Heat inhibition of reticulocyte protein synthesis. Evidence for a mechanism independent of the hemin-controlled repressor.

Incubation of rabbit reticulocytes at 45 degrees C results in a prompt but reversible decrease in protein synthesis and a concomitant conversion of polyribosomes to smaller aggregates. These effects occur even in the presence of 100 micrometer hemin in the incubation medium. There is also inhibition of heme synthesis but this occurs at a later time than the effect on protein synthesis. The inhibtion of heme synthesis results from a decrease in activity of beta-aminolevulinic acid synthetase. This decrease of heme synthesis appears to be secondary to the inhibition of protein synthesis with resultant accumulation of intramitochondrial heme (which will decrease beta-aminolevulinic acid synthetase activity). An inhibitor of reticulocyte cell-free protein synthesis formed in the postribosomal supernatants of cells incubated at both 45 and 37 degrees C but not at 0 degrees C. No temporal or quantitative differences in the amount of this inhibitor from cells treated at either 37 or 45 degrees C was apparent. The inhibitor was not found in the fraction where the hemin-controlled repressor is isolated. It is concluded that heat inactivation of intact reticulocyte protein synthesis does not depend upon a decrease in heme synthesis, heme concentration or generation of the hemin-controlled repressor. Furthermore, it appears that the inhibitor formed in the post-ribosomal supernatant cannot be the sole cause of the heat inhibition of protein synthesis.

Iron requirement for isolated rat liver mitochondrial protein synthesis.

Isolated rat liver mitochondrial protein synthesis was severly inhibited by alpha, alpha-dipyridyl (a ferrous iron-chelating agent), chloramphenicol and hemin (10(-7) M or greater). In contrast, gamma, gamma-dipyridyl (a non-iron-chelating analogue of alpha, alpha-dipyridyl), cycloheximide and lower concentrations of hemin were non-inhibitory. The inhibitory action of alpha, alpha-dipyridyl was reversed by addition of Fe(NH4)2(SO4)2 while ZnCl2, CuCl2 and CoCl2 were ineffective. Hemin, however, did not protect against the alpha, alpha-dipyridyl inhibition of mitochondrial protein synthesis. These results indicate that ferrous iron is required for mitochondrial protein synthesis and suggests that it is through a mechanism independent of hemin concentration.

Age-related decline in the biosynthesis of mitochondrial inner membrane proteins.

Isolated mitochondria from rat livers of various ages show a gradual decline in the rate of inner membrane-matrix protein synthesis with advancing age of the animal. Rats at 112-120 weeks synthesize these proteins at only 40% of the rate of 2-8-week-old animals. The initial rates of incorporation of label were 145 cpm/mg/minute for the "old" animals, and 320 cpm/mg/minute for the "young" animal. No difference in either amino acid pool size or leakage of label through the mitochondrial membrane was detected in the two age groups. Treatment of the mitochondria with ferrous ammonium sulphate produced a 1.62 fold increase in mitochondrial protein synthesis in the young animal but not in the old. Hemin treatment produced a similar effect. These results suggest that the decrease in delta-aminolevulinic acid synthase activity seen with age (Paterniti et al., 1978) may be due to a decrease in the synthesis of mitochondrial inner membrane proteins.

Evaluation of frequency of isolation and trends in antibiotic resistance among Campylobacter isolates over 11 year period.

OBJECTIVE: To analyze frequency of isolation and trends in antibiotic resistance among Campylobacter isolates over 11 year period in Microbiology Laboratory, Aga Khan University from the year 1992-2002. METHODS: Total 52,777 stool specimens were processed during the study period. Enteric pathogens isolated from 8,483 stool samples were further analyzed for frequency of isolation and antimicrobial resistance. Statistical Analysis was done by using descriptive statistics of SPSS version 10. Values were expressed as percentages, mean and rates. RESULTS: Campylobacter species were third in frequency of isolation with an isolation rate of 24.8%. C. jejuni was the predominant pathogen followed by C.coli. Isolation rate of Campylobacter was higher (45.7%) among children under 2 years of age as compared to other age groups. A steady rise in resistance among Campylobacter isolates against ampicillin; tetracycline and ofloxacin has been noted whereas resistance against erythromycin remained fairly low. CONCLUSION: The isolation of Campylobacter is higher from stool specimens of children of less than two years of age rendering Campylobacteriosis to be an important cause of gastroenteritis in pediatric population. This study also demonstrates a steady rise in antibiotic resistance in Campylobacter isolates especially against quinolones with fall in resistance against erythromycin throughout the study period.

Regulation of mitochondrial protein synthesis at the polyribosomal level.

Polysomes consisting of two to eight monosomes were isolated from yeast mitochondria by lysing the mitochondria with Triton X-100 and centrifugation in a 20 to 40% linear sucrose gradient. When yeast spheroplasts were pulse-labeled with [3H]-Leucine in the presence of cycloheximide to block cytoplasmic protein synthesis, radioactivity which was trichloroacetic acid-precipitable was present mainly in the polysome region. Incorporation of leucine was blocked by erythromycin, a specific inhibitor of mitochondrial protein synthesis. Release of radioactivity to the top of the gradient resulted from treating labeled polysomes with either puromycin or ribonuclease (in the latter case with the breakdown of polysomes), indicating that the radioactivity was present in nascent polypeptide chains. Yeast cells were grown in chloramphenicol for 3 hours and in fresh medium for 1 hour and then pulse-labeled with either [3H]leucine or [14C]formate. Three parameters showed a 2-fold increase in cells grown in chloramphenicol prior to pulse labeling: the polysome to monosome ratio, the amount of labeled precursor incorporated into proteins, and the rate of polypeptide chain initiation as judged by the formation of fMet-puromycin. Conversely, these parameters were all decreased approximately 50% in cells treated with cycloheximide prior to pulse labeling. Mitochondria were also isolated from cells previously grown in chloramphenicol or cycloheximide and incubated in vitro with [3H]leucine under optimal conditions. Acid-precipitable radioactivity in the polysome region was increased 3-fold in mitochondria from cells grown previously in chloramphenicol and decreased 75% in those grown in cycloheximide. Furthermore, chain initiation was deomonstrated in the isolated mitochondria by formation of fMet-puromycin. The rate of chain initiation in vitro was increased 2-fold in mitochondria isolated from chloramphenicol-treated cells.

Ethanol inhibition of rabbit reticulocyte haem synthesis at the level of delta-aminolaevulinic acid synthetase.

Ethanol inhibition of rabbit reticulocyte synthesis occurs as a result of a decrease in haem synthesis. The present study therefore was undertaken in order to localize the inhibitory site of ethanol on the haem biosynthetic pathway. Ethanol (0.05--0.15 M) inhibition of reticulocyte protein synthesis was prevented by simultaneous incubation with 0.025--1 mM delta-aminolaevulinic acid (ALA). Ethanol inhibited both 14C-glycine and 14C-ALA incorporation into haem. However, the extent of haem formation with 14C-ALA as substrate in the presence of ethanol was still equal to that when 14C-glycine was used. These data suggest that ethanol inhibits the haem synthetic pathway at several loci, but that the decrease in haem synthesis, responsible for the decrease in protein synthesis, is due to the inhibition at the rate-limiting enzyme, delta-aminolaevulinic acid synthetase (ALA-S). To confirm this, ALA-S activity was then directly measured in intact reticulocytes, and it was shown that ethanol indeed inhibited its activity. The inhibition of ALA-S was prevented by 10(-4) M dibutyryl cyclic AMP (db cAMP) or theophylline, agents which elevate intracellular cAMP and which have previously been shown to prevent and reverse ethanol inhibition of haem and protein synthesis. Thus, it appears that cAMP protects against ethanol toxicity by preventing inhibition of ALA-S.

Induction of liver cell haem oxygenase in iron-overloaded rats.

Rats were chronically iron-overloaded by intraperitonel injections of iron-dextran. Electron microscopy revealed that the excess iron was deposited in ferritin-like particles packed in lysosomes and scattered in hepatic cytoplasm. No mitochondrial iron deposition or damage was seen. Furthermore, mitochondrial preparations from chronically iron-overloaded animals were found to be contaminated with lysosomes, which could explain previously reported increases in mitochondrial iron by chemical analysis. Mitochondrial function, as measured by cytochromes a-a3, b and c concentrations as well as activity of the rate-limiting enzyme of haem synthesis, delta-aminolaevulinate synthetase, was not diminished by chronic iron-overloading. Microsomal haem was decreased by 30% at the time that haem oxygenase, the rate-limiting enzyme of haem degradation, was increased approx. 3-fold. Animals were given a single intraperitoneal injection of iron-dextran and the activities of delta-aminolaevulinate synthetase and haem oxygenase were measured over 24 h. delta-Aminolaevulinate synthetase activity increased approx. 2-fold in these acutely iron-overloaded rat livers, but at a time after the increase in haem oxygenase. These results suggest that an early consequence of excess iron in liver is acceleration of the rate of haem degradation, possible by haem oxygenase.

Control of delta-aminolaevulinate synthase and haem oxygenase in chronic-iron-overloaded rats.

The hepatic porphyrias are inborn errors of porphyrin and haem biosynthesis characterized biochemically by excessive excretion of delta-aminolaevulinate (ALA), porphobilinogen and other intermediates in haem synthesis. Clinical evidence has implicated iron in the pathogenesis of several types of genetically transmitted diseases. We investigated the role of iron in haem metabolism as well as its relationship to drug-mediated induction of ALA synthase and haem oxygenase in acute and chronic iron overload. Acute iron overload in rats resulted in a marked increase in hepatic haem oxygenase that was associated with a decrease in cytochrome P-450 and an increase in ALA synthase activity. Aminopyrine N-demethylase and aniline hydroxylase activities, which are dependent on the concentration of cytochrome P-450, were also decreased. In contrast, in chronic-iron-overloaded rats, there was an adaptive increase in haem oxygenase activity and an increase in ALA synthase that was associated with normal concentrations of microsomal haem and cytochrome P-450. The induction of ALA synthase in chronic iron overload was enhanced by phenobarbital and allylisopropylacetamide, in spite of the fact that these agents did not increase haem oxygenase activity. Small doses of Co2+ were potent inducers of the haem oxygenase in chronic-iron-overloaded, but not in control, animals. We conclude that increased hepatic cellular iron may predispose certain enzymes of haem synthesis to induction by exogenous agents and thereby affect drug-metabolizing enzyme activities.

Solubilization and partial purification of heme oxygenase from rat liver.

Hepatic microsomal heme oxygenase was solubilized, partially purified, and characterized from Co2+-treated rats. The enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis exhibited a minimum molecular weight of greater than or equal to 68,000. The solubilized enzyme was totally devoid of contamination with cytochrome P-450 or b5. The requirement for reduced pyridine nucleotides was absolute, and ascorbate could not support heme oxidative activity. However, both TPNH and DPNH could serve as electron donors, with TPNH being more effective. The presence of an appropriate flavoprotein reductase was essential for heme oxidation. The enzyme had an apparent Km of 40 micrometer, a pH optimum of 7.5, and lost substantial activity upon freezing and thawing. Methemoglobin was 30% as effective a substrate for the enzyme as was heme. Free porphyrins could not serve as substrates for the enzyme. The activity of the enzyme was inhibited by HgCl2, p-chloromercuribenzoate, iodoacetamide, mercaptoethanol, and dithiothrietol indicating that free -SH group(s) is necessary for enzyme activity.

Some properties of a partially purified inhibitor(s) of protein synthesis from rat-liver mitochondria.

We have previously described an inactive inhibitor of protein synthesis from rat liver mitochondria and its activation by brief incubation with N-ethylmaleimide [Wu, J. M. and Ibrahim, N.G. (1980) FEBS Lett. 119, 25-28]. To study the mode of action of the mitochondrial translational inhibitor (MTI), the relative distribution of monosomes and polysomes in rabbit reticulocyte lysates has been analysed by sucrose gradient centrifugation. These studies show that MTI causes a significant decrease in the amount of polysomes with marginal effect on the polysome profile. Under identical experimental conditions, addition of partially purified heme-regulated inhibitor results in a complete disaggregation of polysomes. Studies with micrococcal-nuclease-treated rabbit reticulocyte lysates suggest that the primary target of MTI is the inactivation of globin mRNA with relatively little effect on other components of the translational machinery. The inhibitor also degrades poly(U), vesicular stomatis virus mRNA, reovirus mRNA, but appears to be inactive against poly(A), Escherichia coli 16S rRNA, HeLa cell rRNA or chick embryo DNA. Chromatography of MTI on heparin-agarose results in the resolution of at least two inhibitory activities. The first inhibitory activity (eluted with 250 mM KCl) can be reversed (50-70%) with high concentrations of glucose 6-phosphate or MgGTP (0.7 mM or 3.3 mM) whereas the second inhibitory activity can only be partially reversed with poly(A)-rich RNA.