A Kinesin-5, Cin8, Recruits Protein Phosphatase 1 to Kinetochores and Regulates Chromosome Segregation.

Kinesin-5 is a highly conserved homo-tetrameric protein complex responsible for crosslinking microtubules and pushing spindle poles apart. The budding yeast Kinesin-5, Cin8, is highly concentrated at kinetochores in mitosis before anaphase, but its functions there are largely unsolved. Here, we show that Cin8 localizes to kinetochores in a cell-cycle-dependent manner and concentrates near the microtubule binding domains of Ndc80 at metaphase.

How the kinetochore couples microtubule force and centromere stretch to move chromosomes.

The Ndc80 complex (Ndc80, Nuf2, Spc24 and Spc25) is a highly conserved kinetochore protein essential for end-on anchorage to spindle microtubule plus ends and for force generation coupled to plus-end polymerization and depolymerization. Spc24/Spc25 at one end of the Ndc80 complex binds the kinetochore. The N-terminal tail and CH domains of Ndc80 bind microtubules, and an internal domain binds microtubule-associated proteins (MAPs) such as the Dam1 complex.

A quantitative description of Ndc80 complex linkage to human kinetochores.

The Ndc80 complex, which mediates end-on attachment of spindle microtubules, is linked to centromeric chromatin in human cells by two inner kinetochore proteins, CENP-T and CENP-C. Here to quantify their relative contributions to Ndc80 recruitment, we combine measurements of kinetochore protein copy number with selective protein depletion assays. This approach reveals about 244 Ndc80 complexes per human kinetochore (∼14 per kinetochore microtubule), 215 CENP-C, 72 CENP-T and only 151 Ndc80s as part of the KMN protein network (1:1:1 Knl1, Mis12 and Ndc80 complexes).

The architecture of CCAN proteins creates a structural integrity to resist spindle forces and achieve proper Intrakinetochore stretch.

Constitutive centromere-associated network (CCAN) proteins, particularly CENP-C, CENP-T, and the CENP-H/-I complex, mechanically link CENP-A-containing centromeric chromatin within the inner kinetochore to outer kinetochore proteins, such as the Ndc80 complex, that bind kinetochore microtubules. Accuracy of chromosome segregation depends critically upon Aurora B phosphorylation of Ndc80/Hec1. To determine how CCAN protein architecture mechanically constrains intrakinetochore stretch between CENP-A and Ndc80/Hec1 for proper Ndc80/Hec1 phosphorylation, we used super-resolution fluorescence microscopy and selective protein depletion.

Collaborative practices in unscheduled emergency care: role and impact of the emergency care practitioner--quantitative findings.

Objective: To identify collaborative instances and hindrances and to produce a model of collaborative practice.

Methods: A 12 month (2005-6) mixed methods clinical case study in a large UK ambulance trust. Collaboration was measured through direct observational ratings of communication skills, teamwork and leadership with 24 multiprofessional emergency care practitioners (ECPs); interviews with 45 ECPs and stakeholders; and an audit of 611 patients.

Collaborative practices in unscheduled emergency care: role and impact of the emergency care practitioner--qualitative and summative findings.

Objective: To identify collaborative instances and hindrances and to produce a model of collaborative practice.

Methods: A 12-month (2005-2006) mixed methods clinical case study was carried out in a large UK ambulance trust. Collaboration was measured through direct observational ratings of communication skills, teamwork and leadership with 24 multi-professional emergency care practitioners (ECPs), interviews with 45 ECPs and stakeholders, and an audit of 611 patients

Results: Using a generic qualitative approach, observational records and interviews showed that ECPs' numerous links with other professions were influenced by three major themes as follows.

The training/service continuum: exploring the training/service balance of senior house officer activities.

Background: The continued reduction in junior doctors' hours in the UK has made it necessary to re-examine traditional assumptions about the synonymous natures of training and service. This paper researches senior house officers' (SHOs) perceptions of 'service' and 'training', with reference to where they place activities along the training/service continuum, and the factors that lead them to classify these activities in the way they do.

Methods: Questionnaires were sent to all identified SHOs at Plymouth Hospitals NHS Trust (40% response rate).

Your hand in glove selection.

Supplies that are purchased for facility-wide use can have a sizeable impact on a hospital's bottom line as well as patient and employee safety. Here, review how to maximize your purchasing power when selecting medical gloves.

Prognostic influence on survival of increased ornithine decarboxylase activity in human breast cancer.

Although considerable experimental evidence suggests an important role of polyamines in breast cancer biology, compelling supportive data in patients are lacking. To address this issue, we measured ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase, and spermidine/spermine acetyltransferase (the three key polyamine metabolic enzymes) in a cohort of 50 primary human breast cancers and related their levels of activity to disease-free survival and overall survival. The major finding of our study was that ODC activity level was a negative independent prognostic factor for both end points.

Isolation and characterization of human breast cancer cells overexpressing S-adenosylmethionine decarboxylase.

We report the first successful isolation and initial characterization of S-adenosylmethionine decarboxylase (SAMDC)-overexpressing cells using a transfection approach. Stably transfected MCF-7 breast cancer overproducing SAMDC (approximately 5-fold) manifested reduced ornithine decarboxylase while levels of N'-spermidine/spermine acetyltransferase were variably increased. Analysis of cellular polyamine profile showed that spermine was selectively increased (approximately 80%), while spermidine and putrescine levels were reduced (approximately 48% and approximately 15% of control, respectively).

Biochemical and growth-modulatory effects of the new S-adenosylmethionine decarboxylase inhibitor CGP 48664 in malignant and immortalized normal human breast epithelial cells in culture.

CGP 48664 [4-aminoindanon-1-(2'-amidino)hydrazone dihydrochloride monohydrate] is a newly introduced inhibitor of S-adenosylmethionine decarboxylase (SAMDC) with increased selectivity of action and reduced toxicity. We analyzed the biochemical and antiproliferative effects of this compound in a panel of hormone-dependent (3 clones of MCF-7, T47D) and -independent (MDA-MB-231, BT-20) human breast cancer cell lines in culture. For comparison, we also tested its effects in the spontaneously immortalized human breast epithelial cell line MCF-10A.

Safe care of the latex-sensitive trauma patient.

Latex protein allergies are becoming more commonplace in the healthcare setting and pose a challenge to first responders and trauma resuscitation team members. The authors present an overview of latex sensitivity and its prevalence and outline signs and symptoms. They also identify commonly used assessments and treatment tools that may contain latex, and suggest possible substitutes.

Hormonal regulation of insulin-like growth factor II and insulin-like growth factor binding protein expression by breast cancer cells in vivo: evidence for stromal epithelial interactions.

Insulin-like growth factors (IGFs) I and II are potent mitogens for breast cancer cells. Their proliferative activity is likely to be influenced by their binding proteins (IGFBPs), a family of newly identified proteins. We report here on the in vivo hormonal regulation of mRNAs for IGF-II and IGFBPs in the N-nitrosomethylurea-induced rat mammary tumor, a well-established model of hormone-responsive mammary cancer.

Health care and higher education. The time diary approach for management development.

Discusses a project which aimed to determine the employee development needs of Plymouth Polytechnic and Derriford Hospital NHS senior technicians. Using the time diary approach, it revealed employer and employee variance in opinions over desired managerial competences and variance in the training needs for the two organizations. In the case of NHS technicians it was possible to define areas where provision of a new training scheme would be beneficial.

Synchronization of breast cancer cell proliferation in vivo by combined hormonal and polyamine manipulation.

Optimal synchronization of breast cancer cell proliferation by hormonal means may be limited by cellular heterogeneity in sensitivity to the multistep activation of growth following initial hormone binding to the receptor. We hypothesized that induced synchronous growth may be improved by combined manipulation of the polyamine (PA) pathway since we have previously shown that PAs are distal effectors of hormonal action on proliferation in breast cancer. To test our hypothesis, we induced an initial phase of hormone and PA depletion (castration plus administration of the PA synthesis inhibitor alpha-difluoromethylornithine) in rats bearing N-nitrosomethylurea induced mammary tumors.

Role of polyamines in the growth of hormone-responsive and -resistant human breast cancer cells in nude mice.

Recent in vitro data suggest that at least some hormone-independent breast cancer cells exhibit increased polyamine biosynthesis and resistance to antipolyamine therapy. To address this issue under conditions of in vivo growth, we tested the antiproliferative effect of the polyamine synthetic inhibitor alpha-difluoromethyl-ornithine (DFMO) on hormone-dependent (MCF-7) and -independent (MDA-MB-231, BT-20) breast cancer cell lines growing in nude mice. We observed that DFMO significantly inhibited the growth of established tumors to a similar extent in all cell lines, even though tumor regression was only observed with MCF-7 cells.

Expression of messenger RNA for insulin-like growth factors and insulin-like growth factor binding proteins by experimental breast cancer and normal breast tissue in vivo.

We report the expression of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) by breast cancer cells and normal breast tissue in vivo. N-nitrosomethyl-urea (NMU)-induced rat mammary tumors synthesize mRNAs for IGF-II and IGFBP-2, -3, and -4. In contrast, normal lactating breast contains only IGFBP-2 and IGF-II messages; IGFBP-3 and -4 mRNAs are absent in this tissue.

Role of transforming growth factor-alpha (TGF-alpha) in basal and hormone-stimulated growth by estradiol, prolactin and progesterone in human and rat mammary tumor cells: studies using TGF-alpha and EGF receptor antibodies.

The biological role of transforming growth factor-alpha (TGF-alpha) in basal and hormone-stimulated proliferation of primary human and rat mammary tumor cells was studied using antibodies against TGF-alpha and its receptor. A monoclonal antibody, MAb-425 against human EGF receptor was added to in vitro soft agar, clonogenic cultures of human breast carcinoma cells under basal and estradiol(E2)-stimulated conditions. The antibody had an antagonist effect on colony growth in 4 of 10 tumors and an agonist effect in 4 (72 and 153% of control).

Selectivity of polyamine involvement in hormone action on normal and neoplastic target tissues of the rat.

The present experiments were designed to evaluate the polyamine involvement in hormonal actions on proliferation and receptor content of neoplastic tissue (hormone-responsive breast cancer) as well as on growth of normal endocrine target tissue (uterus) in the same animals. Administration of estradiol and perphenazine (to stimulate endogenous prolactin release) stimulated N-nitrosomethyl-urea (NMU)-induced rat mammary tumor growth following ovariectomy-induced tumor regression. Such hormonal activation of breast cancer growth was completely abolished by treatment with alpha-difluoromethyl-ornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase, which lowered tumor content of polyamines.

Kinetic and morphometric responses of heterogeneous populations of NMU-induced rat mammary tumor cells to hormone and antipolyamine therapy in vivo.

The present experiments were designed to evaluate in vivo the differential sensitivity of tumor cell subpopulations to hormone and polyamine manipulations using the hormone-responsive N-nitrosomethyl-urea (NMU)-induced rat mammary tumor. NMU tumor bearing rats were randomly assigned to control, ovariectomy, alpha-difluoromethyl-ornithine (DFMO) administration (an inhibitor of polyamine biosynthesis), or combination treatment, and were sacrificed on day 2, 4, or 7. The proportion of different cells was estimated by morphometric analysis and their replicative activities by [3H]-thymidine autoradiography.

Role of insulin-like growth factor-related peptides in the estradiol-, prolactin-, and progesterone-stimulated growth of N-nitrosomethylurea-induced rat mammary tumors in soft agar.

The present experiments were designed to test the role of insulin-like growth factor-related peptides (IGF-RPs) in hormonally stimulated N-nitrosomethylurea (NMU)-induced mammary tumor colony formation in soft agar. To evaluate production of IGF-RP by NMU cells, we tested the abilities of a monoclonal antibody directed against insulin-like growth factor I (alpha sm 1.20B) and of a polyclonal antibody raised against the insulin-like growth factor I (Ab 134) to inhibit the colony-stimulating effects of conditioned media (CM) obtained from estradiol (E2)-, prolactin (PRL)-, and progesterone (Pg)-treated NMU-induced rat mammary tumors.

Role of transforming growth factor-alpha-related peptides in the autocrine/paracrine control of experimental breast cancer growth in vitro by estradiol, prolactin, and progesterone.

We have recently suggested that estradiol (E2), prolactin (oPrl), and progesterone (Pg) support the growth of the hormone-responsive N-nitrosomethylurea (NMU) rat mammary tumor in soft agar through autocrine/paracrine mechanisms. To gain insight into the nature of these hormonally regulated growth factors, we tested the ability of two monoclonal antibodies (MAb-425 and 528) directed against the epidermal growth factor receptor (EGF-R) to inhibit the colony-stimulating effects of conditioned media (CM) obtained from E2, oPrl, and Pg-treated NMU rat mammary tumors. Since both MAbs are specific for human EGF-R, MCF-7 breast cancer cells grown in soft agar in the absence of serum were used as our indicator system.