Differential induction of inositol phosphate metabolism by three adipokinetic hormones

Sheep naturally infected by visna-maedi virus often develop a chronic interstitial lung disease characterized by an alveolitis comprising lymphocytes, neutrophils and macrophages. The alpha chemokine interleukin-8 (IL-8) was detected in cell free bronchoalveolar lavage fluid from naturally infected animals, confirmed by RT-PCR, presenting typical lesions of maedi and elevated total alveolar cell counts. No detectable IL-8 was found in the fluid obtained from uninfected animals. IL-8 concentration in alveolar fluid is correlated with alveolar neutrophil counts. Bronchoalveolar lavage cells from infected animals were found to contain a large amount of IL-8 mRNA and may contribute to IL-8 production. In situ hybridization showed that macrophages were the predominant cell type expressing IL-8 mRNA. Sustained production of IL-8 by alveolar macrophages during visna-maedi infection could suffice for neutrophil attraction to the alveoli, and may contribute to the development of lesions.     

Gender differences in hepatic induction and inhibition of theophylline pharmacokinetics and metabolism

This investigation examined whether or not a gender-related effect on theophylline pharmacokinetics and metabolism exists in smoking and nonsmoking subjects with and without the concurrent administration of cimetidine. With a crossover design, 28 healthy men and premenopausal women (14 in each group) were administered intravenous aminophylline before (phase 1) and during (phase 2) a 10-day course of cimetidine, 300 mg four times a day. Statistically significant gender- and smoking-related effects were seen for theophylline half-life and clearance in phase 1, but only the smoking effect was statistically significant for these pharmacokinetic parameters in phase 2. Urinary metabolite excretion patterns demonstrated a greater percentage of excretion of 3-methylxanthine in female smokers than in male smokers in phase 1. Time-averaged urinary clearance of 1,3-dimethyluric acid was greater in female smokers than in male smokers. In phase 2, a significant reduction in the clearance of 1,3-dimethyluric acid was noted in female smokers but not in any other group. The results of our investigation suggest that a significant difference in the pharmacokinetics of theophylline exists that is related to gender and smoking status. Theophylline metabolism also seems to vary according to these factors. Administration of cimetidine results in a significant reduction in clearance of theophylline and abolishes the gender differences but not the smoking differences.     

Differential in vitro hepatic and intestinal metabolism of ifosfamide in the rat

Influence of chitosan molecular weight on drug loading and drug release of drug-loaded chitosan microspheres was studied. Chitosans of 70,000 (LC), 750,000 (MC), and 2,000,000 (HC) molecular weight were employed alone or as mixtures (HC/LC 1:1-1:2 w/w). Ketoprofen (ket) was chosen as the model drug to be encapsulated. Microspheres characterized by different theoretical polymer/drug ratios were prepared (2:1, 1:1, 1:2 w/w). Satisfactory ket contents were obtained for all batches of chitosan microspheres with the theoretical polymer/drug ratio 1:2 w/w; microspheres made of HC/LC (1:2 w/w) were characterized by good drug content and encapsulation efficiency independent by polymer/drug ratio. Prepared chitosan microparticulate delivery systems can modulate ket release within 48 hr. Microspheres consisting of HC/LC (1:2 w/w) were the most suitable formulation in controlling drug release.     

Selenium regulation of hepatic heme metabolism: induction of delta-aminolevulinate synthase and heme oxygenase

Selenium was found to be a novel regulator of cellular heme methabolism in that the element induced both the mitochondrial enzyme delta-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating); EC 2-3-1-37] and the microsomal enzyme heme oxygenase [heme, hydrogen-donor:oxygen oxidoreductase(alpha-methene-oxidizing, hydroxylating); EC 1-14-99-3] in liver. The effect of selenium on these enzyme activities was prompt, reaching a maximum within 2 hr after a single injection. Other changes in parameters of hepatic heme metabolism occurred after administration of the element. Thirty minutes after injection the cellular content of heme was significantly increased; however, this value slightly decreased below control values within 2 hr, coinciding with the period of rapid induction of heme oxygenase. At later peroids heme content returned to normal values. Selenium treatment caused only a slight decrease in microsomal cytochrome P-450 content. However, drug-metabolizing activity was severely inhibited by higher doses of the element. Unlike other inducers of delta-aminolevulinate synthase, which as a rule are also porphyrinogenic agents, selenium induction of this enzyme was not accompanied by an increase in the cellular content of prophyrins. When rats were pretreated with selenium 90 min before administration of heme, a potent inhibitor of delta-aminolevulinate synthase production, the inhibitory effect of heme of formation of this mitochondrial enzyme was completely blocked. Selenium, at high concentrations in vitro, was inhibitory to delta-aminolevulinate synthase activity. It is postulated that selenium may not be a direct inducer of heme oxygenase as is the case with trace metals such as cobalt, but may mediate an increase in heme oxygenase through increased production and cellular availability of "free" heme, which results from the increased heme synthetic activity of hematocytes. Subsequently, the increased heme oxygenase activity is in turn responsible for the lack of increase in the microsomal heme content, thus maintaining heme levels at normal values despite the highly increased activities of both heme oxygenase and delta-aminolevulinate synthase. It is further suggested that the increase in delta-aminolevulinate synthase activity is not due to a decreased rate of enzyme degradation or an activation of preformed enzyme, but to increased rate of synthesis of enzyme protein. Although selenium in trace amounts has been postulated to be involved in microsomal electron transfer process, the data from this study indicate that excess selenium can substantially inhibit microsomal drug metabolism.     

Differential induction of systemic resistance in Arabidopsis by biocontrol bacteria

Selected nonpathogenic, root-colonizing bacteria are able to elicit induced systemic resistance (ISR) in plants. To elucidate the molecular mechanisms underlying this type of systemic resistance, an Arabidopsis-based model system was developed in which Pseudomonas syringae pv. tomato and Fusarium oxysporum f. sp. raphani were used as challenging pathogens. In Arabidopsis thaliana ecotypes Columbia and Landsberg erecta, colonization of the rhizosphere by P. fluorescens strain WCS417r induced systemic resistance against both pathogens. In contrast, ecotype RLD did not respond to WCS417r treatment, whereas all three ecotypes expressed systemic acquired resistance upon treatment with salicylic acid (SA). P. fluorescens strain WCS374r, previously shown to induce ISR in radish, did not elicit ISR in Arabidopsis. The opposite was found for P. putida strain WCS358r, which induced ISR in Arabidopsis but not in radish. These results demonstrate that rhizosphere pseudomonads are differentially active in eliciting ISR in related plant species. The outer membrane lipopolysaccharide (LPS) of WCS417r is the main ISR-inducing determinant in radish and carnation, and LPS-containing cell walls also elicit ISR in Arabidopsis. However, mutant WCS417rOA-, lacking the O-antigenic side chain of the LPS, induced levels of protection similar to those induced by wild-type WCS417r. This indicates that ISR-inducing bacteria produce more than a single factor that trigger ISR in Arabidopsis. Furthermore, WCS417r and WCS358r induced protection in both wild-type Arabidopsis and SA-nonaccumulating NahG plants without activating pathogenesis-related gene expression. This suggests that elicitation of an SA-independent signaling pathway is a characteristic feature of ISR-inducing biocontrol bacteria.     

Inactivation of rifampin by Nocardia brasiliensis

Rifampin was glycosylated by a pathogenic species of Nocardia, i.e., Nocardia brasiliensis. The structures of two glycosylated compounds (RIP-1 and RIP-2) isolated from the culture broth of the bacterium were determined to be 3-formyl-23-(O-[beta-D-glucopyranosyl])rifamycin SV and 23-(O-[beta-D-glucopyranosyl])rifampin, respectively. Both compounds lacked antimicrobial activity against other gram-positive bacteria as well as the Nocardia species.     

Ascorbic acid and hepatic drug metabolism

Previous in vivo studies indicate that hepatic microsomal drug metabolism decreases in ascorbic acid deficiency and is augmented when high supplements of the vitamin are given to guinea pigs. Kinetic studies with O-demethylase indicate no significant change in the apparent Km of p-nitroanisole in normal, ascorbic acid-deficient animals, or in animals given high supplements of ascorbic acid. The decrease in drug metabolism activity caused by ascorbic acid deficiency is not due to increased lipid peroxidation, nor was phosphatidyl choline significantly altered quantitatively or qualitatively in microsomes from ascorbic acid-deficient animals. Microsomal cytochrome P-450 prepared from ascorbic acid-deficient livers is less stable to sonication, dialysis and treatment with metal chelators. The decrease in cytochrome P-450 and O-demethylase activity associated with dialysis could be prevented by the addition of ascorbic acid. The molar ratio of microsomal ascorbic acid to cytochrome P-450 (plus P-420) is in the order of 2:1. This ratio is maintained during ascorbic acid deficiency in liver and adrenal tissue, during dialysis, on storage and with a partial purification of the cytochrome, which suggests a close association between ascorbic acid and the cytochrome. In addition, ascorbic acid protects cytochrome P-450 and aniline hydroxy lase activity from inhibition by ferrous iron chelators such as alpha, alpha'-dipyridyl. The chelator binds to cytochrome P-450 and prevents formation of the reduced cytochrome P-450-CO spectrum; it in turn gives a reduced spectrum with the cytochrome at 450 nm. These studies suggest that there is an interaction between ascorbic acid and cytochrome P-450 involving the reduced form of the heme iron.     

Differential induction of apoptosis by Fas-Fas ligand interactions in human monocytes and macrophages

PURPOSE: To evaluate the differences in accommodative function between subjects with emmetropia and those with late-onset myopia (LOM). METHODS: This study suggests a modified model of static accommodation, in which an accommodative sensory gain as a linear operator is added to simulate the sensory part of the system. Results derived from the model show that the sensory part not only affects the slope of the accommodative response function but also increases the system's effective threshold (ET) to the blur signal. This method expands the utility of using the control model to evaluate accommodation behavior. Thirteen emmetropic and 10 LOM subjects participated in this study. The subject's accommodative responses to one-, two-, three-, and four-diopter stimuli were measured by the Canon R-1 optometer, and the differences in dark focus, the slope of the accommodative response function, and the ET were compared between the emmetropic and the LOM subjects. RESULTS: The results show that although the dark-focus values and the slopes of the accommodative response function are not significantly different in emmetropia and LOM, the ETs are significantly different. CONCLUSIONS: The higher ET found among subjects with LOM suggests that either the blur (or the error) signal is degraded significantly in the sensory part of the system, the dead space as an internal threshold of the system is high, or both factors are important. On the basis of further analysis of the data, we speculate that the sensory system in LOM subjects was less sensitive to blur than that of the emmetropic subjects.     

14C]7-ethoxycoumarin metabolism by precision-cut rat hepatic slices

It is recognized that iodine-123-labelled 15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (123I-BMIPP) slowly washes out of the myocardium. The mechanism for the washout was investigated in normal rat hearts by analyses of the subcellular distribution and lipid classes based on the BMIPP metabolism. Rat hearts were excised at 1-120 min after intravenous injection of 123I-BMIPP. After counting the radioactivity, the hearts were digested with Nagarse and homogenized, and then fractionated into the cytosolic, mitochondrial, microsomal and crude nuclear fractions by centrifugations. The radioactivity of each fraction was counted, and the lipid classes were analysed by radio-thin-layer chromatographic and high-performance liquid chromatographic methods. The heart uptake of 123I-BMIPP was maximal at 5 min (6.81%+/-0.36% ID/g), and 41% of the radioactivity disappeared within 120 min. The myocardial radioactivity was immediately distributed into the cytosolic, mitochondrial, microsomal and crude nuclear fractions. The distribution (%) of each fraction was almost identical from 5 min through 120 min. The cytosolic fraction was always the major site of radioactivity deposition (60%), and the time-activity curve of the cytosolic fraction paralleled that of the whole heart throughout the 120-min study period. In the cytosolic fraction, most of the radioactivity was incorporated into the triglyceride class, and the rest was present in the free fatty acid, phospholipid (phosphatidylcholine) and diglyceride classes. In the mitochondrial fraction, the radioactivity was mostly incorporated into the phospholipid class (phosphatidylethanolamine), followed by free fatty acids. The final metabolite of 123I-BMIPP, 123I-p-iodophenylacetic acid (123I-PIPA), initially appeared in the mitochondrial fraction as early as 1 min, and subsequently in the cytosolic fraction at 5 min. Another intermediary metabolite, 123I-p-iodophenyldodecanoic acid (123I-PIPC12), was found only in the mitochondrial fraction after 5 min. In conclusion, the slow washout kinetics of 123I-BMIPP from the myocardium mainly reflects the turnover rate of the triglyceride pool in the cytosol. The BMIPP metabolism, i.e. initial alpha-oxidation followed by subsequent cycles of beta-oxidation, was confirmed in vivo. The participation of the mitochondria in the metabolism was also proven.     

Differential cerebrovascular responsiveness in spontaneously hypertensive rats following antihypertensive treatment with clonidine and verapamil

Numerous studies have been reported examining the effects of antihypertensive treatment on peripheral vascular responsiveness in spontaneously hypertensive rats (SHR). This study was conducted to determine the effects of chronic treatment with 2 antihypertensive agents on cerebrovascular responsiveness in male SHR and Wistar-Kyoto (WKY) rats. SHR and WKY (3-4 weeks old) received either placebo, clonidine (CLON, 10 mg pellet) or verapamil (VER, 5 mg pellet). Vascular reactivity studies on the basilar artery, using standard smooth muscle bath techniques, were conducted following 6 weeks of treatment. Both CLON and VER significantly attenuated the rise in blood pressure in SHR. Basilar artery responsiveness to KCl, serotonin (5-HT), and calcium were significantly increased whereas responses to acetylcholine (ACH), isoproterenol (ISO) and sodium nitroprusside (SNP) were significantly reduced in SHR compared to WKY. CLON had no effect on basilar artery responsiveness to either the contractile or relaxation agents in SHR. However, although responses to KCl, 5-HT and calcium were not affected by VER in SHR, VER significantly increased the responses to ACH, ISO and SNP. Neither CLON nor VER treatment affected basilar artery responsiveness to any of the agents in WKY. These data demonstrate that, even though CLON and VER have similar antihypertensive effects, differential effects of the 2 agents on cerebrovascular responsiveness in the SHR are apparent. This would suggest that the vascular effects of VER and CLON are dependent upon the mechanism of action of the agents and not simply due to prevention of the elevation in blood pressure.     

Acute modulation of rat hepatic lipid metabolism by sulphur-substituted fatty acid analogues

A single oral dose of two 3-thia (3-thiadicarboxylic and tetradecylthioacetic acids) and of 4-thia (tetradecylthiopropionic acid) fatty acids were administered to normolipidemic rats and their effects on lipid metabolism over a 24 hr period were studied. All three thia fatty acids could be detected in plasma 2 hr after treatment. Tetradecylthioacetic and tetradecylthiopropionic acids were detected in different hepatic lipid fractions but were incorporated mainly into hepatic phospholipids. Two hours after administration hepatic mitochondrial beta-oxidation and the total liver level of long-chain fatty acyl-CoA increased with a concomitant decrease in saturated fatty acids, total hepatic malonyl-CoA and plasma triacylglycerol levels in the 3-thia fatty acid groups. Tetradecylthiopropionic acid administration caused a decrease in mitochondrial beta-oxidation and an increase in plasma triacylglycerol at 24 hr. The activities of key lipogenic enzymes were unaffected in all treatment groups. Plasma cholesterol level was reduced only at 8 hr in 3-thiadicarboxylic acid treated rats although 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase was suppressed already at 2, 4, 8 and 12 hr. The results show that thia fatty acids are rapidly absorbed and are systemically available after oral administration but the 3-thia fatty acids reached systemic circulation more slowly and less completely than the 4-thia fatty acid. Very low levels of the thia fatty acids are detected in plasma 24 hr after a single administration. They are incorporated into all hepatic lipid classes, especially phospholipids. Rapid incorporation of a non beta-oxidizable thia fatty acid into hepatic lipids may cause a diversion of other fatty acids from glycerolipid biosynthesis to mitochondrial beta-oxidation. Stimulation of mitochondrial beta-oxidation and suppression of HMG-CoA reductase are primary events, occurring within hours, after 3-thia fatty acid administration. The hypotriglyceridemic effect of the 3-thia fatty acids observed at 2-4 hr is independent of the activities of key lipogenic and triacylglycerol synthesising enzymes.     

Differential induction of metallothionein synthesis by interleukin-6 and tumor necrosis factor-alpha in rat tissues

Metallothionein (MT) synthesis induced by the inflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF), was studied in vivo. Administration of recombinant human IL-6 or TNF to rats caused the acute phase responses including rapid decreases in plasma zinc (Zn), and increases in plasma copper (Cu) and ceruloplasmin. Hepatic concentration of MT-I, one of MT isoforms, began to increase within 3 h after the injection of IL-6 or TNF. In IL-6-treated rats, MT-I concentration in liver reached a maximum level at 12 h and decreased with a transient rebound, whereas, in TNF-treated rats, a high level of MT-I lasted for about 48 h. MT-II, the other MT isoform, was induced more than MT-I in liver by both cytokines. MT-I was also induced in lung and heart by TNF, but little by IL-6. The data suggest that IL-6 may be responsible for MT synthesis in liver, whereas TNF may be responsible not only in liver but also in lung and heart. Furthermore plasma concentration of MT did not always reflect the enhanced concentration of MT by TNF and IL-6 in liver, suggesting involvement of many factors influencing plasma MT levels. The interrelation between IL-6 and TNF for MT synthesis has also been discussed.     

Mechanism of induction of rat hepatic CYP2B and 3A by the pesticide methoxychlor

The S-phase which assesses tumor proliferation has been considered to be an independent prognostic factor for breast carcinoma. Quantitative analysis of MIB-1 immunoreactivity is a newly recognized method of determining cellular proliferation that offers some advantages over flow cytometry when limited tumor tissue is available. However, it has been controversial whether there is a significant correlation between MIB-1 immunostaining and S-phase in defining proliferation activity in breast cancer. In order to explore the usefulness of MIB-1 as an additional proliferation parameter and a potential prognostic factor for breast cancer, we analyzed 94 cases of invasive ductal carcinoma of the breast by both flow cytometry (for S-phase and DNA ploidy) and quantitative MIB-1 immunohistochemical analysis using formalin-fixed paraffin-embedded tissue. MIB-1 staining was quantitatively analyzed by image analysis and by visual scoring. Forty-six cases were diploid by flow, while the remaining 48 cases were aneuploid tumors. T-test results indicated that S-phase means were significantly greater (p = 0.0001) in aneuploid cases (mean = 18) compared to diploid cases (mean = 7). MIB-1 means were also greater in aneuploid patients, but these differences were only marginally significant (p = 0.05). S-phase was positively correlated with MIB-1 (r = 0.36, p = 0.003 for image analysis and r = 0.34, p = 0.001 for visual scoring). ROC curve analysis indicated that MIB-1 quantitation is a good predictor of high S-phase (i.e., > 10 percent) in aneuploid cases. A MIB-1 cutoff value of 25 percent for image analysis achieved 82 percent specificity and 80 percent sensitivity for aneuploid high S-phase, while a MIB-1 cutoff value of 40 percent for visual scoring was 73 percent specific and 85 percent sensitive. However, in diploid cases, no comparable MIB-1 cutoffs could be achieved for detecting high S-phase. In summary, our study demonstrated that aneuploid breast carcinomas proliferate more aggressively than diploid tumors. Although linear correlation between MIB-1 and S-phase was weak, MIB-1 was considered to be a good predictor of high S-phase in aneuploid breast cancer patients, possibly due to a threshold effect. Image analysis and visual scoring of MIB-1 immunoreactivity appeared to be comparable in analyzing proliferative activity in breast cancer. Thus MIB-1 assessed by visual scoring may be a less expensive alternative to image analysis.     

In vivo and in vitro regulation of hepatic glucagon receptor mRNA concentration by glucose metabolism

We have recently cloned the murine glucagon receptor (GR) gene and shown that it is expressed mainly in liver. In this organ, the glucagon-GR system is involved in the control of glucose metabolism as it initiates a cascade of events leading to release of glucose into the blood stream, which is a main feature in several physiological and pathological conditions. To better define the metabolic regulators of GR expression in liver we analyzed GR mRNA concentration in physiological conditions associating various glucose metabolic pathways in vivo and in vitro in the rat and in the mouse. First, we report that the concentration of the GR mRNA progressively increased from the first day of life to the adult stage. This effect was abolished when newborn rodents were fasted. Second, under conditions where intrahepatic glucose metabolism was active such as during fasting, diabetes, and hyperglycemic clamp, the concentration of GR mRNA increased independent of the origin of the pathway that generated the glucose flux. These effects were blunted when hyperglycemia was corrected by phlorizin treatment of diabetic rats or not sustained during euglycemic clamp. In accordance with these observations, we demonstrated that the glycolytic substrates glucose, mannose, and fructose, as well as the gluconeognic substrates glycerol and dihydroxyacetone, increased the concentration of GR mRNA in primary cultures of hepatocytes from fed rats. Glucagon blunted the effect of glucose without being dominant. The stimulatory effect of those substrates was not mimicked by the nonmetabolizable carbohydrate L-glucose or the glucokinase inhibitor glucosamine or when hepatocytes were isolated from starved rats. In addition, inhibitors of gluconeogenesis and lipolysis could decrease the concentration of GR mRNA from hepatocytes of starved rats. Combined, these data strongly suggest that glucose flux in the glycolytic and gluconeogenic pathways at the level of triose intermediates could control expression of GR mRNA and participate in controlling its own metabolism.     

Differential metabolism of exogenous platelet-activating factor by glandular epithelial and stromal cells of rabbit endometrium

Significant changes in platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) concentration have been observed in rabbit endometrium during the preimplantation period, but, under in vitro conditions, constitutive PAF biosynthesis by isolated endometrial tissues was not easily demonstrable. Relative changes in enzymes involved in the synthesis and metabolism of PAF in the tissues may account for this disparity. In addition, during this period of preimplantation, marked changes in PAF receptor concentration have been noted. The present study examines the factors that may modulate the metabolism of exogenous [3H]PAF in the endometrium of rabbits on day 6 of pregnancy. Since preferential [3H]PAF binding in situ by the glandular epithelial, but not by the stromal, cells was demonstrated, their cell-specific metabolism of exogenous [3H]PAF was also examined. After entry into the endometrial cell, [3H]PAF was rapidly metabolized by the sequential action of cytosolic Ca(2+)-independent acetylhydrolase to [3H]lyso-PAF and this was in turn acylated by membrane-associated transacylase to [3H]alkylacyl-glycerylphosphorylcholine. PAF resynthesis was not observed and, in stromal cells, there was a significant build-up of [3H]lyso-PAF, suggesting that lyso-PAF:acetyl-CoA acetyl-transferase may be a limiting factor. In the glandular epithelial cells, however, there was a significant accumulation of a neutral lipid without a significant build-up of [3H]lyso-PAF or [3H]PAF. The neutral lipid co-migrated with the product of phospholipase C-catalysed metabolism of PAF and authentic 1-O-hexadecyl-2-acetyl-glycerol. In addition, the elution times of phospholipase C digestion of C18 PAF and the neutral lipid produced by cellular metabolism of [3H]PAF, determined by gas chromatography/flame ionization detection, were similar. It seems that it is the synthesis of the neutral lipid from reacetylated [3H]lyso-PAF that prevented [3H]PAF accumulation under in vitro conditions. This is the first documentation of the synthesis of this lipid in the mammalian uterus. The lipid may serve as the precursor for de novo PAF synthesis in the glandular epithelial cells during endometrial proliferation.