A gene for bovine pancreatic trypsin inhibitor (BPTI) was fused to the coding sequence for the Escherichia coli alkaline phosphatase signal peptide and expressed in E. coli under the control of the alkaline phosphatase promoter. When induced in phosphate-depleted medium such cells produced a trypsin inhibitor that was indistinguishable from native, properly folded BPTI. In particular, the BPTI produced by E. coli had three disulfide bonds that appeared to be identical to those found in native BPTI, as assayed by sensitivity to iodoacetate, dithiothreitol, and urea. This expression/secretion system will make possible the production of variant BPTI molecules, thus allowing the perturbing effects of amino acid substitutions on BPTI folding, structure, and function to be assessed. 相似文献
The cystine-rich antifreeze polypeptides (AFP) from sea raven were fractionated by reverse-phase high performance liquid chromatography into several components, with SR2 (Mr 17,000) as the major AFP. Sea raven AFP cDNA clones were isolated from a liver cDNA library using a synthetic oligonucleotide, and the identity of one of the clones, C2-1, was confirmed by hybridization selection and cell-free translation. C2-1 encodes a pre-AFP of 195 amino acids with no evidence of any profragments. Comparison of the deduced amino acid sequence with partial peptide sequences from SR2 showed substitutions in at least four amino acid positions, suggesting that C2-1 cDNA codes for a minor component. Both the primary and the predicted secondary structures of sea raven AFP are completely different from those of other fish AFP. This further confirms that sea raven AFP belongs to a different class of antifreezes. The high frequency of reverse turns and the presence of paired hydrophilic amino acids in these structures are striking features of the protein and may contribute to their antifreeze action. 相似文献
Lipoxin A (5,6,15L-trihydroxy-7,9,11,13-eicosatetraenoic acid) and lipoxin B (5D,14,15-trihydroxy-6,8,10,12-eicosatetraenoic acid), two newly isolated compounds derived from the oxygenation of arachidonic acid in human leukocytes, inhibit the cytotoxic activity of human natural killer (NK) cells. Dose-response studies showed that both lipoxin A and lipoxin B inhibit, at submicromolar concentrations (ID50 10(-7) M), NK cell activity assayed against K562 target cells. Prostaglandin E2 (PGE2) also inhibited cytotoxicity, whereas both 15-HETE (5(S)-hydroxy-5,8,11,13-eicosatetraenoic acid) and leukotriene B4 (synthetic and biologically derived) were ineffective. PGE2 stimulated a time- and dose-dependent increase in intracellular cAMP, which was accompanied by a decrease in NK target cell binding. Lipoxin A and lipoxin B did not elevate intracellular cAMP, nor did they inhibit target cell binding. Together these findings suggest that lipoxin A and lipoxin B abrogate NK cell cytotoxicity at a step distal to target effector cell recognition. In contrast, PGE2 appears to exert its effect, at least in part, on cytotoxicity indirectly by decreasing the binding between target and effector cells (in vitro). Moreover, they suggest that novel oxygenated derivatives of arachidonic acid (i.e., lipoxin A, lipoxin B) may regulate the activities of NK cells. 相似文献
Male Long-Evans rats were injected with 0, 1, 3, or 6 mg/kg of cadmium chloride on the first day of life. Animals free of morphological stigmata at weaning were selected for study. Tissue concentrations of cadmium and operant behavior under various fixed-ratio (FR) schedules of reinforcement were evaluated when these rats were adults. Dose-related increases in cadmium were present in the brains, livers, and kidneys. Dose-related differences in behavior were most evident during the transition from fixed ratio 25 (FR 25 or 25 responses/reinforcer) to FR 75. An inverted U describes the relationship between response output during the transition to FR 75 and cadmium chloride dose response output increased at 3 mg/kg and decreased at 6 mg/kg. The rate decreases were not correlated with weight loss that appeared after some of the animals exposed to 6 mg/kg reached 60 days of age. Challenge doses of d-amphetamine revealed no interaction between neonatal exposure to cadmium and d-amphetamine. The occurrence of alterations in operant behavior in animals that appeared normal on a number of preweaning evaluations suggests that operant behavior in transition was sensitive to subtle effects not observed with other commonly used tests. The data provide evidence for delayed effects in the adult that are due to neonatal exposure to cadmium. 相似文献
The capsular polysaccharide of Klebsiella SK1 was investigated by methylation analysis, Smith degradation, and 1H NMR spectroscopy. The oligosaccharides (P1 and P2) obtained by bacteriophage ΦSK1 degradation of the polymer were studied by methylation analysis, and 1D- and 2D-NMR spectroscopy. The resulting data showed that the patent repeating unit is a branched pentasaccharide having a structure identical to the revised structure recently proposed for Klebsiella serotype K8 capsular polysaccharide. The 2D-NMR data showed that one third of the glucuronic acid residues in the SK1 polymer are acetylated at O-2, O-3, or O-4. FABMS studies confirmed the presence of monoacetylated glucuronic acid residues. Thus, the relationship between the Klebsiella K8 and SK1 polymers is akin to that found for Klebsiella polysaccharides K30 and K33, which have been typed as serologically distinct yet their structures differ only in the degree of acetylation. 相似文献
A proteolytically modified form of the Escherichia coli single-stranded DNA-Binding protein (SSB) has been crystallized from 15% saturated sodium citrate. Crystals as large as 1.0 mm x 0.3 mm x 0.2 mm were obtained and these diffract beyond 3A resolution. X-ray photographic analysis demonstrated a rhombohedral unit cell of space group R3 with an equivalent triple centered hexagonal unit cell having dimensions of a = b = 62.9A and c = 264.3A. These crystals were judged to be adequate for a three dimensional structure determination. 相似文献
Rats were fasted or fasted and refed simple purified diets so the effects of individual carbohydrates or fats could be studied. Freshly isolated hepatocytes from these animals were used to measure both apoE synthesis and mRNA levels so any changes in apoE synthesis that might occur without changes in its mRNA could be detected. Some of these experiments were done with both sexes. Both fasting and fasting and refeeding a 60% glucose fat-free diet significantly increased spoE synthesis. However, cyclic AMP is not likely to rapidly mediate the effect of fasting since dibutyryl cAMP slightly lowered (rather than increased) apoE synthesis and mRNA when injected into rats for 4.5 h. Dietary fat had no effect either in the absence of carbohydrate or when consumption of carbohydrate was constant in pair-fed rats. ApoE mRNA levels remained normal for 4 days in primary hepatocytes cultured in medium that had only amino acids as an energy source. Added hormones or fructose had no significant effect. Thus, only fasting and fasting and refeeding glucose were able to significantly change apoE synthesis or mRNA levels. Synthesis of apoE may be regulated to increase when apoE is secreted with very low density lipoprotein or when apoE in secreted high density lipoprotein is needed to acquire cholesteryl esters for the synthesis of bile salts and acids by liver. 相似文献