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PubMed1988

A simple technique for quantitation of low levels of DNA damage in individual cells.

Human lymphocytes were either exposed to X-irradiation (25 to 200 rads) or treated with H2O2 (9.1 to 291 microM) at 4 degrees C and the extent of DNA migration was measured using a single-cell microgel electrophoresis technique under alkaline conditions. Both agents induced a significant increase in DNA migration, beginning at the lowest dose evaluated. Migration patterns were relatively homogeneous among cells exposed to X-rays but heterogeneous among cells treated with H2O2. An analysis of repair kinetics following exposure to 200 rads X-rays was conducted with lymphocytes obtained from three individuals. The bulk of the DNA repair occurred within the first 15 min, while all of the repair was essentially complete by 120 min after exposure. However, some cells demonstrated no repair during this incubation period while other cells demonstrated DNA migration patterns indicative of more damage than that induced by the initial irradiation with X-rays. This technique appears to be sensitive and useful for detecting damage and repair in single cells.

DNA↗
PubMed1976

Influenza A virus interaction with murine lymphocytes. I. The influence of influenza virus A/Japan 305 (H2N2) on the pattern of migration of recirculating lymphocytes.

The effect of influenza virus A/Japan 305 (H2N2) on the path of migration of recirculating lymphocytes has been studied. 51Cr-labeled rat thoracic duct lymphocytes (TDL) were incubated with virus at 37 degrees C for 1 hr and then infused i.v. into syngeneic recipients which were killed 1 hr later. Virus-treated TDL accumulated in the liver and their recovery in lymph nodes and spleen was severely reduced. Changes in lymphocytes induced by virus developed rapidly and were evident after incubation for only 15 min. UV-irradiated virus altered the pattern of lymphocyte localization but attachment of heat-inactivated virus to lymphocytes in vitro had no effect on their distribution in vivo. Evidence was obtained that some virus-treated TDL, initially sequestered in the liver, subsequently recovered their ability to circulate normally. Recovery was not complete and a population of cells failed to regain their ability to home into lymph nodes. Evidence is also presented demonstrating that influenza virus affected the homing properties of both T and B cells. It is suggested that aberrations in lymphocyte homing were mediated by the viral neuraminidase which induces changes in the cell membrane leading to their accumulation in the liver.

Animals↗
PubMed1997

Pulsed-field gel electrophoretic migration of DNA broken by X irradiation during DNA synthesis: experimental results compared with Monte Carlo calculations.

Synchronous CHO cells were X-irradiated in G1 or mid-S phase with 30-750 Gy, and then the size distribution of DNA molecules resulting from DNA double-strand breaks (DSBs) was studied by pulsed-field gel electrophoresis (PFGE). Cells irradiated in S phase also were pulse-labeled with [3H]dThd for 15 min to compare the migration patterns of replicating DNA with those of DNA mass, measured by imaging with a CCD camera. When cells were irradiated immediately after pulse labeling, a large amount of the 3H-labeled replicating DNA was trapped in the plug, i.e. > 90% for doses < 100 Gy. As the dose increased, the percentage trapped decreased, i.e. to approximately 50% for 750 Gy. The same results were observed for DNA mass when cells were irradiated in S phase, except that much less of the DNA was trapped, i.e. approximately 60% for 70-100 Gy, which produced approximately 2-Mbp molecules, compared to approximately 10% for 750 Gy, which produced approximately 0.3-Mbp molecules. These results and the migration patterns of DNA released into the lane indicated that large molecules are trapped more readily than small molecules because they contain more replicating regions (bands with bubbles) of DNA than small molecules. Our interpretation is that as the dose increases, a greater fraction of the breaks occur between the replicating bands, thus releasing linear molecules that are not replicating. The relatively small amount of 3H-labeled replicating DNA that is released from the PFGE plug migrates aberrantly, with a small amount migrating like linear G1-phase molecules and a large amount, depending on dose, migrating much more slowly than the DNA mass from cells irradiated in G1 or S phase. To explain these results, a Monte Carlo computer program was written to introduce DSBs randomly into DNA that is configured according to a model of DNA replication that is developed in a related study (Dewey and Albright, Radiat. Res. 148, 421-434, 1997). In relating the experimental observations to the results of the Monte Carlo calculations, we assumed that (a) molecules containing replication bubbles with and without forks are trapped in the PFGE plug, (b) linear molecules and molecules with replication forks only that are < or = 8 Mbp are released into the lane, and (c) molecules having replication forks migrate more slowly than linear molecules.

Animals↗
PubMed1990

Axenic culture and characterization of Giardia ardeae from the great blue heron (Ardea herodias).

Trophozoites of Giardia ardeae were obtained from the great blue heron (Ardea herodias) and established in axenic culture using the TYI-S-33 medium. The generation time in culture for G. ardeae was 22-25 hr, which was 3-fold longer than for Giardia duodenalis (WB strain). A morphological comparison of trophozoites in the original intestinal isolate to those grown in culture revealed that they were identical for the following characteristics: a pyriform-shaped body, a ventral adhesive disc with a deep notch in the posterior border, teardrop-shaped nuclei, pleomorphism in median body structure ranging from a round-oval appearance (Giardia muris type) to that of a clawhammer (G. duodenalis type), and a single caudal flagellum on the right side (as viewed dorsally) with the left one being rudimentary. Analysis of the chromosomal migration patterns was performed by orthogonal-field-alternation gel electrophoresis and demonstrated that the pattern for G. ardeae was distinctly different from that for G. duodenalis (Portland 1-CCW strain). Bacterial symbionts were seen attached to trophozoites in the original isolate but could not be detected in cultured trophozoites using scanning electron microscopy, fluorescence light microscopy using the Hoechst 33258 dye for DNA localization, or by standard microbiological techniques using nonselective media for growing aerobic or anaerobic bacteria. This study demonstrated that avian-derived Giardia could be grown in axenic culture; based on morphological criteria and chromosomal migration patterns, that G. ardeae should be considered a distinct species; and that rationale for determining Giardia spp., based on median body structure alone, should no longer be considered adequate for classification at the species level.

Animals↗
PubMed1992

Different extracellular domains of the neural cell adhesion molecule (N-CAM) are involved in different functions.

The neural cell adhesion molecule (N-CAM) engages in diverse functional roles in neural cell interactions. Its extracellular part consists of five Ig-like domains and two fibronectin type III homologous (type III) repeats. To investigate the functional properties of the different structural domains of the molecule in cell interactions and signal transduction to the cell interior, we have synthesized, in a bacterial expression system, the individual domains and tandem sets of individual domains as protein fragments. These protein fragments were tested for their capacity to influence adhesion and spreading of neuronal cell bodies, promote neurite outgrowth, and influence cellular migration patterns from cerebellar microexplants in vitro. Ig-like domains I and II and the combined type III repeats I-II were most efficient for adhesion of neuronal cell bodies, when coated as substrates. Neurite outgrowth was best on the substrate-coated combined type III repeats I-II, followed by the combined Ig-like domains I-V and Ig-like domain I. Spreading of neuronal cell bodies was best on substrate-coated combined type III repeats I-II, followed by Ig-like domain I and the combined Ig-like domains I-V. The cellular migration pattern from cerebellar microexplant cultures plated on a mixture of laminin and poly-L-lysine was modified by Ig-like domains I, III, and IV, while Ig-like domains II and V and the combined type III repeats I-II did not show significant modifications, when added as soluble fragments. Outgrowth of astrocytic processes from the explant core was influenced only by Ig-like domain I. Metabolism of inositol phosphates was strongly increased by Ig-like domain I and less by the Ig-like domains II, III, IV, and V, and not influenced by the combined type III repeats I-II. Intracellular concentrations of Ca2+ and pH values were increased only by the Ig-like domains I and II. Intracellular levels of cAMP and GMP were not influenced by any protein fragment. These experiments indicate that different domains of N-CAM subserve different functional roles in cell recognition and signal transduction, and are functionally competent without nervous system-derived carbohydrate structures.

Animals↗
PubMed1990

[Epidemiological study of group C rotavirus].

By electron microscopy survey of acute gastroenteritis of children in Matsuyama, rotaviruses were detected in 561 of 2479 fecal samples obtained between October, 1984 and September, 1988, in which 60 atypical and 259 typical rotaviruses, so far tested, were detected by polyacrylamide gel electrophoresis (PAGE) of viral RNA. Out of 60 atypical rotaviruses, 5 were observed in 1985, 7 in 1986, none in 1987 and 48 in 1988. These atypical rotaviruses were morphologically indistinguishable from typical ones and showed very similar RNA migration patterns of PAGE to those of group C rotavirus. Furthermore, one atypical virus (86-542) reacted with antiserum against a porcine group C rotavirus in immune electron microscopy, while it did not react with anti-group A rotavirus serum. On the other hand, hyperimmune guinea pig antiserum against 86-542 reacted with a couple of atypical rotavirus including viruses isolated in 1988, so far tested, but did not react with any of the typical viruses. These findings showed that atypical rotaviruses which were endemic in Matsuyama city in 1988 were defined as group C rotavirus. Moreover, these group C rotaviruses showed two kind of RNA migration patterns in PAGE, which clearly discriminated the virus were isolated before and after 1987. Epidemiological features of group C rotavirus were as follows. Children from whom group C rotavirus was isolated were older than those from of whom group A rotavirus was isolated. The epidemic season was in February through April, compared to December through March in group A virus. Retrospective seroepidemiological study by immune adherence hemagglutination test (IAHA) using the purified 86-542 virus as antigen indicated that 15 of 78 sera of children obtained in 1971 already possessed IAHA antibody against group C rotavirus.

Adolescent↗
PubMed1993

Prolonged isoproterenol treatment alters immunoregulatory cell traffic and function in the rat.

The mechanisms by which the sympathetic nervous system modulates functional parameters of the immune system have not been fully defined. In vivo, acute and chronic beta 2-adrenergic stimulation have been shown to have dramatic but opposing effects on circulating lymphocyte number and subset distribution in humans. In vitro studies have suggested impairment of numerous lymphocyte effector functions in the presence of catecholamines. To better define the effects of short and long term beta-adrenergic stimulation on lymphocyte migration patterns between circulating and splenic pools, as well as function, we infused rats with either low or high doses of isoproterenol over various time intervals. Specifically, we determined the number and subset composition of peripheral blood and splenic lymphocytes in addition to assessing responsivity to the T cell mitogen concanavalin A and antibody production. As a measure of in vivo lymphocyte proliferation, we also monitored the effect of these infusions on the number of peripheral blood lymphocytes and splenocytes and the splenic weight. Isoproterenol led to dose-dependent, short-lived decrements in mitogen-induced lymphocyte proliferation with concomitant decreases in circulating and splenic lymphocyte number in a subset-specific manner. Analysis of the distribution of circulating and splenic subtypes with isoproterenol treatment suggests definite but limited influences on beta-adrenergic stimulation on immunoregulatory cell traffic. We conclude that beta-adrenergic stimulation in the rat model leads to a dose-dependent, transient effect on lymphocyte proliferation and migration patterns. The relative lack of functional beta-receptors on rat lymphocytes and the propensity for early sustained receptor desensitization on exposure to agonist may account for qualitative differences seen between rats and humans.

Animals↗
PubMedDate not supplied

[Territorial mobility in medical education in Mexico].

OBJECTIVE: This work aimed to determine the migration patterns of medical students within Mexico. MATERIALS AND METHODS: We obtained the places of origin of graduates and the states where they registered their medical degrees at the Ministry of Education General Registry of Professions (SEP), between 1970-1974, 1980-1984 and 1985-1989. Data were organized as follows: Attracting foci, sending foci, transition foci, and important migrant flows. RESULTS: The concentration of medical human resource development, is reflected by the existence of a few attracting foci (D.F., Jalisco, Nuevo Leon, and to a less extent, Puebla and Michoacan). Also, we observed the persistence of traditional sending foci (Guanajuato, Chiapas, Colima, Campeche). However, some important changes occurred throughout the study period, namely, a decrease of the migratory mobility of university students. During 1970-1974, almost half of them (47%) obtained their degrees outside their place of origin; during 1980-1984 this figure decreased to 34% and during 1985-1989, it decreased further to 30.6%. Second, the participation of D.F. as a main human resource development center diminished; from 59% to 40% during 1970-1974, it went down, to 30% in the following quinquennium, while it increased in Jalisco, Michoacan and Nuevo Leon. CONCLUSIONS: The establishment of medical schools in almost every Mexican state has had a central role in the migration patterns of medical students. Nevertheless, our results show that there are other reasons accounting for the persistence of the concentration of medical human resources development in main cities of the nation such as Guadalajara and Monterrey.

Education, Medical↗
PubMed1996

Sites of origin and patterns of migration of vasotocin/mesotocin neurons in developing brain of the chick.

Vasotocin/mesotocin (VT/MT) producing neurons are known to migrate extensively during development of the hypothalamus. Birthdating studies as well as immunohistochemical studies suggested the possibility that VT/MT producing neurons originate from specific sites of the neural tube. Furthermore, a relationship between the site of origin and the eventual fate of VT/MT cells has been suggested. This study proposes to identify the sites of origin of VT/MT cells and to establish whether magnocellular and parvocellular VT/MT, and neuromodulatory and neurosecretory VT/MT arise from common or different areas of the developing neural tube. To do so, the embryological distribution of VT/MT producing neurons of the chick was studied with immunohistochemistry. Analysis of the youngest brains in which VT/MT cells could be detected (embryonic day 7.25, E7.25) suggested the presence of two separate sites of origin. The first site was located in the hypothalamic anlage, next to the third ventricle, and the second in the mesencephalon, next to the fourth ventricle. Three-dimensional reconstructions of the location of VT/MT cells throughout development substantiated the hypothesis that diencephalic VT/MT cells originate from the first site while mesencephalic ones originate from the second site. Mesencephalic VT/MT producing cells were confined to the nucleus of Edinger-Westphal and were only detectable during a brief period in development (E7.25-E10). Diencephalic VT/MT producing neurons were noted to form two main paths from their sites of origin to the rostral diencephalon. Quantitative analysis confirmed this caudal to rostral displacement. Magnocellular and parvocellular VT/MT+ cells were intermingled at the diencephalic site of origin as well as in the migratory paths. Neuromodulatory and neurosecretory VT/MT cells of the diencephalon appeared to be derived from a common diencephalic site of origin. These studies support the hypothesis that while specific groups of progenitors may be important in allowing their offspring to produce VT/MT, they do not appear to influence the morphological attributes (magnocellular vs. parvocellular), nuclear locations, or functional characteristics of these cells.

Animals↗
PubMed1995

Cell lineage and patterns of migration in the developing cortex.

Knowledge of cell lineage in the cortex is important for understanding normal development as well as brain malformations. We studied cell lineage in rats by injecting a library of up to 3400 retroviruses, distinguishable by PCR analysis and encoding alkaline phosphatase, at E14-19. Histological analysis at P15 revealed normal cell morphology and allowed identification of about 80% of all labelled cells. PCR amplification of DNA tags allowed clonal analysis. Cortical cells labelled at E15 formed clustered or widespread clones with equal frequency. Clustered clones contained one to four cells within about 1 mm that had similar morphology and laminar location. However, 48% of cortical clones contained multiple cell types with widely different locations (2.1-6.7 mm; mean, 3.8 mm). Widespread clones contained two to four 'subunits' (one to five neurons each), spaced at apparent intervals of 2-3 mm, with each subunit morphologically indistinguishable from a clustered clone. Distinct subunits in the same clone usually differed in laminar location suggesting sequential formation. Clones labelled at E17 contained fewer neurons and up to two subunits. Clustered clones seem to be produced by stationary progenitors, whereas progenitors of clusters may themselves be produced by migratory, multipotential cells.

Animals↗
PubMed1989

Patterns of migration indicate sexual transmission of HTLV-I infection in non-pregnant women in Papua New Guinea.

The prevalence of infection with human T-cell leukaemia virus (HTLV-I) was studied in Madang Province on the north coast of Papua New Guinea. Serum specimens collected from non-pregnant women in 17 villages were tested for anti-HTLV-I by gelatin particle agglutination screening and confirmed by immunofluorescence and Western blotting. Overall, 13.9% of subjects were antibody-positive, with the prevalence of antibodies varying from less than 10% to 30% in villages situated less than 10 km apart. Two groups of migrant women were identified, and in both a parity-related increase in antibody prevalence which occurred only after marriage suggested that the predominant mode of transmission in migrant women was sexual. There was no parity-associated increase in anti-HTLV-I in indigenous women, and in contrast to migrant women, nulliparous indigenous women had a high prevalence of antibody (16.8% vs. 0%; p = 0.005). Vertical transmission cannot be excluded in indigenous women. No correlation was detected between the prevalence of anti-HTLV-I and a variety of indices of malarial infection.

Emigration and Immigration↗
PubMed1996

A comparison of the patterns of migration and the destinations of homotopically transplanted neonatal subventricular zone cells and heterotopically transplanted telencephalic ventricular zone cells.

The cells arising in the anterior part of the subventricular zone (SVZa) migrate along a well-demarcated pathway which lacks radial glial fibers to the olfactory bulb where they differentiate into interneurons of the granule cell layer or glomerular layer (Luskin, 1993, Neuron 11, 173). To analyze the mechanisms underlying this highly directed migration, we have compared the migratory behavior of unmanipulated SVZa-derived cells to that of homotopically transplanted SVZa cells and of heterotopically transplanted telencephalic ventricular zone (VZ) cells that ordinarily migrate in association with radial glial fibers. To identify the phenotype of the SVZa progenitor cells prior to their transplantation, we characterized them in vitro using cell type-specific markers. After 1 day in culture nearly all the SVZa cells were stained with TuJ1, a neuron-specific marker; only an occasional cell exhibited a glial phenotype as judged by the presence of GFAP-immunoreactivity. This indicates that SVZa cells express a neuronal phenotype. To reveal the spatiotemporal distribution of homotopically transplanted neonatal SVZa cells in a host brain, dissociated SVZa cells from Postnatal Day 0 (P0)-P2 animals were labeled with the lipophilic dye PKH26 or the cell proliferation marker BrdU and implanted into the SVZa of host animals of the same age. Within the first week after transplantation there were vast numbers of labeled cells throughout the pathway. Over the next 2 weeks the labeled cells migrated into the overlying cellular layer of the olfactory bulb and began to differentiate, and within 4 weeks the transplanted cells had reached their final positions in the granule cell and glomerular layers of the olfactory bulb in the same proportions as for unmanipulated SVZa-derived cells. While en route to the olfactory bulb the homotopically transplanted cells never strayed from the migratory pathway. In contrast, heterotopically transplanted VZ cells from the embryonic telencephalon did not undergo migration although they did differentiate. These results demonstrate that the homotopically transplanted SVZa-derived cells adopt a mode of migration indistinguishable from that ordinarily utilized by SVZa-derived neurons and that the VZ cells are unable to decipher the same set of guidance cues.

Animals↗
PubMed1995

Phenotypic analysis of migrant, efferent lymphocytes after implantation of cold preserved, peripheral nerve allografts.

Cold-preservation of peripheral nerve allografts in vitro (3 weeks, 5 degrees C) was performed to determine its effect on local lymphocyte migration patterns in vivo. Lymphocyte migration was assessed by continuously monitoring the cell output in the regional lymph for nearly 1 month. Cold-preservation delayed or prevented the typical biphasic increase in efferent lymphocyte output observed after fresh allograft implantation. It also decreased the output of activated lymphocytes (CD 5 and MHC class II positive) compared with that seen in the fresh allograft response. These changes suggest that the host immune response to preserved nerve allografts is altered over a prolonged period in vivo (3 weeks). Cold-preservation may be a useful method of reducing allograft immunogenicity, thereby limiting systemic immunosuppression requirements for the successful clinical utilization of peripheral nerve allografts.

Animals↗
PubMed1996

Altered patterns of migration of cytokine-producing T lymphocytes in skin-grafted naive or immune mice following in vivo administration of anti-VCAM-1 or -ICAM-1.

Naive or preimmunized (to B10.BR or BALB.k) C3H/HeJ mice received skin grafts from multiple minor histoincompatible B10.BR or BALB.k mice following antigen-specific portal venous (p.v.) pretransplant transfusion, a protocol known to produce prolongation of graft survival in naive animals. In addition, groups of mice received intravenous (i.v.) infusion following transplantation with a mixture of monoclonal antibodies (mAb) to vascular adhesion molecule-1L: very late activation antigen-4 (VCAM-1:VLA-4) or intracellular adhesion molecule-1:lymphocyte function-associated antigen-3 (ICAM-1:LFA-1). Cells were harvested from different tissues of the grafted mice at various times post grafting. RNA was extracted and analysed, using polymerase chain reaction, for expression of different cytokines potentially involved in the regulation of graft rejection [interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumour necrosis factor-alpha, interferon-gamma and transforming growth factor-beta]. In addition, using limiting dilution analysis, we investigated the frequency of allo-specific and third-party reactive cells producing IL-2 and IL-4 in vitro in different tissues of grafted mice following these treatments. The mAb treatment protocol which produced optimum increases in graft survival in naive versus immune mice was different, with anti-LFA-1:ICAM-1 superior for naive mice compared with anti-VLA-4:VCAM-1, and vice versa for immune animals. However, in each case, increased survival was associated with increases local to the graft in the frequency of occurrence of antigen-specific type-2 cytokine-producing cells.

Animals↗