Citations

Background— Onset of myocardial relaxation is highly energy dependent. Perfusion and therefore energy substrate delivery are predominantly reduced in the subendocardial myocardium in the early stages of progressive ischemia. We hypothesized that delayed onset of subendocardial diastolic thinning will functionally identify regionally hypoperfused resting myocardium. Methods and Results— Progressive left anterior descending coronary artery stenosis was induced by an ameroid occluder and maintained for 1 or 2 weeks (end point) in 12 dogs. M-mode tissue Doppler images of the anterior apical and middle segments (testing region) and middle inferior segment (control region) were acquired selectively in the subendocardium and subepicardium. The time to the onset of thinning was measured with the use of tissue Doppler velocity (TOTv) and a thickness function (TOTt). At the end point in the testing region, myocardial flow was significantly lower in the subendocardial layer ( P <0.05) in all animals, whereas viability staining showed preserved transmural viability in 10 dogs and thin subendocardial necrosis in 2 dogs. Both TOTv and TOTt were significantly ( P <0.01) prolonged in the testing region. The mean difference between subendocardial and subepicardial TOTv values versus that in the control region identified the ischemic region, even when only dogs with hypoperfused but transmurally viable myocardium were considered ( P <0.05). Systolic and diastolic myocardial velocities did not identify subendocardial hypoperfusion. Conclusions— In resting myocardium subtended to progressive coronary stenosis, a delayed onset of subendocardial thinning suggests an early stage of hypoperfusion, before the development of local wall motion abnormalities.

Mutations of c-KIT causing spontaneous activation of the KIT receptor kinase are associated with sporadic adult human mastocytosis (SAHM) and with human gastrointestinal stromal tumors. We have classified KIT-activating mutations as either “enzymatic site” type (EST) mutations, affecting the structure of the catalytic portion of the kinase, or as “regulatory” type (RT) mutations, affecting regulation of an otherwise normal catalytic site. Using COS cells expressing wild-type or mutant KIT, 2 compounds, STI571 and SU9529, inhibited wild-type and RT mutant KIT at 0.1 to 1 μM but did not significantly inhibit the Asp816Val EST mutant associated with SAHM, even at 10 μM. Using 2 subclones of the HMC1 mast cell line, which both express KIT with an identical RT mutation but which differ in that one also expresses the Asp816Val EST mutation, both compounds inhibited the RT mutant KIT, thereby suppressing proliferation and producing apoptosis in the RT mutant-only cell line. Neither compound suppressed activation of Asp816Val EST mutant KIT, and neither produced apoptosis or significantly suppressed proliferation of the cell line expressing the Asp816Val mutation. These studies suggest that currently available KIT inhibitors may be useful in treating neoplastic cells expressing KIT activated by its natural ligand or by RT activating mutations such as gastrointestinal stromal tumors but that neither compound is likely to be effective against SAHM. Furthermore, these results help establish a general paradigm whereby classification of mutations affecting oncogenic enzymes as RT or EST may be useful in predicting tumor sensitivity or resistance to inhibitory drugs.

Heat shock protein (hsp)90 functions in a complex chaperoning pathway where its activity is modulated by ATP and by interaction with several co-chaperones. One co-chaperone, p23, binds selectively to the ATP-bound state of hsp90. However, the isolated ATP-binding domain of hsp90 does not bind p23. In an effort to identify the p23-binding domain, we have constructed a series of hsp90 deletion mutants fused with glutathione- S -transferase (GST). Full-length GST-hsp90 is able to bind p23, and also, to chaperone assembly of progesterone receptor complexes. Truncations from the C terminus of GST-hsp90 reveal a C-terminal boundary for the p23-binding domain at approximately residue 490. This fragment contains, in order, the ATP-binding domain, a highly charged region, and 203 residues beyond the charged region. p23 binding is unaffected by deletion of the charged region, indicating that two noncontiguous regions of hsp90 are involved in p23 binding. These regions are only effective when hsp90 is in a dimeric state as shown by loss of p23 binding upon removal of GST or as shown by use of FK506-binding protein12-hsp90 constructs that form dimers and bind p23 only in the presence of a bivalent drug. Thus, p23 binding requires an hsp90 dimer with close proximity between N-terminal regions of hsp90 and a conformation specified by ATP.