Enhancement of low-energy electron emission in 2D radioactive films.

High-energy radiation has been used for decades; however, the role of low-energy electrons created during irradiation has only recently begun to be appreciated. Low-energy electrons are the most important component of radiation damage in biological environments because they have subcellular ranges, interact destructively with chemical bonds, and are the most abundant product of ionizing particles in tissue. However, methods for generating them locally without external stimulation do not exist. Here, we synthesize one-atom-thick films of the radioactive isotope (125)I on gold that are stable under ambient conditions. Scanning tunnelling microscopy, supported by electronic structure simulations, allows us to directly observe nuclear transmutation of individual (125)I atoms into (125)Te, and explain the surprising stability of the 2D film as it underwent radioactive decay. The metal interface geometry induces a 600% amplification of low-energy electron emission (<10 eV; ref. ) compared with atomic (125)I. This enhancement of biologically active low-energy electrons might offer a new direction for highly targeted nanoparticle therapies.

Salivary peptide tyrosine-tyrosine 3-36 modulates ingestive behavior without inducing taste aversion.

Hormone peptide tyrosine-tyrosine (PYY) is secreted into circulation from the gut L-endocrine cells in response to food intake, thus inducing satiation during interaction with its preferred receptor, Y2R. Clinical applications of systemically administered PYY for the purpose of reducing body weight were compromised as a result of the common side effect of visceral sickness. We describe here a novel approach of elevating PYY in saliva in mice, which, although reliably inducing strong anorexic responses, does not cause aversive reactions. The augmentation of salivary PYY activated forebrain areas known to mediate feeding, hunger, and satiation while minimally affecting brainstem chemoreceptor zones triggering nausea. By comparing neuronal pathways activated by systemic versus salivary PYY, we identified a metabolic circuit associated with Y2R-positive cells in the oral cavity and extending through brainstem nuclei into hypothalamic satiety centers. The discovery of this alternative circuit that regulates ingestive behavior without inducing taste aversion may open the possibility of a therapeutic application of PYY for the treatment of obesity via direct oral application.

Immunological tolerance in vitro: kinetic studies at the cellular level.

When normal mouse spleen, cells in suspension are cultured in vitro in the presence of polymer from S. adelaide flagellin, an immune response can be obtained as measured at the level of single antibody-forming cells. Cultures were stimulated with different doses of antigen, ranging from 0.2 ng to 3 microg/ml of tissue culture fluid and it was found that the peak number of approximately 500 antibody-forming cells per 10(6) harvested cells by day 4 was antigen dose dependent, 2-20 ng/ml being the optimal concentration. When more than 1 microg/ml of polymer from S. adelaide together with either 20 ng/ml of polymer from S. waycross or with 4 x 10(6) sheep erythrocytes were placed in the system, unresponsiveness to S. adelaide, but immunity to the other antigens occurred simultaneously. Cells made immunologically tolerant in vitro to S. adelaide H antigens were transferred into syngeneic lethally irradiated recipients and challenged with the same antigen. The adoptive immune capacity in these mice, as measured at the level of the immunologically competent cell was reduced by 80-90% as compared with relevant controls. Attempts to induce low zone tolerance in vitro were without success. To study the kinetics of tolerance induction in vitro, cells were cultured with tolerogenic doses of antigen for various periods of time, washed, and subsequently cultured with immunogenic doses of antigen for 4 days. It was found, that immunological tolerance may be induced to a significant degree in vitro within a period of 15 min. Similar results were obtained when spleen cells were exposed for various lengths of time to tolerogenic doses of antigen but at a temperature of 4 degrees C instead of 37 degrees C. The results are taken as suggestive evidence that the initial step in tolerance induction is related to the direct interaction between the surface of immune competent cells and antigen molecules.

Specific immune response genes of the guinea pig. I. Dominant genetic control of immune responsiveness to copolymers of L-glutamic acid and L-alanine and L-glutamic acid and L-tyrosine.

The immunogenicity of three random copolymers of amino acids with L-glutamic acid and L-alanine (GA), L-glutamic acid and L-tyrosine (GT), or L-glutamic acid, L-alanine, and L-tyrosine (GAT), administered in complete Freund's adjuvant, was studied in several inbred and random-bred guinea pig strains. The animals were tested for delayed sensitivity and their sera were assayed for the presence of antibody directed against the immunizing polymer. All of the guinea pigs developing delayed hypersensitivity also had significant antibody levels in their sera. Inbred strain 2 guinea pigs responded to immunization with GA, but failed to form detectable responses to GT. Inbred strain 13 animals, on the other hand, responded to GT, but not to GA. The (2 x 13)F(1) hybrids responded to both GA and GT with both delayed hypersensitivity and circulating antibody. Thus, the ability of these inbred guinea pigs to respond immunologically to GA or GT is controlled by distinct autosomal dominant genes. A variable percentage of random-bred guinea pigs, depending on their source as well as their strain, responded to immunization with GA and with GT. All guinea pigs, both inbred and random bred, responded to immunization with GAT. The ability to respond immunologically to GAT, therefore, does not seem to be under simple genetic control. However, the levels of anti-GAT antibody found in the sera of animals lacking the ability to respond to GA were much lower than those detected in GA responder animals.

Specific immune response genes of the guinea pig. V. Influence of the GA and GT immune response genes on the specificity of cellular and humoral immune responses to a terpolymer of L-glutamic acid, L-alanine, and L-tyrosine.

The ability of guinea pigs to make immune responses to the random linear copolymer of L-glutamic acid and L-alanine, GA, and to L-glutamic acid and L-tyrosine, GT, is each controlled by a different immune response gene. On the other hand, the random linear terpolymer of L-glutamic acid, L-alanine, and L-tyrosine, GAT, which contains both GA and GT antigenic determinants, is immunogenic in all guinea pigs. After GAT immunization, all animals develop delayed hvpersensitivity and serum antibody specific for GAT. However, only those guinea pigs possessing the GA immune response gene demonstrate cross-reactive delayed hypersensitivity when challenged with GA. In addition, the anti-GAT antisera produced by those animals having the GA gene contain cross-reacting anti-GA antibodies. The sera from guinea pigs lacking the GA gene have no anti-GA antibody activity. Thus, we have demonstrated that a specific immune response gene controlling responsiveness to a "simple" antigen can determine the specificity of both cellular and humoral immune responses to a more complex antigen.

Solid-pse radioimmunoassay of protein biosynthesis.

Solid-phase radioimmunoassay was used for measuring the rate of radioisotope incorporation into a single protein species. Radioactive antigen was measured by its binding to specific antiserum covalently linked to insoluble bromoacetylcellulose. The insoluble antigen-antibody complex was collected on filters for counting. The assay is performed in about 4 hours as compared with several days for methods based on the precipitin reaction.

Simultaneous study of the metabolic turnover and renal excretion of salivary amylase- 125 I and pancreatic amylase- 131 I in the baboon.

The metabolic turnover of salivary and pancreatic amylase was studied in the baboon, an animal with a serum amylase level and renal clearance of amylase similar to man. Purified amylase was electrolytically iodinated. Although iodinated and uniodinated amylase had similar gel filtration, electrophoretic, enzymatic, glycogen precipitation characteristics, the labeled enzyme was cleared less rapidly by the kidney than was the unlabeled material. However, urinary iodinated amylase which had been biologically screened by the kidney had a renal clearance and serum disappearance rate indistinguishable from unlabeled amylase and thus can serve as a tracer in metabolic turnover studies. Administration of a mixture of salivary amylase-(125)I and pancreatic amylase-(131)I made it possible to simultaneously measure the serum disappearance and renal clearance of these two isoenzymes. The metabolic clearance of both isoenzymes was extremely rapid with half-times of about 130 min. This rapid turnover of serum amylase probably accounts for the transient nature of serum amylase elevation which frequently occurs in pancreatitis. Pancreatic amylase-(131)I was consistently cleared more rapidly (mean clearance ratio: 1.8) by the kidney than was salivary amylase-(125)I. This more rapid renal clearance of pancreatic amylase may help to explain the disproportionate elevation of urinary amylase relative to serum amylase observed in pancreatitis.

Stimulatory effects of induced phagocytosis on the function of isolated thyroid cells.

Stimulation of endocytosis is a very early effect of thyrotropin on thyroid. However, the relationship of the endocytotic process to the many other thyrotropin effects on thyroid is not clearly defined. Since phagocytosis in isolated thyroid cells is a presumed model for in vivo endocytosis of colloid, we induced phagocytosis in isolated thyroid cells by incubating them at 37 degrees C with 0.109-mu diameter polystyrene microbeads; phagocytosis was confirmed in each experiment by electron microscopy and/or spectrophotometric analysis of dioxane cell extracts. Cells incubated with 50-100-mu diameter polystyrene macrobeads (too large to ingest) served as controls. Microbead-induced phagocytosis in isolated thyroid cells was consistently accompanied by increases in: (a) cyclic 3',5'-adenosine monophosphate-(14)C formation from adenine-8-(14)C (66%); (b) iodide-(131)I trapping (40%); (c) protein and RNA synthesis (30%); (d) phospholipogenesis (50%); (e) alpha-aminoisobutyric acid-1-(14)C uptake (15%). 50- to 100-mu diameter polystyrene macrobeads did not influence cell function in any of these experiments. Aminotriazole, 5 x 10(-3) (M), a peroxidase inhibitor, blocked the stimulatory effect of microbead-induced phagocytosis on phospholipogenesis only. These studies indicate that in isolated thyroid cells the phagocytotic process, per se, may alter activity of the membrane-bound adenyl cyclase enzyme. The resultant increase in cyclic 3',5'-adenosine monophosphate may be a triggering mechanism for (some) subsequent metabolic changes occuring during phagocytosis. Since these changes mimic those induced by thyrotropin, it is suggested that a variety of thyrotropin effects on thyroid may be secondary to stimulation of colloid resorption and hormone secretion.

The mechanism of intestinal uptake and transcellular transport of IgG in the neonatal rat.

The transport of immunoglobulins across the intestinal mucosa of neonatal rats provides an excellent model for the study of transcellular protein transport. The mechanism of intestinal uptake and transcellular transport of plasma proteins has been studied in 12-14-day old rats using intraduodenally administered radioiodinated proteins. Appreciable quantities of rat IgG, mouse IgG, rabbit IgG, and all four subclasses of human IgG were taken up by the intestinal wall (19-54% of administered dose at 4 hr) and transported to the animal (10-35% of administered dose at 4 hr). In contrast there was little or no uptake of human IgM, IgA, and IgE and little or no transport of human IgM, IgA, IgD, IgE, albumin, transferrin, and ceruloplasmin. Both the uptake and transport of labeled IgG were significantly inhibited by unlabeled IgG. Further insight into the transport process was obtained from the observation that an appreciable proportion of the label of IgG in intestinal wall homogenates, but not in plasma or intestinal washings, migrated in a sucrose ultracentrifugation gradient much more rapidly than did the administered 7S molecules. This pattern was not observed with other proteins studied. This apparent binding of labeled IgG was also markedly inhibited by unlabeled IgG. In subcellular fractionation studies of intestinal homogenates the complexed labeled IgG was shown to be associated predominantly with cell membrane rather than cell sap fractions. In addition IgG could be shown to bind to purified enterocyte microvillous membranes in vitro. IT IS CONCLUDED THAT IN THE NEONATAL RAT: (a) the major processes involved in both intestinal uptake and transport of IgG are specific and saturable; (b) intestinal transport is associated with complexing of IgG molecules with membranes, most probably with enterocyte microvillous membranes; and (c) the part of the IgG structure involved in this process is probably similar to that involved in the concentration-catabolism effect but is not identical to that mediating other non-antigen combining functions of IgG. Our data are consistent with the existence of specific receptors for IgG on enterocyte microvillous membranes of the neonatal rat. Such receptors would be necessary for the specific uptake and transport of these molecules.

Platelet interaction with polymerizing fibrin.

Interaction of washed pig, rabbit, or human platelets with fibrinogen was studied during its transition to fibrin using photometric, isotopic, and electron microscopic techniques. Untreated fibrinogen and fully polymerized fibrin had no detectable effect on platelets. Fibrinogen, incubated with low concentrations of reptilase or thrombin, formed intermediate products which readily became associated with platelets and caused their aggregation. Neutralization of the thrombin did not prevent this interaction. In the absence of fibrinogen, reptilase did not affect platelets. The interaction of polymerizing fibrin with platelets was accompanied by small losses of platelet constituents (serotonin, adenine nucleotides, platelet factor 4, and lactic dehydrogenase). This loss did not appear to be the result of the platelet release reaction. Inhibitors of the release reaction or of adenosine diphosphate (ADP)-induced aggregation did not prevent the interaction of platelets with polymerizing fibrin. Apyrase or prostaglandin E(1) (PGE(1)) reduced the extent of platelet aggregation by polymerizing fibrin, but the amount of protein associated with platelets was slightly increased. The interaction of polymerizing fibrin with platelets was completely inhibited by ethylenediaminetetraacetate (EDTA) or ethylene glycol bis (beta-aminoethyl ether) N, N,N',N'-tetraacetic acid (EGTA).Fibers formed in solutions of polymerizing fibrin were larger in the presence than in the absence of washed platelets, suggesting that platelets affect fibrin polymerization. The adherence of platelets to polymerizing fibrin may be responsible for the establishment of links between platelets and fibrin in hemostatic plugs and thrombi.

Effects of paraformaldehyde fixation on nicotinic acetylcholine receptor binding in adult and developing rat brain sections.

In vitro receptor autoradiography requires unfixed tissue sections, but incubation and washing procedures often result in substantial tissue damage in sections from developing brain, hindering quantitative and qualitative analysis. Formaldehyde fixation greatly preserves morphology. However, fixation can interfere with pharmacological properties of receptors, increase in non-specific background labeling, or even destroy ligand binding sites. Two mild fixation protocols, 0.2% paraformaldehyde (pFA) and pFA vapor fixation, were compared for their ability to improve tissue morphology in postnatal day 7 (P7) brain slices and maintain binding of [125I]-epibatidine and [125I]-alpha-bungarotoxin to heteromeric and homomeric nicotinic acetylcholine receptors, respectively. Fixation greatly improved the ability of P7 brain slices to withstand incubation and washing procedures during binding, resulting in minimal or no loss of tissue after prior 0.2% pFA or vapor fixation, respectively. In adults, distribution pattern of [125I]-epibatidine was identical in fixed and unfixed slices, with no difference in total and non-specific labeling. Distribution of [125I]-alphaBTX labeling was similarly unaffected by 0.2% pFA fixation, but vapor fixation increased total and non-specific binding signal. Thus, mild fixations greatly improve tissue quality during receptor binding procedures and can preserve pharmacological properties of nicotinic acetylcholine receptors. However, different receptors or ligands might exhibit differential sensitivity to fixation protocols.

In vivo distribution of avidin-conjugated MX35 and (211)At-labeled, biotinylated poly-L-lysine for pretargeted intraperitoneal α-radioimmunotherapy.

PURPOSE: Avidin-coupled monoclonal antibody MX35 (avidin-MX35) and astatine-211-labeled, biotinylated, succinylated poly-l-lysine ((211)At-B-PL(suc)) were administered in mice to assess potential efficacy as an intraperitoneal (i.p.) therapy for microscopic tumors. We aimed to establish a timeline for pretargeted radioimmunotherapy using these substances, and estimate the maximum tolerable activity. METHODS: (125)I-avidin-MX35 and (211)At-B-PL(suc) were administered i.p. in nude mice. Tissue distributions were studied at various time points and mean absorbed doses were estimated from organ uptake of (211)At-B-PL(suc). Studies of myelotoxicity were performed after administration of different activities of (211)At-B-PL(suc). RESULTS: We observed low blood content of both (125)I-avidin-MX35 and (211)At-B-PL(suc), indicating fast clearance. After sodium perchlorate blocking, the highest (211)At uptake was found in kidneys. Red bone marrow (RBM) accumulated some (211)At activity. Mean absorbed doses of special interest were 2.3 Gy/MBq for kidneys, 0.4 Gy/MBq for blood, and 0.9 Gy/MBq for RBM. An absorbed dose of 0.9 Gy to the RBM was found to be safe. These values suggested that RBM would be the key dose-limiting organ in the proposed pretargeting scheme, and that blood data alone was not sufficient for predicting its absorbed dose. CONCLUSIONS: To attain a favorable distribution of activity and avoid major toxicities, at least 1.0 MBq of (211)At-B-PL(suc) can be administered 24 hours after an i.p. injection of avidin-MX35. These results provide a basis for future i.p. therapy studies in mice of microscopic ovarian cancer.