A rapid,sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots

A rapid, sensitive method has been developed to detect antibody-antigen complexes on “Western blots.” The methods of H. Towbin, T. Staehlin, and J. Gordon were used to separate and blot the antigens onto nitrocellulose. The remaining sites of attachment were blocked and the nitrocellulose was washed with polyoxyethylenesorbitan monolaurate (Tween 20). The blot was then reacted with the antiserum or hybridoma supernate to be tested. After the antigen-antibody reaction was completed, the blot was washed and treated with anti-antibody which has been conjugated to alkaline phosphatase. The alkaline phosphatase was detected by the reduction of the tetrazolium salt to diformazan by the hydrogen ions released in the formation of indigo by the reaction of the phosphatase on the indoxyl phosphate. The advantages of this method over previously described techniques are (1) use of Tween 20 allows the blot to be stained with Coomassie blue, (2) the substrates of the alkaline phosphatase reaction are stable for long periods of time, (3) the reaction products form an intense blue color which does not fade, (4) the resolution is extremely good with little to no band broadening, (5) the reaction is sensitive to picogram quantities of antigen, and (6) the reaction is quantitative.     

Breeding-site productivity of the swamp forest mosquitoes Mansonia bonneae and Ma.dives in Sarawak, East Malaysia

Studies on larval population densities and adult emergence rates of the Brugian filariasis vectors Mansonia bonneae Edwards and Ma. dives Schiner were conducted in freshwater swamp forest bordering the Sadong River, Serian District, Sarawak, East Malaysia, during 1984-85. Three species of aquatic host-plants in the Family Araceae were identified as supporting immature stages of the Ma. bonneae/dives complex. Proportions of positive plants were 4.7%, 6.5% and 3.4% with 6.4 +/- 2.6, 7.3 +/- 2.8 and 10.1 +/- 1.1 larvae per positive plant, respectively, for the plant species Homalomena cordata Schott, H. rostrata Griffiths and Hydrostemma motleyi (Hook. f.) Mabberley. These data indicate no significant preferences between the three types of host-plant. Detailed monitoring of the host-plant H. cordata revealed no significant monthly fluctuations in larval density per plant nor the proportion of positive plants. 11.6% of larvae were Ma. dives and 88.4% were Ma. bonneae. Mean daily yields of Ma. bonneae/dives adults per square metre of H. cordata vegetated water surface were 0.45 males plus 0.57 females during the wet season (December-February) compared with 0.2 males plus 0.31 females during the dry season (June-August). Thus output of adults per plant was approximately halved, and suitable breeding areas were further reduced, during the dry season. By extrapolation from these rates, a crude mean estimate for productivity of Ma. bonneae/dives females is 1.6 million per hectare per annum in swamp forest habitats vegetated with any of the host-plants studied.     

Assembly of a functional F1 of the proton-translocating ATPase of Escherichia coli

Assembly of the F1 portion of the proton-translocating ATPase of Escherichia coli was examined in vivo. Analysis of strains lacking genes which specify the Fo polypeptides a, b, and c showed that the F1 subunits were able to assemble into a complex in the absence of the Fo subunits. In addition we have investigated the effects of mutations in the individual genes which specify the F1 polypeptides on the assembly process. Mutations of the uncA(alpha), uncG(gamma), or uncD(beta) genes result in a defective assembly of the F1 complex. In contrast, mutations in the uncH(delta) or uncC(epsilon) genes did not prevent assembly of the core alpha beta gamma complex. In these cases, however, the partial F1 complexes were incapable of restoring energy-linked functions to F1-depleted membranes.     

In vivo cellular responses to electrophoretically applied dynorphin in the rat hippocampus

Recent immunohistochemical and radioimmunochemical observations have demonstrated a differential distribution of immunoreactive dynorphin (DYN) in rat brain. The presence of DYN immunoreactivity in a major intrinsic fiber pathway within the rat hippocampus (the mossy fiber system) has led us to evaluate the possible role of DYN and other closely related peptides in this structure. Single cell activity and hippocampal field potentials have been recorded from the CA1-CA3 cellular fields in halothane or urethane anesthetized rats. DYN, DYN1-13, DYN1-8, and alpha-neo-endorphin had an excitatory effect on most CA1-CA3 neurons encountered as has been previously observed for opiates and other opioid peptides. This response could be blocked by naloxone or by co-administration of Mg++ ion suggesting an indirect (synaptic) mechanism of excitation similar to that hypothetized for enkephalin. A significant number of CA3 neurons, however, exhibited a non-naloxone sensitive inhibitory response to DYN, related opioid peptides, and the kappa agonist WIN 35-197 (ethylketocyclazocine). Field potential analysis of CA1-CA3 neuronal responses to mossy fiber activation also indicated an excitatory, Mg++ reversible, action of iontophoretically applied DYN. These observations support our cytochemical and assay studies indicating diverse opioid systems within the rat hippocampus. In addition, these functional studies are congruent with other evidence suggesting multiple opioid mechanisms in this structure.     

Radiation inactivation target size of rat adipocyte glucose transporters in the plasma membrane and intracellular pools

The in situ assembly states of the glucose transport carrier protein in the plasma membrane and in the intracellular (microsomal) storage pool of rat adipocytes were assessed by studying radiation-induced inactivation of the D-glucose-sensitive cytochalasin B binding activities. High energy radiation inactivated the glucose-sensitive cytochalasin B binding of each of these membrane preparations by reducing the total number of the binding sites without affecting the dissociation constant. The reduction in total number of binding sites was analyzed as a function of radiation dose based on target theory, from which a radiation-sensitive mass (target size) was calculated. When the plasma membranes of insulin-treated adipocytes were used, a target size of approximately 58,000 daltons was obtained. For adipocyte microsomal membranes, we obtained target sizes of approximately 112,000 and 109,000 daltons prior to and after insulin treatment, respectively. In the case of microsomal membranes, however, inactivation data showed anomalously low radiation sensitivities at low radiation doses, which may be interpreted as indicating the presence of a radiation-sensitive inhibitor. These results suggest that the adipocyte glucose transporter occurs as a monomer in the plasma membrane while existing in the intracellular reserve pool either as a homodimer or as a stoichiometric complex with a protein of an approximately equal size.     

Susceptibility of neuroactive peptides to aminopeptidase digestion is related to molecular size.

Peptide fragments derived from the NH₂-terminus of corticotropin were found to exhibit widely differing degrees of stability to degradation by aminopeptidase M. Corticotropin itself was 135 times more stable than its NH₂-terminal pentapeptide, and similar differences in stability were observed with peptides derived from the B-chain of bovine insulin. Enkephalin linked covalently to the A-chain of bovine insulin was at least 100 times more stable than the pentapeptide. The results demonstrate that the molecular size of a peptide is one factor that determines its NH₂-terminal stability.