Ferrochelatase activities in patients with erythropoietic protoporphyria and their families

Ferrochelatase, estimated as zinc chelatase, was measured in the lymphocytes of 30 patients with erythropoietic protoporphyria (EPP), in 35 first- or second-degree relatives of patients with EPP, and in 50 healthy controls. In 30 EPP patients the zinc chelatase level (mean +/- standard deviation, SD) was 0.45 +/- 0.10 nmol of zinc protoporphyrin per hour per milligram of protein, in 14 EPP carriers the zinc chelatase level (mean +/- SD) was 0.42 +/- 0.09 and in 50 healthy controls the zinc chelatase level (mean +/- SD) was 0.84 +/- 0.27. All patients with EPP were also demonstrated to have an elevated protoporphyrin level in their red blood cells: the erythrocyte protoporphyrin levels were as follows EPP patients (mean +/- SD) 1300 +/- 758 nmol protoporphyrin/dl, EPP carriers (mean +/- SD) 60 +/- 24, and healthy controls (mean +/- SD) 50 +/- 25 (P < 0.001 for EPP patients compared to controls and EPP carriers). The families of 12 out of 15 EPP patients were examined with respect to the mode of inheritance of the disorder. Of 35 relatives, 14 were carriers of EPP, as characterized by reduced zinc chelatase activity in lymphocytes and by a normal protoporphyrin level in red blood cells. None of the 14 EPP carriers had presented with clinical symptoms of EPP. The mod of inheritance was autosomal dominant in seven of the 12 examined families, and autosomal recessive in two. In two families only one parent could be investigated, but we nevertheless concluded that the inheritance was autosomal dominant. Inheritance in one EPP family could not be elucidated as both parents showed normal zinc chelatase levels and did not demonstrate abnormal erythrocyte protoporphyrin levels.     

Inhibition of diabetic nephropathy by a GH antagonist: a molecular analysis

Streptozotocin-treated C57B1/SJL mice developed glomerular hypertrophy and light microscopic lesions mimicking human diabetic glomerulosclerosis. In contrast, there were no glomerular hypertrophy and lesions in diabetic mice transgenic (TG) for a mutated growth hormone (bGH-G119K) that competes with native endogenous GH and results in dwarfism. We examined the molecular events underlying these findings. The non-transgenic (non-TG) diabetic mouse glomeruli had an increase in mRNA coding for alpha 1IV collagen, laminin B1, TGF-beta 1, 72 kDa collagenase, and TIMP-3. In contrast, glomerular type IV collagen and laminin B1 mRNA levels were normal in diabetic TG dwarf mice. However, the 72 kDa gelatinase, TIMP-3, and TGF-beta 1 mRNAs were elevated in the diabetic dwarfs. Type IV collagen and laminin accumulated in the glomeruli of diabetic non-TG, but not of diabetic dwarf mice, by immunofluorescence microscopy, confirming the mRNA data. GH binding protein mRNA levels were comparable in glomeruli from dwarf and non-TG mice, both diabetic and non-diabetic. We did not detect GH receptor mRNA in glomeruli. These data suggest that diabetic glomerulosclerosis is associated with an increase in type IV collagen and laminin synthesis, and that these changes do not occur in mice transgenic for bGH119K, a functional antagonist of GH. The increase of 72 kDa gelatinase, TIMP-3 and TGF-beta 1 mRNAs, independent of GH, suggested that these changes induced by hyperglycemia were not sufficient for the induction of glomerulosclerosis.     

Functional analysis of peptide motif for RNA microhelix binding suggests new family of RNA-binding domains

RNA microhelices that recreate the acceptor stems of transfer RNAs are charged with specific amino acids. Here we identify a two-helix pair in alanyl-tRNA synthetase that is required for RNA microhelix binding. A single point mutation at an absolutely conserved residue in this motif selectively disrupts RNA binding without perturbation of the catalytic site. These results, and findings of similar motifs in the proximity of the active sites of other tRNA synthetases, suggest that two-helix pairs are widespread and provide a structural framework important for contacts with bound RNA substrates.     

Disturbances of the blood-brain barrier without expression of amyloid precursor protein- containing neuritic clusters or neuronal loss during late stages of thiamine deficiency in guinea pigs

Generalized oxidative deficits associated with experimental thiamine deficiency (TD) lead to selective neurodegeneration. In mouse brain, TD produces region-specific breach of the blood-brain barrier (BBB), neuronal loss and an accumulation of amyloid precursor protein (APP) in abnormal neurites. The APP-laden abnormal neurites within the damaged areas of mouse brain aggregate into neuritic clusters which strikingly resemble the neuritic component of Alzheimer amyloid plaques. However, amyloid beta-peptide (Abeta) immunoreactivity has not been demonstrated in these neuritic clusters, possibly because the Abeta region of APP in mice contains three amino acid substitutions as compared with the amino acid sequence of human Abeta. In contrast, the guinea pig nucleic acid sequence is more related to the human sequence and the Abeta region is identical in sequence to that of human APP. Thus, the current studies tested whether the presence of an authentic Abeta fragment of APP (i.e., identical to that of man) might make guinea pigs more vulnerable to the development of Abeta-containing neuritic clusters following TD. During late stages of TD, BBB abnormalities, manifested by immunoglobulin G (IgG) extravasation and increased NADPH diaphorase reactivity in microvessels, occurred in brain areas known to be damaged by TD in mice. However, despite the prolonged thiamine deprivation and the advanced neurological symptoms of guinea pigs, no significant neuronal loss or altered APP/Abeta immunostaining occurred in any brain region. Microglial activation, another early marker of damage in mice, was not evident in thiamine-deficient guinea pig brain. Ferritin immunoreactivity and iron deposition in oligodendrocytes within areas of BBB abnormalities were either slightly enhanced or unchanged as compared to controls. This is the first report of brain abnormalities in the guinea pig model of dietary and pyrithiamine-induced TD. The results demonstrate species differences in the response to TD-induced damage, and further support the role of BBB and nitric oxide in the initial events in TD pathology.     

Lack of local suppression in orally tolerant CD8-deficient mice reveals a critical regulatory role of CD8+ T cells in the normal gut mucosa

We found that feeding keyhole limpet hemocyanin (KLH) to CD8-deficient (CD8-/-) mice induced oral tolerance that was comparable in both magnitude and quality to that induced in wild-type (wt) mice. The tolerance was dose dependent, and only higher doses of KLH caused significant reduction in specific Ab and T cell responses. Both Th1 and Th2 CD4+ T cell functions were affected. Feeding KLH together with cholera toxin (CT) adjuvant, however, abrogated the induction of oral tolerance equally well in CD8-/- and wt mice. On the contrary, CT adjuvant was unable to abrogate already established oral tolerance in both CD8-/- and wt mice. Most importantly, whereas Ag feeding induced hyporesponsiveness in systemic as well as in local gut IgA responses in wt mice, a lack of local suppression was evident in orally tolerant CD8-/- mice following oral immunizations. Thus, contrary to the situation in wt mice, Ag feeding induces systemic, but not local, gut IgA hyporesponsiveness in CD8-/- mice, suggesting that CD8+ T cells in the normal gut mucosa exert an important down-regulatory function. In wt mice the local suppression extended to an unrelated Ag, OVA, given together with KLH and CT adjuvant, i.e., bystander suppression. Based on these results we propose that tolerance induced by feeding Ag is highly compartmentalized, requiring CD8+ T cells for local suppression of IgA responses, whereas systemic tolerance may affect CD4+ T cells of both Th1 and Th2 types independently of CD8+ T cells. Finally, the adjuvant effect of CT abrogates induction, but not established, oral tolerance through a mechanism that does not require CD8+ T cells.     

Prediction of major depression and dysthymia from CES-D scores among ethnic minority adolescents

Two tumour cell clones, 6D1 and 4C2 cells, which are defective both in the major histocompatibility gene complex (MHC) class I expression and in the endogenous antigen presentation, are recovered with interferon (IFN)-gamma treatment. The present study describes the ultrastructure of these cells by using scanning and transmission electron microscopy in relation to the effect of IFN-gamma treatment. The general morphology of these cells was found to be similar to each other and comparable to that of a tumour cell clone, 4A1 cells, of the same origin, normal in MHC class I expression; they exhibited a fibroblast-like appearance and had many blebs on all the cell surfaces, with desmosome-like junctions between cells. On IFN-gamma treatment, surface fine blebs appeared less, and mitochondria became more densely stained. Expression of MHC class I molecules on the cell surface was much higher in the IFN-gamma treated 6D1 and 4C2 cells than in untreated cells, when estimated by immunoelectron microscopy. The addition of an epitope peptide to these cells did not enhance the class I expression, which differed from other antigen presentation-defective cells such as RMA-S cells, nor change the cell surface morphology.     

cDNA cloning and characterization of a new human microsomal NAD+-dependent dehydrogenase that oxidizes all-trans-retinol and 3alpha-hydroxysteroids

We report the cDNA sequence and catalytic properties of a new member of the short chain dehydrogenase/reductase superfamily. The 1134-base pair cDNA isolated from the human liver cDNA library encodes a 317-amino acid protein, retinol dehydrogenase 4 (RoDH-4), which exhibits the strongest similarity with rat all-trans-retinol dehydrogenases RoDH-1, RoDH-2, and RoDH-3, and mouse cis-retinol/androgen dehydrogenase (相似文献   

Nuclear estradiol receptor in the adult rat uterus: a new exchange assay

A protamine exchange assay has been developed to measure uterine nuclear estrogen receptor in mature rats exposed to estradiol (E). After ovariectomized-adrenalectomized mature rats are injected with E, estrogen receptor (RnE) is extracted from uterine nuclei with 0.6 M potassium chloride, diluted, and quantitatively precipitated with protamine sulfate. The precipitate is subjected to a ligand exchange with radiolabeled estradiol (E), with or without unlabeled diethylstilbesterol, to determine nonspecific binding. At 37 degrees C complete exchange of E for E in RnE is observed at 2.5 h; virtually no receptor degradation occurs up to at least 5 h. Exchange does not occur at 4 degrees C. Using the protamine assay, the depletion of cytoplasmic estrogen receptor (Rc) and the accumulation of RnE were studied at various doses of E at specific time points. Increasing doses of E result in a decrease of Rc with an equal increase of RnE. At the highest dose of E (10 mug) Rc is completely depleted within 10 min, by 6 h it is 25% replenished, and by 24 h returns to slightly above control levels. Within 10 min after the injection, RnE increases to 80-90% of the original cytoplasmic level of receptor (approximately 2-3 pmol/mg of DNA or approximately 1.5 pmol/100 mg of uterus). At 6 h RnE is 75% depleted and it is completely absent at 24 h. The protamine assay permits precise quantitative studies of nuclear estrogen receptor and avoids the problems of receptor degradation and excessive nonspecific binding often found in exchange reactions at elevated temperatures.     

4-[(1H-imidazol-4-yl) methyl] benzamidines and benzylamidines: novel antagonists of the histamine H3 receptor

A series of amidine substituted phenyl-, benzyl-, and phenethylimidazoles based on the known H3 agonist SK&F 91606 (4) has been synthesized and tested as ligands for the histamine H3 receptor. Insertion of a phenyl ring between the imidazole ring and the amidine moiety produces antagonists. The benzyl series was found to be the most potent and was further investigated. Compounds 9c and 18 (entries 5 and 12, Table 1) are potent ligands for the H3 receptor with K(i) values of 16 nM and 7.2 nM respectively. In vivo, both compounds were shown to be equipotent to thioperamide (2), the standard H3 antagonist.