Synthesis and properties of malic acid-containing functional polymers.

Poly-L-lactides containing beta-alkyl alpha-malate-units were prepared by ring-opening copolymerizations of L-lactide with 3-(s)-[(benzyloxycarbonyl)methyl]- (BMD) and 3-(s)-[(dodecyloxycarbonyl)methyl]-1,4-dioxane-2,5-diones (DMD). The solution-cast films of these copolymers were alkali-treated to form a carboxyl-functionalized surface on which cell-binding Arg-Gly-Asp tripeptide (RGD) was immobilized with dicyclohexylcarbodiimide as coupling agent. For the copolymer of L-lactide and BMD the benzyl groups were removed by catalytic hydrogenolysis to obtain a fully carboxyl-functionalized copolymer (PLGM), and RGD was immobilized on the surface of its cast film. All the RGD-immobilized films thus prepared exhibited improved cell attachment compared with the original films. The cell attachment increased with increasing amount of immobilized RGD, which depended on the composition of the alpha-malate units in the copolymer. The RGD-immobilized PLGM films were degraded rapidly during the cell culture, while the RGD-immobilized films of the beta-alkyl alpha-malate-containing polymers survived the cell culture with little degradation. The rate of hydrolysis increased with increasing content of alpha-malate units for both series, depending on the structure of the protecting groups of the beta-carboxyl. These results suggest that the RGD-immobilized polymers could be a new class of functional bioresorbable polymer having improved cell-attachment and adjustable hydrolysis rate.     

Stimulant effect of nitric oxide generator and roxatidine on mucin biosynthesis of rat gastric oxyntic mucosa.

Although the involvement of nitric oxide (NO) in an increasing gastric mucus metabolism has been reported, information on whether or not its activation is limited to the specific mucus-producing cells is lacking. In this paper, we report the effect of the exogenous NO-donor, isosorbide dinitrate (ISDN), and second-generation histamine H2 receptor antagonist roxatidine (2-acetoxy-N-(3-[m-(1-piperidinylmethyl)phenoxy]propyl)acetamide hydrochloride) which is demonstrated to accelerate the mucin metabolism mediated by endogenous NO, on the mucin biosynthesis in distinct sites and layers of the rat gastric mucosa using an organ culture technique. Radiolabeled mucin was obtained from the tissue of full-thickness and the deep corpus layer, and the antrum of the rat stomach incubated for 5 hr with [3H]glucosamine(GlcN) in vitro. With the addition of ISDN to the culture medium, 3H-labeled mucin in the full-thickness corpus mucosa increased to 124-145% of the control (p<0.05), but not in the antrum. This stimulation of the mucin synthesis disappeared by the removal treatment of the surface mucous cell layer which has immunoreactivity of neuronal NO synthase. Similarly, roxatidine stimulated the mucin biosynthesis in the full-thickness corpus mucosa, but not in the gland mucous cell layer. These results suggest that the stimulation of the mucin biosynthesis mediated by NO is restricted to the surface mucous cells of the rat gastric oxyntic mucosa.     

High-resolution structure of the conger eel galectin, congerin I, in lactose-liganded and ligand-free forms: emergence of a new structure class by accelerated evolution.

BACKGROUND: Congerin I is a member of the galectin (animal beta-galactoside-binding lectin) family and is found in the skin mucus of conger eel. The galectin family proteins perform a variety of biological activities. Because of its histological localization and activity against marine bacteria and starfish embryos, congerin I is thought to take part in the eels' biological defense system against parasites. RESULTS: The crystal structure of congerin I has been determined in both lactose-liganded and ligand-free forms to 1. 5 A and 1.6 A resolution, respectively. The protein is a homodimer of 15 kDa subunits. Congerin I has a beta-sheet topology that is markedly different from those of known relatives. One of the beta-strands is exchanged between two identical subunits. This strand swap might increase the dimer stability. Of the known galectin complexes, congerin I forms the most extensive interaction with lactose molecules. Most of these interactions are substituted by similar interactions with water molecules, including a pi-electron hydrogen bond, in the ligand-free form. This observation indicates an increased affinity of congerin I for the ligand. CONCLUSIONS: The genes for congerin I and an isoform, congerin II, are known to have evolved under positive selection pressure. The strand swap and the modification in the carbohydrate-binding site might enhance the cross-linking activity, and should be the most apparent consequence of positive selection. The protein has been adapted to functioning in skin mucus that is in direct contact with surrounding environments by an enhancement in cross-linking activity. The structure of congerin I demonstrates the emergence of a new structure class by accelerated evolution under selection pressure.     

Study of the relationship between sticky human serum IgA1 and its O-glycan glycoform.

The high-affinity IgA1 toward jacalin was mostly composed of aggregated IgA1 and abundantly contained the asialo disaccharide, Galbeta1,3GalNAc, in the O-linked oligosaccharide in the hinge region [Journal of Biochemistry 120, 92-97 (1996)]. Meanwhile, the removal of sialic acid from IgA1 accelerated the aggregation of the IgA1 molecule [J. Am. Soc. Nephrol. 9, 2048-2054 (1998)]. In order to examine the nature of such a sticky IgA1, affinity chromatography using asialo-IgA1 (deSIgA1)-Sepharose was carried out. Seventeen percent of normal human serum IgA1, 27% of asialo-IgA1 (IgA1-S), and 48% of asialo-, agalacto-IgA1 (IgA1-SG) were bound to the column. Removal of the N-acetylgalactosamine residue from IgA1-SG resulted in a decreasing affinity toward deSIgA1-Sepharose. Thus, the binding ability toward the column was the highest for the IgA1-SG among the deglycosylated IgA1s. On the other hand, heat treatment of IgA1 accelerated the aggregation but decreased its binding ability toward the column. Such heat denaturation probably destroys the structure of the binding site. Since the enzymatic removal of the N-glycan sugar chains did not induce the aggregation and exhibited no effect on the binding, the incomplete O-linked sugar chain on the hinge portion should be directly related to the sticky characteristics of the IgA1 molecule. The binding was non-covalent and not strong because the asialo-, agalacto-hinge glycopeptide was eluted slightly slower than the native one from the column and the bound IgA1 was dissociated in the presence of 1 M NaCl.     

Rat gastric mucins recognized by monoclonal antibodies RGM21 and HIK1083: isolation of mucin species characteristic of the surface and glandular mucosa.

Whole mucins and reduced subunits were extracted from the corpus of the rat stomach. After purification by Sepharose CL-4B chromatography followed by cesium trifluoroacetate equilibrium centrifugation, they were analyzed by Sepharose CL-2B chromatography, rate-zonal sedimentation centrifugation, and Q-Sepharose chromatography. Monoclonal antibodies RGM21 and HIK1083, which histochemically stained mucins in the surface and glandular mucosa of the rat stomach, respectively, were used to detect the site-specific mucins. Although RGM21- and HIK1083-reactive mucins both had a multimerized structure, the density and size of both the whole mucins and reduced subunits differed, thus indicating the presence of distinct mucin species in the surface and glandular mucosa. The mucin subunits were separated into four fractions, UB, B1, B2a, and B2b, by Q-Sepharose chromatography. HIK1083 reacted mainly with UB, while RGM21 reacted with B1, B2a, and B2b. These results, combined with dot-blot, amino acid, and carbohydrate composition analyses, showed that the surface mucins may consist of three kinds of subunits. In contrast, the glandular mucins may consist of one kind of subunit which differs from that of surface mucins.     

Rab11BP/Rabphilin-11, a downstream target of rab11 small G protein implicated in vesicle recycling.

Rab11 small G protein has been implicated in vesicle recycling, but its upstream regulators or downstream targets have not yet been identified. We isolated here a downstream target of Rab11, named rabphilin-11, from bovine brain. Moreover, we isolated from a rat brain cDNA library its cDNA, which encoded a protein with a M(r) of 100,946 and 908 amino acids (aa). Rabphilin-11 bound GTP-Rab11 more preferentially than GDP-Rab11 at the N-terminal region and was specific for Rab11 and inactive for other Rab and Rho small G proteins. Both GTP-Rab11 and rabphilin-11 were colocalized at perinuclear regions, presumably the Golgi complex and recycling endosomes, in Madin-Darby canine kidney cells. In HeLa cells cultured on fibronectin, both the proteins were localized not only at perinuclear regions but also along microtubules, which were oriented toward membrane lamellipodia. Treatment of HeLa cells with nocodazole caused disruption of microtubules and dispersion of GTP-Rab11 and rabphilin-11. Overexpression of the C-terminal fragment of rabphilin-11 (aa 607-730), lacking the GTP-Rab11 binding domain, in HeLa cells reduced accumulation of transferrin at perinuclear regions and cell migration. Rabphilin-11 turned out to be a rat counterpart of recently reported bovine Rab11BP. These results indicate that rabphilin-11 is a downstream target of Rab11 which is involved in vesicle recycling.     

Cecropin D‐like antibacterial peptides from the sphingid moth,Agrius convolvuli

Two major antibacterial peptides were isolated and purified from immunized larval hemolymph of Agrius convolvuli. Acid extraction, gel filtration, ultrafiltration, and reversed‐phase FPLC were used for purification of peptides. These peptides had similar molecular mass and amino acid composition. Moreover, 21 of the first 23 N terminal residues were identical. The peptides were highly homologous with cecropin D in size and primary sequence, and named Agrius cecropin D1 and D2. The molecular masses of Agrius cecropin D1 and D2 were 3,879.39 and 3,839.27, respectively. In antibacterial and hemolytic assays, Agrius cecropin D showed potent antibacterial activities against a panel of Gram positive and negative bacteria without hemolytic activity against human red blood cells. Notably, our antibacterial assay revealed Agrius cecropin D possessed stronger or at least equivalent activities against B. megaterium than cecropin A. It suggests that Agrius cecropin D, which has an alternative structure from cecropin D, could be the model for the development of peptide antibiotics. Arch. Insect Biochem. Physiol. 41:178–185, 1999. © 1999 Wiley‐Liss, Inc.     

Isolation and identification of a humanTRPV1 activating compound from soy sauce

Transient receptor potential vanilloid 1 (TRPV1) was identified as a receptor of capsaicin, which is a pungent ingredient in hot red peppers. Due to its relevance for nociception, a physiological and pharmacological study of TRPV1 has also been developed. Therefore, it is important to enrich scientific knowledge regarding the TRPV1 activating or inhibiting compounds. In this study, we fractionated soy sauce based on the human TRPV1 (hTRPV1) activity using column chromatography and purified 5-(9H-pyrido[3,4-b]indol-1-yl)-2-furanmethanol (perlolyrine) as an hTRPV1-activating compound. Additionally, perlolyrine activates the human transient receptor potential ankyrin 1 (hTRPA1). The EC₅₀ of hTRPV1 and hTRPA1 were 2.87 and 1.67 μmol L^?1, respectively. HPLC quantification of soy sauces showed that they contain 2.22–12.13 μmol L^?1 of perlolyrine. The sensory evaluation revealed that perlolyrine has taste modification effect. The results of this study, for the first time, suggest that perlolyrine induces the activation of hTRPV1 and hTRPA1.     

An expanded latch-bridge model of protein kinase C-mediated smooth muscle contraction.

A thin-filament-regulated latch-bridge model of smooth muscle contraction is proposed to integrate thin-filament-based inhibition of actomyosin ATPase activity with myosin phosphorylation in the regulation of smooth muscle mechanics. The model included two latch-bridge cycles, one of which was identical to the four-state model as proposed by Hai and Murphy (Am J Physiol Cell Physiol 255: C86-C94, 1988), whereas the ultraslow cross-bridge cycle has lower cross-bridge cycling rates. The model-fitted phorbol ester induced slow contractions at constant myosin phosphorylation and predicted steeper dependence of force on myosin phosphorylation in phorbol ester-stimulated smooth muscle. By shifting cross bridges between the two latch-bridge cycles, the model predicts that a smooth muscle cell can either maintain force at extremely low-energy cost or change its contractile state rapidly, if necessary. Depending on the fraction of cross bridges engaged in the ultraslow latch-bridge cycle, the model predicted biphasic kinetics of smooth muscle mechanics and variable steady-state dependencies of force and shortening velocity on myosin phosphorylation. These results suggest that thin-filament-based regulatory proteins may function as tuners of actomyosin ATPase activity, thus allowing a smooth muscle cell to have two discrete cross-bridge cycles with different cross-bridge cycling rates.     

The effects of influenza vaccination of health care workers in nursing homes: insights from a mathematical model

Background

Annual influenza vaccination of institutional health care workers (HCWs) is advised in most Western countries, but adherence to this recommendation is generally low. Although protective effects of this intervention for nursing home patients have been demonstrated in some clinical trials, the exact relationship between increased vaccine uptake among HCWs and protection of patients remains unknown owing to variations between study designs, settings, intensity of influenza seasons, and failure to control all effect modifiers. Therefore, we use a mathematical model to estimate the effects of HCW vaccination in different scenarios and to identify a herd immunity threshold in a nursing home department.

Methods and Findings

We use a stochastic individual-based model with discrete time intervals to simulate influenza virus transmission in a 30-bed long-term care nursing home department. We simulate different levels of HCW vaccine uptake and study the effect on influenza virus attack rates among patients for different institutional and seasonal scenarios. Our model reveals a robust linear relationship between the number of HCWs vaccinated and the expected number of influenza virus infections among patients. In a realistic scenario, approximately 60% of influenza virus infections among patients can be prevented when the HCW vaccination rate increases from 0 to 1. A threshold for herd immunity is not detected. Due to stochastic variations, the differences in patient attack rates between departments are high and large outbreaks can occur for every level of HCW vaccine uptake.

Conclusions

The absence of herd immunity in nursing homes implies that vaccination of every additional HCW protects an additional fraction of patients. Because of large stochastic variations, results of small-sized clinical trials on the effects of HCW vaccination should be interpreted with great care. Moreover, the large variations in attack rates should be taken into account when designing future studies.