Specific activity of LHRH and TRH degrading enzymes in various tissues of normal and castrated male rats.

The chlorophyll composition and Hill activity of the leaf, developing seed parts and pod have been studied in three species of legumes, $\text{Lathyrus latifolius}$, $\text{Pisum sativum}$ and $\text{Vicia faba}$. The studies indicate that in all the species, the level of chlorophyll, on mg/g fresh weight basis, is maximum in the leaf. However, Hill activity studies show that the cotyledonary chloroplasts in all the cases have a higher Hill activity than the leaf chloroplasts. Thus, the Hill activities of the cotyledonary chloroplasts are 340% of the leaf chloroplasts in $\text{Lathyrus}$$\text{latifolius}$, 144% of the leaf chloroplasts in $\text{Pisum}$$\text{sativum}$ and 200% of the leaf chloroplasts in $\text{Vicia}$$\text{faba}$.     

Differential development of cholinergic neurons from cranial and trunk neural crest cells in vitro

Several studies have suggested that the development of cholinergic properties in cranial parasympathetic neurons is determined by these cells' axial level of origin in the neural crest. All cranial parasympathetic neurons normally derive from cranial neural crest. Trunk neural crest cells give rise to sympathetic neurons, most of which are noradrenergic. To determine if there is an intrinsic difference in the ability of cranial and trunk neural crest cells to form cholinergic neurons, we have compared the development of choline acetyltransferase (ChAT)-immunoreactive cells in explants of quail cranial and trunk neural crest in vitro. Both cranial and trunk neural crest explants gave rise to ChAT-immunoreactive cells in vitro. In both types of cultures, some of the ChAT-positive cells also expressed immunoreactivity for the catecholamine synthetic enzyme tyrosine hydroxylase. However, several differences were seen between cranial and trunk cultures. First, ChAT-immunoreactive cells appeared two days earlier in cranial than in trunk cultures. Second, cranial cultures contained a higher proportion of ChAT-immunoreactive cells. Finally, a subpopulation of the ChAT-immunoreactive cells in cranial cultures exhibited neuronal traits, including neurofilament immunoreactivity. In contrast, neurofilament-immunoreactive cells were not seen in trunk cultures. These results suggest that premigratory cranial and trunk neural crest cells differ in their ability to form cholinergic neurons.     

Evidence for a role of helix IV in connecting cation- and sugar-binding sites of Escherichia coli melibiose permease

To improve the structural organization model of melibiose permease, we assessed the individual contributions of the N-terminal tryptophans to the transporter fluorescence variations induced by the binding of cations and beta-configured sugars, by replacement of the six N-terminal tryptophans by phenylalanines and the study of the signal changes. Only two mutations, W116F located in helix IV and W128F located in the cytoplasmic loop 4-5, impair permease activity. The intrinsic fluorescence spectroscopy analysis of the other mutants suggests that W54, located in helix II, W116, and W128 are mostly responsible for the cation-induced fluorescence variations. These tryptophans, W116 and W128, would also be responsible for the beta-galactoside-induced fluorescence changes observed in the N-terminal domain of the transporter. The implication of W116 and W128 in both the cation- and beta-galactoside-induced fluorescence variations led us to investigate in detail the effects of their mutations on the functional properties of the permease. The results obtained suggest that the domains harboring the two tryptophans, or the residues themselves, play a critical role in the mechanism of Na(+)/sugar symport. Taken together, the results presented in this paper and previous results are consistent with a fundamental role of helix IV in connecting cation- and sugar-binding sites of the melibiose permease.     

Secondary structure components and properties of the melibiose permease from Escherichia coli: a fourier transform infrared spectroscopy analysis

The structure of the melibiose permease from Escherichia coli has been investigated by Fourier transform infrared spectroscopy, using the purified transporter either in the solubilized state or reconstituted in E. coli lipids. In both instances, the spectra suggest that the permease secondary structure is dominated by alpha-helical components (up to 50%) and contains beta-structure (20%) and additional components assigned to turns, 3(10) helix, and nonordered structures (30%). Two distinct and strong absorption bands are recorded at 1660 and 1653 cm(-1), i.e., in the usual range of absorption of helices of membrane proteins. Moreover, conditions that preserve the transporter functionality (reconstitution in liposomes or solubilization with dodecyl maltoside) make possible the detection of two separate alpha-helical bands of comparable intensity. In contrast, a single intense band, centered at approximately 1656 cm(-1), is recorded from the inactive permease in Triton X-100, or a merged and broader signal is recorded after the solubilized protein is heated in dodecyl maltoside. It is suggested that in the functional permease, distinct signals at 1660 and 1653 cm(-1) arise from two different populations of alpha-helical domains. Furthermore, the sodium- and/or melibiose-induced changes in amide I line shape, and in particular, in the relative amplitudes of the 1660 and 1653 cm(-1) bands, indicate that the secondary structure is modified during the early step of sugar transport. Finally, the observation that approximately 80% of the backbone amide protons can be exchanged suggests high conformational flexibility and/or a large accessibility of the membrane domains to the aqueous solvent.     

Molecular interaction between organophosphorus acid anhydrolase and diisopropylfluorophosphate

Organophosphorus acid anhydrolases (OPAA; E.C.3.1.8.2) are a class of enzymes that hydrolyze a variety of toxic acetylcholinesterase-inhibiting organophosphorus (OP) compounds, including pesticides and fluorine-containing chemical nerve agents. In this paper, subphase conditions have been optimized to obtain stable OPAA Langmuir films, and the diisopropylfluorophosphate (DFP) hydrolysis reaction catalyzed by OPAA in aqueous solution and at the air-water interface was studied. OPAA-DFP interactions were investigated utilizing different spectroscopic techniques, that is, circular dichroism and fluorescence in aqueous solution and infrared reflection absorption spectroscopies at the air-water interface. The characterization of OPAA and its secondary structure in aqueous solution and as a monolayer at the air-water interface in the absence and in the presence of DFP dissolved in aqueous solution or in the aqueous subphase demonstrated significantly distinctive features. The research described herein demonstrated that OPAA can be used in an enzyme-based biosensor for DFP detection.     

Nucleotide sequence and analysis of conjugative plasmid pVT745

The complete nucleotide sequence and genetic map of pVT745 are presented. The 25-kb plasmid was isolated from Actinobacillus actinomycetemcomitans, a periodontal pathogen. Two-thirds of the plasmid encode functions related to conjugation, replication, and replicon stability. Among potential gene products with a high degree of similarity to known proteins are those associated with plasmid conjugation. It was shown that pVT745 derivatives not only mobilized a coresident nontransmissible plasmid, pMMB67, but also mediated their own conjugative transfer to different A. actinomycetemcomitans strains. However, transfer of pVT745 derivatives from A. actinomycetemcomitans to Escherichia coli JM109 by conjugation was successful only when an E. coli origin of replication was present on the pVT745 construct. Surprisingly, 16 open reading frames encode products of unknown function. The plasmid contains a conserved replication region which belongs to the HAP (Haemophilus-Actinobacillus-Pasteurella) theta replicon family. However, its host range appears to be rather narrow compared to other members of this family. Sequences homologous to pVT745 have previously been detected in the chromosomes of numerous A. actinomycetemcomitans strains. The nature and origin of these homologs are discussed based on information derived from the nucleotide sequence.     

Chemical modifications of the Na+-H+ antiport in Escherichia coli membrane vesicles

The effects of chemical modifications of the Na+-H+ antiport in Escherichia coli have been analyzed by studying the resulting variations of the energy-dependent, downhill Na+ efflux from membrane vesicles. The histidyl reagent diethylpyrocarbonate (EtO)2C2O3 prevents the activation of the Na+ efflux mechanism by delta microH+ or its components. Inactivation of the antiporter by (EtO)2C2O3 is completely reversed by hydroxylamine. The data suggest that histidine residues are involved in the molecular mechanism of the Na+-H+ antiport. In contrast, no conclusive evidence suggesting participation of carboxylic, tyrosine or sulfhydryl residues in the Na+-H+ exchange reaction has been obtained.