Avidin-HRP conjugates in biotin-avidin immunoenzyme cytochemistry

Summary Avidin-HRP conjugates were prepared, analysed and tested for avidin-biotin immunocytochemistry. Suitable biotinylation of enzymes, antigens and antibody was obtained by reacting biotin at equimolar ratio to epsilon aminogroups in proteins. The avidin-biotin interaction was used for immunocytochemical detection of phenomena in the field of immunology, i.e. immune complex trapping, specific antibody forming cells and in serology for the cytochemical detection of human auto-antibodies to basement membrane components. Avidin-HRP conjugation using the two step glutaraldehyde method gave a very small amount of monomeric, low molecular weight conjugate with excellent performance. Avidin-HRP conjugation using the periodate method was modified at two points. The first modification concerns the molar ratio of avidin to HRP in the reaction mixture which was brought to about equimolarity. The second modification concerns the periodate concentration which was decreased five fold, ten fold and twenty fold. Decreasing the periodate concentration decreased the amount of polymeric conjugate. Optimal amounts of monomeric, low molecular weight conjugate were obtained with a ten fold decrease of the periodate concentration. Comparable cytochemical results were obtained with monomeric conjugates obtained using both preparation methods.In honour of Professor P. van Duijn     

Elucidation of a minimal immunoreactive site of vertebrate calmodulin

The heptapeptide AsnTyrGluGluPheValGlnNH₂ corresponding to residues 137–143 of vertebrate calmodulin is as immunoreactive as the entire 148-residue protein. A reproducible and rapid procedure for producing antisera against vertebrate calmodulin has been previously described (L. J. Van Eldik and D. M. Watterson (1981) J. Biol. Chem.256, 4205–4210). Most of the antisera elicited by this method react with a major immunoreactive region (residues 127–144) in the COOH-terminal domain of vertebrate calmodulin. In this report, the minimum segment of calmodulin required for reactivity with an antiserum that readily distinguishes various types of calmodulins is defined. These studies demonstrate that a linear segment of seven amino acid residues shows a competition curve in radioimmunoassay resembling the competition curve of intact calmodulin. This heptapeptide is the smallest calmodulin segment and the only sevenresidue segment in the 135–145 region that shows quantitative immunoreactivity with the anti-calmodulin serum. These data demonstrate that this heptapeptide is a major immunoreactive site of calmodulin. However, when this immunoreactive site heptapeptide is conjugated to a carrier and injected into rabbits, it does not elicit antisera that react with the native protein. These studies demonstrate that quantitative immunoreactivity of antisera produced in animals can be found in small peptide segments and that, for calmodulin, the requirements for production of anti-peptide antibodies that react with the native protein molecule are not as simple as surface exposure of the peptide region.     

Chromosome transfer and R-prime plasmid formation mediated by plasmid pULB113 (RP4::mini-Mu) in Alcaligenes eutrophus CH34 and Pseudomonas fluorescens 6.2.

Plasmid pULB113 (RP4::mini-Mu), which contains the mini-Mu transposon, promoted both homologous and heterologous gene transfer from Pseudomonas fluorescens 6.2 and Alcaligenes eutrophus CH34. Homologous gene transfer in P. fluorescens 6.2 and A. eutrophus CH34 occurred at a frequency of 10(-4) to 10(-5), and recombinants inherited unselected recessive markers, suggesting a process of chromosome mobilization. Loci involved in autotrophic growth were among those transferred in A. eutrophus. In heterospecific matings, markers were transferred from P. fluorescens to A. eutrophus, Salmonella typhimurium LT2, and Escherichia coli, from A. eutrophus to P. fluorescens, and from Erwinia carotovora subsp. chrysanthemi to A. eutrophus. Heterospecific matings resulted in the formation of R-prime plasmids at frequencies of 10(-7) to 10(-4) per transferred plasmid. When S. typhimurium was the recipient, we observed R-prime plasmids with both restriction-proficient and restriction-deficient strains, although restriction markedly affected the frequency of transfer of pULB113. R-prime plasmids were quite stable, but lost the transposed marker more easily in a rec+ background than in a recA background, suggesting excision of transposed material by reciprocal recombination between flanking copies of mini-Mu. R-prime plasmids could be transferred easily into different recipients and were used in complementation studies. PstI restriction digests of four R-prime plasmids carrying P. fluorescens 6.2 DNA showed a number of additional bands, suggesting that several genes were transposed together with the selected marker on the plasmid.     

5-hydroxytryptamine and precapillary vessels

The complexity of the vascular effects of 5-hydroxytryptamine (5-HT) is illustrated by differences in sensitivity to the amine among arterial tissues of different origin. The interaction of 5-HT with 5-HT2 receptors is inhibited by specific antagonists such as ketanserin and methysergide. Such compounds also inhibit the contractile responses to endogenous 5-HT released from aggregating platelets. The vasodilator component of the response to 5-HT can be unmasked in the presence of serotonergic blockade, provided the antagonist used has no partial agonistic properties. 5-HT augments (amplifies) the vasoconstrictor responses to adrenergic and nonadrenergic neurohumoral mediators. The amplifying effect of the monoamine is prevented by 5-HT2-serotonergic antagonists such as ketanserin.     

Should Individual Fitness Increase with Heterozygosity?

Natural selection influences not only gamete frequencies in populations but also the multilocus fitness structures associated with segregating gametes. In particular, only certain patterns of multilocus fitnesses are consistent with the maintenance of stable multilocus polymorphisms. This paper offers support for the proposition that, at stable, viability-maintained, multilocus polymorphisms, the fitness of a genotype tends to increase with the number of heterozygous loci it contains. Average fitness always increases with heterozygosity at stable product equilibria (i.e., those without linkage disequilibrium) maintained by either additive or multiplicative fitness schemes. Simulations suggest that it "generally" increases for arbitrary fitness schemes. The empirical literature correlating allozyme heterozygosity with fitness-correlated traits is discussed in the light of these and other theoretical results.     

Genetic Effects of Uv Irradiation on Excision-Proficient and -Deficient Yeast during Meiosis

The lethal and recombinational responses to ultraviolet light irradiation (UV) by excision-proficient (RAD⁺) and deficient strains (rad1) of Saccharomyces cerevisiae has been examined in cells undergoing meiosis. Cells that exhibit high levels of meiotic synchrony were irradiated either at the beginning or at various times during meiosis and allowed to proceed through meiosis. Based on survival responses, the only excision repair mechanism for UV damage available during meiosis is that controlled by the RAD1 pathway. The presence of pyrimidine dimers at the beginning of meiosis does not prevent cells from undergoing meiosis; however, the spore products exhibit much lower survival than cells from earlier stages of meiosis. The reduced survival is probably due to effects of UV on recombination. Meiotic levels of gene conversion are reduced only two to three times in these experiments; however, intergenic recombination is nearly abolished after a dose of 4 J/m ² to the rad1 strain. Exposure to 25 J/m² had little effect on the wild-type strain. Since normal meiotic reciprocal recombination is generally considered to involve gene conversion-type intermediates, it appears that unrepaired UV damage dissociates the two processes. These results complement those obtained with the mei-9 mutants of Drosophila which also demonstrate a dissociation between gene conversion and reciprocal recombination. These results are consistent with molecular observations on the UV-irradiated rad1 strain in that there is no excision of pyrimidine dimers or exchange of dimers during meiosis.     

Evidence for androgen independence of male mounting behavior in white-crowned sparrows (Zonotrichia leucophrys gambelii)

Previous experiments have shown that expression of mounting behavior in sexually inexperienced, adult male white-crowned sparrows does not require elevated plasma levels of androgen; adult males maintained on nonstimulatory short days mount sexually receptive females. The experiments reported here demonstrate that (1) sexually inexperienced, prepubertal males maintained on nonstimulatory short days show very low mounting rates in response to female sexual displays; (2) these males exhibit high mounting rates when exposed to stimulatory long days but androgen treatment on short days is ineffective in stimulating high mounting rates; and (3) prepubertal castration has no effect on the expression of mounting behavior by photostimulated adult males. Thus, there is no evidence that mounting behavior of adult male white-crowned sparrows depends on androgen.     

Paleolimnology of the Peten Lake District,Guatemala

Long-term changes of sedimentary particle-size distribution in two tropical lowland lakes were compared with changes of human population sizes, estimated archaeologically, in the drainage basins. Mean particle size of silt and clay fractions (<64 µm) varied between 3 and 15 µm. High positive skewness and kurtosis of the distributions were associated with smaller particle sizes; hence small mean size resulted from greater influx of small particles while influx of larger particles was probably constant. An inverse correlation between mean particle size and human population size is interpreted to mean that disturbance-induced erosion results in delivery of very fine inorganic particles at higher rates. Within any one basin, particle-size stratigraphy is more precisely related to archaeological time periods than is pollen stratigraphy. An absolute chronology still eludes us, owing to the failure of ¹⁴C dating of calcareous, colluvial sediments, but our relative chronology is now more precise than before. If certain assumptions about past hydrologic relations can be met, particle-size analysis is a way of comparing the histories of geographically very different lakes, including lakes from tropical, temperate, and arctic regions.     

Specific detection of N-acetylglucosamine-containing oligosaccharide chains on ovine submaxillary asialomucin

Human milk beta-N-acetylglucosaminide beta 1 leads to 4-galactosyltransferase (EC 2.4.1.38) was used to galactosylate ovine submaxillary asialomucin to saturation. The major [14C]galactosylated product chain was obtained as a reduced oligosaccharide by beta-elimination under reducing conditions. Analysis by Bio-Gel filtration and gas-liquid chromatography indicated that this compound was a tetrasaccharide composed of galactose, N-acetylglucosamine and reduced N-acetylgalactosamine in a molar ratio of 2:0.9:0.8. Periodate oxidation studies before and after mild acid hydrolysis in addition to thin-layer chromatography revealed that the most probable structure of the tetrasaccharide is Gal beta 1 leads to 3([14C]Gal beta 1 leads to 4GlcNAc beta 1 leads to 6)GalNAcol. Thus it appears that Gal beta 1 leads to 3(GlcNAc beta 1 leads to 6)GalNAc units occur as minor chains on the asialomucin. The potential interference of these chains in the assay of alpha-N-acetylgalactosaminylprotein beta 1 leads to 3-galactosyltransferase activity using ovine submaxillary asialomucin as an acceptor can be counteracted by the addition of N-acetylglucosamine.