The adenosine analog tubercidin inhibits glycolysis in Trypanosoma brucei as revealed by an RNA interference library.

We used an RNA interference (RNAi) library in a forward genetic selection to study the mechanism of toxicity of tubercidin (7-deazaadenosine) to procyclic Trypanosoma brucei. Following transfection of cells with an RNAi-based genomic library, we used 5 microm tubercidin to select a drug-resistant cell line. Surprisingly, we found in these resistant cells that the hexose transporters had been silenced. We subsequently found that silencing of hexokinase, a glycolytic enzyme, also yielded tubercidin-resistant parasites. These observations suggested that glycolysis could be a target of tubercidin action and that RNAi silencing of glycolytic enzymes was gradual enough to allow the parasites to adapt to alternative sources of energy. Indeed, adaptation of procyclic trypanosomes to a glucose-independent metabolism by reduction of glucose in the culture medium caused tubercidin resistance. High pressure liquid chromatography analysis of glycolytic intermediates from parasites treated with tubercidin showed a dose-dependent increase in concentration of 1,3-bisphosphoglycerate, a substrate of phosphoglycerate kinase. Furthermore, tubercidin triphosphate inhibited recombinant T. brucei phosphoglycerate kinase activity in vitro with an IC50 of 7.5 microm. We conclude that 5 microm tubercidin kills trypanosomes by targeting glycolysis, especially by inhibition of phosphoglycerate kinase.

A potential role of the cytoskeleton of Saccharomyces cerevisiae in a functional organization of glycolytic enzymes.

Numerous individual enzymes participate in a given synthetic or degradative pathway in which the product of one reaction becomes the substrate for the subsequent enzyme. This raises the question of whether the product of one 'soluble' enzyme diffuses freely through the available cell volume, where it accidentally collides with the subsequent 'soluble' enzyme. Alternatively, enzymes acting in a given pathway may be organized in ordered structures, metabolons. Certain glycolytic enzymes have been shown to co-localize with the cytoskeleton in mammalian cells. We deleted genes coding for proteins associated with the cytoskeleton of Saccharomyces cerevisiae: TPM1 coding for tropomyosin, SAC6 for fimbrin and CIN1 for a microtubule-associated protein. Single deletions or deletions of two such genes had no effect on the specific activities of glycolytic enzymes, or on the rates of glucose consumption and ethanol production. However, the concentrations of glycolytic metabolites during a switch from a gluconeogenic mode of metabolism, growth on an ethanol medium, to glycolysis after glucose addition showed transient deviations from the normal change in metabolite concentrations, as observed in wild type cells. However, all metabolites in mutant strains reached wild-type levels within 2-4 h after the shift. Only ATP levels remained low in all but the tmp1-Delta-sac6-Delta double mutant strains. These observations can be interpreted to mean that metabolic reorganization from a gluconeogenic to a glycolytic metabolism is facilitated by an intact cytoskeleton in yeast.

A study on the complexes between human erythrocyte enzymes participating in the conversions of 1,3-diphosphoglycerate.

The ability of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzing the reaction of 1,3-diphosphoglycerate synthesis in human erythrocytes to form complexes with enzymes which use this metabolite as substrate (3-phosphoglycerate kinase (3-PGK) or 2,3-diphosphoglycerate mutase (2,3-DPGM)) was studied. It was found that highly active 2,3-DPGM can be extracted from human erythrocyte hemolysates in a complex with GAPDH adsorbed on Sepharose-bound anti-GAPDH antibodies at pH 6.5, the molar ratio being one 2,3-GPGM subunit per subunit of GAPDH. No complexation was, however, detected at pH 8.0. The opposite was true for the interaction between GAPDH and 3-PGK, which could be observed at pH 8.0. In experiments carried out at pH 7.4, both GAPDH x 2,3-DPGM and GAPGH x 3-PGK complexes were detected. The Kd values of the complexes determined with purified enzyme preparations were in the range 2.40-2.48 microM for both the GAPDH x 2,3-DPGM and GAPGH x 3-PGK enzyme pairs, when titrations of GAPDH covalently bound to CNBr-activated Sepharose were performed by the soluble 2,3-DPGM or 3-PGK. If, however, GAPDH adsorbed on the specific antibodies covalently bound to Sepharose was used in the titration experiments, the Kd for the GAPDH x 2,3-DPGM complex was found to be 0.54 microM, and the Kd for the GAPDH x 3-PGK complex was 0.49 microM. The concentration of 2,3-diphosphoglycerate determined after 1 h of incubation of erythrocytes in the presence of glucose was found to increase 1.5-fold if the incubation was carried out at pH 6.5, but did not change upon incubation at pH 8.0. On the other hand, the concentration of 3-phosphoglycerate after incubation at pH 8.0 was twice as large as that found after incubation at pH 6.5. The results are interpreted on the hypothesis that specific protein-protein interactions between GAPDH and 2,3-DPGM or between GAPDH and 3-PGK may play a role in determining the fate of 1,3-diphosphoglycerate produced in the GAPDH-catalyzed reaction.

Evaluation of canine red blood cells stored in a saline, adenine, and glucose solution for 35 days.

An additive solution for the storage of red blood cells was evaluated for use in dogs. Blood collected from 6 dogs was processed into packed red blood cells and stored for 35 days in the additive solution Nutricel (Miles, Inc, Pharmaceutical Division, West Haven, CT). Packed red blood cells stored in citrate-phosphate-dextrose-adenine (CPDA-1; Fenwal Laboratories, Baxter Health Care Corp, Deerfield, IL) also were evaluated for comparison. Red blood cell 2,3-diphosphoglycerate (2,3-DPG) concentration, adenosine triphosphate (ATP) concentration, percentage hemolysis, and pH were determined. The red blood cell post-transfusion viability (PTV) after 35 days of storage was assessed with both single-labeled chromium 51 (51Cr) and double-labeled technetium 99m/chromium 51 (99mTc/51Cr) techniques. Mean ATP concentration and percentage hemolysis of the cells stored in Nutricel were 1.1 mumol/g hemoglobin (Hb) and 0.28% respectively and did not differ significantly (P < .05) from the values of 1.0 mumol/g Hb and 0.33% from the CPDA.1-stored red blood cells. The mean pH of red blood cells stored in Nutricel was 6.19, which was significantly lower than the pH of 6.47 for cells stored in CPDA-1. The mean 2,3-DPG concentration of red blood cells stored in Nutricel was significantly higher at 10.1 mumol/g Hb than the 2,3-DPG concentration of 3.4 mumol/g Hb for cells stored in CPDA-1. The mean PTV of canine red blood cells stored in Nutricel for 35 days was 85% with 51Cr and 90% with 99mTc/51Cr. This was significantly higher than the mean PTVs of 38% and 36% for the CPDA-1 stored cells as assessed with 51Cr and 99mTc/51Cr techniques, respectively. It was concluded that 35-day-old canine red blood cells stored in Nutricel are of acceptable quality for transfusion purposes.

Substrate utilization by Rana ridibunda erythrocytes.

Various monosaccharides, including ribose, mannose, galactose, and urea, in combination with glucose, were studied to determine their efficacy in supporting the formation of pyruvate, lactate, 2,3-diphosphoglycerate and ATP in Rana ridibunda erythrocytes. Lactate formation was found to increase during the course of incubation in the presence of all the substrates. None of the studied substrates maintained cellular ATP levels. About 0.36 mumole of lactic acid per hour was produced for each mumol of ribose that was metabolized. The presence of 1 mM Na-iodoacetate accelerated the loss of ATP and lactate in the presence of either glucose or ribose. Additionally, ouabain suppressed lactate formation from ribose alone, as well as in combination with glucose. From the metabolic substrates studied, ribose was shown to be the most efficient substrate to support Rana ridibunda erythrocyte metabolism. Mannose, galactose and urea may also be used as alternative metabolic substrates by Rana ridibunda erythrocytes.

Evaluation of an additive solution for preservation of canine red blood cells.

The effect of an additive preservative solution on canine red blood cell posttransfusion viability (PTV) and on selected canine red blood cell biochemical parameters was studied. One unit (450 mL) of blood was collected from 6 clinically normal dogs into the anticoagulant citrate phosphate dextrose, centrifuged, and the plasma removed. The red blood cells were then suspended in 100 mL of a saline, adenine, dextrose, and mannitol solution and stored at 4 degrees C. Aliquots were removed for study at 1, 10, 20, 30, 37, and 44 days. The 24-hour PTV of autologous red blood cells was determined using a sodium chromate (51Cr) label. Red blood cell concentrations of 2,3-diphosphoglycerate (2,3-DPG), adenosine-5'-triphosphate (ATP), and pH were also determined. Canine red blood cell PTV, pH, ATP, and 2,3-DPG concentrations decreased during storage (P < .05). The PTV decreased from 94% using day 1 red blood cells to 80% and 75% using day 37 and day 44 red blood cells, respectively (P < .05). Although the mean PTV of the day 44 stored units equaled the Food and Drug Administration (FDA) minimum standard for human red blood cells, the PTV was substandard in 75% of the day 44 units. The FDA standard was exceeded in 83% of the day 37 units. It was concluded that 37-day-old canine red blood cells preserved with a saline, adenine, dextrose, and mannitol solution are of acceptable quality for transfusion.

Decreased deformability of red cells in refractory anemia and the abnormality of the membrane skeleton.

Rheological characteristics of red cells in two patients with refractory anemia (with single chromosomal abnormality of 20q- or 13q-, respectively) were investigated with the hematological and biochemical properties. (1) Whole blood viscosity was remarkably increased, and the red cell deformability was greatly impaired (the impairments were prominent in patient with 20q-). (2) The hematocrit of both patients was about half of the normal value. Remarkable anisocytosis with elliptocytes and poikilocytes was observed in the patient with 20q-, but the anisocytosis was not so prominent in the patient with 13q-. (3) 2,3-diphosphoglycerate content in red cells was markedly increased in both patients, but adenylate content was not. (4) The red cells were slightly resistant to osmotic hemolysis, but they were not heat-labile. (5) Structural abnormality of spectrin was suggested from the impaired dimer-dimer association in red cell membrane and from the different susceptibility of spectrin to tryptic digestion. In conclusion, the rheological impairments and the abnormal shape of red cells in refractory anemia probably originated from the structural abnormality of cytoskeletal proteins in membrane, and the functional and structural abnormality may be different among patients.

Is the higher incidence of ischemic disease in patients with hypertension and diabetes related to intracellular depletion of high energy metabolites?

To study mechanisms underlying ischemia in hypertension and non-insulin dependent diabetes mellitus (NIDDM), 31P-magnetic resonance spectroscopy was used to evaluate adenosine triphosphate and 2,3 diphosphoglycerate (2,3 DPG) levels in erythrocytes of control (n = 21), hypertensive (n = 22), and NIDDM (n = 10) subjects. Compared to adenosine triphosphate levels in controls (2.22 +/- 0.10 mM), both hypertensive (1.89 +/- 0.10 mM, sig = 0.05 versus normal) and NIDDM subjects (1.57 +/- 0.13 mM, sig = 0.05 versus normal) exhibited lower values. NIDDM subjects also displayed suppressed levels of 2,3 DPG (6.84 +/- 0.48 mM, sig = 0.05 versus normal and EH), compared to hypertensives (8.34 +/- 0.27 mM). These data suggest cellular energy metabolism is disrupted in hypertension and NIDDM. Both conditions may thereby sensitize tissues to ischemic damage, lower adenosine triphosphate levels by decreasing energy reserves, and lower 2,3 DPG levels by inhibiting hemoglobin-oxygen dissociation.

Cyclic 2,3-diphosphoglycerate as a component of a new branch in gluconeogenesis in Methanobacterium thermoautotrophicum delta H.

A unique compound, cyclic 2,3-diphosphoglycerate (cDPG), is the major soluble carbon and phosphorus solute in Methanobacterium thermoautotrophicum delta H under optimal conditions of cell growth. It is a component of an unusual branch in gluconeogenesis in these bacteria. [U-13C]acetate pulse-[12C]acetate chase methodology was used to observe the relationship between cDPG and other metabolites (2-phosphoglycerate and 2,3-diphosphoglycerate [2-PG and 2,3-DPG, respectively]) of this branch. It was demonstrated that cells could grow exponentially under conditions in which 2-PG and 2,3-DPG, rather than cDPG, were the major solutes. While the total concentration of these three phosphorylated molecules was maintained, rapid interconversion of 13C label among them was observed. Label flow from 2-PG to 2,3-DPG to cDPG to polymer is the usual direction in this pathway in exponentially growing cells, while the reverse reactions sometimes predominate in the stationary phase. Evidence of the presence of a polymeric compound in this pathway was provided by 13C nuclear magnetic resonance (one-dimensional and two-dimensional INADEQUATE) studies of solubilized cell debris.

Responses of myocardial high energy phosphates and wall thickening to prolonged regional hypoperfusion induced by subtotal coronary stenosis.

The response of the myocardium to prolonged or chronic ischemia may differ from the well documented changes that occur acutely subsequent to the onset of hypoperfusion. Therefore, we have examined in an instrumented canine model and using spatially localized spectroscopy to achieve transmural differentiation, the myocardial HEP and Pi levels as well as wall thickening in situ during prolonged ischemia induced by sustained coronary artery stenosis. The results demonstrate that subtotal coronary artery occlusion causes immediate and transmurally inhomogeneous decreases in the myocardial HEP content and increase in the Pi/CP ratio; however, during prolonged mild hypoperfusion, metabolic changes occur which lead to statistically significant recovery of CP (but not ATP) and disappearance of Pi despite the persistence of reduced blood flow and oxygen supply. Upon release of the occlusion, the previously ischemic muscle recovered blood flow, and some (but not all) of its preischemic contractile function without parallel changes in the HEP levels. It is concluded that normal HEP and Pi levels cannot be equated with either the absence of underperfusion or insensitivity of NMR spectroscopy to ischemia. Rather, it is imperative that both functional and spectroscopic measurements are performed simultaneously to distinguish between ischemic myocardium which is adapted versus unadapted to the hypoperfusion.

Direct measurement of spin-lattice relaxation times of phosphorus metabolites in human myocardium.

T1 values of phosphorus metabolites visible in human cardiac 31P-MR spectra were determined in 12 volunteers at 1.5 T. Consecutive spectra were acquired with varying pulse repetition time (TR) from 1.6 to 24 s; volume selection was achieved with ISIS. T1's of creatine phosphate (CP), [gamma-P], [alpha-P], and [beta-P]ATP, 2-3 diphosphoglycerate, and phosphodiesters were 6.1 +/- 0.5, 5.4 +/- 0.5, 5.5 +/- 0.5, 5.8 +/- 1.0, 7.6 +/- 1.0, and 5.0 +/- 1.0 s, respectively. CP/ATP ratios showed little change with varying TR; linear regression of CP/ATP vs TR was of borderline significance (r = 0.28, P = 0.06). T1's for CP and ATP were also determined in standard solution (20 mM CP, 10 mM ATP) yielding T1CP of 8.7 +/- 0.2 and T1[gamma-P]-ATP of 9.9 +/- 0.7 s. Thus, T1's for CP and ATP were similar at 1.5 T in both human heart and standard solution. In human cardiac 31P-MR spectra, CP/ATP ratios may need little correction for partial saturation.

31P NMR spectroscopy of the human heart at 4 T: detection of substantially uncontaminated cardiac spectra and differentiation of subepicardium and subendocardium.

31P NMR spectroscopy of the human heart was undertaken at 4 T to investigate whether spectra localized exclusively to the myocardium can be obtained. Utilizing a Fourier series window approach to spectroscopic imaging, we find that at least two layers across the anterior left ventricle wall can be detected, with voxel sizes of about 8 cm3.

The effect of ammonium, phosphate, potassium, and hypotonicity on stored red cells.

The use of an experimental solution that maintained high ATP levels and produced greater than 70 percent viability of stored red cells (RBCs) for up to 18 weeks has been described by other investigators. This solution differed markedly from conventional storage media in that it lacked sodium; contained high concentrations of potassium, ammonium, and phosphate; and was hypotonic. It was not clear which feature or features were responsible for the observed effects. The effects of ammonium, phosphate, potassium, and hypotonicity on stored RBCs have been examined. It was determined that ammonium and phosphate were the important factors in ATP maintenance. The biochemical mechanism by which ammonium acts was studied. In fresh human blood samples, ammonium was found to relieve phosphofructokinase from inhibition by increased ATP concentrations, to have no significant effect on adenine phosphoribosyl transferase activity, and, unexpectedly, to increase the activity of AMP deaminase. Despite prolonged ATP maintenance by ammonium and phosphate-containing storage media, satisfactory viability of RBCs stored up to 77 days was not demonstrated in the rabbit transfusion model.

An evaluation of new processing protocols for in vivo NMR spectroscopy.

In vivo NMR spectroscopy is often complicated with problems of low signal-to-noise, poor resolution, undefined peak shapes, and nonlinear baselines despite the efforts of investigators to optimize their experiments. Several data processing options are available to spectroscopists to enhance resolution and signal-to-noise and/or to flatten baselines. There is some question about how these processing protocols affect quantitative information. This paper evaluates five different processing protocols for their ability to extract quantitative information from a set of nonideal spectra. Three of the protocols involve recently developed statistical signal processing methods, maximum entropy Fourier spectral deconvolution, linear prediction singular value decomposition, and baseline deconvolution. These protocols are compared with the conventional processing methods of convolution difference and zeroing initial data points of the FID. The methods are evaluated by use of a quantitative 31P model sample and also are demonstrated on surface coil 31P data.

Structural characteristics of methanogenic cofactors in the non-methanogenic archaebacterium Archaeoglobus fulgidus.

Archaeoglobus fulgidus is an extremely thermophilic, sulphate reducing archaebacterium thought to represent a biochemical missing-link between sulphur-metabolizing bacteria and methanogenic bacteria. Whereas the phylogenetic position of A.fulgidus is closer to the sulphur-metabolizing bacteria, there is a partial overlap in the biochemical machinery of A.fulgidus with both groups of bacteria. In particular, the presence of a number of aberrant cofactors up to now thought to be involved exclusively in the process of methanogenesis in methanogenic archaebacteria, i.e. coenzyme F420, methanofuran and methanopterin, has been indicated by previous studies. Here we present evidence for the structural identity of the methanopterin cofactor of A.fulgidus with the methanopterin isolated from Methanobacterium thermoautotrophicum and show that this non-methanogenic bacterium contains two as yet unknown analogues of coenzyme F420. The levels of the various cofactors were determined in cultures grown either on formate or lactate as the carbon source and sulphate or thiosulphate as the sulphur source.

Sickle cell disease in permanent residents of mountain and low altitudes in Saudi Arabia.

Report of a comparative study of sickle cell disease in permanent residents of mountain and low altitudes in south-western Saudi Arabia. The ambient oxygen tensions at these altitudes are 14 and 19 kPa (112 and 144 mmHg) respectively. The frequencies of sicklaemic-related illness requiring medical intervention, and hospitalisation due to crisis and complications of the disease, were about twice as great in highlanders as in lowlanders. The incidence and severity of the complications were similar in both locations. No splenic syndrome was observed in those with the disease or trait in either location. Haemoglobin concentration was 10% greater in mountain normals than in their lowland counterparts; the corresponding figure for sicklers was 5%. Erythrocyte 2,3 diphosphoglycerate concentration was 13% greater in mountain than valley patients; the corresponding figure for normals was 4%. We propose that the elevated diphosphoglycerate in mountain patients might contribute to their higher frequency of sicklaemic illness as well as partially blunting their erythropoietic drive.

Hemoglobin oxygen dissociation (P50) in bronchopulmonary dysplasia.

A study was devised to determine the P50 in infants with bronchopulmonary dysplasia (BPD). Other factors such as red blood cell 2,3-diphosphoglycerate (2,3-DPG) level proportions of adult (HbA) to fetal (HbF) hemoglobins which could affect P50 were also measured. Fourteen infants with clinical and radiological signs of BPD with a mean post-conceptional age of 42.2 +/- 4.7 weeks born at a mean gestational age of 29.3 +/- 2.0 weeks were evaluated. The percentage of HbF determined in 8 infants was 40.1 +/- 20.3% and the mean 2,3-DPG concentrations was 13.1 +/- 2.2 mumol/g Hb. The P50 was 25.1 +/- 2.7 mm Hg (range 18-29.5 mm Hg). When a HbO2 curve was established based on a large volume of blood consisting of adult blood and newborn cord blood mixed to attain a P50 of 25.1 mm Hg, the PaO2 at 90% O2 saturation was 52 mm Hg. Since there can be a wide range in HbO2 in infants with chronic BPD, pulse oximetry remains the most prudent method of monitoring oxygen therapy in BPD infants.

Vanadium(IV)-stimulated hydrolysis of 2,3-diphosphoglycerate.

Vanadium(IV) stimulates the hydrolysis of 2,3-diphosphoglycerate at 23 degrees C. The pH optimum is 5.0. Reactions were analyzed by enzymatic and phosphate release assays. The products of 2,3-diphosphoglycerate hydrolysis are inorganic phosphate and 3-phosphoglycerate. The reaction is inhibited by high concentrations of 2,3-diphosphoglycerate and an equation has been formulated that describes the kinetic constants for this reaction at pH 7. The possible relevance of the reaction to the therapeutic lowering by vanadium(IV) of red cell 2,3-diphosphoglycerate in sickle-cell disease is discussed.

A follow-up on functional parameters of blood stored in ACD at +4 degrees C.

The main functional parameters of blood stored at +4 degrees C in ACD, according to the common transfusional practice, have been carefully followed in the course of 40 days. The expected depletion of DPG takes place within 10 days, but apparently, no increase of the Hb affinity towards oxygen is observed in this period (or later), because pH lowering acts in the opposite direction during the same time. However, the intrinsic increased affinity of Hb is promptly revealed if the "actual" pHs are corrected at the standard value of 7.4, and/or are extrapolated at this pH from Bohr effect.

The bottom and top system: a new technique for blood component preparation and storage.

A new, automated technique for the preparation of blood components is described. A system of 3 or 4 integrally connected plastic containers (Optipac) is handled by a new type of extractor (Optipress). The container in which the blood is collected has an outlet at the top and another at the bottom. After normal centrifugation to obtain separation of the blood components, these are squeezed out from the top and bottom simultaneously under control of a photocell. The primary separation step results in three components: a leukocyte-poor red-cell suspension in SAGM medium, CPD plasma, and a buffy-coat preparation. The system has been tested in two laboratories (lab A and lab B). A 'heavy-spin' centrifugation to obtain a maximum yield of cell-poor plasma gave the best removal of leukocytes from the red cells; the remaining leukocyte content was 0.46 +/- 0.25 (lab A) and 0.5 +/- 0.4 (lab B) x 10(9)/red-cell unit. Platelet concentrates can be prepared either the normal way via platelet-rich plasma or from buffy coat. Red-cell 24-hour autologous posttransfusion survival using labeling with 51Cr was 87.5 +/- 4.1% (lab A) after 35 days, and 84.2 +/- 4.2% (lab A) and 77.5 +/- 1.5% (lab B) after 42 days. Red-cell morphology and fluidity compared favorably to previous studies using the same additive solution in traditional plastic-bag systems. The total adenine nucleotide concentration was maintained normal for 42 days.(ABSTRACT TRUNCATED AT 250 WORDS)