Functional analysis of nucleosome assembly protein, NAP-1. The negatively charged COOH-terminal region is not necessary for the intrinsic assembly activity.

A nucleosome assembly protein (NAP-1) of Saccharomyces cerevisiae facilitates the association of histones with DNA to form nucleosomes in vitro at physiological ionic conditions. The cloned gene was expressed in Escherichia coli using a T7 expression system, and the protein (417 amino acid residues) was purified by Mono Q column chromatography. Various deletion fragments of NAP-1 protein were also produced, and their nucleosome assembly activity was examined by supercoiling assay. The internal fragment containing the residues 43-365 was necessary and sufficient for the activity, and a long stretch of negatively charged region near the carboxyl terminus was dispensable. This minimal size fragment could form the 12 S NAP-1-histone complex as the whole protein could, whereas deleted fragments on either side could bind with core histones only to form aggregates.     

Effects of ghrelin on neuronal activity in the ventromedial nucleus of the hypothalamus in infantile rats: An in vitro study

Ghrelin is an endogenous ligand for the growth hormone (GH) secretagogue (GHS) receptor (GHS-R) and a potent stimulant for GH secretion even in infantile rats before puberty. Although the ventromedial nucleus of the hypothalamus (VMH) might be a site of action for ghrelin to induce GH release, the electrophysiological effect of ghrelin on VMH neurons in infantile rats remains to be elucidated. Thus, the purpose of the present study was to investigate the effect of ghrelin on VMH neurons using hypothalamic slices of infantile rats. Ghrelin excited a majority of VMH neurons in a concentration-dependent manner. VMH neurons that were excited by GH releasing peptide-6 (GHRP-6), a synthetic GHS, were also excited by ghrelin and vice versa. Repeated application of ghrelin to the same VMH neuron decreased progressively the excitatory responses depending on the number of times it was administered. The excitatory effect of ghrelin on VMH neurons in normal artificial cerebrospinal fluid (ACSF) persisted in low Ca²⁺-high Mg²⁺ ACSF. The present results indicate that (1) ghrelin excites a majority of VMH neurons dose-dependently and postsynaptically and (2) the excitatory effects of ghrelin are mimicked by GHRP-6 and desensitized by repeated applications of ghrelin.     

Sequential polypeptides: 5. Statistical mechanical analysis of helix-coil transition in sequential polypeptides

A statistical mechanical theory of the helix-coil transition in sequential polypeptides is developed assuming that the statistical weights of the Zimm-Bragg parameters of a given residue depend on the type of adjacent residues. In the case of a sequential polypeptide consisting of two kinds of residues, the theory describes the helix- coil transition of the polypeptide in terms of the Zimm-Bragg parameters associated with the corresponding residues. The theory is then used to determine this parameter, as a function of temperature, from experimental data for transition temperature as a function of solvent composition, for a series of sequential polypeptides consisting of Glu(OBzl) and Lys(Chz) residues in mixtures of dichloroacetic acid and 1,2-dichlorethane. This parameter is then combined with the Zimm-Bragg parameters for the parent homopolypeptides, and the theory used to predict helix coil transition curves which are in good agreement with the experimental ones for the sequential polypeptides studied.     

Adaptive Shifts in the Regulation System of Seed Yield in Japanese Species of Epimedium (Berberidaceae)

Abstract Total seed yield per plant in one season was differentiated neither between Epimedium diphyllum and the E. grandiflorum complex ( E. grandiflorum and E. sempervirens ), nor between these two groups and one of their hybrid derived species, E. trifoliatobinatum . Total ovules per plant and seed-set rate per capsule ( SR ) did not vary greatly between these species, and seed weight ( SW ) was almost the same between them. The number of flowers per inflorescence ( F ) was also constant. However, the remaining reproductive component characters, ovule number per ovary ( O ) and inflorescence number per plant ( I ), were differentiated between the three taxa. These two characters were negatively correlated and a trade-off relationship occurred under the constant total seed yield (= O × F × I × SR × SW ). Ovule number per ovary was highly correlated with spur length of the flower. During the course of the hybrid speciation of E. trifoliatobinatum , selection pressure by pollinators on intermediate spur length seems to have favored plants with an intermediate ovule number. On the other hand, this selection pressure counteracted the increase of the inflorescence number under the trade-off. The resultant seed yield of E. trifoliatobinatum did not differ from that of the parental species, but the pattern of ovule allocation to ovaries (capsules) was altered.     

Structural characterization and dynamics of globotetraosylceramide in vascular endothelial cells under TNF-α stimulation

In several vascular inflammatory reactions (i.e. immunity and thrombosis) inflammatory mediators lead to the activation of vascular endothelial cells (EC). To date, a number of functional molecules induced on the surface of activated-EC have been identified. We report here that Globotetraosylceramide (Gb4), a glycosphingolipid expressed in EC, is a novel inducible molecule on EC activated by TNF-α. The cell surface expression of Gb4 is increased in a time-dependent manner under TNF-α stimulation, which shows distinct expression kinetics of major proteins induced by TNF-α on EC. MALDI-TOF-MS analysis revealed that the enhanced Gb4 predominantly contains C24:0 fatty acid in the ceramide moiety. Isolated caveolae/lipid raft-enriched detergent insoluble membrane domains in activated-EC predominantly contain this molecular species of Gb4. Gb4 containing C16:0 fatty acid in the ceramide moiety, which is known to constitute the major species of Gb4 in plasma, is also found as a major molecular species in EC. These observations indicate that Gb4, especially with very long fatty acid, is enhanced in EC during its inflammatory reaction, and suggest the potential utility of Gb4 as a biomarker for monitoring inflammation status of EC involving its related diseases.     

Immunological detection of a cysteine protease in the skin and other tissues

Summary Monospecific antibody directed to cysteine protease of 2-day-old rat epidermis recently characterized as being different from the proteases previously reported was produced in rabbits. By immunofluorescence microscopy and immunoperoxidase staining with an avidin-biotin-peroxidase method the protease was found to be present in the epidermis of rodents of different ages as well as that of humans, but not in the dermis. The staining in germinative cells was more intense than in cells in the superficial layers. It appeared as irregular patches in the nuclei and stained more diffusely in the cytoplasm where small granular components, strongly stained, were identified. The staining patterns in granular cells showed accumulation of the antigen in a granular form. The morphology and distribution of granules resembled those of keratohyalin-like granules in the nucleus and dense homogenous deposits in the cytoplasm. In cornified cells the reaction product was localized by the plasma membrane where concentration of the dense homogenous deposits occurred, suggesting that the cysteine protease is one component of the unique and characteristic structure of differentiated keratinocytes. In addition, the cysteine protease antigen having the same molecular weight as the epidermal enzyme was detected in liver, kidney and lung indicating a wider tissue distribution of the protease. The significance of the protease in regulation of cellular functions remains to be investigated.     

High-performance anion-exchange chromatography of ultraviolet-absorbing constituents of human urine

A 100-μl urine sample was chromatographed on a column packed with a strongly basic macroreticular anion-exchange resin (Diaion CDR-10, 5– μm diameter with a nominal 35% cross linkage). The elution was performed with a linear acetate gradient from 0 to 6.0 M at an average flow-rate of 0.72 ml/min and at an average pressure of 104 kg/cm². The relative standard deviation of retention times and peak height was ± 4% or less. The properties of the macroreticular anion-exchange resin, the effect of the particle size, the pH of acetate buffers, and the effect of the flow-rate of the eluent on the separation were investigated. Thirty three components of urine were then resolved and named.