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Inorganic pyrophosphate is identified as the major product of photophosphorylation by isolated chromatophores from Rhodospirillum rubrum in the absence of added nucleotides.
A pancreatic ribonuclease digest of carbon-14-labeled Satellite Tobacco Necrosis Virus RNA was fractionated, according to charge, by column chromatography. Individual fractions were dephosphorylated with alkaline phosphomonoesterase and rechromatogramed. The fraction originally containing oligonucleotides with seven negative charges separated into two components corresponding to five and two negative charges, respectively, and therefore must have contained a terminal trinucleotide 5'-pyrophosphate, in addition to the internal hexanucleotides. Other fractions when similarly treated were found to contain only internal oligonucleotides.
The mode of action of 3,5-dihydroxy-4-ethyl-trans-stilbene (ES), an antibiotic produced by Xenorhabdus luminescens symbiotically associated with an entomopathogenic nematode, was investigated. ES was active against gram-positive and a number of gram-negative bacteria. In susceptible bacteria this antibiotic caused the inhibition of total RNA synthesis and, to a lesser extent, protein synthesis. At or above MICs, ES triggered a substantial accumulation of an intracellular regulatory compound, guanosine-3',5'-bis-pyrophosphate (ppGpp). This response was also noticed in species of bacteria which have previously not been shown to use ppGpp as a regulatory molecule. The involvement of ppGpp in antibiotic action was confirmed by using an isogenic stringent and a relaxed pair of Escherichia coli strains. The fact that the accumulation of ppGpp was correlated with the susceptibility of various gram-positive and gram-negative bacteria to ES suggests that this nucleotide is involved in the regulation of RNA synthesis and growth in all these microorganisms. Thus, inhibition of RNA synthesis via an increase in ppGpp concentrations may represent a mechanism that is prevalent among most bacteria and one that could be exploited for achieving a rapid inhibition of bacterial growth.
The effects of two pH levels (5.55 or 5.85) in combination with 0.4% sodium acid pyrophosphate (SAPP), NaH2PO4 X H2O, Na2HPO4 X 7H2O, or NaCl on the growth and toxicity of Clostridium botulinum 52A were studied. Absorbancy measurements at 630 nm, microscopic observations, and the mouse bioassay procedure were used to observe the effects. At pH 5.55 and 5.85 most control cultures exhibited toxicity when cell lysis began. Vegetative cell development was normal (4 micron long; 1 micron wide). SAPP-containing (0.4%) treatment cultures displayed similar growth and lysis but no or delayed (48 h) toxicity. Cells grown in the SAPP treatment culture were longer and wider (6 micron long; 1.5 micron wide) than in most other treatment cultures. Trypsinization of nontoxic supernatants from 0.4% SAPP resulted in toxicity. Addition of 0.4% SAPP to toxic C. botulinum supernatant delayed but did not prevent death of mice. The addition of various levels of SAPP to toxic supernatants resulted in a decrease in zone size with an increase in the level of SAPP (9 mm with 0.4% SAPP to 7 mm with 1.0% SAPP), using a dual substrate protease assay. A decrease in the zone size also occurred with the supernatant from cultures grown in the presence of SAPP and with Bacillus polymyxa protease dilutions containing 0.4% SAPP. Results suggest that the actual production or function of the protease responsible for toxin activation may have been inhibited by the presence of SAPP.
Analyses of survival data of a mixture of Salmonella spp. at fixed temperatures between 55 degrees C (131 degrees F) and 71.1 degrees C (160 degrees F) in ground beef matrices containing concentrations of salt between 0 and 4.5%, concentrations of sodium pyrophosphate (SPP) between 0 and 0.5%, and concentrations of sodium lactate (NaL) between 0 and 4.5% indicated that heat resistance of Salmonella increases with increasing levels of SPP and salt, except that, for salt, for larger lethalities close to 6.5, the effect of salt was evident only at low temperatures (<64 degrees C). NaL did not seem to affect the heat resistance of Salmonella as much as the effects induced by the other variables studied. An omnibus model for predicting the lethality for given times and temperatures for ground beef matrices within the range studied was developed that reflects the convex survival curves that were observed. However, the standard errors of the predicted lethalities from this models are large, so consequently, a model, specific for predicting the times needed to obtained a lethality of 6.5 log(10), was developed, using estimated results of times derived from the individual survival curves. For the latter model, the coefficient of variation (CV) of predicted times range from about 6 to 25%. For example, at 60 degrees C, when increasing the concentration of salt from 0 to 4.5%, and assuming that the concentration of SPP is 0%, the time to reach a 6.5-log(10) relative reduction is predicted to increase from 20 min (CV = 11%) to 48 min (CV = 15%), a 2.4 factor (CV = 19%). At 71.1 degrees C (160 degrees F) the model predicts that more than 0.5 min is needed to achieve a 6.5-log(10) relative reduction.
Cholesterol inhibits isopentenyl Delta(3),Delta(2)-isomerase of Mycoplasma laidlawii in an apparently competitive fashion. The conversion of mevalonic acid to isopentenyl pyrophosphate is slightly stimulated. Organisms grown in the presence of mevalonic-2-(14)C acid contain small amounts of radio-label in nucleic acid and protein fractions. Most of the label is found in the lipids and is reduced dramatically in organisms grown with cholesterol. No significant accumulation of phosphorylated intermediates of polyterpene biosynthesis was observed in cells or culture supernatant fluid. All of the radioactivity appearing in the nucleic acid fraction occurs in the minor nucleoside, isopentenyl adenosine, of the transfer ribonucleic acid. The necessity for synthesis by the organisms of this minor nucleoside from mevalonic acid may explain the site of enzyme inhibition by cholesterol of polyterpene biosynthesis.
Membranes from Saccharomyces cerevisiae protoplasts were fractionated on a continuous sucrose gradient. Six bands were obtained, which contained altogether about 15% of the total cell protein. From their densitites, their behavior in the presence and absence of Mg2+ ions, and the distribution of marker enzymes, it was possible to identify fractions enriched in rough and smooth endoplasmic reticulum and in mitochondria. All glycosyl transfer reactions investigated where dolichyl phosphates served as glycosyl acceptors or where dolichyl phosphate- and pyrophosphate-activated sugars served as glycosyl donors showed the highest specific activity and up to 75% of the total activity in the endoplasmic reticulum. This was the case for the reactions involved in the formation of O-glycosidic as well as N-glycosidic linkages in yeast glycoprotein biosynthesis. Membrane fractions enriched in plasmalemma contained less than 3% of the corresponding activities.
Mycoplasma sp., strain J, which is unable to convert mevalonic acid to isopentenyl pyrophosphate (IPP) will grow with IPP as a substitute for sterol. (14)C-labeled IPP is incorporated into its unsaponifiable lipid. This unsaponifiable lipid was separated into three (14)C-labeled fractions, all of which possess spectral and chromatographic characteristics of polyterpenes.
G4 and p13(II) are accessory proteins encoded by the X region of bovine leukemia virus and human T-cell leukemia virus type 1 (HTLV-1), respectively. Disruption of the G4 and p13(II) open reading frames interferes with viral spread in animal model systems, indicating that the corresponding proteins play a key role in viral replication. In addition, G4 is oncogenic in primary cell cultures and is absolutely required for efficient onset of leukemogenesis in sheep. To gain insight into the function of these proteins, we utilized the yeast two-hybrid system to identify protein partners of G4. Results revealed that G4 interacts with farnesyl pyrophosphate synthetase (FPPS), a protein involved in the mevalonate/squalene pathway and in synthesis of FPP, a substrate required for prenylation of Ras. The specificity of the interaction was verified by glutathione S-transferase (GST) pull-down assays and by coimmunoprecipitation experiments. Furthermore, confocal microscopy showed that the subcellular localization of G4 was profoundly affected by FPPS. The G4 protein itself was not prenylated, at least in rabbit reticulocyte lysate-based assays. The domain of G4 required for binding to FPPS was restricted to an amphipathic alpha-helix rich in arginine residues. Subtle mutation of this alpha-helix abrogated G4 oncogenic potential in vitro, providing a biological relevance for FPPS-G4 complex formation in cells. Finally, HTLV-1 p13(II) was also found to specifically interact with FPPS (in yeast as well as in GST pull-down assays) and to colocalize with G4 in mitochondria, suggesting a functional analogy between these oncoviral accessory proteins. Identification of FPPS as a molecular partner for p13(II) and G4 accessory proteins opens new prospects for treatment of retrovirus-induced leukemia.
A variety of rat tissues were screened at low stringency with a rat farnesyl pyrophosphate (FPP) synthetase cDNA. In testis, an FPP synthetase-related RNA was detected that was larger than the liver FPP synthetase mRNA and was present at very high levels comparable with liver FPP synthetase RNA levels obtained from rats fed diets supplemented with cholestyramine and mevinolin. Sequence analysis of testis cDNA clones, together with primer extension and S1 nuclease experiments, indicated that testis FPP synthetase transcripts contain an extended 5' untranslated region. The 5' extension contained one or two out-of-frame upstream ATGs, depending on the site of transcription initiation. Protein in vitro translation studies indicated that the extended 5' untranslated region may play a role in regulating the translation of the FPP synthetase polypeptide in rat testis. Southern blot analysis with a probe containing both testis and liver 5' untranslated sequences provided evidence that both liver and testis transcripts derive from the same gene. The data suggest that an upstream testis-specific promoter results in the abundant production of FPP synthetase transcripts that are translated at low efficiency; another promoter functions in liver and other somatic tissues and directs the regulated synthesis of shorter discrete transcripts.
OBJECTIVES: To examine the genetic contribution to common, apparently sporadic, radiographic knee chondrocalcinosis (CC) and pyrophosphate arthropathy (PA). METHOD: (1). DESIGN: radiographic sibling study. Comparison of the prevalences of knee CC and PA in siblings of index cases with PA with those in the community. (2). SUBJECTS: 80 index cases with PA listed for total knee replacement; 122 of their siblings aged >or=40 years; and 1729 participants from community knee pain surveys who had undergone knee radiographs. (3). MAIN OUTCOME MEASURE: odds ratios of knee CC and PA in siblings versus community participants. RESULTS: The prevalence of knee CC was 13% (15/116) in the siblings and 6.9% (119/1727) in the community participants. The adjusted odds ratio (aOR) was 1.2, 95% confidence interval (CI) 0.6 to 2.3. The main risk factors for knee CC were age, knee pain, and knee OA. The prevalence of knee PA was 7% (9/122) in the siblings and 3.4% (59/1729) among the community participants (aOR = 1.1, 95% CI 0.4 to 2.7). The main risk factors for PA were age and knee pain. The age, sex, and knee pain standardised prevalence of PA in the Nottingham community aged >or=40 was 2.40%. CONCLUSION: The risk of knee CC and PA in siblings of index cases with PA is no higher than that in the general population. Although rare familial CC is recognised, this study suggests that no major genetic predisposition to CC occurs in common symptomatic knee OA.
Using a quantitative method, scintigraphy of SI joints was performed by means of 99m technetium pyrophosphate in 21 patients with definite ankylosing spondylitis, in 17 control patients, and in 26 patients 'at risk', i.e. patients with complaints of back pain of the inflammatory type where on clinical grounds there was a possibility of sacroiliitis developing but with normal x-ray findings of the SI joints. Radioisotope uptake was higher in the ankylosing spondylitis group than in the other two groups, although the difference was not statistically significant with regard to the group 'at risk'. The high variance in the three groups considerably reduces the diagnostic value of the examination. In the ankylosing spondylitis group no correlation was found between radioisotope uptake and age, duration of disease, erythrocyte sedimentation rate, or radiological stage of scaroiliitis. Since the specificity and sensitivity of scintiscanning are lower than that of clinical and radiological diagnosis of the disease, we conclude that scintigraphy is not very helpful in the early diagnosis of sacroiliitis, at least by the techniques used here.
105 consecutive patients who presented to a rheumatologist because of joint disease and who also had evidence of deposition of calcium pyrophosphate dihydrate (CPD) were studied clinically and radiologically. There were 76 women (mean age 73) and 29 men (mean age 62). Of only 18 patients below the age of 60 at presentation 12 were men. The majority of the younger male group suffered from acute attacks of synovitis, and had no clinical or radiological evidence of joint damage. In contrast the older female group had widespread destructive changes. Associated joint disease included generalised osteoarthritis (45), rheumatoid arthritis (8), joint hypermobility (13), previous knee surgery (8), and gout 92). Sixteen patients had received long-term steroid therapy. Severe destructive joint changes were seen in 16 patients. The radiological features in those with rheumatoid arthritis by ARA criteria were atypical. The relationship between CPD deposition and arthritis is discussed in the light of these findings.
The relationship between osteoarthritic change and amyloid degeneration was studied in 116 joint capsules from osteoarthritic hip joints. Twenty-seven (23%) contained amyloid deposits. Twenty-eight joint capsules showed chondroid metaplasia in the fibrous part. Significantly more amyloid degeneration was found among these. Six joint capsules contained pyrophosphate crystals. Five of these were positive for amyloid. Chronic inflammation of the joint capsule was found in 80% of the cases and was negatively correlated with amyloid degeneration, which is in good agreement with the biphasic theory for the pathogenesis of amyloid. Further investigation is necessary to decide how chondroid metaplasia takes part in the production of amyloid.
The activities of erythrocyte phosphoribosyl pyrophosphate (PRPP) synthetase and glutathione reductase (GTR) were studied in 26 patients with primary gout who were receiving no treatment or treatment with either allopurinol or azapropazone, and compared with the activity in a group of healthy controls. The activity of PRPP synthetase was significantly higher in the gout group and was not influenced by either drug. No significant difference in the activity of GTR was observed. The failure of either drug to suppress the increased activity of PRPP synthetase associated with gout is discussed.
With the use of 99mTc-labelled stannous pyrophosphate scans positive for myocardial infarction were obtained from 28 patients in the acute stage of the disease. In some cases the scan was positive when the initial electrocardiogram gave equivocal results. Negative scans were obtained from a control group of patients and from eight patients in hospital with chest pain but with no other evidence of recent myocardial infarction.
Technetium-99m stannous pyrophosphate scintiscanning was performed in 22 patients with radiographically detected calcification within the cardiac silhouette. All but one of these scintigrams showed a localised area of increased activity similar to that ordinarily seen in acute myocardial infarction. Scintiscans in 3 patients after removal of the calcified aortic valve reverted to negative. It was concluded that this technique for acute infarct detection may yield false positive results in the presence of cardiac calcification.
An apparently new species of Clostridium was isolated from mud samples obtained from a stagnant lake near Athens, Georgia. These organisms were able to utilize inorganic pyrophosphate as a source of energy for growth, a phenomenon representing the simplest ATP-generating system in the biological world.