Autocrine loop between TGF-beta1 and IL-1beta through Smad3- and ERK-dependent pathways in rat pancreatic stellate cells

Pancreatic stellate cells (PSCs) are activated during pancreatitis and promote pancreatic fibrosis by producing and secreting ECMs such as collagen and fibronectin. IL-1 has been assumed to participate in pancreatic fibrosis by activating PSCs. Activated PSCs secrete various cytokines that regulate PSC function. In this study, we have examined IL-1 secretion from culture-activated PSCs as well as its regulatory mechanism. RT-PCR and ELISA have demonstrated that PSCs express IL-1 mRNA and secrete IL-1 peptide. Inhibition of TGF-₁ activity secreted from PSCs by TGF-₁-neutralizing antibody attenuated IL-1 secretion from PSCs. Exogenous TGF-₁ increased IL-1 expression and secretion by PSCs in a dose-dependent manner. Adenovirus-mediated expression of dominant-negative (dn)Smad2/3 expression reduced both basal and TGF-₁-stimulated IL-1 expression and secretion by PSCs. Coexpression of Smad3 with dnSmad2/3 restored IL-1 expression and secretion by PSCs, which were attenuated by dnSmad2/3 expression. In contrast, coexpression of Smad2 with dnSmad2/3 did not alter them. Furthermore, inhibition of IL-1 activity secreted from PSCs by IL-1-neutralizing antibody attenuated TGF-₁ secretion from PSCs. Exogenous IL-1 enhanced TGF-₁ expression and secretion by PSCs. IL-1 activated ERK, and PD-98059, a MEK1 inhibitor, blocked IL-1 enhancement of TGF-₁ expression and secretion by PSCs. We propose that an autocrine loop exists between TGF-₁ and IL-1 in activated PSCs through Smad3- and ERK-dependent pathways. fibrosis; cytokine; chronic pancreatitis     

Protective effect of grape seed polyphenols against high glucose-induced oxidative stress

We aimed to clarify whether grape seed polyphenols (GSPs) are candidates therapeutic agents against diabetes mellitus, and to determine what degree of GSP oligomerization has the most potent efficacy. We studied the protective effects of various molecular weight GSPs (monomer, oligomer, polymer, and oligonol) on high glucose-induced cytotoxicity. In the present study, a high concentration of glucose (30 mM) induced cytotoxicity and oxidative stress (reactive oxygen species and nitric oxide) in cultured LLC-PK1 cells, but treatment with GSPs, especially oligomer GSPs, had potent protective effects against high glucose-induced oxidative stress. In addition, high glucose induced nuclear translocation of nuclear factor-kappa B, and increased expression of cyclooxygenase-2, inducible nitric oxide synthase, and bax, but GSP treatment inhibited them. These results indicate that GSPs have protective effects against high glucose-induced cytotoxicity, and among them, oligomer GSPs have more potent effects than other GSPs (monomer, polymer, and oligonol) on high glucose-induced renal cell damage.     

In vitro study of the proteolytic degradation of Antheraea pernyi silk fibroin

In this study, Antheraea pernyi silk fibroin (Ap-SF) films were incubated with Protease Type XXI from Streptomyces griseus, at 37 degrees C, to investigate the degradation behavior in an in vitro model system. The enzyme-resistant fractions of Ap-SF films and the soluble peptides formed by proteolytic degradation were collected at specified times, from 1 to 17 days, and analyzed by high performance liquid chromatography, differential scanning calorimetry, FT-Raman, and FT-IR spectroscopy. Proteolysis resulted in extensive weight loss and progressive fragmentation of films, especially at long degradation times. A range of soluble peptides was formed by proteolysis. By high performance-size exclusion chromatography it was found that their average molecular weight changed with the time of incubation. The chemical analysis of the enzyme-resistant fraction of Ap-SF films at different times of degradation indicated that the proteolytic attack preferentially occurred in the less ordered Gly rich sequences and that the contribution of the Ala rich crystalline regions to the composition of biodegraded films became progressively larger. Accordingly, DSC and spectroscopic results showed an enhancement of the crystalline character of the biodegraded films. From the behavior of the most important thermal transitions, it was deduced that the alpha-helix domains probably represent the most enzyme-resistant fraction. The in vitro approach used in the present study seems to be a valid tool for studying the rate and mechanism of degradation of Ap-SF films and of other biopolymers of potential biomedical utility.     

Conservation and divergence on plant seed 11S globulins based on crystal structures

The crystal structures of two pro-11S globulins namely: rapeseed procruciferin and pea prolegumin are presented here. We have extensively compared them with the other known structures of plant seed 11S and 7S globulins. In general, the disordered regions in the crystal structures among the 11S globulins correspond to their five variable regions. Variable region III of procruciferin is relatively short and is in a loop conformation. This region is highly disordered in other pro-11S globulin crystals. Local helical and strand variations also occur across the group despite general structure conservation. We showed how these variations may alter specific physicochemical, functional and physiological properties. Aliphatic hydrophobic residues on the molecular surface correlate well with T_m values of the globulins. We also considered other structural features that were reported to influence thermal stability but no definite conclusion was drawn since each factor has additive or subtractive effect. Comparison between proA3B4 and mature A3B4 revealed an increase in r.m.s.d. values near variable regions II and IV. Both regions are on the IE face. Secondary structure based alignment of 11S and 7S globulins revealed 16 identical residues. Based on proA3B4 sequence, Pro60, Gly128, Phe163, Phe208, Leu213, Leu227, Ile237, Pro382, Val404, Pro425 and Val 466 are involved in trimer formation and stabilization. Gly28, Gly74, Asp135, Gly349 and Gly397 are involved in correct globular folding.     

Fluorescence imaging using a fluorescent protein with a large Stokes shift

Keima is a far-red fluorescent protein endowed with a large Stokes shift. It absorbs light maximally at around 440 nm and emits maximally at around 620 nm. While the original Keima is obligately tetrameric (tKeima), the dimeric and monomeric versions (mKeima and dKeima, respectively) have been generated. More recently, a tandem dimer of Keima (tdKeima) has been developed as the brightest version. Here we describe examples, which show the usefulness of Keima for dual-color fluorescence imaging technologies, such as fluorescence cross-correlation spectroscopy (FCCS) and two-photon laser scanning microscopy (TPLSM). Keima can be used in conjunction with existing fluorescent proteins in which the Stokes shift is much smaller, with the idea that while two fluorescent proteins are excited by a single laser each will fluoresce a different color.     

Taste development from health education among schoolchildren: a two-year intervention study

It is necessary to develop a system of nutritional education which can be understood among schoolchildren who have not yet received a basic education. In the present study, we conducted an educational program for lower-grade schoolchildren, which contained dish selection, an agricultural experience, a cooking experience, and a lecture on digestive absorption. We evaluated the effect of this program on development by measuring taste sensitivity regarding sweet, sour, salty and bitter tastes. For the baseline period, there was no significant difference between the intervention school and the control school in each variable. At follow-up periods, both the intervention and the control schools showed an increasing sense of taste. In the intervention school, development of sensitivity to the sweet, the sour, and the bitter taste was significant. In the control school, development of sensitivity to the sweet and the bitter taste was significant. The increases in the sense of the sour and the bitter tastes and the sum of the four tastes for the intervention subjects were significantly larger than comparable values for the control subjects. These results suggest that the development of taste sensitivity is affected by nutritional education for lower-grade elementary schoolchildren.     

Structure and laminin-binding specificity of the NtA domain expressed in eukaryotic cells

Agrin is a key organizer for postsynaptic differentiation at the neuromuscular junction (NMJ). This activity requires the binding of agrin to the synaptic basal lamina via its N-terminal (NtA) domain. It has been suggested that this binding is mediated by conserved amino acids in the gamma 1 chain of laminin. Here, we report the crystal structure of chicken NtA expressed in eukaryotic HEK293 cells. In contrast to the previously published structure [Stetefeld, J., Jenny, M., Schulthess, T., Landwehr, R., Schumacher, B., Frank, S., Ruegg, M.A., Engel, J., Kammerer, R.A., 2001. The laminin-binding domain of agrin is structurally related to N-TIMP-1. Nat. Struct. Biol., 8, 705-709.], which was derived from the NtA domain expressed in E. coli, the new data show that the N-terminal tail region (amino acid residues Asn1-Arg5) is highly structured. Moreover, the disulfide bridge between Cys2 and Cys74 was also present. In addition, we show that the binding of NtA requires the gamma 1 chain of laminin and is not greatly affected by the composition of beta chains. These results confirm a model of the NtA-laminin complex where conserved amino acids in the gamma 1 chain are prerequisite for the binding to agrin and they further emphasize that the source of protein can be critical in structure determination.     

The effect of the amplitude of motor action on anticipatory postural adjustments.

The purpose of this study was to determine whether changes in the amplitude of a motor action triggering the same perturbation affect anticipatory postural adjustments (APAs). Healthy subjects performed releases of the same load with shoulder abduction movements of different amplitudes. Changes in the electrical activity of trunk and leg muscles, as well as displacements of the center of pressure were recorded. Generally, there were no differences in anticipatory activity of muscles and displacements of the center of pressure between series of load releases induced by motor actions of different amplitudes. We suggest that the CNS arranges APAs based on the magnitude of the perturbation if the same muscle groups generate motor actions of different amplitudes.