Isolation and inhibition of a trypsin-like activity from larvae of corn borer (Ostrinia nubilalis) using reverse micelles

Summary Endoproteinase(s) was isolated from a freeze-dried powder of larvae of Ostrinia nubilalis using reverse micellar solutions. The inhibition of proteinase was studied in reverse micelles with commercial Bowman-Birk soybean trypsin inhibitor and three trypsin inhibitors recently isolated from ripe cruciferous seeds.     

Lymphocyte movements and interactions in microgravity

Cell-cell contacts and the formation of aggregates play an important role in the mitogen induced in-vitro activation of lymphocytes. The fact that the formation of cell aggregates is only slightly reduced in microgravity suggests that cells are moving and interacting also in space. Direct evidence was obtained for the first time in an experiment performed on a sounding rocket flight, where the movements and interactions of free-floating, non activated cells could be observed in real time in microgravity. In an experiment performed on the IML-2 mission in July 1994, the movements and interactions of human lymphocytes during activation with the mitogen Con A were studied in real time in microgravity.     

Carbohydrate receptor-mediated gene transfer to human T leukaemic cells

The mucin-type carbohydrate Tn cryptantigen (GalNAc1-O-Ser/Thr,where GalNAc is N-acetyl-D-galactosamine) is expressed in manycarcinomas, in haemopoietic disorders including the Tn syndrome,and on human immunodeficiency virus (HIV) coat glycoproteins,but is not expressed on normal, differentiated cells becauseof the expression of a Tn-processing galactosyltransferase.Using Jurkat T leukaemic cells which express high levels ofTn antigen due to deficient Tn galactosylation, we have establishedthe Tn antigen-mediated gene transfer and demonstrate the considerableefficiency of this approach. We used poly(L-lysine) conjugatesof the monoclonal antibody 1E3 directed against the Tn antigento deliver the luciferase and ß-galactosidase reportergenes to Jurkat cells by receptor-mediated endocytosis. Additionof unconjugated 1E3 reduced transfection efficiency in a concentration-dependentmanner and incubation with free GalNAc abolished DNA transfercompletely, indicating that gene delivery is indeed mediatedby the Tn antigen. Pre-treatment of Jurkat cells with Vibriocholerae sialidase, which uncovers additional Tn antigens, resultedin an improvement of gene transfection. Both human and chickenadenovirus particles attached to the DNA/polylysine complexstrongly augmented transgene expression. When the ß-galactosidase(lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis,up to 60% of the cells were positive in the cytochemical stainusing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside(X-gal) as a chromogenic substrate. The efficiency of the transferrinreceptor-mediated DNA uptake into Jurkat cells was comparativelylow, although these cells were shown to express considerableamounts of transferrin receptor. We show here that a mucin-typecarbohydrate antigen mediates highly efficient DNA uptake byendocytosis into Jurkat T cells. This method represents a 50-foldimprovement of Jurkat cell transfection efficiency over otherphysical gene transfer techniques. Specific gene delivery toprimary cancer cells exhibiting Tn epitopes may especially bedesirable in immunotherapy protocols. adenovirus endocytosis gene transfer T cell Tn antigen     

Anterograde trafficking of Toll-like receptors requires the cargo sorting adaptors TMED-2 and 7

Toll-Like Receptors (TLRs) play a pivotal role in immunity by recognising conserved structural features of pathogens and initiating the innate immune response. TLR signalling is subject to complex regulation that remains poorly understood. Here we show that two small type I transmembrane receptors, TMED2 and 7, that function as cargo sorting adaptors in the early secretory pathway are required for transport of TLRs from the ER to Golgi. Protein interaction studies reveal that TMED7 interacts with TLR2, TLR4 and TLR5 but not with TLR3 and TLR9. On the other hand, TMED2 interacts with TLR2, TLR4 and TLR3. Dominant negative forms of TMED7 suppress the export of cell surface TLRs from the ER to the Golgi. By contrast TMED2 is required for the ER-export of both plasma membrane and endosomal TLRs. Together, these findings suggest that association of TMED2 and TMED7 with TLRs facilitates anterograde transport from the ER to the Golgi.     

Early detection of cell metabolism with a silicon microsensor

A silicon microsensor (ISFET — Ion Sensitive Field Effect Transistor) has been used to detect the metabolism of a cell population cultured on a coverslip and positioned close to the sensor surface. The system output is analyzed as a function of cell density.     

Spectrophotometric measurement of pyruvate dehydrogenase complex activity in cultured human fibroblasts

A spectrophotometric assay for the pyruvate dehydrogenase complex (PDHC) has been adapted for use with cultured human firbroblasts. It is a coupled enzyme assay utilizing pigeon liver arylamine acetyltransferase to measure the acetyl-CoA produced by PDHC. Activity is proportional to fibroblasts protein and to tine and depends completely on added pyruvate, CoA and NAD. In extracts in which PDHC had been activated (dephosphorylated) by the method of Sheu et al. (Sheu, R.K.-F., Hu, C.C. and Utter, M.F. (1981) J. Clin. Invest. 67, 1463–1471), activities in control cell lines are 5–50 fold higher than in earlier reports. Low activity has been demonstrated in a line previously eported to be PDHC-deficient.     

Low molecular weight kininogen binds to platelets to modulate thrombin-induced platelet activation

The kininogens, high molecular weight kininogen (HK) and low molecular weight kininogen (LK), are multifunctional, single-gene products that contain bradykinin and identical amino-terminal heavy chains. Studies were performed to determine if LK would bind directly to platelets. 125I-LK specifically bound to gel-filtered platelets in the presence of 50 microM Zn2+. HK effectively competed with 125I-LK for the same binding site (Ki = 27 +/- 9 nM, n = 5). Similarly, the Ki for LK inhibition of 125I-LK binding was 12 +/- 1 nM (n = 3). Albumin, fibrinogen, factor XIII, and kallikrein did not inhibit 125I-LK binding to unstimulated platelets. 125I-LK (66 kDa) was not cleaved upon binding to platelets. The binding of 125I-LK to unstimulated platelets was found to be fully reversible by the addition of a 50 molar excess of unlabeled LK at both 10 and 20 min. LK binding to platelets was saturable with an apparent Kd of 27 +/- 2 nM (mean +/- S.E., n = 9) and 647 +/- 147 binding sites/platelet. Both LK and HK at plasma concentrations inhibited thrombin-induced platelet aggregation. LK and HK at about 5% of plasma concentration also inhibited thrombin-induced secretion of both stirred and unstirred platelets. Both kininogens were found to be noncompetitive inhibitors of proteolytically active thrombin binding to platelets. The kininogens did not inhibit D-phenylalanyl-prolyl-arginine chloromethyl ketone-treated thrombin from binding to platelets. These studies indicated that both kininogens have a region on their heavy chain which allows them to bind to platelets. Further, kininogen binding by its heavy chain modulates thrombin activation of platelets since it prevents proteolytically active thrombin from binding to its receptor.     

Amino acid sequence of k Sci, the Bence Jones protein isolated from a patient with light chain deposition disease

Light chain Sci was isolated from the urine of a patient affected by light chain deposition disease with an apparent exclusive localization to the kidney. Sci protein is an intact light chain: it consists of 214 amino acid residues and has an Mr of 23.65. Its complete primary structure has been determined by sequence analysis of the corresponding tryptic peptides and by partially sequencing the intact protein. Sequence comparison shows that Sci protein is strictly related to the light chains of kIIIa family (88% structural identity) which are usually expressed in autoimmune rheumatoid syndromes. Computer graphics model suggests a perturbation in k Sci three-dimensional structure due to the unusual replacement of residues 53 and 77.     

The bovine plasma retinol-binding protein. Amino acid sequence, interaction with transthyretin, crystallization and preliminary X-ray data

1. The primary structure of bovine plasma retinol-binding protein (RBP) has been determined and found to be more than 90% identical to human and rabbit RBPs, and more than 80% identical to rat RBP. Main changes in amino acid sequence are observed in two regions on the surface of the protein molecule (residues 138-148 and 169-183). 2. The interactions of bovine RBP with bovine and human transthyretins were investigated using the technique of fluorescence polarization. Bovine RBP was able to form high affinity complexes (K'd = 0.34 +/- 0.02 microM) with both bovine and human transthyretins, displaying a stoichiometry of approximately 2 molecules RBP/molecule transthyretin in both cases. The sites that participate in protein-protein interactions are thus very similar, and this tends to exclude the involvement of the superficial regions more significantly substituted in mammalian RBPs (residues 138-151 and 167-183) in the protein-protein recognition. 3. Bovine RBP has been crystallized (space group P2(1)2(1)2(1), with a = 4.61 nm, b = 4.91 nm, c = 7.61 nm) and the crystals are suitable for high-resolution X-ray diffraction studies.     

Non-enzymatic and microsome-dependent binding of polycyclic hydrocarbons to DNA and polynucleotides

The binding of tritium-labelled 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (BP) and 3-methylcholanthrene (MCA) to DNA or polynucleotides in vitro was re-examined both in the presence and in the absence of rat liver or human placental microsomes.A high level of non-enzymatic binding was evident when thymus DNA was used as acceptor. This non-enzymatic binding made it difficult to determine the effect of microsomes, except in the case of BP when induced rat microsomes were used. Better results were obtained using polynucleotides: a definite microsome-dependent binding occurred between all the polynucleotides and all the hydrocarbons tested.No clear evidence of binding catalysed by microsomes from human placenta was found except in polynucleotide-BP interactions: further studies are required to completely evaluate the ability of such nucleic acid-microsomal system for testing in vitro possible oncogenic substances in animals and humans.