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Demonstration of physical interactions between consecutive enzymes of the citric acid cycle and of the aspartate-malate shuttle. A study involving fumarase, malate dehydrogenase, citrate synthesis and aspartate aminotransferase.

By means of covalently immobilized fumarase and mitochondrial or cytoplasmic malate dehydrogenase we were able to detect physical interactions between different enzymes of the citric acid cycle (fumarase with malate dehydrogenase, malate dehydrogenase with citrate synthase and fumarase with citrate synthase) and between the enzymes of both mitochondrial and cytoplasmic halves of the aspartate-malate shuttle (aspartate amino-transferase and malate dehydrogenase). The interactions between fumarase and malate dehydrogenase were also investigated by immobilizing one enzyme indirectly through antibodies bound to Sepharose-protein A. Our results are consistent with a model in which maximally four molecules of malate dehydrogenase are bound to one fumarase molecule. This complex is able to bind either citrate synthase or aspartate aminotransferase. We propose that these enzymes bind alternatively, in order to allow the cell to perform citric acid cycle or shuttle reactions, according to its needs. The physiological meaning and implications on the regulation of metabolism of the existence of a large citric acid cycle/malate-aspartate shuttle multienzyme complex are discussed.

Animals↗

Effects of phenobarbital on saccharin and citric acid intake in fluid deprived rats.

Rats were adapted to a 23 hr fluid deprivation schedule. Every third day animals were offered either 0.125 percent Na saccharin or 0.2 percent citric acid solutions in place of water during the 1 hr drinking session. Sodium phenobarbital was administered subcutaneously, 40 mg/kg, 15 min prior to drinking. Results indicate that the drug increases saccharin and citric acid consumption following the injections. No decreases in intakes of saccharin and citric acid occurred on subsequent postdrug days and the amounts of fluid consumed on these days were comparable to the baseline predrug days intakes.

Animals↗

Regulation by zinc and adenosine 3',5'-cyclic monophosphate of growth and citric acid accumulation in Aspergillus niger.

The citric acid fermentation by Aspergillus niger is divided into two consecutive phases, growth phase when the cells proliferate but do not accumulate citrate, followed by an accumulating phase when they excrete citrate but do not proliferate. The phase alternative was controlled by the concentration of zinc: high zinc (2 muM) maintained growth phase, and a zinc 'deficiency' apparently signaled the ransition to the accumulating phase. Cyclic AMP affected the rates of growth and acidogenesis when added to cultures growing at low but not at high zinc: that is, cAMP did not induce the phase transition, zinc deficiency did, Cyclic AMP enhanced growth early in the fermentation, but at later stages the response of the mycelia to cAMP changed, and then cAMP inhibited growth. When citrate eventually began to accumulate cAMP augmented its sypthesis. The growth and acidogenic responses were quite specific to cAMP, and were sensitive to concentrations of about 1 muM. Cyclic AMP also either promoted or retarded the appearance of an unidentified yellow pigment. It is proposed that the growth and accumulating phases are distinct differentiated states, at least with respect to cAMP metabolism.

Adenine Nucleotides↗

Citric acid (solution R) irrigation in the treatment of refractory infection (struvite) stone disease: is it useful?

OBJECTIVE: Citric acid, in varying concentrations, has been used in the dissolution treatment of struvite renal calculi. Solution R (Uro-trainer), which contains 6% citric acid, is a solution licensed for use in the management of struvite stone disease in the UK. We report our experience. METHODS: 23 kidneys in 22 patients (10 male and 12 female patients, mean age 45, range 15-60 years) underwent solution R irrigation following debulking of the stone with percutaneous nephrolithotomy (n = 20), ureteroscopy and shock wave lithotripsy (n = 2) combined with open procedures (n = 4) between 1994 and 1998. The original stone configuration consisted of 14 staghorn, 4 partial staghorn and 5 large burden stones. Irrigation was performed through a nephrostomy tube (n = 20) or in a retrograde fashion (n = 3) using a closed infusion pump system (40 ml/h). The response to treatment was checked using a nephrostogram and/or plain X-ray. RESULTS: In 6 (26%) kidneys, after an average duration of 2 (1-5) days, irrigation had to be abandoned due to loin pain, leak or sepsis. The average duration of irrigation was 6.4 (1-20) days. At the end of irrigation, a total of 4 (17.4%) kidneys had complete radiographic clearance, while the stone was reduced to calyceal dust in 3 (13%). Partial response was seen in 11 (47.8%) and no response in 5 (21.8%) kidneys. Following additional alternative intervention(s) in 6 (26%) kidneys (4 with partial and 2 with no response) further clearance was achieved in 3 (13%) and calyceal dust status in 3 (13%). The response was better if the stone was reduced to less than 10mm prior to irrigation. At the mean follow-up of 2.44 (1-4) years, of 13 kidneys with stone clearance or calyceal dust, 9 suffered recurrence or re-growth, 5 of which required further interventions. Only 4 (17.4%) of 23 kidneys remained stone free. CONCLUSIONS: In patients with complex stone disease, adjuvant solution R irrigation can reduce the stone burden. There is a considerable potential for side effects necessitating close monitoring for sepsis and electrolyte abnormalities. However, the overall success rate for solution R is limited.

Adolescent↗

Cementogenesis and soft tissue attachment after citric acid treatment in a human. An electron microscopic study.

The four maxillary incisors and two maxillary premolars of a 25-year-old male patient were used to study epithelial and connective tissue attachment 67 days and 164 days after flap surgery and cutting of an horizontal intradentinal groove near the buccal cervical region. Three teeth were topically conditioned for 3 minutes with citric acid pH = 1. The three other teeth were used as controls. The histologic examination was carried out in double-blind conditions; the examiners did not know which specimens were acid treated until the end of the study. Two of the three cases treated with citric acid showed improved healing conditions, when compared to the controls; a more coronal position of the epithelial attachment in the dentin nick as well as a relatively important gain in connective tissue attachment. Two types of connective tissue attachment were observed. The first consisted of an attachment to dentin, without cementum formation and was characterized by a mineralization of decalcified dentin collagen spliced with collagen, newly secreted by fibroblasts. The second type involved cementum formation. Topical citric acid treatment, however, can not be considered as a completely reliable clinical procedure since in one experimental case the type of attachment observed was not better than that seen in the control.

Adult↗

[Structural changes in Candida lipolytica cells during citric acid biosynthesis].

The techniques of light and electron microscopy were used to study structural changes in Candida lipolytica cells related to limitation of their growth and a nitrogen source in medium with glucose and hexadecane and transition of the culture to the production of citric acids. Changes in the content of protein in the cell and in the activities of enzymes of the citrate and glyoxylate cycles located in mitochondria and peroxysomes of the yeast were investigated at the same time. The results characterizing cytological changes in the cell of C. lipolytica in the conditions of citric acid biosynthesis fit in well with the data on protein content and enzyme activities in the cells obtained by biochemical analysis. The transition of the yeast cells to the overproduction of citric acids has been shown to be related to profound rearrangements of the cell: the inhibition of protein synthesis; changes in the structure of the cytoplasm; the appearance of vacuoles of an irregular shape, often in contact with the mitochondria; changes in the mitochondrial and peroxysomal apparatus; the accumulation of lipids. It has been demonstrated once again that the peroxysomal apparatus of the yeast cell has a relationship with the metabolism of n-alkanes.

Candida↗

[Modification of the micromethod of estimation of citric acid in the bone tissue].

A modified method is developed for estimation of citric acid in bone tissue. Citrate was isolated from acid bone hydrolysate without a preliminary deproteinization; then it was oxidized and converted into pentabromaceton by treatment of citric acid with bromine. The content of pentabromaceton was estimated by a photometric method. The method was highly sensitive and reproductible.

Acetone↗

Role of bradykinin and tachykinins in the potentiation by enalapril of coughing induced by citric acid in pigs.

Angiotensin-converting enzyme (ACE) inhibitors are among the first-choice drugs for treating hypertension and congestive heart disease. It has been reported, however, that these drugs could induce chronic cough and airway hyperresponsiveness. The aim of this work was to assess in pigs the effects of bradykinin and tachykinins on citric-acid-induced coughing after ACE inhibitor pretreatment. Coughing was induced by challenging pigs with an aerosol of 0.8 M citric acid over 15 min. Coughs were counted by a trained observer for 30 min. The animals underwent two cough induction tests two days apart (days 1 and 3), the first being taken as a control. All drugs were injected intravenously 30 min before the second challenge. In the control group, no difference was observed between days 1 and 3. The ACE inhibitor enalapril (7.5 and 15 microg/kg) caused the cough frequency to increase significantly. In contrast, a dose-related decrease was observed with Hoe140 (icatibant), a bradykinin B2 receptor antagonist (0.5 and 1 mg/kg). When both drugs were administered simultaneously (15 microg/kg for enalapril and 1 mg/kg for Hoe140), a significant increase was observed as compared with the control value obtained on day 1. When enalapril was combined with the three tachykinin receptor antagonists SR 140333 (NK1 receptor antagonist), SR 48968 (NK2 receptor antagonist) and SR 142801 (NK3 receptor antagonist), a significant decrease was observed as compared with control value obtained on day 1; the percentage of variation was also significantly different as compared with those observed in enalapril groups at both doses. These data suggest that ACE-inhibitor-induced enhancement of the cough reflex is mainly due to tachykinins and not to bradykinin in our pig model. Bradykinin, however, plays a major role in coughing induced by citric acid alone.

Adrenergic beta-Antagonists↗

The effect of massage treatment of prostatic congestion on the prostatic size and secretion of citric acid.

In this study, 25 men, referred to our clinic for diagnosis and therapy of infertility were included. All had enlarged prostates. They were given 10 sessions of prostatic massage during 3--4 weeks and the fluid expressed was analysed for citric acid. The hypertrophy was seen to recede in almost all cases. Citric acid concentrations fell in only 6/25 cases analysed. In all the others, values did not fall and remained relatively stable. There was no apparent relationship between reduction of prostatic volume and the pattern of citric acid secretion.

Adult↗

Preparation of levoglucosan by pyrolysis of cellulose and its citric acid fermentation.

Levoglucosan (LG), 1,6-anhydro-beta-D-glucopyranose, was produced by pyrolysis of cellulose. A response surface method was used to optimize the reaction parameters: X1, temperature; X2, time required for heating cellulose from room temperature to the designed pyrolysis temperature; and X3, vacuum, and a Box-Behnken design was employed for this purpose. The optimal temperature and time were found to be 388 degrees C and 26.2 min by fixing the vacuum at 1 mm Hg. The levoglucosan prepared was fermented to citric acid by Aspergillus niger CBX-209, which was a mutant derived by gamma-ray mutagenesis of the parent strain CBX-2. The mutant could produce increasing citric acid with increasing LG purity and had a citric acid yield of 87.5% when using purified levoglucosan as the sole carbon source in a 5 day fermentation period.

Aspergillus niger↗

Demineralization of caries-affected transparent dentin by citric acid: an atomic force microscopy study.

OBJECTIVES: This study determined recession rates of peritubular dentin and intertubular dentin in citric acid solution (0.018M, pH=2.5) for caries-affected transparent dentin, one of the major components of dentin substrate as altered by caries, with comparisons made with non-carious dentin. METHODS: Transparent dentin was identified by sagittally sectioning five obviously carious teeth. Sections were then cut through the transparent dentin area perpendicular to the course of the dentinal tubules. Polished dentin samples of the transparent dentin and non-carious dentin were prepared with an internal reference layer and studied at specific intervals for citric acid etching in an atomic force microscope (AFM). RESULTS: At baseline, transparent dentin was identified by dentinal tubules that were largely occluded with mineral deposits that on etching proved to be acid resistant. Peritubular dentin etched rapidly and linearly over time until it could no longer be measured, yielding etching rates for transparent dentin that could not be distinguished from normal dentin. The normal and transparent intertubular dentin surfaces began etching at nearly the same rate, but then surface recession stabilized after less than 1 microm depth change for both dentin types. SIGNIFICANCE: Most previous studies have focused on demineralization and bonding to normal dentin, although many bonding procedures involve altered dentin substrates, such as those modified by caries. In this study, peritubular and intertubular dentin from normal and caries-affected dentin exhibited similar behavior. The major difference was the presence of acid resistant mineral in most of tubule lumens in the transparent dentin.

Acid Etching, Dental↗

The effectiveness of different sweeteners in suppressing citric acid sourness.

The exact mechanism that causes taste suppression in a perceptually heterogeneous mixture, and the locus of that mechanism, are as yet unknown. The present study was designed to explore the idea that mixture suppression is a perceptual phenomenon and not the result of physical, chemical, or receptor-substance interactions. An investigation was carried out as to whether perceptually similar taste stimuli give rise to the same sensory interactions when mixed with a substance of a different taste quality. In the first study, five different sweeteners (sucrose, fructose, aspartame, saccharin, and sorbitol) were matched in perceived sweetness intensity, in order to obtain five perceptually similar stimuli. Every equisweet sweetener concentration was mixed with each of four citric acid concentrations. In a second study, the sourness-suppressing effects of two sweeteners, sucrose and aspartame, were compared at four different concentration levels. Sourness scale values of unmixed citric acid, the unmixed sweeteners, and the citric acid/sweetener mixtures were assessed with a functional measurement approach in combination with a two-stimulus procedure. The equisweet sweeteners were equally effective in suppressing the perceived sourness intensity of citric acid over the concentration range used. The side tastes of the sweeteners, if present, did not have a substantial effect on the degree of sourness suppression.

Adult↗

Kinetic studies on citric acid production by Aspergillus niger. I. Phases of mycelium growth and product formation.

The physiology and growth kinetics of Aspergillus niger B-64-5 in shaken culture were studied, five successive phases of citric acid fermentation being distinguished. Growth was found to be diauxic, spore germination being followed by an exponential growth phase, then a period of growth disturbance, and last of all a phase of secondary growth, characterized by a constant coefficient of growth decline appeared. The citric acid production started in the phase of growth disturbance. The chief increase in citric acid concentration took place in the production phase, i.e. in the last fermentation phase.

Aspergillus↗

Bioelectrochemical determination of citric acid in real samples using a fully automated flow injection manifold.

An enzymic method for the determination of citric acid in fruits, juices and sport drinks is proposed. The method is based on the action of the enzymes citrate lyase, oxaloacetate decarboxylase and pyruvate oxidase, which convert citric acid into H2O2 with the latter being monitored amperometrically with a H2O2 probe. The enzymes pyruvate oxidase and oxaloacetate decarboxylase were immobilized. A multi-membrane system, consisting of a cellulose acetate membrane for the elimination of interferants, an enzymic membrane and a protective polycarbonate membrane were placed on a Pt electrode and used with a fully automated flow injection manifold. Several parameters were optimized, resulting in a readily constructed and reproducible biosensor. Interference from various compounds present in real samples was minimized. Calibration graphs were linear over the range 0.01-0.9 mM pyruvate, 0.015-0.6 mM oxaloacetate and 0.015-0.5 mM citrate. The throughput was 30 samples h-1 with an RSD of 1.0% (n = 8); the mean relative error was 2.4% compared with a standard method. The recovery was 96-104%. A 8-10% loss of the initial activity of the sensor was observed after 100-120 injections.

Beverages↗

Induction of complement sensitivity in Escherichia coli by citric acid and low pH.

AIMS: The lytic functions of the complement system play an important role in the control of Gram-negative infections. Complement-resistant Escherichia coli LP1395 (O18) grown under normal conditions can survive the bactericidal action of complement present in human serum. Towards elucidating the mechanisms of complement resistance, the resistance of E. coli LP1395 grown under conditions of low pH and in the presence of citric acid was tested. METHODS AND RESULTS: E. coli LP1395 becomes sensitive to complement after growth in the presence of citric acid at pH 5. Complement resistance could be restored when the cells were transferred to pH 7 media. However, this recovery was greatly impaired when the cells were transferred to pH 7 media with chloramphenicol. This implies that protein synthesis may be involved in complement resistance. The cells exposed to citric acid at pH 5 showed no indication of a generalized outer membrane (OM) permeability when compared with those grown under normal conditions in terms of sensitivity to lysozyme, uptake of lipophilic dye, or sensitivity to a number of antibiotics. CONCLUSION: Complement-resistant LP1395 may acquire a sensitivity to complement due not to a generalized disruption of the OM barrier, but possibly to the alteration of the activity of one or more normal complement resistance factors. SIGNIFICANCE AND IMPACT OF THE STUDY: The elucidation of the mechanisms of complement resistance of Gram-negative pathogens would bring important information about bacterial infections. Complement resistance factors could also be potential targets in antimicrobial therapies.

1-Naphthylamine↗

Citric acid cycle as a "one-step" reaction.

One of the most familiar metabolic pathways is the citric acid cycle. This reaction sequence is characterized by intermediates which can be detected in aqueous solution. The recent success in isolating an enzyme cluster with five cycle activities suggests that the cyclic sequence should find a counterpart in a highly organized enzyme system for effective catalysis. In the present article, a new way of looking at this complex reaction sequence is proposed utilizing the hydrophobicity of active sites to define the entrance or exit of substrates as a first or last reaction step. With this view, highly reactive intermediates can be formulated which are able to react in a quasi "one step" mechanism to stable end-products. It is possible to derive the known intermediates of the citric acid cycle by a transition from a proposed hydrophobic catalytic site into the aqueous phase. A variety of biochemical concepts in use ("energy-rich"-bonds, group transfer, substrate-channelling, transporters, etc.) can be understood to be derived from methodology commonly in use. The proposed reaction mechanism demands a specific organization of the proteins forming the enzymatic activity(ies). Isolated and purified enzymes should, in consequence, be seen as isolation products, differing from their functionally organized form in a living cell. In this paper, a new concept is proposed by which a variety of observations and concepts can be unified.

Binding Sites↗

Human clinical and histologic repair responses following the use of citric acid in periodontal therapy.

Seven blocks containing one tooth each were obtained from two patients undergoing treatment for periodontitis. Sixteen weeks prior to block removal, these sites were treated by periodontal flap surgery. In six teeth a citric acid solution (pH = 1) was applied for 2 minutes to the planed root surfaces. The seventh tooth received identical periodontal therapy except that saline was applied to the planed root for 2 minutes. At the end of the experimental period, the blocks were removed and prepared for histologic study. In five out of six citric acid treated specimens, no evidence was observed suggesting that citric acid applications either initiated or accelerated cementogenesis or functional connective tissue attachment at root surfaces previously exposed to periodontal pockets. In one specimen, repair cementum was seen crestally to the alveolar margin. This cementum was deposited on the surface of acellular cementum. Unfortunately, however, the specimen does not allow us to date the deposition of the repair cementum. It thus remains questionable whether this cemental deposition is indeed a repair response to the specific treatment performed.

Aged↗

Aconitase and citric acid fermentation by Aspergillus niger.

In view of the often-cited theory that citric acid accumulation is caused by an inhibition of aconitase activity, the equilibrium of the reaction of aconitase was investigated by comparing in vivo steady-state concentrations of citrate and isocitrate in Aspergillus niger grown under various citric acid-producing conditions. With the equilibrium catalyzed by the A. niger enzyme in vitro, similar values were obtained. The validity of our in vivo measurements was verified by the addition of the aconitase inhibitor fluorocitrate, which appreciably elevated the citrate:isocitrate ratio. The results strongly argue against an inhibition of aconitase during citric acid fermentation.

Aconitate Hydratase↗