Nucleophosmin protein expression level, but not threonine 198 phosphorylation, is essential in growth and proliferation.

Nucleophosmin (NPM), an oligomeric phosphoprotein and nucleolar target of the ARF tumor suppressor, contributes to several critical cellular processes. Previous studies have shown that the human NPM's phosphorylation by cyclin E-cyclin-dependent kinase 2 (cdk2) on threonine (Thr) 199 regulates its translocation from the centrosome during cell cycle progression. Given our previous finding that ARF directly binds NPM, impeding its transit to the cytoplasm and arresting cells before S-phase entry, we hypothesized that ARF might also inhibit NPM phosphorylation. However, ARF induction did not impair phosphorylation of the cdk2 target residue in murine NPM, Thr198. Furthermore, phosphorylation of Thr198 occurred throughout the cell cycle and was concomitant with increases in overall NPM expression. To investigate the cell's presumed requirement for NPM-Thr198 phosphorylation in promoting the processes of growth and proliferation, we examined the effects of a non-phosphorylatable NPM mutant, T198A, in a clean cell system in which endogenous NPM had been removed by RNA interference. Here, we show that the T198A mutant is fully capable of executing NPM's described roles in nucleocytoplasmic shuttling, ribosome export and cell cycle progression. Moreover, the proliferative defects observed with stable NPM knockdown were restored by mutant NPM-T198A expression. Thus, we demonstrate that the reduction in NPM protein expression blocks cellular growth and proliferation, whereas phosphorylation of NPM-Thr198 is not essential for NPM's capacity to drive cell cycle progression and proliferation.

Structural valve deterioration in mitral replacement surgery: comparison of Carpentier-Edwards supra-annular porcine and perimount pericardial bioprostheses.

BACKGROUND: Bioprostheses preserved with glutaraldehyde, both porcine and pericardial, have been available as second-generation prostheses for valve replacement surgery. The performance with regard to structural valve deterioration with the Carpentier-Edwards supra-annular (CE-SAV) porcine bioprosthesis and the Carpentier-Edwards Perimount (CE-P) pericardial bioprosthesis (Baxter Healthcare Corp, Edwards Division, Santa Ana, Calif) was evaluated to determine whether there was a difference in mitral valve replacement. METHODS: The CE-SAV bioprosthesis was implanted in 1266 overall mitral valve replacements (isolated mitral, 1066; mitral in multiple, 200) and the CE-P bioprosthesis in 429 overall mitral valve replacements (isolated mitral, 328; mitral in multiple, 101). The mean age of the CE-SAV population was 64.2 +/- 12.2 years and that of the CE-P population, 60.7 +/- 11.7 years (P =.0001). For the study, structural valve deterioration was diagnosed at reoperation for explantation. RESULTS: The freedom from structural valve deterioration was evaluated to 10 years, and the freedom rates reported are at 10 years. For the overall mitral valve replacement groups, the actuarial freedom from deterioration was significant (P =.0001): CE-P > CE-SAV for 40 years or younger, 80% versus 60%; 41 to 50 years, 91% versus 61%; 51 to 60 years, 84% versus 69%; 61 to 70 years, 95% versus 75%. The older than 70-year group was 100% versus 92% (no significant difference). The actual freedom from structural valve deterioration also demonstrated the same pattern at 10 years: 40 years or younger, CE-P 82% versus CE-SAV 68%; 41 to 50 years, 92% versus 70%; 51 to 60 years, 90% versus 80%; 61 to 70 years, 97% versus 88%; and older than 70 years, 100% versus 97%. The independent risk factors of structural valve deterioration for the overall mitral valve replacement group were age and age groups and prosthesis type (CE-SAV > CE-P). The prosthesis type either in isolated replacement or in multiple replacement was not predictive of structural valve deterioration. The pathology of structural valve deterioration was different: 70% of CE-P failures were due to calcification and 57% of CE-SAV failures were due to combined calcification and leaflet tear. CONCLUSION: The actuarial and actual freedom from structural valve deterioration, diagnosed at reoperation, is greater at 10 years for CE-P than for CE-SAV bioprostheses. The mode of failure is different, and the cause remains obscure. Long-term evaluation is recommended, because the different modes of failure may alter the clinical performance by 15 and 20 years.

Pentoxifylline decreases cyclosporine-induced renal endothelin release and vasoconstriction.

Cyclosporine (CSA) causes an acute vasoconstriction of renal artery and a significant increase in renal endothelin release. Pentoxifylline, a vasodilator, has been suggested to prevent CSA toxicity. To study the effect of pentoxifylline treatment on CSA-related vasoconstriction and endothelin release, a model of renal autoperfusion in the dog was used. Oral pentoxifylline at the dose of 400 mg three times daily for 3 days was given to 15 dogs. Pure powder CSA (10 mg) was injected into the isolated renal artery perfused at constant flow; changes in perfusion pressure reflected variations in vascular resistance. In the pentoxifylline-treated group (15 dogs), the infusion of CSA caused an average increase of 27 +/- 8 mm Hg in renal perfusion pressure, compared with 60 +/- 10 mm Hg in a control group of 8 untreated animals (p < or = 0.05). Plasma concentration of endothelin in the renal vein increased from an average of 1.2 +/- 0.2 pg/mL before to 2.4 +/- 0.5 pg/mL after CSA administration (p < or = 0.05) in the control group, whereas it did not change significantly in the pentoxifylline-treated group. Thus, oral pretreatment with pentoxifylline significantly decreased the renal vasoconstriction and endothelin release due to CSA administration.

Mechanism of hind limb vasoconstriction due to cyclosporin A in the dog.

Cyclosporin A (CSA) causes an acute vasoconstriction of hind limb arterial vessels. To determine the mechanism of action of CSA on the peripheral arterial bed, studies were performed on the isolated femoral artery perfused at constant flow in 61 dogs. Changes in femoral perfusion pressure reflected variations in vascular resistance. Pure powder CSA was dissolved in autologous blood and injected at doses of 1, 5, 10, and 20 mg. Infusions of 1 and 5 mg CSA caused nonsignificant mean increases of 4 +/- 2 mm Hg (95% confidence interval [CI], 0-8; p > 0.05) and 10 +/- 4 mm Hg (95% CI, 0-21; p > 0.05) in femoral perfusion pressure, with CSA blood levels in the femoral vein averaging 40 +/- 16 and 126 +/- 50 nmol/l, respectively, at the end of the injections. Infusions of 10 and 20 mg CSA caused significant increases in femoral perfusion pressure averaging of 8 +/- 3 mm Hg (95% CI, 1-14; p < 0.05) and 20 +/- 4 mm Hg (95% CI, 11-29; p < 0.05) in femoral perfusion pressure. CSA blood levels at the end of injections averaged 271 +/- 99 and 431 +/- 146 nmol/l, respectively, in the femoral vein. Blockade of alpha-adrenergic receptors with phentolamine and surgical lumbar sympathectomy decreased significantly the CSA vasoconstrictive effect in peripheral arterial vessels, with increases in perfusion pressure averaging 29 +/- 5 mm Hg before and 14 +/- 3 mm Hg after phentolamine (p < 0.05) and 30 +/- 2 mm Hg before and 8 +/- 2 mm Hg after sympathectomy (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Relative influence of carotid baroreceptors and muscle receptors in the control of renal and hindlimb circulations.

In vagotomized dogs, a comparison was made of the relative ability of the carotid baroreceptors and of the receptors in skeletal muscles to cause constriction of the renal and hindlimb resistance vessels. With kidney and hindlimb perfused at constant pressure a decrease in pressure in the carotid sinuses from 250 to 40-45 mm Hg (1 mm Hg = 133 N/m2) caused the respective blood flows to increase by 19 +/- 6% and 80 +/- 4% (mean +/- SE), and stimulating muscle receptors with capsaicin caused a further decrease of 49 +/- 9% and 4 +/- 2%, respectively. With perfusion at constant flow, the baroreflex caused an increase of 34 +/- 4 mm Hg in the renal perfusion pressure and of 99 +/- 10 mm Hg in the hindlimb; capsaicin caused further increases of 203 +/- 17 and 35 +/- 9 mm Hg; respectively. These responses were abolished by sympathectomy. Capsaicin injection increased mean renal sympathetic nerve activity by 111 +/- 16% over the maximal impulse frequency recorded when the carotid sinus pressure was 40-45 mm Hg. Thus, withdrawal of the restraint exerted by the carotid baroreceptors on the pool of central neurons controlling the vascular beds of the hindlimb and kidney leads to near maximal constriction of the resistance vessels in the former bu not the latter; with strong activation of muscle receptors, near maximal constriction occurs in both beds.

Effect of hypoxia on vascular responses to the carotid baroreflex.

The effect of systemic hypoxia on the vascular responses to the carotid baroreflex was studied in anesthetized, vagotomized, artificially ventilated dogs. One hindlimb, kidney, gracilis muscle, and paw were perfused at constant flow, and neurograms were obtained from renal sympathetic fibers. Bilateral carotid occlusions were performed while the animal was breathing a mixture of air and O2 (mean arterial PO2 = 106 mmHg) and again during ventilation with 10% O2 (PO2 = 40 mmHg). With occlusion, the average increase in mean aortic pressure was 36 mmHg greater during hypoxia than during normoxia and the increase in renal perfusion pressure was 87 mmHg greater; the increase in hindlimb perfusion pressure was identical in both situations. Hypoxia did not change the reflex response of the paw to carotid occlusion and increased that of the muscle vessels by only 10%; the increase in renal sympathetic activity averaged 56 plus or minus 10% more with hypoxia than with normoxia. When the carotid chemoreceptors were destroyed, the greater increase in aortic and renal pressure response to carotid occlusion during hypoxia as compared to normoxia was abolished. Thus systemic hypoxia markedly potentiates the reflex renal constriction caused by the baroreflex, and this effect is due to the carotid chemoreceptor afferent input.