Lizard Tail Extracts Exert Protective Effect Against Oxidative Stress-Induced Cellular Senescence in Human Fibroblasts.

Lizards are the evolutionarily closest animals to humans among the self-renewable species. Recent reports show that lizard tail extracts (LTE) inhibit the proliferation and angiogenesis of cancer cells but do not show any toxicity against human fibroblast cells. Nevertheless, few scientific studies investigated the effects of LTE on the treatment of skin diseases, especially oxidative stress aging. Therefore, we explored the effect of LTE on the anti-aging activity of human fibroblasts. We confirmed the anti-aging effect of LTE by SA-ß-galactosidase staining. In addition, the hydrogen peroxide-induced reactive oxygen species (ROS) were decreased by the LTE, as measured by staining with the 2',7'-dichlorofluorescein diacetate reagent. We performed Western blot analysis to examine the signaling pathways. In conclusion, the LTE can prevent cellular senescence through the suppression of ROS and the downregulation of p21.

Polarization-sensitive sum-frequency generation microscopy of collagen fibers.

Point-scanning sum-frequency generation (SFG) microscopy enables the generation of images of collagen I fibers in tissues by tuning into specific vibrational resonances of the polypeptide. It is shown that when collagen-rich tissues are visualized near the 2954 cm(-1) stretching vibration of methylene groups, the SFG image contrast is higher compared to the contrast seen in nonresonant second-harmonic generation (SHG) imaging. Polarization and spectrally resolved analysis of the SFG signal as a function of fiber orientation in the CH-stretching range of the vibrational spectrum enabled a comparative characterization of the achiral tensor elements of collagen's second-order susceptibility. This analysis reveals that selected on-resonance tensor elements are enhanced over other elements, giving rise to a much stronger anisotropy ρ of the signal for SFG (ρ ≈ 15) compared to SHG (ρ ≈ 3). The improved anisotropy of the vibrationally resonant signal contributes to the higher contrast seen in the SFG tissue images.

Regressing amphibian tail as a model for the cadherin/beta-catenin complex disruption and glycosylation alteration during epithelial apoptosis.

Epidermis is one of the many tissues that are resorbed during metamorphosis in the regressing tail of amphibian tadpoles. Apoptotic mechanisms play an important role in this process. In this study, loss of intercellular contacts and alterations in plasma membrane glycosylation were observed during apoptosis. The cadherin/beta-catenin complex represents one of the major adhesive systems in multiple epithelial tissues. Here, we analysed the fate of cadherin/beta-catenin complex and alterations of plasma membrane glycoconjugate compositions in apoptotic epithelial cells. Our results showed that the cadherin molecules were cleaved into extracellular and beta-catenin associated cytosolic domains by an intracellular mechanism. However, the extracellular domains were probably removed completely by matrix metalloproteinases. Lectin histochemistry studies suggested that mannose and alpha(2-->6) linked (but not alpha(2-->3) linked) sialic acids were major sugar motifs in plasma membranes of apoptotic tadpole epithelial cells. Although previous studies indicated reduced levels of sialic acid residues during apoptosis, elevated Sambucus nigra agglutinin (SNA) reactivity might be due to the degradation of high molecular weight glycoproteins (probably including cadherin) that masked the SNA-binding residues of the plasma membrane prior to apoptosis.

Evaluation of tail biopsy collection in laboratory mice (Mus musculus): vertebral ossification, DNA quantity, and acute behavioral responses.

A preferred method to genotype genetically engineered mice is through collection of distal tail tissue (tail biopsy) followed by DNA isolation. Currently, general or local anesthesia (or both) is recommended for biopsy after 3 wk of age, the time after which tail vertebrae are considered to be ossified. Our objective was to rigorously evaluate vertebral development, DNA content, and acute behavioral responses at different ages by harvesting tail biopsies of different lengths. We evaluated laboratory mice from 5 inbred strains and 1 outbred stock at each of 12 ages (3 to 42 d of age). Biopsies of 5-, 10-, and 15-mm lengths were obtained. Vertebrae were graded according to level of ossification by using complementary modalities of high-resolution microradiography, microcomputed tomography, and histology. Vertebral development progressed at different rates among the strains, with mature tail vertebrae containing endplates detectable in the tail of some strains by 10 d of age. Within the distal 2 mm of tail, end plates were not identified before 21 d of age. DNA yield (DNA weight/tissue weight) was greatest from the 5-mm biopsy harvest. Acute behavioral responses to biopsy varied by age and strain, and these differences were associated with vertebral maturation. Vertebral development progressed most rapidly in C57BL/6 mice, which also demonstrated the highest response rate to biopsy, whereas BALB/c mice had slower vertebral development and were less responsive. These findings support the collection of minimal lengths of tail tissue from mice at ages younger than 17 d, unless anesthesia or analgesia is provided.

[Establishing the method of collagen-gel droplet embedded culture drug sensitivity test with rat tail collagen].

OBJECTIVE: To explore the clinical application of collagen-gel droplet embedded-culture drug sensitivity test (CD-DST) in the malignant tumors. METHODS: CD-DST was established with rat tail collagen. Three pancreatic cancer cell lines, surgical resection specimens including 15 cases of pancreatic cancer and 10 cases of gastrointestinal cancer were examined using CD-DST. RESULTS: The overall achievement ratio of CD-DST for clinical tumor specimens was 80% (20/25). In vitro chemosensitivities of pancreatic carcinoma cells to 5-FU, gemcitabine and oxaliplatin were lower than those of gastrointestinal carcinoma. CONCLUSIONS: CD-DST with rat tail collagen is a valuable chemosensitivity testing method for malignant tumors. It can be used to realize individualized anticancer chemotherapy.

A chemical and genetic approach to the mode of action of fumagillin.

Previous mode of action studies identified methionine aminopeptidase 2 (MetAP-2) as the target of the antiangiogenic natural product fumagillin and its drug candidate analog, TNP-470. We report here that TNP-470-mediated MetAP-2 inhibition blocks noncanonical Wnt signaling, which plays a critical role in development, cell differentiation, and tumorigenesis. Consistent with this finding, antisense MetAP-2 morpholino oligonucleotide injection in zebrafish embryos phenocopies gastrulation defects seen in noncanonical Wnt5 loss-of-function zebrafish mutants. MetAP-2 inhibition or depletion blocks signaling downstream of the Wnt receptor Frizzled, but upstream of Calmodulin-dependent Kinase II, RhoA, and c-Jun N-terminal Kinase. Moreover, we demonstrate that TNP-470 does not block the canonical Wnt/beta-catenin pathway. Thus, TNP-470 selectively regulates noncanonical over canonical Wnt signaling and provides a unique means to explore and dissect the biological systems mediated by these pathways.

Thermal denaturation of UV-irradiated wet rat tail tendon collagen.

The thermal helix-coil transition of UV irradiated collagen in rat tail tendon has been investigated by differential scanning calorimetry. During UVB irradiation the tendons were immersed in water to keep the collagen fibers in a fully hydrated condition at all times. UV irradiation induced changes in collagen which caused both stabilization and destabilization of the triple helix in fibers. The helix-coil transition for non-irradiated collagen occurred near 64 degrees C, for irradiated 1 and 3 h at 66 and 67 degrees C, respectively. After irradiating for longer times (20-66 h) the helix-coil transition peak occurred at much lower temperatures. The peak was very broad and suggested that collagen was reduced by UV to different polypeptides of different molecular weight and different lower thermal stabilities. It was caused by the disruption of a network of hydrogen-bonded water molecules surrounding the collagen macromolecule.

Functional relationships among selenium concentrations in the diet, target tissues, and nondestructive tissue samples of two species of snakes.

Nondestructive sampling methods, such as removal of feathers for contaminant analysis, are desirable in ecological monitoring programs that seek to minimize the impacts of harvesting organisms. Although many reptiles are declining worldwide, nondestructive sampling techniques seldom have been employed for assessing contaminant exposure in these organisms. In this study, we examined the utility of nondestructive tissue sampling for assessing Se exposure in reptiles. We describe the functional relationships among dietary Se concentrations, target tissue Se concentrations, and Se concentrations in nondestructive tissue samples (blood and tail tissue biopsy) in two species of snakes that had been exposed to Se under very different experimental protocols. Using nonlinear regression, we found strong positive correlations (r2 > 0.92) in all comparisons among Se concentrations in nondestructive tissues, diet, and target tissues. Moreover, equations describing these relationships can be used to estimate concentrations of Se in diet and target organs, from known concentrations of Se in nondestructive tissue samples. Although the current paucity of toxicity data on reptiles precludes tests of our models, we demonstrate how the equations describing these relationships might be used to make predictions about Se accumulation in target organs for risk assessment. Future studies on reptiles that examine these relationships under different Se exposure conditions, and those that document physiological responses of reptiles to various concentrations of Se, will help to refine our models and test their efficacy for predicting health risk.

Cyclosporin A inhibits thyroid hormone-induced shortening of the tadpole tail through membrane permeability transition.

Regression of the tadpole tail through muscule cell apoptosis is one of the most spectacular events in amphibian metamorphosis. Accumulated evidence has shown that mitochondrial membrane permeability transition (MPT) plays a crucial role in apoptosis. Previously we reported that cyclosporin A (CsA) suppressed 3,5,3'-triiodothyronine (T(3))-induced mitochondrial swelling, which was coupled with cytochrome c (Cyt.c) release through MPT [Comp. Biochem. Phys. 130 (2001) 411-418]. To further clarify the mechanism of tadpole metamorphosis, the present study investigates the effect of CsA on T(3) induced tadpole tail shortening. A low concentration of T(3) (5 x 10(-8) M) was found to induce a shortening of stage X Rana rugosa tadpole tails, accompanied by an increase in caspase-3- and -9 like protease activity, as well as an increase in DNA-fragmentation and ladder formation, while CsA was seen to suppress the effects of T(3). The stage X tadpole tail was found to express Bax mRNA and this expression was not affected by T(3) treatment. CsA, on the other hand, proved to have a slightly supressive effection on Bax expression. 20 microM T(3) as well as 50 microM Ca(2+) induced swelling in mitochondria isolated from the liver of R. rugosa resulting in the release of apoptosis related substances, and the released fraction activated cytosolic caspase-3 and -9 in the presence of dATP. This result indicated that Cyt.c might be released from mitochondria by treatment with T(3) through both direct and indirect action of T(3). From these results and other data it was concluded that mitochondrial MPT plays an important role in T(3)-induced apoptosis in the tadpole tail, resulting in tail shortening, and CsA was seen to suppress the effects of T(3).

Viscoelastic properties of collagen: synchrotron radiation investigations and structural model.

Collagen type I is the most abundant structural protein in tendon, skin and bone, and largely determines the mechanical behaviour of these connective tissues. To obtain a better understanding of the relationship between structure and mechanical properties, tensile tests and synchrotron X-ray scattering have been carried out simultaneously, correlating the mechanical behaviour with changes in the microstructure. Because intermolecular cross-links are thought to have a great influence on the mechanical behaviour of collagen, we also carried out experiments using cross-link-deficient tail-tendon collagen from rats fed with beta-APN, in addition to normal controls. The load-elongation curve of tendon collagen has a characteristic shape with, initially, an increasing slope, corresponding to an increasing stiffness, followed by yielding and then fracture. Cross-link-deficient collagen produces a quite different curve with a marked plateau appearing in some cases, where the length of the tendon increases at constant stress. With the use of in situ X-ray diffraction, it was possible to measure simultaneously the elongation of the collagen fibrils inside the tendon and of the tendon as a whole. The overall strain of the tendon was always larger than the strain in the individual fibrils, which demonstrates that some deformation is taking place in the matrix between fibrils. Moreover, the ratio of fibril strain to tendon strain was dependent on the applied strain rate. When the speed of deformation was increased, this ratio increased in normal collagen but generally decreased in cross-link-deficient collagen, correlating to the appearance of a plateau in the force-elongation curve indicating creep. We proposed a simple structural model, which describes the tendon at a hierarchical level, where fibrils and interfibrillar matrix act as coupled viscoelastic systems. All qualitative features of the strain-rate dependence of both normal and cross-link-deficient collagen can be reproduced within this model. This complements earlier models that considered the next smallest level of hierarchy, describing the deformation of collagen fibrils in terms of changes in their molecular packing.

Effects of collagen IV and laminin on the reconstruction of human oral mucosa.

To investigate the effects of basement membrane proteins on the reconstruction of mucosa equivalent, oral mucosa substitute were cultured on (1) type I collagen gels, (2) type IV collagen-coated type I collagen gels, (3) laminin-coated type I collagen gels, and (4) type I collagen gels containing both type IV collagen and laminin. H/E and PAS staining showed that the characteristics of the oral mucosa were preserved under all the experimental conditions. However, the basal keratinocytes appeared cuboidal when the type I collagen gels were coated with type IV collagen plus laminin. The expression of the differentiation markers was similar, but weak staining of filaggrin, K13, and involucrin was observed with the type IV collagen plus laminin coating. Furthermore, electron microscopy revealed that the size of the basal keratinocytes was relatively small and uniform when both type IV collagen and laminin were used. These findings suggested that these two major basement membrane proteins are important in the process of differentiation in mucosal keratinocytes.

Characterization of a metalloproteinase: a late stage specific gelatinase activity in the sea urchin embryo.

We have partially purified and characterized an 87 kDa gelatinase activity expressed in later stage sea urchin embryos. Cleavage activity was specific for gelatin and no cleavage of sea urchin peristome type I collagen, bovine serum albumin or casein was detected. Magnesium and Zn2+ inhibited the gelatinase and Ca2+ protected against inhibition. Ethylenediamine tetracetic acid, ethylenebisoxyethylenenitriol tetraacetic acid and 1,10-phenanthroline were inhibitory, suggesting that the gelatinase is a Ca(2+)- and Zn(2+)-dependent metalloproteinase. No inhibition was detected with serine or cysteine protease inhibitors and the vertebrate matrix metalloproteinase (MMP) inhibitor, Batimastat, was also ineffective. The vertebrate MMP activator p-aminophenylmercuric acetate was without effect. These results allow us to identify both similarities and differences between echinoderm and vertebrate gelatinases.

Light-sensitive response in melanophores of Xenopus laevis: II.Rho is involved in light-induced melanin aggregation.

Melanophores of the isolated tail fin of the Xenopus tadpole aggregate melanin granules in response to light. This aggregation was found to be inhibited by subcutaneous injection of exoenzyme C3 of Clostridium botulinum. A 26 kDa protein in homogenate obtained from the Xenopus tail fin was ADP-ribosylated by exoenzyme C3. This reaction was inhibited effectively by a monoclonal antibody, anti-Rho mab A5. raised against the small GTP-binding protein Rho. The extent of ADP-ribosylation depended on light and guanine nucleotide. Incubation under illumination partly reduced ADP-ribosylation and the reduction was restored by addition of guanine nucleotide during incubation. These findings suggest that Rho is involved in the photo-sensitive melanophore response as a signal transducer linking photo-stimuli to melanin granule translocation with Xenopus melanophores.

Molecular markers of differentiation in Caenorhabditis elegans obtained by promoter trapping.

Differentiation of specific cell types during animal development can be detected by monitoring expression of appropriate genes. For this study, six different beta-galactosidase expression patterns which can be used as differentiation markers in the nematode Caenorhabditis elegans are described. An earlier promoter trap screen identified pools of recombinant plasmids which gave patterns of beta-galactosidase expression when used to transform C. elegans. Each recombinant plasmid contained a random fragment of C. elegans genomic DNA fused upstream of a promoterless lacZ gene. Six of these pools were chosen, and individual pattern-producing plasmids within these pools were identified. The expression patterns have been characterized more thoroughly than in the original screen, thereby providing molecular markers for differentiation of several cell types. Many of the expression patterns involve more than one cell type. The genomic origin of the inserts of active plasmids were determined through localization on the physical genome map.