Biomedical subjects

K A Kozlowski

Publications and source records attributed to K A Kozlowski.

3 recordsLinked to original sources

PubMed2000

Myofibrillar disruption in hypocontractile myocardium showing perfusion-contraction matches and mismatches.

Chronically instrumented dogs underwent 2- or 5-h regional reductions in coronary flow that were followed, respectively, by balanced reductions in myocardial contraction and O(2) consumption ("hibernation") and persistently reduced contraction despite normal myocardial O(2) consumption ("stunning"). Previously unidentified myofibrillar disruption developed during flow reduction in both experimental models and persisted throughout the duration of reperfusion (2-24 h). Aberrant perinuclear aggregates that resembled thick filaments and stained positively with a monoclonal myosin antibody were present in 34 +/- 3.8% (SE) and 68 +/- 5.9% of "hibernating" and "stunned" subendocardial myocytes in areas subjected to flow reduction and in 16 +/- 2.5% and 44 +/- 7.4% of subendocardial myocytes in remote areas of the same ventricles. Areas of myofibrillar disruption also showed glycogen accretion and unusual heterochromatin clumping adjacent to the inner nuclear envelope. The degrees of flow reduction employed were sufficient to reduce regional myofibrillar creatine kinase activity by 25-35%, but troponin I degradation was not evident. The observed changes may reflect an early, possibly reversible, phase of the myofibrillar loss characteristic of hypocontractile myocardium in patients undergoing revascularization.

Animals↗

PubMed1989

Active and inactive forms of the transition-state analog protease inhibitor leupeptin: explanation of the observed slow binding of leupeptin to cathepsin B and papain.

Leupeptin and similar peptide argininal (arginine aldehyde) transition-state analog protease inhibitors exist in three covalent forms in aqueous solution, the leupeptin hydrate (IH), a cyclic carbinolamine form (IC) generated by the addition of the guanidino epsilon N to the aldehydic carbon, and the free aldehyde form (IA). 1H NMR in D2O show their equilibrium concentrations to be 42, 56, and 2% for IH, IC (R and S enantiomers), and IA. The rates of conversion of (formula; see text) were determined by 1H NMR in D2O by trapping IA with semicarbazide. Application of a deuterium isotope effect of 2.8 led to rate constants in H2O for kC of 0.092 min-1 and kD of 0.73 min-1. The equilibrium concentration of IA and rates for kC and kD are then used to explain the lag phase in the inhibition of cathepsin B and papain by leupeptin. Two circumstances are observed. (i) At micromolar concentrations of leupeptin and papain the binding of leupeptin is biphasic with rate constants identical to kD and kC. (ii) At more dilute nanomolar concentrations of total leupeptin and proteases, the observed lag phase for approach to steady-state inhibition (with rate constant k') is now explained by the low values of the koff rate constants (0.072 min-1 for cathepsin B and 0.024 min-1 for papain) together with the extremely low concentrations of the active inhibitor form IA, with k' = kon[IA] + koff. While kon[IA] is slow, the second-order rate constant kon is found to be quite fast, 1.2 x 10(7) M-1 s-1 for cathepsin B and 1.8 x 10(7) M-1 s-1 for papain. Thus, the binding of leupeptin to cathepsin B and papain may show a lag phase, but this is not due to slow binding.

Algorithms↗

PubMed1984

Tumor cell proteinase visualization and quantification using a fluorescent transition-state analog probe.

The fluorescent proteinase transition-state analog inhibitor, dansyl-L-argininal (DnsArgH), may be a selective probe of cysteine and serine-type proteinases in a fibrosarcoma tumor cell line (HSDM1C1). DnsArgH binds with high affinity to proteinases because of its transition-state analog properties, and on association it gives a dramatically increased fluorescent yield. The DnsArgH binding is inhibited by the serine proteinase inhibitor diisopropyl fluorophosphate and by the cysteine proteinase inhibitor p-chloromercuribenzoate. The fluorescence emission appears at its maximum steady-state yield immediately on addition of DnsArgH to the HSDM1C1 fibrosarcoma cells. The immediacy of the DnsArgH reaction supports the contention that DnsArgH binding may be to cell surface-associated proteinases. Quantification of the cell proteinase concentration, by comparison of the fluorescence yield obtained from DnsArgH interactions with bovine trypsin and papain, indicates 10(-15) to 10(-16) mol of proteinase per HSDM1C1 cell. In fluorescence microscopy, DnsArgH fluorescence appears distributed throughout the fibrosarcoma cell without association to organelles. DnsArgH fluorescence from normal fibroblast controls (IMR-90) was found to be substantially lower than in the transformed fibrosarcoma cells, supporting a hypothesis that proteinases have a role in malignancy.

Arginine↗