scholarly journals A novel 205-kilodalton testis-specific serine/threonine protein kinase associated with microtubules of the spermatid manchette.

1993 ◽  
Vol 13 (12) ◽  
pp. 7625-7635 ◽  
Author(s):  
P D Walden ◽  
N J Cowan
Keyword(s):  
Protein Complex ◽  
Catalytic Domain ◽  
Signal Transduction Pathway ◽  
Kinase Domain ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Serine Threonine Kinase ◽  
Expression Clone

To identify proteins which interact with and potentially modulate the function of microtubules during spermatogenesis, we prepared a total testis MAP (microtubule-associated protein) antiserum and used it to isolate cDNA clones from a mouse testis cDNA expression library. Antibodies affinity purified by using one expression clone recognized a 205-kDa protein, termed MAST205, which colocalizes with the spermatid manchette. Sequencing of full-length cDNA clones encoding MAST205 revealed it to be a novel serine/threonine kinase with a catalytic domain related to those of the A and C families. The testis-specific MAST205 RNA increases in abundance during prepuberal testis development, peaking at the spermatid stage. The microtubule-binding region of MAST205 occupies a central region of the molecule including the kinase domain and sequences C terminal to this domain. Binding of MAST205 to microtubules requires interaction with other MAPs, since it does not bind to MAP-free tubulin. A 75-kDa protein associated with immunoprecipitates of MAST205 from extracts of both whole testis and testis microtubules becomes phosphorylated in in vitro kinase assays. This 75-kDa substrate of the MAST205 kinase may form part of the MAST205 protein complex which binds microtubules. The MAST205 protein complex may function to link the signal transduction pathway with the organization of manchette microtubules.

scholarly journals Optimized heterologous expression of the human zinc enzyme glyoxalase I

1996 ◽  
Vol 314 (2) ◽  
pp. 463-467 ◽  
Author(s):  
Marianne ÖM ◽  
Bengt MANNERVIK
Keyword(s):  
Culture Medium ◽  
Functional Characterization ◽  
Zinc Salt ◽  
Glyoxalase I ◽  
Kinetic Properties ◽  
Coding Region ◽  
Expression Clone ◽  
High Level ◽  
Inhibition Studies

DNA coding for human glyoxalase I was isolated from a HeLa cell cDNA library by means of PCR. The deduced amino acid sequence differs from previously isolated sequences in that a glutamic acid replaces an alanine in position 111. This variant cDNA may represent the more acidic isoform of glyoxalase I originally identified at the protein level. An expression clone was constructed for high-level production of glyoxalase I in Escherichia coli. For optimal yield of the recombinant protein, silent random mutations were introduced in the cDNA coding region. Antisera against human glyoxalase I were used to select a high-level expression clone. This clone afforded 60 mg of purified enzyme per litre of culture medium. Addition of a zinc salt to the culture medium was essential to obtain an active enzyme and a stoicheiometric metal content. The functional characterization of the recombinant enzyme included determination of kinetic constants for methylglyoxal, phenylglyoxal and p-phenylphenylglyoxal, as well as inhibition studies. The kinetic properties of recombinant glyoxalase I were indistinguishable from those of the enzyme purified from human tissues.

Generation of Expression Clone Set for Functional Proteomics of Human Gastric and Liver Cancers

2009 ◽  
Vol 234 (10) ◽  
pp. 1220-1229
Author(s):  
Nang-Soo Oh ◽  
Ji-Seon Park ◽  
Yeo-Jin Jeon ◽  
Jung-Hwa Oh ◽  
So-Young Jeong ◽  
...  
Keyword(s):  
Functional Proteomics ◽  
Expression Clone ◽  
Liver Cancers

Role of glial fibrillary acidic protein expression in the biology of human glioblastoma U-373MG cells

1998 ◽  
Vol 89 (6) ◽  
pp. 997-1006 ◽  
Author(s):  
Katrina G. Murphy ◽  
James D. Hatton ◽  
Hoi Sang U
Keyword(s):  
Population Doubling ◽  
Cell Behavior ◽  
Glial Tumor ◽  
Invasive Potential ◽  
Content Type ◽  
Expression Clone ◽  
Gfap Expression ◽  
Tumor Cell Behavior ◽  
The Relationship ◽  
Fine Print

Object. The relationship between glial fibrillary acidic protein (GFAP) expression and glial tumor cell behavior has not been well defined. The goal of this study was to examine this relationship further. Methods. To investigate the relationship between GFAP expression and glial tumor cell behavior, the authors isolated clones from the human glioblastoma cell line, U-373MG, according to their level of GFAP expression. Immunochemical analysis demonstrated that one clone had consistently low GFAP expression (approximately 93% of cells were GFAP negative), whereas a second clone had consistently high GFAP expression (approximately 80% of the cells were GFAP positive). The structure, population doubling time, saturation density, anchorage-independent growth, migratory rate, and invasive potential of these two clones were determined in relation to their level of GFAP expression. Morphologically, both clones were composed of ameboid as well as stellate components. Although the population doubling times of the two clones were equally rapid, the clone with low GFAP expression demonstrated a slightly higher saturation density compared with the clone with high GFAP expression. In an anchorage-independent environment (soft agar), a greater difference in growth characteristics was noted between the two clones: the high-expression clone formed more colonies and these colonies were compact, well defined, and spherical, whereas the low-expression clone formed predominantly smaller, two-dimensional colonies with vague boundaries and isolated cells or groups of cells at the periphery. In contrast to these minor differences between the clones, the low-expression clone showed a markedly increased migratory rate and invasive potential compared with the high-expression clone. Therefore, the clone with reduced GFAP expression appeared more aggressive, demonstrating decreased contact inhibition, increased migratory rate, and increased invasive potential. Conclusions. These results suggest a direct correlation between GFAP expression and some measures of aggressive tumor growth and transformation properties.

scholarly journals Virus-Specific Read-Through Codon Preference Affects Infectivity of Chimeric Cucumber Green Mottle Mosaic Viruses Displaying a Dengue Virus Epitope

2009 ◽  
Vol 2009 ◽  
pp. 1-8 ◽  
Author(s):  
Pak-Guan Teoh ◽  
Aik-Seng Ooi ◽  
Sazaly AbuBakar ◽  
Rofina Yasmin Othman
Keyword(s):  
Dengue Virus ◽  
Viral Population ◽  
Wild Type ◽  
Reading Frame ◽  
Recombinant Viruses ◽  
Codon Preference ◽  
Expression Clone ◽  
Protein Binding Region ◽  
Virus Epitope

ACucumber green mottle mosaic virus(CGMMV) was used to present a truncated dengue virus type 2 envelope (E) protein binding region from amino acids 379 to 423 (EB4). The EB4 gene was inserted at the terminal end of the CGMMV coat protein (CP) open reading frame (ORF). Read-through sequences of TMV or CGMMV, CAA-UAG-CAA-UUA, or AAA-UAG-CAA-UUA were, respectively, inserted in between the CP and the EB4 genes. The chimeric clones, pRT, pRG, and pCG+FSRTRE, were transcribed into full-length capped recombinant CGMMV transcripts. Only constructs with the wild-type CGMMV read-through sequence yielded infectious viruses following infection of host plant, muskmelon (Cucumis melo) leaves. The ratio of modified to unmodified CP for the read-through expression clone developed was also found to be approximately 1:1, higher than what has been previously reported. It was also observed that infectivity was not affected by differences in pI between the chimera and its wild counterpart. Analysis of recombinant viruses after 21-days-postinculation (dpi) revealed that deletions occurred resulting in partial reversions of the viral population to near wild type and suggesting that this would be the limiting harvest period for obtaining true to type recombinants with this construct.

scholarly journals From genome to proteome: developing expression clone resources for the human genome

2006 ◽  
Vol 15 (suppl_1) ◽  
pp. R31-R43 ◽  
Author(s):  
Gary Temple ◽  
Philippe Lamesch ◽  
Stuart Milstein ◽  
David E. Hill ◽  
Lukas Wagner ◽  
...  
Keyword(s):  
Human Genome ◽  
Expression Clone

A novel 205-kilodalton testis-specific serine/threonine protein kinase associated with microtubules of the spermatid manchette

1993 ◽  
Vol 13 (12) ◽  
pp. 7625-7635
Author(s):  
P D Walden ◽  
N J Cowan
Keyword(s):  
Protein Complex ◽  
Catalytic Domain ◽  
Signal Transduction Pathway ◽  
Kinase Domain ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Serine Threonine Kinase ◽  
Expression Clone

To identify proteins which interact with and potentially modulate the function of microtubules during spermatogenesis, we prepared a total testis MAP (microtubule-associated protein) antiserum and used it to isolate cDNA clones from a mouse testis cDNA expression library. Antibodies affinity purified by using one expression clone recognized a 205-kDa protein, termed MAST205, which colocalizes with the spermatid manchette. Sequencing of full-length cDNA clones encoding MAST205 revealed it to be a novel serine/threonine kinase with a catalytic domain related to those of the A and C families. The testis-specific MAST205 RNA increases in abundance during prepuberal testis development, peaking at the spermatid stage. The microtubule-binding region of MAST205 occupies a central region of the molecule including the kinase domain and sequences C terminal to this domain. Binding of MAST205 to microtubules requires interaction with other MAPs, since it does not bind to MAP-free tubulin. A 75-kDa protein associated with immunoprecipitates of MAST205 from extracts of both whole testis and testis microtubules becomes phosphorylated in in vitro kinase assays. This 75-kDa substrate of the MAST205 kinase may form part of the MAST205 protein complex which binds microtubules. The MAST205 protein complex may function to link the signal transduction pathway with the organization of manchette microtubules.

scholarly journals Podoplanin: a Prognostic Marker and a Potential Target for Therapy in Bladder Cancer

2020 ◽  
Vol 70 (12) ◽  
pp. 4393-4399
Keyword(s):  
Bladder Cancer ◽  
Tumor Cells ◽  
Lymphatic Vessels ◽  
Lymphatic Invasion ◽  
High Rate ◽  
Invasive Bladder Cancer ◽  
Expression Clone ◽  
Response To Chemotherapy ◽  
Keywords Bladder Cancer ◽  
Podoplanin Expression

Invasive bladder cancer is a frequent neoplastic diseases, and despite progresses made in early diagnosis and surgical procedures, the outcome of patients is characterized by high rate of mortality. This is mainly due to the lack of response to chemotherapy and radiotherapy, and other new resources are limited. In the present work we analyzed 50 consecutive cases of invasive bladder cancer treated by open surgery. Specimens were processed using standard histological procedures, including establishment of the histological diagnosis and grading. Additional slides were stained for podoplanin expression, clone D2-40 to investigate the structure and number of lymphatic vessels. We found lymphatic vessels in both intra- and peritumor areas, showing significant differences in morphology and number. Lymphatic invasion by tumor cells was higher in peritumor than in intratumor vessels. Lymphatic microvessel density correlated with stage and grade of the tumor. Of interest is the expression of podoplanin by tumor cells of urothelial carcinoma in about 14% of the cases, with strong correlation with grading. Based on our results, expression of podoplanin in invasive bladder cancer might indicate three potential targets: lymphatics, myofibroblasts, and tumor cells in selected cases. Keywords: bladder cancer, immunohistochemistry, podoplanin, therapeutic target

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