Dual functions of autophagy in the response of breast tumor cells to radiation

In MCF-7 breast tumor cells, ionizing radiation promoted autophagy that was cytoprotective; pharmacological or genetic interference with autophagy induced by radiation resulted in growth suppression and/or cell killing (primarily by apoptosis). The hormonally active form of vitamin D, 1,25D₃, also promoted autophagy in irradiated MCF-7 cells, sensitized the cells to radiation and suppressed the proliferative recovery that occurs after radiation alone. 1,25D₃ enhanced radiosensitivity and promoted autophagy in MCF-7 cells that overexpress Her-2/neu as well as in p53 mutant Hs578t breast tumor cells. In contrast, 1,25D₃ failed to alter radiosensitivity or promote autophagy in the BT474 breast tumor cell line with low-level expression of the vitamin D receptor. Enhancement of MCF-7 cell sensitivity to radiation by 1,25D₃ was not attenuated by a genetic block to autophagy due largely to the promotion of apoptosis via the collateral suppression of protective autophagy. However, MCF-7 cells were protected from the combination of 1,25D₃ with radiation using a concentration of chloroquine that produced minimal sensitization to radiation alone. The current studies are consistent with the premise that while autophagy mediates a cytoprotective function in irradiated breast tumor cells, promotion of autophagy can also confer radiosensitivity by vitamin D (1,25D₃). As both cytoprotective and cytotoxic autophagy can apparently be expressed in the same experimental system in response to radiation, this type of model could be utilized to distinguish biochemical, molecular and/or functional differences in these dual functions of autophagy.     

A human skin equivalent model that mimics the photoproduction of vitamin D3 in human skin

Summary A human skin equivalent was prepared by culturing human keratinocytes on the surface of nylon filtration meshes containing human skin fibroblasts and by growing the epidermal cells at the air-liquid interface. This human skin equivalent model was used to mimic the photoproduction of vitamin D₃ in human skin. It was found that the concentration of 7-dehydrocholesterol and its photoconversion to previtamin D₃ and its subsequent thermal isomerization to vitamin D₃ in the human skin equivalent was essentially identical to that of human skin. The 7-dehydrocholesterol content in the skin equivalent and human skin was 2187±296 and 2352±320 ng/cm², respectively. The percentage of the major photoproducts of 7-dehydrocholesterol in the skin equivalent following ultraviolet B radiation (0.5 J/cm²) was 35% previtamin D₃, 29% lumisterol, and 6% tachysterol; 30% remained as 7-dehydrocholesterol. Similarly, in human skin they were 36%, 29%, 7%, and 28%, respectively. After incubation at 37°C for 30 min, 11% and 12% of the previtamin D₃ had thermally isomerized to vitamin D₃ in the skin equivalent and human skin. In conclusion, compared with cultured keratinocytes or fibroblasts, the human skin equivalent model provides a superior in vitro system that better mimics the physiology and biochemistry of the photosynthesis of vitamin D₃ in human skin.     

Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes

Human skin is body’s vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC₅₀ values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H₂O₂) induced RBC haemolysis (IC₅₀ 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H₂O₂-induced damage; inhibited H₂O₂ induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations.     

PPARγ inhibits inflammatory reaction in oxidative stress induced human diploid fibloblast

The ageing of an inevitable life function is an unavoidable regressive physical process. Peroxisome proliferator‐activated receptors (PPARs) are members of the nuclear hormone receptor family. PPARγ plays an important role in regulating several metabolic pathways. Recently, PPARγ has been implicated in inflammatory responses and age‐related diseases. The aim of this study was to determine the anti‐inflammatory reaction of PPARγ in an induced ageing progress. The late passage of human diploid fibroblasts (HDF), an in vitro ageing model, reveals the biological index materials of ageing. Aged cells showed decreased PPARγ expression and elevated levels of intracellular adhesion molecule‐1 (ICAM‐1), an inflammatory molecule. To induce the aged cell phenotype, the middle stage of HDF cells (PD31) were induced stress induced premature senescence (SIPS) with 200 µM H₂O₂ for 2 h. SIPS‐HDF cells showed high levels of ICAM‐1, extracellular signal regulated kinase (ERK1/2) activity and matrix metallomatrix protease (MMP‐2, ‐9) activity, and low levels of PPARγ expression. A reconstitution of SIPS HDF cells with Ad/PPARγ resulted in the downregulation of ICAM‐1, ERK1/2, MMP‐2 and ‐9, and normalized growth of SIPS‐HDF cells. Moreover, PPARγ in aged HDF cells reduced pro‐inflammatory molecules and eliminated the formation of reactive oxygen species (ROS) through the ERK1/2 pathway. These results strongly suggest that PPARγ plays a key role in age‐related inflammation and may have clinical applications as a molecular target in the treatment of age‐related inflammation. Copyright © 2010 John Wiley & Sons, Ltd.     

1,25-Dihydroxyvitamin D3 and agents that increase intracellular adenosine 3′, 5′-monophosphate synergistically inhibit fibroblast proliferation

Summary Agents that increase intracellular cAMP (cAMP elevating agents) and 1,25(OH)₂D₃ inhibit the proliferation of many cell types. We investigated the combined effect of 1,25(OH)₂D₃ and cAMP elevating agents on exponentially growing mouse 3T3 fibroblasts. The following cAMP elevating agents were used: theophylline and pentoxyfilline, which inhibit cAMP-dependent phosphodiesterase; prostaglandin E₂ which activates adenylate cyclase by a receptor-mediated mechanism; forskolin, which directly stimulates adenylate cyclase; and the cell permeable cAMP analogs 8-bromo cAMP and N⁶ benzoyl cAMP. 1,25(OH)₂D₃ and cAMP elevating agents were added to exponentially growing fibroblasts cultured in 96-well microtiter plates and cell number was monitored 3–7 d later. 1,25(OH)₂D₃ and the cAMP elevating agents as single agents inhibited the growth of the 3T3 cells. The combined treatment of the fibroblasts with 1,25(OH)₂D₃ and the cAMP elevating agents resulted in an antiproliferative effect that was more than additive. The synergistic interaction depended on the dose of 1,25(OH)₂D₃ and was apparent already at 10⁻⁸ M of the hormone. The specificity of the effect of 1,25(OH)₂D₃ was demonstrated by the finding that 24,25-dihydroxyvitamin D₃, a vitamin D metabolite with low affinity for the vitamin D receptor, did not affect the antiproliferative effect of cAMP elevating agents. From the synergistic interaction between 1,25(OH)₂D₃ and the cell permeable cAMP analogs, we infer that the site of interaction between the two signaling pathways is distal to the cAMP generating and degrading machinery.     

Summer/winter differences in the serum 25-hydroxyvitamin D3 and parathyroid hormone levels of Japanese women

Serum 25-hydroxyvitamin D₃ [25(OH)D₃] is produced in the skin in response to exposure to ultraviolet radiation, and is a good indicator of vitamin D nutritional status. The aim of this study was to determine summer/winter differences in serum 25(OH)D₃ and parathyroid hormone (PTH) in Japanese women and how the summer and winter values are related. The subjects were 122 healthy Japanese women aged 45–81 years (average age: 65.7 years). They were medically examined twice, in September 1997 and February 1999. Serum 25(OH)D₃ and intact PTH were determined by high-performance liquid chromatography and a two-site immunoradiometric assay respectively. Lifestyle information was obtained through an interview. The seasonal differences (winter minus summer) in 25(OH)D₃ [Δ25(OH)D₃] and intact PTH concentrations were –18.8 nmol/l (SD 19.2, P<0.0001) and 0.98pmol/l (SD 1.02, P<0.0001) respectively. The correlation coefficient between summer (x) and winter (y) 25(OH)D₃ levels was 0.462 (P<0.0001), with a linearly fitted line of y=0.42x+26.4. This relationship was interpreted as subjects with higher summer 25(OH)D₃ values having greater reductions in winter 25(OH)D₃ concentrations. There were inter-individual differences in Δ25(OH)D₃, although the summer and winter 25(OH)D₃ concentrations were well-correlated. Since Δ25(OH)D₃ was not associated with any of the lifestyle factors, seasonal differences in the 25(OH)D₃ concentrations of an individual appeared to reflect her ability to produce 25(OH)D₃ photochemically in the skin. Sun bathing would be a less effective means of attaining adequate vitamin D nutritional status in a person with a small seasonal difference in 25(OH)D₃, i.e., one with a low 25(OH)D₃ level. Received: 17 December 1999 / Revised: 24 April 2000 / Accepted: 10 May 2000     

瘢痕疙瘩中TIEG1的高表达

转化生长因子-β1(TGF-β1/Smads)信号转导通路的持续激活是瘢痕疙瘩形成的重要机制.研究发现这条通路重要的负反馈调节信号分子Smad7表达明显下调,Smad2/3的磷酸化水平和蛋白质量并无明显改变.但是,Smad7下调的机制尚不清楚.采用生物信息学方法对Smad7的启动子进行分析;用RT-PCR和蛋白质印迹分别检测了正常皮肤、正常瘢痕及瘢痕疙瘩组织中的Sp1样转录因子TIEG1mRNA及蛋白质的表达水平;体外培养正常皮肤、正常瘢痕及瘢痕疙瘩成纤维细胞,检测TIEG1 mRNA及蛋白的表达水平.研究结果显示,Smad7启动子上有Sp1的位点,TIEG1 mRNA及蛋白质水平在瘢痕疙瘩组织及瘢痕疙瘩成纤维细胞中表达明显高于正常瘢痕和正常皮肤(P<0.05).说明瘢痕疙瘩中TIEG1可能是Smad7下调的重要原因,有必要进一步研究TIEG1对Smad7的调控作用机制.     

Dramatic Increase in Oxidative Stress in Carbon-Irradiated Normal Human Skin Fibroblasts

Skin complications were recently reported after carbon-ion (C-ion) radiation therapy. Oxidative stress is considered an important pathway in the appearance of late skin reactions. We evaluated oxidative stress in normal human skin fibroblasts after carbon-ion vs. X-ray irradiation. Survival curves and radiobiological parameters were calculated. DNA damage was quantified, as were lipid peroxidation (LPO), protein carbonylation and antioxidant enzyme activities. Reduced and oxidized glutathione ratios (GSH/GSSG) were determined. Proinflammatory cytokine secretion in culture supernatants was evaluated. The relative biological effectiveness (RBE) of C-ions vs. X-rays was 4.8 at D₀ (irradiation dose corresponding to a surviving fraction of 37%). Surviving fraction at 2 Gy (SF2) was 71.8% and 7.6% for X-rays and C-ions, respectively. Compared with X-rays, immediate DNA damage was increased less after C-ions, but a late increase was observed at D_10% (irradiation dose corresponding to a surviving fraction of 10%). LPO products and protein carbonyls were only increased 24 hours after C-ions. After X-rays, superoxide dismutase (SOD) activity was strongly increased immediately and on day 14 at D_0% (irradiation dose corresponding to a surviving fraction of around 0%), catalase activity was unchanged and glutathione peroxidase (GPx) activity was increased only on day 14. These activities were decreased after C-ions compared with X-rays. GSH/GSSG was unchanged after X-rays but was decreased immediately after C-ion irradiation before an increase from day 7. Secretion of IL-6 was increased at late times after X-ray irradiation. After C-ion irradiation, IL-6 concentration was increased on day 7 but was lower compared with X-rays at later times. C-ion effects on normal human skin fibroblasts seemed to be harmful in comparison with X-rays as they produce late DNA damage, LPO products and protein carbonyls, and as they decrease antioxidant defences. Mechanisms leading to this discrepancy between the two types of radiation should be investigated.     

Lipid nano-bubble combined with ultrasound for anti-keloids therapy

Keloids were characterized by excessive growth of fibrous tissues, and shared several pathological characteristics with cancer. They did put physical and emotional stress on patients in that keloids could badly change appearance of patients. N-(4-hydroxyphenyl) retinamide (4HPR) showed cytotoxic activity on a wide variety of invasive-growth cells. Our work was aim to prepare N-(4-hydroxyphenyl) retinamide-loaded lipid microbubbles (4HPR-LM) combined with ultrasound for anti-keloid therapy. 4HPR-loaded liposomes (4HPR-L) were first prepared by film evaporation method, and then 4HPR-LM were manufactured by mixing 4HPR-L and perfluoropentane (PFP) with ultrasonic cavitation method. The mean particle size and entrapment efficiency 4HPR-LM were 113?nm and 95%, respectively. The anti-keloids activity of 4HPR-LM was assessed with BALB/c nude mice bearing subcutaneous xenograft keloids model. 4HPR-LM, combined with ultrasound, could significantly induce apoptosis of keloid fibroblasts in vitro and inhibited growth of keloids in vivo. Thus, 4HPR-LM could be considered as a promising agent for anti-keloids therapy.     

Protease Activated Receptor-1 Deficiency Diminishes Bleomycin-Induced Skin Fibrosis

Accumulating evidence shows that protease-activated receptor-1 (PAR-1) plays an important role in the development of fibrosis, including lung fibrosis. However, whether PAR-1 also plays a role in the development of skin fibrosis remains elusive. The aim of this study was to determine the role of PAR-1 in the development of skin fibrosis. To explore possible mechanisms by which PAR-1 could play a role, human dermal fibroblasts and keratinocytes were stimulated with specific PAR-1 agonists or antagonists. To investigate the role of PAR-1 in skin fibrosis, we subjected wild-type and PAR-1-deficient mice to a model of bleomycin-induced skin fibrosis. PAR-1 activation leads to increased proliferation and extra cellular matrix (ECM) production, but not migration of human dermal fibroblasts (HDF) in vitro. Moreover, transforming growth factor (TGF)-β production was increased in keratinocytes upon PAR-1 activation, but not in HDF. The loss of PAR-1 in vivo significantly attenuated bleomycin-induced skin fibrosis. The bleomycin-induced increase in dermal thickness and ECM production was reduced significantly in PAR-1-deficient mice compared with wild-type mice. Moreover, TGF-β expression and the number of proliferating fibroblasts were reduced in PAR-1-deficient mice although the difference did not reach statistical significance. This study demonstrates that PAR-1 contributes to the development of skin fibrosis and we suggest that PAR-1 potentiates the fibrotic response mainly by inducing fibroblast proliferation and ECM production.     

Reactivation of UV and γ-inactivated herpes virus in BHK cells

The DNA-repair capabilities of baby hamster kidney (BHK) cells were investigated by comparing the reactivation of irradiated herpes simplex virus type I (HSV1) in BHK cells with its reactivation in mouse fibroblasts and in normal and repairdeficient human diploid fibroblasts. BHK cells were found to have an intermediate ability to reactive UV-irradiated HSV1 (the viral D_o was 14 J/m²) relative to normal human fibroblasts (viral D_o = 19 J/m²) and xeroderma pigmentosum (XP) group A cells (viral D_o = 4.5 J/m²). With mouse L929 cells as the host, the response of the UV-irradiated virus was biphasic with D_os of 4.6 and 30 J/m² for the low- and high-dose components respectively. In contrast to the response following UV radiation, γ-irradiated HSV1 was similarly reactivated by BHK and normal human cells (the D_os for the irradiated virus in BHK and CRl 1106 were 55 and 51 krad, respectively, whereas xeroderma pigmentosum cells were slightly less efficient in the repair of γ-irradiated virus (D_o = 45 krad). UV irradiation of BHK host cells 0–48 h prior to infection enhanced the reactivation of UV-irradiated HSV.     

Repair of 8-methoxypsoralen photoinduced cross-links in yeast

Summary The repair of interstrand cross-links induced by 8-methoxypsoralen plus UVA (365 nm) radiation DNA was analyzed in diploid strains of the yeast Saccharomyces cerevisiae. The strains employed were the wild-type D₇ and derivatives homozygous for the rad18-1 or the rad3-12 mutation. Alkaline step-elution and electron microscopy were performed to follow the process of induction and removal of photoinduced crosslinks. In accordance with previous reports, the D₇ rad3-12 strain failed to remove the induced lesions and could not incise cross-links. The strain D₇ rad18-1 was nearly as efficient in the removal of 8-MOP photoadducts after 2 h of post-treatment incubation as the D₇ RAD⁺ wild-type strain. However, as demonstrated by alkaline step-elution and electron microscopic analysis, the first incision step at DNA cross-links was three times more effective in D₇ rad18-1 than in D₇ RAD⁺. This is consistent with the hypothesis that the RAD18 gene product is involved in the filling of gaps resulting from persistent non-informational DNA lesions generated by the endonucleolytic processing of DNA cross-links. Absence of this gene product may lead to extensive strand breakage and decreased recognition of such lesions by structural repair systems.     

Downregulation of PLK4 expression induces apoptosis and G0/G1‐phase cell cycle arrest in keloid fibroblasts

ObjectivesKeloids are benign fibroproliferative tumors that display many cancer‐like characteristics, such as progressive uncontrolled growth, lack of spontaneous regression, and extremely high rates of recurrence. Polo‐like kinase 4 (PLK4) was recently identified as a master regulator of centriole replication, and its aberrant expression is closely associated with tumorigenesis. This study aimed to investigate the expression and biological role of PLK4 in the pathogenesis of keloids.Materials and MethodsWe evaluated the expression of PLK4 in keloids and adjacent normal skin tissue samples. Then, we established PLK4 knockdown and overexpression cell lines in keloid fibroblasts (KFs) and normal skin fibroblasts (NFs), respectively, to investigate the roles of PLK4 in the regulation of proliferation, migration, invasion, apoptosis, and cell cycle in KFs. Centrinone B (Cen‐B), a highly selective PLK4 inhibitor, was used to inhibit PLK4 activity in KFs to evaluate the therapeutic effect on KFs.ResultsWe discovered that PLK4 was overexpressed in keloid dermal samples and KFs compared with adjacent normal skin samples and NFs derived from the same patients. High PLK4 expression was positively associated with the proliferation, migration, and invasion of KFs. Furthermore, knockdown of PLK4 expression or inhibition of PLK4 activity by Cen‐B suppressed KF growth, induced KF apoptosis via the caspase‐9/3 pathway, and induced cell cycle arrest at the G0/G1 phase in vitro.ConclusionsThese findings demonstrate that PLK4 is a critical regulator of KF proliferation, migration, and invasion, and thus, Cen‐B is a promising candidate drug for keloid treatment.

Keloids are benign fibroproliferative tumors that display many cancer‐like characteristics, such as progressive uncontrolled growth, lack of spontaneous regression, and extremely high rates of recurrence. Polo‐like kinase 4 (PLK4) was recently identified as a master regulator of centriole replication, and its aberrant expression is closely associated with tumorigenesis. This study aimed to investigate the expression and biological role of PLK4 in the pathogenesis of keloids. Here, we discovered that PLK4 is a potential target for the treatment of keloids. PLK4 was overexpressed in keloid dermal samples and keloid fibroblasts (KFs) compared with adjacent normal skin samples and normal skin fibroblasts derived from the same patients. High PLK4 expression was positively associated with the proliferation, migration, and invasion of KFs. Furthermore, knockdown of PLK4 expression or inhibition of PLK4 activity by a highly selective inhibitor, centrinone B (Cen‐B), suppressed KF growth, induced KF apoptosis via the caspase‐9/3 pathway, and induced cell cycle arrest at the G0/G1 phase via the p53/p21/Cyclin D1 pathway in vitro. These findings demonstrate that PLK4 is a critical regulator of KF proliferation, migration, and invasion, and thus, Cen‐B is a promising candidate drug for keloid treatment.     

Radiation Resistance of the Microflora Associated with Amniotic Membranes

Summary Amniotic membrane is widely used in the treatment of burn wounds and ulcers of various etiology. As it comes into contact with open wounds, it needs to be perfectly sterile to avoid the transmission of any disease. Accordingly, amniotic membrane needs to bear a high sterility assurance level (SAL). Conventionally, a radiation dose of 25 kGy is the generally accepted dose for sterilization. But to keep intact the biomechanical and other properties, it is sometimes proposed to use a lower dose without compromising an SAL of 10⁻⁶. The initial microbial contamination level and the radiation resistance of the contaminants determine the dose required for sterilization. The microbial species associated with the amniotic membrane from about 70 different batches were isolated. Twenty-two representative bacterial isolates were characterized and tested for survival in an incremental series of radiation doses from 0.5 to 5.0 kGy. The radiation decimal reduction dose (D₁₀) values for the strains were determined. Relatively higher D₁₀ values were recorded for the gram-positive isolates. The D₁₀ values of microbial isolates ranged from 0.16 to 1.3 kGy, and most resistant Bacillus strain had a D₁₀ value of 2.1 kGy. The radiation dose necessary to achieve an SAL of 10⁻⁶ was calculated based on the D₁₀values of the isolated strains. For a bioburden of 1000 Bacillus organism, the sterilization dose of 18.9 kGy is obtained. However, based on the experimental determination of D₁₀ of the radiation-resistant reference strain Bacillus pumilus, the adequate dose for radiation sterilization is found to be 19.8 kGy if bioburden level of 1000 is granted. The results substantiate that radiation dose of 25 kGy assures sterilization of amniotic membranes with bioburden level of 1000 colony forming units.     

A method for deriving net primary productivity and component respiratory fluxes from tower-based eddy covariance data: a case study using a 17-year data record from a Douglas-fir chronosequence

Conventional gap‐filling procedures for eddy covariance (EC) data are limited to calculating ecosystem respiration (R_E) and gross ecosystem productivity (P_G) as well as missing values of net ecosystem productivity (F_NEP). We develop additional postprocessing steps that estimate net primary productivity (P_N), autotrophic (R_a), and heterotrophic respiration (R_h). This is based on conservation of mass of carbon (C), Monte Carlo (MC) simulation, and three ratios: C use efficiency (CUE, P_N to P_G), R_a to R_E, and F_NEP to R_E. This procedure, along with the estimation of F_NEP, R_E, and P_G, was applied to a Douglas‐fir dominated chronosequence on Vancouver Island, British Columbia, Canada. The EC data set consists of 17 site years from three sites: initiation (HDF00), pole/sapling (HDF88), and near mature (DF49), with stand ages from 1 to 56 years. Analysis focuses on annual C flux totals and C balance ratios as a function of stand age, assuming a rotation age of 56 years. All six C balance terms generally increased with stand age. Average annual P_N by stand was 213, 750, and 1261 g C m⁻² yr⁻¹ for HDF00, HDF88, and DF49, respectively. The canopy compensation point, the year when the chronosequence switched from a source to a sink of C, occurred at stand age ca. 20 years. HDF00 and HDF88 were strong and moderate sources (F_NEP=−581 and −138 g C m⁻² yr⁻¹), respectively, while DF49 was a moderate sink (F_NEP=294 g C m⁻² yr⁻¹) for C. Differences between sites were greater than interannual variation (IAV) within sites and highlighted the importance of age‐related effects in C cycling. The validity of the approach is discussed using a sensitivity analysis, a comparison with growth and yield estimates from the same chronosequence, and an intercomparison with other chronosequences.     

Possible origin of extremely high contents of vitamin D3 in some kinds of fish liver

• 1.1. Comparative studies on the possible origin of extremely high contents of vitamin D₃ in some kinds of fish liver were performed.
• 2.2. Neither photochemical formation of vitamin D₃ in fish skin by solar radiation of 7-dehydrocholesterol (7-DHC) nor nonphotochemical enzymatic formation of vitamin D₃ from 7-DHC in fish liver was demonstrated as the origin of vitamin D₃.
• 3.3. On the other hand, when bastard halibuts and carps were farmed from fingerlings to adults with feedstuff's containing vitamin D₂ or D₃, significant amounts of the vitamins were accumulated in the fish liver.
• 4.4. The contents of vitamins D₂ and D₃ in bastard halibut liver increased according to the duration of farming and dose responses of the vitamins in carp liver were observed.
• 5.5. Significant amounts of vitamins D₂ and D₃ in phytoplankton and vitamin D₃ in Zooplankton and small fish were detected.
• 6.6. Therefore, we have concluded that the most probable origin of vitamin D₃ in fish liver is a result of food chains from plankton.

     

Genetic control of repair of radiation damage produced under euoxic and anoxic conditions in diploid yeastSaccharomyces cerevisiae

Summary Diploid wild type yeast strains 211, X2180 and the radiation sensitive mutantsrad2, 6, 9, 18, 50–55, and57 were exposed to cobalt-60 gamma radiation, in the presence and absence of oxygen, in order to identify the RAD loci involved in the repair of sublethal damage (SLD), recovery from potentially lethal damage (PLD) and oxygen enhancement ratio (OER). Response of wild type and mutants were compared in terms of survival curve parameters D_q, D₁₀, D₁, and D₀. As compared to wild type the mutants showed increased sensitivity to radiation lethality, both under euoxic and hypoxic conditions, as judged by the reduction in D_q and D₀ values. OER was reduced in therad2, 9, 18, 50, 51, and57 mutants indicating that these genes could be associated with the repair of gamma radiation damage produced under hypoxic condition.Shoulder (D_q) a measure of the ability of the cells to repair SLD, was reduced in therad6, 9, 18, 50, 53, and57 strains and was almost absent in therad51, 52, 54, and55 mutants. The ability to recover from PLD was equal to that of wild type strain in therad2, 6, 9, and18 strains, reduced in therad53, 55, and57 strains and was absent in therad50–52 and54 strains. In the mutants with liquid holding recovery ability, the extent of recovery from PLD produced under euoxic and hypoxic conditions was the same. These observations suggest that different groups of loci are involved in the control of different repair processes and that the expression of therad50–57 loci play a very important role in the repair of ionising radiation damage.On the basis of the liquid holding recovery data presented here and the observations made by others it is suggested that the unrepaired DSB constitute the PLD and that the repair of DSB involves recombination between homologous chromosomes.     

Activity of 25-Hydroxylase in Human Gingival Fibroblasts and Periodontal Ligament Cells

Background

We previously demonstrated that 25-hydroxyvitamin D₃ concentrations in gingival crevicular fluid are 300 times higher than those in the plasma of patients with aggressive periodontitis. Here we explored whether 25-hydroxyvitamin D₃ can be synthesized by periodontal soft tissue cells. We also investigated which of the two main kinds of hydroxylases, CYP27A1 and CYP2R1, is the key 25-hydroxylase in periodontal soft tissue cells.

Methodology/Principal Findings

Primary cultures of human gingival fibroblasts and periodontal ligament cells from 5 individual donors were established. CYP27A1 mRNA, CYP2R1 mRNA and CYP27A1 protein were detected in human gingival fibroblasts and periodontal ligament cells, whereas CYP2R1 protein was not. After incubation with the 25-hydroxylase substrate vitamin D₃, human gingival fibroblasts and periodontal ligament cells generated detectable 25-hydroxyvitamin D₃ that resulted in the production of 1α,25-dihydroxyvitamin D₃. Specific knockdown of CYP27A1 in human gingival fibroblasts and periodontal ligament cells using siRNA resulted in a significant reduction in both 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ production. Knockdown of CYP2R1 did not significantly influence 25-hydroxyvitamin D₃ synthesis. Sodium butyrate did not influence significantly CYP27A1 mRNA expression; however, interleukin-1β and Porphyromonas gingivalis lipopolysaccharide strongly induced CYP27A1 mRNA expression in human gingival fibroblasts and periodontal ligament cells.

Conclusions

The activity of 25-hydroxylase was verified in human gingival fibroblasts and periodontal ligament cells, and CYP27A1 was identified as the key 25-hydroxylase in these cells.     

Kinetic Modeling of Amino Acid Oxidation Catalyzed by a New D‐Amino Acid Oxidase from Arthrobacter protophormiae

Amino acid oxidases, which enantiospecifically catalyze the oxidative deamination of either D‐ or L‐amino acids, belong to the class of oxidoreductases functioning with a tightly bound cofactor. This cofactor favors industrial applications of D‐amino acid oxidases (D‐AAO). Hence, the enzyme is very important for the industrial application in the purification and determination of certain amino acids. In developing the enzyme‐catalyzed reaction for large‐scale production, modeling of the reaction kinetics plays an important role. Therefore, the subject of this study was the kinetics of the oxidative deamination, a very complex reaction system, which is catalyzed by D‐AAO from Arthrobacter protophormiae using its natural substrate D‐methionine and the aromatic amino acid 3,4‐dihydroxyphenyl‐D‐alanine (D‐DOPA). The kinetic parameters determined by the measurement of the initial rate and nonlinear regression were verified in batch reactor experiments by comparing calculated and experimental concentration‐time curves. It was found that the enzyme is highly specific towards D‐methionine (K_m = 0.24 mM) and not as specific to D‐DOPA as a substrate (K_m = 9.33 mM). The enzyme activity towards D‐methionine ( = 3.01 U/mL) was approx. seven times higher than towards D‐DOPA ( = 20.01 U/mL). The enzyme exhibited no activity towards L‐methionine and L‐DOPA. Batch and repetitive batch experiments were performed with both substrates in the presence and in the absence of catalase for hydrogen peroxide decomposition. Their comparison made it possible to conclude that hydrogen peroxide has no negative influence on the enzyme activity.