An enzymatic assay for the determination of α-amylase in serum was developed which employed a soluble substrate, maltoheptaose, and a coupled enzymatic indicator reaction consisting of α-glucosidase and the hexokinase—glucose-6-phosphate dehydrogenase system. We used high-performance liquid chromatography (HPLC) to establish the action pattern of maltoheptaose under the test conditions: (A) the action pattern of α-amylase alone, (B) that of the combined action of α-amylase and α-glucosidase. Conducive to this effort was: the availability of pure maltoheptaose and human pancreatic α-amylase; the development of an adequate procedure for sample pretreatment (partition chromatography on a mixed-bed ion exchanger) and of an HPLC system for separation of substrate and reaction products without interference from by-products of the assay (partition chromatography on a cation-exchange column with acetonitrile—water); and the use of a new, very sensitive refractometric detector revealing sugar amounts as low as 40 ng.We derived the following stoichiometric equations: The standard deviation of the rate coefficients is about 5%. 相似文献
Interleukin-1 is a cytokine involved in the immune response to infection and inflammation as well as a growth promotor for several cell types. Interleukin-1-like immunoreactive material has been found in the nervous system. We now show that antisera, which blocked the T-cell proliferative effects of interleukin-1 alpha, decreased neuronal cell counts (to 40% of control) in dissociated spinal cord cultures derived from fetal mice. This neuronal loss was prevented by addition of interleukin-1 alpha, and to a lesser extent by interleukin-1 beta. Exogenous interleukin-1 alpha increased the survival of neurons when added to cultures in which the electrical activity was blocked with tetrodotoxin, whereas no such cytokine-related increase in neuronal survival was observed in electrically active cultures. The antiserum-induced death could also be prevented by cotreatment of the cultures with 0.1 nM vasoactive intestinal peptide, a substance that induces the secretion of neuronal trophic factors from nonneuronal spinal cord cells and thereby increases neuronal survival in electrically inactive cultures. These studies indicate that the cytokine interleukin-1, or an immunologically cross-reactive protein, can increase neuronal survival. 相似文献
Full-thickness human cadaver skin was maintained on the dorso-lateral thoracic region of hairless mice whose immune rejection mechanism was suppressed using anti-mouse-thymocyte globulin. The bacterial profile of the pregrafted skin did not differ significantly from the normal human microflora. In contrast, the murine skin exhibited quantitative and qualitative differences from the human flora, in particular by the complete absence of Propionibacterium acnes, the dominant bacterium on sebum-rich areas of human skin. The normal microbial profile of the human grafts was maintained throughout the experimental period despite the novel environmental milieu. There was little contamination of the grafts from the normal murine flora. It was concluded that the grafted human skin would provide a realistic model for studying the ecology of human cutaneous micro-organisms. 相似文献
Summary Restriction maps of several recombinant plasmids representing a section of the E. coli K12 chromosome 35,000 bp in size with the genes phoA, proC and phoB were prepared. The orientation of phoA and the exact position of its N-terminal end on this map were determined by identifying a subfragment which carried the phoA promoter and by determining the nucleotide sequence of a 160 bp portion of this subfragment comprising the codons for the N-terminal end of pre-alkaline phosphatase. From this DNA sequence the leader sequence of alkaline phosphatase which consists of 21 amino acids was derived. 相似文献
Diets rich in polyunsaturated fatty acids lower plasma HDL cholesterol concentrations when compared to diets rich in saturated fatty acids. We investigated the mechanistic basis for this effect in the hamster and sought to determine whether reduced plasma HDL cholesterol concentrations resulting from a high polyunsaturated fat diet are associated with a decrease in reverse cholesterol transport. Animals were fed semisynthetic diets enriched with polyunsaturated or saturated fatty acids for 6 weeks. We then determined the effect of these diets on the following parameters: 1) hepatic scavenger receptor B1 (SR-BI) mRNA and protein levels, 2) the rate of hepatic HDL cholesteryl ester uptake, and 3) the rate of cholesterol acquisition by the extrahepatic tissues (from de novo synthesis, LDL and HDL) as a measure of the rate of reverse cholesterol transport. Compared to saturated fatty acids, dietary polyunsaturated fatty acids up-regulated hepatic SR-BI expression by approximately 50% and increased HDL cholesteryl ester transport to the liver; as a consequence, plasma HDL cholesteryl ester concentrations were reduced. Although dietary polyunsaturated fatty acids increased hepatic HDL cholesteryl ester uptake and lowered plasma HDL cholesterol concentrations, there was no change in the cholesterol content or in the rate of cholesterol acquisition (via de novo synthesis and lipoprotein uptake) by the extrahepatic tissues.These studies indicate that substitution of polyunsaturated for saturated fatty acids in the diet increases SR-BI expression and lowers plasma HDL cholesteryl ester concentrations but does not affect reverse cholesterol transport. 相似文献
With the aim of investigating the mechanisms that maintain auxin homeostasis in plants, we have monitored the net uptake and metabolism of exogenously supplied indole-3-acetic acid (IAA) and naphthalene-1-acetic acid (NAA) in seedlings of wild type and the IAA-overproducing mutant sur1 of Arabidopsis thaliana . Tritiated IAA and NAA entered the seedling tissues within minutes and were mostly accumulated as metabolites, probably amino acid and sugar conjugates. The mutant seedlings were marked by a strong increase of [3H]IAA metabolism and a reduction of the accumulation levels of both free [3H]IAA and [3H]NAA. The same characteristics were observed in wild-type seedlings grown on 5 μ M picloram. We measured [3H]NAA uptake in the presence of high concentrations of unlabeled NAA or the auxin efflux carrier inhibitor naphthylphthalamic acid (NPA). This abolished the difference in free [3H]NAA accumulation between the mutant or picloram-treated seedlings and wild-type seedlings. These data indicated that active auxin efflux carriers were present in Arabidopsis seedling tissues. Picloram-treated seedlings and seedlings of the IAA-overproducing mutant sur1 displayed increased auxin efflux carrier activity as well as elevated conjugation of IAA. There is previous evidence to suggest that conjugation is a means to remove excess IAA in plant cells. Here, we discuss the possibility of efflux constituting an additional mechanism for regulating free IAA levels in the face of an excess auxin supply. 相似文献
At least 50 different mutations in the presenilin 1 gene have been shown to cause early onset familial Alzheimer's disease. Although presenilin 1 has an obvious role in the pathogenesis of Alzheimer's disease, its function is still unknown. In the present study, the occurrence and distribution of presenilin 1 mRNA was examined in rat peripheral organs as well as in the brain by in situ hybridization histochemistry, using a radiolabelled oligonucleotide probe. In comparison to the brain, a high presenilin 1 mRNA expression was found in the testis, kidney, spleen, adrenal gland and thymus. It was also observed in skeletal muscle, liver, small intestine and lung, whereas no presenilin 1 could be detected in the heart, spinal cord and pancreas. Since presenilin 1 mRNA was found to be abundant in peripheral tissues which apparently are not affected in Alzheimer's disease, additional functions of presenilin 1 are suggested, unrelated to its role in the pathological processes of the disease. 相似文献
Cell plasticity is a crucial hallmark leading to cancer metastasis. Upregulation of Rho/ROCK pathway drives actomyosin contractility, protrusive forces, and contributes to the occurrence of highly invasive amoeboid cells in tumors. Cancer stem cells are similarly associated with metastasis, but how these populations arise in tumors is not fully understood. Here, we show that the novel oncogene RASSF1C drives mesenchymal‐to‐amoeboid transition and stem cell attributes in breast cancer cells. Mechanistically, RASSF1C activates Rho/ROCK via SRC‐mediated RhoGDI inhibition, resulting in generation of actomyosin contractility. Moreover, we demonstrate that RASSF1C‐induced amoeboid cells display increased expression of cancer stem‐like markers such as CD133, ALDH1, and Nanog, and are accompanied by higher invasive potential in vitro and in vivo. Further, RASSF1C‐induced amoeboid cells employ extracellular vesicles to transfer the invasive phenotype to target cells and tissue. Importantly, the underlying RASSF1C‐driven biological processes concur to explain clinical data: namely, methylation of the RASSF1C promoter correlates with better survival in early‐stage breast cancer patients. Therefore, we propose the use of RASSF1 gene promoter methylation status as a biomarker for patient stratification. 相似文献