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J Lips

Publications and source records attributed to J Lips.

13 recordsLinked to original sources
PubMed2002

Induction of DNA breaks and apoptosis in crosslink-hypersensitive V79 cells by the cytostatic drug beta-D-glucosyl-ifosfamide mustard.

To study molecular aspects of cytotoxicity of the anticancer drug beta-D-glucose-ifosfamide mustard we investigated the potential of the agent to induce apoptosis and DNA breakage. Since beta-D-glucose-ifosfamide mustard generates DNA interstrand crosslinks, we used as an in vitro model system a pair of isogenic Chinese hamster V79 cells differing in their sensitivity to crosslinking agents. CL-V5B cells are dramatically more sensitive (30-fold based on D(10) values) to the cytotoxic effects of beta-D-glucose-ifosfamide mustard as compared to parental V79B cells. After 48 h of pulse-treatment with the agent, sensitive cells but not the resistant parental line undergo apoptosis and necrosis, with apoptosis being the predominant form of cell death (70 and 20% of apoptosis and necrosis, respectively). Apoptosis increased as a function of dose and was accompanied by induction of DNA double-strand breaks in the hypersensitive cells. Furthermore, a strong decline in the level of Bcl-2 protein and activation of caspases-3, -8 and -9 were observed. The resistant parental cells were refractory to all these parameters. Bcl-2 decline in the sensitive cells preceded apoptosis, and transfection-mediated overexpression of Bcl-2 protected at least in part from apoptosis. From the data we hypothesize that non-repaired crosslinks induced by beta-D-glucose-ifosfamide mustard are transformed into double-strand breaks which trigger apoptosis via a Bcl-2 dependent pathway.

Animals↗
PubMed2001

Repair of O(6)-methylguanine is not affected by thymine base pairing and the presence of MMR proteins.

Methylation at the O(6)-position of guanine (O(6)-MeG) by alkylating agents is efficiently removed by O(6)-methylguanine-DNA methyltransferase (MGMT), preventing from cytotoxic, mutagenic, clastogenic and carcinogenic effects of O(6)-MeG-inducing agents. If O(6)-MeG is not removed from DNA prior to replication, thymine will be incorporated instead of cytosine opposite the O(6)-MeG lesion. This mismatch is recognized and processed by mismatch repair (MMR) proteins which are known to be involved in triggering the cytotoxic and genotoxic response of cells upon methylation. In this work we addressed three open questions. (1) Is MGMT able to repair O(6)-MeG mispaired with thymine (O(6)-MeG/T)? (2) Do MMR proteins interfere with the repair of O(6)-MeG/T by MGMT? (3) Does MGMT show a protective effect if it is expressed after replication of DNA containing O(6)-MeG? Using an in vitro assay we show that oligonucleotides containing O(6)-MeG/T mismatches are as efficient as oligonucleotides containing O(6)-MeG/C in competing for MGMT repair activity, indicating that O(6)-MeG mispaired with thymine is still subject to repair by MGMT. The addition of MMR proteins from nuclear extracts, or of recombinant MutSalpha, to the in vitro repair assay did not affect the repair of O(6)-MeG/T lesions by MGMT. This indicates that the presence of MutSalpha still allows access of MGMT to O(6)-MeG/T lesions. To elucidate the protective effect of MGMT in the first and second replication cycle after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment, MGMT transfected CHO cells were synchronized and MGMT was inactivated by pulse-treatment with O(6)-benzylguanine (O(6)-BG). Thereafter, the recovered cells were treated with MNNG and subjected to clonogenic survival assays. Cells which expressed MGMT in the first and second cell cycle were more resistant than cells which expressed MGMT only in the second (post-treatment) cell cycle. Cells which did not express MGMT in both cell cycles were most sensitive. This indicates that repair of O(6)-MeG can occur both in the first and second cell cycle after alkylation protecting cells from the killing effect of the lesion.

Animals↗
PubMed2001

BER, MGMT, and MMR in defense against alkylation-induced genotoxicity and apoptosis.

Methylating carcinogens and cytostatic drugs induce different methylation products in DNA. In cells not expressing the repair protein MGMT or expressing it at a low level, O6-methylguanine is the major genotoxic, recombinogenic, and apoptotic lesion. Genotoxicity and apoptosis triggered by O6-methylguanine require mismatch repair (MMR). In cells expressing O6-methylguanine-DNA methyl transferase (MGMT) at a high level or for agents producing low amounts of O6-methylguanine, N-alkylations become the major genotoxic lesions. N-Alkylations are repaired by base excision repair (BER). In mammalian cells, naturally occurring mutants of BER have not been detected, which points to the importance of BER for viability. In order to ascertain the role of BER in cellular defense, BER was modulated either by transfection or mutational inactivation. It has been shown that overexpression of N-methylpurine-DNA glycosylase (MPG) does not protect, but rather sensitizes cells to SN2 agents. This has been interpreted in terms of an imbalance in BER. Regarding abasic site endonuclease (APE), transient but not stable overexpression of the enzyme was achieved upon transfection in CHO cells, which indicates that unphysiologic APE levels are not tolerated by the cell. Besides the repair function, APE (alias Ref-1) exerts redox capability by which the activity of various transcription factors is modulated. Therefore, it is possible that stable overexpression of mammalian APE impairs transcriptional regulation of genes, whereas transient overexpression may exert some protective effect. DNA polymerase beta (Pol beta) transfection was ineffective in conferring resistance to methylmethane sulfonate (MMS). On the other hand, Pol beta-deficient cells proved to be highly sensitive to methylation-induced chromosomal aberrations and reproductive cell death. The dramatic hypersensitivity in the killing response is largely due to induction of apoptosis. Obviously, nonrepaired BER intermediates are clastogenic and act as a strong trigger of the apoptotic pathway. The elements of this pathway are currently under investigation.

Alkylation↗
PubMed2001

DNA double-strand breaks trigger apoptosis in p53-deficient fibroblasts.

DNA double-strand breaks (DSBs) are induced by ionizing radiation (IR) and various radiomimetic agents directly, or indirectly as a consequence of DNA repair, recombination and replication of damaged DNA. They are ultimately involved in the generation of chromosomal aberrations and were reported to cause genomic instability, gene amplification and reproductive cell death. To address the question of whether DSBs act as a trigger of apoptosis, we induced DSBs by means of restriction enzyme electroporation and compared the effect with IR in mouse fibroblasts that differ in p53 status [wild-type (+/+) versus p53-deficient (-/-) cells]. We show that (i) electroporation of PVU:II is highly efficient in the induction of DSBs, (ii) electroporation of PVU:II increases the rate of apoptosis, but not of necrosis in p53-/- cells, (iii) treatment with gamma-rays induces both apoptosis and necrosis in p53-/- cells, (iv) the frequency of DSBs correlates with the yield of apoptosis and (v) both PVU:II and gamma-ray treatment reduce the level of anti-apoptotic Bcl-2 protein in p53-/- cells whereas the level of Bax remains unaltered. Cells expressing wild-type p53 were more resistant than p53-deficient cells as to the induction of apoptosis and did not show Bcl-2 decline upon treatment with PVU:II and gamma-rays. The data provide evidence that blunt-ended DSBs induced by restriction enzyme PVU:II act as a highly efficient trigger of apoptosis, but not of necrosis. This process is related to Bcl-2 decline and does not require p53.

Animals↗
PubMed2000

Development of membrane extraction with a sorbent interface-micro gas chromatography system for field analysis.

The commercially available portable gas chromatographs have a rather limited scope of applications, typically allowing analysis of gaseous samples only, and having relatively poor sensitivity. Combination of those instruments with modern sampling/sample preparation techniques can remedy these problems. A Chrompack micro-GC system equipped with a thermal conductivity detector has been coupled to membrane extraction with a sorbent interface (MESI). The sorbent trap has replaced the GC injector. The design of the trap was also modified in order to enhance the preconcentration of analytes. The use of a thin flat sheet membrane reduces the response time, and decreases the memory effect of the system. Rapid separation times were achieved, and the sensitivity was significantly improved. MESI enables semi-continuous monitoring of both gaseous and aqueous samples, owing to the selectivity of the membrane material. The system does not use moving parts, therefore being reliable. The sensitivity of the micro-GC system was increased by a factor of more than 100 by the addition of the MESI system, even with a preconcentration time as short as 1 min. Chloroform, having a concentration lower than 1 ppb, was detected in tap water. A cup system was used to allow headspace sampling of volatile organic compounds from aqueous matrices, keeping the membrane away from interfering species that could be present in water, and improving the mass transfer. A linear calibration line was obtained, and the estimated limit of detection was 60 ppt. This represents a great improvement for the sensitivity of the micro-GC system.

Chromatography, Gas↗
PubMed2000

Effect of ischemic pretreatment on heat shock protein 72, neurologic outcome, and histopathologic outcome in a rabbit model of spinal cord ischemia.

OBJECTIVE: In the present study, we investigated the effect of ischemic pretreatment on heat shock protein 72 concentration and neurologic and histopathologic outcome after transient spinal cord ischemia. METHODS: In 28 New Zealand White rabbits, an aortic occlusion device was placed infrarenally. The animals were randomly assigned to 2 groups: ischemic pretreatment (n = 14 animals) and control (n = 14 animals). The duration of ischemic pretreatment was 6 minutes. After 24 hours, the aorta was occluded for 26 minutes in both groups of animals. Neurologic function was assessed 24 and 48 hours after the definite ischemic insult. At 48 hours, the animals were killed for histopathologic evaluation of the spinal cord. In a separate set of animals, heat shock protein 72 levels were determined in the lumbar spinal cord after both a 6- and 10-minute ischemic period, with the use of a Western blot analysis. RESULTS: No significant difference in neurologic outcome between the groups was observed at 24 and 48 hours. The incidence of paraplegia and severe paresis at 48 hours was 79% in the control group and 92% in the ischemic pretreatment group. There was no difference in histopathologic scores between the groups. Heat shock protein 72 could be clearly detected 1 and 2 days after 6- or 10-minute periods of spinal cord ischemia. CONCLUSIONS: In the present rabbit study, ischemic pretreatment could not induce tolerance against a moderately severe spinal cord ischemic insult, despite increased heat shock protein 72 levels after the preconditioning stimulus.

Animals↗
PubMed2000

Neuroprotective effects of riluzole and ketamine during transient spinal cord ischemia in the rabbit.

BACKGROUND: Massive release of central excitatory neurotransmitters is an important initial step in ischemic neuronal injury, and modification of this process may provide neuroprotection. We studied the protective effects of the voltage-dependent sodium channel antagonist riluzole and the N-methyl-d-aspartate receptor antagonist ketamine on hind limb motor function and histopathologic outcome in an experimental model of spinal cord ischemia. METHODS: Temporary spinal cord ischemia was induced by 29 min of infrarenal balloon occlusion of the aorta in 60 anesthetized New Zealand white rabbits. Animals were randomly assigned to one of four treatment groups (n = 15 each): group C, saline (control); group R, riluzole, 8 mg/kg intravenously; group K, ketamine, 55 mg/kg intravenously; group RK, riluzole and ketamine. After reperfusion, riluzole treatment was continued with intraperitoneal infusions. Normothermia (38 degrees C) was maintained during ischemia, and rectal temperature was assessed before and after intraperitoneal infusions. Neurologic function, according to Tarlov's criteria, was evaluated every 24 h, and infarction volume and the number of eosinophilic neurons and viable motoneurons in the lumbosacral spinal cord was evaluated after 72 h. RESULTS: Neurologic outcome was better in groups R and RK than in groups C and K. All animals in group C (100%) and all animals but one in group K (93%) were paraplegic 72 h after the ischemic insult versus 53% in group R and 67% in group RK (P < 0.01 each). More viable motoneurons were present in groups R and RK than in controls (P < 0.05). CONCLUSIONS: The data indicate that treatment with riluzole can increase the tolerance of spinal cord motoneurons to a period of normothermic ischemia. Intraischemic ketamine did not provide neuroprotection in this model.

Animals↗
PubMed2000

Effects of nucleus prepositus hypoglossi lesions on visual climbing fiber activity in the rabbit flocculus.

The caudal dorsal cap (dc) of the inferior olive is involved in the control of horizontal compensatory eye movements. It provides those climbing fibers to the vestibulocerebellum that modulate optimally to optokinetic stimulation about the vertical axis. This modulation is mediated at least in part via an excitatory input to the caudal dc from the pretectal nucleus of the optic tract and the dorsal terminal nucleus of the accessory optic system. In addition, the caudal dc receives a substantial GABAergic input from the nucleus prepositus hypoglossi (NPH). To investigate the possible contribution of this bilateral inhibitory projection to the visual responsiveness of caudal dc neurons, we recorded the climbing fiber activity (i.e., complex spikes) of vertical axis Purkinje cells in the flocculus of anesthetized rabbits before and after ablative lesions of the NPH. When the NPH ipsilateral to the recorded flocculus was lesioned, the spontaneous complex spike firing frequency did not change significantly; but when both NPHs were lesioned, the spontaneous complex spike firing frequency increased significantly. When only the contralateral NPH was lesioned, the spontaneous complex spike firing frequency decreased significantly. Neither unilateral nor bilateral lesions had a significant influence on the depth of complex spike modulation during constant velocity optokinetic stimulation or on the transient continuation of complex spike modulation that occurred when the constant velocity optokinetic stimulation stopped. The effects of the lesions on the spontaneous complex spike firing frequency could not be explained when only the projections from the NPH to the inferior olive were considered. Therefore we investigated at the electron microscopic level the nature of the commissural connection between the two NPHs. The terminals of this projection were found to be predominantly GABAergic and to terminate in part on GABAergic neurons. When this inhibitory commissural connection is taken into consideration, then the effects of NPH lesions on the spontaneous firing frequency of floccular complex spikes are qualitatively explicable in terms of relative weighting of the commissural and caudal dc projections of the NPH. In summary, we conclude that in the anesthetized rabbit the inhibitory projection of the NPH to the caudal dc influences the spontaneous firing frequency of floccular complex spikes but not their modulation by optokinetic stimulation.

Action Potentials↗
PubMed1999

Development of spinal cord ischemia after clamping of noncritical segmental arteries in the pig.

BACKGROUND: Blood flow to the thoracolumbar spinal cord is thought to be critically dependent on the arteria radicularis magna. We investigated whether spinal cord blood supply becomes dependent on other, noncritical, segmental arteries if spinal cord perfusion pressure (SCPP) is decreased. The SCPP is equal to the mean arterial pressure (MAP) minus the cerebrospinal fluid (CSF) pressure (SCCP = MAP - CSF). METHODS: The thoracoabdominal aorta was exposed in 10 pigs. Functional integrity of spinal cord motor pathways was assessed with myogenic motor-evoked potentials after transcranial electrical stimulation (tc-MEPs). Using this technique, a group of segmental arteries not critical for spinal cord blood supply was identified. Before, during, and after clamping of the noncritical segmental arteries, spinal cord ischemia was produced by decreasing SCPP by means of increasing CSF pressure, and the SCPP threshold at which tc-MEPs showed evidence of spinal cord ischemia was determined. Ischemic SCPP thresholds, obtained during and after clamping of the noncritical segmental arteries, were compared with the ischemic threshold obtained before clamping (control value). RESULTS: Before noncritical segmental arteries were clamped, ischemic tc-MEP changes occurred when the SCPP was below 15 +/- 5 (SD) mm Hg. With a total of 9 +/- 3 (SD) segmental arteries clamped, the ischemic SCPP threshold was 48 +/- 14 mm Hg (p < 0.01). After the release of all clamps, ischemia occurred at a SCPP of 15 +/- 5 (SD) mm Hg. CONCLUSIONS: In this porcine experiment, clamping of originally noncritical segmental arteries significantly reduced the tolerance of the spinal cord to a decrease in SCPP.

Animals↗
PubMed1999

The influence of regional spinal cord hypothermia on transcranial myogenic motor-evoked potential monitoring and the efficacy of spinal cord ischemia detection.

OBJECTIVE: Myogenic motor-evoked responses to transcranial electrical stimulation (transcranial myogenic motor-evoked potentials) can rapidly detect spinal cord ischemia during thoracoabdominal aortic aneurysm repair. Recent evidence suggests that regional spinal cord hypothermia increases spinal cord ischemia tolerance. We investigated the influence of subdural infusion cooling on transcranial myogenic motor-evoked potential characteristics and the time to detect spinal cord ischemia in 6 pigs. METHODS: Regional hypothermia was produced by subdural perfusion cooling. A laminectomy and incision of the dura were performed at L2 to advance 2 inflow catheters at L4 and L6, to cool the lumbar subdural space with saline solution. Two temperature probes were advanced at L3 and L5, and 1 cerebrospinal fluid pressure line was advanced at L4. Spontaneous cerebrospinal fluid outflow was allowed. Spinal cord ischemia was produced by clamping a set of critical lumbar arteries, previously identified by transcranial myogenic motor-evoked potentials and lumbar artery clamping. The time between the onset of ischemia and detection with transcranial myogenic motor-evoked potentials (amplitude < 25%) was determined at cerebrospinal fluid temperatures of 37 degrees C and 28 degrees C. Thereafter, the influence of progressive cerebrospinal fluid cooling on transcranial myogenic motor-evoked potential amplitude and latency was determined. RESULTS: The time necessary to produce ischemic transcranial myogenic motor-evoked potentials, after the clamping of critical lumbar arteries, was not affected at moderate subdural hypothermia (3.8 +/- 0.9 min) compared with subdural normothermia (3.2 +/- 0.5 min; P =.6). Thereafter, progressive cooling resulted in a transcranial myogenic motor-evoked potential amplitude increase at 28 degrees C to 30 degrees C and was followed by a progressive decrease. Response amplitudes decreased below 25% at 14.0 degrees C +/- 1.1 degrees C. The influence of cerebrospinal fluid temperature on transcranial myogenic motor-evoked potential amplitude was best represented by a quadratic regression curve with a maximum at 29.6 degrees C. In contrast, transcranial myogenic motor-evoked potential latencies increased linearly with decreasing subdural temperatures. CONCLUSIONS: Detection of spinal cord ischemia with transcranial myogenic motor-evoked potentials is not delayed at moderate subdural hypothermia in pigs. At a cerebrospinal fluid temperature of 28 degrees C, transcranial myogenic motor-evoked potential amplitudes are increased. Further cerebrospinal fluid temperature decreases result in progressive amplitude decreases and latency increases.

Animals↗
PubMed1999

Spatiotemporal distribution of dying neurons during early mouse development.

Apoptosis is a critical cellular event during several stages of neuronal development. Recently, we have shown that biotinylated annexin V detects apoptosis in vivo in various cell lineages of a wide range of species by binding to phosphatidylserines that are exposed at the outer leaflet of the plasma membrane. In the present study, we tested the specificity by which annexin V binds apoptotic neurons, and subsequently investigated developmental cell death in the central and peripheral nervous system of early mouse embryos at both the cellular and histological level, and compared the phagocytic clearance of apoptotic neurons with that of apoptotic mesodermal cells. Our data indicate: (i) that biotinylated annexin V can be used as a sensitive marker that detects apoptotic neurons, including their extensions at an early stage during development; (ii) that apoptosis plays an important part during early morphogenesis of the central nervous system, and during early quantitative matching of brain-derived neurotrophic factor and neurotrophic factor 3 responsive postmitotic large clear neurons in the peripheral ganglia with their projection areas; and (iii) that apoptotic neurons are removed by a process that differs from classical phagocytosis of non-neuronal tissues.

Animals↗
PubMed1997

Novel preconcentration technique for on-line coupling to high-speed narrow-bore capillary gas chromatography: sample enrichment by equilibrium (ab)sorption. II. Coupling to a portable micro gas chromatograph.

The technique of equilibrium (ab)sorption has been proven to be a powerful method for preconcentration of gaseous samples for high-speed narrow-bore capillary gas chromatography (GC) in general and field-portable GC instruments, often referred as micro GCs, in particular. Using a simple experimental set-up equipped with an open-tubular enrichment column it is possible to produce a homogeneously enriched sample plug, allowing reproducible injections of an enriched sample into the micro GC. Using a non-polar trapping column enrichment factors found for n-alkanes in the range of C7 to C10 ranged from 15 to 150 and agree well with calculated values. Using a highly retentive Thermocap column, the enrichment factor observed for heptane was above 500. As the use of this new preconcentration method requires only minimum modification of the micro GC, the chromatographic performance of the instrument was not compromised by direct coupling to the preconcentration device. Examples of on-line enrichment with portable micro GC analysis of VOCs from air are shown. These examples clearly demonstrate the potentials of the new method in field analysis.

Absorption↗