The Hopscotch Jak kinase requires the Raf pathway to promote blood cell activation and differentiation in Drosophila.

Cytokines regulate the development and differentiated functions of hematopoietic cells by activating multiple signaling pathways, including the Jak-Stat pathway, the PI3-kinase pathway, and the Ras/Raf pathway. While the Jak-Stat interaction has been extensively studied, the relationship between this pathway and other cytokine-induced signaling pathways is not fully understood. In Drosophila melanogaster, mutations that result in hyperactivity of the Jak kinase Hopscotch (Hop) cause an activation of the larval blood cell encapsulation response, including blood cell aggregation and differentiation of plasmatocytes into apparent lamellocytes. Here, we demonstrate that Hop requires the activity of the Raf pathway to promote the activation response of larval plasmatocytes, and provide evidence to suggest that the Hop and D-Raf proteins physically interact. We also show that basal level activity of the Raf pathway is required for the accumulation of circulating blood cells.

Primary intracerebral mesenchymal chondrosarcoma with rhabdomyosarcomatous differentiation: case report and literature review.

Intracranial chondrosarcoma (Ch-S) is a slow-growing, locally recurrent, malignant cartilaginous tumour of the skull base. Intracranial mesenchymal chondrosarcoma (MsCh-S) is a rarer, more malignant variant associated with the supratentorial meninges. Only seven cases of Ch-S, and six of MsCh-S, that were primarily intraparenchymal in origin have been reported. Moreover, no case of intracranial Ch-S or MsCh-S has been reported in which rhabdomyosarcomatous differentiation was prominent. A 17-year-old Asian girl presented with a 4-week history of occipital headache, vomiting and paraesthesia in the left hand. She was drowsy with a left hemiparesis and had a dilated right pupil with bilateral papilloedema. CT demonstrated a large, partly calcified, contrast-enhancing mass in the right temporo-parietal region with oedema and midline shift. Through a large craniotomy, a tense brain was encountered with no apparent cortical abnormality. Despite a radical tumour excision, with excellent initial clinical recovery, a local recurrence rapidly occurred within weeks prior to the administration of any radiotherapy. Initial histopathological examination revealed a primary MsCh-S with osseous and rhabdomyosarcomatous differentiation, with an indistinct margin. After a second radical excision, a second recurrence rapidly occurred; however, this proved excessively vascular and inoperable. Radiotherapy was declined and death followed within 3 weeks. This is the seventh case of primary intracerebral MsCh-S to be reported and the first to demonstrate rhabdomyosarcomatous differentiation. It was characterized clinically by rapid, local recurrence with increased vascularity.

Contrasting effects of HSP72 expression on apoptosis in human umbilical vein endothelial cells and an angiogenic cell line, ECV304.

The effect of overexpression of heat shock protein (HSP)72 on apoptosis induced by different stimuli in human umbilical vein endothelial cells (HUVECs) and the angiogenic cell line, ECV304, was studied. Transient overexpression of HSP72 was achieved using an adenoviral vector (Advhsp72) and apoptosis was induced by heat shock, tumour necrosis factor (TNF)-alpha with cycloheximide (CHX), lipopolysaccharide (LPS) with TNF-alpha and verocytotoxin (VT). Apoptosis induced by heat shock was reduced by HSP72 expression. However, HSP72 expression in HUVECs increased apoptosis induced by TNF-alpha/CHX, LPS and VT measured by flow cytometric analysis of propidium iodide (PI)-stained permeabilized cells. In contrast, apoptosis in ECV304 induced by the same stimuli was reduced by HSP72 expression. No difference was seen in cells transduced with a control adenoviral vector expressing beta-galactosidase. These data imply that induction of HSP72 in cells modulates responses to apoptotic stimuli, but that the nature of the response varies with the cell type. However, it is clear that in situations where apoptosis may be part of a pathological process, HSP72 induction, for example by reperfusion injury, may exacerbate the process.

Anticoagulant therapy and referral form. Haemostasis and Thrombosis Task Force of the British Committee for Standards in Haematology.

Many hospitals use the recommendations contained in the British Committee for Standards in Haematology anticoagulant guideline documents to develop local protocols for anticoagulant management. A combined anticoagulant treatment chart and referral form has been produced to help incorporate the recently updated recommendations for oral anticoagulation into day-to-day practice.

Oral anticoagulation management in primary care with the use of computerized decision support and near-patient testing: a randomized, controlled trial.

BACKGROUND: There is increased pressure on primary care physicians to monitor oral anticoagulation. OBJECTIVE: To test the null hypothesis that oral anticoagulation care can be provided at least as well in primary care through a nurse-led clinic, involving near-patient testing and computerized decision support software, compared with routine hospital management based on a variety of clinical outcome measures. METHODS: A randomized, controlled trial in 12 primary care practices in Birmingham, England (9 intervention and 3 control). Two control populations were used: patients individually randomly allocated as controls in the intervention practices (intrapractice controls) and all patients in control practices (interpractice controls). Intervention practices' patients were randomized to the intervention (practice-based anticoagulation clinic) or control (hospital clinic) group. The main outcome measure was therapeutic control of the international normalized ratio. RESULTS: Three hundred sixty-seven patients were recruited (122 intervention patients, 102 intrapractice control patients, and 143 interpractice control patients). Standard measures of control of the international normalized ratio (point prevalence) showed no significant difference between the intervention and control groups. Data on proportion of time spent in the international normalized ratio range showed significant improvement for patients in the intervention group (paired t test, P =.008). CONCLUSIONS: Nurse-led anticoagulation clinics can be implemented in novice primary care settings by means of computerized decision support software and near-patient testing. Care given by this model is at least as good as routine hospital follow-up. The model is generalizable to primary health care centers operating in developed health care systems.

Is the international normalised ratio (INR) reliable? A trial of comparative measurements in hospital laboratory and primary care settings.

AIM: To determine the reliability of international normalised ratio (INR) measurement in primary care by practice nurses using near patient testing (NPT), in comparison with results obtained within hospital laboratories by varied methods. METHODS: As part of an MRC funded study into primary care oral anticoagulation management, INR measurements obtained in general practice were validated against values on the same samples obtained in hospital laboratories. A prospective comparative trial was undertaken between three hospital laboratories and nine general practices. All patients attending general practice based anticoagulant clinics had parallel INR estimations performed in general practice and in a hospital laboratory. RESULTS: 405 tests were performed. Comparison between results obtained in the practices and those in the reference hospital laboratory (gold standard), which used the same method of testing for INR, showed a correlation coefficient of 0.96. Correlation coefficients comparing the results with the various standard laboratory techniques ranged from 0.86 to 0.92. It was estimated that up to 53% of tests would have resulted in clinically significant differences (change in warfarin dose) depending upon the site and method of testing. The practice derived results showed a positive bias ranging from 0.28 to 1.55, depending upon the site and method of testing. CONCLUSIONS: No technical problems associated with INR testing within primary care were uncovered. Discrepant INR results are as problematic in hospital settings as they are in primary care. These data highlight the failings of the INR to standardise when different techniques and reagents are used, an issue which needs to be resolved. For primary care to become more involved in therapeutic oral anticoagulation monitoring, close links are needed between hospital laboratories and practices, particularly with regard to training and quality assurance.

Structural analysis of the lymphocyte-specific kinase Lck in complex with non-selective and Src family selective kinase inhibitors.

BACKGROUND: The lymphocyte-specific kinase Lck is a member of the Src family of non-receptor tyrosine kinases. Lck catalyzes the initial phosphorylation of T-cell receptor components that is necessary for signal transduction and T-cell activation. On the basis of both biochemical and genetic studies, Lck is considered an attractive cell-specific target for the design of novel T-cell immunosuppressants. To date, the lack of detailed structural information on the mode of inhibitor binding to Lck has limited the discovery of novel Lck inhibitors. RESULTS: We report here the high-resolution crystal structures of an activated Lck kinase domain in complex with three structurally distinct ATP-competitive inhibitors: AMP-PNP (a non-selective, non-hydrolyzable ATP analog); staurosporine (a potent but non-selective protein kinase inhibitor); and PP2 (a potent Src family selective protein tyrosine kinase inhibitor). Comparison of these structures reveals subtle but important structural changes at the ATP-binding site. Furthermore, PP2 is found to access a deep, hydrophobic pocket near the ATP-binding cleft of the enzyme; this binding pocket is not occupied by either AMP-PNP or staurosporine. CONCLUSIONS: The potency of staurosporine against Lck derives in part from an induced movement of the glycine-rich loop of the enzyme upon binding of this ligand, which maximizes the van der Waals interactions present in the complex. In contrast, PP2 binds tightly and selectively to Lck and other Src family kinases by making additional contacts in a deep, hydrophobic pocket adjacent to the ATP-binding site; the amino acid composition of this pocket is unique to Src family kinases. The structures of these Lck complexes offer useful structural insights as they demonstrate that kinase selectivity can be achieved with small-molecule inhibitors that exploit subtle topological differences among protein kinases.

A laboratory model of toxin-induced hemolytic uremic syndrome.

BACKGROUND: Verocytotoxin-producing (Shiga-like toxin-producing) Escherichia coli infection is the principal cause of hemolytic uremic syndrome (HUS). The pathogenesis is unclear, and there is a need for animal models. These are impeded by the different distribution of verocytotoxin receptors between species. We have circumvented this restriction using ricin, which gains entry into cells via various galactose receptors. Like verocytotoxin, ricin specifically cleaves a single adenine from ribosomal RNA. METHODS: Rats were given ricin at a dose of 6.7 micrograms/100 g body wt, with or without lipopolysaccharide at 10 micrograms/100 g body wt. Lipopolysaccharide alone or saline were used as controls. Changes in glomerular filtration rate, hematological parameters, histology, and plasma cytokine concentrations were measured. RESULTS: Extensive glomerular thrombosis, pyknotic nuclei, and an infiltration of ED1-positive cells into glomeruli were observed eight hours after an injection of ricin. Other vascular beds were unaffected. Histologic changes were preceded by oliguric renal failure, hemolysis, and thrombocytopenia. Ricin produced a rise in plasma concentrations of monocyte chemotactic protein-1, > tumor necrosis factor-alpha, > interleukin-1 beta, > interleukin-6. Interferon-gamma showed a small increase at the end of the experiment. CONCLUSIONS: Ricin induces glomerular thrombotic microangiopathy, closely resembling that which occurs in verocytotoxin-producing E. coli-induced HUS. As in HUS, high concentrations of proinflammatory cytokines are present, which are probably a result of cytokine superinduction by the toxin.

New approaches to the delivery of anticoagulant services.

Anticoagulant services are changing in response to the increasing demands on the service. New approaches to the delivery of the service are evolving with more local delivery of services and a shift in the service from secondary to primary care. This change has been assisted by the development of near patient testing devices and the use of computerized anticoagulant decision support systems that are increasingly used in both secondary and primary care. The evolving role of the clinical nurse/pharmacist in the provision of this service is an important development enabling more rapid discharge of patients and the provision of local delivery of service.

Use of different histomorphometric parameters in the routine assessment of human skeletal muscle biopsies.

In the diagnosis of muscle biopsies it has become traditional to identify any changes in fibre size by measuring minimum fibre diameter (dmin), as past technical constraints prevented the routine measurement of other parameters. The advent of user-friendly computerised image analysis, however, has facilitated such measurements. We examined a total of 39 skeletal muscle biopsies, including normal, myopathic, myositic and neuropathic cases, to determine whether improved discrimination between normal and abnormal histology was now possible on the basis of fibre cross-sectional areas (CSA). Using a semi-automated image analysis system, measurements of dmin, CSA and fibre perimeter were made. In all case groups, the skew of the frequency distribution of area was greater than that of dmin, moreover the difference was more marked in the abnormal cases. As expected, the mean values of dmin and area were reduced in all abnormal cases. As expected, the mean values of dmin and area were reduced in all abnormal cases. The range of their values, however, was reduced in the myopathic and neuropathic cases and increased in the myositic cases. In conclusion, fibre area is a more valuable discriminator between normal and abnormal skeletal muscle than is dmin.

The cytokine-activated tyrosine kinase JAK2 activates Raf-1 in a p21ras-dependent manner.

JAK2, a member of the Janus kinase superfamily was found to interact functionally with Raf-1, a central component of the ras/mitogen-activated protein kinase signal transduction pathway. Interferon-gamma and several other cytokines that are known to activate JAK2 kinase were also found to stimulate Raf-1 kinase activity toward MEK-1 in mammalian cells. In the baculovirus coexpression system, Raf-1 was activated by JAK2 in the presence of p21ras. Under these conditions, a ternary complex of p21ras, JAK2, and Raf-1 was observed. In contrast, in the absence of p21ras, coexpression of JAK2 and Raf-1 resulted in an overall decrease in the Raf-1 kinase activity. In addition, JAK2 phosphorylated Raf-1 at sites different from those phosphorylated by pp60v-src. In mammalian cells treated with either erythropoietin or interferon-gamma, a small fraction of Raf-1 coimmunoprecipitated with JAK2 in lysates of cells in which JAK2 was activated as judged by its state of tyrosine phosphorylation. Taken together, these data suggest that JAK2 and p21ras cooperate to activate Raf-1.

Susceptibility of AML cells to in vitro apoptosis correlates with heat shock protein 70 (hsp 70) expression.

Acute myeloid leukaemia (AML) cells from some individuals rapidly undergo apoptosis during in vitro culture. We have analysed this mode of cell death in AML cells harvested from patients at initial presentation and during subsequent treatment/relapse. Using flow cytometric analysis of propidium iodide-stained cells and quantitation of the subdiploid apoptotic peak, we observed that leukaemic cells from patients with AML displayed a heterogenous susceptibility to apoptosis in terms of the rate of accumulation of apoptotic cells. After 48 h incubation in the absence of added serum or exogenous growth factors the percentage of apoptotic cells ranged from 3% to 99%. This susceptibility to apoptosis correlated significantly with intracellular expression of hsp70 (P = 0.009), but not hsp90, and was also associated with the presence of p53 and low levels of expression of bcl-2.

Use of interferon-2 alpha in the treatment of cryoglobulinaemic leg ulcers.

We report three cases of leg ulcers complicating cryoglobulinaemia which resisted conventional therapy but responded to interferon-2 alpha. All three cases improved with the complete resolution of painful vasculitic lesions and healing of deep ulcers. The dose of interferon to produce ulcer healing was determined by individual response with escalation of the daily dose to 4.5 x 10(6) units per day required in one case. A lower dose of maintenance interferon could be used to prevent subsequent relapses of disease.