Developmental and genetic mosaic analysis of Drosophila m-dy mutants: Tissue foci for behavioral and morphogenetic defects

Mutants of the Drosophila miniature-dusky (m-dy) gene complex display morphogenetic phenotypes (miniature or dusky) caused by a change in the size and/or shape of the epidermal cells comprising the adult wing. In addition to a dusky phenotype, certain Andante-type mutants also exhibit lengthened circadian periods for two different behavioral rhythms. If the latter phenotype results from a direct effect on the circadian pacemaker, the Andante function should be required within the brain. In order to define the tissues that require the morphogenetic and behavioral functions, we have carried out a genetic mosaic analysis. This study demonstrates that normal wing morphogenesis is entirely dependent on the genotype of wing cells. Furthermore, temperature-shift experiments with a temperature-sensitive dy mutant indicate that the morphogenetic function is required during adult development, and after the cessation of wing epidermal cell proliferation. At this time in development, a columnar epithelium in the developing wing becomes flattened into the mature wing blade, and we postulate that the cell-size defect of m-dy mutants results from an alteration of this mor-phogenetic process. In contrast to the wing mor-phogenesis phenotype, the characterization of locomotor activity in mosaic adults revealed a strong correlation between the head genotype and the Andante circadian-period phenotype. This result indicates that neural tissues mediate the rhythm function. Thus, the behavioral and morphogenetic functions require gene expression in distinct tissues. Furthermore, the behavioral results are consistent with a requirement for Andante function within circadian pacemaker neurons. © 1995 Wiley-Liss, Inc.     

Immunochemical and immunohistological expression of Lewis histo-blood group antigens in small intestine including individuals of the Le(a+b+) and Le(a−b−) nonsecretor phenotypes

Histological samples and total non-acid glycosphingolipids were prepared from small intestine of human cadavers with the Le(a+b+) and Le(a–b–) nonsecretor phenotypes and contrasted with the more common Lewis phenotypes. Glycolipid fractions were analysed by thin-layer chromatography and tested for Lewis activity with monoclonal antibodies reactive to Lewis epitopes. Paraffin-embedded small intestine sections were also fluorescently immunostained with anti-Lewis antibodies. Unlike the common Lewis positive phenotypes, we were immunochemically able to demonstrate the copresence of large amounts of Le^a and Le^b glycolipids in the Le(a+b+) sample. In addition we demonstrated increased formation of extended Lewis structures in this phenotype. By immunohistochemistry Le^a, Le^b and type 1 precursor chain epitopes could be demonstrated in the brush border. These results show that the expression of the Le(a+b+) phenotype at the erythrocyte phenotyping level parallels the small intestinal expression of this phenotype, and the patterns of Lewis antigen expressions are unique to this phenotype. By immunohistochemistry and immunochemistry we also demonstrated the presence of trace amounts of Lewis active glycoconjugates in the small intestine of the Le(a–b–) nonsecretor and Le(a+b–) samples. In the Le(a–b–) nonsecretor Le^a and Le^b activity was absent and type 1 precursor was present in brush border, while Le^b activity was immunohistologically demonstrated in the Golgi apparatus of the deep glands. Trace amounts of both Le^a and Le^b glycolipids were identified in this sample. In parallel trace Le^b activity could also be detected in the glycolipids of the Le(a+b–) sample and could be immunohistologically demonstrated to be fully expressed in occasional cells in the deep glands of the small intestine, a pattern quite dissimilar to that of the Le(a–b–) nonsecretor. The results in this paper show that the expression of Lewis glycoconjugates in the small intestine parallel the expression of Lewis erythrocyte phenotypes. However, inappropriate Lewis activity is also seen in individuals of other phenotypes and the mechanisms by which these Lewis antigens are made appears to be different for different phenotypes.Abbreviations FITC fluorescein isothiocyanate - HPLC high-performance liquid chromatography - NeuAc N-acetyl-d-neuraminic acid - RBC red blood cell - TLC thin-layer chromatography - TRITC tetramethyl rhodamine isothiocyanate     

Expression of Lewis histo-blood group glycolipids in the plasma of individuals of Le(a+b+) and partial secretor phenotypes

Red cell Lewis antigens are carried by glycosphingolipids passively absorbed from plasma. Plasma was collected from a spectrum of individuals with normal and unusual Lewis/secretor phenotypes in order to investigate the glycolipid basis for the unusual phenotypes. Samples were obtained from: a Le(a+b–) ABH nonsecretor who secreted Lewis substances; a Le(a+b–) partial secretor; Le(a+b+) partial secretors; Le(a+b+) secretors; and a full range of normal Lewis/secretor phenotypes as controls. The Le(a+b+) samples represented Polynesian, Asian and Réunion Island ethnic backgrounds. Nonacid glycolipids were prepared, separated by thin-layer chromatography, and then immunostained with potent monoclonal antibodies of known specificity. Despite different serological profiles of the Le(a+b–) and Le(a+b+) Polynesian samples, their plasma glycolipid expressions were very similar, with both Le^a and Le^b co-expressed. The copresence of Le^a and Le^b in Le(a+b+) samples is in marked contrast to Caucasians with normal Lewis phenotypes, who have predominantly either Le^a or Le^b. These results suggest that there is a range of the secretor transferases in different individuals, possibly due to different penetrance or to several weak variants. We also show that Lewis epitopes on longer and/or more complex core chains appear to be predominant in the Polynesian Le(a+b+) samples. The formation of these extended glycolipids is compatible with the concept that in the presence of reduced secretor fucosyltransferase activity, increased elongation of the precursor chain occurs, which supports the postulate that fucosylation of the precursor prevents or at least markedly reduces chain elongation.Abbreviations CBA chromatogram binding assay - TLC thin-layer chromatography     

On some formulas in a partnership model from the perspective of a semi-Markov process

Many deterministic models of sexually transmitted diseases, as well as population models in general, contain elements of stochastic or statistical reasoning. An example of such a model is that of Dietz and Hadeler (1988) concerning sexually transmitted diseases in which there is partnership formation and dissolution. Among the interesting formulas in this paper, which enter into the analysis of the model, are those for the expected number of partners a male or female has during a lifetime. To a probabilist such formulas suggest the possibility that some stochastic process may be constructed so as to yield these formulas as well as others that may be of interest. The principal purpose of this paper is to demonstrate that such a stochastic process does indeed exist in the form of a three state semi-Markov process in continuous time with stationary laws of evolution and with a one-step density matrix determined by four parameters which were interpreted as constant latent risk functions in the classical theory of competing risks. This construction of a semi-Markov process not only provides a framework for the systematic derivation of the formulas of Dietz and Hadeler but also suggests pathways,for extensions to the age-dependent case.This research was partially supported by NATO Grant D.890350     

The pharmacophore of the human C5a anaphylatoxin.

We have determined which amino acids contribute to the pharmacophore of human C5a, a potent inflammatory mediator. A systematic mutational analysis of this 74-amino acid protein was performed and the effects on the potency of receptor binding and of C5a-induced intracellular calcium ion mobilization were measured. This analysis included the construction of hybrids between C5a and the homologous but unreactive C3a protein and site-directed mutagenesis. Ten noncontiguous amino acids from the structurally well-defined 4-helix core domain (amino acids 1-63) and the C-terminal arginine-containing tripeptide were found to contribute to the pharmacophore of human C5a. The 10 mostly charged amino acids from the core domain generally made small incremental contributions toward binding affinity, some of which were independent. Substitutions of the C-terminal amino acid Arg 74 produced the largest single effect. We also found the connection between these 2 important regions to be unconstrained.     

Self-splicing group I and group II introns encode homologous (putative) DNA endonucleases of a new family.

A new family of protein domains consisting of 50-80 amino acid residues is described. It is composed of nearly 40 members, including domains encoded by plastid and phage group I introns; mitochondrial, plastid, and bacterial group II introns; eubacterial genomes and plasmids; and phages. The name "EX1HH-HX3H" was coined for both domain and family. It is based on 2 most prominent amino acid sequence motifs, each encompassing a pair of highly conserved histidine residues in a specific arrangement: EX1HH and HX3H. The "His" motifs often alternate with amino- and carboxy-terminal motifs of a new type of Zn-finger-like structure CX2,4CX29-54[CH]X2,3[CH]. The EX1HH-HX3H domain in eubacterial E2-type bacteriocins and in phage RB3 (wild variant of phage T4) product of the nrdB group I intron was reported to be essential for DNA endonuclease activity of these proteins. In other proteins, the EX1HH-HX3H domain is hypothesized to possess DNase activity as well. Presumably, this activity promotes movement (rearrangement) of group I and group II introns encoding the EX1HH-HX3H domain and other gene targets. In the case of Escherichia coli restrictase McrA and possibly several related proteins, it appears to mediate the restriction of alien DNA molecules.     

Evaluation of the role of State transitions in determining the efficiency of light utilisation for CO2 assimilation in leaves

Wheat leaves were exposed to light treatments that excite preferentially Photosystem I (PS I) or Photosystem II (PS II) and induce State 1 or State 2, respectively. Simultaneous measurements of CO₂ assimilation, chlorophyll fluorescence and absorbance at 820 nm were used to estimate the quantum efficiencies of CO₂ assimilation and PS II and PS I photochemistry during State transitions. State transitions were found to be associated with changes in the efficiency with which an absorbed photon is transferred to an open PS II reaction centre, but did not correlate with changes in the quantum efficiencies of PS II photochemistry or CO₂ assimilation. Studies of the phosphorylation status of the light harvesting chlorophyll protein complex associated with PS II (LHC II) in wheat leaves and using chlorina mutants of barley which are deficient in this complex demonstrate that the changes in the effective antennae size of Photosystem II occurring during State transitions require LHC II and correlate with the phosphorylation status of LHC II. However, such correlations were not found in maize leaves. It is concluded that State transitions in C₃ leaves are associated with phosphorylation-induced modifications of the PS II antennae, but these changes do not serve to optimise the use of light absorbed by the leaf for CO₂ assimilation.Abbreviations Fm, Fo, Fv maximal, minimal and variable fluorescence yields - Fm, Fv maximal and variable fluorescence yields in a light adapted state - LHC II light harvesting chlorophyll a/b protein complex associated with PS II - q_P photochemical quenching - A₈₂₀ light-induced absorbance change at 820 nm - _{PS I}, _{PS II} relative quantum efficiencies of PS I and PS II photochemistry - _{CO 2} quantum yield of CO₂ assimilation     

ATP-induced Secretion in PC12 Cells and Photoaffinity Labeling of Receptors

Abstract— Secretion of catecholamines by rat PC12 cells is strongly stimulated by extracellular ATP via a P₂-type pur-inergic receptor. ATP-induced norepinephrine release was inhibited 80% when extracellular Ca²⁺ was absent. Only four nucleotides, ATP, ATPγS, benzoylbenzoyl ATP (BzATP), and 2-methylthio-ATP, gave substantial stimulation of norepinephrine release from PC12 cells. ATP-induced secretion was inhibited by Mg²⁺, and this inhibition was overcome by the addition of excess ATP suggesting that ATP^4-was the active ligand. ATP-induced secretion of catecholamine release was enhanced by treatment of cells with pertussis toxin or 12- O -tetradecanoylphorbol 13-acetate. The stimulatory effects of 12- O -tetradecanoyl-phorbol 13-acetate and pertussis toxin on norepinephrine release were additive. After brief exposure of intact cells to the photoaffinity analog, [α-³²P]BzATP, two major proteins of 44 and 50 kDa and a minor protein of 97 kDa were labeled. An excess of ATP-γS and BzATP but not GTP blocked labeling of the proteins by [³²P]BzATP. Labeling of the 50-kDa protein was more sensitive to competition by 2-methylthio-ATP than the other labeled proteins, suggesting that the 50-kDa protein represents the P₂ receptor responsible for ATP-stimulated secretion in these cells.