Carnitine resembles choline in the induction of cholinesterase,acid phosphatase,and phospholipase C and in its action as an osmoprotectant in Pseudomonas aeruginosa

The present study demonstrates that under conditions of iso or hyperosmolarity, P. aeruginosa utilized carnitine as the carbon, nitrogen or carbon and nitrogen sources. As occurred in the case of choline, the bacteria synthesized cholinesterase (ChE), acid phosphatase (Ac.Pase) and phospholipase C (PLC) under any of these conditions and in the presence of high or low P_i concentrations.Carnitine acted as an osmoprotectant when the cells were grown in the presence of preferred carbon and nitrogen sources and high NaCl concentrations. Under these conditions the three enzyme activities were not produced.The osmotically stressed bacteria grown under any of the above conditions accumulated betaine. Its presence indicated that carnitine may be metabolized by P. aeruginosa to produce betaine which could account for the induction of the three enzyme activities or its action as an osmoprotectant.The phosphatidylcholine encountered in the host cell membranes allows the bacteria to obtain free choline by the coordinated action of PLC and Ac.Pase. Since the consequence of this action may be cell disruption, the increase of free carnitine in the natural environment of the bacteria is also possible. These two compounds, choline and carnitine, acting in conjunction or separately, may increase the production of PLC and Ac.Pase activities by P. aeruginosa and thus enhance the degradative effect upon the host cells.     

Mutational Events in the Triplo- and Haplo-Lethal Region (83de) of the DROSOPHILA MELANOGASTER Genome

The Drosophila melanogaster genome contains a single region (at 83DE on the polytene chromosome map) for which both heterozygous deficiency and heterozygous duplication are inviable. Seven EMS-induced mutations have been recovered that are viable in combination with a duplication of this region. Two classes of mutations are reported: (1) Mutations that allow survival of flies with either a duplication or a normal third chromosome. These mutations retain Ki, a closely linked marker on the mutagenized chromosome. They fail to complement, and one has been mapped to the vicinity of 83DE. (2) Mutations that allow survival only in heterozygous combination with a duplication and have lost the Ki marker. These mutations represent new deletions of the dose-sensitive information. The possible structural organization of the 83DE region is discussed in light of these two classes of mutations.     

Male-Specific Lethal Mutations of DROSOPHILA MELANOGASTER

A total of 7,416 ethyl methanesulfonate (EMS)-treated second chromosomes and 6,212 EMS-treated third chromosomes were screened for sex-specific lethals. Four new recessive male-specific lethal mutations were recovered. When in homozygous condition, each of these mutations kills males during the late larval or early pupal stages, but has no detectable effect in females. One mutant, mle^ts, is a temperature sensitive allele of maleless, mle (Fukunaga, Tanaka and Oishi 1975), while the other three mutants identify two new loci: male-specific lethal-1 (msl-1) (two alleles) at map position 2-53.3 and male-specific lethal-2 (msl-2) at 2-9.0.——The male-specific lethality associated with these mutants is not related to the sex per se of the mutant flies, since sex-transforming genes fail to interact with these mutations. Moreover, the presence or absence of a Y chromosome in males or females has no influence on the male-specific lethal action of these mutations. Finally, no single region of the X chromosome, when present as a duplication, is sufficient to rescue males from the lethal effects of msl-1 or msl-2. These results suggest that the number of complete X chromosomes determines whether a fly homozygous for a male-specific lethal mutation lives or dies.     

On the interaction of bovine pancreatic trypsin inhibitor with maxi Ca(2+)-activated K+ channels. A model system for analysis of peptide-induced subconductance states.

Bovine pancreatic trypsin inhibitor (BPTI) is a 58-residue basic peptide that is a representative member of a widely distributed class of serine protease inhibitors known as Kunitz inhibitors. BPTI is also homologous to dendrotoxin peptides from mamba snake venom that have been characterized as inhibitors of various types of voltage-dependent K+ channels. In this study we compared the effect of DTX-I, a dendrotoxin peptide, and BPTI on large conductance Ca(2+)-activated K+ channels from rat skeletal muscle using planar bilayer methodology. As previously found for DTX-I (1990. Neuron. 2:141-148), BPTI induces the appearance of distinct subconductance events when present on the internal side of maxi K(Ca) channels. The single channel kinetics of substate formation follow the predictions of reversible binding of the peptide to a single site or class of sites with a Kd of 4.6 microM at 0 mV and 50 mM symmetrical KCl. The apparent association rate of BPTI binding decreases approximately 1,000-fold per 10-fold increase in ionic strength, suggestive of a strong electrostatic interaction between the basic peptide and negative surface charge in the vicinity of the binding site. The equilibrium Kd for BPTI and DTX-I is also voltage dependent, decreasing e-fold per 30 mV of depolarization. The unitary subconductance current produced by BPTI binding exhibits strong inward rectification in the presence of symmetrical KCl, corresponding to 15% of open channel current at +60 mV and 70% of open state at -40 mV. In competition experiments, the internal pore-blocking ions, Ba2+ and TEA+, readily block the substate with the same affinity as that for blocking the normal open state. These results suggest that BPTI does not bind near the inner mouth of the channel so as to directly interfere with cation entry to the channel. Rather, the mechanism of substate production appears to involve a conformational change that affects the energetics of K+ permeation.     

Choline and betaine as inducer agents of Pseudomonas aeruginosa phospholipase C activity in high phosphate medium

In Pseudomonas aeruginosa, choline or betaine employed as the sole carbon and nitrogen source in a high phosphate medium induced a phospholipase C and an acid phosphatase activity but not an alkaline phosphatase activity. The P. aeruginosa strain utilized in this work does not possess a constitutive phospholipase C, since under culture conditions identical to those utilized by other authors (J. Bacteriol. 93, 670-674 (1967) and J. Bacteriol. 150, 730-738 (1982), our phospholipase C proved to be an inorganic phosphate-repressible enzyme. These findings enable us to conclude that although the phosphate control for the synthesis of phospholipase C may exist, it is expressed only under certain favorable culture conditions.     

Analysis of the blocking activity of charybdotoxin homologs and iodinated derivatives against Ca2+-Activated K+ channels

Summary Two charybdotoxin peptides were purified from venom of the Israeli scorpion,Leiurus quinquestriatus hebraeus. Microsequencing of the most abundant toxin, ChTX-Lq1, revealed identity with the 37-residue peptide previously sequenced by Gimenez-Gallego et al. [Gimenez-Gallego, G., et al.,Proc. Natl. Acad. Sci. USA 85:3329–3333 (1988)]. Sequence data on the minor peptide, ChTX-Lq2, showed substantial homology to ChTX-Lq1 with differences observed at eight positions. These two charybdotoxin sequences, along with that of noxiustoxin, define a distinct family of scorpion peptide toxins with activity against K⁺ channels. Both charybdotoxin homologs inhibited Ca²⁺-dependent K⁺ efflux from human erythrocytes with similar potency,K _0.5-40nm. In planar bilayer assays of single K(Ca) channels from rat muscle, ChTX-Lq1 and ChTX-Lq2 blocked with intrinsicK _d's of 1.3 and 43nm, respectively, in the presence of 50mm external KCl. A new application of dwell-time histogram analysis of single-channel blocking events was used to characterize the kinetic homogeneity of toxin samples and the blocking kinetics of ChTX derivatives. The lower blocking affinity of ChTX-Lq2 was the combined result of a faster dissociation rate and a slower association rate as compared to ChTX-Lq1. The blocking activity of two mono-iodinated derivatives of ChTX-Lq1 was also analyzed. Blocked dwell-time histograms of the iodinated peptides were characterized by predominately brief (0.2–2 sec) blocking events in comparison to the native toxin (20 sec). Histogram analysis revealed that mono-iodination of ChTX-Lq1 impairs blocking activity by adverse effects on both dissociation and association rate constants. Frequency density histograms of single channel blocking events provide a sensitive assay of toxin purity suitable for quantitating structure-activity relationships of charybdotoxin derivatives.