Measurement of solar UV radiation in Antarctica with collagen sheets.

Collagen sheets were used in a unique evaluation method to examine skin damage caused by ultraviolet (UV) light of short wavelength during a season of the Antarctic ozone hole. The collagen sheets were exposed outdoors for 25 and 50 d, in the spring when the ozone hole was formed and in the ozone-hole-free autumn. Extracts from the exposed collagen sheets were analyzed for total protein and terminal amino acid concentrations as an index of collagen fragmentation. The results show that the amount of extractable collagen and terminal amino acid concentration in the spring exposure were approximately double and five times higher, respectively, when compared with those in the autumn exposure. During the ozone hole occurrence, the terminal amino acid concentration of the extracted collagen was about five times higher when exposure lasted 50 d from mid-September to the end of October compared to when exposure lasted 25 d from mid-September to early October. This result could be attributed to a limited amount of short-wavelength UV radiation reaching the ground surface as a result of the low height of the sun in September, when the ozone hole occurred. In fact, UV radiation measurements taken at Syowa Station indicate that short-wavelength UV radiation in the range 290-295 nm was not detected until approximately 1-2 months after the beginning of the ozone hole occurrence.

Cloning of a novel collagenase gene from the gram-negative bacterium Grimontia (Vibrio) hollisae 1706B and its efficient expression in Brevibacillus choshinensis.

The collagenase gene was cloned from Grimontia (Vibrio) hollisae 1706B, and its complete nucleotide sequence was determined. Nucleotide sequencing showed that the open reading frame was 2,301 bp in length and encoded an 84-kDa protein of 767 amino acid residues. The deduced amino acid sequence contains a putative signal sequence and a zinc metalloprotease consensus sequence, the HEXXH motif. G. hollisae collagenase showed 60 and 59% amino acid sequence identities to Vibrio parahaemolyticus and Vibrio alginolyticus collagenase, respectively. In contrast, this enzyme showed < 20% sequence identity with Clostridium histolyticum collagenase. When the recombinant mature collagenase, which consisted of 680 amino acids with a calculated molecular mass of 74 kDa, was produced by the Brevibacillus expression system, a major gelatinolytic protein band of ~ 60 kDa was determined by zymographic analysis. This result suggested that cloned collagenase might undergo processing after secretion. Moreover, the purified recombinant enzyme was shown to possess a specific activity of 5,314 U/mg, an ~ 4-fold greater activity than that of C. histolyticum collagenase.

Enhancement of procollagen biosynthesis by p180 through augmented ribosome association on the endoplasmic reticulum in response to stimulated secretion.

A coiled-coil microtubule-bundling protein, p180, was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported a novel role for p180 in the trans-Golgi network (TGN) expansion following stimulated collagen secretion. Here, we show that p180 plays a key role in procollagen biosynthesis and secretion in diploid fibroblasts. Depletion of p180 caused marked reductions of secreted collagens without significant loss of the ER membrane or mRNA. Metabolic labeling experiments revealed that the procollagen biosynthetic activity was markedly affected following p180 depletion. Moreover, loss of p180 perturbs ascorbate-stimulated de novo biosynthesis mainly in the membrane fraction with a preferential secretion defect of large proteins. At the EM level, one of the most prominent morphological features of p180-depleted cells was insufficient ribosome association on the ER membranes. In contrast, the ER of control cells was studded with numerous ribosomes, which were further enhanced by ascorbate. Similarly biochemical analysis confirmed that levels of membrane-bound ribosomes were altered in a p180-dependent manner. Taken together, our data suggest that p180 plays crucial roles in enhancing collagen biosynthesis at the entry site of the secretory compartments by a novel mechanism that mainly involves facilitating ribosome association on the ER.

Tracing conformational transition of abnormal prion proteins during interspecies transmission by using novel antibodies.

Conformational differences in abnormal prion proteins (PrP(Sc)) have been postulated to produce different prion phenotypes. During the interspecies transmission of prions, the conformation of PrP(Sc) may change with passage; however, little is known about the mechanism of PrP(Sc) transition. In this study, novel PrP(Sc)-specific monoclonal antibodies (mAbs) were developed that could detect the PrP(Sc) of mouse but not that of sheep. By using these mAbs, we attempted to examine PrP(Sc) accumulated in mice inoculated with sheep scrapie serially up to five passages. The presence of PrP(Sc) in the mice was confirmed at all passages; however, mAb-bound PrP(Sc) conformer was detected only from the third passage onward. The generated mAb enabled tracing of a particular conformer during adaptation in sheep-to-mice transmission of prion, suggesting that the conformational transition of PrP(Sc) was caused by propagation of this conformer. Such mAbs capable of discriminating conformational differences may allow us to address questions concerning PrP(Sc) conformation and strain diversity.

Delay of cell cycle progression and induction death of cancer cells on type I collagen fibrils [corrected].

Here, we report the behavior of three kinds of human cancer cell lines (Caco-2, MCF-7, HT-1080) on type I collagen substrates, which are in two-dimensional coated collagen or three-dimensional fibrils form. All tested cells on coated collagen adhered and proliferated. However, in the case of collagen fibrils, the proliferation of cancer cells was suppressed. Furthermore, Akt activation, which is known as a cell-survival signal, was inhibited in cells on collagen fibrils. But the activation of ERK1/2 was not completely inhibited. In Caco-2 cells, delay of cell cycle progression and cell death occurred at the same time. Thus, cell division and cell death occurred at equivalent rates on the collagen fibrils, and cell growth seemed to be stopped. These results imply that the fibril form of collagen plays a potential role in inhibiting the growth of cancer cells.

Expansion of the trans-Golgi network following activated collagen secretion is supported by a coiled-coil microtubule-bundling protein, p180, on the ER.

A coiled-coil endoplasmic reticulum (ER) protein, p180, was originally reported as a ribosome-binding receptor on the rough ER and is highly expressed in secretory tissues. Recently, we reported new functions of p180 as a microtubule-bundling protein on the ER. Here, we investigated the specific roles of p180 in the Golgi complex organization following stimulated collagen secretion. Targeted depletion of p180 by siRNA transfection caused marked reduction of TGN, while other marker levels for the cis or medial Golgi were not markedly changed. Ascorbate stimulation resulted in trans-Golgi network (TGN) expansion to the periphery in control cells that is characterized by both increased membrane amounts and extended shape. In contrast, loss of p180 resulted in retraction of the TGN regardless of ascorbate stimulation. The TGN developed to the periphery along stabilized microtubule bundles, and overexpression of MTB-1 fragment caused dominant-negative phenotypes. Once disorganized, the retracted TGN did not recover in the absence of p180 despite elevated acetylated tubulin levels. TGN46 and p180 were co-distributed in epithelial basal layer cells of human mucosal and gastrointestinal tissues. Taken together, we propose a novel function of p180-abundant ER on the TGN expansion, both of which are highly developed in various professional secretory cells.

Mammalian arylsulfatase A functions as a novel component of the extracellular matrix.

Inherited deficiency for arylsulfatase (Ars) leads to lysosomal storage of sulfated compounds and to serious diseases such as growth retardation, heart failure, and demyelination in the central nervous system. Ars has been regarded as a lysosomal enzyme because of its hydrolytic activity on synthetic aromatic substrates and the lysosomal localization of its enzymatic activity. We previously demonstrated that a large portion of the mammalian arylsulfatase A (ArsA) protein exists on the cell surface of vascular endothelial cells, suggesting that ArsA plays a role in the components of the extracellular matrix. Here we show that ArsA functions as a substrate on which cells adhere and form protrusions. Coating culture plates with recombinant mouse ArsA (rmArsA) stimulates adhesion of human microvascular endothelial cells to the plate followed by the formation of cell protrusions as well as lamellipodia. rmArsA affects the architecture of the cytoskeleton, with a high density of actin filaments localized to peripheral regions of the cells and the extension of bundles of microtubules into the tips of cellular protrusions. rmArsA also affects the distribution pattern of the cell adhesion-associated proteins, integrin α2ß1, and paxillin. rmArsA seems to modulate signaling of basic fibroblast growth factor (bFGF) stimulating cytoskeletal rearrangement. We also show that rmArsA tightly binds to sulfated polysaccharides. We suggest that mammalian ArsA plays a role as a novel component of the extracellular matrix. This viewpoint of Ars could be very useful for clarifying the mechanisms underpinning syndromes caused by the deficiency of the function of Ars genes.

p180 is involved in the interaction between the endoplasmic reticulum and microtubules through a novel microtubule-binding and bundling domain.

p180 was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum membrane, although its precise role in animal cells has not yet been elucidated. Here, we characterized a new function of human p180 as a microtubule-binding and -modulating protein. Overexpression of p180 in mammalian cells induced an elongated morphology and enhanced acetylated microtubules. Consistently, electron microscopic analysis clearly revealed microtubule bundles in p180-overexpressing cells. Targeted depletion of endogenous p180 by small interfering RNAs led to aberrant patterns of microtubules and endoplasmic reticulum in mammalian cells, suggesting a specific interaction between p180 and microtubules. In vitro sedimentation assays using recombinant polypeptides revealed that p180 bound to microtubules directly and possessed a novel microtubule-binding domain (designated MTB-1). MTB-1 consists of a predicted coiled-coil region and repeat domain, and strongly promoted bundle formation both in vitro and in vivo when expressed alone. Overexpression of p180 induced acetylated microtubules in cultured cells in an MTB-1-dependent manner. Thus, our data suggest that p180 mediates interactions between the endoplasmic reticulum and microtubules mainly through the novel microtubule-binding and -bundling domain MTB-1.

An advantageous method utilizing new homogenizing device BioMasher and a sensitive ELISA to detect bovine spongiform encephalopathy accurately in brain tissue.

A new screening method was developed to detect bovine spongiform encephalopathy (BSE). This method is advantageous because it has a simpler and safer protocol than commercial kits. A new device was developed for this method; it was named the BioMasher, to homogenize brain tissue by passing it through a porous rigid polypropylene filter. In this system, a purification step was eliminated in the sample preparation. Thus, the time needed for sample pretreatment is substantially shortened, and the risk of infection during sample processing is effectively reduced. Monoclonal antibodies to prion protein were created and used to construct a sensitive sandwich enzyme-linked immunosorbent assay system. The sensitivity of this assay kit using frozen BSE-positive brain is comparable or more sensitive than commercial kits. Moreover, the detection sensitivity for deteriorated samples, which were kept at 37 degrees C for 1 day, is 10- to 30-fold more sensitive than a commercial kit.

Effects of ingestion of collagen peptide on collagen fibrils and glycosaminoglycans in the dermis.

In order to investigate the effects of collagen peptide ingestion on fibroblasts and the extracellular matrix in the dermis, collagen peptide was administered orally to pigs at 0.2 g/kg body weight/d for 62 d, and its effects were compared with those of lactalbumin and water controls. Fibroblast density, and diameter and density of collagen fibrils were significantly larger in the collagen peptide group than in the lactalbumin and water control groups. The two major components of dermal glycosaminoglycans, hyaluronic acid and dermatan sulfate, which are present in the inter-fibrillar space, did not differ significantly among the three groups. However, the ratio of dermatan sulfate, which is derived from fibril-bound decorin, was largest in the collagen peptide group. These results suggest that ingestion of collagen peptide induces increased fibroblast density and enhances formation of collagen fibrils in the dermis in a protein-specific manner.

Cloning and nucleotide sequence of a novel 28-kDa protein from the mantle muscle of the squid Todarodes pacificus with homology to tropomyosin.

In recent studies, we found autodegradation of collagen from the mantle muscle of the squid Todarodes pacificus and also that the 28- and 25-kDa proteins are closely related to this phenomenon [Connect. Tissue Res. 45 (2004) 109-121]. We obtained partial sequences of three internal portions of this protein, which suggested that 25-kDa protein is a partially degraded form of the 28-kDa protein. We determined the full cDNA sequence of this protein by the degenerate polymerase chain reaction (PCR) using the information of amino acid sequences. The deduced amino acid sequence corresponding to the 212-bp cDNA contained all of the amino acid identified from the 28-kDa protein. Rapid amplification of cDNA ends (RACE) and squid mantle muscle RNA allowed cloning of the full 522-bp sequence, corresponding to a protein of 174 amino acids. A database search indicated that this is a new protein that shares 27-34% identity with tropomyosins from various animals. Structural prediction suggested that it possesses heptad repeats that form coiled-coil structures. We expressed a recombinant protein encoded by the 212-bp cDNA in Escherichia coli and used it to generate a polyclonal antibody. Western blotting with this antibody showed that the 28-kDa protein is expressed in fin, tentacle, and mantle muscle, but not in liver.

Effects of ingestion of collagen peptide on collagen fibrils and glycosaminoglycans in Achilles tendon.

In order to investigate whether the oral ingestion of collagen peptide affects the extracellular matrix of tendon, two doses (0.2 g/kg and 1.0 g/kg body weight) were orally administered daily for 56 d to a rabbit, and both the size of collagen fibrils and the amount of glycosaminoglycans in the Achilles tendon were measured in comparison with those in a rabbit fed with a control protein, lactalbumin, or water alone. Ingestion of collagen peptide or lactalbumin induced a significant increase in collagen fibril diameter and a decrease in fibril density except for a high dose of lactalbumin compared with the water control. A histogram pattern of fibril diameter in a high dose of collagen peptide showed a peak at 160-180 nm, which was not observed in other groups. However the percentage of diameters over 200 nm was the lowest in this group but highest in the low-dose group of collagen peptide. The mean fibril diameter and mass average diameter of a high dose of collagen peptide were significantly smaller than those in a low dose. The amount of dermatan sulphate increased in the high-dose groups, while the amount of hyaluronic acid decreased in rabbits fed with collagen peptide or lactalbumin at either dose. These results suggest that the ingestion of collagen peptide affects the size of collagen fibrils and composition of glycosaminoglycans in the Achilles tendon and thus may improve the mechanical properties of the Achilles tendon.

Collagen of chronically inflamed skin is over-modified and upregulates secretion of matrix metalloproteinase 2 and matrix-degrading enzymes by endothelial cells and fibroblasts.

In order to investigate the properties of collagen in chronically inflamed tissue, we isolated collagen from the ear skin of mice with chronic contact dermatitis and examined its biochemical characteristics and the functions that regulate the secretion of matrix metalloproteinase 2 and collagen-degrading enzymes from endothelial cells and fibroblasts. Collagen in skin with chronic contact dermatitis comprised 60% type I collagen and 40% type III collagen, which latter is higher than the content of type III collagen in control skin (35%). The denaturation temperature was higher (42 degrees C) than that of control skin (39 degrees C). The alpha2 chain of type I collagen was over-hydroxylated at both proline and lysine residues. Segment-long-spacing crystallites of type I collagen were unusually connected in tandem. Collagen of chronically inflamed skin was less susceptible to matrix metalloproteinase 2 after heat denaturation. Endothelial cells and fibroblasts secreted an increased amount of matrix metalloproteinase 2 when cultured on a gel formed from the collagen of chronically inflamed skin. Collagen-degrading activity secreted from fibroblasts was also upregulated when cells were in contact with collagen of chronically inflamed skin. These results suggest that the collagen in chronically inflamed tissue has altered biochemical characteristics and functions, which may affect the pathogenesis of the chronic skin disease.

Cultured human corneal endothelial cell transplantation with a collagen sheet in a rabbit model.

PURPOSE: To evaluate the function of cultured human corneal endothelial cells (HCECs) in vivo and the feasibility of HCEC transplantation with a collagen sheet as the substitute carrier of HCECs. METHODS: Adult human donor cornea derived from cultured HCECs was labeled with the fluorescent tracker DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) and seeded on a collagen sheet. The pump function of the HCEC sheet was evaluated by measurement of the potential difference and short-circuit current. A 6-mm sclerocorneal incision and Descemetorhexis were performed on rabbit eyes. The HCECs on a collagen sheet was brought into the anterior chamber and fixed to the posterior stroma (HCEC group). Rabbit corneas with collagen sheet transplantation after Descemetorhexis (collagen group) and with only Descemetorhexis (no-transplantation group) were the control. Each group, observed for 28 days after surgery, underwent histologic and fluorescence microscopic examinations. RESULTS: Pump function parameters of the HCEC sheets were 76% to 95% of those of human donor corneas. Mean corneal thickness in the HCEC group was significantly less than in the collagen and no-transplantation groups 1, 3, 7, 14, 21, and 28 days (P < 0.05) after surgery. DiI-labeled cells were spread over the rear corneal surface in the HCEC group. Marked stromal edema was present in the collagen and no-transplantation groups with hematoxylin-eosin staining, but none in the HCEC group with collagen sheets bearing monolayer cells. CONCLUSIONS: The findings indicate that cultured HCECs transplanted from adult human donor cornea by means of a collagen sheet can retain their function of corneal dehydration in a rabbit model and suggest the feasibility of transplantation for CEC dysfunction using cultured HCECs with a collagen sheet.

Developmental Changes in the Activity of Xanthine Dehydrogenase and closely Related Enzyme in Chick Tissues: (chick tissues/hatching/xanthine dehydrogenase/preexisting enzyme).

Xanthine-oxidizing activities in the chick tissues were measured by a couple of assay procedures during development of chick embryo. With the usual assay using pterine and NAD+ , no detectable level of XDH activity was observed in the liver and little in the duodenum before hatching, whereas an appreciable activity was detected in the kidney of chick embryo. When assayed with xanthine and dichlorophenol indophenol, an XDH-like enzyme activity was significantly detected in the embryonic liver, while no further enhancement of the activity was detected in the kidney and duodenum. Electrophoregrams obtained with samples from various developmental stages, followed by activity staining with tetrazolium dye, supported the above results and revealed that the embryonic XDH-like enzyme is not distinguishable from XDH of adult tissue in molecular size. This XDH-like enzyme, pre-existing in the liver before hatching, however, exhibited no cross reaction with antibody against intact XDH. The nature of this material was discussed in comparison with deflavinated XDH.

Subunits of Xanthine Dehydrogenase and Their Differential Appearance in Chick Tissues During Development: (chick tissues/hatching/xanthine dehydrogenase/subunits/preexisting enzyme).

Xanthine dehydrogenase (XDH) from adult chick liver comprises two polypeptide chains of different size in a molar ratio of 1: 1. The molecular weights of these subunits were estimated to be 155K (α) and 135K (ß) daltons (1). However, XDH isolated from the liver of newly hatched chick was not found to represent the equimolar ratio of these two subunits; that is, the amount of subunit ß was lower than that of subunit α. While examamining electrophoretically the change in the amounts of these subunits in the liver, the subunit α was found to appear earlier in the embryonic stage, but ß only after hatching. In the kidney, however, both subunits were detected before hatching, being consistent with the fact that XDH exists before hatching in the kidney. The two subunits also appeared differentially in the kidney; i.e., subunit α appeared earlier than subunit ß. In either tissue, the rate of increase in XDH activity corresponded to that of subunit ß. Thus, the synthesis of two subunits of XDH are separately regulated at least until just after hatching.

Size control of decorin dermatan sulfate during remodeling of collagen fibrils in healing skin.

Recently it has been reported that the molecular size of decorin dermatan sulfate (DS) was increased in healing skin after hapten application and that the elongated DS was distributed in enlarged interfibrillar space among thin collagen fibrils in situ. Here we show that such modulation of the length of decorin DS is temporary. Although the size of decorin DS was evidently increased on day 15, it decreased to almost normal size on day 35 when the altered disaccharide composition of DS was also recovered. Electron microscopic observation revealed that elongated decorin DS was localized among thin collagen fibrils packed loosely in hapten-treated skin on day 15. In contrast, decorin DS of normal size was distributed among thick collagen fibrils packed tightly on day 35. These results suggest that size control of decorin DS plays important roles in organization of collagen fibrils into bundles by regulating interfibrillar space in healing skin, particularly in maturation of collagen fibrils through shortening of decorin DS in later stages of healing.

Expression of cellular prion-related protein by murine Langerhans cells and keratinocytes.

Transmissible spongiform encephalopathies are characterized by the accumulation of a proteinase-resistant isoform of the cellular prion-related protein (PrP(c)) within the central nervous system (CNS). The accumulation of scrapie-associated PrP (PrP(Sc)) within cells of the lymphoreticular system prior to its accumulation in the CNS is regarded as important for the development of neurological diseases after peripheral inoculation. Little, however, is known as to which cells are the targets for peripheral inoculation. Here, the presence of PrP(c) on murine Langerhans cells (LC), dendritic cells in the skin and mucosa, and keratinocytes (KC) is demonstrated by immunohistochemical staining, Western-blotting and FACS analysis. The expression of PrP(c) mRNA in freshly purified LC and KC was also detected by reverse transcriptase-polymerase chain reaction. The expression of PrP(c) on LC was slightly increased during culture. These data suggest that LC and KC may be the targets for peripheral infection with prions.

Degradation of fodrin by m-calpain in fibroblasts adhering to fibrillar collagen I gel.

When skin fibroblasts were cultured on fibrillar collagen I gel, we observed rapid degradation of talin, fodrin and ezrin, which are well-known calpain substrates. The protease m-calpain was activated only in cells adhering to fibrillar collagen, whereas micro-calpain was activated in cells adhering to monomeric or fibrillar collagen at the same level. The calpain inhibitor Z-Leu-Leu-aldehyde inhibited degradation of fodrin, but not talin. Degradation of fodrin, alpha-actinin and ezrin was prevented by over-expression of dominant negative m-calpain. However, over-expression of calpastatin, an endogenous calpain inhibitor, had no effect the degradation of these three proteins. These results suggest that m-calpain is responsible for degradation of their membrane proteins via adhesion to fibrillar collagen I gel.

Improved RNA extraction method using the BioMasher and BioMasher power-plus.

The BioMasher is a disposable homogenizer that was developed to homogenize bovine brain tissue for bovine spongiform encephalopathy diagnosis. Capable of preventing the biohazard risk from infectious samples, it also prevents cross-contamination among samples. The BioMasher is thus widely used in biochemical research, especially for RNA extraction. Here, we tested a novel BioMasher application for RNA extraction from animal and plant tissues. We also developed a grinding machine specific for the BioMasher, named the BioMasher Power-Plus. We developed RNA extraction protocols using the BioMasher combined with the BioMasher Power-Plus. We compared RNA extraction efficiency of the BioMasher with that of the FastPrep and the glass homogenizer. Though the RNA extraction efficiency by the BioMasher was nearly equivalent to that of the FastPrep and the glass homogenizer, sample preparation time was shorter for the BioMasher. The utility of RNA extraction by the BioMasher was examined in mouse, rat, and tomato tissue samples. In the rodent tissues, the highest extraction efficiency of total RNA was from liver, with lowest efficiency from fibrous tissues such as muscle. The quality of extracted total RNA was confirmed by agarose gel electrophoresis which produced highly visible clear bands of 18S and 28S rRNAs. Reproducibility among different operators in RNA extraction from tomato roots was improved by using the BioMasher Power-Plus. The BioMasher and BioMasher Power-Plus provide an effective and easy homogenization method for total RNA extraction from some rodent and plant tissues.