ABSTRACT
OBJECTIVES: The antibacterial activity of two different formulations of a chlorhexidine/thymol varnish should be elucidated in vitro and in vivo. METHODS: The agar diffusion assay with Cervitec(®) and CervitecPlus(®) and three reference strains each of streptococci, lactobacilli, actinomyces and periodontal pathogens was performed. In a split-mouth study, 40 volunteers applied the test (CervitecPlus(®), solvent water and ethanol) and control (Cervitec(®), solvent ethyl acetate) varnish at buccal recessions of premolar teeth at baseline as well as after two, four and seven days. Supra- and subgingival plaques were collected 2 weeks before baseline and at the screening appointments. Supragingival plaque was analysed for mutans streptococci and lactobacilli and subgingival samples for Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis and Porphyromonas intermedia. Friedman/Wilcoxon tests and U-test were used for statistical analysis (P < 0.05). RESULTS: Most reference strains were susceptible with inhibition zones (mm) as follows: Cervitec(®)/CervitecPlus(®) streptococci 27 ± 1.7/21.3 ± 2.5, lactobacilli 26 ± 9.2/23.7 ± 4.9, actinomyces 36.3 ± 6.6/27.3 ± 1.5, periodontal pathogens 18.7 ± 7.6/18 ± 1.7. Both varnishes reduced significantly the counts of mutans streptococci and lactobacilli in the patients. However, no significant differences were found between test and control sides at any time. The total counts of periodontal pathogens were low. A tendency to higher counts of A. actinomycetemcomitans at the control side could be shown; the test side did not harbour significantly higher counts. CONCLUSION: Both varnishes may influence the plaque formation and reduce mutans streptococci in supragingival plaque.
Subject(s)
Anti-Infective Agents, Local/therapeutic use , Bacteria/drug effects , Chlorhexidine/therapeutic use , Dental Plaque/microbiology , Thymol/therapeutic use , Actinomyces/drug effects , Adult , Aggregatibacter actinomycetemcomitans/drug effects , Anti-Infective Agents, Local/administration & dosage , Bacterial Load/drug effects , Bicuspid/microbiology , Biofilms/drug effects , Chemistry, Pharmaceutical , Chlorhexidine/administration & dosage , Dental Plaque/prevention & control , Drug Combinations , Female , Follow-Up Studies , Fusobacterium nucleatum/drug effects , Humans , Lactobacillus/drug effects , Lacticaseibacillus casei/drug effects , Lactobacillus plantarum/drug effects , Male , Middle Aged , Porphyromonas gingivalis/drug effects , Streptococcus mutans/drug effects , Streptococcus sanguis/drug effects , Streptococcus sobrinus/drug effects , Thymol/administration & dosageABSTRACT
AIMS: Fructose-2,6-bisphosphate, a key regulator of glycolysis, is synthesized and degraded by four different isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB1-4). The PFKFB3 isozyme is upregulated in several human tumours. Six alternatively spliced variants of PFKFB3 mRNA are known in humans (UBI2K1-6). Here, we studied the role of the PFKFB3 splice variants in human astrocytic gliomas. METHODS: We analysed the PFKFB3 splice variants in 48 astrocytic gliomas by RT-PCR and real-time PCR. The effect of transient and stable overexpression of the PFKFB3 isoforms was studied in U87 glioblastoma cells by MTT, cell counting, clone formation assay and metabolic measurements. RESULTS: UBI2K5 and UBI2K6 are the predominant splice variants in rapidly proliferating high-grade astrocytomas while the expression of UBI2K3 and UBI2K4 is mainly restricted to low-grade astrocytomas and nonneoplastic brain tissue. Overexpression of UBI2K5 or UBI2K6 in the U87 glioblastoma cell line enhances the glycolytic flux but does not affect cell growth. In contrast, overexpression of UBI2K4 reduces cell viability and anchorage-independent growth of U87 cells. The UBI2K4 mRNA level is downregulated in astrocytic gliomas with increasing malignancy grade. Moreover, the UBI2K4 mRNA level correlates with growth rate of several human cancer cell lines derived from different tissue types. CONCLUSIONS: Our results demonstrate that the splice variant UBI2K4 impedes the tumour cell growth and might serve as a tumour suppressor in astrocytic tumours.
Subject(s)
Astrocytoma/metabolism , Brain Neoplasms/metabolism , Glioblastoma/metabolism , Phosphofructokinase-2/metabolism , Brain/metabolism , Cell Count , Cell Line, Tumor , Cell Proliferation , Cell Survival , Down-Regulation , Humans , Neoplasm Staging , Phosphofructokinase-2/genetics , Polymerase Chain Reaction , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , TransfectionABSTRACT
Information concerning phenotypic heterogeneity of Streptococcus mutans in carious dentin is sparse. Matrix-assisted laser-desorption/ionization-time-of-flight mass-spectrometry (MALDI-TOF-MS) facilitates the phenotypic differentiation of bacteria to the subspecies level. To verify a supposed influence of restorative treatment on the phenotypic heterogeneity of S. mutans, we isolated and compared a total of 222 S. mutans strains from dentin samples of 21 human deciduous molars during caries excavation (T(1)) and 8 wks (T(2)) after removal of the temporary restoration. Phenotypic heterogeneity was determined by MALDI-TOF-MS and hierarchical clustering. Thirty-six distinct S. mutans phenotypes could be identified. Although indistinguishable phenotypes were found in the same teeth at T(1) and T(2), as well as in different teeth of individual participants, the phenotypic heterogeneity increased significantly, from 1.4 phenotypes per S. mutans-positive dentin sample at T(1) to 2.2 phenotypes at T(2). We attribute this to an adaptation of S. mutans to the modified environment under the restoration following caries excavation.
Subject(s)
Dentin/microbiology , Genetic Heterogeneity , Streptococcus mutans/genetics , Adaptation, Physiological/genetics , Child , Colony Count, Microbial , Dental Caries/microbiology , Dental Caries/therapy , Dental Cavity Preparation/methods , Dental Restoration, Temporary , Follow-Up Studies , Glass Ionomer Cements , Glutamic Acid/therapeutic use , Humans , Leucine/therapeutic use , Lysine/therapeutic use , Molar/microbiology , Phenotype , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Streptococcus mutans/classification , Tooth, Deciduous/microbiologyABSTRACT
INTRODUCTION: To facilitate the identification of anaerobes cultivated from periodontal disease, whole cell bacterial identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was evaluated. METHODS: A total of 84 strains (nine reference strains and 75 recent clinical isolates from 33 patients with aggressive periodontitis) previously identified with phenotypic methods were used. All the references and 10 clinical isolates belonging to the same species as the reference strains were genotypically identified by sequence analysis of the 16S ribosomal RNA gene. All the strains were then analyzed using MALDI-TOF-MS. RESULTS: The reference strains of anaerobic bacteria used showed characteristic MALDI-TOF-MS spectra with peaks between m/z 2000 and up to about m/z 13,000. On visual inspection, the similarity of spectra produced by strains of a single genus could be recognized. Obvious differences between spectra produced by strains of different species were also easily noticed. The reproducibility of the method was proved by the similarity of spectra belonging to the same species. The spectra of the Prevotella intermedia strains identified with MALDI clustered together and clustered separately from the spectra of Prevotella nigrescens, proving that MALDI-TOF-MS is an accurate method that is capable of separating these two species. The quality of clustering was characterized by calculating an inconsistency coefficient (Mathworks:/Matlab Reference Manual v2007a/, Statistical toolbox). CONCLUSION: Our results suggest that MALDI-TOF-MS might become a useful method for the identification of anaerobic bacteria, especially for those that cannot be readily identified by biochemical analysis. It may become an attractive system even for the routine identification of clinical isolates.
Subject(s)
Bacteria, Anaerobic/classification , Biofilms/classification , Mouth/microbiology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Actinomyces/classification , Adult , Bacteroides/classification , Fusobacterium nucleatum/classification , Genotype , Humans , Peptostreptococcus/classification , Periodontitis/microbiology , Phenotype , Porphyromonas gingivalis/classification , Prevotella intermedia/classification , Prevotella nigrescens/classification , RNA, Ribosomal, 16S/analysisABSTRACT
Fructose-1,6-bisphosphatase (FBPase) deficiency (OMIM 229700) has been characterized as the cause of life-threatening hypoglycaemia and lactic acidaemia following prolonged fasting. The patient, an adult African-American woman, presented during the second trimester of her first pregnancy with recurrent episodes of lactic acidaemia and hypoglycaemia. She had recently been admitted to a nearby intensive care unit after presentation with profound hypoglycaemia and lactic acidosis, and was found to be pregnant. The history was remarkable for approximately 30 hospitalizations for hypoglycaemia and acidosis. She had previously undergone liver biopsy at another centre and was diagnosed with a 'glycogen storage disease', although no enzyme testing had been done for confirmation. Based on clinical symptoms, a diagnosis of FBPase deficiency was accomplished through gene sequencing, which revealed homozygosity for a panethnic, common mutation, 960/961insG in exon 7. The availability of mutation testing facilitated the confirmation of FBPase deficiency in this patient, obviating liver biopsy for enzyme activity confirmation. The patient underwent three successful pregnancies by strict compliance with dietary management, including nocturnal uncooked cornstarch to manage hypoglycaemia. The pregnancies were complicated by mild gestational diabetes, increased cornstarch requirements, and hypoglycaemia at the time of discharge from the hospital. The three infants had normal birth weights and experienced no complications during the neonatal period. The patient subsequently developed sensorineural hearing loss and early-onset cognitive impairment, despite compliance with the monitoring and treatment of hypoglycaemia. The experience with multiple pregnancies in this FBPase-deficient patient provides insight into the management of hypoglycaemia in inherited disorders of gluconeogenesis.
Subject(s)
Fructose-1,6-Diphosphatase Deficiency/diet therapy , Pregnancy Complications/diet therapy , Adult , Exons , Female , Fructose-1,6-Diphosphatase Deficiency/diagnosis , Fructose-1,6-Diphosphatase Deficiency/enzymology , Fructose-Bisphosphatase/genetics , Genetic Testing , Gravidity , Humans , Live Birth , Mutation , Pregnancy , Pregnancy Complications/diagnosis , Pregnancy Complications/enzymologyABSTRACT
Viridans streptococci (VS) are responsible for several systemic diseases, such as endocarditis, abscesses, and septicemia. Unfortunately, species identification by conventional methods seems to be more difficult than species identification of other groups of bacteria. The aim of the present study was to evaluate the use of cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) for the rapid identification of 10 different species of VS. A total of 99 VS clinical isolates, 10 reference strains, and 20 strains from our in-house culture collection were analyzed by MALDI-TOF-MS. To evaluate the mass-spectrometric discrimination results, all strains were identified in parallel by phenotypic and genotypic methods. MALDI-TOF-MS identified 71 isolates as the mitis group, 23 as the anginosus group, and 5 as Streptococcus salivarius. Comparison of the species identification results obtained by the MALDI-TOF-MS analyses and with the phenotypic/genotypic identification systems showed 100% consistency at the species level. Thus, MALDI-TOF-MS seems to be a rapid and reliable method for the identification of species of VS from clinical samples.
Subject(s)
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Streptococcal Infections/microbiology , Viridans Streptococci/chemistry , Viridans Streptococci/classification , Bacterial Typing Techniques , Humans , Polymerase Chain Reaction , Sensitivity and Specificity , Sequence Analysis, DNAABSTRACT
AIM: This was to study the longitudinal assessment of caries activity of Streptococcus sobrinus (SS) positive children during their mixed dentition. METHODS: The occurrence of mutans streptococci (MS) in plaque and saliva was determined in a representative sample of 55 children aged 8 to 12 years over a period of 4 years. A total of 708 bacterial strains was isolated which were identified as MS or SS. Caries activity (DeltaD(1-4)MFS) as well as plaque and gingival inflammation were recorded. RESULTS: During the period of observation 52 of the 55 children harboured MS; 12 of these children were SS positive. SS was not permanently detectable and 3 of the children were MS and SS negative. SS was not found without the presence of MS. Children that were infected with both SS and MS showed a slightly higher increase in caries compared with children that were infected exclusively by MS (DeltaD(1,2)MFS 6.2 vs. 3.0 and DeltaD(3,4)MFS 5.3 vs. 3.8) over the period of 4 years. An SS infection accelerated the increase of DeltaD(3,4)MFS significantly by a factor of 4 one year after its detection, whereas the DeltaD(1,2)MFS was 3 times as high during the period of infection. CONCLUSION: The findings suggest that an SS infection represents an important additional risk factor for dental caries due to its obvious aggravating of caries activity.
Subject(s)
Dental Caries/microbiology , Streptococcus sobrinus/isolation & purification , Child , Cohort Studies , Colony Count, Microbial , DMF Index , Dental Caries Susceptibility , Dental Plaque/microbiology , Dental Plaque Index , Dentition, Mixed , Follow-Up Studies , Gingivitis/microbiology , Humans , Longitudinal Studies , Periodontal Index , Risk Factors , Saliva/microbiology , Streptococcus mutans/isolation & purification , Streptococcus sobrinus/physiology , Tooth, Deciduous/microbiologyABSTRACT
Adenosine 5'-monophosphate (AMP) inhibits muscle fructose 1,6-bisphosphatase (FBPase) about 44 times stronger than the liver isozyme. The key role in strong AMP binding to muscle isozyme play K20, T177 and Q179. Muscle FBPase which has been mutated towards the liver enzyme (K20E/T177M/Q179C) is inhibited by AMP about 26 times weaker than the wild-type muscle enzyme, but it binds the fluorescent AMP analogue, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP), similarly to the wild-type liver enzyme. The reverse mutation of liver FBPase towards the muscle isozyme significantly increases the affinity of the mutant to TNP-AMP. High affinity to the inhibitor but low sensitivity to AMP of the liver triple mutant suggest differences between the isozymes in the mechanism of allosteric signal transmission.
Subject(s)
Adenosine Monophosphate/pharmacology , Fructose-Bisphosphatase/antagonists & inhibitors , Fructose-Bisphosphatase/genetics , Muscles/enzymology , Adenosine Monophosphate/analogs & derivatives , Allosteric Regulation/genetics , Amino Acid Sequence , Amino Acids/genetics , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Liver/enzymology , Molecular Sequence Data , MutationABSTRACT
It is difficult to distinguish mutans streptococci on the species level, and even more so on the subspecies level. Intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (ICM) was applied to reference strains of five of the species of the mutans group (Streptococcus criceti, Streptococcus downei, Streptococcus mutans, Streptococcus ratti, Streptococcus sobrinus), nonmutans streptococci (Streptococcus oralis, Streptococcus mitis, Streptococcus salivarius, and Streptococcus sanguinis), and 177 mutans streptococci isolated from saliva of 10 children. From the analysis of the reference strains, readily distinguishable ICM mass spectra were obtained for the different species. Based on multivariate statistical analysis, a correct and unambiguous assignment was made of the spectra of 159 isolated mutans streptococci to S. mutans and 16 isolates to S. sobrinus. Two isolates were sorted out and were identified by sequencing of their 16S rRNA genes as Streptococcus anginosus. In addition, ICM indicated a misclassification for some reference strains (AHT, V 100 and E 49) and re-classified AHT and E 49 as S. ratti and V 100 as S. sobrinus. This was confirmed by 16S rDNA sequencing. Based on a statistical similarity analysis of the spectra of reference strains and a quantitative assessment of the reproducibility of ICM, the isolates identified as either S. mutans or S. sobrinus were phenotyped on the subspecies level. In the population of the clinical isolates, 14 unambiguously different S. mutans and three different S. sobrinus phenotypes were detected. ICM proved to be a powerful tool for a differentiation of mutans streptococci down to the subspecies level.
Subject(s)
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Streptococcus/classification , Child , Humans , Phenotype , RNA, Ribosomal, 16S/genetics , Reproducibility of Results , Saliva/microbiology , Sequence Analysis, DNA , Sequence Analysis, RNA , Streptococcus/genetics , Streptococcus anginosus/classification , Streptococcus anginosus/genetics , Streptococcus mitis/classification , Streptococcus mitis/genetics , Streptococcus mutans/classification , Streptococcus mutans/genetics , Streptococcus oralis/classification , Streptococcus oralis/genetics , Streptococcus sobrinus/classification , Streptococcus sobrinus/geneticsABSTRACT
OBJECTIVE: Primary myoadenylate deaminase deficiency (MADD) is probably the most frequent inborn metabolic myopathy with a prevalence of up to 2%. It is the result of mutations in the AMPDI gene, the most common of which is a C34-T transition in exon 2. The importance of the more rare mutation G468-T in exon 5 is uncertain. Primary objective was to elucidate the clinical significance of the enzyme disorder, which remains unclear since its first description in 1978. We further examined the existence of an association of MADD with other muscle disorders, such as malignant hyperthermia and rhabdomyolysis, as was suspected in earlier studies. MATERIAL AND METHODS: In a large collection of 1673 muscle biopsies that had been stored deep frozen we identified 33 cases of primary MADD, 12 of which without any other coinciding muscle diseases, by histochemical, biochemical and molecular genetic examinations. Clinical and laboratory data was collected. By additional examination of randomly chosen blood samples we identified one person carrying the rare compound heterozygosity C34-T/ G468-T, who was examined in clinical respects and a muscle biopsy was taken. RESULTS: As underlying mutation, the most common transition C34-T/C 143-T was detected in 33 cases. One patient carried the compound heterozygosity C34-T/G468-T. The overall frequency of MADD in the contingent was 1.8%. Only three patients out of 12 with isolated primary MADD suffered from muscle complaints, one of whom did not experience the typical symptoms of exercise related myalgia, muscle cramps and weakness as described by Fishbein. The patient carrying C34-T/G468-T was a fully healthy female. She had never experienced any muscle complaints. Any association with other neuromuscular disorders, if not completely ruled out, was found to be very unlikely. CONCLUSION: The results suggest that MADD itself is unlikely to be solely responsible for the manifestation of muscular symptoms. It is probable that either the loss of a compensation mechanism or coexistent disturbances in muscle metabolism which are unidentified so far are required for the emergence of complaints.
Subject(s)
AMP Deaminase/deficiency , AMP Deaminase/genetics , Metabolism, Inborn Errors/genetics , Muscle, Skeletal/enzymology , Muscular Diseases/genetics , Mutation/genetics , Adult , Aged , DNA Mutational Analysis , Exons/genetics , Female , Heterozygote , Humans , Male , Malignant Hyperthermia/enzymology , Malignant Hyperthermia/genetics , Metabolism, Inborn Errors/enzymology , Metabolism, Inborn Errors/pathology , Middle Aged , Muscle, Skeletal/pathology , Muscular Diseases/enzymology , Muscular Diseases/pathologyABSTRACT
AIMS: This study was to develop a simple and reliable method for quantifying Microthrix parvicella 16S rRNA gene copies and its application to activated sludge samples collected from wastewater treatment plants (WWTP) with and without foaming problems. METHODS AND RESULTS: The relative frequency of M. parvicella was determined by combining real-time PCR assays for quantification of total bacterial 16S rRNA gene copies and M. parvicella 16S rRNA gene copies. The developed method was applied to analyse 32 activated sludge samples obtained from German WWTP. The level of M. parvicella 16S rRNA gene copies in the 18 nonfoaming samples was below 3% of the total number of 16S rRNA gene copies and in the range of 0-18% for the 14 foaming samples. CONCLUSIONS: The described method allows reliable monitoring of the amount of M. parvicella in activated sludge samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The described method may become an important component of a warning system for forthcoming bulking and foaming episodes.
Subject(s)
Actinobacteria/isolation & purification , Polymerase Chain Reaction/methods , RNA, Ribosomal, 16S/analysis , Sewage/microbiology , Actinobacteria/genetics , Gene Dosage , Waste Disposal, Fluid/standardsABSTRACT
Saliva samples from 16 children with current caries activity were investigated for Streptococcus mutans using three different PCR techniques, and the results were compared with those of selective cultivation on mitis salivarius agar with bacitracin (MSB) (I, II: LightCycler - competitive PCR end-point analysis; III: LightCycler - kinetic real-time analysis; IV, V: block cycler - competitive PCR end-point analysis; VI: cultivation on MSB agar). In groups I, III, IV and VI the saliva samples were analyzed directly. A DNA preparation before PCR with added competitors was carried out in groups II and V to exclude the influence of PCR inhibitors. The coefficients of correlation ranged from 0.97 to 0.98 among the competitive PCR methods, 0.8 to 0.85 for competitive vs. real-time PCR and 0.5 to 0.65 for PCR vs. cultivation methods. Competitive PCR on the real-time instrument was found to be more rapid than, comparably sensitive to, but less reproducible than competitive PCR on a block cycler.
Subject(s)
Dental Caries/microbiology , Polymerase Chain Reaction/methods , Saliva/microbiology , Streptococcus mutans/isolation & purification , Bacteriological Techniques , Child , Colony Count, Microbial , DNA, Bacterial/analysis , Dental Caries Activity Tests , Humans , Reproducibility of Results , Sensitivity and Specificity , Statistics, Nonparametric , Streptococcus mutans/geneticsABSTRACT
Alzheimer's disease is associated with markedly impaired cerebral glucose metabolism as detected by reduced cortical desoxyglucose utilization, by altered activities of key glycolytic enzymes or by reduced densities of cortical glucose transporter subtypes. To determine whether formation and/or deposition of beta-amyloid plays a role in the pathology of glucose metabolism, transgenic Tg2576 mice that overexpress the Swedish mutation of the human amyloid precursor protein and demonstrate a progressive, age-related cortical and hippocampal deposition of beta-amyloid plaques, were used to study expression and activity of key enzymes of brain glycolysis (phosphofructokinase, PFK) and glyconeogenesis (fructose1,6-bisphosphatase; FbPase). Quantitative RT-PCR revealed high expression levels of both C- and M-type PFK mRNA in non-transgenic mouse cerebral cortex, whilst there was little expression of the L-type. In 24-month-old transgenic Tg2576 mouse cortex, but not in 7-, 13-, and 17-month-old mice, the copy number of PFK-C mRNA was significantly reduced in comparison to non-transgenic littermates, while the mRNA level of the other PFK isoforms and FbPase did not differ between transgenic and non-transgenic tissue samples. In situ hybridization in brain sections from aged Tg2576 mice revealed reduced PFK-C mRNA expression in beta-amyloid plaque-associated neurons and upregulation in reactive astrocytes surrounding beta-amyloid deposits. The decreased PFK-C protein level detected by Western analysis in cerebral cortical tissue from 24-month-old transgenic Tg2576 mice was accompanied by reduced enzyme activity of PFK in comparison to non-transgenic littermates. Our data demonstrate that impairment of cerebral cortical glucose metabolism occurs only due to the long-lasting high beta-amyloid burden. This results from a reduction in glycolytic activity in beta-amyloid plaque-associated neurons and a concomitant upregulation in reactive, plaque-surrounding astrocytes.
Subject(s)
Alzheimer Disease/metabolism , Cerebral Cortex/metabolism , Fructose-Bisphosphatase/metabolism , Glucose/metabolism , Phosphofructokinase-1/metabolism , Plaque, Amyloid/metabolism , Age Factors , Alzheimer Disease/enzymology , Amyloid beta-Protein Precursor/genetics , Animals , Blotting, Western , Cerebral Cortex/enzymology , Disease Models, Animal , Fructose-Bisphosphatase/genetics , Immunohistochemistry , In Situ Hybridization , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation , Phosphofructokinase-1/genetics , Plaque, Amyloid/enzymology , Polymerase Chain Reaction , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Phosphorylation of yeast 6-phosphofructo-2-kinase and its role for the regulation of glycolysis under hypoosmotic conditions were investigated. 6-Phosphofructo-2-kinase was found to be phosphorylated in vitro by protein kinase C at serine 652 and thereby inactivated. Protein phosphatase 2A reversed the phosphorylative inhibition of the enzyme. When yeast cells were shifted to hypotonic media, 6-phosphofructo-2-kinase was found to be phosphorylated and inactivated. Under in vivo conditions, two phosphate residues were incorporated into the enzyme. One of them is bound to serine 652, indicating that this modification was probably caused by yeast protein kinase C1. The second phosphate is bound to Ser8 within the N-terminal peptide T(1-41) which contains several serine residues but no protein kinase C recognition sequence. Site-directed mutagenesis confirmed that the phosphorylation of serine 652 but not the N-terminal modification is responsible for the in vivo inactivation of 6-phosphofructo-2-kinase. The obtained results suggest that the phosphorylation of 6-phosphofructo-2-kinase mediates a response of the cells to an activation of the hypoosmolarity MAP kinase pathway. Via a suppression of glycolysis, the inactivation of 6-phosphofructo-2-kinase is expected to be responsible for the observed accumulation of glucose 6-phosphate, an essential precursor of the cell wall glucans, and the decrease of glycerol, an important osmolyte.
Subject(s)
Glycolysis/physiology , Phosphofructokinase-2/metabolism , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , Binding Sites , Cloning, Molecular , Glucose-6-Phosphate/analysis , Glycerol/analysis , In Vitro Techniques , Mass Spectrometry , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptide Fragments/metabolism , Phosphofructokinase-2/chemistry , Phosphoprotein Phosphatases/metabolism , Phosphorylation , Plasmids , Protein Kinase C/metabolism , Protein Phosphatase 2 , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Serine/chemistry , Trypsin/metabolismABSTRACT
425 strains of mutans streptococci and 12 reference strains were investigated by membrane fatty acid spectra (MFAS) and peroxidase reaction (PR) after aerobic and anaerobic incubation. 423 strains were identified as Streptococcus mutans. The remaining 2 strains were identified as Streptococcus sobrinus. The PR of 29 strains was doubtful; immediately after anaerobic incubation a negative PR changed into a slightly positive PR. To test the diagnostic value of PR the strains were additionally investigated by means of species-specific polymerase chain reactions (PCR). The species-specific PCRs were developed on the basis of the respective genes of 16S rRNA of the pathogens S. mutans and S. sobrinus. Specificity and sensitivity were tested on reference strains (n = 17) and negative control strains (n = 39). The results of this investigation showed that an anaerobic incubation regime could lead to false-positive (S. mutans) or false-negative (S. sobrinus) PR. The 425 MS strains were classified as either S. mutans (n = 420) or S. sobrinus (n = 5). The findings on the reference strains required a reclassification of S. mutans V 100 into S. sobrinus V 100. Summarising, it is possible now to differentiate strains of mutans streptococci by MFAS and PR after aerobic incubation.
Subject(s)
Horseradish Peroxidase , Streptococcus mutans/classification , Streptococcus sobrinus/classification , Aerobiosis , Anaerobiosis , Child , Chromatography, Gas , Culture Media , Dental Plaque/microbiology , Electrophoresis, Agar Gel , False Negative Reactions , False Positive Reactions , Fatty Acids/analysis , Humans , Membrane Lipids/analysis , Polymerase Chain Reaction , Predictive Value of Tests , RNA, Ribosomal, 16S/genetics , Sensitivity and Specificity , Species Specificity , Streptococcus mutans/genetics , Streptococcus sobrinus/geneticsABSTRACT
Changes in the metabolic activity within the brain of patients suffering from Alzheimer's disease (AD) were investigated and compared with biochemical alterations in the hippocampus induced by fimbria/fornix transection in the rat. The deafferentation of the hippocampus results in a degeneration of cholinergic septo-hippocampal terminals accompanied by a persistent decrease of choline acetyltransferase (ChAT) and acetylcholine esterase (AChE) activities similar to the cholinergic malfunction in AD. In the animal model the [3H]-cytochalasin B binding to the glucose transporters was elevated up to the day 7 after surgery as was the activity of the phosphofructokinase (PFK) on day 3. A reactive astrogliosis could be evidenced by the upregulation of glial fibrillary acidic protein (GFAP). An increase of the PFK activity was also found in AD being accompanied by enhanced level of GFAP as well. A higher concentration of mRNA for all three isoenzymes of PFK was shown by reverse transcription (RT)-real time polymerase chain reaction (PCR) amplification. However, the pattern of PFK isoenzyme proteins and mRNAs did neither change in diseased human nor in the lesioned rat brain. The activities of the mitochondrial enzymes pyruvate dehydrogenase complex (PDHC) and cytochrome c oxidase (CO) were diminished in the lesioned rat hippocampus on day 7 as well as in AD brain. Subcellular fractionation showed that the activity of these enzymes was affected in the synaptosomal as well as in the extrasynaptosomal mitochondria indicating a loss of neuronal input and also a vulnerability of intrinsic hippocampal neurons and/or non-neuronal cells. The recovery of the mitochondrial enzyme activity in the animal model at later post lesion intervals may be the result of compensatory responses of surviving cells or of sprouting of other non-affected inputs. It is concluded that common metabolic mechanisms may underlie the concurrent degenerative and repair processes in the denervated hippocampus and the diseased Alzheimer brain.
Subject(s)
Alzheimer Disease/metabolism , Basal Nucleus of Meynert/cytology , Diagonal Band of Broca/cytology , Energy Metabolism/physiology , Hippocampus/cytology , Acetylcholinesterase/metabolism , Afferent Pathways , Animals , Astrocytes/chemistry , Astrocytes/metabolism , Basal Nucleus of Meynert/metabolism , Choline O-Acetyltransferase/metabolism , Cholinergic Fibers/metabolism , Denervation , Diagonal Band of Broca/metabolism , Disease Models, Animal , Electron Transport Complex IV/metabolism , Fornix, Brain/cytology , Fornix, Brain/metabolism , Fornix, Brain/surgery , Gene Expression Regulation, Enzymologic , Glial Fibrillary Acidic Protein/analysis , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Glycolysis/physiology , Hippocampus/metabolism , Humans , Male , Mitochondria/enzymology , Phosphofructokinase-1/genetics , Phosphofructokinase-1/metabolism , Pyruvate Dehydrogenase Complex/metabolism , RNA, Messenger/analysis , Rats , Rats, WistarABSTRACT
Fructose-1,6-bisphosphatase is one of the key enzymes in the gluconeogenic pathway predominantly occurring in liver, kidney and muscle. In the brain, fructose-1,6-bisphosphatase has been suggested to be an astrocyte-specific enzyme but the functional importance of glyconeogenesis in the brain is still unclear. To further elucidate the cellular source of fructose-1,6-bisphosphatase in the brain, non-radioactive in situ hybridizations were performed using digoxigenin-labeled RNA probes based on the sequence of recently cloned rat liver and muscle fructose-1,6-bisphosphatase cDNAs. In situ hybridization using a riboprobe for the liver isoform revealed a location of the hybridization signal mainly in neurons, while rat muscle fructose-1,6-bisphosphatase mRNA was detected in both neurons and astrocytes in the hippocampal formation and in layer I of the cerebral cortex.RT-PCR using RNA preparations of rat astrocytes, neurons, and adult whole brain demonstrated a localization of liver fructose-1,6-bisphosphatase mRNA isoform in neurons but not in astrocytes. The muscle fructose-1,6-bisphosphatase mRNA isoform could be detected by RT-PCR in total rat brain, astrocytic, and neuronal mRNA preparations. The isoforms of fructose-1,6-bisphosphatase mRNA seemingly demonstrate a distinct cellular expression pattern in rat brain suggesting a role of glyconeogenesis in both neurons and glial cells.
Subject(s)
Cholinergic Fibers/enzymology , Fructose-Bisphosphatase/genetics , Gluconeogenesis/physiology , Isoenzymes/genetics , Prosencephalon/metabolism , Animals , Astrocytes/chemistry , Astrocytes/enzymology , Denervation , Fructose-Bisphosphatase/metabolism , Gene Expression Regulation, Enzymologic , Glial Fibrillary Acidic Protein/analysis , In Situ Hybridization , Isoenzymes/metabolism , Male , Neurons/enzymology , Prosencephalon/cytology , RNA Probes , RNA, Messenger/analysis , Rats , Rats, WistarABSTRACT
A cDNA encoding fructose-1,6-bisphosphatase (FBPase) was isolated from mouse liver RNA. The cDNA encodes a polypeptide of 338 amino acids (36.9 kDa). The liver and muscle FBPase isoenzymes of the mouse show positional identities of 69% at the cDNA level and 72% at the protein primary structure level. Starting from genomic YAC libraries and based upon the cDNA sequence all functional parts of the mouse liver FBPase gene (including exon-intron boundaries) were PCR-amplified and sequenced. The 5'-flanking regions of the liver and muscle FBPase genes were compared and showed no sequence similarity. Both genes are co-localized at chromosome 13B3-C1. The transcriptional start site was assigned to a guanine 118 bases before the start codon in the liver FBPase gene. An analysis of the steady state mRNA levels of liver and muscle FBPase in various mouse tissues was performed by Northern blotting and RT/PCR.