Disruption of phosphofructokinase activity and aerobic glycolysis in human bronchial epithelial cells by atmospheric ultrafine particulate matter.

Exposure to ambient ultrafine particulate matter (UPM) causes respiratory disorders; however, the underlying molecular mechanisms remain unclear. In this study, we synthesized simulated UPM (sUPM) with controlled physicochemical properties using the spark-discharge method. Subsequently, we investigated the biological effects of sUPM using BEAS-2B human bronchial epithelial cells (HBECs) and a mouse intratracheal instillation model. High throughput RNA-sequencing and bioinformatics analyses revealed that dysregulation of the glycolytic metabolism is involved in the inhibited proliferation and survival of HBECs by sUPM treatment. Furthermore, signaling pathway and enzymatic analyses showed that the treatment of BEAS-2B cells with sUPM induces the inactivation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB, also known as AKT), resulting in the downregulation of phosphofructokinase 2 (PFK2) S483 phosphorylation, PFK enzyme activity, and aerobic glycolysis in HBECs in an oxidative stress-independent manner. Additionally, intratracheal instillation of sUPM reduced the phosphorylation of ERK, AKT, and PFK2, decreased proliferation, and increased the apoptosis of bronchial epithelial cells in mice. The findings of this study imply that UPM induces pulmonary toxicity by disrupting aerobic glycolytic metabolism in lung epithelial cells, which can provide novel insights into the toxicity mechanisms of UPM and strategies to prevent their toxic effects.

Hexokinase and phosphofructokinase activity and intracellular distribution correlate with aggressiveness and invasiveness of human breast carcinoma.

Glycolytic enzymes, such as hexokinase and phosphofructokinase, have been reported to be upregulated in many cancer types. Here, we evaluated these two enzymes in 54 breast cancer samples collected from volunteers subjected to mastectomy, and the results were correlated with the prognosis markers commonly used. We found that both enzymes positively correlate with the major markers for invasiveness and aggressiveness. For invasiveness, the enzymes activities increase in parallel to the tumor size. Moreover, we found augmented activities for both enzymes when the samples were extirpated from patients presenting lymph node involvement or occurrence of metastasis. For aggressiveness, we stained the samples for the estrogen and progesterone receptors, HER-2, p53 and Ki-67. The enzyme activities positively correlated with all markers but Ki-67. Finally, we conclude that these enzymes are good markers for breast cancer prognosis.

Techniques to monitor glycolysis.

An increased flux through glycolysis supports the proliferation of cancer cells by providing additional energy in the form of ATP as well as glucose-derived metabolic intermediates for nucleotide, lipid, and protein biosynthesis. Thus, glycolysis and other metabolic pathways that control cell proliferation may represent valuable targets for therapeutic interventions and diagnostic procedures. In this context, the measurement of glucose uptake and lactate excretion by malignant cells may be useful to detect shifts in glucose catabolism, while determining the activity of rate-limiting glycolytic enzymes can provide insights into points of metabolic regulation. Moreover, metabolomic studies can be used to generate large, integrated datasets to track changes in carbon flux through glycolysis and its collateral anabolic pathways. As discussed here, these approaches can reveal and quantify the metabolic alterations that underlie malignant cell proliferation.

Extraction and detection of mRNA from horsehair.

After RNA extraction from horsehair shafts and roots, the mRNAs of beta-actin, muscle-type phosphofructokinase, and transforming growth factor-beta1 were detected by reverse transcription polymerase chain reaction assay. Low amounts of RNA were present in the horsehair. These specific mRNA transcripts were readily detected when more than three hair roots were used. However, detection of the mRNA transcripts was difficult in the hair shaft. These findings indicate that the small amounts of residual RNA in horsehair roots can be utilized as samples for molecular biological analysis.

Expression of caveolin-1 in lymphocytes induces caveolae formation and recruitment of phosphofructokinase to the plasma membrane.

Compartmentation of carbohydrate metabolism has been shown in a wide range of tissues including reports of one compartment of glycolysis associated with the plasma membrane of cells. However, only in the erythrocyte has the physical basis for plasma membrane-associated glycolytic pathway been established. We have previously found that phosphofructokinase (PFK) appeared to colocalize with the fairly ubiquitous plasma membrane protein caveolin-1 (CAV-1), consistent with a role for CAV-1 as an anchor for glycolysis to the plasma membrane. To test the hypothesis that CAV-1 functions as a scaffolding protein for PFK, we transfected human lymphocytes (a cell without CAV-1 expression) with human CAV-1 cDNA. We demonstrate that expression of CAV-1 in lymphocytes results in the formation of caveolae at the plasma membrane and affects the subcellular localization of PFK by recruiting PFK to the plasma membrane. Targeting of PFK by CAV-1 also was validated by the significant colocalization between the proteins after transfection, which resulted in a correlation of 0.97 +/- 0.004 between the two fluorophores. This finding is significant in as much as it illustrates the CAV-1 feasibility of generating binding sites for glycolytic enzymes on the plasma membrane. We therefore conclude that CAV-1 functions as a scaffolding protein for PFK and that this may contribute to the elucidation of the basis for carbohydrate compartmentation to the plasma membrane in a wide variety of cell types.

Muscular adaptations to computer-guided strength training with eccentric overload.

AIMS: In order to investigate the muscular adaptations to a novel form of strength training, 18 male untrained subjects performed 4 weeks of low resistance-high repetition knee extension exercise. METHODS: Nine of them trained on a conventional weight resistance device (Leg curler, CON/ECC group), with loads equivalent to 30% of the concentric one-repetition maximum (1RM) for both the concentric and eccentric phase of movement. The other nine trained on a newly developed computer-driven device (CON/ECC-OVERLOAD group) with the concentric load equivalent to 30% of the concentric 1RM and the eccentric load equivalent to 30% of the eccentric 1RM. RESULTS: Training resulted in significantly (P < or = 0.05) increased peak torque and a tendency (P=0.092) to increased muscle cross-sectional area for the CON/ECC-OVERLOAD but not the CON/ECC group, while strength endurance capacity was significantly (P < or = 0.05) increased in the CON/ECC group only. RT-PCR revealed significantly increased myosin heavy chain (MHC) IIa and lactate dehydrogenase (LDH) A mRNAs, a tendency for increased MHC IIx mRNA (P = 0.056) and high correlations between the changes in MHC IIx and LDH A mRNAs (r=0.97, P=0.001) in the CON/ECC-OVERLOAD group. CONCLUSIONS: These results indicate a shift towards a more type II dominated gene expression pattern in the vasti laterales muscles of the CON/ECC-OVERLOAD group in response to training. We suggest that the increased eccentric load in the CON/ECC-OVERLOAD training leads to distinct adaptations towards a stronger, faster muscle.

Skeletal muscle enzymes as predictors of 24-h energy metabolism in reduced-obese persons.

BACKGROUND: Little is known about the effects of weight loss on the relation between skeletal muscle enzymes and energy metabolism. OBJECTIVE: This study was performed retrospectively to investigate the relation between skeletal muscle enzymes and 24-h energy metabolism in obese persons before and after weight loss. DESIGN: Ten women and 9 men [with body mass indexes (in kg/m(2)) > 30] underwent a 15-wk weight-loss program (-700 kcal/d). Body weight and composition, 24-h energy metabolism (whole-body indirect calorimetry), and maximal activities of phosphofructokinase (EC 2.7.1.11), creatine kinase (CK; EC 2.7.3.2), citrate synthase (CS; EC 4.1.3.7), 3-hydroxyacyl-CoA dehydrogenase (HADH; EC 1.1.1.35), and cytochrome-c oxidase (COX; EC 1.9.3.1) were determined from biopsy samples of the vastus lateralis taken before and after weight loss. RESULTS: Before weight loss, fat-free mass (FFM) was the only predictor of 24-h energy expenditure (R(2) = 0.70, P < 0.001), whereas the cumulative variance in sleeping metabolic rate explained by FFM and fat mass (FM) was 83% (P < 0.001). After weight loss, CS (r = 0.45, P = 0.05) and COX (r = 0.65, P < 0.01) were significantly associated with 24-h energy expenditure, whereas CK (r = 0.53, P < 0.05), CS (r = 0.45, P < 0.05), COX (r = 0.64, P < 0.01), and HADH (r = 0.45, P = 0.05) were all significant correlates of sleeping metabolic rate. After weight loss, FFM, FM, and COX explained 84% (P < 0.01) of the variance in 24-h energy expenditure, whereas FFM, FM, and CK all contributed to the cumulative variance in sleeping metabolic rate explained by this model (R(2) = 0.82, P < 0.05). CONCLUSION: Maximal activities of key skeletal muscle enzymes contribute to the variability in 24-h energy metabolism in reduced-obese persons.

Skeletal muscle changes in patients with obstructive sleep apnoea syndrome.

Previous studies have shown that chronic hypoxia leads to changes in skeletal muscle structure (fibre size and type) and activities of several bioenergetic enzymes. Whether this occurs also in conditions characterised by intermittent hypoxia, such as the obstructive sleep apnoea syndrome (OSAS), is unknown. To explore this possibility, we obtained a needle biopsy of the quadriceps femoris in 12 consecutive stable outpatients with severe OSAS (52 +/- 9 year, apnoea-hypopnoea index 70 +/- 14 h(-1)) (x +/- SD) and in six healthy volunteers (49 +/- 8 year), where we quantified fibre type, size and protein content, as well as phosphofructo-kinase (PFK) and cytochrome oxidase (CytOx) activities. We found that fibre-type distribution was similar in patients and controls. In contrast, the diameter of type II fibres (74 +/- 10 microm vs. 56 +/- 11 microm, P < 0.05) and protein content (100 +/- 14 vs. 88 +/- 8 microg/mg) was higher in patients with OSAS. Likewise, we observed upregulation of CytOx (0.93 +/- 0.38 vs. 0.40 +/- 0.22 microkat/mg protein, P < 0.01) and PFK activities (5.35 +/- 4.8 vs. 1.3 +/- 1.3 microkat/ mg protein, P < 0.05) in patients with OSAS. These results show that, paralleling which occurs in conditions characterised by continuous hypoxia, patients with OSAS (and intermittent hypoxia) also show structural and bioenergetic changes in their skeletal muscle.

Quantification of allosteric influence of Escherichia coli phosphofructokinase by frequency domain fluorescence.

The allosteric properties of the wild-type Escherichia coli phosphofructokinase were compared to the E187A mutant by using frequency-domain techniques. Tryptophan-shifted mutants comprising of double (W311Y/Y55W and W/311F/F188W) and triple (W311Y/Y55W/E187A and W311F/F188W/E187A) amino acid residue changes, which allowed for better fluorescence probing at targeted sites, were also compared to the wild-type and E187A. The additive nature of multiple mutations allowed one to partition the net effect of modifying residue 187. In general, the mutant enzymes displayed greater heterogeneity in sub-state population than did the wild-type enzyme. The semi-cone angle model was used to quantify the extent of depolarization of the fluorophore. Use of the model presupposes that the extent of depolarization directly correlates with the degree of flexibility of the fluorophore. A relationship has been established between the values determined from the semi-cone angle calculations and the thermodynamic components responsible for the allosteric linkage between the regulatory and substrate binding. Coupling interactions giving rise to positive entropy components are manifested by increasing flexibility of the ternary complexes rather than the binary complexes.

A method for activity staining after native polyacrylamide gel electrophoresis using a coupled enzyme assay and fluorescence detection: application to the analysis of several glycolytic enzymes.

We describe a method for the detection of isoforms of several glycolytic enzymes by activity staining after native PAGE. The staining is based on coupled enzyme assays carried out on the gel after electrophoresis and is linked to the disappearance of NADH, which is visualized by fluorescence. This method offers reliable and sensitive detection for phosphoenolpyruvate carboxylase, PPi-dependent phosphofructokinase, and pyruvate kinase from plant tissues. It can be applied to the detection of all enzymes which are normally detected spectrophotometrically using coupled enzyme assays consuming NAD(P)H.