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J C Fleet

Publications and source records attributed to J C Fleet.

33 records · Page 2Linked to original sources
PubMed1994

Identification of osteocalcin mRNA in nonosteoid tissue of rats and humans by reverse transcription-polymerase chain reaction.

Diseased or necrotic tissue can become calcified in a way that resembles bone. We examined soft tissues for the presence and regulation of the mRNA for the bone-associated protein, osteocalcin (OC). RNA was isolated from liver, kidney, lung, brain, muscle, and bone of young (2 months) male SD rats and analyzed for beta-actin, IGF-I, metallothionein IIa, alpha 1 collagen, calbindin-D9k (CaBP), and OC mRNA by reverse transcription-polymerase chain reaction (RT-PCR). All PCR products but CaBP were found in bone; CaBP was present only in duodenum, kidney, and lung. OC product was detected in all tissues; the identity of the PCR product was confirmed by sequencing. Bone OC mRNA levels were calculated to be 1000-fold higher than duodenal levels. Rats fed a 0.8% strontium diet for 7 days to drive down serum 1,25-dihydroxyvitamin D3 levels [1,25(OH)2D3] and then injected with 300 ng 1,25(OH)2D3/100 body weight had increased duodenal CaBP (2.5-fold) and femur OC mRNA (2.2-fold) 24 h after treatment. Duodenal OC mRNA was unchanged. OC mRNA was found in nondiseased human aortae, and the amount of message was elevated in calcified aorta and calcified aortic plaques. These results demonstrate that (1) tissues other than bone have low basal expression of OC mRNA, (2) OC mRNA is not regulated by vitamin D in nonosteoid tissue, and (3) expression of OC mRNA in atherosclerotic aorta reflects a role for bone-forming cells in ectopic bone formation observed in certain disease conditions.

Animals↗
PubMed1994

Identification of calbindin D-9k mRNA and its regulation by 1,25-dihydroxyvitamin D3 in Caco-2 cells.

We have identified and quantified specific mRNAs in the human colonic carcinoma cell line Caco-2 by reverse transcriptase-polymerase chain reaction. Initial examination revealed that like rat duodenal mucosa, Caco-2 cells possessed mRNA for the vitamin D receptor. Using primers for human calbindin we found a 237-bp PCR product in Caco-2 cell RNA, but not from rat duodenal RNA. Primers for rat calbindin did not amplify calbindin mRNA in Caco-2 RNA, confirming a high degree of mismatch between rat and human sequences. 1,25(OH)2 vitamin D3 treatment (10 nM) significantly elevated calbindin mRNA levels 50% by 12 h, with maximal levels occurring by 48 h (fivefold elevation). Increasing concentrations of 1,25(OH)2 vitamin D3 (from 15 pM to 100 nM) caused progressive increases in calbindin mRNA levels following 48 h of treatment. Elevated calbindin mRNA levels were associated with enhancement of transcellular calcium transport. Our results are the first demonstration of vitamin D-regulated calbindin mRNA in a human intestinal cell line.

Animals↗
PubMed1994

New support for a folk remedy: cranberry juice reduces bacteriuria and pyuria in elderly women.

Cranberry juice has developed a following as a simple, nonpharmacologic means to reduce or treat urinary tract infections, yet the scientific basis for such a claim has been lacking. A new study suggests that bacterial infections (bacteriuria) and associated influx of white blood cells into the urine (pyuria) can be reduced by nearly 50% in elderly women who drink 300 mL of cranberry juice cocktail each day over the course of a 6-month study. The results of this study suggest that consumption of cranberry juice is more effective in treating than preventing bacteriuria and pyuria. Along with earlier reports on the ability of cranberry juice to inhibit bacterial adherence to urinary epithelial cells in cell culture, this new work suggests that drinking cranberry juice each day may be clinically useful. Additional work must be conducted, however, to more completely define the efficacy of cranberry juice.

Aged↗
PubMed1994

Growth hormone and parathyroid hormone stimulate intestinal calcium absorption in aged female rats.

Aged (16-month-old) female rats (n = 8/treatment) were injected for 12 days with GH (100 micrograms/100 g x day), PTH (8 micrograms/100 g x day), GH plus PTH, or vehicle (V) in an experiment designed to determine the effects of these hormones on intestinal mineral absorption in senescent rats. PTH and GH increased fractional net calcium absorption to a similar extent (PTH, 1.6-fold; GH, 1.4-fold) even though PTH increased serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] 3.7-fold, and GH had no significant effect. GH plus PTH caused no further increase in serum 1,25-(OH)2D3 above that caused by PTH alone, but resulted in an additive effect on net calcium absorption (2.3-fold increase). PTH and GH also had statistically independent effects on phosphate absorption; magnesium absorption was elevated only by PTH. Duodenal calbindin-D9k levels were increased by GH (from 3.79 +/- 0.72 to 6.98 +/- 0.73 micrograms/mg protein) and PTH (from 3.23 +/- 0.46 to 7.55 +/- 0.75 micrograms/mg protein); PTH plus GH treatment resulted in an additive effect on calbindin-D9k levels. Additional in vitro transport studies in the human intestinal cell line Caco-2 showed that 72 h of pretreatment with the local mediator of GH action, insulin-like growth factor-I (at 10 and 100 ng/ml), stimulated transcellular calcium transport (22% and 44%, respectively) regardless of concomitant 1 nM 1,25-(OH)2D3 pretreatment (80% increase). Our findings suggest a 1,25-(OH)2D3-mediated mechanism for PTH-induced changes in calcium and phosphorus absorption. In contrast, the effects of GH in the senescent rat are independent of changes in circulating 1,25-(OH)2D3 and our data suggest that these effects may be mediated by insulin-like growth factor-I.

Aging↗
PubMed1993

Vitamin D-sensitive and quinacrine-sensitive zinc transport in human intestinal cell line Caco-2.

We studied the mechanism of transepithelial zinc (Zn) transport using monolayers of Caco-2 cells grown on permeable filter supports. 65Zn transport could be fitted to a modified Michaelis-Menten equation, which includes a nonsaturable [linear diffusion constant of nonsaturable component (Kd) = 0.08%.cm-2.90 min-1] and a saturable component [upper well Zn concentration at half Jmax (Kt) = 226 microM and maximal rate of saturable Zn transport (Jmax) = 1.06 nmol.cm-2.90 min-1]. Caco-2 cells contained metal-inducible metallothionein (MT) protein and mRNA as well as mRNA for cysteine-rich intestinal protein (CRIP). Cells pretreated with 10 nM 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] for 3 days transported more Zn (159%) than controls (0.48 +/- 0.02 nmol.cm-2.90 min-1) when each was incubated with 100 microM Zn for 90 min. This effect was significant after 24 h of 1 alpha,25-(OH)2D3 pretreatment and continued to increase up to 72 h, with concomitant increases in MT mRNA levels being observed (4-fold by 24 h, 10-fold by 72 h). MT protein levels were only modestly elevated by 72 h 1 alpha,25(OH)2D3 treatment (from 0.32 +/- 0.04 to 0.45 +/- 0.03 nmol MT/mg protein). CRIP mRNA levels were reduced by 1 alpha,25(OH)2D3 treatment. The lysosome-disrupting agent quinacrine (0.5 mM) inhibited basal Zn transport by 68%, suggesting the possible presence of a lysosome-mediated component for transepithelial Zn transport in Caco-2 cells. 1 alpha,25(OH)2D3-stimulated Zn transport was not affected by quinacrine, suggesting that 1 alpha,25(OH)2D3-induced Zn transport is distinct from the putative lysosome-mediated Zn transport pathway.

Biological Transport↗
PubMed1992

Atherogenic diets enhance endotoxin-stimulated interleukin-1 and tumor necrosis factor gene expression in rabbit aortae.

Cytokine-induced vascular changes may contribute to the atherogenic process. We examined the effect of atherogenic diets on the aortic expression of genes for the cytokines interleukin-1 alpha and beta and tumor necrosis factor alpha. Male New Zealand White rabbits were fed one of the following diets for 10 wk: nonpurified diet, a semipurified diet with 10% corn oil, a semipurified diet with 1% corn oil and 9% partially hydrogenated coconut oil, or the 9% coconut oil diet supplemented with either 0.1, 0.3 or 0.9% added cholesterol (n = 6/group). At 10 wk, 3 rabbits per group received lipopolysaccharide (200 micrograms/kg) intravenously. After 1.5 h the rabbits were killed and their aortae removed and analyzed. Histologic examination showed that the 0.3% and 0.9% cholesterol-fed rabbits developed appreciable aortic lesions. Diet had no major effect on the basal levels of aortic cytokine mRNA as determined by polymerase chain reaction analysis of DNAs. However, aortic tissue from rabbits fed 0.3% and 0.9% cholesterol diets showed significantly enhanced lipopolysaccharide-evoked levels of mRNA encoding interleukin-1 alpha (392 +/- 91 for saturated fat vs. 759 +/- 191 and 800 +/- 120 fmoles/reaction for 0.3% and 0.9% cholesterol), interleukin-1 beta (99 +/- 10 vs. 353 +/- 80 and 355 +/- 86) and tumor necrosis factor alpha (195 +/- 15 vs. 594 +/- 78 and 667 +/- 97). Extracts of aortae from rabbits injected with lipopolysaccharide contained interleukin-1 and tumor necrosis factor alpha activity but differences in biological activity due to diet were not detectable at the 1.5 h time point (chosen for maximal mRNA expression). Increased local cytokine gene expression in response to acute stimulus might influence the evolution of the vascular response to diets rich in cholesterol and saturated fats.

Animals↗
PubMed1991

Interleukin-1 gene expression in rabbit vascular tissue in vivo.

Cultured human vascular endothelial and smooth muscle cells express interleukin-1 (IL-1) genes when exposed to bacterial lipopolysaccharides (LPS) and a variety of inflammatory mediators. Local production of IL-1 may contribute to the pathogenesis of various vascular diseases. Therefore the ability of intact vascular tissue to accumulate IL-1 mRNA and synthesize de novo biologically active IL-1 protein was examined. Escherichia coli LPS (10 micrograms/kg) was administered intravenously to adult rabbits and total RNA was isolated from aortic tissue at various times after LPS injection. In saline-injected rabbits, RNA extracted from the thoracic aorta contained little or no IL-1 message detected by Northern analysis using IL-1 alpha and beta cDNA probes cloned from an LPS-stimulated rabbit splenic macrophage library. Lipopolysaccharide treatment promptly induced transient accumulation of mRNA for IL-1 alpha and IL-1 beta within the aorta (maximal 1-hour after injection). Short-term organoid cultures of rabbit aorta exposed to LPS in vitro synthesized immunoprecipitable IL-1 alpha protein. Extracts of aortic tissue excised 1.5 to 3.0 hours after intravenous LPS administration contained immunoreactive and biologically active IL-1 alpha. Anti-rabbit IL-1 alpha antibody neutralized the biologic activity (more than 90%). Microscopic and immunohistochemical studies did not disclose adherent or infiltrating macrophages in rabbit aorta at the time of maximal IL-1 mRNA accumulation after LPS administration (1.5 hours), indicating that intrinsic vascular wall cells rather than mononuclear phagocytes probably account for the IL-1 activity induced by LPS. In addition, aortic tissue from rabbits fed an atherogenic diet showed an enhanced ability to accumulate IL-1 alpha and beta mRNA and produce immunodetectable protein in response to LPS administration. These studies demonstrate inducible IL-1 gene expression in rabbit vascular tissue in vivo and support a local role for this cytokine in vascular pathophysiology.

Animals↗
PubMed1990

Iron-induced metallothionein in chick liver: a rapid, route-dependent effect independent of zinc status.

The induction of hepatic metallothionein (MT) by the parenteral administration of iron was studied. Iron administered to chicks by intravenous or subcutaneous injection caused a 1.9-fold increase in hepatic MT. In marked contrast, intraperitoneal (ip) Fe resulted in a 10-fold increase, thus demonstrating the importance of the route of metal administration. This route-dependent effect was found to be dose-dependent, with ip injections between 1 and 10 mg Fe/kg resulting in a linear increase in MT and a concomitant reduction in serum zinc concentration and feed intake. High ip doses of Fe resulted in a persistent depression in serum Zn and elevated MT and MTmRNA. Equimolar ip injections of either Zn or Fe showed similar patterns of MTmRNA accumulation. In both cases MTmRNA levels were elevated by 3 h, with a peak at 6 h postinjection (Fe 8-fold, Zn 12-fold above 0 h). Plasma Zn was maximally reduced by Fe at 9 h (60%). The MT induction by Fe, as well as related depression in plasma Zn, was completely inhibited by actinomycin D. Zn depletion eliminated the accumulation of hepatic Zn and MT protein following ip injection of Fe or endotoxin, but not of cadmium, despite marked elevation of hepatic MTmRNA. Our results demonstrate Fe injected into the body cavity of chicks results in a rapid induction of hepatic MT that, like endotoxin induction, is independent of dietary Zn status.

Animals↗
PubMed1990

Time-course studies of pancreatic exocrine damage induced by excess dietary zinc in the chick.

Experiments were conducted to determine the time course of Zn-induced changes in the exocrine status of the chick pancreas. In experiments 1 and 2, chicks killed at intervals after the addition of excess Zn (500 mg/kg as ZnO) to a purified diet containing 70 mg Zn/kg showed a rapid increase in plasma Zn concentration that reached a plateau within 1 h. The pancreatic soluble Zn level increased linearly for 24 h, but then its rate of accumulation diminished. The same pattern of accumulation was shown by pancreatic metallothionein (MT), which correlated highly with pancreatic Zn; that is, MT-associated Zn consistently accounted for 70 to 80% of pancreatic Zn. Plasma amylase activity started to increase 8-10 h after Zn introduction and reached a maximum by 24 h. Two critical levels of pancreatic soluble Zn (1 and 2.7 micrograms Zn/mg of protein, respectively) were observed at which amylase activities increased in the plasma and decreased in the pancreas. In experiment 3, feeding excess Zn reduced the incorporation of [3H]leucine into pancreatic amylase protein. This was associated with a reduction of tracer incorporation into total pancreatic proteins, that is, the fraction that is precipitable by trichloracetic acid (TCA). In experiment 4, the enzyme (or potential) activities of the exportable Zn-containing enzymes procarboxypeptidases A and B and ribonuclease were not affected by excess Zn intake, indicating the selective nature of the effect of excess Zn on pancreatic function.

Amylases↗
PubMed1990

Induction of hepatic metallothionein by intraperitoneal metal injection: an associated inflammatory response.

The nature of hepatic metallothionein (MT) induction by several metals and its relationship to an inflammatory response was studied in chicks. Intraperitoneal (ip) injection of chromium (Cr), managanese, and iron (Fe) caused a much greater increase in hepatic MT (10.2-, 9.0-, and 6.8-fold) compared with cobalt and nickel (2.5- and 2.9-fold); thus not all transition metals are effective. Cr3+ caused markedly greater hepatic MT accumulation than Cr6+, suggesting that the ionic nature of the metal is an important factor. Small organic complexes of Fe (ferrous gluconate or lactate, 6.2-fold) caused significantly greater accumulation of hepatic MT than ferric dextran (1.4-fold), a large organic aggregate. In vitro data from chick hepatocytes and/or fibroblasts clearly indicated that Fe does not effect the induction of MT directly. The role of inflammation, as measured by recruitment of peritoneal exudate cells (PEC), was examined. Endotoxin (LPS), Sephadex (S), and Fe elicited significant elevations in PEC number at 24 h posttreatment (S), and Fe elicited significant elevations in PEC number at 24 h posttreatment (S = Fe greater than LPS much greater than control). The percentage of heterophils but not macrophages was significantly correlated with the accumulation and induction of hepatic MT. In a similar experiment with Cr, we demonstrated that Cr3+ but not Cr6+ stimulated MT messenger RNA accumulation and concomitant hetereophil infiltration at 3 h after injection. Our results indicate that the induction of hepatic MT by the parenteral administration of a number of metals is dependent on the chemical nature of the metal and is associated with an inflammatory response.

Animals↗
PubMed1989

Metalloforms of metallothionein induced by parenteral copper: the influence of route of administration.

Previous results regarding the two metalloforms of MT which accumulate in chick liver following the parenteral administration (ip) of copper were discussed. One metalloform, which is exclusively zinc, was suggested to reflect the marked accumulation of hepatic zinc following copper injection. The present report shows that there is a marked difference in hepatic zinc accumulation if copper is administered iv. Under these conditions there is virtually no change in hepatic zinc and thus MT produced under these conditions appears to contain only copper. We suggest the the changes in zinc metabolism as effected by copper when given intraperitoneally reflect a secondary response analogous to that observed when iron is similarly administered.

Animals↗
PubMed1988

The toxicity of parenteral copper in the chick: dependence on route of administration.

Preliminary work in our laboratory suggested that the route of parenteral copper administration influences the acute toxicity of copper (Cu). The present work examined the effects of Cu given either intraperitoneally (i.p.) or intravenously (i.v.) on mortality, feed intake, peritoneal fluid and hepatic accumulation of Cu and zinc (Zn) in chicks. We also studied the potential interaction of prior Zn loading on these parameters. Four-wk-old chicks were pretreated by injection (subcutaneous) with Zn acetate (5 mg Zn/kg) or sodium acetate 24 h prior to Cu treatments. Copper as Cu acetate was given either i.p. or i.v. at a dose of 1.84 mg Cu/kg. Mortality was markedly greater in those chicks given Cu i.p. compared to those given Cu i.v. (46.1% vs. 3.9%). Zinc pretreatment had no effect on any of the parameters associated with copper treatments. The accumulation (24 h) of copper in either total hepatic tissue or cytosol was comparable for both routes of injection. However, marked differences in Cu-induced hepatic Zn accumulation were observed. Massive peritoneal fluid (41% of total plasma volume) was observed 1 to 3 h following the administration of Cu i.p. None was observed in those given Cu i.v. Thus, this effect was associated with the route-dependent mortality observed in the present study. We suggest that our data highlight the gratuitous effects of reactive agents given intraperitoneally.

Animals↗
PubMed1988

Tissue-specific accumulation of metallothionein in chickens as influenced by the route of zinc administration.

The effect of the route of zinc (Zn) administration on the induction of metallothionein (MT) in various tissues of chicks was examined. Four-week-old, male chicks were assigned to one of four treatments: 5 mg Zn/kg intraperitoneal (i.p.) injection, 5 mg Zn/kg intravenous (i.v.) injection, 16 mg Zn oral (O) dose or a saline control (C). Chicks were fasted overnight, treated and killed 24 h later. 109Cd radioassay analysis of liver (L), kidney (K) and pancreas (P) showed a significant elevation of MT in all tissues except K of i.v. chicks. Comparing tissue MT accumulation within treatments showed that L was induced to a greater extent than P for the i.p. treatment (P/L ratio = 0.69 +/- 0.04), while the reverse effect was seen for both O (1.51 +/- 0.10) and i.v. (1.67 +/- 0.14) chicks, reflecting greater P than L accumulation. Zn injected i.p. did not result in significantly greater total peritoneal exudate cell (PEC) or macrophage (M) numbers than saline-injected controls. Sephadex, while causing massive increases in PEC and M, did not induce tissue MT, demonstrating the lack of correlation between PECs or Ms and MT accumulation. Feed intake by chicks during the 24-h period following i.p. Zn treatment was only 30% of that by controls. A subsequent experiment demonstrated that a similar restriction in feed intake increased L but not P MT. This increase accounted for 17.8% of the L induction due to i.p. Zn injection. This does not fully account for the reduction in P/L that is characteristic of i.p. treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1988

The ontogeny and induction by zinc of hepatic chick embryo metallothionein.

To avoid the maternal influences inherent in mammalian models, the chick embryo was examined for the effects of development on the ontogeny of hepatic metallothionein (MT) induction. Livers from embryos were examined for total and cytosolic zinc (Zn) and copper (Cu), and cytosolic MT on 12, 14, 16, and 18-21 days of incubation (DI) as well as 1, 7, and 14 days posthatch (ph). Cytosolic Zn levels fell, from a high on 12 DI (45.04 +/- 1.05 ng/micrograms DNA), to 25.79 +/- 1.05 ng/micrograms DNA on 19 DI and then increased to 44.73 +/- 3.47 ng/micrograms DNA by 1 day ph. Cytosolic Cu followed a reverse pattern, with a high of 24.10 +/- 0.74 ng/microgram DNA on 16 DI. Both 109Cd radioassay analysis and gel filtration chromatography of hepatic cytosols showed a developmental pattern for MT similar to that of cytosolic Zn. These results are in contrast to the developmental pattern of the rat. To establish if yolk Zn levels limit hepatic MT in the 1-day neonate, the yolks of unincubated fertile eggs were supplemented with 26, 52, or 78% of the endogenous Zn (768 micrograms/yolk). As a result, hepatic MT of 1-day neonates increased by 3.9-, 4.7-, and 7.1-fold, respectively. Thus the initial level of Zn in the yolk has a significant impact on the final concentration of MT in neonatal liver. A study of MT induction in the 18-DI embryo revealed that yolk sac administration of Zn (550, 2750, 4950 micrograms/yolk) increased hepatic MT by 5.8-, 23-, and 39-fold, respectively. This demonstrates the inducibility of MT even at the point of minimum endogenous MT (18 DI). In summary, our results show that (1) a marked difference exists between the developmental patterns of MT in avian and mammalian species, and (2) chick embryo hepatic MT is highly responsive to exogenous Zn introduced into the yolk.

Animals↗