bluuebunny/crossref_metadata_2025 huggingface dataset:
To 5D and Beyond: Quantitative Fluorescence Microscopy in the Postgenomic Era
Authors: Andrews Paul D. , Harper Ian S. , Swedlow Jason R.
Published: March 2003

Digital fluorescence microscopy is now a standard technology for assaying molecular localisation in cells and tissues. The choice of laser scanning (LSM) and wide‐field microscopes (WFM) largely depends on the type of sample, with LSMs performing best on thick samples and WFMs performing best on thin ones. These systems are increasingly used to collect large multidimensional datasets. We propose a unified image structure that considers space, time, and fluorescence wavelength as integral parts of the image. Moreover, the application of fluorescence imaging to large‐scale screening means that large datasets are now routinely acquired. We propose that analysis of these data requires querying tools based on relational databases and describe one such system.
CK2 and GAK/auxilin2 Are Major Protein Kinases in Clathrin‐Coated Vesicles
Authors: Korolchuk Viktor I. , Banting George
Published: March 2003

Several peripheral membrane proteins associated with clathrin‐coated vesicles (CCVs) are reversibly phosphorylated, but it is not clear precisely which protein kinases are involved. In order to address this question directly, we have isolated highly purified CCVs from porcine brain. The peripheral membrane proteins have been removed and assayed for kinase activity using the CCV peripheral membrane proteins as substrate. The major kinase activity identified has a molecular mass of 40 kDa, is inhibited by known specific inhibitors of the protein kinase CK2 and is recognised by an antibody specific to CK2. We show that CK2 is responsible for the phosphorylation of the majority of CCV‐associated proteins that are subject to phosphorylation. Intriguingly, CK2 is inactive when associated with CCVs but becomes active once the clathrin coat has been removed. The medium subunit of the AP2 adaptor complex (μ2) is not a substrate for CK2, but is phosphorylated by a second kinase that we show to be cyclin G‐associated kinase (GAK/auxilin2). Unlike the situation for the CK2 substrates, μ2 is a substrate for GAK/auxilin2, both in intact CCVs and in solution. In addition, we show that the ‘stripped’ CCV membranes that remain once the peripheral membrane proteins have been removed from CCVs inhibit CK2 but not GAK/auxilin2 activity.
Induction of Autophagy Causes Dramatic Changes in the Subcellular Distribution of GFP‐Rab24
Authors: Munafó Daniela B. , Colombo María I.
Published: March 2003

Rab GTPases comprises a large family of proteins, with more than 50 gene products localized in distinct subcellular compartments. Rab24 is a member of this family whose function is not presently known. In order to elucidate the role of this protein we have generated a GFP‐tagged Rab24 and studied the distribution of this chimera by fluorescence microscopy. GFP‐Rab24 showed a perinuclear reticular localization that often encircled the nucleus. This reticular pattern partially overlapped with ER markers, cis‐Golgi, and the ER‐Golgi intermediate compartment. Surprisingly, when GFP‐Rab24‐transfected cells were starved to induce autophagy the distribution of the protein changed dramatically. GFP‐Rab24 localized in large dots, cup‐shaped structures and ring‐shaped vesicles. Some of these vesicles were labeled with monodansylcadaverine , a specific autophagosome marker. In the presence of vinblastine, an agent that induces the formation of very large autophagic vesicles, GFP‐Rab24 accumulated in the large vacuoles that were also labeled by monodansylcadaverine. Furthermore, Rab24 colocalized with LC3, a mammalian homolog of the yeast protein Apg8/Aut7, an essential gene for autophagy. This is the first report indicating that Rab24 localizes on autophagosomes, suggesting that this Rab protein is involved in the autophagic pathway.
The Transitional ER Defines a Boundary for Quality Control in the Secretion of tsO45 VSV Glycoprotein
Authors: Mezzacasa Anna , Helenius Ari
Published: March 2003

Quality control in the secretory pathway limits forward transport of newly synthesized cargo proteins to those that have acquired their fully folded conformation. To determine which organelles participate in this conformation‐dependent sorting process, we analyzed the trafficking of the temperature‐sensitive, thermo‐reversible folding mutant of vesicular stomatitis virus glycoprotein (tsO45 G protein) in VERO cells. Using temperature blocks, the G protein could be localized to the ER (39.5 °C), to the vesiculo‐tubular clusters (VTCs, 15 °C), and to the trans‐Golgi network (TGN, 20 °C). To localize the G protein specifically to ER exit sites, we incubated cells at 10 °C. The exit sites contained Sec13p, a COPII component, and were devoid of calnexin and other ER chaperones. We found that if the G protein in the exit sites was misfolded by a temperature shift from 10 °C to 39.5 °C, it failed to enter the VTCs. Instead, it was returned to the reticular ER where it associated with calnexin. However, if the G protein was in the VTCs or beyond, its folding status no longer affected further transport. The observations indicate that quality control took place in the ER and in the ER transitional elements, but not in the VTCs or the Golgi complex. The results provide a way to discriminate biochemically between exit sites and VTCs, two related structures that are difficult to distinguish from each other.
The clinical and microbiological effects of a novel acidified sodium chlorite mouthrinse on oral bacterial mucosal infections
Authors: Fernandes‐Naglik L , Downes J , Shirlaw P , Wilson R , Challacombe SJ , Kemp GK , Wade WG
Published: March 2003

Acidified sodium chlorite mouthrinses have been shown to have equivalent anti‐plaque activity to those containing chlorhexidine, the current ‘gold standard’. In this study, sodium chlorite mouthrinses (ASC) acidified with either malic or gluconic acids were compared to each other and with a chlorhexidine rinse and sterile water for their effect on salivary bacterial counts. Sixteen subjects participated in the study, which had a cross‐over Latin square design. In a second study, a sodium chlorite/ gluconic acid rinse was compared with chlorhexidine for its clinical and microbiological effects in 36 patients with oral mucosal infections. The sodium chlorite rinses acidified with malic and gluconic acids and the chlorhexidine rinse caused significant reductions in salivary bacterial counts up to 7 h after a single rinse compared with water. There were no significant differences between the three active treatments. In the mucosal infection study, there was a significant reduction in erythema in the chlorhexidine group compared with the ASC group. Patients who received the ASC rinse reported significantly less discomfort following treatment than those receiving the chlorhexidine rinse.Staphylococcus aureuscounts were significantly reduced in the group who received the sodium chlorite rinse. There were no other significant differences between the treatments. ASC appears to be an effective alternative to chlorhexidine mouthrinse.
Oral mucosal immunity and HIV infection: current status
Authors: Challacombe SJ , Sweet SP
Published: March 2003

There is a paradox that profound HIV‐induced immunodeficiency is present systemically, whereas the majority of infections associated with HIV disease are present or initiated at mucosal surfaces. There is therefore a need to understand both specific and non‐specific mechanisms of mucosal protection against HIV and its copathogens. The majority of HIV infections occur as a result of the passage of virus across mucosal membranes. Resistance to HIV infection at mucosal surfaces may be related to HIV‐specific CD8+T cell responses in some individuals and may be the basis for protective vaccine design. However, T‐cells, macrophages and dendritic cells in mucosa may be a portal of entry for HIV. Transcytosis of HIV can occur from the mucosal to the submucosal surface and vice versa, and may be inhibited by mucosal immunoglobulins and neutralizing IgA within epithelial cells. HIV‐induced alterations to oral epithelial cells, together with impairment of mucosal CD4+T‐cells and consequent altered cytokine secretion, may contribute to secondary infections. It also appears that HIV infection is associated with decreased salivary IgA levels, although a dichotomy between IgA concentrations in saliva and serum has been reported. Mucosal antibody responses, however, seem to be maintained. Considerable attention has been given to the possibility of mucosal immunization against HIV and there is evidence that secretory IgA antibody is neutralizing to different HIV strains. In addition to specific immune factors, it is likely that innate non‐specific factors may be significant in protecting mucosal surfaces, including lactoferrin, secretory leukocyte protease inhibitor, mucins, proline rich proteins and cystatins. These may be useful candidate virucides in topical preparations. Thus humoral, cellular and innate immune mechanisms, as well as lymphocyte–epithelial interactions, may all be impaired at mucosal surfaces as a result of HIV infection and may contribute to the susceptibility of mucosa to infective processes.
Consensus guidelines on quantifying HIV‐related oral mucosal disease
Authors: Flint S , Glick M , Patton L , Tappuni A , Shirlaw P , Robinson P
Published: March 2003

The workshop agreed that the development of consensus quantification protocols was desirable, but that this whole field was underdeveloped, to date. The working goals were therefore redefined(vide infra). Three possible protocols were presented and discussed relating to oral hairy leukoplakia, oral ulceration (NOS) and oral candidiasis. There was agreement that different data‐set protocols would have to be developed for specific HIV‐related mucosal diseases. Furthermore, the data set should be consistent, standardized and disaggregated. In this way, with everyone working to the same standards of outcome, data from geographically, nationally, socially or culturally different areas could be readily compared. A future meeting to generate the disease‐specific protocols would, most likely, be needed.
Oral and dental care and treatment protocols for the management of HIV‐infected patients
Authors: Shirlaw PJ , Chikte U , MacPhail L , Schmidt‐Westhausen A , Croser D , Reichart P
Published: March 2003

This paper describes the workings of the workshop dedicated to oral and dental care and treatment protocols for the management of HIV‐infected patients. The questions addressed were: 1) What are the current ethical issues in dental care of HIV patients, do they need to be addressed? 2) Do we need to modify the dental care we give HIV‐positive patients? 3) When is it necessary to give antibiotic prophylaxis to HIV‐positive patients? 4) What is the evidence for the effective treatment of oral lesions associated with HIV? 5) What is the most successful palliative treatment for KS? 6) Can we provide clinical treatment that has a scientific basis rather being trial based? 7) Is ddI + hydroxy‐urea an effective African alternative to HAART? 8) What is the influence of protease inhibitors and HAART on the excretion of HIV in saliva? 9) What is the effect of anti‐HIV therapy on the oral mucosa and oral health? This workshop did not fully cover the issue of ddI and hydroxy‐urea as an alternative HIV therapy as this was considered to be the remit of general physicians caring for patients with HIV and AIDS rather than that of oral health care workers.
The enigmatic mechanism of irradiation‐induced damage to the major salivary glands
Authors: Nagler RM
Published: March 2003

Irradiation is a central treatment modality administered for head and neck malignancies. Its major and most devastating side‐effect is an induced damage to the major salivary glands. This article aims at suggesting a comprehensive explanation for the underlying mechanism of this damage, which has been considered as enigmatic throughout the 90 years since it was first described in 1911. The mechanism suggested is based on the considerable literature concerning this enigma in rat salivary glands. According to this proposed mechanism, the irradiation results in a sublethal DNA damage, which manifests and becomes lethal at a delayed phase. Thus, when the acinar progenitor cells are going through a reproductive phase when parenchylmal replenishment is required, they die. The injurious agents, which result in this delayed reproductive cell death, appear to be highly redox‐active transition metal ions, such as iron and copper. These metal ions, which seem to be associated with secretion granules, are not necessarily contained within the granules as previously suggested, but rather are probably located at sites more proximal to the DNA.
Fungal infections associated with HIV infection
Authors: Samaranayake LP , Fidel PL , Naglik  , Sweet SP , Teanpaisan R , Coogan MM , Blignaut E , Wanzala P
Published: March 2003

Oral candidiasis is perhaps the commonest infection seen in HIV disease. The aim of this workshop was to provide a sketch of the multifarious aspects of the disease from a global perspective. To this end the panellists addressed issues such as the virulence of Candida, emergence of antifungal resistance, management of candidiasis and other exotic, oral mycotic diseases. An all‐pervasive theme was the dramatic differences in the management of fungal infections consequential to the availability (or the lack) of anti‐HIV drugs in the developed and the developing world. Further, the social stigmata associated with the HIV disease in many developing regions in Africa and Asia appears to modify the therapeutic strategies. Additionally, the lesser‐known regional variations in the disease manifestations and therapeutic approaches were stark. Further work is direly needed to address these issues.
The rational use of fluoride toothpaste
Authors: Davies R. M. , Ellwood R. P. , Davies G. M.
Published: March 2003

Abstract:  Well‐formulated fluoride toothpastes are clinically proven to prevent and control dental caries. They may also be a risk factor in the aetiology of dental fluorosis. This review considers the available evidence to support the appropriate use of fluoride toothpaste to maximise the benefit and minimise the risk. Three factors have an important influence on the anticaries efficacy of fluoride toothpaste, namely concentration, frequency of brushing and postbrushing rinsing behaviour. The evidence suggests that low‐fluoride (<600 ppm F) toothpastes provide less caries protection than standard (1000 ppm F) or high (1500 ppm F) concentration formulations. However, low‐fluoride toothpastes are appropriate for very young children (under 7 years) at low caries risk, particularly if living in fluoridated areas. For other young children, higher concentrations of fluoride should be used. Brushing should be recommended twice daily, whilst rinsing with large volumes of water should be discouraged. Small amounts of toothpaste are comparable in efficacy to large amounts. The risk of fluorosis is associated with the ingestion of high doses of fluoride during tooth development and consequently only young children are at risk. The variability in the dose of fluoride ingested is mainly a function of the amount used, less so its concentration. To minimise fluorosis risk, parents should be advised to use only a pea‐sized amount of toothpaste and encourage spitting out of excess. It is concluded that by using fluoride toothpastes appropriately, the benefits can be maximised and the risks of fluorosis minimised.
Risk of Hip Fracture in Disabled Community‐Living Older Adults
Authors: Walter Louise C. , Lui Li‐Yung , Eng Catherine , Covinsky Kenneth E.
Published: March 2003

OBJECTIVES:To determine the rate of hip fracture and risk factors associated with hip fractures in disabled older persons who enroll in the Program of All‐Inclusive Care for the Elderly (PACE), a program providing comprehensive care to community‐living nursing‐home‐eligible persons.

DESIGN:Prospective cohort study between January 1990 and December 1997.

SETTING:The twelve PACE demonstration sites: San Francisco, California; Columbia, South Carolina; Detroit, Michigan; Denver, Colorado; East Boston, Massachusetts; El Paso, Texas; Milwaukee, Wisconsin; Oakland, California; Portland, Oregon; Rochester, New York; Sacramento, California; and the Bronx, New York.

PARTICIPANTS:Five thousand one hundred eighty‐seven individuals in PACE; mean age 79, 71% female, 49% white, 47% with dementia.

MEASUREMENTS:Functional status, cognitive status, demographics, and comorbid conditions were recorded on all the participants, who were tracked for occurrence of a hip fracture. The goals were to determine the rate of hip fracture and identify risk factors.

RESULTS:Two hundred thirty‐eight hip fractures (4.6%) occurred during follow‐up. The rate of hip fracture was 2.2% per person‐year. Four independent predictors of hip fracture were identified using Cox proportional hazard analysis: age of 75 and older (adjusted hazard ratio (HR) = 2.0, 95% confidence interval (CI) = 1.4–2.8); white ethnicity (HR = 2.1, 95% CI = 1.6–2.8); ability to transfer independently to and from bed, chair, and toilet (HR = 3.0, 95% CI = 1.2–7.2); and five or more Short Portable Mental Status Questionnaire errors (HR = 1.6, 95% CI = 1.3–2.1). The incidence of hip fracture ranged from 0.5% per person‐year in persons with zero to one independent risk factors to 4.7% per person‐year in those with all four independent risk factors.

CONCLUSIONS:The rate of hip fracture in this cohort of disabled community‐living older adults was similar to that reported in nursing home cohorts. Older age, white race, ability to transfer independently, and cognitive impairment were independent predictors of hip fracture. Persons with these risk factors should be targeted for preventive interventions, which should include strategies for making transferring safer.
5-Aminoimidazole-4-carboxamide riboside induces apoptosis in Jurkat cells, but the AMP-activated protein kinase is not involved
Authors: LÓPEZ José M. , SANTIDRIÁN Antonio F. , CAMPÀS Clara , GIL Joan
Published: March 2003

5-Aminoimidazole-4-carboxamide (AICA) riboside, a precursor of purine nucleotide biosynthesis, induces apoptosis in Jurkat cells. Incorporation of AICAriboside into the cells is necessary for this effect since addition of nitrobenzylthioinosine, a nucleoside-transport inhibitor, completely protects Jurkat cells from apoptosis. Adenosine, but not other nucleosides, also protects Jurkat cells from AICAriboside-induced apoptosis. The apoptotic effect is caspase-dependent since caspases 9 and 3 are activated and the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) blocks apoptosis. Furthermore, AICAriboside induces mitochondrial cytochrome c release. AICAriboside, when phosphorylated to AICAribotide (ZMP), is a specific activator of the AMP-activated protein kinase (AMPK) in certain cell types. However, AICAriboside does not activate AMPK in Jurkat cells. Moreover, 5-iodotubercidin, an inhibitor of AICAriboside phosphorylation, does not inhibit apoptosis in Jurkat cells. These results indicate that AICAriboside induces apoptosis independently of ZMP synthesis and AMPK activation in Jurkat cells.
Sphingomonas paucimobilis beta-glucosidase Bgl1: a member of a new bacterial subfamily in glycoside hydrolase family 1
Authors: MARQUES Ana Rita , COUTINHO Pedro M. , VIDEIRA Paula , FIALHO Arsénio M. , SÁ-CORREIA Isabel
Published: March 2003

The Sphingomonas paucimobilis β-glucosidase Bgl1 is encoded by the bgl1 gene, associated with an 1308bp open reading frame. The deduced protein has a potential signal peptide of 24 amino acids in the N-terminal region, and experimental evidence is consistent with the processing and export of the Bgl1 protein through the inner membrane to the periplasmic space. A His6-tagged 44.3kDa protein was over-produced in the cytosol of Escherichia coli from a recombinant plasmid, which contained the S. paucimobilis bgl1 gene lacking the region encoding the putative signal peptide. Mature β-glucosidase Bgl1 is specific for aryl-β-glucosides and has no apparent activity with oligosaccharides derived from cellulose hydrolysis and other saccharides. A structure-based alignment established structural relations between S. paucimobilis Bgl1 and other members of the glycoside hydrolase (GH) family 1 enzymes. At subsite —1, the conserved residues required for catalysis by GH1 enzymes are present in Bgl1 with only minor differences. Major differences are found at subsite +1, the aglycone binding site. This alignment seeded a sequence-based phylogenetic analysis of GH1 enzymes, revealing an absence of horizontal transfer between phyla. Bootstrap analysis supported the definition of subfamilies and revealed that Bgl1, the first characterized β-glucosidase from the genus Sphingomonas, represents a very divergent bacterial subfamily, closer to archaeal subfamilies than to others of bacterial origin.
Oestrogen Receptors, Receptor Variants and Oestrogen Actions in the Hypothalamic‐Pituitary Axis
Authors: Shupnik M. A.
Published: March 2003

Information on oestrogen action has grown exponentially in the past decade, and recent studies have begun to define the mechanism of ligand‐dependent activation and cell‐specific effects. Oestrogen‐mediated gene transcription in a specific tissue depends on several factors, the most important of which is the presence of at least one of the two nuclear oestrogen receptor (ER) isoforms, ERα and ERβ. The presence and levels of specific ER isoform variants, along with receptor coactivator, corepressor and integrator proteins, directly modulate overall nuclear ER activity. The structure of the ligand, including both physiological oestrogens and synthetic oestrogen receptor modulators, influences ER interactions with these other proteins and thus determines the biological response. Furthermore, peptide and neurotransmitter‐stimulated intracellular signalling pathways activate specific enzyme cascades and may modify the receptors and their interacting proteins, resulting in either independent or ligand‐enhanced ER‐mediated responses. Finally, several rapid effects of oestrogen probably occur at the membrane through nongenomic pathways that may or may not require the same ER proteins that are found in the nucleus. This review concentrates on the pituitary‐hypothalamic axis and the genomic effects of oestrogen, and discusses the current knowledge of each of these factors in determining oestrogen actions in the neuroendocrine system.
Sleep Restriction Alters the Hypothalamic‐Pituitary‐Adrenal Response to Stress
Authors: Meerlo P. , Koehl M. , Van Der Borght K. , Turek F. W.
Published: March 2003

Chronic sleep restriction is an increasing problem in many countries and may have many, as yet unknown, consequences for health and well being. Studies in both humans and rats suggest that sleep deprivation may activate the hypothalamic‐pituitary‐adrenal (HPA) axis, one of the main neuroendocrine stress systems. However, few attempts have been made to examine how sleep loss affects the HPA axis response to subsequent stressors. Furthermore, most studies applied short‐lasting total sleep deprivation and not restriction of sleep over a longer period of time, as often occurs in human society. Using the rat as our model species, we investigated: (i) the HPA axis activity during and after sleep deprivation and (ii) the effect of sleep loss on the subsequent HPA response to a novel stressor. In one experiment, rats were subjected to 48 h of sleep deprivation by placing them in slowly rotating wheels. Control rats were placed in nonrotating wheels. In a second experiment, rats were subjected to an 8‐day sleep restriction protocol allowing 4 h of sleep each day. To test the effects of sleep loss on subsequent stress reactivity, rats were subjected to a 30‐min restraint stress. Blood samples were taken at several time points and analysed for adrenocorticotropic hormone (ACTH) and corticosterone. The results show that ACTH and corticosterone concentrations were elevated during sleep deprivation but returned to baseline within 4 h of recovery. After 1 day of sleep restriction, the ACTH and corticosterone response to restraint stress did not differ between control and sleep deprived rats. However, after 48 h of total sleep deprivation and after 8 days of restricted sleep, the ACTH response to restraint was significantly reduced whereas the corticosterone response was unaffected. These results show that sleep loss not only is a mild activator of the HPA axis itself, but also affects the subsequent response to stress. Alterations in HPA axis regulation may gradually appear under conditions of long total sleep deprivation but also after repeated sleep curtailment.
Changes in total leaf nitrogen and partitioning of leaf nitrogen drive photosynthetic acclimation to light in fully developed walnut leaves
Authors: Frak E. , Le Roux X. , Millard P. , Dreyer E. , Jaouen G. , Saint‐Joanis B. , Wendler R.
Published: March 2003

Comprehensive studies on the processes involved in photosynthetic acclimation after a sudden change in light regime are scarce, particularly for trees. We tested (i) the ability of photosynthetic acclimation in the foliage of walnut trees growing outdoors after low‐to‐high and high‐to‐low light transfers made early or late in the vegetation cycle, and (ii) the relative importance of changes in total leaf nitrogen versus changes in the partitioning of leaf nitrogen between the different photosynthetic functions during a 2 month period after transfer. Changes in maximum carboxylation rate, light‐saturated electron transport rate, respiration rate, total leaf nitrogen, ribulose 1·5‐bisphosphate carboxylase/oxygenase (Rubisco) and total chlorophylls were surveyed before and after the change in light regime. Respiration rate acclimated fully within 1 week of transfer, and full acclimation was observed 1 month after transfer for the amount of Rubisco. In contrast, total nitrogen and photosynthetic capacity acclimated only partially during the 2 month period. Changes in photosynthetic capacity were driven by changes in both total leaf nitrogen and leaf nitrogen partitioning. The extent of acclimation also depended strongly on leaf age at the time of the change in light regime.
Effect of alterations in cuticular wax biosynthesis on the physicochemical properties and topography of maize leaf surfaces
Authors: Beattie G. A. , Marcell L. M.
Published: March 2003

The leaf surface properties of 11 cuticular wax mutants of maize were characterized, and this information was used to identify the quantitative relations among distinct leaf surface traits. Compared with the wild‐type maize, these mutants were reduced 3–24% in their leaf surface hydrophobicity, 20–88% in the mass of cuticular waxes on their leaves, and 52–94% in the percentage of planar leaf surface area covered with epicuticular crystalline waxes. They also differed in the presence and abundance of the epicuticular crystalline waxes in each of seven structural classes. With the exception of one mutant, the mass of cuticular waxes produced by these mutants was positively correlated with the number of epicuticular crystalline waxes per unit area on their leaves. Furthermore, an increase of 0·4 mg of cuticular wax per gram of leaf (dry weight) was associated with a 1% increase in leaf surface area covered by epicuticular crystalline waxes, and this 1% increase was associated with a 2° increase in the contact angle of a water droplet on the leaf surface. Linear differences in the leaf surface hydrophobicity were associated with exponential differences in the mass of the cuticular waxes produced. Quantitative knowledge of these leaf surface properties is highly relevant to the interactions of leaves with environmental factors such as microbes, insects, agricultural chemicals, and pollutants.
Prostaglandin D2, its metabolite 15‐d‐PGJ2, and peroxisome proliferator activated receptor‐γ agonists induce apoptosis in transformed, but not normal, human T lineage cells
Authors: Harris Sarah G. , Phipps Richard P.
Published: March 2003

Prostaglandin D2 (PGD2) is abundantly produced by mast cells, platelets, and alveolar macrophages and has been proposed as a key immunoregulatory lipid mediator. 15‐Deoxy‐Δ12,14‐PGJ2 (15‐d‐PGJ2), a key PGD2 metabolite, is under intense study as a potential anti‐inflammatory mediator. Little is known about PGD2 or the role of 15‐d‐PGJ2, if any, in regulating the activities of human T lineage cells. In this report we demonstrate that both PGD2 and 15‐d‐PGJ2 have potent antiproliferative effects, and in fact kill human T lymphocyte lines derived from malignant cells by an apoptotic mechanism. Interestingly, normal human T cells were not similarly affected. Although the T lymphocyte lines express mRNA for the PGD2 receptor (DP‐R), a potent DP receptor agonist, BW245C, did not inhibit the proliferation or viability of the cells, suggesting an alternative mechanism of action. PGD2 and 15‐d‐PGJ2 can bind to the peroxisome proliferator activated receptor‐γ (PPAR‐γ) which is implicated in lipid metabolism and apoptosis. Exposure to synthetic PPAR‐γ ligands (e.g. ciglitazone, troglitazone) mimicked the inhibitory responses of PGD2 and 15‐d‐PGJ2, and induced apoptosis in the transformed T cells consistent with a PPAR‐γ‐dependent mechanism. These observations suggest that PPAR‐γ ligands (which may include PGD2) provide strong apoptotic signals to transformed, but not normal T lymphocytes. Thus, the efficacy of utilizing PPAR‐γ and its ligands as therapeutics for human T cell cancers needs to be further evaluated.
CXCR4 expression on monocytes is up‐regulated by dexamethasone and is modulated by autologous CD3+ T cells
Authors: Caulfield Jason , Fernandez Maria , Snetkov Vladimir , Lee Tak , Hawrylowicz Catherine
Published: March 2003

Chemokines and their receptors regulate cell migration to sites of inflammation. The glucocorticoid dexamethasone has potent anti‐inflammatory effects, yet paradoxically up‐regulates expression of some cytokine receptors. We have examined the effects of dexamethasone on chemokine receptor expression. Using an RNase protection assay, we show that dexamethasone up‐regulates human peripheral blood mononuclear cell (PBMC) expression of CXCR4 mRNA. Flow cytometric analysis demonstrated that increased expression of CXCR4, but not CXCR1 and CXCR2, occurred on both monocytes and CD3+ T cells in PBMC mixed cultures. A stromal‐derived factor (SDF)‐1α‐mediated calcium influx was detected on monocytes. Basal levels of CXCR4 expression on purified monocytes were lower when compared with monocytes in mixed PBMC cultures. Co‐culture of monocytes with purified CD3+ T cells led to enhanced basal expression of CXCR4 on monocytes. The use of transwells to partition CD3+ T cells resulted in increased CXCR4 expression on monocytes, suggesting that CD3+ T‐cell derived soluble factors regulate CXCR4 expression.
Brief and repeated noise exposure produces different morphological and biochemical effects in noradrenaline and adrenaline cells of adrenal medulla
Authors: Gesi Marco , Lenzi Paola , Alessandrì Maria G. , Ferrucci Michela , Fornai Francesco , Paparelli Antonio
Published: March 2003

Exposure to stressful stimuli is known to activate the peripheral sympathetic nervous system and the adrenal gland. In this study, we evaluated the effects of single or repeated bouts of exposure to a readily measurable stressful stimulus (loud noise) on the catecholamine content and ultrastructure of the rat adrenal medulla. In particular, we measured tissue levels of dopamine, noradrenaline, adrenaline and metabolites. In parallel studies, we evaluated the fine ultrastructure of catecholamine cells, including a detailed study of catecholamine granules and a morphometric analysis of adrenaline and noradrenaline medullary cells. Animals were exposed either to a single (6 h) session of loud (100 dBA) noise, or to this noise stimulus repeated every day for 21 consecutive days. There was a marked correlation between biochemical indexes of catecholamine activity and the ultrastructural morphometry of specific catecholamine granules. Exposure to loud noise for 6 h induced a parallel increase in dopamine, noradrenaline, adrenaline and their metabolites, a polarization and an increased numerical density of noradrenaline and adrenaline granules in the cells. After repeated noise exposure, noradrenaline levels were significantly higher than in controls, and adrenaline decreased significantly. In addition, adrenaline cells also exhibited ultrastructural alterations consisting of wide homogeneous cytoplasmic areas and large, pale vesicles.
Social organization of cooperatively breeding long‐tailed tits: kinship and spatial dynamics
Authors: Hatchwell B. J. , Anderson C. , Ross D. J. , Fowlie M. K. , Blackwell P. G.
Published: March 2003

Long‐tailed tits Aegithalos caudatus L. are cooperative breeders in which breeders that have failed in their own breeding attempt become helpers at the nest of relatives. We investigated the effects of kinship on the spatial dynamics of non‐breeding flocks of long‐tailed tits in order to determine the information available on the kinship of other members of the population from their use of home ranges.

A novel method of home range analysis was devised based on ‘convex hull peeling’. This method takes into account the dispersion of all fixes within a home range and permits the quantitative analysis of home range use. In addition, the method allows the extent of overlap between adjacent home ranges to be determined and the use of those areas to be investigated.

Non‐breeding flocks of long‐tailed tits were composed mainly of relatives, but also included unrelated immigrants. Flock ranges were large and there was extensive overlap between adjacent flocks.

The degree of range overlap was significantly affected by the relatedness of flocks. If two flocks contained close relatives they were more likely to overlap than two flocks containing non‐relatives. Moreover, the amount of overlap was significantly greater for two adjacent related flocks than for two adjacent unrelated flocks.

The use of overlapping areas of non‐breeding ranges of long‐tailed tit flocks was also influenced significantly by relatedness. Overlapping flocks that were unrelated to each other usually avoided areas of overlap, while related flocks did not generally show such avoidance behaviour.

Kinship has significant effects on the spatial dynamics of non‐breeding flocks of long‐tailed tits and therefore flock behaviour can provide information on the relatedness of other members of the population that might be important for helping decisions in this cooperatively breeding species.
Costs and benefits of prey specialization in a generalist insect predator
Authors: Rana J. S. , Dixon A. F. G. , Jarošík V.
Published: March 2003

There are very few studies on prey specialization in predatory insects. Of the prey that the larvae of the generalist ladybird beetle Adalia bipunctata regularly feed on in the field, some are more suitable as food than others. A laboratory experiment was undertaken to determine whether it is possible to select for improved performance of this insect predator on a ‘poor quality’ prey, and the cost, if any, of such specialization.

The ladybird performed better when reared on the pea aphid Acyrthosiphon pisum than on the black bean aphid Aphis fabae. Over the course of six generations of selection there was a significant increase in performance on both species of aphid, especially the black bean aphid.

Ladybirds previously selected for five generations for better performance on the black bean aphid performed significantly worse when reared on pea aphid compared with those reared continuously for six generations on pea aphids, and vice versa. That is, specialization on one species of aphid resulted in a poorer performance on another.

If, as reported here, the specialization on one kind of prey generally entails a trade‐off in performance on another, then the interactions between insect predators and their prey are more homologous to those of herbivorous insects and plants than previously thought. The significance of this for prey specialization in ladybird beetles is discussed.
The life‐history basis of latitudinal diversity gradients: how do species traits vary from the poles to the equator?
Authors: Cardillo Marcel
Published: March 2003

1.Latitudinal variation among species in life‐history traits is often suggested to contribute to high tropical species richness. However, traditional methods of analysing such variation rarely control for phylogeny and latitudinal range overlap between species, potentially giving misleading results.

2.Using a method of pairwise independent contrasts which overcomes these problems, I tested for latitudinal variation among bird species in a number of traits which have been linked, theoretically or empirically, with both latitude and species richness.

3.This method indicates strong support for Rapoport’s Rule and decreasing clutch size towards the equator in both hemispheres, but only partial support for decreasing body size and ecological generalism towards the equator.

4.Indirect measures of sexual selection (sexual dichromatism and size dimorphism) show no variation with latitude; an apparent increase in dichromatism towards the equator is shown to be an artefact of phylogeny.

5.Many of the associations between life history and latitude were not detected by traditional cross‐species analyses, highlighting the importance of incorporating phylogeny and overlap in studies of geographical life‐history variation. Establishing associations between life‐history traits and latitude does not prove, but is a necessary prerequisite for, a link between these traits and latitudinal diversity gradients.
Shared predators and indirect trophic interactions: lemming cycles and arctic‐nesting geese
Authors: Bêty Joël , Gauthier Gilles , Korpimäki Erkki , Giroux Jean‐François
Published: March 2003

We investigated the hypothesis that cyclic lemming populations indirectly affect arctic‐nesting greater snow geese (Anser caerulescens atlanticus L.) through the behavioural and numerical responses of shared predators.

The study took place on Bylot Island in the Canadian High Arctic during two lemming cycles. We recorded changes in foraging behaviour and activity rate of arctic foxes, parasitic jaegers, glaucous gulls and common ravens in a goose colony during one lemming cycle and we monitored denning activity of foxes for 7 years. We also evaluated the total response of predators (i.e. number of eggs depredated).

Arctic foxes were more successful in attacking lemmings than goose nests because predators were constrained by goose nest defence. Predators increased their foraging effort on goose eggs following a lemming decline.

Activity rates in the goose colony varied 3·5‐fold in arctic foxes and 4·8‐fold in parasitic jaegers, and were highest 2 and 3 years after the lemming peak, respectively. The breeding output of arctic foxes appeared to be driven primarily by lemming numbers.

Predators consumed 19–88% of the annual goose nesting production and egg predation intensity varied 2·7‐fold, being lowest during peak lemming years. Arctic foxes and parasitic jaegers were the key predators generating marked annual variation in egg predation.

Our study provides strong support for short‐term, positive indirect effects and long‐term, negative indirect effects of lemming populations on arctic‐nesting geese. The outcome between these opposing indirect effects is probably an apparent competition between rodents and many terrestrial arctic‐nesting birds.
Optical far‐field microscopy of single molecules with 3.4 nm lateral resolution
Authors: Bloeß A. , Durand Y. , Matsushita M. , Van Dermeer H. , Brakenhoff G. J. , Schmidt J.
Published: March 2003

Optical far‐field imaging of single molecules in a frozen solution at 1.2 K with a lateral resolution of 3.4 nm is reported. The mechanical stability of the fluorescence microscope, especially of the low‐temperature insert, allows for the localization of fluorescing molecules with a reproducibility of better than 5 nm within observation times up to 10 min. For observation times of 9 h the reproducibility of the lateral position is limited to about 20 nm due to mechanical drift. Lateral position and orientation of 314 single molecules, present within the confocal detection volume of ~10 µm3, are obtained. The possibility to correct for mechanical drift by monitoring the position of a spatial reference in the sample is demonstrated.
3D in‐vivo X‐ray microtomography of living snails
Authors: Postnov A. , De clerck N. , Sasov A. , Van dyck D.
Published: March 2003

In this paper we report the first in‐vivo scanning of living snails by desktop X‐ray microtomograph with a resolution up to 10 m. Consecutive cross‐sections were acquired without destroying the specimen. Subsequently, 3D images were reconstructed. The results clearly demonstrate the possibilities of in‐vivo scanning. Processes of growth and regeneration of living snails were visualized over a period of time.
Evidence that glucokinase regulatory protein is expressed  and interacts with glucokinase in rat brain
Authors: Alvarez Elvira , Roncero Isabel , Chowen Julie A. , Vázquez Patricia , Blázquez Enrique
Published: March 2003

Our previous description of functional glucokinase isoforms in the rat brain has opened new questions concerning the presence of glucokinase regulatory protein in the brain and the functional role of its interactions with glucokinase. In this study, we found glucokinase regulatory protein mRNA in rat brain, pancreatic islets and liver. In addition, we found two other variant splicing isoforms, both identified in hypothalamus, pancreatic islets and liver. In situ hybridization studies revealed the presence of glucokinase regulatory protein mRNA, the highest number of positive cells being found in the paraventricular nucleus of the hypothalamus. Glucokinase regulatory protein gene expression gave rise to a protein of 69 kDa mainly in nuclear and soluble cell fractions. Glutathione S‐transferase protein fused either to rat liver or human pancreatic islet glucokinase were able to precipitate glucokinase regulatory protein from liver or hypothalamic extracts in the presence of fructose‐6‐phosphate, the amount of protein co‐precipitated being decreased with fructose‐1‐phosphate. These findings suggest that the presence of glucokinase and glucokinase regulatory protein in the rat brain would facilitate the adaptation of this organ to fluctuations in blood glucose concentrations, and both proteins may participate in glucose‐sensing and metabolic regulation in the central nervous system.
Characterization of an N‐terminal secreted domain  of the type‐1 human metabotropic glutamate receptor  produced by a mammalian cell line
Authors: Selkirk Julie V. , Challiss R. A. John , Rhodes Andrew , McIlhinney R. A. Jeffrey
Published: March 2003

A Chinese hamster ovary cell line has been established which secretes the N‐terminal domain of human mGlu1 receptor. The secreted protein has been modified to contain a C‐terminal hexa‐histidine tag and can be purified by metal‐chelate chromatography to yield a protein with an apparent molecular weight of 130 kDa. Following treatment with dithiothreitol the apparent molecular weight is reduced to 75 kDa showing that the protein is a disulphide‐bonded dimer. N‐terminal protein sequencing of both the reduced and unreduced forms of the protein yielded identical sequences, confirming that they were derived from the same protein, and identifying the site of signal‐peptide cleavage of the receptor as residue 32 in the predicted amino acid sequence. Endoglycosidase treatment of the secreted and intracellular forms of the protein showed that the latter was present as an endoglycosidase H‐sensitive dimer, indicating that dimerization is taking place in the endoplasmic reticulum. Characterization of the binding of [3H]quisqualic acid showed that the protein was secreted at levels of up to 2.4 pmol/mL and the secreted protein has a Kd of 5.6 ± 1.8 nm compared with 10 ± 1 nm for baby hamster kidney (BHK)‐mGlu1α receptor‐expressing cell membranes. The secreted protein maintained a pharmacological profile similar to that of the native receptor and the binding of glutamate and quisqualate were unaffected by changes in Ca2+ concentration.
Modulation of the GABAA‐gated chloride channel  by reactive oxygen species
Authors: Sah Renu , Galeffi Francesca , Ahrens Rebecca , Jordan Gwendolyn , Schwartz‐Bloom Rochelle D.
Published: March 2003

The accumulation of reactive oxygen species during cellular injury leads to oxidative stress. This can have profound effects on ionic homeostasis and neuronal transmission. γ‐Aminobutyric acid (GABA) neurotransmission is sensitive to reactive oxygen species, but most studies have indicated that this is due to alterations in GABA release. Here, we determined whether reactive oxygen species can alter GABAA receptor‐gated Cl– channels in the adult hippocampus. First, we measured the effects of hydrogen peroxide on intracellular Cl– using UV laser scanning confocal microscopy and the Cl–‐sensitive probe, 6‐methoxy‐N‐ethylquinolium iodide (MEQ). Superfusion of adult rat hippocampal slices with hydrogen peroxide for 10 min decreased MEQ fluorescence (elevation in [Cl–]i) significantly in area CA1 pyramidal cell soma. Alterations in [Cl–]i were prevented by the vitamin E analog Trolox, an antioxidant that scavenges free radicals. After exposure of slices to hydrogen peroxide, the ability of the GABA agonist muscimol to increase [Cl–]i was attenuated. To determine if GABAA receptors were sensitive to oxidative insults, the effect of hydrogen peroxide on the binding of [35S]t‐butylbicyclophosphorothionate (TBPS) to GABA‐gated Cl– channels was measured using receptor autoradiography and homogenate binding assays. Hydrogen peroxide inhibited [35S]TBPS binding in a regionally selective manner, with the greatest inhibition in cerebral cortex, hippocampus and striatum, areas␣vulnerable to oxidative stress. Similarly, xanthine and xanthine oxidase, which generate superoxide radicals, reduced [35S]TBPS binding in these regions. The effect of hydrogen peroxide on [35S]TBPS binding was non‐competitive and was prevented by Trolox and the iron chelator, deferoxamine. We conclude that reactive oxygen species may compromise GABAA‐mediated neuronal inhibition via interaction with pre and postsynaptic sites. A reduction in GABAA‐gated Cl– channel function during periods of oxidative stress may contribute to the development of neuronal damage.
Hydrocortisone decreases retinal endothelial cell water and solute flux coincident with increased content and decreased phosphorylation of occludin
Authors: Antonetti David A. , Wolpert Ellen B. , DeMaio Lucas , Harhaj Nicole S. , Scaduto Russell C.
Published: March 2003

Corticosteroids provide an effective treatment to reduce edema for conditions in which the blood–brain or blood–retinal barrier is compromised. However, little is known about the mechanism by which these hormones affect endothelial cell function. We hypothesized that hydrocortisone would reduce transport of water and solutes across bovine retinal endothelial cell (BREC) monolayers coincident with changes to the tight junction protein occludin. Treatment of BREC with 103 nm hydrocortisone for two days significantly decreased water and solute transport across cell monolayers. Immunoblot analysis of occludin extracted in SDS or urea based buffers revealed a 1.65‐ or 2.57‐fold increase in content, respectively. A similar two‐fold increase in occludin mRNA was observed by real‐time PCR. Immunocytochemistry revealed hydrocortisone dramatically increased both occludin and ZO‐1 staining at the cell border. Additionally, 4 h of hydrocortisone treatment significantly reduced occludin phosphorylation. To our knowledge, this is the first example of a regulated decrease in occludin phosphorylation associated with increased barrier properties. In conclusion, hydrocortisone directly affects retinal endothelial cell barrier properties coincident with changes in occludin content, phosphorylation and tight junction assembly. Localized hydrocortisone therapy may be developed as a treatment option for patients suffering from retinal edema due to diabetes.