A simple method for the determination of affinity and binding site concentration in receptor binding studies

In ligand binding studies, it is often difficult to apply kinetic analyses because of an uncertainty in experimental data obtained at high ligand concentrations. Under such circumstances, K_d value (an index of the affinity) and the binding site concentration may be estimated more accurately from the binding of a fixed concentration of labelled ligand observed in the presence of various concentrations of the non-labelled ligand, if the fraction of both labelled and non-labelled ligand bound is small. When there is no cooperative effect of the ligand binding, the K_d value may be calculated by subtracting the concentration of the labelled drug from the concentration of the non-labelled drug to cause a 50% reduction of the saturable binding of the labelled drug. From above values, the binding site concentration may be calculated. The proposed method is capable of examining the cooperativity of the ligand binding, the labelled drug concentration and the specific radioactivity of the labelled drug and does not require large amounts of the labelled drug.     

Complex Segregation Analysis on Sib-Pairs Data

Using sib-pairs and parent-pairs data, a quantitative trait can be tested for the existence of a major locus under the mixed model of Morton and MacLean (1974). The basic idea is to obtain the two conditional likelihoods for sib-pair differences and parent-pair differences provided that it is known which sib-pairs or parent-pairs have the same effect at the major locus. Two conditions are introduced to obtain two recursive computer algorithms that distinguish the sib-pairs or parent-pairs having the same effect at the major locus. This method has the advantage of reducing complicated computations involving maximum likelihood estimates from nuclear families. A simulation experiment is performed to illustrate the method and its results are discussed.     

Visualization of DC-SIGN-Mediated Entry Pathway of Engineered Lentiviral Vectors in Target Cells

Dendritic cells (DCs) are potent antigen-presenting cells and therefore have enormous potential as vaccine targets. We have previously developed an engineered lentiviral vector (LV) that is pseudotyped with a mutated Sindbis virus glycoprotein (SVGmu), which is capable of targeting DCs through Dendritic Cell-specific ICAM3-grabbing Nonintegrin (DC-SIGN), a receptor that is predominantly expressed by DCs. In this study, we aimed to elucidate the internalization and trafficking mechanisms of this viral vector system through direct visualization of GFP-Vpr-tagged viral particles in target DCs, which was further corroborated by drug inhibition and dominant-negative mutants of cellular proteins that regulate the endocytic traffic. We demonstrated that our engineered LVs enter the cell via receptor-mediated clathrin- and dynamin-dependent endocytosis. Microtubule networks were also involved in a productive infection. Viral vector fusion was low-pH-dependent and occurred in the early endosomal stage of the intracellular transport. Autophagy was also examined for its effect on transduction efficiency, and we observed that enhanced autophage activity reduced vector infectivity, while suppressed autophagy boosted transduction efficiency. This study shed some light on the internalization and trafficking mechanisms of DC-directed LVs and offers some strategies to further improve the efficiency of LV-mediated gene therapy.     

Progranulin compensates for blocked IGF-1 signaling to promote myotube hypertrophy in C2C12 myoblasts via the PI3K/Akt/mTOR pathway

It is well known that growth hormone (GH)-induced IGF-1 signaling plays a dominant role in postnatal muscle growth. Our previous studies have identified a growth factor, progranulin (PGRN), that is co-induced with IGF-1 upon GH administration. This result prompted us to explore the function of PGRN and its association with IGF-1. In the present study, we demonstrated that, similar to IGF-1, PGRN can promote C2C12 myotube hypertrophy via the PI(3)K/Akt/mTOR pathway. Moreover, PGRN can rescue the muscle atrophy phenotypes in C2C12 myotube when IGF-1 signaling is blocked. This result shows that PGRN can substitute for IGF-1 signaling in the regulation of muscle growth. Our findings provide new insights into IGF-1-modulated complicated networks that regulate muscle growth.     

Increased urinary phosphate excretion in pseudohypoparathyroidism type II with long-term treatment with phosphodiesterase inhibitor.

A 58-year-old woman was diagnosed to have pseudohypoparathyroidism (PHP) type II because of the absence of an increase of urinary phosphate secretion, despite a marked increase in urinary cAMP excretion on the Ellsworth-Howard test. We treated the patient with a cyclic-nucleotide phosphodiesterase inhibitor, theophylline, resulting in increased urinary phosphate and cAMP excretions. Dibutyl cAMP administration induced the increase in the urinary phosphate excretion. In this case, the unresponsiveness of the urinary phosphate secretion to cAMP was recovered by a high dose of cAMP or long-term administration of a phosphodiesterase inhibitor. These data imply that cAMP responsiveness to renal tubular phosphate reabsorption should be more strictly elucidated in the patient with PHP type II.     

Directionality in the mechanical response to substrate vibration in a treehopper (Hemiptera: Membracidae: Umbonia crassicornis)

The use of substrate vibrations in communication and predator-prey interactions is widespread in arthropods. In many contexts, localization of the vibration source plays an important role. For small species on solid substrates, time and amplitude differences between receptors in different legs may be extremely small, and the mechanisms of vibration localization are unclear. Here we ask whether directional information is contained in the mechanical response of an insect's body to substrate vibration. Our study species was a membracid treehopper (Umbonia crassicornis) that communicates using bending waves in plant stems. We used a bending-wave simulator that allows precise control of the frequency, intensity and direction of the vibrational stimulus. With laser-Doppler vibrometry, we measured points on the substrate and on the insect's thorax and middle leg. Transfer functions showing the response of the body relative to the substrate revealed resonance at lower frequencies and attenuation at higher frequencies. There were two modes of vibration along the body's long axis, a translational and a rotational mode. Furthermore, the transfer functions measured on the body differed substantially depending on whether the stimulus originated in front of or behind the insect. Directional information is thus available in the mechanical response of the body of these insects to substrate vibration. These results suggest a vibration localization mechanism that could function at very small spatial scales.     

Variations of Some Enzyne and Coenzyme Concentrations in Nornal Human Erythrocytes Fractionated by Velocity Sedimentation

Abstract

The concentrations of glucose-6-phosphate dehydrogenase, adenosine triphosphate and 2,3-diphosphoglycerate were measured in various fractions of normal human erythrocytes separated by velocity sedimentation in isotonic saline and sucrose buffer based on their tendency to aggregate. The levels of 2,3-diphosphoglycerate varied most drastically in fresh erythrocytes, being lowest in fast sedimenting cells and highest in slowly sedimenting cells. This result implies that the 2,3-diphosphoglycerate concentrations is not constant in all normal human erythrocytes and that 2,3-diphosphoglycerate may be involved in the mechanism of red cell clumping in vitro.     

Eighty years of Chinese Association for Physiological Sciences in retrospect

The Chinese Association for Physiological Sciences （CAPS） celebrated her 80th anniversary last November. Eighty years ago, Dr. Robert K. S. Lim, Professor of Physiology at the Peking Union Medical College, in association with Dr. Wu Xian （Professor of Biochemistry） and Dr. B. E. Read （Professor of Pharmacology）, sponsored the establishment of the Chinese Physiological Society. They held the inaugural meeting in Beijing on February 27, 1926. This event was an important landmark in the history of the development of physiological sciences in China.