Detailed Morphologic and Immunohistochemical Characterization of Myomectomy and Hysterectomy Specimens From Women With Hereditary Leiomyomatosis and Renal Cell Carcinoma Syndrome (HLRCC).

Hereditary leiomyomatosis and renal cell carcinoma syndrome (HLRCC), caused by a germline mutation in the fumarate hydratase (FH) gene, predisposes patients to uterine and cutaneous smooth muscle tumors and an aggressive type of renal cell carcinoma. Almost all women with HLRCC develop symptomatic uterine leiomyomas resulting in surgery at young ages, presenting an ideal opportunity for early detection of these patients and the implementation of surveillance measures for renal cell carcinoma. FH-deficient uterine leiomyomas can show characteristic morphologic features (FH-d morphology) that have been previously described. Immunohistochemistry (IHC) for FH can also be helpful in detecting FH deficiency in leiomyomas, which manifests as complete loss of staining for FH. However, the distribution and topography of FH-d morphology and FH loss by IHC in the context of multiple leiomyomas in patients with HLRCC has not been evaluated. The aim of this study is to describe in detail the clinical and pathologic characteristics of uterine leiomyomas from women with HLRCC. Six patients with proven FH germline mutations were included. All available slides were reviewed and FH IHC staining was performed on multiple blocks when possible. Clinical data were extracted from online medical records. All 6 patients presented with symptomatic uterine fibroids and underwent myomectomy (age 24 to 36 y), followed by hysterectomy in 2 patients (age 31 and 40 y). Specimens showed conventional leiomyomas, cellular leiomyomas and leiomyomas with bizarre nuclei. FH-d morphology was present in leiomyomas from all patients and was typically observed as a diffuse finding in the majority of slides across different leiomyoma types. FH-d morphology was absent in some leiomyoma sections from one patient and the morphologic features were focal and subtle in leiomyomas from 2 patients. Both hysterectomy specimens were also notable for showing scattered irregular tongues and nodules of smooth muscle proliferation (leiomyomatosis-like) in the background myometrium. Immunohistochemical staining of multiple slides per patient for FH showed either retained staining in all sections (2/6 cases), loss of staining in all sections (1 case) or variable staining across different leiomyomas (3 cases). In conclusion, patients with HLRCC undergo surgery at young ages for highly symptomatic uterine leiomyomas. FH-d morphology is usually a diffuse and well developed finding across different leiomyomas but may be absent or focal and subtle. FH IHC can show variable results and presence of retained FH staining should not be used to exclude the possibility of HLRCC. Referral for genetic counselling and testing should be considered in a young patient with uterine leiomyomas showing FH-d morphology even if immunohistochemical staining for FH is retained.

The utility of SDHB and FH immunohistochemistry in patients evaluated for hereditary paraganglioma-pheochromocytoma syndromes.

A significant portion of paragangliomas (PGL) and pheochromocytomas (PCC) occur in patients with hereditary PGL/PCC syndromes, including those with germline mutations in succinate dehydrogenase (SDHx) subunit genes. Recently, germline fumarate hydratase (FH) mutations have been identified in a subset of PGL/PCC, and patients with hereditary leiomyomatosis and renal cell carcinoma (HLRCC) may have an increased risk of developing PGL/PCC. SDHB immunohistochemistry (IHC) has previously been shown to be useful for identifying SDHx-deficient PGL/PCC, however, FH IHC has never been explored in these tumors. Thus, we characterized SDHB and FH IHC in a large cohort of PGL/PCC patients (n = 41) at our institution who were evaluated for hereditary PGL/PCC syndromes. Overall, there was strong, positive correlation between germline SDHx subunit gene mutation status and SDHB IHC status (rφ = 0.77; P < .0001), with high corresponding sensitivity, specificity, positive predictive value, and negative predictive value (95.0%, 81.8%, 82.6%, and 94.7%, respectively). Although SDHB loss by IHC was highly correlated with germline SDHx gene mutations, its utility in this population was dependent on clinicopathologic context: while all head and neck PGL patients with SDHB-deficient tumors had germline SDHx gene mutations, only a small subset (25.0%) of PCC patients with SDHB-deficient tumors harbored a germline SDHx gene mutation. Finally, although our cohort contained only one HLRCC patient, their tumor was FH-deficient by IHC, and all other PGL/PCC showed retained FH IHC. Thus, in the appropriate clinical setting, SDHB and FH IHC may be useful for identifying PGL/PCC patients for Medical Genetics evaluation.

Cancer-associated fibroblasts enhance pancreatic cancer cell invasion by remodeling the metabolic conversion mechanism.

We investigated the mechanism of cancer-associated fibroblasts (CAFs) in promoting the invasion and metastasis of pancreatic cancer cells in a non-vascular manner. We verified the original generation of isolated cultured CAFs and normal fibroblasts (NFs) based on the expression of α-SMA and vimentin, and we examined the cell glycolysis level through glucose consumption and lactate production experiments. The mRNA and protein expression of CAF glycolytic enzymes, lactate dehydrogenase and pyruvate kinase m2, were examined by RT-PCR and western blotting, respectively. In vitro culture first-generation pancreatic CAFs were collected and cultured together with pancreas cancer BxPc-3 and Panc-1 cells. Cell invasion and migration were assessed using a Transwell assay and scratch test, respectively. Mitochondrial activity was assessed by experimentally determining oxidative phosphorylation (OP) activity. The aerobic oxidation index of cancer cells was also examined. Succinate dehydrogenase, fumarate hydratase (FH), and monocarboxylate transporter 1 (MCT1) expression were examined using an MCT1-specific inhibitor to remove 'tumor-stromal' metabolic coupling to observe the influence of cell interstices on pancreas cancer progression. First-generation isolated cultured CAFs and NFs both grew well, and showed active proliferation. Glucose absorption and lactate production were significantly enhanced in CAFs compared with that in NFs. PCR and western blotting showed that the lactate dehydrogenase and pyruvate kinase m2 mRNA and protein expression levels were increased in the CAFs. After indirect co-culture, OP was increased in the BxPc-3 and Panc-1 cells; correspondingly, succinate dehydrogenase, FH and MCT expression were increased. After the MCT1-specific inhibitor removed 'tumor-stromal' metabolic coupling, the migration and invasion abilities of the pancreatic cancer cells were decreased. Pancreatic CAFs can alter metabolism as well as communicate with and respond to cancer cell migration and invasion. This may be an important mechanism for promoting tumor progression in a non-vascular manner in the tumor microenvironment. The mechanism by which CAFs reshape metabolic transition requires further analysis.

Fumarate Production by Torulopsis glabrata: Engineering Heterologous Fumarase Expression and Improving Acid Tolerance.

Fumarate is a well-known biomass building block compound. However, the poor catalytic efficiency of fumarase is one of the major factors preventing its widespread production. To address this issue, we selected residues 159HPND162 of fumarase from Rhizopus oryzae as targets for site-directed mutagenesis based on molecular docking analysis. Twelve mutants were generated and characterized in detail. Kinetic studies showed that the Km values of the P160A, P160T, P160H, N161E, and D162W mutants were decreased, whereas Km values of H159Y, H159V, H159S, N161R, N161F, D162K, and D162M mutants were increased. In addition, all mutants displayed decreased catalytic efficiency except for the P160A mutant, whose kcat/Km was increased by 33.2%. Moreover, by overexpressing the P160A mutant, the engineered strain T.G-PMS-P160A was able to produce 5.2 g/L fumarate. To further enhance fumarate production, the acid tolerance of T.G-PMS-P160A was improved by deleting ade12, a component of the purine nucleotide cycle, and the resulting strain T.G(△ade12)-PMS-P160A produced 9.2 g/L fumarate. The strategy generated in this study opens up new avenues for pathway optimization and efficient production of natural products.

Light inhibition of fumarase in Arabidopsis leaves is phytochrome A-dependent and mediated by calcium.

Inhibition of fumarase activity in the light has been studied in Arabidopsis in relation to the involvement of phytochrome. Using knockout phytochrome mutants, we observed that the main regulator of FUM1 gene transcription, encoding the mitochondrial form of fumarase, is phytochrome A. The active form of phytochrome A suppressed FUM1 expression, while the expression of the FUM2 gene encoding the cytosolic form of fumarase was unaffected both in darkness and in light. The nuclear concentration of Ca(2+) was modulated by red and far-red light. We suggest that the signal transduction mechanism operates via Ca(2+) activation of expression of the gene encoding the transcription factor PIF3, which binds to promoters of phytochrome-regulated genes and inhibits FUM1 expression.

C4-dicarboxylic acid production by overexpressing the reductive TCA pathway.

As C4-dicarboxylic acids could replace C4-petrochemicals, the reductive tricarboxylic acid (TCA) pathway was overexpressed in Pichia pastoris for production of the C4-dicarboxylic acids. Three expression cassettes which carried the pyruvate carboxylase gene (pc), the cytoplasmic malate dehydrogenase gene (mdh1) and the retargeted fumarase gene (Tfum) were integrated into the chromosomal DNA of P. pastoris GS115 alone or jointly. Multicopy integrations were screened using quantitative PCR for C4-dicarboxylic acid overaccumulation. The results showed that the highest titer in 96 h of fumaric, malic and succinic acid (0.76, 42.28 and 9.42 g l(-1)) was obtained by co-expression of pc and mdh1 in P. pastoris. This is the first report about multiple genes engineered in P. pastoris for C4-dicarboxylic acid production. The strain Pp-PC-MDH1, moreover, has a significant potential to produce malic acid in aerobic conditions.

Identification of a novel fumarase C from Streptomyces lividans TK54 as a good candidate for L-malate production.

Fumarase is a key enzyme that catalyzes the reversible hydration of fumarate to L-malate in the tricarboxylic acid cycle. This reaction has been extensively utilized for industrial applications in producing L-malate. In this study, a fumarase C gene from Streptomyces lividans TK54 (slFumC) was cloned and expressed as a fused protein (SlFumC) in Escherichia coli. The molecular mass of SlFumC was about 49 kDa determined by SDS-PAGE. Kinetic studies showed that the K m value of SlFumC for L-malate increased by approximately 8.5-fold at pH 6.5 (6.7 ± 0.81 mM) to 8.0 (57.0 ± 1.12 mM), which was higher than some known fumarases. The catalytic efficiency (k cat) and the specific activity increased by about 9.5-fold at pH 6.5 (65 s(-1)) to 8.0 (620 s(-1)) and from 79 U/mg at pH 6.5 to 752 U/mg at pH 8.0, respectively. Therefore, SlFumC may acquire strong catalytic ability by increasing pH to partially compensate for the loss of substrate affinity. The enzyme also showed substrate inhibition phenomenon, which is pH-dependent. Specific activity of SlFumC was gradually enhanced with increasing phosphate concentrations. However, no inhibition was observed at high concentration of phosphate ion, which was distinctly different in case of other Class II fumarases. In industrial process, the reaction temperatures for L-malate production are usually set between 40 and 60 °C. The recombinant SlFumC displayed maximal activity at 45 °C and remained over 85 % of original activity after 48 h incubation at 40 °C, which was more thermostable than other fumarases from Streptomyces and make it an efficient enzyme for use in the industrial production of L-malate.

Regulatory role of cAMP receptor protein over Escherichia coli fumarase genes.

Escherichia coli expresses three fumarase genes, namely, fumA, fumB, and fumC. In the present study, catabolite repression was observed in the fumA-lacZ and fumC-lacZ fusion strains, but not in the fumB-lacZ fusion strain. The Crp-binding sites in fumA and fumC were identified using an electrophoretic mobility shift assay and footprint analysis. However, the electrophoretic mobility shift assay did not detect band shifts in fumB. Fnr and ArcA serve as transcription regulators of fumarase gene expression. In relation to this, different mutants, including Δcya, Δcrp, Δfnr, and ΔarcA, were used to explore the regulatory role of Crp over fumA and fumC. The results show that Crp is an activator of fumA and fumC gene expression under various oxygen conditions and growth rates. ArcA was identified as the dominant repressor, with the major repression occurring at 0-4% oxygen. In addition, Fnr was confirmed as a repressor of fumC for the first time. This study elucidates the effects of Crp on fumarase gene expression.

Stimulation of Erwinia sp. fumarase and aspartase synthesis by changing medium components.

The optimal concentrations of nutrient medium components, aeration conditions, and pH providing for maximum biomass yields, as well as fumarase and L-aspartase activities, during submerged cultivation of Erwinia sp. were determined. The data showed that different concentrations of carbon source (molasses) and pH of the nutrient medium were required to reach the maximum fumarase and L-aspartase activities. Calculations performed by application of the additive lattice model suggested that the combination of these optimized factors would result in 3.2-, 3.4-, and 3.8-fold increases as compared to the experimental means in Erwinia sp. biomass, and L-aspartase and fumarase activities, respectively. The conditions of the fumaric acid biotransformations into L-malic and L-aspartic acids were optimized on the basis of intact Erwinia sp. cells, a fumarase and L-aspartase producer. In the cases of fumarate transformation into L-malic acid and of fumarate transformation into L-aspartic acids, fumarase and L-aspartase activities increased 1.5- and 1.7-fold, respectively. The experimental data were consistent with these estimates to 80% accuracy. In comparison with the additive lattice model, the application of polynomial nonlinear model allowed the between-factor relations to be considered and analyzed, which resulted in 1.1-, 1.27-, and 1.1-fold increases in Erwinia sp. biomass and fumarase and L-aspartase activities for the case of cultivation. In the case of fumarate transformation into L-malic acid, this model demonstrated a 1.7-fold increase in fumarase activity, whereas during fumarate transformation into L-aspartic acid no significant change in aspartase activity was observed.

Oxygen- and growth rate-dependent regulation of Escherichia coli fumarase (FumA, FumB, and FumC) activity.

Escherichia coli contains three biochemically distinct fumarases which catalyze the interconversion of fumarate to L-malate in the tricarboxylic acid cycle. Batch culture studies indicated that fumarase activities varied according to carbon substrate and cell doubling time. Growth rate control of fumarase activities in the wild type and mutants was demonstrated in continuous culture; FumA and FumC activities were induced four- to fivefold when the cell growth rate (k) was lowered from 1.2/h to 0.24/h at 1 and 21% O(2), respectively. There was a twofold induction of FumA and FumC activities when acetate was utilized instead of glucose as the sole carbon source. However, these fumarase activities were still shown to be under growth rate control. Thus, the activity of the fumarases is regulated by the cell growth rate and carbon source utilization independently. Further examination of FumA and FumC activities in a cya mutant suggested that growth rate control of FumA and FumC activities is cyclic AMP dependent. Although the total fumarase activity increased under aerobic conditions, the individual fumarase activities varied under different oxygen levels. While FumB activity was maximal during anaerobic growth (k = 0.6/h), FumA was the major enzyme under anaerobic cell growth, and the maximum activity was achieved when oxygen was elevated to 1 to 2%. Further increase in the oxygen level caused inactivation of FumA and FumB activities by the high oxidized state, but FumC activity increased simultaneously when the oxygen level was higher than 4%. The same regulation of the activities of fumarases in response to different oxygen levels was also found in mutants. Therefore, synthesis of the three fumarase enzymes is controlled in a hierarchical fashion depending on the environmental oxygen that the cell encounters.

Induction of the soxRS regulon of Escherichia coli by superoxide.

The soxRS regulon orchestrates a multifaceted defense against oxidative stress, by inducing the transcription of approximately 15 genes. The induction of this regulon by redox agents, known to mediate O-2 production, led to the view that O-2 is one signal to which it responds. However, redox cycling agents deplete cellular reductants while producing O-2, and one may question whether the regulon responds to the depletion of some cytoplasmic reductant or to O-2, or both. We demonstrate that raising [O-2] by mutational deletion of superoxide dismutases and/or by addition of paraquat, both under aerobic conditions, causes induction of a member of the soxRS regulon and that a mutational defect in soxRS eliminates that induction. This establishes that O-2, directly or indirectly, can cause induction of this defensive regulon.

Molecular characterization of potato fumarate hydratase and functional expression in Escherichia coli.

The tricarboxylic acid cycle enzyme fumarase (fumarate hydratase; EC 4.2.1.2) catalyzes the reversible hydration of fumarate to L-malate. We report the molecular cloning of a cDNA (StFum-1) that encodes fumarase from potato (Solanum tuberosum L.). RNA blot analysis demonstrated that StFum-1 is most strongly expressed in flowers, immature leaves, and tubers. The deduced protein contains a typical mitochondrial targeting peptide and has a calculated molecular mass of 50.1 kD (processed form). Potato fumarase complemented a fumarase-deficient Escherichia coli mutation for growth on minimal medium that contains acetate or fumarate as the sole carbon source, indicating that functional plant protein was produced in the bacterium. Antiserum raised against the recombinant plant enzyme recognized a 50-kD protein in wild-type but not in StFum-1 antisense plants, indicating specificity of the immunoreaction. A protein of identical size was also detected in isolated potato tuber mitochondria. Although elevated activity of fumarase was previously reported for guard cells (as compared with mesophyll cells), additional screening and genomic hybridization data reported here do not support the hypothesis that a second fumarase gene is expressed in potato guard cells.

Functional significance of the Cu,ZnSOD in Escherichia coli.

Diethyl dithiocarbamate (DDC) was used to inhibit the copper- and zinc- containing superoxide dismutase (Cu,ZnSOD) of Escherichia coli in order to expose its physiological function. DDC inhibited the aerobic growth of a sodA sodB mutant much more than it did the growth of a SOD-replete parental strain and this inhibitory effect was oxygen-dependent. The SOD mimic MnTMPyP markedly diminished the growth inhibitory effect of DDC. Transfer of the sodA sodB strain from anaerobic to aerobic conditions induced fumarase C, which is a member of the soxRS regulon. DDC augmented this induction. These results indicate that the Cu,ZnSOD provides a defense against oxidative stress, which is more important to the sodA sodB mutant than to its SOD-replete parental strain.

A single base-pair change (ATG-->ATC) nullifies the activity of cytosolic fumarase in Saccharomyces cerevisiae.

A respiratory defective pet mutant (W303 delta FUM1) of Saccharomyces cerevisiae deficient in fumarase was transformed with the plasmid construct pG5/ST7. This plasmid contains the entire FUM1 gene with only a single base pair change (ATG-->ATC) confined to the putative second inframe translation initiation codon. While transformation of the fumarase deficient mutant with pG5/ST7 resulted in an elevation of fumarase activity in the mitochondria of the transformed strain, fumarase activity in the cytosol remained negligible his result indicated that the cytosolic fumarase isoenzyme is synthesized exclusively from the second translation initiation codon of FUM1. Results of RACE-PCR of the 5' ends of FUM1 transcripts confirmed that the FUM1 gene synthesizes two distinct species of transcripts. These data provide strong evidence for the synthesis and targeting of two fumarase isomers.

The fumR gene encoding fumarase in the filamentous fungus Rhizopus oryzae: cloning, structure and expression.

The filamentous fungus Rhizopus oryzae (Ro) is known for its ability to overproduce and accumulate high levels of fumaric acid (FA) under stress conditions. In order to study the molecular mechanisms involved in the increased biosynthesis of FA, the gene (designated fumR) encoding Ro fumarase was cloned and analysed for its structure and expression. Nucleotide (nt) sequence and comparison of the fumR product with fumarases from various sources established that fumR contains nine introns and encodes a deduced product of 494 amino acids (aa), related to class-II fumarases. A fumarase protein of 50 kDa was immuno-detected in crude Ro extracts. Primer extension experiments mapped the 5' end of the fumR RNA 159 nt upstream from the putative translation start codon. Both primer extension and Northern analysis showed the existence of one transcript of fumR. The level of fumR RNA increased in cells producing FA under stress conditions (high carbon and low nitrogen levels in the medium), suggesting that transcriptional regulation of fumR might be involved in the overproduction and accumulation of FA by Ro cells under stress conditions. The possibility that additional mechanisms are responsible for this phenomenon is discussed.

A cationic manganic porphyrin inhibits uptake of paraquat by Escherichia coli.

A manganic porphyrin (MnTMPyP), which catalyzed the dismutation of O2-, facilitated the aerobic growth of a sodA sodB strain of Escherichia coli and protected a superoxide dismutase (SOD)-competent parental strain against paraquat. Surprisingly, the latter effect was more complete than the former and the mimic could block the inductions of fumarase C and of glucose 6-phosphate dehydrogenase by paraquat, even though SOD could not. An explanation for these apparent paradoxes was found in the ability of MnTMPyP to inhibit the uptake of paraquat by E. coli. MnTMPyP was accumulated by E. coli until its intracellular concentration was 20-fold greater than the extracellular concentration. This happened in a glucose plus salts medium, but not from a rich LB medium. MnTMPyP was bound onto cellular macromolecules and was maintained in the reduced state within E. coli. The free form of the reduced MnTMPyP was autoxidizable, but the bound form was not. Consequently, the free form could catalyze the oxidation of ascorbate, while the bound form did not.

The single translation product of the FUM1 gene (fumarase) is processed in mitochondria before being distributed between the cytosol and mitochondria in Saccharomyces cerevisiae.

The yeast mitochondrial and cytosolic isoenzymes of fumarase, which are encoded by a single nuclear gene (FUM1), follow a unique mechanism of protein subcellular localization and distribution. Translation of all FUM1 messages initiates only from the 5'-proximal AUG codon and results in a single translation product that contains the targeting sequence located within the first 32 amino acids of the precursor. All fumarase molecules synthesized in the cell are processed by the mitochondrial matrix signal peptidase; nevertheless, most of the enzyme (80 to 90%) ends up in the cytosol. The translocation and processing of fumarase are cotranslational. We suggest that in Saccharomyces cerevisiae, the single type of initial translation product of the FUM1 gene is first partially translocated, and then a subset of these molecules continues to be fully translocated into the organelle, whereas the rest are folded into an import-incompetent state and are released by the retrograde movement of fumarase into the cytosol.

Effects of paraquat on Escherichia coli: sensitivity to small changes in pH of the medium--a cautionary note.

Uric acid appears to protect Escherichia coli against the growth-inhibiting effect of paraquat, but this is actually due to acidification of the medium and does not occur when the pH of the medium is readjusted to neutrality. Any compound which lowers the pH of the medium will thus diminish the effect of paraquat on E. coli, whether that effect is inhibition of growth or adaptive induction of members of the soxRS regulon.

Changes in enzyme activities in skin fibroblasts derived from persons of various ages.

We examined antioxidant enzyme activities (catalase, glutathione peroxidase, and superoxide dismutase) in cultured skin fibroblasts (passage number 2-3) derived from 30 persons of various ages. With increasing ages, catalase activity decreased, glutathione peroxidase activity increased slightly, and superoxide dismutase activity was unchanged. After UVA irradiation (4.8 joule/cm2) of the fibroblasts, only catalase activity decreased by 70%. This suggests that catalase may play an important role in the aging of human skin fibroblasts.