Growth factor regulation of insulin-like growth factor (IGF) binding proteins (IGFBP) and preadipocyte differentiation in porcine stromal-vascular cell cultures

The influence of anti-IGF-1 and anti-transforming growth factor beta (TGF-beta) neutralizing antibodies on preadipocyte differentiation and secretion of IGFBPs was examined in serum free porcine stromal-vascular cultures. Cultures were stained for morphological analysis and conditioned media were collected for: TGF-beta determination by ELISA, IGF-1 by RIA, and IGFBP analysis by ligand blotting. After 6 d of treatment, anti-TGF-beta increased fat proportions by 2.7 fold compared to controls. Anti-IGF-1 decreased fat cell proportions by 14-fold. Anti-TGF-beta increased concentrations of IGF-1 5.8-fold and IGFBP-2 and IGFBP-3 by 8- and 7-fold in conditioned media whereas IGFBP-4 decreased 5-fold. Anti-IGF-1 increased concentrations of IGFBP-2 and 3 by 9- and 35-fold, respectively. TGF-beta increased concentrations of IGFBP-1, 2 and 3 by 3-fold, 18-fold and 3-fold, respectively, after 9 d in culture (6 d of treatment). There was no change in TGF-beta levels in anti-IGF-1 treated cultures compared to controls. Control antibodies and negative controls had no effect. These results provide evidence that endogenously produced IGF-1 and TGF-beta has a major influence on preadipocyte differentiation in serum free media by modulating IGFBP production/secretion.     

Uveitis and central nervous system vasculitis

The purpose of this double-blind study was to investigate the influence of adding a quercetin-containing supplement to the diet on plasma quercetin status, serum/platelet fatty acid levels and risk factors for heart disease. Healthy men and women with cholesterol levels of 4.0-7.2 mmol/L, consumed four capsules daily of either a quercetin-containing supplement (1.0 g quercetin/d) or rice flour placebo for 28 d. Quercetin intakes were approximately 50-fold greater than the dietary intakes associated with lower coronary heart disease mortality on the basis of epidemiologic studies. Subjects consuming quercetin-containing capsules had plasma quercetin concentrations approximately 23-fold higher than those of subjects consuming the control capsules. Quercetin supplementation did not modify serum total, LDL or HDL cholesterol or triglyceride levels. There were also no alterations of other cardiovascular disease or thrombogenic risk factors, including platelet aggregation, platelet thromboxane B2 production, blood pressure or resting heart rate. Furthermore, there was no effect on the levels of (n-6) or (n-3) polyunsaturated fatty acids in serum or platelet phospholipids. In conclusion, supplementation with quercetin-containing capsules markedly enhanced the plasma quercetin concentration but had no effect on other cardiovascular or thrombogenic risk factors.     

Combined nicotinic and muscarinic blockade in elderly normal volunteers: cognitive, behavioral, and physiologic responses

1-, 3-, and 6-nitrobenzo[a]pyrene (nitro-BaP) are environmental contaminants that can be metabolized to genotoxic derivatives by either nitroreduction or ring-oxidation. In this study, we examined the types of mutations produced by the primary nitroreduced metabolites, 1-, 3-, and 6-nitroso-BaP (NO-BaP) in the hprt gene of Chinese hamster ovary cells. RNA from 6-thioguanine-resistant mutants was reverse-transcribed to cDNA and the hprt coding sequence was amplified and sequenced. The mutational patterns produced by the three compounds exhibited extensive similarities: 1) base pair substitutions accounted for 67% (28/42) of 1-NO-BaP, 51% (26/51) of 3-NO-BaP, and 50% (11/22) of 6-NO-BaP mutations; 19-36% of the mutations were exon deletions and 14-18% were frameshifts; 2) most (64-84%) of the simple base pair substitutions occurred at G:C, mainly G:C-->T:A and G:C-->C:G transversions; 3) 98% (46/47) of the simple base pair substitutions at G:C had the mutated dG on the non-transcribed strand and 81% (38/47) were located with the mutated dG flanked 3' by at least one purine; and 4) most simple base pair substitutions (48/62, 77%) occurred in exons 2, 3, and 8 of the hprt gene. Although there were no significant differences among the mutation profiles of the NO-BaPs, a significant difference did exist between the mutation pattern produced by 3-NO-BaP and the mutation pattern previously determined for the ring-oxidized product of 3-nitro-BaP metabolism, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9, 10-tetrahydro-3-nitrobenzo[a]pyrene. This observation indicates that differences in the structures of closely related adducts can be important enough to have an effect on mutation profiles.     

Temporal expression of pro-inflammatory cytokines and inducible nitric oxide synthase in experimental acute Chagasic cardiomyopathy

Pelizaeus-Merzbacher disease (PMD) is a dysmyelinating disease resulting from mutations, deletions, or duplications of the proteolipid protein (PLP) gene. Distinguishing features of PMD include pleiotropy and a range of disease severities among patients. Previously, we demonstrated that, when expressed in transfected fibroblasts, many naturally occurring mutant PLP alleles encode proteins that accumulate in the endoplasmic reticulum and are not transported to the cell surface. In the present communication, we show that oligodendrocytes in an animal model of PMD, the msd mouse, accumulate Plp gene products in the perinuclear region and are unable to transport them to the cell surface. Another important aspect of disease in msd mice is oligodendrocyte cell death, which is increased by two- to threefold. We demonstrate in msd mice that this death occurs by apoptosis and show that at the time oligodendrocytes die, they have differentiated, extended processes that frequently contact axons and are expressing myelin structural proteins. Finally, we define a hypothesis that accounts for pathogenesis in most PMD patients and animal models of this disease and, moreover, can be used to develop potential therapeutic strategies for ameliorating the disease phenotype.     

Virtual colonoscopy: imaging features with colonoscopic correlation

BACKGROUND: We determined whether activation of the nitric oxide/cyclic guanosine monophosphate pathway by sodium nitroprusside (SNP) protects hearts subjected to cardioplegic arrest and prolonged hypothermic storage. METHODS: Isolated rat hearts arrested with St. Thomas' II cardioplegia and stored at 3 degrees +/- 1 degree C for 8 hours were reperfused at 37 degrees C in Langendorff (10 minutes) and working (60 minutes) modes. RESULTS: During reperfusion, left ventricular work was depressed in stored hearts relative to fresh hearts. When present during arrest, storage, and both reperfusion phases, SNP (200 mumol/L) improved work to values close to those in fresh hearts. When added only during the 10-minute period of Langendorff reperfusion, SNP also improved the subsequent recovery of work. This effect was antagonized by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Poststorage coronary perfusion was not increased by SNP. CONCLUSIONS: The ability of SNP to enhance recovery independent of changes in coronary perfusion and in an ODQ-sensitive manner suggests that SNP-induced protection is due to activation of the myocardial nitric oxide/cyclic guanisine monophosphate pathway. These results suggest that supplementing cardioplegic solutions with SNP, administering SNP during early reperfusion, or both may offer additional means to improve donor heart preservation.     

Molecular and cellular changes in vein grafts: influence of pulsatile stretch

Sudden cardiac death is frequent in patients with dilated cardiomyopathy. To assess the risk of an arrhythmic event is still difficult. Here the analysis of the heart rate variability offers new possibilities. METHOD: 25 patients (18 males, 7 females, age 53 +/- 9 yrs) with dilated cardiomyopathy were included in the study. Analysis of heart rate variability assessed by time- and frequency-domain measures was determined from Holter recording. The mean follow-up was 18 +/- 5 months. RESULTS: 6 patients died (5 of sudden cardiac death, 1 of heart failure), 1 patient with an implanted defibrillator received an adequate shock. Parameters influenced by low- and mid-frequent oscillations of the heart rate were significantly lower in patients who died suddenly or had adequate shocks. The best predictive parameter was the s.d.RR: all patients with an s.d.RR < 50 ms had lethal arrhythmias whereas the s.d.RR of the surviving patients was > or = 50 ms. No significant difference was found or high frequency parameters, which are mainly influenced by parasympathetic activity. CONCLUSION: The analysis of heart rate variability is of prognostic relevance in patients with dilated cardiomyopathy. Especially the s.d.RR is able to identify patients with a high risk of a sudden cardiac death.     

Periodic orbits: a new language for neuronal dynamics

A new nonlinear dynamical analysis is applied to complex behavior from neuronal systems. The conceptual foundation of this analysis is the abstraction of observed neuronal activities into a dynamical landscape characterized by a hierarchy of "unstable periodic orbits" (UPOs). UPOs are rigorously identified in data sets representative of three different levels of organization in mammalian brain. An analysis based on UPOs affords a novel alternative method of decoding, predicting, and controlling these neuronal systems.     

Oligoclonality of Vdelta1 and Vdelta2 cells in human peripheral blood mononuclear cells: TCR selection is not altered by stimulation with gram-negative bacteria

Despite the enormous potential repertoire of gammadelta T cells, there are several observations which suggest that the expressed gammadelta repertoire in the periphery of normal individuals is often quite restricted. To assess selective expansions among gammadelta T cells from both adult and newborn blood samples, PBMC from 12 normal adults and cord blood from 15 normal newborns were analyzed for TCRDV1 and TCRDV2 junctional diversity by CDR3 size spectratyping and single-strand conformational polymorphism. Although TCRBV usage showed extensive heterogeneity in adults and newborns, both populations often showed CDR3 region restriction for TCRDV1 and TCRDV2. Analysis of the CDR3 spectratype patterns of newborn twins suggested that clonal selection for TCRDV is independent of genetic background. The possible role of Gram-negative bacteria in driving selective responsiveness of gammadelta T cells in PBMCs from adults was examined by in vitro stimulation with Escherichia coli and Pseudomonas aeruginosa. Donors whose TCRDV repertoire was highly clonal in the unstimulated blood cells showed the same predominant clones among the bacteria-stimulated cultures. In individuals whose gammadelta T cells were less restricted, in vitro stimulation did not select for clonality; rather, the TCRDV repertoires were similar before and after bacterial stimulation. Together, these data indicate that gammadelta T cells are often clonally restricted in adults as well as in newborns and suggest that the prominent stimulatory activity of Gram-negative bacteria does not by itself account for the restriction or diversity of the gammadelta T cell repertoire.     

Compartmentation of [14C]glutamate and [14C]glutamine oxidative metabolism in the rat hippocampus as determined by microdialysis

Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free-moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect 14CO2 generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min-fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced 14C-amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [14C]glutamate and [14C]glutamine were 59 +/- 18 and 2.1 +/- 0.5 dpm/pmol, respectively. The initial infused specific activities for [U-14C]glutamate and [U-14C]glutamine were 408 +/- 8 and 387 +/- 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [14C]glutamine in the interstitial space when [14C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.     

Hybrid bilayer membranes in air and water: infrared spectroscopy and neutron reflectivity studies

In this report we describe the fabrication and characterization of a phospholipid/alkanethiol hybrid bilayer membrane in air. The bilayer is formed by the interaction of phospholipid with the hydrophobic surface of a self-assembled alkanethiol monolayer on gold. We have characterized the resulting hybrid bilayer membrane in air using atomic force microscopy, spectroscopic ellipsometry, and reflection-absorption infrared spectroscopy. These analyses indicate that the phospholipid added is one monolayer thick, is continuous, and exhibits molecular order which is similar to that observed for phospholipid/phospholipid model membranes. The hybrid bilayer prepared in air has also been re-introduced to water and characterized using neutron reflectivity and impedance spectroscopy. Impedance data indicate that when moved from air to water, hybrid bilayers exhibit a dielectric constant and thickness that is essentially equivalent to hybrid bilayers prepared in situ by adding phospholipid vesicles to alkanethiol monolayers in water. Neutron scattering from these samples was collected out to a wave vector transfer of 0.25 A(-1), and provided a sensitivity to changes in total layer thickness on the order of 1-2 A. The data confirm that the acyl chain region of the phospholipid layer is consistent with that observed for phospholipid-phospholipid bilayers, but suggest greater hydration of the phospholipid headgroups of HBMs than has been reported in studies of lipid multilayers.