Germinal and non-germinal lines in the ovotestis of Helix aspersa: a survey

Summary The study of gonadal organogenesis and differentiation by means of light and electron microscopy suggested the following in Helix aspersa: (1) the distal parts of the acini have components of mesodermal origin, whereas the neck and efferent duct comprise ectodermal elements; (2) a segregation of a germinal line occurs early, during the embryonic life; (3) in juvenile and adult animals, male and female cells arise from a germinal ring located at the base of the acinar neck. Apart from developing oocytes, the epithelium lining the distal region of the acini consists of somatic cells (Sertoli and follicle cells).     

Receptor inactivation by dye-neuropeptide conjugates. 3. Comparative binding of dye-neuropeptide conjugates to FMRFamide receptors of Helix aspersa and Loligo pealei

Three neuropeptide analogues of FMRFamide (FMRFa) were covalently attached to a tethered derivative of methylene blue to form dye-neuropeptide conjugates. The comparative binding of the latter to FMRFa receptors was subsequently examined in both Helix aspersa (circumesophageal ganglia) and squid (optic lobe membrane). In Helix, the FMRFa analogue CFMRFamide (CFMRFa) inhibited the specific binding of the FMRFa ligand [¹²⁵I]daYFnLRFa in a dose-dependent manner. Az-CFMRFa, one of the dye-neuropeptide conjugates, also dose-dependently inhibited the specific binding of [¹²⁵I]daYFnLRFa. Moreover, their potencies equaled or exceeded that of FMRFamide. In squid, the binding of CFMRFa and FMRFa was similar. However, the dye-neuropeptide conjugate (IC₅₀ of 14 nM) was about 44-fold less potent than FMRFa. The conjugates were synthesized as part of a study seeking to target and inactivate preselected receptors with heretofore unattainable selectivity and permanence.     

Effectors of metabolic depression in an estivating pulmonate snail (Helix aspersa): whole animal and in vitro tissue studies

 We have examined metabolic depression in the land snail (Helix aspersa) during estivation, and have developed a tissue model of metabolic depression using an in vitro mantle preparation. The metabolic rate of H. aspersa is depressed by 84% in vivo within 4 weeks of onset of estivation, and this metabolic depression is accompanied by a decrease in haemolymph PO₂ and pH, and an increase in haemolymph PCO₂. The in vitro mantle preparation has a stable O₂ consumption and energy charge, and an energy charge similar to that of mantle in vivo. The in vitro mantle is an O₂-conforming tissue, with VO₂ varying curvilinearly with PO₂. Consequently, we have developed a mathematical method of calculating tissue VO₂ at any PO₂. These calculations show that under appropriate incubation conditions of pH and PO₂, the mantle from estivating animals shows a stable in vitro metabolic depression of 48% compared to mantle from control snails. The extrinsic effects of PO₂ and pH account for 70% of the total in vitro metabolic depression of mantle tissue; intrinsic effectors contribute a further 30%. Accepted April 26, 1996     

Local helix content in an alanine-rich peptide as determined by the complete set of 3JHNα coupling constants

Summary Alanine-rich peptides serve as models for exploring the factors that control helix structure in peptides and proteins. Scalar CH-NH couplings (³J_HN) are an extremely useful measure of local helix content; however, the large alanine content in these peptides leads to significant signal overlap in the CH region of ¹H 2D NMR spectra. Quantitative determination of all possible ³J_HN values is, therefore, very challenging. Szyperski and co-workers [(1992) J. Magn. Reson., 99, 552–560] have recently developed a method for determining ³J_HN from NOESY spectra. Because ³J_HN may be determined from 2D peaks outside of the CH region, there is a much greater likelihood of identifying resolved resonances and measuring the associated coupling constants. It is demonstrated here that ³J_HN can be obtained for every residue in the helical peptide Ac-(AAAAK)₃A-NH₂. The resulting ³J_HN profile clearly identifies a helical structure in the middle of the peptide and further suggests that the respective helix termini unfold via distinct pathways.Abbreviations ³J_HN three-bond CH-NH scalar coupling constant - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser spectroscopy - COSY two-dimensional correlated spectroscopy - DQF-COSY two-dimensional double-quantum-filtered correlated spectroscopy - TOCSY two-dimensional total correlation spectroscopy To whom correspondence should be addressed.Deceased March 5, 1996.     

Dynamic stiffness and crossbridge action in muscle

Small sinusoidal vibrations at 300 Hz were applied to frog sartorius muscle to measure the dynamic stiffness (Young's modulus) throughout the course of tetanus. For a peak-to-peak amplitude of 0.4% the dynamic Young's modulus increased from 1.5×10⁵ Nm^–2 in the resting state to 2×10⁷ Nm^–2 in tetanus. After correction for the external connective tissue, the dynamic Young's modulus of the muscle was almost directly proportional to the tension throughout the development of tetanus. The ratio of dynamic Young's modulus to tensile stress thus remained constant (with a value at 300 Hz of approximately 100), consistently with Huxley and Simmons' identification of the crossbridges as the source of both tension and stiffness.For a single crossbridge the ratio of stiffness to tension was 8.2×10⁷ m^–1 at 300 Hz; it is deduced from literature data that the limiting value at high frequencies is about 1.6×10⁸ m^–1. This ratio is interpreted on Harrington's (1971) model to show that crossbridge action can be explained by a helix-coil transition of about 80 out of the 260 residues in each S-2 myosin strand. It is also shown that a helix-coil model can account for the observed rapid relaxation of muscle without invoking any complex behaviour of the crossbridge head.     

Genotoxicity of Nicotiana tabacum leaves on Helix aspersa

Tobacco farmers are routinely exposed to complex mixtures of inorganic and organic chemicals present in tobacco leaves. In this study, we examined the genotoxicity of tobacco leaves in the snail Helix aspersa as a measure of the risk to human health. DNA damage was evaluated using the micronucleus test and the Comet assay and the concentration of cytochrome P450 enzymes was estimated. Two groups of snails were studied: one fed on tobacco leaves and one fed on lettuce (Lactuca sativa L) leaves (control group). All of the snails received leaves (tobacco and lettuce leaves were the only food provided) and water ad libitum. Hemolymph cells were collected after 0, 24, 48 and 72 h. The Comet assay and micronucleus test showed that exposure to tobacco leaves for different periods of time caused significant DNA damage. Inhibition of cytochrome P450 enzymes occurred only in the tobacco group. Chemical analysis indicated the presence of the alkaloid nicotine, coumarins, saponins, flavonoids and various metals. These results show that tobacco leaves are genotoxic in H. aspersa and inhibit cytochrome P450 activity, probably through the action of the complex chemical mixture present in the plant.     

Experimental hybridization between two sub-species of snails (Helix aspersa aspersa and Helix aspersa maxima): consequences for fertility,survival and growth

Summary

The experimental laboratory pairing of hermaphroditic snails of different sub-species, Helix aspersa aspersa (H.a.a) and H. aspersa maxima (H.a.m.), made it possible to obtain both reciprocal hybrids for the first time and to describe their growth. The relatively small number of matings observed (20–25%) was partly responsible for the small number of clutches obtained (10–13%). The cross H.a.a.M × H.a.m.F is more fertile than the reciprocal. The observed tendency to reproductive isolation (behavioural and anatomical) suggests that H.a.a. and H.a.m. should be considered as two sub-species. Hybridization is not accompanied by a beneficial heterosis effect since significant juvenile mortality and an increased variation in growth were observed. The small size of H.a.a. is a dominant characteristic, whereas the colour of the mantle edge in the Fl individuals is intermediate between the black of H.a.m. and the white of H.a.a. The Fl generation is fertile and gives F2 snails with an adult size close to that of H.a.a. but slower growing. This approach allows investigations into the mechanisms underlying the reproductive isolation of parental sub-species.     

Antiamoebin I in methanol solution: rapid exchange between right-handed and left-handed 3(10)-helical conformations

Antiamoebin I (Aam-I) is a membrane-active peptaibol antibiotic isolated from fungal species belonging to the genera Cephalosporium, Emericellopsis, Gliocladium, and Stilbella. Antiamoebin I has the amino acid sequence: Ac-Phe(1)-Aib-Aib-Aib-Iva-Gly-Leu-Aib(8)-Aib-Hyp-Gln-Iva-Hyp-Aib-Pro-Phl(16). By using the uniformly (13)C,(15)N-labeled sample of Aam-I, the set of conformationally dependent J couplings and (3h)J(NC) couplings through H-bonds were measured. Analysis of these data along with the data on magnetic nonequivalence of the (13)C(beta) nuclei (Deltadelta((13)C(beta))) in Aib and Iva residues allowed us to draw the univocal conclusion that the N-terminal part (Phe(1)-Gly(6)) of Aam-I in MeOH solution is in fast exchange between the right-handed and left-handed 3(10)-helical conformations, with an approximately equal population of both states. An additional conformational exchange process was found at the Aib(8) residue. The (15)N-NMR-relaxation and CD-spectroscopy measurements confirmed these findings. Molecular modeling and Monte Carlo simulations revealed that both exchange processes are correlated and coupled with significant hinge-bending motions around the Aib(8) residue. Our results explain relatively low activity of Aam-I with respect to other 15-amino acid residue peptaibols (for example, zervamicin) in functional and biological tests. The high dynamic 'propensity' possibly prevents both initial binding of the antiamoebin to the membrane and subsequent formation of stable ionic channels according to the barrel-stave mechanism.     

Crystal structure of Salmonella typhimurium 2-methylisocitrate lyase (PrpB) and its complex with pyruvate and Mg(2+)

Propionate metabolism in Salmonella typhimurium occurs via 2-methylcitric acid cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate, is catalysed by 2-methylisocitrate lyase (PrpB). Here we report the X-ray crystal structure of the native and the pyruvate/Mg(2+) bound PrpB from S. typhimurium, determined at 2.1 and 2.3A, respectively. The structure closely resembles that of the Escherichia coli enzyme. Unlike the E. coli PrpB, Mg(2+) could not be located in the native Salmonella PrpB. Only in pyruvate bound PrpB structure, Mg(2+) was found coordinated with pyruvate. Binding of pyruvate to PrpB seems to induce movement of the Mg(2+) by 2.5A from its position found in E. coli native PrpB. In both the native enzyme and pyruvate/Mg(2+) bound forms, the active site loop is completely disordered. Examination of the pocket in which pyruvate and glyoxalate bind to 2-methylisocitrate lyase and isocitrate lyase, respectively, reveals plausible rationale for different substrate specificities of these two enzymes. Structural similarities in substrate and metal atom binding site as well as presence of similar residues in the active site suggest possible similarities in the reaction mechanism.