Biomedical subjects

K Ostwald

Publications and source records attributed to K Ostwald.

4 recordsLinked to original sources

PubMed1994

A method for protein assay in Laemmli buffer.

A simple, cheap, and rapid assay for soluble and membrane-bound protein solubilized in Laemmli sodium dodecyl sulfate sample buffer is described. Following the addition of trichloroacetic acid to a final concentration of 24%, the induced turbidity was measured at 570 nm in a microplate reader, after an incubation period of approximately 10-30 min. The assay is suitable for measuring bovine serum albumin at a concentration of 15 to 500 micrograms/ml in Laemmli buffer, corresponding to 2 to 75 micrograms protein per well on the microtiter plate. Other nonproteinaceous macromolecules in the sample may introduce an error.

Animals↗

PubMed1994

Subcellular distribution of calpain and calpastatin immunoreactivity and fodrin proteolysis in rabbit hippocampus after hypoxia and glucocorticoid treatment.

Rabbits were subjected to hypoxia (5% O2) for up to 90 min and allowed to recover for a maximum of 4 days. Hippocampus homogenate was assayed for fodrin breakdown product (BDP). After separation into a nuclear and mitochondrial fraction (NMF), a membrane and microsomal fraction (MMF), and a cytosolic fraction (CF), samples were assayed for mu-calpain, m-calpain, and calpastatin immunoreactivity. Calpain and calpastatin immunoreactivity decreased in the NMF and CF but increased in the MMF during hypoxia and short-term recovery. This translocation occurred in parallel with the increase in fodrin BDP. Because the increase in the MMF was not large enough to explain the decrease in the other two fractions, it was assumed that the translocation and activation was accompanied by a reduction in the total amounts of calpains and calpastatin. Glucocorticoid pretreatment (beta-methasone, 0.4 mg x kg-1 x day-1) for 7 days produced a decrease in the ratio of activated mu-calpain in all three fractions in nearly all samples before, during, and after hypoxia, compared with untreated animals. Glucocorticoid pretreatment also prevented the increase in fodrin BDP that occurred in untreated animals during hypoxia and short-term recovery, indicating impairment of calpain activation.

Animals↗

PubMed1993

Upregulation of calpain activity in neonatal rat brain after hypoxic-ischemia.

Neonatal rats were subjected to transient cerebral hypoxic-ischemia (unilateral occlusion of the common carotid artery plus 7.7% O2 for 2 h) and allowed to recover for 0 min, 30 min or 20 h. The calpain and calpastatin activities were assayed in subcellular fractions of the ipsilateral, hypoxic-ischemic and the contralateral, hypoxic hemisphere. An upregulation of calpain activity occurred in the hypoxic hemisphere, both in the major, cytosolic fraction and in the hypotonic, membrane associated fraction (110% and 133% of controls, respectively). The hypoxic-ischemic hemisphere displayed a decrease in calpain activity in the cytosolic fraction but an increase in the hypotonic fraction (90% and 111% of controls, respectively). The changes in calpastatin activity were less pronounced. This indicates that an upregulation of calpain activity occurs in parallel with development of hypoxic-ischemic damage. However, this upregulation is not necessarily coupled to development of injury as lesions are not seen in the hypoxic hemisphere.

Animals↗

PubMed1991

Changes in brain calpain activity as a result of in vitro ischemia and pH alterations.

Calpains and calpastatin in the brain of the rabbit were examined in experimental situations that could mimic some features of brain ischemia. Incubations of bisected brains in saline at 39 degrees C for 0.5, 1, or 1.5 h resulted in a decreased calpain I activity in the cytosol and in an increased hydrophobicity of cytosolic calpain II activity. Incubation of brain homogenates at different pH levels demonstrated an almost-complete transfer of calpains from the cytoplasmic compartment to the membranes when pH was lowered from 6 to 5. At pH values lower than 5, the total calpain activity (soluble plus membrane-bound) markedly decreased. No significant changes of calpastatin activity or its subcellular distribution was found following incubation of the homogenates at different pH levels.

Animals↗