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R Rozental

Publications and source records attributed to R Rozental.

At least 19 recordsLinked to original sources
PubMed2001

Renal morphology in connexin43 knockout mice.

Connexins (Cx) are a family of proteins that constitute the intercellular membrane channels of gap junctions. These junctions permit intercellular movement of ions and other molecules between cells, a property vital to organogenesis. Cx43 is a member of the family of channel-forming proteins that are essential for cell-cell communication of developmental signals. Studies demonstrate that Cx43 is observed in mesenchymal cells of 12-day gestation mouse kidney, a crucial period of renal development. In order to study the significance of Cx43 on renal developmental morphology, we evaluated the kidneys of embryos lacking the gene encoding for Cx43. Polymerase chain reaction (PCR) from tail specimens identified wild-type (WT), heterozygote (HT) and knockout (KO) progeny. In situ RT-PCR displayed abundant Cx43 staining in glomeruli, vasculature, and tubules in kidneys obtained from WT progeny. In contrast, Cx43 expression was completely absent in kidneys isolated from the KO. Renal histology in all three groups displayed no significant differences. Renal size was similar and there was no evidence of dysplasia or cyst formation in the KO. Our results indicate that absence of Cx43, heretofore considered essential for renal development, does not affect early renal morphological development.

Animals↗
PubMed2000

Introduction

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Journal Article↗
PubMed2000

Gap junctions in the nervous system.

Synapses are classically defined as close connections between two nerve cells or between a neuronal cell and a muscle or gland cell across which a chemical signal (i.e., a neurotransmitter) and/or an electrical signal (i.e., current-carrying ions) can pass. The definition of synapse was developed by Charles Sherrington and by Ramon y Cajal at the beginning of this century and refined by John Eccles and Bernard Katz 50 years later; in this collection of papers, the definition of synapses is discussed further in the chapter by Mike Bennett. who provided the first functional demonstration of electrical transmission via gap junction channels between vertebrate neurons. As is evidenced by the range of topics covered in this issue, research dealing with gap junctions in the nervous system has expanded enormously in the past decade, major findings being that specific cell types in the brain expresses specific types of connexins and that expression patterns coincide with tissue compartmentalization and function and that these compartments change during development.

Animals↗
PubMed2000

Temporal expression of neuronal connexins during hippocampal ontogeny.

Communication through gap junction channels provides a major signaling mechanism during early brain histogenesis, a developmental time during which neural progenitor cells are inexcitable and do not express ligand-gated channel responses to the major CNS neurotransmitters. Expression of different gap junction types during neurogenesis may therefore define intercellular pathways for transmission of developmentally relevant molecules. To better understand the molecular mechanism(s) by which growth and differentiation of neurons are modulated by gap junction channels, we have been examining the developmental effects of a specific set of cytokines on differentiation and gap junction expression in a conditionally immortalized mouse embryonic hippocampal neuronal progenitor cell line (MK31). When multipotent MK31 cells are in an uncommitted state, they uniformly express the neuroepithelial intermediate filament class VI marker, nestin, are strongly coupled by gap junctions composed of connexin43 (Cx43) and express connexin45 (Cx45) at the mRNA level. As these cells undergo neuronal lineage commitment and exit from cell cycle, they begin to express the early neurofilament marker, NF66, and coupling strength and expression of Cx43 begin to decline with concurrent expression of other connexin proteins, including Cx26, Cx33, Cx36, Cx40 and Cx45. Terminal neuronal differentiation is heralded by the expression of more advanced neurofilament proteins, increased morphologic maturation, the elaboration of inward currents and action potentials that possess mature physiological properties, and changing profiles of expression of connexin subtypes, including upregulation of Cx36 expression. These important developmental transitions are regulated by a complex network of cell cycle checkpoints. To begin to examine the precise roles of gap junction proteins in traversing these developmental checkpoints and in thus regulating neurogenesis, we have focused on individual members of two classes of genes involved in these seminal events: ID (inhibitor of differentiation)-1 and GAS (growth arrest-specific gene)5. When MK31 cells were maintained in an uncommitted state, levels of ID-1 mRNA were high and GAS5 transcripts were essentially undetectable. Application of cytokines that promote neuronal lineage commitment and cell cycle exit resulted in down-regulation of ID-1 and upregulation of GAS5 transcripts, whereas additional cytokine paradigms that promoted terminal neuronal differentiation resulted in the delayed down-regulation of GAS5 mRNA. Stable MK31 transfectants were generated for ID-1 and GAS5. In basal conditions, cellular proliferation was enhanced in the ID-1 transfectants and inhibited in the GAS5 transfectants when compared with control MK31 cells. When cytokine-mediated neurogenesis was examined in these transfected cell lines, constitutive expression of ID-1 inhibited and constitutive expression of GAS5 enhanced initial and terminal stages of neuronal differentiation, with evidence that terminal neuronal maturation in both transfectant lines was associated with decreased cellular viability, possibly due to the presence of conflicting cell cycle-associated developmental signals. These experimental reagents will prove to be valuable experimental tools to help define the functional interrelationships between changing profiles of connexin protein expression and cell cycle regulation during neuronal ontogeny in the mammalian brain. The present review summarizes the current state of research involving the temporal expression of such connexin types in differentiating hippocampal neurons and speculates on the possible role of these intercellular channels in the development and plasticity of the nervous system. In addition, we describe the functional properties and expression pattern of the newly discovered neuronal-specific gap junctional protein, Cx36, in the developing mouse fetal hippocampus and in the rat retina and brain.

Animals↗
PubMed2000

Gap junctions: the "kiss of death" and the "kiss of life".

Cells expressing herpes simplex-thymidine kinase (HSV-tk) can be killed "in vitro" within 5 days of treatment with 20 microM ganciclovir (GCV) and transmit this toxicity to adjacent cells lacking HSV-tk; this phenomenon was termed "bystander effect" or "kiss of death". On testing a large number of cell lines in vitro, a wide range of sensitivity to GCV-mediated bystander killing has been reported. Although intercellular transfer of GCV metabolites through gap junction channels seems to be a likely mechanism for the "kiss of death", some studies suggest that other pathways may contribute to induced apoptosis of neighboring cells. To further investigate the mechanism underlying cell death mediated by HSV-tk and to evaluate the efficacy of gap junction channels formed by different connexins in this process, we have stably transfected a virtually uncoupled mouse neuroblastoma cell line (N2A cells) with different connexin-types expressed by neural cells (Cx32, Cx37, Cx40, Cx43) and co-cultured these cells with N2A cells stably transfected with Cx37 and HSV-tk. Here, we confirm our previous studies and those of others that the extent of cell death and sensitivity to GCV depend on the degree of connexin expression in transfectants. Further, we show that the bystander effect also depends on which connexin is expressed; reported disparities regarding the extent of GCV-mediated cellular apoptosis are likely due both to the degree of functional coupling and the type of connexin expressed. These results support the notion that gap junction hemichannels formed of certain connexins are more likely than others to pair functionally with Cx37, and suggest co-transfection strategies that might prove effective in sensitizing tumor cell populations to GCV. In addition, potential applications are discussed for use of the "good Samaritan effect", a mechanism by which bystander cells have been suggested to prevent cytotoxicity.

Animals↗
PubMed2000

Gap junctions in the cardiovascular and immune systems.

Gap junctions are clusters of intercellular channels directly connecting the cytoplasm of adjacent cells. These channels are formed by proteins named connexins and are present in all metazoan organisms where they serve diverse functions ranging from control of cell growth and differentiation to electric conduction in excitable tissues. In this overview we describe the presence of connexins in the cardiovascular and lympho-hematopoietic systems giving the reader a summary of the topics to be covered throughout this edition and a historical perspective of the discovery of gap junctions in the immune system.

Animals↗
PubMed1999

Functional properties of channels formed by the neuronal gap junction protein connexin36.

The expression and functional properties of connexin36 (Cx36) were examined in two communication-deficient cell lines (N2A-neuroblastoma and PC-12 cells) transfected with Cx36 and in hippocampal neurons that express the connexin endogenously. Transfected cells expressed the expected 2.9 kb Cx36 transcript and Cx36 immunoreactivity, whereas nontransfected cells were devoid of Cx36. The relationship between steady-state junctional conductance (g(j)) and transjunctional voltage was well described by a two-state Boltzmann equation. The half-inactivation voltage (V(0)), the ratio of minimal to maximal g(j) (g(min)/g(max)), and the equivalent gating charge were +/- 75 mV, 0.55, and 1.75, respectively, indicating that Cx36 exhibits very low voltage sensitivity. Conductance of single Cx36 channels measured with patch pipettes containing 130 mM CsCl was 10-15 pS (n = 15 cell pairs); despite this low unitary conductance, Cx36 channels were permeable to the dye Lucifer yellow. Hippocampal neurons expressed Cx36 both in vivo and in culture. The electrophysiological properties of channels in cultured hippocampal neurons were similar to those of the channels expressed by the transfected cell lines, and the neuronal channels were similarly permeable to Lucifer yellow. The unique combination of weak voltage sensitivity, small unitary conductance, and permeation by anions as large as second messenger molecules endows Cx36 gap junction channels with properties well suited for mediating flexible electrical and biochemical interactions between neurons.

Animals↗
PubMed1999

A simple RT-PCR-based strategy for screening connexin identity.

Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx) proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s) can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain) that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins.

Animals↗
PubMed1999

Disorders of calcium metabolism at various times after renal transplantation.

OBJECTIVES: Increased parathyroid hormone (PTH) production and related defects of calcium-phosphorus metabolism could persist even after successful kidney transplantation. Much more serious long term consequences after the transplantation are bone defects caused by immunosuppressive drugs. Many authors consider steroid therapy as one of the factors that maintain this process. Our study aimed to investigate calcium-phosphorus and bone pathological features during the various post transplantation periods, using non-invasive bone research methods (bone ultrasound structurally-densitometric analysis), and also to analyse the risk of hyperparathyroidism and steroid therapy in the development of post transplantation osteopathy. METHODS: 52 patients after successful kidney transplantation were investigated. All patients were divided in three groups according to the time after transplantation. 1st group-patients in the earlier post transplantation period, up to 1 year (n = 12); 2nd group-patients in the period from 1 to 5 years after transplantation (n = 25); 3rd group-patients in later post transplantation period (more than 5 years after the transplantation, n = 15). RESULTS: 8 patients from the 1st group (66.7%), 18 patients from the 2nd group (72%) and 8 patients from the 3rd group (53.3%) had an increased level of serum creatinine. The level of corrected serum Ca was increased (p < 0,05) in the first year after the transplantation. Hypercalcaemia was noted in 5 patients (41.7%) from the 1st group, in 3 patients (12%) from the 2nd group and in 2 patients (13.3%) from the 3rd group. Urine Ca level was lower (p < 0.05) in patients with post transplantation period over 5 years. Serum iPTH level as well as the level of osteocalcin was higher in all groups. The highest iPTH and osteocalcin level (p < 0.05) were observed during the first post transplantation year, but in the later post transplantation period they had a tendency to decrease, but never reached the norm for healthy subjects even in later post transplantation period. The decreased speed of ultrasound in the trabecular bones and osteopenia were noted in 6 patients from the 1st group (50%), osteoporosis -- in 1 patient from the 1st group(8.3%). In the 2nd group 8 patients had osteopenia (32%) and 1 patient had osteoporosis (4%), and in the 3rd group 7 patients had osteopenia (46.7%) and 4 patients -- osteoporosis (26.7%). A negative correlation was noted between patient age and speed of sound in all patient populations (r = -0.39, p<0,01), both in the early post transplantation period (r = -0.67, p<0.01), and during the period 1-5 years after transplantation (r = -0.5, p <0.01). The whole patient population showed negative correlation (r = -0.28, p<0.05) between Z-score and time after the transplantation. Z-score negatively correlates with a cumulative steroid dose in all investigated patients groups(r = -0.35, p<0.02). CONCLUSIONS: Disorders of calcium metabolism and immunosuppression related bone disease are the most common complications after transplantation, especially in patients with an impaired graft function. The mild hyperparathyroidism is usually noted in these patients at various times after transplantation. We also can note hypocalciuria in the later post transplantation period in these patients, which is based on the parathyroid glands hyperfunction and on the negative effects of the steroid therapy. The cumulative steroid dose and patient age are the determining factors for the development of osteopenia in transplantation patients at the stage of 5 or more years after transplantation.

Adult↗
PubMed1998

Changes in the properties of gap junctions during neuronal differentiation of hippocampal progenitor cells.

The cellular mechanisms that regulate progenitor cell lineage elaboration and maturation during embryonic development of the mammalian brain are poorly understood. Conditionally immortalized mouse hippocampal multipotent progenitor cells (MK31 cells) were found to be strongly coupled by gap junctions comprising connexin 43 (Cx43) during early neuronal ontogeny; the presence of this Cx type was confirmed by electrophysiological, molecular biological, and immunocytochemical assays. However, as progenitor cells underwent intermediate stages of neuronal differentiation under the influence of interleukin 7 (IL-7) alone or terminal differentiation after composite exposure to basic fibroblast growth factor, IL-7, and transforming growth factor alpha, coupling strength and the level of Cx43 expression declined. An additional population of junctional channels with distinct properties was detected at an intermediate stage of neuronal differentiation. Reverse transcription-PCR assays detected mRNA encoding Cx40 in IL-7-treated cells and Cx33 after both treatment conditions. Because functional channels in exogenous expression systems are not formed by pairing Cx40 with Cx43 or by pairing Cx33 with itself or additional connexins, these experimental observations raise the possibility that the progressive loss of coupling during differentiation of neural progenitor cells may involve downregulation of Cx43 coupled with potentiation of expression of Cx33 and Cx40. Furthermore, continued expression of Cx43 in differentiating neuroblasts could mediate intercellular communication between neuronal precursor cells and astrocytes by direct signaling via homotypic gap junction channels.

Animals↗
PubMed1998

EDITORIAL.

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Journal Article↗
PubMed1998

Purification of human fetal hippocampal neurons by flow cytometry for transplantation.

We have established primary cultures, highly enriched in neurons, from the hippocampus of human fetal brains at 20-23 gestational weeks. More than 80% of cells were viable when seeded. Neurons were isolated from primary cultures by flow cytometry to a high degree of purity, as demonstrated by immunocytochemical staining. FACS scanning analysis using a DNA-staining dye showed that hippocampal neurons did not divide in culture. To demonstrate that FACS-sorted neurons can be transplanted and integrated into the host brain, neuron-enriched primary culture from human fetal striatum was infected with a viral-mediated vector containing a reporter gene, beta-galactosidase. Striatal neurons were subsequently purified by flow cytometry and transplanted into the striatum of rats. Following transplantation, the rat brains were processed for beta-galactosidase histochemistry and electron microscopy. Beta-galactosidase expression indicates that transplanted human neurons survived in the host and were metabolically active. The transplanted neurons received synaptic inputs, as judged from the presence of presynaptic terminals on their surface. Our study demonstrates connectivity between transplanted human fetal primary neurons and host tissue at the ultrastructural level. Our results support the feasibility of ultimately transplanting neurons into humans as a possible treatment for recovery of the nervous system (e.g., neurodegenerative diseases).

Animals↗
PubMed1998

Treatment of end-stage renal disease in central and eastern Europe: overview of current status and future needs.

The situation of end-stage renal disease (ESRD) patients in central and eastern Europe was very poor for many years during the so called socialistic era. Economical and political liberation resulted in the significant growth of renal replacement facilities in this region. The number of hemodialysis units increased significantly (56%) during the period 1990-1996, and the number of patients treated with this modality has risen by 75%. More dramatic progress was achieved in peritoneal dialysis. The number of units performing this method of renal replacement therapy (RRT) increased by 277% and the number of patients by more than 300%. Not only quantitative but also qualitative changes were observed. More modern hemodialysis machines installed in the vast majority of units allow for the performance of bicarbonate dialysis, controlled ultrafiltration, and sodium profile modeling. Also, a wider choice of biocompatible dialyzers has become available during the last few years. The number of centers performing renal transplantation has increased significantly, but the number of renal transplants has not followed this progress. Despite all the progress, further development of all RRT methods is necessary to achieve acceptance rates comparable to those observed in developed countries.

Europe↗
PubMed1996

Age dependence of tolerance to anoxia and changes in cytosolic calcium in rabbit renal proximal tubules.

Calcium(Ca2+)-dependent processes mediate, in part, anoxic cell injury. These may account for the difference in sensitivity to anoxia between certain immature and mature renal cells. To address this question, we studied the effects of anoxia on cytosolic free Ca2+ concentration ([Ca2+]i), cell integrity, and transport functions in microdissected proximal convoluted tubules (PCT) of < 3-week-old (newborn) and > 12-week-old (adult) rabbits. Tubules were loaded with 10 microM fura-2 AM by incubation for 60 min at 37 degrees C, and then superfused with isosmotic saline solution gassed with either 95%O2-5%CO2 (control group) or 95%N2-5%CO2 (anoxia group) for 30 min. [Ca2+]i was measured ratiometrically; cell damage was assessed by nuclear binding of propidium iodide (PI). Anoxia resulted in a fourfold increase in [Ca2+]i in adult tubules (from resting values of 245 +/- 10 to 975 +/- 100 nM, P < 0.001), whereas in newborn tubules the rise was significantly less (from resting values of 137 +/- 5 to 165 +/- 5 nM, P < 0.001 between anoxic groups). Transient exposure to 100 mM potassium chloride, which depolarizes the PCT cells, induced increases in [Ca2+]i from baseline, to 920 +/- 90 nM in tubules from adult and to 396 +/- 16 nM in those from newborn rabbits (P < 0.001 between age groups). After exposure to ligands such as parathyroid hormone (PTH) and ATP, [Ca2+]i increased in both newborn and adult tubules, but to lower levels in newborn tubules. The response to PTH and ATP was transient in both age groups, [Ca2+]i returning to baseline levels after 2 min. Following anoxia, tubules from adult animals exhibited staining of all cell nuclei by 1 min exposure to PI, indicative of gross permeabilization of the cells. Nuclei of anoxic immatures tubules did not stain with PI. The sodium-dependent uptakes of a glucose analogue (14C-alpha-methyl-glucopyranoside) and phosphate (32Pi) were preserved in agarose-filled tubules of newborns after anoxia, whereas in those of adults recovery from anoxia was associated with drastic reduction in the uptake of these solutes. Overall, our results suggest that: (1) during anoxia, cell Ca2+ rises to critical levels in PCTs of adults compared with those of < 3-week-old animals, (2) Ca2+ influx occurs via a pathway activated by exposure to high [K+]o, presumably voltage-sensitive Ca2+ channels or reversal of Na(+)-Ca2+ exchange, (3) these pathways are either less active or less abundant in proximal tubules of newborn compared with adult rabbits, and (4) secondary active transport activity and cellular integrity are well preserved after anoxia in PCT cells of newborn but not of adult rabbits.

Adenosine Triphosphate↗