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This article describes how conductors of the top performing groups and music education faculty at one university collaborated to create a Family Concert Series for parents and children of all ages, including infants in arms. Recognizing the conflict between “The first three years of life are the most important for educating a young child in music” and “Children under the age of six are not allowed to attend the concert,” the Family Concert Series is an attempt to resolve the issue. The Family Concerts require a minimum of extra work for conductors and are becoming a curriculum component for university music education students. This article also includes strategies that teachers might use in classrooms with recorded music when live music is not available.

“Let’s do it again,” shout the children as they complete an activity in music class. A casual observer would be aware that the children are fully engaged but may not be sure of the lesson focus. Is it to help children learn a new orchestra piece? To teach about the beat? To teach form? To teach rhythm? To teach children to read a music score? To teach them how to manage themselves in space? To help children practice thinking and problem solving? This article explores the power of movement to teach multiple music concepts, problem solving, and social skills simultaneously and effectively.

This article explores reading at its most fundamental: the interpretation of marks for the eye that represent musical sounds. An examination of young children’s created symbols for songs suggests that they perceive music in meaningful units rather than individual notes. In one class, arbitrary symbols that represent chunks or units of sound were used to encourage 3- and 4-year-old children to read music. The results of children’s behaviors and the musical growth potential of such an approach is outlined.

This article reports on a learner-centered kindergarten music program at Gan Michael Kindergarten in Israel. Actual experiences with stories and pictures provide examples of young children engaged in music making as performers, composers, and analytical listeners. The stories include teacher thoughts and reflections, providing a model of teacher as researcher and learner. What can we learn from these stories?

In many schools, classroom teachers are responsible for the music experiences of young children. Children may learn songs, but may not learn how to sing. This article outlines simple teaching strategies to help young children develop listening and vocal habits leading to beautiful singing. The article discusses how the kindergarten classes at one elementary school succeeded, and includes recordings of children’s singing.

In 1979, MENC President James Mason announced the intention to create a national council on elementary general music education to be organized within the framework of MENC. This action was the first important step toward creating what became the Society for General Music. This article is a brief history of the Society, including description of events leading to its formation, and contributions the Society has made to general music education.

The 4-year-olds gathered on the rug in response to the teacher’s singing cue (see Figure 1). Savannah, however, got distracted by a tambourine and spontaneously began tapping the rhythm while the rest of us sang (see Figure 2). This is not an unusual event when music is a regular part of the preschool environment. But it always feels like a small miracle when—without formal instruction in meter, beat, rhythm, or tambourine playing—a young child, caught up in the excitement of the moment, performs them all. Savannah was demonstrating examples of emerging music literacy. But of equal interest is that at the same time, each tap on the tambourine represented one syllable in language (syllabication). The heavier and lighter taps represented stress patterns in language. In one simple, spontaneous act, a 4-year-old child was demonstrating not only emerging music literacy but also emerging language literacy. And it all began with a song. A careful study of folk songs is revealing. Generally, it is the language that determines how the music will go. The rhythm, stress patterns, and phrases of the words dictate the rhythm, meter, and phrases of the music. Often the vocal inflections of the spoken words are imitated in the pitch direction of the melody. At the same time, the music intensifies the language. Each syllable of every word is strengthened by the music. The music also gives power to the meaning of the words. The children demonstrate their awareness of meaning by responding in movement. The phrase structure of the words is intensified by the musical phrases, and the natural inflections of the words are often enhanced by the melodic direction. Think of traditional children’s songs such as “Teddy Bear” or “The Wheels on the Bus.” Speak the words and notice the natural rhythm of the words. The rhythm of the song reflects the spoken language. The flow of the language is also represented in the music. “Wheels” has more emphasis than “the” when spoken. The pick-up in the music emphasizes that stress pattern. Notice that the vocal inflection when you say “teddy bear” descends or “the wheels” ascends or “round and round” descends. The melodies take the same spoken shape. Finally, consider the vocabulary and meaning taught through the singing and acting out of the words. One might say that song is simply language intensified. Helping early childhood teachers understand that singing is language development as well as music development may encourage them to provide more singing experiences with those for whom they care. Songs can also be a bridge to reading. Just as reading stories to children prepares them to understand that words can be written, so exposing them to actual music symbols helps them come to understand that songs can be written. But because the song is really language enhanced by music, beginning symbols for songs provide opportunity for language reading. One of the many ways to encourage “reading” of songs is to combine pictures, icons, and words on a mat for child exploration. The first “Teddy Bear” mat (Figure 3) provides an example. After children know the song well, show them the mat asking them what they notice. Recognize each response, and then sing the song while following the symbols. Place the mat in the free-play General Music Today Volume 21 Number 2 Winter 2008 35-38 © 2008 MENC: The National Association for Music Education 10.1177/1048371308317050

Aminoflavone (4H-1-benzopyran-4-one, 5-amino-2-(4-amino-3-fluorophenyl)-6,8-difluoro-7-methyl; NSC 686288) demonstrates differential antiproliferative activity in the National Cancer Institute's anticancer drug screen. We demonstrate here that MCF-7 human breast cancer cells are sensitive to aminoflavone both in vitro and when grown in vivo as xenografts in athymic mice. As previous work has indicated that aminoflavone requires metabolic activation by cytochrome P450 1A1 (CYP1A1), we investigated the effect of aminoflavone on CYP1A1 expression and on the aryl hydrocarbon receptor (AhR), a transcriptional regulator of CYP1A1. In aminoflavone-sensitive but not aminoflavone-resistant cells, the drug caused a 100-fold induction of CYP1A1 mRNA and a corresponding increase in ethoxyresorufin-O-deethylase activity. An AhR-deficient variant of the MCF-7 breast carcinoma, AHR100, with diminished CYP1A1 inducibility, exhibits cellular resistance to aminoflavone and is refractory to CYP1A1 mRNA induction by the drug. The increase in CYP1A1 mRNA in the aminoflavone-sensitive MCF-7 breast tumor cell results from transcriptional activation of xenobiotic-responsive element (XRE)–controlled transcription. Aminoflavone treatment causes a translocation of the AhR from the cytoplasm to the nucleus with subsequent formation of AhR-XRE protein DNA complexes. In contrast to the aminoflavone-sensitive MCF-7 cells, the resistant cell lines (MDA-MB-435, PC-3, and AHR100) demonstrated constitutive nuclear localization of AhR. Additionally, aminoflavone failed to induce ethoxyresorufin-O-deethylase activity, CYP1A1 transcription, AhR-XRE complex formation, and apoptosis in aminoflavone-resistant cells. These results suggest that the cytotoxicity of aminoflavone in a sensitive breast tumor cell line is the result of the engagement of AhR-mediated signal transduction.

This article discusses the creative, educational, and musical growth that comes from manipulating pieces of a music game. The importance of child-centered play is discussed, with a focus on children using icons to change the melodic and word order of a song. They then “read” the icons to sing their new creation. The music manipulatives are the creation of Barbara Andress.

This article explores ways for music teachers to influence music making in the home. Often preschool music programs include parents in the music education process, but when children enter school, the parent connection is not usually continued with the same intensity. This article will serve as a catalyst for further conversations on ways to include parents as partners in our music education process.

The tricyclic compound 2,5-bis(5-hydroxymethyl-2-thienyl)furan (NSC 652287) has shown a highly selective pattern of differential cytotoxic activity in the tumor cell lines comprising the National Cancer Institute (NCI) Anticancer Drug Screen. The mechanism underlying the selective cytotoxicity is unknown. We hypothesized that differential sensitivity to the compound observed in several renal tumor cell lines could be the result of selective accumulation or differential metabolism of this agent. We demonstrated here that the capacity of certain renal cell lines to accumulate and retain the compound, determined by accumulation of [14C]NSC 652287-derived radioactivity and by flow cytometric determination of unlabeled compound, paralleled the sensitivity of the renal cell lines to growth inhibition by NSC 652287: A-498 > TK-10 >> ACHN approximately/= to UO-31. The ability of the cell lines to metabolize [14C]NSC 652287 to a reactive species capable of binding covalently to cellular macromolecules also directly correlated with sensitivity to the compound. Different patterns of metabolites were generated by relatively more drug-sensitive cell lines in comparison with drug-resistant cell lines. The metabolizing capacity for NSC 652287 was localized primarily to the cytosolic (S100) fraction. The rate of metabolism in the cytosolic fraction from the most sensitive renal cell line, A-498, was faster than that observed in the cytosolic fractions from the other, less sensitive cell lines. The data support the hypothesis that both selective cellular accumulation and the capacity to metabolize NSC 652287 to a reactive species by certain renal carcinoma cell types are the basis for the differential cytotoxicity of this compound class.

In his book Emotional Intelligence (1995), Daniel Goleman describes a research study conducted at Stanford University. The test went something like this: Four-year-old Jeremy is ushered into a room and invited to sit at a table. The researcher tells Jeremy that he forgot something and will be right back. On the table is a marshmallow. Jeremy is told that if he can leave the marshmallow there until the researcher returns, he will be given two marshmallows. But if he eats the one marshmallow while the researcher is gone, he will not get another one. Jeremy was one of over 500 four-yearolds who took this test at a Stanford University campus preschool. Some children (about two thirds) managed to wait, using strategies such as covering their eyes, talking to themselves, playing games, and even trying to go to sleep in order to avoid eating the marshmallow. In the end, they got the two-marshmallow reward. Others would grab and eat the one marshmallow, usually within seconds after the researcher left the room. The children’s behaviors fell into two general categories— those who acted on impulse, and those who were able to delay gratification. About 10 years later, a follow-up study was done with these same children, and 3 years after that, another follow-up study was conducted. What the researchers found was dramatic. Those who were able to delay gratification at age four were more selfassertive, more personally and socially competent, more able to cope with difficulties in life, and more able to cope under stress as adolescents. They were also better students, scored significantly higher on SAT tests, were able to communicate better, able to resolve conflict peacefully, and felt better about themselves. The marshmallow grabbers were more troubled, stubborn, and frustrated with life, had lower self-images, were mistrustful, more jealous, and more likely to get into fights. They did not do as well in school and seemed unable to resolve conflict or reason through problems. In short, what the research showed was that the ability of a four-yearold to control impulse and delay gratification is a very powerful predictor of life success. According to Phil Peake, who analyzed the data, the ability to delay gratification at age four is twice as powerful a predictor of SAT scores as is an IQ test (Goleman, 1995, p. 82). As I read this study, I reflected on how many songs seem to invite children to delay gratification. Music exists in time. One cannot get to the end of the song before going through the song. Is this significant for helping children wait? And can the way we present a singing game activity intensify the delay of what might be a gratifying moment at the end of the song? Can music teach delayed gratification? Think of the rhyme “Patty Cake,” for example. The most exciting part of the rhyme comes when we “throw it in the oven.” Babies who have played patty cake with a loving adult many times may even raise their arms for that special part before it comes in anticipation, they get so excited. But the rhyme won’t allow that part to come too soon. In fact, the rhyme intensifies the excitement when performed with musical expression. It begins soft and gently with “Patty cake, patty cake, baker’s man.” The voice may get a little bit louder and faster when saying “Bake me a cake as fast as you can.” But then, just when it feels like something is about to happen, the voice gets very soft, the actions are slow, and the quiet energy holds the infant tight to “Roll it and pat it.” A crescendo in the voice slowly grows as the voice rises to a higher pitch, and slows way down on “Mark it with a B.” The “B” has a long fermata as the adult looks excitedly in the baby’s face. The tension is strong as it finally explodes into “And throw it in the oven ...” The moment we have been waiting for finally arrives and it is all the more satisfying because of the long musical journey getting there. Finally, a warm cuddle at the end, “... for baby and me,” brings the experience to a satisfying close. Many of the bouncing songs such as “Trot Trot to Boston” provide the same kind of experience, especially when sung musically. “Trot trot to Boston to buy a loaf of bread” is sung softly while gently bouncing baby to General Music Today Volume 22 Number 2 January 2009 27-29 © 2009 MENC: The National Association for Music Education 10.1177/1048371308328383

9-Methoxy-N2-methylellipticinium acetate (MMEA) was preferentially cytotoxic to human brain tumor cell lines in the in vitro primary screen of the U.S. National Cancer Institute. In the present study, the average intracellular accumulation of radioactivity derived from [14C]MMEA concentrations that were selectively cytotoxic to sensitive brain tumor cell lines was nearly 4-fold greater than in human tumor cell lines derived from the lung, kidney, ovary and colon. The extent of peak cellular accumulation of [14C]MMEA-derived radioactivity, achieved after 10-15 hr of drug exposure, was correlated positively with relative MMEA cytotoxicity in brain tumor cell lines (r2 = 0.963). A similar correlation (r2 = 0.967) was observed in selected non-brain tumor cell lines but required substantially higher (18-fold) concentrations of MMEA. [14C]MMEA radioactivity accumulation by a selected glioblastoma cell line occurred via an energy-requiring system that was predominantly sodium and pH independent.

Gene expression data, collected from ASPS tumors of seven different patients and from one immortalized ASPS cell line (ASPS-1), was analyzed jointly with patient ASPL-TFE3 (t(X;17)(p11;q25)) fusion transcript data to identify disease-specific pathways and their component genes. Data analysis of the pooled patient and ASPS-1 gene expression data, using conventional clustering methods, revealed a relatively small set of pathways and genes characterizing the biology of ASPS. These results could be largely recapitulated using only the gene expression data collected from patient tumor samples. The concordance between expression measures derived from ASPS-1 and both pooled and individual patient tumor data provided a rationale for extending the analysis to include patient ASPL-TFE3 fusion transcript data. A novel linear model was exploited to link gene expressions to fusion transcript data and used to identify a small set of ASPS-specific pathways and their gene expression. Cellular pathways that appear aberrantly regulated in response to the t(X;17)(p11;q25) translocation include the cell cycle and cell adhesion. The identification of pathways and gene subsets characteristic of ASPS support current therapeutic strategies that target the FLT1 and MET, while also proposing additional targeting of genes found in pathways involved in the cell cycle (CHK1), cell adhesion (ARHGD1A), cell division (CDC6), control of meiosis (RAD51L3) and mitosis (BIRC5), and chemokine-related protein tyrosine kinase activity (CCL4).

In vitro growth of alveolar soft part sarcoma (ASPS) and establishment of an ASPS cell line, ASPS-1, are described in this study. Using a recently developed xenograft model of ASPS derived from a lymph node metastasis, organoid nests consisting of 15 to 25 ASPS cells were isolated from ASPS xenograft tumors by capture on 70 μm filters and plated in vitro. After attachment to the substratum, these nests deposited small aggregates of ASPS cells. These cells grew slowly and were expanded over a period of 3 years and have maintained characteristics consistent with those of both the original ASPS tumor from the patient and the xenograft tumor including (1) presence of the alveolar soft part locus-transcription factor E3 type 1 fusion transcript and nuclear expression of the alveolar soft part locus-transcription factor E3 type 1 fusion protein; (2) maintenance of the t(X;17)(p11;q25) translocation characteristic of ASPS; and (3) expression of upregulated ASPS transcripts involved in angiogenesis (ANGPTL2, HIF-1-α, MDK, c-MET, VEGF, and TIMP-2), cell proliferation (PRL, PCSK1), metastasis (ADAM9), as well as the transcription factor BHLHB3 and the muscle-specific transcripts TRIM63 and ITGβ1BP3. This ASPS cell line forms colonies in soft agar and retains the ability to produce highly vascularized ASPS tumors in NOD.SCID/NCr mice. Immunohistochemistry of selected ASPS markers on these tumors indicated similarity to those of the original patient tumor as well as to the xenografted ASPS tumor. We anticipate that this ASPS cell line will accelerate investigations into the biology of ASPS including identification of new therapeutic approaches for treatment of this slow growing soft tissue sarcoma.

With microtubule-associated proteins (MAPs) BeSO 4 and MgSO 4 stimulated tubulin polymerization as compared to a reaction mixture without exogenously added metal ion, while beryllium fluoride had no effect (E. Hamel et al. , 1991, Arch. Biochem. Biophys. 286, 57–69). Effects of both cations were most dramatic at GTP concentrations in the same molar range as the tubulin concentration. We have now compared effects of beryllium and magnesium on tubulin-nucleotide interactions in both unpolymerized tubulin and in polymer. Polymer formed with magnesium had properties similar to those of polymer formed without exogenous cation, except for a 20% lower stoichiometry of exogenous GTP incorporated into the latter. In both polymers the incorporated GTP was hydrolyzed to GDP. Stoichiometry of GTP incorporation into polymers formed with beryllium or magnesium was identical, but much of the GTP in the beryllium polymer was not hydrolyzed. The beryllium polymer was more stable than the magnesium polymer. Beryllium also differed from magnesium in only weakly enhancing the binding of GTP in the exchangeable site of unpolymerized tubulin, while neither cation affected GDP exchange at the site. If both cations were present in a reaction mixture, polymer stability was little changed from that of the beryllium polymer, but most of the GTP incorporated into polymer was hydrolyzed. Six additional metal salts (AlCl 3 , CdCl 2 , CoCl 2 , MnCl 2 , SnCl 2 , and ZnCl 2 ) also stimulated MAP-dependent tubulin polymerization, but enhanced polymer stability did not correlate with polymer GTP content. We postulate that enhanced polymer stability is a consequence of cation binding directly to tubulin and/or polymer while deficient GTP hydrolysis in the presence of beryllium, as well as aluminum and tin, is a consequence of tight binding of cation to GTP in the exchangeable site.

In vivo growth of alveolar soft part sarcoma (ASPS) was achieved using subcutaneous xenografts in sex-matched nonobese diabetic severe combined immunodeficiency mice. One tumor, currently at passage 6, has been maintained in vivo for 32 months and has maintained characteristics consistent with those of the original ASPS tumor including (1) tumor histology and staining with periodic acid Schiff/diastase, (2) the presence of the ASPL-TFE3 type 1 fusion transcript, (3) nuclear staining with antibodies to the ASPL-TFE3 type 1 fusion protein, (4) maintenance of the t(X;17)(p11;q25) translocation characteristic of ASPS, (5) stable expression of signature ASPS gene transcripts and finally, the development and maintenance of a functional vascular network, a hallmark of ASPS. The ASPS xenograft tumor vasculature encompassing nests of ASPS cells is highly reactive to antibodies against the endothelial antigen CD34 and is readily accessible to intravenously administered fluorescein isothiocyanate-dextran. The therapeutic vulnerability of this tumor model to antiangiogenic therapy, targeting vascular endothelial growth factor and hypoxia-inducible factor-1 alpha, was examined using bevacizumab and topotecan alone and in combination. Together, the 2 drugs produced a 70% growth delay accompanied by a 0.7 net log cell kill that was superior to the antitumor effect produced by either drug alone. In summary, this study describes a preclinical in vivo model for ASPS which will facilitate investigation into the biology of this slow growing soft tissue sarcoma and demonstrates the feasibility of using an antiangiogenic approach in the treatment of ASPS.

Alveolar soft part sarcoma (ASPS), a rare soft tissue sarcoma, is characterized by a chromosomal translocation der(17)t(X;17)(p11;q25) resulting in the production of 2 fusion proteins encoded by regions of the genes for alveolar soft part locus (ASPL) and the transcription factor E3 (TFE3). In this study, polyclonal antibodies were generated to 25 mer peptides encompassing the junctional regions of ASPL-TFE3 type 1 and ASPL-TFE3 type 2. The specificity of the affinity purified antibodies for the synthetic peptides and recombinant expressed ASPL-TFE3 type 1 and ASPL-TFE3 type 2 proteins was evaluated by enzyme-linked immunosorbent assay and was highly fusion type specific. Immunohistochemical staining of formalin-fixed, paraffin-embedded ASPS tumors with the fusion-specific antibodies resulted in intense nuclear staining and differentiation between tumors that express the type 1 protein and tumors that express the type 2 protein. These antibodies will be useful for the differential diagnosis of type 1 and type 2 ASPS and also in the detection of the fusion proteins in biochemical and cell biologic investigations.

We have developed a rapid, sensitive, and inexpensive method for measuring the cellular protein content of adherent and suspension cultures in 96-well microtiter plates. The method is suitable for ordinary laboratory purposes and for very large-scale applications, such as the National Cancer Institute's disease-oriented in vitro anticancer-drug discovery screen, which requires the use of several million culture wells per year. Cultures fixed with trichloroacetic acid were stained for 30 minutes with 0.4% (wt/vol) sulforhodamine B (SRB) dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and protein-bound dye was extracted with 10 mM unbuffered Tris base [tris (hydroxymethyl)aminomethane] for determination of optical density in a computer-interfaced, 96-well microtiter plate reader. The SRB assay results were linear with the number of cells and with values for cellular protein measured by both the Lowry and Bradford assays at densities ranging from sparse subconfluence to multilayered supraconfluence. The signal-to-noise ratio at 564 nm was approximately 1.5 with 1,000 cells per well. The sensitivity of the SRB assay compared favorably with sensitivities of several fluorescence assays and was superior to those of both the Lowry and Bradford assays and to those of 20 other visible dyes. The SRB assay provides a colorimetric end point that is nondestructive, indefinitely stable, and visible to the naked eye. It provides a sensitive measure of drug-induced cytotoxicity, is useful in quantitating clonogenicity, and is well suited to high-volume, automated drug screening. SRB fluoresces strongly with laser excitation at 488 nm and can be measured quantitatively at the single-cell level by static fluorescence cytometry.

The aminoflavone analogue (AF) exhibits antitumor activity in vitro, particularly against neoplastic cells of renal origin. We identified cellular correlates of responsiveness to AF in continuous human tumor renal cell carcinoma lines and in tumor cell isolates, termed renal carcinoma cell strains, from patients with clear cell and papillary renal neoplasms.In vitro antiproliferative activity of AF was evaluated using the sulforhodamine B protein dye assay. In vivo antitumor activity of the drug was determined in mice bearing xenografts. Covalent binding of AF/metabolite(s) was assessed following exposure of cells to AF for 16 hours. CYP1A1 and CYP1B1 mRNA and apoptosis were quantitated by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively.AF produced total growth inhibition in vitro in 3 of 6 human tumor renal cell lines at concentrations of 90 to 400 nM. In vivo treatment of mice bearing xenografts of the Caki-1 renal cell carcinoma, sensitive to AF in vitro, resulted in significant antitumor activity, including tumor-free survivors. Studies in 13 renal cell strains isolated from patients with clear cell (9) or papillary (4) renal cell carcinoma indicated that 3 of 4 papillary strains were sensitive to AF compared with 2 of 9 clear cell strains. AF sensitive renal cell lines and strains exhibited induction of CYP1A1 and CYP1B1 gene expression, increased covalent binding of AF metabolite(s) and apoptosis.AF has noteworthy antitumor activity against certain human tumor renal cell lines in vitro and in vivo, which correlates with drug metabolism to covalently binding metabolites after CYP1A1 and CYP1B1 gene expression. We hypothesize that it leads to apoptosis induction. AF sensitive renal cell strains are predominantly of the papillary histological type. These results are limited by the small numbers of cell lines and cell strains but they are suggestive of the need for further testing in larger collections of cell strains.

This article discusses song prompts as a way to encourage children to sing during exploratory play. A song prompt for “I Had a Cat” is included for educators to try in their own classrooms or preschools. Educators are invited to share ideas they have used that encourage children to sing during free play.

KRN5500 is a semisynthetic spicamycin analogue consisting of a seven-carbon amino sugar linked to a C 14 unsaturated fatty acid through glycine and to the amino group of adenine. The drug inhibits cell growth potently and has antitumor activity in in vivo models. The mechanism of the antiproliferative effect of KRN5500 remains to be elucidated. We have found that acute exposure of drug-sensitive HT-29 colon adenocarcinoma cells to the drug results initially in swelling of the Golgi apparatus. Continuous exposure to the drug resulted in the emergence of a resistant population of cells characterized by numerous intracellular vacuoles. These KRN5500-resistant tumor cells exhibited increased staining with the Golgi stain NBD C 6 –ceramide and the ER–Golgi fluorescent dye BODIPY–brefeldin A, which, unlike the parental drug-sensitive cells, was dispersed throughout the cytoplasm. Marker enzymes associated with the ER (glucose 6-phosphatase) and cis-Golgi (GalNAc transferase) were elevated >2-fold and nearly 4-fold, respectively, in drug-resistant cell lines while the trans-Golgi marker enzyme, galactosyltransferase, was not. The additional findings that the KRN5500-resistant cells have a >2-fold elevation in ERGIC-53, a cis-Golgi marker protein of the ER–Golgi intermediate compartment (ERGIC), as well as increased 58K, a 58-kDa microtubule-binding protein with formiminotransferase cyclodeaminase activity, and tubulin indicate that the cellular secretory pathway is a primary determinant of sensitivity to KRN5500, as resistance to this agent corresponds with accumulation of several components relatable to ER and cis-Golgi function. Further support for this conclusion is provided by studies which demonstrate that KRN5500 alters the distribution of newly synthesized carcinoembryonic antigen within the secretory pathway, including arrest of this N-glycosylated protein in the Golgi of LS-174T colon carcinoma cells.

Proprotein convertases (PCs) are a family of calcium-dependent serine endoproteases which mediate the proteolytic cleavage, at pairs of dibasic amino acids, of many precursor proteins, including growth factors and pro-hormones, into their functionally active forms. One family member, PCSK1, was identified as a potential therapeutic target in ASPS. This was based on gene expression profiling of 4 primary and 3 metastatic ASPS tumors (BMC Cancer 9:22, 2009). Affymetrix 133 Plus 2 arrays revealed that PCSK1, but not PCSK2 or furin, was significantly up-regulated (range 18.6 to 131 fold) relative to universal RNA, a multi-organ pool of non-tumor RNA's. PCSK1 expression was confirmed by immunohistochemistry and Western Blot analysis in patient tumors, in an ASPS xenograft model, and in a cell line developed from the xenograft tumor (ASPS-1; J Pediatr. Hematol. Oncol., in press 2010) as well as in tumors produced by ASPS-1 in immunocompromised mice. ASPS-1 was utilized, in-vitro, to evaluate a panel of PCSK1 siRNA's, the most potent of these produced a 75% reduction in the PCSK1 RNA transcript, reduced expression of PCSK1 protein and produced a 50% reduction in ASPS-1 growth. As part of an effort to develop new therapeutic approaches toward the treatment of ASPS we have implemented a high-throughput screening effort to identify small molecule inhibitors of PCSK1. Utilizing recombinant PCSK1 and the fluorescent substrate (Pyr-Arg-Thr-Lys-Arg-MCA) a high-throughput screen of a small molecule library (∼15 K compounds) was initiated. Results of a pilot screen of ∼2400 compounds at concentrations ranging from 0.1 to 100 uM yielded several inhibitors of PCSK1 which were retested in a 20 concentration assay to confirm activity. Preliminary analysis of structure-activity relationships identified several active inhibitor chemotypes, comparable in potency to the known peptide PCSK1 inhibitor, Ac-Leu-Leu-Arg-Val-Lys-Arg-NH2, containing aryl groups linked by aliphatic chains containing peptide bonds. Such compounds may act as substrate mimetic inhibitors and will be further evaluated for inhibition of growth of ASPS-1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1621. doi:10.1158/1538-7445.AM2011-1621

Alveolar soft part sarcoma (ASPS) is a rare soft tissue sarcoma primarily affecting young children and adolescents. ASPS is characterized by a nonreciprocal chromosomal translocation, der(17)t(X;17)(p11;q25), in which the N-terminal region of the alveolar soft part locus (ASPL) gene, located at 17q25, is fused to the C-terminal region of the transcription factor E3 (TFE3), located at Xp11. Two alternative fusion junctions have been observed resulting in the expression of 2 distinct fusion transcripts, ASPL-TFE3 type 1 and 2, and their respective proteins which function as hybrid transcription factors. Recently we have described the first xenograft model of ASPS, established from a lymph node metastasis derived from a female patient expressing the ASPL-TFE3 type 1 fusion, in NOD.SCID mice (J. Pediatr. Hematol. Oncol 31:561, 2009) and an ASPS cell line, ASPS-1, derived from it (Proc. Amer. Assoc, Cancer Res. 50:978, 2009). We report here our efforts to target the ASPL-TFE3 type 1 gene fusion (CGG GAG CGG CTG CCT GTG TCA) in ASPS-1 cells using siRNA directed against its nucleotide sequence and compared results with siRNA directed against the ASPL-TFE3 type 2 gene fusion (CGG ATT GAT GAT GTC ATT GAT). ASPL-TFE3 type 1 and 2 siRNA were evaluated in vitro in ASPS-1 cells, which express the ASPL-TFE3 type 1 gene fusion and protein, at concentrations ranging from 1-50 nM and treatment times ranging from 3-12 days. These results indicate that the ASPL-TFE3 type 1 siRNA, but not the ASPL-TFE3 type 2 siRNA or a control siRNA not known to target any human gene, was effective in reducing the ASPL-TFE3 type 1 fusion transcript, the ASPL-TFE3 type 1 fusion protein and retarding the growth of ASPS-1 cells. Importantly the ASPL-TFE3 type 1 siRNA down regulated several signature ASPS genes (BMC Cancer 9:22, 2009) including those involved in angiogenesis of this highly vascular tumor (ANGPTL2, MDK, c-MET, and TIMP-2). Additionally, PCSKI, a proprotein convertase responsible for processing of neuropeptides, growth factors and receptors, and several tissue restricted markers including ITGB1BP3, was strikingly down regulated. Together these results suggest that the ASPL-TFE3 fusion gene and its fusion protein play an important role in pathways involved in the growth of ASPS and present potentially exploitable targets for development of novel therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3415.

Carlier et al. (1988, Biochemistry 27, 3555–3559; 1989, Biochemistry 28, 1783–1791) described enhancement of tubulin polymerization and stabilization of glycerol-induced microtubules by BeF 3 − (by addition of both BeSO 4 and NaF to reaction mixtures). We were able to confirm the stabilization of glycerol-induced polymer reported by these workers, provided Mg 2+ was also present in the reaction. When we examined polymerization dependent on microtubule-associated proteins (MAPs), however, we obtained very different results. BeF 3 − had no significant effect on this reaction, or the polymer formed, under any condition examined. Lower concentrations of BeSO 4 alone, in contrast to a negligible effect in glycerol, enhanced polymerization with MAPs provided the concentrations of both Mg 2+ and GTP were low; and Be 2+ stabilized the polymer, if the GTP concentration was low, at both low and high Mg 2+ concentrations. Higher concentrations of BeSO 4 precipitated tubulin, an effect which was not affected by Mg 2+ , partially prevented but not reversed by MAPs, and prevented or reversed by either NaF or nucleotides at adequate concentrations. These results suggest that Be 2+ binds at site(s) distinct from Mg 2+ site(s), and that partial occupancy of these site(s) at lower Be 2+ concentrations enhances tubulin polymerization and polymer stability, while extensive occupancy at higher Be 2+ concentrations results in tubulin precipitation. Effects of Be 2+ and BeF 3 − on polymerization dependent on dimethyl sulfoxide or glutamate were also evaluated. The dimethyl sulfoxide system displayed properties similar to those of the glycerol system, while the glutamate system was similar to the MAPs system.

Preschoolers need music! Susan Kenney of the music department of Brigham Young University, Provo, Utah, discusses some effective ways to meet this need in day-care settings.

The effects of 0.3 mg/kg haloperidol (H) on the acquisition and maintenance of footshock escape behavior of rats in a one meter runway was investigated. In the acquisition phase, a group (N=6) given H before testing (HB) showed severely retarded acquisition and performance of the escape response, as compared with a group (N=6) given H after testing (HA). When the HB and HA treatments were reversed for the groups behavioral performance was initially unaffected. At first, the HA group switched to the HB condition continued to exhibit rapid escape behavior and the HB group switched to the HA treatment continued to have slow escape behavior. Over the course of 8 days of testing, however, the performances of the two groups gradually reversed. After completion of this testing the HB and HA treatments again were switched and the animals were tested for both avoidance and escape behavior. Again, the performance of the animals initially did not change after the treatment switch, but with repeated testing and treatments, the avoidance and escape behavior of the HB group slowed substantially and that of the HA group accelerated markedly. These findings support previous observations that over learned behaviors are much less sensitive to disruption by haloperidol treatment than behaviors which are undergoing learning. The important contribution of the present study was in demonstrating that this insensitivity is a transitional, transient phenomenon and that with chronic treatment and testing, over learned behaviors can be strongly affected by haloperidol. This observation indicates that the study of the effects of haloperidol on over learned behavior may provide a useful animal behavior model to investigate the important clinical issue of delayed onset of efficacy with neuroleptic drugs.

The present report presents evidence that operant conditioning processes may contribute to hypokinetic effects in rats induced by repeated haloperidol (HAL) treatments. Twenty-four male Sprague-Dawley rats were subdivided into three equal groups and given 9 daily injections of either 0.1 or 1.0 mg/kg HAL, i.p., or the lactic acid vehicle 1 h before testing of motoric behavior. The effects of the HAL were dependent upon dose and the test situation. In an open field, 1.0 HAL produced a complete akinesia whereas 0.1 HAL initially had no effect, but with repeated treatments the 0.1-HAL-treated rats became hypokinetic. When the animals were tested for their response to enter a dark compartment, the 2 doses had equivalent effects. Initially, HAL had no effect, but with repeated treatments, latencies to enter the dark compartment increased at the equivalent rates for the 0.1 and 1.0 HAL groups. When conditioned drug effects were assessed following withdrawal, only in drug environment situations which operant behavior occurred initially did hypokinesia persist. These results are relevant to understanding the delayed onset of action and antipsychotic effects of neuroleptic drugs.

Background Alveolar soft-part sarcoma (ASPS) is an extremely rare, highly vascular soft tissue sarcoma affecting predominantly adolescents and young adults. In an attempt to gain insight into the pathobiology of this enigmatic tumor, we performed the first genome-wide gene expression profiling study. Methods For seven patients with confirmed primary or metastatic ASPS, RNA samples were isolated immediately following surgery, reverse transcribed to cDNA and each sample hybridized to duplicate high-density human U133 plus 2.0 microarrays. Array data was then analyzed relative to arrays hybridized to universal RNA to generate an unbiased transcriptome. Subsequent gene ontology analysis was used to identify transcripts with therapeutic or diagnostic potential. A subset of the most interesting genes was then validated using quantitative RT-PCR and immunohistochemistry. Results Analysis of patient array data versus universal RNA identified elevated expression of transcripts related to angiogenesis (ANGPTL2, HIF-1 alpha, MDK, c-MET, VEGF, TIMP-2), cell proliferation (PRL, IGFBP1, NTSR2, PCSK1), metastasis (ADAM9, ECM1, POSTN) and steroid biosynthesis (CYP17A1 and STS). A number of muscle-restricted transcripts (ITGB1BP3/MIBP, MYF5, MYF6 and TRIM63) were also identified, strengthening the case for a muscle cell progenitor as the origin of disease. Transcript differentials were validated using real-time PCR and subsequent immunohistochemical analysis confirmed protein expression for several of the most interesting changes (MDK, c-MET, VEGF, POSTN, CYP17A1, ITGB1BP3/MIBP and TRIM63). Conclusion Results from this first comprehensive study of ASPS gene expression identifies several targets involved in angiogenesis, metastasis and myogenic differentiation. These efforts represent the first step towards defining the cellular origin, pathogenesis and effective treatment strategies for this atypical malignancy.