Biomedical subjects

T Tachovsky

Publications and source records attributed to T Tachovsky.

4 recordsLinked to original sources

PubMed1995

Western blotting and isoform analysis of cathepsin D from normal and malignant human breast cell lines.

Cathepsin D from normal (Hs578Bst) and malignant (MCF7, MDA-MB-231) breast cell lines has been characterized with regard to its kinetic properties, activity levels, precursor and processed M(r) forms, and isoform composition. Normal cell cathepsin D appears to have a more neutral pH optimum (pH 3.5) than the cancer cell line (pH 3.0-3.2) and greater activity between pH values of 4.0 to 4.5. The two cancer cell lines have approximately 1.5 to 2.0-fold increased total acid protease activity and 2 to 3-fold increased pepstatin-inhibitable protease activity (i.e. cathepsin D) when compared to the normal breast cell line. Western blotting indicates that a major processed form of cathepsin D for all three cell lines occurs at 31 kDa. The cancer cell lines contain significant amounts of cathepsin D precursors of 47 and 42 kDa whereas the normal cell line contains little if any of these precursors. Isoelectric focusing indicates that the normal cell line contains approximately 50% of its total acid protease activity at pIs above 4 whereas the cancer cell lines contain 70-80% of their protease activity at such pIs. In addition, the cancer cell lines contain two to three major isoforms between pIs of 5.5 and 6.3 which were not present in the normal cell line. The isoforms from pI values of 5.5 to 7.3 for all three cell lines are 100% pepstatin-inhibitable. In addition, Western blot analysis indicates that these isoforms contain the processed 31 kDa form of cathepsin D. The combined results indicate that the two breast cancer cell lines are similar to biopsied malignant breast tissue in exhibiting altered acid protease isoform profiles with increased relative amounts of pepstatin-inhibitable and immunoreactive acid protease activity (cathepsin D) compared to normal breast tissue or cells.

Aspartic Acid Endopeptidases↗

PubMed1994

Western blotting and enzymatic activity analysis of cathepsin D in breast tissue and sera of patients with breast cancer and benign breast disease and of normal controls.

Increased total antigen amounts of cathepsin D in breast tissue have been reported to be associated with increased disease recurrence, more frequent metastasis, and increased mortality in breast cancer patients. In the present study, Western blotting analysis has been used for the first time to determine the relative amounts of precursor and processed forms of cathepsin D in sera and breast tissue of patients with breast cancer, benign breast disease, and normal controls. Sera gave similar blots for breast cancer patients and controls with two major forms of cathepsin D (M(r) 52,000 and 27,000). Malignant breast tissue contained the two forms of cathepsin D found in sera and an additional M(r) 31,000 form which was found in significantly increased (P < 0.001) relative amounts in breast tissue from 43 breast cancer patients [24 +/- 12% (SD)] when compared to 51 benign breast disease patients (13 +/- 8.9%) and 23 normal controls (1.8 +/- 4.4%). Preliminary analysis of subgroups of benign breast disease patients suggested no significant difference (P = 0.41) in relative amounts of the M(r) 31,000 form of cathepsin D between proliferative-type and non-proliferative-type fibrocystic breast disease. A cathepsin D assay has been optimized for human breast tissue and used to demonstrate for the first time significantly increased (P < 0.001) amounts of pepstatin-inhibitable, cathepsin D-specific activity in breast tissue from 36 breast cancer patients (2.2 +/- 1.4 units/mg of protein) when compared to 47 benign breast disease patients (0.63 +/- 0.43) and 23 normal controls (0.24 +/- 0.21). Preliminary analysis of subgroups of benign breast disease patients suggested no significant difference (P = 0.21) in pepstatin-inhibitable, cathepsin D-specific activity between proliferative-type and nonproliferative-type fibrocystic breast disease. The positive correlation (r = 0.82) of increased amounts of the M(r) 31,000 form of cathepsin D and increased pepstatin-inhibitable, cathepsin D enzymatic activity in malignant breast tissue suggests that the M(r) 31,000 form is the proteolytically active form of the enzyme which may be involved in the development and/or metastatic spread of breast cancer.

Adult↗

PubMed1979

Immunoglobulin elution from multiple sclerosis brain.

Immunoglobulin (Ig) was eluted from multiple sclerosis (MS) brain tissue. Razor thin slices of white matter from 10 g of MS and control brains were washed with 5 liters of phosphate-buffered saline (PBS), then treated for 90 sec with acetic acid, pH 2.5, containing Pepstatin and epsilon-aminocaproic acid. The protein concentrations of the PBS washes and neutralized acid eluates were determined, and the eluates were assayed for Ig by competitive microradioimmunoassay using rabbit anti-human F(ab1)2. Successive PBS washes reduced extracellular protein to a very low level. Equivalent quantities of protein were recovered from 7 MS and 6 non-MS brain samples after PBS washing and acetic acid elution. However, the amount of protein needed for 50% inhibition of [125I]IgG binding to anti-human F(ab1)2 was significantly less in MS brain than in non-MS brain (P less 0.05). The Ig in brain eluates was present in the void volume of a DEAE cellulose column. The techniques described above facilitate the isolation and characterization of cell-surface Ig in MS brain.

Brain↗

PubMed1976

Experimental parainfluenza type 1 virus-induced encephalopathy in adult mice. Pathogenesis of chronic degenerative changes in the CNS.

The pathogenicity of the 6/94 strain of parainfluenza type 1 virus, originally isolated from multiple sclerosis brain, was studied in adult mouse brains. Intracerebral inoculation of the virus caused mononuclear cell infiltration in the form of perivascular cuffing and a diffuse exudation into the parenchymal tissue, preferentially in cerebral white matter, that resulted in marked degeneration over a 90-day observation period. Immunofluorescent staining revealed viral antigen in the ependymal lining cells only during the first seven days after infection. No correlation was found between the severity of the brain lesions and the level of circulating antiviral antibody, preexisting or newly produced.

Animals↗