Stereoselectivity of the membrane potential-generating citrate and malate transporters of lactic acid bacteria.

The citrate transporter of Leuconostoc mesenteroides (CitP) and the malate transporter of Lactococcus lactis (MleP) are homologous proteins that catalyze citrate-lactate and malate-lactate exchange, respectively. Both transporters transport a range of substrates that contain the 2-hydroxycarboxylate motif, HO-CR(2)-COO(-) [Bandell, M., et al. (1997) J. Biol. Chem. 272, 18140-18146]. In this study, we have analyzed binding and translocation properties of CitP and MleP for a wide variety of substrates and substrate analogues. Modification of the OH or the COO(-) groups of the 2-hydroxycarboxylate motif drastically reduced the affinity of the transporters for the substrates, indicating their relevance in substrate recognition. Both CitP and MleP were strictly stereoselective when the R group contained a second carboxylate group; the S-enantiomers were efficiently bound and translocated, while the transporters had no affinity for the R-enantiomers. The affinity of the S-enantiomers, and of citrate, was at least 1 order of magnitude higher than for lactate and other substrates with uncharged R groups, indicating a specific interaction between the second carboxylate group and the protein that is responsible for high-affinity binding. MleP was not stereoselective in binding when the R groups are hydrophobic and as large as a benzyl group. However, only the S-enantiomers were translocated by MleP. CitP had a strong preference for binding and translocating the R-enantiomers of substrates with large hydrophobic R groups. These differences between CitP and MleP explain why citrate is a substrate of CitP and not of MleP. The results are discussed in the context of a model for the interaction between sites on the protein and functional groups on the substrates in the binding pockets of the two proteins.     

Graphical representation of the salient conformational features of protein residues.

A composite plot for depicting in two dimensions the conformation and the secondary structural features of protein residues has been developed. Instead of presenting the exact values of the main- and side-chain torsion angles (φ, psi and chi(1)), it indicates the region in the three-dimensional conformational space to which a residue belongs. Other structural aspects, like the presence of a cis peptide bond and disulfide linkages, are also displayed. The plot may be used to recognize patterns in the backbone and side-chain conformation along a polypeptide chain and to compare protein structures derived from X-ray crystallography, NMR spectroscopy or molecular modelling studies and also to highlight the effect of mutation on structure.     

Variable modes of inheritance of morphometrical traits in hybrids between Drosophila melanogaster and Drosophila simulans.

We investigated body-size inheritance in interspecific sterile hybrids by crossing a Drosophila simulans strain with 13 strains of Drosophila melanogaster, which were of various origins and chosen for their broad range of genetic variation. A highly significant parent-offspring correlation was observed, showing that the D. melanogaster genes for size are still expressed in a hybrid background. Superimposed on to this additive inheritance, the size of hybrids was always less than the mid-parent value. This phenomenon, which at first sight might be described as dominance or overdominance, is more precisely interpreted as a consequence of a hybrid breakdown, that is, a dysfunction of the parental genes for size when put to work together. This interpretation is enforced by the fact that phenotypic variability was much more prevalent in hybrids than in parents. We also analysed body pigmentation inheritance in the same crosses and got a very different picture. There was no increase in the phenotypic variance of F(1) hybrids and only a low parent-offspring correlation. Apparent overdominance could be observed but in opposite directions, with no evidence of hybrid breakdown. Our data point to the possibility of analysing a diversity of quantitative traits in interspecific hybrids, and indicate that breakdown might be restricted to some traits only.     

Ventilatory responses to specific CNS hypoxia in sleeping dogs.

Our study was concerned with the effect of brain hypoxia on cardiorespiratory control in the sleeping dog. Eleven unanesthetized dogs were studied; seven were prepared for vascular isolation and extracorporeal perfusion of the carotid body to assess the effects of systemic [and, therefore, central nervous system (CNS)] hypoxia (arterial PO(2) = 52, 45, and 38 Torr) in the presence of a normocapnic, normoxic, and normohydric carotid body during non-rapid eye movement sleep. A lack of ventilatory response to systemic boluses of sodium cyanide during carotid body perfusion demonstrated isolation of the perfused carotid body and lack of other significant peripheral chemosensitivity. Four additional dogs were carotid body denervated and exposed to whole body hypoxia for comparison. In the sleeping dog with an intact and perfused carotid body exposed to specific CNS hypoxia, we found the following. 1) CNS hypoxia for 5-25 min resulted in modest but significant hyperventilation and hypocapnia (minute ventilation increased 29 +/- 7% at arterial PO(2) = 38 Torr); carotid body-denervated dogs showed no ventilatory response to hypoxia. 2) The hyperventilation was caused by increased breathing frequency. 3) The hyperventilatory response developed rapidly (<30 s). 4) Most dogs maintained hyperventilation for up to 25 min of hypoxic exposure. 5) There were no significant changes in blood pressure or heart rate. We conclude that specific CNS hypoxia, in the presence of an intact carotid body maintained normoxic and normocapnic, does not depress and usually stimulates breathing during non-rapid eye movement sleep. The rapidity of the response suggests a chemoreflex meditated by hypoxia-sensitive respiratory-related neurons in the CNS.     

Modified moving average analysis of T-wave alternans to predict ventricular fibrillation with high accuracy.

T-wave alternans is a marker of cardiac electrical instability with the potential for arrhythmia risk stratification. The modified moving average method was developed to measure alternans in settings with artifacts, noise, and nonstationary data. Algorithms were developed and performance characteristics were validated with simulated electrocardiograms (ECGs). Experimental laboratory ECGs with dynamically changing alternans values were analyzed. Alternans values estimated by modified moving average analysis correlated strongly with input alternans values (r(2) = 0.9999). Rapidly changing alternans levels and phase reversals did not perturb the measurement. When heart rate was increased from 60 to 180 beats/min, with T-wave alternans apex moving from 237 to 103 ms after the R wave, the measured alternans peak varied <5% from input value. Simulated 50- to 1,000-microV motion artifact spikes typical of treadmill ECGs produced inaccuracies <2%. Alternans values in experimental laboratory study using standard electrodes tracked vulnerability to myocardial ischemia-induced ventricular fibrillation with 100% sensitivity and specificity at a cut point of 0.75 mV. Modified moving average analysis is a robust method that precisely measures T-wave alternans in settings with artifacts, noise, and nonstationary data typical of clinical ECGs and yields an accurate estimate of risk for ventricular fibrillation.     

Comparison of the roles of neurohormones in the regulation of blood distribution from the hearts of American and Japanese lobsters

The decapod cardiovascular system consists of a single ventricle that pumps blood into seven arteries; previous work has shown that the outflow distribution patterns of intact animals are variable. In the present study, flow recordings were made from pairs of arteries in semi-isolated hearts whilst different cardioactive hormones were infused into the heart. Each hormone (5-hydroxytryptamine, octopamine, dopamine, proctolin and F1) changed the outflow pattern, heart rate and ventricular pressure in a unique way. The probable sites of hormone action are the cardioarterial valves located at the origin of each artery except one, the dorsal abdominal. Outflow from the dorsal abdominal is controlled downstream by valves located at the origin of the segmental lateral arteries. The responses to a particular hormone were sometimes different between the hearts of American and Japanese lobsters. Accepted: 11 May 1998