bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure, linkage mapping and expression of the heart‐type fatty acid‐binding protein gene (fabp3 ) from zebrafish (Danio rerio)
Authors: Liu Rong‐Zong , Denovan‐Wright Eileen M. , Wright Jonathan M.
Published: July 2003

We have determined the cDNA nucleotide sequence, deduced the amino acid sequence and defined the gene structure for the cellular heart‐type (H‐FABP) or fatty acid‐binding protein 3 (FABP3) from zebrafish. The zebrafish FABP3 exhibited the greatest amino acid sequence identity to fish and mammalian heart‐type FABPs. 3′ RACE and 5′ RLM‐RACE mapped two alternative polyadenylation sites and three transcription start sites, respectively. Southern blot and hybridization analysis indicated that a single fabp3 gene exists in the zebrafish genome. The zebrafish fabp3 gene consists of four exons interrupted by three introns with identical exon/intron structure and coding capacity with that of orthologous mammalian H‐FABP genes. Radiation hybrid mapping assigned the zebrafish fabp3 gene to linkage group 19 of the zebrafish genome. Comparative genomic analysis revealed conserved syntenies of the zebrafish fabp3 gene and the orthologous human and mouse fabp3 genes. Northern blot analysis detected an mRNA transcript of 780 nucleotides. In situ hybridization of the zebrafish fabp3‐specific oligonucleotide probe to tissue sections of adult zebrafish revealed that the fabp3 mRNA was localized in the ovary and liver, but not in the heart, muscle or brain as reported for the mammalian fabp3 gene transcript. RT‐PCR, however, detected zebrafish fabp3 mRNA in all the tissues examined. Emulsion autoradiography further revealed that the zebrafish fabp3 mRNA was most abundant in primary growth stage (stage I) oocytes and decreased during the oocyte growth phase. The fabp3 mRNA levels were reduced and restricted to the ooplasm of cortical alveolus stage (stage II) oocytes, and nearly undetectable in stage III and matured oocytes. Inspection of the 5′ upstream sequence of the zebrafish fabp3 gene revealed a number of cis elements that may be involved in the expression of the zebrafish fabp3 gene in oocytes and liver.
Comparative analysis of the site‐specific N‐glycosylation of human lactoferrin produced in maize and tobacco plants
Authors: Samyn‐Petit Bénédicte , Wajda Dubos Jean‐Pierre , Chirat Frédéric , Coddeville Bernadette , Demaizieres Grégory , Farrer Sybille , Slomianny Marie‐Christine , Theisen Manfred , Delannoy Philippe
Published: July 2003

We have compared the site‐by‐site N‐glycosylation status of human lactoferrin (Lf) produced in maize, a monocotyledon, and in tobacco, used as a model dicotyledon. Maize and tobacco plants were stably transformed and recombinant Lf was purified from both seeds and leaves. N‐glycopeptides were generated by trypsin digestion of recombinant Lf and purified by reverse‐phase HPLC. The N‐glycosylation pattern of each site was determined by mass spectrometry. Our results indicated that the N‐glycosylation patterns of recombinant Lf produced in maize and tobacco share common structural features. In particular, both N‐glycosylation sites of each recombinant Lf are mainly substituted by typical plant paucimannose‐type N‐glycans, with β1,2‐xylose and α1,3‐linked fucose at the proximal N‐acetylglucosamine. However, tobacco Lf shows a significant amount of processed N‐glycans with one or two β1,2GlcNAc linked to the trimannose core, which are weakly expressed in maize Lf. Finally, no Lewisa epitope was observed on tobacco Lf.
Assembly of collagen types II, IX and XI into nascent hetero‐fibrils by a rat chondrocyte cell line
Authors: Fernandes Russell J. , Schmid Thomas M. , Eyre David R.
Published: July 2003

The cell line, RCS‐LTC (derived from the Swarm rat chondrosarcoma), deposits a copious extracellular matrix in which the collagen component is primarily a polymer of partially processed type II N‐procollagen molecules. Transmission electron microscopy of the matrix shows no obvious fibrils, only a mass of thin unbanded filaments. We have used this cell system to show that the type II N‐procollagen polymer nevertheless is stabilized by pyridinoline cross‐links at molecular sites (mediated by N‐ and C‐telopeptide domains) found in collagen II fibrils processed normally. Retention of the N‐propeptide therefore does not appear to interfere with the interactions needed to form cross‐links and mature them into trivalent pyridinoline residues. In addition, using antibodies that recognize specific cross‐linking domains, it was shown that types IX and XI collagens, also abundantly deposited into the matrix by this cell line, become covalently cross‐linked to the type II N‐procollagen. The results indicate that the assembly and intertype cross‐linking of the cartilage type II collagen heteropolymer is an integral, early process in fibril assembly and can occur efficiently prior to the removal of the collagen II N‐propeptides.
Identification of different isoforms of eEF1A in the nuclear fraction of human T‐lymphoblastic cancer cell line specifically binding to aptameric cytotoxic GT oligomers
Authors: Dapas Barbara , Tell Gianluca , Scaloni Andrea , Pines Alex , Ferrara Lino , Quadrifoglio Franco , Scaggiante Bruna
Published: July 2003

GT oligomers, showing a dose‐dependent cytotoxic effect on a variety of human cancer cell lines, but not on normal human lymphocytes, recognize and form complexes with nuclear proteins. By working with human T‐lymphoblastic CCRF‐CEM cells and by using MS and SouthWestern blotting, we identified eukaryotic elongation factor 1 alpha (eEF1A) as the main nuclear protein that specifically recognizes these oligonucleotides. Western blotting and supershift assays confirmed the nature of this protein and its involvement in forming a cytotoxicity‐related complex (CRC). On the contrary, normal human lymphocytes did not show nuclear proteins able to produce CRC in a SouthWestern blot. Comparative bidimensional PAGE and Western‐blotting analysis for eEF1A revealed the presence of a specific cluster of spots, focusing at more basic pH, in nuclear extracts of cancer cells but absent in those of normal lymphocytes. Moreover, a bidimensional PAGE SouthWestern blot demonstrated that cytotoxic GT oligomers selectively recognized the more basic eEF1A isoform expressed only in cancer cells. These results suggest the involvement of eEF1A, associated with the nuclear‐enriched fraction, in the growth and maintenance of tumour cells, possibly modulated by post‐translational processing of the polypeptide chain.
Solution structure of the matrix attachment region‐binding domain of chicken MeCP2
Authors: Heitmann Björn , Maurer Till , Weitzel Joachim M. , Strätling Wolf H. , Kalbitzer Hans Robert , Brunner Eike
Published: July 2003

Methyl‐CpG‐binding protein 2 (MeCP2) is a multifunctional protein involved in chromatin organization and silencing of methylated DNA. MAR‐BD, a 125‐amino‐acid residue domain of chicken MeCP2 (cMeCP2, originally named ARBP), is the minimal protein fragment required to recognize MAR elements and mouse satellite DNA. Here we report the solution structure of MAR‐BD as determined by multidimensional heteronuclear NMR spectroscopy. The global fold of this domain is very similar to that of rat MeCP2 MBD and MBD1 MBD (the methyl‐CpG‐binding domains of rat MeCP2 and methyl‐CpG‐binding domain protein 1, respectively), exhibiting a three‐stranded antiparallel β‐sheet and an α‐helix α1. We show that the C‐terminal portion of MAR‐BD also contains an amphipathic helical coil, α2/α3. The hydrophilic residues of this coil form a surface opposite the DNA interface, available for interactions with other domains of MeCP2 or other proteins. Spectroscopic studies of the complex formed by MAR‐BD and a 15‐bp fragment of a high‐affinity binding site from mouse satellite DNA indicates that the coil is also involved in protein·DNA interactions. These studies provide a basis for discussion of the consequences of six missense mutations within the helical coil found in Rett syndrome cases.
Physicochemical characterization and biological activity of a glycoglycerolipid from Mycoplasma fermentans
Authors: Brandenburg Klaus , Wagner Frauke , Müller Mareike , Heine Holger , Andrä Jörg , Koch Michel H. J. , Zähringer Ulrich , Seydel Ulrich
Published: July 2003

We report a comprehensive physicochemical characterization of a glycoglycerolipid from Mycoplasma fermentans, MfGl‐II, in relation to its bioactivity and compared this with the respective behaviors of phosphatidylcholine (PC) and a bacterial glycolipid, lipopolysaccharide (LPS) from deep rough mutant Salmonella minnesota strain R595. The β⇆α gel‐to‐liquid crystalline phase transition behavior of the hydrocarbon chains with Tc = 30 °C for MfGl‐II as well as for LPS exhibits high similarity between the two glycolipids. A lipopolysaccharide‐binding protein (LBP)‐mediated incorporation into negatively charged liposomes is observed for both glycolipids. The determination of the supramolecular aggregate structure confirms the existence of a mixed unilamellar/cubic structure for MfGl‐II, similar to that observed for the lipid A moiety of LPS. The biological data clearly show that MfGl‐II is able to induce cytokines such as tumor necrosis factor‐α (TNF‐α) in human mononuclear cells, although to a significantly lower degree than LPS. In contrast, in the Limulus amebocyte lysate test, MfGl‐II is completely inactive, and in the CHO reporter cell line it does not indicate any reactivity with the Toll‐like receptors TLR‐2 and ‐4, in contrast to control lipopeptides and LPS. These data confirm the applicability of our conformational concept of endotoxicity to nonlipid A structures: an amphiphilic molecule with a nonlamellar cubic aggregate structure corresponding to a conical conformation of the single molecules and a sufficiently high negative charge density in the backbone.
Moult cycle‐related changes in biological activity of moult‐inhibiting hormone (MIH) and crustacean hyperglycaemic hormone (CHH) in the crab, Carcinus maenas
Authors: Chung J. Sook , Webster Simon G.
Published: July 2003

The currently accepted model of moult control in crustaceans relies entirely on the hypothesis that moult‐inhibiting hormone (MIH) and crustacean hyperglycaemic hormone (CHH) repress ecdysteroid synthesis of the target tissue (Y‐organ) only during intermoult, and that changes in synthesis and/or release of these neurohormones are central to moult control. To further refine this model, we investigated the biological activities of these neuropeptides in the crab Carcinus maenas, at the target tissue, receptor and cellular level by bioassay (inhibition of ecdysteroid synthesis), radioligand (receptor) binding assays, and second messenger (cGMP) assays, at defined stages of the moult cycle. To investigate possible moult cycle‐related changes in neuropeptide biosynthesis, steady‐state transcript levels of both neuropeptide mRNAs were measured by quantitative RT‐PCR, and stored neuropeptide levels in the sinus gland were quantified during intermoult and premoult. The results show that the most important level of moult control lies within the signalling machinery of the target tissue, that expression and biosynthesis of both neuropeptides is constant during the moult cycle, and are not central to the currently accepted model of moult control.
Magnesium balance in adult cats fed a dry food rich in lactose
Authors: Beynen A. C. , Yu S.
Published: July 2003

The question addressed was whether lactose consumption would stimulate apparent magnesium absorption in adult cats. In a cross‐over study, eight cats were fed dry diets without or with 10% lactose, which was added at the expense of the glucose component. Urine and faeces were quantitatively collected and the balance of magnesium, calcium and phosphorus was determined. Lactose feeding did not affect mineral absorption. The dry matter content of faeces was unchanged after lactose consumption. Urinary excretion of galactose represented 0.2% of the intake. It is concluded that lactose ingestion at a level of 1.2 g/kg body weight has no effect on mineral absorption and on the consistency of faeces in adult cats.
Taurine concentrations in animal feed ingredients; cooking influences taurine content
Authors: Spitze A. R. , Wong D. L. , Rogers Q. R. , Fascetti A. J.
Published: July 2003

The aim of this study was to determine the taurine content in a variety of animal feeds. There is very little information on the taurine content of ingredients used in home‐prepared diets for dogs and cats, and foods fed to wild animals in captivity. This study reports the taurine content of both common and alternative feed ingredients, and compares taurine loss as a result of different methods of food preparation. Foods were selected based on their use in commercial and home‐prepared diets. Animal muscle tissue, particularly marine, contained high taurine concentrations. Plant products contained either low or undetectable amounts of taurine. The amount of taurine that remained in a feed ingredient after cooking depended upon the method of food preparation. When an ingredient was constantly surrounded by water during the cooking process, such as in boiling or basting, more taurine was lost. Food preparation methods that minimized water loss, such as baking or frying, had higher rates of taurine retention.
Short‐term effect of acarbose on specific intestinal disaccharidase activities and hyperglycaemia in CBA diabetic mice
Authors: Juretić D. , Bernik Š. , Čop L. , Hadžija M. , Petlevski R. , Lukač‐Bajalo J.
Published: July 2003

The purpose of this study was to examine the short‐term effects of 75, 100 and 150 mg of acarbose mixed in 100 g standard laboratory chow on specific intestinal disaccharidase activities and on hyperglycaemia in diabetic CBA strain mice on standard diet. The small intestine was excised and divided into three segments, from pylorus to duodenum, and two equal lengths of the jejunum and ileum of control and diabetic mice with or without added acarbose. Specific maltase and sucrase activities were determined using maltose and sucrose as substrates respectively. Increased specific activities of maltase and sucrase were detected in the intestines of CBA mice on standard laboratory diet seven days after alloxan‐induced diabetes. Feeding for 7 days with 75, 100 or 150 mg acarbose uniformly mixed in 100 g standard laboratory chow, induced a decrease in the specific maltase and sucrase activities, compared with diabetic mice on standard laboratory diet. Feeding with 75 mg acarbose mixed in 100 g standard laboratory chow caused a statistically significant decrease of maltase in the duodenum and of sucrase in duodenum and jejunum, without a antihyperglycaemic effect. Feeding with 100 or 150 mg caused statistically significant decreases in specific maltase and sucrase activities in duodenum, jejunum and ileum. An antihyperglycaemic effect was observed only in the group of diabetic mice fed with 100 mg acarbose. This indicates that the antihyperglycaemic effect of acarbose involves factors other than these, related only to its inhibitory effect on disaccharidase activities.
Effect of apparent ileal digestible lysine to energy ratio on performance of finishing pigs at different dietary metabolizable energy levels
Authors: Ettle T. , Roth‐Maier D. A. , Roth F. X.
Published: July 2003

A study with a total of 96 crossbred barrows and gilts fed ad libitum in a body weight range of 56–110 kg was carried out in order to investigate whether the optimum ratio of apparent ileal digestible lysine : energy (ME) depends on the energy density of the diet. Dietary treatments were ratios of 0.34, 0.42, 0.50 and 0.58 g digestible lysine/MJ ME either at an energy density of 13 or 14 MJ ME. Body weight gain as well as feed and ME conversion rate were improved when lysine : ME ratio increased from 0.34 to 0.50, whereby all parameters showed a significant ratio × sex interaction. Feed intake and feed conversion rate were higher at a dietary energy density of 13 MJ ME than at an energy density of 14 MJ ME but energy density did not influence daily growth rate, ME intake and ME conversion rate. Fat area above eye muscle and meat : fat ratio were lower and eye muscle area and lean percentage were higher at a ratio of 0.42 compared with a ratio of 0.34. Daily feed intake, body weight gain, feed conversion rate and parameters of fatness were higher in barrows than in gilts. Conversely, gilts had higher eye muscle area and lean percentage. Optimum ratio for body weight gain, feed and ME conversion rate calculated by exponential regression analysis were 0.42–0.43 in barrows. In gilts, feed and ME conversion rate were optimized at a ratio of 0.53 and 0.54. Because growth showed a linear response to increasing digestible lysine : ME ratios, optimum ratio for daily gain in gilts is considered to be at least 0.58.
Zur Notwendigkeit eines Überschusses in der Stickstoffbilanz im Pansen von Kühen mit hoher Milchleistung
Authors: Kluth H. , Engelhard T. , Rodehutscord M.
Published: July 2003

Die Proteinversorgung der Milchkuh basiert in Deutschland auf den Kenngrößen des nutzbaren Rohproteins am Darm (nXP) und der ruminalen N‐Bilanz (RNB). Praxisübliche Empfehlungen weisen eine positive RNB von bis zu 50 g N/d aus. Dieser Überschuß ist aus wissenschaftlicher Sicht in Frage zu stellen. Es sollte daher geklärt werden, ob bei Kühen mit einer sehr hohen Milchleistung eine ausgeglichene RNB ausreichend ist. Es wurden 3 Rationen mit gleichen Gehalten an Nettoenergie für Laktation (NEL) (7,2 MJ/kg Trockensubstanz; T) und nXP (173 g/kg T) in der Höhe der RNB variiert. Die Grundfutter (Mais‐, Gras‐, Lieschkolbenschrot‐ und Biertrebersilage) blieben im Rationsanteil unverändert. Die Variation im RNB‐Gehalt wurde entweder durch den Einsatz von Harnstoff oder durch Variation beim Einsatz von Proteinträgern vorgenommen. Alle Rationen wurden als TMR vorgelegt. Je Behandlung wurden mindestens 35 Milchkühe vom 21.‐98. Laktationstag geprüft. Die Tiere wurden im Laufstall gehalten und dreimal täglich gemolken. Zusätzlich wurde der ME‐Gehalt der Rationen in einer Verdaulichkeitsbestimmung mit Hammeln ermittelt und eine chemische Fraktionierung des Rohproteins vorgenommen. Die Mittelwerte für die drei Behandlungen betrugen für die Futteraufnahme 20,1 bis 21,7 kg T/Tag, für die Milchmenge 43,0 bis 44,0 kg/Tag und für die Eiweißmenge 1,39 bis 1,42 kg/Tag. In keinem der untersuchten Kriterien ergab sich ein signifikanter Einfluss der Behandlung. Es wird geschlussfolgert, dass eine positive RNB in der Milchkuhfütterung nicht notwendig ist, wenn die nXP‐Versorgung bedarfsdeckend ist.
The rat as a model animal for the growing pig in determining ileal amino acid digestibility in soya and milk proteins
Authors: Rutherfurd S. M. , Moughan P. J.
Published: July 2003

The growing pig is an established model animal for humans to determine protein digestibility to the end of the small intestine. An ileal amino acid digestibility assay based on the laboratory rat offers an alternative. True ileal amino acid digestibility in high quality refined protein sources was determined and compared between the laboratory rat and the growing pig. Both species of animals were fed their daily ration in nine separate meals. Ileal digesta were collected at slaughter and chromic oxide was used as an indigestible marker. For several of the amino acids examined, there were no significant (p < 0.05) differences between the two species for true ileal amino acid digestibility, but for aspartic acid, serine, cysteine, methionine, tyrosine, phenylalanine and histidine, significant (p < 0.05) interspecies differences were found. The overall average amino acid digestibility, for all amino acids and all diets, was 95.8% for the pig and 96.1% for the rat. Cysteine digestibility was highly variable between individual animals fed the same diet. The extent of amino acid digestion and absorption appears to be broadly similar across the two species, but some important differences in digestibility were recorded.
Reproductive performance of rats supplemented with L‐carnitine
Authors: Brandsch C. , Eder K.
Published: July 2003

It has been shown previously that supplementation of sows with l‐carnitine increases their reproduction performance. The current study was carried out to investigate if feeding of l‐carnitine also affects the reproduction performance of rats. Thirty female rats at 4 weeks of age were divided into two groups. The rats were fed diets with or without l‐carnitine (1 g/kg diet) over a period of 34 weeks. After 8 weeks of feeding, the female rats were mated the first time. Two more reproductive cycles followed, with 3‐week intervals in between. Body weight development of the females was similar in both groups during the whole experimental period. Number of pregnancies and number of total rat pups, pups born alive and stillborn pups were not influenced by l‐carnitine. There was also no influence of dietary l‐carnitine on the body weight of individual pups and the litter weights at birth. Weight development of litters differed between both groups on several days, but no uniform effect of l‐carnitine was observed. Body weight development of weaned rats fed a commercial diet was different between both groups, but only in one reproduction cycle. In conclusion, this study shows that l‐carnitine supplementation does not improve the reproductive performance of rats.
Molecular basis of opioid dependence: role of signal regulation by G‐proteins
Authors: Tso Prudence H , Wong Yung H
Published: July 2003

1. Morphine and opiate narcotics are potent analgesics that have a high propensity to induce tolerance and physical dependence following their repeated administration.

2. The molecular basis of opiate dependence has not been completely elucidated, although the participation of opioid receptors is a prerequisite. Cellular dependence on opioids is believed to result from the chronic stimulation of opioid‐regulated signalling networks.

3. As G‐protein‐coupled receptors, the opioid receptors must rely on heterotrimeric G‐proteins for signal transduction. Recent advances in our understanding of G‐protein signalling have unveiled novel signalling molecules and mechanisms, some of which may be intricately involved in the manifestation of opiate dependence.

4. In the present review, we will attempt to trace chronic opioid signals along elaborate G‐protein‐regulated pathways.
Pressure‐independent enhancement of cardiac hypertrophy in atrial natriuretic peptide–deficient mice
Authors: Feng Ji An , Perry Gilbert , Mori Tatsuhiko , Hayashi Tetsuya , Oparil Suzanne , Chen Yiu‐Fai
Published: July 2003

1. Homozygous deletion of the pro‐atrial natriuretic peptide (Nppa) gene (ANP–/–) has been associated with both cardiac hypertrophy and salt‐sensitive hypertension in mice, suggesting that cardiac hypertrophy in ANP–/– mice may be related, at least in part, to increased afterload.

2. To test the hypothesis that cardiac hypertrophy in ANP–/– mice is independent of blood pressure, male ANP–/– and wild‐type ANP+/+ mice were fed a low (0.05%) or basal (0.55%) NaCl diet. Five weeks later, mean arterial pressure (MAP) was measured in conscious mice; the whole heart, atria, left and right ventricles (LV and RV, respectively), brain, lung, kidney, liver and spleen were weighed and fixed for histological analysis. Separate groups of mice were subjected to echocardiographic examination under tribromoethanol anaesthesia.

3. Mean arterial pressure and atrial, LV and RV mass were greater in ANP–/– mice than in ANP+/+ mice fed the basal salt diet. Salt depletion equalized MAP in the two genotypes, but did not alter the relative cardiac hypertrophy in ANP–/– mice. The ANP–/– mice had significant LV cardiomyocyte hypertrophy when fed either basal or low‐salt diets.

4. Left ventricle chamber dimensions did not differ between genotypes, but were significantly reduced in mice fed the low‐salt diet; LV posterior wall and septal thickness were greater in ANP–/– than ANP+/+ mice and were not altered by diet, indicating a concentric pattern of LV hypertrophy in ANP–/– mice. Left ventricle function (cardiac output, stroke volume, ejection fraction, circumferential wall stress and velocity of circumferential wall shortening) did not differ between strains on either diet; circumferential wall stress was reduced in the low‐salt groups; other functional parameters were not altered by diet.

5. These findings indicate that ANP deletion results in cardiomyocyte hypertrophy and biventricular hypertrophy independent of blood pressure, supporting the concept that ANP has direct antihypertrophic effects in the heart.
Cardiovascular status following combined angiotensin‐converting enzyme and AT1 receptor inhibition in conscious spontaneously hypertensive rats
Authors: Duke Lisa M , Paull Jeremy RA , Widdop Robert E
Published: July 2003

1. Combined treatment of spontaneously hypertensive rats (SHR) with AT1 receptor antagonists and angiotensin‐converting enzyme (ACE) inhibitors has been shown to reduce mean arterial pressure (MAP) more than monotherapy with either agent. The aims of the present study were to investigate the effects of chronic dual renin–angiotensin system (RAS) inhibition using non‐hypotensive doses of the AT1 receptor antagonist candesartan cilexetil and the ACE inhibitor perindopril on cardiovascular function and structure.

2. Adult male SHR, aged 15 weeks, were divided into four groups: (i) candesartan cilexetil (0.5 mg/kg per day in drinking water); (ii) perindopril (0.3 mg/kg per day in drinking water); (iii) combined treatment (dual RAS inhibition); or (iv) the appropriate vehicle (0.1% ethanol/0.1% polyethylene glycol/1.5 mmol/l sodium bicarbonate dissolved in water for candesartan cilexetil; distilled water for perindopril). Systolic blood pressure was measured weekly using the tail‐cuff method and urinary microalbuminuria was measured fortnightly.

3. After 4 weeks, rats were instrumented for intravenous drug administration and measurement of MAP. At this time, the cardiovascular effects of angiotensin (Ang) I and AngII (5–20 ng) and sodium nitroprusside (SNP) and acetylcholine (ACh; 1–5 µg) were assessed. In addition, left ventricular : bodyweight and media : lumen ratios were determined as indices of cardiac and vascular hypertrophy, respectively.

4. Candesartan cilexetil and perindopril alone had minimal effect on MAP when measured both directly and indirectly, whereas direct MAP was significantly decreased in the combined treatment group (131 ± 6 mmHg; P < 0.05) compared with the vehicle group (156 ± 9 mmHg). Pressor responses to AngI were significantly decreased in all groups compared with the vehicle‐treated group and pressor responses to AngII were significantly decreased in the candesartan cilexetil‐treated (P < 0.01) and combined treatment groups (P < 0.01) compared with the vehicle‐treated group. Depressor responses to ACh and SNP were not significantly affected by any of the antihypertensive therapies compared with vehicle‐treated SHR.

5. Vascular hypertrophy was significantly decreased in the candesartan cilexetil and combined groups compared with the vehicle‐treated group, whereas cardiac hypertrophy was reduced, with the rank order of effect being: dual RAS inhibition > perindopril > candesartan cilexetil. Urinary albumin tended to decrease with dual RAS inhibition, but was not significantly affected by this short‐term treatment.

6. These results demonstrate the efficacy of low‐dose dual RAS inhibition as an antihypertensive modality, at least in SHR, not only in reducing arterial pressure, but also in improving cardiovascular structure.
Impact of extracellular matrix and strain on proliferation of bovine airway smooth muscle
Authors: Bonacci John V , Harris Trudi , Stewart Alastair G
Published: July 2003

1. The proliferation of airway smooth muscle (ASM) and collagen deposition are major contributors to airway remodelling in asthma that may be an important component of airway hyperresponsiveness. The ratio of collagen to laminin in asthma is increased as a result of fibrosis.

2. We investigated the effects of the extracellular matrix proteins laminin and collagen type I, as well as airway cell elongation (mechanical strain), on the proliferative responses of cultured bovine ASM cells and the impact of these biomechanical influences on glucocorticoid antimitogenic actions.

3. Bovine ASM cells were cultured onto a flexible silastic membrane coated with laminin or collagen. Cells were pretreated with dexamethasone (100 nmol/L) prior to incubation with the mitogen basic fibroblast growth factor (bFGF) for 72 h, at which time cells were enumerated. Cells were either subjected to mechanical strain (cell elongation) by stretching the flexible silastic membrane or were grown under static conditions.

4. Culture on collagen without elongation enhanced ASM proliferation compared with cells grown on laminin. Cells grown on laminin and subjected to 4% elongation did not respond to the mitogenic actions of bFGF.

5. Dexamethasone‐mediated inhibition of bFGF‐induced proliferation was unaffected by the extracellular matrix on which cells were seeded, or the degree of cell elongation.

6. These data support the hypothesis that the asthmatic airway wall microenvironment (collagen, reduced airway strain) enhances the proliferation of ASM cells.
Purinoceptor‐mediated contractility of the perfused uterine vasculature of the guinea‐pig: influence of oestradiol and pregnancy
Authors: Haynes John M , Pennefather Jocelyn N , Sikorski Bogdan
Published: July 2003

1. The effects of ATP, the stable ATP analogues α,β‐methylene ATP (α,β‐mATP), 2‐methylthioATP (2meSATP) and adenosine tetraphosphate (ATP4), the pyrimidine nucleotide uridine 5′‐triphosphate (UTP) and the α1‐adrenoceptor agonist phenylephrine were examined on the isolated perfused uterine vasculature of dioestrous, oestradiol‐treated, dexamethasone‐treated and late‐pregnant guinea‐pigs.

2. The α1‐adrenoceptor agonist phenylephrine elicited concentration‐dependent vasoconstriction from preparations of perfused uterine vasculature from dioestrous, estradiol‐treated and late‐pregnant guinea‐pigs. The mean maximal response to phenylephrine was unaffected by treatment of dioestrus guinea‐pigs with oestradiol or dexamethasone, but was reduced in preparations from late‐pregnant animals.

3. In perfused uterine arteries from dioestrous animals, the pyrimidine UTP, but not ATP4 and ATP, elicited vasoconstrictor responses. In preparations from oestradiol‐treated animals, all three agonists elicited vasoconstriction, with a rank order of potency of ATP4 = UTP ≫ ATP, whereas in preparations from late‐pregnant animals this order of potency was ATP4 ≫ UTP = ATP. In preparations from dexamethasone‐treated animals, the vasoconstriction was similar to that seen in dioestrous animals. Vasoconstrictor responses to ATP4 were significantly greater in preparations of uterine vasculature from oestradiol‐treated and pregnant animals than in preparations from dioestrous animals or dexamethasone‐treated animals.

4. In preparations from dioestrous, oestradiol‐treated, pregnant and dexamethasone‐treated animals, α,β‐mATP was approximately two to three orders of magnitude more potent than 2meSATP. Compared with preparations from dioestrous animals, the maximal responses to α,β‐mATP were significantly greater in tissues from oestradiol‐treated and pregnant animals. In preparations from dioestrous animals, the P2 purinoceptor antagonist suramin (100 µmol/L) inhibited the responses to α,β‐mATP, but not those to ATP4.

5. The present study has demonstrated that pregnancy, but not the steroid treatment of dioestrous guinea‐pigs with oestradiol or dexamethasone, reduces the sensitivity of the guinea‐pig isolated perfused uterine vasculature to phenylephrine. In contrast, preparations from pregnant or oestradiol‐treated guinea‐pigs respond to ATP4 and to α,β‐mATP with significantly greater constrictions than those of dioestrous or dexamethasone‐treated animals. These data indicate that the sensitivity of the uterine vasculature to adrenoceptor and purinoceptor agonists is differentially regulated by oestradiol and pregnancy, but not by the synthetic glucocorticoid dexamethasone.
Apoptosis is involved in the cardiac damage induced by sinoaortic denervation in rats
Authors: Tao Xia , Zhang Shu‐Hui , Chu Zheng‐Xu , Su Ding‐Feng
Published: July 2003

1. The arterial baroreflex plays an important role in the maintenance of the stability of blood pressure. Sinoaortic denervation (SAD) produces severe organ damage in rats. The present study was designed to investigate whether apoptosis, which is a ubiquitous physiological mode of cell death distinct from cell mortality induced by injury and necrosis, is involved in SAD‐induced cardiac damage.

2. Male Sprague‐Dawley rats (10 weeks old) were used. Rats underwent SAD (n = 9) or sham operation (n = 10). Sixteen weeks after operation, the heart tissues were taken for investigations including electron microscopy, immunohistochemistry, terminal deoxynucleotidyl transferase (TdT)‐mediated dUTP nick‐end labelling (TUNEL) and reverse transcription–polymerase chain reaction (RT‐PCR).

3. Cardiac hypertrophy and fibrosis was found in SAD rats. The number apoptotic cardiomyocytes was increased in SAD rats compared with sham‐operated rats.

4. The expression of Bcl‐2mRNA and protein (an inhibitory factor of apoptosis) in cardiomyocytes was decreased in SAD rats. In contrast, the expression of Bax, Fas and Fas ligand mRNA and proteins (promoters of apoptosis) in cardiomyocytes was significantly increased in SAD rats.

5. In conclusion, the present study reveals a high level of apoptosis in cardiomyocytes in SAD rats. It is proposed that apoptosis is involved in SAD‐induced cardiac damage.
Haemodynamic action of B‐type natriuretic peptide substantially outlasts its plasma half‐life in conscious dogs
Authors: Thomas Colleen J , Woods Robyn L
Published: July 2003

1. The objective of the present study was to determine the plasma half‐life of B‐type natriuretic peptide (BNP) in conscious dogs after intravenous administration and to compare this with its haemodynamic effects. In six chronically instrumented dogs, plasma BNP concentrations were measured under basal conditions, during a constant infusion of canine BNP‐32 (10 pmol/kg per min; 25 min) to steady state and at nominated time points up to 75 min after stopping the infusion. Concomitant, continuous measurements of mean arterial blood pressure (MAP), heart rate (HR), central venous pressure (CVP) and mesenteric blood flow (MBF) were obtained.

2. Baseline plasma BNP levels were 15.0 ± 2.3 fmol/mL and rose approximately 10‐fold to 159 ± 23 fmol/mL after 20–25 min BNP infusion. When the infusion was turned off, plasma BNP levels declined in a biphasic manner, with an initial half‐life of 1.57 ± 0.14 min and a terminal half‐life of 301 ± 85 min. The metabolic clearance rate of BNP was 2.29 ± 0.34 L/min.

3. The infusion of BNP reduced MAP (approximately 10%), CVP (approximately 65%) and MBF (approximately 25%), whereas haematocrit (approximately 4%) and mesenteric vascular resistance (MVR) increased (approximately 40%; all P < 0.05). Plasma BNP levels returned to baseline by 20 min after BNP infusion had been stopped, whereas none of the haemodynamic variables returned to normal by this time. Mean arterial pressure returned to resting levels within 10–15 min after plasma BNP returned to normal. However, CVP, haematocrit and MBF remained substantially below baseline values for more than 20 min after circulating BNP levels had returned to pre‐infusion levels. Of these, only mesenteric vascular changes were returned to baseline within 60 min of plasma BNP levels normalizing.

4. These results demonstrate that the removal of BNP from the canine circulation is rapid, similar to observations made regarding the metabolism of circulating atrial natriuretic peptide in dogs. The half‐life of BNP in dogs was shorter than that in rats, sheep or humans. However, the haemodynamic actions of BNP substantially outlasted its plasma half‐life. Whether this disparity in plasma level and haemodynamic activity of BNP reflects long‐lasting activation of second messenger systems or slow recovery from the hydraulic changes at the capillary level, reflected in the haematocrit and CVP, remains to be answered.
Effect of Centella asiatica on cognition and oxidative stress in an intracerebroventricular streptozotocin model of Alzheimer's disease in rats
Authors: Veerendra Kumar MH , Gupta YK
Published: July 2003

1. Centella asiatica, an Indian medicinal plant, has been described as possessing central nervous system activity, such as improving intelligence. In addition, we have demonstrated that C. asiatica has cognitive‐enhancing and anti‐oxidant properties in normal rats. Oxidative stress or an impaired endogenous anti‐oxidant mechanism is an important factor that has been implicated in Alzheimer's disease (AD) and cognitive deficits seen in the elderly.

2. Intracerebroventricular (i.c.v.) streptozotocin (STZ) in rats has been likened to sporadic AD in humans and the cognitive impairment is associated with free radical generation in this model. Therefore, in the present study, the effect of an aqueous extract of C. asiatica (100, 200 and 300 mg/kg for 21 days) was evaluated in i.c.v. STZ‐induced cognitive impairment and oxidative stress in rats.

3. Male Wistar rats were injected with STZ (3 mg/kg, i.c.v.) bilaterally on the days 1 and 3. Cognitive behaviour was assessed using passive avoidance and elevated plus‐maze paradigms on the days 13, 14 and 21. Rats were killed on the day 21 for estimation of oxidative stress parameters (malondialdehyde (MDA), glutathione, superoxide dismutase and catalase) in the whole brain upon completion of the behavioural task.

4. Rats treated with C. asiatica showed a dose‐dependent increase in cognitive behaviour in both paradigms. A significant decrease in MDA and an increase in glutathione and catalase levels were observed only in rats treated with 200 and 300 mg/kg C. asiatica.

5. The present findings indicate that an aqueous extract of C. asiatica is effective in preventing the cognitive deficits, as well as the oxidative stress, caused by i.c.v. STZ in rats.
Myocardial adenosine does not correlate with the protection mediated by ischaemic or pharmacological preconditioning in rat heart
Authors: Vasara Eleni , Katharou Ioanna , Lazou Antigone
Published: July 2003

1. We tested the hypothesis that ischaemic preconditioning of the rat heart activates cardiovascular adenosine formation to provide enhanced cardioprotection.

2. Rat isolated perfused hearts were either non‐preconditioned, preconditioned with 5 min ischaemia or treated for 5 min with the α1‐adrenoceptor agonist phenylephrine (50 µmol/L) before being subjected to 30 min sustained ischaemia followed by 30 min reperfusion. Isolated cardiomyocytes were either non‐preconditioned, subjected to 10 min simulated ischaemia or treated for 10 min with phenylephrine (50 µmol/L) before being subjected to 30 min simulated ischaemia. Functional recovery of hearts and cell viability were used as indices of the effects of ischaemia.

3. Myocardial adenosine, as well as intracellular pH, was determined at the end of the preconditioning period and at 10, 20 and 30 min of sustained ischaemia. Intracellular pH was also determined during the reperfusion.

4. Ischaemic or pharmacological preconditioning with phenylephrine correlated with an improved functional recovery of perfused hearts during reperfusion and increased cell viability during ischaemia.

5. In perfused hearts, ischaemic preconditioning resulted in increased adenosine production in the myocardium during the following sustained ischaemia. However, in isolated cardiomyocytes, adenosine levels during sustained ischaemia were lower in ischaemically preconditioned cells compared with the respective non‐preconditioned cardiomyocytes.

6. The increase in adenosine production was not observed in hearts preconditioned with phenylephrine instead of transient ischaemia. Similarly, pharmacological preconditioning resulted in decreased adenosine levels during sustained ischaemia in isolated cardiomyocytes.

7. Intracellular pH was preserved during ischaemia to the same extent in both ischaemically or pharmacologically preconditioned hearts and cardiomyocytes, indicating that less acidosis during ischaemia is related to protection.

8. Taken together, the results suggest that cardioprotection does not necessarily correlate with increased adenosine production. Thus, adenosine concentration is not crucial to the beneficial effects of preconditioning in rat heart.
Placental endothelial nitric oxide synthase localization and expression in normal human pregnancy and pre‐eclampsia
Authors: Orange SJ , Painter D , Horvath J , Yu B , Trent R , Hennessy A
Published: July 2003

1. The aim of the present study was to investigate whether pre‐eclampisa, a state of placental hypoxia, is associated with placental abnormalities in the amount, distribution and expression of enothelial nitric oxide synthase (eNOS).

2. Localization and intensity of eNOS was determined by immunohistochemistry using an antibody specific for eNOS. The amount of eNOS mRNA expression was determined by reverse transcription–polymerase chain reaction (RT‐PCR) and the densitometry of gel bands was expressed as a ratio of the band density of the housekeeping gene β2‐microglobulin.

3. Endothelial NOS staining was localized to syncytiotrophoblast cells within the villi and decidual trophoblast cells. It was not present in the endothelium of terminal villous vessels. There was no significant difference in eNOS villous or decidual staining intensity between normal pregnancy (NP; n = 12), pre‐eclampsia (n = 14), or gestational hypertension (GH; n = 4). Staining for eNOS was not significantly different in the decidua compared with the villi in NP, GH or pre‐eclampsia. Within the decidua, the depth of eNOS staining was similar in NP, pre‐eclampisa and GH.

4. There was no significant difference in eNOS mRNA expression between NP (0.70 ± 0.11), pre‐eclampsia (0.5 ± 0.07) or GH (0.69 ± 0.26).

5. These findings suggest that the amount of eNOS in the placenta is not deficient in pre‐eclampsia, excluding a possible pathogenic role for eNOS in this disease. Furthermore, placental hypoxia, which is associated with pre‐eclampsia, did not induce an upregulation of eNOS
Role of copper in the relaxant action of S‐nitrosothiols in the rat anococcygeus muscle
Authors: Ng Chi Wai , Najbar‐Kaszkiel Alicja T , Li Chun Guang
Published: July 2003

1. The effects of copper chelators were investigated on the relaxant actions of the S‐nitrosothiols S‐nitrosoglutathione (GSNO) and S‐nitroso‐N‐acetyl‐d,l‐penicillamine (SNAP), the non‐S‐nitrosothiol nitric oxide (NO) donor sodium nitroprusside (SNP), free radical NO (NO·) and the nitrergic neurotransmitter in rat isolated anococcygeus muscle.

2. Cumulative additions of GSNO (0.01–100 µmol/L), SNAP (0.001–10 µmol/L), SNP (0.001–1 µmol/L) and NO· (0.5–5 µmol/L) and electrical field stimulation (EFS; 1–5 Hz, 10 s) of nitrergic nerves in preparations precontracted with guanethidine (10–30 µmol/L) and clonidine (0.01–0.3 µmol/L) produced concentration‐dependent relaxations.

3. The Cu[I] chelator neocuproine (10–30 µmol/L) produced concentration‐dependent inhibitions of the relaxations to GSNO and SNAP. At 30 µmol/L, neocuprinone had no effect on relaxations to SNP (0.001–1 µmol/L), NO· (0.5–5 µmol/L) or EFS (1–5 Hz, 10 s).

4. The Cu[II] chelator cuprizone (30 µmol/L) slightly and significantly enhanced relaxations to GSNO and NO·, but had no effect on relaxations to SNAP, SNP or EFS.

5. In conclusion, the results indicate that Cu[I], but not Cu[II], may be involved in the relaxant actions of GSNO and SNAP in the rat anococcygeus muscle.
Effects of mild aerobic physical exercise on membrane fluidity of erythrocytes in essential hypertension
Authors: Tsuda Kazushi , Yoshikawa Akiyoshi , Kimura Keizo , Nishio Ichiro
Published: July 2003

1. The present study was undertaken to investigate the effects of aerobic physical exercise on membrane function in mild essential hypertension.

2. Hypertensive patients were divided into an exercise group (n = 8) and a non‐exercise (control) group (n = 8). Physical exercise within the intensity of the anaerobic threshold level was performed twice a week for 6 months. Membrane fluidity of erythrocytes was examined by means of electron paramagnetic resonance (EPR) and spin‐labelling methods before and after the trial period in both groups.

3. After physical exercise, blood pressure decreased significantly.

4. Compared with the non‐exercise group, in the exercise group both the order parameter (S) and the peak height ratio (ho/h‐1) in the EPR spectra of erythrocytes were significantly reduced (S, 0.717 ± 0.004 vs 0.691 ± 0.008, respectively (n = 8), P < 0.05; ho/h‐1, 5.38 ± 0.06 vs 4.89 ± 0.06, respectively (n = 8), P < 0.05). These findings indicated that exercise increased membrane fluidity and improved the membrane microviscosity of erythrocytes.

5. There was no direct correlation between blood pressure reduction and the exercise‐induced increase in membrane fluidity of erythrocytes.

6. In the non‐exercise (control) group, blood pressure and membrane fluidity were not changed after a 6 month follow‐up period.

7.  The results show that aerobic physical exercise increased erythrocyte membrane fluidity and improved the rigidity of cell membranes in hypertensive patients. The improvement of rheological properties of erythrocytes may explain, in part, the cellular mechanisms for the beneficial effects of physical exercise in hypertension.
Magnolol protects against cerebral ischaemic injury of rat heatstroke
Authors: Chang CP , Hsu YC , Lin MT
Published: July 2003

1. Free radicals mediate cerebral ischaemic injury associated with heatstroke. Magnolol, an active component of Magnolia officinalis, is 1000‐fold more potent than α‐tocopherol in inhibiting lipid peroxidation in rat mitochondria. The aim of the present study was to ascertain whether magnolol attenuated cerebral ischaemic injury and free radical formation associated with heatstroke.

2. Urethane‐anaesthetized rats were exposed to heat stress (ambient temperature 42°C) to induce heatstroke. Controlled rats were exposed to 24°C. Mean arterial pressure, cerebral perfusion pressure and cerebral blood flow after the onset of heatstroke were all significantly lower than in control rats. However, colonic temperature, intracranial pressure, heart rate, cerebral free radicals, lipid peroxidation and the neuronal damage score were greater after the onset of heatstroke.

3. Magnolol (20 or 40 mg/kg, i.v.) significantly attenuated the heatstroke‐induced hyperthermia, arterial hypotension, intracranial hypertension, cerebral ischaemia and neuronal damage and increased free radical formation and lipid peroxidation in the brain. The extracellular concentrations of ischaemic (e.g. glutamate and lactate/pyruvate ratio) and damage (e.g. glycerol) markers in the corpus striatum were increased after the onset of heatstroke. Magnolol significantly attenuated the increase in striatal ischaemia and damage markers associated with heatstroke.

4. Thus, it appears that magnolol has impressive effects against heatstroke reactions.