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Biomedical subjects

A Manzano

Publications and source records attributed to A Manzano.

18 recordsLinked to original sources
PubMed2006

PFKFB3 gene silencing decreases glycolysis, induces cell-cycle delay and inhibits anchorage-independent growth in HeLa cells.

The high rate of glycolysis despite the presence of oxygen in tumor cells (Warburg effect) suggests an important role for this process in cell division. The glycolytic rate is dependent on the cellular concentration of fructose 2,6-bisphosphate (Fru-2,6-P2), which, in turn, is controlled by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2). The ubiquitous PFK-2 isoenzyme (uPFK-2, alternatively named UBI2K5 or ACG) coded by the pfkfb3 gene is induced by different stimuli (serum, progesterone, insulin, hypoxia, etc.) and has the highest kinase/phosphatase activity ratio amongst all PFK-2 isoenzymes discovered to date, which is consistent with its role as a powerful activator of glycolysis. uPFK-2 is expressed in brain, placenta, transformed cells and proliferating cells. In the present work, we analyze the impact of small interfering RNA (siRNA)-induced silencing of uPFK-2 on the inhibition of cell proliferation. HeLa cells treated with uPFK-2 siRNA showed a decrease in uPFK-2 RNA levels measured at 24h. uPFK-2 protein levels were severely depleted at 48-72h when compared with cells treated with an unrelated siRNA, correlating with decreased glycolytic activity, Fru-2,6-P2, lactate and ATP concentrations. These metabolic changes led to reduced viability, cell-cycle delay and an increase in the population of apoptotic cells. Moreover, uPFK-2 suppression inhibited anchorage-independent growth. The results obtained highlight the importance of uPFK-2 on the regulation of glycolysis, on cell viability and proliferation and also on anchorage-independent growth. These data underscore the potential for uPFK-2 as an effective tumor therapeutic target.

Cell Cycle↗
PubMed2001

The human ubiquitous 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene (PFKFB3): promoter characterization and genomic structure.

A DNA fragment containing 1.5 kb of the 5'-flanking region of the human ubiquitous PFKFB3 gene, coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, was cloned and its promoter activity was examined. The 5' flanking region contains a TATA box-like and GC-rich sequences, yielding several potential Specific protein (Sp-1) and activator protein (AP)-2 binding sites. Putative regulatory motifs for E-box, nuclear factor (NF)-1 and progesterone response element were also found by computer assisted analysis. Transient expression assays of truncated promoter-reporter constructs in HeLa cells showed that this gene is induced by phorbol esters (PDB) and cyclic-AMP-dependent protein kinase signal activation. Furthermore, the genomic organization of the PFKFB3 gene is reported. This gene spans more than 26 kb containing at least 16 exons that accounts for the two reported isoforms, inducible and ubiquitous, generated through alternative splicing of exon 15.

1-Methyl-3-isobutylxanthine↗
PubMed2001

PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2,6-bisphosphate.

Fructose-2,6-bisphosphate is responsible for mediating glucagon-stimulated gluconeogenesis in the liver. This discovery has led to the realization that this compound plays a significant role in directing carbohydrate fluxes in all eukaryotes. Biophysical studies of the enzyme that both synthesizes and degrades this biofactor have yielded insight into its molecular enzymology. Moreover, the metabolic role of fructose-2,6-bisphosphate has great potential in the treatment of diabetes.

Animals↗
PubMed2000

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase expression in rat brain during development.

This study reports the expression of the ubiquitous 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene (PFKFB3) (PFK-2/FBPase-2) in different stages of rat brain development. Northern blot and RT-PCR analysis demonstrated that ubiquitous PFK-2/FBPase-2 is expressed in rat brain from embryonic to adult life and shows a transient increase 1 day before birth, coincident with the maximum concentration of Fru-2,6-P(2) and PFK-2 activity. The levels of brain PFK-2/FBPase-2 gene expression as well as the enzymatic activity and the concentration of Fru-2,6-P(2) appear to be remarkably constant during adult life, without significant differences in the brain hippocampus, cortex, cerebellum or striatum areas.

Aging↗
PubMed2000

Overexpression of fructose 2,6-bisphosphatase decreases glycolysis and delays cell cycle progression.

The ability to overexpress 6-phosphofructo-2-kinase/fructose 2, 6-bisphosphatase (PFK-2)/(FBPase-2) or a truncated form of the enzyme with only the bisphosphatase domain allowed us to analyze the relative role of the kinase and the bisphosphatase activities in regulating fructose 2,6-bisphosphate (Fru-2,6-P(2)) concentration and to elucidate their differential metabolic impact in epithelial Mv1Lu cells. The effect of overexpressing PFK-2/FBPase-2 resulted in a small increase in the kinase activity and in the activity ratio of the bifunctional enzyme, increasing Fru-2,6-P(2) levels, but these changes had no major effects on cell metabolism. In contrast, expression of the bisphosphatase domain increased the bisphosphatase activity, producing a significant decrease in Fru-2,6-P(2) concentration. The fall in the bisphosphorylated metabolite correlated with a decrease in lactate production and ATP concentration, as well as a delay in cell cycle. These results provide support for Fru-2,6-P(2) as a regulator of glycolytic flux and point out the role of glycolysis in cell cycle progression.

Adenosine Triphosphate↗
PubMed2000

Hepatic preconditioning preserves energy metabolism during sustained ischemia.

We evaluated the possibility that ischemic preconditioning could modify hepatic energy metabolism during ischemia. Accordingly, high-energy nucleotides and their degradation products, glycogen and glycolytic intermediates and regulatory metabolites, were compared between preconditioned and nonpreconditioned livers. Preconditioning preserved to a greater extent ATP, adenine nucleotide pool, and adenylate energy charge; the accumulation of adenine nucleosides and bases was much lower in preconditioned livers, thus reflecting slower adenine nucleotide degradation. These effects were associated with a decrease in glycogen depletion and reduced accumulation of hexose 6-phosphates and lactate. 6-Phosphofructo-2-kinase decreased in both groups, reducing the availability of fructose-2, 6-bisphosphate. Preconditioning sustained metabolite concentration at higher levels although this was not correlated with an increased glycolytic rate, suggesting that adenine nucleotides and cAMP may play the main role in the modulation of glycolytic pathway. Preconditioning attenuated the rise in cAMP and limited the accumulation of hexose 6-phosphates and lactate, probably by reducing glycogen depletion. Our results suggest the induction of metabolic arrest and/or associated metabolic downregulation as energetic cost-saving mechanisms that could be induced by preconditioning.

Adenosine Diphosphate↗
PubMed1999

Cloning, expression and chromosomal localization of a human testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene.

6-Phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2/FBPase-2) is a bifunctional enzyme responsible for the synthesis and breakdown of Fru-2,6-P2, a key metabolite in the regulation of glycolysis. Several genes encode distinct PFK-2/FBPase-2 isozymes that differ in their tissue distribution and enzyme regulation. In this paper, we present the isolation of a cDNA from a human testis cDNA library that encodes a PFK-2/FBPase-2 isozyme. Sequencing data show an open reading frame of 1407 nucleotides that codifies for a protein of 469 amino acids. This has a calculated molecular weight of 54kDa and 97% similarity with rat testis PFK-2/FBPase-2, with complete conservation of the amino acid residues involved in the catalytic mechanism. Fluorescence in-situ hybridization (FISH) localized testis PFK-2/FBPase-2 gene (PFKFB4) in human chromosome 3 at bands p21-p22. A Northern blot analysis of different rat tissues showed the presence of a 2.4-kb mRNA expressed specifically in testis. In mammalian COS-1 cells, the human testis cDNA drives expression of an isozyme with a molecular weight of 55kDa. This isozyme shows clear PFK-2 activity. Taken together, these results provide evidence for a new PFK-2/FBPase-2 gene coding for a human testis isozyme.

Amino Acid Sequence↗
PubMed1998

Effect of starvation on gene expression of regulatory enzymes of glycolysis/gluconeogenesis in genetically obese (fa/fa) Zucker rats.

OBJECTIVE: To study the mechanism that controls fructose-2,6-bisphosphate (Fru-2,6-P2) accumulation, as well as the mRNAs levels of the glycolytic/gluconeogenic regulatory enzymes in the livers of fed and starved lean (fa/-) and obese (fa/fa) Zucker rats. DESIGN: Rats were fed a standard chow or deprived of food for 24 h. SUBJECTS: Male lean (fa/-) and genetically obese (fa/fa) rats (nine weeks old). MEASUREMENTS: Fru-2,6-P2 concentration, 6-phosphofructo-2-kinase (PFK-2), glucokinase (GK), pyruvate kinase (PK) activities and the mRNA levels of GK, PFK-2, L-type pyruvate kinase, fructose-1,6-bisphosphatase (FBPase-1) and phosphoenolpyruvate carboxykinase (PEPCK) were analyzed. RESULTS: PFK-2/FBPase-2 mRNA decreased during starvation in both fa/- and fa/fa animals. Although PFK-2/FBPase-2 mRNA levels were similar in fed lean and obese rats, PFK-2 concentration and activity were higher in fed obese than in fed lean animals, which might explain the high concentration of Fru-2,6-P2 observed in obese animals. During starvation, PFK-2 protein concentration decreased, correlating with the enzymatic activity and Fru-2,6-P2 levels. The activities of GK and L-pyruvate kinase (L-PK) also increased in fed obese (fa/fa) rats compared with fed lean (fa/-) animals, but decreased during starvation. The mRNA levels of glycolytic enzymes in fed obese rats were similar (PFK-2) or higher than (GK, L-PK) in fed lean animals. During starvation, they decreased in lean and obese rats with one important exception, GK mRNA remained high in obese animals. The mRNA of gluconeogenic enzymes remained constant (FBPase-1) or increased (PEPCK) during fasting. CONCLUSION: The changes observed might be explained by the hyperinsulinaemia observed in the liver of obese rats, which might lead to the stimulation of glycolysis and lipogenesis.

Animals↗
PubMed1998

Molecular cloning, expression, and chromosomal localization of a ubiquitously expressed human 6-phosphofructo-2-kinase/ fructose-2, 6-bisphosphatase gene (PFKFB3).

We report the identification of a human 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase gene (PFKFB3) isolated from a human fetal brain cDNA library. The gene was localized to 10p15-->p14 by fluorescence in situ hybridization. The entire cDNA (4,322 bp) codes for a polypeptide of 520 amino acid residues (molecular weight, 59.571 kDa). Structural analysis showed the presence of a kinase domain located at the amino terminus and a bisphosphatase domain at the carboxy terminus, characteristic of previously described 6-phosphofructo-2-kinase/fructose 2, 6-bisphosphatase isozymes. In addition, a phosphorylation site for cAMP-dependent protein kinase was found at the carboxy terminus. Northern blot analysis showed the presence of a unique 4.8-kb mRNA expressed in the different tissues studied. In mammalian COS-1 cells, this cDNA drives the expression of an active isozyme. Taken together, these results identify the presence of a gene coding for a human 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase isozyme which is ubiquitously expressed.

Animals↗
PubMed1997

[Evaluation of hospital emergency service attendance by patients from a basic health district].

OBJECTIVES: To find the sociodemographic characteristics of patients registered at a Health Centre (HC) who attended a hospital Casualty Department, their reasons for attendance, features of their use of facilities and how justified their attendance was. DESIGN: Descriptive study. SETTING: Urban Health Centre, Llefià (Badalona), between February and June, 1995. PARTICIPANTS: We used a questionnaire composed by the research team to survey 360 patients over 14 who attended the HC with a report from a Casualty Department. MEASUREMENTS AND RESULTS: The variables recorded were: sociodemographic characteristics, reasons for attending Casualty, whether they attended on their own initiative, whether they knew about non-hospital Emergencies, the hospital, day and time, chronic pathology, diagnosis and destination on discharge from Casualty, length of stay and justification. 53.7% were women. 57.8% were married. 95% lived with a partner or intimate family member, 56.9% had completed primary education and 39.4% had jobs. 41% suffered a chronic pathology. Monday was the busiest day and physical trauma was the most frequent diagnosis on discharge. We considered 54.7% of all the consultations non-justified emergencies. CONCLUSIONS: There is a high percentage of users who attend Casualty on their own initiative. Most of the emergencies are unjustified.

Adolescent↗
PubMed1997

Serum-liposome interaction is an oxygen-dependent process.

Measurements of heat dissipation, oxygen concentration and average vesicle size were correlated to study the effect of serum components on different types of liposome. The results indicate that the interaction between serum components and liposomes is exothermic and oxygen dependent, and leads to disruption of vesicles. The dependence of this effect on serum concentration, vesicle surface charge and type of liposome was also evaluated. Serum components did not produce any effect on conventional liposomes in the absence of oxygen. Moreover, in hypoxic conditions the serum-liposome interaction was delayed. Both results suggest that this interaction is an oxygen-dependent event. Finally, we confirmed that sterically stabilised liposomes remain unalterated in the presence of serum.

Animals↗
PubMed1996

Effects of taxol on isolated rat hepatocyte metabolism.

Taxol is a natural product, isolated from Taxus brevifolia, with increasing clinical applications because of its potent antitumor activity. Although it is mainly metabolized in the liver, its effect on hepatocyte metabolism has been scarcely investigated. In this study, the response of isolated rat hepatocytes to taxol was evaluated by correlating the changes observed in global indexes such as viability and heat dissipation with those produced in oxygen consumption and intracellular metabolites (ATP, fructose 2,6-bisphosphate, and lactate). The results indicate that taxol reduces aerobic metabolism, which induces an insufficient increase of the ATP via anaerobic glycolysis. Moreover, incubations of isolated mitochondria with taxol indicate that the respiratory chain is directly affected by this drug.

Animals↗
PubMed1995

[Difficulties in carrying out preventive activities in primary care].

OBJECTIVE: To find the difficulties perceived by Primary Care health professionals in integrating preventive activities into their daily practice. DESIGN: A descriptive study using a survey. SETTING: Barcelonès Nord and Maresme health region. PARTICIPANTS: Health professionals from primary care teams. RESULTS: The overall coverage of the survey was 47.7%. Pressure from patients or lack of time obtained the highest score (6.2), followed by the absence of a procedure or programme (4.5), absence of economic incentives (4.4) and the lack of material resources (4.2). The lowest scores corresponded to training (3.4) and motivation (2.9). CONCLUSIONS: Primary Care occupies a strategic position for carrying out preventive activities and health promotion. But in practice health professionals encounter various obstacles. Lack of time and forgetting preventive activities are basic factors. The registration sheets or other specific devices to act as reminders, together with procedures or programmes involving the joint participation of all the team members, can help resolve these problems. There is a clear need to concentrate on those problems which the professionals directly involved are most concerned with.

Adult↗