HCV E2蛋白诱导的体液免疫及CTL应答研究

应用PCR方法扩增出HCVE2基因编码417a．a-750a．a的DNA片段,克隆到原核表达载体pQE30 LacZ启动子下游,转化JM109菌株。在JM109菌株中诱导表达出N端含6个组氨酸的E2融合蛋白,用Ni-NTA-Superflow亲和层析柱纯化作为抗原免疫实验兔和BALB／c鼠。定期取兔血,采用间接ELISA方法检测兔子体内针对E2的抗体水平和维持规律。结果显示,距初次免疫14d兔子体内已有抗体产生,直至免疫第55d抗体水平持续上升,之后抗体水平保持稳定,抗体滴度达到1：3200。六周后,取鼠脾脏制各淋巴细胞,定向刺激扩增后与经过重组真核表达质粒pCE2转染的P815细胞作用,利用LDH释放试验检测作用效果。在E：T＝200：1的情况下,杀伤率超过30％。这些结果表明工程菌株表达的HCVE2蛋白具有良好的免疫原性,可以诱发免疫实验动物机体产生较高滴度的抗体及特异性CTL应答。由此我们认为E2蛋白是发展HCV预防工程蛋白疫苗的合适候选者。     

Antioxidant and anti‐inflammatory effects of the monocarbonyl curcumin analogs B2BRBC and C66 in monocrotaline‐induced right ventricular hypertrophy

For 22 days after monocrotaline injection two groups of rats received either of the monocarbonyl curcumin analogs (2E,6E)‐2,6‐bis(2‐bromobenzylidene)cycloxehanone (B2BrBC) and (2E,6E)‐2,6‐bis([2‐tri?uoromethyl]benzylidene)cyclohexanone (C66), and their right ventricle parameters were compared to those from the control and the monocrotaline injected animals. B2BrBC and C66 treatments did not prevent the monocrotaline‐induced right ventricular hypertrophy but attenuated the changes in antioxidant enzyme activities and reduced inflammation. The level of thiol‐based nonenzymatic antioxidants did not change in the function of monocrotaline or curcumin analogs treatment. However, due to its stronger antioxidant properties, only B2BrBC treatment was effective in the reduction of monocrotaline‐associated lipid peroxidation. The obtained results suggest that increasing the levels of antioxidant enzymes may not be sufficient to reduce oxidative stress and chronic inflammation optimally and our current study supports the potential of compounds with more than one beneficial biological activity as a promising treatment against the progression of cardiac hypertrophy.     

Enzymatic preparation of high-specific-activity β-d-[6,6′-H]fructose-2,6-bisphosphate: Application to a sensitive assay for fructose-2,6-bisphosphatase

β-d-Fructose-2,6-bisphosphate (Fru-2,6-P₂) is an important regulator of eukaryotic glucose homeostasis, functioning as a potent activator of 6-phosphofructo-1-kinase and inhibitor of fructose-1,6-bisphosphatase. Pharmaceutical manipulation of intracellular Fru-2,6-P₂ levels, therefore, is of interest for the treatment of certain diseases, including diabetes and cancer. [2-³²P]Fru-2,6-P₂ has been the reagent of choice for studying the metabolism of this effector molecule; however, its short half-life necessitates frequent preparation. Here we describe a convenient, economical, one-pot enzymatic preparation of high-specific-activity tritium-labeled Fru-2,6-P₂. The preparation involves conversion of readily available, carrier-free d-[6,6′-³H]glucose to [6,6′-³H]Fru-2,6-P₂ using hexokinase, glucose-6-phosphate isomerase, and 6-phosphofructo-2-kinase. The key reagent in this preparation, bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from human liver, was produced recombinantly in Escherichia coli and purified in a single step using an appendant C-terminal hexa-His affinity tag. Following purification by anion exchange chromatography using triethylammonium bicarbonate as eluant, radiochemically pure [6,6′-³H]Fru-2,6-P₂ having a specific activity of 50 Ci/mmol was obtained in yields averaging 35%. [6,6′-³H]Fru-2,6-P₂ serves as a stable, high-specific-activity substrate in a facile assay capable of detecting fructose-2,6-bisphosphatase in the range of 10⁻¹⁴ to 10⁻¹⁵ mol, and it should prove to be useful in many studies of the metabolism of this important biofactor.     

Resolution and characterization of multiple cytosolic phosphatases capable of hydrolyzing fructose-1,6-bisphosphate in spinach and soybean leaves

The apparent activity of cytoplasmic fructose bisphosphatase (EC 3.1.3.11) in crude extracts of spinach ( Spinacia oleracea L.) and soybean ( Glycine max [L.] Merr.) leaves was only partially dependent on Mg²⁺. At least two major non-chloroplastic fructose bisphosphatases that differed in dependence on Mg²⁺ were chromatographically resolved from spinach leaves. The Mg²⁺-dependent enzyme had an apparent Michaelis constant of 4 μM for fructose-1,6-P₂, was highly specific, and was strongly inhibited by fructose-2,6-P₂. Enzyme activity was inhibited by physiological levels of fructose-6-P.
Both species also contained at least one major enzyme, the activity of which was independent of Mg²⁺. These enzymes had pH optima near neutrality, Michaelis constants of 25 to 30 μM for fructose-1,6-P₂, and were inhibited by AMP. Although hexose monophosphates were not metabolized, the enzymes were not specific for fructose-1,6-P₂: phosphate was released from phosphoenolpyruvate and ribulose-1, 5-P₂, and with fructose-1,6-P₂, as substrate, Pi release was about 1.5-fold greater than fructose-6-P production. It is concluded that only the Mg²⁺-dependent fructose bisphosphatase, previously characterized, functions in the photosynthetic sucrose formation pathway. Inhibition of the Mg²⁺-dependent enzyme by fructose-6-P may be involved in regulation of sucrose formation.     

Fructose 2, 6-Bisphosphate in Hypoglycemic Rat Brain

Abstract: Fructose 2,6-bisphosphate has been studied during hypoglycemia induced by insulin administration (40 IU/kg). No changes in content of cerebral fructose 2,6-bisphosphate were found in mild hypoglycemia, but the level of this compound was markedly decreased in hypoglycemic coma and recovered after 30 min of glucose administration. To correlate a possible modification of the concentration of the metabolite with selective regional damage occurring during hypoglycemic coma, we have analyzed four cerebral areas (cortex, striatum, cerebellum, and hippocampus). Fructose 2,6-bisphosphate concentrations were similar in the four areas analyzed; severe hypoglycemia decreased levels of the metabolite to the same extent in all the brain areas studied. The decrease in content of fructose 2,6-bisphosphate was not always accompanied by a parallel decrease in ATP levels, a result suggesting that the low levels of the bisphosphorylated metabolite during hypoglycemic coma could be due to the decreased 6-phosphofructo-2-kinase activity, mainly as a consequence of the fall in concentration of its substrate (fructose 6-phosphate). These results suggest that fructose 2,6-bisphosphate could play a permissive role in cerebral tissue, maintaining activation of 6-phosphofructo-l-kinase and glycolysis.     

Rat liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase: a review of relationships between the two activities of the enzyme

Both the synthesis and the degradation of Fru-2,6-P2 are catalyzed by a single enzyme protein; ie, the enzyme is bifunctional. This protein, which we have designated 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase is an important enzyme in the regulation of hepatic carbohydrate metabolism since its activity determines the steady-state concentration of fructose 2,6-P2, an activator of 6-phosphofructo 1-kinase and an inhibitor of fructose 1,6-bisphosphatase. Regulation of the bifunctional enzyme in intact cells is a complex function of both covalent modification via phosphorylation/dephosphorylation and the influence of substrates and low molecular weight effectors. Recent evidence suggests that both reactions may proceed by two-step transfer mechanisms with different phosphoenzyme intermediates. The enzyme catalyzes exchange reactions between ADP and ATP and between fructose 6-P and fructose 2,6-P2. A labeled phosphoenzyme is formed rapidly during incubation with [2-32P]Fru-2,6-P2. The labeled residue has been identified as 3-phosphohistidine. However, it was not possible to demonstrate significant labeling of the enzyme directly from [gamma-32P]ATP. These results can be most readily explained in terms of two catalytic sites, a kinase site whose phosphorylation by ATP is negligible (or whose E-P is labile) and a fructose 2,6-bisphosphatase site which is readily phosphorylated by fructose 2,6-P2. Additional evidence in support of two active sites include: limited proteolysis with thermolysin results in loss of 6-phosphofructo 2-kinase activity and activation of fructose 2,6-bisphosphatase, mixed function oxidation results in inactivation of the 6-phosphofructo 2-kinase but no affect on the fructose 2,6-bisphosphatase, N-ethylmaleimide treatment also inactivates the kinase but does not affect the bisphosphatase, and p-chloromercuribenzoate immediately inactivates the fructose 2,6-bisphosphatase but not the 6-phosphofructo 2-kinase. Our findings indicate that the bifunctional enzyme is a rather complicated enzyme; a dimer, probably with two catalytic sites reacting with sugar phosphate, and with an unknown number of regulatory sites for most of its substrates and products. Three enzymes from Escherichia coli, isocitric dehydrogenase kinase/phosphatase, glutamine-synthetase adenylyltransferase, and the uridylyltransferase for the regulatory protein PII in the glutamine synthetase cascade system also catalyze opposing reactions probably at two discrete sites. All four enzymes are important in the regulation of metabolism and may represent a distinct class of regulatory enzymes.     

Studies on the stimulation of gluconeogenesis and lipolysis by glucagon and epinephrine in isolated rat Kupffer cells.

Kupffer cells were isolated by collagenease-pronase treatment. Activity and leakage of GOT, GPT, LDH, GlDH and of nucleotide pyrophosphatase were measured and compared to parenchymal cells. In addition, the effects of glucagon and epinephrine on gluconeogenesis and lipolysis were studied. Both glucagon and epinephrine stimulated gluconeogenesis from lactate and alanine. The epinephrine response, however, was far greater than that of glucagon. Additional studies showed a 50% stimulation of lipolysis by epinephrine with triolein and tripalmitin as substrates. No stimulation of lipolysis was observed with glucagon.     

Hormonal control of fructose 2,6-bisphosphate concentration and of phosphofructokinase 2 in the rat liver during development

In fetal rat liver the concentration of fructose 2,6-bisphosphate is decreased by administration of glucagon. The glucagon effect, i.e., the phosphorylation state of phosphofructokinase 2, dominates over the substrate supply. Insulin was found to increase fructose 2,6-bisphosphate only when exogenous glucose is supplied simultaneously. The total activity of phosphofructokinase 2 exhibits remarkable developmental changes. It is high at term, moderate in the fetal as well as in the mature organ, and low during suckling. The level of the enzyme during development is controlled by pancreatic and adrenal hormones.     

The kinase activity of human brain 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is regulated via inhibition by phosphoenolpyruvate

The two enzymatic activities of the highly conserved catalytic core of 6PF2K/Fru-2,6-P₂ase are thought to be reciprocally regulated by the amino- and carboxy-terminal regions unique to each isoform. In this study, we describe the recombinant expression, purification, and kinetic characterization of two human brain 6PF2K/Fru-2,6-P₂ase splice variants, HBP1 and HBP2. Interestingly, both lack an arginine which is highly conserved among other tissue isoforms, and which is understood to be critical to the fructose-2,6-bisphosphatase mechanism. As a result, the phosphatase activity of both HBP isoforms is negligible, but we found that it could be recovered by restoration of the arginine by site directed mutagenesis. We also found that AMP activated protein kinase and protein kinases A, B, and C catalyzed the phosphorylation of Ser-460 of HBP1, and that in addition both isoforms are phosphorylated at a second, as yet undetermined site by protein kinase C. However, none of the phosphorylations had any effect on the intrinsic kinetic characteristics of either enzymatic activity, and neither did point mutation (mimicking phosphorylation), deletion, and alternative-splice modification of the HBP1 carboxy-terminal region. Instead, these phosphorylations and mutations decreased the sensitivity of the 6PF2K to a potent allosteric inhibitor, phosphoenolpyruvate, which appears to be the major regulatory mechanism.     

Pyrophosphate functions as phosphoryl donor with UDP-glucose-treated mammalian phosphofructokinase

CGP 28392, a novel compound structurally related to the dihydropyridine Ca2+-entry blockers, causes a dose-dependent increase in intracellular free Ca2+ in human platelets, as measured with the Quin-2 Ca2+ indicator, with a semimaximal effective concentration of 2.2 X 10(-7) M. This effect occurs in a concentration range in which CGP 28392 competes for specific [3H]nitrendipine binding in guinea pig heart membranes. It can be inhibited by nitrendipine. The data presented furnish direct evidence of the Ca2+-entry-stimulating properties of CGP 28392 and indicate the presence of dihydropyridine-susceptible structures in human platelets.     

Subunit interactions in pig-kidney fructose-1,6-bisphosphatase: Binding of substrate induces a second class of site with lowered affinity and catalytic activity

Background

Fructose-1,6-bisphosphatase, a major enzyme of gluconeogenesis, is inhibited by AMP, Fru-2,6-P₂ and by high concentrations of its substrate Fru-1,6-P₂. The mechanism that produces substrate inhibition continues to be obscure.

Methods

Four types of experiments were used to shed light on this: (1) kinetic measurements over a very wide range of substrate concentrations, subjected to detailed statistical analysis; (2) fluorescence studies of mutants in which phenylalanine residues were replaced by tryptophan; (3) effect of Fru-2,6-P₂ and Fru-1,6-P₂ on the exchange of subunits between wild-type and Glu-tagged oligomers; and (4) kinetic studies of hybrid forms of the enzyme containing subunits mutated at the active site residue tyrosine-244.

Results

The kinetic experiments with the wild-type enzyme indicate that the binding of Fru-1,6-P₂ induces the appearance of catalytic sites with lower affinity for substrate and lower catalytic activity. Binding of substrate to the high-affinity sites, but not to the low-affinity sites, enhances the fluorescence emission of the Phe219Trp mutant; the inhibitor, Fru-2,6-P₂, competes with the substrate for the high-affinity sites. Binding of substrate to the low-affinity sites acts as a “stapler” that prevents dissociation of the tetramer and hence exchange of subunits, and results in substrate inhibition.

Conclusions

Binding of the first substrate molecule, in one dimer of the enzyme, produces a conformational change at the other dimer, reducing the substrate affinity and catalytic activity of its subunits.

General significance

Mimics of the substrate inhibition of fructose-1,6-bisphosphatase may provide a future option for combatting both postprandial and fasting hyperglycemia.     

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog skeletal muscle: purification,kinetics and immunological properties

Fructose 2,6-bisphosphate is the most potent activator of 6-phosphofructo-1-kinase, a key regulatory enzyme of glycolysis in animal tissues. This study was prompted by the finding that the content of fructose 2,6-bisphosphate in frog skeletal muscle was dramatically increased at the initiation of exercise and was closely correlated with the glycolytic flux during exercise. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, the enzyme system catalyzing the synthesis and degradation of fructose 2,6-bisphosphate, was purified from frog (Rana esculenta) skeletal muscle and its properties were compared with those of the rat muscle type enzyme expressed in Escherichia coli using recombinant DNA techniques. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was purified 5600-fold. 6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities could not be separated, indicating that the frog muscle enzyme is bifunctional. The enzyme preparation from frog muscle showed two bands on sodium dodecylsulphate polyacrylamide gel electrophoresis. The minor band had a relative molecular mass of 55800 and was identified as a liver (L-type) isoenzyme. It was recognized by an antiserum raised against a specific amino-terminal amino acid sequence of the L-type isoenzyme and was phosphorylated by the cyclic AMP-dependent protein kinase. The major band in the preparations from frog muscle (relative molecular mass = 53900) was slightly larger than the recombinant rat muscle (M-type) isoenzyme (relative molecular mass = 53300). The pH profiles of the frog muscle enzyme were similar to those of the rat M-type isoenzyme, 6-phosphofructo-2-kinase activity was optimal at pH 9.3, whereas fructose-2,6-bisphosphatase activity was optimal at pH 5.5. However, the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle differed from other M-type isoenzymes in that, at physiological pH, the maximum activity of 6-phosphofructo-2-kinase exceeded that of fructose-2,6-bisphosphatase, the activity ratio being 1.7 (at pH 7.2) compared to 0.2 in the rat M-type isoenzyme. 6-Phosphofructo-2-kinase activity from the frog and rat muscle enzymes was strongly inhibited by citrate and by phosphoenolpyruvate whereas glycerol 3-phosphate had no effect. Fructose-2,6-bisphosphatase activity from frog muscle was very sensitive to the non-competitive inhibitor fructose 6-phosphate (inhibitor concentration causing 50% decrease in activity = 2 mol · l^-1). The inhibition was counteracted by inorganic phosphate and, particularly, by glycerol 3-phosphate. In the presence of inorganic phosphate and glycerol 3-phosphate the frog muscle fructose-2,6-bisphosphatase was much more sensitive to fructose 6-phosphate inhibition than was the rat M-type fructose-2,6-bisphosphatase. No change in kinetics and no phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was observed after incubation with protein kinase C and a Ca²⁺/calmodulin-dependent protein kinase. The kinetics of frog muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, although they would favour an initial increase in fructose 2,6-bisphosphate in exercising frog muscle, cannot fully account for the changes in fructose 2,6-bisphosphate observed in muscle of exercising frog. Regulatory mechanisms not yet studied must be involved in working frog muscle in vivo.Abbreviations BSA bovine serum albumin - Ca/CAMK Ca²⁺/calmodulin-dependent protein kinase (EC 2.7.1.37) - CL anti-l-type PFK-21 FBPase-2 antiserum - DTT dithiothreitol - EP phosphorylated enzyme intermediate - FBPase-2 fructose-2,6-bisphosphatase (EC 3.1.3.46) - F2,6P₂ fructose 2,6-bisphosphate - I_0,5 inhibitor concentration required to decrease enzyme activity by 50% - MCL-2 anti-PFK-2/FBPase-2 antiserum - M_r relative molecular mass - PEG polyethylene glycol - PFK-1 6-phosphofructo-1-kinase (EC 2.7.1.11) - PKF-2 6-phosphofructo-2-kinase (EC 2.7.1.105) - PKA protein kinase A = cyclic AMP-dependent protein kinase (EC 2.7.1.37) - PKC protein kinase C (EC 2.7.1.37) - SDS sodium dodecylsulphate - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - U unit of enzyme activity     

剪接因子异质核糖核蛋白A2B1在癌细胞和衰老细胞中的相反表达趋势及其对癌细胞衰老的调控

剪接因子异质核糖核蛋白A2/B1(HNRNPA2B1)与人类及小鼠的寿命相关,并在多个癌症的病程进展中发挥重要的作用.然而,HNRNPA2B1能否在细胞衰老这一与个体衰老和抑制癌症密切相关的生物学过程中发挥作用尚不清楚.本研究发现,HNRNPA2B1在多个癌症体系中呈显著上调表达趋势,而在多个细胞衰老体系中则呈显著下调...     

甲型H5N1流感病毒M2与HA双基因载体疫苗在小鼠体内的免疫学评价

高致病性H5N1亚型禽流感病毒 (AIV) 严重威胁到人类健康,因此研制高效、安全的禽流感疫苗具有重要意义。以我国分离的首株人H5N1亚型禽流感病毒 (A/Anhui/1/2005) 作为研究对象,PCR扩增基质蛋白2 (M2) 和血凝素 (HA) 基因全长开放阅读框片段,构建共表达H5N1亚型AIV膜蛋白基因 M2和HA的重组质粒pStar-M2/HA。此外,还通过同源重组以293细胞包装出表达M2基因的重组腺病毒Ad-M2以及表达HA基因的重组腺病毒Ad-HA。用间接免疫荧光 (IFA) 方法检测到了各载体上插入基因的表达。按初免-加强程序分别用重组质粒pStar-M2/HA和重组腺病毒Ad-HA+Ad-M2免疫BALB/c小鼠,共免疫4次,每次间隔14 d。第1、3次用DNA疫苗,第2、4次用重组腺病毒载体疫苗,每次免疫前及末次免疫后14 d采集血清用于检测体液免疫应答,末次免疫后14 d采集脾淋巴细胞用于检测细胞免疫应答。血凝抑制 (HI) 实验检测到免疫后小鼠血清中的HI活性。ELISA实验检测到免疫后小鼠血清中抗H5N1亚型流感病毒表面蛋白的IgG抗体。ELISPOT实验检测到免疫后小鼠针对M2蛋白和HA蛋白的特异性细胞免疫应答。流感病毒M2与HA双基因共免疫的研究,为研究开发新型重组流感疫苗奠定了基础。     

E2F-1- and E2Ftr-mediated apoptosis: the role of DREAM and HRK

E2F-1-deleted mutant, 'truncated E2F' (E2Ftr, E2F-1[1-375]), lacking the carboxy-terminal transactivation domain, was shown to be more potent at inducing cancer cell apoptosis than wild-type E2F-1 (wtE2F-1; full-length E2F-1). Mechanisms by which wtE2F-1 and E2Ftr induce apoptosis, however, are not fully elucidated. Our study demonstrates molecular effects of pro-apoptotic BH3-only Bcl-2 family member Harakiri (Hrk) in wtE2F-1- and E2Ftr-induced melanoma cell apoptosis. We found that Hrk mRNA and Harakiri (HRK) protein expression was highly up-regulated in melanoma cells in response to wtE2F-1 and E2Ftr overexpression. HRK up-regulation did not require the E2F-1 transactivation domain. In addition, Hrk gene up-regulation and HRK protein expression did not require p53 in cancer cells. Hrk knockdown by Hrk siRNA was associated with significantly reduced wtE2F-1- and E2Ftr-induced apoptosis. We also found that an upstream factor, 'downstream regulatory element antagonist modulator' (DREAM), may be involved in HRK-mediated apoptosis in response to wtE2F-1 and E2Ftr overexpression. DREAM expression levels increased following wtE2F-1 and E2Ftr overexpression. Western blotting detected increased DREAM primarily in dimeric form. The homodimerization of DREAM resulting from wtE2F-1 and E2Ftr overexpression may contribute to the decreased binding activity of DREAM to the 3'-untranslated region of the Hrk gene as shown by electromobility shift assay. Results showed wtE2F-1- and E2Ftr-induced apoptosis is partially mediated by HRK. HRK function is regulated in response to DREAM. Our findings contribute to understanding the mechanisms that regulate wtE2F-1- and E2Ftr-induced apoptosis and provide insights into the further evaluation of how E2Ftr-induced apoptosis may be used for therapeutic gain.     

Cloning and expression of novel isoforms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from bovine heart

Distinct 6-phosphofructo-2-kinase (PFK-2)/fructose 2,6-bisphosphatase (FBPase-2) cDNAs were cloned from bovine heart, showing that PFK-2/FBPase-2 gene B, which contains 16 exons, codes for at least five mRNAs. Three of them (B1, B2, B4) could encode the 58,000-M_r isozyme. In B2 mRNA, exon 15 encodes four more residues than in Bl. In B4 mRNA, exon 15 encodes six more residues than in B1, butexon 16 (20 residues) is missing. B3 mRNA corresponds to the 54,000-M_r isozyme. It lacks exon 15 and also differs from the other mRNAs in the 5' noncoding region. B5 mRNA encodes a truncated form. When expressed in E. coli, the recombinant isoforms corresponding to all these mRNAs except B5 exhibited PFK-2 activity.     

Hypoxic regulation of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene family (PFKFB-1-4) expression in vivo

When oxygen becomes limiting, cells shift primarily to a glycolytic mode for generation of energy. A key regulator of glycolytic flux is fructose-2,6-bisphosphate (F-2,6-BP), a potent allosteric regulator of 6-phosphofructo-1-kinase (PFK-1). The levels of F-2,6-BP are maintained by a family of bifunctional enzymes, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB or PFK-2), which have both kinase and phosphatase activities. Each member of the enzyme family is characterized by their phosphatase:kinase activity ratio (K:B) and their tissue-specific expression. Previous work demonstrated that one of the PFK-2 isozyme genes, PFKFB-3, was induced by hypoxia through the hypoxia-inducible factor-1 (HIF-1) pathway. In this study we examined the basal and hypoxic expression of three members of this family in different organs of mice. Our findings indicate that all four isozymes (PFKFB-1-4) are responsive to hypoxia in vivo. However, their basal level of expression and hypoxia responsiveness varies in the different organs studied. Particularly, PFKFB-1 is highly expressed in liver, heart and skeletal muscle, with the highest response to hypoxia found in the testis. PFKFB-2 is mainly expressed in the lungs, brain and heart. However, the highest hypoxia responses are found only in liver and testis. PFKFB-3 has a variable low basal level of expression in all organs, except skeletal muscle, where it is highly expressed. Most importantly, its hypoxia responsiveness is the most ample of all three genes, being strongly induced in the lungs, liver, kidney, brain, heart and testis. Further studies showed that PFKFB-1 and PFKFB-2 were highly responsive to hypoxia mimics such as transition metals, iron chelators and inhibitors of HIF hydroxylases, suggesting that the hypoxia responsiveness of these genes is also regulated by HIF proteins. In summary, our data demonstrate that PFK-2 genes are responsive to hypoxia in vivo, indicating a physiological role in the adaptation of the organism to environmental or localized hypoxia/ischemia.     

Switches in 6-phosphofructo-2-kinase isoenzyme expression during rat sperm maturation

Here we analyzed Pfkfb3 and Pfkfb4 gene expression in rat testis development, isolated testicular cells and spermatozoa. Real time RT-PCR analysis during testis development showed the maximum expression of Pfkfb3 in pre-puber samples and of Pfkfb4 in adult samples. Western blot analysis showed that uPFK-2 protein, a product of Pfkfb3 gene, was present in all the cell types forming the seminiferous epithelium (Sertoli, interstitial and spermatogenic cells). In contrast, tPFK-2, a product of Pfkfb4 gene, was restricted to spermatogenic cells. Confocal analyses by indirect immunofluorescence also corroborated this expression pattern. Immunoblotting studies of isolated spermatozoa demonstrated the presence of uPFK-2 only in immature sperm and once spermatozoa became fully functional this isozyme was replaced by the testicular isozyme tPFK-2. Moreover, immunostaining confirmed that tPFK-2 was localized mainly in the acrosomal region of the sperm head and in the mid-piece of the flagellum, where other spermatogenic cell-specific glycolytic enzymes have been found.     

Effects of vanadate on 6-phosphofructo 2-kinase activity and fructose 2,6-bisphosphate levels in cultured rat hepatocytes

The presence of vanadate in primary cultures of rat hepatocytes produced a significant increase in the concentration of fructose 2,6-bisphosphate and in the activity of 6-phosphofructo 2-kinase. Compared with insulin, vanadate had a more potent action on the metabolite increase, but a similar effect on the 6-phosphofructo 2-kinase activity. Both the insulin- and the vanadate-dependent enhancements of 6-phosphofructo 2-kinase were inhibited by cycloheximide which specifically blocks protein synthesis on the translational level, suggesting that the increase of the enzyme activity was due to induction rather than to a change in the catalytic activity.