Energy dependence of protein synthesis by isolated cestode mitochondria.

Mitochondria of helminth parasites produce energy in a number of ways that vary from species to species, and, in general, do not possess a classical tricarboxylic acid (Krebs) cycle. The process of protein synthesis, which is energy dependent, is not well understood in this organelle. However, protein synthesis in helminth mitochondria seems to serve the purpose to the desired level despite its limited capacity to produce ATP compared to its mammalian counterpart. Data presented here demonstrate that helminth mitochondria can synthesize proteins in vitro and the process can be supported by energy supplied exogenously by the cytosol, by an ATP generating system utilizing a substrate such as malate, or by ATP itself.

Identification, cloning, and sequencing of the immunoglobulin A1 protease gene of Streptococcus pneumoniae.

The pneumococcus expresses a protease that hydrolyzes human immunoglobulin A1 (IgA1). A gene for IgA1 protease was identified from a plasmid library of pneumococcal DNA because of the effect of its overexpression on the colony morphology of Streptococcus pneumoniae. The deduced 1,964-amino-acid sequence is highly homologous to that of the IgA1 protease from Streptococcus sanguis. The similarity to the S. sanguis enzyme and the presence of a putative zinc-binding site suggest that the pneumococcal enzyme is a metalloprotease. The two streptococcal sequences differ in a hydrophilic region with 10 tandem repeats of a 20-mer in S. sanguis, which is replaced by a similar but less repetitive sequence in S. pneumoniae. Antiserum reactive with the pneumococcal IgA1 protease was used to demonstrate that the majority of the protein is cell associated. The expression and function of this gene were confirmed by insertional mutagenesis. Interruption of the chromosomal gene resulted in loss of expression of an approximately 200-kDa protein and complete elimination of detectable IgA1 protease activity.

Relationship between phase variation in colony morphology, intrastrain variation in cell wall physiology, and nasopharyngeal colonization by Streptococcus pneumoniae.

Streptococcus pneumoniae undergoes phase variation in colony morphology, which has been implicated as a factor in the pathogenesis of pneumococcal disease. Phenotypic differences between opaque and transparent colony forms correlate with differences in rates of autolysis. This study examined whether differences in autolysis are caused by differences in expression of the major amidase, LytA, or the structure of its peptidoglycan substrate. No significant difference was detected by high-pressure liquid chromatography analysis of stem peptides released after treatment of purified peptidoglycan with amidase. Differences in the rate of digestion of purified cell walls, furthermore, did not correlate with susceptibility to autolysis. Lower levels of autolysis in opaque variants, however, was associated with decreased levels of immunodetectable LytA on colony immunoblots and Western blots (immunoblots). Diminished cell-surface-associated LytA in opaque variants was also demonstrated by whole-cell inhibition enzyme-linked immunosorbent assay. Since transparent variants have been shown both to colonize the nasopharynx more efficiently in an animal model and to express more surface-exposed LytA, it was determined whether LytA contributes to colonization in a neonatal rat model of pneumococcal carriage. Defined mutants in the lytA gene were used to show that there was no significant contribution by LytA to nasopharyngeal colonization in this model. Although the expression of LytA was shown to undergo phase variation in association with colony morphology, lytA mutants are still capable of phenotypic variation in colony morphology, which suggests that other factors are responsible for intrastrain differences which affect colonization.

Neonatal phenobarbital-induced persistent alterations in plasma testosterone profiles and testicular function.

Daily sc injections of phenobarbital at anticonvulsant therapeutic doses for the rat (40 mg/kg) for the first 7 days of life resulted in below normal levels of serum testosterone from around birth to before puberty, normal levels during puberty and above normal levels of the androgen after puberty and in adulthood. Cluster analysis of the plasma testosterone secretory profiles obtained at 15-min intervals from phenobarbital-treated rats at 65 and 165 days of age revealed a significant increase in both the peak amplitudes and their durations resulting in a 100% increase in the amount of hormone secreted during the peak periods. In general, most of the rats (control and experimental) secreted testosterone as two large peaks, each 3 to 4 hr in duration, during the 10-hr lights-on collection period. In addition to permanently disrupting the ultradian profiles of plasma testosterone, neonatal exposure to the barbiturate altered testicular responsiveness to steroidogenic regulatory agents. That is, neonatal exposure to phenobarbital enhanced the responsiveness to exogenous hCG as measured by an above-normal increase in testosterone concentration. Moreover, phenobarbital-induced reductions in serum testosterone levels were delayed in adult rats neonatally exposed to the barbiturate. Whereas a single challenge dose of phenobarbital (1 or 10 mg/kg) reduced serum testosterone concentrations in control animals by almost 80% within 3 hr, a decline in serum androgen levels in the neonatally phenobarbital exposed males was not observed until 12 hr after the challenge dose. These results indicate that postpartum exposure to therapeutic levels of phenobarbital can permanently disrupt testosterone secretory profiles and alter pathways regulating testicular steroidogenesis.

Cation-induced efflux of calcium from the mitochondria of Hymenolepis diminuta.

We have previously reported that Ca2+ influx into the mitochondria of Hymenolepis diminuta, a rat intestinal cestode, takes place through an electrophoretic uniport system. Sodium and lithium were found to induce efflux of 45Ca2+ from the mitochondria of H. diminuta. The two cations induced the efflux in a hyperbolic and linear fashion, respectively. The efflux as well as an exchange of external Ca2+ with internal 45Ca2+ was inhibited by lanthanum. The type of Ca2+ transport system in the cestode organelle has been discussed and compared with that of the host (mammalian) counterpart.

Ca2+/Mg(2+)-dependent ATPase activity in Hymenolepis diminuta mitochondria.

Ca2+ and Mg2+ caused a concentration-dependent activation of ATP hydrolysis by mitochondrial membranes of Hymenolepis diminuta, a rat intestinal cestode. Ca2+ was the more potent, but Mg2+ the more effective. The Lineweaver-Burk plot yielded Km and Vmax values of 1.15 nM and 217.4 nmol Pi min-1 mg-1 protein for Ca(2+)-dependent activity, and 1.86 mM and 333.3 nmol Pi min-1 mg-1 protein for Mg(2+)-dependent activity, respectively. Neither Na+ nor K+, nor a combination of the two cations, induced the hydrolysis of ATP. Ouabain, a specific inhibitor of Na+/K+ ATPase, did not affect the rate of ATP hydrolysis induced by Mg2+ alone or in combination with Na+ or K+. The membrane-bound enzyme was not affected by neuraminidase and concanavalin A. Ca2+ and Mg2+ also induced appreciable hydrolysis of other nucleoside triphosphates by the membranes. Some known anthelmintics, e.g. niclosamide, praziquantel and mebendazole, had no effect on ATPase activities. In addition to other compounds including respiratory inhibitors and uncouplers of phosphorylation, ruthenium red, which blocks Ca2+ influx into the cestode mitochondria, had no influence on the rate of ATP hydrolysis induced by the cations. Triton X-100 was found most suitable for solubilization of both activities. The differences between cestode ATPase and its mammalian counterpart have been discussed.

Effect of cations and anthelmintics on enzymes of respiratory chains of the cestode Hymenolepis diminuta.

The enzymes involved in the catabolism of malate namely fumarate reductase, NADH oxidase, "malic" enzyme, succinate dehydrogenase and fumarase as well as NADPH:NAD transhydrogenase, which is involved in the electron transport chain, were studied in Hymenolepis diminuta, a rat intestinal tapeworm. Among cations, K+ had no effect on any enzyme whereas Ca2+ and Mg2+ showed an increase or decrease of varying degrees of different enzyme activities. Most of the compounds, which have been synthesized by the Central Drug Research Institute, Lucknow (India) and found to possess some anthelmintic properties, strongly inhibited the above enzymes except malic enzyme.

Effect of antibiotics on protein synthesis in filarial parasites.

Acanthocheilonema viteae, Litomosoides carinii and Setaria cervi were found to actively synthesize proteins in vitro. Different centrifugation fractions and their TCA-precipitable fractions were assessed for the distribution of newly synthesized proteins. Penicillin and streptomycin inhibited the process in A. viteae. The synthesis in S. cervi was susceptible to puromycin, chloramphenicol, cycloheximide, neomycin and polymyxin B. The process in L. carinii was strongly blocked by puromycin while chloramphenicol had no significant effect.

In vitro protein synthesis in Hymenolepis diminuta, a rat intestinal cestode.

Helminth parasitic infestations are of great concern to the health of man and other animals. Limited studies have been carried out on the process of protein synthesis which, apart from having academic importance, could possibly provide a target for drug attack. In the present study distribution pattern of newly synthesized proteins in Hymenolepis diminuta, a rat intestinal cestode, has been worked out. The worms absorbed and incorporated about 21% and 3.8% of the amino acids added to the incubation medium, respectively. In other words, 18.3% of the absorbed amino acids was incorporated into proteins. Absorbed amino acids were distributed between washed cell debris (unbroken cells, nuclei, etc.) and crude extract in a ratio of 1:12. Cytosolic, microsomal and mitochondrial fractions received 72%, 3.2% and 6.6% amino acids, respectively. The mitochondrial membranes and the matrix shared equally the absorbed amino acids. The distribution pattern of amino acid incorporation was, however, different from that of absorption. The incorporation ratio between washed cell debris and crude extract was 1:3.7. The cytosolic, microsomal and mitochondrial fractions received 32.8%, 8.6% and 18.4% of the incorporated amino acids, respectively. Within mitochondria incorporation was more in membranes than in the matrix. The ratios of incorporated versus free-pool amino acids in different fractions varied widely from 1:0.54 to 1:11.