Biopharmaceutical production: Applications of surface plasmon resonance biosensors

Surface plasmon resonance (SPR) permits the quantitative analysis of therapeutic antibody concentrations and impurities including bacteria, Protein A, Protein G and small molecule ligands leached from chromatography media. The use of surface plasmon resonance has gained popularity within the biopharmaceutical industry due to the automated, label free, real time interaction that may be exploited when using this method. The application areas to assess protein interactions and develop analytical methods for biopharmaceutical downstream process development, quality control, and in-process monitoring are reviewed.     

Insulin-like growth factor (IGF)-like peptide and 20-hydroxyecdysone regulate the growth and development of the male genital disk through different mechanisms in the silkmoth,Bombyx mori

It is well established that ecdysteroids play pivotal roles in the regulation of insect molting and metamorphosis. However, the mechanisms by which ecdysteroids regulate the growth and development of adult organs after pupation are poorly understood. Recently, we have identified insulin-like growth factor (IGF)-like peptides (IGFLPs), which are secreted after pupation under the control of 20-hydroxyecdysone (20E). In the silkmoth, Bombyx mori, massive amounts of Bombyx-IGFLP (BIGFLP) are present in the hemolymph during pupal-adult development, suggesting its importance in the regulation of adult tissue growth. Thus, we hypothesized that the growth and development of adult tissues including imaginal disks are regulated by the combined effects of BIGFLP and 20E. In this study, we investigated the growth-promoting effects of BIGFLP and 20E using the male genital disks of B. mori cultured ex vivo, and further analyzed the cell signaling pathways mediating hormone actions. We demonstrate that 20E induces the elongation of genital disks, that both hormones stimulate protein synthesis in an additive manner, and that BIGFLP and 20E exert their effects through the insulin/IGF signaling pathway and mitogen-activated protein kinase pathway, respectively. These results show that the growth and development of the genital disk are coordinately regulated by both BIGFLP and 20E.     

GRF is a highly potent activator of adenylate cyclase in the normal human, bovine and rat pituitary: interaction with somatostatin

The effect of GRF adenylate cyclase activation was studied in normal human, bovine and rat pituitary tissues. Human GRF (hGRF) activates adenylate cyclase in normal human pituitary membrane preparations in a concentration dependent manner (ED5 0 = 10(-11) M). In bovine pituitary cells hGRF stimulates GH secretion into the medium (ED5 0 = 7 X 10(-12) M) and activates adenylate cyclase (ED5 0 = 10(-11) M). In normal rat pituitary cells in monolayer culture, rat GRF (rGRF) stimulates adenylate cyclase (ED5 0 = 3 X 10(-11) M). In normal human pituitary membrane preparations and in normal rat pituitary cells in culture, somatostatin inhibits GRF-stimulated adenylate cyclase in a non-competitive manner, while it does not affect basal (i.e. non-stimulated) adenylate cyclase levels. VIP, a peptide which is structurally homologous to hGRF and rGRF is a weak GRF-agonist and activates adenylate cyclase in human and rat pituitary preparations at concentrations greater than 10 nM.     

Carotid ligation alters cholecystokinin-8 (CCK-8) immunoreactivity in gerbil brains

The unilateral or bilateral carotid arteries were ligated in gerbils used as a model of cerebral ischemia. The effect of different times of bilateral ischemia on the content of CCK-8 in fore regions of gerbil brain and the effect of 30 min of unilateral ischemia on the content of CCK-8 of the same regions in gerbils with or without neurological signs were observed. Our results show that the content of CCK-8 of cortex, basal ganglia, thalamus and hypothalamus decreased significantly. But, in brain stem it remained basically unchanged no matter whether the ischemia was unilateral or bilateral. This suggests that there is a close relationship between CCK-8 and cerebral ischemia, and raises the possibility that CCK-8 may be involved in cerebral ischemia through a yet unclear mechanism.     

免疫学方法作为早期诊断高血压患者肾损害的探讨

为了探讨高血压患者血压与肾脏损害的关系 ,分别以RIA和ELISA共同检测尿视黄醇结合蛋白、尿微量白蛋白、尿 β2 微球蛋白和血中半胱氨酸蛋白酶抑制物C。结果显示高血压组血CystatinC、尿RBP、mALB、β2 MG均显著高于对照组 (p <0 .0 1) ,且随病程的延长而有逐渐升高的趋势。血压昼夜节律异常组的血CystatinC、尿RBP、mALB和 β2 MG也明显高于血压昼夜节律正常组 (p <0 .0 1) ,提示高血压所致的肾损害不仅与血压增高程度有关 ,而且与血压在较高水平持续时间有关。结论 :高血压病引起的肾小球、肾小管损害 ,以检测CystatinC、RBP、mALB、β2 MG可作为早期诊断肾脏损害的敏感指标     

Regional Distribution of Neurotensin in Human Brain

Abstract: Neurotensin (NT) is an endogenous neuropeptide that is active in many preclinical screening tests for neuroleptic drugs. Using a radioimmunoassay, we have studied the regional distribution of NT in postmortem human brain and in cerebrospinal fluid. Highest levels were present in the hypothalamus, substantia nigra, and limbic areas, whereas much lower amounts were found in the cortex and striatum. The chromatographic properties of hypothalamic immunoreactivity on ion-exchange and high pressure liquid chromatography were similar to those of the synthetic tridecapeptide. We conclude that NT is present in human brain with a distribution resembling that seen in other species, such as rat and monkey.     

α2-Macroglobulin-proteinase complexes in cystic fibrosis serum Analysis by monoclonal antibodies and receptor-mediated endocytosis by normal human fibroblasts

α₂-Macroglobulin complexed to proteinases activated during clotting of cystic fibrosis and control sera was quantitated with the complex-specific monoclonal antibody F₂B₂. Similar amounts of α₂-macroglobulin complexes (between 40 and 90 μg/ml) were generated in cystic fibrosis and control sera. Endocystosis of the complexes by normal fibroblasts was compared to the amount of complexes detected by the F₂B₂-radioimmunoassay. Normal uptake was observed with 13 out of 14 cystic fibrosis sera. One cystic fibrosis serum showed strongly reduced endocytosis of the complexes. Complexes isolated from this serum on immobilized F₂B₂ failed to inhibit binding of purified α₂-macroglobulin-trypsin to its receptor, demonstrating deficient receptor-binding of these complexes. The low complexes could not be distinguised from complexes isolated from control or other cystic fibrosis sera by isoelectric focusing, rate electrophoresis or SDS-polyacrylamide gel electrophoresis.     

Nitric oxide/cGMP pathway components in the Leydig cells of the human testis

In this study we sought to determine whether the main components of the nitric oxide (NO) pathway are localized within the Leydig cells of the human testis and whether the soluble guanylyl cyclase (sGC), the enzyme that accounts for NO effects, is functionally active in these cells. Using an amplified immunocytochemical technique, immunoreactivity for nitric oxide synthase (NOS-I), sGC and cyclic guanosine monophosphate (cGMP) was detected within the cytoplasm of human Leydig cells. Distinct differences in staining intensity were found between individual Leydig cells, between cell groups and between Leydig cells of different patients. By means of a specific cGMP-RIA, a concentration-dependent increase in the quantity of cGMP was measured in primary cultures of human Leydig cells following exposure to the NO donor sodium nitroprusside. In addition, NOS-I immunoreactivity was seen in Sertoli cells, whereas cGMP and sGC immunoreactivity was found in Sertoli cells, some apically situated spermatids and residual bodies of seminiferous tubules. Dual-labelling studies and the staining of consecutive sections showed that there are several populations of Leydig cells in the human testis. Most cells were immunoreactive for NOS-I, sGC and cGMP, but smaller numbers of cells were unlabelled by any of the antibodies used, or labelled for NOS-I or cGMP alone, for sGC and cGMP, or for NOS-I and sGC. These results show that the Leydig cells possess both the enzyme by which NO is produced and the active enzyme which mediates the NO effects. There are different Leydig cell populations that probably reflect variations in their functional (steroidogenic) activity. Received: 27 March 1996 / Accepted: 14 July 1996