BPR1J-097, a novel FLT3 kinase inhibitor, exerts potent inhibitory activity against AML.

BACKGROUND: Activating mutations of Fms-like tyrosine kinase 3 (FLT3) constitute a major driver in the pathogenesis of acute myeloid leukaemia (AML). Hence, pharmacological inhibitors of FLT3 are of therapeutic interest for AML. METHODS: The effects of inhibition of FLT3 activity by a novel potent FLT3 inhibitor, BPR1J-097, were investigated using in vitro and in vivo assays. RESULTS: The 50% inhibitory concentration (IC(50)) of BPR1J-097 required to inhibit FLT3 kinase activity ranged from 1 to 10 nM, and the 50% growth inhibition concentrations (GC(50)s) were 21±7 and 46±14 nM for MOLM-13 and MV4-11 cells, respectively. BPR1J-097 inhibited FLT3/signal transducer and activator of transcription 5 phosphorylation and triggered apoptosis in FLT3-driven AML cells. BPR1J-097 also showed favourable pharmacokinetic property and pronounced dose-dependent tumour growth inhibition and regression in FLT3-driven AML murine xenograft models. CONCLUSION: These results indicate that BPR1J-097 is a novel small molecule FLT-3 inhibitor with promising in vivo anti-tumour activities and suggest that BPR1J-097 may be further developed in preclinical and clinical studies as therapeutics in AML treatments.

The stage-dependent inhibitory effect of porcine follicular cells on the development of preantral follicles.

The objective of this study was to examine the effects of follicular cells on the in vitro development of porcine preantral follicles. In Experiment 1, one preantral follicle alone (Trt 1) was cocultured with a follicle of the same size with oocytes (Trt 2) or without oocytes (Trt 3). Preantral follicles cultured alone in vitro for 12 days had greater follicle diameters (1017 +/- 96 microm versus 706 +/- 69 or 793 +/- 72 microm, P < 0.05), growth rates (201 +/- 0.3 versus 103 +/- 0.2 or 128 +/- 0.2, P < 0.05) and oocyte survival rates (73% versus 48, or 25%, P < 0.05) than other groups. The inhibitory effects of follicle cells on the growth of preantral follicles and oocyte survival rates were not enhanced by the addition of oocytectomized preantral follicles (Experiment 2). Follicles were cocultured with different sources of follicular cells in other experiments. Coculture with cumulus cells enhanced oocyte survival compared to the control (without coculture) and mural follicular cell groups (Experiment 3). The growth and survival rates of oocytes collected from the group of follicles cocultured with cumulus cells from large antral follicles (>3 mm) were greater (P < 0.05) than those from small antral follicles (<3 mm), or than the control group (without cumulus cells, experiment 4). No significant differences in the follicular diameters (674 +/- 30 microm versus 638 +/- 33 and 655 +/- 28 microm) and growth rate (105% versus 94 and 105%) were observed among the preantral follicles of the different treatments (P > 0.05). Taken together, coculture with the cells from large antral follicles (>3 mm) exerted a significant positive effect on oocyte survival. The growth and oocyte survival of preantral follicle cocultured with the same size of follicles (with or without oocyte) were inhibited. Growth and survival rates of preantral follicles and oocytes are improved by coculturing them with the cumulus cells derived from larger antral follicles.

Combination of a new composite biocampatible skin graft on the neodermis of artificial skin in an animal model.

INTRODUCTION: There have been very limited and inconsistent attempts at combining the cultured epidermal autograft (CEA) with the neodermis of artificial skin (Integra). The reasons for this remain unknown. The basement membrane proteins of conventional CEA sheets are easily damaged by the dispase treatment during the harvesting of the CEA from the culture flask. The damage of the basement membrane proteins may affect the anchorage of CEA onto the neodermis of Integra. A new Composite Biocompatible Skin Graft (CBSG) was recently developed. METHODS: Composite biocompatible skin graft consists of autologous keratinocytes cultivated on a pliable hyaluronate-derived membrane (Laserskin)which has been pre-seeded with allogenic dermal fibroblasts. Basement membrane proteins of CBSG are protected from the dispase treatment because the keratinocytes are directly seeded onto Laserskin. The engraftment of CBSG was evaluated on 20 wounds of 10 rats. Integrawas grafted on two freshly excised full-thickness wounds (3cm in diameter) in the dorsum of each animal. A polypropylene ring was applied to each wound to prevent the migration of epithelium from the edges. Composite Biocompatible Skin Graft was used to cover the neodermis of Integra after the silicone membrane was removed 14-21 days postgrafting. RESULTS: Fourteen (70%) of 20 skin biopsies taken at day 21 from the centre of the grafted wounds revealed regenerated epithelium. CONCLUSION: A feasible delivery system of cultured keratinocytes onto theneodermis of Integra is demonstrated in this animal -experiment.

The efficacy of collagen dermis membrane and fibrin on cultured epidermal graft using an athymic mouse model.

A human skin substitute consisting of human cultured keratinocytes, collagen dermis, and fibrin was evaluated in athymic mice. Eighty athymic mice were divided randomly into four groups. A 1.5x1.5-cm full-thickness wound defect was created on the back of each athymic mouse under anesthesia. These wounds were covered by sheets of cultured epidermal graft (group A), cultured epidermal graft with collagen dermis and fibrin (group B), cultured epidermal graft with collagen dermis (group C), or cultured epidermal graft with fibrin (group D). The grafts were secured and kept moist by specially designed saline gauze chambers. The take rates of the cultured graft with more than 50% of the wound covered were 65%, 15%, 50%, and 45% respectively. Group B had a significantly lower graft take rate, however the difference was not significant among groups A, C, and D. Light microscopy of biopsies of the grafted sites at 12 days showed complete epithelialization. The incidence of discharge from wound beds in groups A, B, C, and D was 0%, 15%, 15%, and 10% respectively. The results suggest that cultured cells are best grafted directly onto the wound bed or in combination with either a thin layer of collagen or fibrin but not both because the collagen dermal membrane and the fibrin together may impose too great a diffusion barrier for the cultured cell graft to become vascularized.

Development and evaluation of a new composite Laserskin graft.

BACKGROUND: Tremendous effort has been made to improve the graft take rate of cultured epidermal autograph. The purpose of this study is to develop and evaluate a new composite Laserskin graft (CLSG) as a human skin substitute for wound resurfacing. METHODS: The seeding efficacy of cultured keratinocytes on plain Laserskin was compared with the 3T3 cell-seeded Laserskin and allogenic fibroblast-populated Laserskin. Three different types of CLSG, 2 cm in diameter each, were prepared and tested in rats. Type A CLSG consisted of proliferative allogenic rat fibroblasts on both sides of the Laserskin with autologous keratinocytes also on the upper side. Fibroblasts and keratinocytes were seeded only on the upper side of the Laserskin in type B CLSG. Keratinocytes alone were seeded on plain Laserskin in type C CLSG. Type B CLSG consisting of autologous keratinocytes and autologous dermal fibroblasts was tested on five selected wounds (5x5 cm each) of a patient with full-thickness burn. In another burn patient, type B CLSG consisting of autologous keratinocytes and allogenic dermal fibroblasts was grafted onto three wounds (5x5 cm each). RESULTS: The seeding efficacy of human keratinocytes on plain Laserskin increased from 75% to 95% when proliferative allogenic fibroblasts were grown as a feeder layer on the Laserskin. The seeding efficacy of rat keratinocytes increased from 36% to 88% in the presence of a proliferative allogenic fibroblast feeder layer, whereas human/rat keratinocytes had respective seeding efficacy of 98%/91% on Laserskin preseeded with mitomycin C-treated 3T3 cells. Skin biopsies of grafted type A CLSG on day 14 after grafting showed complete epithelialization without severe inflammation in 16 of 20 (80%) grafted surgical wounds in rats. There were eight (40%) and seven (35%) "takes" of the CLSG in types B and C, respectively. The infection rate in type B CLSG was two (10%). There was one (5%) infection in types A and C. The respective take rates on the two patients grafted with type B CLSG were 60% and 100%. CONCLUSION: The animal experiment and the preliminary clinical data showed that the CSLGs consisting of autologous keratinocytes and of autologous/allogenic fibroblasts are good human skin substitutes in terms of durability, biocompatibility, high seeding efficacy for keratinocytes, high graft take rate, and low infection rate.

PAK promotes morphological changes by acting upstream of Rac.

The serine/threonine kinase p21-activated kinase (PAK) has been implicated as a downstream effector of the small GTPases Rac and Cdc42. While these GTPases evidently induce a variety of morphological changes, the role(s) of PAK remains elusive. Here we report that overexpression of betaPAK in PC12 cells induces a Rac phenotype, including cell spreading/membrane ruffling, and increased lamellipodia formation at growth cones and shafts of nerve growth factor-induced neurites. These effects are still observed in cells expressing kinase-negative or Rac/Cdc42 binding-deficient PAK mutants, indicating that kinase- and p21-binding domains are not involved. Furthermore, lamellipodia formation in all cell lines, including those expressing Rac binding-deficient PAK, is inhibited significantly by dominant-negative RacN17. Equal inhibition is achieved by blocking PAK interaction with the guanine nucleotide exchange factor PIX using a specific N-terminal PAK fragment. We conclude that PAK, via its N-terminal non-catalytic domain, acts upstream of Rac mediating lamellipodia formation through interaction with PIX.

Extraction of perceptually salient contours by striate cortical networks.

We present a cortical-based model for computing the perceptual salience of contours embedded in noisy images. It has been suggested that horizontal intra-cortical connections in primary visual cortex may modulate contrast detection thresholds and pre-attentive "pop-out". In our model, horizontal connections mediate context-dependent facilitatory and inhibitory interactions among oriented cells. Strongly facilitated cells undergo temporal synchronization; and perceptual salience is determined by the level of synchronized activity. The model accounts for a range of reported psychophysical and physiological effects of contour salience. In particular, the model proposes that intrinsic properties of synchronization account for the increased salience of smooth, closed contours. Application of the model to real images is demonstrated.

Coarse-grain parallel computing for very large scale neural simulations in the NEXUS simulation environment.

We describe a neural simulator designed for simulating very large scale models of cortical architectures. This simulator, NEXUS, uses coarse-grain parallel computing by distributing computation and data onto multiple conventional workstations connected via a local area network. Coarse-grain parallel computing offers natural advantages in simulating functionally segregated neural processes. We partition a complete model into modules with locally dense connections--a module may represent a cortical area, column, layer, or functional entity. Asynchronous data communications among workstations are established through the Network File System, which, together with the implicit modularity, decreases communications overhead, and increases overall performance. Coarse-grain parallelism also benefits from the standardization of conventional workstations and LAN, including portability between generations and vendors.

Regulation of insulin-like growth factor binding protein-1 during the 24-hour metabolic clock and in response to hypoinsulinemia induced by fasting and Sandostatin in normal women.

OBJECTIVE: To establish the relation of insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 (IGFBP-1) with 24-hour metabolic excursions in normal healthy women and in response to acute interruption of metabolic homeostasis by hypoinsulinemia. METHODS: Hourly blood samples during the 24-hour metabolic clock were obtained from seven normally cycling women. Uniform dietary composition (50% carbohydrate, 35% fat, and 15% protein) and timing of meals (8 AM, 12 PM, and 6 PM) were prescribed. Daytime hypoinsulinemia was induced by omitting meals and by Sandostatin (100 micrograms) administration. Changes in serum levels of glucose, insulin, cortisol, IGF-I, and IGFBP-1 were measured. RESULTS: The diurnal pattern of serum IGFBP-1 levels during the 24-hour metabolic clock was characterized by a rapid fall during the feeding phase of the day and a progressive 3.5-fold rise during nocturnal fasting; IGF-I levels were unchanged. Changes in IGFBP-1 levels were in parallel to those of cortisol and were inversely related to increases in glucose (80%) and insulin (tenfold) levels after each meal and to their decline during nocturnal fasting. Daytime fasting and administration of Sandostatin were accompanied by rapid and sustained increases in IGFBP-1 when insulin levels declined to 54 +/- 20 pmol/L. CONCLUSIONS: With constant levels of IGF-I, the diurnal rhythm of IGFBP-1 may subserve a physiologic function by coordinating insulin and IGF-I action with substrate availability. Fluctuations of insulin levels during the 24-hour metabolic clock in normal women appear to serve as a signal, with an inhibitory effect on IGFBP-1 production when levels are above 70 pmol/L and a stimulatory effect at levels below 70 pmol/L. These findings provide a basis for future investigations in women with nutritionally related reproductive disorders.

The premenstrual syndrome. Effects of "medical ovariectomy".

In a crossover study conducted over a six-month period in eight patients with well-characterized premenstrual syndrome, physical and behavioral symptoms were relieved by daily administration of an agonist of gonadotropin-releasing hormone. The reversible "medical ovariectomy" attained with this agonist suggests that it may be an effective and rational treatment for this distressing syndrome in the short term. Whether prolonged therapy would be safe and effective, or even necessary, remains to be determined.

Hormonal changes following hypophysectomy in humans.

PIP: Pituitary function was studied before and after surgery in 1 patient who underwent chryohypophysectomy and 5 patients who underwent transsphenoidal excision hypophysectomy for metastatic breast cancer ablation therapy. Changes in serum luteinizing hormone, follicle stimulating hormone, growth hormone, thyrotropin stimulating hormone, pituitary prolactin (PRL), and steroid hormone levels of estrone, estradiol, testosterone, and rostenedione, and dehydroepiandrosterone levels were determined. All of the above were assessed before and from 6 weeks to 10 months after hypophysectomy. Pituitary hormone release in response to sequential stimuli of arginine infusion, thyrotropic releasing factor (TRF), and luteinizing hormone-releaseing factor was analyzed. In 3 patients, hypophysectomy was considered incomplete because of the presence of measurable amounts of circulating pituitary hormones and their release in response to stimuli, although all levels except PRL were markedly reduced. In 3 patients, hypophysectomy was considered nearly complete. Basal PRL levels remained unchanged whether the procedure was considered complete or not. PRL release was absent in response to TRF or arginine stimulation in 4 patients with substantial residual pituitary function. Hypophysectomy was followed by a marked reduction in circulating levels of estrogens and androgens. The assessment of quantitative release of pituitary hormones in response to stimuli is an improved direct measure of residual functioning pituitary tissue.^ieng