Characterisation of ACP5 missense mutations encoding tartrate-resistant acid phosphatase associated with spondyloenchondrodysplasia.

Biallelic mutations in ACP5, encoding tartrate-resistant acid phosphatase (TRACP), have recently been identified to cause the inherited immuno-osseous disorder, spondyloenchondrodysplasia (SPENCD). This study was undertaken to characterize the eight reported missense mutations in ACP5 associated with SPENCD on TRACP expression. ACP5 mutant genes were synthesized, transfected into human embryonic kidney (HEK-293) cells and stably expressing cell lines were established. TRACP expression was assessed by cytochemical and immuno-cytochemical staining with a panel of monoclonal antibodies. Analysis of wild (WT) type and eight mutant stable cell lines indicated that all mutants lacked stainable enzyme activity. All ACP5 mutant constructs were translated into intact proteins by HEK-293 cells. The mutant TRACP proteins displayed variable immune reactivity patterns, and all drastically reduced enzymatic activity, revealing that there is no gross inhibition of TRACP biosynthesis by the mutations. But they likely interfere with folding thereby impairing enzyme function. TRACP exists as two isoforms. TRACP 5a is a less active monomeric enzyme (35kD), with the intact loop peptide and TRACP 5b is proteolytically cleaved highly active enzyme encompassing two subunits (23 kD and 16 kD) held together by disulfide bonds. None of the mutant proteins were proteolytically processed into isoform 5b intracellularly, and only three mutants were secreted in significant amounts into the culture medium as intact isoform 5a-like proteins. Analysis of antibody reactivity patterns revealed that T89I and M264K mutant proteins retained some native conformation, whereas all others were in "denatured" or "unfolded" forms. Western blot analysis with intracellular and secreted TRACP proteins also revealed similar observations indicating that mutant T89I is amply secreted as inactive protein. All mutant proteins were attacked by Endo-H sensitive glycans and none could be activated by proteolytic cleavage in vitro. In conclusion, determining the structure-function relationship of the SPENCD mutations in TRACP will expand our understanding of basic mechanisms underlying immune responsiveness and its involvement in dysregulated bone metabolism.

Transition Prediction in Hypersonic Boundary Layers Using Receptivity and Freestream Spectra.

Boundary-layer transition in hypersonic flows over a straight cone can be predicted using measured freestream spectra, receptivity, and threshold values for the wall pressure fluctuations at the transition onset points. Simulations are performed for hypersonic boundary-layer flows over a 7-degree half-angle straight cone with varying bluntness at a freestream Mach number of 10. The steady and the unsteady flow fields are obtained by solving the two-dimensional Navier-Stokes equations in axisymmetric coordinates using a 5th-order accurate weighted essentially nonoscillatory (WENO) scheme for space discretization and using a third-order total-variation-diminishing (TVD) Runge-Kutta scheme for time integration. The calculated N-factors at the transition onset location increase gradually with increasing unit Reynolds numbers for flow over a sharp cone and remain almost the same for flow over a blunt cone. The receptivity coefficient increases slightly with increasing unit Reynolds numbers. They are on the order of 4 for a sharp cone and are on the order of 1 for a blunt cone. The location of transition onset predicted from the simulation including the freestream spectrum, receptivity, and the linear and the weakly nonlinear evolutions yields a solution close to the measured onset location for the sharp cone. The simulations overpredict transition onset by about twenty percent for the blunt cone.

Receptivity and Forced Response to Acoustic Disturbances in High-Speed Boundary Layers.

Supersonic boundary-layer receptivity to freestream acoustic disturbances is investigated by solving the Navier-Stokes equations for Mach 3.5 flow over a 7 deg half-angle cone. The freestream disturbances are generated from a wavy wall placed at the nozzle wall. The freestream acoustic disturbances radiated by the wavy wall are obtained by solving the linearized Euler equations. The results show that no noticeable instability modes are generated when the acoustic disturbances impinge the cone obliquely. The results show that the perturbations generated inside the boundary layer by the acoustic disturbances are the response of the boundary layer to the external forcing. The amplitude of the forced disturbances inside the boundary layer are about 2.5 times larger than the incoming field for zero azimuthal wave number, and they are about 1.5 times for large azimuthal wave numbers.

Possible role of poly(ADP-ribose) polymerase in pathological and physiological cardiac hypertrophy.

The present study was designed to investigate the effect of 5-aminoisoquinoline, a specific poly(ADP-ribose) polymerase (PARP) inhibitor, in partial abdominal aortic constriction (PAAC) for 4 weeks it induced pathological and chronic swimming training (CST) for 8 weeks it induced physiological cardiac hypertrophy. 5-Aminoisoquinoline (0.3 mg/kg/day and 3 mg/kg/day, i.p.) treatment was started 3 days before PAAC and CST, and it was continued for 4 weeks after PAAC and 8 weeks after initiation of CST. The left ventricular (LV) function and LV hypertrophy were assessed by measuring LVDP, dp/dtmax, dp/dtmin, ratio of LV weight to body weight (LVW/BW), LV wall thickness (LVWT), LV collagen content, LV protein content, and LV RNA concentration. Further, venous pressure (VP) and mean arterial blood pressure (MABP) were recorded. The PAAC, but not CST, produced LV dysfunction by decreasing LVDP, dp/dtmax, dp/dtmin, and increasing LV collagen content. Further, PAAC and CST were noted to produce LV hypertrophy by increasing LVW/BW, LVWT, LV protein content, and LV RNA concentration. Moreover, in contrast to CST, PAAC significantly increased VP and MABP. The 5-aminoisoquinoline, a potent selective inhibitor of PARP, significantly attenuated PAAC-induced LV dysfunction, LV hypertrophy, increase in VP and MABP. On the other hand, treatment with 5-aminoisoquinoline did not modulate CST-induced physiological cardiac hypertrophy. These results implicate PARP in PAAC-induced LV dysfunction and pathological cardiac hypertrophy. However, PARP may not be involved in CST-induced physiological cardiac hypertrophy.

Design, synthesis, and evaluation of novelly substituted benzimidazole compounds as angiotensin II receptor antagonists.

5-Nitrobenzimidazole derivatives with varying substituents at 2-position have been designed, synthesized, and evaluated for angiotensin II antagonistic activity. A drug-receptor interaction model has been proposed.