Biomedical subjects

M S Campbell

Publications and source records attributed to M S Campbell.

At least 19 recordsLinked to original sources

PubMed2006

Intra-arterial reteplase compared to urokinase for thrombolytic recanalization in acute ischemic stroke.

BACKGROUND AND PURPOSE: Reteplase (RP) and urokinase (UK) are being used "off-label" to treat acute ischemic stroke. The safety and efficacy of intra-arterial RP or UK in the treatment of acute ischemic stroke, however, has yet to be proved. We aim to evaluate the safety and efficacy of RP compared with UK in acute ischemic stroke patients with large vessel occlusion. METHODS: Retrospective analysis was conducted of cases from a prospectively collected stroke data base on consecutive acute ischemic stroke patients with large vessel occlusion by digital subtraction angiography treated with intra-arterial RP or UK. Thrombolytic dosage, recanalization rate, intracerebral hemorrhage (ICH), mortality, and outcome were determined. RESULTS: Thirty-three patients received RP and 22 received UK (mean doses, 2.5 +/- 1.4 mg and 690,000 +/- 562,000 U, respectively). Vascular occlusions included 9 basilar arteries (BAs), 7 internal carotid arteries (ICAs), and 17 middle cerebral arteries (MCAs) with RP and 9 BAs, 4 ICAs, and 9 MCAs with UK. Median baseline National Institutes of Health Stroke Scales were as follows: 16 (range, 5-25; 81% > or = 10) with RP and 17 (range, 6-38; 85% > or =10) with UK. Mean time from symptom onset to thrombolytic initiation: 333 +/- 230 minutes with RP and 343 +/- 169 minutes with UK. Recanalization rates were as follows: 82% with RP and 64% with UK (P = .13). Symptomatic ICH rates were as follows: 12% with RP and 4.5% with UK (P = .50). The mortality rate was 24% with RP and 27% with UK (P = .8). CONCLUSION: Although limited in statistical power, our study suggests that, although IA thrombolysis with RP shows a trend for higher recanalization rates and hemorrhage rates, IA thrombolysis with RP is not significantly different in recanalization, outcome, mortality, and ICH compared with that of UK or rates reported with IA pro-UK.

Acute Disease↗

PubMed2004

Postpartum cerebral angiopathy: atypical features and treatment with intracranial balloon angioplasty.

Postpartum cerebral angiopathy (PCA) is an uncommon cause of ischemic and hemorrhagic stroke in young women. It is usually clinically benign and not relapsing. We describe a patient with nonhemorrhagic PCA who had an atypical progressive neurological deficit from bilateral hemisphere watershed ischemia despite treatment with aggressive medical therapy and intracranial balloon angioplasty.

Adult↗

PubMed2004

Review article: the evolving role of liver biopsy.

Liver biopsy is traditionally the 'gold standard' for the evaluation of liver diseases. There are several situations in which its role is being challenged. In hepatitis C, liver biopsy helps assess prognosis and treatment candidacy. An important exception is genotype 2 or 3 because treatment is more likely to succeed and therapy is relatively short in duration. For hepatitis B, liver biopsy gives some prognostic information, but serologic tests and hepatic biochemical tests are the primary determinants of treatment candidacy. Non-alcoholic fatty liver disease can be accurately diagnosed without a liver biopsy and, furthermore, there are no specific therapies available. The role of liver biopsy to assess methotrexate-associated hepatotoxicity remains controversial. Finally, patients with focal liver lesions usually do not require biopsy and, in the case of hepatocellular carcinoma, biopsy carries a risk of needle-track seeding. In short, the need for liver biopsy depends on the specific situation and should be performed when there is sufficient uncertainty about diagnosis, severity of disease, prognosis, and treatment decisions.

Biopsy↗

PubMed2001

Mitotic checkpoint proteins HsMAD1 and HsMAD2 are associated with nuclear pore complexes in interphase.

Mad1 was first identified in budding yeast as an essential component of the checkpoint system that monitors spindle assembly in mitosis and prevents premature anaphase onset. Using antibodies to the human homologue of Mad1 (HsMAD1), we have begun to characterize this protein in mammalian cells. HsMad1 is found localized at kinetochores in mitosis. The labeling is brightest in prometaphase and is absent from kinetochores at metaphase and anaphase. In cells where most chromosomes have reached the metaphase plate, those aligned at the plate show no labeling while remaining, unaligned chromosomes are still brightly labeled. We find HsMad1 associated with HsMad2. Association with p55CDC, a protein previously shown to bind HsMad2, was not detected. Surprisingly, unlike any other known mitotic checkpoint proteins, HsMad1 and HsMAD2 were found localized at nuclear pores throughout interphase. This was confirmed by co-labeling with an antibody to known nuclear pore complex proteins and by their co-purification with enriched nuclear envelope fractions. HsMad1 was identified serendipitously by its binding to a viral protein, HTLV-1 Tax, which affects transcription of viral and human proteins. The localization of HsMad1 to nuclear pore complexes suggests an alternate, non-mitotic role for the Mad1/Tax interaction in the viral transformation of cells.

Amino Acid Sequence↗

PubMed2000

Infectious cDNA clones of Langat tick-borne flavivirus that differ from their parent in peripheral neurovirulence.

Tick-borne flavivirus strain Langat TP21 (LGT TP21) recovered from ticks, is naturally attenuated for humans but retains demonstrable neurovirulence and peripheral virulence ("neuroinvasiveness") for mice. Previously a mutant, strain E5, less virulent for mice was derived from LGT TP21. Multiple attempts to prepare a full-length infectious TP21 cDNA from cDNA fragments cloned in E. coli were uniformly unsuccessful. A more informative sequence than that obtained from these cloned cDNA fragments and similar E5 cDNA fragments was derived from RT-PCR fragments that had not been cloned in E. coli. Comparison of the RT-PCR consensus sequence of TP21 and E5 identified only seven amino acid differences that might be responsible for the observed difference in virulence of these strains for mice. Eleven independent infectious cDNA clones of TP21 were recovered using two overlapping long RT-PCR fragments. Importantly, low-titered virus used to prepare cDNA as template for PCR was harvested early in the growth cycle to minimize the frequency of deletion mutants that accumulated late in infection. The four analyzed rescued clones exhibited clone-specific minimal divergence from the consensus sequence but this limited variation was associated with diminished peripheral virulence for immunocompetent mice. Manipulation of these clones should facilitate elucidation of LGT virulence.

Amino Acid Substitution↗

PubMed2000

Kinetochore "memory" of spindle checkpoint signaling in lysed mitotic cells.

The spindle checkpoint prevents errors in mitosis. Cells respond to the presence of kinetochores that are improperly attached to the mitotic spindle by delaying anaphase onset. Evidence suggests that phosphorylations recognized by the 3F3/2 anti-phosphoepitope antibody may be involved in the kinetochore signaling of the spindle checkpoint. Mitotic cells lysed in detergent in the absence of phosphatase inhibitors rapidly lose expression of the 3F3/2 phosphoepitope. However, when ATP is added to lysed and rinsed mitotic cytoskeletons, kinetochores become rephosphorylated by an endogenous, bound kinase. Kinetochore rephosphorylation in vitro produced the same differential phosphorylation seen in appropriately fixed living cells. In chromosomes not yet aligned at the metaphase plate, kinetochores undergo rapid rephosphorylation, while those of fully congressed chromosomes are under-phosphorylated. However, latent 3F3/2 kinase activity is retained at kinetochores of cells at all stages of mitosis including anaphase. This latent activity is revealed when rephosphorylation reactions are carried out for extended times. The endogenous, kinetochore-bound kinase can be chemically inactivated. Remarkably, a soluble kinase activity extracted from mitotic cells also caused differential rephosphorylation of kinetochores whose endogenous kinase had been chemically inactivated. We suggest that, in vivo, microtubule attachment alters the kinetochore 3F3/2 phosphoprotein, causing it to resist phosphorylation. This kinetochore modification is retained after cell lysis, producing a "memory" of the in vivo phosphorylation state.

Animals↗

PubMed2000

Emergency endovascular treatment of cerebral sinus thrombosis with a rheolytic catheter device.

Severe thrombosis of the superior sagittal, transverse, and straight sinuses developed in a 53-year-old woman. This resulted in extensive multifocal hemorrhagic venous infarction and severe intracranial hypertension refractory to intensive management. Endovascular therapy using a rheolytic catheter device in combination with a small amount of fibrinolytic agent led to rapid normalization of the intracranial pressure, allowing optimization of the cerebral perfusion pressures and was followed by steady, albeit protracted, clinical improvement. The patient not only survived but also left the hospital with minimal neurologic deficit. The rheolytic catheter endovascular treatment is, in the opinion of the authors, the treatment of choice for patients with life-threatening cerebral sinus thrombosis.

Angioplasty, Balloon↗

PubMed2000

Elective stenting of symptomatic basilar artery stenosis.

BACKGROUND AND PURPOSE: Percutaneous angioplasty of the intracranial arteries still carries the risk of dissection, with acute closure and embolization. Stenting has been shown to improve the safety and durability of angioplasty in every circulatory bed in which it has been applied. However, stenting of the intracranial arteries has been limited by the availability of stents that can be reliably deployed intracranially. METHODS: Twelve patients underwent elective stenting of the basilar artery after episodes of vertebrobasilar ischemia. In all patients, either medical therapy had failed or the patient had a contraindication for long-term anticoagulation. Information from independent neurological examinations, quantitative angiography, and clinical follow-up was collected. Differences between pretreatment and posttreatment degree of stenoses were subjected to 1-way ANOVA for repeated measures. RESULTS: There were 10 men and 2 women, all white, aged 40 to 82 years (mean age, 62.6 years). Stent placement was successful in all patients, leading to statistically significant changes in the degree of stenosis, from 71. 4% (range, 53% to 90%) to 10.3% (range, 0% to 36%) (P<0.0001). There were no deaths, stent thromboses, perforations, ruptures, or myocardial infarctions. Clinical follow-up was available for 0.5 to 16 months (mean, 5.9 months). One patient had nonspecific symptoms, and another had a transient ischemic attack. All other patients remained asymptomatic. CONCLUSIONS: Elective stenting of the basilar artery is feasible, with minimal risk to the patient. Its impact on long-term stroke prevention and its durability are unknown and will require further study.

Adult↗

PubMed2000

Elective stenting of symptomatic middle cerebral artery stenosis.

Percutaneous balloon angioplasty has been found to be useful for the treatment of intracranial atherosclerotic arterial stenosis. Nonetheless, an ongoing risk of this procedure is arterial dissection, which increases the hazards of acute closure, stroke, and restenosis. Stenting of the intracranial vasculature recently has been shown to be feasible in a variety of circumstances. To our knowledge, however, stenting of the middle cerebral artery has not been possible until now primarily because of difficulty with tracking stents across the carotid siphon. We describe the successful treatment of a symptomatic middle cerebral artery stenosis achieved using a balloon-expandable flexible coronary stent.

Angioplasty, Balloon↗

PubMed1999

HRad17 colocalizes with NHP2L1 in the nucleolus and redistributes after UV irradiation.

The rad17 gene of Schizosaccharomyces pombe plays an important role as a checkpoint protein following DNA damage and during DNA replication. The human homologue of S. pombe rad17, Hrad17, was recently identified, but its function has not yet been established. Using the yeast two-hybrid system, we determined that HRad17 can interact with a nucleolar protein, NHP2L1. This interaction was also demonstrated biochemically, in human cells. Immunofluorescence studies revealed that HRad17 and NHP2L1 colocalize to the nucleolus, and immunogold labeling further resolved the location of NHP2L1 to the dense fibrillar component of the nucleolus. Interestingly, the localization of HRad17 in the nucleolus was altered in response to UV irradiation. These results provide some insight into the DNA damage and replication checkpoint mechanisms of HRad17.

Amino Acid Sequence↗

PubMed1999

HRad17, a human homologue of the Schizosaccharomyces pombe checkpoint gene rad17, is overexpressed in colon carcinoma.

Using the palindromic PCR-cDNA display method, we have cloned a novel gene overexpressed by human colon carcinoma relative to normal colon. Among normal tissues examined, only testis expresses it at a high level. Sequence analysis revealed its extensive homology with checkpoint genes rad17 of Schizosaccharomyces pombe and RAD24 of Saccharomyces cerevisiae. This novel gene designated as hRad17 is localized to chromosome 5q12,13.1, a region known to be deleted in a variety of human cancers. Promoter region and one pseudogene of hRad17 have been identified. Whereas the increased expression of hRad17 by human colon carcinomas may be related to the known resistance of these cells to DNA-damaging agents during therapy, the deletion of hRad17 in a variety of cancers may predispose them to increased rate of mutation and heightened sensitivity to DNA-damaging agents, including radiation and anticancer drugs.

Animals↗

PubMed1999

Extracranial vertebral artery stent placement: in-hospital and follow-up results.

OBJECT: The authors summarize their experience with stent deployment in the treatment of vertebrobasilar artery (VBA) insufficiency. This is an underdiagnosed condition, and the incidence of significant vertebral artery (VA) stenosis has been underappreciated. Medical therapy has been the mainstay of treatment because of the high rate of morbidity associated with surgical correction of VA stenosis. Recently, some authors have reported acceptable results with the use of percutaneous transluminal angioplasty, but this technique has significant weaknesses such as elastic recoil and problems in achieving safe treatment of dissections. METHODS: The authors investigated the feasibility, safety, and outcome of VA stent placement in 50 patients in whom 55 vessels were treated using stents. Technical success was achieved in 54 (98%) of 55 vessels, with no procedure-related complications. However, one patient (2%) died of nonneurological causes, and one (2%) suffered a stroke that occurred within the 30-day postprocedural period and was related to a complicated coronary intervention. Clinical follow-up review performed at a mean of 25 +/- 10 months revealed two patients (4%) with recurrence of VBA symptoms. Six-month angiographic follow up was completed in 90% of eligible patients, with a 10% incidence of restenosis as defined by greater than 50% luminal narrowing. CONCLUSIONS: Vertebral artery stent placement is feasible in patients who have significant VA stenosis, with predictably good angiographically demonstrated and clinical results. The 6-month angiographically confirmed restenosis rate appears to be low, as does the clinical recurrence rate. This technique solves the problems of elastic recoil and the treatment of angioplasty-induced dissections. Further prospective comparison with medical preventive strategies is warranted.

Adult↗

PubMed1998

Tension-sensitive kinetochore phosphorylation in vitro.

Many cells have a checkpoint that detects a single misattached chromosome and delays anaphase, allowing time for error correction. Detection probably depends on tension-sensitive kinetochore protein phosphorylation. Somehow, mechanical tension, or some consequence of tension, produces a chemical change, dephosphorylation. The mechanism of tension-mediated dephosphorylation can be approached using an in vitro system. Earlier work showed that the kinetochores of washed chromosomes from a mammalian cell line can be phosphorylated in vitro simply by incubation with ATP and a phosphatase inhibitor. We confirm this for chromosomes from insect meiotic cells. Thus, kinetochores of washed chromosomes from diverse sources contain a complete phosphorylation system: a kinase, a phosphatase and the substrate protein(s). We show that phosphorylation in vitro is sensitive to tension, as it is in living cells. This makes the conditions required for phosphorylation in vitro relevant to the process in living cells. The phosphatase is ruled out as the tension-sensitive component in vitro, leaving either the kinase or the substrate as the sensitive component. We show that a kinase extracted from mammalian cells in mitosis phosphorylates the kinetochores of insect meiotic chromosomes very effectively. The mammalian kinase under-phosphorylates the kinetochore of the insect's X-chromosome, just as the native insect kinase does. This provides a clue to the evolution of a chromosome that is not detected by the checkpoint. The mammalian kinase is not tightly bound to the chromosome and thus functions primarily in solution. This suggests that the substrate's phosphorylatable groups are freely available to outside constituents, e.g. regulators, as well as to the kinetochore's own kinase and phosphatase.

Adenosine Triphosphate↗

PubMed1997

MPM-2 antibody-reactive phosphorylations can be created in detergent-extracted cells by kinetochore-bound and soluble kinases.

The MPM-2 antibody labels mitosis-specific and cell cycle-regulated phosphoproteins. The major phosphoproteins of mitotic chromosomes recognized by the MPM-2 antibody are DNA topoisomerase II (topoII) alpha and beta. In immunofluorescence studies of PtK1 cytoskeletons, prepared by detergent lysis in the presence of potent phosphatase inhibitors, the MPM-2 antibody labels phosphoproteins found at kinetochores, chromosome arms, midbody and spindle poles of mitotic cells. In cells extracted without phosphatase inhibitors, labeling of the MPM-2 antibodies at kinetochores is greatly diminished. However, in cytoskeletons this epitope can be regenerated through the action of kinases stably bound at the kinetochore. Various kinase inhibitors were tested in order to characterize the endogenous kinase responsible for these phosphorylations. We found that the MPM-2 epitope will not rephosphorylate in the presence of the broad specificity kinase inhibitors K-252a, staurosporine and 2-aminopurine. Several other inhibitors had no effect on the rephosphorylation indicating that the endogenous MPM-2 kinase at kinetochores is not p34cdc2, casein kinase II, MAP kinase, protein kinase A or protein kinase C. The addition of N-ethylmaleimide inactivated the endogenous kinetochore kinase; this allowed testing of several purified kinases in the kinetochore rephosphorylation assay. Active p34cdc2-cyclin B, casein kinase II and MAP kinase could not generate the MPM-2 phosphoepitope. However, bacterially expressed NIMA from Aspergillus and ultracentrifuged mitotic HeLa cell extract were able to catalyze the rephosphorylation of the MPM-2 epitope at kinetochores. Furthermore, fractionation of mitotic HeLa cell extract showed that kinases that create the MPM-2 epitope at kinetochores and chromosome arms are distinct. Our results suggest that multiple kinases (either soluble or kinetochore-bound), including a homolog of mammalian NIMA, can create the MPM-2 phosphoepitope. The kinetochore-bound kinase that catalyzes the formation of the MPM-2 phosphoepitope may play an important role in key events such as mitotic kinetochore assembly and sister chromatid separation at anaphase.

Adenosine Triphosphate↗

PubMed1995

Stability of nuclear segments in human neutrophils and evidence against a role for microfilaments or microtubules in their genesis during differentiation of HL60 myelocytes.

The nucleus of the mature human neutrophil is segmented into three to five interconnected lobes. The physiological purpose of this segmentation is unknown, as is the mechanism by which the lobes are formed during differentiation. Using video observation of migrating human neutrophils simultaneously illuminated for fluorescence and phase-contrast microscopy, we analyzed nuclear movements with respect to cell shape changes. The number of nuclear lobes and their relative size remained constant during observation (up to 1 h). The thin connecting segments between the lobes elongated and attenuated extensively but never separated. Electron microscopic analysis of neutrophil nuclei revealed no specialized nuclear or cytoplasmic structures in the vicinity of connecting segments. With fluorescence in situ hybridization of whole chromosome probes, we determined that chromosomes are randomly distributed among neutrophil nuclear lobes. HL60 cells are a human myelocytic line that, with retinoic acid treatment, segment their nuclei and differentiate into neutrophil-like cells over several days. Using a rapidly responding variant line termed HL60/S4 (Cancer Res. 52, 949-954), we found that segmentation could be induced within 24 h. We tested the role of cytoskeletal elements in the process of nuclear segmentation. Neither the microtubule inhibitor nocodazole nor the microfilament inhibitor cytochalasin D prevented nuclear segmentation. Together, our studies suggest that nuclear lobes in neutrophils are relatively stable structures that are not generated by microtubule- or microfilament-dependent forces.

Actin Cytoskeleton↗

PubMed1995

Cell-cycle-regulated localization of tyrosine and threonine phosphoepitopes at the kinetochores of mitotic chromosomes.

We have detected novel phosphotyrosine epitopes at the kinetochores of mitotic chromosomes in rat kangaroo PtK1 and mouse P388D1 tissue culture cells. Immunofluorescence labeling of detergent-resistant cytoskeletons reveals that these phosphotyrosine epitopes are tightly bound at the centrosomes and kinetochores of mitotic cells. These phosphoepitopes are found at the kinetochores during only prophase and prometaphase. Inclusion of a mixture of phosphatase inhibitors in the cell extraction procedure was necessary to preserve these previously undetected phosphotyrosine epitopes. The use of the phosphatase inhibitor mixture also improved the detection of the centrosome and kinetochore antigens recognized by the monoclonal antibody MPM-2. The MPM-2 antibody labels a subset of phosphothreonine-containing antigens found primarily during M phase. Ultrastructural immunolabeling studies indicated that both the phosphotyrosine and the MPM-2 phosphoepitopes were contained in both the outer and the inner dense plaques of the kinetochore. We developed large-scale chromosome isolation procedures designed to maintain chromosome protein phosphorylation. Immunoblot analysis revealed that the phosphotyrosine and MPM-2 antibodies recognized a number of chromosomal proteins, some of which were concentrated in the chromosome scaffold fraction prepared by nuclease digestion and salt extraction of whole chromosomes. The strictly regulated appearance of the phosphotyrosine and MPM-2 epitopes at the kinetochores of chromosomes during various stages of mitosis suggests that these phosphoepitopes may be involved in signal transduction pathways controlling kinetochore assembly and function during mitosis.

Animals↗

PubMed1995

Microinjection of mitotic cells with the 3F3/2 anti-phosphoepitope antibody delays the onset of anaphase.

The transition from metaphase to anaphase is regulated by a checkpoint system that prevents chromosome segregation in anaphase until all the chromosomes have aligned at the metaphase plate. We provide evidence indicating that a kinetochore phosphoepitope plays a role in this checkpoint pathway. The 3F3/2 monoclonal antibody recognizes a kinetochore phosphoepitope in mammalian cells that is expressed on chromosomes before their congression to the metaphase plate. Once chromosomes are aligned, expression is lost and cells enter anaphase shortly thereafter. When microinjected into prophase cells, the 3F3/2 antibody caused a concentration-dependent delay in the onset of anaphase. Injected antibody inhibited the normal dephosphorylation of the 3F3/2 phosphoepitope at kinetochores. Microinjection of the antibody eliminated the asymmetric expression of the phosphoepitope normally seen on sister kinetochores of chromosomes during their movement to the metaphase plate. Chromosome movement to the metaphase plate appeared unaffected in cells injected with the antibody suggesting that asymmetric expression of the phosphoepitope on sister kinetochores is not required for chromosome congression to the metaphase plate. In antibody-injected cells, the epitope remained expressed at kinetochores throughout the prolonged metaphase, but had disappeared by the onset of anaphase. When normal cells in metaphase, lacking the epitope at kinetochores, were treated with agents that perturb microtubules, the 3F3/2 phosphoepitope quickly reappeared at kinetochores. Immunoelectron microscopy revealed that the 3F3/2 epitope is concentrated in the middle electronlucent layer of the trilaminar kinetochore structure. We propose that the 3F3/2 kinetochore phosphoepitope is involved in detecting stable kinetochore-microtubule attachment or is a signaling component of the checkpoint pathway regulating the metaphase to anaphase transition.

Anaphase↗

PubMed1986

The overexpression and complete amino acid sequence of Escherichia coli 3-dehydroquinase.

The enzyme 3-dehydroquinase was purified in milligram quantities from an overproducing strain of Escherichia coli. The amino acid sequence was deduced from the nucleotide sequence of the aroD gene and confirmed by determining the amino acid composition of the overproduced enzyme and its N-terminal amino acid sequence. The complete polypeptide chain consists of 240 amino acid residues and has a calculated subunit Mr of 26,377. Transcript mapping revealed that aroD is a typical monocistronic gene.

Amino Acid Sequence↗