Binding of a distamycin-ellipticine hybrid molecule to DNA and chromatin: spectroscopic, biochemical, and molecular modeling investigations.

A bifunctional molecule in which an ellipticine chromophore is attached to a distamycin residue via a diaminopropyl tether has been designed and synthesized in the expectation of creating a hybrid molecule capable of bidentate binding to DNA by both intercalation and minor-groove interactions. The strength and mode of binding to DNA of this conjugate have been studied by means of circular and linear dichroism as well as by stopped-flow kinetics and measurements of reactivity toward a chemical probe. The results converge to reveal that the ellipticine moiety of the hybrid largely dominates the binding reaction with DNA. In the presence of chromatin, the hybrid molecule binds preferentially to the internucleosomal DNA, a preference dictated by its intercalating chromophore. Theoretical computations were performed on the comparative complexation energies of distamycin, the ellipticine derivative, and the hybrid ligand with a B-representative octanucleotide, d(GCATATGC)2. The best binding configuration of the ellipticine derivative locates its aminoalkyl side chain in the minor groove where distamycin is also present. The molecular modeling analysis fully supports the involvement of a bimodal binding process for the hybrid and reveals that the binding of the conjugate to DNA favors a pronounced bending toward the minor groove. This effect is attributed to intercalation of the ellipticine chromophore. An interesting link is established between the DEPC reactivity experiments and the theoretical computations, suggesting that DEPC can be used as a probe for drug-induced DNA bending. On the basis of these results, we propose the design of a new hybrid ligand bearing an additional positively-charged amidine side chain to confer higher DNA-binding affinity.     

Specific binding sites on human phagocytic blood cells for Gly-Leu-Phe and Val-Glu-Pro-Ile-Pro-Tyr, immunostimulating peptides from human milk proteins.

Two immunostimulating peptides were isolated from human milk proteins by enzymatic digestion, the tripeptide GLF and the hexapeptide VEPIPY. These peptides increased the phagocytosis of human and murine macrophages and protected mice against Klebsiella pneumoniae infection. The present study showed that this activity may be correlated to the presence of specific binding sites on human blood phagocytic cells. The receptor molecules implicated were different for the two peptides. [3H]GLF specifically bound to PMNL and monocytes, whereas [3H]VEPIPY only bound to monocytes. The leukemic promyelocytic cell line HL-60 differentiated into granulocytes or into macrophages (depending on inducer used) coroborated these results. Specific binding of [3H]GLF on plasma membrane preparations of human PMNL (20 degrees C) was saturable and Scatchard analysis indicated two classes of binding sites: high-affinity sites of Kd 2.3 +/- 1.0 nM and Bm 60 +/- 9 fmol/mg protein and low-affinity sites of Kd 26.0 +/- 3.5 nM and Bm 208 +/- 45 fmol/mg protein. [3H]GLF binding was inhibited in a concentration-dependent manner by various analogous peptides, such as LLF, GLY, LLY and RGDGLF, but not by RGD, RGDS, VEPIPY and the chemotactic peptide f-Met-Leu-Phe (f-MLF). Only at high concentrations the direct analog MLF competed with labeled GLF. An important inhibitory effect was also observed with C1q component of the complement whereas C3 and BSA were uneffective. Specific binding of [3H]VEPIPY on monocyte membranes (20 degrees C) was saturable and Scatchard analysis was consistent with one class of binding sites of Kd 3.7 +/- 0.3 nM and Bm 150 +/- 6 fmol/mg protein.     

Absence of fodrin (spectrin-like protein) under the pseudopod membrane in stimulated thyroid cells

A fodrin-like protein purified from porcine thyroid cells and characterized by its properties identical to those of pig brain spectrin (F. Regnouf et al., Eur. J. Biochem. 153, 313-319 (1985)) has been localized by immunofluorescence and electron immunocytochemistry in porcine and rat thyroid. Fodrin-like polypeptides were detected in subplasmalemmal meshworks of microfilaments attached to isolated or in situ plasma membranes. In resting cells, fodrin was found under apical and basolateral membrane domains, whereas it was always absent under the pseudopod membrane domain induced by acute TSH stimulation in vitro, using monolayers of porcine cultured cells attached to collagen permeable substrates, as well as in vivo, using rats intravenously treated with TSH. Thyroid fodrin could be involved in exocytosis and membrane stabilization which occurs during the formation of pseudopods induced by TSH stimulation.     

Development of cytochrome b558 and oxidative metabolism in human granulocytes, monocytes and during differentiation of HL-60 and U 937 cells

The development of cytochrome b558 (Cyt b) as determined spectrophotometrically, was investigated in human polymorphonuclear neutrophils (PMN), monocytes (MN) and during differentiation of HL-60 and U 937 cells induced by retinoic acid (RA) alone or in combination with IFN gamma. O2- release in response to a panel of stimulating agents, ie latex particles, opsonised zymosan, PMA, Con A and fMLP, was monitored by lucigenin-amplified chemiluminescence (CL). In parallel the expression of myeloperoxidase (MPO) was investigated and its catalytic activity on H2O2 related to luminol-amplified CL responses. In mature PMN and MN phagocytes, regardless of the stimulating agent, the O2- production is closely related to Cyt b but not to MPO specific contents. In differentiated HL-60 and U 937 cells, the oxidative metabolism increases in parallel with Cyt b specific contents, both being enhanced by the addition of IFN gamma to the RA treatment. However, marked differences in the O2- production intensities are observed depending on the stimulating agent tested and the state of differentiation considered. The PMA-stimulated O2- production is rather low ie 100 and 20 times less in granulocytic HL-60 and monocyto-macrophagic U 937 cells than in PMN and MN respectively. Latex, zymosan and Con A stimulated responses are close to those of MN, in monocyte-macrophagic U 937 cells. In conclusion, these data show that during differentiation; 1), Cyt b plays a critical role in O2- production; 2), the pathways leading to NADPH oxidase activation are diversely modulated following phagocyte differentiation with IFN gamma and/or with RA.     

Influence of the Molecular Structure of Steroids on Their Ability to Interrupt Gap Junctional Communication

17β-estradiol propionate was found to reduce the gap junctional communication in a concentration range similar to that of testosterone propionate, in primary cultures of rat Sertoli cells and cardiac myocytes. Uncoupling was reversible on washing out and occurred without concomitant rise in the intracellular calcium concentration. Esterification was a prerequisite for the activity of extracellularly applied steroid compounds (for example, testosterone was ineffective even at external concentrations up to 100 μm, whereas its intracellular application at 1 μm totally interrupted intercellular communication), but their uncoupling efficiency did not depend on the nature of the ester chain nor on its position on the steroid nucleus. The derivatives of two other androgen hormones (derivatives of the androstane nucleus) were also efficient as junctional uncouplers. Among five steroid molecules belonging to the pregnane family, only one (pregnanediol diacetate) interrupted the junctional communication. Neither cholic acid nor cholesteryl acetate or ouabain showed this effect. Altogether, no correlation with the presence or position of double bonds nor with the trans- or cis-fusion of the A and B rings could be recognized. These results suggest that this reversible, nondeleterious uncoupling effect of steroids is independent of the shape of the molecules and is more probably related to their size and liposolubility, that condition their insertion into the lipid bilayer. Their incorporation into the membrane could disturb the activity of the membrane proteins by a physical mechanism. Received: 10 April 1995/Revised: 27 October 1995     

Intraspecific larval competition in two solitary parasitoids, Apoanagyrus (Epidinocarsis) lopezi and Leptomastix dactylopii

Analysis of total aromatic amino acid (free and bound) in some cucumber accessions selected previously for resistance to western flower thrips, Frankliniella occidentalis (Pergande) [Thysanoptera: Thripidae], indicated that low concentrations of these essential nutrients, relative to total leaf protein, were correlated with a reduction in damage by the insect. Further analysis of samples of four important horticultural crops (lettuce, tomato, pepper and cucumber) with unknown levels of resistance to thrips showed a significant genotypic variation in the concentrations of total aromatic amino acids relative to the total leaf protein. Accessions from each crop with low or high concentrations of aromatic amino acids in proteins were exposed to thrips larvae. Regression analysis showed a highly significant positive correlation between aromatic amino acid concentration in leaf protein and thrips damage, regardless of crop species. It is concluded that higher concentrations of aromatic amino acids in plant proteins are important for successful thrips development. These results provide plant breeders with a promising tool for indirect selection without using undesirable insect bioassays.     

Identification of four novel splice site mutations in the ornithine transcarbamylase gene

Ornithine transcarbamylase (OTC) deficiency, the most common inborn error of the urea cycle, shows X-linked inheritance with frequent new mutations. Using polymerase chain reaction (PCR) amplification of the individual exons including adjacent intron sequences followed by direct sequencing of the amplimers we identified four new mutations affecting donor splice sites of introns 2, 5, 6, and 8. The mutation at the first position of intron 2 was a G to A exchange associated with acute neonatal hyperammonemia in a male patient at the age of 5 months. A G to C substitution in intron 5 was detected in a boy who developed 2 days after birth hypotonia, and respiratory distress, followed by severe hyperammonemia and terminal coma. The intron 6 mutation, a G to T substitution, was detected in a girl presenting with first episodes of vomiting and agitation at the age of 2 months. The mutation in intron 8, also a G to T transition, caused fatal hyperammonemia and early death at the age of 15 days in a male patient. We present four donor splice site mutations resulting in severe neonatal or very early onset of the disease in three boys and in one female patient. As the GT dinucleotide of the 5 donor splice site is invariant and required for correct splicing the described mutations may lead to improperly spliced mRNAs and aberrant gene products.     

Calamintha cretica (Lamiaceae), a Cretan endemic: Distribution and essential oil composition

The distribution of Calamintha cretica , a taxon restricted on the massif of Levka Ori (White Mountains W. Crete, Greece), is presented. The essential oils of three populations were examined by means of GC and GC-MS. The essential oil yield varied from 0.5% to 1.9%, whereas the major compounds were in all cases piperitenone oxide (26.4–41.3%) and piperitone oxide (33.8–59.9%). Like all other Calamintha taxa examined to date, it is a species rich in p -menthane compounds. The results are further discussed in relation to their chemotaxonomic value.