The fine needle aspiration of translocation sarcomas.

INTRODUCTION: Soft tissue sarcomas comprise a heterogeneous group of clinically aggressive cancers that are often hard to classify on limited cytological samples. "Translocation sarcomas" (TS) are a diverse subset of such cancers, different from pleomorphic sarcomas, and characterised by unique single chromosomal translocations in each sarcoma subtype. Interestingly, despite their high-grade biological behaviour, TS have deceptively monotonous and bland cytomorphology, therefore creating diagnostic issues on limited samples. MATERIALS AND METHODS: A retrospective search was conducted of the cytopathology archives of The Johns Hopkins Hospital revealing 147 translocation sarcoma cases over a 25-year period. RESULTS: The common morphological denominators for most translocation sarcomas were: hypercellularity, cellular monotony, mostly discohesive and single cells, round-to-oval or short spindled cells and a lack of necrosis. The exceptions were an inflammatory myofibroblastic tumour, in which cellular monotony was not present owing to the prominence of lymphocytes and plasma cells, and low-grade fibromyxoid sarcoma, in which the specimens were generally hypocellular. Ancillary testing, especially immunoperoxidase staining, was often required for primary lesions. CONCLUSION: Distinct morphological clues and subsequent ancillary testing (particularly immunoperoxidase staining) provide an accurate diagnosis on cytological interpretation of both, primary and recurrent/metastatic lesions.

A G/C element mediates repression of the SM22alpha promoter within phenotypically modulated smooth muscle cells in experimental atherosclerosis.

A hallmark of smooth muscle cell (SMC) phenotypic switching in atherosclerotic lesions is suppression of SMC differentiation marker gene expression. Yet little is known regarding the molecular mechanisms that control this process. Here we show that transcription of the SMC differentiation marker gene SM22alpha is reduced in atherosclerotic lesions and identify a cis regulatory element in the SM22alpha promoter required for this process. Transgenic mice carrying the SM22alpha promoter-beta-galactosidase (beta-gal) reporter transgene were crossed to apolipoprotein E (ApoE)-/- mice. Cells of the fibrous cap, intima, and underlying media showed complete loss of beta-gal activity in advanced atherosclerotic lesions. Of major significance, mutation of a G/C-rich cis element in the SM22alpha promoter prevented the decrease in SM22alpha promoter-beta-gal reporter transgene expression, including in cells that compose the fibrous cap of the lesion and in medial cells in proximity to the lesion. To begin to assess mechanisms whereby the G/C repressor element mediates suppression of SM22alpha in atherosclerosis, we tested the hypothesis that effects may be mediated by platelet-derived growth factor (PDGF)-BB-induced increases in the G/C binding transcription factor Sp1. Consistent with this hypothesis, results of studies in cultured SMCs showed that: (1) PDGF-BB increased expression of Sp1; (2) PDGF-BB and Sp1 profoundly suppressed SM22alpha promoter activity as well as smooth muscle myosin heavy chain promoter activity through mechanisms that were at least partially dependent on the G/C cis element; and (3) a short interfering RNA to Sp1 increased basal expression and attenuated PDGF-BB induced suppression of SM22alpha. Together, these results support a model whereby a G/C repressor element within the SM22alpha promoter mediates transcriptional repression of this gene within phenotypically modulated SMCs in experimental atherosclerosis and provide indirect evidence implicating PDGF-BB and Sp1 as possible mediators of these effects.

L-type voltage-gated Ca2+ channels modulate expression of smooth muscle differentiation marker genes via a rho kinase/myocardin/SRF-dependent mechanism.

Vascular smooth muscle cell (SMC) contraction is mediated in part by calcium influx through L-type voltage-gated Ca2+ channels (VGCC) and activation of the RhoA/Rho kinase (ROK) signaling cascade. We tested the hypothesis that Ca2+ influx through VGCCs regulates SMC differentiation marker expression and that these effects are dependent on RhoA/ROK signaling. Depolarization-induced activation of VGCCs resulted in a nifedipine-sensitive increase in endogenous smooth muscle myosin heavy chain (SMMHC) and SM alpha-actin expression and CArG-dependent promoter activity, as well as c-fos promoter activity. The ROK inhibitor, Y-27632, prevented depolarization-induced increase in SMMHC/SM alpha-actin but had no effect on c-fos expression. Conversely, the Ca2+/calmodulin-dependent kinase inhibitor, KN93, prevented depolarization-induced increases in c-fos expression with no effect on SMMHC/SM alpha-actin. Depolarization increased expression of myocardin, a coactivator of SRF that mediates CArG-dependent transcription of SMC marker gene promoters containing paired CArG cis regulatory elements (SMMHC/SM alpha-actin). Both nifedipine and Y-27632 prevented the depolarization-induced increase in myocardin expression. Moreover, short interfering RNA (siRNA) specific for myocardin attenuated depolarization-induced SMMHC/SM alpha-actin transcription. Chromatin immunoprecipitation (ChIP) assays revealed that depolarization increased SRF enrichment of the CArG regions in the SMMHC, SM alpha-actin, and c-fos promoters in intact chromatin. Whereas Y-27632 decreased basal and depolarization-induced SRF enrichment in the SMMHC/SM alpha-actin promoter regions, it had no effect of SRF enrichment of c-fos. Taken together, these results provide evidence for a novel mechanism whereby Ca2+ influx via VGCCs stimulates expression of SMC differentiation marker genes through mechanisms that are dependent on ROK, myocardin, and increased binding of SRF to CArG cis regulatory elements.

Quality of care in family planning clinics in Jamaica. Do clients and providers agree?

This paper uses data from 199 providers and 20 simulated clients collected at 50 public sector and Non Governmental Organization (NGO) health facilities islandwide in 1995 to compare the two groups' views on quality of care of family planning services. Each of the five components of quality of care studied can be improved in Jamaica. Nearly two-thirds of the simulated clients felt able to freely choose a contraceptive method; however, more adequate and appropriate information needs to be imparted to clients through improved counselling, including promotion of dual method use (against STD/HIV/AIDS and conception). The requirement that a woman must be menstruating to receive services has inadvertently resulted in many clients going away empty-handed (without counselling or condoms) when they visit family planning clinics. While providers generally treat clients well, training and service delivery practices need to be revised to improve the technical competence of providers. All of the providers would recommend these clinics to others, compared to a little over half of the simulated clients. Both the providers and simulated clients said that privacy should be strengthened, particularly in small facilities in rural areas. Many of these aspects of quality of care are being improved in Jamaica's public sector health facilities. Managers can learn more about quality of care by seeking the knowledge, opinions and experiences of both providers and clients.

Quality of care in family planning clinics in Jamaica. Do clients and providers agree?

This paper uses data from 199 providers and 20 simulated clients collected at 50 public sector and Non Governmental Organization (NGO) health facilities islandwide in 1995 to compare the two groups' views on quality of care of family planning services. Each of the five components of quality of care studied can be improved in Jamaica. Nearly two-thirds of the simulated clients felt able to freely choose a contraceptive method; however, more adequate and appropriate information needs to be imparted to clients through improved counselling, including promotion of dual method use (against STD/HIV/AIDS and conception). The requirement that a woman must be menstruating to receive services has inadvertently resulted in many clients going away empty-handed (without counselling or condoms) when they visit family planning clinics. While providers generally treat clients well, training and service delivery practices need to be revised to improve the technical competence of providers. All of the providers would recommend these clinics to others, compared to a little over half of the simulated clients. Both the providers and simulated clients said that privacy should be strengthened, particularly in small facilities in rural areas. Many of these aspects of quality of care are being improved in Jamaica's public sector health facilities. Managers can learn more about quality of care by seeking the knowledge, opinions and experiences of both providers and clients.

A prodrug approach to the design of cRaf1 kinase inhibitors with improved cellular activity.

Earlier we reported potent cRaf1 kinase inhibitors with a key acidic phenol pharmacophore that had, at best, adequate cellular efficacy. To improve the cellular potency, phenol isosteres and prodrugs were investigated. Many phenol isosteres were synthesized and tested, but failed to provide adequate enzyme potency. A prodrug approach resulted in a 2- to 17-fold improvement over the parent compound in cell-based efficacy.

The discovery of potent cRaf1 kinase inhibitors.

A series of benzylidene-1H-indol-2-one (oxindole) derivatives was synthesized and evaluated as cRaf-1 kinase inhibitors. The key features of the molecules were the donor/acceptor motif common to kinase inhibitors and a critical acidic phenol flanked by two substitutions. Diverse 5-position substitutions provided compounds with low nanomolar kinase enzyme inhibition and inhibited the intracellular MAPK pathway.

Accuracy of strategies for monitoring fetal heart rate in labor.

We investigated whether the accuracy of auscultation could be improved with the use of a heart rate meter. Six fetal heart rate (FHR) traces were presented in a random sequence to 30 subjects using a customized computer program in each of three modalities: auscultation by counting alone, auscultation with the aid of an FHR meter, and visual assessment. The following characteristics were assessed: baseline rate, baseline variability, periodic change, and interpretation of the trace. For baseline rate, counting was associated with consistent underestimation of the FHR, which became more evident as the heart rate increased. The variation observed with each method was greatest with counting. For baseline variability, the proportion of correct responses using a meter was comparable to visual assessment, whereas counting was significantly less effective in 4 of 6 traces. For periodic change, the use of a meter was superior to counting in 4 of 6 traces, but both were inferior to visual assessment in 4 of 6 traces. In the interpretation of the trace, the use of a meter was again superior to counting, but both were inferior to visual assessment. Discrepancies were most marked in the least reassuring traces. Assessment of the FHR is significantly more accurate with the aid of a heart rate meter, and reduces interobserver variation.

A scintillation proximity assay for the Raf/MEK/ERK kinase cascade: high-throughput screening and identification of selective enzyme inhibitors.

We have developed a quantitative scintillation proximity assay (SPA) that reproduces the Raf/MEK/ERK signal transduction pathway. The components of this assay include human cRaf1, MEK1, and ERK2 and a biotinylated peptide substrate for ERK2. cRaf1 was expressed as a his-tagged protein in insect cells in an active form. MEK1 and ERK2 were expressed in Escherichia coli as glutathione S-transferase (GST)-fusion proteins in their inactive forms. ERK2 was removed from the GST portion of the fusion protein by cleavage with thrombin protease. When the purified components are incubated together, cRaf-1 phosphorylates and activates MEK1, MEK1 phosphorylates and activates ERK2, and ERK2 phosphorylates the peptide, biotin-AAATGPLSPGPFA. Phosphorylation of the peptide using [gamma-33P]ATP is detected following binding to streptavidin-coated SPA beads. The assay detects inhibitors of cRaf1, MEK1, or ERK2, and has been used to screen large numbers of compounds. The specific target of inhibition was subsequently identified with secondary assays described herein.

Assessing family planning service-delivery practices: the case of private physicians in Jamaica.

This report presents the results of a study of the family planning service-delivery practices of private physicians in Jamaica. All 367 private physicians in Jamaica who offer family planning services, counseling, or referral were included in the survey. The study revealed that a client seeking services might be given a method by one provider and not by another, and that the methods clients use are likely to be influenced by the providers' preferences. Private physicians in Jamaica are in need of access to current international guidance on contraceptive methods and service practices.

PIP: In 1993 a group of 367 physicians participated in a survey of family planning service-delivery practices. 76% of them were male with an average tenure of 16 years as doctors. 95% worked in urban areas, and 98% counseled their clients, although 1/4 of them received no training in counseling. Only 25% of them knew of The Family Planning Service Delivery Manual guidelines, and only 27% of those who had heard of the manual ever used it. They stated that women could use Depo-Provera after age 20, but 42% of the doctors would require women to have 2 children before using this method. The guidelines recommended the IUD to women who were at least 27 years old after they had had at least 1 child. 27 years was the average recommended age for female sterilization and 31 years for male sterilization. The average systolic and diastolic blood pressure points noted by the physicians were almost the same for both the oral contraceptive pill and Depo-Provera: an average systolic pressure of 138 and diastolic pressure of 89. They were generally interested in the patient's age, smoking habits, any cardiovascular problems, varicose veins, abnormal vaginal bleeding, and blood pressure. In addition, anemia, pelvic inflammatory disease, and sexually transmitted diseases were important considerations for IUD use but were mentioned by only 12%, 41%, and 82% of the doctors, respectively. 68% of them required tests for male sterilization compared to 97% for IUD use. More than 20% of them required urinalysis before prescribing a contraceptive method. The average number of follow-up visits for combined oral contraceptive users was 2.7 in the 1st year and 1.4 in the 2nd year. 67% of the physicians recommended a rest from at least 1 contraceptive method. More than 1 in 10 of all physicians cited opposition to Depo-Provera (12% mostly for safety reasons) and natural family planning (13% for efficacy reasons).

Quality of care among Jamaican private physicians offering family planning services.

The National Family Planning Board is the agency of Government empowered to prepare, carry out and promote family planning programs in Jamaica. The Board has prioritized the expansion and sustainability of family planning services in large part through encouraging the participation of the private sector. To enhance the availability, acceptability and effectiveness of private physician family planning services, information was collected on the service practices of 90% of physicians, through face to face interviews. Bruce's framework was used to evaluate the findings of the study. The study indicated that: A wide variety of contraceptives are available - Basic equipment and adequate supplies are in place for the provision of services - Provider bias, inappropriate contraindicators and process and scheduling hurdles exist. The major recommendations relate to the: Revision of norms and guidelines for all contraceptives - Continuation of contraceptive technology updates for private physicians - Revision of legal/regulatory barriers which restrict access to some contraceptives for certain target groups.

p56lck phosphorylation by Ca2+/calmodulin-dependent protein kinase type II.

Ca2+/calmodulin-dependent protein kinases are implicated in regulating the Ca2+ signaling involved in T cell activation and in thymocyte selection. One of the earliest events in signaling through the T cell antigen receptor is activation of the protein tyrosine kinase p56lck. Following T cell activation or signaling through the IL-2 receptor, Ca(2+)-mediated phosphorylation of p56lck occurs on serine/threonine residues. Isoforms of the multifunctional Ca2+/calmodulin-dependent protein kinases, CaM kinase-II and CaM kinase-Gr are found in human T lymphocytes. CaM kinase-II, but not CaM kinase-Gr, phosphorylates the T cell tyrosine kinase p56lck in vitro. Tryptic phosphopeptide maps indicate that CaM kinase-II phosphorylates p56lck on multiple sites in vitro. Kinase assays of p56lck modified by CaM kinase-II indicate that CaM kinase-II modification does not appreciably affect p56lck phosphotransfer activity.

Site and consequences of the autophosphorylation of Ca2+/calmodulin-dependent protein kinase type "Gr".

CaM kinase-Gr is a Ca2+/calmodulin-dependent protein kinase that is enriched in brain and thymus. The enzyme was isolated from rat cerebellum, which contained alpha (M(r) 65,000) and beta (M(r) 67,000) polypeptides, and rat forebrain, which contained only the alpha polypeptide. Both enzyme preparations readily underwent autophosphorylation with dramatic up-regulation of their Ca2+/calmodulin-dependent, as well as-independent, activity. Autophosphorylation also caused a characteristic retardation in the electrophoretic gel mobility of the alpha and beta polypeptides. Treatment of autophosphorylated CaM kinase-Gr with acid phosphatase fully dephosphorylated the enzyme and reversed the changes in electrophoretic migration of both polypeptides. Phosphopeptide mapping indicated that the alpha and beta polypeptides were phosphorylated on identical or homologous sites, which probably induces similar structural and catalytic modifications in the two polypeptides. The actual site(s) of autophosphorylation was determined by the purification and amino acid sequencing of tryptic peptides from 32P-labeled CaM kinase-Gr. The major site of autophosphorylation was localized to a novel N-terminal domain, which is rich in Ser/Thr/Pro residues. The functional and structural studies on CaM kinase-Gr autophosphorylation imply that the enzyme is comprised of two regulatory domains, one on either side of a catalytic domain, followed by a C-terminal, putative association domain. The properties of such a structural model are discussed.

Quantitative analysis of SH2 domain binding. Evidence for specificity and competition.

We report the development of a quantitative assay for measuring SH2 domain binding in vitro. Using this assay we have analyzed the binding of purified recombinant SH2 domains from ras GTPase activating protein (GAP) and the 85-kDa subunit of phosphatidylinositol 3-kinase (p85) to proteins from epidermal growth factor-stimulated and v-src-transformed cells. The purified recombinant SH2 domains from GAP and p85 bind to the tyrosine phosphorylated epidermal growth factor receptor with nanomolar affinities. Moreover, competition studies suggest that these two proteins bind to equivalent or overlapping sites on this receptor. In v-src-transformed cells the purified recombinant SH2 domains from GAP and p85 bind to distinct but overlapping sets of proteins.

Activation of casein kinase II by sphingosine.

Sphingosine activates casein kinase II in the presence of endogenous substrates as well as a synthetic peptide substrate. The activation response occurred between 12 and 25 micrograms/ml sphingosine and exhibited positive cooperativity with a Hill coefficient of 3.0. Sphingosine not only increased the Vmax of casein kinase II but decreased the Km(app) for the peptide substrate from 0.5 to 0.08 mM. In contrast, the Km(app) for MgCl2 was increased from 0.12 to 0.7 mM. Consequently, sphingosine altered significantly several parameters which determine casein kinase II activity. The effect of sphingosine was relatively specific, inasmuch as related lipids were less potent activators or largely ineffective in stimulating casein kinase II. On the other hand, the effect of sphingosine itself could be potentiated or inhibited by other lipids. Ceramide and sphingosylphosphorylcholine augmented the sphingosine effect. Phospholipids alone did not alter the activity of casein kinase II significantly, but abolished enzyme activation by sphingosine with different potencies (phosphatidylserine greater than phosphatidylethanolamine greater than phosphatidylinositol greater than phosphatidylcholine). Moreover, the sphingosine effect could be abrogated by KCI and NaCl, which alone are known to induce enzyme activation and dissociation of aggregated casein kinase II protein; LiCl and NH4Cl also inhibited the sphingosine effect. Polyamines, known activators of casein kinase II, partially mimicked the effect of sphingosine on endogenous polypeptide phosphorylation but failed to do so with the peptide substrate. These observations demonstrate that sphingosine is a potent activator of casein kinase II. The potential pharmacological and physiological modulation of casein kinase II by sphingoid bases is discussed.

Phosphorylation of a Ras-related GTP-binding protein, Rap-1b, by a neuronal Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr.

A neuron-specific Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr, phosphorylates selectively a Ras-related GTP-binding protein (Rap-1b) that is enriched in brain tissue. The phosphorylation reaction achieves a stoichiometry of about 1 and involves a serine residue near the carboxyl terminus of the substrate. Both CaM kinase Gr and cAMP-dependent protein kinase, but not CaM kinase II, phosphorylate identical or contiguous serine residues in Rap-1b. The rate of phosphorylation of Rap-1b by CaM kinase Gr is enhanced following autophosphorylation of the protein kinase. Other low molecular weight GTP-binding proteins belonging to the Ras superfamily, including Rab-3A, Rap-2b, and c-Ha-ras p21, are not phosphorylated by CaM kinase Gr. The phosphorylation of Rap-1b itself can be reversed by an endogenous brain phosphoprotein phosphatase. These observations provide a potential connection between a neuronal Ca2(+)-signaling pathway and a specific low molecular weight GTP-binding protein that may regulate neuronal transmembrane signaling, vesicle transport, or neurotransmitter release.

The effect of sodium chloride concentration, water content, and protein on the gas chromatographic headspace analysis of ethanol in plasma.

The authors used gas chromatographic headspace analysis to study the sodium chloride concentration dependence of the partitioning of acetonitrile, ethanol, n-propanol, and t-butanol from water and plasma to headspace vapor. Increasing the sodium chloride concentration caused logarithmic increases in the partitioning. At 25 degrees C the slopes (log10[peak height]/mol sodium/L) obtained with the use of water or plasma were as follows: acetonitrile, 0.064 (0.059); ethanol, 0.126 (0.125); n-propanol, 0.152 (0.149); and t-butanol, 0.200 (0.183). Differences in water content between the two liquids may contribute to the small differences in the regression data. More importantly, saturation with sodium chloride at 25 degrees C produced solutions with different sodium molarities: 5.2 mol/L for water and 4.8 mol/L for plasma. This difference in salt concentration at saturation and the volatile dependent slopes can account for a large part of the error in plasma ethanol concentrations when measured with the use of aqueous external standardization and internal standardization with any of the other volatiles. Deproteinization of the plasma abolished the liquid phase-dependent differences in saturated salt concentration and partitioning.

A synaptosomal protein kinase is regulated by phosphatidylinositol 4-phosphate and Mg2+.

Triton X-100 extracts of purified rat brain synaptosomes exhibited marked phosphorylation of an endogenous Mr 87,000 polypeptide following chromatography on DEAE-cellulose. The protein kinase catalyzing this reaction was insensitive to cyclic AMP, Ca2+, calmodulin, and phorbol esters. However, phosphatidylinositol 4-phosphate (PIP) proved to be a potent inhibitor of the Mr 87,000 polypeptide phosphorylation at submicromolar concentrations, whereas phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol were less potent inhibitors. Unsaturated fatty acids could also mimic the effects of PIP at levels above 4 micrograms/ml. The inhibitory effect of PIP largely reflected a profound increase in the apparent Km for Mg2+ such that increasing Mg2+ levels could partially offset the action of PIP. The PIP-sensitive protein kinase was enriched in hypotonic lysates of synaptosomes from which it was partially purified by DEAE-cellulose, hydroxylapatite, and gel permeation chromatography. This purification separated the enzyme from its Mr 87,000 substrate; however, the presence of this polypeptide in heat-inactivated alkali extracts of rat brain provided an exogenous source of substrate which could be used to assay enzyme activity. The relevance of these data to a possible role for PIP and Mg2+ in cellular signaling is discussed.

The effect of biologic specimen type on the gas chromatographic headspace analysis of ethanol and other volatile compounds.

Gas chromatographic analyses of 37 degrees C headspace vapors above liquid phases saturated with sodium chloride demonstrated that the partitioning of isopropanol, n-propanol, and t-butanol from blood, plasma, or serum to headspace vapor was greatly reduced relative to that observed with the use of water as the liquid phase. The partitioning of ethanol and acetone was moderately reduced with these specimens relative to water, while no effect was seen with methanol or acetonitrile. Normal urine only slightly affected relative partitioning, and vitreous humor had no effect. The partitioning reductions observed with blood were not affected by changing the equilibration temperature to 25 degrees C, by lengthening the equilibration time to three days, nor by changing the concentration of the volatiles in the liquid phase. The use of saturated sodium sulfate enhanced the differences in partitioning observed between water and blood. Only with substantial dilution (5:1) of blood specimens was the effect abolished.