Fluorescence Polarization of αs1, κ-Caseins and αs1-κ-Casein Complex

Conjugates of α^s1-,κ-caseins and α^s1-,κ-casein complex were prepared with dimethylaminonaphthalenesulfonate and pyrenebutyrate. Their fluorescence lifetimes and the rotational relaxation times were measured by single photon counting technique and fluorescence depolarization technique, respectively. Both dimethylaminonaphthalenesulfonate and pyrenebutyrate conjugates had more than two lifetimes and the longer lifetime of pyrenebutyrate conjugates was near 140 nsec.

The rotational relaxation time of pyrenebutyrate α^s1-,κ-casein complex was smaller than that of pyrenebutyrate κ-casein polymer, which suggested that the complex formation of α^s1- and κ-casein polymers led to dissociation of the κ-casein polymer.

Changes of the rotational relaxation time as a function of weight ratio of α^s1- and κ-casein polymers (α^s1/κ) showed the specific variation and it was suggested that 4 moles of α^s1-κ-casein complex were formed from one mole of κ-casein polymer.     

Analysis of Factors That Determine Weight Gain during Smoking Cessation Therapy

Cigarette smokers are generally known to gain weight after quitting smoking, and such weight gain is thought to contribute to the worsening of glucose tolerance. While smoking cessation therapy such as nicotine replacement is useful to minimize post-cessation weight gain, substantial gain occurs even during the therapy. The purpose of the present study was to identify factors associated with weight gain during smoking cessation therapy. We evaluated 186 patients(132 males and 54 females)who visited our outpatient clinic for smoking cessation, and successfully achieved smoking abstinence. We performed gender-adjusted regression analysis for the rate of BMI increase from the beginning of cessation to 3 months after initiation. Furthermore, we performed multivariate analysis to investigate factors that determine the BMI increase after smoking cessation. The mean BMI significantly (p<0.0001) increased from 23.5±3.6 kg/m² at the initial consultation to 23.9±3.8 kg/m² at 3 months after the start of therapy. There was no significant difference in the extent of BMI increase between nicotine patch and varenicline therapy groups. Factors significantly correlated with the %BMI increase at 3 months after the start of therapy were triglyceride (p = 0.0006, βa = 0.260), high-density lipoprotein cholesterol (p = 0.0386, βa = −0.168), daily cigarette consumption (p = 0.0385, βa = 0.154), and the Fagerström Test for Nicotine Dependence (FTND) score (p = 0.0060, βa = 0.203). Stepwise multivariate analysis demonstrated that triglyceride and the FTND score were the factors determining the post-cessation BMI increase and that the FTND score was the strongest one. The present study demonstrated that smokers with a high FTND score are more likely to gain weight during smoking cessation therapy. Thus, smokers with a high nicotine dependency may require intervention against weight gain in the cessation clinic.     

Dynamic Changes of CD44 Expression from Progenitors to Subpopulations of Astrocytes and Neurons in Developing Cerebellum

We previously reported that CD44-positive cells were candidates for astrocyte precursor cells in the developing cerebellum, because cells expressing high levels of CD44 selected by fluorescence-activated cell sorting (FACS) gave rise only to astrocytes in vitro. However, whether CD44 is a specific cell marker for cerebellar astrocyte precursor cells in vivo is unknown. In this study, we used immunohistochemistry, in situ hybridization, and FACS to analyze the spatial and temporal expression of CD44 and characterize the CD44-positive cells in the mouse cerebellum during development. CD44 expression was observed not only in astrocyte precursor cells but also in neural stem cells and oligodendrocyte precursor cells (OPCs) at early postnatal stages. CD44 expression in OPCs was shut off during oligodendrocyte differentiation. Interestingly, during development, CD44 expression was limited specifically to Bergmann glia and fibrous astrocytes among three types of astrocytes in cerebellum, and expression in astrocytes was shut off during postnatal development. CD44 expression was also detected in developing Purkinje and granule neurons but was limited to granule neurons in the adult cerebellum. Thus, at early developmental stages of the cerebellum, CD44 was widely expressed in several types of precursor cells, and over the course of development, the expression of CD44 became restricted to granule neurons in the adult.     

Mutant analyses reveal different functions of fgfr1 in medaka and zebrafish despite conserved ligand-receptor relationships

Medaka (Oryzias latipes) is a small freshwater teleost that provides an excellent developmental genetic model complementary to zebrafish. Our recent mutagenesis screening using medaka identified headfish (hdf) which is characterized by the absence of trunk and tail structures with nearly normal head including the midbrain-hindbrain boundary (MHB). Positional-candidate cloning revealed that the hdf mutation causes a functionally null form of Fgfr1. The fgfr1hdf is thus the first fgf receptor mutant in fish. Although FGF signaling has been implicated in mesoderm induction, mesoderm is induced normally in the fgfr1hdf mutant, but subsequently, mutant embryos fail to maintain the mesoderm, leading to defects in mesoderm derivatives, especially in trunk and tail. Furthermore, we found that morpholino knockdown of medaka fgf8 resulted in a phenotype identical to the fgfr1hdf mutant, suggesting that like its mouse counterpart, Fgf8 is a major ligand for Fgfr1 in medaka early embryogenesis. Intriguingly, Fgf8 and Fgfr1 in zebrafish are also suggested to form a major ligand-receptor pair, but their function is much diverged, as the zebrafish fgfr1 morphant and zebrafish fgf8 mutant acerebellar (ace) only fail to develop the MHB, but develop nearly unaffected trunk and tail. These results provide evidence that teleost fish have evolved divergent functions of Fgf8-Fgfr1 while maintaining the ligand-receptor relationships. Comparative analysis using different fish is thus invaluable for shedding light on evolutionary diversification of gene function.     

Empirical Bayes inference of pairwise F(ST) and its distribution in the genome

Populations often have very complex hierarchical structure. Therefore, it is crucial in genetic monitoring and conservation biology to have a reliable estimate of the pattern of population subdivision. F(ST)'s for pairs of sampled localities or subpopulations are crucial statistics for the exploratory analysis of population structures, such as cluster analysis and multidimensional scaling. However, the estimation of F(ST) is not precise enough to reliably estimate the population structure and the extent of heterogeneity. This article proposes an empirical Bayes procedure to estimate locus-specific pairwise F(ST)'s. The posterior mean of the pairwise F(ST) can be interpreted as a shrinkage estimator, which reduces the variance of conventional estimators largely at the expense of a small bias. The global F(ST) of a population generally varies among loci in the genome. Our maximum-likelihood estimates of global F(ST)'s can be used as sufficient statistics to estimate the distribution of F(ST) in the genome. We demonstrate the efficacy and robustness of our model by simulation and by an analysis of the microsatellite allele frequencies of the Pacific herring. The heterogeneity of the global F(ST) in the genome is discussed on the basis of the estimated distribution of the global F(ST) for the herring and examples of human single nucleotide polymorphisms (SNPs).     

Thrombomodulin is a clock-controlled gene in vascular endothelial cells

Cardiovascular diseases are closely related to circadian rhythm, which is under the control of an internal biological clock mechanism. Although a biological clock exists not only in the hypothalamus but also in each peripheral tissue, the biological relevance of the peripheral clock remains to be elucidated. In this study we searched for clock-controlled genes in vascular endothelial cells using microarray technology. The expression of a total of 229 genes was up-regulated by CLOCK/BMAL2. Among the genes that we identified, we examined the thrombomodulin (TM) gene further, because TM is an integral membrane glycoprotein that is expressed primarily in vascular endothelial cells and plays a major role in the regulation of intravascular coagulation. TM mRNA and protein expression showed a clear circadian oscillation in the mouse lung and heart. Reporter analyses, gel shift assays, and chromatin immunoprecipitation analyses using the TM promoter revealed that a heterodimer of CLOCK and BMAL2 binds directly to the E-box of the TM promoter, resulting in TM promoter transactivation. Indeed, the oscillation of TM gene expression was abolished in clock mutant mice, suggesting that TM expression is regulated by the clock gene in vivo. Finally, the phase of circadian oscillation of TM mRNA expression was altered by temporal feeding restriction, suggesting TM gene expression is regulated by the peripheral clock system. In conclusion, these data suggest that the peripheral clock in vascular endothelial cells regulates TM gene expression and that the oscillation of TM expression may contribute to the circadian variation of cardiovascular events.     

Age-Associated Different Transcriptome Profiling in Zebrafish and Rats: an Insight into the Diversity of Vertebrate Aging

Marine Biotechnology - Most mammals, including humans, show obvious aging phenotypes, for example, loss of tissue plasticity and sarcopenia. In this regard, fish can be attractive models to study...     

Evaluation of the Duty Ratio of the Bacterial Flagellar Motor by Dynamic Load Control

The bacterial flagellar motor is one of the most complex and sophisticated nanomachineries in nature. A duty ratio D is a fraction of time that the stator and the rotor interact and is a fundamental property to characterize the motor but remains to be determined. It is known that the stator units of the motor bind to and dissociate from the motor dynamically to control the motor torque depending on the load on the motor. At low load, at which the kinetics such as proton translocation speed limits the rotation rate, the dependency of the rotation rate on the number of stator units N implies D: the dependency becomes larger for smaller D. Contradicting observations supporting both the small and large D have been reported. A dilemma is that it is difficult to explore a broad range of N at low load because the stator units easily dissociate, and N is limited to one or two at vanishing load. Here, we develop an electrorotation method to dynamically control the load on the flagellar motor of Salmonella with a calibrated magnitude of the torque. By instantly reducing the load for keeping N high, we observed that the speed at low load depends on N, implying a small duty ratio. We recovered the torque-speed curves of individual motors and evaluated the duty ratio to be 0.14 ± 0.04 from the correlation between the torque at high load and the rotation rate at low load.