Heat treatment of Corynebacterium ammoniagenes leads to aeration dependent accumulation of 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate.

2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEC) identified as a new bacterial oxidative stress substance (Ostrovsky D. et al. (1993) Biochem. J., 295, 901-902) was shown to accumulate in Corynebacterium (Brevibacterium) ammoniagenes cells aerobically cultivated in peptone-yeast extract-glucose broth on heating for 1 hour at 45 degrees C. The enzyme(s) responsible for MEC biosynthesis is evidently oxidized for activation and is completely loosing its activity on anaerobic incubation at this temperature in an hour. Salt stress or drying did not provoke the MEC biosynthesis.

1H and 13C NMR study of the molecular interaction mechanism between chloramphenicol and human serum albumin.

Molecular mechanism of the interaction between human serum albumin and cloramphenicol was studied by 1H and 13C NMR-spectroscopy. It was found that the main role belongs to [formula: see text] groups of chloramphenicol. A schematic model of the complex-formation between serum albumin and chloramphenicol was proposed.

Bacteria and pesticides: a new aspect of interaction--involvement of a new biofactor.

Positively charged hydrophobic pesticides of the dipyridyl family [diquat, paraquat, benzylviologen (BV++), etc.] were shown to provoke accumulation of 2-methylbutane-1,2,3,4-tetraol-2,4- cyclopyrophosphate in the cells Corynebacterium (Brevibacterium) ammoniagenes while neutral dipyridyls were not. Hydrophobicity was also an important factor in this phenomenon. Of the other pesticides tested, only linuron was effective. BV++ also induced biosynthesis of the compound in Rhodococcus rhodochrous, Rh.ruber, Rh.sp. (Nocardia corynebacteroides). These microorganisms as well as most of the previously identified oxidative stress activated producers of this new cyclopyrophosphate were able to synthesize free radical generating compounds. The microorganisms concerned belong mainly to the order Actinomycetales.

Bacterial oxidative stress substance spontaneously recyclizes to form 2-methylbutane-1,2,3,4-tetraol-1,2-cyclophospho-4-phosphate.

The cells of Corynebacterium (Brevibacterium) ammonia-genes cultivated in a medium supplemented with diquat or benzylviologen accumulate 2-methylbutane-1,2,3,4-tetraol-2,4- cyclopyrophosphate as revealed by 31P-NMR spectroscopy. On heating at 120 degrees C for 30 min the cells still maintain a substantial portion of this compound and acquire new cyclic phosphates characterized by 31P-NMR chemical shifts of +17.3 and +20 p.p.m. The +17.3 p.p.m. component was isolated from the preparation of the purified cyclopyrophosphate kept for some time at pH above 7 and it was shown to be 2-methylbutane-1,2,3,4-tetraol-1,2,- cyclophospho-4-phosphate on the grounds of two-dimensional NMR spectroscopy.

The ability of bacteria to synthesize a new cyclopyrophosphate correlates with their tolerance to redox-cycling drugs: on a crossroad of chemotherapy, environmental toxicology and immunobiochemical problems.

Many redox-cyclers were recently shown to induce, in some bacterial species, large-scale biosynthesis of a new 2-methylbutan-1,2,3,4-tetraol-2,4-cyclopyrophosphate believed to be involved in anti-stress reactions. In the present study Mycobacterium smegmatis, Micrococcus luteus and Brevibacterium ammoniagenes were shown to begin synthesis of the new cyclopyrophosphate when cultivated in a medium containing furacilin or furadonin (widely used nitrofuran antibacterial drugs) and to maintain close to normal growth rates, whereas Staphylococcus aureus, Bacillus subtilis and Escherichia coli were inhibited by the drugs and were unable to synthesize the cyclopyrophosphate compound. Preferential binding of Mg2+ and Cd2+ with one or other phosphoryl groups of the cyclopyrophosphate, which was indicated by selective changes of 31P-NMR chemical shifts and intramolecular hydrogen bonding, is suggested as a reason for this selectivity.

General features of systemic effects of murine leukemias on phosphate metabolism in liver studied by 31P NMR.

31P NMR was used to study the systemic effects of a tumor on a host organism by monitoring the phosphate metabolite content in freshly excised mouse liver at 0-4 degrees C and in ethanolic liver extracts of animals suffering from La, L1210 and P388 leukemias and Ehrlich ascites tumor (EAT). The progression of murine leukemia is characterized by increases in the intensities of the resonances of Pi and phosphomonoesters (PME), in particular, phosphorylethanolamine, in liver; phosphodiester (PDE) signals increase two- to four-fold during the period of rapid tumor growth and decline to undetectable levels in the terminal stage. There were no reliable alterations detected in the ATP content and intracellular pH throughout the course of the leukemia. The kinetics of intracellular phosphates are similar in various kinds of leukemia but quite different in EAT. The reduction of inoculum causes the appearance of maxima in the Pi and PME profiles in the latent period of La leukemia, but the profiles of liver PDE considered from the end of the latent period are independent of inoculum. Possible mechanisms for the changes in PDE concentrations and their biochemical role are discussed. NMR spectroscopy of liver may be used to indirectly monitor the progression of tumors unavailable for direct NMR assay.

Synthesis of a new organic pyrophosphate in large quantities is induced in some bacteria by oxidative stress.

Brevibacterium ammoniagenes and Micrococcus luteus were shown to synthesize up to 50 mM of a novel substance, 2-methylbutan-1,2,3,4-tetraol 2,4-cyclopyrophosphate, in response to oxidative stress created by benzyl viologen and other redox mediators under aerobic conditions. The substance, which represents greater than 50% of the extractable phosphorus, is suggested to play a role as a bacterial antistressor and is thought to be a product of condensation of two molecules of phosphoenolpyruvate whose accumulation is prompted by conversion of intracellular NADPH into an oxidized form.

A new cyclopyrophosphate as a bacterial antistressor?

In a number of bacteria an unusual glycosyl pyrophosphate (31P NMR signal chemical shift at about -15 ppm) was detected when the cells were subjected to oxidative stress. This substance from Brevibacterium ammoniagenes has now been identified as 2-methyl-butan-1,2,3,4,-tetraol-2,4-cyclopyrophosphate, which is accumulated in the cell under certain conditions in concentrations of of about 50 mM. It is now suggested that this compound is the long sought after bacterial antistressor.

1H NMR urinalysis in glomerulonephritis: a new prognostic criterion.

The applicability of 1H NMR urinalysis to glomerulonephritis was investigated: storage of urine at -18 degrees C and its subsequent lyophilization were shown to cause no loss of available biochemical information. Proteinuria was demonstrated not to interfere with the breadth and intensities of 1H NMR signals of low-molecular-weight metabolites in urine. The urine content of patients with glomerulonephritis was shown to be independent of exogenous factors, whereas these factors did affect the content of the urine of healthy volunteers. Statistical analysis of 1H NMR data for 52 patients with glomerulonephritis and 8 healthy volunteers on a pseudo-nephrological diet allowed the confirmation of the obligatory tubular interstitial changes that accompany the disease progression. It was established that severe tubular interstitial distortions can be revealed in patients with clinically preserved kidney function who display early stages of the glomerulonephritis, whereas these distortions can be practically negligible even at the terminal phase of uremia. Among patients with clinically preserved kidney function, especially those with nephrotic variant of the disease, isolated tubular and papilla distortions, as well as simultaneous tubular interstitial changes, were discovered, and this complicated the prognosis of glomerulonephritis progression. Thus, a new criterion to unmask a group at risk of rapid progression to uremia can be proposed, justifying an early prescription of immune suppressive drugs or other adequate treatment to these patients.

Phosphorus-containing metabolites in anthracycline-resistant murine leukemia P388 cells.

The method of 31P nuclear magnetic resonance was used to study in vivo the level of phosphorus-containing metabolites in P388 leukemia cells sensitive or resistant to rubomycin (daunomycin) and its nitroxyl analog-emoxyl. It was shown that decreased content of phosphomonoesters (PME) is characteristic of the resistant strains in comparison with the parent cells. Rubomycin and emoxyl were established not to affect practically the pool of phosphorus-containing metabolites in the cells of the resistant strains, but caused considerable increase of PME level in the cells of the parent strain.

NMR estimation of protective effect of insulin on mouse liver with epinephrine-induced metabolic lesions.

In order to study the effects of epinephrine and insulin on liver metabolism, measurements of cellular phosphates and intracellular pH by 31PNMR, of glycogen by 13C NMR and of lactate by 1H NMR were performed in freshly dissected mouse liver at 0-4 degrees C and in ethanolic liver extracts. The injection of epinephrine hydrochloride (0.1 mL of 0.1% solution i.p. per mouse) caused remarkable changes in liver metabolic profiles which were expressed most distinctly in 15-30 min and could not be attributed solely to epinephrine-induced hyperglycemia. Among these metabolic changes are falls in the levels of ATP and uridine diphosphate sugars by 60-70%, possibly related to glycogen depletion, and intracellular acidification by 0.5 units attributed to the release of protons during hydrolysis of ATP rather than to accumulation of lactate in anaerobic glycolysis. Insulin injected prior to epinephrine (4 units i.p.) markedly suppressed epinephrine-induced metabolic alterations, although the effect of the combination of insulin and epinephrine was not the sum of the separate effects of these hormones. The maximum protective effect of insulin was reached when insulin was injected 15 min prior to epinephrine. The results obtained demonstrate the applicability of NMR for evaluating the protective activity of modifiers at various extreme exposures.

23Na NMR measurement of the maximal rate of active sodium efflux from human red blood cells.

The method for 23Na NMR measurement of the maximal rate of active Na+ efflux from human red blood cells (RBC) is proposed. The nonpenetrating paramagnetic shift reagent (SR) bis(tripolyphosphate)dysprosium(III) complex is used to distinguish extracellular Na+ ions from intracellular. RBC are proved to retain their physiological activity in the presence of SR. Intracellular Na+ is shown to be 100% NMR visible. The levels of intracellular and extracellular Na+ and K+ ions are changed to decrease their concentration gradients across the erythrocyte membrane to make active Na+ efflux the only 23Na NMR measurable process; so the integrated areas of intra- and extracellular Na+ peaks remain invariant throughout the incubation period in the presence of 0.25 mM ouabain, a specific inhibitor of Na+, K+-ATPase. The accuracy of the proposed technique is evaluated to be 10%. The maximal Na+ efflux is determined to be 10.1 +/- 1.0 mM/h/liter of cells.

Nuclear magnetic resonance imaging of human lymph nodes.

MR-imaging of surgically excised enlarged and non-enlarged human lymph nodes was studied in patients with and without malignant metastatic lymphadenopathy. NMR tomograms were performed at frequency of 200 MHz using the CPMG sequence. Space resolution was up to 0.1 mm. "In vitro" NMR-tomograms of both non-enlarged and enlarged lymph nodes disclosed tiny foci of metastatic deposits. It is proposed that use of T2 differences for localization of a pathologic focus is an effective mode of visualization during NMR tomography at high frequency.