bluuebunny/crossref_metadata_2025 huggingface dataset:
A statin‐based inhibitor of lymphocyte function antigen‐1 protects against ischemia/reperfusion‐induced leukocyte adhesion in the colon
Authors: Wan Min Xiu , Schramm Rene , Klintman Daniel , Welzenbach Karl , Weitz‐Schmidt Gabriele , Thorlacius Henrik
Published: September 2003

Statins are mainly used to control hypercholesterolemia; however, recent studies have also ascribed anti‐inflammatory effects to the statins. LFA703 is a novel statin‐derived compound, which potently inhibits lymphocyte function antigen‐1 (LFA‐1, CD11a/CD18) but does not affect HMG‐CoA reductase activity.

The objective of this study was to examine the anti‐inflammatory mechanisms of LFA703 in ischemia/reperfusion (I/R)‐induced leukocyte–endothelium interactions in the colon. For this purpose, the superior mesenteric artery was occluded for 30 min and leukocyte responses were analyzed in colonic venules after 120 min of reperfusion in mice using inverted intravital fluorescence microscopy.

First, the inhibitory mechanisms of LFA703 on leukocyte adhesion were investigated in vitro using a mouse CD4+8+ thymocyte cell line. Immunoneutralization of LFA‐1 and ICAM‐1 abolished leukocyte adhesion, whereas inhibition of VLA‐4 had no effect in this in vitro assay. Indeed, it was found that LFA703 dose‐dependently reduced LFA‐1‐dependent leukocyte adhesion to mouse endothelial cells in vitro with an IC50 of 3.2 μM.

I/R caused an increase in leukocyte rolling and adhesion in colonic venules. Immunoneutralization of LFA‐1 significantly reduced I/R‐induced leukocyte adhesion by 89% in colonic venules. In contrast, I/R‐provoked leukocyte rolling was insensitive to inhibition of LFA‐1 function.

Administration of 30 mg kg−1 of LFA703 decreased reperfusion‐induced leukocyte adhesion by more than 91%, while the level of leukocyte rolling was unchanged, suggesting that LFA703 effectively blocked LFA‐1‐dependent firm adhesion of leukocyte in the colon. However, LFA703 did not decrease the expression of LFA‐1 on circulating leukocytes.

This study demonstrates that LFA‐1 is indeed a critical adhesion molecule in mediating postischemic leukocyte adhesion in the colon. Moreover, this is the first study showing that a statin‐based synthetic compound has the capacity to abolish LFA‐1‐dependent leukocyte adhesion in I/R. These novel findings may have great implications in the clinical treatment of conditions associated with I/R‐induced tissue injury, such as organ transplantation, trauma and major surgery.

British Journal of Pharmacology (2003) 140, 395–401. doi:10.1038/sj.bjp.0705432
Involvement of EDHF in the hypotension and increased gastric mucosal blood flow caused by PAR‐2 activation in rats
Authors: Kawabata Atsufumi , Nakaya Yumiko , Kuroda Ryotaro , Wakisaka Mariko , Masuko Takashi , Nishikawa Hiroyuki , Kawai Kenzo
Published: September 2003

Agonists for protease‐activated receptor‐2 (PAR‐2) cause hypotension and an increase in gastric mucosal blood flow (GMBF) in vivo. We thus studied the mechanisms underlying the circulatory modulation by PAR‐2 activation in vivo, especially with respect to involvement of endothelium‐derived hyperpolarizing factor (EDHF).

Arterial blood pressure and GMBF were measured in anesthetized rats in vivo. Vascular relaxation was assessed in the precontracted rat gastric arterial rings in vitro.

The PAR‐2‐activating peptide SLIGRL‐NH2 and/or trypsin, administered i.v., produced largely NO‐independent hypotension and increase in GMBF accompanied by decreased gastric mucosal vascular resistance (GMVR) in rats.

Combined administration of apamin and charybdotoxin, but not each of them, specifically abolished the hypotension, increased GMBF and decreased GMVR caused by the PAR‐2 agonists.

In the isolated rat gastric artery, SLIGRL‐NH2 elicited endothelium‐dependent relaxation even in the presence of an NO synthase inhibitor and indomethacin, which was abolished by apamin plus charybdotoxin.

Our data suggest involvement of apamin/charybdotoxin‐sensitive K+ channels in the PAR‐2‐triggered hypotension and increased GMBF, predicting a role of EDHF‐like factors.

British Journal of Pharmacology (2003) 140, 247–254. doi:10.1038/sj.bjp.0705433
Staurosporine induces rapid homotypic intercellular adhesion of U937 cells via multiple kinase activation
Authors: Cho Jae Youl , Katz David R , Chain Benjamin M
Published: September 2003

Staurosporine is a broad‐specificity kinase inhibitor, which has acted as lead compound for the development of some novel cytotoxic compounds for treatment of cancer. This study investigates the unexpected observation that staurosporine can also induce homotypic cellular aggregation.

In this study, staurosporine is shown to activate rapid homotypic aggregation of U937 cells, at concentrations below those required to induce cell death. This activity is a particular feature of staurosporine, and is not shared by a number of other kinase inhibitors. The proaggregating activity of staurosporine is inhibited by deoxyglucose, cytochalasin B and colchicine. Staurosporine‐induced aggregation can be distinguished from that induced by the phorbol 12‐myristate 13‐acetate by faster kinetics and insensitivity to cycloheximide. Staurosporine induces translocation of conventional and novel, but not atypical isoforms of protein kinase C (PKC). Aggregation induced by staurosporine is inhibited by a number of inhibitors of PKC isoforms, and by inhibitors of protein tyrosine kinases. Staurosporine also induces rapid phosphorylation of ERK and p38, and inhibitors of both these enzymes block aggregation.

Staurosporine induces dysregulated activation of multiple kinase signaling pathways in U937 cells, and the combined activity of several of these pathways is essential for the induction of aggregation.

British Journal of Pharmacology (2003) 140, 269–276. doi:10.1038/sj.bjp.0705436
Agonist actions of dihydroergotamine at 5‐HT2B and 5‐HT2C receptors and their possible relevance to antimigraine efficacy
Authors: Schaerlinger B , Hickel P , Etienne N , Guesnier L , Maroteaux L
Published: September 2003

The pharmaceutical compound, dihydroergotamine (DHE) is dispensed to prevent and reduce the occurrence of migraine attacks. Although still controversial, the prophylactic effect of this drug is believed to be caused through blockade and/or activation of numerous receptors including serotonin (5‐HT) receptors of the 5‐HT2 subtype.

To elucidate if 5‐HT2 receptors (5‐HT2Rs) may be involved in DHE prophylactic effect, we performed investigations aimed to determine the respective pharmacological profile of DHE and of its major metabolite 8′‐hydroxy‐DHE (8′‐OH‐DHE) at the 5‐HT2B and 5‐HT2CRs by binding, inositol triphosphate (IP3) or cyclic GMP (cGMP) coupling studies in transfected fibroblasts.

DHE and 8′‐OH‐DHE are competitive compounds at 5‐HT2B and 5‐HT2CRs. 8′‐OH‐DHE interaction at (5‐HT2BRs) was best fitted by a biphasic competition curve and displayed the highest affinity with a Ki of 5 nM. These two compounds acted as agonists for both receptors in respect to cGMP production with pEC50 of 8.32±0.09 for 8′‐OH‐DHE at 5‐HT2B and 7.83±0.06 at 5‐HT2CRs.

Knowing that the antimigraine prophylactic effect of DHE is only observed after long‐term treatment, we chronically exposed the recombinant cells to DHE and 8′‐OH‐DHE. The number of 5‐HT2BR‐binding sites was always more affected than 5‐HT2CRs. At 5‐HT2BRs, 8′‐OH‐DHE was more effective than DHE, with an uncoupling that persisted for more than 40 h for IP3 or cGMP. By contrast, the 5‐HT2CR coupling was reversible after either treatment.

Chronic exposure to 8′‐OH‐DHE caused a persistent agonist‐mediated desensitisation of 5‐HT2B, but not 5‐HT2CRs. This may be of relevance to therapeutic actions of the compound.

British Journal of Pharmacology (2003) 140, 277–284. doi:10.1038/sj.bjp.0705437
Spinal administration of lipoxygenase inhibitors suppresses behavioural and neurochemical manifestations of naloxone‐precipitated opioid withdrawal
Authors: Trang Tuan , Sutak Maaja , Quirion Remi , Jhamandas Khem
Published: September 2003

This study investigated the role of spinal lipoxygenase (LOX) products in the induction and expression of opioid physical dependence using behavioural assessment of withdrawal and immunostaining for CGRP and Fos protein expression in the spinal cord.

Administration of escalating doses (5–50 mg kg−1; i.p.) of morphine for 5 days markedly elevated CGRP‐like immunoreactivity in the dorsal horn of the rat spinal cord. Naloxone (2 mg kg−1; i.p.) challenge precipitated a robust withdrawal syndrome that depleted CGRP‐like immunoreactivity and increased the number of Fos‐like immunoreactive neurons in the dorsal horn.

Intrathecal administration of NDGA (10, 20 μg), a nonselective LOX inhibitor, AA‐861 (1.5, 3 μg), a 5‐LOX selective inhibitor, or baicalein (1.4, 2.8 μg), a 12‐LOX selective inhibitor, concurrently with systemic morphine for 5 days or as a single injection immediately preceding naloxone challenge, blocked the depletion of CGRP‐like immunoreactivity, prevented increase in the number of Fos‐like immunoreactive neurons in the dorsal horn, and significantly attenuated the morphine withdrawal syndrome.

The results of this study suggest that activity of LOX products, at the spinal level, contributes to the expression of opioid physical dependence, and that this activity may be expressed through increased sensory neuropeptide release.

British Journal of Pharmacology (2003) 140, 295–304. doi:10.1038/sj.bjp.0705440
Effects of amitriptyline and fluoxetine upon the in vitro proliferation of tumor cell lines
Authors: Volpe Donna A. , Ellison Christopher D. , Parchment Ralph E. , Grieshaber Charles K. , Faustino Patrick J.
Published: September 2003

Previous publications have suggested that commonly prescribed antidepressants have the potential to stimulate the proliferation of extant tumors in human and rodent in vivo and in vitro models. The direct effects of amitriptyline and fluoxetine were evaluated in assays that detect different aspects of proliferative responses at pharmacologically relevant drug concentrations. Three in vitro assays of cellular proliferation and clonal growth were used with human (MCF7, PA‐1 and LS174T) and murine (B16.f10, C‐3 and B16.f1) tumor cell lines. The cells were exposed to amitriptyline or fluoxetine (0.001–100 μM) for different time periods (1–7 days) and at varying serum concentrations (0.1–15%). Amitriptyline and fluoxetine failed to significantly stimulate tumor cell proliferation, DNA synthesis, or colony formation. Both drugs inhibited B16.f10 colony growth at concentrations above 5 μM along with significant suppression of DNA synthesis in B16.f10 and C‐3 cells at 30 μM. Although there were generally no effects on cell proliferation by the drugs in the microtiter tetrazolium assay, several rare instances of stimulation were noted. Amitriptyline and fluoxetine were consistent in their lack of effect or inhibition with the human or murine tumor cell lines in conventional in vitro assays of cell proliferation and clonogenicity in optimal or suboptimal culture conditions.
siRNA agents inhibit oncogene expression and attenuate human tumor cell growth
Authors: Yin James Q. , Gao Jingsong , Shao Ronguang , Tian Wang‐Ni , Wang Jianping , Wan Yinsheng
Published: September 2003

Cancer is a genomic functional disease with features of oncogene activation and tumor suppressor inactivation. These genomic features have resulted in the limited effectiveness of conventional therapies and therefore forced considerable efforts to explore new types of anticancer agents. It has been clear that chemically synthesized or in vivo‐expressed short interfering RNA (siRNA) can specifically and effectively direct homology‐dependent post‐transcriptional gene silencing. In the present study, we intended to investigate whether siRNA could suppress the proliferation of human cancer cells through interfering oncogene activities and recovering the functions of tumor‐suppressor gene. Single siRNA or combinatorial siRNAs were successfully transfected into HeLa cells, lung adenocarcinoma cells, hepatoma cells, ovarian carcinoma cells, and melanoma cells with cationic lipid complexes. These siRNA molecules not only specifically knocked down their cognate targets such as bcl‐2, cdk‐2, mdm‐2, pkc‐alpha, tgf‐beta1, H‐ras, vegf, and GFP mRNAs, but also effectively suppressed the proliferation of cancer cells to different extents. These data suggest that (1) all these human cancer cells preserve RNAi machinery; (2) chemically synthesized and vector‐driven siRNAs can be incorporated into intrinsic RNAi system for silencing target mRNA molecules; and (3) the combination of different siRNAs inhibits the growth and proliferation of cancer cells.
Low‐dose total body irradiation augments the therapeutic effect of interleukin‐2 in a mouse model for metastatic malignant melanoma
Authors: Safwat Akmal , Aggerholm Ninna , Roitt Ivan , Overgaard Jens , Hokland Marianne
Published: September 2003

Background:  Low‐dose total body irradiation (LTBI) is known for its antitumor immune modulatory effects. Moreover, there is theoretical ground suggesting that combining LTBI with interleukin‐2 (IL‐2) will have a synergistic immune‐mediated antitumor effect. However, the use of LTBI in combination with IL‐2 or other forms of immunotherapy has not been tested before. Aim of the work: To test the efficacy of combining LTBI and IL‐2 in controlling lung metastases in a murine model for malignant melanoma compared to IL‐2 alone. Material and methods: Ten‐week‐old female C57BL/6 mice were inoculated intravenously (on day 0) with 1 million B16F1 malignant melanoma cells. The mice received either no treatment (control group), LTBI alone (single fraction of 0.75 Gy), IL‐2 treatment alone (30,000 CU × 2 daily for 5 consecutive days), or a combination of LTBI and IL‐2. LTBI was given on day +7 and IL‐2 treatment started day +8. On day +14, the mice were sacrificed and the lungs were removed and analyzed for tumor burden. Lung sections were also tested for tumor‐infiltrating cells using immunohistochemical staining. Peripheral blood and splenic cells were collected and tested for the percentage of the various lymphocytic subsets using immunostaining and flow cytometry. Results: Tumor burden expressed as the percentage of lung area occupied with metastases (±1 SD), was the same in the control group (8.1 ± 4.9%), and in the group receiving LTBI alone (8.3 ± 4.5%). Tumor burden was reduced to 6.4%(±3.4%) in the IL‐2 alone group (P = 0.3) and further reduced to 3%(±1%) in the combined treatment group (P = 0.004). The difference in tumor burden between the IL‐2 alone group and the combined treatment group was statistically significant (P = 0.006). The combined treatment caused a significant increase in the number of natural killer (NK) cells and macrophages infiltrating the metastatic sites. This was associated with a significant increase in the percentage of CD122+(IL‐2Rβ) cells and NK cells in both peripheral blood and spleens. Conclusion: We conclude that combining LTBI and IL‐2 treatment is synergistic and therapeutically more effective than IL‐2 alone. The data points to NK cells and macrophages as likely major effectors of the synergistic outcome of the combined treatment. This observation may have important clinical implications in the treatment of patients with metastatic malignant melanoma.
Effects of etherlipid analogs on cell membrane functions
Authors: Berkovic Dinko , Wernicke Jan Heino , Fleer Eduard A. M.
Published: September 2003

Hexadecylphosphocholine and other etherlipid‐derived substances show a pronounced antiproliferative activity on neoplastic cells and a broad spectrum of other biological effects on many cell types in vitro and in vivo. Though the precise molecular mechanism by which these etherlipid analogs act still remains unresolved, it seems clear that it most probably involves some essential function of the cell membrane.

We investigated the effect of different etherlipids with and without cytotoxic activity in etherlipid‐susceptible and ‐resistant tumor cell lines on three important membrane functions. We observed various inhibitory activities on endocytosis and the uptake of small precursor molecules as sugars, amino acids, and alcohols by toxic and nontoxic substances in resistant as well as susceptible cells. There was no correlation between the antiproliferative characteristics of the compounds and the effects on these membrane transport functions. Furthermore, the substances reduced the number of membrane tumor necrosis factor‐α receptors regardless of their antiproliferative properties. The results of these investigations suggest that etherlipid analogs may interfere with many membrane functions in an unspecific manner. Therefore, many of the previously reported biological effects of etherlipids have to be viewed under a different light. Future investigation on these compounds should always contain appropriate control substances and cell models to really prove the specificity of the observed effects.
Protein–protein interactions involving inducible nitric oxide synthase
Authors: Zhang W. , Kuncewicz T. , Yu Z.‐Y. , Zou L. , Xu X. , Kone B. C.
Published: September 2003

Aim:  Nitric oxide (NO) is a signaling and effector molecule that contributes to multiple physiological and pathophysiological processes in the kidney, vasculature, and other tissues. High output NO generation by inducible NO synthase (iNOS) participates in host defense against pathogens and contributes to tissue injury during inflammatory states. Because of its potent reactivity and diffusibility, NO generation by iNOS is subject to multiple levels of regulation, inculding transcriptional, translational, and post‐translational controls, inculding protein‐protein interactions. This review examines the experimental basis for these protein‐protein interactions and their known and potential importance for kidney and vascular physiology.

Methods:  Analysis of the biomedical literature in the area.

Results:  iNOS interacts with the inhibitory molecules Kalirin and NOS‐associated protien 1.10 kd (NAP110), which inhibit iNOS homodimerization, as well as activator proteins, the Rac‐GTPases. Interactions with caveolin‐1 control the intracellular locale and degradation of iNOS in tumor cells. In polarized epithelial cells, associations of iNOS with the scaffolding protien EBP50 position iNOS in the apical membrane near key ion transport proteins that also interact with EPB50. In addition, protein‐protein interactions of proteins governing iNOS transcription function to specify activation or suppression of iNOS induction by cytokines.

Conclusion:  Interactions of iNOS with a diverse group of heterologous proteins provides a selective mechanism to control the activity, spatial distribution, and proximity of iNOS to intended targets, while potentially limiting autotoxicity to the iNOS‐expressing cell.
Facilitation of renal autoregulation by angiotensin II is mediated through modulation of nitric oxide
Authors: Guan Z. , Willgoss D. A. , Matthias A. , Manley S. W. , Crozier S. , Gobe G. , Endre Z. H.
Published: September 2003

Aims: This study was designed to investigate the influence of angiotensin II (Ang II) and nitric oxide (NO) on autoregulation of renal perfusion.

Methods: Autoregulation was investigated in isolated perfused kidneys (IPRK) from Sprague‐Dawley rats during stepped increases in perfusion pressure.

Results: Ang II (75–200 pM) produced dose‐dependent enhancement of autoregulation whereas phenylephrine produced no enhancement and impaired autoregulation of GFR. Enhancement by Ang II was inhibited by the AT1 antagonist, Losartan, and the superoxide scavenger, Tempol. Under control conditions nitric oxide synthase (NOS) inhibition by 10 μm N‐omega‐nitro‐ιl‐arginine methyl ester (ιl‐NAME) facilitated autoregulation in the presence of non‐specific cyclooxygenase (COX) inhibition by 10 μm indomethacin. Both COX and combined NOS/COX inhibition reduced the autoregulatory threshold concentration of Ang II. Facilitation by 100 pm Ang II was inhibited by 100 μm frusemide. Methacholine (50 nm) antagonised Ang II‐facilitated autoregulation in the presence and absence of NOS/COX inhibition. Infusion of the NO donor, 1 μm sodium nitroprusside, inhibited ιl‐NAME enhancement of autoregulation under control conditions and during Ang II infusion.

Conclusions: The results suggest than an excess of NO impairs autoregulation under control conditions in the IPRK and that endogenous and exogenous NO, vasodilatory prostaglandins and endothelium‐derived hyperpolarizing factor (EDHF) activity antagonise Ang II‐facilitated autoregulation. Ang II also produced a counterregulatory vasodilatory response that included prostaglandin and NO release. We suggest that Ang II facilitates autoregulation by a tubuloglomerular feedback–dependent mechanism through AT1 receptor‐mediated depletion of nitric oxide, probably by stimulating generation of superoxide.
Effects of exercise on platelet and aortic functions in aged rats
Authors: Tanaka Y. , Hashimoto M. , Masumura S.
Published: September 2003

Aim and methods:  To assess age‐ and exercise‐related changes in platelet aggregation, we measured the magnitude of platelet aggregation with a four‐channel aggregometer, plasma and aortic polyunsaturated fatty acids by gas chromatography and related prostanoids with a reagent kit in young and aged non‐exercised and in aged exercised rats.

Results:  Platelet aggregation in platelet‐rich plasma induced by ADP (5 μm) in the primary wave increased with age. In the non‐exercised groups, the basal levels of thromboxane B2 in platelet‐rich plasma increased in aged rats compared with young rats. In aged exercised rats, the basal levels of 6‐keto‐prostaglandin F1α in platelet‐rich plasma were stimulated and those of thromboxane B2 were depressed, compared with non‐exercised aged rats. The plasma levels of eicosapentaenoic acid and docosahexaenoic acid increased with age. Only aortic eicosapentaenoic acid in the aged group increased by exercise. In the aged non‐exercised and exercised groups, the aortic, but not the plasma, levels of eicosapentaenoic acid correlated inversely with the basal levels of thromboxane B2 in platelet‐rich plasma (r = −0.53, P < 0.05) and associated negatively with the magnitudes of platelet aggregation induced by ADP (5 μm) (r = −0.47, P < 0.05).

Conclusion:  These findings suggest that exercise in aged rats increases aortic eicosapentaenoic acid concentrations, which in turn depress the basal levels of thromboxane, B2 in platelet‐rich plasma to modulate platelet aggregation.
Endotoxin‐induced shock in the pig – limited effects of low and high concentrations of inhaled nitric oxide
Authors: Middelveld R. J. M. , Alving K.
Published: September 2003

Aim:  This study was carried out to study the prophylactic effects of inhalation of nitric oxide (NO) before and during the induction of endotoxic shock.

Methods:  Eighteen anaesthetized pigs received an infusion of 10–20 μg kg−1 endotoxin during 2 h after pre‐treatment with the cortisol‐synthesis inhibitor metyrapone. Three groups were tested (n = 6 each) and received 0, 0.2 or 20 ppm inhaled NO from 30 min before start of endotoxin infusion until 4 h after start of endotoxin. Both 0.2 and 20 ppm NO were able to improve blood gas values.

Results:  Area above curve values of arterial P2/FiO2 from 0 to 4 h were 0.83 ± 0.09 kPa h (control), 0.78 ± 0.22 (0.2 ppm NO, non‐significant) and 0.31 ± 0.06 (20 ppm NO, P < 0.01, Mann–Whitney U‐test, compared to control). Area under curve values of PCO2 from 0 to 4 h were 3.96 ± 0.66 kPa h (control), 1.20 ± 0.46 (0.2 ppm NO, P < 0.05, Mann–Whitney U‐test, compared to control) and 2.78 ± 1.06 (20 ppm NO group, non‐significant). The increase in pulmonary arterial pressure (PAP) was partly prevented by 20 ppm NO, but not by 0.2 ppm NO at 4 h. Inhaled NO did not affect the levels of BAL fluid total protein, tumour necrosis factor‐α, interleukin‐8 and neutrophil counts.

Conclusions:  The addition of a high (20 ppm), but not a low (0.2 ppm), concentration of NO to the inhaled air during endotoxin shock improves arterial oxygen tension and reduces pulmonary artery pressure. Neither dose affects lung mechanics or inflammatory indices, in spite of being given prophylactically.
A physiological role for protoporphyrin IX photodynamic action in the rat Harderian gland?
Authors: Cui Z. J. , Zhou Y. D. , Satoh Y. , Habara Y.
Published: September 2003

Aims: The lipid‐secreting exocrine Harderian gland contains a large amount of porphyrins (mainly protoporphyrin IX, PPIX) in the glandular cells, the physiological significance of which is rather poorly understood.

Methods: In the present study, the possibility of using Fura‐2 to measure intracellular calcium ([Ca2+]c) changes in these cells was assessed.

Results: It was found that when Fura‐2‐loaded cells were excited by light at 340/380 nm, [Ca2+]cincreased spontaneously, indicating a photodynamic action powered by light at 340/380 nm. In contrast, with the visible spectrum calcium probe Fluo‐3 (λex = 475 nm), carbachol at 10 μminduced [Ca2+]cincrease; [Ca2+]cdid not change without carbachol stimulation. Brief illumination with light at 340/380 nm induced a large [Ca2+]cincrease in Fluo‐3‐loaded cells. Photodynamic stimulation of [Ca2+]cincrease was confirmed with an exogenous photosensitizer sulphonated aluminium phthalocyanine (SALPC) and visible light (>580 nm). The wavelength‐dependence of the [Ca2+]cincrease correlates well with the excitation spectrum of the isolated Harderian glandular cells.

Conclusion:These data suggest that PPIX present in rat Harderian glandular cells plays the role of a photosensitizer which upon activation by UVA and blue components of daylight and subsequent singlet oxygen generation, triggers [Ca2+]cincrease and secretory response. The PPIX photodynamic action may also play a potential role in photic entrainment of the central circadian clock.
NOS 3 subcellular localization in the regulation of nitric oxide production
Authors: Sullivan J. C. , Pollock J. S.
Published: September 2003

Endothelium‐derived nitric oxide (NO) is a key signalling molecule in the maintenance of cardiovascular health. Endothelial NO synthase (NOS 3), which catalyses the formation of NO, is targeted to the plasma membrane by dual acylation. In vitro studies suggest that membrane localization of NOS 3 is an important regulatory element of NO production. Dysfunction of the vascular endothelium and a decrease in NO bioavailability is associated with the development and progression of a number of cardiovascular diseases, including hypertension. Our laboratory has previously published that in salt‐dependent hypertension there is an altered localization of NOS 3, with an increase in cytosolic expression. These data have led us to question whether the increased cytosolic NOS 3 expression is a form of compensation for endothelial dysfunction in hypertension, or an indicator and contributing factor to endothelial dysfunction. This review will outline the importance of subcellular localization in the regulation of NOS 3 in vitro, the role of NOS 3 in endothelial dysfunction associated with salt‐dependent hypertension, and the potential physiological consequences of altered NOS 3 localization in vivo.
From molecules to mammals: what's NOS got to do with it?
Authors: Mungrue I. N. , Bredt D. S. , Stewart D. J. , Husain M.
Published: September 2003

Nitric oxide synthases (NOSs) generate nitric oxide (NO) and the by‐product l‐citrulline, via the catalytic combination of l‐arginine and molecular oxygen. In mammals, there are three NOS genes: nNOS (NOS1), iNOS (NOS2) and eNOS (NOS3). The molecular structure, enzymology and pharmacology of these enzymes have been well defined, and reveal critical roles for the NOS system in a variety of important processes. The studies of NOS enzymes using knockout and transgenic mouse models have provided an invaluable contribution, highlighting critical roles in neuronal, renal, pulmonary, gastro‐intestinal, skeletal muscle, reproductive and cardiovascular biology. This review will outline the data gleaned from complementary knockout and transgenic over‐expression models in mice, and focus on the interactions between NOS enzymes and pathophysiology of the vascular system. These studies are a paradigm for the near future, which will involve the translation of an enormous amount of genomic data into physiological insights that penetrate the realms of both health care and biology.
Caveolae, caveolin and cav‐p60 in smooth muscle and renin‐producing cells in the rat kidney
Authors: Voldstedlund M. , Thuneberg L. , Tranum‐Jensen J. , Vinten J. , Christensen E. I.
Published: September 2003

Aims: In vascular smooth muscle cells caveolae are important for signalling mechanisms regulating vascular contraction. In smooth muscle layer of the renal afferent arteriole juxtaglomerular cells (JG cells) are non‐contractile renin producing cells that have the capacity to change their phenotype into smooth muscle cells and back again by metaplastic transformation.

Signalling mechanisms in JG cells are not fully understood and we therefore investigated if caveolae were present, and thereby could be involved as integrators of cellular signalling in both of these phenotypes of smooth muscle cells.

Methods: Using electron microscopy we compared the number of caveolae in JG cells and smooth muscle cells in the afferent arteriole of the rat kidney. The expression of caveolin and cav‐p60 was examined using a combination of immunogold electron microscopy and immunofluorescence microscopy.

Results: We found that JG cells have sixfold less caveolae per cell surface sectional length than smooth muscle cells. The expression of cavolin‐1 and cav‐p60 correlated with the number of caveolae. An examination of the general distribution of caveolae, cav‐p60 and caveolins in the rat kidney showed that cav‐p60, like caveolin‐1, is a specific maker of caveolae.

Conclusion: The number of caveolae in JG cells is very low, and this makes it unlikely that caveolae are of major importance for the renin secretion specific for JG cells.
Muscle fibre size and capillarity in Korean diving women
Authors: Bae K. A. , An N. Y. , Kwon Y. W. , Kim C. , Yoon C. S. , Park S. C. , Kim C. K.
Published: September 2003

Aim:  Effects of prolonged habitual cold‐water immersion on fibre size and capillarity in vastus lateralis muscle were studied in human beings. The hypothesis tested in the present study was that cold acclimatized human skeletal muscle would have reduced muscle fibre size and higher capillarity, favouring the idea of efficacy of recruitment under cold environment.

Methods:  Ten women breath‐hold divers (BHDs) and 10 active women (controls CONs) participated in this study. Muscle biopsy was obtained from vastus lateralis and determined fibre type composition and capillary density.

Results: A major finding was that all BHDs revealed a markedly smaller cross‐sectional area (CSA) in all fibre types than the CONs, or even than any other morphological data reported in previous investigations. Furthermore, mean CSA of type II fibre (range 1205–2766 μm2) was much smaller than type I fibre (2343–4327 μm2). The number of capillaries per fibre in different fibre types in the BHDs was higher than in the CONs (P < 0.001), and diffusional area was smaller in type II fibres than in type I fibres (P < 0.001). The BHDs and the CONs have similarity in the percentage of type I fibres, but type II fibre was predominant in both groups. Interestingly the proportion of type IIx fibre in the BHDs was higher (31%) than in the CONs (22%). No significant difference was found in the thigh circumference between the groups.

Conclusion:  The present study demonstrates that prolonged habitual cold‐water immersion may induce a decrease in fibre size and an increase in capillarity in human skeletal muscle.
Lack of efficacy of ‘naked’ small interfering RNA applied directly to rat brain
Authors: Isacson R. , Kull B. , Salmi P. , Wahlestedt C.
Published: September 2003

The intrastriatal infusions of ‘naked’ small interfering RNA (siRNA) targeted to dopamine D1 receptors (1.0–10.0 nmol over 3 days) did not reduce dopamine D1 receptor messenger RNA levels or receptor protein, assessed by [125I] SCH 23982 binding in intact rats. This was in contrast to results in vitro where a 76% reduction in dopamine D1 receptor ligand binding could be observed.

Conclusion:

The results suggest that synthetic siRNA, when applied directly to rat brain, is not capable of inducing RNA interference.
Role of Ca2+‐activated Cl− current during proarrhythmic early afterdepolarizations in sheep and human ventricular myocytes
Authors: Verkerk A. O. , Tan H. L. , Kirkels J. H. , Ravesloot J. H.
Published: September 2003

Objectives:  The proarrhythmic early afterdepolarizations (EADs) during phase‐2 of the cardiac action potential (phase‐2 EADs) are associated with secondary Ca2+‐release of the sarcoplasmic reticulum. This makes it probable that the Ca2+‐activated Cl− current [ICl(Ca)] is present during phase‐2 EADs. Activation of ICl(Ca) during phase‐2 of the action potential will result in an outwardly directed, repolarizing current and may thus be expected to prevent excessive depolarization of phase‐2 EADs. The present study was designed to test this hypothesis.

Methods and Results:  The contribution of ICl(Ca) during phase‐2 EADs was studied in enzymatically isolated sheep and human ventricular myocytes using the patch‐clamp methodology. EADs were induced by a combination of a low stimulus frequency (0.5 Hz) and exposure to 1 μm noradrenaline. In sheep myocytes, the ICl(Ca) blocker 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonic acid (DIDS, 0.5 mm) abolished phase‐1 repolarization of the action potential in all myocytes tested. This indicates that ICl(Ca) is present in all sheep myocytes. However, DIDS had no effect on phase‐2 EAD characteristics. In human myocytes, DIDS neither affected phase‐1 repolarization nor phase‐2 EAD characteristics.

Conclusion:  In sheep ventricular myocytes, but not in human ventricular myocytes, ICl(Ca) contributes to phase‐1 repolarization of the action potential. In both sheep and human myocytes, ICl(Ca) plays a limited role during phase‐2 EADs.
Trafficking and activation of eNOS in epithelial cells
Authors: Ortiz P. A. , Garvin J. L.
Published: September 2003

In mammalian cells, formation of nitric oxide (NO) is catalysed by a family of enzymes termed NO synthases (NOS). There are three isoforms of this enzyme, NOS I, II and III. NOS III was originally cloned and identified in endothelial cells; thus this isoform is commonly called endothelial NOS (eNOS). The physiological role of NO produced by eNOS has been documented in most organs, including the brain, lung, cardiovascular system, kidney, liver, gastrointestinal tract and reproductive organs. The bioavailability of NO in these tissues is determined by the balance between its rate of production and degradation. The rate of NO production by eNOS is ultimately dependent on the activity of the enzyme. In the past years, co‐ and post‐translational modifications such as myristoylation, palmitoylation, phosphorylation, protein–protein interactions and subcellular localization have been shown to play an important role in determining eNOS activity. In order to maintain specificity, the production of most signalling molecules occurs in an organized spatial and temporal pattern. Spatial localization of eNOS has been shown to be regulated by different mechanisms that control its targeting from the Golgi apparatus to the plasma membrane, correct compartmentalization within the membrane, and internalization from the plasma membrane to the cytoplasm after activation. Thus, regulated localization and trafficking of eNOS may be essential in regulating enzyme activity and maintaining the spatial and temporal organization of NO signalling in different cell types.
Identification and physical mapping of genes expressed in the corpus luteum in cattle1
Authors: Bønsdorff T. , Eggen A. , Gautier M. , Åsheim H.‐C. , Rønningen K. , Lingaas F. , Olsaker I.
Published: September 2003

A representational difference analysis was performed to identify genes expressed in the corpus luteum of cattle. The corpus luteum is an ovarian structure that is essential for the establishment and maintenance of pregnancy. Knowledge of gene expression and function of corpus luteum will be important to improve fertility in humans and domestic animals. Housekeeping genes were removed from the corpus luteum representation (tester) using skeletal muscle as the subtracting agent (driver). A total of 80 clones of the final subtraction product were analysed by sequencing and 11 new bovine gene sequences were identified (pBTCL1–11). The sequences were mapped to segments of 10 different chromosomes using a somatic cell hybrid panel and a radiation hybrid panel. With one exception the locations are in agreement with published comparative maps of cattle and man. Expression in corpus luteum was verified by RT–PCR for all the 11 clones.
Chromosomal mapping, sequence and transcription analysis of the porcine fertilin beta gene (ADAM2)1
Authors: Day A. E. , Quilter C. R. , Sargent C. A. , Mileham A. J.
Published: September 2003

Fertilin beta (ADAM2) forms a part of the heterodimeric surface protein fertilin, found on the plasma membrane of mammalian sperm, and has been implicated in the process of sperm–egg fusion. Analysis of cDNA products obtained from adult porcine testis mRNA has presented a sequence corresponding to 2620 bp of the ADAM2 gene. This sequence contained an open reading frame encoding a 735‐amino acid protein and homologous to ADAM2 genes known in other mammalian species. Polymerase chain reaction (PCR) analysis of genomic DNA showed that the 2620 bp of cDNA sequence comprises at least 21 exons and spans approximately 76 kb of genomic DNA, with its size and structure being relatively conserved between mouse, human and pig. Fluorescence in situ hybridization was used to map ADAM2 to chromosome 15 of the pig, using a bacterial artificial chromosome clone from the PigE BAC library. This finding is consistent with comparative mapping experiments performed between pig and human chromosomes. Analysis of nine mRNA samples, by reverse transcriptase‐PCR, from different porcine tissues has also suggested that expression of ADAM2 is limited to the testis, a finding that is consistent with other mammalian species.
Radiation‐induced changes in oral carcinoma cells – a multiparametric evaluation
Authors: Bindu L. , Balaram P. , Mathew A. , Remani P. , Bhattathiri V. N. , Nair M. K.
Published: September 2003

The aim of this study was to see whether serial cytological evaluation of various cellular abnormalities in tumours from patients receiving fractionated radiotherapy can predict radio‐response in oral carcinoma. Cytological assessment was carried out in scrape smears collected prior to and during the course of radiotherapy in 68 patients with squamous cell carcinoma of the oral cavity planned for radical radiotherapy with accelerated fraction schedule. Smears were evaluated for a set of 15 radiation‐induced cellular abnormalities. The relationship between the cellular alterations and the cumulative radiation dose was analysed by Kruskal–Wallis one‐way anova. The results showed that among the various quantifiable changes that occur in irradiated cancer cells, karyolysis, karyorrhexis, pyknosis, cytolysis, multinucleation, micronucleation and nuclear budding show significant increase depending on the dose of radiation. The radio‐resistant group of patients exhibited a lesser degree of change compared with the radio‐sensitive group. This suggests that radio‐resistance may be due to the defective induction of cell damage and that these cytological features may have potential use as predictive markers of radio‐sensitivity in oral carcinoma.
Interlaboratory reproducibility of atypical squamous cells of undetermined significance report: a national survey
Authors: Confortini M. , Carozzi F. , Dalla Palma P. , Ghiringhello B. , Parisio F. , Prandi S. , Ronco G. , Ciatto S. , Montanari G. ,   GISCi Working Group for Cervical Cytology
Published: September 2003

The study was aimed at assessing interlaboratory reproducibility in the reporting of cervical smears in the atypical squamous cells of undetermined significance (ASCUS) category. A set of 50 selected slides circulated among 89 laboratories, currently involved in population‐based screening programmes for cervical cancer, which provided a diagnostic report according to four main reporting categories based on the 1991 Bethesda system. Interlaboratory agreement was determined according to kappa (K) statistics: overall and weighted K values were determined for each laboratory and for single reporting categories. The results showed a very low reproducibility for the ASCUS category. This finding supports the Bethesda system 1991 recommendation to limit the use of this reporting category and suggests that the clinical response to ASCUS reports should be decided locally, based on the observed positive predictive value for cervical intraepithelial neoplasia 2 or more severe lesions.
Rapid pre‐screening: a validated quality assurance measure in cervical cytology
Authors: Smith J. , Nicholas D. , Boyd K. , Deacon‐Smith R.
Published: September 2003

We present the results of 3 years' experience of rapid pre‐screening in cervical cytology. In our laboratory we rapidly pre‐screen all smears. The performance of each primary screener can be assessed. In addition, the relative sensitivity and specificity of each rapid pre‐screener can itself be continuously monitored using the final report as a yardstick. In our laboratory individual sensitivity of rapid pre‐screening for the detection of high‐grade abnormalities was in the range of 44–90% with an overall laboratory sensitivity of 69%. Specificity was in the range of 94–99% with an overall laboratory specificity of 98%. Rapid pre‐screening allows checking of the checkers and pathologists and tends to promote uniformity in the assessment of smear adequacy. This form of continuous quality assurance is practical, convenient and acceptable to staff.
Cervical screening in Luxembourg: 1990–1999
Authors: Scheiden R. , Wagener C. , Knolle U. , Wehenkel A. , Dippel W. , Capesius C.
Published: September 2003

For quality assurance purposes, the results of the 1990's obtained by the National Cervical Cancer Screening Programme (NCCSP) launched in 1962 were reviewed. The positive cytodiagnosis, the histologically verified in situ and invasive cervical cancers and the mortality rates were reported.
Atypical squamous cells of undetermined significance: audit and the impact of potential litigation. Retrospective review of 682 cases
Authors: Scheiden R. , Wagener C. , Knolle U. , Dippel W. , Capesius C.
Published: September 2003

For quality assurance purposes, the frequency of ‘abnormal’ cytological diagnoses of the non‐systematic National Cervical Cancer Screening Programme (NCCSP) was evaluated. In 1999, an unexpected high number of Class (Cl) III cases (i.e. atypical squamous cells of undetermined significance) was reported. The cytological and histological results were reviewed in order to detect a possible cause for this threefold increase. The abnormal Papanicolaou (PAP) smears examined by conventional methods from 1 January 1990 to 31 December 2002 were analysed. The smears of 682 cases diagnosed in 1999 with a Cl III category were reviewed in 2000 and correlated with the available histological diagnoses provided by the Central Department of Pathology.

Of the 682 Cl III cases, 176 cases (26.1%) had no follow‐up, 314 cases (46.0%) had repeat cytology and 192 cases (28.2%) an histological correlate corresponding to 90 (46.9%) benign lesions, 78 (40.6%) squamous intraepithelial lesions, two (1%) invasive cervical cancers (one squamous and one glandular). Twenty‐two Cl III cases (11.5%) were histologically within normal limits. Retrospective smear review confirmed 330 Cl III diagnoses (48.3%), 127 cases (18.6%) were recategorized as Cl IIIG (i.e. atypical glandular cells of undetermined significance), 22 cases (3.2%) as Cl IIID (i.e. mild to moderate dysplasia) and six cases (0.9%) as Cl IVa (i.e. severe dysplasia and/or carcinoma in situ). A total of 197 original Cl III cases had to be reclassified in the Cl II category (28.9%), only two cases showing mild and moderate dysplasia on histology. Thus, 195 cases (28.6%) comprised cytological overdiagnoses. The Cl III category being, by definition, a delicate and often subjective diagnosis, all external influences such as pressure of litigation should be avoided to reduce cytological overdiagnoses as a result of an unnecessary ‘fear‐factor’.
Subtyping borderline nuclear changes: is it of value in predicting high‐grade cervical intraepithelial neoplasia on histology?
Authors: Shanbhag S. , Roberts M. , Johnson S. J. , Wadehra V.
Published: September 2003

The aim was to determine the association between the subtypes of borderline nuclear changes (BNC) in cervical smears and high‐grade cervical intraepithelial neoplasia (HCIN). BNC was reported in 23 236 smears received in our laboratory over a 7‐year period, 3278 patients were referred for colposcopy. Analysis was restricted to 2007 cases, which fitted the criteria of: (1) consistent subtyping of borderline change and (2) cervical histology result within 12 months of the last abnormal smear. BNC was reported in six categories and correlated with histology. Atypia bordering on dyskaryosis, atypical metaplastic cells and endocervical atypia, were associated with HCIN in 25%, 25.4% and 23.8% of cases, respectively. Dyskeratosis and koilocytotic atypia were associated with HCIN in 19.2% and 13.7% of cases, respectively. Some subtypes of borderline change are more frequently associated with HCIN. The difference is not sufficient to dictate clinical management.
The ASCUS : SIL ratio and the reference laboratory pathologist
Authors: Wachtel M. S. , Dahm P. F.
Published: September 2003

The atypical squamous cells of undetermined significance (ASCUS) : squamous intraepithelial lesion (SIL) ratio was proposed to monitor laboratory use of the ASCUS diagnosis. This study addresses problems associated with comparing pathologists by this means. An intuitive example showed the ASCUS : SIL ratio depends on the prevalence of smears from patients who actually have SIL. In this study of 2000 cervical smears, each of five pathologists made 400 diagnoses. Differences among proportions of SIL diagnoses were statistically significant; differences among proportions of ASCUS diagnoses were not. Had an ASCUS : SIL ratio upper limit of 3.0 been used, two pathologists would have been misidentified as having high ASCUS diagnosis rates. Unlike the situation for laboratories, potential variability in SIL prevalence requires caution in the use of this ratio in assessing pathologists. An alternative measure that is independent of prevalence, the ASCUS : SIL odds ratio, is posited.
Implementation of liquid‐based cytology in the screening programme against cervical cancer in the County of Funen, Denmark, and status for the first year
Authors: Hoelund B.
Published: September 2003

As one of the first laboratories in Denmark (and Scandinavia), we have gradually implemented liquid‐based cytology into the screening programme against cervical cancer in the County of Funen since 1 June 2001. This paper describes the course of the implementation period in the different steps in the screening programme, and the preliminary results obtained after the first year. We conclude that the new technique has improved the specimen and diagnostic quality. As a result of the reduction of the screening time, the workload in the laboratory is in balance although we have introduced a rapid review as a quality control. Besides, the reduction in the number of repeated cytological tests and follow‐up visits at the gynaecologist means a saving for the screening programme as a whole. From our point of view the implementation of ThinPrep method is economically neutral.
Cytopathology training in the United States – a personal view
Authors: Clark I. , Zakowski M.
Published: September 2003

In this review we present an outline of cytopathology training in the United States, for a non‐US readership.