Non-core region modulates interleukin-11 signaling activity: generation of agonist and antagonist variants

Human interleukin-11 (hIL-11) is a pleiotropic cytokine administered to patients with low platelet counts. From a structural point of view hIL-11 belongs to the long-helix cytokine superfamily, which is characterized by a conserved core motif consisting of four α-helices. We have investigated the region of hIL-11 that does not belong to the α-helical bundle motif, and that for the purpose of brevity we have termed "non-core region." The primary sequence of the interleukin was altered at various locations within the non-core region by introducing glycosylation sites. Functional consequences of these modifications were examined in cell-based as well as biophysical assays. Overall, the data indicated that the non-core region modulates the function of hIL-11 in two ways. First, the majority of muteins displayed enhanced cell-stimulatory properties (superagonist behavior) in a glycosylation-dependent manner, suggesting that the non-core region is biologically designed to limit the full potential of hIL-11. Second, specific modification of a predicted mini α-helix led to cytokine inactivation, demonstrating that this putative structural element belongs to site III engaging a second copy of cell-receptor gp130. These findings have unveiled new and unexpected elements modulating the biological activity of hIL-11, which may be exploited to develop more versatile medications based on this important cytokine.     

Microtubule dynamics regulate cyclic stretch-induced cell alignment in human airway smooth muscle cells

Microtubules are structural components of the cytoskeleton that determine cell shape, polarity, and motility in cooperation with the actin filaments. In order to determine the role of microtubules in cell alignment, human airway smooth muscle cells were exposed to cyclic uniaxial stretch. Human airway smooth muscle cells, cultured on type I collagen-coated elastic silicone membranes, were stretched uniaxially (20% in strain, 30 cycles/min) for 2 h. The population of airway smooth muscle cells which were originally oriented randomly aligned near perpendicular to the stretch axis in a time-dependent manner. However, when the cells treated with microtubule disruptors, nocodazole and colchicine, were subjected to the same cyclic uniaxial stretch, the cells failed to align. Lack of alignment was also observed for airway smooth muscle cells treated with a microtubule stabilizer, paclitaxel. To understand the intracellular mechanisms involved, we developed a computational model in which microtubule polymerization and attachment to focal adhesions were regulated by the preexisting tensile stress, pre-stress, on actin stress fibers. We demonstrate that microtubules play a central role in cell re-orientation when cells experience cyclic uniaxial stretching. Our findings further suggest that cell alignment and cytoskeletal reorganization in response to cyclic stretch results from the ability of the microtubule-stress fiber assembly to maintain a homeostatic strain on the stress fiber at focal adhesions. The mechanism of stretch-induced alignment we uncovered is likely involved in various airway functions as well as in the pathophysiology of airway remodeling in asthma.     

Dominant induction of vaccine antigen-specific cytotoxic T lymphocyte responses after simian immunodeficiency virus challenge

Cytotoxic T lymphocyte (CTL) responses are crucial for the control of human and simian immunodeficiency virus (HIV and SIV) replication. A promising AIDS vaccine strategy is to induce CTL memory resulting in more effective CTL responses post-viral exposure compared to those in natural HIV infections. We previously developed a CTL-inducing vaccine and showed SIV control in some vaccinated rhesus macaques. These vaccine-based SIV controllers elicited vaccine antigen-specific CTL responses dominantly in the acute phase post-challenge. Here, we examined CTL responses post-challenge in those vaccinated animals that failed to control SIV replication. Unvaccinated rhesus macaques possessing the major histocompatibility complex class I haplotype 90-088-Ij dominantly elicited SIV non-Gag antigen-specific CTL responses after SIV challenge, while those induced with Gag-specific CTL memory by prophylactic vaccination failed to control SIV replication with dominant Gag-specific CTL responses in the acute phase, indicating dominant induction of vaccine antigen-specific CTL responses post-challenge even in non-controllers. Further analysis suggested that prophylactic vaccination results in dominant induction of vaccine antigen-specific CTL responses post-viral exposure but delays SIV non-vaccine antigen-specific CTL responses. These results imply a significant influence of prophylactic vaccination on CTL immunodominance post-viral exposure, providing insights into antigen design in development of a CTL-inducing AIDS vaccine.     

5-(1,3-Benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazole derivatives as potent and selective transforming growth factor-β type I receptor inhibitors

A series of 5-(1,3-benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazole derivatives was synthesized as transforming growth factor-β (TGF-β) type I receptor (also known as activin-like kinase 5 or ALK5) inhibitors. These compounds were evaluated for their ALK5 inhibitory activity in an enzyme assay and for their TGF-β-induced Smad2/3 phosphorylation inhibitory activity in a cell-based assay. As a representative compound, 16i was a potent and selective ALK5 inhibitor, exhibiting a good enzyme inhibitory activity (IC₅₀ = 5.5 nM) as well as inhibitory activity against TGF-β-induced Smad2/3 phosphorylation at a cellular level (IC₅₀ = 36 nM). Furthermore, the topical application of 3% 16i lotion significantly inhibited Smad2 phosphorylation in Mouse skin (90% inhibition compared with vehicle-treated animals).     

The CatSper channel: a polymodal chemosensor in human sperm

The sperm-specific CatSper channel controls the intracellular Ca(2+) concentration ([Ca(2+)](i)) and, thereby, the swimming behaviour of sperm. In humans, CatSper is directly activated by progesterone and prostaglandins-female factors that stimulate Ca(2+) influx. Other factors including neurotransmitters, chemokines, and odorants also affect sperm function by changing [Ca(2+)](i). Several ligands, notably odorants, have been proposed to control Ca(2+) entry and motility via G protein-coupled receptors (GPCRs) and cAMP-signalling pathways. Here, we show that odorants directly activate CatSper without involving GPCRs and cAMP. Moreover, membrane-permeable analogues of cyclic nucleotides that have been frequently used to study cAMP-mediated Ca(2+) signalling also activate CatSper directly via an extracellular site. Thus, CatSper or associated protein(s) harbour promiscuous binding sites that can host various ligands. These results contest current concepts of Ca(2+) signalling by GPCR and cAMP in mammalian sperm: ligands thought to activate metabotropic pathways, in fact, act via a common ionotropic mechanism. We propose that the CatSper channel complex serves as a polymodal sensor for multiple chemical cues that assist sperm during their voyage across the female genital tract.     

Quality assessment for processed and sterilized bone using Raman spectroscopy

To eliminate the potential for infection, many tissue banks routinely process and terminally sterilize allografts prior to transplantation. A number of techniques, including the use of scanning electron microscopy, bone graft models, and mechanical property tests, are used to evaluate the properties of allograft bone. However, as these methods are time consuming and often destroy the bone sample, the quality assessment of allograft bones are not routinely performed after processing and sterilization procedures. Raman spectroscopy is a non-destructive, rapid analysis technique that requires only small sample volumes and has recently been used to evaluate the mineral content, mineral crystallinity, acid phosphate and carbonate contents, and collagen maturity in human and animal bones. Here, to establish a quality assessment method of allograft bones using Raman spectroscopy, the effect of several common sterilization and preservation procedures on rat femoral bones were investigated. We found that freeze-thawing had no detectable effects on the composition of bone minerals or matrix, although heat treatment and gamma irradiation resulted in altered Raman spectra. Our findings suggest Raman spectroscopy may facilitate the quality control of allograft bone after processing and sterilization procedures.     

Uniaxial cyclic stretch increases glucose uptake into C2C12 myotubes through a signaling pathway independent of insulin-like growth factor I

Insulin-like growth factor I (IGF-I), an autocrine/paracrine growth factor involved in myogenesis, has rapid effects on muscle metabolism. In a manner analogous to insulin and mechanical stimuli such as stretch, IGF-I stimulates glucose transport through recruitment of glucose transporters to surface membranes in skeletal muscles. It is known that IGF-I is secreted from skeletal muscle cells in response to stretch. Therefore, we examined whether IGF-I is involved in the mechanism by which mechanical stretch regulates glucose transport using cultured C2C12 myotubes. IGF-I increased 2-deoxy- D-glucose (2-DG) uptake, and this created an additive effect with mechanical stretch, suggesting that these stimuli enhance glucose transport through different mechanisms. In fact, IGF-I-stimulated 2-DG uptake was not blocked by dantrolene (an inhibitor of Ca (2+)release from sarcoplasmic reticulum), whereas the stretch-stimulated effect was abolished. Conversely, the IGF-I-stimulated 2-DG uptake was prevented by phosphatidylinositol 3-kinase inhibitor wortmannin, which did not prevent the stretch-stimulated 2-DG uptake. In addition, experiments using media conditioned by stretched myotubes indicated that a mechanically induced release of locally acting autocrine/paracrine growth factors was not sufficient for induction of 2-DG uptake. Thus, our results demonstrate that mechanical stretch signaling for glucose transport is independent of the mechanism through which IGF-I increases this transport.     

Embryogenesis and swimming behavior of Medaka fish in JUSTSAP STARS Program (STS-107).

JUSTSAP (Japan-US Science, Technology and Space Application Program) Medaka fish experiment was carried out as a part of STARS (Space Technology and Research for Student) experiment, a space shuttle mission, STS-107 in January 2003. Four eggs laid on earth under artificially controlled environment were put in a closed ecological system, AHAB (Aquatic Habitat), and launched by Space Shuttle Columbia. For the control experiment, four eggs were put in the AHAB and remained on the ground. There was no remarkable difference in the time course of the development. In ground experiment embryos were observed to rotate in the egg membrane, whereas in flight unit they did not rotate. One egg hatched out on L (Launch) +8 days in flight unit. Four eggs hatched out in ground unit. Fry in flight unit was observed to face its back usually to the camera with little swimming movement. Fry in ground unit were observed to move actively and also to control their posture with respect to gravity vector.