 8q24 encompassing the MYC locus is amplified in prostate cancer, but this occurs mostly in advanced disease suggesting that MYC alterations occur late in prostate cancer. In contrast, MYC mRNA is elevated in most prostate cancers, even those of relatively low stage and grade (eg Gleason score 6) suggesting that MYC plays a role in initiation. However, since MYC protein levels are tightly regulated, elevated MYC mRNA does not necessarily imply elevated MYC protein. Thus, it is critical to determine whether MYC protein is elevated in human prostate cancer, and if so, at what stage of the disease this elevation occurs. Prior studies of MYC protein localization have been hampered by lack of suitable antibodies and controls. We utilized a new anti-MYC antibody coupled with genetically defined control experiments to localize MYC protein within human tissue microarrays consisting of normal, atrophy, PIN, primary adenocarcinoma, and metastatic adenocarcinoma. Nuclear overexpression of MYC protein occurred frequently in luminal cells of PIN, as well as in most primary carcinomas and metastatic disease. MYC protein did not correlate with gain of 8q24, suggesting alternative mechanisms for MYC overexpression. These results provide evidence that upregulation of nuclear MYC protein expression is a highly prevalent and early change in prostate cancer and suggest that increased nuclear MYC may be a critical oncogenic event driving human prostate cancer initiation and progression.

Nuclear MYC protein overexpression is an early alteration in human prostate carcinogenesis.

Activation of the MYC oncoprotein is among the most ubiquitous events in human cancer. MYC functions in part as a sequence-specific regulator of transcription. Although early searches for direct downstream target genes that explain MYC's potent biological activity were met with enthusiasm, the postgenomic decade has brought the realization that MYC regulates the transcription of not just a manageably small handful of target genes but instead up to 15% of all active loci. As the dust has begun to settle, two important concepts have emerged that reignite hope that understanding MYC's downstream targets might still prove valuable for defining critical nodes for therapeutic intervention in cancer patients. First, it is now clear that MYC target genes are not a random sampling of the cellular transcriptome but instead fall into specific, critical biochemical pathways such as metabolism, chromatin structure, and protein translation. In retrospect, we should not have been surprised to discover that MYC rewires cell physiology in a manner designed to provide the tumor cell with greater biosynthetic properties. However, the specific details that have emerged from these studies are likely to guide the development of new clinical tools and strategies. This raises the second concept that instills renewed optimism regarding MYC target genes. It is now clear that not all MYC target genes are of equal functional relevance. Thus, it may be possible to discern, from among the thousands of potential MYC target genes, those whose inhibition will truly debilitate the tumor cell. In short, targeting the targets may ultimately be a realistic approach after all.

Emerging Concepts in the Analysis of Transcriptional Targets of the MYC Oncoprotein: Are the Targets Targetable?

QSAR models for analogs of antiplasmodial artemisinin compounds were established, based on atomic net charges by using multivariate adaptive regression splines (MARS) in comparison with some other methods such as multiple linear regression, alternating conditional expectations and projection pursuit regression. The established models were then evaluated by an Anova decomposition procedure so that the effects of each predictor (additive or interaction) could be viewed graphically, facilitating the interpretation of the underlying relationship. It was found that the QSARs derived from the MARS method are the most satisfactory predictive models, and that the artemisinin pharmacophore identification is in agreement with previous experimental findings.

Using multivariate adaptive regression splines to QSAR studies of dihydroartemisinin derivatives.

Why Cancer & Metabolism? Why now?

Despite Vietnam's large population, there are only about 366 pathologists in the country. Most work in big cities or provincal hospitals, not rural areas. Pathologists face difficulties in daily practice and in consulting colleagues. To improve practice in remote areas, people have to post paraffin blocks and microscope slides between cities or abroad to obtain an expert's diagnosis. An alternative, sending images by internet, is less effective, because it's static.As a result, telepathology is being developed in Vietnam, starting at Bachmai Hospital in 2011 with IMCJ (Japan), showing immediately an improvement in diagnosis. After a few trials, it was postponed because Bachmai didn't have an adequate telepathological system.Also in 2011, Cho-Ray Hospital developed a system, assisted by Mita Hospital (Japan) holding consultations regularly till the present. About 50 cases have been done including cancer of gastric, breast, cervical, lung, with 97% diagnosis in agreement. Diagnostic quality is maintained, or improved by informatics. Specific case images are automatically stored on a central server in Japan permitting pathologists and students to have continuous access for training and education. Efficient results are obvious, economically, timewise and in security of records. Telepathology, therefore, should be widely applied and strategically developed in sustainable and long-term approach in Vietnam.

Article	Year
Disassembly and gross structure of particulate aminoacyl-tRNA synthetases from rat liver. Isolation and the structural relationship of synthetase complexes. The Journal of biological chemistry\| PubMed ID: 447654	1979
Maximum in the stability of the amorphous state in the system water--glycerol--ethanol. Cryobiology\| PubMed ID: 487853	1979
Karyoplasmic interaction selection strategy: a general strategy to detect protein-protein interactions in mammalian cells. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 1387709	1992
Physical and biochemical characterization of a purified arginyl-tRNA synthetase-lysyl-tRNA-synthetase complex from rat liver. Biochemistry\| PubMed ID: 7082655	1982
Characterization of a homogeneous arginyl- and lysyl-tRNA synthetase complex isolated from rat liver. Arginyl- and lysyl-tRNA synthetases contain carbohydrates. Biochemistry\| PubMed ID: 7138839	1982
Purification and characterization of lysyl-tRNA synthetase after dissociation of the particulate aminoacyl-tRNA synthetases from rat liver. The Journal of biological chemistry\| PubMed ID: 7372681	1980
A humoral factor in the plasma of dogs with hemorrhagic hypotension causing histopathologic changes when infused into normals. The Journal of trauma\| PubMed ID: 7463533	1981
Overexpression of cyclin D1 in the Dami megakaryocytic cell line causes growth arrest. Blood\| PubMed ID: 7795236	1995
TaqI polymorphism of the human MXI1 gene. Human molecular genetics\| PubMed ID: 7881441	1994
Nuclear MYC protein overexpression is an early alteration in human prostate carcinogenesis. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc\| PubMed ID: 18567993	2008
Emerging Concepts in the Analysis of Transcriptional Targets of the MYC Oncoprotein: Are the Targets Targetable? Genes & cancer\| PubMed ID: 21533016	2010
Using multivariate adaptive regression splines to QSAR studies of dihydroartemisinin derivatives. European journal of medicinal chemistry\| PubMed ID: 22026935	1996
Why Cancer & Metabolism? Why now? Cancer & metabolism\| PubMed ID: 24280107	2013
Computational Pathology and Telepathology: SY05-2 PATHOLOGY AND TELEPATHOLOGY IN VIETNAM. Pathology\| PubMed ID: 25188195	2014

Van Dang

Biography

JoVE Journal Publications

Other Publications