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P Maddox

Publications and source records attributed to P Maddox.

At least 19 recordsLinked to original sources
PubMed2005

Roles of polymerization dynamics, opposed motors, and a tensile element in governing the length of Xenopus extract meiotic spindles.

Metaphase spindles assemble to a steady state in length by mechanisms that involve microtubule dynamics and motor proteins, but they are incompletely understood. We found that Xenopus extract spindles recapitulate the length of egg meiosis II spindles, by using mechanisms intrinsic to the spindle. To probe these mechanisms, we perturbed microtubule polymerization dynamics and opposed motor proteins and measured effects on spindle morphology and dynamics. Microtubules were stabilized by hexylene glycol and inhibition of the catastrophe factor mitotic centromere-associated kinesin (MCAK) (a kinesin 13, previously called XKCM) and destabilized by depolymerizing drugs. The opposed motors Eg5 and dynein were inhibited separately and together. Our results are consistent with important roles for polymerization dynamics in regulating spindle length, and for opposed motors in regulating the relative stability of bipolar versus monopolar organization. The response to microtubule destabilization suggests that an unidentified tensile element acts in parallel with these conventional factors, generating spindle shortening force.

Adenylyl Imidodiphosphate↗
PubMed2004

Bipolarization and poleward flux correlate during Xenopus extract spindle assembly.

We investigated the mechanism by which meiotic spindles become bipolar and the correlation between bipolarity and poleward flux, using Xenopus egg extracts. By speckle microscopy and computational alignment, we find that monopolar sperm asters do not show evidence for flux, partially contradicting previous work. We account for the discrepancy by describing spontaneous bipolarization of sperm asters that was missed previously. During spontaneous bipolarization, onset of flux correlated with onset of bipolarity, implying that antiparallel microtubule organization may be required for flux. Using a probe for TPX2 in addition to tubulin, we describe two pathways that lead to spontaneous bipolarization, new pole assembly near chromatin, and pole splitting. By inhibiting the Ran pathway with excess importin-alpha, we establish a role for chromatin-derived, antiparallel overlap bundles in generating the sliding force for flux, and we examine these bundles by electron microscopy. Our results highlight the importance of two processes, chromatin-initiated microtubule nucleation, and sliding forces generated between antiparallel microtubules, in self-organization of spindle bipolarity and poleward flux.

Animals↗
PubMed2001

Merotelic kinetochore orientation is a major mechanism of aneuploidy in mitotic mammalian tissue cells.

In mitotic cells, an error in chromosome segregation occurs when a chromosome is left near the spindle equator after anaphase onset (lagging chromosome). In PtK1 cells, we found 1.16% of untreated anaphase cells exhibiting lagging chromosomes at the spindle equator, and this percentage was enhanced to 17.55% after a mitotic block with 2 microM nocodazole. A lagging chromosome seen during anaphase in control or nocodazole-treated cells was found by confocal immunofluorescence microscopy to be a single chromatid with its kinetochore attached to kinetochore microtubule bundles extending toward opposite poles. This merotelic orientation was verified by electron microscopy. The single kinetochores of lagging chromosomes in anaphase were stretched laterally (1.2--5.6-fold) in the directions of their kinetochore microtubules, indicating that they were not able to achieve anaphase poleward movement because of pulling forces toward opposite poles. They also had inactivated mitotic spindle checkpoint activities since they did not label with either Mad2 or 3F3/2 antibodies. Thus, for mammalian cultured cells, kinetochore merotelic orientation is a major mechanism of aneuploidy not detected by the mitotic spindle checkpoint. The expanded and curved crescent morphology exhibited by kinetochores during nocodazole treatment may promote the high incidence of kinetochore merotelic orientation that occurs after nocodazole washout.

Anaphase↗
PubMed2001

Drosophila APC2 and Armadillo participate in tethering mitotic spindles to cortical actin.

Proper positioning of mitotic spindles ensures equal allocation of chromosomes to daughter cells. This often involves interactions between spindle and astral microtubules and cortical actin. In yeast and Caenorhabditis elegans, some of the protein machinery that connects spindles and cortex has been identified but, in most animal cells, this process remains mysterious. Here, we report that the tumour suppressor homologue APC2 and its binding partner Armadillo both play roles in spindle anchoring during the syncytial mitoses of early Drosophila embryos. Armadillo, alpha-catenin and APC2 all localize to sites of cortical spindle attachment. APC2-Armadillo complexes often localize with interphase microtubules. Zeste-white 3 kinase, which can phosphorylate Armadillo and APC, is also crucial for spindle positioning and regulates the localization of APC2-Armadillo complexes. Together, these data suggest that APC2, Armadillo and alpha-catenin provide an important link between spindles and cortical actin, and that this link is regulated by Zeste-white 3 kinase.

Actins↗
PubMed2001

The effect of stopping smoking on cervical Langerhans' cells and lymphocytes.

OBJECTIVE: To investigate the effects of stopping smoking on cervical Langerhans' cells and lymphocytes. DESIGN: Prospective intervention study. SETTING: A large family planning clinic in central London. POPULATION: Women volunteers prepared to attempt to give up smoking for six months. Their most recent cervical smear showed no abnormality greater than mild dyskaryosis. METHODS: The women were seen at three-month intervals for six months. Reduction in smoking was assessed by self-reporting and validated by salivary cotinine concentrations. Colposcopy and a biopsy of a normal area were performed at the first and last visits. Any area of abnormality was also biopsied at the final visit. Langerhans' cells and lymphocytes were counted. MAIN OUTCOME MEASURES: Proportional changes in counts of Langerhans' cells and lymphocytes with reduction in smoking. RESULTS: Reduction in smoking by 20 to 40 cigarettes per day was significantly associated with a reduction of between 6% and 16% in counts of Langerhans cells, CD8 and total lymphocytes. Heavy smoking was significantly associated (P = 0.02) with an increased chance of persistent human papillomavirus infection. The presence of candida was associated with significantly higher counts of between 41% and 47% in total lymphocytes and CD8 lymphocytes. In contrast, the presence of anaerobic vaginosis was associated with significantly lower counts of between 16% and 30% in Langerhans cells, CD4 and CD8 lymphocytes. CONCLUSIONS: This large intervention study has demonstrated a clear relationship between reduction in smoking and changes in cervical immune cell counts. Future studies need to take into account cytokine interactions, which recent studies suggest may be significant in the immune response to both human papillomavirus and cervical intraepithelial neoplasia and the ever-increasing complexity of the cell-mediated immune system of the cervix.

Adult↗
PubMed2000

The role of the proteins Kar9 and Myo2 in orienting the mitotic spindle of budding yeast.

BACKGROUND: Two genetic 'pathways' contribute to the fidelity of nuclear segregation during the process of budding in the yeast Saccharomyces cerevisiae. An early pathway, involving Kar9p and other proteins, orients the mitotic spindle along the mother-bud axis. Upon the onset of anaphase, cytoplasmic dynein provides the motive force for nuclear movement into the bud. Loss of either pathway results in nuclear-migration defects; loss of both is lethal. Here, to visualize the functional steps leading to correct spindle orientation along the mother-bud axis, we imaged live yeast cells expressing Kar9p and dynein as green fluorescent protein fusions. RESULTS: Transport of Kar9p into the bud was found to require the myosin Myo2p. Kar9p interacted with microtubules through the microtubule-binding protein Bim1p and facilitated microtubule penetration into the bud. Once microtubules entered the bud, Kar9p provided a platform for microtubule capture at the bud cortex. Kar9p was also observed at sites of microtubule shortening in the bud, suggesting that Kar9p couples microtubule shortening to nuclear migration. CONCLUSIONS: Thus, Kar9p provides a key link between the actin cytoskeleton and microtubules early in the cell cycle. A cooperative mechanism between Kar9p and Myo2p facilitates the pre-anaphase orientation of the spindle. Later, Kar9p couples microtubule disassembly with nuclear migration.

Actins↗
PubMed2000

Coordinated spindle assembly and orientation requires Clb5p-dependent kinase in budding yeast.

The orientation of the mitotic spindle along a polarity axis is critical in asymmetric cell divisions. In the budding yeast, Saccharomyces cerevisiae, loss of the S-phase B-type cyclin Clb5p under conditions of limited cyclin-dependent kinase activity (cdc28-4 clb5Delta cells) causes a spindle positioning defect that results in an undivided nucleus entering the bud. Based on time-lapse digital imaging microscopy of microtubules labeled with green fluorescent protein fusions to either tubulin or dynein, we observed that the asymmetric behavior of the spindle pole bodies during spindle assembly was lost in the cdc28-4 clb5Delta cells. As soon as a spindle formed, both poles were equally likely to interact with the bud cell cortex. Persistent dynamic interactions with the bud ultimately led to spindle translocation across the bud neck. Thus, the mutant failed to assign one spindle pole body the task of organizing astral microtubules towards the mother cell. Our data suggest that Clb5p-associated kinase is required to confer mother-bound behavior to one pole in order to establish correct spindle polarity. In contrast, B-type cyclins, Clb3p and Clb4p, though partially redundant with Clb5p for an early role in spindle morphogenesis, preferentially promote spindle assembly.

CDC28 Protein Kinase, S cerevisiae↗
PubMed2000

Dynamic positioning of mitotic spindles in yeast: role of microtubule motors and cortical determinants.

In the budding yeast Saccharomyces cerevisiae, movement of the mitotic spindle to a predetermined cleavage plane at the bud neck is essential for partitioning chromosomes into the mother and daughter cells. Astral microtubule dynamics are critical to the mechanism that ensures nuclear migration to the bud neck. The nucleus moves in the opposite direction of astral microtubule growth in the mother cell, apparently being "pushed" by microtubule contacts at the cortex. In contrast, microtubules growing toward the neck and within the bud promote nuclear movement in the same direction of microtubule growth, thus "pulling" the nucleus toward the bud neck. Failure of "pulling" is evident in cells lacking Bud6p, Bni1p, Kar9p, or the kinesin homolog, Kip3p. As a consequence, there is a loss of asymmetry in spindle pole body segregation into the bud. The cytoplasmic motor protein, dynein, is not required for nuclear movement to the neck; rather, it has been postulated to contribute to spindle elongation through the neck. In the absence of KAR9, dynein-dependent spindle oscillations are evident before anaphase onset, as are postanaphase dynein-dependent pulling forces that exceed the velocity of wild-type spindle elongation threefold. In addition, dynein-mediated forces on astral microtubules are sufficient to segregate a 2N chromosome set through the neck in the absence of spindle elongation, but cytoplasmic kinesins are not. These observations support a model in which spindle polarity determinants (BUD6, BNI1, KAR9) and cytoplasmic kinesin (KIP3) provide directional cues for spindle orientation to the bud while restraining the spindle to the neck. Cytoplasmic dynein is attenuated by these spindle polarity determinants and kinesin until anaphase onset, when dynein directs spindle elongation to distal points in the mother and bud.

Anaphase↗
PubMed2000

Surgical treatment of carcinoid tumours.

Carcinoid tumours are the most common endocrine tumour of the gut comprising 75% of appendicular tumours and a third of ileal tumours. They are also an incidental finding in 1% of post mortems. Carcinoid syndrome however is rare and it is estimated that two cases would appear in a quarter of a million population over a decade. We have reviewed a consecutive series of carcinoid tumours accrued from 1976-1999 in order to further study the natural history of the disease and also to examine the relationship between pathological tumour size, extent of disease and prognosis for each tumour site. In this 19 year period 145 patients were treated for the disorder. The bronchus was the commonest site (35.9%) with appendix (24.8%) and ileal (13.8%) carcinoids comprising the majority of known primaries. Overall midgut carcinoids comprised 40.7% of the series with the carcinoid syndrome present in 20.3%. A significant group was constituted by those patients presenting with metastatic disease and an uncertain site of primary tumour (9.7%). Metastatic disease was not found on presentation or follow up in any patient with a foregut primary tumour less than 2 cm diameter or a midgut primary tumour less than 1 cm diameter; a direct correlation of size and metastases being found above these levels. Overall survival for all groups was 60% at 5 years with the best prognosis being seen in patients with bronchial or appendicular carcinoids. Other clinical features, pathology, extent of disease and prognosis of carcinoid tumour will be discussed along with the role of current management strategies.

Adult↗
PubMed1999

Microtubule dynamics from mating through the first zygotic division in the budding yeast Saccharomyces cerevisiae.

We have used time-lapse digital imaging microscopy to examine cytoplasmic astral microtubules (Mts) and spindle dynamics during the mating pathway in budding yeast Saccharomyces cerevisiae. Mating begins when two cells of opposite mating type come into proximity. The cells arrest in the G1 phase of the cell cycle and grow a projection towards one another forming a shmoo projection. Imaging of microtubule dynamics with green fluorescent protein (GFP) fusions to dynein or tubulin revealed that the nucleus and spindle pole body (SPB) became oriented and tethered to the shmoo tip by a Mt-dependent search and capture mechanism. Dynamically unstable astral Mts were captured at the shmoo tip forming a bundle of three or four astral Mts. This bundle changed length as the tethered nucleus and SPB oscillated toward and away from the shmoo tip at growth and shortening velocities typical of free plus end astral Mts (approximately 0.5 micrometer/min). Fluorescent fiduciary marks in Mt bundles showed that Mt growth and shortening occurred primarily at the shmoo tip, not the SPB. This indicates that Mt plus end assembly/disassembly was coupled to pushing and pulling of the nucleus. Upon cell fusion, a fluorescent bar of Mts was formed between the two shmoo tip bundles, which slowly shortened (0.23 +/- 0.07 micrometer/min) as the two nuclei and their SPBs came together and fused (karyogamy). Bud emergence occurred adjacent to the fused SPB approximately 30 min after SPB fusion. During the first mitosis, the SPBs separated as the spindle elongated at a constant velocity (0.75 micrometer/min) into the zygotic bud. There was no indication of a temporal delay at the 2-micrometer stage of spindle morphogenesis or a lag in Mt nucleation by replicated SPBs as occurs in vegetative mitosis implying a lack of normal checkpoints. Thus, the shmoo tip appears to be a new model system for studying Mt plus end dynamic attachments and much like higher eukaryotes, the first mitosis after haploid cell fusion in budding yeast may forgo cell cycle checkpoints present in vegetative mitosis.

Cell Fusion↗
PubMed1999

Differential expression of keratins 10, 17, and 19 in normal cervical epithelium, cervical intraepithelial neoplasia, and cervical carcinoma.

AIM: To examine the value of immunohistochemistry in defining a keratin profile to aid cervical histopathological diagnosis. METHODS: Immunohistochemical localisation of keratins 17, 10, and 19 was studied in 268 cervical biopsies from 216 women including normal epithelia (with and without human papilloma virus), low and high grade cervical intraepithelial neoplasia, and invasive carcinoma. The percentage of positive immunostaining was scored using a Kontron MOP videoplan image analyser. RESULTS: All major categories of cervical epithelia expressed these keratins to varying degrees. The median percentage of immunostaining for keratin 10 was 40% in normal tissue compared with just 1% in invasive carcinoma (p < 0.0001). The medians for keratin 17 were 0% in the normal group and 80% in carcinomas (p < 0.0001). By contrast, there was no significant difference in staining for keratin 19. Using a combination of the keratin 10 and 17 percentages, it was possible to separate the carcinomas from the benign conditions with a sensitivity of 100% and a specificity of 93%. Further analyses within the groups revealed more extensive staining for keratins 10 and 19 in reserve cell hyperplasia, immature squamous metaplasia, and congenital transformation zone. CONCLUSIONS: The morphological variety within the cervix is reflected, in part, by distinct keratin patterns. There are striking differences in the patterns of keratins 10 and 17 between infiltrating squamous carcinoma and normal cervical epithelia.

Biomarkers, Tumor↗
PubMed1998

Involvement of an actomyosin contractile ring in Saccharomyces cerevisiae cytokinesis.

In Saccharomyces cerevisiae, the mother cell and bud are connected by a narrow neck. The mechanism by which this neck is closed during cytokinesis has been unclear. Here we report on the role of a contractile actomyosin ring in this process. Myo1p (the only type II myosin in S. cerevisiae) forms a ring at the presumptive bud site shortly before bud emergence. Myo1p ring formation depends on the septins but not on F-actin, and preexisting Myo1p rings are stable when F-actin is depolymerized. The Myo1p ring remains in the mother-bud neck until the end of anaphase, when a ring of F-actin forms in association with it. The actomyosin ring then contracts to a point and disappears. In the absence of F-actin, the Myo1p ring does not contract. After ring contraction, cortical actin patches congregate at the mother-bud neck, and septum formation and cell separation rapidly ensue. Strains deleted for MYO1 are viable; they fail to form the actin ring but show apparently normal congregation of actin patches at the neck. Some myo1Delta strains divide nearly as efficiently as wild type; other myo1Delta strains divide less efficiently, but it is unclear whether the primary defect is in cytokinesis, septum formation, or cell separation. Even cells lacking F-actin can divide, although in this case division is considerably delayed. Thus, the contractile actomyosin ring is not essential for cytokinesis in S. cerevisiae. In its absence, cytokinesis can still be completed by a process (possibly localized cell-wall synthesis leading to septum formation) that appears to require septin function and to be facilitated by F-actin.

Actins↗
PubMed1998

Costs of care associated with non-small-cell lung cancer in a commercially insured cohort.

PURPOSE: To examine the cost of incident cases of non-small-cell lung cancer (NSCLC) in a commercially insured cohort. METHODS: Claims from Virginia Blue Cross and Blue Shield (BCBS) beneficiaries with lung cancer from 1989 to 1991 were merged with records from the Virginia Cancer Registry (VCR). Data from the VCR identified incident cases, stage, and type of cancer at diagnosis. Costs for all medical care included insurance payment, copayments, and deductibles for 2 years after diagnosis or until death. RESULTS: Three hundred forty-nine incident NSCLC patients were evaluated. The mean 2-year cost for each patient after diagnosis or until death was $47,941 (95% confidence interval, $43,758 to $52,124). Total average costs and hospital days were significantly lower for local disease ($37,514, 21.2 days), but were similar for regional ($52,797, 30.0 days) and distant ($49,382, 33.0 days) disease. Hospital days accounted for 48% and hospital-based claims for 70% of costs. Initial treatments, which included radiation, unadjusted for stage, had the lowest survival rates and the highest costs, and were associated with the most hospital days. Initial stage, race, gender, and age were not predictors of total 2-year costs. The independent predictors of total 2-year costs were type of treatment: any radiation therapy, any surgery, or any chemotherapy (all, P < .001). Inpatient hospital days was only a modest predictor of costs after adjusting for type of treatment. Patients who survived less than 1 year spent 30.5 days in hospital and had an average cost of $47,280. CONCLUSION: The direct health care costs of younger NSCLC patients care are substantial. These results should serve as a benchmark for future comparisons as the United States market shifts to managed care.

Blue Cross Blue Shield Insurance Plans↗
PubMed1997

CENP-E function at kinetochores is essential for chromosome alignment.

CENP-E is a kinesin-like protein that binds to kinetochores and may provide functions that are critical for normal chromosome motility during mitosis. To directly test the in vivo function of CENP-E, we microinjected affinity-purified antibodies to block the assembly of CENP-E onto kinetochores and then examined the behavior of these chromosomes. Chromosomes lacking CENP-E at their kinetochores consistently exhibited two types of defects that blocked their alignment at the spindle equator. Chromosomes positioned near a pole remained mono-oriented as they were unable to establish bipolar microtubule connections with the opposite pole. Chromosomes within the spindle established bipolar connections that supported oscillations and normal velocities of kinetochore movement between the poles, but these bipolar connections were defective because they failed to align the chromosomes into a metaphase plate. Overexpression of a mutant that lacked the amino-terminal 803 amino acids of CENP-E was found to saturate limiting binding sites on kinetochores and competitively blocked endogenous CENP-E from assembling onto kinetochores. Chromosomes saturated with the truncated CENP-E mutant were never found to be aligned but accumulated at the poles or were strewn within the spindle as was the case when cells were microinjected with CENP-E antibodies. As the motor domain was contained within the portion of CENP-E that was deleted, the chromosomal defect is likely attributed to the loss of motor function. The combined data show that CENP-E provides kinetochore functions that are essential for monopolar chromosomes to establish bipolar connections and for chromosomes with connections to both spindle poles to align at the spindle equator. Both of these events rely on activities that are provided by CENP-E's motor domain.

Animals↗