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131.
Arulkumaran S Ramprasath VR Shanthi P Sachdanandam P 《Molecular and cellular biochemistry》2006,291(1-2):77-82
Cancer prevention and treatment using phytochemicals have attracted increased interest. Recent studies have shown that Semecarpus anacardium Linn nut milk extract (SA), a promising antioxidant and anticancer drug, exerts its anticancer effect through reducing or quenching reactive oxygen species under different conditions. The present study examined whether Phyllanthus emblica Linn fruit, rich in vitamin C content synergistically in combination can enhance both the antioxidant and anticancer activity of S. anacardium nut milk extract in 7, 12-dimethyl benz[a]anthracene (DMBA)-induced experimental mammary carcinoma in rat model. Female Sprague Dawley rats of 180 ± 10g were categorized into six groups. Three groups were administered DMBA (25mg/rat, orally) dissolved in olive oil to induce mammary carcinoma. One of these groups received Kalpaamruthaa (KA) (300mg/kg b.wt, orally) and other group received SA (200mg/kg b.wt, orally) for 14 days after 90 days of DMBA induction. A vehicle treated control and drug control groups were also included. The mitochondrial fraction of untreated DMBA-induced mammary gland showed 2.61-fold increase in lipid peroxidation level and abnormal changes in the activities/levels of mitochondrial enzymic (superoxide dismutase, glutathione peroxidase and glutathione reductase) and non-enzymic (glutathione, vitamin C and vitamin E) antioxidants were observed. DMBA treated rats also showed decline in the activities of mitochondrial enzymes such as succinate dehydrogenase, malate dehydrogenase, α-ketoglutarate dehydrogenase and isocitrate dehydrogenase. In contrast, rats treated with Kalpaamruthaa showed normal lipid peroxide level and antioxidant defenses. The results of the present study highlight the improved antioxidant property of KA than sole treatment of S. anacardium nut milk extract. 相似文献
132.
Viswanathan M Solomon SP Tsuchida N Selvam GS Shanmugam G 《Indian journal of experimental biology》2006,44(2):115-119
Hypermethylation of promoter regions leading to inactivation of tumor suppressor genes is a common event in the progression of several tumor types. We have employed a novel restriction digestion based multiplex PCR assay to analyse the methylation status of promoter regions of tumor suppressor genes (p16, hMLH1, MGMT and E-cadherin) in sporadic breast carcinomas of Indian women. The present results indicated the absence of hypermethylation in promoter region of p16 and MGMT genes. However, 6 of the 19 (31.6%) sporadic breast carcinomas showed hypermethylation in the promoters of two of the genes analysed; three in hMLH1 and another three in E-cad. Since our earlier studies have shown lack of genetic alterations such as missense mutations and deletions in the tumor associated genes-p16, ras and p14ARF in sporadic breast tumors, the epigenetic alterations of the two genes reported in the present study could be of interest and might be among the events in the genesis/progression of sporadic breast carcinomas. 相似文献
133.
Characterization of the siderophore of Francisella tularensis and role of fslA in siderophore production
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We determined that LVS and Schu S4 strains of the human pathogen Francisella tularensis express a siderophore when grown under iron-limiting conditions. We purified this siderophore by conventional column chromatography and high-pressure liquid chromatography and used mass spectrometric analysis to demonstrate that it is structurally similar to the polycarboxylate siderophore rhizoferrin. The siderophore promoted the growth of LVS and Schu S4 strains in iron-limiting media. We identified a potential siderophore biosynthetic gene cluster encoded by fslABCD in the F. tularensis genome. The first gene in the cluster, fslA, encodes a member of the superfamily of nonribosomal peptide synthetase-independent siderophore synthetases (NIS synthetases) characterized by the aerobactin synthetases IucA and IucC. We determined that fslA is transcribed as part of an operon with downstream gene fslB and that the expression of the locus is induced by iron starvation. A targeted in-frame nonpolar deletion of fslA in LVS resulted in the loss of siderophore expression and in a reduced ability of F. tularensis to grow under conditions of iron limitation. Siderophore activity and the ability to grow under iron limitation could be regained by introducing the fslA(+) gene on a complementing plasmid. Our results suggest that the fslA-dependent siderophore is important for survival of F. tularensis in an iron-deficient environment. 相似文献
134.
The outer membrane channel TolC is a key component of multidrug efflux and type I secretion transporters in Escherichia coli. Mutational inactivation of TolC renders cells highly susceptible to antibiotics and leads to defects in secretion of protein toxins. Despite impairment of various transport functions, no growth defects were reported in cells lacking TolC. Unexpectedly, we found that the loss of TolC notably impairs cell division and growth in minimal glucose medium. The TolC‐dependent phenotype was further exacerbated by the loss of ygiB and ygiC genes expressed in the same operon as tolC and their homologues yjfM and yjfC located elsewhere on the chromosome. Our results show that this growth deficiency is caused by depletion of the critical metabolite NAD+ and high NADH/NAD+ ratios. The increased amounts of PspA and decreased rates of NADH oxidation in ΔtolC membranes indicated stress on the membrane and dissipation of a proton motive force. We conclude that inactivation of TolC triggers metabolic shutdown in E. coli cells grown in minimal glucose medium. The ΔtolC phenotype is partially rescued by YgiBC and YjfMC, which have parallel functions independent from TolC. 相似文献
135.
Out of forty rhizosphere soils collected from semi arid tropical regions of Andhra Pradesh, India, 30 gave positive enrichments for heliobacteria. These bacteria were recognized by the presence of bacteriochlorophyll-g together with endospores in the initial enrichments. Using group specific primers of 16S rRNA gene, ten monoheliobacterial cultures were sequenced. They were finally grouped into 3 clusters based on the 16S rRNA gene similarity. Based on a few phenotypic characters, in addition to genetic characterization, we identified them as potential novel species and the 16S rRNA gene sequences were deposited with EMBL. 相似文献
136.
Shanmugam Achiraman Govindaraju Archunan Ponnirul Ponmanickam Soundrapandian Kannan 《Theriogenology》2010,74(3):345-353
In our previous investigations [1], urine of female mice contained specific compounds, namely isocroctylhydrazine, 4-methyl-2-heptanone, and azulene during proestrus, whereas during estrus it contained 1-H-cyclopop.e.azulene, caryophyllene, and copanene. Furthermore, 1-iodo-2 methyl undecane (1I2MU), present during both proestrus and estrus, was regarded as a putative estrus-specific chemo-signal [1]. The primary objective of the present study was to determine the estrogen-dependency of the above-mentioned compounds, including 1I2MU. Furthermore, the effect of these compounds on pre-mating behavior, e.g., sniffing, licking, and grooming, were recorded to determine their role as sex pheromones. Based on gas chromatography linked mass spectrometry (GC-MS) of urine samples, profiles in oophorectomized female mice had 14 major peaks. Furthermore, neither 1I2MU (nor other estrus-specific compounds) were detected in the urine of these mice, although they were detected in urine of proestrus and estrus mice. In addition, 1I2MU was not detected in urine of prepubertal mice. It was noteworthy that both 1I2MU and 4-methyl-2-heptanone reappeared in estrogen-treated females. Based on pre-mating behavioral analysis, 1I2MU was the compound most preferred by males. In conclusion, production of 1I2MU was estrogen-dependent in females, and it enhanced reproductive activities in males. 相似文献
137.
Xueli Zhang K. T. Shanmugam Lonnie O. Ingram 《Applied and environmental microbiology》2010,76(8):2397-2401
The fermentative metabolism of Escherichia coli was reengineered to efficiently convert glycerol to succinate under anaerobic conditions without the use of foreign genes. Formate and ethanol were the dominant fermentation products from glycerol in wild-type Escherichia coli ATCC 8739, followed by succinate and acetate. Inactivation of pyruvate formate-lyase (pflB) in the wild-type strain eliminated the production of formate and ethanol and reduced the production of acetate. However, this deletion slowed growth and decreased cell yields due to either insufficient energy production or insufficient levels of electron acceptors. Reversing the direction of the gluconeogenic phosphoenolpyruvate carboxykinase reaction offered an approach to solve both problems, conserving energy as an additional ATP and increasing the pool of electron acceptors (fumarate and malate). Recruiting this enzyme through a promoter mutation (pck*) to increase expression also increased the rate of growth, cell yield, and succinate production. Presumably, the high NADH/NAD+ ratio served to establish the direction of carbon flow. Additional mutations were also beneficial. Glycerol dehydrogenase and the phosphotransferase-dependent dihydroxyacetone kinase are regarded as the primary route for glycerol metabolism under anaerobic conditions. However, this is not true for succinate production by engineered strains. Deletion of the ptsI gene or any other gene essential for the phosphotranferase system was found to increase succinate yield. Deletion of pflB in this background provided a further increase in the succinate yield. Together, these three core mutations (pck*, ptsI, and pflB) effectively redirected carbon flow from glycerol to succinate at 80% of the maximum theoretical yield during anaerobic fermentation in mineral salts medium.Renewable bioenergy offers the potential to solve many environmental problems associated with petroleum-based fuels and chemicals. Biodiesel is produced by reacting vegetable oil or animal fat with alcohol (methanol or ethanol) and used as a transportation fuel in many countries (33). Glycerol is formed as an abundant waste product with limited commercial uses. As the worldwide production of biodiesel continues to increase, the development of effective uses for glycerol may prove essential for the economics and competitiveness of the biodiesel industry. The value of glycerol waste from biodiesel is similar to that of sugars currently used to produce fuel ethanol. Bioconversion of glycerol to higher-value products that replace petroleum, such as polymers, surfactants, solvents, and chemical intermediates, represents an opportunity to decrease waste and improve the economics of the biodiesel industry (5).Many previous investigations have focused on the fermentative production of 1,3-propanediol (1,3-PD) from glycerol (2, 26, 35). Microorganisms including Klebsiella (14), Citrobacter (6), Enterobacter (1), Lactobacillus (29), and Clostridium (10, 28) have the native ability to ferment glycerol into this product. Dupont and Genencor have commercialized a 1,3-PD-based polyester, a condensation product of 1,3-PD and terephthalic acid using glucose as the feedstock. Potential demand for this polymer is estimated to be 1 billion to 2 billion pounds per year over the next 10 years (26). Other investigations of glycerol fermentation have described the production of hydrogen and ethanol (15), polyhydroxyalkanoates (PHAs) (20, 27), glyceric acid (13), and small amounts of succinate (21).Succinic acid is currently used as a specialty chemical in the agricultural, food, and pharmaceutical industries (24, 34). It has also been identified by the U.S. Department of Energy as one of the top 12 building block chemicals (31) because it can be converted into a wide variety of products, including green solvents, pharmaceutical products, and biodegradable plastics (24, 34). Succinate is primarily produced from petroleum-derived maleic anhydride. Recent increases in the petroleum price have generated considerable interest in the fermentative production of succinate from sugars using either natural succinate-producing rumen bacteria or metabolically engineered Escherichia coli strains (24, 36, 38). Succinate can also be produced from glycerol by rumen bacteria, such as Anaerobiospirillum succiniciproducens (21). However, these strains require complex nutrients that increase costs of production, purification, and waste treatment.E. coli has been previously engineered for the commercial production of 1,3-PD from sugars by Dupont and Genecor (26). It is an excellent organism for biotechnology applications but was long thought incapable of anaerobic growth on glycerol (23). Recent studies demonstrated that E. coli can ferment glycerol anaerobically (8, 11, 25, 33), and a new model was proposed for glycerol fermentation (11). In this model, glycerol is metabolized through the glycerol dehydrogenase (encoded by gldA) and dihydroxyacetone kinase (encoded by dhaKLM) pathway with the production of ethanol and acetate as primary fermentation products (11). Small amounts of succinate and 1,2-propanediol were also produced. Native genes encoding glycerol dehydrogenase and dihydroxyacetone kinase were expressed from a plasmid to increase the rates of glycerol metabolism and ethanol production (32). Succinate production has also been increased by expressing Clostridium freundii dihydroxyacetone kinase (encoded by dhaKL) (11). However, neither of these enhanced pathways would appear suitable for efficient succinate production due to the absence of net ATP production and the requirement for phosphoenolpyruvate as a phosphoryl donor for dihydroxyacetone, limiting the carboxylation of this intermediate (Fig. (Fig.11).Open in a separate windowFIG. 1.Glycerol uptake and fermentation by E. coli. (A) Native E. coli pathways. Bold black arrows represent dominant fermentation reactions prior to engineering; thin black arrows represent minor fermentation reactions. GlpK and GlpD are thought to function primarily during aerobic metabolism. Pathways are based on current reviews in EcoSal (3, 4, 22), data available in Ecocyc (19), and primary literature (11, 12, 18, 25, 30). (B) Engineered pathway for the fermentative metabolism of glycerol to succinate. Bold black arrows represent the engineered reactions for glycerol fermentation to succinate as the dominant product; thin black arrows represent minor fermentation reactions in the engineered strain. Dashed arrows represent reactions that are not functional due to deletions in ptsI and pflB. Deleted genes are marked with a black X. In native E. coli strains, phosphoenolpyruvate carboxykinase functions during gluconeogenesis to produce phosphoenolpyruvate. Mutational activation of the pck gene (denoted pck*) allows this enzyme to function in the reverse direction and to serve as the dominant carboxylation step, conserving energy as ATP. With this engineered pathway, competing needs for PEP have been eliminated and net ATP production has been increased. PEP is boxed to indicate a common pool. Abbreviations: DHA, dihydroxyacetone; DHAP, dihydroxyacetone 3-phosphate; PEP, phosphoenolpyruvate; G3P, glycerol 3-phosphate; GA3P, glyceraldehydes 3-phosphate.Previous studies in our laboratory (16, 17, 36, 38) have engineered E. coli ATCC 8739 for the efficient production of succinate from glucose by recruiting genes from alternative pathways (36, 38). In this paper, we report the use of a similar approach to engineer strains for succinate production from glycerol in mineral salts medium. 相似文献
138.
Qingzhao Wang Mark S. Ou Y. Kim L. O. Ingram K. T. Shanmugam 《Applied and environmental microbiology》2010,76(7):2107-2114
During anaerobic growth of Escherichia coli, pyruvate formate-lyase (PFL) and lactate dehydrogenase (LDH) channel pyruvate toward a mixture of fermentation products. We have introduced a third branch at the pyruvate node in a mutant of E. coli with a mutation in pyruvate dehydrogenase (PDH*) that renders the enzyme less sensitive to inhibition by NADH. The key starting enzymes of the three branches at the pyruvate node in such a mutant, PDH*, PFL, and LDH, have different metabolic potentials and kinetic properties. In such a mutant (strain QZ2), pyruvate flux through LDH was about 30%, with the remainder of the flux occurring through PFL, indicating that LDH is a preferred route of pyruvate conversion over PDH*. In a pfl mutant (strain YK167) with both PDH* and LDH activities, flux through PDH* was about 33% of the total, confirming the ability of LDH to outcompete the PDH pathway for pyruvate in vivo. Only in the absence of LDH (strain QZ3) was pyruvate carbon equally distributed between the PDH* and PFL pathways. A pfl mutant with LDH and PDH* activities, as well as a pfl ldh double mutant with PDH* activity, had a surprisingly low cell yield per mole of ATP (YATP) (about 7.0 g of cells per mol of ATP) compared to 10.9 g of cells per mol of ATP for the wild type. The lower YATP suggests the operation of a futile energy cycle in the absence of PFL in this strain. An understanding of the controls at the pyruvate node during anaerobic growth is expected to provide unique insights into rational metabolic engineering of E. coli and related bacteria for the production of various biobased products at high rates and yields.In Escherichia coli as well as in other aerobic organisms, sugars such as glucose are metabolized in two separate steps: glycolysis, which converts glucose to pyruvate, and tricarboxylic acid (TCA) cycle enzymes, which oxidize acetyl coenzyme A (acetyl-CoA) to CO2 (5, 9). The pyruvate dehydrogenase complex (PDH) connects the glycolytic reactions to TCA cycle enzymes by catalyzing the production of acetyl-CoA from pyruvate. Because of its unique central role in metabolism, PDH is regulated at both the genetic and the biochemical level (7, 12, 27, 33, 34). The NADH generated during the complete oxidation of sugar is reoxidized to NAD+ by O2 through the respiratory electron transport pathway with accompanying energy production (11). Optimum coupling of these enzyme reactions helps to maintain the internal ratios of [NADH] to [NAD+] (redox balance) and of [ATP] to [ADP] plus [AMP] in order to support growth at the highest rate.The absence of O2 or another external electron acceptor during the growth of E. coli (anaerobic conditions) forces the bacterium to minimize the contribution of the TCA cycle enzymes to biosynthesis from catabolism (4, 14). Under these conditions, pyruvate or acetyl-CoA derived from pyruvate serves as the electron acceptor (reduced to lactate or ethanol, respectively) to maintain the redox balance. The enzymes responsible for redox balance in anaerobic E. coli are pyruvate formate-lyase (PFL), lactate dehydrogenase (LDH), and alcohol/aldehyde dehydrogenase (adhE; ADH-E). The main products of the fermentation of E. coli are a mixture of organic acids, such as acetate, lactate, and formate, in addition to ethanol (2, 4). Succinate, derived from phosphoenolpyruvate (PEP), is a minor product of fermentation and normally accounts for less than 5% of the total products produced from glucose by the culture.Anaerobic growth of E. coli, compared to aerobic growth, is also limited by energy, leading to an increase in glycolytic flux (19). The conversion of pyruvate to acetate and ethanol yields an additional ATP per glucose, suggesting that this would be the preferred route for pyruvate oxidation during anaerobic growth. This is accomplished by the PFL-dependent production of acetyl-CoA and further conversion to acetate (Fig. (Fig.1).1). This preference for PFL has been demonstrated `with several bacteria under carbon limitation conditions imposed either in a chemostat or in the presence of a poor carbon source (10, 20, 23). This additional ATP also elevates the ATP yield per glucose to 3, with an increase in the growth rate, and has been shown to be essential for the anaerobic growth of E. coli in xylose-mineral salts medium (13). The absence of this third ATP in a pfl mutant has been reported to increase glycolytic flux to lactate to compensate for this decrease in ATP yield per glucose (39). However, the flow of pyruvate carbon to acetate is tempered by the need to maintain redox balance, and this is achieved by the conversion of a second acetyl-CoA to ethanol by ADH-E. Under conditions of energy excess due to a declining growth rate, lactate production is expected to support redox balance maintenance without the additional ATP from the PFL-ADH-E pathway (Fig. (Fig.1).1). The production of this mixture of products in an appropriate ratio helps to maintain the redox balance under anaerobic conditions while also maximizing the ATP yield per glucose to support high growth rates and cell yields.Open in a separate windowFIG. 1.Anaerobic metabolic pathways of E. coli carrying the lpd101 mutation (PDH*).No PDH-based fermentation reaction to ethanol that can also help maintain cellular redox balance in an anaerobic cell has evolved in E. coli or other closely related bacteria. PDH activity is inhibited by NADH, normally found at higher levels in anaerobically growing cultures than in aerobic cultures (12, 18, 34, 35). Based on genome sequences available in GenBank, the genes encoding the components of PDH are not found in strictly anaerobic bacteria.We have recently described a mutation (lpd101) in the dihydrolipoamide dehydrogenase (LPD) of the PDH that allowed the enzyme to function in anaerobic cells (designated PDH* here) (17, 18). With this altered PDH*, an anaerobic cell can have three different pathways for pyruvate metabolism (Fig. (Fig.1).1). The three main enzymes that utilize pyruvate as a substrate, PDH*, PFL, and LDH, have different apparent Km values for pyruvate (0.4, 2.0, and 7.2 mM, respectively) (1, 18, 37, 41). PDH requires NAD+ for activity (apparent Km, 0.07 mM), while LDH is dependent on NADH (apparent Km, 0.2 mM) as the second substrate (18, 37).The PDH* serves as the first enzyme in a pathway that oxidatively decarboxylates pyruvate to acetyl-CoA and NADH, followed by reduction of the acetyl-CoA by alcohol dehydrogenase to ethanol in a two-step process using 2 NADHs (Fig. (Fig.1).1). The NADH produced during the conversion of glucose to acetyl-CoA dictates that the acetyl-CoA generated by PDH be used for redox balance (ethanol) and not for ATP generation (acetate), unless some of the NADH is used for biosynthesis by the growing cell (17). PDH* and LDH serve essentially the same physiological role in the cell, oxidizing NADH to support continued operation of glycolysis, although it is not readily apparent with PDH*. Although PDH* contributes to an increase in NADH pool, the redox balance is still maintained by coupling PDH* to NADH-dependent reduction of acetyl-CoA to ethanol by ADH-E (Fig. (Fig.1).1). This potential competition between LDH and PDH has been eliminated in the wild type through inhibition of the activity of PDH by NADH (12, 18, 32). However, the in vivo role of PDH* in a mutant that has all three pathways has not been investigated, since the flow of pyruvate through any of the three reactions during growth and postgrowth fermentation of sugars to products is expected to be dependent on the redox state, the ATP requirement, and other physiological conditions of the anaerobic cell. Using a combination of metabolic flux analysis and mutations in one or more of the genes encoding these enzymes, we have evaluated the flow of pyruvate carbon among the three potential pathways. The results are presented in this communication. 相似文献
139.
Leprosy is an infectious disease caused by Mycobacterium leprae. M. leprae has undergone a major reductive evolution leaving a minimal set of functional genes for survival. It remains non-cultivable. As M. leprae develops resistance against most of the drugs, novel drug targets are required in order to design new drugs. As most of the essential genes mediate several biosynthetic and metabolic pathways, the pathway predictions can predict essential genes. We used comparative genome analysis of metabolic enzymes in M. leprae and H. sapiens using KEGG pathway database and identified 179 non-homologues enzymes. On further comparison of these 179 non-homologous enzymes to the list of minimal set of 48 essential genes required for cell-wall biosynthesis of M. leprae reveals eight common enzymes. Interestingly, six of these eight common enzymes map to that of peptidoglycan biosynthesis and they all belong to Mur enzymes. The machinery for peptidoglycan biosynthesis is a rich source of crucial targets for antibacterial chemotherapy and thus targeting these enzymes is a step towards facilitating the search for new antibiotics. 相似文献