Physiological Heterogeneity Research Articles

Volatile Anesthetics and the Long QT Syndrome

*University Hospital Carl Gustav Carus, Technical University Dresden, Dresden, Germany. stefan.rasche@uniklinikum-dresden.deWith much interest we read the article by Susan J. Kies et al. regarding patients with congenital long QT syndrome (LQTS). We congratulate the authors on their excellent review but would like to discuss several aspects. As stated in the introduction, patients with LQTS often show a “delayed cellular repolarization and heterogeneity in dispersion of repolarization, which … can lead to early after-depolarization, further dispersion of repolarization, and the formation of reentry circuits.”It becomes increasingly apparent that the QT interval prolongation per se is not the crucial pathology in LQTS. The delayed cellular repolarization in LQTS represents either impaired rapid or slow delayed rectifier potassium currents (iKr or iKs) or inappropriately inactivated sodium currents (iNa). The main arrhythmogenic substrate resulting from these altered ion currents is an increase in transmural repolarization heterogeneity. This heterogeneity favors the development of torsade de pointes, which is triggered by early after-depolarizations. More than 300 mutations in six genes encoding cardiac ion channel subunits and ankyrin B have been identified in patients with LQTS. According to the affected ion channel subunit, LQTS is classified into six subtypes with partially different clinical courses and triggers of torsade de pointes. Hence, the clinical and electrophysiologic presentations of the syndrome are considerably heterogeneous, and the effects of different drugs may be unpredictable.The QT interval obtained by a 12-lead electrocardiogram is only a rough measure of the repolarization time. Diagnosis based solely on summation vectors projected to the body surface is therefore neither sensitive nor specific. Accordingly, studies that only focus on drug effects on the QT interval may produce premature conclusions regarding potential safety or risk of these drugs in LQTS. This explains the contradicting results of numerous investigations, which report different effects of drugs on QT interval. Kies et al. recommend isoflurane as volatile anesthetic of choice in LQTS patients. In our opinion, to date, such recommendation cannot be supported. The reported effects of volatile anesthetics on QT interval are inconsistent or even conflicting. Only few studies have focused on QT heterogeneity or ion channel physiology, and it seems that all volatile anesthetics—including isoflurane—interact directly with cardiac delayed rectifier potassium channels.8,9,10,11 We therefore propose to avoid this class of anesthetics and would prefer propofol as the anesthetic of choice until more information is available from pharmacologic studies that focus on ion channel physiology and transmural heterogeneity of repolarization. These studies should ideally differentiate between LQTS subtypes.*University Hospital Carl Gustav Carus, Technical University Dresden, Dresden, Germany. stefan.rasche@uniklinikum-dresden.de

Modulation of neurons in the paraventricular thalamic nucleus by α2 adrenoceptor agonists: evidence for physiological and morphological heterogeneity

The paraventricular thalamic nucleus (PVT) receives dense noradrenergic input, but little is known about α2 adrenoceptors (ARs) in this nucleus. We have investigated effects of the agonist α-methyl-norepinephrine (m-NE) on PVT neurons in vitro. Based on their physiological and morphological characteristics, three distinct classes of PVT neurons have been identified. The first class exhibits membrane hyperpolarization on stimulation with m-NE (0.05–25 µM). This hyperpolarizing effect is observed in the presence of tetrodotoxin (TTX; 0.5–1 µM), blocked by yohimbine (1 µM) and mimicked by clonidine (10 µM), which indicates that it is mediated by postsynaptic α2 ARs. Further experiments indicate that it is mediated through an increase in G protein-coupled K+ conductance. In a second class of neurons, m-NE (0.05–25 µM) induces a slow membrane depolarization that is mimicked by phenylephrine (5 µM) and blocked by prazosin (75 nM), which indicates the involvement of α1 ARs. The third class of neurons is insensitive to m-NE (5–25 µM), and has a lower input resistance and a larger dendritic tree compared to the two other classes. The three types of neurons differ in their resting properties, and their firing patterns are changed by m-NE. These findings indicate anatomical and functional specialization of PVT neurons.

Additional morphological and physiological heterogeneity within the midgut of larval Aedes aegypti (Diptera: Culicidae) revealed by histology, electrophysiology, and effects of Bacillus thuringiensis endotoxin

Analysis of larval Aedes aegypti midgut using scanning electron microscopy, nuclear and mitochondrial dyes, response to Bacillus thuringiensis israelensis CryIVB toxin, and electrophysiology is described. The anterior ventriculus (“stomach”) region is found to have much lower mitochondrial densities than other midgut regions. The transitional region is distinguished by apical surface architecture, and by region-specific effects of CryIVB endotoxin. In this region CryIVB causes holes ranging from 1.0 to 7.0 μm in diameter (mean 3.3 ± 0.53 μm, N = 12), blisters 16.9 ± 1.54 μm in diameter ( N = 10), and separation of adjacent cells. The holes are not consistent with damage due to the colloid osmotic lysis model of δ-endotoxin activity. The posterior ventriculus possesses a distinctive cellular architecture consisting of hemispherical, domed apical membranes surrounded by deep clefts. Functional and morphological heterogeneity is revealed within the posterior ventriculus, with the anterior end dominating the electrical profile of isolated, perfused preparations and showing the greatest response to serotonin. Hyperpolarization of the transepithelial potential by serotonin occurred in conjunction with a decrease in the space constant lambda, ruling out closure of ion channels as the mechanism of action of serotonin.

Global analysis of proteins synthesized by Mycobacterium smegmatis provides direct evidence for physiological heterogeneity in stationary-phase cultures.

We have characterized the induction kinetics of approximately 1,700 proteins during entry into and survival in carbon-starved stationary phase by Mycobacterium smegmatis. Strikingly, among the patterns of expression observed were a group of proteins that were expressed in exponential-phase cultures and severely repressed in 48-h stationary-phase cultures (Spr or stationary-phase-repressed proteins) but were synthesized again at high levels in > or =128-day stationary-phase cultures (Spr(128) proteins). A number of Spr(128) proteins were identified, and they included the heat shock protein DnaK, the tricarboxylic acid cycle enzyme succinyl coenzyme A synthase, a FixA-like flavoprotein, a single-stranded DNA binding protein, and elongation factor Tu (EF-Tu). The identification of EF-Tu as an Spr(128) protein is significant, as ribosomal components are known to be expressed in a growth rate-dependent way. We interpreted these data in terms of a model whereby stationary-phase mycobacteria comprise populations of cells that differ in both their growth status and gene expression patterns. To investigate this further, we constructed gene fusions between the rpsL gene promoter (which heads the Mycobacterium smegmatis operon encoding the tuf gene encoding EF-Tu) or the rrnA promoter gene and an unstable variant of green fluorescent protein. While the majority of cells in old stationary-phase cultures had low levels of fluorescence and so rpsL expression, a small but consistently observed population of approximately 1 in 1,000 cells was highly fluorescent. This indicates that a small fraction of the cells was expressing rpsL at high levels, and we argue that this represents the growing subpopulation of cells in stationary-phase cultures.

Genetic Diversity of Viable, Injured, and Dead Fecal Bacteria Assessed by Fluorescence-Activated Cell Sorting and 16S rRNA Gene Analysis

A novel approach combining a flow cytometric in situ viability assay with 16S rRNA gene analysis was used to study the relationship between diversity and activity of the fecal microbiota. Simultaneous staining with propidium iodide (PI) and SYTO BC provided clear discrimination between intact cells (49%), injured or damaged cells (19%), and dead cells (32%). The three subpopulations were sorted and characterized by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene amplicons obtained from the total and bifidobacterial communities. This analysis revealed that not only the total community but also the distinct subpopulations are characteristic for each individual. Cloning and sequencing of the dominant bands of the DGGE patterns showed that most of clones retrieved from the live, injured, and dead fractions belonged to Clostridium coccoides, Clostridium leptum, and Bacteroides. We found that some of the butyrate-producing related bacteria, such as Eubacterium rectale and Eubacterium hallii, were obviously viable at the time of sampling. However, amplicons affiliated with Bacteroides and Ruminococcus obeum- and Eubacterium biforme-like bacteria, as well as Butyrivibrio crossotus, were obtained especially from the dead population. Furthermore, some bacterial clones were recovered from all sorted fractions, and this was especially noticeable for the Clostridium leptum cluster. The bifidobacterial phylotypes identified in total samples and sorted fractions were assigned to Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium pseudocatenulatum, and Bifidobacterium bifidum. Phylogenetic analysis of the live, dead, and injured cells revealed a remarkable physiological heterogeneity within these bacterial populations; B. longum and B. infantis were retrieved from all sorted fractions, while B. adolescentis was recovered mostly from the sorted dead fraction.

New aspects of vulnerability in heterogeneous models of ventricular wall and its modulation by loss of cardiac sodium channel function

This numerical study quantified the vulnerable period (VP) in heterogeneous models of the cardiac ventricular wall and its modulation by loss of cardiac sodium channel function (NaLOF). According to several articles, NaLOF prolongs the VP and therefore increases the risk of re-entrant arrhythmias, but the studies used uniform models, neglecting spatial variation of action potential duration (APD). Here, physiological transmural heterogeneity was introduced into one-dimensional cables of the Luo-Rudy model cells. Based on the results with paired S1-S2 stimulation, a generalised formula for the VP was proposed that takes into account APD dispersion, and new phenomena pertaining to the VP are described that are not present in homogeneous excitable media. Under normal conditions, the vulnerable period in the heterogeneous cable with M cells was in the range of 0-21 ms, depending on S2 localisation, but only 2.4 ms throughout the uniform fibre. Unidirectional propagation induced during the VP could be antegrade or retrograde, depending on the localisation of the test stimulus and cable parameters, but, in a uniform model, it was always in the retrograde direction. Reduced sodium channel conductance from control 16 mS microF(-1) to 4 mS microF(-1) decreased the maximum VP to 11 ms in the heterogeneous cable, but increased the VP to 3 ms in the homogeneous model. It was concluded that realistic models of cardiac vulnerability should take into account spatial variations of cellular refractoriness. Several new qualitative and quantitative aspects of the VP were revealed, and the modulation of the VP by NaLOF differed significantly in heterogeneous and homogeneous models.

Physiology of Neuronal Subtypes in the Respiratory–Vocal Integration Nucleus Retroamigualis of the Male Zebra Finch

Learned vocalizations, such as bird song, require intricate coordination of vocal and respiratory muscles. Although the neural basis for this coordination remains poorly understood, it likely includes direct synaptic interactions between respiratory premotor neurons and vocal motor neurons. In birds, as in mammals, the medullary nucleus retroambigualis (RAm) receives synaptic input from higher level respiratory and vocal control centers and projects to a variety of targets. In birds, these include vocal motor neurons in the tracheosyringeal part of the hypoglossal motor nucleus (XIIts), other respiratory premotor neurons, and expiratory motor neurons in the spinal cord. Although various cell types in RAm are distinct in their anatomical projections, their electrophysiological properties remain unknown. Furthermore, although prior studies have shown that RAm provides both excitatory and inhibitory input onto XIIts motor neurons, the identity of the cells in RAm providing either of these inputs remains to be established. To characterize the different RAm neuron types electrophysiologically, we used intracellular recordings in a zebra finch brain stem slice preparation. Based on numerous differences in intrinsic electrophysiological properties and a principal components analysis, we identified two distinct RAm neuron types (types I and II). Antidromic stimulation methods and intracellular staining revealed that type II neurons, but not type I neurons, provide bilateral synaptic input to XIIts. Paired intracellular recordings in RAm and XIIts further indicated that type II neurons with a hyperpolarization-dependent bursting phenotype are a potential source of inhibitory input to XIIts motor neurons. These results indicate that electrically distinct cell types exist in RAm, affording physiological heterogeneity that may play an important role in respiratory-vocal signaling.

Geographic patterns and pathogenetic implications of IGHV gene usage in chronic lymphocytic leukemia: the lesson of the IGHV3-21 gene

AbstractWe studied immunoglobulin variable heavy-chain (IGHV) repertoire and mutational status in 553 patients with chronic lymphocytic leukemia (CLL) from the Mediterranean area to gain insight into the potential pathogenetic role of antigenic stimulation. The most commonly represented IGHV genes mirrored the usage of normal B cells, with the exception of IGHV1-18, IGHV3-30.3, and IGHV4-59 that were underrepresented. The IGHV3-21 gene, frequently expressed in Northern European CLL, was present only in 16 cases (2.9%). Based on HCDR3 cluster analysis, cases using IGHV3-21 could be grouped in 2 subsets of similar frequency. The first one (7 of 16 cases) carried a similar HCDR3 amino acid sequence (common-HCDR3 subset), virtually identical to the Scandinavian IGHV3-21 CLL. These cases used the IGHJ6 gene; 4 of 7 were unmutated; 6 of 7 carried the Vλ2-14 (IGLV3-21) light-chain gene with a similar LCDR3. All expressed CD38 and had a progressive disease. The second subset (9 of 16) was characterized by heterogeneous HCDR3 rearrangements (nonhomogeneous-HCDR3 subset), diverse IGHJ and IGV light-chain gene usage, variable IGHV mutational status (5 of 9 unmutated), variable CD38 expression, and variable clinical course (4 of 9 progressed). The first subset suggests a potential antigenic element rarely encountered in the Mediterranean area, possibly responsible for a negative outcome. The second subset may reflect the physiologic heterogeneity of expression of IGHV3-21 rearrangements in the normal repertoire and is characterized by a variable clinical outcome. (Blood. 2005;105:1678-1685)

Heterogeneous GABAA receptor subunit expression in pediatric epilepsy patients

The gamma-amino-butyric acid type A receptors (GABAAR) are a heteropentameric receptor complex, composed of 16 possible subunits in various combinations, forming a ligand-gated ion channel. Subunit composition is the primary determinant of GABAAR physiology and pharmacology. Here we have measured mRNA levels for 16 GABAAR subunits in isolated dentate granule neurons (DGN) from eight pediatric patients undergoing resective surgery for intractable epilepsy. We found tightly correlated expression of a subset of GABAAR subunit mRNAs within a single DGN (alpha1, gamma1, and gamma2; alpha4, alpha5, and beta2; alpha4 and beta3). Analysis of inter-patient variability (ANOVA) of eleven highly expressed GABAAR subunit mRNAs found seven of the subunits varied between patients, as did whole cell GABAAR currents. Due to inter-patient differences, there is heterogeneity in DGN GABAAR subunit mRNA and physiology within pediatric epilepsy patients. Patient-specific GABAAR expression might contribute to variability in anti-epileptic drug efficacy, side-effect profiles, and seizure susceptibility.

Amaurose congénitale de Leber : le point sur l’hétérogénéité génétique, actualisation de la définition clinique

Leber congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies, responsible for congenital blindness. Disease-associated mutations have been hitherto reported in seven genes. These genes are all expressed preferentially in the photoreceptor cells or the retinal pigment epithelium, but they are involved in strikingly different physiologic pathways, resulting in an unforeseeable pathophysiologic variety. This broad genetic and physiologic heterogeneity, which could greatly increase in the coming years, hinders molecular diagnosis in LCA patients. Genotyping is, however, required to establish genetically defined subgroups of patients ready for therapy. Here we report a comprehensive mutational analysis of all the known genes in 179 unrelated LCA patients, including 52 familial and 127 sporadic (27/127 consanguineous) cases. Mutations were identified in 47.5% of patients. GUCY2D accounted for by far the largest part of the LCA cases in our series (21.2%), followed by CRB1 (10%), RPE65 (6.1%), RPGRIP1 (4.5%), AIPL1 (3.4%), TULP1 (1.7%) and CRX (0.6%). The clinical history of all patients with mutations was carefully revisited in the search for phenotype variations. Genotype-phenotype correlations were found that made it possible to divide patients into two main groups. The first one includes patients whose symptoms fit the traditional definition of LCA, i.e., congenital or very early cone-rod dystrophy, while the second group gathers patients affected with severe yet progressive rod-cone dystrophy. In addition, objective ophthalmologic data subdivided each group into two subtypes. Based on these findings, we have drawn decisional flowcharts directing the molecular analysis of LCA genes in a given case. These flowcharts will hopefully lighten the onerous task of genotyping new patients, but only if the most precise clinical history since birth is available.

Monitoring population dynamics of the thermophilic Bacillus licheniformis CCMI 1034 in batch and continuous cultures using multi-parameter flow cytometry

Multi-parameter flow cytometry was used to monitor the population dynamics of Bacillus licheniformis continuous cultivations and the physiological responses to a starvation period and a glucose pulse. Using a mixture of two specific fluorescent stains, DiOC 6(3) (3,3′-dihexylocarbocyanine iodide), and PI (propidium iodide), flow cytometric analysis revealed cell physiological heterogeneity. Four sub-populations of cells could be easily identified based on their differential fluorescent staining, these correspond to healthy cells (A) stained with DiOC 6(3); cells or spores with a depolarised cytoplasmic membrane (B), no staining; cells with a permeabilised depolarised cytoplasmic membrane (C), stained with PI; and permeablised cells with a disrupted cytoplasmic membrane ‘ghost cells’ (D), stained with both DiOC 6(3) and PI. Transmission electron micrographs of cells starved of energy showed different cell lysis process stages, highlighting ‘ghost cells’ which were associated with the double stained sub-population. It was shown, at the individual cell level, that there was a progressive inherent fluctuation in physiological heterogeneity in response to changing environmental conditions. All four sub-populations were shown to be present during glucose-limited continuous cultures, revealing a higher physiological stress level when compared with a glucose pulsed batch. A starvation period (batch without additional nutrients) increased the number of cells in certain sub-populations (cells with depolarised cytoplasmic membranes and cells with permeabilised depolarised cytoplasmic membranes), indicating that such stress may be caused by glucose limitation. Such information could be used to enhance process efficiency.

Impact of methicillin resistance on outcome of Staphylococcus aureus ventilator-associated pneumonia.

The impact of methicillin resistance on morbidity and mortality of patients suffering from severe Staphylococcus aureus infections remains highly controversial. We analyzed a retrospective cohort of 97 patients with methicillin-susceptible and 74 patients with methicillin-resistant Staphylococcus aureus ventilator-associated pneumonia (VAP). Initial empiric antibiotic therapy was appropriate for every patient. Patients with methicillin-resistant Staphylococcus aureus VAP were older, had higher disease-severity scores, and had been on mechanical ventilation longer at onset of VAP. Factors associated with 28-day mortality retained by multivariate logistic regression analysis were: age (odds ratio [OR] = 1.05, 95% confidence interval [CI], 1.02-1.08, p = 0.001) and Day 1 organ dysfunctions or infection (ODIN) score (OR = 1.90, 95% CI, 1.31-2.78, p = 0.001), but not methicillin resistance (OR = 1.72, 95% CI, 0.73-4.05, p = 0.22). The percentages of infection relapse or superinfection did not differ significantly between the two patient groups. In conclusion, after controlling for clinical and physiologic heterogeneity between groups, methicillin resistance did not significantly affect 28-day mortality of patients with Staphylococcus aureus VAP receiving appropriate antibiotics.

Leber congenital amaurosis: comprehensive survey of the genetic heterogeneity, refinement of the clinical definition, and genotype-phenotype correlations as a strategy for molecular diagnosis.

Leber congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies, responsible for congenital blindness. Disease-associated mutations have been hitherto reported in seven genes. These genes are all expressed preferentially in the photoreceptor cells or the retinal pigment epithelium but they are involved in strikingly different physiologic pathways resulting in an unforeseeable physiopathologic variety. This wide genetic and physiologic heterogeneity that could largely increase in the coming years, hinders the molecular diagnosis in LCA patients. The genotyping is, however, required to establish genetically defined subgroups of patients ready for therapy. Here, we report a comprehensive mutational analysis of the all known genes in 179 unrelated LCA patients, including 52 familial and 127 sporadic (27/127 consanguineous) cases. Mutations were identified in 47.5% patients. GUCY2D appeared to account for most LCA cases of our series (21.2%), followed by CRB1 (10%), RPE65 (6.1%), RPGRIP1 (4.5%), AIPL1 (3.4%), TULP1 (1.7%), and CRX (0.6%). The clinical history of all patients with mutations was carefully revisited to search for phenotype variations. Sound genotype-phenotype correlations were found that allowed us to divide patients into two main groups. The first one includes patients whose symptoms fit the traditional definition of LCA, i.e., congenital or very early cone-rod dystrophy, while the second group gathers patients affected with severe yet progressive rod-cone dystrophy. Besides, objective ophthalmologic data allowed us to subdivide each group into two subtypes. Based on these findings, we have drawn decisional flowcharts directing the molecular analysis of LCA genes in a given case. These flowcharts will hopefully lighten the heavy task of genotyping new patients but only if one has access to the most precise clinical history since birth.

Manipulating cardiac contractility in heart failure: data from mice and men.

In the year 1990, cardiovascular disease surpassed infectious diseases as the leading cause of death worldwide, and by the year 2020 it is predicted to be the leading cause of all disability.1,2 In the United States, cardiovascular disease has been estimated to account for 44% of the nation’s mortality and is a leading cause of morbidity.3,4 Heart failure afflicts an estimated 5 million Americans, with ≈400 000 new individuals diagnosed each year at an annual cost of more than $20 billion.4–6 The alarming reality behind these statistics is our current lack of an effective therapy to repair or otherwise reverse severe forms of cardiac dysfunction and pathological remodeling associated with heart failure. Typically, heart failure is the final culmination of protracted disease states precipitated by underlying hypertension, ischemic disease and atherosclerosis, valvular insufficiency, viral myocarditis, or mutations in genes encoding sarcomeric proteins.6 Given these diverse etiologies, it is not surprising that the final phenotypic manifestations of heart failure can also vary considerably, although dilated cardiomyopathy is the most common. This syndrome is characterized by a progressive loss in contractility and ejection fraction, ventricular chamber dilatation, ventricular wall thinning, increased peripheral vascular resistance, and dysregulated fluid homeostasis. The predominant therapeutic strategy used over the past two decades for treating such patients has been based in pharmacological manipulation of cardiac contractility.7–9 Initially, positive inotropic agents were used as a means of enhancing cardiac pump function aimed at alleviating congestive symptomology. However, use of positive inotropes is now indicated only as a means of acutely bridging patients in severe heart failure because these agents actually worsen prognosis in individuals with somewhat more stable heart failure.8 More recently, pharmacological blockade of β-adrenergic receptors has emerged as the favored treatment for individuals in heart failure. β-Adrenergic receptor antagonists initially …

Chromatin condensation and DNA damage of human epididymal spermatozoa in obstructive azoospermia

Obstructive azoospermia (OA) includes patients with different aetiologies of obstruction such as congenital bilateral absence of vas deferens (CBAVD), post-vasectomy/failed vaso–vasostomy, or unknown (idiopathic). The frequency of DNA-damaged spermatozoa and abnormal chromatin condensation in epididymal spermatozoa of OA patients was investigated for the different aetiology subgroups. DNA damage in caput epididymis spermatozoa was assessed with the TdT-UTP nick-end labelling (TUNEL) assay and chromatin condensation status was measured with chromomycin A3 (CMA3) stain ( n = 60 patients). All epididymal sperm samples showed high levels of TUNEL positivity (mean and SD, 43 ± 17%). CMA3 rates showed large variation within the samples (mean and SD, 53 ± 21%, range 7–97%); however, a significant difference in chromatin condensation was found between the different aetiologies of obstruction. The highest percentage of CMA3 positivity was found in the CBAVD and idiopathic groups (60 ± 17 and 65 ± 18% respectively) compared with the post-vasectomy samples (37 ± 17%) or ejaculated spermatozoa from normozoospermic males (29 ± 12%). In conclusion, despite the normal testicular histology in OA men, chromatin condensation differed between OA patients. The physiological heterogeneity found in chromatin condensation between patient subgroups is probably influenced by the aetiology of the obstruction.

Regional myocardial function in healthy adults: assessment through tissue Doppler echocardiography.

To characterize left ventricular regional myocardial function through tissue Doppler echocardiography in healthy adults and to assess the influence of aging in this function. In 45 healthy volunteers divided in two groups (< 45 and > 45 years old) we assessed longitudinal and radial regional function (velocities, times intervals and velocity-time integrals). Data were compared in each group and between groups. Systolic function: a). longitudinal: higher velocities and integrals in lateral and inferior walls and in basal segments, with a trend to reduction of these parameters with aging; b). radial: higher basal velocities, no significant change with aging. Diastolic function: a). longitudinal: higher velocities in lateral and inferior walls and in basal segments. With aging e and e/a velocities and integrals decreased, a increased and older individuals showed lower percentage of segments with e/a >1; b). radial: aging was associated with lower e and higher a velocities. 1). Tissue Doppler echocardiography detects physiological differences between regional myocardial function of different ventricular segments, in velocities, times intervals and integrals, with physiological heterogeneity and asynchrony; 2). Many of these data are age dependent; 3). Our data contribute to define normal values, and may become useful when compared with data from populations with heart diseases.

Heterogeneity of microvascular endothelial cells isolated from human term placenta and macrovascular umbilical vein endothelial cells

The present study compares some phenotypic and physiologic characteristics of microvascular and macrovascular endothelial cells from within one human organ. To this end microvascular endothelial cells from human full-term placenta (PLEC) were isolated using a new method and compared with macrovascular human umbilical vein endothelial cells (HUVEC) and an SV40-transformed placental venous endothelial cell line (HPEC-A2). PLEC were isolated by enzymatic perfusion of small placental vessels, purified on a density gradient and cultured subsequently. Histological sections of the enzyme-treated vessels showed a selective removal of the endothelial lining in the perfused placental cotyledons. The endothelial identity of the cells was confirmed by staining with the endothelial markers anti-von Willebrand factor, Ulex europaeus lectin and anti-QBEND10. The cells internalized acetylated low-density lipoprotein and did not show immunoreactivity with markers for macrophages, smooth muscle cells and fibroblasts. The spindle-shaped PLEC grew in swirling patterns similar to that described for venous placental endothelial cells. However, scanning electron microscopic examination clearly showed that PLEC remained elongated at the confluent state, in contrast to the more polygonal phenotype of HPEC-A2 and HUVEC that were studied in parallel. The amount of vasoactive substances (endothelin-1,2, thromboxane, angiotensin II, prostacyclin) released into the culture medium and the proliferative response to cytokines was more similar to human dermal microvessels (MIEC) derived from non-fetal tissue than to HUVEC. Potent mitogens such as vascular endothelial growth factors (VEGF121, VEGF165) and basic fibroblast growth factor (FGF-2) induced proliferation of all endothelial cell types. Placental growth factors PIGF-1 and PIGF-2 effectively stimulated cell proliferation on PLEC (142 +/- 7% and 173 +/- 10%) and MIEC (160 +/- 20% and 143 +/- 28%) in contrast to HUVEC (9 +/- 8% and 15 +/- 20%) and HPEC-A2 (15 +/- 7% and 24 +/- 6%) after 48 h incubation time under serum-free conditions. These data support evidence for (1) the microvascular identity of the isolated PLEC described in this study, and (2) the phenotypic and physiologic heterogeneity of micro- and macrovascular endothelial cells within one human organ.

Neurons with distinctive firing patterns, morphology and distribution in laminae V-VII of the neonatal rat lumbar spinal cord.

It is generally accepted that neurons in the ventral spinal grey matter, a substantial proportion of which can be regarded as constituents of the spinal motor apparatus, receive and integrate synaptic inputs arising from various peripheral, spinal and supraspinal sources. Thus, a profound knowledge concerning the integrative properties of interneurons in the spinal ventral grey matter appears to be essential for a fair understanding of operational principles of spinal motor neural assemblies. Using the whole cell patch clamp configuration in a correlative physiological and morphological experimental approach, here we demonstrate that the intrinsic membrane properties of neurons vary widely in laminae V-VII of the ventral grey matter of the neonatal rat lumbar spinal cord. Based on their firing patterns in response to depolarizing current steps, we have classified the recorded neurons into four categories: 'phasic', 'repetitive', 'single' and 'slow'. Neurons with firing properties characteristic of the 'phasic', 'repetitive' and 'single' cells have previously been reported also in the superficial and deep spinal dorsal horn, but this is the first account in the literature in which 'slow' neurons have been recovered and described in the spinal cord. The physiological heterogeneity in conjunction with the morphological correlation and distribution of neurons argues that different components of motor neural assemblies in the spinal ventral grey matter possess different signal processing characteristics.

Bone as an effect compartment : models for uptake and release of drugs.

"Bone-seeking agents" are drugs characterised by high affinity for bone, and are disposed in bone for prolonged periods of time while maintaining remarkably low systemic concentrations. As a consequence, the bone becomes a reservoir for bone-seeking agents, and a site of both desirable and adverse effects, depending on the pharmacological activities of the specific agent. For some agents, significant systemic effects may also be produced following their prolonged release from bone, a process that is governed mostly by the rate of bone remodelling. This review covers the pharmacokinetic and pharmacodynamic features of bone-seeking agents with different pharmacological properties, including drugs (bisphosphonates, drug-bisphosphonate conjugates, radiopharmaceuticals and fluoride), bone markers (tetracycline, bone imaging agents) and toxins (lead, chromium, aluminium). In addition, drugs that do not possess bone-seeking properties but are used for therapy of bone diseases (such as antibacterials for treatment of osteomyelitis) are discussed, along with targeting of these drugs to the bone by conjugation to bone-seeking agents, local delivery systems, and other approaches. The pharmacokinetic and pharmacodynamic behaviour of bone-seeking agents is extremely complex due to heterogeneity in bone morphology and physiology. This complexity, accompanied by difficulties in human bone research caused by ethical and other limitations, gave rise to modelling approaches to study bone drug disposition. This review describes the pharmacokinetic models that have been proposed to describe the pharmacokinetic behaviour of bone-seeking agents and predict bone concentrations of these agents for different doses and patient populations. Models of different types (compartmental and physiologically based) and of different complexity have been applied, but their relevance to drug effects in the bone tissue is limited since they describe the behaviour of the "average" drug molecule. Understanding of the cellular and molecular processes responsible for the heterogeneity of bone tissue will provide better comprehension of the influence of microenvironment on drug bone disposition and the resulting pharmacological response.

Organization of the ectostriatum based on afferent connections in the zebra finch ( Taeniopygia guttata)

In birds with laterally-located eyes, such as zebra finches and pigeons, the tectofugal visual pathway is the most prominent route from the retina to the telencephalon. However, little is known about exactly how the visual information is processed in this pathway, especially at the core region of the ectostriatum (Ec) in the telencephalon. In order to reveal a detailed organization of Ec, we decided to systematically analyze the afferent connections of Ec by injecting small amounts of sensitive tracers (biotinylated dextran amine and cholera toxin subunit B) selectively into different regions of Ec and the thalamic center of the tectofugal pathway (the nucleus rotundus, Rt). The present study revealed a clearer picture of the organization of Ec subdivisions than previously known. The present results showed that the anterior portion of Rt sent a heavy projection to the ventral region of the anterior Ec, whereas the more caudal subdivisions of Rt sent projections to more caudal and dorsal portions in Ec. The results suggest that Ec subdivisions appear to be arranged along an axis ‘rotated’ in the anterior direction, almost parallel to other major telencephalic laminae. These results may clarify the physiological and chemical heterogeneity of Ec found in the previous studies. The present findings also provide an insight into the possible organization of a visual processing center in a non-mammal.