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PubMed1991

Hypertrophy of isolated adult feline heart cells following beta-adrenergic-induced beating.

Catecholamine-induced beating and myocardial hypertrophy were evaluated in isolated adult feline cardiomyocytes maintained in culture for up to 30 days. Adult feline cardiomyocytes were used in this study because they displayed several unique characteristics that facilitated assessment of factors regulating cardiomyocyte hypertrophy in vitro. These characteristics included the following. 1) A single heart provides a high yield of 20-40 x 10(6) calcium-tolerant rod-shaped myocytes. 2) In culture, isolated adult feline cardiomyocytes maintain a stable population of differentiated myocytes that could be maintained without the dramatic loss of cell number, DNA content, or cell structure seen in adult rat cardiomyocyte cultures. 3) Cultured feline cardiomyocytes remained quiescent in culture unless appropriately stimulated to begin beating. 4) Sustained regular beating activity could be readily initiated up to 3 wk in culture by addition of 1 x 10(-5) M isoproterenol, other beta-adrenergic agonists, or agents known to elevate adenosine 3',5'-cyclic monophosphate. Beating could be maintained indefinitely in the presence of isoproterenol, but ceased upon removal of isoproterenol from the medium. Initiation of beating in 7-day-old cultures resulted in a profound restructuring of cardiomyocyte morphology compared with quiescent cultures. Beating heart cells were 66% larger with increased protein content, and they had significantly greater development of striated myofibrillar structure than quiescent myocytes at the same age in culture. We conclude that maintenance of an organized myofibrillar structure in cultured adult cardiac myocytes requires activation of intrinsic beating. Cardiomyocyte hypertrophy also develops following beta-adrenergic activation of beating, but it is unclear whether beating per se is required for inducing hypertrophy in isolated adult cardiomyocytes in vitro.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Isolation and morphology of calcium-tolerant feline ventricular myocytes.

A technique has been developed for isolating a high yield of Ca2+-tolerant rod-shaped myocytes from the right and left ventricles of cat myocardial tissue. Myocytes were prepared by retrograde perfusion of the coronary arteries via the aorta with a nominally Ca2+-free (20-30 microM) modified Krebs-Henseleit buffer containing 0.12% collagenase. After exposure to physiological levels of Ca2+ (1-2.5 mM), the cells retained rod-shaped morphology, exhibited clear cross striations, and excluded the dye trypan blue (0.4%). Initial percents of viable Ca2+-tolerant rod-shaped cells were 58.6 +/- 3.4 (SE) and 51.8 +/- 3.5 for right and left ventricular cells, respectively. Viability studies demonstrated that these values decreased approximately 10% at the conclusion of a 2-h incubation in 1 mM Ca2+. The total numbers of rod-shaped myocytes obtained were 4.48 X 10(7) and 3.89 X 10(7) in nominal (8-10 microM) and 1 mM Ca2+-containing buffer, respectively. A total of 3.44 +/- 0.40 X 10(6) rod-shaped Ca2+-tolerant myocytes was initially isolated per gram of tissue wet weight. Measurements of cell length, width, and sarcomere length demonstrated no significant differences between right and left ventricular cells suspended in nominal (8-10 microM) and 1 mM Ca2+-containing buffer. No significant difference was found in the percent of binucleate cells when right and left ventricular myocytes were compared. These results demonstrate that a stable population of Ca2+-tolerant myocytes with similar morphological characteristics can be isolated from the right and left ventricles of cat myocardium.

Action Potentials↗
PubMedDate not supplied

Risk for relatives of patients with multiple sclerosis in central Sardinia, Italy.

Multiple sclerosis (MS) is a chronic, inflammatory, disabling disease of the central nervous system, known for its complex interplay between genetic and environmental factors. We used life table techniques to calculate age-adjusted recurrence risks for different categories of relatives of MS patients from Central Sardinia (Italy), a genetically homogeneous, stable population with a high degree of consanguinity. We included 313 probands and a total of 12,717 relatives in the analysis. The overall age-adjusted recurrence risk for relatives of MS probands is 1.90% [95% confidence interval (CI): 1.57-2.30]. The age-adjusted recurrence risk in parents was 1.26% (95% CI 0.60-2.63), in children 2.33% (95% CI 0.09-5.56), in sibs 4.76% (95% CI 3.57-6.32), in second-degree relatives 0.72% (95% CI 0.42-1.22), and in third-degree relatives 1.79% (95% CI 1.27-2.51). The sex of the probands (male) and of the relatives (female), and the number of affected relatives in the family significantly increase the risk of MS in relatives.

Confidence Intervals↗
PubMed1982

Cytogenetic surveillance of spontaneous abortions.

During the five years of a cytogenetic study on spontaneous abortions in Hawaii there have been significant increases in the frequencies of triploidy of maternal origin and trisomy 16, a significant decrease in sex chromosome monosomy, and variation without trend in the frequency of tetraploidy. Attempts to explain these results in terms of technical factors or changes in maternal and gestational age have been unsuccessful, as have preliminary attempts to identify viral, pharmacological, or other causes. Apparently the etiology of chromosomal abnormality is variable over a few years even in a stable population. Research to identify these causes is continuing on the supposition that variable risks can be reduced.

Abortion, Spontaneous↗
PubMed1990

Dental caries changes in the Scottish Isle of Lewis.

The Scottish Isle of Lewis has an island community which traditionally is a stable population that has little migration of its school children. The island has low levels of fluoride (less than 0.1 ppm) in the drinking water supplies, but the island inhabitants have the availability of all UK commodities. The island has well detailed dental data going back to 1937. The present study (1987) examined dental caries and oral hygiene status in the school community between 1984 and 87 and compares the findings to the previous examinations completed by the same survey team. The caries levels declined during the period 1984-87, but not as rapidly as the decline reported in the 1970s. The main reason for the continued decline in dental caries appears to be the availability of fluoride dentifrices.

Adolescent↗
PubMed2006

Functional relevance of Golgi- and plasma membrane-localized endothelial NO synthase in reconstituted endothelial cells.

OBJECTIVE: We have previously shown in COS-7 cells that targeting of endothelial NO synthase (eNOS) to the Golgi or plasma membrane (PM) regulates the mechanism and degree of eNOS activation. However, little is known about the functional significance of eNOS targeting in endothelial cells (ECs). The goal of the current study was to isolate these 2 pools of enzyme in ECs and determine their functional significance in response to agonist stimulation and manipulation of membrane cholesterol levels. METHODS AND RESULTS: Using an RNA interference strategy, we generated stable populations of ECs that had >90% inhibition of eNOS expression and lacked the ability to produce NO. Reconstitution of these eNOS "knockdown" ECs with Golgi- and PM-targeted eNOS restored the ability of ECs to produce NO. Calcium-dependent agonists were the more efficient stimulus for the PM-restricted eNOS in ECs. In contrast, Golgi eNOS was less responsive to both calcium- and Akt-dependent agonists. eNOS restricted to the PM was more sensitive to manipulation of membrane cholesterol levels and was significantly attenuated by modified low-density lipoprotein. CONCLUSIONS: Within ECs, the PM is the most efficient location to produce NO but is more vulnerable to cholesterol levels and modified low-density lipoprotein.

Animals↗
PubMed1976

Characterization of immunocompetent cells recovered from the respiratory tract and tracheobronchial lymph node of normal guinea pigs.

To gain insight into the structure of the lung's immune system, the identity and function of immunocompetent cells in the broncho-alveolar air spaces and tracheobronchial lymph nodes of normal guinea pigs were examined. Guinea pig thymus-derived (T) lymphocytes (mediators of cellular immunity) were identified by their ability to form rosettes with rabbit erythrocytes (E rosettes). Bone marrow-derived (B) lymphocytes (mediators of humoral immunity) were identified by their ability to form rosettes with sheep erythrocytes sensitized with antibody and complement (EAC rosettes) and by the presence of surface immunoglobulin, detected by direct immunofluorescence. Total lung lavage yielded 14+/-5.0 X 10(6) (mean +/- SD) cells. There were 68+/-5 per cent cells of the monocyte-macrophage series as judged by morphology, the ingestion of latex particles, and the uptake of neutral red; 20+/-9 per cent were eosinophils, and 12+/-5 per cent were lymphocytes. Stable populations of T and B lymphocytes were recovered from guinea pig airways and tracheobronchial lymph nodes. T cells represented 76 per cent of lymphocytes from the airways and 64 per cent of lymphocytes in tracheobronchial lymph node; B cells equalled 14 per cent and 30 per cent, respectively. The functional potential of T cells present in the lung and tracheobronchial lymph node was demonstrated by their proliferative response to phytohemagglutinin. These results indicated that, although macrophages are the predominant cell type, viable T and B lymphocytes are present in the guinea pig broncho-alveolar air space. The composition of these cells is comparable to what has been observed in normal human lungs and provides preliminary evidence that the guinea pig may be a useful model for humans. This report also describes a method using 2 vital stains whereby macrophages, lymphocytes, and rosetted lymphocytes can be distinguished in unseparated cell suspensions prepared from bronchial aspirates.

Animals↗
PubMed1992

A rat alveolar type II cell line developed by adenovirus 12SE1A gene transfer.

The regulation of pulmonary alveolar type II cell proliferation and differentiation is poorly understood and has been difficult to study, in part due to lack of proliferation, cellular heterogeneity, and phenotypic instability of type II cells in primary culture. To develop a stable population of homogeneous cells capable of proliferation, we transfected type II cells isolated from the lungs of neonatal rats with an immortalizing oncogene, adenovirus 12SE1A, using a retroviral vector. Individual clones were isolated, screened for cytokeratin expression, and further characterized. One of the 12SE1A expressing clones, E1A-T2, has epithelial features such as cytokeratin expression and tight junctions, and coexpresses vimentin. E1A-T2 rapidly proliferate when grown in 10% fetal bovine serum, and slow their growth at confluence. A labeling index of greater than 90% during a 24-h pulse of [3H]thymidine reflects a uniform population of proliferating cells. E1A-T2 can be grown and passed in 0.4% fetal bovine serum, suggesting the production of an autocrine growth factor(s). The type II cell Maclura pomifera agglutinin (MPA)-binding glycoprotein, MPA-gp200, appears to be expressed in an incompletely glycosylated form, whereas other features of differentiated type II cells, such as lamellar bodies, surfactant protein A, and a high percentage of saturated phosphatidylcholine, are absent. Homogeneous, clonally derived type II cell lines, such as E1A-T2 may retain sufficient type II cell features of interest to test new hypotheses relating to cell proliferation and differentiation otherwise not feasible using primary cultures of type II cells.

Adenoviridae↗
PubMed1985

Use of an X-linked human neutrophil marker to estimate timing of lyonization and size of the dividing stem cell pool.

In families with X-linked chronic granulomatous disease (CGD), heterozygous females have two stable populations of polymorphonuclear leukocytes (PMN) in their blood; one normal, the other, deficient in oxygen metabolism. The two types of PMN can be distinguished by the ability or lack of ability to reduce nitroblue tetrazolium dye. The variation in the percent normal PMN among 11 CGD heterozygotes was shown to follow a binomial distribution based on eight independent trials and a chance of success of 50%. This is consistent with the occurrence of X-chromosome inactivation (lyonization) when eight embryonic founder cells for the hematopoietic system are present. Serial determinations of the percent normal PMN in individual heterozygotes showed very limited variability (standard deviations ranged from 2.0% to 5.2%) most of which could be ascribed to experimental error. An estimate of the remaining variation (residual variance) was introduced into a well-known formula to calculate the appropriate number of pluripotent stem cells necessary to support hematopoiesis and a figure exceeding 400 was obtained. Thus, the data indicate that in humans there is a highly polyclonal system of hematopoiesis.

Clone Cells↗
PubMed1996

GPI-anchored complement regulatory proteins in seminal plasma. An analysis of their physical condition and the mechanisms of their binding to exogenous cells.

We analyzed and compared the properties of three glycosylphosphatidylinositol (GPI)-anchored proteins. CD59, CD55 (both C regulators), and CDw52, and of the transmembrane C regulator CD46 in seminal plasma (SP). We demonstrated previously that anchor-intact SP CD59 is present on the membranes of vesicles (prostasomes) and that cells acquire this protein during incubation with SP. We now report that this acquisition is due partly to adherence of prostasomes to cells and partly to a second mechanism which may involve micellar intermediates. Using fluorescent labeling, ultracentrifugation, and density gradient centrifugation, virtually all CD46 was present on prostasomes whereas CD59, CD55, AND CDw52 were also detected in a form which remained in the 200,000 g supernatant and equilibrated at higher density than prostasomes in gradients. All three GPI-linked proteins eluted at high molecular mass during size exclusion chromatography of this nonprostasome fraction. As documented by videomicroscopy and biochemical analysis, cells acquired new copies of the GPI-linked proteins during incubation with the nonprostasome fraction as well as with prostasomes. These data demonstrate the presence in SP of a stable population of membrane-free, GPI-linked proteins available for transfer to cells. Binding of these proteins to spermatozoa and pathogens in SP may confer new properties on their membranes including increased resistance to C attack. Finally, our data raise the possibility that lipid-associated GPI-linked proteins may be suitable for therapeutic applications.

Animals↗
PubMed1985

Acute renal failure: the tip of the iceberg?

This study reports the experience, during a six-year period, of the Aberdeen Renal Unit in the treatment of patients with acute renal failure. The combination of a relatively stable population base and a single regional dialysis centre has allowed the incidence of acute renal failure to be assessed. Approximately 30 patients per million population were dialysed annually for acute renal failure; 69 per cent of these patients (20.5 per million population per year) were dialysed for acute reversible intrinsic renal failure (ARIRF) and mortality in this group was 44 per cent. Patients with more severe disease at the time of presentation to the renal unit, as defined by a clinical severity score, had significantly reduced survival rates. However, it was not possible to predict the outcome in individual cases; ten of 24 patients with clinical severity scores which indicated a poor prognosis survived the period of oliguria and were discharged from hospital. The fact that other renal units dialyse fewer patients per million population per year for ARIRF probably reflects a reluctance to refer patients whose general condition appears poor. As the overall mortality rate reported in this study does not differ significantly from rates reported previously from centres treating a smaller proportion of patients, such decisions may not be correct. It is well known that facilities in Britain for treating patients with end-stage renal disease are inadequate; it now appears likely that some patients who might benefit from acute dialysis are being denied treatment for a potentially reversible disease process.

Acute Kidney Injury↗
PubMed2001

The paired availability design for historical controls.

BACKGROUND: Although a randomized trial represents the most rigorous method of evaluating a medical intervention, some interventions would be extremely difficult to evaluate using this study design. One alternative, an observational cohort study, can give biased results if it is not possible to adjust for all relevant risk factors. METHODS: A recently developed and less well-known alternative is the paired availability design for historical controls. The paired availability design requires at least 10 hospitals or medical centers in which there is a change in the availability of the medical intervention. The statistical analysis involves a weighted average of a simple "before" versus "after" comparison from each hospital or medical center that adjusts for the change in availability. RESULTS: We expanded requirements for the paired availability design to yield valid inference. (1) The hospitals or medical centers serve a stable population. (2) Other aspects of patient management remain constant over time. (3) Criteria for outcome evaluation are constant over time. (4) Patient preferences for the medical intervention are constant over time. (5) For hospitals where the intervention was available in the "before" group, a change in availability in the "after group" does not change the effect of the intervention on outcome. CONCLUSION: The paired availability design has promise for evaluating medical versus surgical interventions, in which it is difficult to recruit patients to a randomized trial.

Clinical Trials as Topic↗
PubMed2002

Heparin-binding epidermal growth factor-like growth factor stimulates androgen-independent prostate tumor growth and antagonizes androgen receptor function.

Peptide growth factors have been implicated in progression of prostate cancer (PCa) to the androgen-independent state; however, much of the evidence linking diffusible mitogens and survival factors to this process remains circumstantial. Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a prostate stroma-derived factor, promotes survival, proliferation, and neuroendocrine differentiation of androgen-dependent LNCaP PCa cells in vitro. To test whether sustained exposure to HB-EGF can confer an androgen-independent phenotype, we generated stable populations of LNCaP cells that express constitutively a secreted form of HB-EGF (LNCaP/sHB). LNCaP/sHB cells proliferated more rapidly under androgen-depleted conditions in vitro and formed larger tumors with higher frequency in intact and castrated severe combined immunodeficient mice, in comparison to control cells. LNCaP/sHB tumors also expressed higher levels of the neuroendocrine marker, neuron-specific enolase, compared with control tumors. In castrates, increased neuron-specific enolase expression in LNCaP/sHB tumors was associated with reduced androgen receptor (AR) levels. In vitro, AR protein levels were reduced in LNCaP/sHB cells, and in transient transfection assays using an androgen-responsive promoter (mouse mammary tumor virus-long terminal repeat), LNCaP/sHB cells showed reduced sensitivity to dihydrotestosterone compared with controls. This is the first demonstration that continuous exposure of AR-positive PCa cells to a single growth factor can promote an androgen-independent phenotype in vivo. These findings also emphasize the potential role of pathways other than the AR axis in acquisition of androgen independence.

Androgen Receptor Antagonists↗
PubMed1991

Apical cell shape changes during Drosophila imaginal leg disc elongation: a novel morphogenetic mechanism.

Imaginal discs of Drosophila are simple epithelial tissues that undergo dramatic changes in shape during metamorphosis, including elongation to form adult appendages such as legs and wings. We have examined the cellular basis of leg disc morphogenesis by staining filamentous actin to outline cell boundaries in discs and observing cell shapes with scanning confocal laser microscopy (SCLM). Surprisingly, we found that prior to the onset of morphogenesis, cells in the dorsal-lateral regions of leg discs are compressed in the proximal-distal axis and greatly elongated circumferentially. These cells are also asymmetric in the apical-basal axis, being more elongated in the apical-most region of the cell than they are subapically, and frequently contacting different sets of neighbors apically and basally. Elongated cells were first observed in early third instar discs, and persisted through several rounds of cell division as the discs matured. During appendage elongation in vivo and trypsin-accelerated elongation in vitro, these highly asymmetric cells became isometric. As the apical cell profiles changed shape, apical and basal cell contacts came into register. Measurements of apical cell dimensions suggest that changes in cell shape account for most of the elongation in the basitarsal and tibial leg segments between 0 and 6 h after puparium formation (AP). The conversion of a stable population of anisometric cells to isometric dimensions constitutes a novel mechanism for altering the proportions of an epithelial sheet during development.

Animals↗
PubMed1977

Influence of the time in culture on cellular and neuronal properties of neuroblastoma x glioma hybrid cells. With an appendix, mathematical description of the kinetics of the loss in cell volume.

The stability of a clonal mouse neuroblastoma x rat glioma hybrid cell line was examined. Cell volume and cellular content of DNA and protein were measured as functions of the passage number. They decreased with the number of serial subcultivations. Cellular volume was linearly related to cellular DNA and protein. Thus, measurements of cell volume can be used to monitor the loss of DNA from hybrid cells. After about 60 passages a stable population of hybrid cells arose, as judged by the constancy of cellular volume and by the decreased coefficient of variation of the cell volume distribution. A mathematical model for the kinetics of the simultaneous loss of cellular volume, DNA and protein is introduced. Several neuronal properties were investigated. The specific activity of the neurotransmitter enzyme choline acetyltransferase decreased by more than 50% during 56 passages. After 70 subcultivations, the hybrid cells were still capable of extending processes, action potentials could still be elicited electrically or by iontophoretic application of acetylcholine, and the cells still responded to prostaglandin E1 as they do at low passage number.

Action Potentials↗
PubMed2002

Antiangiogenic agents targeting vascular endothelial growth factor and its receptors in clinical development.

There is ample therapeutic opportunity for the use of antiangiogenic inhibitors in the clinic, as there are several human diseases that are dependent upon angiogenesis [1]. However, no disease has attracted as much attention as a target for antiangiogenic therapy as malignant disorders. There is a vast amount of literature acting as proof-of-principle for the use of angiogenic inhibitors as effective agents for blocking tumour-induced angiogenesis and subverting tumour growth and disease dissemination. One of the unique attractions of targeting tumour angiogenesis is that vascular endothelial cells are a genetically stable population in which acquisition of therapeutic resistance might be less efficient than in genetically unstable tumour cells [2,3]. This review covers inhibitors that target the tumour angiogenic agent vascular endothelial growth factor and its receptors as one such antiangiogenic approach. Many agents in this class are in clinical trials with limited reports of toxicity and some early evidence of clinical benefit.

Angiogenesis Inhibitors↗
PubMed2006

Mechanical unfolding of RNA: from hairpins to structures with internal multiloops.

Mechanical unfolding of RNA structures, ranging from hairpins to ribozymes, using laser optical tweezer experiments have begun to reveal the features of the energy landscape that cannot be easily explored using conventional experiments. Upon application of constant force (f), RNA hairpins undergo cooperative transitions from folded to unfolded states whereas subdomains of ribozymes unravel one at a time. Here, we use a self-organized polymer model and Brownian dynamics simulations to probe mechanical unfolding at constant force and constant-loading rate of four RNA structures of varying complexity. For simple hairpins, such as P5GA, application of constant force or constant loading rate results in bistable cooperative transitions between folded and unfolded states without populating any intermediates. The transition state location (DeltaxFTS) changes dramatically as the loading rate is varied. At loading rates comparable to those used in laser optical tweezer experiments, the hairpin is plastic, with DeltaxFTS being midway between folded and unfolded states; whereas at high loading rates, DeltaxFTS moves close to the folded state, i.e., RNA is brittle. For the 29-nucleotide TAR RNA with the three-nucleotide bulge, unfolding occurs in a nearly two-state manner with an occasional pause in a high free energy metastable state. Forced unfolding of the 55 nucleotides of the Hepatitis IRES domain IIa, which has a distorted L-shaped structure, results in well-populated stable intermediates. The most stable force-stabilized intermediate represents straightening of the L-shaped structure. For these structures, the unfolding pathways can be predicted using the contact map of the native structures. Unfolding of a RNA motif with internal multiloop, namely, the 109-nucleotide prohead RNA that is part of the 29 DNA packaging motor, at constant value of rf occurs with three distinct rips that represent unraveling of the paired helices. The rips represent kinetic barriers to unfolding. Our work shows 1), the response of RNA to force is largely determined by the native structure; and 2), only by probing mechanical unfolding over a wide range of forces can the underlying energy landscape be fully explored.

Computer Simulation↗
PubMedDate not supplied

Observations on the distribution of anophelines in Suriname with particular reference to the malaria vector Anopheles darlingi.

A study was made on the distribution of anophelines in Suriname with special emphasis on the principal malaria vector Anopheles darlingi and on the occurrence of other possible vector species. Peridomestic human bait collections of adult mosquitoes and collections of larvae were made in many localities with a recent history of malaria transmission. Stable populations of An. darlingi were only found in the interior, south of the limit of tidal influence, due to year-round availability of breeding habitats in quiet partly sunlit places in flooded forest areas and along river banks. In the area with tidal movement of the rivers, breeding is limited to flooded areas in the wet season. Anopheles darlingi was only incidentally collected in low densities. In the interior, malaria transmission occurred in all places where An. darlingi was found. The absence of malaria transmission along the Upper Suriname River could be explained by the absence of An. darlingi. In the malaria endemic areas, An. darlingi was the most numerous mosquito biting on man. In the tidal region, malaria outbreaks are infrequent and might be explained by the temporary availability of favourable breeding habitats for An. darlingi. However, evidence is insufficient to incriminate An. darlingi as the vector of malaria in this region and the possible vectorial role of other anophelines is discussed.

Animals↗