Progression of flagellar stages during artificially delayed motility initiation in sea urchin sperm

Transition from immotile to motile flagella may involve a series of states, in which some of regulatory mechanisms underlying normal flagellar movement are working with others being still suppressed. To address ourselves to the study of starting transients of flagella, we analyzed flagellar movement of sea urchin sperm whose motility initiation had been retarded in an experimental solution, so that we could capture the instance at which individual spermatozoa began their flagellar beating. Initially straight and immotile flagella began to shiver at low amplitude, then propagated exclusively the principal bend (P bend), and finally started stable flagellar beating. The site of generation of the P bend in the P-bend propagating stage varied in position in the basal region up to 10 microm from the base, indicating that the ability of autonomous bend generation is not exclusively possessed by the very basal region but can be unmasked throughout a wider region when the reverse bend (R bend) is suppressed. The rate of change in the shear angle, the curvature of the R bend and the frequency and regularity of beating substantially increased upon transition from P-bend propagating to full-beating, while the propagation velocity of bends remained unchanged. These findings indicate that artificially delayed motility initiation may accompany sequential modification of the motile system and that mechanisms underlying flagellar motility can be analyzed separately under experimentally retarded conditions.     

Odontoblasts induced from mesenchymal cells of murine dental papillae in three-dimensional cell culture

In an organ culture system under a three-dimensional microenvironment that provides the conditions needed for odontoblast differentiation, a row of odontoblasts can be induced (Kikuchi et al. 1996, 2001). Therefore, in a newly designed three-dimensional cell culture system that fulfils the conditions necessary for odontoblast differentiation (Kikuchi et al. 2002), we examined whether dental papilla cells in rat mandibular incisors could differentiate into tubular dentine-forming cells. In our previously established organ culture system, CM-Dil-labeled cells that were microinjected into isolated dental papillae were replaced by a row of odontoblasts. In a three-dimensional cell culture system, which consists of two kinds of type I collagen in the upper layer over multi-layered cells seeded onto collagen containing Matrigel in the lower layer and which acts as a structural meshwork, dental papilla cells were incubated as multi-layered cells in an artificial extracellular matrix (ECM). The cells aggregated to form a cell mass and invaginated as a cell mass into the ECM. The cells also extended fine fibrillar processes into the ECM. With regard to invagination, the proteolytic activities of matrix metalloproteinase-2 (MMP-2)/membrane type 1-matrix metalloproteinase (MT 1-MMP) were observed on the outer multi-layers of cells within a cell mass adjacent to the ECM. The cell mass progressively shrank to about one-half to one-third of its original diameter and was organized as a tissue surrounded by a newly secreted ECM, like dental pulp-dentine. The cells adjacent to the secreted ECM were constructed as a row of polarized columnar cells. They extended slender processes into the new ECM, which is characteristic of tubular matrix. Dentine sialophosphoprotein (DSPP) and dentine matrix protein 1 (DMP 1) genes, which are specific for odontoblast differentiation, were expressed in an aggregated cell mass where tubular matrix-forming cells were induced. Furthermore, the tubular matrix became mineralized under prolonged culture. These results imply that the putative progenitor cells/stem cells residing in dental papillae can differentiate into odontoblasts under appropriate conditions in vitro.     

Formation mechanism of 2,6-dimethyl-2,6-octadienes from thermal decomposition of linalyl beta-D-glucopyranoside

Thermally decomposed products of (+/-)-linalyl beta-D-glucoside were analyzed by GC and GC/MS. 2,6-dimethyl-2,6-octadienes produced by mild pyrolysis of linalyl beta-D-glucopyranoside under a vacuum were detected and characterized by MS and NMR spectroscopy. This suggests that 2,6-dimethyl-2,6-octadienes are produced during thermal decomposition of the glucoside via proton transfer from the anomeric position to C-6 in the aglycon moiety. A stable isotope labeling experiment directly indicated the new reaction mechanism.     

Does seed production of spring ephemerals decrease when spring comes early?

To predict the effect of global warming on plant reproductive success, seed-sets of spring ephemerals were compared between a year of extremely warm spring (2002) and normal years at cool-temperate deciduous forests in northern Japan. The spring of 2002 was the warmest in the last 40years and most spring-ephemeral plants bloomed 7–17days earlier than usual. The seed-set of bumblebee-pollinated Corydalis ambigua drastically decreased in 2002 in every population. The small bee-pollinated Gagea lutea also significantly decreased in 2002. However, the seed-sets of two fly pollinated species, Adonis ramosa and Anemone flaccida, were not influenced by early flowering. These results indicat that the effect of global warming on seed production of spring ephemerals differs between species depending on the type of pollinators, and that bee-pollinated species can have serious impacts on reproductive success as a result of climate change.     

Acute and chronic effects of FR-149175, a beta 3-adrenergic receptor agonist, on energy expenditure in Zucker fatty rats

Clinical therapies for both obesity and obese non-insulin-dependent diabetes mellitus require maintenance of reduced body weight after the initial successful reduction resulting from calorie control, exercise, or medication. Although beta(3)-adrenergic receptor (beta(3)-AR) agonists have been shown to stimulate whole body energy expenditure and lipid mobilization, whether stimulatory effects on oxygen consumption and lipolysis are influenced by chronic exposure to agonists has not been fully characterized. We therefore examined the acute and chronic effects of FR-149175, a selective beta(3)-AR agonist, on whole body oxygen consumption in genetically obese Zucker fatty rats. Chronic treatment with FR-149175 caused a decrease in both body weight gain and white fat pad weight at doses that induced lipolysis in acute treatment (1 and 3.2 mg/kg p.o.). Single administration of FR-149175 (0.1, 1, and 3.2 mg/kg p.o.) dose dependently increased whole body oxygen consumption. Repetitive administration did not cause attenuation of the thermogenic response at lower doses (0.1 and 1 mg/kg 2 times daily), whereas the highest dose (3.2 mg/kg 2 times daily) induced a progressive increase in oxygen consumption. PCR analyses of retroperitoneal white adipose tissue indicated little or no change in beta(3)-AR mRNA levels. Uncoupling protein 1 gene expression increased at 1 mg/kg, and drastic upregulation was detected at 3.2 mg/kg. FR-149175 also increased HSL mRNA levels in a dose-related manner, whereas there was no effect on genes involved in beta-oxidation. These results support that the thermogenic effect of beta(3)-AR agonists is not attenuated by chronic exposure to agonists.     

Plant-specific microtubule-associated protein SPIRAL2 is required for anisotropic growth in Arabidopsis

In diffusely growing plant cells, cortical microtubules play an important role in regulating the direction of cell expansion. Arabidopsis (Arabidopsis thaliana) spiral2 (spr2) mutant is defective in directional cell elongation and exhibits right-handed helical growth in longitudinally expanding organs such as root, hypocotyl, stem, petiole, and petal. The growth of spr2 roots is more sensitive to microtubule-interacting drugs than is wild-type root growth. The SPR2 gene encodes a plant-specific 94-kD protein containing HEAT-repeat motifs that are implicated in protein-protein interaction. When expressed constitutively, SPR2-green fluorescent protein fusion protein complemented the spr2 mutant phenotype and was localized to cortical microtubules as well as other mitotic microtubule arrays in transgenic plants. Recombinant SPR2 protein directly bound to taxol-stabilized microtubules in vitro. Furthermore, SPR2-specific antibody and mass spectrometry identified a tobacco (Nicotiana tabacum) SPR2 homolog in highly purified microtubule-associated protein fractions from tobacco BY-2 cell cultures. These results suggest that SPR2 is a novel microtubule-associated protein and is required for proper microtubule function involved in anisotropic growth.     

Thresholds of color attribute differences in detecting camouflaged textures

We examined the threshold at which a camouflaged color texture pattern (target) embedded in a surrounding colored texture pattern (background) was discriminated by making the difference between their color distributions serve as a cue. The texture consisted of 900 colored disks. The color applied to the disk was chosen from a normal distribution with the mean and the standard deviation set beforehand. The mean of the background's distribution was a standard achromatic color set at L*=40, u*=0, and v*=0 of CIELUV. In experiment 1, the mean of the target's color distribution was shifted from the background's one. The threshold for the mean of the target's color distribution depended on the standard deviation and increased as the standard deviation became bigger. In experiment 2, the standard deviation of the target's color distribution was shifted. There was the slight dependence of threshold of the standard deviation of the target's distribution on that of the background's distribution. In experiment 3, both of the mean and the standard deviation of the target's color distribution were shifted at the same time. The threshold was not determined by each of the mean and the standard deviation independently. There seemed to be some compensating contribution between them to each other. The threshold could be characterized by Doyle metric or modified Doyle metric.     

Determination of trivalent methylated arsenicals in rat urine by liquid chromatography-inductively coupled plasma mass spectrometry after solvent extraction

A method for the determination of trivalent arsenicals in urine was examined. Trivalent arsenicals, extracted as complexes with diethylammonium diethyldithiocarbamate (DDDC) into carbon tetrachloride, were determined by liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS). The trivalent methylated arsenicals monomethylarsonous acid (MMA(III)), dimethylarsinous acid (DMA(III)), and trimethylarsine (TMA) were detected in urine of rats that had received dimethylarsinic acid (DMA(V)) or monomethylarsonic acid (MMA(V)) at concentration of 200 microg ml(-1) in drinking water for 24 weeks. This method is the first to permit quantification of trivalent methylated arsenicals in urine without significant changes in concentration during storage or pretreatment.     

Proteomic signature of human cancer cells

We assessed proteomic profiles as biomarkers for monitoring cell phenotypes. Protein expression profiles were obtained by fluorescence two-dimensional difference gel electrophoresis (2-D-DIGE), in which quantitative ability is improved by labeling proteins with fluorescent dyes prior to electrophoresis. Integrated protein spot intensities were analyzed by a statistical approach. The proteomic data of two groups of cell lines: (1) adenocarcinoma (AC) cell lines derived from lung, pancreas and colon tissues and (2) lung cancer cell lines with different histological backgrounds, including AC, squamous cell carcinoma and small cell carcinoma, were assessed on the basis of prior biological information. Hierarchical clustering analysis and principal component analysis were used to divide the cell lines into subgroups on the basis of similarities between their protein expression profiles. The majority of cell lines were grouped according to their organ of origin or histological background. A machine-learning algorithm selected 32 protein spots that were responsible for the classification. The results indicate that proteomic data generated by 2-D-DIGE can provide a signature of essential cell phenotypes, suggesting that it might be possible to apply this technique to developing tumor markers that could identify the organ of origin of metastatic tumors and contribute to the differential diagnosis of lung cancer.     

Synthesis and biological evaluation of novel curcumin analogs as anti-cancer and anti-angiogenesis agents

A series of novel curcumin analogs were synthesized and screened for anti-cancer and anti-angiogenesis activities at Emory University and at the National Cancer Institute (NCI). These compounds are symmetrical alpha,beta-unsaturated and saturated ketones. The majority of the analogs demonstrated a moderate degree of anti-cancer activity. Compounds 10, 11, and 14 exhibited a high degree of cytotoxicity in the NCI in vitro anti-cancer cell line screen. In addition, this screen revealed that these compounds inhibit tumor cell growth with a higher potency than the commonly used chemotherapeutic drug, cisplatin. In independent in vitro screens conducted at Emory, the same compounds plus 4, 5, 8, 9, and 13 exhibited a high degree of cytotoxicity to tumor cells. Analogs that were effective in the anti-cancer screens were also effective in in vitro anti-angiogenesis assays. Compounds 4, 9, 11, and 14 were most effective in the anti-angiogenesis assays run at Emory. In the assays conducted by the NCI, compound 14 was almost as potent as the anti-angiogenic drug TNP-470, which has undergone clinical trials. Based on the favorable in vitro anti-cancer and anti-angiogenesis results with 14, further in vivo tests were conducted. This compound effectively reduced the size of human breast tumors grown in female athymic nude mice and showed little toxicity. This data, coupled with the remarkable in vitro data, suggests that compound 14 may potentially be an effective chemotherapeutic agent. As a follow-up, a 3D quantitative structure relationship based on 14 has been developed. It shows a cross-validated r2(q2) and a predictive r2(p2) = 0.71. COMPARE analysis suggests the compound to be a possible RNA/DNA antimetabolite, but also implies that the compound's cytotoxicity may arise from a presently unknown mechanism.