Measurement of the Membrane Potential of Isolated Nerve Terminals by the Lipophilic Cation [3H]Triphenylmethylphosphonium Bromide

Abstract: The lipophilic cation [³H]triphenylrnethylphosphonium bromide ([³H]TPMP⁺) was investigated as a measure of the membrane potential of synaptosomes. Conditions under which [³H]TPMP⁺ achieved an equilibrium distribution were tested. The toxicity of TPMP has been studied relative to its inhibitory effects on [³H]y-aminobutyric acid ([³H]GABA) transport. In some experiments the distribution of ⁸⁶RbZ⁺ and [³H]TPMP⁺ was changed upon incubation in the presence of elevated levels of K⁺, ouabain, or KCN, or at 0°C in a way that would be expected from the membrane potential. In normal incubation conditions a membrane potential of ∼−60 mv was calculated.     

Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease

The Bacillus subtilis nprE gene lacking its own promoter sequence was inserted in the lactococcal expression vector pMG36e. Upon introduction of the recombinant plasmid into Lactococcus lactis subsp. lactis strain MG1363, neutral protease activity could be visualized by the appearance of large clearing zones around colonies grown on milk agar plates. By measuring the activities of the neutral protease and the intracellular enzyme lactate dehydrogenase in culture supernatants and cell fractions, it was demonstrated that the neutral protease was actively secreted into the growth medium. This was corroborated by using the Western blot (immunoblot) technique, which showed the presence of the mature form of the neutral protease in the culture supernatant. On the basis of these results, it is concluded that the B. subtilis neutral protease gene was expressed in L. lactis and that the gene product was secreted into the growth medium and was apparently correctly processed to produced a biologically active protein. The secretion of this particular enzyme may be helpful in achieving accelerated cheese ripening.     

A semiautomated fluorometric determination of 5-hydroxyindoles in the nanogram range

Automated determinations of 5-hydroxytryptamine and its main metabolite, 5-hydroxyindoleacetic acid, have been described (Technicon autoanalyzer). The determinations are based on an extraction procedure from deproteinized tissue extracts or cerebrospinal fluid by means of butanolheptane mixtures. The indoles are transferred from the organic phase to a water phase and determined fluormetrically with the cysteine-o-phthaldialdehyde method. The method is highly sensitive: solutions containing 1 ng/ml can be reproducibly determined. Twenty samples per hour can be passed through the system. The determination of both 5-hydroxyindoles is performed with the same manifold.     

Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis.

A pair of vectors for expression of heterologous genes in Lactococcus lactis was constructed. In addition to an origin of replication that has a broad host range, these vectors contain a multiple cloning site flanked by gene expression signals originating from L. lactis subsp. cremoris Wg2. The two vectors, about 3.7 kilobase pairs in size, differ only in the type of antibiotic resistance they confer to their hosts. pMG36 carries a kanamycin resistance marker, which was replaced by an erythromycin resistance marker in pMG36e. As an example of the use of these vectors, the hen egg white lysozyme-coding sequence was inserted. A fusion protein of the expected size was detected in a transformed L. lactis subsp. lactis strain by using Western blotting (immunoblotting).     

Development of a beta-galactosidase alpha-complementation system for molecular cloning in Bacillus subtilis

A versatile beta-galactosidase alpha-complementation system for Bacillus subtilis was developed, which can be used for molecular cloning in this Gram+ organism. The cloning system, which is based on the highly efficient host-vector system 6GM-pHP13, offers several advantages over previously described systems: (1) convenient direct selection of recombinants; (2) the cloning of large heterologous DNA fragments with high efficiency; and (3) the availability of six unique target sites: SphI, NdeI, NheI, BamHI, SmaI and EcoRI.     

Cloning and characterization of the tetracycline resistance determinant of and several promoters from within the conjugative transposon Tn919

Tn919 is a 15- to 16-kilobase (kb) tetracycline resistance conjugative transposon that was originally isolated from Streptococcus sanguis FC1. The tetracycline resistance determinant (tet) was found on a 4.2-kb HindII fragment by in vitro deletion analysis. This fragment was subcloned to a pWV01 origin capable of directing replication in Escherichia coli, Bacillus subtilis, and Streptococcus lactis, and expression was observed in all three genera. In all cases, expression was weaker when only the 4.2-kb cloned fragment rather than the full transposon was present. The resistance gene is of the streptococcal tetM class and codes for a protein of approximately 70 kilodaltons. The restriction map resembles that of the tetM gene of Tn1545 (P. Martin, P. Trieu-Cuot, and P. Courvalin, Nucleic Acids Res. 14:7047-7058, 1986), which codes for a protein of 72.5 kilodaltons. A number of transposon-derived promoter-bearing fragments were also cloned and sequenced. These closely resemble the consensus sequence of E. coli and B. subtilis promoters. Fusion experiments with a truncated lacZ gene indicate the possibility of an open reading frame for one of the promoters.     

Signal peptidase I of Bacillus subtilis: patterns of conserved amino acids in prokaryotic and eukaryotic type I signal peptidases.

Signal peptidases (SPases) remove signal peptides from secretory proteins. The sipS (signal peptidase of subtilis) gene, which encodes an SPase of Bacillus subtilis, was cloned in Escherichia coli and was also found to be active in E.coli. Its overproduction in B.subtilis resulted in increased rates of processing of a hybrid beta-lactamase precursor. The SipS protein consisted of 184 amino acids (mol. wt 21 kDa). The protein showed sequence similarity with the leader peptidases of E.coli and Salmonella typhimurium, and the mitochondrial inner membrane protease I of Saccharomyces cerevisiae. Patterns of conserved amino acids present in these four proteins were also detected in the Sec11 subunit of the SPase complex of S.cerevisiae and the 18 and 21 kDa subunits of the canine SPase complex. Knowledge of the sequence of SipS was essential for the detection of these similarities between prokaryotic and eukaryotic SPases. The data suggest that these proteins, which have analogous functions, belong to one class of enzymes, the type I SPases.