山药化学成分的研究

从胡椒科植物山蒟中分得两个新木脂素类成分(Ia)和(Ⅱ),以及巴豆环氧素(Crorepoxide)(Ⅲ)和β-谷甾醇(Ⅳ)。经光谱分析及化学鉴定,(Ia)、(Ⅱ)为新结构,分别命名为山蒟素 B(hancinone)、山蒟素 C(hancinone C)。     

Pregnancy diagnosis in zoo animals by estrogen determination in feces

Estrogen concentration in feces was investigated in five different herbivorous species of zoo animals. Using a nonspecific estrogen radioimmunoassay, in four species (red buffalo, yak, Grevy's zebra, and Nubian ibex) pregnancy was revealed by measuring estrogen concentration in feces. In hippopotamus, the levels of fecal estrogens were not different between pregnant and nonpregnant animals.     

Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning

We have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase [Van den Bosch, H., Aarsman, A. J., DeJong, G. N., & Van Deenen, L. M. (1973) Biochim. Biophys. Acta 296, 94-104].     

Expression of rat transforming growth factor alpha mRNA during development occurs predominantly in the maternal decidua.

Previous studies have shown that transforming growth factor alpha is expressed during rodent development. To establish the site(s) of transforming growth factor alpha mRNA expression during rat embryogensis, we performed in situ hybridization and Northern blot analyses on samples of embryonic and maternal tissues at various gestational ages. Our results indicate that the high levels of transforming growth factor alpha mRNA that are observed during early development are the result of expression in the maternal decidua and not in the embryo. Decidual expression appears to be induced after implantation, peaks at day 8, and then slowly declines through day 15 at which time the decidua is being resorbed. Expression of transforming growth factor alpha mRNA is highest in that region of the decidua adjacent to the embryo and is low or nondetectable in the uterus, placenta, and other maternal tissues. The developmentally regulated expression of transforming growth factor alpha mRNA in the decidua, together with the presence of epidermal growth factor receptors in this tissue, suggests that transforming growth factor alpha stimulates proliferation locally through an autocrine mechanism. Since epidermal growth factor receptors are present in the embryo and placenta, transforming growth factor alpha produced in the decidua may also act on these tissues through paracrine or endocrine mechanisms.     

Effect of the uvs-2 allele of Neurospora crassa on the mutagenic potency of two N-hydroxylaminopurines and 2-aminopurine in the ad-3 forward-mutation test

The mutagenic potencies of 3 purine analogs were determined in the ad-3 forward-mutation test in growing cultures of heterokaryon 59 (H-59), a nucleotide excision repair-deficient (uvs-2/uvs-2) 2-component heterokaryon of Neurospora crassa. Two N-hydroxylaminopurines, 2-amino-6-N-hydroxylaminopurine (AHA) and 6-N-hydroxylaminopurine (HAP), were potent and strong mutagens, respectively, whereas 2-aminopurine (AP) was a moderate mutagen. Dose-response curves showed that AHA and HAP were about equally mutagenic at low doses but that AHA was more mutagenic than HAP at high doses. Comparison of these results in H-59 with our earlier results in heterokaryon 12 (H-12) of N. crassa, which is identical to H-59 except for being DNA-repair-proficient (uvs-2+/uvs-2+), shows that the defect in nucleotide excision repair due to uvs-2 has little or no effect on the mutagenic potencies of these 3 purine analogs. Therefore, the nucleotide excision-repair pathway in N. crassa that is deficient in H-59 does not appear to have a major role in the repair of pre-mutational lesions induced by these 3 purine analogs. On the other hand, based on the controls of these experiments, the frequency of spontaneous ad-3 mutants was 4 greater in H-59 than in H-12. This result suggests that the nucleotide excision-repair pathway in N. crassa that is inactivated by the uvs-2 mutation has a major role in the repair of lesions that would lead to spontaneous mutation at the ad-3+ region if they were not repaired.     

Qualitative and quantitative comparison of brain proteins in Alzheimer's disease

In human brain extracts, most proteins of pathological interest in Alzheimer's disease are insoluble and their analysis is often performed on denatured and reduced samples by immunoblotting after electrophoresis on polyacrylamide gel in presence of sodium dodecyl sulfate. Because we needed to accurately compare the concentration of several proteins in brain extracts to investigate the etiology of the disease, the quantitative aspect of immunoblotting was assessed and the results compared for a soluble component with those obtained by electroimmunoassay. Glial fibrillary acidic protein (GFAP) and Tau proteins were analysed by immunoblotting in brain homogenates treated with the Laemmli sample buffer from 10 control and 25 Alzheimer's disease brains. The linearity of densitometric measures of dilutions for one given sample was demonstrated. A 8 to 16-fold GFAP increase in Alzheimer brain was established. With regard to Tau proteins it was possible to show the presence of two pathological Tau variants (Tau 64 and 69) in all the Alzheimer brain homogenates, furthermore, the amount of Tau 64 and 69 was proportional to the presence of neurofibrillary degeneration. As far as alpha 1-antichymotrypsin is concerned, we showed, in a second set of brain samples (14 control and 12 Alzheimer brains), discrepancies between the results obtained by immunoblotting and by electroimmunoassay while for a given sample linearity of immunoblotting measures of dilutions of this sample was demonstrated. Quantitation by immunoblotting of such components which can be quantified using other procedures is uncertain whereas the interest of immunoblotting is undoubted for the insoluble proteins in the brain extracts.     

Ouabain prevents loss of autoregulation in rat pial arterioles caused by reoxygenation after a brief hypoxic episode

Pial arteriolar diameter changes inversely with changes in systemic arterial blood pressure. Such changes are consistent with autoregulatory functions. These responses are reduced by a brief period of hypoxia followed by reoxygenation. By using an open cranial window preparation we assessed the changes in pial arteriolar diameters during blood pressure changes in rats induced by hemorrhage and reinfusion of blood, before and after a brief period of hypoxia. The slopes of the changes in pial arteriolar diameter as a function of mean arterial blood pressure were -0.47 +/- 0.26 micron/mmHg (mean +/- SD; 1 mmHg = 133.3 Pa) before hypoxia and -0.11 +/- 0.23 micron/mmHg after hypoxia in the untreated rats. In ouabain-treated rats, corresponding slopes were -0.42 +/- 0.24 and -0.46 +/- 0.22 micron/mmHg. The observed protective effects of ouabain might be a blockade of the Na-K pump in the sarcolemma of the vascular smooth muscle.     

Cellular retinoic acid-binding protein and the role of retinoic acid in the development of the chick embryo

The distribution of cellular retinoic acid-binding protein (CRABP) in four stages of chick development is described using an affinity-purified antibody against rat CRABP. CRABP is the protein to which retinoic acid (RA) binds when it enters cells and may reflect the requirement of those cells for RA. We found several discrete cell populations which showed high levels of immunoreactivity. Some were in the neural tube such as the commissural neurons and the dorsal roof plate. Some were of neural crest origin such as the dorsal root ganglia, sensory axons, sympathetic ganglia, and enteric ganglia. The remaining populations were certain connective tissue cells, limb bud cells, and the myotome. These results suggest that certain organ systems, particularly the nervous system, have a requirement for RA during development and they may further our understanding of the teratogenic effects of retinoids on the embryo.     

Studies on the structure of NADH: ubiquinone oxidoreductase complex: topography of the subunits of the iron-sulfur protein component

Resolution of the mitochondrial NADH:ubiquinone oxidoreductase complex (Complex I) by chaotropic agents result in the separation of three building blocks of the enzyme, designated FP (flavoprotein), IP (iron-sulfur protein), and HP (hydrophobic protein). FP contains three subunits of Mr 51, 24, and 9 kDa; one FMN; and two iron-sulfur clusters. Immunochemical studies with monospecific antibodies to the FP subunits have indicated that all three subunits of FP protrude from the inner mitochondrial membrane on the matrix side, whereas no reactive epitopes from these subunits were found exposed on the cytosolic side [A.-L. Han, T. Yagi, and Y. Hatefi (1988) Arch. Biochem. Biophys. 267, 490-496]. IP contains six subunits of Mr 75, 49, 30, 18, 15, and 13 kDa and four iron-sulfur clusters. In the present study, immunochemical experiments (enzyme-linked immunosorbent assays and 125I-protein A labeling) were carried out with monospecific antibodies to the above IP subunits and with bovine Complex I, submitochondrial particles, mitoplasts, and intact mitochondria as sources of antigens. Results have indicated that all six IP subunits protrude from the inner mitochondrial membrane into the matrix, and that the 75-kDa subunit, and possibly the 15-kDa subunit, protrude in mitoplasts from the cytosolic side as well. No epitopes reactive toward the monospecific antibodies to the 49-, 30-, 18-, and 13-kDa subunits were detected in mitoplasts.     

Resonance Raman studies of Escherichia coli sulfite reductase hemoprotein. 2. Fe4S4 cluster vibrational modes

Resonance Raman (RR) spectra from the hemoprotein subunit of Escherichia coli sulfite reductase (SiR-HP) are examined in the low-frequency (200-500 cm-1) region where Fe-S stretching modes are expected. In spectra obtained with excitation in the siroheme Soret or Q bands, this region is dominated by siroheme modes. Modes assignable to the Fe4S4 cluster are selectively enhanced, however, with excitation at 488.0 or 457.9 nm. The assignments are confirmed by observation of the expected frequency shifts in SiR-HP extracted from E. coli grown on 34S-labeled sulfate. The mode frequencies and isotopic shifts resemble those seen in RR spectra of other Fe4S4 proteins and analogues, but the breathing mode of the cluster at 342 cm-1 is higher than that observed in the other species. Spectra of various ligand complexes of SiR-HP reveal only slight sensitivity of the cluster terminal ligand modes to the presence of exogenous heme ligands, at variance with a model of ligand binding in a bridged mode between heme and cluster. Close examination of RR spectra obtained with siroheme Soret-band excitation reveals additional 34S-sensitive features at 352 and 393 cm-1. These may be attributed to a bridging thiolate ligand.