Wild-type IDH1 Knockout Leads to G0/G1 Arrest, Impairs Cancer Cell Proliferation, Altering Glycolysis, and the TCA Cycle in Colon Cancer.

The isocitrate dehydrogenase (IDH), which participates in the TCA cycle, is an important key enzyme in regulating cell metabolism. The effect of the metabolic IDH enzyme on cancer pathogenesis has recently been shown in different types of cancer. However, the role of wild-type (wt) IDH1 in the development of colon cancer is still unknown. Our study investigated the role of the IDH1 enzyme in key hallmarks of colon cancer using various methods such as wound healing, cell cycle, colony formation ability, invasion, and apoptosis analysis. Furthermore, cell metabolism was investigated by pyruvate analysis, dinitrosalicylic acid, and HPLC methods. In addition, CRISPR/Cas9 tool was utilized to knockout the IDH1 gene in colon adenocarcinoma cells (SW620). Further studies were performed in two isogenic IDH1 KO clones. Our findings in both clones suggest that IDH1 KO results in G0/G1 arrest, and reduces proliferation by approximately twofold compared to IDH1 WT cells. In addition, the invasion, migration, and colony formation abilities of IDH1 KO clones were significantly decreased accompanied by significant morphological changes. In the context of metabolism, intracellular glucose, pyruvate, αKG, and malate levels were decreased, while the intracellular citrate level was increased in IDH1 KO clones as compared to IDH1 WT cells. Our results reveal that wt IDH1 knockout leads to a decrease in the aggressive features of colon cancer cells. In conclusion, we reported that wt IDH1 has an effective role in colon cancer progression and could be a potential therapeutic target.

The elevated D-2-hydroxyglutarate level found as a characteristic metabolic change of colon cancer in both in vitro and in vivo models.

The D-2-hydroxyglutarate (D-2-HG), whose normal cellular concentration is low, can be accumulated 10-100 times natural levels in some cancer types and participates in the carcinogenesis process. D-2-HG is produced by different pathways specific to cancer type. In this study, the level of significant metabolites produced in some metabolic pathways related to D-2-HG in the energy metabolism was determined in colon adenocarcinoma cell lines at different stages. Then, the differences in TCA and Cori cycle, glutaminolysis, and Glycolysis were investigated in the brain, colon, liver, and tumor tissues extracted from xenograft models. The levels of glucose, pyruvate, lactate, all TCA cycle intermediates, and D-2-HG were determined by the HPLC analysis, DNS method, and pyruvate assay. The intracellular D-2-HG level was found at 22.6 µmol/mg in primary (Caco-2) and 152.6 µmol/mg in metastatic (SW620) colon adenocarcinoma cells, whereas it could not be detected in colon epithelial cell line (CCD-18Co). In the xenograft models, D-2-HG could not be detected in CCD-18Co colon and brain tissues, whereas it was produced in Caco-2 and SW620 tissues. Most importantly, the level of D-2-HG was 7.4 and 19.9-fold increased in Caco-2 and SW620 tumor tissues compared to healthy tissue, respectively. In addition, the D-2-HG production pathways were investigated. The results revealed that the carbon source of D-2-HG is glucose, and the imbalance of wt-IDH1/2 enzymes plays a role in its production. Overall, the in vitro and in vivo results show that the enhanced production of endogenous D-2-HG is a characteristic change in the metabolism of colon cancer.

Could lysosomal acid lipase enzyme activity be used for clinical follow-up in cryptogenic cirrhosis?

BACKGROUND: Cholesterol ester storage disease (CESD) is one of the rare causes that should be kept in mind in the etiology of cirrhosis. Recent studies detected that significantly reduced lysosomal acid lipase deficiency enzyme (LAL) in patients with cryptogenic cirrhosis (CC). Moreover, studies have evaluated that LAL activity is as effective as scoring systems in assessing the severity of cirrhosis. In this study, we aimed to investigate the CESD with LAL level and mutation analysis of LIPA gene in patients diagnosed with CC and to compare LAL activities between patients with CC and healthy volunteers. METHODS: Laboratory parameters and cirrhosis stage (CHILD and MELD) were recorded for the patient group included in the study. In addition, blood samples were taken from each case included in the study for LAL activity determination and LIPA gene analysis. RESULTS: A statistically significant decrease in LAL activity was found in patients diagnosed with CC compared to the healthy group. LIPA gene analysis did not detect CESD in any patient group. Correlation analysis showed a positive correlation between LAL activity and white blood cell and platelet counts in both healthy volunteers and CC patient groups. In the univariate and multivariate logistic regression analysis of the parameters associated with the MELD of ≥10 in patients with CC, significant relationship was found between the MELD of ≥10 and the LAL activity. DISCUSSION: In our study, LAL activity was significantly lower in CC patients than in the normal population. LAL activity level appears to be a parameter that can be used to assess the severity of cirrhosis.

Synthesis of Murrayaquinone-A Derivatives and Investigation of Potential Anticancer Properties.

A series of novel murrayaquinone a derivatives were synthesized and their anti-cancer activity were evaluated on healthy colon cell lines (CCD-18Co), primary (Caco-2) and metastatic (DLD-1) colon cancer cell lines. The results showed that the cytotoxicity of murrayaquinone molecules is significantly high even in micromolar levels. The DNA binding, cell cycle arrest and metabolic activity studies of these molecules were also carried out and the results showed that these molecules induce apoptosis. In conclusion, the data support further studies on murrayaquinone derivatives toward selection of a candidate for cancer treatment.

Screening organometallic thiophene containing thiosemicarbazone ruthenium (II/III) complexes as potential anti-tumour agents.

The new ruthenium (III) complex has been synthesized and characterized by elemental analysis, FT-IR, UV-Vis, EI-Mass, EPR spectroscopy, and magnetic susceptibility measurement. Cytotoxic effects of organoruthenium (II/III) complexes 1a, 1b, and 2a, and their ligands (TSC1 and TSC2) in cultured human ovarian (A2780, SKOV-3, and OVCAR-3) and colon (DLD, CCD18Co, and Caco-2) cells have been investigated comparing reactivity of the Ru (II/III) complexes and their free TSC ligands. The complexes exhibit higher cytotoxicity in three cancer cell lines than in normal cells. The binding with CT-DNA and BSA of the all complexes were weak compared with their ligand in spite of the cellular uptake of these complexes into the cytoplasm and then nucleus while their cytotoxic effects were vice versa. All the results showed that Complex 1b has more efficient cytotoxicity on the colon cancer cells than ovarian cancer cells. However, Complex 2a is a better drug candidate especially for antitumor therapy of metastasized ovarian cancer.

The Investigation of the Antitumor Agent Toxicity and Capsaicin Effect on the Electron Transport Chain Enzymes, Catalase Activities and Lipid Peroxidation Levels in Lung, Heart and Brain Tissues of Rats.

Cisplatin is one of the most active cytotoxic agents in cancer treatment. To clarify the interaction with mitochondria, we hypothesize that the activities of mitochondrial electron transport chain (ETC) enzymes succinate dehydrogenase (SDH) and cytochrome c oxidase (COX), nucleotide levels, as well as levels of catalase (CAT) enzyme and membrane lipid peroxidation (LPO) can be affected by cisplatin. There was a significant decrease of both SDH and COX activities in the lung, heart, and brain tissues at the 1st day after cisplatin exposure, and the observed decreased levels of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) in comparison with the control could be because of cisplatin-induced mitochondrial dysfunction. The investigations suggested that cisplatin inhibits SDH, COX, and ATP synthase. The higher LPO level in the studied tissues after 1 and 4 days post-exposure to cisplatin compared to control can be inferred to be a result of elevated electron leakage from the ETC, and reactive oxygen species (ROS) can lead to wide-ranging tissue damage such as membrane lipid damage. Consequently, it was observed that capsaicin may have a possible protective effect on ETC impairment caused by cisplatin. The activities of SDH and COX were higher in heart and brain exposed to cisplatin + capsaicin compared to cisplatin groups, while LPO levels were lower. The investigated results in the cisplatin + capsaicin groups suggested that the antioxidant capacity of capsaicin scavenges ROS and prevents membrane destruction.

Exosomes released from cisplatin-resistant ovarian cancer cells modulate the reprogramming of cells in tumor microenvironments toward the cancerous cells.

Exosomes released from cancer cells are involved in the reorganization of the tumor microenvironment which is the essential aspect of cancer pathogenesis. The intercommunications between cancer cells and diverse cell types in the microenvironment are accomplished by exosomes in ovarian cancer. Internalization pathway, intracellular fate, and biological functions in recipient cells mediated by exosomes released from cisplatin-resistant A2780cis have been studied. Also, histopathological evaluation of tumor, ovary, liver tissues and lymph nodes in vivo studies have been performed. The recipient cells internalized the exosomes via active uptake mechanisms, as shown by confocal microscopy. However, inhibitor studies and flow cytometry analysis showed that each recipient cell line used different uptake pathways. Also, confocal microscopy imaging indicated that the internalized exosomes trapped in the endosomes or phagosomes were distributed to the different cellular compartments including ER, Golgi, and lysosome. The transfer of exosomal oncogenic cargo into the cells modified the intracellular signaling of recipient cells including invasion and metastasis by Boyden-Chamber assay, proliferation by ATP analysis, epithelial-mesenchymal transition (EMT) markers at protein and mRNA levels by western blotting and real-time PCR, and protein kinases in the phospho-kinase array. This remodeling contributed to the initiation of carcinogenesis in ovarian epithelial and peritoneal mesothelial cells, and the progression of carcinogenesis in ovarian cancer cells. In addition, intraperitoneal tumor model studies show that exosomes released from cisplatin-resistant A2780cis cells may play role in the enlargement of lymph nodes, and tumor formations integrated with the liver, attached to the stomach and in the ovarian tissues.

The variation of antioxidant defense system of Streptomyces sp. M4018 with respect to carbon sources.

The effect of glycerol, glucose, and starch as carbon sources on the antioxidant defense system such as superoxide dismutase (SOD) and catalase (CAT) activities, pyruvate levels, and membrane lipid peroxidation (LPO) levels of Streptomyces sp. M4018, after isolation from the rhizosphere samples of Colutea arborescens and identification as a strain of S. hiroshimensis based on phenotypic and genotypic characteristics, were investigated. As an antioxidant defense enzyme, SOD activities increased up to 20 g/L of glycerol and 15 g/L of starch, while they showed negative correlation with glucose concentration. CAT activity variations of glycerol- and glucose-supplemented mediums showed significant positive correlations with the trend of SOD activities. However, CAT activity, in contrast to SOD, in Streptomyces sp. M4018 tended to decrease as the starch concentration increased. The production of pyruvate increased with respect to glycerol and starch up to 15 g/L, while it was positively correlated with glucose concentration. The highest pyruvate production was seen at 20 g/L glucose. Membrane LPO levels were negatively correlated with the activities of SOD and CAT enzymes, and the minimum LPO level was determined at 5 g/L of glucose, where SOD and CAT activities reached their maximum levels. Nevertheless, the higher SOD and CAT activities in a wider range of incubation period compared to the beginning by resulting in insignificant increases in membrane LPO levels showed the unusual antioxidant response capacities of the in Streptomyces sp. M4018 against the potentially deleterious effects of reactive oxygen species (ROS) for glycerol, glucose, and starch as carbon sources.

Half-Sandwich Arene Ruthenium(II) Thiosemicarbazone Complexes: Evaluation of Anticancer Effect on Primary and Metastatic Ovarian Cancer Cell Lines.

In this study, we describe the synthesis, characterization and antiproliferative activity of three organo-ruthenium(II) half-sandwich complexes [RuCl(η6-p-cym)(N,S-L)]Cl (I, II, and III). To form these complexes, three thiosemicarbazone ligands (TSCs) were synthesized; L = 5-nitro-2-carboxyaldehyde-thiophen-N-methyl-thiosemicarbazone, (L1); 2-acetyl-5-bromo-thiophen-N-methyl-thiosemicarbazone, (L2) and 2-acetyl-5-bromo-thiophen-N,N-dimethyl-thiosemicarbazone, (L3). The isolated compounds were analyzed using spectroscopic techniques such as elemental analysis, conductance measurements, FT-IR, 1H NMR spectroscopy, MALDI-TOF mass spectrometry, and single-crystal XRD. Our results demonstrated that the synthesized thiosemicarbazone ligands (TSCs) are bound to the metal ion as a bidentate ligand that coordinates through the thiocarbonyl sulfur and azomethine nitrogen atoms in all complexes (I, II, and III). The X-ray crystal structures of L1 and L2 revealed that both compounds are crystallized in the triclinic crystal system with space group P-1. The biological potency of newly synthesized TSC ligands (L1, L2, and L3) and their corresponding ruthenium complexes (I, II, and III) were investigated on human primary ovarian (A2780) and human metastatic ovarian (OVCAR-3) cell lines. To get detailed information respecting antitumor properties, cytotoxicity, DNA/BSA binding affinity, cellular uptake, DNA binding competition, and trans-epithelial resistance measurement assays were performed. Our results demonstrate that newly synthesized ruthenium(II) complexes possess potential biological activity. Moreover, we observe that the ruthenium complexes reported here show anticancer activity on primary (A2780) and metastatic (OVCAR-3) ovarian cancer cells.

Synthesis and characterization of thiosemicarbazone-functionalized organoruthenium (II)-arene complexes: Investigation of antitumor characteristics in colorectal cancer cell lines.

In the current study, organoruthenium(II)-arene complexes (I-IV) have been prepared by the reaction of [{(η6-p-cym)RuCl}2(µ-Cl)2] with new thiosemicarbazones (TSC1-4).The isolate was analyzed using elemental analysis, FT-IR, 1H and 13C NMR spectroscopy and single-crystal XRD. Subsequently, the complexes and TSC ligands were assessed for anticancer properties in vitro against three different colorectal cancer stage's cell lines (Caco-2, DLD-1, and SW620) and a noncancerous cell line (CCD18Co). The complexes (I-IV) showed higher cytotoxicity with low IC50 values as 0.1-0.33 µM in colorectal cell lines except for SW620 (47.4-84.20 µM) than in a noncancerous cell. Complex I was 2.8 and 24.5-fold more active against Caco-2 and DLD-1 than CCD18Co, respectively. The complexes (I-IV) accumulated at a high concentration in the cell nuclei and caused cell cycle arrest by affecting the G0/G1 and/or G2/M phase and showed high binding affinity with CT-DNA (Kb = 104 M-1). The expression of Caspase-3 and Caspase-9 apoptosis-related protein levels was slightly upregulated and Atg5 autophagy-related protein level was clearly downregulated according to control and 5-FU-treated cells after complex I and II treatment. Furthermore, it was observed that cytotoxicity of the complexes is decreased while cancer progresses. Altogether, this study indicates that all organoruthenium (II)-arene complexes (particularly complex I) can be a promising alternative to platinum counterparts in cancer treatment.

Flavonoids showed anticancer effects on the ovarian cancer cells: Involvement of reactive oxygen species, apoptosis, cell cycle and invasion.

Flavonoids have been recently identified as a potential anticancer agent against various human epithelial cancers. In this study, the elucidation of mechanisms underlying the anticancer effects of the apigenin, luteolin and myricetin will be new knowledge about preventive strategies against epithelial ovarian cancer in which the effect of flavonoids is still unclear. The cytotoxic effect of flavonoids was assessed by MTT analysis of the ovarian cancer cells (A2780, OVCAR-3 and SKOV-3) in comparison to the ovarian epithelial cells (OSE). The intracellular reactive oxygen species (ROS) generation, malondialdehyde (MDA) and protein carbonyl levels, caspase-3 and -9 activities were evaluated using fluorescence spectrometry. Apoptosis and cell cycle arrest, and cell invasion were measured by flow cytometry and Boyden chamber assay, respectively. MTT analysis showed that flavonoids selectively decreased the cell viability of cancer cells. Furthermore, the intracellular ROS generation was induced or scavenged by flavonoids depending on the structural differences. The flavonoids increased MDA levels due to the disruption of the membrane. Caspase activities indicated that flavonoids activated the extrinsic apoptotic pathway when ROS was scavenged. In contrast, the induced intracellular ROS generation resulted in the activation of the intrinsic apoptotic pathway. In addition, the cell cycle was arrested in different cell cycle phases and cell invasion on the collagen was disrupted by flavonoids. The anticancer activities of apigenin, luteolin and myricetin were attributed to the alterations of ROS signaling, and as well as the induction of apoptosis, cell cycle arrest and abrogation of the invasion. The present study may uncover new strategies for ovarian cancer therapy.

Crystal structures, spectroscopic properties of new cobalt(II), nickel(II), zinc(II) and palladium(II) complexes derived from 2-acetyl-5-chloro thiophene thiosemicarbazone: Anticancer evaluation.

The reactions of cobalt(II), nickel(II), zinc(II) chlorides and [Pd(DMSO)2Cl2] with 2-acetyl-5-chloro-thiophene thiosemicarbazone (HL) leads to the formation of a series of new complexes: [CoCl2(S-HL)2], 1; [Ni(N,S-L)2], 2 [ZnCl2(S-HL)2], 3 and [PdCl2(N,S-HL)], 4. All the complexes have been characterized by elemental analysis, IR, LC-MS. 1H and 13C NMR spectroscopy have been performed for Zn(II) and Pd(II) complexes. The crystal structures of the complexes 1-3 have been determined by single-crystal X-ray diffraction methods. The compounds, (1) and (3), crystallized in the monoclinic crystal system with C2/c space group. In both complexes, the metal centers are four-coordinated in a distorted tetrahedral configuration by two sulfur atoms from two thiosemicarbazone ligands and two Cl anions. The crystal structure of (2) consists of monomeric entities where the nickel(II) ion exhibit distorted square planar geometry. The coordination geometry around nickel ion is four-coordinate with four atoms of the two chelating thiosemicarbazone ligands which are in cis position. The τ4 value of 0.255 obtained from the τ4 analysis of complex (2) shows that the four-coordinate geometry around the central nickel ion is close to square planar. Complex (4) is mononuclear, the central ion is coordinated through the sulfur and the azomethine nitrogen atom of neutral ligand. The cytotoxic effects of all complexes were analyzed for three cancer cell lines, Caco-2, DLD-1, and SW620 compared to normal colon epithelial cell line, CCD18Co. Complex (4) is more active against DLD-1, SW620 and Caco-2 than CCD18Co. The efficiency of complex (4) is more effective in aggressive cancer cell lines. Therefore, it can be used as a new chemotherapeutic, especially in the treatment of resistant cancer types caused by long-term use of platinum-based drugs.

Replacement of Soybean Meal with Animal Origin Protein Meals Improved Ramoplanin A2 Production by Actinoplanes sp. ATCC 33076.

Ramoplanin A2 is the last resort antibiotic for treatment of many high morbidity- and mortality-rated hospital infections, and it is expected to be marketed in the forthcoming years. Therefore, high-yield production of ramoplanin A2 gains importance. In this study, meat-bone meal, poultry meal, and fish meal were used instead of soybean meal for ramoplanin A2 production by Actinoplanes sp. ATCC 33076. All animal origin nitrogen sources stimulated specific productivity. Ramoplanin A2 levels were determined as 406.805 mg L(-1) in fish meal medium and 374.218 mg L(-1) in poultry meal medium. These levels were 4.25- and 4.09-fold of basal medium, respectively. However, the total yield of poultry meal was higher than that of fish meal, which is also low-priced. In addition, the variations in pH levels, protein levels, reducing sugar levels, extracellular protease, amylase and lipase activities, and intracellular free amino acid levels were monitored during the incubation period. The correlations between ramoplanin production and these variables with respect to the incubation period were determined. The intracellular levels of L-Phe, D-Orn, and L-Leu were found critical for ramoplanin A2 production. The strategy of using animal origin nitrogen sources can be applied for large-scale ramoplanin A2 production.

Role of pyruvate and ascorbate production in regulation of antioxidant enzymes and membrane LPO levels in Fusarium acuminatum.

The role of pyruvate and ascorbate in the regulation of superoxide dismutase (SOD); catalase (CAT); glutathione peroxidase enzymes; and, therefore, membrane lipid peroxidation (LPO) levels in Fusarium acuminatum was investigated in media containing either glycerin or glucose as a carbon source, depending on the incubation period, in the range of 5-25 g/L. Increasing SOD activity between d 9 and 16 of the incubation period showed a positive correlation with a significant increase in pyruvate production up to 15 g/L of glycerin and glucose. In addition, maximum ascorbate production was observed at 15 g/L of glycerin as 82.5+/-2.1 and 20 g/L of glucose as 54+/-1.51, whereas CAT activity decreased with an increased concentration of both carbon sources. When compared with the LPO levels determined in media supplemented with glycerin and glucose, the minimum LPO level was 1.88+/-0.028 nmol of malondialdehyde/g wet wt at 15 g/L of glycerin on d 16, at which it was also observed to have a maximum pyruvate and ascorbate production and SOD, CAT, and GSH-Px activities of 75+/-1.42 microg/mL, 82.5+/-2.1 microg/mL, 32.5+/-0.634 microg/mL, 86.8+/-2.58 IU/mg, and 1.867 IU/mg, respectively. These results indicate that the biosynthesis of pyruvate and ascorbate may be involved in the regulation of antioxidant enzymes, depending on the glycerin and glucose concentrations, and also this defense network was effective in preventing membrane damage from oxidative stress.

The effect of cisplatin toxicity and capsaicin on electron transport chain in liver and kidney of sprague dawley rats.

Cisplatin accumulates in mitochondria, which is a potent and widely used chemotherapeutic agent. In order to clarify the potential effect of cisplatin on electron transport chain (ETC), the variation of succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) enzyme activities, nucleotide levels, as well as catalase (CAT) enzyme and membrane lipid peroxidation (LPO) level with respect to liver and kidney of cisplatin-exposed rats were studied. We found that cisplatin caused significant impairment in the SDH, COX, and CAT activities, and nucleotide levels associated with membrane LPO in isolated mitochondria. It was determined whether capsaicin, as an antioxidant, has a possible protective role on all investigated parameters of liver and kidney induced by cisplatin. The results of capsaicin + cisplatin suggest that capsaicin have antioxidant capacity to scavenge ROS to prevent membrane damage.

The glutathione-related detoxication responses to juvenile and ecdysone hormones in Galleria mellonella.

The effect of 20-hydroxyecdysone (20E) and juvenile hormone (JH) on the glutathione pathway of the greater wax moth Galleria mellonella (Lepidoptera: Pyralidae) was determined by investigating glutathione peroxidase (GSH-Px), glutathione S-transferases (GST), and glutathione reductase (GR) activities as well as reduced and oxidized glutathione (GSH and GSSG) content with respect to developmental stage. The continuous decreases of GSH-Px and GST activities dependent on the growth period of G. mellonella occurred in JH and 20E groups over and under their controls, respectively. While the GR activities of G. mellonella showed increases in young pupa (YP) for both control and in old larvae (OL) for the 20E groups after the minimum at these periods, they also increased after old pupa (OP) for the JH group with a maximum in OL period. Although GR activity levels in the JH group were significantly higher compared with controls and 20E groups up to OP period, the activity levels for the control and 20E groups were higher than those of the JH group at adult (AD) and old pupa (OP) periods, respectively. In spite of increases in the GR activity of 20E and control groups of G. mellonella, decreased GSH and increased GSSG levels were observed at aging period. GSH levels in the JH group reached a maximum at prepupa (PP) and then decreased with non-significant changes from OL to AD period. According to the results, GSH and GSSG levels, as well as GSH/GSSG ratios, were below and over control levels in 20E and JH groups, respectively, during all of the investigated developmental stages. On the contrary, the LPO levels were higher than the control for 20E and lower for the JH groups during the developmental period. These results show that while ecdysone hormone has a negative effect on the glutathione-related detoxication capacity of G. mellonella, the juvenile hormone has a positive effect on this process.

Carbohydrate metabolite pathways and antibiotic production variations of a novel Streptomyces sp. M3004 depending on the concentrations of carbon sources.

To determine the variations of growth, some key enzyme activities such as glucose kinase (GK), glucose-6-phosphate dehydrogenase (G6PDH), α-ketoglutarate dehydrogenase (KGDH), and isocitrate lyase (ICL) besides metabolite levels of pyruvate and antibiotic production of newly isolated Streptomyces sp. M3004 were grown in culture media which contain 10-20 g/l concentration with either glucose or glycerol as carbon source. Biomass and intracellular glucose and glycerol levels of Streptomyces sp. M3004 showed positive correlation with the concentration of these carbon sources, and these levels were higher in glucose compared with the glycerol-supplemented mediums. GK, G6PDH, and KGDH activities showed marked correlation with the concentration of both glucose and glycerol, and the activity levels were 4.14-, 1.47-, and 1.27-fold higher in glucose than glycerol. A key enzyme of the glyoxalate cycle, ICL activities decreased with increasing glucose concentrations from 10 to 20 g/l, but increased up to 15 g/l of glycerol. The positive correlations were also determined between intracellular glucose and glycerol levels besides pyruvate and protein variations with respect to concentrations of the carbon sources. Antibacterial activities of Streptomyces sp. M3004 reached maximum on the stationary phase, while it did not change significantly with respect to glucose and glycerol.

The correlations between TCA-glyoxalate metabolite and antibiotic production of Streptomyces sp. M4018 grown in glycerol, glucose, and starch mediums.

The alterations of organic acids citrate, α-ketoglutarate, succinate, fumarate, malate production together with isocitrate lyase activity as a glyoxalate shunt enzyme, and antibiotic production of Streptomyces sp M4018 were investigated in relation to changes in the glucose, glycerol and starch concentrations (5-20 g/L) after identification as a strain of Streptomyces hiroshimensis based on phenotypic and genotypic characteristics. The highest intracellular citrate and α-ketoglutarate levels in 20 g/l of glucose, glycerol, and starch mediums were 399.47 ± 4.78, 426.93 ± 6.40, 355.84 ± 5.38 ppm and 444.81 ± 5.12, 192.96 ± 2.26, 115.20 ± 2.87 ppm, respectively. The highest succinate, malate, and fumarate levels were also determined in 20 g/l of glucose medium as 548.9 ± 11.21, 596.15 ± 8.26, and 406.42 ± 6.59 ppm and the levels were significantly higher than the levels in glycerol and starch. Extracellular organic acid levels measured also showed significant correlation with carbon source concentrations by showing negative correlation with pH levels of the growth medium. The antibiotic production of Streptomyces sp. M4018 was also higher in glucose medium as was the case also for organic acids when compared with glycerol. On the other hand, there is no production in starch.

In vitro antioxidant properties of Cucurbita pepo L. male and female flowers extracts.

Total antioxidant capacities, 2,2-diphenyl-1-picrylhydrazyl (DPPH.), hydroxyl (HO.), scavenging activities, and total phenolic values were determined in extracts of Cucurbita pepo L. female and male flowers. Powdered C. pepo L. samples were extracted in aqueous ethyl acetate (EA: W1, 17:3), ethanol (E), and water (W) by agitating in magnetic stirrer for 80 degrees C, 15 min and also by in aqueous ethyl acetate (EA: W2, 17:3) at 25 degrees C, 15 min. DPPH., HO. scavenging capacities and total phenolic values of C. pepo L. female and male were higher in EA:W2 than in other extracts. In addition, all determined antioxidant capacities of female were significantly higher than male.

Functions of antioxidant enzyme activities on the membrane bound total sialic acid and lipid peroxidation level in F. equiseti and F. acuminatum.

The variations of membrane bound total sialic acid (TSA) and lipid peroxidation level dependent on the antioxidant enzyme activities such as Superoxide Dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GSH-Px) have been studied in yeast extract supplemented medium. The maximum SOD and CAT activities of F. equiseti tended to increase with raises of yeast extract concentration up to 25 g/L where they were determined to be 78.6 +/- 0.96 and 312.7 +/- 5.6 IU/mg. On the other hand, SOD and CAT activities in F. acuminatum significantly increased with the rise of yeast extract concentration up to 10 g/L (p < 0.01) and maximum activities were observed at this concentration as 36.3 +/- 0.54 and 115.3 +/- 2.19 IU/mg on the 12th day incubation. Other H2O2 scavenger enzyme, GSH-Px activities of F. equiseti and F. acuminatum were reached the maximum at 5 and 25g/L yeast extract and determined as 5.06 +/- 0.04 and 4.74 +/- 0.09 IU/mg, respectively. TSA level showed positive correlation with SOD and CAT activities while LPO levels variations negatively correlated. The results may indicate that these antioxidant enzymes also appeared to be involved in protecting membrane bound sialic acids as well as membrane lipid of the fungus from exogenous reactive oxygen species.