Effects of the intermediate retinoid metabolite retinal on the pattern of the dental lamina in vitro

Retinoids have important roles in pattern formation during embryonic development and might act as endogenous morphogens. They are necessary for normal odontogenesis and excess retinol alters the pattern of odontogenesis producing supernumerary buds of the dental lamina in the diastema region of the mouse mandible. Because the metabolism of retinoids in the developing mandible is unknown, the effects of retinal (an intermediate metabolite in the local conversion of retinol to retinoic acid) on the patterning of odontogenesis were examined. Retinal produces supernumerary buds and enhanced epithelial proliferation in day-9 mandibles in vitro. The endogenous levels of retinal in the mandible at the time of initiation of odontogenesis were also measured by high-performance liquid chromatography. Retinal was detected only at day 10 and not at later stages of development. Local metabolism of this intermediate retinoid may be a rate-determining step in the production of active retinoid metabolites that may control the pattern of the dentition, which is established at the time of the appearance of the dental lamina at embryonic day 12.     

Proton NMR studies of cytochrome c peroxidase mutant N82A: hyperfine resonance assignments, identification of two interconverting enzyme ofecies, quantitating the rate of interconversion, and determination of equilibrium constants

The cyanide-ligated form of the baker's yeast cytochrome c peroxidase mutant bearing the mutation Asn82-->Ala82 ([N82A]CcPCN) has been studied by proton NMR spectroscopy. This mutation alters an amino acid that forms a hydrogen bond to His52, the distal histidine residue that interacts in the heme pocket with heme-bound ligands. His52 is a residue critical to cytochrome c peroxidase's normal function. Proton hyperfine resonance assignments have been made for the cyanide-ligated form of the mutant by comparison with 1-D and NOESY spectra of the wild-type native enzyme. For [N82A]CcPCN, proton NMR spectra reveal two significant phenomena. First, similar to results published for the related mutant [N82D]CcPCN [Satterlee, J. D., et al. (1994) Eur. J. Biochem. 244, 81-87], for Ala82 mutation disrupts the hydrogen bond between His52 and the heme-ligated CN. Second, four of the 24 resolved hyperfine-shifted resonances are doubled in the mutant enzyme's proton spectrum, leading to the concept that the heme active site environment is dynamically microheterogeneous on a very localized scale. Two magnetically inequivalent enzyme forms are detected in a pure enzyme preparation. Varying temperature causes the two enzyme forms to interconvert. Magnetization transfer experiments further document this interconversion between enzyme forms and have been used to determine that the rate of interconversion is 250 (+/- 53) s-1. The equilibrium constant at 20 degrees C is 1.5. Equilibrium constants have been calculated at various temperatures between 5 and 29 degrees C leading to the following values: delta H = 60 kJ mol-1; delta S = 0.20 kJ K-1 mol-1.     

BMP-2 and BMP-4, but not BMP-6, increase the number of adrenergic cells which develop in quail trunk neural crest cultures

Isolated rat liver mitochondria were incubated under various metabolic conditions to determine their membrane potential (MMP) as measured continuously by a tetraphenylphosphonium (TPP+)-selective electrode. By flow cytometry, a parallel analysis of fluorescence emissions observing single mitochondria stained with the lipophilic cation 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) revealed linear correlation between the median orange fluorescence (FL2) due to J-aggregate formations and MMP values measured by TPP+. No correlation was detected with the green fluorescence (FL1) emission. A significantly higher correlation appeared between the FL2/FL1 ratio and MMP values. Within the same mitochondrial population, cytofluorimetric analysis revealed the presence of various classes of organelles with different MMP, whose distribution was dependent on metabolic condition. The highest functional heterogeneity was found in deenergized mitochondria, while the highest homogeneity was observed during the first phase of the phosphorylative process. Thus, these data suggest that the cytofluorimetric use of JC-1 provides direct experimental evidence for the hypothesis of functional mitochondrial heterogeneity, at least with respect to their membrane potential.     

X-linked juvenile retinoschisis: localization between (DXS1195, DXS418) and AFM291wf5 on a single YAC

We studied 17 pedigrees with 108 affected males with X-linked juvenile retinoschisis (RS; McKusick No. 31270) and have analyzed all of the known polymorphic markers in the RS region of Xp22.1-p22.2 between DXS987 and DXS41. By haplotype analyses we found 7 individuals who showed crossovers in this interval surrounding RS. We previously reported AFM291wf5 as the centromeric boundary, and this remains unchanged in the present study. A new recombination was identified on the telomeric side at (DXS1195, DXS418). Our data support the locus order Xpter--(DXS987, DXS207, DXS1053, DXS43)--(DXS1195, DXS418)--(RS, DXS257, DXS999)--(AFM291wf5, DXS443)--DXS1052--(DXS1226, DXS274, DXS41)--Xcen; loci grouped in parentheses could not be mutually ordered by our genetic data. Physical mapping has indicated a distance of at most 900-1,000 kb between (DXS1195, DXS418) and AFM291wf5. No recombination was observed between RS and DXS257 which lies in our new interval of interest, but one critical individual was not informative with this marker. Our data now define the smallest RS inclusion interval. This interval is contained on a single YAC from which we have identified expressed sequences as candidate genes for RS.     

A comparison of the W-stapled ileal reservoir with hand-sewn reservoirs for orthotopic bladder replacement

Activated calpain I immunoreactivity (76 kDa band) was detected in membranes prepared from rat brain hippocampal slices using a polyclonal antiserum raised against an N-terminus peptide of the cleaved subunit of calpain I. While basal levels of activated calpain I were stable over incubation times, 1 nM dopamine (DA) produced an initial 32% increase (5 min) in the 76 kDa protein followed by a 53% decrease in this band at 20 min of incubation. The DA-induced changes in activated calpain I immunoreactivity were blocked by the calpain inhibitor peptide, N-acetyl-Leu-Leu-norleucinal(100 microM) or by EGTA. Basal levels of the 76 kDa band were not affected by the calpain inhibitor. These changes in activated calpain I, elicited by DA, are in accord with the DA-induced decreases in the levels of the calpain substrate, gamma PKC (Yurko-Mauro and Friedman; J Cell Biochem [Abstr] 180:80, 1994; J Neurochem 65: 1622-1630, 1995) and suggest that DA activates this Ca(++)-dependent protease in its regulation of neuronal signal transduction.     

Detection and quantification of endolymphatic hydrops in the guinea pig cochlea by magnetic resonance microscopy

The experiments carried out on the model of immobilization stress permit establishing the antiulcer effect of preventive introduction of sodium succinate solution, milk serum solution and solution of milk serum with sodium succinate. A decrease in the degree and number of ulcers and in the integrative indices characterizing the ulcer process after introduction of the studied drugs was accompanied by a fall of the intensity of lipid peroxidation processes.     

Kinetic analysis of the in vitro inhibition, aging, and reactivation of brain acetylcholinesterase from rat and channel catfish by paraoxon and chlorpyrifos-oxon

In rats, the phosphorothionate insecticide parathion exhibits greater toxicity than chlorpyrifos, while in catfish the toxicities are reversed. The in vitro inhibition of brain acetylcholinesterase (AChE) by the active metabolites of the insecticides and the rates at which these inhibitor-enzyme complexes undergo reactivation/ aging were investigated in both species. Rat AChE was more sensitive to inhibition than catfish AChE as demonstrated by greater bimolecular rate constants (ki) in rats than in catfish. In both species, chlorpyrifos-oxon yielded higher ki's than paraoxon. The higher association constant (KA) of chlorpyrifos-oxon than paraoxon in both species and the lack of significant differences in the phosphorylation constants (kp) suggest that association of the inhibitor with AChE is the principal factor in the different potencies between these two inhibitors. In catfish, the ki of chlorpyrifos-oxon was 22-fold greater than that of paraoxon, while in rats it was 9-fold greater, suggesting that target site sensitivity is an important factor in the higher toxicity of chlorpyrifos to catfish but not in the higher toxicity of parathion to rats. No spontaneous reactivation of phosphorylated catfish AChE occurred and there were no differences in the first oder aging constants (ka) between compounds. For phosphorylated rat AChE, there were no differences in the first order reactivation constants (kr) but the ka for chlorpyrifos-oxon was significantly greater than that for paraoxon. This difference suggests that the steric positioning of the diethyl phosphate in the esteratic site is not the same between the two compounds, leading to differences in aging.