Molecular View of the Interaction between iota-Carrageenan and a Phospholipid Film and Its Role in Enzyme Immobilization

Proteins incorporated into phospholipid Langmuir-Blodgett (LB) films are a good model system for biomembranes and enzyme immobilization studies. The specific fluidity of biomembranes, an important requisite for enzymatic activity, is naturally controlled by varying phospholipid compositions. In a model system, instead, LB film fluidity may be varied by covering the top layer with different substances able to interact simultaneously with the phospholipid and the protein to be immobilized. In this study, we immobilized a carbohydrate rich Neurospora crassa alkaline phosphatase (NCAP) in monolayers of the sodium salt of dihexadecylphosphoric acid (DHP), a synthetic phospholipid that provides very condensed Langmuir films. The binding of NCAP to DHP Langmuir-Blodgett (LB) films was mediated by the anionic polysaccharide iota-carrageenan (iota-car). Combining results from surface isotherms and the quartz crystal microbalance technique, we concluded that the polysaccharide was essential to promote the interaction between DHP and NCAP and also to increase the fluidity of the film. An estimate of DHP:iota-car ratio within the film also revealed that the polysaccharide binds to DHP LB film in an extended conformation. Furthermore, the investigation of the polysaccharide conformation at molecular level, using sum-frequency vibrational spectroscopy (SFG), indicated a preferential conformation of the carrageenan molecules with the sulfate groups oriented toward the phospholipid monolayer, and both the hydroxyl and ether groups interacting preferentially with the protein. These results demonstrate how interfacial electric fields can reorient and induce conformational changes in macromolecules, which may significantly affect intermolecular interactions at interfaces. This detailed knowledge of the interaction mechanism between the enzyme and the LB film is relevant to design strategies for enzyme immobilization when orientation and fluidity properties of the film provided by the matrix are important to improve enzymatic activity.

Insertion/deletion polymorphism of the angiotensin converting enzyme gene and loss of the insertion allele in aldosterone-producing adenoma

The genetic mechanisms responsible for the formation of adrenocortical adenomas which autonomously produce aldosterone are largely unknown, The adrenal renin-angiotensin system has been implicated in the pathophysiology of these tumours, Angiotensin-converting enzyme (ACE) catalyses the generation of angiotensin II, and the insertion/deletion (I/D) polymorphism of the ACE gene regulates up to 50% of plasma and cellular ACE variability in humans. We therefore examined the genotypic and allelic frequency distributions of the ACE gene I/D polymorphism in 55 patients with aldosterone-producing adenoma, APA, (angiotensin-unresponsive APA n = 28, angiotensin-responsive APA n = 27), and 80 control subjects with no family history of hypertension, We also compared the ACE gene I/D polymorphism allelic pattern in matched tumour and peripheral blood DNA in the 55 patients with APA, The frequency of the D allele was 0.518 and 0.512 and the I allele was 0.482 and 0.488 in the APA and control subjects respectively, Genotypic and allelic frequency analysis found no significant differences between the groups, Examination of the matched tumour and peripheral blood DNA samples revealed the loss of the insertion allele in four of the 25 patients who were heterozygous for the ACE I/D genotype. The I/D polymorphism of the ACE gene does not appear to contribute to the biochemical and phenotypic characteristics of APA, however, the deletion of the insertion allele of the ACE gene I/D polymorphism in 16% of aldosterone-producing adenomas may represent the loss of a tumour suppressor gene/s or other genes on chromosome 17q which may contribute to tumorigenesis in APA.

A fructan: fructan fructosyltransferase activity from Lolium rigidum

Fructan:fructan fructosyltransferase (FFT) activity was purified about 300-fold from leaves of Lolium rigidura Gaudin by a combination of affinity chromatography, gel filtration, anion exchange and isoelectric focusing. The FFT activity was free of sucrose:sucrose fructosyltransferase and invertase activities. It had an apparent pI of 4.7 as determined by isoelectric focusing, and a molecular mass of about 50000 (gel filtration). The FFT activity utilized the trisaccharides 1-kestose and 6(G)-kestose as sole substrates, but was not able to use 6-kestose as sole substrate. The FFT activity was not saturated when assayed at concentrations of 1-kestose, 6(G)-kestose or (1,1)-kestotetraose of up to 400 mM The rate of reaction of the FFT activity was most rapid when assayed with 1-kestose and was less rapid when assayed with 6(G)-kestose, (1,1)-kestotetraose or (1,1,1)-kestopentaose. The FFT activity when assayed at a relatively high concentration of enzyme activity (approximately equivalent to about half the activity in crude extracts per gram fresh mass) did not synthesize fructan of degree of polymerization > 6, even during extended assays of up to 10 h. When assayed with a combination of 1-kestose and uniformly labelled [C-14]sucrose as substrates, the major reaction was the transfer of a fructosyl residue from 1-kestose to sucrose resulting in the re-synthesis of 1-kestose. Tetrasaccharide and 6(G)-kestose were also synthesized. When assayed with 6(G)-kestose and [C-14]sucrose as substrates, the major reaction of the FFT activity was the synthesis of tetrasaccharide. However, some synthesis of 1-kestose and re-synthesis of 6(G)-kestose also occurred. When 6, kestose was the sole substrate for the FFT activity, synthesis of tetrasaccharide was 2.7 to 3.4-fold slower than when 1-kestose was used as the sole substrate. Owing to differences in the fructan:sucrose fructosyltransferase activity of the FFT with each of the trisaccharides, net synthesis of tetrasaccharide by the FFT was altered significantly in the presence of sucrose. The magnitude of this effect depended on the concentration of the trisaccharides. In the presence of sucrose, 6(G)-kestose could be a substrate of equivalent importance to 1-kestose for synthesis of tetrasaccharide.

High sucrose intake in rats is associated with increased ACE2 and angiotensin-(1-7) levels in the adipose tissue

Sucrose-fed rats, a model of metabolic syndrome, are characterized by insulin resistance, obesity, hypertension, and high plasma levels of triacylglycerols and angiotensin II (Ang II). However, whether tissue renin-angiotensin system (RAS) is altered in metabolic syndrome is unclear. To study this issue, food ad libitum and water (C) or 20% sucrose solution (SC) were given to adult male Wistar rats, for 30 days. Body weight (BW), blood pressure (BP), epididymal adipose tissue (EPI) mass, rate of in vivo fatty acid (FA) synthesis in EPI, circulating glucose, insulin, leptin, angiotensins I and II, triacylglycerols, and plasma renin (PRA) and angiotensin-converting enzyme (ACE) activities were evaluated. In kidneys and EPI, gene and protein expression of type 1 (AT(1)) and 2 (AT(2)) Ang II receptors, ACE, angiotensinogen (ACT) as well as protein expression of angiotensin-converting enzyme 2 (ACE2) were determined. In both tissues, Ang I, Ang II and Ang-(1-7) contents were also measured by HPLC. In SC rats higher BP, EPI mass, circulating triacylglycerols, insulin, leptin, PRA and, Ang II were found. In EPI, the rate of in vivo FA synthesis was associated with increased Ang-(1-7), protein expression of AT(1) and AT(2) receptors, ACE2, ACT, and gene expression of ACT although a reduction in ACE activity and in adipose Ang I and Ang II contents was observed. In kidneys, AT(1) and AT(2), ACE and ACT gene and protein expression as well as protein expression of ACE2 were unaltered while Ang II, Ang-(1-7) and ACE activity increased. These RAS component changes seem to be tissue specific and possibly are related to enhancement of FA synthesis, EPI mass and hypertension. (C) 2010 Elsevier B.V. All rights reserved.

Characterisation of five missense mutations in the cystathionine beta-synthase gene from three patients with B-6-nonresponsive homocystinuria

Homocystinuria, due to a deficiency of the enzyme cystathionine beta-synthase (CBS), is an inborn error of sulphur-amino acid metabolism, This is an autosomal recessive disease which results in hyperhomocysteinaemia and a wide range of clinical features, including optic lens dislocation, mental retardation, skeletal abnormalities and premature thrombotic events, We report the identification of 5 missense mutations in the protein-coding region of the CBS gene from 3 patients with pyridoxine-nonresponsive homocystinuria. Reverse-transcription PCR was used to amplify CBS cDNA from each patient and the coding region was analysed by direct sequencing, The mutations detected included 3 novel (1058C --> T, 992C --> A and 1316G --> A) and 2 previously identified (430G --> A and 833C --> T) base alterations in the CBS cDNA, Each of these mutations predicts a single amino acid substitution in the CBS polypeptide, Appropriate cassettes of patient CBS cDNA, containing each of the above defined mutations, were used to replace the corresponding cassettes of normal CBS cDNA sequence within the bacterial expression vector pT7-7. These recombinant mutant and normal CBS constructs were expressed in Escherichia coli cells and the catalytic activities of the mutant proteins were compared with normal. All of the mutant proteins exhibited decreased catalytic activity in vitro, which confirmed the association between the individual mutation and CBS dysfunction in each patient.

Aerobic Exercise Training-Induced Left Ventricular Hypertrophy Involves Regulatory MicroRNAs, Decreased Angiotensin-Converting Enzyme-Angiotensin II, and Synergistic Regulation of Angiotensin-Converting Enzyme 2-Angiotensin (1-7)

Aerobic exercise training leads to a physiological, nonpathological left ventricular hypertrophy; however, the underlying biochemical and molecular mechanisms of physiological left ventricular hypertrophy are unknown. The role of microRNAs regulating the classic and the novel cardiac renin-angiotensin (Ang) system was studied in trained rats assigned to 3 groups: (1) sedentary; (2) swimming trained with protocol 1 (T1, moderate-volume training); and (3) protocol 2 (T2, high-volume training). Cardiac Ang I levels, Ang-converting enzyme (ACE) activity, and protein expression, as well as Ang II levels, were lower in T1 and T2; however, Ang II type 1 receptor mRNA levels (69% in T1 and 99% in T2) and protein expression (240% in T1 and 300% in T2) increased after training. Ang II type 2 receptor mRNA levels (220%) and protein expression (332%) were shown to be increased in T2. In addition, T1 and T2 were shown to increase ACE2 activity and protein expression and Ang (1-7) levels in the heart. Exercise increased microRNA-27a and 27b, targeting ACE and decreasing microRNA-143 targeting ACE2 in the heart. Left ventricular hypertrophy induced by aerobic training involves microRNA regulation and an increase in cardiac Ang II type 1 receptor without the participation of Ang II. Parallel to this, an increase in ACE2, Ang (1-7), and Ang II type 2 receptor in the heart by exercise suggests that this nonclassic cardiac renin-angiotensin system counteracts the classic cardiac renin-angiotensin system. These findings are consistent with a model in which exercise may induce left ventricular hypertrophy, at least in part, altering the expression of specific microRNAs targeting renin-angiotensin system genes. Together these effects might provide the additional aerobic capacity required by the exercised heart. (Hypertension. 2011;58:182-189.).

AP-1 and Egr DNA-binding activities are increased in rat brain during ethanol withdrawal

The DNA-binding activities of AP-1 and Egr proteins were investigated in nuclear extracts of rat brain regions during ethanol withdrawal. Both DNA-binding activities were transiently elevated in the hippocampus and cerebellum 16 h after withdrawal. In the cerebral cortex, AP-1 and Egr DNA-binding activities increased at 16 h and persisted until 32 and 72 h, respectively. The AP-1 DNA-binding activities in all regions at all times after withdrawal were composed of FosB, c-Jun, JunB, and JunD. c-Fos was detected at all times in the cerebral cortex, at 16 h only in the hippocampus, and from 16 to 72 h in the cerebellum. Withdrawal severity did not affect the composition of the AP-1 DNA-binding activities. Two Egr DNA-binding activities were present in the cortex and hippocampus. The faster-migrating complex predominated in hippocampus, and only the slower-migrating complex (identified as Egr-1) was present in the cerebellum. The increase in DNA-binding activity of immediate early gene-encoded transcription factors supports their proposed role in initiating a cascade of altered gene expression underlying the long-term neuronal response to ethanol withdrawal.

Differential expression of Egr-1-like DNA-binding activities in the naive rat brain and after excitatory stimulation

Egr-1 and related proteins are inducible transcription factors within the brain recognizing the same consensus DNA sequence. Three Egr DNA-binding activities were observed in regions of the naive rat brain. Egr-1 was present in all brain regions examined. Bands composed, at least in part, of Egr-2 and Egr-3 were present in different relative amounts in the cerebral cortex, striatum, hippocampus, thalamus, and midbrain. All had similar affinity and specificity for the Egr consensus DNA recognition sequence. Administration of the convulsants NMDA, kainate, and pentylenetetrazole differentially induced Egr-1 and Egr-2/3 DNA-binding activities in the cerebral cortex, hippocampus, and cerebellum. All convulsants induced Egr-1 and Egr-2 immunoreactivity in the cerebral cortex and hippocampus. These data indicate that the members of the Egr family are regulated at different levels and may interact at promoters containing the Egr consensus sequence to fine tune a program of gene expression resulting from excitatory stimuli.

Centella asiatica water extract inhibits iPLA(2) and cPLA(2) activities in rat cerebellum

Centella asiatica (L.) Urb an is distributed widely in South America and Asia and is known as a therapeutic agent in folk medicine, capable of improving memory and treating several neurological disorders. Asiaticoside is one of the compounds found in C asiatica leaves that is suggested to be responsible for its pharmacological potential. Phospholipase A(2) (PLA(2)) is a group of enzymes that has abnormal activity in the central nervous system in some neuropsychiatric diseases. In this work, the asiaticoside present in C asiatica water extract was quantified by HPLC analysis. We also evaluated the activity of subtypes of PLA(2) in cerebellar samples from rats after C asiatica water extract treatment using a radioenzymatic assay. Asiaticoside was the major compound (84%) found in Centella water extract. We found a dose-dependent inhibitory effect of C asiatica water extract on the activity of Ca(2+)-independent PLA(2) (iPLA(2)) and cytosolic PLA(2) (cPLA(2)). The inhibition of these enzymes in the brain suggests that C asiatica may be useful to treat conditions associated with increased PLA(2) activity in the brain, such as epilepsy, stroke, multiple sclerosis and other neuropsychiatric disorders. (C) 2008 Elsevier GmbH. All rights reserved.

Landholder Attitudes to and Participation in Farm Forestry Activities in Sub-tropical and Tropical Eastern Australia

‘This book is a landmark opening and first attempt at such a process for defining farm forestry, as well as making a contribution to small-scale forestry.’