Disappearance of cyclin A correlates with permanent withdrawal of cardiomyocytes from the cell cycle in human and rat hearts.

The regulated expression of cyclins controls the cell cycle. Because cardiomyocytes in adult mammals withdraw permanently from the cell cycle and thus cannot regenerate after injury, we examined cyclin expression during development by comparing cyclin A-E mRNA levels in fetal and adult human hearts. Cyclin B mRNA was detectable in adult hearts, although at a level markedly lower than that in fetal hearts. Levels of cyclin C, D1, D2, D3, and E mRNA were essentially identical in the two groups. In contrast, cyclin A mRNA was undetectable in adult hearts whereas cyclin A mRNA and protein were readily detectable in fetal hearts and cardiomyocytes, respectively. We then measured cyclin A mRNA and protein levels in rat hearts at four stages of development (fetal and 2, 14, and 28 d). Cyclin A mRNA and protein levels decreased quickly after birth (to 37% at day 2) and became undetectable within 14 d, an observation consistent with reports that cardiomyocytes stop replicating in rats by the second to third postnatal week. This disappearance of cyclin A gene expression in human and rat hearts at the time cardiomyocytes become terminally differentiated suggests that cyclin A downregulation is important in the permanent withdrawal of cardiomyocytes from the cell cycle.

Hypoxia induces severe right ventricular dilatation and infarction in heme oxygenase-1 null mice.

Heme oxygenase (HO) catalyzes the oxidation of heme to generate carbon monoxide (CO) and bilirubin. CO increases cellular levels of cGMP, which regulates vascular tone and smooth muscle development. Bilirubin is a potent antioxidant. Hypoxia increases expression of the inducible HO isoform (HO-1) but not the constitutive isoform (HO-2). To determine whether HO-1 affects cellular adaptation to chronic hypoxia in vivo, we generated HO-1 null (HO-1(-/-)) mice and subjected them to hypoxia (10% oxygen) for five to seven weeks. Hypoxia caused similar increases in right ventricular systolic pressure in wild-type and HO-1(-/-) mice. Although ventricular weight increased in wild-type mice, the increase was greater in HO-1(-/-) mice. Similarly, the right ventricles were more dilated in HO-1(-/-) mice. After seven weeks of hypoxia, only HO-1(-/-) mice developed right ventricular infarcts with organized mural thrombi. No left ventricular infarcts were observed. Lipid peroxidation and oxidative damage occurred in right ventricular cardiomyocytes in HO-1(-/-), but not wild-type, mice. We also detected apoptotic cardiomyocytes surrounding areas of infarcted myocardium by terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) assays. Our data suggest that in the absence of HO-1, cardiomyocytes have a maladaptive response to hypoxia and subsequent pulmonary hypertension. J.Clin. Invest. 103:R23-R29 (1999).

The ATF site mediates downregulation of the cyclin A gene during contact inhibition in vascular endothelial cells.

Contact inhibition mediates monolayer formation and withdrawal from the cell cycle in vascular endothelial cells. In studying the cyclins--key regulators of the cell cycle--in bovine aortic endothelial cells (BAEC), we found that levels of cyclin A mRNA decreased in confluent BAEC despite the presence of 10% fetal calf serum. We then transfected into BAEC a series of plasmids containing various lengths of the human cyclin A 5' flanking sequence and the luciferase gene. Plasmids containing 3,200, 516, 406, 266, or 133 bp of the human cyclin A promoter directed high luciferase activity in growing but not confluent BAEC. In contrast, a plasmid containing 23 bp of the cyclin A promoter was associated with a 65-fold reduction in activity in growing BAEC, and the promoter activities of this plasmid were identical in both growing and confluent BAEC. Mutation of the activating transcription factor (ATF) consensus sequence at bp -80 to -73 of the cyclin A promoter decreased its activity, indicating the critical role of the ATF site. We identified by gel mobility shift analysis protein complexes that bound to the ATF site in nuclear extracts from growing but not confluent BAEC and identified (with antibodies) ATF-1 as a binding protein in nuclear extracts from growing cells. Also, ATF-1 mRNA levels decreased in confluent BAEC. Taken together, these data suggest that the ATF site and its cognate binding proteins play an important role in the downregulation of cyclin A gene expression during contact inhibition.

Impaired abdominal wall development and deficient wound healing in mice lacking aortic carboxypeptidase-like protein.

Aortic carboxypeptidase-like protein (ACLP) is a member of a diverse group of proteins that contain a domain with similarity to that of the Dictyostelium discoideum protein discoidin I. The discoidin domain has been identified in mammalian milk fat globule membrane proteins, blood coagulation factors, and receptor tyrosine kinases, where it may facilitate cell aggregation, adhesion, or cell-cell recognition. Here we show that ACLP is a secreted protein that associates with the extracellular matrix (ECM). During mouse embryogenesis, ACLP is abundantly expressed in the ECM of collagen-rich tissues, including the vasculature, dermis, and the developing skeleton. We deleted the ACLP gene in mice by homologous recombination. The majority of ACLP(-/-) mice die perinatally due to gastroschisis, a severe disruption of the anterior abdominal wall and herniation of the abdominal organs. ACLP(-/-) mice that survived to adulthood developed nonhealing skin wounds. Following injury by a dermal punch biopsy, ACLP(-/-) mice exhibited deficient wound healing compared with controls. In addition, dermal fibroblasts isolated from ACLP(-/-) 18.5-day-postconception embryos exhibited a reduced proliferative capacity compared with wild-type cells. These results indicate that ACLP is an ECM protein that is essential for embryonic development and dermal wound healing processes.

Prevention of hypoxia-induced pulmonary hypertension by enhancement of endogenous heme oxygenase-1 in the rat.

We investigated the role of heme oxygenase (HO)-1 in the development of hypoxia-induced pulmonary hypertension. HO catalyzes the breakdown of heme to the antioxidant bilirubin and the vasodilator carbon monoxide. Hypoxia is a potent but transient inducer of HO-1 in vascular smooth muscle cells in vitro and in the lung in vivo. By using agonists of HO-1, we sustained a high expression of HO-1 in the lungs of rats for 1 week. We report that this in vivo enhancement of HO-1 in the lung prevented the development of hypoxic pulmonary hypertension and inhibited the structural remodeling of the pulmonary vessels. The mechanism(s) underlying this effect may involve a direct vasodilating and antiproliferative action of endogenous carbon monoxide, as well as an indirect effect of carbon monoxide on the production of vasoconstrictors. These results provide evidence that enhancement of endogenous adaptive responses may be used to prevent hypoxia-induced pulmonary hypertension.

Cell refractive index for cell biology and disease diagnosis: past, present and future.

Cell refractive index is a key biophysical parameter, which has been extensively studied. It is correlated with other cell biophysical properties including mechanical, electrical and optical properties, and not only represents the intracellular mass and concentration of a cell, but also provides important insight for various biological models. Measurement techniques developed earlier only measure the effective refractive index of a cell or a cell suspension, providing only limited information on cell refractive index and hence hindering its in-depth analysis and correlation. Recently, the emergence of microfluidic, photonic and imaging technologies has enabled the manipulation of a single cell and the 3D refractive index of a single cell down to sub-micron resolution, providing powerful tools to study cells based on refractive index. In this review, we provide an overview of cell refractive index models and measurement techniques including microfluidic chip-based techniques for the last 50 years, present the applications and significance of cell refractive index in cell biology, hematology, and pathology, and discuss future research trends in the field, including 3D imaging methods, integration with microfluidics and potential applications in new and breakthrough research areas.

Influence of suture on bronchial anastomosis in growing puppies.

A comparison study of synthetic nonabsorbable suture (nylon or Prolene) with absorbable suture (Dexon-S or Vicryl) for bronchoplasty was performed in growing puppies. The experiments (n = 15) were followed up for 185 to 381 days (average 323.8 days). Bronchoscopic and bronchographic studies were done at intervals. No stenosis was observed in the group receiving absorbable suture (n = 8), and the anastomoses grew proportionately with the proximal and distal bronchi. In the group receiving nonabsorbable suture (n = 7), variable stenoses developed; two moderate and two severe stenoses were observed during the early healing stage by bronchoscopic examination. In the sequential bronchograms, an increase of stenosis was noted in two dogs. The results suggest that absorbable suture is superior to nonabsorbable suture in pediatric bronchoplasty.

Cardiopulmonary exercise testing after single and double lung transplantation.

The cardiopulmonary response to exercise was investigated in six single and six double lung transplant recipients using a three-minute incremental work rate protocol on a cycle ergometer. Maximum VO2 averaged 44.2 +/- 9.2 percent and 48.5 +/- 5.0 percent of predicted maximal VO2 in the single and double lung transplant groups, respectively. No evidence of ventilatory limitation to exercise was found in either group. Circulatory factors that may have limited exercise capacity included anemia and submaximal heart rates. There was a strong correlation between VO2/kg at venous blood lactate level of 2.2 mEq/L and vital capacity/body surface area in the single, but not in the double, lung recipients. Maximum VO2 in these lung transplant recipients was comparable to previously published values in heart-lung transplant recipients. The factors that limit maximum exercise capacity after lung transplantation deserve further study.

A reevaluation of heparin requirements for cardiopulmonary bypass.

We wished to determine if reduction in the standard heparin administration for cardiopulmonary bypass could be accomplished safely with the use of membrane oxygenators. An experimental study was designed to evaluate two different heparin administration protocols for cardiopulmonary bypass with hollow-fiber membrane oxygenators. Two groups of six pigs were submitted to hypothermic cardiopulmonary bypass (28 degrees C) for 3 hours, then rewarmed, decannulated, and reassessed after 1 hour. In group I (control) heparin was administered to maintain the activated clotting time in excess of 450 seconds; in group II activated clotting time was maintained between 250 and 300 seconds. The mean total heparin administered was 41,000 units in group I and 25,000 units in group II. Concentration of coagulation factors II, V, and VIII, fibrinogen, and platelet count were determined before, during, and 1 hour after bypass. No significant difference in any of these coagulation parameters was observed between the groups. The performance of the oxygenators was similar in both groups, with no evidence of thrombosis. Thus reduced heparin administration, enough to keep activated clotting time between 250 and 300 seconds, was not related either to major coagulation factors and platelet consumption or to derangements in the oxygenator's performance.

The effect of ischemic time and temperature on lung preservation in a simple ex vivo rabbit model used for functional assessment.

Despite 25 years of research in lung transplantation, little progress has been made in methods for improving lung preservation. To evaluate many factors that may affect lung function after preservation, we have developed a simple, reliable, and inexpensive animal model. This consists of an isolated rabbit lung preparation perfused with blood and ventilated with a closed-circuit system. Heart-lung blocks were harvested and the left lung was assessed after ligation of the right pulmonary artery and right main-stem bronchus. On completion of a storage period, the left lung was ventilated and perfused with fresh rabbit venous blood at a rate of 40 ml/min for 10 minutes. Assessment of lung function included gas analysis of infused and effluent blood, oxygen uptake, mean pulmonary artery perfusion pressure, and mean airway pressure. A control group and six preservation groups were evaluated, each with different storage temperatures (38 degrees C, 34 degrees C, 23 degrees C, 15 degrees C, 10 degrees C, and 4 degrees C). For each temperature, ischemic periods ranging from 1 to 30 hours were studied. In the control group, the lungs were assessed immediately after being harvested. In the preservation groups, the lungs were kept partially inflated, stored at a predetermined temperature by immersion, and later assessed after variable ischemic periods. Our studies demonstrated the following: (1) The degree of impaired lung function produced by ischemia is reflected by a decrease in oxygen uptake and in oxygen tension of the effluent pulmonary venous blood and an increase in pulmonary artery perfusion pressure; (2) hypothermia improves ischemic tolerance; (3) preservation of lung at 10 degrees C is superior to preservation at 15 degrees C and 4 degrees C. This screening model has allowed evaluation of independent multiple factors and methods pertinent to lung preservation and enables one to assess lung function reliably and rapidly.

Influence of temperature of flushing solution on lung preservation.

For lung transplantation the technique of flushing the donor pulmonary vascular bed may provide advantages in lung preservation such as rapid cooling and washout of blood. However, rapid cooling of the ischemic lung may also produce adverse effects. The aim of this study was to compare methods of cold flushing and topical cooling, and to evaluate the effect of temperature of the flushing solution on lung preservation. A total of 25 rabbit lungs were studied. Using an ex vivo rabbit lung model, postischemic function was assessed by the ability of the lung to oxygenate perfused blood and by measurement of pulmonary artery and airway pressures. The lungs in group I were preserved with simple immersion at 10 degrees C for 30 hours. The lungs in groups II through V were flushed with solution containing phosphate-buffered dextran (LPD) at different temperatures (groups II and IV, 10 degrees C; groups III and V, 23 degrees C) and stored at 10 degrees C for various ischemic periods (groups II and III, 30 hours; groups IV and V, 36 hours). Pulmonary vascular resistance during flushing at 10 degrees C was significantly higher than that at 23 degrees C (p < 0.001). Flushing resulted in better preservation than topical hypothermia. Flushing at 23 degrees C resulted in superior postischemic function compared with flushing at 10 degrees C. We conclude that in lung preservation, uniform flushing with LPD solution improves the ischemic tolerance as compared with topical hypothermia, and that flushing with solutions at too low temperatures may have adverse effects on lung preservation.

Production and reversibility of right ventricular hypertrophy and right heart failure in dogs.

Combined heart-lung transplantation has been used for end-stage primary pulmonary hypertension. Experience with single-lung transplantation for other conditions suggested that associated severe right ventricular dysfunction resulting from increased afterload would recover after placement of a satisfactory lung allograft. Early experience with the application of single-lung transplantation for pulmonary hypertension supports this contention. We devised a reversible canine model of chronic progressive pressure-overloaded right heart failure by pulmonary artery banding to study the echocardiographic, hemodynamic, and pathological reversibility of the failing right heart. Clinical right heart failure was defined as the development of ascites and pleural effusions. Right heart failure developed in 23 dogs 67 to 348 days after banding, and they were divided into two groups to determine its early and long-term effects. Group 1 dogs (n = 11) were either sacrificed immediately after the onset of right heart failure (n = 5) or unbanded (n = 6); group 2 dogs (n = 12) were maintained in right heart failure for 3 months and then either sacrificed (n = 6) or unbanded. Unbanded dogs in both groups were observed for 4 additional months before sacrifice. A control group of 6 normal dogs was sacrificed for pathological comparisons. After unbanding, the right ventricular systolic pressure fell from 97 +/- 17 mm Hg (group 1) and 88 +/- 31 mm Hg (group 2) to 44 +/- 11 mm Hg and 47 +/- 13 mm Hg, respectively. Despite this persistent gradient across the pulmonary artery, echocardiographic and hemodynamic measures of right ventricular function returned to normal, albeit more slowly in the group 2 dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic variation at nine STR loci in populations from the Philippines and Thailand living in Taiwan.

In order to apply a set of nine STR loci and the amelogenin locus in forensic testing, we have performed a population study on individuals from the Philippines and Thailand living in Taiwan (273 Philippine and 146 Thai individuals were typed by commercially available kits and an automated sequencer). A total of 73 alleles for all systems for both populations could be observed in these two populations. No new intermediate fragments were found. Allele frequencies showed no deviation from Hardy-Weinberg equilibrium. The mean exclusion power (MEP) ranged from 0.327 (TPOX) to 0.706 (FGA), the discriminating power (DP) ranged from 0.790 (TPOX) to 0.963 (FGA) for Philippinos, MEP ranged from 0.247 (TPOX) to 0.723 (FGA), DP ranged from 0.761 (TPOX) to 0.968 (FGA) for Thais, the combined MEP is > 0.9988 and the combined DP is > 0.9999999993 for both Philippinos and Thais.

[The comparison of the binding of Ru486 to progesterone receptor of pituitary and endometrium in ovariectomized rat].

Following intramuscular injections of increasing doses of Ru486 (0.1 mg/kg-2 mg/kg), free progesterone binding sites remaining in pituitary and endometrium in estrogen-primed OVX rats decreased. Thirty minutes after administration of Ru486 (2 mg/kg) the binding sites of progesterone receptor in both tissues showed a significant initial decrease, and then gradually reached the lowest level in 2 h. Afterwards the levels of progesterone receptor began to increase. The results suggested that Ru486 could interact with progesterone receptor in both tissues. However, the binding ability of Ru486 for different tissue was not exactly the same, and Ru486 binding to the progesterone receptor in endometrium might be easier.

[The effect of RU486 on contractility of rabbit oviduct smooth muscle].

An investigation on the effect of RU486 on isolated ovicluct smooth muscle contraction of pseudopregant rabbits was carried out by using the mechanical-electrical transducer of Galson recorder. The results showed that: (1) RU486 acted directly on the oviduct smooth muscle by increasing contractile frequency, without changing tension and amplitude. This effect is similar to that found in the in vivo experiment. (2) RU486 partially inhibited Ca(2+)-induced smooth muscle contraction and also showed significant synergistic effect of Verapamil and antagonistic effect of NE on muscle contractility. However, RU486 is unable to exert any effect on Forskolin-stimulated contraction. Thus it appears that the RU486 effect on oviduct smooth muscle contraction is a result of affecting intracellular free calium, due possibly to interference of Ca2+ influx and/or endoplasmic reticulum Ca2+ release and Ca(2+)-Ip3 transducing mechanism.

Is child maltreatment a leading cause of delinquency?

This article explores the assumption that child maltreatment is a major cause of juvenile delinquency. Although this relationship is widely accepted as fact, the research that exists in the literature so far is inconclusive at best, and at worst, deeply flawed. For too long, juvenile justice resources have been channeled into the apprehension and processing of youthful offenders, while relatively few dollars have gone into the research necessary to formulate a coherent strategy for solving the juvenile crime problem. Without funding for this much-needed research, the money we do spend on the control and prevention of juvenile crime is blindly spent.

Positron emission tomography in bronchioloalveolar carcinoma of the lung.

OBJECTIVES: This study assessed the maximum standard uptake value of positron emission tomography-computed tomography in patients of pulmonary adenocarcinoma with bronchioloalveolar carcinoma features and whether SUVmax correlates with pathological status, lymph node metastasis, and prognosis. METHODS: We retrospectively reviewed 674 patients diagnosed with non-small-cell lung cancer between January 2002 and June 2009. Patients with clinical stage I-II disease underwent a preoperative PET-CT scan followed by anatomic resection. We reviewed the clinical features of 209 patients with an average follow-up of 87 months. RESULTS: We analyzed clinical variables for 40 patients with BAC features and 169 patients without BAC features. Age, sex, location, and number of dissected lymph nodes, carcinoembryonic antigen level, and lymphovascular invasion had no difference between the two groups. Compared with non-BAC patients, patients with BAC features had a lower SUVmax (2.51 ± 2.02 vs 4.98 ± 4.03, p < 0.001), lower ratio of SUVmax (1.10 ± 0.34 vs 1.22 ± 0.27, p = 0.014), better tumor differentiation (p < 0.001), and smaller tumor size (2.30 ± 1.41 vs 2.97 ± 1.71, p < 0.03). The negative prediction rate was 87.08% for N2 and 80.80% for N1 disease. All patients in the BAC group were alive after the operation. The five-year survival rate of patients without BAC features was 71.2%. CONCLUSIONS: Preoperative SUVmax of PET-CT was more accurate at predicting negative N2 than N1 disease. BAC is associated with markedly better prognosis compared with invasive adenocarcinoma and may be cured with surgical resection Aggressive surgical resection is recommended even for patients with false-negative N2 disease.