Structure analysis of trypsin inhibitors isolated from Cucurbitacae seeds. Circular dichroism studies

Structure analysis jointly based on circular dichroism and Chou and Fasman analysis has been performed on a homologous group of 12 squash proteinase inhibitors. The analysis favours a structure incorporating 40–45% helix which is tightly crosslinked by three disulphide bonds. The proposed structure is consistent with the previously published CD and ¹H n.m.r. spectra. The amino acid which forms the centre of the interaction between trypsin and the inhibitor, arginine⁵ or lysine⁵, is located on the N-terminus of one of the helical segments. The basic residue is orientated away from the rest of the structure and thus is ideally situated for interaction with the confined space of the trypsin active site.     

Complete amino acid sequence of arachin subunit of molecular weight 21000

A subunit of molecular weight 21 000 from arachin, the major peanut protein, was isolated in pure form and primary structure was determined. The subunit was fragmented with CNBr, trypsin, and NBS; the fragments were separated and isolated by PAGE, gel filtration, Dowex treatment, and paper electrophoresis, and Edman degradation on each fragment, including the intact subunit, was performed. The PTH-amino acids thus obtained were identified by UV spectroscopy and TLC. The complete sequence of 176 residues was established by overlapping technique.     

Diagnosis of Cystic Fibrosis, by Assay of Immuno-Reactive Trypsin in a Newborn Infant with Jejuno-lleal Atresias

ABSTRACT. A newborn infant with ante-natally ultrasonically diagnosed small bowel obstruction was found to have two isolated jejunal atresias at laparotomy. Assay of immuno-reactive trypsin enabled an early diagnosis of cystic fibrosis to be made, later confirmed by an elevated sweat sodium.     

臭氧和固定化微生物对黄腐酸氯化前,后致突变性的影响

为探索降低饮水致突变活性的途径，通过Ａｍｅｓ试验，对经臭氧和／或固定化微生物预处理的黄腐酸氯化前后形成的非挥发性有机物致突变性进行对比研究。研究表明，黄腐酸直接氯化后形成的ＮＶＯＣ致突变性为阳性，主要形成移码突变型直接致突变性；其它处理方式形成的ＮＯＶＣ致突变性为阴性。     

Effects of Proteolytic Enzyme Inhibitors on the Nasal Absorption of Vasopressin and an Analogue

Proteolytic enzyme inhibitors were examined as absorption enhancers for the nasal delivery of vasopressin (AVP) and desmopressin (l-d-8-DAVP) in rats. Aprotinin, soybean trypsin inhibitor, and camostat mesilate were used as enzyme inhibitors. The nasal absorption of AVP and l-d-8-DAVP was evaluated by measuring its antidiuretic effect. Nasal administration of AVP (0.005 IU/kg) or l-d-8-DAVP alone (2.5 ng/kg) produced a small antidiuretic effect. Coadministration with aprotinin (1000 and 10000 KIU/kg) or soybean trypsin inhibitor (1.25 and 6.25 mM) did not change the antidiuretic effect. However, coadministration with camostat mesilate (1 to 50 mM) significantly increased the antidiuretic effect and, thus, the nasal absorption of AVP and l-d-8-DAVP. The activities of aminopeptidase, cathepsin-B, and trypsin in the nasal mucosal tissue of rats were 7 nmol/min/mg protein, 0.7 nmol/min/mg protein, and 4.6 pmol/min/mg protein, respectively. Aprotinin and soybean trypsin inhibitor inhibited only the trypsin activity, whereas camostat mesilate inhibited aminopeptidase and trypsin activities. Aprotinin (MW 6500) and soybean trypsin inhibitor (MW 8000), with relatively high molecular weights, may not permeate into the nasal mucosal tissue. In contrast, camostat mesilate is slowly absorbed (8%/hr) and could inhibit the proteolytic activity in the nasal mucosa, resulting in enhanced nasal absoprtion of AVP and l-d-8-DAVP.     

Advances in the diagnosis and management of cystic fibrosis

Cystic fibrosis (CF), the most common lethal genetic disease affecting Caucasians, is a multi-system illness, most frequently characterized by childhood chronic obstructive pulmonary disease, pancreatic exocrine insufficiency, and abnormal sweat electrolyte concentrations. The diagnosis of CF is based on a combination of the above clinical findings and/or a positive family history of the illness in conjunction with an abnormal sweat test. The quantitative pilocarpine iontophoresis test is the sole acceptable method for diagnostic confirmation of the clinical suspicion of CF. A recent advance in the diagnosis of CF has been in the development of methods for neonatal detection. The immunoreactive trypsinogen (IRT) detection test is practical, adaptable to large scale screening of dried neonatal blood spots, relatively inexpensive, and promising for the detection of newborns with CF who have pancreatic insufficiency. However, the reliability and validity of this method have not yet been adequately established. Major advances in the treatment of patients with CF have emerged in the last decades, particularly in supportive pulmonary and nutritional care.     

Treatment of isolated fat cells with trypsin inhibits the effects of insulin on incorporation of leucine into protein

Summary Incorporation of 0.1 mM³H-leucine into protein by isolated fat cells, with pyruvate as the only extracellular carbohydrate, was stimulated by: a) insulin, 0.1–4 ng/ml, or b) a mixture of 19 amino acids, 0.05–5 mM. Treatment of fat cells with trypsin, under conditions known to reduce binding of insulin by their plasma membranes, abolished stimulation of incorporation by the hormone but not by the amino acid mixture. These results imply that the actions of insulin on incorporation of amino acids into protein are initiated by interaction with plasma membranes.A preliminary report of this work was presented on June 22, 1972, at the IVth International Congress of Endocrinology, Washington, D.C. (abstract No. 423).     

Anti-ageing and rejuvenating effects of quercetin

Homeostasis is a key feature of the cellular lifespan. Its maintenance influences the rate of ageing and it is determined by several factors, including efficient proteolysis. The proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. Alterations of proteasome function have been recorded in various biological phenomena including ageing and replicative senescence. Proteasome activities and function are decreased upon replicative senescence, whereas proteasome activation confers enhanced survival against oxidative stress, lifespan extension and maintenance of the young morphology longer in human primary fibroblasts. Several natural compounds possess anti-ageing/anti-oxidant properties. In this study, we have identified quercetin (QUER) and its derivative, namely quercetin caprylate (QU-CAP) as a proteasome activator with anti-oxidant properties that consequently influence cellular lifespan, survival and viability of HFL-1 primary human fibroblasts. Moreover, when these compounds are supplemented to already senescent fibroblasts, a rejuvenating effect is observed. Finally, we show that these compounds promote physiological alterations when applied to cells (i.e. whitening effect). In summary, these data demonstrate the existence of naturally occurring anti-ageing products that can be effectively used through topical application.     

水凝胶包埋酶(单凝胶和双网络凝胶)及磁性粒子固定化酶的制备及评价(英文)

酶固定化过程中,固定化酶的方法及其载体的选择是酶固定化过程的关键因素,适宜的固定化法和良好的载体微环境对酶活保持率提高和稳定性增强尤其重要。在本研究中,利用96孔微分析板评价了用于高通量筛选的水凝胶包埋酶(单凝胶和双网络凝胶)及磁性粒子固定化酶的制备方法及其对酶活性(保存时间,精度和重现性)的影响。胰蛋白酶(trypsin)成功地包埋在单凝胶和双网络凝胶中并且固定在磁性粒子上,然而,包埋在单凝胶和双网络凝胶,或固定在磁性粒子上的胃蛋白酶无法与底物反应。在对酶的适应性方面,与双网络凝胶比较,单凝胶和磁性粒子固定化法更加优越,适用于多种酶(如:胰蛋白酶,葡糖苷酸酶,CYP1A1)的固定。然而,我们也发现浸置后,以单凝胶包埋的固定酶有较多的损失。双网络凝胶包埋法只限于包埋胰蛋白酶,无法用于包埋其它酶,例如葡糖苷酸酶、CYP1A1和胃蛋白酶,因为双网络凝胶包埋法会由于丙烯酰胺和过硫酸胺的存在而使酶失去活性。在三种酶固定方法中,磁性粒子固定酶的方法能够最好地保留酶活性。另外,磁性粒子固定酶的稳定性比其他两种方法好,存放一周后胰蛋白酶和葡糖苷酸酶的活性没有任何下降。其次,磁性粒子固定法的重复利用重现性也良好。此外,尽管双网络凝胶法包埋酶的种类有限,但是我们认为通过改变丙烯酰胺等载体的选择和设计,改进载体微环境,可以使包埋酶的效率得到提高。     

比较四种消化方法对神经干细胞传代增殖及活性的影响

目的：观察4种消化方法对体外培养的神经干细胞（N SC s）增殖及活性的影响。方法：原代培养新生大鼠海马N SC s,采用机械消化、0.25%单纯胰酶、0.25%胰酶＋0.02%EDTA和0.02%EDTA 4种不同消化方法对N SC s球进行传代培养,经四唑盐比色法（M TT法）法检测不同方法对N SC s活性及增殖能力的变化。结果：与机械消化相比,0.25%单纯胰酶组,0.25%胰酶＋EDTA组消化后的大鼠海马N SC s球长时间不聚集,细胞增殖停顿,而单纯EDTA组则明显提高了N SC s的增殖及活性,其余各组差异无统计学意义。结论：随培养时间延长,单纯0.02%EDTA组能够显著提高体外培养的大鼠海马N SC s的增殖水平及活力,且对细胞的后续损伤相对较小。