A microassay for proteases using succinylcasein as a substrate.

A photometric assay for proteases has been developed. A chemically modified casein whose amino groups were succinylated was used as a substrate. After incubation with trypsin, chymotrypsin, thermolysin, and subtilisin, the extent of hydrolysis of the substrate was determined with trinitrobenzene sulfonate (TNBS). The whole procedure of the assay was performed in the microtiter plate wells and the increase in the absorbance resulting from the reaction between TNBS and newly formed amino groups in the substrate was able to be determined with a high sensitivity by a microtiter plate reader, enabling the simultaneous measurement of a number of samples. Application of this method to the measurement of proteolytic activity contained in the protein extract of Tapes philippinarum is demonstrated.     

Electrotransformation of intact cells ofBrevibacterium flavum MJ-233

Summary Electroporation allowed transformation of intact cells ofBrevibacterium flavum MJ-233. The two plasmids used for electroporation were pCRY2 (6.3 kilobases) and pCRY3 (8.2 kilobases). Both plasmids contain the chloramphenicol-resistance gene and the autonomous replication origin inB. flavum MJ-233. The efficiency of electrotransformation was optimal with cells harvested at the middle log phase of growth, and was imporved by the addition of 1.0U/ml of penicillin G to the culture medium. The optimum yield of transformants per g DNA was 5×10⁴ when the cell suspension was pulsed at a cell density of 1×10¹⁰/ml and at a DNA amount of 1.0g.     

Biological and physicochemical characterization of recombinant human erythropoietins fractionated by Mono Q column chromatography and their modification with sialytransferase

Ten erythropoietin (EPO) fractions differing in sialic acid content, ranging from 9.5 to 13.8 mol mol^–1 of EPO, were obtained from baby hamster kidney cell-derived recombinant human EPO by Mono Q column chromatography. The mean pI values of the EPO fractions determined by IEF-gel electrophoresis systematically shifted from 4.11 to 3.31, coinciding with the sialic acid content, without a change in the constitution of asialo N-linked oligosaccharides of each fraction. Although a linear relationship between thein vivo bioactivity and the sialic acid content of the fractionated, samples was observed until 12.1 mol mol^–1 of EPO, there was no further increase in their activity over 12.4 mol mol^–1 of EPO. On the other hand, an inverse relationship between thein vitro bioactivity and sialic acid content of EPO was observed. Also, we showed that thein vivo bioactivity of some fractions with low sialic acid contents was increased after treatment with 2,6-sialyltransferase, but thein vivo bioactivity of the other fractions with high sialic acid contents was either decreased or not affected.Abbreviations EPO erythropoietin - rHuEPO recombinant human erythropoietin - hCG human chorionic gonadotropin - BHK baby hamster kidney - CHO Chinese hamster ovary - NeuAc N-acetyl neuraminic acid - Gal galactose - HRCs hemolyser-resistant cells - WST-1 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Na - IEF isoelectric focusing - pI isoelectric point     

Characterization of peroxidases in luminol chemiluminescence coupled with copper-catalysed oxidation of cysteamine

Hydrogen peroxide formed during the course of the copper(II)-catalysed oxidation of cysteamine with oxygen was continuously determined by a peroxidase (POD)-catalysed luminol chemiluminescence (CL) method. Horseradish peroxidase (HRP), lactoperoxidase (LPO) and Arthromyces ramosus peroxidase (ARP) were used as a CL catalyst. The respective PODs gave specific CL intensity-time profiles. HRP caused a CL delay, and ARP gave a time-response curve which followed the production rate of H₂O₂. LPO gave only a weak CL flash which decayed promptly. These differences of CL response curves could be explained in terms of the different reactivities of PODs for superoxide anion and the different formation rate of luminol radicals in the peroxidation of luminol catalysed by POD.     

Striatal L-DOPA Decarboxylase Activity in Parkinson's Disease In Vivo: Implications for the Regulation of Dopamine Synthesis

Abstract: L-DOPA is a large neutral amino acid subject to transport out of, as well as into, brain tissue. Competition between dopamine synthesis and L-DOPA egress from striatum must favor L-DOPA egress if decarboxylation declines relatively more than transport in Parkinson's disease. To test this hypothesis, we injected patients with Parkinson's disease with a radidabeled analogue of L-DOPA and recorded regional brain radioactivity as a function of time by means of positron emission tomography. We simultaneously estimated the activity of the decarboxylating enzyme and the amino acid transport. In the striatum of patients, we found the L-DOPA decarboxylase activity to be reduced in the head of the caudate nucleus and the putamen. However, the rate of egress of the DOPA analogue was unaffected by the disease and thus inhibited dopamine synthesis more than predicted in the absence of L-DOPA egress.     

Purification and Heterogeneous Localization of Sarcophaga Lectin Receptor on the Surface of Imaginal Discs of Sarcophaga peregrina (Flesh Fly)

Previously, we reported autocrine involvement of Sarcophaga lectin in the development of Sarcophaga imaginal discs (Kawaguchi et al. , Dev. Biol. 144 , 86–93 (1991)). In this study, we purified Sarcophaga lectin binding protein from the membrane fraction of cultured embryonic cells of Sarcophaga to near homogeneity and raised a monoclonal antibody against it. Histochemical analysis using the monoclonal antibody revealed that this binding protein is distributed heterogeneously on the surface of leg imaginal discs. This binding protein was especially clearly localized in the central region of the basal side of leg discs which forms the junction between the leg and body, suggesting the participation of Sarcophaga lectin in morphogenesis of the basal region of the developing leg.     

Genetics and biology of the sawfly,Athalia rosae (Hymenoptera)

Hymenopteran insects are a unique group of animals in which arrhenotokous reproduction (haploid males develop from unfertilized eggs) is a rule. Males produce sperm through a non-reductional maturation division. A sawfly species,Athalia rosae ruficornis Jakovlev (Tenthredinidae, Symphyta, Hymenoptera), has been introduced as a new experimental material for studies on genetics and developmental biology. Basic features relating to the potential usefulness of the species in elucidating some of the important genetic and developmental biological problems are described.     

Stimulation of endothelial cell migration in culture by ladsin,a laminin-5-like cell adhesion protein

Summary Ladsin is a laminin-like cell-adhesive scatter factor with potent cell motility-stimulating ability and was purified from serum-free conditioned medium of a malignant human gastric adenocarcinoma cell line STKM-1. To test its possible role in tumor angiogenesis, we investigated its effect on primary culture of endothelial cells (human umbilical vein endothelial cells) and endothelial cell line ECV304 in this study. Cell adhesion and motility effects of ladsin were observed in both types of endothelial cells. In cell-attachment assay, ladsin interacted with integrin α3β1 that was expressed on the endothelial cell surface. In Boyden chambers, ladsin stimulated both directed and random migration of ECV304 cells. Ladsin induced repair of artificial wounds generated in ECV304 cell monolayers by stimulating cell migration. Ladsin did not affect the growth rate of ECV304 cells at a low cell density but significantly increased the saturation cell density. These results suggest that ladsin may be involved in the adhesion and migration of endothelial cells under some physiological and pathological conditions.     

Regulation of DOPA Decarboxylase Activity in Brain of Living Rat

Abstract: To test the hypothesis that l -DOPA decarboxylase (DDC) is a regulated enzyme in the synthesis of dopamine (DA), we developed a model of the cerebral uptake and metabolism of [³H]DOPA. The unidirectional blood-brain clearance of [³H]DOPA ( K ^D₁) was 0.049 ml g⁻¹ min⁻¹. The relative DDC activity ( k ^D₃) was 0.26 min⁻¹ in striatum, 0.04 min⁻¹ in hypothalamus, and 0.02 min⁻¹ in hippocampus. In striatum, 3,4-[³H]dihydroxyphenylacetic acid ([³H]DOPAC) was formed from [³H]DA with a rate constant of 0.013 min⁻¹, [³H]homovanillic acid ([³H]HVA) was formed from [³H]DOPAC at a rate constant of 0.020 min⁻¹, and [³H]HVA was eliminated from brain at a rate constant of 0.037 min⁻¹. Together, these rate constants predicted the ratios of endogenous DOPAC and HVA to DA in rat striatum. Pargyline, an inhibitor of DA catabolism, substantially reduced the contrast between striatum and cortex, in comparison with the contrast seen in autoradiograms of control rats. At 30 min and at 4 h after pargyline, k ^D₃ was reduced by 50% in striatum and olfactory tubercle but was unaffected in hypothalamus, indicating that DDC activity is reduced in specific brain regions after monoamine oxidase inhibition. Thus, DDC activity may be a regulated step in the synthesis of DA.     

Cloning of cDNA for Vitellogenin of the Parasitoid Wasp, Pimpla nipponica (Hymenoptera: Apocrita: Ichneumonidae): Vitellogenin Primary Structure and Evolutionary Considerations

The cDNA for vitellogenin (Vg) of the parasitoid wasp Pimpla nipponica (Hymenoptera: Apocrita) was cloned and sequenced.¹ The deduced amino acid sequence with 1807 residues was obtained. The N-terminal 20 amino acids chemically determined for vitellin (Vn) agreed completely with the deduced 20 amino acids that follow the 16 amino acid residues for putative signal peptide. The cDNA clone for the Vg of the turnip sawfly Athalia rosae (Hymenoptera: Symphyta), previously obtained and partially sequenced, was also completely sequenced and the amino acid sequence deduced. Amino acid sequences were compared between these two species and also with known Vg sequences from other insects. Common to all these insects is the presence of two long regions with relatively well-conserved amino acid sequences, one near the N-terminal extending 267–282 residues (including two cysteines at conserved locations), and the other starting at position 450 to 655 and extending 279–283 residues, and of a region at the C-terminal extending some 200 residues (about 250 in Aedes aegypti due to the presence of a serine-rich stretch) with 10 cysteines at conserved locations. A molecular phylogenetic tree was constructed.