bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a protein Ser/Thr kinase cascade that regulates essential transcriptional activators in Myxococcus xanthus development
Authors: Nariya Hirofumi , Inouye Sumiko
Published: September 2005

Pkn8 is a membrane‐associated protein Ser/Thr kinase (PSTK) of Myxoccocus xanthus that was previously found to associate with a novel cytoplasmic kinase, Pkn14. In the present study, MrpC, an essential transcription factor for fruA expression during fruiting body development, was identified using a genomic yeast two‐hybrid screen with Pkn14 as bait. Our biochemical studies demonstrated that purified Pkn8 and Pkn14 are active kinases and that Pkn8 is able to phosphorylate Pkn14 that forms a tetramer via its C‐terminal 41 residues. Moreover, Pkn14 phosphorylated purified MrpC, indicating that Pkn8 is a Pkn14 kinase and Pkn14 is an MrpC kinase. The pkn8 and pkn14 deletion strains (Δpkn8 and Δpkn14) developed into fruiting bodies significantly faster than that of the parent strain, DZF1. While mrpC expression was at a low level in DZF1 during vegetative growth, it was highly elevated in Δpkn8 and Δpkn14 during vegetative growth and development. Furthermore, FruA, usually induced at 6 h of development, was instead detected at the early stationary phase and accumulated faster during development in Δpkn8 and Δpkn14. Therefore, the developmental phenotype of Δpkn8 and Δpkn14 seems to be due to untimely FruA production mediated by elevated levels of MrpC in Δpkn8 and Δpkn14 during vegetative growth. As pkn14 expression was increased at the mid‐ and late‐log. phases in DZF1 but decreased during development, the Pkn8–Pkn14 kinase cascade appears to negatively regulate mrpC expression by phosphorylating MrpC during vegetative growth. This is the first demonstration of a functional PSTK cascade in prokaryotes. mrpC expression has been proposed to be activated by MrpA and MrpB which belong to a two‐component His‐Asp phosphorelay signal transduction system and that MrpC autoregulate its own expression (Sun H. and Shi W., 2001 J Bacteriol 183: 4786–4795). Therefore, M. xanthus seems to utilize both eukaryotic PSTK cascade and prokaryotic His‐Asp phosphorelay system to precisely regulate mrpC expression with specific timing during development.
The transcriptome response of Neisseria gonorrhoeae to hydrogen peroxide reveals genes with previously uncharacterized roles in oxidative damage protection
Authors: Stohl Elizabeth A. , Criss Alison K. , Seifert H. Steven
Published: September 2005

Symptomatic gonococcal infection, caused by the pathogen Neisseria gonorrhoeae (Gc), is characterized by the influx of polymorphonuclear leukocytes (PMNs) to the site of infection. Although PMNs possess several mechanisms of oxidative killing, intact Gc can be found associated with PMNs, suggesting that gonococcal defences against oxidative stress are crucial for its ability to evade killing by PMNs. We used microarrays to identify genes that were differentially expressed after transient exposure of Gc to hydrogen peroxide (H2O2). Of the 75 genes found to be upregulated after H2O2 treatment, over one‐quarter, including two of the most highly upregulated genes (NGO1686 and NGO554), were predicted to encode proteins with unknown functions. Further characterization of a subset of these upregulated genes demonstrated that NGO1686, a putative zinc metalloprotease, protects against oxidative damage caused by both H2O2 and cumene hydroperoxide, and that NGO554, a Gc‐specific protein, acts to protect against damage caused by high levels of H2O2. Our current study also ascribes a role in H2O2 damage protection to recN, a gene previously characterized for its role in DNA repair. A PMN survival assay demonstrated that the recN and NGO1686 mutants were more susceptible to killing than the parent strain FA1090. These results define for the first time the robust transcriptional response to H2O2 by this strict human pathogen and underscore the importance of this system for survival to host defences.
Fluorescence thiol modification assay: oxidatively modified proteins in Bacillus subtilis
Authors: Hochgräfe Falko , Mostertz Jörg , Albrecht Dirk , Hecker Michael
Published: September 2005

Oxidatively modified thiol groups of cysteine residues are known to modulate the activity of a growing number of proteins. In this study, we developed a fluorescence‐based thiol modification assay and combined it with two‐dimensional gel electrophoresis and mass spectrometry to monitor the in vivo thiol state of cytoplasmic proteins. For the Gram‐positive model organism Bacillus subtilis our results show that protein thiols of growing cells are mainly present in the reduced state. Only a few proteins were found to be thiol‐modified, e.g. enzymes that include oxidized thiols in their catalytic cycle. To detect proteins that are particularly sensitive to oxidative stress we exposed growing B. subtilis cells to diamide, hydrogen peroxide or to the superoxide generating agent paraquat. Diamide mediated a significant increase of oxidized thiols in a variety of metabolic enzymes, whereas treatment with paraquat affected only a few proteins. Exposure to hydrogen peroxide forced the oxidation especially of proteins with active site cysteines, e.g. of cysteine‐based peroxidases and glutamine amidotransferase‐like proteins. Moreover, high levels of hydrogen peroxide were observed to influence the isoelectric point of proteins of this group indicating the generation of irreversibly oxidated thiols. From the overlapping set of oxidatively modified proteins, also enzymes necessary for methionine biosynthesis were identified, e.g. cobalamin‐independent methionine synthase MetE. Growth experiments revealed a methionine limitation after diamide and hydrogen peroxide stress, which suggests a thiol‐oxidation‐dependent inactivation of MetE. Finally, evidence is presented that the antibiotic nitrofurantoin mediates the formation of oxidized thiols in B. subtilis.
Agrobacterium tumefaciens oncogenic suppressors inhibit T‐DNA and VirE2 protein substrate binding to the VirD4 coupling protein
Authors: Cascales Eric , Atmakuri Krishnamohan , Liu Zhenying , Binns Andrew N. , Christie Peter J.
Published: September 2005

Agrobacterium tumefaciens uses a type IV secretion (T4S) system composed of VirB proteins and VirD4 to deliver oncogenic DNA (T‐DNA) and protein substrates to susceptible plant cells during the course of infection. Here, by use of the Transfer DNA ImmunoPrecipitation (TrIP) assay, we present evidence that the mobilizable plasmid RSF1010 (IncQ) follows the same translocation pathway through the VirB/D4 secretion channel as described previously for the T‐DNA. The RSF1010 transfer intermediate and the Osa protein of plasmid pSa (IncW), related in sequence to the FiwA fertility inhibition factor of plasmid RP1 (IncPα), render A. tumefaciens host cells nearly avirulent. By use of a semi‐quantitative TrIP assay, we show that both of these ‘oncogenic suppressor factors’ inhibit binding of T‐DNA to the VirD4 substrate receptor. Both factors also inhibit binding of the VirE2 protein substrate to VirD4, as shown by coimmunoprecipitation and bimolecular fluorescence complementation assays. Osa fused to the green fluorescent protein (GFP) also blocks T‐DNA and VirE2 binding to VirD4, and Osa‐GFP colocalizes with VirD4 at A. tumefaciens cell poles. RSF1010 and Osa interfere specifically with VirD4 receptor function and not with VirB channel activity, as shown by (i) TrIP and (ii) a genetic screen for effects of the oncogenic suppressors on pCloDF13 translocation through a chimeric secretion channel composed of the pCloDF13‐encoded MobB receptor and VirB channel subunits. Our findings establish that a competing plasmid substrate and a plasmid fertility inhibition factor act on a common target, the T4S receptor, to inhibit docking of DNA and protein substrates to the translocation apparatus.
A novel bacterial signalling system with a combination of a Ser/Thr kinase cascade and a His/Asp two‐component system
Authors: Lux Renate , Shi Wenyuan
Published: September 2005

Prokaryotes and eukaryotes have long been thought to use very different types of kinases (the His kinases of the ‘bacterial’ two‐component systems versus the ‘eukaryotic’ Ser/Thr/Tyr kinases) to carry out signal transduction. This paradigm no longer holds true, because both systems are now found together in an increasing number of prokaryotic organisms and ‘two‐component’ His kinase are present in eukaryotes. Pioneering work on bacterial protein serine threonine kinases (PSTKs) has been performed in Myxococcus xanthus, a soil bacterium with a complex life cycle that possesses orthologues of signalling‐related kinases ‘typical’ of both the prokaryotic and the eukaryotic kingdoms. In the work reported in this volume of Molecular Microbiology, Nariya and Inouye describe a PSTK cascade that modulates the biochemical activity of MrpC, a CRP‐like transcriptional regulator for essential developmental signalling pathways in M. xanthus whose transcription is under the control of a two‐component system. This is the first report of both a functional PSTK cascade in bacteria and the use of both PSTK and two‐component systems to control a single complex bacterial signalling event.
Evidence for functional overlap among multiple bacterial cell division proteins: compensating for the loss of FtsK
Authors: Geissler Brett , Margolin William
Published: September 2005

In Escherichia coli, at least 12 proteins colocalize to the cell midpoint, assembling into a membrane‐associated protein machine that forms the division septum. Many of these proteins, including FtsK, are essential for viability but their functions in cell division are unknown. Here we show that the essential function of FtsK in cell division can be partially bypassed. Cells containing either the ftsA R286W mutation or a plasmid carrying the ftsQAZ genes suppressed a ftsK44(ts) allele efficiently. Moreover, ftsA R286W or multicopy ftsQAZ, which can largely bypass the requirement for the essential cell division gene zipA, allowed cells with a complete deletion of ftsK to survive and divide, although many of these ftsK null cells formed multiseptate chains. Green fluorescent protein (GFP) fusions to FtsI and FtsN, which normally depend on FtsK to localize to division sites, localized to division sites in the absence of FtsK, indicating that FtsK is not directly involved in their recruitment. Cells expressing additional ftsQ, and to a lesser extent ftsB and ftsN, were able to survive and divide in the absence of ftsK, although cell chains were often formed. Surprisingly, the cytoplasmic and transmembrane domains of FtsQ, while not sufficient to complement an ftsQ null mutant, conferred viability and septum formation in the absence of ftsK. These findings suggest that the N‐terminal domain of FtsK is normally involved in stability of the division protein machine and shares functional overlap with FtsQ, FtsB, FtsA, ZipA and FtsN.
Ketolide resistance in Streptococcus pyogenes correlates with the degree of rRNA dimethylation by Erm
Authors: Douthwaite Stephen , Jalava Jari , Jakobsen Lene
Published: September 2005

Macrolide and ketolide antibiotics inhibit protein synthesis on the bacterial ribosome. Resistance to these antibiotics is conferred by dimethylation at 23S rRNA nucleotide A2058 within the ribosomal binding site. This form of resistance is encoded by erm dimethyltransferase genes, and is found in many pathogenic bacteria. Clinical isolates of Streptococcus pneumoniae with constitutive erm(B) and Streptococcus pyogenes with constitutive erm(A) subtype (TR) are resistant to macrolides, but remain susceptible to ketolides such as telithromycin. Paradoxically, some strains of S. pyogenes that possess an identical erm(B) gene are clinically resistant to ketolides as well as macrolides. Here we explore the molecular basis for the differences in these streptococcal strains using mass spectrometry to determine the methylation status of their rRNAs. We find a correlation between the levels of A2058‐dimethylation and ketolide resistance, and dimethylation is greatest in S. pyogenes strains expressing erm(B). In constitutive erm strains that are ketolide‐sensitive, appreciable proportions of the rRNA remain monomethylated. Incubation of these strains with subinhibitory amounts of the macrolide erythromycin increases the proportion of dimethylated A2058 (in a manner comparable with inducible erm strains) and reduces ketolide susceptibility. The designation ‘constitutive’ should thus be applied with some reservation for most streptococcal erm strains. One strain worthy of the constitutive designation is S. pyogenes isolate KuoR21, which has lost part of the regulatory region upstream of erm(B). In S. pyogenes KuoR21, nucleotide A2058 is fully dimethylated under all growth conditions, and this strain displays the highest resistance to telithromycin (MIC > 64 µg ml−1).
Subcutaneous Angiolipoma of Abdomen in a Golden Hamster (Mesocrietus auratus)
Authors: Kondo H. , Sato T. , Shibuya H. , Onuma M.
Published: September 2005

This is a single case report of an angiolipoma located in the subcutis of a 2‐year‐old golden hamster. The histological appearance of the tumour resembled that described in other species. The hamster died 1 month following removal of tumour and a necropsy was not performed. This is apparently the first recorded case of angiolipoma in a hamster.
Dual‐energy X‐ray Absorptiometry in Canine Legg‐Calvé‐Perthes Disease
Authors: Isola M. , Zotti A. , Carnier P. , Baroni E. , Busetto R.
Published: September 2005

Radiographic examination and subsequent dual‐energy X‐ray absorptiometry scans of the hips were performed in seven dogs with monolateral Legg‐Perthes‐Calvé disease to quantify bone changes produced by osteonecrosis in the proximal femur on the affected and unaffected side. All dogs were found to be affected with grade 2 and 3 of the radiographic classification proposed by Ljunggren [Acta Orthop. Scand.(1967) vol. 95]. Bone mineral density (g/cm2) of the femoral neck and proximal femoral metaphysis were evaluated on the affected and unaffected side; we detected no differences in bone mineral density for both regions of interest within the population studied in relation to gender, body weight and side analysed nor between the affected and the unaffected limb. We therefore assume that radiographic areas of decreased density in the proximal femoral epiphysis during chronic stages of Legg‐Calvé‐Perthes disease do not alter the global mineral content of the scanned region.
Locomotive Disorders Associated with Sow Mortality in Danish Pig Herds
Authors: Kirk R. K. , Svensmark B. , Ellegaard L. P. , Jensen H. E.
Published: September 2005

Given the increase in sow mortality in Danish pig herds over the last 10 years, we have investigated the causes of spontaneous death and killing of sows, especially those due to locomotive disorders. A total of 265 culled sows (93 spontaneously dead sows and 172 killed sows) from 10 different herds were sent for complete necropsy, and grouped according to primary diagnoses. From 35 of these sows, tissue for histological evaluation and samples for bacteriological cultivation were also collected, and secondary diagnoses and body condition were recorded. The main causes for killing were related to the locomotive system (72%), arthritis (24%) and fractures (16%) being the most frequent lesions. The main causes of spontaneous death were related to the gastrointestinal system and spleen (45%) and the reproductive system (24%). Arthrosis was a secondary diagnosis in 88% of the killed sows and in 92.5% of the spontaneously dead sows. Arcanobacterium pyogenes was the main cause of arthritis. The killed sows had significantly lower body condition compared with spontaneously dead sows. The first two parities and the time around farrowing were associated with the highest mortality in both groups. Hopefully, these observations can guide breeding units attempting to monitor, control and reduce sow mortality.
Plasma Concentrations of 15‐Ketodihydro‐PGF2α, Cortisol and Progesterone during Manual Twin Reduction in Thoroughbred Mares
Authors: Veronesi M. C. , Faustini M. , Villani M. , Kindahl H. , Galeati G. , Battocchio M.
Published: September 2005

The aim of the present study was to highlight the effect of two different techniques of one embryo crushing on some hormonal changes. Ten twinning mares were submitted to the mobile or fixed manual crushing of one blastocyst within day 19 after the last mating. Blood sample was collected from 20 min before to 90 min, 24 and 72 h after the procedure was performed to analyse 15‐ketodihydro‐PGF2α, cortisol and progesterone plasma concentrations. Singleton pregnancy diagnosis was checked 72 h after crushing and at term of pregnancy. Because the unwanted crushing of both embryos occurred in one mare during the attempt of manual separation of the twins, that mare was not included in the evaluation of crushing‐induced hormonal changes. No significant differences in hormonal concentrations were observed after one embryo crushing and also when the effect of the mobile (n = 6) or fixed (n = 3) technique was specifically evaluated. When the effect of the two techniques on each post‐crushing sampling time hormonal levels was analysed, only a higher cortisol level 30 min after the fixed compared with the mobile technique was observed. The crushing performed within 19 days of gestation does not induce significant changes in 15‐ketodihydro‐PGF2α, cortisol or progesterone plasma concentrations. When the fixed technique was performed, only a temporary higher cortisol concentration was seen 30 min after crushing, suggesting that the fixed technique might be responsible for a slight level of stress for the mare.
A Rare Case of Odontogenic Myxoma in a Dog
Authors: Gupta K. , Singh A. , Sood N. , Mohindroo J. , Sood N. K.
Published: September 2005

A case of odontogenic myxoma in an old Doberman bitch involving the periodontal region of the mandible was recorded. Grossly, the tumour mass was soft and slimy, having brownish‐black foci on cut surface. Histopathologically, the tumour predominantly consisted of mesenchyme‐like cells with prominent nuclei and nucleoli. The tumour had abundant ground substance, rich in acid mucopolysaccharides.
Carboplatin Sustained Delivery System Using Injectable Microspheres
Authors: Manunta M. L. , Gavini E. , Chessa G. , Passino E. S. , Careddu G. M. , Giua S. , Mollica A. , Demontis M. P. , Leoni A. , Muzzetto P.
Published: September 2005

A controlled carboplatin delivery system using biodegradable polymer has been used in this study. The purpose was to evaluate the local and systemic effects of injectable, biodegradable microspheres containing carboplatin when injected as aqueous suspension subcutaneously in rats. Biocompatibility and toxicity of empty microspheres and microspheres loaded with carboplatin were evaluated by clinical and histological examination. The diffusion of carboplatin in tissues and time of drug release were evaluated by platinum determination in plasma and tissues over the time. The results of the study suggest that microspheres provide a sustained slow release of carboplatin and that multiple inoculations of microspheres containing drug and no evidence of local or systemic toxicity is found. This device may be useful in the treatment of solid tumours.
Renal (Uremic) Encephalopathy in a Goat
Authors: Radi Z. A. , Thomsen B. V. , Summers B. A.
Published: September 2005

Renal encephalopathy was diagnosed in a 2‐year‐old male boar goat with a history of chronic weight loss and ataxia. Histopathological examination of the brain revealed a striking myelin vacuolation distributed mainly in two patterns: (i) along the junction of the neocortex and corona radiata, and (ii) in the bundles of the internal capsule as it dissects through the basal nuclei. The kidneys had diffuse severe tubular and glomerular necrosis and degeneration. The neural lesions are consistent with renal (uremic) encephalopathy. To the authors’ knowledge, this is the first report of renal encephalopathy in a goat.
Prevalence of Osseous Fragments in Distal and Proximal Interphalangeal, Metacarpo‐ and Metatarsophalangeal and Tarsocrural Joints of Hanoverian Warmblood Horses
Authors: Stock K. F. , Hamann H. , Distl O.
Published: September 2005

The prevalence of osseous fragments in different limb joints was analysed in 3749 young Warmblood riding horses, which were pre‐selected for sale at auction from 1991 to 1998 by the Association of Hanoverian Warmblood Breeders (Verband hannoverscher Warmblutzüchter e.V.) in Verden (Aller), Germany. The evaluation of these horses was based on a standardized radiological examination, including the distal interphalangeal (DIJ), the proximal interphalangeal (PIJ), the metacarpo‐ and metatarsophalangeal (MC/MTJ) and the tarsocrural joints (TCJ). In 32% of all horses at least one osseous fragment was found on these radiographs. TCJ were affected in 9.6% of the horses and MC/MTJ in 20.7%. The percentage of horses affected in the MTJ was significantly higher (13.7%) than that of horses affected in the MCJ (9.5%). The prevalences of osseous fragments were considerably lower in DIJ (4.5%) and PIJ (0.9%). Osseous fragments only occurred either in DIJ or PIJ or MC/MTJ or TCJ in 87% of the affected horses. Depending on the joint type, osseous fragments were found in analogous joints in the front and the hind limbs in up to 26% of the affected horses, whilst bilateral affection occurred in between 10% and 46% of the horses. Distribution of osseous fragments in the limb joints investigated here did not differ significantly between male and female horses. There was a significant increase in the percentage of horses with osseous fragments in MC/MTJ during the study period. The results of this study indicate the need to act against the high prevalences of osseous fragments in the limb joints of young Warmblood riding horses.
Characterization of the Cytokine Pattern of Porcine Bone Marrow‐Derived Cells Treated with 1α,25(OH)2D3
Authors: Sipos W. , Duvigneau J. C. , Schmoll F. , Exel B. , Hofbauer G. , Baravalle G. , Hartl R. T. , Dobretsberger M. , Pietschmann P.
Published: September 2005

The biologically active form of vitamine D3 [1α,25(OH)2D3] has recently been described not only to influence bone metabolism but also to exert immunomodulating activities, which may have an impact on bone formation/resorption as well. In this study, we analysed the effects of 1α,25(OH)2D3 on the cytokine pattern of porcine bone marrow‐derived cells from piglets aged 1–3 weeks. After culture for 1 week, the number of osteoclasts was determined, with tartrate‐resistant acid phosphatase (TRAP)‐positive, multinucleated cells being considered osteoclasts. Cultured bone marrow cell‐derived mRNA was subjected to semiquantitative RT‐PCR specific for a panel of porcine cytokines (IL‐1α, IL‐6, IL‐8, IL‐10, and TNF‐α). In addition, an immunofluorescence analysis using anti‐porcine mAbs specific for IL‐1β, IL‐2, IL‐4, IL‐6, IL‐12, TNF‐α, and IFN‐γ was performed. In order to prove the existence of a porcine homologue of the receptor activator of NF‐κB ligand (RANKL) bone marrow cell‐ as well as porcine white blood cell‐derived mRNA was investigated by RT‐PCR using primer pairs specific for murine RANKL. Cell culture supernatant was analysed for soluble RANKL by means of an ELISA designed for quantification of human RANKL. By means of RT‐PCR, expression of IL‐1α, IL‐6, IL‐8, IL‐10 and TNF‐α mRNA could be found in cells cultured with and without 1α,25(OH)2D3. Immunofluorescence analysis revealed that IL‐1, IL‐6, and TNF‐α were produced by both stromal cells and osteoclasts. Besides its known osteoclastogenic effects, 1α,25(OH)2D3 tended to downregulate the respective cytokines, but significantly upregulated RANKL expression. The homology between the porcine RANKL‐specific sequence and the corresponding human RANKL sequence was 79%. The data found support the idea that porcine bone marrow cell cultures may provide a suitable alternative to murine systems in human osteological research.
Daily Rhythms of Blood Pressure, Heart Rate, and Body Temperature in Fed and Fasted Male Dogs
Authors: Piccione G. , Caola G. , Refinetti R.
Published: September 2005

Daily or circadian rhythmicity in physiological processes has been described in a large number of species of birds and mammals. However, in dogs, most studies have either failed to detect rhythmicity or have found that rhythmicity reflects merely an acute exogenous effect of feeding rather than an autonomous rhythmic process. In the present study, we investigated the rhythmicity of body temperature, blood pressure, and heart rate in dogs fed daily as well as in dogs deprived of food for 60 h. Our results document clear rhythmicity in all three parameters and demonstrate that the rhythmicity is independent of the feeding schedule. The failure of various previous investigations to document daily rhythmicity in dogs is probably due to lack of experimental rigour rather than to weakness of daily rhythmicity in dogs.
Current models of human prostate contractility
Authors: Haynes John M , Ventura Sabatino
Published: September 2005

1. The human prostate is a compact gland contributing to seminal fluid. With increasing age, most humans will develop benign prostatic hyperplasia, a condition of prostatic enlargement and contractility that leads to occlusion of the urethra. Over many years, investigators have used a variety of animal and cell culture models to elucidate some of the contractile and proliferative mechanisms that may be associated with the development of this condition.

2. This review briefly assesses the current state of knowledge of the mechanisms underlying human prostatic contractility and compares it with that of animal and cell culture models. It is not intended as a comprehensive methodological review, nor is it intended to indicate our preferences for either model. Our aim is to correlate findings from animal and cell culture models with the current understanding of human prostate contractility.

3. We hope that the present review will increase awareness of the suitability of the current models in developing our understanding of benign prostatic hyperplasia.
The anti‐diabetic drug miglitol is protective against anginal ischaemia through a mechanism independent of regional myocardial blood flow in the dog
Authors: Uno Yoshihiro , Minatoguchi Shinya , Arai Masazumi , Wang Ningyuan , Chen Xue‐Hai , Hashimoto Kazuaki , Lu Cuanjiang , Takemura Genzou , Fujiwara Hisayoshi
Published: September 2005

1. In the present study, we attempted to clarify whether the antidiabetic drug miglitol, an α‐glucosidase inhibitor, has a protective effect against anginal ischaemia. We had reported previously that miglitol reduces myocardial infarct size through inhibition of glycogenolysis during ischaemia in rabbits. However, the effect of miglitol on anginal ischaemia remains unknown.

2. In open‐chest beagle dogs with a severely stenosed left anterior descending coronary artery, an epicardial electrode was attached to the surface of the risk area of the left ventricle and a microdialysis probe was implanted into the myocardium to measure ST segment changes and interstitial lactate accumulation. The first episode of anginal ischaemia was induced by atrial pacing and phenylephrine infusion (50–100 µg/min) for 10 min. The second episode of anginal ischaemia was induced 210 min after the first episode. Miglitol (10 mg/kg, i.v.) was administered to the miglitol group (n = 10) 30 min before the second episode of anginal ischaemia, whereas saline was administered to the control group (n = 10). Regional myocardial blood flow was measured using coloured microspheres.

3. There was no significant difference in regional myocardial blood flow in the risk and non‐risk areas between the first and second episodes of anginal ischaemia and between the miglitol and control groups. During the first and second episodes of anginal ischaemia, the ST segment was decreased to a similar extent in the control group. Although ST segment depression during the first episode of anginal ischaemia was similar in both groups, ST segment depression during the second episode of anginal ischaemia was significantly attenuated in the miglitol‐treated group compared with the control group (1.3 ± 0.4 vs 2.2 ± 0.4 mV, respectively). Miglitol significantly attenuated myocardial interstitial lactate accumulation in the risk area.

4. In conclusion, in the present study miglitol improved ST segment depression and attenuated the accumulation of myocardial interstitial lactate during anginal ischaemia without altering regional myocardial blood flow. Miglitol has an anti‐anginal ischaemia effect via a mechanism that is independent of regional myocardial blood flow.
Antiviral activities of extracts and selected pure constituents of Ocimum basilicum
Authors: Chiang Lien‐Chai , Ng Lean‐Teik , Cheng Pei‐Win , Chiang Win , Lin Chun‐Ching
Published: September 2005

1. Ocimum basilicum (OB), also known as sweet basil, is a well known medicinal herb in traditional Chinese medicine preparations. In the present study, extracts and purified components of OB were used to identify possible antiviral activities against DNA viruses (herpes viruses (HSV), adenoviruses (ADV) and hepatitis B virus) and RNA viruses (coxsackievirus B1 (CVB1) and enterovirus 71 (EV71)).

2. The results show that crude aqueous and ethanolic extracts of OB and selected purified components, namely apigenin, linalool and ursolic acid, exhibit a broad spectrum of antiviral activity. Of these compounds, ursolic acid showed the strongest activity against HSV‐1 (EC50 = 6.6 mg/L; selectivity index (SI) = 15.2), ADV‐8 (EC50 = 4.2 mg/L; SI = 23.8), CVB1 (EC50 = 0.4 mg/L; SI = 251.3) and EV71 (EC50 = 0.5 mg/L; SI = 201), whereas apigenin showed the highest activity against HSV‐2 (EC50 = 9.7 mg/L; SI = 6.2), ADV‐3 (EC50 = 11.1 mg/L; SI = 5.4), hepatitis B surface antigen (EC50 = 7.1 mg/L; SI = 2.3) and hepatitis B e antigen (EC50 = 12.8 mg/L; SI = 1.3) and linalool showed strongest activity against AVD‐II (EC50 = 16.9 mg/L; SI = 10.5).

3. No activity was noted for carvone, cineole, β‐caryophyllene, farnesol, fenchone, geraniol, β‐myrcene and α‐thujone.

4. The action of ursolic acid against CVB1 and EV71 was found to occur during the infection process and the replication phase.

5. With SI values greater than 200, the potential use of ursolic acid for treating infection with CVB1 and EV71 merits further investigation.
Expression and functional role of the RhoA/Rho‐kinase pathway in rat coeliac artery
Authors: Teixeira Cleber E , Jin Liming , Ying Zhekang , Palmer Trenis , Priviero Fernanda BM , Webb R Clinton
Published: September 2005

1. Rho‐kinase (ROK) stimulation represents a key step in the maintenance of agonist‐induced contraction, an effect counteracted by nitric oxide (NO) released from the endothelium. The aim of the present study was to characterize the involvement of ROK in smooth muscle contraction of the rat coeliac artery using functional and expression studies.

2. Rings of rat coeliac artery were mounted in 5 mL myographs containing warmed and oxygenated Krebs' solution. Rings were connected to isometric transducers and data were recorded in a PowerLab system (ADInstruments, Colorado Springs, CO, USA). After a 60 min equilibration period, preparations were precontracted with phenylephrine (1 µmol/L). Endothelial integrity was assessed by treating the vessels with acetylcholine (1 µmol/L). Expression of ROKα, ROKβ and RhoA was analysed using western blot, whereas Rho guanine nucleotide exchange factors (RhoGEF) were measured at the mRNA level.

3. The addition of Y‐27632 (0.01–30 µmol/L) caused sustained relaxation of rings contracted with phenylephrine (PE; 1 µmol/L), with intact or denuded endothelium (pEC50 = 6.38 ± 0.03 and 5.65 ± 0.02, respectively). NG‐Nitro‐l‐arginine methyl ester (100 µmol/L) or 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (10 µmol/L), but not indomethacin (10 µmol/L), caused marked rightward shifts of the concentration–response curves to Y‐27632. The contractile response to KCl (80 mmol/L) was significantly reduced by Y‐27632, with a maximal inhibition of 57 ± 6%. Nifedipine (0.1–100 nmol/L) fully blocked KCl‐evoked contractions, but only marginally affected those in response to PE (27 ± 2% maximal inhibition). At 1 µmol/L, Y‐27632 also significantly enhanced relaxations to sodium nitroprusside (SNP; 0.0001–1 µmol/L).

4. At 1 µmol/L, SNP (but not 1 µmol/L Y‐27632) significantly elevated the cGMP content above basal levels. Coincubation with SNP and Y‐27632 increased cGMP levels, but the results were not significantly different from those in the presence of SNP alone.

5. Western blot analysis revealed the protein expression of RhoA, ROKα and ROKβ. The PDZ‐RhoGEF, p115RhoGEF and leukaemia‐associated RhoGEF (LARG) mRNA expression in coeliac artery was visualized by electrophoresis on agarose gels.

6. The results clearly demonstrate a role for the RhoA/ROK signalling pathway in the regulation of rat coeliac artery smooth muscle contraction. The findings of the present study suggest that endogenous nitric oxide‐induced relaxation is mediated, in part, by inhibition of RhoA/ROK signalling in this tissue.
HIGH‐FAT DIET PREVENTS CARDIAC HYPERTROPHY AND IMPROVES CONTRACTILE FUNCTION IN THE HYPERTENSIVE DAHL SALT‐SENSITIVE RAT
Authors: Okere Isidore C , Chess David J , McElfresh Tracy A , Johnson Janean , Rennison Julie , Ernsberger Paul , Hoit Brian D , Chandler Margaret P , Stanley William C
Published: September 2005

1. The role that dietary lipid and plasma fatty acid concentration play in the development of cardiac hypertrophy in response to hypertension is not clear.

2. In the present study, we treated Dahl salt‐sensitive rats with either normal chow (NC), normal chow with salt added (NC + salt) or a diet high in long‐chain saturated fatty acids with added salt (HFD + salt). Cardiac function was assessed by echocardiography and left ventricular (LV) catheterization.

3. The HFD + salt group had significantly higher plasma free fatty acid concentrations and myocardial triglyceride content compared with the NC + salt group, but did not upregulate the activity of the fatty acid oxidation enzyme medium chain acyl‐coenzyme A dehydrogenase. Systolic blood pressure was elevated to a similar extent in the NC + salt and HFD + salt groups compared with the NC group. Although LV mass was increased in the NC + salt group compared with the NC group, LV mass in the HFD + salt group did not differ from that of the NC group and was significantly lower than that in the NC + salt group.

4. There was no evidence of cardiac dysfunction in the NC + salt group compared with the NC group; however, high fat feeding significantly increased LV contractile performance (e.g. increased cardiac output and peak dP/dt).

5. In conclusion, the HFD + salt diet prevented the hypertrophic response to hypertension and improved the contractile performance of the heart. It remains to be determined whether preventing cardiac hypertrophic adaptations would be deleterious to the heart if the hypertensive stress is maintained long term.
Cooling‐induced contraction of the rat gastric fundus: Mediation via transient receptor potential (TRP) cation channel TRPM8 receptor and Rho‐kinase activation
Authors: Mustafa S , Oriowo MA
Published: September 2005

1. Cooling has been shown to induce contractions of several smooth muscles in vitro. However, the mechanism involved in the response is not yet known. In the present study, we investigated the possible involvement of transient receptor potential (TRP) cation channel TRPM8 receptors and the Rho‐kinase pathway in cooling‐induced contraction of the rat fundus.

2. Cooling‐induced contractions were inversely proportional to temperature. Contractions were significantly reduced (by 65.6 ± 2.4%; P < 0.05) in a Ca2+‐free (1 mmol/L EGTA) medium, but were not significantly inhibited by nifedipine (10−6 mol/L).

3. Capsazepine (3 × 10−6 and 3 × 10−5 mol/L), a TRPM8 receptor antagonist, inhibited cooling‐induced contraction of the rat gastric fundus.

4. The Rho‐kinase inhibitor Y‐27632 concentration‐dependently inhibited cooling‐induced contraction of the gastric fundus, producing approximately 90% inhibition at a concentration of 10−5 mol/L. Contractions were also inhibited by genistein (3 × 10−5 mol/L), a tyrosine kinase inhibitor, but not by GF 109203X (10−7 mol/L), a protein kinase C inhibitor.

5. Using reverse transcription–polymerase chain reaction techniques, it was observed that the mRNA for the TRPM8 receptor and Rho‐kinase were expressed in the rat gastric fundus.

6. These results would suggest that cooling‐induced contraction of the rat fundus is mediated by activation of TRPM8 receptors via a mechanism involving activation of Rho‐kinase.
Pathophysiological levels of the obesity related peptides resistin and ghrelin increase adhesion molecule expression on human vascular endothelial cells
Authors: Skilton Michael R , Nakhla Shirley , Sieveking Daniel P , Caterson Ian D , Celermajer David S
Published: September 2005

1. In the present study, we sought to determine whether physiological or pathophysiological concentrations of obesity related peptides influence the key early atherogenic events of monocyte adhesion to endothelial cells and adhesion molecule expression using primary human cells.

2. Human umbilical vein endothelial cells were grown to confluence and human monocytes were obtained by elutriation. Adhesion was assessed by automated cell counting and cell adhesion molecule expression (E‐selectin, intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1)) was assayed by ELISA.

3. Experimental conditions included untreated control, ghrelin (100, 150, 450 and 1350 pmol/L), resistin (15, 40 and 100 ng/mL) and combined leptin and insulin (combinations of 30 and 120 pmol/L insulin and 5, 50 and 500 ng/mL leptin).

4. Both resistin and ghrelin produced modest but significant increases in VCAM‐1 expression (110 ± 4 and 117 ± 13% compared with controls, respectively; both P ≤ 0.01). Ghrelin also increased ICAM‐1 expression (119 ± 17% of control; P ≤ 0.01).

5. However, despite these increases in adhesion molecule expression, neither ghrelin nor resistin altered monocyte adhesion values.

6. Neither leptin nor insulin altered monocyte adhesion to endothelial cells or cell adhesion molecule expression.

7. Pathophysiologically relevant concentrations of ghrelin and resistin, within the range of concentrations exhibited by patients with anorexia nervosa or the Prader–Willi syndrome and type 2 diabetes, respectively, increase endothelial cell adhesion molecule expression, possibly contributing to increased atherosclerosis risk in such subjects.
Tetramethylpyrazine downregulates angiotensin II‐induced endothelin‐1 gene expression in vascular endothelial cells
Authors: Lee Wen‐Sen , Yang Hung‐Yu , Kao Pai‐Feng , Liu Ju‐Chi , Chen Cheng‐Hsien , Cheng Tzu‐Hurng , Chan Paul
Published: September 2005

1. Tetramethylpyrazine (TMP) is one of the active ingredients of the Chinese herb Ligusticum wallichii Franchat. It is well documented that TMP exerts a cardiovascular protective effect. The aims of the present study were to examine whether TMP alters angiotenisn (Ang) II‐induced endothelin (ET)‐1 gene expression and to identify the putative underlying signalling pathways in vascular endothelial cells.

2. Cultured vascular endothelial cells were pre‐incubated with TMP, stimulated with AngII and ET‐1 gene expression was then examined. The effects of TMP pretreatment on AngII‐induced extracellular signal‐regulated kinase (ERK) phosphorylation were investigated to elucidate the intracellular mechanism responsible for the effects of TMP on ET‐1 gene expression.

3. Tetramethylpyrazine inhibited AngII‐induced ET‐1 gene expression, as revealed by nothern blotting and a promoter activity assay. Tetramethylpyrazine also inhibited the AngII‐induced increase in intracellular reactive oxygen species (ROS), as measured by the redox sensitive fluorescent dye 2′ 7′‐dichlorofluorescin diacetate and ERK phosphorylation.

4. In summary, we have demonstrated, for the first time, that TMP inhibits AngII‐induced ROS generation, ERK phosphorylation and ET‐1 gene expression in vascular endothelial cells. Thus, the present study delivers important new insights into the molecular pathways that may contribute to the proposed beneficial effects of TMP in the cardiovascular system.
Upregulated function of phosphatidylinositol‐3‐kinase in genetically hypertensive rats: a moderator of arterial hypercontractility
Authors: Northcott Carrie A , Hayflick Joel , Watts Stephanie W
Published: September 2005

1. The growth enzyme phosphatidylinositol 3‐kinase (PI3K) was recently implicated in the mediation of arterial spontaneous tone, an event observed in arteries from hypertensive, but not normotensive, subjects that contributes to changes in total peripheral resistance in the hypertensive state. We have shown this occurrence in experimentally induced models of hypertension. However, because the majority of hypertension is genetically based, it is important to demonstrate a similar change in genetically hypertensive animals.

2. Aorta from spontaneously hypertensive rats (SHR; systolic blood pressure = 183 ± 4 mmHg) and Wistar Kyoto (WKY) rats (115 ± 2 mmHg) were isolated for the measurement of isometric contractile force. Aorta from SHR displayed small increases (approximately 5% maximum phenylephrine (PE)‐induced contraction) in spontaneous tone, whereas aorta from WKY rats displayed none. The non‐selective PI3K inhibitor LY294002 (20 µmol/L) and the selective inhibitor of the p110δ catalytic subunit of PI3K IC87114 (20 µmol/L) caused a fall of basal tone in SHR aorta (20 ± 7 and 24 ± 6% of the initial PE contraction, respectively), but did not alter tone in arteries from WKY rats. LY294002, but not IC87114, normalized the increased potency of noradrenaline (NA) observed in aorta from SHR (–log EC50 values for NA in the presence of vehicle in WKY rats and SHR 7.5 ± 0.1 and 7.8 ± 0.1, respectively (P < 0.05); –log EC50 values for NA in the presence of LY294002 in WKY rats and SHR 7.0 ± 0.1 and 7.0 ± 0.1, respectively).

3. Biochemical expression of the p110 catalytic and p85 regulator subunits of PI3K in western analyses revealed no difference in expression of the regulatory p85α or p110α protein subunits between WKY rats and SHR; p110γ was not detected. In contrast, p110δ expression was increased greater than 30% in aorta from SHR compared with WKY rats (827.6 ± 88.5 vs 576.8 ± 53.4 arbitrary densitometry units, respectively). Immunohistochemical analyses revealed expression of the p110δ isoform in the smooth muscle of arteries.

4. These data underscore the relevance of an enzyme historically classified as one committed to growth/anti‐apoptosis in modifying contractility and supports involvement of PI3K in genetically based hypertension.
Remodelling of mesenteric arteries in genetically hypertensive rats cross‐fostered from birth to normotensive Wistar rats
Authors: Ledingham Janet M , Ashton Nick
Published: September 2005

1. The effects of cross‐fostering genetically hypertensive (GH) with normotensive (N) Wistar rats on the structure of mesenteric resistance arteries (MRA) in young (6 week old) and adult (18 week old) rats were investigated to see whether the abnormal remodelling known to exist in GH rats could be prevented by changing the maternal environment.

2. Genetically hypertensive and normotensive rat pups were reared either by their natural mothers or a foster mother of the opposite strain (NX and GHX) with fostering done within 24 h of birth.

3. Blood pressure (BP) was measured from age 6–18 weeks; at 6 and 18 weeks MRA structure was assessed.

4. At the time of death, intra‐arterial mean BP was measured (via the femoral artery), after which MRA were fixed by perfusion (at the systolic BP of the rat) via the abdominal aorta, first with 75% Tyrode's solution containing heparin and papaverine, followed by 2% glutaraldehyde in 75% Tyrode's solution. Arteries were dissected out, processed and embedded in Technovit (Heraeus Kulzer, Werheim, Germany) and serial sections were cut and stained with Giemsa.

5. Stereological techniques were used to determine media width, lumen diameter and medial cross‐sectional area (CSA); in addition, the ratio of media width to lumen diameter was calculated. Smooth muscle cell density was also calculated.

6. In MRA from 6‐week‐old rats, GH rats compared with N rats had increased media width and CSA and an increased ratio of media width to lumen diameter.

7. There were no significant changes in structure in the GHX group compared with GH rats. The NX group compared with N rats had increased media width and CSA and lumen diameter, but no change in the ratio of media width to lumen diameter. Smooth muscle cell density, reduced in GH compared with N rats, was increased (P < 0.001) in the NX group, but not changed in the GHX group compared with GH rats.

8. In 18‐week‐old GH rats compared with N rats, the MRA had a decreased media width and medial CSA and smaller lumen diameter, but there was no change in the ratio of media width to lumen diameter.

9. In the GHX group compared with GH rats, media width and CSA were reduced; in the NX group compared with N rats, media width was increased, lumen decreased and the ratio of media width to lumen diameter increased. Smooth muscle cell density was increased (P < 0.001) in the GHX group, but not in the NX group.

10. Changing the maternal environment significantly affected BP in GHX and NX groups up to 9–10 weeks of age but, in adult rats, the BP differences were no longer present. Thus, structural changes were seen at 6 weeks of age in MRA from NX rats and also at 18 weeks in GHX and NX rats (even though the BP differences were no longer significant); structural remodelling occurred independently of BP.
Renal haemodynamics and function in weanling rats treated with enalapril from birth
Authors: Guron Gregor
Published: September 2005

1. Inhibition of the renin–angiotensin system (RAS) during kidney development produces chronic alterations in renal morphology and function that have been characterized in detail in adult animals. The aim of the present study was to determine the consequences of neonatal angiotensin‐converting enzyme (ACE) inhibition on renal haemodynamics and function in rats at a much earlier age, namely 3–4 weeks.

2. Male Wistar pups received daily intraperitoneal injections of enalapril (10 mg/kg) or isotonic saline from birth until 24–28 days of age, when renal haemodynamics and function were assessed using clearance techniques under pentobarbital anaesthesia.

3. Enalapril‐treated rats showed significant reductions in glomerular filtration rate (GFR; −44 ± 6%; P < 0.05), effective renal plasma flow (ERPF; −33 ± 6%; P < 0.05) and filtration fraction (−16 ± 3%; P < 0.05) compared with saline‐treated controls. Although mean arterial pressure tended to be lower in enalapril‐treated rats, this group demonstrated a significant increase in renal vascular resistance compared with control rats (RVR; 46 ± 6 vs 32 ± 3 mmHg/mL per·min per g·kidney weight, respectively; P < 0.05). In enalapril‐treated rats, urine osmolality was reduced (−59 ± 5%; P < 0.05) and urine flow rate and fractional urinary excretion rates of sodium and potassium were markedly elevated compared with controls (P < 0.05). Enalapril‐treated rats showed severe renal histological abnormalities, including wall thickening of cortical arterioles, papillary atrophy and tubulointerstitial alterations, mimicking those described previously in similarly treated rats examined in adulthood.

4. In conclusion, neonatal ACE inhibition in rats induces pronounced alterations in renal haemodynamics and function, characterized by reductions in ERPF and GFR, increased RVR and impaired tubular sodium and water reabsorption, which are evident at weaning.