The mechanical properties of simple epithelial keratins 8 and 18: discriminating between interfacial and bulk elasticities

The abundance and cytoplasmic organization of keratin filaments enables them to contribute to the maintenance of structural integrity in epithelial tissues. Co-polymers of the type II keratin 8 and type I keratin 18 form the major intermediate filament network in simple epithelia. We investigated the mechanical properties of K8-K18 filament suspensions using rheological assays in conjunction with light and electron microscopy. Suspensions of K8-K18 filaments behave like a viscoelastic solid under standard assembly conditions. Bulk elasticity is weakly dependent on deformation frequency but is very sensitive to the concentration (G' approximately C1.5) and size of individual keratin polymers, in agreement with recent models of semiflexible-polymer physics. K8-K18 filaments can self-organize to form a bundled network that exhibits gel-like mechanical properties. In all cases the mechanical properties of the suspensions correlate with the structural features of individual polymers, as seen under light and electron microscopy. Importantly, these bulk viscoelastic properties of K8-K18 filaments are revealed only when interfacial elastic effects are minimized by the application of phospholipids at the air-liquid interface. Suspensions of K5-K14 and vimentin filaments also exhibit interfacial elasticity, which distorts the interpretation of the viscoelastic moduli as determined by standard rheometry. The potential for modulation of mechanical properties through self-organization may be a general property of keratin polymers and contribute to their organization and function in vivo.     

Spatial heterogeneity of TNF-alpha-induced T cell migration to colonic mucosa is mediated by MAdCAM-1 and VCAM-1

Relatively little is known about how recirculation of lymphocytes through the inflamed intestinal mucosa is regulated. The aim of this study was to investigate the dynamic process of T lymphocyte-endothelial cell adhesion in TNF-alpha-challenged murine colonic mucosa by intravital microscopy. T lymphocytes from spleen (SPL) and intestinal lamina propria (LPL) were fluorescence labeled, and their adhesion to microvessels in the colonic mucosa was observed. In TNF-alpha (25 microg/kg)-stimulated colonic venules, an enhanced adhesion of SPL and LPL was demonstrated, with dominant recruitment of LPLs. The magnitude of the increased LPL adhesion was more significant in the colon than in the small intestine. These T lymphocyte interactions in the colonic mucosa were significantly reduced by blocking MAbs against either mucosal addressin cell adhesion molecule-1 (MAdCAM-1), VCAM-1, alpha(4)-integrin, or beta(7)-integrin but not by anti-ICAM-1. Immunohistochemistry revealed significant MAdCAM-1 expression in the lamina propria and VCAM-1 expression in the submucosa of TNF-alpha-treated colon. Spatial heterogeneity of MAdCAM-1 and VCAM-1 activation following TNF-alpha challenge may promote specific T lymphocyte recruitment in the inflamed colonic mucosa.     

Effect ofNocardia rubra cell-wall skeleton on cancer prevention in humans

Summary The retired workers at the chemical weapons plant in Japan are regarded as a high-risk group for cancers. Under the Cancer Preventive Program,Nocardia rubra cell-wall skeleton (N-CWS) was administered to 80 workers directly involved in the production of sulfur mustard and 66 workers engaged in work related to sulfur mustard production. Untreated workers whose age, sex, duties and duration of work at this factory were individually matched to the N-CWS-treated workers were used as controls. During a 4.5-year observation period, development of cancers was found in 7 treated workers and 17 untreated controls. After elimination of the influence of the difference in smoking level, the incidence of subjects who developed cancers was compared statistically between the N-CWS-treated workers and the untreated controls and a significant suppression of development of cancers was noted in the N-CWS-treated workers. Thus, it was concluded that the administration of N-CWS could prevent cancer development in humans.     

Enhanced anti-rotavirus action of human cystatin C by site-specific glycosylation in yeast

The cDNA encoding human cystatin C (HCC) was subjected to site-specific substitution of alanine for serine at the position 37, to obtain the Asn(35)-Lys(36)-Ser(37) sequence that is a signal for asparagine-linked (N-linked) glycosylation of protein in eukaryotes, and was transformed into Pichia pastoris X33. As a result, 1.2 mg/L oligomannosyl HCC with a carbohydrate chain of Man(10)GlcNAc(2) was produced by the Pichia transformant. The oligomannosyl HCC was more stable at the low ionic strength condition of 50 mM potassium phosphate buffer, pH 7.0, than the wild-type. In addition, the oligomannosylation substantially improved the molecular stability of cystatin against an aspartic proteinase, cathepsin D, in which the susceptibility decreased to less than 50% of nonglycosylated one. The anti-rotavirus activity of HCC was substantially enhanced by the site-directed glycosylation using the yeast expression system. A MA-104 cell line was used as a host cell for human rotavirus type-2 Wa strain in this study, to which both the wild-type and oligomannosyl HCCs did not show cytotoxicity at a concentration of 100 mug/mL. More than 80% viability of the host cell infected with 1.0 x 10(5) PFU/mL of rotavirus was conserved under the condition coexisting with 75 mug/mL of the oligomannosyl HCC, which was 15.2% higher than that of wild-type HCC. Thus, the in vitro anti-rotavirus assay indicated that the supplement of a proper amount of the oligomannosyl HCC could be used as an anti-rotavirus agent.     

Demonstration of functional role of TECK/CCL25 in T lymphocyte-endothelium interaction in inflamed and uninflamed intestinal mucosa

It has recently been suggested that C-C chemokines may play a role in the organ-specific homing of lymphocytes, but there is not enough in vivo evidence in intestinal mucosa. The aim of this study was to examine whether thymus-expressed chemokine (TECK)/CCL25 and its ligand CCR9 are involved in T-lymphocyte interaction with microvessels of murine intestinal mucosa. T lymphocytes from the small intestine were fluorescence labeled, and their adhesion to mucosal microvessels was observed by intravital microscopy. Lamina proprial lymphocytes (LPL) and intraepithelial lymphocytes (IEL) adhered to both the small intestine and colon, and desensitization of CCR9 with TECK/CCL25 or anti-TECK/CCL25 antibody significantly inhibited these adhesions only in small intestine. At both sites, TNF-alpha significantly increased LPL adhesion but not IEL adhesion. Desensitization of CCR9 or anti-TECK/CCL25 antibody also attenuated the TNF-alpha-induced LPL adhesion in the small intestine. Increased expression of TECK/CCL25 by TNF-alpha was observed in the lamina propria of small intestine. TECK/CCL25 may thus play an important role in the adherence of mucosal lymphocytes to the microvessels of the small intestine but not the colon under uninflamed as well as inflamed conditions.     

Evaluation of microsatellite markers in association studies: a search for an immune-related susceptibility gene in sarcoidosis

Association studies using linkage disequilibrium (LD) between candidate loci and nearby markers have been proposed to identify susceptibility genes for complex diseases. We analyzed polymorphisms of microsatellites (MSs) and LD patterns of the regions in which candidate genes related to the Th1 immune response have been annotated and attempted to identify a susceptibility gene for sarcoidosis in a marker-based association study. Nineteen MSs were identified in six Th1-related genes (IFNGR1, IFNGR2, IL12RB1, IL12RB2, STAT1 and STAT4) and then eight were further characterized as useful polymorphic markers. Most of these MSs showed LD with single nucleotide polymorphisms (SNPs) on both 5 and 3 ends of these candidate genes, in which r² values between at least one of the MS marker alleles and the SNPs were higher than 0.1. A significant association with one MS allele near STAT4 was shown and a cluster of SNPs in LD with the MS marker was associated with sarcoidosis. These results suggest that association studies using not only SNPs but also multi-allelic MS within or near candidate loci would be useful markers to search for a disease susceptibility gene, especially in populations with unknown LD structure.     

Melanoidin, a food protein-derived advanced maillard reaction product, suppresses Helicobacter pylori in vitro and in vivo

BACKGROUND: Extracellular urease proteins located on the surface of Helicobacter pylori are gastric mucin-targeted adhesins, which play an important role in infection and colonization to the host. In this study we have determined the inhibitory activity of a variety of melanoidins, protein-derived advanced Maillard reaction products, ubiquitously found in heat-treated foods, on urease-gastric mucin adhesion. In addition, we have determined the anticolonization effect of melanoidin I, prepared by the Maillard reaction between casein and lactose, in an animal model and in human subjects infected with this bacterium. METHODS: The inhibitory activity of each compound was determined by a competitive binding assay of labeled gastric mucin to plate-immobilized urease. Melanoidin I was used in an in vivo trial using euthymic hairless mice as an infection model. Melanoidin I was consumed for 8 weeks by subjects infected with H. pylori. The [(13)C] urease breath test and H. pylori-specific antigen in the stool (HpSA) test were performed on subjects at week 0 and week 8. RESULTS: A variety of food protein-derived melanoidins strongly inhibited urease-gastric mucin adhesion in the concentration range of 10 micro g/ml to 100 micro g/ml. In particular, melanoidin I significantly (p <.05) suppressed colonization of H. pylori in mice when given for 10 weeks via the diets. Eight weeks daily intake of 3 g melanoidin I significantly (p <.05) decreased the optical density of HpSA in subjects. CONCLUSION: Foods containing protein-derived melanoidins may be an alternative to antibiotic-based therapy to prevent H. pylori that combines safety, ease of administration and efficacy.     

The contraction-induced increase in Achilles tendon moment arm: A three-dimensional study

The present study aimed to re-examine the influence of the isometric plantarflexors contraction on the Achilles tendon moment arm (ATMA) and the factors influencing the ATMA in three-dimensions. A series of coronal magnetic resonance images of the right ankle were recorded at foot positions of 10° of dorsiflexion, neutral position, and 10° of plantarflexion for the rest condition and the plantarflexors contraction condition at 30% maximal voluntary effort. The shortest distance between the talocrural joint axis and the line of action of the Achilles tendon force projected to the orthogonal plane of the talocrural joint axis was determined as the ATMA. The ATMA determined in the contraction condition was significantly greater by 8 mm than that determined in the rest condition. The talocrural joint axis was displaced anteriorly by 3 mm and distally by 2 mm due to the muscle contraction. As the same time, the line of action of the Achilles tendon force was displaced posteriorly by 5 mm and medially by 2 mm. These linear displacements of the talocrural joint axis and the line of action of the Achilles tendon force accounted for the difference in the ATMAs between the two conditions by 35.9 and 62.4%, respectively. These angular displacements accounted for the total of 0.4% increase in the ATMA. These results confirm the previous findings reported in two-dimensional studies and found that the linear displacement of the line of action of the Achilles tendon force is the primary source of the contraction-induced increase in the ATMA.     

Reversal effect of the genetic background on some small-eye mutants,with special reference to gene action of morphogenetic mutants inDrosophila

The facet number of heterozygous females of theBar-eye mutant ofDrosophila melanogaster varies from ±154 to ±665 according to the full eye strains combined with the mutant. TheBar character is commonly recognized as a semidominant, but its manifestation varies from nearly complete dominance to recessiveness according to the mode of the heterozygous combination. Virtually the same is true of the mutantLobe ² which is usually acknowledged as a complete dominant, but may be manifested as an incompletely dominant gene. Thew ^e andb gp strains decrease the eye size of the heterozygotes withBar, but increase the eye size of the heterozygotes withLobe ².Co-isogenic strains ofBar, bar-3 andeyeless ² mutants with Oregonisogenic increase the facet number to degrees 1.4–1.8 times of the original strains. However, the co-isogenicLobe ² strain with the same Oregonisogenic strain reduces the facets completely, and makes virtually all individuals eye-less. These observations indicate that the same genetic background may give an opposite effect to different small-eye mutants. Analysis was carried out for modifying genes ofbar-3. Modifiers were detected on all large chromosomes, besides a recessive major gene located on the 2nd chromosome of Oregon-isogenic strain.A part of this work was performed in the Division of Biology, California Institute of Technology, Pasadena.