Splenic and peritoneal B-1 cells differ in terms of transcriptional and proliferative features that separate peritoneal B-1 from splenic B-2 cells.

B-1 cells constitute a distinct B cell subset with characteristic phenotypic and functional features. B-1 cells are highly represented among peritoneal lymphocytes; substantial numbers of B-1 cells are also located within splenic tissue. Here a number of differences in transcription factor and gene expression were identified that separate peritoneal B-1 and splenic B-2 cells, and then splenic B-1 cells obtained from immunoglobulin transgenic mice were tested for these parameters. Splenic B-1 cells resembled splenic B-2 cells rather than peritoneal B-1 cells in terms of nuclear expression of DNA-binding STAT3, CREB, and PU.1, with respect to transcriptional activation of IL-10, and in the failure to enter cell cycle in response to PMA. Splenic B-1 cells (B-1S) appear to constitute a unique population of B-1 cells, which, while sharing with peritoneal B-1 cells (B-1P) certain phenotypic features, differ from them in transcription factor and gene expression and in signaling for cell cycle progression.

Metabolism of carnitine in phenylacetic acid-treated rats and in patients with phenylketonuria.

The effect of metabolites accumulating in phenylketonuria (PKU) was investigated on carnitine metabolism in rats and in patients with PKU. Of phenylacetic acid (PEAA), phenylpyruvic acid and homogentisic acid the PEAA was found to be the most effective in inhibiting carnitine biosynthesis in rats. Following 60 min, a single intraperitoneal dose of PEAA the relative conversion rate, i. e. the hydroxylation, of tracer [Me-(3)H]butyrobetaine to [Me-(3)H]carnitine decreased from 62.2+/-6.00% to 39.4+/-5.11% (means+/-S.E.M., P<0.01) in the liver, in the only organ doing this conversion in rats. The conversion of loading amount of unlabeled butyrobetaine to carnitine was also markedly reduced. The impaired hydroxylation of butyrobetaine was reflected by a reduced free and total carnitine levels in the liver and a reduced total carnitine concentration in the plasma. PEAA decreased the hepatic level of glutamic acid and alpha-ketoglutaric acid (alpha-KG), suggesting a mechanism for the reduced flux through the butyrobetaine hydroxylase enzyme, because alpha-KG is an obligatory co-enzyme. In the plasma and urine of PKU patients on unrestricted diet, markedly decreased total carnitine levels were detected. In the liver of PEAA-treated rats and urine of PKU patients, a novel carnitine derivative, phenacetyl-carnitine was verified by HPLC and gas chromatography-mass spectrometry.

Molecular mechanism of the short-term cardiotoxicity caused by 2',3'-dideoxycytidine (ddC): modulation of reactive oxygen species levels and ADP-ribosylation reactions.

The short-term cardiac side effects of 2',3'-dideoxycytidine (ddC, zalcitabine) were studied in rats in order to understand the biochemical events contributing to the development of ddC-induced cardiomyopathy. In developing animals, ddC treatment provoked a surprisingly rapid appearance of cardiac malfunctions characterized by prolonged RR, PR, and QT intervals and J point depression. The energy metabolism in the heart was compromised, characterized by a decreased creatine phosphate/creatine ratio (from 2.05 normal value to 0.75) and a decreased free ATP/ADP ratio (from 332 normal value to 121). The activity of respiratory complexes (NADH: cytochrome c oxidoreductase and cytochrome oxidase) also decreased significantly. Southern blot and polymerase chain reaction analysis did not show deletions or a decrease in the quantity of mitochondrial DNA (mtDNA) deriving from ddC-treated rat hearts, indicating that under our experimental conditions, ddC-induced heart abnormalities were not the direct consequence of mtDNA-related damage. The ddC treatment of rats significantly increased the formation of reactive oxygen species (ROS) in heart and skeletal muscle as determined by the oxidation of non-fluorescent dihydrorhodamine123 to fluorescent rhodamine123 and the oxidation of cellular proteins determined from protein carbonyl content. An activation of the nuclear poly-(ADP-ribose) polymerase (EC 2.4.2.30) and an increase in the mono-ADP-ribosylation of glucose-regulated protein and desmin were observed in the cardiac tissue from ddC-treated animals. A decrease in the quantity of heat shock protein (HSP)70s was also detected, while the level of HSP25 and HSP60 remained unchanged. Surprisingly, ddC treatment induced a skeletal muscle-specific decrease in the quantity of three proteins, one of which was identified by N-terminal sequencing as myoglobin, and another by tandem mass spectrometer sequencing as triosephosphate isomerase (EC 5.3.1.1). These data show that the short term cardiotoxicity of ddC is partially based on ROS-mediated signalling through poly- and mono-ADP-ribosylation reactions and depression of HSP70 levels, whose processes represent a new mtDNA independent mechanism for ddC-induced cell damage.

Enhanced ADP-ribosylation and its diminution by lipoamide after ischemia-reperfusion in perfused rat heart.

Poly-ADP-ribose polymerase (PARP) is considered to play an important role in oxidative cell damage. We assumed that ischemia-reperfusion resulting from the increasing reactive oxygen species (ROS) can lead to the activation of endogenous mono- and poly-ADP-ribosylation reactions and that the reduction of ROS level by lipoamide, a less known antioxidant, can reverse these unfavorable processes. Experiments were performed on isolated Langendorff hearts subjected to 60-min ischemia followed by reperfusion. ROS, malondialdehyde, deoxyribonucleic acid (DNA) breaks, and NAD+ content were assayed in the hearts, and the ADP-ribosylation of cytoplasmic and nuclear proteins were determined by Western blot assay. Ischemia-reperfusion caused a moderate (30.2 +/- 8%) increase in ROS production determined by the dihydrorhodamine 123 method and significantly increased the malondialdehyde production (from < 1 to 23 +/- 2.7 nmol/ml), DNA damage (undamaged DNA decreased from 71 +/- 7% to 23.1 +/- 5%), and NAD+ catabolism. In addition, ischemia-reperfusion activated the mono-ADP-ribosylation of GRP78 and the self-ADP-ribosylation of the nuclear PARP. The perfusion of hearts with lipoamide significantly decreased the ischemia-reperfusion-induced cell membrane damage determined by enzyme release (LDH, CK, and GOT), decreased the ROS production, reduced the malondialdehyde production to 5.5 +/- 2.4 nmol/ml, abolished DNA damage, and reduced NAD+ catabolism. The ischemia-reperfusion-induced activation of poly- and mono-ADP-ribosylation reactions were also reverted by lipoamide. In isolated rat heart mitochondria, dihydrolipoamide was found to be a better antioxidant than dihydrolipoic acid. Ischemia-reperfusion by ROS overproduction and increasing DNA breaks activates PARP leading to accelerated NAD+ catabolism, impaired energy metabolism, and cell damage. Lipoamide by reducing ROS levels halts PARP activation and membrane damage and improves the recovery of postischemic myocardium.

A novel gene coding for a Fas apoptosis inhibitory molecule (FAIM) isolated from inducibly Fas-resistant B lymphocytes.

The sensitivity of primary splenic B cells to Fas-mediated apoptosis is modulated in a receptor-specific fashion. Here we used a differential display strategy to detect cDNAs present in B cells rendered Fas resistant but absent in those rendered Fas sensitive. This led to the cloning and characterization of a novel 1.2-kb gene that encodes a Fas apoptosis inhibitory molecule (FAIM). faim-transfected BAL-17 B lymphoma cells were less sensitive by half or more to Fas-mediated apoptosis than were vector-transfected controls, using Fas ligand-bearing T cells or a cytotoxic anti-Fas antibody to trigger Fas, and this was associated with inhibition of Fas- induced poly-ADP ribose polymerase (PARP) cleavage. In primary B cells, the time course of faim mRNA and FAIM protein expression correlated with the induction of Fas resistance by surface (s)Ig engagement. Thus, FAIM is an inducible effector molecule that mediates Fas resistance produced by sIg engagement in B cells. However, faim is broadly expressed in various tissues and the faim sequence is highly conserved evolutionarily, suggesting that its role extends beyond lymphocyte homeostasis. As FAIM has no significant regions of homology to other gene products that modulate Fas killing, it appears to represent a distinct, new class of antiapoptotic protein.

Role of reactive oxygen species and poly-ADP-ribose polymerase in the development of AZT-induced cardiomyopathy in rat.

The short term cardiac side-effects of AZT (3'-azido-3'-deoxythymidine, zidovudine) was studied in rats to understand the biochemical events contributing to the development of AZT-induced cardiomyopathy. Developing rats were treated with AZT (50 mg/kg/day) for 2 wk and the structural and functional changes were monitored in the cardiac muscle. AZT treatment provoked a surprisingly fast appearance of cardiac malfunctions in developing animals characterized by prolonged RR, PR and QT intervals and J point depression. Electron microscopy showed abnormal mitochondrial structure but the cardiomyocyte had normal myofibers. The AZT treatment of rats significantly increased ROS and peroxynitrite formation in heart tissues as determined by the oxidation of nonfluorescent dihydrorhodamine123 and dichlorodihydro-fluorescein diacetate (H2DCFDA) to fluorescent dyes, and induced single-strand DNA breaks. Lipid peroxidation and oxidation of cellular proteins determined from protein carbonyl content were increased as a consequence of AZT treatment. Activation of the nuclear poly-ADP-ribose polymerase and the accelerated NAD+ catabolism were also observed in AZT-treated animals. Western blot analysis showed that mono-ADP-ribosylation of glucose regulated protein (GRP78/BIP) was enhanced by AZT treatment, that process inactivates GRP78. In this way moderate decrease in the activity of respiratory complexes was detected in the heart of AZT-treated animals indicating a damaged mitochondrial energy production. There was a significant decrease in creatine phosphate concentration resulting in a decrease in creatine phosphate/creatine ratio from 2.08 to 0.58. ATP level remained close to normal but the total extractable ADP increased with 45%. The calculated free ATP/ADP ratio decreased from 340 to 94 in the heart of AZT-treated rats as a consequence of increased free ADP concentration. It was assumed that the increased free ADP in AZT-treated cardiomyocyte may help cells to compensate the defective ATP production in damaged mitochondria by activating the ATP synthesis in undamaged mitochondria. Southern blot analysis did not show decreased quantity of mtDNA deriving from AZT-treated rat hearts indicating that under our experimental conditions AZT-induced heart abnormalities are not the direct consequence of the mtDNA depletion. These data show that ROS-mediated oxidative damages, activated ADP-ribosylation reactions and accelerated NAD+ catabolism play basic roles in the development of AZT-induced cardiomyopathy in our animal model and indicated that these ROS-mediated processes can be important factors in the development of myopathy and cardiomyopathy in zidovudine-treated AIDS patients.

Remineralization by fluoride enhanced with calcium and phosphate ingredients.

The effectiveness of fluoride ions provided by toothpastes and mouthrinses in promoting remineralization can be limited by the low concentrations of calcium and phosphate ions in saliva. The purpose of this study was to determine whether improved remineralization can be obtained from toothpastes or mouthrinses that simultaneously deliver fluoride, calcium, and phosphate ions from dual-dispensing systems. Enamel specimens with artificial lesions between 60 and 90 microns deep were cycled 15 times through demineralization for 30 minutes, treated for 5 minutes with an experimental or control fluoride toothpaste or mouthrinse, and remineralized for 60 minutes. In the toothpaste study, surface hardness increased by 11.5 +/- 9.2 and 2.7 +/- 3.6 Vickers hardness units, and enamel fluoride content was 5984 +/- 521 ppm and 3971 +/- 531 ppm for the experimental and control fluoride toothpastes, respectively. Remineralization was confirmed by x-ray microradiography. In the mouthrinse study, surface hardness increased by 8.8 +/- 7.7 and 2.2 +/- 3.7 Vickers hardness units, and enamel fluoride content was 6111 +/- 1078 ppm and 3160 +/- 364 ppm for the experimental and control fluoride mouthrinses, respectively. Use of a non-fluoride control mouthrinse led to a decrease in surface hardness of 3.7 +/- 5.2 Vickers hardness units despite a fluoride content of 402 ppm. The results demonstrate that calcium and phosphate supplementation in a toothpaste or mouthrinse can improve remineralization and increase fluoride uptake.

Measurement of enamel remineralization using microradiography and confocal microscopy. A correlational study.

Tooth minerals are lost and regained constantly in a normal, human oral environment. Different methods have been developed to measure this gain and loss in enamel minerals; however, these methods deal with different problems, such as being time consuming or involving the use of X-rays. The aim of this study was to determine if remineralization measured in a thin enamel section (TS) by transversal microradiography (MR) can be reliably monitored by measuring lesion parameters (area, total and average dye fluorescence) on the same TS or on half a tooth (HT) with confocal laser scanning microscopy (CLSM). Thirty-six human enamel specimens were demineralized for 96h, and then half of each specimen was covered with an acid-resistant nail varnish. Specimens were divided into three groups (12/group) and subjected for 20 days to a cyclic remineralization regimen with consisted daily of a 4-hour acid challenge, four 1-min treatment periods with 0, 250 or 1,100 ppm F dentifrice slurries (1:2; dentifrice:water) and 20 h in pooled, human saliva, at room temperature. Specimens were cut and analyzed by MR, then stained with a fluorescent dye (0.1mM rhodamine B) for 1 h and analyzed using CLSM. Both MR and CLSM detected significantly greater remineralization (p<0.05) in the specimens treated with the fluoride-containing dentifrices than in the specimens treated with 0 ppm F. Significant differences were detected between specimens treated witht the fluoride-containing dentifrices by MR and CLSM (HT area and total fluorescence). Statistically significant (p<0.05) Pearson correlation coefficients were calculated between the MR and CLSM data: difference in MR mineral content (DeltaM) versus HT lesion area = 0.71; DeltaM versus HT total fluorescence = 0.70; DeltaM versus HT average fluorescence = 0.61; DeltaM versus TS lesion area = 0.88; DeltaM versus TS total fluorescence = 0.63, and DeltaM versus TS average fluorescence = 0.40. It is concluded that confocal microscopy in either TS or HT may provide valid surrogates (area and total fluorescence) for MR measurements in enamel remineralization studies.

An in situ interproximal model for studying the effect of fluoride on enamel.

This crossover study determined the ability of an interproximal, intra-oral model to demonstrate a fluoride dose response to 0-, 250- and 1,100-ppm fluoride (sodium fluoride) dentifrices with respect to fluoride uptake into, and remineralization of, incipient subsurface enamel lesions. Following a 1 week 'lead in' period during which 30 panelists were randomly assigned to use one of the products, two enamel specimens with artificial carious lesions were mounted into a specially designed functional partial denture worn by each panelist. Panelists continued to brush three times daily with their test dentifrice for 4 weeks, after which the specimens were removed and analyzed for fluoride uptake and remineralization. The procedure was repeated until each panelist had followed all three treatment regimens. Fluoride analyses were performed using a microdrill biopsy technique, and mineral content changes were determined by transverse microradiography. Fluoride uptake data were significantly different (p < 0.01) for all three products with the effect of 1,100 ppm F > 250 ppm F > placebo. The 1,100 ppm F dentifrice also effected significantly greater remineralization (p < 0.01) than did the 250-ppm-F or placebo dentifrices. Relative efficacy of the three fluoride dentifrices tested in this study was similar to that established in a clinical trial, and, therefore, supports the use of this model for in situ studies of the effects of fluoride-containing products on enamel lesions.

Intracellular signaling for inducible antigen receptor-mediated Fas resistance in B cells.

CD40 ligand-activated B cells are sensitive targets for CD4+ Th1 effector cells that kill in a Fas-dependent fashion. Susceptibility to apoptosis is counteracted by Ag receptor binding that produces a state of resistance to Fas engagement in otherwise sensitive targets. In the present study, protection from Th1-mediated apoptosis was found to be induced by protein kinase C and calcium signals, which in combination mimicked the level of Fas resistance produced by surface Ig engagement. Signaling for Fas resistance did not alter Fas expression. Furthermore, B cells that were protected against Th1-mediated apoptosis were also resistant to apoptosis mediated by soluble, rFas ligand. Taken together, these results indicate that signaling for protection against Fas-mediated apoptosis does not depend on alteration of the interaction between B cell target and Th1 effector populations. Instead, surface IgM-derived protein kinase C and calcium signals appear to produce an intracellular change in the Fas signaling pathway that develops over a period of hours and interferes with the apoptotic process through a mechanism that depends on protein synthesis.

Validation and assessment of a conversational interaction intervention.

Multiple measures of social behavior were obtained in order to (a) evaluate the effects of a systematic replication of a conversational interaction intervention, (b) identify critical target behaviors, (c) select participants likely to benefit from intervention, and (d) assess the social and experimental validity of the intervention. Combined measures (e.g., performance screening, assessing students' goals, behavioral ratings) corroborated the decision to introduce multiple-exemplar, self-instructional training to 4 students with moderate mental retardation who were observed to have limited social interaction with their peers at school. Findings indicated that the procedure was effective at increasing empirical indicators of participants' generalized conversational interaction and that behavioral change was validated socially by ratings of peers and important others.

Spatially Resolved Observation of Static Magnetic Flux States in YBa2Cu3O7-dgr Grain Boundary Josephson Junctions.

With low-temperature scanning electron microscopy, the magnetic flux states in high critical temperature Josephson junctions have been imaged. The experiments were performed with YBa(2)Cu(3)O(7-delta) thin-film grain boundary Josephson junctions fabricated on [001] tilt SrTiO(3) bicrystals. For applied magnetic fields parallel to the grain boundary plane, which correspond to local maxima of the magnetic field dependence of the critical current, the images clearly show the corresponding magnetic flux states in the grain boundary junction. The spatial modulation of the Josephson current density by the external magnetic field is imaged directly with a spatial resolution of about 1 micrometer.

Amalgam retention using pins, boxes, and Amalgambond.

The purpose of this study was to evaluate and compare the retentive strength of amalgam to tooth structure by utilizing pins, boxes, Amalgambond adhesive only, and Amalgambond adhesive plus retentive boxes. There was no statistical difference (P = 0.05) in retentive strength of the amalgam between the group utilizing pins alone and the group utilizing Amalgambond plus retentive boxes. However, both of these sample groups demonstrated statistically greater retentive strengths than either the boxes only or Amalgambond only groups.

A three-rooted mandibular second premolar.

If an endodontic problem is diagnosed in a mandibular premolar, and root canal therapy is provided, it must be considered that second and third canals might exist. Although the existence of a third canal would be rare, a thorough evaluation may help to prevent future complications. In the case presented here, failure to recognize a third canal might have resulted in incomplete instrumentation and canal obturation, and endodontic failure. Variations in root canal morphology and the number of canals might not have been detected even during close inspection of the pulp chamber floor. Therefore, the dentist must have a thorough knowledge of root canal morphology and its variations.

A possible mechanism in arterial wall for mediation of sex difference in atherosclerosis.

Female rabbits on an atherogenic diet were treated with cottonseed oil (control), tamoxifen, testosterone, or progesterone. After 10 weeks the rabbits were killed, the aortas quickly removed, graded for atherosclerosis, and incubated with [14C]proline to determine collagen and elastin synthesis. Rabbits treated with testosterone and progesterone had the greatest degree of atherosclerosis, the highest DPM in hydroxyproline of collagen and elastin, and the greatest accumulation of collagen and elastin in the aorta. Tamoxifen-treated rabbits had less incorporation of radioactivity. In separate experiments aortas of similarly treated rabbits were analyzed for estradiol and progesterone receptor density. These receptors were found to be present, and progesterone and testosterone administration caused a translocation of progesterone receptors from cytosol to nucleus. Results are consistent with the hypothesis that sex hormones can affect the development of atherosclerosis through a direct effect of the hormones on arterial wall to alter collagen and elastin synthesis, the effect being mediated through hormone receptors in the wall.

Effects of estradiol and progesterone on the increased synthesis of collagen in atherosclerotic rabbit aortas.

The effects of sex hormones on atherosclerosis and collagen synthesis in blood vessels were studied in rabbits fed an atherogenic diet. Ovariectomy without hormone replacement resulted in significantly greater degree of atherosclerosis and collagen synthesis in the aortic arch when compared with the intact state. The administration of estradiol to ovariectomized rabbits resulted in a degree of atherosclerosis and collagen synthesis similar to that of intact rabbits. Ovariectomized rabbits administered progesterone on the other hand resembled the ovariectomized rabbits without hormone replacement. Specific activity of hydroxyproline in individual specimens of the arch correlated strongly with degree of atherosclerosis. An analysis of regional variation demonstrated declining values for collagen synthesis in the caudal direction along the aorta. Collagen synthesis was much higher in the portal vein as compared with the inferior vena cava. Plasma cholesterol values were higher in the ovariectomized rabbits than in the intact rabbits. Tissue deposition of cholesterol paralleled the degree of atherosclerosis.

Diet- and hormone-induced lipid deposition in rat kidney: correlation with systolic blood pressure.

The influence of estradiol on deposition of cholesterol in tissues of ovariectomized rats on normal and high lipid diets was studied. Concomitantly the influence of a contraceptive steroid combination was studied in a similar manner in intact rats. It was found that the high lipid diet resulted in increased deposition of cholesterol in aorta, heart, liver and kidney. The presence of either endogenous or exogenous hormones accentuated the deposition of cholesterol in the kidney and resulted in significantly higher systolic blood pressures in these rats. In the rats on a high lipid diet, the concentration of cholesterol in the kidney correlated positively with systolic blood pressure. It is concluded that estrogen and high lipid diet exert a synergistic effect on deposition of cholesterol in kidney. The positive correlation between kidney cholesterol concentration and systolic blood pressure suggests a possible role for kidney lipid deposition in the hypertensive effect of estrogens.