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Osteosarcomas account for about 20% of all primary bone neoplasms, and affect predominantly adolescents. Important prognostic factors include the presence or absence of metastases at the time of presentation and tumor response to neoadjuvant chemotherapy. Biological markers predicting metastases and chemoresistance are not well characterized. cDNA microarray analysis enables one to examine the expression of thousands of genes in tumor samples. We performed cDNA microarray analysis of histologically low and high grade areas in osteosarcomas to identify gene expression patterns which may depict aggressive behavior. Microarray analysis with 1.2 K cancer array revealed many differentially expressed genes (both upregulated and downregulated), in histologically high grade tumor samples as compared with a low grade sample. Selected up and down regulated markers in the high grade sarcomas were tested in a group of high grade osteosarcomnas (OS) with varying responses to chemotherapy. Of the multiple markers analyzed, ezrin, a member of the ERM family of membrane-cytoskeleton linkers showed an expression pattern statistically significant between tumors with good response to chemotherapy compared with tumors with poor response (p = 0.036). We discuss our findings, with current review of literature.

The phenomenon of dedifferentiation typically occurs in soft tissue sarcomas where a low grade or well-differentiated tumor shows an abrupt transformation to a high-grade sarcoma without lineage specificity. The biological behavior and metastatic potential of these tumors is dictated by the dedifferentiated phenotype. Tumor material was available from two dedifferentiated liposarcomas. We performed cDNA microarray analysis of a dedifferentiated liposarcoma in which the atypical lipomatous/well-differentiated and dedifferentiated portions were grossly distinct, to find differentially expressed genes in the dedifferentiated component compared to the well-differentiated component. There were 100 differentially expressed genes, both up- and down-regulated in the high grade sarcoma. In addition, we performed RT-PCR on selected genes in both cases to confirm the microarray findings. We discuss the expression patterns of these genes in comparison to other studies in the literature.

The term peripheral arterial disease (PAD) is often used to describe atherosclerosis involving the arteries supplying the lower extremities. Potentially modifiable factors that predispose to the development and progression of both symptomatic and asymptomatic PAD include smoking, diabetes mellitus, hyperlipidemia, and hypertension. Since the same risk factors for PAD predispose to the development of systemic atherosclerosis, identification of PAD increases the likelihood of coexistent coronary heart and cerebrovascular disease. Even after adjustment for risk factors, PAD appears to increase the risk for ischemic manifestations involving these other vascular territories with about a 2-fold increase in myocardial infarction and perhaps stroke. The most dramatic consequence of PAD is impaired survival with a 2- to 3-fold increased risk of 5- to 10-year mortality. While the adverse cardiovascular and cerebrovascular complications are highest for persons with more severe PAD, there is still a significant risk in persons with mild and even asymptomatic disease. The focus in the management of PAD should be on early diagnosis and efforts to reduce the risk of adverse events including risk factor modification and antiplatelet therapy.

Adult T-cell leukemia (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) are associated with Human T-cell lymphotropic virus type 1 (HTLV-1) infection. The viral transactivator, Tax is able to mediate the cell cycle progression by targeting key regulators of the cell cycle such as p21/waf1, p16/ink4a, p53, cyclins D1–3/cdk complexes, and the mitotic spindle checkpoint MAD apparatus, thereby deregulating cellular DNA damage and checkpoint control.

New Horizons Bands are wind and percussion bands designed for both novice and former musicians over the age of 50. This article describes the Iowa City, Iowa, program from the director's observations and from excerpted comments by participants. Not only does the band program fulfill musical aspirations, but it also provides meaningful interpersonal relationships for the adult learners and their families, the college-aged instructors, and the director.

This article briefly reviews some basic issues of adult development and measures used in assessing quality of life as a background for reviewing music studies with healthy older adults. Research shows that music activities (both music listening and music making) can influence older adults' perceptions about the quality of their lives. Some research has examined the effects of music listening on biological markers of health and subjective perceptions of well-being. Other studies on the psychological and social benefits associated with music making activities have demonstrated that participants often place considerable value on these "nonmusical" benefits of music activity. In 1900, approximately 4% of the United States population was over the age of 65. By 2010, as the baby boom generation begins to turn 65, this proportion is expected to exceed 13% (Hooyman & Kiyak, 1996). Not only are there proportionally more senior Americans, it is more commonplace to discuss the concept of adult development, rather than assume that all development plateaus after adolescence, or worse, is merely a narrative of decline. Old misconceptions that stereotyped the older adult as frail, uninterested in learning or relearning skills, prone to passive activities, and unable or unwilling to strive for excellence, have gradually given way to images of vigorous individuals interested in active lifestyles during their retirement years. Accompanying this shift in attitudes has been an increased focus on improving the quality of life for older adults instead of merely trying to extend the quantity or length of life. Historically, music educators and researchers have concentrated on the teaching and learning of music in children. In an era when older adults are beginning to outnumber young people in the United States, it is important for music researchers, educators and therapists to examine ways that music can enhance the quality of life in senior adults. Several music educators have reported on healthy older adults enjoying a variety of music making experiences, including choral groups (Darrow, Johnson, & Ollenberger, 1994; Frego, 1995), wind bands (Coffman & Levy, 1997; Ernst E Senate Hearings, 1992). Several concepts have been used interchangeably with the term quality of life, including life satisfaction, meaning of life, meaning in life, sense of purpose, successful aging, well-being (mental, emotional, social, spiritual), and wellness. Some of these terms are closely linked to indicators of social and economic indicators of success, while other terms connote mental and physical health concerns that go beyond the mere absence of disease and disability. A useful list of 15 factors that contribute to quality of life was arranged by Flanagan (1982, p. 57) into five groups: Physical and material well-being: 1. Material comforts-desirable home, food, conveniences, security 2. Health and personal comforts Relations with other people: 3. Relationships with relatives 4. Having and rearing children 5. Close relationships with spouse or member of opposite sex 6. Close friends-sharing views, interests, activities Social, community, and civic activities: 7. Helping and encouraging others 8. Participating in governmental and local affairs Personal development and fulfilment: 9. Learning, attending school, improving understanding 10. Understanding yourself and knowing your assets and limitations 11. Work that is interesting, rewarding, worthwhile 12. Expressing yourself in a creative manner Recreation: 13. Socializing with others 14. Reading, listening to music, or watching sports, other entertainment 15. Participation in active recreation (p. …

The synaptotagmin (Syt) family of proteins contains tandem C2 domains, C2A and C2B, which bind membranes in the presence of Ca(2+) to trigger vesicle fusion during exocytosis. Despite recent progress, the role and extent of interdomain interactions between C2A and C2B in membrane binding remain unclear. To test whether the two domains interact on a planar lipid bilayer (i.e., experience thermodynamic interdomain contacts), diffusion of fluorescent-tagged C2A, C2B, and C2AB domains from human Syt7 was measured using total internal reflection fluorescence microscopy with single-particle tracking. The C2AB tandem exhibits a lateral diffusion constant approximately half the value of the isolated single domains and does not change when additional residues are engineered into the C2A-C2B linker. This is the expected result if C2A and C2B are separated when membrane-bound; theory predicts that C2AB diffusion would be faster if the two domains were close enough together to have interdomain contact. Stopped-flow measurements of membrane dissociation kinetics further support an absence of interdomain interactions, as dissociation kinetics of the C2AB tandem remain unchanged when rigid or flexible linker extensions are included. Together, the results suggest that the two C2 domains of Syt7 bind independently to planar membranes, in contrast to reported interdomain cooperativity in Syt1.

IL-10 is overexpressed in human chronic lymphocytic leukemia (CLL), and is an autocrine growth factor involved in the development of malignant B1 clones in NZB mice, a murine model for CLL. Antisense IL-10 oligonucleotide treatment induces apoptosis and cell cycle disruption in these cells both in vitro and in vivo. In addition, NZB IL-10 knock-out mice fail to develop the B-1 clones. Dampening of IL-10 protein production via antisense IL-10 oligonucleotide treatment is correlated with decreased p27/Kip1 protein expression which results in increased cyclin D2, cyclin E and cyclin A associated kinase activity. The action of the antisense oligonucleotides is through alterations in cell cycle regulation, resulting in accelerated cell cycle progression, a G2/M block which culminates in apoptosis induction in the malignant cells. This implies that the role of IL-10 as an autocrine growth factor in malignant B-1 cells lies in its ability to inhibit apoptosis induction through the maintenance of sustainable cell cycle progression in malignant cells.

The transcription factor B-cell-specific activator protein (BSAP) plays an important role in B-cell development. We explored the involvement of BSAP in the growth regulation of malignant B-1 cells derived from the NZB murine model of human chronic lymphocytic leukemia. BSAP protein was found in normal B-2 cells, elevated in normal B-1 cells, and highest in NZB malignant B-1 cells. When these malignant B-1 cells were treated with antisense oligonucleotides for BSAP, their growth was inhibited with a G2/M phase arrest. In contrast, B cell lines that did not appear to be of B-1 origin (IgG+/B220+/BSAPlow) were unaffected by treatment with antisense BSAP oligonucleotides. Centrifugal elutriation experiments showed that BSAP mRNA was expressed at the highest levels in the G2/M phases in malignant B-1 cells. Treatment with demecolcine (Colcemid), a known mitotic blocker, resulted in a decrease in the level of BSAP gene expression in malignant B-1 cells, further demonstrating links between BSAP expression and successful G2/M transition in the cell cycle. These data suggest a correlation between BSAP and the development of B-1 malignancy, perhaps through the regulation of cell-cycle progression.

p53/56(lyn) is a member of the src family that is predominantly expressed in hematopoietic cells and is thought to play a role in cellular proliferation. In this study, we demonstrate the participation of p53/56(lyn) in 1,25-dihydroxyvitamin D(3) (1, 25D(3))-induced growth arrest in HL60 cells. We show that the mRNA and protein levels of p53/56(lyn) are markedly elevated after 1, 25D(3) treatment, which is accompanied by an increase of p53/56(lyn) kinase activity. We also demonstrate that treatment with p53/56(lyn) antisense oligodeoxynucleotides reverses the 1,25D(3)-induced G1/S block, and results in an accumulation of cells with S-phase DNA content. BrdU pulse-chase experiments reveal that this accumulation results from an increased proportion of cells actively synthesizing DNA, which are inhibited from exiting the S-phase compartment. These results indicate that upregulation of p53/56(lyn) contributes significantly to the G1/S growth arrest induced by 1,25D(3) in HL60 cells and thus its activation may be a desirable outcome of chemotherapeutic regimens.

Measurements of submicron aerosol mass and the mass of major ionic components have been made over the past 5 years on cruises in the Pacific and Southern Oceans and at monitoring stations across North America (Barrow, Alaska; Cheeka Peak, Washington; Bondville, Illinois; and Sable Island, Nova Scotia). Reported here are submicron concentrations of aerosol mass, nonsea salt (nss) sulfate, sea salt, methanesulfonate, other nss inorganic ions, and residual, or chemically unanalyzed, mass. Residual mass concentrations are based on the difference between simultaneously measured aerosol mass and the mass of the major ionic components. A standardized sampling protocol was used for all measurements making the data from each location directly comparable. For the Pacific and Southern Oceans, concentrations of the chemical components are presented in zonally averaged 20° latitude bins. For the monitoring stations, mean concentrations are presented for distinct air mass types (marine, clean continental, and polluted based on air mass back trajectories). In addition, percentile information for each chemical component is given to indicate the variability in the measured concentrations. Mean nss sulfate submicron aerosol mass fractions for the different latitude bins of the Pacific ranged from 0.14±0.01 to 0.34±0.03 (arithmetic mean±absolute uncertainty at the 95% confidence level). The lowest average value occurred in the 40°–60°S latitude band where nss sulfate concentrations were low due to the remoteness from continental sources and sea salt concentrations were relatively high. Mean nss sulfate aerosol mass fractions were more variable at the monitoring stations ranging from 0.13±0.004 to 0.65±0.02. Highest values occurred in polluted air masses at Bondville and Sable Island. Sea salt mean mass fractions ranged between 0.20±0.02 and 0.53±0.03 at all latitude bands of the Pacific (except 20°–40°N where the residual mass fraction was relatively high) and at Barrow. The concentration of residual mass was significant at the 95% confidence level at all stations and all Pacific latitude bands (assuming that all errors were random and normally distributed and contamination of the samples did not occur beyond that accounted for by storage and transport uncertainties). Mean residual mass fractions ranged from 0.09±0.07 to 0.74±0.04.

We have previously shown that apoptosis induced in thymocytes by dexamethasone or teniposide (VM-26) could be inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and sangivamycin, both relatively specific inhibitors for protein kinase C, but not by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a more specific inhibitor for cAMP-dependent protein kinases. Apoptosis in this model system was not blocked by EGTA and no increase in cytosolic Ca2+ was observed during apoptosis induced by either dexamethasone or VM-26, suggesting that this kinase was Ca2+-independent. In the present study, we demonstrate that addition of 10 microM sangivamycin to thymocyte cultures up to 2 h after addition of either inducer resulted in virtually complete inhibition of apoptosis. Addition of 10 microM sangivamycin at 3 or 4 h after addition of inducer resulted in partial inhibition of apoptosis. Computerized image analysis of two-dimensional PAGE analyses of whole-cell lysates demonstrated that treatment of mouse thymocytes with VM-26 resulted in a limited number of de novo phosphorylation events within 1 h of treatment. The most prominent phosphorylation events associated with VM-26-induced apoptosis were that two intracellular protein species (Protein 1: m.w. = 22.9 kDa, pI, 5.11; and Protein 2: m.w. = 22.9 kDa, pI, 4.98). Similar phosphorylation events were seen in cells treated with dexamethasone. Finally, Western blot analysis suggests that de novo protein phosphorylation induced by VM-26 is on serine/threonine residues. These results provide further evidence that the mechanism of VM-26-induced apoptosis of murine thymocytes involves the action of one or more serine/threonine kinases.

Butyrolactone I [α-oxo-β-( p -hydroxyphenyl)-γ-( p -hydroxy- m -3,3-dimethylallyl-benzyl)-γ-methoxycarbonyl-γ-butyrolactone] is produced as a secondary metabolite by Aspergillus terreus . Because small butyrolactone-containing molecules act as self-regulating factors in some bacteria, the effects of butyrolactone I on the producing organism were studied; specifically, changes in morphology, sporulation, and secondary metabolism were studied. Threefold or greater increases in hyphal branching (with concomitant decreases in the average hyphal growth unit), submerged sporulation, and secondary metabolism were observed when butyrolactone I was added to cultures of A. terreus . Among the secondary metabolites whose production was increased by this treatment was the therapeutically important compound lovastatin. These findings indicate that butyrolactone I induces morphological and sporulation changes in A. terreus and enhances secondary metabolite production in a manner similar to that previously reported for filamentous bacteria.

Drug resistance that occurs during cancer chemotherapy has been a major problem in controlling neoplastic progression. To study the cellular mechanisms of acquired drug resistance we developed 1,25-dihydroxyvitamin D3 (1,25D3)-resistant sublines of promyelocytic leukemia HL60 cells which have increased proliferation rates (Exp. Cell Res., 224, 312, 1996; Cancer Res., 50, 5513, 1996). We report here that the resistant sublines display varying degrees of shortening of the G1 phase as compared to the parental HL60-G cells. Protein levels of cyclins E, D1, D2 and D3 are elevated in these resistant cell lines, and cyclin D1 is especially high in 40AF cells, which has the shortest G1 length. The protein levels of cyclin-dependent kinase (Cdk)2, Cdk4 and Cdk6 are not altered in the resistant sublines. Both Cdk2 and Cdk6-associated kinase activites are increased in the resistant sublines, but not Cdk4 kinase activity. Protein levels of p27Kip1 are not consistently altered in the resistant sublines as compared to the parental HL60-G cells, but are reduced relative to HL60-G cells arrested by 96 h treatment with 1,25D3. Interestingly, the resistant cell lines constitutively express high levels of retinoblastoma protein (pRb), and pRb is highly phosphorylated, indicating that the G1 cyclin/Cdk complexes in the resistant cells are physiologically active. The results suggest that the increased activity of cyclin D/Cdk6, and perhaps cyclin E/Cdk2, lead to rapid hyperphosphorylation of pRb and consequently a shorter early G1 phase, and that in the resistant cells the increased ratio of cyclin E to p27Kip1 results in activation of Cdk2 and contributes to the abrogation of the 1,25D3-induced block to the S phase entry. Additionally, it is apparent that constitutively increased levels of pRb are compatible with increased rates of cell proliferation.

This report describes a murine monoclonal IgM antibody, 6E3.C4, induced by retrovirus infection of BALB/ c-H-2b mice which inhibits mitogen stimulation of both mouse and human lymphocytes in vitro. The molecule bound by this antibody appears to be an activation antigen since binding is upregulated by mitogen stimulation. Analysis of the epitope bound by mAb 6E3.C4 revealed that it is associated with a 52-kDa protein with a pI of approximately 5.7 as determined by Western blot analysis. A protein expressing this or a cross-reactive epitope was isolated and determined to be alpha-tubulin by amino acid sequencing. Reactivity with purified alpha-tubulin confirms this identification. These findings suggest a potential role for a cell surface molecule that is either alpha-tubulin or a cross-reactive molecule in the activation processes of T cells.

Emissions factors (EFs) for gas and sub-micron particle-phase species were measured in intercepted plumes as a function of vessel speed from an underway research vessel, the NOAA Ship Miller Freeman, operating a medium-speed diesel engine on low-sulfur marine gas oil. For many of the particle-phase species, EFs were determined using multiple measurement methodologies, allowing for an assessment of how well EFs from different techniques agree. The total sub-micron PM (PM1) was dominated by particulate black carbon (BC) and particulate organic matter (POM), with an average POM / BC ratio of 1.3. Consideration of the POM / BC ratios observed here with literature studies suggests that laboratory and in-stack measurement methods may over-estimate primary POM EFs relative to those observed in emitted plumes. Comparison of four different methods for black carbon measurement indicates that careful attention must be paid to instrument limitations and biases when assessing EFBC. Particulate sulfate (SO42−) EFs were extremely small and the particles emitted by Miller Freeman were inefficient as cloud condensation nuclei (CCN), even at high super saturations, consistent with the use of very low sulfur fuel and the overall small emitted particle sizes. All measurement methodologies consistently demonstrate that the measured EFs (fuel mass basis) for PM1 mass, BC and POM decreased as the ship slowed. Particle number EFs were approximately constant across the speed change, with a shift towards smaller particles being emitted at slower speeds. Emissions factors for gas-phase CO and formaldehyde (HCHO) both increased as the vessel slowed, while EFs for NOx decreased and SO2 EFs were approximately constant.

Telomere integrity is critical for telomere function and genomic stability. We previously demonstrated that non-erythroid α-spectrin (αIISp) is present in mammalian cell nuclei where it is important in repair of DNA interstrand cross-links (ICLs) and chromosome stability. We now demonstrate that αIISp is also important for telomere maintenance after ICL damage. It localizes to telomeres in S phase after ICL damage where it has enhanced association with TRF1 and TRF2 and is required for recruitment of the ICL repair protein, XPF, to damage-induced foci at telomeres. In telomerase-positive normal cells depleted of αIISp by siRNA or in Fanconi anemia, complementation group A (FA-A) cells, where αIISp levels are 35–40% of normal, ICL damage results in failure of XPF to localize to telomeres, markedly increased telomere dysfunction-induced foci, followed by catastrophic loss of telomeres. Restoration of αIISp levels to normal in FA-A cells corrects these deficiencies. Our studies demonstrate that αIISp is critical for repair of DNA ICLs at telomeres, likely by facilitating the recruitment of repair proteins similar, but not identical, to its proposed role in repair of DNA ICLs in genomic DNA and that this function in turn is critical for telomere maintenance after DNA ICL damage.

Biological organisms and their component organs, tissues and cells have unique electrical impedance properties. Impedance properties often change with changes in structure, composition, and metabolism, and can be indicative of the onset and progression of disease states. Over the past 100 years, instruments and analytical methods have been developed to measure the impedance properties of biological specimens and to utilize these measurements in both clinical and basic science settings. This chapter will review the applications of impedance measurements in the biomedical sciences, from whole body analysis to impedance measurements of single cells and cell monolayers, and how cellular impedance measuring instruments can now be used in high throughput screening applications.

The role of a 150-kD SR-cyclophilin (NK-TR1) in monocyte differentiation was investigated. Using an antipeptide monoclonal antibody, we have detected NK-TR1 in human peripheral blood monocytes and HL-60 cells. Unstimulated monocytes showed a low intracellular level of NK-TR1 protein that increased over 3 days of lipopolysaccharide + interferon-gamma treatment, consistent with the kinetics of monocyte differentiation. Normal HL-60 cells also had a low level of NK-TR1 protein, and exposure to 1.25% dimethyl sulfoxide (DMSO) resulted in a marked transient increase in expression that returned to basal levels before the development of granulocyte differentiation-associated biochemical changes. Phorbol myristate acetate, a promoter of monocytic differentiation in HL-60 cells, also caused a significant increase in NK-TR1 over basal levels. Transfection of a vector expressing NK-TR1 antisense RNA into HL-60 cells suppressed DMSO-mediated growth arrest. In addition, the development of a more mature phenotype, as measured by expression of CD16, and the ability to reduce nitroblue tetrazoleum dye was inhibited in transfectants when compared with controls. These results are consistent with the hypothesis that the NK-TR1 gene product is required for the progression towards a mature differentiated phenotype.

Synaptotagmin (Syt) triggers Ca(2+)-dependent membrane fusion via its tandem C2 domains, C2A and C2B. The 17 known human isoforms are active in different secretory cell types, including neurons (Syt1 and others) and pancreatic β cells (Syt7 and others). Here, quantitative fluorescence measurements reveal notable differences in the membrane docking mechanisms of Syt1 C2A and Syt7 C2A to vesicles comprised of physiological lipid mixtures. In agreement with previous studies, the Ca(2+) sensitivity of membrane binding is much higher for Syt7 C2A. We report here for the first time that this increased sensitivity is due to the slower target membrane dissociation of Syt7 C2A. Association and dissociation rate constants for Syt7 C2A are found to be ~2-fold and ~60-fold slower than Syt1 C2A, respectively. Furthermore, the membrane dissociation of Syt7 C2A but not Syt1 C2A is slowed by Na(2)SO(4) and trehalose, solutes that enhance the hydrophobic effect. Overall, the simplest model consistent with these findings proposes that Syt7 C2A first docks electrostatically to the target membrane surface and then inserts into the bilayer via a slow hydrophobic mechanism. In contrast, the membrane docking of Syt1 C2A is known to be predominantly electrostatic. Thus, these two highly homologous domains exhibit distinct mechanisms of membrane binding correlated with their known differences in function.

This article contextualizes what community music ensembles mean, and then provides examples of them in practice. It proposes three broad perspectives of community music: (1) community music as the “music of a community”; (2) community music as “communal music-making”; and (3) community music as an active intervention between a music leader or leaders and participants. The article suggests that participating in community ensembles is much more than just music-making—it can have life-changing effects.

Because conditions of culture in vitro cannot be identical to those in the living system, cell lines cultured in vitro are referred to as ‘model systems’. Not all cell types are capable of differentiation in vitro. Stem cells are of various types, including embryonic stem cells (ESC), mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), cancer/leukaemia stem cells (CSCs/LSCs), etc. Procedures for studying differentiation in model systems depend on the type of culture that can be employed. Techniques employed include liquid suspension cultures, monolayer cultures and complex culture systems involving semisolid matrices or liquid overlay methods. Cells derived from various tissues can be maintained in culture known as ‘primary culture’. Only a rare human cell in a culture can undergo changes in its molecular machinery known as ‘immortalisation’ which allow it to propagate indefinitely. Recent developments include systems optimised for stem cell differentiation. Such in vitro culture techniques have applications to the study of differentiation in basic research and clinical applications. Key Concepts: Mammalian cells derived from many tissues, the subject of this article, can propagate outside of the body. Stem cells can differentiate, but provide a reservoir for development of functional cells of various types which is known as ‘lineage expansion’. Stem cells give rise to progenitor cells (PCs) which can differentiate into mature cells, but have a limited repertoire of lineages of differentiation. Mature cells can be induced to have the capabilities of stem cells and PCs by introduction of four or less transcription factors (TFs), for example, Oct4, Sox2, Klf4 and c-Myc, under defined conditions. The cell environment – other cells, extracellular material – can favour lineage expansion into mature, functional cells and is called a ‘niche’. Keywords: differentiation; induction of differentiation; assessment of differentiation; cell cycle; culture conditions

Synaptotagmin (Syt) triggers Ca2+-dependent membrane fusion during secretion via its tandem C2 domains, termed C2A and C2B. The seventeen known human isoforms are active in different secretory cell types, including neurons (SytI and others) and pancreatic β cells (SytVII and others). Here, quantitative fluorescence measurements reveal notable differences in the membrane docking affinities, kinetics, and molecular driving forces for C2A and C2B domains from SytI and SytVII, using vesicles comprised of physiological target lipid mixtures. In agreement with previous studies, the Ca2+ sensitivity of membrane binding is greater for both domains from SytVII than for their counterparts in SytI. We demonstrate that for C2A, this increased sensitivity is due to a stronger SytVIIC2A membrane interaction, which involves substantial contribution from the hydrophobic effect. Association and dissociation rate constants for both SytVII domains are found to be significantly slower than their counterparts in SytI. For SytVIIC2A, the dissociation rate constant is ∼50-fold slower than SytIC2A and is reminiscent of the cPLA2C2 domain that is known to insert deeply into membranes. Addition of sodium sulfate decreases the dissociation rate of SytVIIC2A but not SytIC2A, further indicating that hydrophobic contacts play a major role in SytVIIC2A membrane docking. Thus, SytVIIC2A docks to membranes via both hydrophobic and electrostatic interactions, while the membrane docking interaction of SytIC2A is predominantly electrostatic. The inclusion of phosphatidylinositol-4,5-bisphosphate (PIP2) in membrane mixtures leads to increased affinity and slower dissociation for both C2B domains, but has minimal effects on C2A domains. Overall, highly homologous domains from these two proteins exhibit distinct mechanisms of membrane binding that may reflect their functions in different cell types.

The effects of NG-monomethyl-L-arginine (L-NMMA) on total finger and forearm, and dorsal finger and forearm skin, blood flows were studied in the basal state and during reflex sympathetic vasoconstriction in normal subjects. Total flows were measured by venous occlusion plethysmography and skin flows by laser-Doppler flowmetry (LDF). L-NMMA in doses of 2, 4, and 8 microM/min given by constant infusion via a brachial artery catheter significantly decreased finger blood flow, forearm blood flow, and vascular conductances. At 8 microM/min, total finger blood flow decreased 38.4% and forearm blood flow decreased 24.8%. Dorsal finger and forearm skin LDF were also significantly decreased (25 and 37% at 8 microM/min). Body cooling significantly decreased finger blood flow (73.6%), vascular conductance, and finger LDF (59.7%). L-NMMA had no effect on total finger blood flow or dorsal finger LDF during body cooling. Nitric oxide or related compounds contribute to the basal dilator tone of the dorsal finger and forearm skin but not during reflex sympathetic vasoconstriction.

The supernatant of Mycoplasma arginini-infected murine L5178Y T lymphoma cell cultures (SN-L51) synergizes with small concentrations of IFN-gamma to activate murine peritoneal, thioglycollate-elicited macrophages (M phi) to exhibit cytostatic activity against tumor cells. Treatment of M phi with IFN-gamma and SN-L51 sequentially, but not in the reverse order, activates M phi, which indicates that SN-L51 contains a M phi-triggering factor (MTF). MTF activity could be inhibited by small concentrations of prostaglandin E2, but not by polymyxin B. M phi activated by IFN-gamma plus MTF produce cytostatic effects on tumor cells through a nitric oxide-dependent pathway. MTF activity in SN-L51 is associated with infection of L5178Y cells by M. arginini. Mycoplasma-free L5178Y cells do not produce MTF activity, infection of these L5178Y cells with M. arginini generates the activity, and supernatants of pure M. arginini cultures contain MTF activity. MTF activity is thermostable and resistant to acid, dilute alkali, proteases, and nucleases. MTF was partially purified by ammonium sulfate precipitation, chromatography, electrophoresis, and electroelution. On 12.5% SDS-urea gels, MTF activity migrated with a molecular mass of 2.5 to 4 kDa. MTF activity and the silver staining of this band was resistant to proteinase K; however, Coomassie staining of this band was abolished by proteinase K. The combined data suggest that MTF is either a stable peptide or a peptide linked to lipid or carbohydrate.

In this study, we compared the efficiency, reliability, validity, and motivational benefits of computerized-adaptive and self-adapted music-listening tests (referred to hereafter as CAT and SAT, respectively). Junior high school general music students completed a tonal memory CAT, a tonal memory SAT, standardized music aptitude and achievement tests; and questionnaires assessing test anxiety, demographics, and attitudes about the CAT and SAT. Standardized music test scores and music course grades served as criterion measures in the concurrent validity analysis. Results showed that the SAT elicited more favorable attitudes from examinees and yielded ability estimates that were higher and less correlated with test anxiety than did the CAT. The CAT, however, required fewer items and less administration time to match the reliability and concurrent validity of the SAT and yielded higher levels of reliability and concurrent validity than the SAT when test length was held constant. These results reaffirm importa...

We previously investigated the role of an aprotinin-binding protein (ADR) in the initiation of DNA replication in isolated quiescent nuclei. In the present study, we have used a cell-free DNA replication system to test the ability of plasmid vectors which contain sequences from the human ribosomal RNA gene to serve as replicative templates in vitro when exposed to ADR-containing preparations. Significant dTTP incorporation was seen using DNA from either a 7-kb sequence in the 5' spacer region (CHE) or a 7-kb sequence which begins near the end of the 28S coding region and extends into the 3' spacer region (ADBB), while sequences from other regions of the rRNA gene mediated little or no dTTP incorporation. The characteristics of plasmid-directed dTTP incorporation indicate that most incorporation is due to DNA replication and not repair or damage-initiated processes. To conclusively demonstrate origin-dependent replication in the plasmid system and to further map replication origins, an approach was developed using ddGTP to restrict the length of daughter strands followed by hybridization of these replication products to restriction fragments spanning the putative origin region. This approach allowed us to identify replication origin activity apart from parent strand repair or synthesis initiated at random damaged sites. One of the origins was localized to a 1375-bp fragment within the 5' spacer region, and this fragment contains sequences homologous to those found in other replication origins.

The epipodophyllotoxins, etoposide (VP-16) and teniposide (VM-26), inhibit topoisomerase II activity by stabilization of the cleavable complex between the enzyme and DNA and formation of protein-bound double-stranded DNA breaks. While it is thought that these agents are cytotoxic by preventing cells from completing the S phase or undergoing mitosis, recent evidence suggests that these agents are also potent inducers of programmed cell death or apoptosis in both normal and malignant cells. We have examined the intracellular pathway leading to epipodophyllotoxin-induced apoptosis in normal mouse thymocytes. Epipodophyllotoxin-induced apoptosis may proceed via a mechanism that is independent of inhibition of topoisomerase activity per se because novobiocin and coumermycin, which inhibit the ATPase subunit of topoisomerase II, were relatively inefficient inducers of apoptosis in these cells, under conditions where strong apoptosis by the epipodophyllotoxins and dexamethasone could be observed. In addition, camptothecin, which inhibits topoisomerase 1 by stabilization of the cleavable complex between that enzyme and DNA, was also a poor inducer of apoptosis in these cells. Our data suggest that epipodophyllotoxin-induced mouse thymocyte apoptosis, like that induced by dexamethasone, proceeds via a mechanism that involves protein kinase C (PKC) or a similar enzyme. Apoptosis induced by VM-26 or by dexamethasone was inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (H7), an inhibitor of both PKC and cAMP-dependent protein kinases, but was relatively unaffected by N -(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a more specific inhibitor of cAMP-dependent protein kinases. A more specific inhibitor of PKC, sangivamycin, also inhibited both VM-26-induced and dexamethasone-induced apoptosis. Both VM-26- and dexamethasone-induced apoptosis were unaffected by EGTA, a calcium (Ca 2+ ) chelator, under conditions that inhibited apoptosis induced by the Ca 2+ ionophore A23187. Moreover, while strong increases in intracellular Ca 2+ were observed in thymocytes treated with A23187, we failed to detect increases in intracellular Ca 2+ in cells induced to apoptose with either VM-26 or dexamethasone within the first 2 hr of culture. These results suggest that in mouse thymocytes there are at least two intracellular pathways leading to apoptosis: one, utilized by glucocorticoid and the epipodophyllotoxins, that proceeds in the absence of detectable increases in intracellular Ca 2+ and possibly requires a novel Ca 2+ -independent PKC-like enzyme and another, utilized by Ca 2+ ionophores, that is at least partially dependent on increased intracellular Ca 2+ .

Cytosolic extracts from actively dividing lymphoid cells have been shown to induce DNA synthesis in isolated, quiescent nuclei. An initiating factor in such extracts (activator of DNA replication; ADR) is a >90-kDa aprotinin-binding protein whose activity is inhibitable not only by aprotinin, but also by several other protease inhibitors as well. Although cytosol from nonproliferating lymphocytes is devoid of ADR activity, we have shown that these preparations can be induced to express ADR activity by brief exposure to a membrane-enriched fraction of spontaneously proliferating MOLT-4 cells via a kinase-dependent mechanism. In the present study, we examine the role of tyrosine kinases in this process. Three inhibitors of tyrosine kinases (genistein, kaempferol, and quercetin) can inhibit the in vitro generation of ADR activity. In vitro generation of ADR activity is associated with the de novo phosphorylation of several proteins, many of which are detectable using anti-phosphotyrosine monoclonal antibodies. ADR itself may be tyrosine phosphorylated in active form as immunoprecipitation using such monoclonal antibodies leads to the depletion of its activity. Moreover, immunoprecipitation results in the removal of several de novo tyrosine-phosphorylated proteins, including species at approximately 122, 105, 93, 86, 79, and 65 kDa. A subset of de novo-phosphorylated proteins, migrating at approximately 105, 93, and 70 kDa, also bound to aprotinin, suggesting that at least one of these proteins may represent ADR itself.

Isolated cell nuclei were used as the source of template DNA to investigate the role of a cytosolic aprotinin-binding protein (ADR) in the initiation of eukaryotic DNA replication. Computerized image cytometry demonstrated that the DNA content of individual nuclei increased significantly following incubation with ADR-containing preparations, and the extent of DNA synthesis is consistent with that allowed by the limiting concentration of dTTP. Thus, dTTP incorporation into isolated nuclei represents DNA synthesis and not parent strand repair. We found that dTTP incorporation into the isolated nuclei is dependent on DNA polymerase alpha (a principal polymerase in DNA replication) but that DNA polymerase beta (a principal polymerase in DNA repair processes) does not play a significant role in this system. Finally, neither aprotinin nor a previously described cytosolic ADR inhibitor can block the replication of nuclease-treated calf thymus DNA, while both strongly inhibit replication of DNA in isolated nuclei. This result, coupled with the relative ineffectiveness of nuclease-treated DNA compared with nuclear DNA to serve as a replicative template in this assay, argues against a significant contribution from repair or synthesis which initiates at a site of DNA damage. These data indicate that ADR-mediated incorporation of 3H-dTTP into isolated nuclei results from DNA replicative processes that are directly relevant to in vivo S phase events.

Experts believe that comparative-effectiveness research (CER) can substantially reduce future health care spending and improve the quality of care.1,2 Their analyses indicate that CER can control costs if its results are used to inform coverage, payment, and cost-sharing policies that provide incentives for appropriate and cost-effective care.1,2 But the proposed approach to CER in the United States would constrain these uses of the research, to avoid any implication that health care will be rationed. Though the word elicits fear and opposition, “rationing” is simply the equitable, or rational, distribution of resources; it involves delivering health care services according . . .

The purpose of this study was to examine the feasibility of applying the mathematics of information theory to the analysis of musical originality. Originality can be thought of, in information theory, as entropy, the degree of freedom a composer has in selecting musical symbols to convey a message. Theoretically, original music has high information, or low predictability. Seventh grade students (N = 34) enrolled in a nine-week general music course were asked to create a composition on a MIDI keyboard prior to receiving course instruction and once again at the end of the course. The pitch content of pre- and post-instruction compositions were analysed for levels of entropy. Results showed that post-instruction compositions exhi- bited significantly higher pitch entropy, indicating greater freedom of choice and less predictability in pitch choices, despite a sizeable reduction in the average length of compositions.

We present a study of the decay {ital J}/{psi}{r arrow}{gamma}{eta}{pi}{sup +}{pi}{sup {minus}} using the Mark III detector at the SLAC {ital e}{sup +}{ital e{minus}} storage ring SPEAR. A partial-wave amplitude analysis is performed on the {eta}{pi}{sup +}{pi}{sup {minus}} system in the mass region from 1.2 to 1.5 GeV. We observe two {eta}{pi}{sup +}{pi}{sup {minus}} resonances, each decaying via {ital a}{sub 0}(980){pi}. We identify one as the axial-vector meson {ital f}{sub 1}(1285) and the other as a pseudoscalar resonance at 1.40 GeV.

The adherence of cells to microvascular endothelium is important in a number of processes, including inflammatory responses and metastasis. It has been demonstrated that in human models, cytokines such as TNF, IL-1, IFN-gamma increase the adhesiveness of endothelium for cells of the immune and inflammatory system by stimulating the expression of cell adhesion molecules on endothelial cell surfaces. We and others have shown similar cytokine-induced endothelial adhesiveness for tumor cells in murine and human models. In contrast to the effect of those modulators, transforming growth factor-beta (TGF-beta) has been shown to inhibit the binding of human neutrophils and T lymphocytes to human endothelium, although the mechanism of TGF-beta action remains unknown. Little is known about the effect of TGF-beta on tumor cell-endothelial interaction. In the present study, we demonstrate that TGF-beta inhibits basal and TNF-enhanced binding of murine P815 mastocytoma cells to murine microvascular endothelium (MME). The alterations in MME mediated by TGF-beta, also lead to the inhibition of adherence of murine splenocytes, thymocytes, and human lymphoblastoid cells but do not inhibit adherence of murine B16 melanoma cells. The effect of TGF-beta is transient and inhibition of the endothelial adhesive phenotype is strongest 12 to 24 h after addition of the factor to MME. The TGF-beta-mediated inhibition of P815 basal binding to endothelium is dependent on protein synthesis because cycloheximide reverses the TGF-beta effect. TGF-beta does not appear to activate classical signal transduction pathways. Inhibitors of G proteins do not abolish TGF-beta action, protein kinase C and protein kinase A activators elicit an effect opposite to that of the factor, TGF-beta does not increase intracellular cAMP levels, and finally calcium-mobilizing agents do not mimic, but rather inhibit the effect of TGF-beta. However, TGF-beta-mediated inhibition of both basal binding and TNF-enhanced P815 binding to MME is completely abolished in the presence of the protein phosphatase inhibitor okadaic acid which suggests that TGF-beta may elicit its effect by stimulating protein phosphatase activity.

We present an analysis of J ψ →γf 1 (1285) , f 1 (1285)→ π + π − π + π − , using the Mark III detector at SPEAR, based on 5.8 × 10 6 produced J ψ events. We measure B( J ψ →γf 1 (1285) , f 1 (1285)→ π + π − π + π − )=(4.8±1.3±0.9) × 10 −5 . We obtain a new measurement of the absolute branching ratio of J ψ →γf 1 (1285) . The mixing angle of the f 1 (1285) and the f 1 (1420) in the 1 ++ nonet is determined.

A lovastatin-hyperproducing culture ofAspergillus terreus was shown to produce several co-metabolites extracted from whole broth. The predominant co-metabolite was the benzophenone, sulochrin, reported to arise from a polyketide biosynthetic pathway. This compound was targeted for elimination by classical mutagenesis and screening. A surface culture method employing microtiter, plates was used to ferment mutants for the primary screen. Qualitative determinations of lovastatin and sulochrin production were achieved by high-performance thin-layer chromatography. A mutant, strain AH6, which produced lovastatin titers equivalent to the parent culture and no detectable sulochrin was isolated. In addition, a lovastatin-hyperproducing mutant designated CB4 was capable of producing 16% more lovastatin and 30% less sulochrin than the parent culture in shake flask fermentations. In a pilot-scale 250-gallon fermentation, strain CB4 gave a 20% increase in lovastatin titer while producing 83% less sulochrin than the parent culture.

We report a high statistics study of the inclusive decay properties of the charmed D0 and D+ mesons, and the first inclusive study of the charmed Ds+. The data are collected at s =3.77 GeV and s =4.14 GeV with the Mark III detector at the e+e− storage ring SPEAR. For each charmed meson species, the charged-particle multiplicity, the strangeness content of the final state, and the average π0 multiplicity are determined.

Absolute branching fractions for the {ital D}{sub {ital l}3} and {ital D}{sub {ital l}4} decays {ital D}{sup +}{r arrow}{ital {bar K}} {sup 0}{ital e+}{nu}{sub {ital e}}, {ital D}{sup +}{r arrow}{ital {bar K}} {sup 0}{mu}{sup +}{nu}{sub {mu}}, {ital D}{sup +}{r arrow}{ital {bar K}} {sup *0}{ital e+}{nu}{sub {ital e}}, and {ital D}{sup 0}{r arrow}{ital K}{sup *{minus}}{ital e+}{nu}{sub {ital e}} are determined using completely reconstructed {ital D{bar D}} events at the {psi}(3770). Reconstructed {ital D}{sup 0}{r arrow}{ital K}{sup {minus}}{ital e+}{nu}{sub {ital e}} decays are used to determine the pole mass of the {ital f}{sub 4}({ital q}{sup 2}) form factor. Resonant {ital K}{sup *} production dominates the process {ital D}{r arrow}{ital K}{pi}{ital e}{nu}; the {ital K}{sup *} polarization is measured. Limits on several Cabibbo-suppressed channels are evaluated. A global fit imposing isospin symmetry is performed to the measured exclusive and inclusive semileptonic {ital D}{sup 0} and {ital D}{sup +} branching fractions to obtain an improved set of branching fractions.

For a number of years, the sense of hearing has been granted particular attention as an important factor in the perception and conceptualization of time (Rileigh & Odom, 1972). In fact, some have labeled hearing as the “time sense,” suggesting that we learn much about the sequence of events through time as we hear a stream of auditory cues. Furthermore, the ability to perceive changes in frequency and melodic patterns has been typically assigned to the auditory system. Complicating this picture is the belief that sense of time and the ability to perceive complex rhythmic and melodic patterns develop with increasing maturation and exposure to auditory information. If these theories are indeed correct, are persons who have early and severe hearing losses at risk for achieving these very basic perceptual skills? If so, what interventions need to be undertaken? To date, a relatively small body of research exists comparing the rhythmic and melodic perception of hearing-impaired persons with normally hearing individuals. The bulk of studies have been conducted with young children of elementary school age, and the greater number of extant studies examine rhythmic perception. In a study comparing hard-of-hearing children with normally hearing children (Sterritt, Camp, & Lippman, 1966), nine children with hearing losses and nine normally hearing children reproduced temporal patterns created by an above-threshold tone or by a flashing light on a telegraph key. The authors concluded that the hard-of-hearing children’s performance on a rhythmic reproduction task was poorer than that of normally hearing children. This decrement was attributed to early auditory deprivation, which the authors believed influenced perception for not only auditory patterns but also for visual temporal patterns. Davis and Hardick (1981) noted that for children with severe and profound hearing losses, the developmental history for auditory perception is believed to begin at the age the children are aided; children of identical chronological ages may have different length auditory histories. In a later test of rhythmic perception (Rileigh & Odom, 1972), deaf and normally hearing subjects were asked to reproduce visually-produced rhythmic patterns that varied in length and complexity. The outcome suggested that a developmental continuum of skills relating to rhythmic perception existed, with hearing-impaired subjects demonstrating a later mastery of more complex patterns than the normally hearing peers. In other words, rather than being deviant in rhythmic perception, children with hearing losses may develop similar rhythmic skills at a later age. Limited exposure to auditory signals might be one explanation for this delay.

Chitinase 3-Like-1 (CHI3L1) is a secreted 40 kDa glycoprotein that is upregulated in a number of human cancers and in non-neoplastic disease states characterized by chronic inflammation and tissue remodeling. Increased serum levels of CHI3L1 parallel disease severity, poorer prognosis, and shorter survival in many human neoplasias, including cancers of the breast, colon, prostate, ovaries, brain, thyroid, lung, and liver. Increased serum CHI3L1 also correlates with disease severity in rheumatoid arthritis, osteoarthritis, liver fibrosis, inflammatory bowel disease, and bacterial septicemia. CHI3L1 is a rheumatoid arthritis (RA) autoantigen, and MHC complexes containing specific CHI3L1 peptides have been found in RA patients; however, intranasal introduction of these same CHI3L1 peptides can induce tolerance towards them. CHI3L1 is a nonhydrolytic member of the human chitinase family that binds chitin tightly and heparin at lower affinity. Interactions with type I collagen, CHI3L1's only known protein-binding partner, helps regulate collagen fibril formation. The principal sources of CHI3L1 are activated macrophages and chondrocytes, neutrophils, and some tissue and tumor cells. CHI3L1 can act as a fibroblast mitogen and can activate several signaling pathways, however, no cell surface-binding partner for CHI3L1 has been identified. The ability of CHI3L1 to bind both proteins and carbohydrates allows potential interactions with a variety of cell-surface and extracellular-matrix proteins, proteoglycans, and polysaccharides, and thus CHI3L1 can interface between proteomics and glycomics.

Results from a partial-wave analysis of the decay {ital J}/{psi}{r arrow}{gamma}{ital K}{sub {ital S}}{sup 0}{ital K{plus minus}}{pi}{sup {minus plus}} in the {ital K}{sub {ital S}}{sup 0}{ital K}{sup {plus minus}}{pi}{sup {minus plus}} invariant-mass range 1.35--1.6 GeV/{ital c}{sup 2} are presented. Within the isobar model, the data in this mass range are best described by a mixture of 0{sup {minus}+} and 1{sup ++} amplitudes, corresponding to the {ital K}{sup *}{ital {bar K}}+c.c ({ital P} wave), {ital K}{sup *}{ital {bar K}}+c.c ({ital S} wave), and {ital a}{sub 0}(980) {pi} ({ital S} wave) channels. These results show that {eta}(1430) is not a {ital J}{sup {ital P}{ital C}}=0{sup {minus}+} {ital a}{sub 0}(980){pi} resonance, but a mixture of overlapping states.