Ribonucleic acid–deoxyribonucleic acid hybridization in aqueous solutions and in solutions containing formamide

Hybridization in 6xSSC (SSC, 0.15m-sodium chloride-0.015m-sodium citrate) at 66 degrees C was compared with hybridization in formamide-6xSSC (1:1, v/v) at 35 degrees C. As expected, the RNA hybridization potential was labile in the former system and stable in the latter. DNA retention by filters was poor in the formamide system, but could be improved. Several other properties of the hybridization reaction were explored and it was concluded that the formamide system is generally superior.     

Role of fatty acid uptake and fatty acid β-oxidation in mediating insulin resistance in heart and skeletal muscle

Fatty acids are a major fuel source used to sustain contractile function in heart and oxidative skeletal muscle. To meet the energy demands of these muscles, the uptake and β-oxidation of fatty acids must be coordinately regulated in order to ensure an adequate, but not excessive, supply for mitochondrial β-oxidation. However, imbalance between fatty acid uptake and β-oxidation has the potential to contribute to muscle insulin resistance. The action of insulin is initiated by binding to its receptor and activation of the intrinsic protein tyrosine kinase activity of the receptor, resulting in the initiation of an intracellular signaling cascade that eventually leads to insulin-mediated alterations in a number of cellular processes, including an increase in glucose transport. Accumulation of fatty acids and lipid metabolites (such as long chain acyl CoA, diacylglycerol, triacylglycerol, and/or ceramide) can lead to alterations in this insulin signaling pathway. An imbalance between fatty acid uptake and oxidation is believed to be responsible for this lipid accumulation, and is thought to be a major cause of insulin resistance in obesity and diabetes, due to lipid accumulation and inhibition of one or more steps in the insulin-signaling cascade. As a result, decreasing muscle fatty acid uptake can improve insulin sensitivity. However, the potential role of increasing fatty acid β-oxidation in the heart or skeletal muscle in order to prevent cytoplasmic lipid accumulation and decrease insulin resistance is controversial. While increased fatty acid β-oxidation may lower cytoplasmic lipid accumulation, increasing fatty acid β-oxidation can decrease muscle glucose metabolism, and incomplete fatty acid oxidation has the potential to also contribute to insulin resistance. In this review, we discuss the proposed mechanisms by which alterations in fatty acid uptake and oxidation contribute to insulin resistance, and how targeting fatty acid uptake and oxidation is a potential therapeutic approach to treat insulin resistance.     

Dual localization of β-glucuronidase and acid phosphatase in lysosomes and in microsomes

Summary The dual localization of certain hydrolases in lysosomes and in endoplasmic reticulum as studied in enzyme staining reactions is now supported by cytobiochemical studies on mouse liver and kidney -glucuronidase and acid phosphatase. Use was made of the renal -glucuronidase response to endogenous androgen for both studies. Accordingly, sucrose homogenates were prepared of liver and kidney of male BALB/C mice previously injected with gonadotrophin along with control animals receiving saline instead. The homogenates were subjected to differential ultracentrifugation yielding six fractions. These were characterized as to their organelle composition by measurements of marker enzymes and by observations with the electron microscope. In all subcellular fractions, -glucuronidase was uniformly increased 5 to 8 times over the corresponding control value and, in fractions rich in lysosomes, this enzyme was easily released by alternate freezing and thawing. On the other hand, the microsomal -glucuronidase and acid phosphatase enzymes were not liberated by freezing and thawing nor were they after treatment with 0.1 % Triton X-100 and by employing other reagents and conditions which are known to release lysosomal enzymes. In contrast to microsomal acid phosphatase, microsomal -glucuronidase activity could be liberated by treatment with hyaluronidase. This soluble -glucuronidase showed the same optimum pH, Michaelis Constant and heat inactivation behavior as the lysosomal -glucuronidase prepared by freezing and thawing treatment. These observations define two populations of microsomal vesicles each identifiable by an individual membrane-associated acid hydrolase. One of these -glucuronidase, increases in specific activity in the animal on androgens and is released by hyaluronidase and the other, acid phosphatase, does not respond to androgen and is not released by hyaluronidase. There would appear to be a variety of mechanisms by which hydrolases enter into association with the membranes of the endoplasmic reticulum and from there, a variety of routes to the lysosomes. A comment is made concerning the question of acid phosphatases and -glucuronidase as enzyme markers for lysosomes in mouse kidney.Aided in part by Research Grant, P-106, of the American Cancer Society, Inc., New York, and by U.S.P.H.S. Grant CA-07538 and by a Research Career Award, CA-K6-18453 to William H. Fishman.     

Relationship between transport and metabolism of α-naphthaleneacetic acid, β-naphthaleneacetic acid and α-decalylacetic acid in segments of Coleus

Summary Transportand metabolism of -naphthaleneacetic acid -naphthaleneacetic acid, and -decalylacetic acid, all labelled with ¹⁴C in the carboxyl, group, were studied. Only -naphthaleneacetic acid is transported in a polar way. Most of the radioactivity in the tissue is in a low molecular form, either free or as immobilization products. The immobilization of -naphthaleneacetic acid is similar to that of -naphthaleneacetic acid. Immobilization of -decalylacetic acid is typically different. Bioassays showed -naphthaleneacetic acid as the sole biologically active component. It is concluded that stereo requirements necessary for biological activity are also required for polar auxin transport. It is further concluded that the observed specificity of the transport system is not related to the formation of immobilization products.     

Effectiveness of indoleacetic acid,indolebutyric acid and naphthaleneacetic acid during adventitious root formation in vitro in Malus ‘Jork 9’

The production of an antifungal spirostanol saponin designated SC-1 has been detected in cell suspension cultures of the Mexican species Solanum chrysotrichum. Batch cultures of a cell suspension obtained from hypocotyl derived calluses of this species were grown for 25 days in shake flasks containing Murashige & Skoog (MS) medium. Throughout the growth cycle, fresh and dry weight, SC-1 yield, and uptake of sucrose, glucose and fructose were determined. The effects of inoculum size and sucrose concentration on the biomass accumulation and synthesis of the active metabolite, were studied. The maximum SC-1 production, above 14 mg.g⁻¹ (which was fifty times that of field grown plants), was reached after 20 days using a 2% inoculum and complete MS medium supplemented with 2 mgl⁻¹ 2,4-D, 2 mg l⁻¹kinetin, and sucrose between 30 and 45 gl⁻¹. . This revised version was published online in June 2006 with corrections to the Cover Date.     

δ-Aminolaevulinic acid and amino acid neurotransmitters

Summary The effects of the porphyrin precursor -aminolaevulinic acid (ALA) on -aminobutyric acid (GABA) and L-glutamate transmitter systems was investigated in rat brain. It was found that ALA inhibited GABA and glutamate uptake and stimulated basal efflux of the amino acids in purified nerve endings. These effects were evident only at relatively high concentrations of ALA (at least 100 M). Such concentrations probably do not occur in the nervous systems of patients suffering from acute porphyria. In addition, it was found that ALA inhibited the stimulated release of GABA from nerve endings probably by acting as an agonist at GABA autoreceptors. This effect was found at very low concentrations of ALA (1 M). It is therefore likely that the neuropsychiatric manifestations of the acute porphyric attack are attributable, to some extent, to reduced GABA release at central synapses.     

Endogenous abscisic acid and 2-trans-abscisic acid in alternate bearing ‘Wilking’ mandarin trees

The relationship of abscisic acid (ABA) and 2-trans-abscisic acid (t-ABA) to alternate bearing has been examined in Wilking mandarin (Citrus reticulata Blanco) trees. Leaves, stems and buds of trees loaded with fruit (on trees) had 4.3, 6.0 and 2.2 fold higher ABA levels than the corresponding organs from off trees. Leaves had higher ABA levels than stems and buds in both on and off trees. t-ABA was non-detectable in Wilking leaf, stem and bud tissue. Amounts of t-ABA not exceeding 40% of the ABA content, were found in Shamouti and Valencia orange buds and in Wilking fruit peel.The elevated levels of ABA in on tree organs may reflect a stress imposed by the fruit overload.     

The influence of melatonin and N-acetylcysteine in δ-aminolevulinic acid and lead induced genotoxicity in lymphocytes in vitro

As is well known from earlier studies, the genotoxic effect of lead exposure was partly attributed to the formation of the highly reactive oxygen metabolites (ROMs) in the blood. However, lead ions have no ability to generate ROMs. Therefore, the recently published studies paid more attention to the role of δ-aminolevulinic acid (ALA) accumulation in lead-induced DNA damage. If the above-mentioned assumptions were taken into consideration, it seemed a reasonable approach to study the possible protective effects of antioxidants against genotoxic effects of lead. According to our results, N-acetylcysteine (NAC) and melatonin (MEL) were able to reduce significantly (p < 0.05) the lead- and ALA-induced sister chromatid exchange frequencies in human lymphocytes in vitro. In spite of a relative reduction in the lead- and ALA-induced micronucleus formation in human lymphocytes, the reduction was not statistically significant (p > 0.05). These results could be evaluated as supportive evidence for the hypothesis that increased antioxidant capacity of cells might fortify the efficiency of protective pathways against cytogenetic damage in lead exposure.     

Organic acid behavior in soils – misconceptions and knowledge gaps

Organic acids have been hypothesized to perform many functions in soil including root nutrient acquisition, mineral weathering, microbial chemotaxis and metal detoxification. However, their role in most of these processes remains unproven due to a lack of fundamental understanding about the reactions of organic acids in soil. This review highlights some of the knowledge gaps and misconceptions associated with the behavior of organic acids in soil with particular reference to low-molecular-weight organic acids (e.g., citrate, oxalate, malate) and plant nutrient acquisition. One major concern is that current methods for quantifying organic acids in soil may vastly underestimate soil solution concentrations and do not reveal the large spatial heterogeneity that may exist in their concentration (e.g., around roots or microbes). Another concern relates to the interaction of organic acids with the soil's solid phase and the lack of understanding about the relative importance of processes such as adsorption versus precipitation, and sorption versus desorption. Another major knowledge gap concerns the utilization of organic acids by the soil microbial community and the forms of organic acids that they are capable of degrading (e.g., metal-complexed organic acids, adsorbed organic acids etc). Without this knowledge it will be impossible to obtain accurate mathematical models of organic acid dynamics in soil and to understand their role and importance in ecosystem processes. Fundamental research on organic acids and their interaction with soil still needs to be done to fully elucidate their role in soil processes.     

Oleanolic acid and ursolic acid as potential inhibitors of human salivary α-amylase: insights from in vitro assays and in silico simulations

It is known that inhibiting α-amylase, an important enzyme in digestion of starch and glycogen, is a useful strategy for treating disorders in carbohydrate uptake. Two natural components distributed in many fruits and plants, oleanolic acid and ursolic acid, are endowed with important pharmacological activities and wide therapeutic possibilities. Until now, only a tiny fraction of their applications have been identified and exploited. Our in vitro inhibition studies demonstrated that oleanolic acid and ursolic acid non-competitively inhibit the activity and function of human salivary α-amylase. The molecular simulations revealed that oleanolic acid and ursolic acid interact with amino acid residues within the binding pocket of human salivary α-amylase, among which the side chain of Arg195 and Asp 197 was supposed to be important in imparting the inhibitory activity of triterpenoids. The present work will provide meaningful information for future development of functional drugs for the treatment of disorders in carbohydrate metabolism.

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Graphical abstract This work is valuable for providing a deeper insight into the interaction mechanism of oleanolic acid and ursolic acid with α-amylase

     

α-Keto acid elongation and glucosinolate biosynthesis in Arabidopsis thaliana

QTL mapping of glucosinolates in a RI population derived from an F₁ hybrid between the Arabidopsis thaliana ecotypes Columbia and Landsberg erecta identified a single major QTL coincident with the GSL-ELONG locus which regulates side chain elongation. Physical mapping and sequencing identified two members of an isopropylmalate synthase-like gene family within the region of maximum LOD score for the QTL and the GSL-ELONG non-recombinant region. These genes are prime candidates for regulating glucosinolate biosynthesis. Received: 19 November 1999 / Accepted: 16 December 1999     

Antioxidant defense and apoptotic effectors in ascorbic acid and β-glycerophosphate-induced osteoblastic differentiation

MC3T3-E1 cells grown in the presence of ascorbic acid and β-glycerophosphate (AA/β-GP) express alkaline phosphatase and produce an extensive collagenous extracellular matrix. Differentiated MC3T3-E1 cells are more sensitive to hydrogen peroxide-induced oxidative stress than undifferentiated cells. In this study, we compared the profile of antioxidant enzymes and molecular markers of apoptosis in undifferentiated and differentiated MC3T3-E1 cells (cell differentiation was induced by treatment with AA/β-GP). Differentiated osteoblasts showed lower expression and activity of catalase, glutathione S-transferase and glutathione peroxidase. The total superoxide dismutase activity and the expression of Cu/Zn superoxide dismutase were also lower, while the expression of Mn superoxide dismutase was higher in differentiated osteoblasts. The level of malondialdehyde, a widely used marker for oxidative stress, was lower in the AA/β-GP group compared with control cells, but this difference was not significant. Western blotting showed that treatment with AA/β-GP increased the Bax/Bcl-2 ratio used as an index of cellular vulnerability to apoptosis. In addition, the activities of caspases 3, 8 and 9 and cleaved poly (ADP) ribose polymerase were significantly higher in differentiated cells. These findings provide new insights into how changes in the activities of major antioxidant enzymes and in the signaling pathways associated with apoptosis may influence the susceptibility of bone cells to oxidative stress.     

Substitution of dietary oleic acid for myristic acid increases the tissue storage of α-linolenic acid and the concentration of docosahexaenoic acid in the brain, red blood cells and plasma in the rat

Various strategies have been developed to increase the cellular level of (n-3) polyunsaturated fatty acids in animals and humans. In the present study, we investigated the effect of dietary myristic acid, which represents 9% to 12% of fatty acids in milk fat, on the storage of α-linolenic acid and its conversion to highly unsaturated (n-3) fatty acid derivatives. Five isocaloric diets were designed, containing equal amounts of α-linolenic acid (1.3% of dietary fatty acids, i.e. 0.3% of dietary energy) and linoleic acid (7.0% of fatty acids, i.e. 1.5% of energy). Myristic acid was supplied from traces to high levels (0%, 5%, 10%, 20% and 30% of fatty acids, i.e. 0% to 6.6% of energy). To keep the intake of total fat and other saturated fatty acids constant, substitution was made with decreasing levels of oleic acid (76.1% to 35.5% of fatty acids, i.e. 16.7% to 7.8% of energy) that is considered to be neutral in lipid metabolism. After 8 weeks, results on physiological parameters showed that total cholesterol and low-density lipoprotein-cholesterol did not differ in the diets containing 0%, 5% and 10% myristic acid, but were significantly higher in the diet containing 30% myristic acid. In all the tissues, a significant increasing effect of the substitution of oleic acid for myristic acid was shown on the level of both α-linolenic and linoleic acids. Compared with the rats fed the diet containing no myristic acid, docosahexaenoic acid significantly increased in the brain and red blood cells of the rats fed the diet with 30% myristic acid and in the plasma of the rats fed the diet with 20% myristic acid. Arachidonic acid also increased in the brain of the rats fed the diet with 30% myristic acid. By measuring Δ6-desaturase activity, we found a significant increase in the liver of the rats fed the diet containing 10% of myristic acid but no effect at higher levels of myristic acid. These results suggest that an increase in dietary myristic acid may contribute in increasing significantly the tissue storage of α-linolenic acid and the overall bioavailability of (n-3) polyunsaturated fatty acids in the brain, red blood cells and plasma, and that mechanisms other than the single Δ6-desaturase activity are involved in this effect.     

Differences in properties between aromatic amino acid: Aromatic keto acid aminotransferases and aromatic amino acid: α-ketoglutarate aminotransferases

Homogenates of rat liver transaminate phenylpyruvate (PP), as well as α-ketoglutarate (α-KG), in the presence of L-tyrosine, 3,4-dihydroxyphenylalanine (L-DOPA) or L-tryptophan. Aminotransferase activity with phenylpyruvate and DOPA, but not with tyrosine, was inhibited by excess phenylpyruvate. Tyrosine and DOPA aminotransferase activities with phenylpyruvate were more heat stable than the corresponding activities with α-ketoglutarate. Aminotransferase activities with phenylpyruvate were not significantly induced following intraperitoneal injections of cortisol, glucagon or serotonin, compared with a 3 to 7-fold increase in the aminotransferase activities with α-ketoglutarate. Tyrosine:phenylpyruvate aminotransferase activity rose 40% at night, compared with a 300% increase in tyrosine:α-ketoglutarate aminotransferase activity. The results suggest that aminotransferases catalysing transfers between aromatic keto acids and aromatic amino acids are separate enzymes from those utilizing α-ketoglutarate as the acceptor keto acid.     

Effects of γ-rays on Poly-L-glutamic acid and Poly-D,L-glutamic acid in the solid state

Summary Poly-L-glutamic acid and poly-D,L-glutamic acid, as models of proteins, were irradiated with⁶⁰Co--radiation in air and under vacuo to examine whether or not the changes caused by the exposure to ionizing radiation depend on the conformations of polypeptides.It was found that theG- values (yield of main-chain scissions per 100 eV of energy absorbed) of both polypeptides are approximately equal for the irradiation in air, while under vacuo theG- value of poly-D,L-glutamic acid is larger than that of poly-L-glutamic acid. This observation for irradiation under vacuo was ascribed to the stabilizing effect of intramolecular hydrogen bond bridges in poly-L-glutamic acid. It was also found that the-helical structure of poly-L-glutamic acid is destroyed by the exposure to ionizing radiation.     

Conversion of 4-aminobutyraldehyde to γ-aminobutyric acid in striatum treated with semicarbazide and kainic acid

4-Aminobutyraldehyde (ABAL) has been shown to cross the blood-brain barrier and to be converted rapidly to -aminobutyric acid (GABA) in various regions of the brain. In this paper, the formation of GABA from ABAL was studied with striatum that had suffered a lesion to GABA synthesis via glutamic acid decarboxylase (GAD). The GABA formation from ABAL was invariably observed in striatum in which GAD was severely inhibited by semicarbazide or kainic acid. Thus, this is another pathway for GABA formation.     

Dietary linoleic acid has no effect on arachidonic acid,but increases n-6 eicosadienoic acid,and lowers dihomo-γ-linolenic and eicosapentaenoic acid in plasma of adult men

High intakes of linoleic acid (LA,18:2n-6) have raised concern due to possible increase in arachidonic acid (ARA, 20:4n-6) synthesis, and inhibition of alpha linolenic acid (ALA, 18:3n-3) desaturation to eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). In healthy men, 10.5% energy compared to 3.8% energy LA with 1% energy ALA increased plasma phospholipid LA and 20:2n-6, the elongation product of LA, and decreased EPA, with no change in ARA. However, LA was inversely related to ARA at both 10.5% energy and 3.8% energy LA, (r=?0.761, r=?0.817, p<0.001, respectively). A two-fold variability in ARA among individuals was not explained by the dietary LA, ARA, ALA, or fish intake. Our results confirm LA requirements for ARA synthesis is low, <3.8% energy, and they suggest current LA intakes saturate Δ-6 desaturation and adversely affect n-3 fatty acid metabolism. Factors other than n-6 fatty acid intake are important modifiers of plasma ARA.     

Does alpha-linolenic acid in combination with linoleic acid influence liver metastasis and hepatic lipid peroxidation in BOP-induced pancreatic cancer in Syrian hamsters?

Some fatty acids are reported to inhibit tumor growth of pancreatic carcinoma. However, it is still unknown if alpha-linolenic acid (ALA) and linoleic acid (LA) inhibit liver metastasis of ductal pancreatic adenocarcinoma. Therefore we studied the effect of these fatty acids on liver metastasis in the animal model of N-nitrosobis(2-oxopropyl)amine (BOP)-induced pancreatic adenocarcinoma in Syrian hamsters. Since lipid peroxidation seems to be involved in carcinogenesis and metastasis, we further analyzed the intrahepatic concentration of thiobarbituric acid-reactive substances (TBARS) and activity of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD).We observed an increase in the incidence and the number of liver metastases in response to the combination of ALA and LA. This was accompanied by a decrease in hepatic GSH-Px activity and an increase in hepatic SOD activity and TBARS concentration. The increase in hepatic lipid peroxidation seems to be one possible mechanism of increasing liver metastasis in this study.