bluuebunny/crossref_metadata_2025 huggingface dataset:
Hormone‐sensitive lipase in skeletal muscle: regulatory mechanisms
Authors: Langfort J. , Donsmark M. , Ploug T. , Holm C. , Galbo H.
Published: July 2003

Aim:  The enzymatic regulation of intramuscular triacylglycerol (TG) breakdown has until recently not been well understood. Our aim was to elucidate the role of hormone‐sensitive lipase (HSL), which controls TG breakdown in adipose tissue.

Methods:  Isolated rat muscle as well as exercising humans were studied.

Results:  The presence of HSL was demonstrated in all muscle fibre types by Western blotting of muscle fibres isolated by collagenase treatment or after freeze‐drying. The content of HSL varies between fibre types, being higher in oxidative than in glycolytic fibres. Analysed under conditions optimal for HSL, neutral lipase activity in muscle can be stimulated by adrenaline as well as by contractions. These increases are abolished by presence of anti‐HSL antibody during analysis. Moreover, immunoprecipitation with affinity‐purified anti‐HSL antibody causes similar reductions in muscle HSL protein concentration and in measured neutral lipase responses to contractions. The immunoreactive HSL in muscle is stimulated by adrenaline via beta‐adrenergic activation of protein kinase A (PKA). From findings in adipocytes it is likely that PKA phosphorylates HSL at residues Ser563, Ser659 and Ser660. Contraction probably also enhances muscle‐HSL activity by phosphorylation, because the contraction‐induced increase in HSL activity is increased by the protein phosphatase inhibitor okadaic acid and reversed by alkaline phosphatase. A novel signalling pathway in muscle by which HSL activity may be stimulated by protein kinase C (PKC) via extracellular signal regulated kinase (ERK) has been demonstrated. In contrast to previous findings in adipocytes, in muscle activation of ERK is not necessary for stimulation of HSL by adrenaline. However, contraction‐induced HSL activation is mediated by PKC, at least partly via the ERK pathway. In fat cells ERK is known to phosphorylate HSL at Ser600. So, phosphorylation of different sites may explain that in muscle the effects of contractions and adrenaline on HSL activity are partially additive. In line with the view that the two stimuli act by different mechanisms, training increases the contraction‐mediated, but diminishes the adrenaline mediated HSL activation in muscle.

Conclusion:  The existence and regulation of HSL in skeletal muscle indicate a role of HSL in muscle TG metabolism.
Molecular regulation of individual skeletal muscle fibre types
Authors: Spangenburg E. E. , Booth F. W.
Published: July 2003

The purpose of this review is to present current understanding of cellular and molecular regulation of fibre type expression in skeletal muscle. Published literature seems to conclusively suggest that muscle fibre type expression is regulated by multiple signalling pathways and transcription factors rather than a single ‘master’ switch or signalling pathway. While the current nomenclature for fibre types is convenient for communication, based upon the evolution of this nomenclature, the prediction that fibre type classifications may change in the future to incorporate post‐genomic information is made. It is predicted that future fibre type classifications could be based upon the contractile‐activity‐induced changes in a common regulatory factor(s) within a subpopulation of genes whose expressions are altered to modify and maintain the new muscle fibre phenotype.
AMPK as a metabolic switch in rat muscle, liver and adipose tissue after exercise
Authors: Ruderman N. B. , Park H. , Kaushik V. K. , Dean D. , Constant S. , Prentki M. , Saha A. K.
Published: July 2003

An increasing body of evidence has revealed that activation of adenosine monophosphate (AMP)‐activated protein kinase (AMPK)‐activated protein kinase increases fatty acid oxidation by lowering the concentration of malonyl coenzyme A (CoA), an inhibitor of carnitine palmitoyl transferase 1. Studies carried out primarily in skeletal muscle suggest that AMPK modulates the concentration of malonyl CoA by concurrently phosphorylating and inhibiting acetyl CoA carboxylase (ACC), the rate limiting enzyme in malonyl CoA synthesis, and phosphorylating and activating malonyl CoA decarboxylase (MCD), an enzyme involved in its degradation. We have recently observed that AMPK and MCD activities are increased and ACC activity diminished in skeletal muscle, liver and, surprisingly, in adipose tissue 30 min following exercise (treadmill run) in normal rats. In liver and adipose tissue these changes were associated with a decrease in the activity of glycerol‐3‐phosphate acyltransferase (GPAT), which catalyses the first committed reaction in glycerolipid synthesis and, which like ACC, is phosphorylated and inhibited by AMPK. Similar changes in ACC, MCD and GPAT were observed following the administration of 5‐aminoimidazole 4‐carboxamide‐riboside (AICAR), further indicating that the exercise‐induced alterations in these enzymes were AMPK‐mediated.

Conclusions:  (1) AMPK plays a major role in regulating lipid metabolism in multiple tissues following exercise. (2) The net effect of its activation is to increase fatty acid oxidation and diminish glycerolipid synthesis. (3) The relevance of these findings to the regulation of muscle glycogen repletion in the post‐exercise state and to the demonstrated ability of AMPK activation to decrease adiposity and increase insulin sensitivity in rodents remains to be determined.
AMP‐activated protein kinase and muscle glucose uptake
Authors: Musi N. , Goodyear L. J.
Published: July 2003

The AMP‐activated protein kinase (AMPK) is an enzyme that is activated in situations where there are changes in the cellular energy status such as muscle contraction and hypoxia. AMPK can also be pharmacologically activated by the compound 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR) and the antidiabetic agent metformin. Several studies support the hypothesis that AMPK plays an important role in the stimulation of muscle glucose uptake by these physiological and pharmacological stimuli. In isolated rat muscles, activation of AMPK is associated with increases in glucose uptake through an insulin‐independent mechanism. Studies done in rodents have shown that the activation of AMPK by AICAR is accompanied by decreases in blood glucose concentrations, in part due to enhanced muscle glucose uptake. Similar to exercise, AICAR not only directly stimulates glucose uptake into the skeletal muscle, but also enhances insulin sensitivity. The activation of AMPK and associated increases in muscle glucose uptake are affected by factors such as glycogen content, exercise training and fibre type. The effects of AMPK on muscle glucose uptake makes this protein a promising pharmacological target for the treatment of type 2 diabetes.
Variants in the aromatase gene and on the Y‐chromosome are not associated with adult height or insulin resistance in a UK population
Authors: Weedon Michael N. , Turner Martina , Knight Beatrice , Clark Penny , Hattersley Andrew T. , Frayling Timothy M.
Published: July 2003

objective  To assess the association of polymorphisms in the aromatase gene (CYP19) and on the Y‐chromosome, with adult height and insulin resistance in a UK Caucasian population, after a recent report indicated these variants explain 4 cm of adult male height variation.

patients and design  We performed an association study using 917 healthy UK Caucasian subjects from the Exeter Family Study, an ongoing consecutive‐birth cohort. Our study had > 85% (95% for the CYP19 variant; 85% for the Y variant) power to detect the association suggested by the previous study.

measurements  Subjects’CYP19 genotype were determined using tetra‐primer PCR, and the Y‐chromosome variant genotype was identified using a restriction fragment length polymorphism (RFLP) method. Trained research nurses were responsible for measurement of height. Fasting insulin concentration was determined by an immunoenzymometric assay.

results  We did not find any evidence for an effect of the CYP19 polymorphism or Y‐RFLP on adult height (P > 0·83 for both variants). In addition, there was no evidence for an effect on insulin resistance in a subset of 416 subjects (P > 0·46).

conclusion  We have not confirmed the initial observation in a larger replication cohort. Our results highlight the importance of replicating initial results from genetic association studies.
Recent concerns surrounding HRT
Authors: Armitage Mary , Nooney Janet , Evans Stephen
Published: July 2003

Millions of women are treated with hormone replacement therapy (HRT) for relief of menopausal symptoms, including vasomotor flushes and sweats for which oestrogen is uniquely and highly effective. Others may continue longer‐term treatment in the hope that HRT will help to prevent chronic disease. The preservation of bone mass with continuing oestrogen therapy and reduction of subsequent risk of fracture is well established. Observational studies of the metabolic and vascular effects of oestrogens have suggested a potential benefit in reducing the risk of vascular disease, but recently published randomized controlled trials demonstrate no evidence of benefit in women with established vascular disease or in apparently healthy women. The increased risks of breast cancer and thromboembolic disease have been confirmed in these trials, with evidence of increased risk of stroke. Observational data suggest there may be a small increased risk of ovarian cancer associated with longer‐term use of HRT. The premature termination of one arm of the Women's Health Initiative randomized controlled trial caused concern among patients, doctors and pharmaceutical companies. There are difficulties in extrapolating the results from trials using a specific HRT product to advise women on the wide range of other hormone products, doses, combinations and routes of administration. However, in the absence of evidence that other products are safer, the data suggest that for many women the risks associated with long‐term use of HRT outweigh the benefits. There are nonhormonal strategies for the prevention and treatment of osteoporosis. HRT is not, and has never been, licensed in the UK for the prevention or treatment of vascular disease, and the data suggesting potential benefit should now be regarded as biased. The absolute incidence of an adverse event is low, and the risk in an individual woman in a single year is very small, but the risks are cumulative over time with long‐term use. The risk–benefit balance of each woman needs regular reappraisal with continued use.
Detection and localization of chemokine gene expression in autoimmune thyroid disease
Authors: Kemp E. Helen , Metcalfe Russell A. , Smith Karen A. , Woodroofe M. Nicola , Watson Philip F. , Weetman Anthony P.
Published: July 2003

objective Autoimmune thyroid disease (Hashimoto's thyroiditis and Graves’ disease) is characterized by lymphocytic infiltration of the thyroid gland. Chemokines are cytokines with chemoattractant properties for a range of immune effector cells and might therefore play a significant role in the initiation and maintenance of the autoimmune process. The aim of this study was to analyse chemokine gene expression in autoimmune thyroid tissue and in cultured thyroid follicular cells (TFC).

design and patients Immunocytochemistry and reverse‐transcriptase polymerase chain reaction (RT–PCR) amplification were used to analyse the expression of monocyte chemoattractant protein (MCP)‐1, RANTES (regulated upon activation, normal T cell expressed and secreted), macrophage inflammatory protein (MIP)‐1α, MIP‐1β, interferon (IFN)‐γ‐inducible protein (IP)‐10 and monokine induced by IFN‐γ (Mig) in thyroid tissue from patients with Hashimoto's thyroiditis (n = 4), Graves’ disease (n = 6) and nonautoimmune multinodular goitre (n = 4). Chemokine gene expression was also examined in cultured TFC by RT–PCR.

results Expression of MCP‐1, RANTES, MIP‐1α, MIP‐1β, IP‐10 and Mig was demonstrated in all Hashimoto's and most Graves’ thyroid specimens but very little expression was detected in the nonautoimmune goitre samples. In thyroid tissue from Graves’ disease patients, positive staining for chemokines was largely restricted to the lymphocytic cell infiltrate. Within thyroid tissue from Hashimoto's patients, there was evidence for the expression of all chemokines by thyroid follicular cells, suggesting a role for local chemokine synthesis by the glandular epithelial cells in the recruitment of inflammatory cells into the gland in autoimmunity. The present work also showed that expression all the chemokine genes analysed could be induced in cultured thyroid cells by IFN‐γ and interleukin (IL)‐1α. Expression of all the chemokines examined was not stimulated by TSH.

conclusion We postulate that TFC may play a role in the pathogenesis of autoimmune thyroid disease as they are able to express the chemokines MIP‐1α, MIP‐1β, MCP‐1, RANTES, IP‐10 and Mig that would promote the infiltration of immune cells into the thyroid gland.
The pituitary–testicular axis in Klinefelter's syndrome and in oligo‐azoospermic patients with and without deletions of the Y chromosome long arm
Authors: Tomasi Paolo A. , Oates Robert , Brown Laura , Delitala Giuseppe , Page David C.
Published: July 2003

objective The most frequent known genetic causes of severe oligospermia (< 5 million sperm/ml) or azoospermia in men are Klinefelter's syndrome (KS), and deletions in the Y chromosome long arm (Yq). We aimed to compare the function of the pituitary–testicular axis in patients with severe oligospermia or azoospermia, idiopathic or associated with Y chromosome deletions or Klinefelter's syndrome (KS) and in control subjects.

patients We studied 47 men with idiopathic oligo‐azoospermia, 42 with Yq deletions (27 AZFc, 13 AZFb and two AZFa) and oligo‐azoospermia, 14 with KS and 39 control subjects (total 143).

measurements We analysed levels of FSH, inhibin‐B, LH, free testosterone and oestradiol in all subjects, and we calculated indexes based on those hormones.

results Inhibin‐B levels were indistinguishable between patients with idiopathic and Y deletion‐associated oligo‐azoospermia, lowest in the Klinefelter's patients and highest in controls. FSH levels followed the reverse pattern: indistinguishable between patients with idiopathic and deletion‐associated oligo‐azoospermia, highest in Klinefelter's patients and lowest in controls. Oestradiol, free testosterone and the derived indeces were not different in subjects with Yq deletions compared to those with idiopathic oligo‐azoospermia. Among the Yq‐deleted patients, no measured or derived parameter differed between the subjects with AZFc deletion and those with AZFb deletion. When non‐KS oligo‐azoospermic patients were classified according to histology [Sertoli cell‐only (SCO), n = 18 or non‐Sertoli cell only (non‐SCO), n= 18] and compared to KS patients, the hormonal pattern did not differ between SCO and non‐SCO subjects, but levels in KS patients were significantly different for FSH, inhibin‐B and the FSH/inhibin‐B ratio. KS patients not only had lower inhibin‐B than SCO and non‐SCO oligo‐azoospermic men, but also higher FSH levels for any given inhibin‐B concentration.

conclusion Our data show that Y‐deleted patients do not have a lesser impairment of Sertoli cell function than patients with idiopathic oligo‐azoospermia, and support the concept that the main determinant of inhibin‐B production is the germ cell mass. Also, our results suggest that one or more other factors, apart from inhibin‐B, may contribute to increased pituitary secretion of FSH in KS patients.
Recombinant human TSH testing is a valuable tool for differential diagnosis of congenital hypothyroidism during l‐thyroxine replacement
Authors: Fugazzola Laura , Persani Luca , Mannavola Deborah , Reschini Eugenio , Vannucchi Guia , Weber Giovanna , Beck‐Peccoz Paolo
Published: July 2003

objective The differential diagnosis of congenital hypothyroidism (CH) is aimed to distinguish transitory from permanent forms, to optimize l‐thyroxine (l‐T4) therapy to replacement or TSH‐suppressive regimens, and to reach accurate definition of the clinical and biochemical phenotype for subsequent genetic investigations and counselling. Therefore, l‐T4 therapy is presently withdrawn in most instances and investigations are performed in a disturbing hypothyroid state.

design The availability of recombinant human TSH (rhTSH) prompted us to assess its efficacy in the differential diagnosis of CH during l‐T4 therapy.

patients and measurements Eight adult patients with permanent CH remained on l‐thyroxine and underwent a new protocol for rhTSH (Thyrogen®) testing with injections [4 g/kg/day intramuscularly (i.m.)] at days 1, 2 and 3. At day 3, 123I was administered and uptake obtained after 2 and 24 h. Serum TSH and thyroglobulin (Tg) levels were measured at days 1–4. Neck ultrasound was carried out in all cases.

results Serum TSH reached levels > 20 mU/l at day 2 and remained above 30 mU/l on days 3 and 4. Stimulation of Tg levels was seen in five patients with peak at day 4. Lingual thyroid was documented at scintigraphy (TS) in three Tg‐responsive patients who were previously diagnosed as having thyroid agenesia. In one patient with dyshormonogenesis and high Tg, TS confirmed the presence of goitre with positive perchlorate test. TS was negative in the remaining four cases. All tests indicated complete agenesia in one, whereas a minimal Tg response was marker of nearly complete agenesia in another. The last two TS‐negative patients had hypoplastic glands at ultrasound, and refractoriness to TSH stimulation was confirmed by absent Tg response.

conclusions We report the first application of rhTSH for differential diagnosis of patients with permanent CH, avoiding the undesirable transient hypothyroidism consequent to l‐T4 withdrawal. The data obtained led to the change of the diagnosis at presentation in 4/8 patients and to a more accurate description of the clinical picture in all patients. The proposed protocol has been proved to cause Tg increases even in the presence of small amounts of responsive thyroid cells. The rhTSH testing led to the desired disease characterization, thus allowing specific management and targeted genetic analyses.
Increased Interleukin‐6 levels in pituitary‐deficient patients are independently related to their carotid intima‐media thickness
Authors: Leonsson M. , Hulthe J. , Johannsson G. , Wiklund O. , Wikstrand J. , Bengtsson B. Å. , Oscarsson J.
Published: July 2003

objective Increased cardiovascular morbidity and mortality has been observed in patients with pituitary deficiency and untreated growth hormone deficiency (GHD). We investigated peripheral inflammatory and fibrinolytic markers and their associations with arterial intima‐media thickness (IMT) in GHD.

design Cross‐sectional study.

patients Thirty‐four patients with GHD, but without cardiovascular disease, were compared to healthy controls matched for age, sex, body mass index (BMI) and smoking habits.

measurements IMT of the common carotid artery, C‐reactive protein (CRP), interleukin‐6 (IL‐6), fibrinogen, plasminogen activator inhibitor‐1 (PAI‐1) activity and tissue plasminogen activator antigen (tPA‐ag) were measured.

results Median IL‐6 concentrations were increased by 208% and 248% in GHD patients compared to BMI‐matched and nonobese controls, respectively. Median CRP and tPA‐ag levels were increased by 237% and 167% in patients compared to nonobese controls, but not significantly different compared to BMI‐matched controls. Plasma levels of fibrinogen and PAI‐1 activity did not differ between groups. Age, low‐density lipoprotein (LDL) cholesterol, tPA‐ag and IL‐6 were positively correlated, and IGF‐I was negatively correlated to IMT in the patient group, but only age and IL‐6 were independently related to IMT.

conclusions IL‐6 concentrations were increased in GHD patients compared to controls and independently related to IMT in patients. This finding may help to explain the variance in IMT and the increased vascular morbidity and mortality in hypopituitary patients with GHD.
The effectiveness of arginine + GHRH test compared with GHRH + GHRP‐6 test in diagnosing growth hormone deficiency in adults
Authors: Popovic Vera , Pekic Sandra , Doknic Mirjana , Micic Dragan , Damjanovic Svetozar , Zarkovic Milos , Aimaretti Gianluca , Corneli Ginerva , Ghigo Ezio , Deiguez Carlos , Casanueva Felipe F.
Published: July 2003

objective The objective of this study is to investigate the performance of two novel tests in diagnosing growth hormone deficiency in adults.

patients Fifty patients presenting with long‐standing hypopituitarism and two control groups consisting of 50 healthy subjects in each group [age‐, sex‐ and body mass index (BMI)‐matched] participated in a multicentre study. All underwent two provocative tests on different days within 3 months apart: (i) a combined GHRH + GHRP‐6 test and (ii) arginine + GHRH test. Main outcome measures were sensitivity, specificity and areas under reciever operating characteristic curves (ROC) for GHRH + GHRP‐6 and arginine + GHRH tests in the diagnosis of GH deficiency in adults.

results A peak GH level after GHRH + GHRP‐6, under 15·0 µg/l had a sensitvity of 94·4% and specificity of 98·8% for diagnosing GHD and area under the ROC curve was 0·99 (95% confidence interval 0·95–0·99). For peak GH levels after arginine + GHRH, a level under 9·6 µg/l had a sensitivity of 88·4% and specficity of 95% and area under the ROC curve of 0·96 (95% confidence interval 0·91–0·98). The relationship between the increment in serum GH following GHRH + GHRP‐6 and arginine + GHRH test was positive, i.e. r = 0·749, P = 0·001.Thus, there was high concordance between both tests.

conclusion Both GHRH + GHRP‐6 and/or arginine + GHRH test may serve as an alternative to the ITT in diagnosing GHD in adults.
High‐risk clinical features predict increased post‐infarction myocardial apoptosis and the benefits as a result of an open infarct‐related artery
Authors: Abbate A. , Biondi‐Zoccai G. G. L. , Bussani R. , Camilot D. , Dobrina A. , Leone A. M. , Baldi F. , Silvestri F. , Biasucci L. M. , Baldi A.
Published: July 2003

Background Infarct‐related artery (IRA) patency after acute myocardial infarction (AMI) is associated with a more favourable clinical course, in particular in patients with high‐risk features. As it has been recently reported that IRA patency is associated with a reduced postinfarction apoptotic rate (AR), the aim of our study was to assess whether IRA status late after AMI had a different impact on AR in high‐ vs. low‐risk patients.

Methods and results Co‐localization of TUNEL and caspase‐3 was used to calculate the AR at the site of infarction at the time of death in 30 subjects. The Norris coronary prognostic index (NI) was calculated (computing age, presence of pulmonary congestion, heart size and history of previous additional AMI) in order to define the patients’ individual risk at the time of hospitalization. According to the NI (≤ 7 vs. > 7), subjects were divided into low and high risk, as NI > 7 carries an approximate threefold higher risk of death. The NI was significantly correlated with the AR at the time of death both in infarct and remote areas. Twenty subjects had IRA occlusion at the time of death, and in these patients AR was significantly higher both in infarct and remote areas (P < 0·001 and P= 0·009 vs. the others, respectively). However the impact of IRA occlusion on AR was significantly different comparing high‐ vs. low‐risk subjects. In particular, AR at the infarct site was 10‐fold higher in the high‐risk subjects with IRA occlusion (26·1%[20·4–28·7%]) vs. those with open IRA (2·3%[0·6–3·5%]; P = 0·002) and was nonsignificantly different in the low‐risk subjects vs. those without IRA occlusion (8·2%[2·5–17·5%] vs. 5·4%[1·5–7·9%]; P = 0·48). Similarly, in the high‐risk subjects, AR in remote areas was significantly greater in cases with occluded vs. open IRA (0·7%[0·4–0·9%] vs. 0·3%[0·3–0·32%]; P= 0·009).

Conclusion A significantly higher AR is associated with IRA occlusion late post AMI in subjects with high‐risk clinical features, and not in low‐risk patients. The diverse impact of IRA occlusion on AR in subjects with different risk profiles may explain the greater benefit associated with coronary reperfusion in high‐risk subjects. The overall lower AR in low‐risk subjects, independently from the IRA status, may be correlated with the better long‐term prognosis after AMI in this case.
Interleukins 1 beta and 6 induce functional alteration of rat colonic motility: an in vitro study
Authors: Natale L. , Piepoli A. L. , De Salvia M. A. , De Salvatore G. , Mitolo C. I. , Marzullo A. , Portincasa P. , Moschetta A. , Palasciano G. , Mitolo‐Chieppa D.
Published: July 2003

Background In rodents, interleukins administration induces intestinal changes similar to those found in inflammatory bowel disease. We investigated the effects of in vivo subchronic treatment with IL‐1 beta and IL‐6 on rat colonic mucosa and circular smooth muscle.

Materials and method We evaluated transmucosal electrical parameters (Ussing chambers) and early changes of in vitro direct contractility induced by carbachol and tachykinins. Alterations in excitatory and inhibitory neurotransmission were studied with electrical field stimulation (EFS).

Results Treatment with interleukins induces inflammation proved by fever, early signs of colonic histological damage and changes in mucosal ion transport. Concentration response‐curve to carbachol was significantly lower in treated rats (P < 0·02) with significant difference in Emax between control (1·67 ± 0·17 g) and treated preparations (1·20 ± 0·13 g) (P < 0·05). Concentration response‐curve to NK2 agonist was significantly lower in the treated rats (P < 0·005) with a significant difference in Emax between the control (0·26 ± 0·04 g) and treated preparations (0·12 ± 0·02 g) (P < 0·02). None of the drugs used induces changes in EC50. The contractile reflex response to electrically induced distension was significantly higher in the treated rats and more reduced after administration of atropine. Adding NK2 receptor antagonist resulted in a further reduction being observed in the treated and control rats (P = NS). Relaxation by EFS on cholinergic tone was not different between treatments, although pretreatment with L‐NNA resulted in greater relaxation in the treated (−21·7%) than in the control rats (−14·8%).

Conclusion Early inflammation induced by a subchronic treatment with ILs causes changes in mucosal ionic transport parameters, a reduction in the direct contractile response, and an alteration in the neurotransmission (by an enhancing cholinergic component) that may affect the physiological pattern of colonic motility and the sensory reflex.
Super‐marathon race increases serum and urinary nitrotyrosine and carbonyl levels
Authors: Radák Z. , Ogonovszky H. , Dubecz J. , Pavlik G. , Sasvari M. , Pucsok J. , Berkes I. , Csont T. , Ferdinandy P.
Published: July 2003

Background In normal conditions, proteins are not present in the urine, however, exercise of long duration could result in proteinurea. Increased levels of reactive oxygen and nitrogen species (RONS) are formed during exhaustive physical exercise and causes alterations to cellular proteins.

Materials In the present study serum and urinary nitrotyrosine and protein carbonyl levels were measured before and after each run of a 4‐day super‐marathon race.

Result Serum nitrotyrosine and protein carbonyl levels increased after the first (93 km) day running and reached a plateau on the second (120 km), third (56 km) and forth (59 km) days of the competition. A significant correlation was found between urinary and serum protein carbonyl and nitrotyrosine levels (r = 0·78, r = 0·71, respectively). A large percentage of urinary proteins were carbonylated and nitrated. Therefore, it appears that clearance of oxidized proteins in certain conditions occurs not only by the proteolytic pathways but also by filtration and urination.

Conclusion Data reveals that exhaustive aerobic exercise causes oxidative stress and increases the nitration and carbonylation of serum proteins. The presence of carbonyl and nitrotyrosine in proteins of the urine might reflect oxidative stress and could serve as a noninvasive diagnostic tool for exercise physiology.
Effects of homocysteine on l‐arginine transport and nitric oxide formation in human platelets
Authors: Leoncini G. , Pascale R. , Signorello M. G.
Published: July 2003

Background Recent evidence indicates that hyperhomocysteinaemia is an independent risk factor for atherosclerosis, thrombosis and other cardiovascular diseases. This may be secondary to impaired fibrinolysis or increased platelet reactivity. Nitric oxide (NO), a product from l‐arginine by NOS and potent antiaggregating agent, plays an important role in the regulation of platelet function.

Design The present study aimed to define the effect of homocysteine on the l‐arginine/NO pathway in human platelets. l‐arginine uptake, NO formation and Ca2+ levels were measured. Moreover the homocysteine effect on platelet activation induced by thrombin was tested.

Results Homocysteine causes a concentration‐dependent inhibition of l‐arginine transport. Results show that homocysteine does not modify the Km parameter, but it significantly decreases the Vmax value. The nitrite and nitrate formation, strictly correlated with the l‐arginine transport, also significantly decreased. In contrast, cNOS activity remained unchanged upon homocysteine treatment. In addition homocysteine in a dose dependent manner increased the intracellular Ca2+ concentration and platelet response to thrombin.

Conclusions Results indicate that the l‐arginine/NO pathway is one of the various targets of homocysteine in human platelets. The increased Ca2+ levels associated with reduced NO formation may generate hyperactivation and may contribute to the thrombogenic processes.
Current Status and Future Directions of Geriatric General Surgery
Authors: Pofahl Walter E. , Pories Walter J.
Published: July 2003

As the population ages and requires more health care, a significant part of this is in surgical services. As elderly patients account for a growing proportion of most surgeons' practices, it is apparent that this patient group has special requirements, differences in outcomes, and different physiology from other patients encountered in the typical surgical practice. The greater frequency of emergency operations in older than in younger patients, with higher morbidity and mortality rates, compounds these differences. This paper reviews the current status of general surgery in older patients with a special focus on emergency procedures, major abdominal surgery, biliary disease, endocrine disease, and breast cancer. Each of these areas is a source of considerable interest to general surgeons.
Geriatric Urology
Authors: Drach George W. , Griebling Tomas L.
Published: July 2003

Urology is one of the surgical specialties that has been most affected by the growing demographic of older adults in the United States. Urological problems are common in elderly people, and care of patients aged 65 and older represents a large proportion of many urologists' practices. However, this has also presented unique challenges to the development of the field of geriatric urology as an identified subspecialty. Until recently, formal educational requirements and opportunities in geriatric urology were limited. In addition, there is a paucity of basic science and clinical research related specifically to urological problems in older adults, despite the growing numbers of patients in this age group. This article reviews recent efforts to increase the activity, content, and visibility of the field of geriatric urology. Curriculum materials have been developed for urology training programs, research needs have been assessed and targeted, and a new professional organization (the Geriatric Urology Society) has been established. Current activities in the field of geriatric urology at the local, regional, and national levels, and goals for the future are reviewed.
Stochastic modelling of nutrient loading and lake ecosystem response in relation to submerged macrophytes and benthivorous fish
Authors: Portielje R. , Rijsdijk R. E.
Published: July 2003

SUMMARY 1. The strong stabilising effect of increased submerged macrophytes (charophytes) and benthivorous fish reduction on the clear water state was shown for shallow Lake Veluwe and Lake Wolderwijd.

2. The first two links in the chain of relationships from external phosphorus (P) loading to in‐lake total‐P concentrations to chlorophyll a concentrations to water transparency, showed a significant correlation with the areal fraction of coverage with charophytes. Higher coverages lead to (i) lower ratios of the in‐lake total‐P concentration compared with the volume weighted average concentration in the inlet water, indicating a higher retention of P in the presence of charophytes (ii) lower chlorophyll a to total‐P ratios, indicating a positive effect of charophytes on top‐down control of algae, and (iii) higher water transparency because of lower algal turbidity. Transparency further improved as a result of benthivorous fish reduction and a significant positive correlation between non‐algal turbidity and benthivorous fish biomass.

3. A model was developed taking into account the inherent variability in precipitation and uncertainties in the empirical relationships determining phosphorus export from stream catchments and other sources and eutrophication variables in the receiving lakes. The model was used to compute (i) probability distributions for in‐lake total‐P, chlorophyll a and Secchi Disc transparency in relation to the coverage with charophytes and benthivorous fish biomass, and (ii) exceedence probabilities with respect to critical values for in‐lake total‐P and water transparency for several management scenarios.

4. The effects of an expected rise in external nutrient loading on the in‐lake total‐P and chlorophyll a concentrations and on water transparency can be compensated for by two proposed control measures: (i) extended treatment at a waste water treatment plant directly discharging into Lake Veluwe, and (ii) diverting the outlet of a stream draining a catchment with high fertilisation. The minimal internal charophyte coverage needed to sufficiently stabilise the clear water state and to meet with the objective of a summer mean water transparency of at least 1 m was estimated at well over 30% of the lake area, while the benthivorous fish stock should be maintained at the present level of c. 20 kg ha−1.
Negative effects of UVB‐irradiated phytoplankton on life history traits and fitness of Daphnia magna
Authors: De Lange Hendrika J. , Van Reeuwijk Paul L.
Published: July 2003

SUMMARY 1. We tested the effect of ultraviolet‐B (UVB)‐irradiated phytoplankton on life history characteristics of Daphnia magna. Two phytoplankton species were used, Chlamydomonas reinhardtii and Cryptomonas pyrenoidifera. The phytoplankton species were cultured under photosynthetically active radiation (PAR) conditions, and under PAR supplemented with ultraviolet‐A and ultraviolet‐B radiation, and fed to Daphnia.

2. Life history traits of Daphnia were negatively affected when fed on UVB‐irradiated Cryptomonas. Size at maturity was depressed and fewer juveniles with lower fitness were produced in the UVB treatments. In the Chlamydomonas experiment, no significant effects were found.

3. The cause of the observed UVB effects is likely to be constraints in food quality. Ultraviolet‐B radiation thus has the potential of inhibiting energy transfer from the first to the second trophic level.
Microhabitat selection by the invasive amphipod Echinogammarus ischnus and native Gammarus fasciatus in laboratory experiments and in Lake Erie
Authors: Van Overdijk Colin D. A. , Grigorovich Igor A. , Mabee Tracy , Ray William J. , Ciborowski Jan J. H. , Macisaac Hugh J.
Published: July 2003

1. The amphipod Echinogammarus ischnus was first reported in the Laurentian Great Lakes during 1995. However, analysis of archived samples revealed the presence of the species from western Lake Erie in 1994 and possibly as early as 1993.

2. Surveys conducted in Lake Erie between 1995 and 1998 revealed that Echinogammarus was the dominant amphipod on rocks covered by Dreissena molluscs compared with those fouled by the filamentous alga Cladophora, while Gammarus fasciatus used both Dreissena and Cladophora substrata extensively.

3. In laboratory habitat selection studies, Echinogammarus chose Dreissena‐ over Cladophora‐encrusted rocks and bare rocks, while Gammarus occupied the more complex substrata equally.

4. Field colonisation experiments demonstrated that the densities of Echinogammarus and Gammarus were positively correlated when the total density of the species was low, in contrast to the large‐scale natural distribution of the species that revealed a strong inverse relationship.

5. The on‐going replacement of Gammarus by Echinogammarus in the Laurentian Great Lakes may be related to the stronger affinity of the latter for substrata fouled by Dreissena, a genus with which it co‐evolved.
Structure and evolution of the luciferin‐regenerating enzyme (LRE) gene from the firefly Photinus pyralis
Authors: Day J. C. , Bailey M. J.
Published: July 2003

To study the structural features of genes for the luciferin‐regenerating enzyme (LRE), the entire gene along with 524 bp of upstream sequence was determined from Photinus pyralis (Coleoptera: Lampyridae). The LRE gene revealed an open reading frame composed of five exons divided by four introns ranging in size from 47 to 904 bp. The deduced LRE amino acid sequence showed identity to senescence marker protein‐30 (SMP30) from a number of insects and mammals including four putative SMP30 sequences from Anopheles gambiae. Gene structure comparisons showed some intron/exon site conservation with A. gambiae and mammalian SMP30 proteins but not Drosophila. LRE and luciferase sequence comparisons revealed two conserved putative luciferin‐binding sites. The evolution of LRE was discussed in relation to its function.
RNase G of Escherichia coli exhibits only limited functional overlap with its essential homologue, RNase E
Authors: Ow Maria C. , Perwez Tariq , Kushner Sidney R.
Published: July 2003

RNase G (rng) is an E. coli endoribonuclease that is homologous to the catalytic domain of RNase E (rne), an essential protein that is a major participant in tRNA maturation, mRNA decay, rRNA processing and M1 RNA processing. We demonstrate here that whereas RNase G inefficiently participates in the degradation of mRNAs and the processing of 9S rRNA, it is not involved in either tRNA or M1 RNA processing. This conclusion is supported by the fact that inactivation of RNase G alone does not affect 9S rRNA processing and only leads to minor changes in mRNA half‐lives. However, in rng rne double mutants mRNA decay and 9S rRNA processing are more defective than in either single mutant. Conversely, increasing RNase G levels in an rne‐1 rng::cat double mutant, proportionally increased the extent of 9S rRNA processing and decreased the half‐lives of specific mRNAs. In contrast, variations in the amount of RNase G did not alter tRNA processing under any circumstances. Thus, the failure of RNase G to complement rne mutations, even when overproduced at high levels, apparently results from its inability to substitute for RNase E in the maturation of tRNAs.
Leptospira spp. possess a complete haem biosynthetic pathway and are able to use exogenous haem sources
Authors: Guégan Rozenn , Camadro Jean‐Michel , Saint Girons Isabelle , Picardeau Mathieu
Published: July 2003

Unlike the spirochetes Borrelia burgdorferi and Treponema pallidum, Leptospira spp. contain genes encoding the enzymes for most biosynthetic pathways. In this study, we describe the first haem biosynthetic pathway genes in the order Spirochaetales. Sequence analysis of the L. interrogans genome shows that all haem biosynthetic genes (hemA, heml, hemB, hemC, hemE, hemN, hemY and hemH) are clustered in a 15 kb region of the CII secondary chromosome. Although no hemD homologue (encoding uroporphyrinogen III synthase) was found in the genome, the L. interrogans hemC gene (encoding porphobilinogen deaminase) was able to restore uroporphyrinogen III synthase activity in an Escherichia coliΔhemD mutant, suggesting that the L. interrogans hemC gene encodes a bifunctional enzyme. Similarly, we show that the L. interrogans hemH gene (encoding ferrochelatase, the terminal enzyme of the haem biosynthetic pathway) is able to complement a ferrochelatase‐defective E. coliΔhemH mutant. Further investigation of ferrochelatases was undertaken in both saprophytic and pathogenic species of Leptospira. Ferrochelatase activity of 2.3 ± 0.1 nmol h−1 mg−1 (in comparison with 0.25 ± 0.02 nmol h−1 mg−1 in E. coli) was found in membrane fractions of pathogenic and saprophytic species, suggesting that ferrochelatase is a membrane‐associated protein. Leptospira biflexa allelic exchange mutants containing an inactivated hemH gene were recovered only when exogenous haemin was present. The results indicate that haem is an essential growth factor for Leptospira, and that these spirochetes are capable of both de novo synthesis or uptake of haem. This may have implications in a better understanding of the pathogenesis of Leptospira.
Immunoreceptor tyrosine‐based activation motif phosphorylation during engulfment of Neisseria gonorrhoeae by the neutrophil‐restricted CEACAM3 (CD66d) receptor
Authors: McCaw Shannon E. , Schneider Jutta , Liao Edward H. , Zimmermann Wolfgang , Gray‐Owen Scott D.
Published: July 2003

Gonorrhea is characterized by a purulent urethral or cervical discharge consisting primarily of neutrophils associated with Neisseria gonorrhoeae. These interactions are facilitated by gonococcal colony opacity‐associated (Opa) protein binding to host cellular CEACAM receptors. Of these, CEACAM3 is restricted to neutrophils and contains an immunoreceptor tyrosine‐based activation motif (ITAM) reminiscent of that found within certain phagocytic Fc receptors. CEACAM3 was tyrosine phosphorylated by a Src family kinase‐dependent process upon infection by gonococci expressing CEACAM‐specific Opa proteins. This phosphorylation was necessary for efficient bacterial uptake; however, a less efficient uptake process became evident when kinase inhibitors or mutagenesis of the ITAM were used to prevent phosphorylation. Ligated CEACAM3 was recruited to a cytoskeleton‐containing fraction, intense foci of polymerized actin were evident where bacteria attached to HeLa‐CEACAM3, and disruption of polymerized actin by cytochalasin D blocked all bacterial uptake by these cells. These data support a model whereby CEACAM3 can mediate the Opa‐dependent uptake of N. gonorrhoeae via either an efficient, ITAM phosphorylation‐dependent process that resembles phagocytosis or a less efficient, tyrosine phosphorylation‐independent mechanism.
Complex regulation of csgD promoter activity by global regulatory proteins
Authors: Gerstel Ulrich , Park Chankyu , Römling Ute
Published: July 2003

The starvation‐induced csgD gene of Salmonella typhimurium encodes for the positive transcriptional regulator of extracellular matrix components curli fimbriae and cellulose. To analyse regulatory elements of csgD promoter (PcsgD) response genetic studies combined with in vitro experiments were performed. Six binding sites (D1 to D6) for OmpR, a transcriptional regulator, were identified by gel shifts and DNase I footprints. While ompR is required for PcsgD expression, binding of OmpR‐P to D2 centred immediately upstream of D1 at position −70.5 is proposed to repress PcsgD activity. The elevated expression of regulated and semiconstitutive PcsgD in response to microaerophilic conditions required integration host factor (IHF). Subsequently, two IHF‐binding sites were identified up‐ and downstream of PcsgD. IHF competes with OmpR‐P for binding at its upstream site IHF1, which overlaps with D3–D6 and thereby modulates the response to microaerophilic conditions. A complex regulatory network involving IHF, H‐NS and OmpR is proposed whereby the nucleo‐complex composition in the csgD–csgBA intergenic region is altered in response to oxygen tension.
Pneumococcal zinc metalloproteinase ZmpC cleaves human matrix metalloproteinase 9 and is a virulence factor in experimental pneumonia
Authors: Oggioni Marco R. , Memmi Guido , Maggi Tiziana , Chiavolini Damiana , Iannelli Francesco , Pozzi Gianni
Published: July 2003

The ZmpC zinc metalloproteinase of Streptococcus pneumoniae, annotated in the type 4 genome as SP0071, was found to cleave human matrix metalloproteinase 9 (MMP‐9). The previously described IgA protease activity was confirmed to be specifically linked to the IgA1‐protease/SP1154 zinc metalloproteinase. MMP‐9 is a protease cleaving extracellular matrix gelatin and collagen and is activated by proteolytic cleavage like most proteases. MMP‐9 is a human protease and is involved in a variety of physiological and pathological matrix degrading processes, including tissue invasion of metastases and opening of the blood–brain barrier. While TIGR4 (serotype 4) and G54 (serotype 19) pneumococcal genome strains have a highly conserved copy of zmpC, the genome of R6 (a derivative of serotype 2 D39 strain) lacks zmpC. Both the analysis for zmpC presence and MMP‐9 cleavage activity in various pneumococcal strains showed correlation of ZmpC with MMP‐9 cleavage activity. When assaying clinical isolates of S. pneumoniae, the zmpC gene was not found in any of the nasal and conjunctival swab isolates, but it was present in 1 out of 13 meningitis isolates and in 6 out of 11 pneumonia isolates. In a murine pneumonia model, infection with a zmpC‐mutant reduced mortality at 3–4 days post‐infection by 75%, when compared with infection with wild‐type strains. These data indicate that the ZmpC pneumococcal protease may play a role in pneumococcal virulence and pathogenicity in the lung.
Mapping of the Plasmodium falciparum multidrug resistance gene 5′‐upstream region, and evidence of induction of transcript levels by antimalarial drugs in chloroquine sensitive parasites
Authors: Myrick Alissa , Munasinghe Anusha , Patankar Swati , Wirth Dyann F.
Published: July 2003

The Plasmodium falciparum multidrug resistance gene, pfmdr1, has been shown to be involved in the mediation of the parasite's response to various antimalarial drugs. Previous studies of pfmdr1 expression have shown that transcript levels are increased in drug‐resistant isolates. However, a detailed examination of the transcriptional regulation of this gene has not been completed. The aim of this study was to map the 5′ UTR of pfmdr1, and to examine the transcriptional profile of the gene in sensitive parasites treated with four different antimalarial drugs. RT‐PCR and 5′‐RACE mapping showed that the 5′ UTR has a length of 1.94 kb. A putative promoter has been identified via transient transfection. Northern analysis revealed a 2.1‐ to 2.7‐fold increase in pfmdr1 expression in 3D7 parasites treated with 50 nM chloroquine for 6 h, confirming results from Serial Analysis of Gene Expression. 3D7 parasites were subsequently treated with experimentally derived IC50 concentrations of mefloquine, quinine and pyrimethamine. pfmdr1 transcript levels specifically increased 2.5‐fold at 6 h in mefloquine‐treated parasites and threefold in parasites treated with quinine for 30 min. There was no evidence of transcript induction in pyrimethamine‐treated parasites. This is the first evidence of induction of pfmdr1 expression in sensitive cells; and suggests a novel method of transcriptional control for this gene.