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J Glass

Publications and source records attributed to J Glass.

At least 19 recordsLinked to original sources
PubMed1992

Kinetic characterization of reductant dependent processes of iron mobilization from endocytic vesicles.

The reductant dependence of iron mobilization from isolated rabbit reticulocyte endosomes containing diferric transferrin is reported. The kinetic effects of acidification by a H(+)-ATPase are eliminated by incubating the endosomes at pH 6.0 in the presence of 15 microM FCCP to acidify the intravesicular milieu and to dissociate 59Fe(III) from transferrin. In the absence of reductants, iron is not released from the vesicles, and iron leakage is negligible. The second-order dependence of rate constants and amounts of 59Fe mobilized from endosomes using ascorbate, ferrocyanide, or NADH are consistent with reversible mechanisms. The estimated apparent first-order rate constant for mobilization by ascorbate is (2.7 +/- 0.4) x 10(-3) s-1 in contrast to (3.2 +/- 0.1) x 10(-4) s-1 for NADH and (3.5 +/- 0.6) x 10(-4) s-1 for ferrocyanide. These results support models where multiple reactions are involved in complex processes leading to iron transfer and membrane translocation. A type II NADH dehydrogenase (diaphorase) is present on the endosome outer membrane. The kinetics of extravesicular ferricyanide reduction indicate a bimolecular-bimolecular steady-state mechanism with substrate inhibition. Ferricyanide inhibition of 59Fe mobilization is not detected. Significant differences between mobilization and ferricyanide reduction kinetics indicate that the diaphorase is not involved in 59Fe(III) reduction. Sequential additions of NADH followed by ascorbate or vice versa indicate a minimum of two sites of 59Fe(III) residence; one site available to reducing equivalents from ascorbate and a different site available to NADH. Sequential additions using ferrocyanide and the other reductants suggest interactions among sites available for reduction. Inhibition of ascorbate-mediated mobilization by DCCD and enhancement of ferrocyanide and NADH-mediated mobilization suggest a role for a moiety with characteristics of a proton pore similar to that of the H(+)-ATPase. These data provide significant constraints on models of iron reduction, translocation, and mobilization by endocytic vesicles.

Animals↗
PubMed1992

Tempol, a stable free radical, is a novel murine radiation protector.

Nitroxide compounds are stable free radicals which were previously investigated as hypoxic cell radiosensitizers. The stable nitroxide 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (Tempol) has recently been shown to protect aerated cells in culture against superoxide generated from hypoxanthine/xanthine oxidase, hydrogen peroxide, and radiation-induced cytotoxicity and to modestly sensitive hypoxic cultured cells. To extend these observations from the cellular level to the whole animal, the toxicity, pharmacology, and in vivo radioprotective effects of Tempol were studied in C3H mice. The maximum tolerated dose of Tempol administered i.p. was found to be 275 mg/kg, which resulted in maximal Tempol levels in whole blood 5-10 min after injection. Mice were exposed to whole-body radiation in the absence or presence of injected Tempol (275 mg/kg) 5-10 min after administration. Tempol treatment provided significant radioprotection (P less than 0.0001); the dose of radiation at which 50% of Tempol-treated mice die at 30 days was 9.97 Gy, versus 7.84 Gy for control mice. Tempol represents a new class of in vivo, non-sulfur-containing radiation protectors. Given the potential for hypoxic radiosensitization and aerobic cell radioprotection, Temporal or other analogues may have potential therapeutic application.

Animals↗
PubMed1992

Measurement of thymidine replacement in patients with high grade gliomas, head and neck tumors, and high grade sarcomas after continuous intravenous infusions of 5-iododeoxyuridine.

Based upon the radiation sensitization properties of the halogenated pyrimidines, 5-iododeoxyuridine (IdUrd) and 5-bromodeoxyuridine, long term i.v. infusions of halogenated pyrimidines in conjunction with fractionated radiation therapy have been evaluated in the treatment of a variety of human malignancies. While clinical studies have attempted to measure the halogenated pyrimidine incorporation, few have successfully related tumor response to the incorporation of IdUrd by the tumor. The present study reports the continuous IdUrd labeling index (number of cells labeled) and the IdUrd corrected replacement (percentage of thymidine replacement in the labeled cells of the population) from the tumors of 17 patients who received continuous infusions of IdUrd (1000 mg/m2/24 h). The tumors treated included four high grade gliomas, five head and neck tumors, four high grade sarcomas, and five other tumors of varying types. Less than 25% of the cells in three of four gliomas incorporated IdUrd after 5-7-day IdUrd infusion time. Corrected replacement for the gliomas ranged from 0 to 4%. In contrast, 63-85% of the cells in the head and neck biopsies were labeled with IdUrd after 3-7-day IdUrd infusions suggesting that these large tumors (3-12 cm diameter) have a high fraction of dividing cells. Corrected replacements values for the head and neck tumor patients ranged from 2.9 to 26.3%. The high grade sarcomas also demonstrated a high percentage of IdUrd labeled cells (57-79%) with three patients having corrected replacements of 7.5-14.2%. The continuous labeling and thymidine replacement data for four patients from whom serial biopsies were taken during IdUrd infusion demonstrated both an increasing IdUrd replacement and continuous labeling index with an increasing duration of IdUrd infusion. The clinical response of both the high grade glioma and head and neck tumor patients indicate that the IdUrd replacement and labeling data may provide some important predictive information with regard to the successful use of the halogenated pyrimidines in clinical radiation trials.

Animals↗
PubMed1992

Topical application of nitroxide protects radiation-induced alopecia in guinea pigs.

We have recently found that treatment of Chinese hamster V79 cells with the stable nitroxide radical TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) afforded significant protection against superoxide, hydrogen peroxide, and X-ray mediated cytotoxicity. Radiation-induced alopecia is a common radiotherapeutic problem. Topical application of TEMPOL was evaluated for possible protective effects against radiation-induced alopecia using guinea pig skin as a model. For single acute X-ray doses up to 30 Gy, TEMPOL, when topically applied 15 min prior to irradiation provided a marked increase in the rate and extent of new hair recovery when compared to untreated skin. TEMPOL was detected in treated skin specimens with electron paramagnetic resonance (EPR) spectroscopy. Similar measurements of blood samples failed to show any signal resulting from topical application, nor could TEMPOL be detected in brain tissue after application on the scalp. TEMPOL represents a new class of compounds with potential for selective cutaneous radioprotection without systemic absorption.

Administration, Topical↗
PubMed1992

The mechanisms of nonheme iron uptake determined in IEC-6 rat intestinal cells.

The mechanisms of iron absorption, which control total body iron stores, are not fully understood. We have defined an in vitro model using rat small intestinal cells (IEC-6) to study nutrient uptake by the intestine and have characterized the mechanisms of nonheme iron uptake in this model. IEC-6 cells were grown to confluency, and then 59Fe solutions were layered on the cells, allowing iron uptake to occur through the apical surface. Iron uptake increased over time and comparable results were seen with either 59Fe(II) or 59Fe(III). Two types of 59Fe(II) binding sites were detected, a high affinity site with a dissociation constant of 1 x 10(-8) mol/L and 5 x 10(5) sites per cell, and a lower affinity site with a dissociation constant of 2 x 10(-6) mol/L and 2.6 x 10(6) sites per cell. The 59Fe uptake was inhibited by low temperature, low and high pH, prior iron loading of the cells, high osmotic concentrations, and by N,N'-dicyclohexylcarbodiimide, tannic acid, bathophenanthroline and colchicine. Uptake was not inhibited by vinblastine or verapamil. Transferrin mRNA was not detected in IEC-6 cells grown under normal growth conditions but could be induced under some other conditions. IEC-6 cell proteins that bind iron were detected in a subcellular vesicle fraction having estimated molecular weights of 120,000, 95,000, 55,000 and 35,000. These studies confirm iron uptake studies by other models and demonstrate that IEC-6 cells possess iron-binding moieties, a regulated iron uptake process, and little or no transcription of the transferrin gene under normal growth conditions.

Animals↗
PubMed1992

The treatment of oligodendrogliomas and mixed oligodendroglioma-astrocytomas with PCV chemotherapy.

Malignant oligodendrogliomas have been shown to be responsive to chemotherapy. The authors administered systemic chemotherapy to seven patients with oligodendroglioma or anaplastic oligodendroglioma, and to 14 with mixed oligodendroglioma-astrocytoma. Fourteen patients underwent chemotherapy before and seven after irradiation. The PCV (procarbazine, methyl-1-(2-chloroethyl)-1-nitrosourea (CCNU), and vincristine) chemotherapy was administered every 6 weeks (42-day cycles) for two to five cycles as follows: CCNU, 110 mg/sq m on Day 1; procarbazine, 60 mg/sq m/day on Days 8 to 21; and vincristine, 1.4 mg/sq m/day on Days 8 and 29. Complete or partial (greater than 50% reduction in tumor mass) responses at 20 to 100+ weeks after treatment were noted in 11 (79%) of the 14 patients treated before irradiation, including two with anaplastic oligodendroglioma and nine with mixed tumors. Complete responses were seen in two patients, one with anaplastic oligodendroglioma and one with a mixed tumor. Partial responses were seen in three of seven patients treated after radiotherapy. Stabilization of tumor growth followed PCV chemotherapy in four patients (two treated before and two after radiotherapy). Tumor growth progressed in two patients during therapy despite an initial response and in two patients despite therapy. The authors conclude that mixed oligodendroglial tumors as well as anaplastic oligodendrogliomas are responsive to PCV chemotherapy.

Adult↗
PubMed1992

Non-transferrin dependent 59Fe uptake in phytohemagglutinin-stimulated human peripheral lymphocytes.

Human peripheral lymphocytes stimulated by phytohemagglutinin (PHA) have been used to demonstrate the characteristics of iron uptake from non-transferrin iron donors. When incubated with 59Fe(II)-ascorbate or 59Fe(III)-nitrilotriacetate (Fe-NTA), stimulated lymphocytes (2 micrograms/ml PHA) showed a tenfold increase in uptake of 59Fe as compared with the resting cells. The uptake of 59Fe from these iron donors was time and concentration dependent, showed saturation kinetics, and was not influenced by the addition of a tenfold excess of unlabeled Fe(III)2-transferrin (Fe-Tf). The amount of 59Fe accumulated from 59Fe-NTA was about one-third of that from 59Fe2-Tf, whereas the uptake of 59Fe from 59Fe-ascorbate was about tenfold higher. When stimulated with varying doses of PHA, lymphocytes showed maximal uptake of 59Fe from 59Fe2-Tf at the concentration of PHA (1 microgram/ml) optimal for 3H-thymidine incorporation. Lymphocytes stimulated with supraoptimal concentrations (5-20 micrograms/ml) of PHA showed slightly less iron uptake from 59Fe-Tf and had a longer transferrin cycle time, as compared with the cells under optimal stimulation. In contrast, the uptake of 59Fe from non-transferrin 59Fe donors in cells stimulated with 5-20 micrograms/ml PHA was greater than that in cells grown with 1 or 2 micrograms/ml PHA. It is suggested that lymphocytes take up iron from non-transferrin iron donors by processes different from the iron uptake pathway used by transferrin.

Biological Transport↗
PubMed1991

Caco-2 cell line: a system for studying intestinal iron transport across epithelial cell monolayers.

Iron transport across polarized intestinal epithelium was studied by using Caco-2 cells grown in bicameral chambers. When cells were grown under conditions of low, normal, or high iron concentration not only was the iron content of the cells markedly altered but the low iron cells exhibited a nearly 2-fold increase in transepithelial electrical resistance (TEER). 59Fe uptake from the apical surface into cells and transport into the basal chamber was affected both by the valency of the iron and the iron status of the cells. Uptake from 59Fe(II)-ascorbate was about 600 pmol 59Fe/h per mg protein, increased about 2-fold in low iron cells, and was about 13-200-fold greater than uptakes from 59Fe(III) chelated to nitrilotriacetic acid, BSA, or citrate. Transport into the basal chamber from 59Fe(II)-ascorbate was 3.7 +/- 1.7 pmol/h per cm2 for Fe-deficient cells vs. 0.72 +/- 0.1 pmol/h per cm2 for normal-Fe cells and from 59Fe(III)-BSA 1.1 +/- 0.2 pmol/h per cm2 vs. 0.3 +/- 0.03 pmol/h per cm2 for deficient vs. normal iron cells, respectively. The greater transport of iron both from Fe(II) and in iron deficient cells supports the use of the Caco-2 cells as a model for iron transport.

Biological Transport↗
PubMed1991

Congenital myopathy with oculo-facial abnormalities (Marden-Walker syndrome).

Marden-Walker syndrome present in the neonatal period is characterized by oculo-facial abnormalities, congenital myopathy, and contractures. A newborn infant with a similar pattern of anomalies is reported, and further evidence for autosomal recessive inheritance as well as new neuropathological findings are included.

Contracture↗
PubMed1991

Abrogation of interferon-induced resistance to interferon-activated major histocompatibility complex-unrestricted killers by treatment of a melanoma cell line with 5-fluorouracil.

Advanced cancer responds clinically to combined therapy with recombinant interferon-alpha and 5-fluorouracil. Although the two agents may interact in the biosynthetic pathway for thymidine, we investigated, as an alternative mechanism, the regulation of susceptibility of the A375 human melanoma to natural killers activated by interferon. A375 were preincubated with 5-fluorouracil, interferon, or both sequentially prior to assay as targets for cell-mediated killing. Pretreatment of A375 with interferon decreased apparent lytic efficiency. 5-Fluorouracil alone increased the susceptibility of A375 to killing. Pretreatment of targets with 5-fluorouracil abrogated the resistance normally induced by interferon pretreatment. Thus, 5-fluorouracil modulates certain immunoregulatory effects of interferon-alpha. Thymidine does not block the effect of 5-fluorouracil. While fluorodeoxyuridine is relatively ineffective in this system, fluorouridine is more effective than 5-fluorouracil in abrogating the effect of interferon. These data suggest important interactions of 5-fluorouracil and interferon in pathways for protein synthesis. It is known that interferon both increases the activity of natural killers and increases resistance of tumors to natural killers. We have shown that 5-fluorouracil, by blocking the resistance, may allow the augmented natural killing to be effective. This observation provides an alternate hypothesis for the clinical activity of 5-fluorouracil and interferon in combination.

Adult↗
PubMed1991

Cl-, Na+, and H+ fluxes during the acidification of rabbit reticulocyte endocytic vesicles.

The ionic fluxes associated with the ATP-dependent acidification of endocytic vesicles were studied in a preparation isolated from rabbit reticulocytes enriched for transferrin-transferrin receptor complexes. No vesicle acidification was observed in the absence of intra- and extravesicular ions (sucrose(in)/sucrose(out), while maximal acidification was observed with NaCl(in)/KCl(out).K+(in) was a poor substitute for Na+(in), and Cl-(out) could be replaced by other anions with the following efficacy of acidification: Cl- greater than Br- greater than I- greater than PO4(3-) greater than gluconate greater than SO4(2-). Flux studies using 36Cl- and 22Na+ showed that the vesicles had a permeability for Cl- and Na+, and that ATP-dependent H+ pumping was accompanied by a net influx of Cl- and a net efflux of Na+ provided that there was a Na+ concentration gradient. After 3 mins, the time necessary to maximal acidification, the electrical charge generated by the entrance of H+ was countered to about 45% by the Cl- influx and to about 42% by the Na+ efflux. These studies demonstrated that both Cl- and Na+ fluxes are necessary for optimal endocytic vesicle acidification.

Adenosine Triphosphatases↗
PubMed1991

Lead, cadmium, and aluminum accumulation in the red swamp crayfish Procambarus clarkii G. collected from roadside drainage ditches in Louisiana.

The concentration of Pb, Cd, and Al in tissues of crayfish Procambarus clarkii were evaluated from several wetland sites located adjacent to roadways and were compared to crayfish harvested from a commercial site free from roadside influences. Abdominal muscle, hepatopancreas, alimentary tract, exoskeleton and blood were analyzed for metal content. Results indicated that levels of contamination obtained in almost all tissues of crayfish from roadside ditches contained significantly higher amounts of metals than those of the commercially harvested control crayfish (p = less than or equal to .05-.001). Detection limits of Pb, Cd, and Al ranged from 0.04 microgram Pb/g to 16.15 micrograms Pb/g, .001 microgram Cd/g to .13 microgram Cd/g, and 1.22 micrograms Al/g to 981 micrograms Al/g, respectively. Concentrations of Pb, Cd, and Al were highest in the hepatopancreas and alimentary tract. High levels of these elements were also detected in the exoskeleton. In contrast, muscle tissue was the least affected tissue. Several significant correlations among concentrations of metals were found when comparing a variety of tissues in Procambarus clarkii.

Aluminum↗
PubMed1991

Kinetics of iron passage through subcellular compartments of rabbit reticulocytes.

The kinetics of the separate processes of Fe2(III)-transferrin binding to the transferrin receptor, transferrin-receptor internalization, iron dissociation from transferrin, iron passage through the membrane, and iron mobilization into the cytoplasm were studied by pulse-chase experiments using rabbit reticulocytes and 59Fe, 125I-labeled rabbit transferrin. The binding of 59Fe-transferrin to transferrin receptors was rapid with an apparent rate constant of 2 x 10(5) M-1 sec-1. The rate of internalization of 59Fe-transferrin was directly measured at 520 +/- 100 molecules of Fe2(III)-transferrin internalized/sec/cell with 250 +/- 43 sec needed to internalize the entire complement of reticulocyte transferrin receptors. Subsequent to Fe2(III)-transferrin internalization the flux of 59Fe was followed through three compartments: internalized transferrin, membrane, and cytosol. A process preceding iron dissociation from transferrin and a reaction involving membrane-associated iron required 17 +/- 2 sec and 34 +/- 5 sec, respectively. Apparent rate constants of 0.0075 +/- 0.002 sec-1 and 0.0343 +/- 0.0118 sec-1 were obtained for iron dissociation from transferrin and iron mobilization into the cytosol, respectively. Iron dissociation from transferrin is the rate-limiting step. An apparent rate constant of 0.0112 +/- 0.0025 sec-1 was obtained for processes involving iron transport through the membrane although at least two reactions are likely to be involved. Based on mechanistic considerations, iron transport through the membrane may be attributed to an iron reduction step followed by a translocation step. These data indicate that the uptake of iron in reticulocytes is a sequential process, with steps after the internalization of Fe2(III)-transferrin that are distinct from the handling of transferrin.

Animals↗
PubMed1991

Transforming growth factor-beta improves healing of radiation-impaired wounds.

Exogenously applied TGF-beta 1 has been shown to increase wound strength in incisional wounds early in the healing process. An impaired wound healing model was first established in guinea pigs by isolating flaps of skin and irradiating the flaps to 15 Gray in one fraction using a 4-MeV linear accelerator. Incisions made 2 d after irradiation were excised 7 d later, and showed decreased linear wound bursting strength (WBS) as compared to non-irradiated control wounds on the contralateral side of each animal (p = 0.001). The effect of TGF-beta on healing of radiation-impaired wounds was studied using this model. Skin on both left and right sides of guinea pigs was irradiated as above. A linear incision was made in each side. Collagen with either 1, 5, or 20 micrograms of TGF-beta was applied to one side prior to closure with staples, whereas the contralateral side received saline in collagen. Wounds given either 1 or 5 micrograms of TGF-beta were found to be stronger than controls at 7 d (p less than 0.05), whereas those receiving the higher 20-micrograms dose were weaker than controls (p less than 0.05). Thus, TGF-beta in lower doses improved healing at 7 d but very large amounts of the growth factor actually impaired healing. In situ hybridization done on wound samples showed increased type I collagen gene expression by fibroblasts in wounds treated with 1 micrograms TGF-beta over control wounds. These results indicate that TGF-beta improved wound healing as demonstrated by increased WBS. This improvement is accompanied by an up-regulation of collagen gene expression by resident fibroblasts.

Animals↗
PubMed1991

Aluminum uptake and toxicity in cultured mouse hepatocytes.

Hepatic aluminum (Al) accumulation in association with hepatobiliary dysfunction has been described in children receiving contaminated parenteral alimentation solutions and in aluminum-overloaded experimental animals. The mechanisms of hepatic Al uptake are not clearly understood, and it is not known whether Al is directly toxic to the hepatic cell or if toxicity occurs from the effect of Al on hepatic iron (Fe) metabolism. Al causes a microcytic hypochromic anemia and concomitant hepatic Al and Fe can accumulate in dialysis patients, suggesting that Al may alter Fe metabolism. Therefore, Al uptake and toxicity were studied in mouse hepatocytes in culture. Al accumulation, cell growth, media hepatic enzyme concentrations, and cell malonyldialdehyde concentrations, a marker of membrane lipid peroxidation, were measured in mouse hepatocytes grown in media containing either Al citrate, transferrin-Al (Tf-Al), or no additions over 96 h. Al uptake occurred only in cells grown in Tf-Al and Al citrate at 24 h and increased linearly achieving cellular concentrations at 96 h of 522 +/- 36 and 186 +/- 12 micrograms/L, respectively, compared with 31 +/- 3 micrograms/L (P less than 0.001) in control media. Inhibition of cell growth occurred at 48, 72, and 96 h (P less than 0.001), and media lactate dehydrogenase and aspartate aminotransferase concentrations increased starting at 48 and 72 h, respectively (P less than 0.001), only in media containing Tf-Al. Cell malonyldialdehyde levels were significantly higher in Tf-Al-loaded mouse hepatocytes compared with control cells at 96 h (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Aluminum↗
PubMed1990

Translational control during the acute phase response. Ferritin synthesis in response to interleukin-1.

Interleukin-1 (IL-1 beta) increases the synthesis of both heavy and light (L)-ferritin subunits when added to human hepatoma cells (HepG2) grown in culture. RNase protection and Northern blot analysis with L-ferritin probes revealed that no changes in L-ferritin mRNA levels occur after cytokine stimulation. However, the induction coincides with an increased association of the L-subunit mRNA with polyribosomes. Since the recruitment of stored ferritin mRNA onto polyribosomes is seen when iron enters the cell, the effect of IL-1 beta on iron uptake was tested and was found to be unaffected by the lymphokine. Neither transferrin receptor mRNA levels nor the number of receptors displayed on the cell surface was affected by IL-1 beta. However, the action of the cytokine on ferritin translation is inhibited by the action of the intracellular iron chelator deferoxamine. These data indicate that IL-1 beta induces ferritin gene expression by translational control of its mRNA. The pathway of induction is different from iron-dependent ferritin gene expression whereas regulation requires the background presence of cellular iron.

Acute-Phase Proteins↗