Potent antiobesity effect of a short-length peptide YY-analogue continuously administered in mice

The gastrointestinal peptide, peptide YY_3–36 (PYY_3–36) and its shorter peptide analogues have been reported to reduce appetite by activating the neuropeptide Y2 receptor (Y2R), which is associated with obesity and other metabolic diseases. A 14-amino acid PYY analogue, Ac-[d-Pro²⁴,Cha^27,28,36,Aib³¹]PYY(23–36) (3), showed high binding affinity and agonist activity for the Y2R, similar to that of PYY_3–36, but had weak anorectic activity upon continuous administration in lean mice. Three amino acid substitutions [Pya(4)²⁶, Aib²⁸, Lys³⁰], which contributed to the decreased hydrophobicity of 3, efficiently increased its anorectic activity. The compound containing these three amino acids, Ac-[d-Pro²⁴,Pya(4)²⁶,Cha^27,36,Aib^28,31,Lys³⁰]PYY(23–36) (22), exerted more potent and durable food intake suppression than that by PYY_3–36 in lean mice, as well as excellent Y2R agonist activity (EC₅₀: 0.20 nM) and good subcutaneous bioavailability (66.6%). The 11-day continuous administration of 22 at 1 mg/kg/day successfully produced antiobese and antidiabetic effects, with more than 20% body weight loss in obese and Type 2 diabetes ob/ob model mice.     

A potent neuromedin U receptor 2-selective alkylated peptide

Neuromedin U (NMU) mediates various physiological functions via NMUR1 and NMUR2 receptors. NMUR2 has been considered a promising treatment option for diabetes and obesity. Although NMU-8, a shorter peptide, has potent agonist activity for both receptors, it is metabolically unstable. Therefore, NMU-8 analogs modified with long-chain alkyl moieties via a linker were synthesized. An octadecanoyl analog (17) with amino acid substitutions [αMePhe¹⁹, Nle²¹, and Arg(Me)²⁴] and a linker [Tra-γGlu-PEG(2)] dramatically increased NMUR2 selectivity, with retention of high agonist activity. Subcutaneous administration of 17 induced anorectic activity in C57BL/6J mice. Owing to its high metabolic stability, 17 would be useful in clarifying the physiological role and therapeutic application of NMU.     

PLASTOCHRON3/GOLIATH encodes a glutamate carboxypeptidase required for proper development in rice

Most aerial parts of the plant body are products of the continuous activity of the shoot apical meristem (SAM). Leaves are the major component of the aerial plant body, and their temporal and spatial distribution mainly determines shoot architecture. Here we report the identification of the rice gene PLASTOCHRON3 ( PLA3 )/ GOLIATH ( GO ) that regulates various developmental processes including the rate of leaf initiation (the plastochron). PLA3 / GO encodes a glutamate carboxypeptidase, which is thought to catabolize small acidic peptides and produce small signaling molecules. pla3 exhibits similar phenotypes to pla1 and pla2 – a shortened plastochron, precocious leaf maturation and rachis branch-to-shoot conversion in the reproductive phase. However, in contrast to pla1 and pla2 , pla3 showed pleiotropic phenotypes including enlarged embryo, seed vivipary, defects in SAM maintenance and aberrant leaf morphology. Consistent with these pleiotropic phenotypes, PLA3 is expressed in the whole plant body, and is involved in plant hormone homeostasis. Double mutant analysis revealed that PLA1 , PLA2 and PLA3 are regulated independently but function redundantly. Our results suggest that PLA3 modulates various signaling pathways associated with a number of developmental processes.     

Specific induction of a 72-kDa heat shock protein protects esophageal mucosa from reflux esophagitis

AimsThe aim of this study is to investigate the expression and cytoprotective function of a 72-kDa heat shock protein (HSP72) using a reflux esophagitis model in rats.Main methodsExpression of HSP60, HSP72, and HSP90 in rat esophageal mucosa was evaluated by Western blot analysis before and after hyperthermia (42.5 °C, 20 min). Rats received the operation to produce reflux esophagitis with or without pretreatment with hyperthermia to induce HSPs. The esophageal mucosal damage was evaluated 12 h after the operation.Key findingsExpression of HSP72 was significantly increased by hyperthermia in rat esophageal mucosa. Reflux esophagitis was dramatically prevented when HSP72 was preinduced by hyperthermia. Furthermore, activation of TNF-α and IL-1β in esophageal mucosa was also suppressed.SignificanceThese results suggested that hyperthermia protects the esophageal mucosa in reflux esophagitis model by inducing HSP72 and suppressing proinflammatory cytokine activation. These findings might suggest that HSP-inducing therapy could be a novel and unique therapy for reflux esophagitis.     

Asymmetric intercellular communication between bone cells: Propagation of the calcium signaling

Bone functional adaptation by remodeling is achieved by harmonized activities of bone cells in which osteocytes in the bone matrix are believed to play critical roles in sensing mechanical stimuli and transmitting signals to osteoclasts/osteoblasts on the bone surface in order to regulate their bone remodeling activities through the lacuno-canalicular network with many slender osteocytic processes. In this study, we investigated the intercellular communication between bone cells, particularly focusing on its directionality, through in vitro observations of the calcium signaling response to mechanical stimulus and its propagation to neighboring cells (NCs). Direct mechanical stimulus was applied to isolated bone cells from chick calvariae, osteocytes (Ocys) and bone surface cells (BSCs) mainly containing osteoblasts, and the percentage of calcium signaling propagation from the stimulated cell to NCs was analyzed. The results revealed that, regardless of the type of stimulated cell, the signaling propagated to BSCs with a significantly higher percentage, implying that calcium signaling propagation between bone cells strongly depends on the type of receiver cell and not the transmitter cell. In addition, in terms of mutual communication between Ocys and BSCs, the percentage of propagation from Ocys to BSCs is significantly higher than that in the opposite direction, suggesting that the calcium signaling mainly propagates asymmetrically with a bias from Ocys in bone matrix to BSCs on bone surfaces. This asymmetric communication between Ocys and BSCs suggests that osteocytic mechanosensing and cellular communications, which significantly affect bone surface remodeling activities to achieve functional adaptation, seem to be well coordinated and active at the location of biologically suitable and mechanically sensitive regions close to the bone surfaces.     

A mathematical model for the distribution of hemodynamic parameters in the human retinal microvascular network

To quantitatively assess the arteriovenous distribution of hemodynamic parameters throughout the microvascular network of the human retina, we constructed a retinal microcirculatory model consisting of a dichotomous symmetric branching system. This system is characterized by a diameter exponent of 2.85, instead of 3 as dictated by Murray’s law, except for the capillary networks. The value of 2.85 was the sum of a fractal dimension (1.70) and a branch exponent (1.15) of the retinal vasculature. Following the feeding artery (central retinal artery), each bifurcation was recursively developed at a distance of an individual branch length [L(r) = 7.4r ^1.15] by a centrifugal scheme. The venular tree was formed in the same way. Using this model, we evaluated hemodynamic parameters, including blood pressure, blood flow, blood velocity, shear rate, and shear stress, within the retinal microcirculatory network as a function of vessel diameter. The arteriovenous distributions of blood pressure and velocity in the simulation were consistent with in vivo measurements in the human retina and other vascular beds of small animals. We therefore conclude that the current theoretical model was useful for quantifying hemodynamics as a function of vessel diameter within the retinal microvascular network.     

A novel method for measuring tension generated in stress fibers by applying external forces

The distribution of contractile forces generated in cytoskeletal stress fibers (SFs) contributes to cellular dynamic functions such as migration and mechanotransduction. Here we describe a novel (to our knowledge) method for measuring local tensions in SFs based on the following procedure: 1), known forces of different magnitudes are applied to an SF in the direction perpendicular to its longitudinal axis; 2), force balance equations are used to calculate the resulting tensions in the SF from changes in the SF angle; and 3), the relationship between tension and applied force thus established is extrapolated to an applied force of zero to determine the preexisting tension in the SF. In this study, we measured tensions in SFs by attaching magnetic particles to them and applying known forces with an electromagnetic needle. Fluorescence microscopy was used to capture images of SFs fluorescently labeled with myosin II antibodies, and analysis of these images allowed the tension in the SFs to be measured. The average tension measured in this study was comparable to previous reports, which indicates that this method may become a powerful tool for elucidating the mechanisms by which cytoskeletal tensions affect cellular functions.     

Site-specific tetrameric streptavidin-protein conjugation using sortase A

Streptavidin is tetrameric protein which has tight and specific biotin binding affinity, and streptavidin modification of proteins or small molecules is widely used for biotechnology tool. Here, we demonstrate site-specific streptavidin-protein conjugation using enzymes. We focused on sortase A, a transpeptidase from Staphylococcus aureus. A streptavidin-tagged LPETG motif (Stav-LPETG) was expressed in Escherichia coli. We achieved soluble streptavidin expression in E. coli without refolding using a cold shock expression system. Then we successfully conjugated Stav-LPETG with pentaglycine-appended green fluorescence protein (Gly5-GFP) or triglycine-appended glucose oxidase (Gly3-GOD) using sortase A. SDS-PAGE analysis showed site-specific tetrameric streptavidin-protein conjugation with the tagged proteins. In addition, the functions of a Stav-GOD conjugate, i.e., biotin-binding and glucose oxidase activity, were significantly higher compared to those of streptavidin-GOD conjugates prepared by chemical modification.