Biomedical subjects

K Radke

Publications and source records attributed to K Radke.

At least 19 recordsLinked to original sources

PubMed2006

Dissemination of bovine leukemia virus-infected cells from a newly infected sheep lymph node.

To investigate the early establishment of bovine leukemia virus (BLV) infection, we injected BLV-infected or mock-infected allogeneic cells into the shoulder of sheep in which an efferent lymphatic duct of the draining prescapular lymph node had been cannulated. Rare mononuclear cells acting as centers of BLV infection in culture were present within 4 to 6 days in efferent lymph and within 6 to 10 days in blood. Soon after BLV injection, immunoglobulin M+ (IgM+) and CD8+ cells increased in efferent lymph and oscillated reciprocally in frequency. CD8+ blasts increased on days 4 to 6, when infectious centers increased 100-fold in lymph. On days 6 and 7, both lymph and blood were enriched with CD8+ cells that were labeled late on day 5 with an intravenous pulse of 5-bromo-2'-deoxyuridine (BrdU). Lymph, but not blood, was enriched with BrdU+ B cells on day 7. Capsid-specific antibodies became detectable in efferent lymph on days 6 to 8 and surface glycoprotein-specific antibodies on day 9, preceding their detection in serum by 9 to 14 days. Systemic dissemination of BLV-infected cells was thus accompanied by an increase in proliferating CD8+ cells and the onset of BLV-specific antibodies in lymph. Infectious centers reached maximum frequencies of 0.2% in lymph by days 11 to 13, and then their frequencies increased by 5- to 40-fold in blood cells, suggesting that many infected blood cells do not recirculate back into lymph. Beginning on days 10 to 13, a subpopulation of B cells having high levels of surface IgM increased sharply in peripheral blood. Such cells were not present in lymph. After a day 16 pulse of BrdU, recently proliferated cells that stained intensely for surface IgM appeared in blood within 15 h. Predominantly B lymphocytes contained the viral capsid protein when lymph and blood cells were cultured briefly to allow BLV expression. However, both early in lymph and later in blood, BrdU+ B cells greatly exceeded productively infected cells, indicating that new BLV infections stimulate proliferation of two different populations of B cells.

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PubMed2001

Blood flow velocities in the basal vein after subarachnoid haemorrhage. A prospective study using transcranial duplex sonography.

BACKGROUND: Early recognition of emerging delayed neurological deficits (DND) in patients after subarachnoid haemorrhage (SAH) is not always possible by transcranial Doppler sonography. Aim of this study was to investigate a) whether determination of blood flow velocities in deep cerebral basal veins can predict DND in these patients b) the correlation of venous flow velocity to cerebral blood flow (CBF). METHODS: a) We prospectively investigated the mean flow velocity in the basal vein (VBVR), in the middle cerebral artery (VMCA) and in the extracranial internal carotid artery (VICA) in 66 patients after spontaneous SAH. Examinations were performed daily during the first 10 days, using transcranial duplex sonography. Thirty-seven patients had VMCA exceeding 120 cm/s. They were categorised in three groups: 1: no delayed neurological deficit; II: transient DND; III: permanent DND or death associated with vasospasm. b) In another group of 14 patients, interdiane variations in global cerebral blood flow (CBF) measured by the Kety-Schmidt-method were correlated with variations in VBVR, VMCA, and VICA. FINDINGS: a) In patients without deficit, VBVR was significantly elevated above normal values the first day (p < 0.05), and days 5 and 6 (p < 0.1) after VMCA exceeding 120 cm/s. In group III (permanent deficit), flow velocities in the BVR were significantly below normal on day 5 (p < 0.05) and 9 (p < 0.1). b) The correlation between changes in VBVR to changes in CBF (r = 0.78, p < 0.001) was closer than between changes in VMCA to the changes in CBF (r = 0.54, p < 0.05). INTERPRETATION: In case of elevated VMCA, patients with higher VBVR seem to have a better outcome. Changes in CBF correlate better with VBVR than with arterial flow velocities.

Adult↗

PubMed2001

Drosophila-raf acts to elaborate dorsoventral pattern in the ectoderm of developing embryos.

In the early Drosophila embryo the activity of the EGF-receptor (Egfr) is required to instruct cells to adopt a ventral neuroectodermal fate. Using a gain-of-function mutation we showed that D-raf acts to transmit this and other late-acting embryonic Egfr signals. A novel role for D-raf was also identified in lateral cell development using partial loss-of-function D-raf mutations. Thus, we provide evidence that zygotic D-raf acts to specify cell fates in two distinct pathways that generate dorsoventral pattern within the ectoderm. These functional requirements for D-raf activity occur subsequent to its maternal role in organizing the anterioposterior axis. The consequences of eliminating key D-raf regulatory domains and specific serine residues in the transmission of Egfr and lateral epidermal signals were also addressed here.

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PubMed2000

The SU and TM envelope protein subunits of bovine leukemia virus are linked by disulfide bonds, both in cells and in virions.

After the polyprotein precursor of retroviral envelope proteins is proteolytically cleaved, the surface (SU) and transmembrane (TM) subunits remain associated with each other by noncovalent interactions or by disulfide bonds. Disulfide linkages confer a relatively stable association between the SU and TM envelope protein subunits of Rous sarcoma virus and murine leukemia virus. In contrast, the noncovalent association between SU and TM of human immunodeficiency virus leads to significant shedding of SU from the surface of infected cells. The SU and TM proteins of bovine leukemia virus (BLV) initially were reported to be disulfide linked but later were concluded not to be, since TM is often lost during purification of SU protein. Here, we show that SU and TM of BLV do, indeed, associate through disulfide bonds, whether the envelope proteins are overexpressed in transfected cells, are produced in virus-infected cells, or are present in newly produced virions.

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PubMed1997

Lack of checkpoint control at the metaphase/anaphase transition: a mechanism of meiotic nondisjunction in mammalian females.

A checkpoint mechanism operates at the metaphase/anaphase transition to ensure that a bipolar spindle is formed and that all the chromosomes are aligned at the spindle equator before anaphase is initiated. Since mistakes in the segregation of chromosomes during meiosis have particularly disastrous consequences, it seems likely that the meiotic cell division would be characterized by a stringent metaphase/ anaphase checkpoint. To determine if the presence of an unaligned chromosome activates the checkpoint and delays anaphase onset during mammalian female meiosis, we investigated meiotic cell cycle progression in murine oocytes from XO females and control siblings. Despite the fact that the X chromosome failed to align at metaphase in a significant proportion of cells, we were unable to detect a delay in anaphase onset. Based on studies of cell cycle kinetics, the behavior and segregation of the X chromosome, and the aberrant behavior and segregation of autosomal chromosomes in oocytes from XO females, we conclude that mammalian female meiosis lacks chromosome-mediated checkpoint control. The lack of this control mechanism provides a biological explanation for the high incidence of meiotic nondisjunction in the human female. Furthermore, since available evidence suggests that a stringent checkpoint mechanism operates during male meiosis, the lack of a comparable checkpoint in females provides a reason for the difference in the error rate between oogenesis and spermatogenesis.

Anaphase↗

PubMed1997

Characterization of maternal and zygotic D-raf proteins: dominant negative effects on Torso signal transduction.

The maternal D-raf serine/threonine kinase acts downstream of Torso (Tor) for specification of cell fates at the embryonic termini. D-raf activity is also required in other signal transduction pathways and consistent with its pleiotropic role, we find accumulation of a 90-kD D-raf protein throughout embryonic development. We also characterize the accumulation of maternal D-raf proteins in 0-2-hr embryos derived from females with germ cells lacking D-raf activity. Accumulation of a 90-kD or truncated mutant D-raf protein is observed for some of these embryos, while others lack the maternal D-raf protein. Then to determine whether rescue of the Tor pathway is influenced by pools of nonfunctional maternal D raf. wild-type D-raf mRNA was injected into embryos that inherit maternal stores of inactive 90-kD of truncated D-raf protein. For embryos lacking the maternal D-raf protein, a high level of terminal rescue is obtained. In contrast, rescue is reduced or not observed for embryos that accumulate mutant maternal D-raf proteins. These findings suggest that mutant forms of D-raf may deplete the embryo of a positive activator and/or form inactive protein complexes that affect rescue of the Tor pathway.

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PubMed1996

Beta 1 integrins regulate axon outgrowth and glial cell spreading on a glial-derived extracellular matrix during development and regeneration.

In the present study we have investigated functional roles for beta 1 integrin receptors in regulating axon outgrowth, and glial cell adhesion and spreading in the Xenopus retina. The XR1 glial cell line, isolated from Xenopus retinal neuroepithelium, deposits a proteinaceous extracellular matrix (ECM) with potent neurite outgrowth promoting activity. To investigate a potential role of the integrins as cellular receptors for these glial cell-derived ECM components, embryonic and regenerating retinal explants were cultured in the presence of polyclonal antibodies directed against the beta 1 integrin receptor complex. The IgGs and Fabs of the anti-beta 1 integrin antibody strongly inhibited ganglion cell axon outgrowth on the glial cell-derived ECM, although axons grew freely across the surfaces of glial cells surrounding the explants. The antibodies also inhibited outgrowth on purified laminin containing substrates in a dose-dependent fashion. In addition, the anti-beta 1 antibodies were effective at inhibiting the spreading of glial cells that migrated out from the embryonic explants, and also inhibited attachment and spreading of Xenopus XR1 glial cells on ECM substrates. These results show that the beta 1 integrins play important functional roles in axon outgrowth during development and regeneration, and also serve in regulating retinal glial cell attachment and spreading in vitro, and thus are likely to play similar roles in vivo.

Animals↗

PubMed1996

Lymphocyte activators elicit bovine leukemia virus expression differently as asymptomatic infection progresses.

Since bovine leukemia virus (BLV) replicates in B-lymphocytes, viral expression and production should respond to agents activating these cells. We asked whether synthesis of BLV capsid (CA) protein and production of infectious virus would increase when peripheral blood mononuclear cells (PBMCs) from infected sheep were stimulated in short-term culture with lipopolysaccharide (LPS), a polyclonal activator of B-cells, and compared its effects with those of phytohemagglutinin (PHA), a lymphocyte activator known to increase BLV expression. LPS treatment of PBMCs from asymptomatic sheep that had been infected for 1-4 years increased the number of cells synthesizing CA protein, the amount of CA protein per cell, and the number of PBMCs acting as infectious centers. LPS derived from several different microbes was effective. During the ensuing 4 years of asymptomatic infection, the number of PBMCs expressing virus under minimal stimulation increased for each animal. The ability of LPS to recruit additional cells to express CA protein remained constant or decreased in magnitude, yet at the same time, lower concentrations of LPS were required to elicit a maximal effect. This suggests that the cellular pathways affected by LPS are endogenously more activated as infection progresses. PHA initially stimulated fewer cells to synthesize BLV CA protein than LPS did although the amount of CA protein synthesized per cell was greater with PHA. As infection progressed, PHA surpassed LPS in the numbers of PBMCs induced to express CA protein. This suggests that the cellular pathways affected by PHA become more responsive to its effects as infection progresses. LPS increased CA expression early and transiently during culture whereas the PHA-mediated increase continued to develop for several days. Thus, LPS increases BLV expression but does so differently than PHA. Moreover, these longitudinal results show that the activation state of BLV-infected cells changes as asymptomatic infection progresses.

Animals↗

PubMed1996

Peripheral blood mononuclear cells from sheep infected with a variant of bovine leukemia virus synthesize envelope glycoproteins but fail to induce syncytia in culture.

Peripheral blood mononuclear cells (PBMCs) infected with the oncogenic retrovirus bovine leukemia virus (BLV) produce virus when cultured briefly. BLV can be transmitted in cocultures to adherent susceptible cells, which become infected, express viral proteins, and fuse into multinucleated syncytia several days later. PBMCs from 3 of 10 BLV-infected sheep displayed a lifelong deficiency in induction of syncytium formation among indicator cells in culture, although large numbers of PBMCs synthesized viral transcripts or capsid protein. Since the infected, syncytium-deficient PBMCs were > or = 97% B cells, the deficiency could not be attributed to altered host cell tropism. The syncytium-deficient phenotype was recapitulated in newly infected sheep, demonstrating that this property is regulated by the viral genotype. The alteration in the BLV genome delayed but did not prohibit the establishment of BLV infection in vivo. Envelope glycoproteins were synthesized in syncytium-deficient PBMCs, translocated to the cell surface, and incorporated into virions. However, monoclonal antibodies specific for the BLV surface glycoprotein did not stain fixed PBMCs of the syncytium-deficient phenotype. Moreover, an animal with syncytium-deficient PBMCs had lower titers of neutralizing antibodies throughout the first 5 years of infection than an animal with similar numbers of infected PBMCs of the syncytium-inducing phenotype. The syncytium-deficient variant productively infected indicator cells at greatly reduced efficiency, showing that the alteration affects an early step in viral entry or replication. These results suggest that the alteration maps in the env gene or in a gene whose product affects the maturation or conformation, and consequently the function, of the envelope protein complex.

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PubMed1993

Symmetrical segregation of potassium channels at cytokinesis.

To determine how voltage-gated ion channels segregate between sibling cells at cytokinesis, we used a whole-cell patch clamp to measure the electrophysiological phenotypes of siblings within 45 min of division. Recently born siblings in an immortalized line of embryonic retinal cells were identified as pairs of spherical cells adhering to one another. All siblings were electrically coupled when cells were simultaneously voltage clamped, whereas nonsiblings were not coupled. Twelve pairs of siblings were electrically isolated by mechanical separation so that their phenotypes could be measured independently. Cells expressed two principal membrane conductances, delayed rectifier-like (IK) and inward rectifier (IK(IR)) potassium currents. Despite qualitative and quantitative variability in IK and IK(IR) expression within the population, each cell of a given pair expressed similar steady-state current densities between -110 and +50 mV. We estimated IK(IR) slope conductance by blocking the current specifically with 5 mM Cs and calculated IK(IR) ratios in siblings and nonsiblings. Three pairs of siblings expressed IK(IR) ratios of approximately 1.2, while ratios in three pairs of adhered nonsiblings varied between 1.6 and 5.4. When currents were sampled continuously through cytokinesis by using the perforated-patch recording mode, current amplitude showed no net change within 30 min of division. Because channel number did not appear to change in siblings during this interval, parental channels were inherited by each daughter in proportion to the area of membrane received. Heterogeneity therefore arises after siblings reenter interphase and is not due to the asymmetrical segregation of channels at cytokinesis.

Animals↗

PubMed1992

Transcription of bovine leukemia virus in peripheral blood cells obtained during early infection in vivo.

Bovine leukemia virus (BLV) is transcriptionally silent in most circulating peripheral blood mononuclear cells (PBMCs) of animals with well-established infections. Using PBMCs from a newly infected sheep, we asked whether viral transcription proceeded differently during the initial months of infection, when the prevalence of BLV-infected cells and the host's immunological response change markedly. Shortly after being injected with BLV, the animal displayed a characteristic, transient increase in PBMCs that transcribed BLV when cultured. Even when transcriptionally competent PBMCs were most prevalent (1.2%), only rare cells in the circulation (1 in 50,000) contained enough BLV transcripts to be identified readily by in situ hybridization. However, at one point several weeks later, some PBMCs appeared to contain small amounts of BLV RNA as soon as they had been purified from blood. Throughout this period, BLV-transcribing PBMCs greatly outnumbered virus-producing cells, which were counted using a new infectious centers assay. Its viscous medium reduced cell to cell contact among PBMCs, enabling increased detection of BLV-producing cells at a time when virus-specific killer cells might be active. Early infection was polyclonal, and most infected PBMCs transcribed BLV upon being cultured. By 2 months after infection, provirus-containing cells were as abundant as they had been earlier, but few cells transcribed BLV. These results suggest that BLV-infected cells are more easily stimulated to transcribe the provirus and produce infectious virus during the early months of a new infection.

Animals↗

PubMed1992

Activation of bovine leukemia virus transcription in lymphocytes from infected sheep: rapid transition through early to late gene expression.

Bovine leukemia virus (BLV) expression is mostly silent in peripheral blood mononuclear cells (PBMCs) of infected animals. However, when infected cells are cultured, they are stimulated to produce virus. We studied viral transcription in PBMCs taken from BLV-infected sheep because the pattern of transcriptional activation in these cells should closely mimic activation of virus expression within mononuclear cells in vivo. BLV transcription was activated as early as 30 min after PBMCs were cultured. Expression was characterized by early and late stages, each distinguished by a unique pattern of cytoplasmic RNAs. In early expression, cytoplasmic viral RNA was exclusively the doubly spliced tax/rex transcript, although all transcripts were present in the nucleus. Early expression gave way rapidly to late expression, in which all viral transcripts accumulated in the cytoplasm. The polyclonal B-cell activator lipopolysaccharide increased the amount of viral RNA by at least twofold but did not alter the pattern of transcription. The transition from early to late expression required new protein synthesis and was blocked by the inhibitor cycloheximide. This requirement reflects the essential role of the viral Rex protein in the transition, but synthesis of cellular factors may be required as well. These results provide the first demonstration of staged viral expression in lymphocytes naturally infected by either BLV or the closely related human T-cell leukemia virus (HTLV) and validate the model of BLV and HTLV gene expression that previously was derived from transfection experiments performed mainly in nonlymphoid cells.

Animals↗

PubMed1991

Clonal cells from embryonic retinal cell lines express qualitative electrophysiological differences.

Cells from the embryonic quail retina were immortalized with the v-mil oncogene and cloned by limiting dilution. Their phenotype was examined using the whole-cell patch clamp method. Three membrane currents, IK(IR), INa and IK, were found at different frequencies within a sample of 170 cells drawn from a large clone. Nearly all combinations of these three markers were found and the frequency of combinations showed that the markers assorted independently. Examination of clones of less than 10 cells showed that heterogeneity originates with a high probability within clones, arguing that chromosomal mutation, for example, is unlikely to account for phenotypic diversity. A possible explanation is that phenotypic differences between cells might reflect the local exchange of instructive signals. If so, then the genes for the three phenotypic markers are controlled independently.

Animals↗

PubMed1991

Episodic occurrence of antibodies against the bovine leukemia virus Rex protein during the course of infection in sheep.

Infection by bovine leukemia virus (BLV) is characterized by a long clinical latency after which some individuals develop B-cell tumors. The contributions of the viral regulatory proteins Tax and Rex during clinical latency and disease are incompletely understood. To learn about Rex expression in the host, we used a sensitive immunoprecipitation assay to detect Rex antibodies throughout the course of BLV infection in sheep. Sixty percent of the infected animals produced Rex antibodies in intermittent episodes. This pattern differed markedly from that of antibodies to virion structural proteins, which were maintained in all animals throughout infection. Only one of two animals that developed tumors had detectable Rex antibodies at the time, although the other had previously demonstrated an especially strong Rex antibody response. We examined the Rex response in the context of BLV infection by comparing it with the frequency of circulating mononuclear blood cells that could transcribe BLV RNA or produce infectious virus. Episodes of Rex antibody occurrence followed some but not all increases in the number of BLV-transcribing cells. Since the appearance of circulating antibodies requires that the intracellular Rex protein be available to serve as antigen, the episodic pattern of occurrence of Rex antibodies could result from intermittent killing by virus-specific cytotoxic cells. Fluctuations in titer that were observed during some episodes of Rex response could be due to antibody retention by antigen present in lymphoid tissue.

Animals↗

PubMed1990

Transient increases of blood mononuclear cells that could express bovine leukemia virus early after experimental infection of sheep.

To investigate the early spread of bovine leukemia virus (BLV) infection in vivo, we enumerated infected mononuclear cells that could express the BLV genome in vitro as they appeared in the peripheral blood of lambs newly injected with the virus. Cells that transcribed viral RNA within a few hours of isolation and cells that produced infectious virus in culture were first detected in very small numbers. Soon afterward, cells that expressed BLV transiently increased to represent 0.2 to 1.5% of the mononuclear cells. The increases occurred within leukocyte populations of normal size and cellular composition. Then, throughout the rest of the first 8 months, sharply reduced numbers of cells transcribed BLV or produced virus. All the infected animals tested by in situ hybridization displayed increased numbers of cells that transcribed BLV RNA, but only two-thirds had large increases of cells that produced infectious BLV in culture. In addition, BLV-transcribing cells exceeded virus-producing cells at most times after infection. These results demonstrate that transient increases of circulating, expression-competent cells characterize the first 3 to 4 months of BLV infection and that the extent of BLV genome expression by cultured mononuclear cells can differ among animals.

Animals↗

PubMed1990

Humoral immune response of experimentally infected sheep defines two early periods of bovine leukemia virus replication.

We have correlated the virus-specific humoral immune response of sheep newly infected with bovine leukemia virus (BLV) with the appearance in their blood of cells that transcribe BLV RNA or produce virus in culture. Neutralizing antibodies and antibodies binding to the viral capsid protein were present in most animals early after infection, often before BLV-expressing cells were first detected in blood. Neutralizing antibodies increased rapidly during the period when the number of cells that expressed BLV was also increasing. However, the titers developed by individual animals were independent of the maximum number of BLV-expressing cells. Antibodies that bound to the viral surface glycoprotein on immunoblots became evident at the same time as large peaks in the numbers of BLV-expressing cells. Despite ensuing sharp drops in BLV-expressing cells, neutralizing titers remained relatively constant through the rest of the first 8 months after infection. Two early phases of BLV replication were thus defined: initial, low-level replication that induced neutralizing and capsid-specific antibodies followed by a second period of intense replication that induced sharp increases in antiviral antibodies and preceded the release of many infected cells into the blood.

Animals↗

PubMed1989

Transcriptional activation of bovine leukemia virus in blood cells from experimentally infected, asymptomatic sheep with latent infections.

Infection by bovine leukemia virus (BLV) is characterized by a long latency period, after which some individuals develop B-cell tumors. The behavior of BLV and related retroviruses during the latency period between initial infection and subsequent tumorigenesis is poorly understood. We used in situ hybridization to detect BLV transcripts in individual peripheral blood mononuclear cells from experimentally infected, asymptomatic sheep with latent infections. Viral RNA was not found in most peripheral blood cells that had been isolated as rapidly as possible from circulating blood, but it was present in rare cells. BLV RNA transcripts increased in a biphasic manner within a few hours after the blood cells were placed in culture. Exposure to fetal bovine serum was identified as the principal cause of this transcriptional activation, which occurred in fewer than 1 in 1,000 cells. Agents known to activate immune cells polyclonally caused a further increase in the number of cells containing BLV RNA within 8 h. In some cases, the numbers of viral transcripts within individual cells also increased. Thus, BLV is not detectably expressed in most resting lymphocytes circulating in the blood, but its transcription is activated by components of fetal bovine serum and can be augmented by molecules that mimic activation of immune cells. This activation, which might occur in lymphoid tissue during an immune response, may lead to the synthesis of viral regulatory proteins that are thought to initiate tumorigenesis through host cell genes.

Animals↗

PubMed1988

A role for the extracellular matrix in retinal neurogenesis in vitro.

The germinal neuroepithelial cells that give rise to the majority of neurons in the vertebrate central nervous system are in contact with the basement membrane that surrounds the neural tube from the very earliest stages. The effect of removing this basement membrane on the organization and proliferative potential of these cells was examined in a new slice culture preparation of developing Rana tadpole retina. The results indicate that the germinal neuroepithelium, like other epithelial tissues, requires contact with a basement membrane for the maintenance of its structure and a normal degree of cell proliferation.

Animals↗