Thermal helix-coil transition in UV irradiated collagen from rat tail tendon

The thermal helix-coil transition in UV irradiated collagen solution, collagen film and pieces of rat tail tendon (RTT) were compared. Their thermal stability’s were determined by differential scanning calorimeter (DSC) and by viscometric measurements. The denaturation temperatures of collagen solution, film and pieces of RTT were different. The helix-coil transition occur near 40°C in collagen solution, near 112°C in collagen film, and near 101°C in pieces of RTT. After UV irradiation the thermal helix-coil transition of collagen samples were changed. These changes depend on the degree of hydratation.     

Identification of an intermediate state in the helix-coil degradation of collagen by ultraviolet light

Differential scanning calorimetry has revealed the presence of a new denaturation endotherm at 32 degrees C following UV irradiation of collagen, compared with 39 degrees C for the native triple helix. Kinetic analyses showed that the new peak was a previously unknown intermediate state in the collagen helix-coil transition induced by UV light, and at least 80% of the total collagen was transformed to random chains via this state. Its rate of formation was increased by hydrogen peroxide and inhibited by free radical scavengers. SDS-polyacrylamide gels showed evidence of competing reactions of cross-linking and random primary chain scission. The cross-linking was evident from initial gelling of the collagen solution, but there was no evidence for a dityrosine cross-link. Primary chain scission was confirmed by end group analysis using fluorescamine. Electron microscopy showed that the segment long spacing crystallites formed from the intermediate state were identical to the native molecules. Clearly, collagen can undergo quite extensive damage by cleavage of peptide bonds without disorganizing the triple helical structure. This leads to the formation of a damaged intermediate state prior to degradation of the molecules to short random chains.     

Thermal behavior of fowl feather keratin

Differential scanning calorimetry (DSC) was applied to elucidate the thermal behavior of fowl feather keratins (barbs, rachis, and calamus) with different morphological features. The DSC curves exhibited a clear and relatively large endothermic peak at about 110-160 degrees C in the wet condition. A considerable decrease in transition temperature with urea and its helical structure content estimated by Fourier transform infrared spectroscopy (FT-IR), and the disappearance of one of the diffraction peaks with heating at 160 degrees C for 30 min, indicated that DSC could be used to evaluate the thermal behavior of keratin. Barbs showed a lower denaturation temperature than rachis and calamus. The pulverized samples showed a slightly higher denaturation temperature than the native samples. In the dry condition, thermal transition occurred in a markedly higher temperature region close to 170-200 degrees C. It is hence concluded that fowl feather keratins have very high thermal stability, and that the elimination of water brings about even greater thermal stability.     

Age-related thermal stability and susceptibility to proteolysis of rat bone collagen.

The shrinkage temperature (Ts) and the pepsin-solubilizability of collagen fibrils in bone matrix obtained from decalcified femur diaphysis from 2-, 5-, 15- and 25-month-old rats were found to decrease with age. Digestion with human fibroblast collagenase dissolved less than half of the collagen, whereas sequential treatment by pepsin followed by collagenase resulted in its complete dissolution. This result shows that collagenase and a telopeptide-cleaving enzyme, when acting in an appropriate sequence, have a great potential for the degradation of bone collagen. The 'melting' profile of the pepsin-solubilized collagen showed a biphasic transition with transition peak at 35.9 degrees C and 40.8 degrees C. With increasing age an increasing proportion of the collagen 'melted' in the transition peak at 35.9 degrees C (pre-transition), and the 'melting' temperature (Tm) of the collagen decreased in parallel with Ts in relation to age. Both Ts and Tm decreased by 3 degrees C in the age span investigated. The age-related change in Ts could therefore be accounted for by the decrease in molecular stability. The collagenase-cleavage products of the bone collagen obtained by the sequential treatment with pepsin and collagenase showed only one peak transition (at 35.1 degrees C), and the Tm for the products was independent of age. The results indicate that the pre-transition for the pepsin-solubilized collagen is due to an age-related decrease in thermal stability may have implications for the mechanical strength and turnover of the bone collagen. In contrast with bone collagen, soft-tissue collagen showed neither the age-dependency of thermal stability nor the characteristic biphasic 'melting' profile.     

UV damage of collagen: insights from model collagen peptides

Fibrils of Type I collagen in the skin are exposed to ultraviolet (UV) light and there have been claims that collagen photo-degradation leads to wrinkles and may contribute to skin cancers. To understand the effects of UV radiation on collagen, Type I collagen solutions were exposed to the UV-C wavelength of 254 nm for defined lengths of time at 4°C. Circular dichroism (CD) experiments show that irradiation of collagen leads to high loss of triple helical content with a new lower thermal stability peak and SDS-gel electrophoresis indicates breakdown of collagen chains. To better define the effects of UV radiation on the collagen triple-helix, the studies were extended to peptides which model the collagen sequence and conformation. CD studies showed irradiation for days led to lower magnitudes of the triple-helix maximum at 225 nm and lower thermal stabilities for two peptides containing multiple Gly-Pro-Hyp triplets. In contrast, the highest radiation exposure led to little change in the T(m) values of (Gly-Pro-Pro)(10) and (Ala-Hyp-Gly)(10) , although (Gly-Pro-Pro)(10) did show a significant decrease in triple helix intensity. Mass spectroscopy indicated preferential cleavage sites within the peptides, and identification of some of the most susceptible sites of cleavage. The effect of radiation on these well defined peptides gives insight into the sequence and conformational specificity of photo-degradation of collagen.     

The differential response of the skin in young and old rats to a combination of X-rays and 'wet' or 'dry' hyperthermia

The left hind feet of groups of female rats aged 7, 14 and 52 weeks were irradiated at three dose levels of X-rays (20, 25 or 30 Gy). Hyperthermia (42.5 degrees C for 1 h) was carried out immediately following irradiation using either 'wet' or 'dry' heat, achieved by immersion in either water or fluorocarbon liquid. The results demonstrated that 'wet' heat produced a consistently greater enhancement of the irradiation damage than 'dry' heat. The thermal enhancement ratio for irradiation plus 'wet' heat was approximately 1.5 and for irradiation plus 'dry' heat it was in the range 1.17 to 1.39. Immersion of the feet in fluorocarbon liquid at 37 degrees C did not significantly modify the irradiation response of the skin. The lower thermal enhancement ratios obtained using immersion in fluorocarbon liquid at 42.5 degrees C are close to those obtained in large animal studies and also similar to the limited amount of data from clinical studies where microwave or ultrasound heating techniques were used. It has been demonstrated that there are large age-related differences in the response of the rat foot skin to irradiation alone. It has also been shown in the present study, using rats of the same age, that the response to irradiation plus hyperthermia was less age dependent. This finding may reflect the differing methods by which damage occurs in tissue after irradiation or hyperthermia.     

Collagen, collagenase and collagenolytic activity in rat Graafian follicles during follicular growth and ovulation

Follicles were dissected from the ovaries of immature rats at intervals after subcutaneous injection of 20 IU of pregnant mare's serum gonadotropin. A surge of luteinizing hormone was observed at 54 h and ovulation occurred at 64-66 h. The follicular volume between 36 and 48 h, then doubled again shortly before ovulation. The collagen content of the follicles increased 3-fold from 35 to 56 h, but decreased significantly (25%) from 61 to 66 h. Follicle homogenates, activated with trypsin or aminophenylmercuric acetate, digested Type I collagen at 28 degrees C to produce typical of a true collagenase. Collagenolytic activity assayed against endogenous collagen at 37 degrees C did not change significantly between 38 and 66 h.     

Importance of amino acid composition to improve skin collagen protein synthesis rates in UV-irradiated mice

Skin collagen metabolism abnormalities induced by ultraviolet (UV) radiation are the major causes of skin photoaging. It has been shown that the one-time exposure of UV irradiation decreases procollagen mRNA expression in dermis and that chronic UV irradiation decreases collagen amounts and induces wrinkle formation. Amino acids are generally known to regulate protein metabolism. Therefore, we investigated the effects of UV irradiation and various orally administered amino acids on skin collagen synthesis rates. Groups of 4-5 male, 8-week-old HR-1 hairless mice were irradiated with UVB (66 mJ/cm2) twice every other day, then fasted for 16 h. The fractional synthesis rate (FSR; %/h) of skin tropocollagen was evaluated by incorporating L-[ring-2H5]-phenylalanine. We confirmed that the FSR of dermal tropocollagen decreased after UVB irradiation. The FSR of dermal tropocollagen was measured 30 min after a single oral administration of amino acids (1 g/kg) to groups of 5-16 UVB-irradiated mice. Branched-chain amino acids (BCAA, 1.34±0.32), arginine (Arg, 1.66±0.39), glutamine (Gln, 1.75±0.60), and proline (Pro, 1.48±0.26) did not increase the FSR of skin tropocollagen compared with distilled water, which was used as a control (1.56±0.30). However, essential amino acids mixtures (BCAA+Arg+Gln, BCAA+Gln, and BCAA+Pro) significantly increased the FSR (2.07±0.58, 2.04±0.54, 2.01±0.50 and 2.07±0.59, respectively). This result suggests that combinations of BCAA and glutamine or proline are important for restoring dermal collagen protein synthesis impaired by UV irradiation.     

Discrete reduction of type I collagen thermal stability upon oxidation

The oxidation of acid-soluble calf skin collagen type I caused by metal-dependent free radical generating systems, Fe(II)/H2O2 and Cu(II)/H2O2, was found to bring down in a specific, discrete way the collagen thermal stability, as determined by microcalorimetry and scanning densitometry. Initial oxidation results in splitting of the collagen denaturational transition into two components. Along with the endotherm at 41 degrees C typical for non-oxidized collagen, a second, similarly cooperative endotherm appears at 35 degrees C and increases in enthalpy with the oxidant concentration and exposure time, while the first peak correspondingly decreases. The two transitions at 35 and 41 degrees C were registered by densitometry as stepwise increases of the collagen-specific volume. Further oxidation results in massive collagen destruction manifested as abolishment of both denaturational transitions. The two oxidative systems used produce identical effects on the collagen stability but at higher concentrations of Cu(II) in comparison to Fe(II). The discrete reduction of the protein thermal stability is accompanied by a decrease of the free amino groups, suggestive of an oxidation attack of the side chains of lysine residues. Since the denaturation temperature of collagen shifts from above to below body temperature (41 degrees C-35 degrees C) upon oxidation, it appears important to account for this effect in a context of the possible physiological implications of collagen oxidation.     

Formation and stability of repairable pyrimidine photohydrates in DNA

Ultraviolet irradiation of poly(dG-dC) and poly(dA-dU) in solution produces pyrimidine hydrates that are repaired by bacterial and mammalian DNA glycosylases [Boorstein et al. (1989) Biochemistry 28, 6164-6170]. Escherichia coli endonuclease III was used to quantitate the formation and stability of these hydrates in the double-stranded alternating copolymers poly(dG-dC) and poly(dA-dU). When poly(dG-dC) was irradiated with 100 kJ/m2 of 254-nm light at pH 8.0, 2.2% of the cytosine residues were converted to cytosine hydrate (6-hydroxy-5,6-dihydrocytosine) while 0.09% were converted to uracil hydrate (6-hydroxy-5,6-dihydrouracil). To measure the stability of these products, poly(dG-dC) was incubated in solution for up to 24 h after UV irradiation. Cytosine hydrate was stable at 4 degrees C and decayed at 25, 37, and 55 degrees C with half-lives of 75, 25, and 6 h. Uracil hydrate produced in irradiated poly(dA-dU) was stable at 4 degrees C and at 25 degrees C and decayed with a half-life of 6 h at 37 degrees C and less than 0.5 h at 55 degrees C. Uracil hydrate and uracil were also formed in irradiated poly(dG-dC). These experiments demonstrate that UV-induced cytosine hydrate may persist in DNA for prolonged time periods and also undergo deamination to uracil hydrate, which in turn undergoes dehydration to yield uracil. The formation and stability of these photoproducts in DNA may have promoted the evolutionary development of the repair enzyme endonuclease III and analogous DNA glycosylase/endonuclease activities of higher organisms, as well as the development of uracil-DNA glycosylase.     

A novel method to prepare size-regulated spheroids composed of human dermal fibroblasts

A simple method to prepare size-regulated spheroids has been successfully developed by combining a temperature responsive polymer, poly-N-isopropyl-acrylamide (PNIPAAm), conjugated with collagen and ultraviolet (UV) irradiation with photomasks. The coating layer composed of PNIPAAm conjugated with collagen functions as a cell substratum at 37 degrees C, then when lowering the temperature of culture medium the cells attached to it detach as a self-supporting sheet. This is because PNIPAAm dissolves into the culture medium below the lower critical solution temperature LCST; about 30 degrees C, but it is insoluble above the LCST. The detached cell sheet forms a multicellular spheroid. On the other hand, UV effectively immobilized collagen in the coating layer because UV generates crosslinkages in collagen molecules. Crosslinkages were quantitatively introduced by controlling the energy of UV-irradiation thus the ability of human dermal fibroblasts to attach to and detach from the surface was tightly controlled. When the collagen content in the coating layer was 9 mug/cm(2) (collagen ratio, 4.5%), UV-irradiation energy of 2000 J/m(2) was suitable to obtain 100% of the attachability and detachability. However, the cells did not attach to the nonirradiated surface at this collagen content because insufficient collagen was immobilized. Using photomakes to apply UV-irradiation, it was possible to obtain cell-adhesive areas(irradiated areas) and nonadhesive areas (nonirradiated areas) on the same surface. Consequently, spheroids of any size and in any number from one dish were prepared. The viability of cells in spheroids 350 mum in diameter was maintained at a high level for 28 days; however, viability of spheroids 800 mum in diameter rapidly decreased for 2 days. The size was very important to maintain the viability. This novel method is useful to develop size-regulated spheroids for different applications; for example, in toxicology tests. (c) 1994 John Wiley & Sons, Inc.     

Influence of x-rays on depolymerization of lathyric collagen fibers reconstituted in vitro]

Irradiation of native collagen from lathyric rats in solution reduces the depolymerisation speed in the cold of fibres formed by gelification at 37 degrees C, rendering it thus comparable to the speed observed with normal collagen. In our test this normalisation does not appear if the collagen is irradiated in the state of gel. These observations and the absence of specific modifications induced by irradiation in the presence of reagents of the aldehydes speak in favour of the interference, on irradiation, of chemical groups different from the physiological aldehyde groups.     

UV‐A/BLUE LIGHT–INDUCED REACTIVATION OF SPORE GERMINATION IN UV‐B IRRADIATED ULVA PERTUSA (CHLOROPHYTA)1

Recent reduction in the ozone shield due to manufactured chlorofluorocarbons raised considerable interest in the ecological and physiological consequences of UV‐B radiation (λ=280–315 nm) in macroalgae. However, early life stages of macroalgae have received little attention in regard to their UV‐B sensitivity and UV‐B defensive mechanisms. Germination of UV‐B irradiated spores of the intertidal green alga Ulva pertusa Kjellman was significantly lower than in unexposed controls, and the degree of reduction correlated with the UV doses. After exposure to moderate levels of UV‐B irradiation, subsequent exposure to visible light caused differential germination in an irradiance‐ and wavelength‐dependent manner. Significantly higher germination was found at higher photon irradiances and in blue light compared with white and red light. The action spectrum for photoreactivation of germination in UV‐B irradiated U. pertusa spores shows a major peak at 435 nm with a smaller but significant peak at 385 nm. When exposed to December sunlight, the germination percentage of U. pertusa spores exposed to 1 h of solar radiation reached 100% regardless of the irradiation treatment conditions. After a 2‐h exposure to sunlight, however, there was complete inhibition of germination in PAR+UV‐A+UV‐B in contrast to 100% germination in PAR or PAR+UV‐A. In addition to mat‐forming characteristics that would act as a selective UV‐B filter for settled spores under the parental canopy, light‐driven repair of germination after UV‐B exposure could explain successful continuation of U. pertusa spore germination in intertidal settings possibly affected by intense solar UV‐B radiation.     

Temperature and the formation of radiation-induced chromosome aberrations. I. The effect of irradiation temperature

Irradiation temperature, changed from 37 degrees C to 4 degrees C, acts as a dose-modifying factor with regard to the dose-yield relationship for dicentric chromosome aberrations in human lymphocytes irradiated with 150 kV X-rays. The temperature dependence of the aberration yield observed at constant dose is S-shaped, with a sharp rise near 15 degrees C from a lower plateau below 12 degrees C to a higher plateau beyond 17 degrees C. The aberration yield is determined by the irradiation temperature, irrespective of fast temperature changes from 4 degrees C to 37 degrees C or from 37 degrees C to 4 degrees C, applied at various delay times before and after irradiation. It is concluded that irradiation temperature influences the formation of chromatin lesions rather than their interaction.     

提高产抗生素链霉菌紫外诱变正变率的研究

将UV诱变了的产抗生素链霉菌（Streptomyces sp.）AP 19-1菌株之孢子,置于适宜的生长温度27℃与接近抑制生长的胁迫温度33℃下培养,结果表明：在33℃下生长获得的子代菌株中,产抗生素水平超过其出发菌株的正向突变体所占的比例,明显比在27℃下培养的高。27℃下培养,正向突变体占总子代菌株数的25.8%,而在33℃下培养则为58.1%。用17种随机引物对出发菌株与UV诱变子代菌株进行总DNA的RAPD测验证明,在接近抑制生长的温度33℃下培养获得的子代,发生在其DNA水平上的变异程度比在27℃的要高得多。这一方法能较大幅度提高链霉菌紫外诱变育种的工作效率,同时也为链霉菌经紫外诱变后突变形成机制的进一步研究提供了新途径。Abstract: UV irradiated spores of Streptomyces sp. AP 19 -1 strain that can produce antibiotics were incubated at 27 ℃, and 33 ℃ which is close to inhibiting growth temperature, respectively. The results showed that there were much more forward mutants, whose level of producing antibiotics is higher than that of original strain, among the offspring of UV irradiated spores grown at 33 ℃, compared to that grown at 27 ℃. The percentage of the forward mutants was 25.8 % at 27 ℃ and 58.1% at 33 ℃. The progeny strains and the original strain were tested by RAPD using total DNA with 17 primers. It was demonstrated that more variations occurred in the chromosomal DNA of the progeny strains grown at 33 ℃ than in that at 27 ℃. This method facilitates increasing the efficiency of induced mutagenesis in breeding and provides a new way to study the mechanisms of mutation formation in UV irradiated Streptomyces sp. cells.     

Phase transition in natural collagen]

The state of water protons and peptide chains in natural collagen was investigated by the method of nuclear magnetic resonance. The study of orientation and temperature dependence of collagen PMR spectra allows us to mage a conclusion about the considerable disorder of mutual orientation in the polypeptide chains at the temperature above -4 degrees C. Under these conditions it was observed that natural collagen was near to the liquid-crystal. The phasic transition at -4 degrees C was discovered, the latter was followed by an abrupt change of the character of water proton mobility and by the change of orientation of collagen fibres order. The thermal effect of transition was measured, the latter presents 18k/gr of the whole specimen.     

In situ control of cell adhesion using photoresponsive culture surface

A photoresponsive culture surface (PRCS) allowing photocontrol of cell adhesion was prepared with a novel polymer material composed of poly(N-isopropylacrylamide) having spiropyran chromophores as side chains. Cell adhesion of the surface was drastically enhanced by the irradiation with ultraviolet (UV) light (wavelength: 365 nm); after subsequent cooling and washing on ice, many cells remained in the irradiated region, whereas most cells were removed from the nonirradiated region. The cell adhesion of the PRCS, which had been enhanced by previous UV irradiation, was reset by the visible light irradiation (wavelength 400-440 nm) and the annealing at 37 degrees C for 2 h. Also it was confirmed that the regional control of cell adhesion was induced several times by repeating the same series of operations. Further, living cell patterning with the 200 microm line width was produced readily by projecting UV light along a micropattern on the PRCS on which the living cells had been seeded uniformly in advance. By using a fluorescent probe that stains living cells only, it was confirmed that the cells maintained sufficient viability even after UV light irradiation followed by cooling and washing.     

Thermally induced alpha-helix to beta-sheet transition in regenerated silk fibers and films

The structure of thin films cast from regenerated solutions of Bombyx mori cocoon silk in hexafluoroisopropyl alcohol (HFIP) was studied by synchrotron X-ray diffraction during heating. A solid-state conformational transition from an alpha-helical structure to the well-known beta-sheet silk II structure occurred at a temperature of approximately 140 degrees C. The transition appeared to be homogeneous, as both phases do not coexist within the resolution of the current study. Modulated differential scanning calorimetry (DSC) of the films showed an endothermic melting peak followed by an exothermic crystallization peak, both occurring near 140 degrees C. Oriented fibers were also produced that displayed this helical molecular conformation. Subsequent heating above the structural transition temperature produced oriented beta-sheet fibers very similar in structure to B. mori cocoon fibers. Heat treatment of silk films at temperatures well below their degradation temperature offers a controllable route to materials with well-defined structures and mechanical behavior.     

Potential repair of Escherichia coli DNA following exposure to UV radiation from both medium- and low-pressure UV sources used in drinking water treatment

The increased use of UV radiation as a drinking water treatment technology has instigated studies of the repair potential of microorganisms following treatment. This study challenged the repair potential of an optimally grown nonpathogenic laboratory strain of Escherichia coli after UV radiation from low- and medium-pressure lamps. Samples were irradiated with doses of 5, 8, and 10 mJ/cm(2) from a low-pressure lamp and 3, 5, 8, and 10 mJ/cm(2) from a medium-pressure UV lamp housed in a bench-scale collimated beam apparatus. Following irradiation, samples were incubated at 37 degrees C under photoreactivating light or in the dark. Sample aliquots were analyzed for up to 4 h following incubation using a standard plate count. Results of this study showed that E. coli underwent photorepair following exposure to the low-pressure UV source, but no repair was detectable following exposure to the medium-pressure UV source at the initial doses examined. Minimal repair was eventually observed upon medium-pressure UV lamp exposure when doses were lowered to 3 mJ/cm(2). This study clearly indicates differences in repair potential under laboratory conditions between irradiation from low-pressure and medium-pressure UV sources of the type used in water treatment.