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PubMed2006

DNA damage evaluated by gammaH2AX foci formation by a selective group of chemical/physical stressors.

It has been reported that the phosphorylated form of histone variant H2AX (gammaH2AX) plays an important role in the recruitment of DNA repair and checkpoint proteins to sites of DNA damage, particularly at double strand breaks (DSBs). Using gammaH2AX foci formation as an indicator for DNA damage, several chemicals/stress factors were chosen to assess their ability to induce gammaH2AX foci in a 24h time frame in a human amnion FL cell line. Two direct-acting genotoxins, methyl methanesulfonate (MMS) and N-ethyl-N-nitrosourea (ENU), can induce gammaH2AX foci formation in a time- and dose-dependent manner. Similarly, an indirect-acting genotoxin, benzo[a]pyrene (BP), also induced the formation of gammaH2AX foci in a time- and dose-dependent manner. Another indirect genotoxin, 2-acetyl-aminofluorene (AAF), did not induce gammaH2AX foci formation in FL cells; however, AAF can induce gammaH2AX foci formation in Chinese hamster CHL cells. Neutral comet assays also revealed the induction of DNA strand breaks by these agents. In contrast, epigenetic carcinogens azathioprine and cyclosporine A, as well as non-carcinogen dimethyl sulfoxide, did not induce gammaH2AX foci formation in FL cells. In addition, heat shock and hypertonic saline did not induce gammaH2AX foci. Cell survival analyses indicated that the induction of gammaH2AX is not correlated with the cytotoxic effects of these agents/factors. Taken together, these results suggest that gammaH2AX foci formation could be used for evaluating DNA damage; however, the different cell types used may play an important role in determining gammaH2AX foci formation induced by a specific agent.

2-Acetylaminofluorene↗

PubMed2003

Regulation by nectin of the velocity of the formation of adherens junctions and tight junctions.

Cadherins are key Ca(2+)-dependent cell-cell adhesion molecules at adherens junctions (AJs) in fibroblasts and epithelial cells, whereas claudins are key Ca(2+)-independent cell-cell adhesion molecules at tight junctions (TJs) in epithelial cells. The formation and maintenance of TJs are dependent on the formation and maintenance of AJs. Nectins are Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules which comprise a family of four members, nectin-1, -2, -3, and -4, and are involved in the formation of AJs in cooperation with cadherins, and the subsequent formation of TJs. We show here that the velocity of the formation of the E-cadherin-based AJs is increased by overexpression of nectin-1 and is reduced by addition of the nectin-1 inhibitors to the medium in L cells stably expressing E-cadherin and Madin-Darby canine kidney cells. Moreover, the velocity of the formation of the claudin-based TJs is increased by overexpression of nectin-1 and is reduced by addition of the nectin-1 inhibitors to the medium in Madin-Darby canine kidney cells. These results indicate that nectins regulate the velocity of the formation of the E-cadherin-based AJs and the subsequent formation of the claudin-based TJs.

Adherens Junctions↗

PubMed2003

Hemozoin formation in malaria: a two-step process involving histidine-rich proteins and lipids.

Major blood stage antimalarial drugs like chloroquine and artemisinin target the heme detoxification process of the malaria parasite. Hemozoin formation reactions in vitro using the Plasmodium falciparum histidine-rich protein-2 (Pfhrp-2), lipids, and auto-catalysis are slow and could not explain the speed of detoxification needed for parasite survival. Here, we show that malarial hemozoin formation is a coordinated two component process involving both lipids and histidine-rich proteins. Hemozoin formation efficiency in vitro is 1-2% with Pfhrp-2 and 0.25-0.5% with lipids. We added lipids after 9h in a 12h Pfhrp-2 mediated reaction that resulted in sixfold increase in hemozoin formation. However, a lipid mediated reaction in which Pfhrp-2 was added after 9h produced only twofold increase in hemozoin production compared to the reaction with Pfhrp-2 alone. Synthetic peptides corresponding to the Pfhrp-2 heme binding sequences, based on repeats of AHHAAD, neither alone nor in combination with lipids were able to generate hemozoin in vitro. These results indicate that hemozoin formation in malaria parasite involves both the lipids and the scaffolding proteins. Histidine-rich proteins might facilitate hemozoin formation by binding with a large number of heme molecules, and facilitating the dimer formation involving iron-carboxylate bond between two heme molecules, and lipids may then subsequently assist the mechanism of long chain formation, held together by hydrogen bonds or through extensive networking of hydrogen bonds.

Acetone↗

PubMed1998

The glutathione dependence of inorganic sulfate formation from L- or D-cysteine in isolated rat hepatocytes.

The GSH dependence of the metabolic pathways involved in the conversion of cysteine to sulfate in intact cells has been investigated. It was found that hepatocyte-catalysed sulfate formation from added L-cysteine did not occur if hepatocyte GSH was depleted beforehand, but was restored when GSH levels recovered. Furthermore, sulfate formation did not recover in GSH-depleted hepatocytes if GSH synthesis was prevented with buthionine sulfoximine. Thiosulfate formation was, however, markedly enhanced in GSH-depleted hepatocytes. These results suggest that thiosulfate is an intermediate in the formation of inorganic sulfate from L-cysteine and that GSH was required for the conversion of thiosulfate to inorganic sulfate. Much less sulfate was formed if the cysteine was replaced with cysteinesulfinate. Furthermore, sulfate formation from L-cysteine was markedly inhibited by the addition of the transaminase inhibitor DL-cycloserine or the gamma-cystathionase inhibitor DL-propargylglycine. The major routes of sulfate formation from L-cysteine therefore seems to involve pathways that do not involve L-cysteinesulfinate. Similar amounts of sulfate were formed from D-cysteine as L-cysteine. Thiosulfate instead of sulfate was also formed in GSH-depleted hepatocytes. However, sulfate formation from D-cysteine differed from L-cysteine in that it was inhibited by the D-aminoacid oxidase inhibitor sodium benzoate and was not affected by transaminase or gamma-cystathionase inhibitors. These results suggest that thiosulfate is an intermediate in sulfate formation from D-cysteine and involves the oxidation of D-cysteine by D-amino acid oxidase to form beta-mercaptopyruvate.

Alkynes↗

PubMed2002

Chymase inhibitors may prevent postoperative adhesion formation.

OBJECTIVE: To clarify the role of chymase produced from mast cells in adhesion formation, we measured chymase activity level and investigated the preventive effect of a chymase inhibitor, Suc-Val-Pro-Phe(p)(OPh)(2), on the postoperative adhesion formation. DESIGN: Prospective randomized study using a surgical model for adhesion formation. SETTING: Clean hamsters in an academic research environment. ANIMAL(S): Sixty-seven female Syrian hamsters. INTERVENTION(S): Hamsters were given a lesion, produced by uterus scraping, and the chymase inhibitor (10 microM) or placebo was injected into the abdomen. Chymase activities in uteri were measured 3 days after the operation, and the scores of adhesion formations were assessed at 2 weeks. MAIN OUTCOME MEASURE(S): Measurement of chymase activity and scoring of adhesion formation were performed. RESULT(S): A significant increase of chymase activity in the injured uterus reduced by treatment with the chymase inhibitor. The scores of adhesion formations in the chymase inhibitor-treated group were significantly decreased in comparison with those in the placebo-treated group. CONCLUSION(S): Chymase contained in mast cells plays an important role in adhesion formation, and a chymase inhibitor may be a useful drug for prevention of adhesion formation.

Animals↗

PubMed1998

Nitric oxide, prostacyclin and cyclic nucleotide formation in externally stented porcine vein grafts.

Non-restrictive, porous, external stents inhibit neointima formation in porcine vein grafts. Since the mechanisms underlying these effects are unknown we investigated the impact of this external stent on factors known to inhibit vascular smooth muscle cell proliferation: prostacyclin (PGI2), nitric oxide (NO), cAMP and cGMP formation in different regions of stented and unstented porcine vein grafts. Paired stented and unstented saphenous vein-carotid artery interposition grafting was carried out in Landrace pigs. One month after surgery, the vessels were excised and the formation of PGI2, cAMP and cGMP determined using radioimmunoassay and nitric oxide synthase (NOS) distribution studied using autoradiography and histochemistry. There were no significant differences between PGI2, cAMP and cGMP (nitroprusside-stimulated) formation in the medial/intimal regions of grafts of stented vein graft and ungrafted saphenous vein whereas all were significantly reduced in unstented vein graft. A23187-stimulated cGMP formation (mediated by NO release) and NOS content was significantly greater in the medial/intimal region of stented and unstented vein graft compared to ungrafted saphenous vein, indicating induction of endothelial NOS (eNOS) in both types of graft. This normalisation of the PGI2-cAMP axis and guanylyl cyclase activity in the medial/intimal region may contribute to the beneficial impact of the external stent on vein graft thickening. The increase in eNOS in both stented and unstented vein grafts mitigates against this isoform as playing a role in mediating the inhibitory effect of the stent on neointima formation. In the adventitia of both stented and unstented grafts there was an increase in PGI2, cAMP and cGMP formation compared to ungrafted saphenous vein, the production being greater in the stented compared to the unstented graft. In the adventitia of stented veini grafts, NOS, detected with NAPDH diaphorase staining, was associated with microvessels as well as with inflammatory cells. Taken together, these data are suggestive of a role for PGI2 and NO in promoting microangiogenesis in the adventitia of stented vein grafts which may in turn minimize graft hypoxia, an established contributory factor to neointima formation.

Animals↗

PubMed2002

An Insight into the pathway of the amyloid fibril formation of hen egg white lysozyme obtained from a small-angle X-ray and neutron scattering study.

It is known that hen egg white lysozyme (HEWL) forms amyloid fibrils. Since HEWL is one of the proteins that have been studied most extensively and is closely related to human lysozyme, the variants of which form the amyloid fibrils that are related to hereditary systemic amyloidosis, this protein is an ideal model to study the mechanism of amyloid fibril formation. In order to gain an insight into the mechanism of amyloid fibril formation, systematic and detailed studies to detect and characterize various structural states of HEWL were conducted. Since HEWL forms amyloid fibrils in highly concentrated ethanol solutions, solutions of various concentrations of HEWL in various concentrations of ethanol were prepared, and the structures of HEWL in these solutions were investigated by small-angle X-ray and neutron scattering. It was shown that the structural states of HEWL were distinguished as the monomer state, the state of the dimer formation, the state of the protofilament formation, the protofilament state, and the state towards the formation of amyloid fibrils. A phase diagram of these structural states was obtained as a function of protein, water and ethanol concentrations. It was found that under the monomer state the structural changes of HEWL were not gross changes in shape but local conformational changes, and the dimers, formed by the association at the end of the long axis of HEWL, had an elongated shape. Circular dichroism measurements showed that the large changes in the secondary structures of HEWL occurred during dimer formation. The protofilaments were formed by stacking of the dimers with their long axis (nearly) perpendicular to and rotated around the protofilament axis to form a helical structure. These protofilaments were characterized by their radius of gyration of the cross-section of 2.4nm and the mass per unit length of 16,000(+/-2300)Da/nm. It was shown that the changes of the structural states towards the amyloid fibril formation occurred via lateral association of the protofilaments. A pathway of the amyloid fibril formation of HEWL was proposed from these results.

Amyloid↗

PubMed1995

Analysis of the kinetics of peritoneal adhesion formation in the rat and evaluation of potential antiadhesive agents.

BACKGROUND: Peritoneal adhesions continue to be a significant cause of postoperative complications. Elucidating the origin of these adhesions has been hampered by the lack of a reproducible animal model. The purpose of this study was to create a standardized model in which a single, specific adhesion could be objectively measured. With this model the kinetics of adhesion formation were then evaluated. A variety of potential antiadhesive agents were then tested and compared. METHODS: In this study a reproducible, quantitative rat model was developed that used uniform defects on the peritoneal wall and cecal surface. The resulting adhesions were subsequently scored, and their strength was measured with a tensiometer. An evaluation of the kinetics of peritoneal adhesion formation was obtained by using a timed removal of silicone elastomer sheeting held between the two injured surfaces. The following antiadhesive agents were evaluated: Ringer's lactate solution; dextran 70 (32%); modified carboxymethylcellulose (1.0% and 2.0%); an absorbable barrier of specially knitted material composed of oxidized regenerated cellulose; fibrin sealant; silicone elastomer film; and expanded polytetrafluoroethylene membrane. RESULTS: Evaluation of the kinetics of peritoneal adhesion formation indicated that the susceptibility for adhesion formation was significantly decreased or eliminated after the first 36 hours. Evaluation of antiadhesion agents indicated that the magnitude of adhesion prevention was directly proportional to the agent's ability to remain at the site of injury during the critical period of adhesion formation. Permanent barriers (silicone elastomer film, expanded polytetrafluoroethylene membrane) provided the greatest antiadhesion effect but were not believed to be ideal agents because they remained at the site of injury well after the critical period of adhesion formation. The incidence of adhesion formation for the other agents was as follows: control (34 of 34), Ringer's lactate (12 of 12), absorbable barrier of knitted cellulose (10 of 10), 32% dextran 70 (8 of 12), 1% carboxymethylcellulose (6 of 12), fibrin sealant (4 of 9), and 2% carboxymethylcellulose (4 of 12). CONCLUSIONS: The efficacy of antiadhesion agents appears to be related to the agent's viscosity, ability to coat the wound surface, and residence time at the site of injury. In this rat model an agent that remained on the injured surfaces for at least 36 hours after injury appeared to be more effective in reducing adhesion formation than an agent with a shorter residence time.

Animals↗

PubMed2002

Chlorination of natural organic matter: kinetics of chlorination and of THM formation.

The kinetics of the formation of trihalomethanes (THMs) and of chlorine consumption for the chlorination of natural organic matter with an excess of chlorine (50 microM > [Cl2]o >210 microM) was investigated. THM precursors could be divided into a fast and a slowly reacting fraction. Long term chlorine demand and the formation of THM could be described by second order kinetics. Rate constants were between 0.01 and 0.03 M(-1) s(-1) in the pH range 7-9 for surface waters and humic materials extracted from surface waters. A groundwater gave a higher rate constant of 0.124 M(-1) s(-1). Resorcinol-type structures were tested with respect to kinetics and yield of THM formation. They could possibly be responsible for the fast reacting THM precursors. which represent 15-30% of the THM precursors of natural waters. Additional classes of compounds that might contribute to the initial THM formation include readily enolizable compounds such as beta-diketones and beta-ketoacids. Experiments with phenol showed that slowly reacting THM precursors may consist of phenolic compounds. The influence of pretreatments (UV/visible irradiation, ozone and chlorine dioxide) on chlorine demand and THM formation from NOM was also studied: UV/visible irradiation does not alter THM formation but leads to a higher chlorine demand. Preoxidation with ozone leads to a lower THM formation with an unaltered chlorine demand and preoxidation with chlorine dioxide reduces THM formation and the chlorine demand.

Chlorine Compounds↗

PubMed1998

Shear-induced platelet activation and platelet microparticle formation in native human blood.

Shear-induced platelet activation and platelet microparticle formation are triggered in native human blood by high arterial shear or by a sudden increase in shear as introduced by a stenosis with potential consequences for collagen-induced platelet thrombus formation. Blood was drawn from healthy volunteers and directly perfused ex vivo over various well-defined eccentric stenoses. Shear-induced platelet activation was determined by using flow cytometry to assess: 1) GPIIb-IIIa activation by fluorescein isothiocyanate (FITC)-labeled Mab PAC-1; and 2) translocation of membrane aminophospholipids (procoagulant activity) by FITC-labeled Annexin V. Microparticle formation was measured by flow cytometry and FITC-labeled Mab Y2/51 directed against GPIIIa. Significant platelet activation and platelet microparticle formation were elicited when the wall shear rate reached 10,500 sec-1 for a period of 0.075 sec. Prolonged exposure to or a rapid increase in shear further enhanced activation and microparticle formation. Shear-induced platelet activation was associated with significantly increased collagen-induced platelet thrombus formation that was insensitive to aspirin ingestion. Exposure of native blood to very high shear thus activates platelets to express GPIIb-IIIa, renders the platelet membrane procoagulant and stimulates microparticle formation. These responses are associated with enhanced collagen-induced thrombus formation by prostaglandin-independent mechanisms.

Adult↗

PubMed1993

Induction of bone formation in rat tail vertebrae by mechanical loading.

We have developed an experimental model in which pins, inserted into the seventh and ninth caudal vertebrae of 13-week-old rats, are used to load the eighth caudal vertebra in compression. Four groups of animals were used in the study: unpinned; animals with pins inserted, but non-loaded; animals loaded once, for 360 cycles at 0.5 Hz; and animals subjected to daily loading for 36 cycles at 0.5 Hz. Pins were immobilised by clamps when not undergoing loading. The animals were killed 9 days after pinning, and the eighth caudal vertebra was subjected to histomorphometric and histodynamic analysis. We found that vertebrae subjected to 36 daily loading cycles showed a 30-fold increase in bone formation compared to non-loaded controls. A single loading regime of 360 cycles was sufficient to increase bone formation 4-fold. Bone formation on trabecular surfaces was of lamellar rather than woven bone and was accompanied by a decrease in indices of bone resorption. Loaded vertebrae also showed substantial periosteal woven bone formation, although a minor degree of periosteal woven bone formation was also seen in one non-loaded pinned control vertebra. Our results suggest that in the rat, as in avian species, short loading regimes are capable of inducing bone formation. The model may assist an analysis of the interactions between bone resorption, bone formation and mechanical stimuli, and may enable identification of the molecular signals that mediate induction of lamellar bone formation on trabecular surfaces.

Animals↗

PubMed2000

Inhibition of benzo[a]pyrene- and 1,6-dinitropyrene-DNA adduct formation in human mammary epithelial cells bydibenzoylmethane and sulforaphane.

Numerous phytochemicals have been examined for their capacity to act as cancer chemopreventive agents. Dibenzoylmethane, a minor constituent of licorice and a compound structurally-related to curcumin, recently was identified as an effective inhibitor of chemically-induced rat mammary DNA-adduct formation and tumorigenesis (Carcinogenesis 19(1998)1039-1043). The present studies were conducted to examine the capacity of dibenzoylmethane to inhibit the formation of DNA adducts following exposure to benzo[a]pyrene (BP) and 1,6-dinitropyrene (1,6-DNP), and to stimulate the expression of glutathione-S-transferase (GST) and NAD(P)H-quinone reductase (QR) proteins in the human mammary epithelial cell line MCF-10F. In addition, the efficacy of dibenzoylmethane as an enzyme inducer and adduct inhibitor was compared with that of sulforaphane, a potent inducer of phase II detoxification enzymes and inhibitor of chemically-induced rat mammary tumorigenesis. Dibenzoylmethane at concentrations from 0.1 M to 2.0 microM inhibited BP-DNA adduct formation by 63 to 81%. Likewise, sulforaphane inhibited BP-DNA adduct formation by 68 to 80% over the same concentration range. DNA adduct formation following exposure to 1,6-DNP was significantly inhibited by 46 to 61% due to dibenzoylmethane treatment (0.1 to 2.0 microM) and 30 to 56% due to sulforaphane treatment at the same concentrations. The expression of QR and GSTP1-1 proteins were increased by 3 to 4-fold and 3 to 5-fold, respectively, for MCF-10F cells treated with sulforaphane (0.5-2.0 microM). Dibenzoylmethane treatment at the same concentrations did not induce GSTP1-1 expression and significantly stimulated QR expression only at the 2.0 microM concentration. These data indicate that human mammary epithelial MCF-10F cells can convert BP and 1,6-DNP to DNA-binding forms, and that DNA adduct formation can be inhibited by the phytochemicals dibenzoylmethane and sulforaphane. The inhibition of BP-DNA and 1, 6-DNP adduct formation by sulforaphane was associated with increases in QR and GST protein expression. The mechanisms underlying the capacity of dibenzoylmethane to inhibit BP-DNA and 1,6-DNP-DNA adduct formation could not be explained by changes in QR or GST expression and remain to be determined. Together these data suggest that dibenzoylmethane and sulforaphane warrant continued evaluation as breast cancer chemopreventive agents.

Anticarcinogenic Agents↗

PubMed1998

Overlapping 3'-end formation signals and ARS elements: tightly linked but functionally separable.

3'-End formation signals are closely associated with autonomous replicating sequences (ARSs) in Saccharomyces cerevisiae in that ARSs frequently contain signals that direct 3'-end formation (Chen et al., 1996). Mutationally-inactivated ARSs that co-reside with 3'-end formation sequences do not disrupt 3'-end formation, thus demonstrating that replication function does not affect termination function. To test the corollary possibility that 3'-end formation is important for replication function, we made point mutations in ARS305 that increase readthrough of the 3'-end formation signals and determined plasmid replication efficiency. Replication efficiency, as assessed by plasmid stability assays, was not altered by mutations affecting 3'-end formation when transcription through the ARS was either absent or highly-induced. Under conditions of high-level transcription through the ARS, the rate of plasmid loss in both wild-type and mutated terminators increased over five-fold from rates observed during transcriptionally repressed conditions. This result indicates that the native 3'-end formation signal is incapable of protecting the replication function when high levels of transcription are directed into the ARS. Thus, the compact nature of the S. cerevisiae genome, rather than a functional inter-dependence, may account for close association of transcription terminators and ARSs.

Base Sequence↗

PubMed2000

Ethyl formate - alternative dispersed solvent useful in preparing PLGA microspheres.

In an effort to substitute methylene chloride with a less toxic solvent, this study was aimed at developing new ethyl formate-based emulsion processes to fabricate poly-D,L-lactide-co-glycolide (PLGA) microspheres. To do so, a polymeric dispersed phase was emulsified in a 1% polyvinyl alcohol aqueous solution at an ethyl formate to aqueous volume ratio of 8:20. Microsphere hardening was then achieved by solvent evaporation and quenching techniques. The average encapsulation efficiency of a model drug progesterone amounted to 95.2+/-2.7%. When the tendency of ethyl formate and methylene chloride to evaporate to air was compared, the evaporation rate of ethyl formate was 2.1 times faster than that of methylene chloride. The ease with which ethyl formate evaporated to air was beneficial in shortening the microsphere hardening step. For the solvent quenching process, only 80 ml of additional water was required to extract ethyl formate to the aqueous phase, due to its considerable water miscibility. In particular, the timing of ethyl formate quenching affected to a great extent dynamic processes of the breakup of elementary microdroplets into smaller ones. Therefore, variations in quenching time affected microsphere characteristics such as the degree of solvation, size distribution, and tendency to aggregate on drying. The results of this study showed that PLGA microspheres were successfully prepared using the new ethyl formate-based processes.

Chemistry, Pharmaceutical↗

PubMedDate not supplied

Actions of the endocrine disruptor methoxychlor and its estrogenic metabolite on in vitro embryonic rat seminiferous cord formation and perinatal testis growth.

The current study examines the actions of methoxychlor and its estrogenic metabolite, 2, 2-bis-(p-hydroxyphenyl)-1, 1, 1-trichloroethane (HPTE), on seminiferous cord formation and growth of the developing rat testis. The developing testis in the embryonic and early postnatal period is likely more sensitive to hormonally active agents than at later stages of development. Embryonic day 13 (E13) testis organ cultures were treated with either 0.2, 2, or 20 microM methoxychlor or 1, 3, 6, 15, 30, or 60 microM HPTE to examine effects on cord formation. No concentration of methoxychlor completely inhibited cord formation. However, cord formation was abnormal with the presence of a reduced number of cords and appearance of "swollen" cords at the 2 and 20 microM concentrations of methoxychlor. The swollen cords were due to an increase in the number of cells in a cord cross section and reduction of interstitial cell numbers between cords. Treatment of embryonic day 13 (E13) testes with HPTE caused abnormal cord formation at the 3 microM and 6 microM concentrations, and completely inhibited cord formation at the 15, 30, and 60 microM concentrations. In addition to the estrogenic metabolite HTPE, methoxychlor can also be metabolized into anti-androgenic compounds. Therefore, to determine the spectrum of potential actions of methoxychlor on testis development, different concentrations of estradiol, testosterone, and an anti-androgen (flutamide) were utilized to determine their effects on E13 testis organ culture morphology. Estradiol (1 microM) and flutamide (0.1microM) both inhibited seminiferous cord formation in E13 testis organ cultures. Therefore, methoxychlor may be acting through the androgen and/or estrogen receptors to elicit its actions on seminiferous cord formation. Reverse transcription polymerase chain reaction (PCR) (RT-PCR) confirmed the presence of estrogen receptor alpha (ERalpha) mRNA from embryonic day 14 (E14) through postnatal day 5 (P5) while estrogen receptor beta (ERbeta) mRNA did not appear until approximately E16 of testis development. Androgen receptor (AR) expression was present from E14 through P5 of testis development, but at apparently reduced levels at E14 and E16. Immunohistochemical analysis localized ERalpha to the cells of the seminiferous cords at E14 though P5 while ERbeta was present in cells of the interstitium at E16 and P0. Androgen receptor was localized to germ and interstitial cells. The effects of methoxychlor, HPTE, estradiol, and testosterone on cell growth of perinatal testes was determined with a thymidine incorporation assay in postnatal day zero (P0) testis cell cultures. Methoxychlor (0.002, 0.02, and 0.2 microM) and HPTE (2 and 20 microM) stimulated thymidine incorporation in P0 testis cell cultures in a similar manner to estradiol (0.01, 0.1, and 1 microM). In addition, testosterone (0.1 microM) also stimulated thymidine incorporation in P0 testis cultures. Observations suggest that methoxychlor and its metabolite HPTE can alter normal embryonic testis development and growth. The actions of methoxychlor and HPTE are likely mediated in part through the steroid receptors confirmed to be present in the developing testis.

Animals↗

PubMed1999

The regulation of oestrone sulphate formation in breast cancer cells.

The formation of oestrone sulphate has been examined in MCF-7 (oestrogen receptor positive, ER+) and MDA-MB-231 (ER negative, ER-) breast cancer cells. Using intact cell monolayers and a physiological substrate concentration, progesterone (1 microM) and dexamethasone (1 microM) both increased oestrone sulphate formation in MCF-7 cells. In MDA-MB-231 cells, dexamethasone, but not progesterone, increased conjugate formation. A number of growth factors, cytokines and human serum albumin (HSA), which have previously been found to regulate oestrogen synthesis, were also examined for their ability to regulate oestrone sulphate formation. In MCF-7 cells epidermal growth factor, acidic and basic fibroblast growth factors, insulin-like growth factor-type I and insulin all stimulated oestrone sulphate formation. The cytokines, tumour necrosis factor alpha (TNFalpha) and interleukin-1beta also increased conjugate formation in the ER+ cells, as did HSA. In contrast, in MDA-MB-231 cells TNFalpha was without effect and HSA inhibited oestrone sulphate formation. The ability to modulate oestrone sulphate formation in ER+ cells may be an important mechanism to limit the availability of oestrogen to interact with the ER.

Breast Neoplasms↗

PubMedDate not supplied

Thrombus organization plays no major role in late neointimal formation after angioplasty in porcine coronary arteries.

Thrombus organization has been suggested to play a major role in late neointimal formation after coronary angioplasty. We sought to describe the time sequence of lesion formation after angioplasty in porcine coronary arteries and to quantify the relation between early thrombosis and late neointimal formation. Deep vessel wall injury was induced by conventional balloon angioplasty in the circumflex (CX) and right coronary (RCA) arteries and by retraction of a chain-encircled balloon in the left anterior descendent artery (LAD). Lesions were assessed by histomorphometry at days 0, 1, 4, 7, 14, 28, and 56 after angioplasty. A response-to-injury index (lesion area/injury length) was determined for each artery. Angioplasty led to rupture/removal of media. Thrombus was present at the exposed adventitia at days 0, 1, and 4. From day 7, neointima was observed on the luminal side of the arterial wall. All thrombus had disappeared at day 28, at which only neointima was observed. Histomorphometry revealed that lesion formation after angioplasty was a gradually increasing process from day 0 to day 28 with no further growth from day 28 to day 56. Maximal thrombus size (day 4, RCA: 0.07+/-0.04 mm, CX: 0.23+/-0.16 mm, LAD: 0.15+/-0.11 mm) was significantly smaller than late neointimal formation (day 28, RCA: 0.68+/-0.18 mm, CX: 0.63+/-0.23 mm, LAD: 0.71+/-0.18 mm) in all three arteries (p < .03). Lesion formation after angioplasty is a gradually increasing process for 4 weeks. Maximal thrombus size is about four times smaller than late neointimal formation. Thus, thrombus organization plays no major role in late neointimal formation.

Angioplasty, Balloon, Coronary↗

PubMed2002

Regulating DeltaFosB expression in adult Tet-Off-DeltaFosB transgenic mice alters bone formation and bone mass.

The DeltaFosB isoforms are naturally occurring AP-1 family members that increase bone volume via a cell-autonomous effect on osteoblastic bone formation. Mice overexpressing DeltaFosB demonstrate a very high level of bone formation, resulting in a progressive osteosclerosis. Despite the linkage of bone formation and resorption in physiological systems, no alteration in bone resorption was detected in mice overexpressing DeltaFosB. To determine whether altering DeltaFosB expression can regulate bone formation independently of bone resorption in adult mice, we used the Tet-Off-inducible transgene system to induce or block transgenic DeltaFosB overexpression and thereby regulate bone formation in vivo. Overexpression of DeltaFosB after skeletal maturity increased trabecular bone volume by increasing bone formation, again without altering bone resorption, indicating that developmental DeltaFosB overexpression is not required for the osteosclerotic phenotype. Similarly, switching off DeltaFosB overexpression after osteosclerosis had developed led to a marked decrease in bone formation and loss of bone mass such that trabecular bone volume approached normal levels. Despite this dramatic reduction, no alteration in bone resorption was detected. These results clearly demonstrate that DeltaFosB regulates bone formation and bone mass in adult mice with no effect on bone resorption.

Animals↗