A review of the USAF/NASA proton bioeffects project: rationale and acute effects.

Ionizing radiation represented one of the important hazards facing the first manned lunar mission. A combined USAF/NASA project was conducted from 1963 through 1969 to estimate the relative biological effectiveness (RBE) of the radiations of space. Approximately 2000 primates (Macaca mulatta) and 5000 mice were irradiated with protons and electromagnetic radiations. The proton energies studied were selected to be representative of the proton spectrum in space. Much of the project was concerned with the use of cyclotrons for proton irradiations and with dosimetry. Biological measurements included clinical findings, physiological changes, hematological changes, histopathology, and mortality. When allowance was made for variation of response as a consequence of depth-dose distribution, the RBE for protons was approximately 1. This was anticipated from earlier theoretical studies and radiation therapy in humans with high-energy charged-particle beams.     

Automated thermometric enzyme immunoassay of human proinsulin produced by Escherichia coli

We have determined and monitored the production and release of human proinsulin by genetically engineered Escherichia coli cells. Several M9 media samples were analyzed sequentially after centrifugation with the aid of a rapid automated flow-through thermometric enzyme-linked immunosorbent assay (TELISA) system. The response time was 7 min after sample injection and a single assay was complete after 13 min. Insulin concentrations in the range of 0.1-50 micrograms/ml could be determined. The TELISA method correlated well with conventional radioimmunoassay determinations. Standard curves were reproducible over a period of several days even when the immobilized antibody column was stored at 25 degrees C in the enzyme thermistor unit. Thus, immediate assay start up was possible.     

A transferrin receptor antibody represents one signal for the induction of IL 2 production by a human T cell line

We previously demonstrated a two-signal requirement for the activation of the human T cell lines Jurkat and HUT 78. Interleukin 2 (IL 2) production by these lines can be induced by phytohemagglutinin (PHA), T3 antibodies, or calcium ionophores, but only in combination with phorbol myristate acetate (PMA). To obtain further information about surface structures involved in T cell activation, we produced a monoclonal antibody that could substitute for PMA in the activation of HUT 78. This antibody, designated J64, induced IL 2 secretion by HUT 78 in combination with PHA, T3 antibodies, or calcium ionophores, however not by itself. J64 also had other PMA-like effects on HUT 78, such as an increase in IL 2 receptor expression and an inhibition of cell growth. J64 was shown to immunoprecipitate the transferrin receptor (TfR). However, it bound to an epitope different from those recognized by other TfR antibodies and different from the transferrin-binding site. In addition, other previously described TfR antibodies did not, like J64, function as activating stimuli for HUT 78. Possible mechanisms for activation signaling in T cells involving the TfR are discussed.     

Novel approach to the study of the antigenicities and receptor functions of carbohydrate chains of glycoproteins

This report describes the construction of neoglycolipids as a novel approach to determining the antigenicities and receptor functions of minute amounts of oligosaccharides derived from glycoproteins. Reduced oligosaccharides are converted into oligosaccharide alditols by controlled selective periodate oxidation and conjugated to phosphatidyl ethanolamine dipalmitoyl by reductive amination. The resulting neoglycolipids can be rendered multivalent by binding to polyvinylchloride or silica plates or they can be incorporated into liposomes and their antigenicities and receptor activities determined in low concentrations by direct binding or inhibition of binding assays. This approach, which has been successfully used with two monoclonal antibodies and a plant lectin, should be widely applicable to the direct analysis of O- and N-glycosidically linked carbohydrate chains of glycoproteins and proteoglycans both as antigens and recognition structures of diverse receptor systems.     

Different modes of ligand binding to the hepatic galactose/N-acetylgalactosamine lectin on the surface of rabbit hepatocytes

A study of the binding of three different 125I-labeled, galactose-terminated ligands to the hepatic galactose/N-acetylgalactosamine-specific lectin found on the surface of rabbit hepatocytes revealed that the different ligands manifest different physical parameters of binding. Asialoorosomucoid (125I-ASOR) binding was best described as involving two independent classes of binding sites on rabbit hepatocytes, with 161 000 sites/cell with a dissociation constant of 0.44 nM and 292 000 sites/cell with a Kd of 9.7 nM. Asialotriantennary glycopeptide purified from human alpha-1 protease inhibitor and modified with tyrosine at the N-terminus to permit radioiodination (TRI) [Lee, Y. C., Townsend, R. R., Hardy, M. R., L?nngren, J., Arnarp, J., Haraldsson, M., & L?nn, H. (1983) J. Biol. Chem. 258, 199-202] was also found to bind to two apparent classes of binding sites but with different binding parameters: 292 000 sites/cell of Kd = 1.47 nM and 982 000 sites/cell of Kd = 25.3 nM. A synthetic ligand, alpha,beta-diaspartamide of tris[(beta-lactosyloxy)methyl](6-aminohexanamido)methane (di-tris-lac) containing six nonreducing galactose residues [Lee, R. T., Lin, P., & Lee, Y. C. (1984) Biochemistry 23, 4255-4261], was found to bind to 817 000 sites/cell of Kd = 0.63 nM and 1.23 X 10(6) sites/cell of Kd = 25.3 nM. Thus, there were many more total binding sites for TRI or di-tris-lac on the surface of rabbit hepatocytes than there were for asialoorosomucoid, although the dissociation constants were similar for all three ligands.(ABSTRACT TRUNCATED AT 250 WORDS)     

Validity of lymphoid cell line for enzymatic studies of GM2-gangliosidosis variant 0 (Sandhoff disease)

A lymphoid cell line established by Epstein-Barr virus (EBV)-transformation of peripheral blood B-lymphocytes from a patient with Sandhoff disease showed a severe deficiency of beta-N-acetylhexosaminidase activity (residual activity around 10% of that in lymphoid cell lines from normals or other lipidotic patients). This residual beta-N-acetylhexosaminidase was completely heat-labile in contrast to that of normals. The molecular forms of residual beta-N-acetylhexosaminidase from Sandhoff lymphoid cell line were separated by Con A-sepharose and electrofocusing. Their properties and electrofocusing profiles were compared to those from Sandhoff fibroblasts and from fetal brain: this comparison permitted to identify the residual molecular forms with Hex S and Hex C. The microheterogeneity of Hex S and Hex C, demonstrated by electrofocusing, was discussed. 2-Acetamido-2-deoxy-D-galactonolactone (GalNAcLone) showed a strong inhibitory effect on lysosomal Hex A, B and S, but only a very slight effect on Hex C. Studies of the inhibition type (competitive on Hex A, B and S and mixed on Hex C) gave some informations about the enzymatic site. Elsewhere, differences in affinity of GalNAcLone for the various isoenzymes could be utilized to define optimal assay conditions for specifically determining Hex C (standard assay containing 400 mumol/l of GalNAcLone). These results demonstrated that EBV-transformed lymphoid cell lines represent an accurate model system for enzymatic studies of Sandhoff disease.     

Gibberellins in immature seeds and dark-grown shoots of Pisum sativum

Gibberellins (GAs) A₁₇, A₁₉, A₂₀, A₂₉, A₄₄, 2OH-GA₄₄ (tentative) and GA₂₉-catabolite were identified in 21-day-old seeds of Pisum sativum cv. Alaska (tall). These GAs are qualitatively similar to those in the dwarf cultivar Progress No. 9 with the exception of GA₁₉ which does not accumulate in Progress seeds. There was no evidence for the presence of 3-hydroxylated GAs in 21 day-old Alaska seeds. Dark-grown shoots of the cultivar Alaska contein GA₁, GA₈, GA₂₀, GA₂₉, GA₈-catabolite and GA₂₉-catabolite. Dark-grown shoots of the cultivar Progress No.9 contain GA₈, GA₂₀, GA₂₉ and GA₂₉-catabolite, and the presence of GA₁ was strongly indicated. Quantitation using GAs labelled with stable isotope showed the level of GA₁ in dark-grown shoots of the two cultivars to be almost identical, whilst the levels of GA₂₀, GA₂₉ and GA₂₉-catabolite were significantly lower in Alaska than in Progress No. 9. The levels of these GAs in dark-grown shoots were 10²- to 10³-fold less than the levels in developing seeds. The 2-epimer of GA₂₉ is present in dark-grown-shoot extracts of both cultivars and is not thought to be an artefact.Abbreviations cv cultivar - GA_n gibberellin A_n - GC gas chromatography - GC-MS combined gas chromatographymass spectrometry - HPLC high-pressure liquid chromatography - KRI Kovats retention index - MeTMSi methyl ester trimethylsilyl ether     

The effect of neurotensin and secretin on gastric acid secretion and mucosal blood flow in man

Neurotensin stimulates pancreatic secretion directly and by potentiating the effect of secretin. Neurotensin also inhibits gastric secretion. Secretin inhibits gastric secretion as well, but whether it also interacts with neurotensin is not known. Secretin is known to inhibit gastric mucosal blood flow (GMBF). The effect of neurotensin on GMBF is not known. Acid secretion (triple lumen perfused orogastric tube) and GMBF ([14C]aminopyrine clearance) were therefore measured in 6 subjects during neurotensin, secretin and neurotensin plus secretin infusions. Neurotensin plus secretin reduced acid secretion by a median 130 (range 34-394) mumol/min which was significantly greater than either neurotensin at 36 (7-67) mumol/min or secretin 54 (20-347) mumol/min alone (P less than 0.05). This effect appeared independent of GMBF. Neurotensin plus secretin reduced GMBF by 14 (12-27) ml/min but not significantly more than neurotensin at 11 (3-20) ml/min or secretin 18 (2-27) ml/min alone. Further, there was no correlation between changes in acid output and GMBF during infusion of the peptides. We conclude that the inhibitory effects of neurotensin and secretin on gastric secretion are at least additive and together they may function as an 'enterogastrone'.     

Cellular location of heat-labile enterotoxin in Escherichia coli.

We demonstrated that both the A and B subunits of heat-labile enterotoxin from Escherichia coli are located in the periplasm. The toxin was shown to form aggregates in Tris-EDTA buffers which are routinely used for isolating membranes. The aggregates pellet upon centrifugation, and this may explain why several previous investigators have concluded that enterotoxin is associated with membranes.