Copper(ii) complexes with alloferon analogues containing phenylalanine H6F and H12F stability and biological activity lower stabilization of complexes compared to analogues containing tryptophan.

Copper(ii) complex formation processes between alloferon 1 (Allo1) (H1 GVSGH6 GQH9 GVH12G) analogues where the phenylalanine residue is introduced in the place of His residue H6F and H12F have been studied by potentiometric, UV-visible, CD and EPR spectroscopic, and MS methods. For the phenylalanine analogues of alloferon 1, complex speciation has been obtained for a 1 : 1, 2 : 1 and 3 : 1 metal-to-ligand molar ratio. At physiological pH and in 1 : 1 metal-to-ligand molar ratio the phenylalanine analogues of alloferon 1 form a CuL complex similar to that of alanine analogues with the 4N{NH2,N1Im,2NIm} coordination mode. The stability of the complexes of the phenylalanine analogues is higher in comparison to those of alanine analogues, but lower in comparison to those containing tryptophan. Injection of Allo12F into insects induced prominent apoptotic changes in all hemocytes. The presence of apoptotic bodies only in the insect hemolymph testifies to the fact that Allo12F is an extremely pro-apoptotic peptide.

Mono- and polynuclear copper(II) complexes of alloferons 1 with point mutations (H6A) and (H12A): stability structure and cytotoxicity.

Mononuclear and polynuclear copper(II) complexes of the alloferons 1 (Allo1) with point mutations (H6A) H(1)GVSGA(6)GQH(9)GVH(12)G-COOH (Allo6A) and (H12A) H(1)GVSGH(6)GQH(9)GVA(12)G-COOH (Allo12A) have been studied by potentiometric, UV-visible, CD, EPR spectroscopic, and mass spectrometry (MS) methods. Complete complex speciation at different metal-to-ligand ratios ranging from 1:1 to 3:1 was obtained. At physiological pH 7.4 and a 1:1 metal-to-ligand molar ratio, the Allo6A and Allo12A peptides form CuL complexes with the 4N {NH2, N(Im)-H(1),2N(Im)} binding mode. The amine nitrogen donor and the imidazole nitrogen atoms (H(9)H(12) or H(6)H(9)) can be considered to be independent metal-binding sites in the species formed for the systems studied. As a consequence, di- and trinuclear complexes for the metal-to-ligand 2:1 and 3:1 molar ratios dominate in solution, respectively. The induction of apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH 7.4 was studied. The biological results show that copper(II) ions in vivo did not cause any apparent apoptotic features. The most active was the Cu(II)-Allo12A complex formed at pH 7.4 with a {NH2, N(Im)-H(1),N(Im)-H(6),N(Im)-H(9)} binding site. It exhibited 123% higher of caspase activity in hemocytes than the native peptide, Allo1.

Double whole-cell patch-clamp characterization of gap junctional channels in isolated insect epidermal cell pairs.

Double whole-cell patch-clamp methods were used to characterize junctional membrane conductances in epidermal cell pairs isolated from the prepupal integument of the flour beetle, Tenebrio molitor. The mean initial junctional conductance in 267 cell pairs was 9.5 +/- 1.0 nS (range 0-95 nS). Well-coupled cell pairs uncoupled spontaneously with a half-time of 7.6 min. Adding 5 mM ATP to the pipette solution stabilized coupling with less than a 50% drop occurring after 30 min. Nonjunctional membrane potential was the major determinant of junctional conductance with transjunctional potential playing a minor role. Junctional conductance approached 0 pA at nonjunctional membrane potentials greater than 0 mV and increased with hyperpolarization. The voltage at half-maximal conductance was -26 mV. The time course of the reversible changes in junctional conductance were slow (< or = 30 sec) with time-dependent decay occurring faster and recovery occurring slower with increasing depolarization. Single gap junctional channel activity was recorded in uncoupling cell pairs and in poorly coupled ATP-stabilized cell pairs. One main single channel conductance was observed in each cell pair. The mean single channel conductances from all cell pairs in this study ranged from 197-347 pS (mean 248 pS). Single channel conductance was linear over the +/- 60 mV transjunctional voltage range tested. A broad range of subconductance states of the main state representing 5% of the total open time of measurable main state events was observed. Single channel activity was strongly dependent on the nonjunctional membrane potential, increasing with hyperpolarization.

Histochemical and biochemical investigations concerning the function of larval oenocytes of Tenebrio molitor L. (Coleoptera, Insecta).

Larval oenocytes of Tenebrio molitor were investigated histochemically. In contrast to the lipid droplets of the fat body, they did not stain wit Sudan black. A positive reaction for lipoproteins appeared only after destructive oxidation with sodium hypochlorite. These lipoproteins are the remnants of degenerated membranes, as revealed by ultrastructural analysis. Polyphenols could be identified in th exocuticle of exuvia, and in the newly formed procuticle. Endocuticle, epidermis and oenocytes showed no staining reaction. In oenocytes a great amount of lipase is also present which could be detected with several Tweens as substrates. The significance of these lipases remains unclear, since only few glycerides are synthesized in the cells, as shown below. They may play a role in the extended membrane turnover observed in this cell type. In vitro incubation of oenocytes of the larval generation demonstrated that 14C-labeled acetate was only incorporated into the paraffin fraction. A negligible amount of the label was found in glycerides; wax esters were free of label. Larval epidermis is also capable of paraffin formation, but only to a small degree. Oenocytes of the imaginal generation located between the sternal epidermis cells of pupae and adults do not synthesize paraffins, but other more polar compounds not yet identified. Labeled waxes in cuticular lipids were detected only when 14C-acetate was injected into whole larvae, and the lipids extracted some hours later. Autoradiographs demonstrated that 14C-acetate was intensively incorporated into larval oenocytes, the rate varying in different cells. Incorporation into the epicuticle, probably into the wax layer, was clearly shown. Cuticulin and dense layer do not show an intensive label. The lamellated cuticle also seems to be impregnated with acetate derivatives.

Intercellular communication in insect development is hormonally controlled.

Cellular coupling in the insect epidermis changes in a characteristic way during metamorphosis. In vitro, beta-ecdysone mimics the initial phase of these changes by increasing electrical coupling. Both adenosine 3',5'-monophosphate (cyclic AMP) and Ca2+ reverse natural and beta-ecdysone-stimulated changes, which suggests that ecdysone could work on communication through changes in cyclic AMP and Ca2+ levels. The transient changes in intercellular communication before metamorphosis may reflect the timing of the signals that trigger proliferation and the generation of new spatial patterns in the epidermis.