DNA protective properties of vanillin against gamma-radiation under different conditions: possible mechanisms

Ionizing radiation is an important genotoxic agent. Protecting against this form of toxicant, especially by a dietary component, has several potential applications. In the present study, we have examined the ability of vanillin (4-hydroxy-3-methoxybenzaldehyde), a naturally occurring food flavouring agent, to inhibit radiation-induced DNA damage measured as strand breaks under in vitro, ex vivo and in vivo conditions besides the possible mechanisms behind the observed protection. Our study showed that there was a concentration-dependent inhibition of the disappearance of super-coiled (ccc) form of plasmid pBR322 (in vitro) upon exposure to 50 Gy of gamma-radiation. Presence of 0.5 mM vanillin has a dose-modifying factor (DMF) of 6.75 for 50% inactivation of ccc form. Exposure of human peripheral blood leucocytes (ex vivo) to gamma-radiation causes strand breaks in the cellular DNA, as assessed by comet assay. When leucocytes were exposed to 2 Gy of gamma-radiation there was an increase in parameters of comet assay such as %DNA in tail, tail length, 'tail moment' and 'Olive tail moment'. The presence of 0.5 mM vanillin during irradiation significantly reduced these parameters. Damage to DNA in mouse peripheral blood leucocytes after whole-body exposure of mice (in vivo) to gamma-radiation was studied at 1 and 2 h post-irradiation. There was recovery of DNA damage in terms of the above-mentioned parameters at 2 h post-irradiation. This was more than that observed at 1 h. The recovery was more in vanillin treated mice. Hence our studies showed that vanillin offers protection to DNA against radiation-induced damage possibly imparting a role other than modulation of DNA repair. To examine the possible mechanisms of radioprotection, in terms of radiation-derived radicals, we carried out the reaction of vanillin with ABTS*(+) radical spectrophotometrically besides with DNA peroxyl and carbonyl radicals by using pulse radiolysis. Our present investigations show that vanillin has ability to protect against DNA damage in plasmid pBR322, human and mouse peripheral blood leucocytes and splenic lymphocytes besides enhancing survival in splenic lymphocytes against gamma-radiation, and that the possible mechanism may involve scavenging of radicals generated during radiation, apart from modulation of DNA repair observed earlier.     

Edaravone protects human peripheral blood lymphocytes from γ-irradiation-induced apoptosis and DNA damage

Radiation-induced cellular injury is attributed primarily to the harmful effects of free radicals, which play a key role in irradiation-induced apoptosis. In this study, we investigated the radioprotective efficacy of edaravone, a licensed clinical drug and a powerful free radical scavenger that has been tested against γ-irradiation-induced cellular damage in cultured human peripheral blood lymphocytes in studies of various diseases. Edaravone was pre-incubated with lymphocytes for 2 h prior to γ-irradiation. It was found that pretreatment with edaravone increased cell viability and inhibited generation of γ-radiation-induced reactive oxygen species (ROS) in lymphocytes exposed to 3 Gy γ-radiation. In addition, γ-radiation decreased antioxidant enzymatic activity, such as superoxide dismutase and glutathione peroxidase, as well as the level of reduced glutathione. Conversely, treatment with 100 μM edaravone prior to irradiation improved antioxidant enzyme activity and increased reduced glutathione levels in irradiated lymphocytes. Importantly, we also report that edaravone reduced γ-irradiation-induced apoptosis through downregulation of Bax, upregulation of Bcl-2, and consequent reduction of the Bax:Bcl-2 ratio. The current study shows edaravone to be an effective radioprotector against γ-irradiation-induced cellular damage in lymphocytes in vitro. Finally, edaravone pretreatment significantly reduced DNA damage in γ-irradiated lymphocytes, as measured by comet assay (% tail DNA, tail length, tail moment, and olive tail moment) (p < 0.05). Thus, the current study indicates that edaravone offers protection from radiation-induced cytogenetic alterations.     

Protection of cellular DNA from γ-radiation-induced damages and enhancement in DNA repair by troxerutin

The effect of troxerutin on γ-radiation-induced DNA strand breaks in different tissues of mice in vivo and formations of the micronuclei were studied in human peripheral blood lymphocytes ex vivo and mice blood reticulocytes in vivo. Treatments with 1 mM troxerutin significantly inhibited the micronuclei induction in the human lymphocytes. Troxerutin protected the human peripheral blood leucocytes from radiation-induced DNA strand breaks in a concentration dependent manner under ex vivo condition of irradiation (2 Gy). Intraperitoneal administration of troxerutin (175 mg/kg body weight) to mice before and after whole body radiation exposure inhibited micronuclei formation in blood reticulocytes significantly. The administration of different doses (75, 125 and 175 mg/kg body weight) of troxerutin 1 h prior to 4 Gy γ-radiation exposure showed dose-dependent decrease in the yield of DNA strand breaks in murine blood leucocytes and bone marrow cells. The dose-dependent protection was more pronounced in bone marrow cells than in blood leucocytes. Administration of 175 mg/kg body weight of the drug (i.p.) 1 h prior or immediately after whole body irradiation of mice showed that the decrease in strand breaks depended on the post-irradiation interval at which the analysis was done. The observed time-dependent decrease in the DNA strand breaks could be attributed to enhanced DNA repair in troxerutin administered animals. Thus in addition to anti-erythrocytic, anti-thrombic, fibrinolytic and oedema-protective rheological activity, troxerutin offers protection against γ-radiation-induced micronuclei formation and DNA strand breaks and enhances repair of radiation-induced DNA strand breaks. (Mol Cell Biochem xxx: 57–68, 2005)     

Protective effect of curcumin on γ-radiation induced DNA damage and lipid peroxidation in cultured human lymphocytes

The present work is aimed at evaluating the radioprotective effect of curcumin, a naturally occurring phenolic compound on γ-radiation induced toxicity. The cellular changes were estimated by using lipid peroxidative indices like thiobarbituric acid reactive substances (TBARS), the antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH). The DNA damage was analysed by using cytokinesis blocked micronucleus assay and dicentric aberration (DC). The γ-radiation at different doses (1, 2 and 4 Gy) were found to significantly increase micronuclei (MN), DC frequencies and TBARS level whereas the levels of GSH and antioxidant enzymes were significantly decreased. The maximum damage to lymphocytes was observed at 4 Gy irradiation. Curcumin pretreatment (1, 5 and 10 μg/ml) significantly decreased the frequency of MN and DC. The levels of TBARS decreased and activities of SOD, CAT and GPx significantly increased along with GSH levels. At 1 Gy irradiation all the concentrations of curcumin (1, 5 and 10 μg/ml) significantly protected the lymphocytes from radiation damage. At 2 Gy irradiation, 5 and 10 μg/ml of curcumin showed significant radioprotection. Since the highest damage was observed at 4 Gy irradiation both 1 and 5 μg/ml of curcumin pretreatment were not sufficient to protect the lymphocytes from radiation damage but 10 μg/ml of curcumin significantly protected the cultured lymphocytes from radiation damage. Thus, pretreatment with curcumin gives protection to lymphocytes against γ-radiation induced cellular damage.     

Gamma ray induced DNA damage in human and mouse leucocytes measured by SCGE-Pro: a software developed for automated image analysis and data processing for Comet assay

The studies reported in this communication had two major objectives: first to validate the in-house developed SCGE-Pro: a software developed for automated image analysis and data processing for Comet assay using human peripheral blood leucocytes exposed to radiation doses, viz. 2, 4 and 8 Gy, which are known to produce DNA/chromosome damage using alkaline Comet assay. The second objective was to investigate the effect of gamma radiation on DNA damage in mouse peripheral blood leucocytes using identical doses and experimental conditions, e.g. lyses, electrophoretic conditions and duration of electrophoresis which are known to affect tail moment (TM) and tail length (TL) of comets. Human and mouse whole blood samples were irradiated with different doses of gamma rays, e.g. 2, 4 and 8 Gy at a dose rate of 0.668 Gy/min between 0 and 4°C in air. After lyses, cells were electrophorased under alkaline conditions at pH 13, washed and stained with propidium iodide. Images of the cells were acquired and analyzed using in-house developed imaging software, SCGE-Pro, for Comet assay. For each comet, total fluorescence, tail fluorescence and tail length were measured. Increase in TM and TL was considered as the criteria of DNA damage. Analysis of data revealed heterogeneity in the response of leucocytes to gamma ray induced DNA damage both in human as well as in mouse. A wide variation in TM and TL was observed in control and irradiated groups of all the three donors. Data were analyzed for statistical significance using one-way ANOVA. Though a small variation in basal level of TM and TL was observed amongst human and mouse controls, the differences were not statistically significant. A dose-dependent increase in TM (P<0.001) and TL (P<0.001) was obtained at all the radiation doses (2–8 Gy) both in human and mouse leucocytes. However, there was a difference in the nature of dose response curves for human and mouse leucocytes. In human leucocytes, a linear increase in TM and TL was observed up to the highest radiation dose of 8 Gy. However, in case of mouse leucocytes, a sharp increase in TM and TL was observed only up to 4 Gy, and there after saturation ensued. In human samples, the dose response of both TM and TL showed best fits with linear model (r_TM=0.999 and r_TL=0.999), where as in mouse, the best fit was obtained with Sigmoid (Boltzman) model. From the present data on leucocytes with increase in TM and TL as the criteria of DNA damage, it appears that mouse is relatively more sensitive to radiation damage than humans.     

Protection of DNA and membrane from gamma radiation induced damage by gallic acid

Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is a naturally occurring plant phenol. In vitro and in vivo studies have shown that this phytochemical protected DNA and membranes against ionizing radiation. Rat liver microsomes and plasmid pBR322 DNA were exposed to various doses of gamma radiation in presence and absence of GA. Exposure of the microsomes to gamma radiation resulted in the formation of peroxides of membrane lipids measured as thiobarbituric acid reactive substances and presence of GA during irradiation prevented the formation of lipid peroxidation. Gamma irradiation of plasmid DNA resulted in induction of strand breaks in DNA resulting in disappearance of the supercoiled (ccc) form. Presence of GA during irradiation protected the DNA from undergoing the strand breaks. In in vivo studies it was found that whole body exposure of mice to gamma radiation (4 Gy) increased the formation of lipid peroxides in various tissues and damage to cellular DNA (as measured by alkaline comet assay) in peripheral blood leucocytes. Administration of GA to mice prior to whole body radiation exposure reduced the peroxidation of lipids and the damage to the cellular DNA indicating in vivo radiation protection of membranes and DNA by GA. (Mol Cell Biochem 278: 111–117, 2005)     

α-, β-, and γ-Tubulin Polymerization in Response to DNA Damage

Microtubules provide structural support for a cell and play key roles in cell motility, mitosis, and meiosis. They are also the targets of several anticancer agents, indicating their importance in maintaining cell viability. We have investigated the possibility that alterations in microtubule structure and tubulin polymerization may be part of the cellular response to DNA damage. In this report, we find that γ-radiation stimulates the production and polymerization of α-, β-, and γ- tubulin in hematopoeitic cell lines (Ramos, DP16), leading to visible changes in microtubule structures. We have found that this microtubule reorganization can be prevented by caffeine, a drug that concomitantly inhibits DNA damage-induced cell cycle arrest and apoptosis. Our results support the idea that microtubule polymerization is an important facet of the mammalian response to DNA damage.     

The mitochondrial DNA polymerase as a target of oxidative damage

The mitochondrial respiratory chain is a source of reactive oxygen species (ROS) that are responsible for oxidative modification of biomolecules, including proteins. Due to its association with mitochondrial DNA, DNA polymerase γ (pol γ) is in an environment to be oxidized by hydrogen peroxide and hydroxyl radicals that may be generated in the presence of iron ions associated with DNA. We tested whether human pol γ was a possible target of ROS with H₂O₂ and investigated the effect on the polymerase activities and DNA binding efficiency. A 1 h treatment with 250 µM H₂O₂ significantly inhibited DNA polymerase activity of the p140 subunit and lowered its DNA binding efficiency. Addition of p55 to the p140 catalytic subunit prior to H₂O₂ treatment offered protection from oxidative inactivation. Oxidatively modified amino acid residues in pol γ  resulting from H₂O₂ treatment were observed in vitro as well as in vivo, in SV40-transfected human fibroblasts. Pol γ was detected as one of the major oxidized mitochondrial matrix proteins, with a detectable decline in polymerase activity. These results suggest pol γ as a target of oxidative damage, which may result in a reduction in mitochondrial DNA replication and repair capacities.     

Investigating DNA damage in tannery workers occupationally exposed to trivalent chromium using comet assay

DNA damage of peripheral lymphocytes in 60 workers occupationally exposed to trivalent chromium [Cr(III)] in a tannery was studied using comet assay. The urinary and blood chromium levels were detected as a biomarker of internal exposure. The 90 subjects were divided into three groups: (i) exposure group I included 30 tannery workers highly exposed to chromium from tanning department; (ii) exposure group II included 30 tannery workers with moderate chromium exposure from finishing department; (iii) control group included 30 individuals without exposure to physical or chemical genotoxic agents. No significant difference was found among the three groups for age and smoking. The results showed that the medians of blood and urinary Cr of two exposure groups were significantly higher than those of control group (P < 0.01). And the medians of blood and urinary Cr of exposure group I were significantly higher than those of exposure group II (P < 0.05 or P < 0.01). The medians of mean tail length (MTL) of the three groups were 5.33 (2.90–8.50), 3.43 (2.31–8.29) and 2.04 (0.09–3.83) μm, respectively; The medians of mean tail moment (MTM) of the three groups were 6.28 (2.14–11.81), 3.41 (1.25–11.07) and 0.53 (0.13–3.29), respectively. The MTL and MTM of two exposure groups were significantly higher than those of control group (P < 0.01). The MTL and MTM of exposure group I were significantly higher than those of exposure group II (P < 0.01). The results of the present investigation suggest that occupational exposure to trivalent chromium can lead to a detectable DNA damage of human peripheral lymphocytes. Moreover, DNA damage was associated with chromium levels in blood. DNA damage may serve as a valuable effective biomarker and total chromium in blood may serve as a useful internal exposure biomarker in the population occupationally exposed to trivalent chromium.     

Lithium Chloride Dependent Glycogen Synthase Kinase 3 Inactivation Links Oxidative DNA Damage,Hypertrophy and Senescence in Human Articular Chondrocytes and Reproduces Chondrocyte Phenotype of Obese Osteoarthritis Patients

Introduction

Recent evidence suggests that GSK3 activity is chondroprotective in osteoarthritis (OA), but at the same time, its inactivation has been proposed as an anti-inflammatory therapeutic option. Here we evaluated the extent of GSK3β inactivation in vivo in OA knee cartilage and the molecular events downstream GSK3β inactivation in vitro to assess their contribution to cell senescence and hypertrophy.

Methods

In vivo level of phosphorylated GSK3β was analyzed in cartilage and oxidative damage was assessed by 8-oxo-deoxyguanosine staining. The in vitro effects of GSK3β inactivation (using either LiCl or SB216763) were evaluated on proliferating primary human chondrocytes by combined confocal microscopy analysis of Mitotracker staining and reactive oxygen species (ROS) production (2'',7''-dichlorofluorescin diacetate staining). Downstream effects on DNA damage and senescence were investigated by western blot (γH2AX, GADD45β and p21), flow cytometric analysis of cell cycle and light scattering properties, quantitative assessment of senescence associated β galactosidase activity, and PAS staining.

Results

In vivo chondrocytes from obese OA patients showed higher levels of phosphorylated GSK3β, oxidative damage and expression of GADD45β and p21, in comparison with chondrocytes of nonobese OA patients. LiCl mediated GSK3β inactivation in vitro resulted in increased mitochondrial ROS production, responsible for reduced cell proliferation, S phase transient arrest, and increase in cell senescence, size and granularity. Collectively, western blot data supported the occurrence of a DNA damage response leading to cellular senescence with increase in γH2AX, GADD45β and p21. Moreover, LiCl boosted 8-oxo-dG staining, expression of IKKα and MMP-10.

Conclusions

In articular chondrocytes, GSK3β activity is required for the maintenance of proliferative potential and phenotype. Conversely, GSK3β inactivation, although preserving chondrocyte survival, results in functional impairment via induction of hypertrophy and senescence. Indeed, GSK3β inactivation is responsible for ROS production, triggering oxidative stress and DNA damage response.     

Anti-inflammatory properties of α- and γ-tocopherol

Natural vitamin E consists of four different tocopherol and four different tocotrienol homologues (α, β, γ, δ) that all have antioxidant activity. However, recent data indicate that the different vitamin E homologues also have biological activity unrelated to their antioxidant activity. In this review, we discuss the anti-inflammatory properties of the two major forms of vitamin E, α-tocopherol (αT) and γ-tocopherol (γT), and discuss the potential molecular mechanisms involved in these effects. While both tocopherols exhibit anti-inflammatory activity in vitro and in vivo, supplementation with mixed (γT-enriched) tocopherols seems to be more potent than supplementation with αT alone. This may explain the mostly negative outcomes of the recent large-scale interventional chronic disease prevention trials with αT only and thus warrants further investigation.     

Poly(ADP-ribose) polymerase-1 is a survival factor for radiation-exposed intestinal epithelial stem cells in vivo

Poly(ADP-ribose) polymerase-1 (PARP-1) is a key enzyme mediating the cellular response to DNA strand breaks. It plays a critical role in genomic stability and survival of proliferating cells in culture undergoing DNA damage. Intestinal epithelium is the most proliferative tissue in the mammalian body and its stem cells show extreme sensitivity to low-level genotoxic stress. We investigated the role of PARP-1 in the in vivo damage response of intestinal stem cells in crypts of PARP-1^–/– and control mice following whole-body γ-irradiation (1 Gy). In the PARP-1^–/– mice there was a significant delay during the first 6 h in the transient p53 accumulation in stem cells whereas an increased number of cells were positive for p21^CIP1/WAF1. Either no or only marginal differences were noted in MDM2 expression, apoptosis, induction of or recovery from mitotic blockage, or inhibition of DNA synthesis. We further observed a dose-dependent reduction in crypt survival measured at 4 days post-irradiation in control mice, and this crypt-killing effect was significantly potentiated in PARP-1^–/– mice. Our results thus establish that PARP-1 acts as a survival factor for intestinal stem cells in vivo and suggest a functional link with early p53 and p21^CIP1/WAF1 responses.     

A γ-aminobutyrate pathway in mammalian kidney cortex

Rat kidney cortex converts l-glutamate to γ-aminobutyrate by a decarboxylation reaction which differs from the corresponding reaction in brain. Renal l-glutamate decarboxylase has two apparent K_m values for glutamate in homogenates (0.4 and 2.5 mM). γ-Aminobutyrate is converted by a transaminase whose capacity appears to exceed the decarboxylase. γ-Aminobutyrate is converted ultimately to succinate and CO₂.γ-Aminobutyrate stimulates respiration of kidney cortex slices in vitro and the compound crosses cell membranes in kidney by a respiration-linked, mediated process.Chronic acidosis lowers renal γ-aminobutyrate in the rat; brain γ-aminobutyrate is unaffected by acidosis. Glutamic acid decarboxylase and γ-aminobutyrate transaminase activities are unchanged in acidosis. α-Methylglutamate, an inhibitor of renal glutaminase, lowers the γ-aminobutyrate and glutamate content of rat kidney in normal and acidotic states. Aminooxyacetic acid in vivo, an inhibitor of γ-aminobutyrate transaminase, causes a striking increase in renal γ-aminobutyrate during chronic acidosis.At concentrations of glutamate in vitro, which are similar to the tissue glutamate content in vivo, the γ-aminobutyrate pathway accounts for approximately one-fourth of glutamate disposal in rat kidney cortex slices.     

Protective effect of an aminothiazole compound against γ-radiation induced oxidative damage

Abstract

Ionizing radiation causes its biological effects mainly through oxidative damage induced by reactive oxygen species. During radiotherapy of cancer, one of the undesirable side-effects is toxicity to normal cells. Compounds with antioxidant activities are being tried as ‘prophylactic radioprotectants’ to overcome this problem. We evaluated the protective effect of an aminothiazole compound, in the form of dendrodoine analogue (DA) originally derived from a marine tunicate, against γ-radiation-induced damage to lipid, protein, and DNA besides its cytotoxicity. Oxidative damage was examined by different biochemcial assays. Our studies reveal that DA gave significant protection, in fairly low concentrations, against damage induced by γ-radiation to rat liver mitochondria, plasmid pBR322 DNA, and mouse splenic lymphocytes in vitro. It also protected against oxidative damage in whole-body irradiated mice exposed to therapeutic dose of radiation (2 Gy) in vivo. Spleen, a major target organ for radiation damage, of the irradiated mice showed significant protection when treated with DA, as examined by histopathology. In conclusion, due to the possible protective effects against normal cells/tissues both in vitro and in vivo, DA shows potential to be a radioprotector for possible use during radiotherapy.     

In vivo repair of methylation damage in Aag 3-methyladenine DNA glycosylase null mouse cells

3-Methyladenine (3MeA) DNA glycosylases initiate base excision repair by removing 3MeA. These glycosylases also remove a broad spectrum of spontaneous and environmentally induced base lesions in vitro. Mouse cells lacking the Aag 3MeA DNA glycosylase (also known as the Mpg, APNG or ANPG DNA glycosylase) are susceptible to 3MeA-induced S phase arrest, chromosome aberrations and apoptosis, but it is not known if Aag is solely responsible for repair of 3MeA in vivo. Here we show that in Aag^–/– cells, 3MeA lesions disappear from the genome slightly faster than would be expected by spontaneous depurination alone, suggesting that there may be residual repair of 3MeA. However, repair of 3MeA is at least 10 times slower in Aag^–/– cells than in Aag^+/+ cells. Consequently, 24 h after exposure to [³H]MNU, 30% of the original 3MeA burden is intact in Aag^–/– cells, while 3MeA is undetectable in Aag^+/+ cells. Thus, Aag is the major DNA glycosylase for 3MeA repair. We also investigated the in vivo repair kinetics of another Aag substrate, 7-methylguanine. Surprisingly, 7-methylguanine is removed equally efficiently in Aag^+/+ and Aag^–/– cells, suggesting that another DNA glycosylase acts on lesions previously thought to be repaired by Aag.     

Selective analogs of 1α,25-dihydroxyvitamin D3 for the study of specific functions of Vitamin D

New analogs of 1α,25-dihydroxyvitamin D₃ synthesized in our research group that show selective activity in vivo are presented along with supporting biological results. Compounds that act preferentially on intestine are 2-(3′-propylidene-19-nor-(20S or 20R))-1α,25-dihydroxyvitamin D₃ and 2-methylene-19-21-dinor-1α,25-dihydroxyvitamin D₃. Compounds that act anabolically to induce bone formation are 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D₃ (2MD), the 2α-methyl derivative, the 26,27-dimethyl derivative, and the 26-dimethylene derivative. Compounds that act preferentially on parathyroid glands are 2-methylene-19-nor-1α-hydroxy-homopregnacalciferol, the 20S-bishomo derivative and the 2-methylene-19,26,27-trinor-1α,25-dihydroxyvitamin D₃. These latter compounds do not elevate serum calcium until doses of the order of >300 μg/kg body weight are used, while parathyroid hormone levels are suppressed at much lower doses. Some of these novel analogs may ultimately be useful as new and safer therapeutic agents. Regardless of their clinical utility, they represent valuable research tools that can be used to study the specific functions of the Vitamin D hormone in vivo.     

Effects of cigarette smoking, XRCC1 genetic polymorphisms, and age on basal DNA damage in human blood mononuclear cells

The aim of this study was to investigate the effects of smoking, polymorphisms of XRCC1 codons 194 and 399, and age on levels of basal DNA damage (as measured by an alkaline comet assay) on mononuclear cells in 122 healthy Japanese workers. In the whole group of 122 individuals, the tail moment (TM) values of current smokers (P < 0.001) or former smokers (P = 0.03) were significantly higher than those of nonsmokers. Individuals bearing the XRCC1 399Gln variant allele showed significant increases in TM values in all subjects or in referent subgroups stratified by age or smoking status except in the current smokers group; in contrast, the TM values of individuals bearing the XRCC1 194Trp variant allele were significantly lower than those of individuals bearing wild-type Arg/Arg genotypes. Furthermore, older subjects (≥47 years old) had significantly higher TM values than younger subjects (<47 years old) in all subjects (P = 0.008). Multiple regression analysis indicated that smoking habits, polymorphisms of XRCC1 codons 194 and 399, and age were important variables affecting individuals basal DNA damage.     

Butanol-extracted lipoteichoic acid induces in vivo leukocyte adhesion

Lipoteichoic acid (LTA), an immunostimulatory component of the cell walls of Gram positive bacteria, has pro-inflammatory effects in vitro and in vivo. However, one in vivo study concluded that LTA had no noticeable effects on leukocyte recruitment. In this study we investigated the effects of highly purified LTA, prepared by butanol extraction (Bu-LTA) at room temperature, on in vivo leukocyte adhesion. Using intravital microscopy we measured adhesion of leukocytes in mesenteric post-capillary venules of rats and mice. Topical superfusion of Bu-LTA (1 μg/ml) in rats significantly (p < 0.05) increased adhesion within 30 min. By contrast, hot phenol-extracted LTA did not increase adhesion. Alkaline hydrolysis of Bu-LTA removed alanine residues and prevented adhesion. Also, pre-administration of anti-rat β₂-integrin antibody abolished Bu-LTA-induced adhesion. Finally, intraperitoneal injection of Bu-LTA (100 μg/ml) into mice also significantly (p < 0.01) increased leukocyte adhesion measured at 60 min. In conclusion, Bu-LTA with intact alanine residues promotes β₂-integrin-dependent leukocyte adhesion in vivo.     

In vitro and in vivo growth and casein gene expression of mouse mammary tumor epithelial cells in response to hormones

Cells from autochthonous mouse mammary carcinomas which display estrogen-independent growth _vivo were studied for their hormonal responses in primary culture. A culture system employing insulin-supplemented, serum-free medium and basement membrane Matrigel as a substratum was used to cultivate tumor cells. The cells did not exhibit in vitro estrogenor prolactin-dependent growth. Primary tumors still displayed a constitutional expression of α-, β-, and γ-casein mRNAs. These messages were dramatically reduced during the culture period. However, seven to eightfold increases in α- and β-casein mRNAs were inducible in the 5-day cultures by treatment with prolactin and hydrocortisone. If the hormones were present through a 2-week culture period, the levels of α-, β-, and γ-casein mRNAs in the cells were maintained and displayed in a time-dependent increase with a peak at 10–14 days. The accumulation of β-casein mRNA in vitro did not require DNA synthesis. Administration of prolactin directly into the growing tumors in vivo could also enhance β-casein mRNA levels in the tumor cells. Morphological studies of the cells cultured in the presence of prolactin and hydrocortisone did not reveal visible changes compared with those without hormonal treatment. Transplantation of tumor cells cultured in the presence or absence of hormones resulted in the development of tumors in mice at approximately the same time. The current studies suggest that the autochthonous mammary tumor cells, independent of estrogen for cell growth, were still inducible for casein gene expression in vitro and in vivo by appropriate hormones. The induction and maintenance of casein messages by a single hormonal treatment did not appear to correlate with morphology and DNA synthesis of cells in vitro or with tumor-producing capacities in vivo.