乳酸脱氢酶实验检测细胞新陈代谢的实验研究

目的 探索LDH实验检测细胞活力的可行性。方法 原代培养骨髓细胞和软骨细胞，用LDH实验测定上述两组细胞的活力，并与镜下活体观察到细胞的生长状况相比较。与目前比较成熟的测定细胞活力的MTS实验的测得的值相比较。结果 LDH实验对上述两组细胞的活力的测定结果与镜下活体观察到的结果相符合。与MTS实验的测得的结果经统计学处理无显著差异。结论 LDH实验可用于细胞活力的直接测定，而对活细胞的生存、繁殖无影响。     

Oxygen-derived free radicals and postischemic myocardial dysfunction ("stunned myocardium")

Experimental studies have demonstrated that myocardium reperfused after reversible ischemia exhibits prolonged depression of contractile function ("stunning"), which is associated with various ultrastructural, biochemical, vascular and other functional abnormalities. Clinical observations suggest that stunning occurs in many situations (for example, rest and exercise-induced angina, myocardial infarction with early reperfusion, open heart surgery, transplantation) and thus may contribute significantly to morbidity among patients with coronary artery disease. In recent years an increasing number of studies have provided indirect evidence that postischemic myocardial dysfunction may be mediated in part by the generation of reactive oxygen species, such as superoxide radical (.O2-), hydrogen peroxide (H2O2) and hydroxyl radical (.OH). Thus, it has been shown that the recovery of the stunned myocardium is enhanced by agents that either scavenge oxygen metabolites, such as superoxide dismutase and catalase, N-2-mercaptopropionylglycine and dimethylthiourea, or prevent their generation, such as allopurinol, oxypurinol and desferrioxamine. More recent experiments utilizing electron paramagnetic resonance spectroscopy have directly demonstrated that reperfusion after a reversible ischemic episode is associated with a burst of free radical production. At present, the evidence supporting the free radical hypothesis is suggestive but not conclusive. Definitive demonstration of the role of oxy-radicals will require careful studies measuring the production of these species in conscious animal models of postischemic dysfunction. If confirmed, the free radical hypothesis will provide not only new important insights into the pathophysiology of ischemic injury, but also a rationale for developing clinically applicable interventions.     

Mutations in the Ca(2+)-sensing receptor gene cause autosomal dominant and sporadic hypoparathyroidism

Parathyroid hormone secretion is negatively regulated by a 7- transmembrane domain, G-protein coupled Ca(2+)-sensing receptor. We hypothesized that activating mutations in this receptor might cause autosomal dominant hypoparathyroidism (ADHP). Consistent with this hypothesis, we identified, in two families with ADHP, heterozygous missense mutations in the Ca(2+)-sensing receptor gene that cosegregated with the disorder. None of 50 normal controls had either mutation. We also identified a de novo, missense Ca(2+)-sensing receptor mutation in a child with severe sporadic hypoparathyroidism. The amino acid substitution in one ADHP family affected the N-terminal, extracellular domain of the receptor. The other mutations involved the transmembrane region. Unlike patients with acquired hypoparathyroidism, patients with these mutations had hypercalciuria even at low serum calcium concentrations. Their greater hypercalciuria presumably reflected activation of Ca(2+)-sensing receptors in kidney cells, where the receptor negatively regulates calcium reabsorption. This augmented hypercalciuria increases the risk of renal complications and thus has implications for the choice of therapy.      

Mosaic pathogenic variants in AKT3 cause capillary malformation and undergrowth

Capillary malformations are slow-flow vascular malformations that affect the microcirculation including capillaries and post capillary venules and can be associated with growth differences. Specifically, the association of capillary malformations with undergrowth is a vastly understudied vascular syndrome with few reports of genetic causes including PIK3CA, GNAQ, and GNA11. Recently, a somatic pathogenic variant in AKT3 was identified in one child with a cutaneous vascular syndrome similar to cutis marmorata telangiectatica congenita, undergrowth, and no neurodevelopmental features. Here, we present a male patient with a capillary malformation and undergrowth due to a somatic pathogenic variant in AKT3 to confirm this association. It is essential to consider that mosaic pathogenic variants in AKT3 can cause a wide spectrum of disease. There is a need for future studies focusing on capillary malformations with undergrowth to understand the underlying mechanism.     

Evidence for an essential role of reactive oxygen species in the genesis of late preconditioning against myocardial stunning in conscious pigs.

Conscious pigs underwent a sequence of 10 2-min coronary occlusions, each separated by 2 min of reperfusion, for three consecutive days (days 1, 2, and 3). On day 1, pigs received an i.v. infusion of a combination of antioxidants (superoxide dismutase, catalase, and N-2 mercaptopropionyl glycine; group II, n = 9), nisoldipine (group III, n = 6), or vehicle (group I [controls], n = 9). In the control group, systolic wall thickening (WTh) in the ischemic-reperfused region on day 1 remained significantly depressed for 4 h after the 10th reperfusion, indicating myocardial "stunning." On days 2 and 3, however, the recovery of WTh improved markedly, so that the total deficit of WTh decreased by 53% on day 2 and 56% on day 3 compared with day 1 (P < 0.01), indicating the development of a powerful cardioprotective response (late preconditioning against stunning). In the anti-oxidant-treated group, the total deficit of WTh on day 1 was 54% less than in the control group (P < 0.01). On day 2, the total deficit of WTh was 85% greater than that observed on day 1 and similar to that observed on day 1 in the control group. On day 3, the total deficit of WTh was 58% less than that noted on day 2 (P < 0.01). In the nisoldipine-treated group, the total deficit of WTh on day 1 was 53% less than that noted in controls (P < 0.01). On days 2 and 3, the total deficit of WTh was similar to the corresponding values in the control group. These results demonstrate that: (a) in the conscious pig, antioxidant therapy completely blocks the development of late preconditioning against stunning, indicating that the production of reactive oxygen species (ROS) on day 1 is the mechanism whereby ischemia induces the protective response observed on day 2; (b) antioxidant therapy markedly attenuates myocardial stunning on day 1, indicating that ROS play an important pathogenetic role in postischemic dysfunction in the porcine heart despite the lack of xanthine oxidase; (c) although the administration of a calcium-channel antagonist (nisoldipine) is as effective as antioxidant therapy in attenuating myocardial stunning on day 1, it has no effect on late preconditioning on day 2, indicating that the ability of antioxidants to block late preconditioning is not a nonspecific result of the mitigation of postischemic dysfunction on day 1. Generation of ROS during reperfusion is generally viewed as a deleterious process. Our finding that ROS contribute to the genesis of myocardial stunning but, at the same time, trigger the development of late preconditioning against stunning supports a complex pathophysiological paradigm, in which ROS play an immediate injurious role (as mediators of stunning) followed by a useful function (as mediators of subsequent preconditioning).     

Dibenzo[a,l]pyrene-induced DNA adduction, tumorigenicity, and Ki-ras oncogene mutations in strain A/J mouse lung

Dibenzo[a,l]pyrene (DB[a,l]P), an environmental polycyclic aromatic hydrocarbon, is the most potent carcinogen ever tested in mouse skin and rat mammary gland. In this study, DB[a,l]P was examined for DNA adduction, tumorigenicity, and induction of Ki-ras oncogene mutations in tumor DNA in strain A/J mouse lung. Groups of mice received a single i.p. injection of 0.3, 1.5, 3.0, or 6.0 mg/kg DB[a,l]P in tricaprylin. Following treatment, DNA adducts were measured at times between 1 and 28 days, while tumors were counted at 250 days and analyzed for the occurrence of point mutations in codons 12 and 61 of the Ki-ras oncogene. DB[a,l]P in strain A/J mouse lung induced six major and four minor DNA adducts. Maximal levels of adduction occurred between 5 and 10 days after injection followed by a gradual decrease. DB[a,l]P-DNA adducts in lung tissue were derived from both anti- and syn-11,12- dihydroxy-13,14-epoxy- 11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE) and both deoxyadenosine (dAdo) and deoxyguanosine (dGuo) residues in DNA as revealed by cochromatography. The major adduct was identified as a product of the reaction of an anti-DB[a,l]PDE with dAdo in DNA. DB[a,l]P induced significant numbers of lung adenomas in a dose- dependent manner, with the highest dose (6.0 mg/kg) yielding 16.1 adenomas/mouse. In tricaprylin-treated control animals, there were 0.67 adenomas/mouse. Based on the administered dose, DB[a,l]P was more active than other environmental carcinogens including benzo[a]pyrene. As a function of time-integrated DNA adduct levels, DB[a,l]P induced lung adenomas with about the same potency as other PAHs, suggesting that the adducts formed by DB[a,l]P are similar in carcinogenic potency to other PAHs in the strain A/J mouse lung model. Analysis of the Ki- ras mutation spectrum in DB[a,l]P-induced lung tumors revealed the predominant mutations to be G-->T transversions in the first base of codon 12, A-->G transitions in the second base of codon 12, and A-->T transversions in the second or third base of codon 61, concordant with the DNA adduct profile.      

Influence of coronary collateral vessels on myocardial infarct size in humans. Results of phase I thrombolysis in myocardial infarction (TIMI) trial. The TIMI Investigators

BACKGROUND. The influence of coronary collateral vessels on infarct size in humans remains controversial, partly because no previous study has examined the impact of collaterals present at the onset of acute myocardial infarction on infarct size. METHODS AND RESULTS. The present study used the data base of the Thrombolysis in Myocardial Infarction (TIMI) Phase I trial to correlate the presence or absence of angiographically documented collaterals in the initial hours of myocardial infarct evolution with the size of the infarct as assessed by serial measurements of serum creatine kinase (CK). To avoid the confounding effects of reperfusion on enzymatic estimates of infarct size, this report is limited to those 125 patients who failed to recanalize at 90 minutes after administration of tissue plasminogen activator or streptokinase. Patients with angiographically documented collaterals (group A, n = 51) had significantly lower values of peak serum CK than patients without collaterals (group B, n = 74) (1,877 +/- 216 versus 2,661 +/- 212 IU/l, respectively [mean +/- SEM], p = 0.004). Similarly, CK-derived infarct size estimates were significantly lower in group A than in group B (20.6 +/- 2.5 versus 31.4 +/- 2.8 CK gram equivalents, p = 0.001). The infarct size observed in patients with collaterals was less for anterior infarctions as well as for infarctions of other locations; thus, the beneficial effects of collaterals were independent of the site of the infarct. In 65 of the 125 patients who failed to reperfuse, left ventricular ejection fraction (LVEF) was assessed by contrast ventriculography both at initial cardiac catheterization (before thrombolytic therapy) and at hospital discharge. Among the patients who had both studies, global LVEF tended to increase from pretreatment to hospital discharge in group A (from 50.6 +/- 1.8% to 53.4 +/- 1.8%, p = 0.10) but decreased in group B patients (from 50.3 +/- 1.8% to 47.8 +/- 1.7%, p = 0.02). At hospital discharge, global LVEF was greater in patients with coronary collaterals (53.5 +/- 1.7% versus 49.6 +/- 1.7%, p = 0.01). CONCLUSIONS. The results demonstrate that, in patients in whom thrombolytic therapy fails to induce reperfusion, the presence of coronary collateral vessels at the onset of myocardial infarction is associated with limitation of infarct size as assessed enzymatically and with improved ventricular function on discharge as assessed by LVEF.     

End-systolic radius to thickness ratio: an echocardiographic index of regional performance during reversible myocardial ischemia in the conscious dog

Regional myocardial dysfunction induced by ischemia is associated with less thickening and a larger ventricular radius at end-systole. Thus, end-systolic radius to thickness ratio measured by echocardiography may provide an accurate index of regional left ventricular function that is totally independent of cardiac motion. To test this hypothesis, a total of 14 transient (less than or equal to 10 minutes) coronary artery occlusions (8 left anterior descending, 6 left circumflex) followed by up to 24 hours of reperfusion were performed in six chronically instrumented conscious dogs providing multiple grades of regional ventricular dysfunction. Regional myocardial thickening fraction was determined with epicardial pulsed Doppler probes and served as an independent standard for comparison with simultaneous echocardiographic measurements. End-systolic radius to thickness ratio and radial shortening fraction were derived from the two-dimensional echocardiographic short-axis view along 12 equidistant radii. In the ischemic zone, percent thickening fraction averaged 22 +/- 5% during baseline, decreased to -4 +/- 4% during occlusion with gradual return to baseline after reperfusion. End-systolic radius to thickness ratio averaged 1.39 +/- 0.25 before coronary occlusion and increased to 2.97 +/- 0.48 during occlusion with a gradual return to baseline values. A significant correlation was found between Doppler-determined thickening fraction measurements and echocardiographic end-systolic radius to thickness ratio as well as radial shortening fraction for absolute values (r = -0.83 and 0.75, respectively; n = 65) and percent change from baseline (r = -0.86 and 0.78, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)