bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Tyrosinase Expression and Activity in Cultured Human Retinal Pigment Epithelial Cells
Authors: ABUL‐HASSAN KHALED , WALMSLEY RICHARD , TOMBRAN‐TINK JOYCE , BOULTON MIKE
Published: March 2003

The purpose of this study was to investigate the regulation of tyrosinase gene expression and activity in cultured human retinal pigment epithelial (RPE) cells. The tyrosinase promoter (Ty.prom) region (400 bp) was PCR amplified and cloned into a modified mammalian expression vector (pcDNA3.1) upstream of a firefly luciferase (Luc) cDNA and was designated ‘pcDNA3.1‐Ty.prom.Luc’. The plasmid was co‐transfected into RPE cells with a second mammalian expression plasmid (pRL‐TK) containing a herpes simplex virus thymidine kinase promoter region upstream of Renilla Luc in a protocol designated the ‘dual luciferase assay’ (DLA). After co‐transfection, cells were treated with a range of potential melanogenic agents; basic fibroblast growth factor (bFGF), methyl methane sulphonate, α‐melanocyte stimulating hormone, verapamil, phorbol myristate acetate, cholera toxin (CT), pigment epithelium derived factor (PEDF), and
l‐tyrosine. The expression of tyrosinase promoter and enzymatic activities were determined 48 hr post‐transfection using the DLA and DOPA oxidase assays, respectively. Tyrosinase activity could not be detected in RPE cells with any of the treatments. Tyrosinase promoter activity was significantly up‐regulated in RPE cells treated with bFGF, PEDF, verapamil, CT and tyrosine compared with control cells. In conclusion, the tyrosinase gene is not only expressed but can be regulated in response to different chemicals in cultured human RPE cells. However, it appears that RPE cells in culture lack a post‐transcriptional and/or translational modification point(s), which are necessary for tyrosinase enzymic activity.
Expression Analysis of a Tyrosinase Promoter Sequence in Zebrafish
Authors: Camp Esther , Badhwar Prerna , Mann Graham J. , Lardelli Michael
Published: March 2003

Sequence comparisons and functional analysis of the 5′ upstream regions of tyrosinase genes have revealed the importance of cis‐regulatory elements acting to control the spatiotemporal expression of tyrosinase in the melanocytes and retinal pigmented epithelium of developing embryos. To date there are no reports addressing the control of tyrosinase gene transcription in zebrafish, a vertebrate model organism of increasing importance. To exploit the tyrosinase gene as a marker in zebrafish we set out to clone its promoter and analyse its regulation during embryogenesis. Amplification of a zebrafish tyrosinase complementary DNA fragment by reverse transcriptase polymerase chain reaction allowed us to isolate and sequence a 1041 nt genomic DNA fragment that includes a transcription initiation site and 73 nt of the open reading frame. Bioinformatic analysis of this genomic sequence revealed five E‐box motifs, including one CATGTG type E‐box present in a putative initiation region. These are conserved positive regulatory elements in vertebrate tyrosinase promoters. We show that a region of 814 nt upstream from the translation start site of the zebrafish tyrosinase gene can drive expression in retinal pigmented epithelium in transiently transgenic zebrafish embryos but that its activity is not restricted to melanin‐producing cells. This region is unable to drive transcription in human melanoma cell lines. Ectopic expression from this zebrafish tyrosinase promoter fragment is probably due to the absence of positive and negative cis‐regulatory elements, such as a tyrosinase distal element, which is known to function as a pigment cell‐specific enhancer.
Nicotine effects on polymorphonuclear cell apoptosis and lipopolysaccharide‐induced monocyte functions. A possible role in periodontal disease?
Authors: Mariggiò M. A. , Guida L. , Laforgia A. , Santacroce R. , Curci E. , Montemurro P. , Fumarulo R.
Published: March 2003

Apoptosis provides a mechanism for clearance of unwanted cells in a variety of situations in which programmed or physiological cell death occurs; but the premature death of defensive cells could promote infection, inflammation and concomitant disease. We detected high values of apoptosis in polymorphonuclear cells (PMN) elicited from crevicular sulci of smokers affected by adult periodontitis. To learn more about the effects of nicotine on the periodontal environment, we studied its ability to modulate the apoptosis of two phagocytic lines, PMN and mononuclear cells, which are continuously recruited from gingival vessels to prevent or control plaque extension. Brief exposure of PMN to nicotine concentrations ranging from 0.01 to 0.3% shortened, in a dose‐dependent relationship, the lag culture time required to observe at fluorescent microscopy the morphological traces of apoptosis. These observations were confirmed by specific tools of apoptosis: DNA fragmentation on gel electrophoresis and expression of the apoptosis‐signaling receptor Fas/Apo‐1. The apoptotic effect excited by nicotine on these first line defensive cells may be an important feature of the pathogenesis of periodontal disease. As for mononuclear leukocytes, nicotine was unable to induce apoptotic modifications on cells observed up to 72 h culture time, but the drug inhibited IL‐1β release and procoagulant activity (PCA) expression. The conflicting role played by these lipopolysaccharide (LPS)‐induced monocyte functions in the inflammatory process is a further intrigue in the mechanism by which nicotine compromises the oral health.
Epidemiology of necrotizing ulcerative gingival lesions in adolescents
Authors: Lopez R , Fernandez O , Jara G , Aelum V. B
Published: March 2003

Objective and background:   Relatively little is known about the epidemiology of necrotizing ulcerative gingival lesions (NUG) in adolescent populations. Most studies have comprised special target groups, such as military recruits, HIV patients, or severely malnourished subjects.

Methods:  A multistage random cluster sampling scheme was used to obtain a sample of 9203 students aged 12–21 years from 98 of a total of 618 high schools in Santiago, Chile. Students were given a clinical oral examination and questionnaire information was obtained on smoking and oral hygiene habits, dental attendance patterns, and diabetic status. The diagnostic criteria for NUG were the presence of necrosis and ulceration of at least one interproximal papillae.

Results:  The estimated prevalence of NUG was 6.7% (95% CI = [6.2; 7.3]). A multivariable logistic regression analysis showed that last seeing a dentist more than 1 year ago (OR = 1.60), or never (OR = 1.93), and reporting diabetes (OR = 2.12) showed a significant positive association with the presence of NUG; whereas neither gender nor smoking were important predictors. Students aged 18‐21 years were more, albeit statistically insignificantly, likely to have NUG than were younger students (OR = 1.40).

Conclusions:  Our observation that reporting to be diabetic was positively associated with the presence of NUG is interesting, as diabetes has never been addressed as a possible risk factor. As the association between diabetes and periodontitis in adults is thought to relate to impaired function of neutrophils, microangiopathy, and impaired wound healing this finding suggests a significant role of the host response also for the occurrence of NUG.
Cytolethal distending toxin of Actinobacillus actinomycetemcomitans
Authors: Tan Kai Soo , Song Keang‐Peng , Ong Grace
Published: March 2003

The cytolethal distending toxin (Cdt) of Actinobacillus actinomycetemcomitans, a periodontal pathogen, is a newly described cytotoxin with immunosuppressive properties, capable of causing cell cycle arrest of lymphocytes. The objectives of this study were to investigate the occurrence of A. actinomycetemcomitans with the cdt genotype in the subgingival plaque of periodontitis patients and to determine the association of this bacterial genotype with periodontal disease.

A total of 146 subgingival plaque samples from periodontitis patients were assayed by the PCR method using oligonucleotide primers targeting the cdt operon of A. actinomycetemcomitans. Primers targeting the leukotoxin gene A (ltxA) of A. actinomycetemcomitans was used to determine the occurrence of the bacteria in the plaque samples at baseline.

At baseline, A. actinomycetemcomitans was detected in 106 out of 146 (73%) diseased sites studied. Among the 106 diseased sites found to harbor A. actinomycetemcomitans, 13 sites were positive for the bacteria with the cdt genotype (12%). Out of the 13 positive sites, 10 sites were obtained from patients diagnosed with aggressive periodontitis (77%).

Thus, A. actinomycetemcomitans with the cdt genetic subtype has low occurrence in the subgingival plaque of periodontitis patients. However, a strong association was observed between the presence of the bacteria and aggressive forms of periodontitis. Thus, the cytotoxic and immunosuppressive properties of A. actinomycetemcomitans Cdt may function to cripple the host immunity and contribute to the pathogenesis of aggressive periodontitis.
Interleukin‐8 and anti‐interleukin‐8 autoantibodies in gingival crevicular fluid from patients with periodontitis
Authors: Kurdowska A. K. , Noble J. M. , Adcock J. E.
Published: March 2003

Interleukin‐8 (IL‐8) is believed to play an important role in the pathogenesis of various forms of periodontitis. In addition, the anti‐IL‐8 autoantibody has been recently recognized as a potent modulator of IL‐8 function. In the current study, the concentrations of IL‐8 and its autoantibody in gingival crevicular fluid from patients with chronic generalized periodontitis were compared to those in gingival crevicular fluid from patients with refractory chronic periodontitis. Gingival crevicular fluids were collected from patients treated in a private periodontal clinic. Nine patients who were identified as having chronic generalized periodontitis and four with refractory chronic periodontitis were selected for the study. Patients included in the latter group had undergone supportive periodontal therapy for more than 10 years, and during that time had experienced many episodes of periodontal destruction. The gingival crevicular fluid concentrations of total protein, IL‐8, free anti‐IL‐8 autoantibody and IL‐8 bound to the autoantibody (anti‐IL‐8:IL‐8 complexes) were examined. There were no differences in concentration of total protein, but significantly higher levels of IL‐8 were detected in patients with chronic generalized periodontitis in comparison to patients with refractory chronic periodontitis (P < 0.05). In addition, anti‐IL‐8:IL‐8 complexes were present in 90% of patients with chronic generalized periodontitis, but in only 50% of patients with refractory chronic periodontitis. The results suggest that elevated concentrations of free and complexed IL‐8 can differentiate patients with chronic generalized periodontitis from patients with refractory chronic periodontitis.
Pharmacological Antagonism of Fumonisin B1 Cytotoxicity in Porcine Renal Epithelial Cells (LLC‐PK1): A Model for Reducing Fumonisin‐Induced Nephrotoxicity in vivo
Authors: He Quanren , Riley Ronald T. , Sharma Raghubir P.
Published: March 2003

Abstract: Fumonisin B1 is a mycotoxin commonly found on corn. It is hepatotoxic and nephrotoxic in domestic and experimental animals, and causes equine leukoencephalomalacia and porcine pulmonary oedema. It is a potent inhibitor of ceramide synthase. Inhibition leads to accumulation of free sphingoid bases in cells and tissues. In pig kidney epithelial cells (LLC‐PK1), fumonisin B1 induces increased tumour necrosis factor α (TNFα) expression independent of the accumulation of sphingoid bases. The objective of this study was to investigate pharmacological approaches for intervening in fumonisin B1 toxicity using the LLC‐PK1 cell model. The toxicity of fumonisin B1 was assayed using cell viability and lactate dehydrogenase (lactate dehydrogenase) release. Pretreatment of cells with myriocin, preventing sphinganine accumulates, prevented the fumonisin B1‐induced decrease in cell viability and increased lactate dehydrogenase release. Modulation of adenosine receptor activity did not reduce the fumonisin B1 cytotoxicity. As with myriocin, silymarin pretreatment prevented the fumonisin B1‐induced effects on cell viability and lactate dehydrogenase release. When added 6 or 24 hr after treatment of cells with fumonisin B1, both myriocin and silymarin reversed the decreased cell viability and suppressed the increased lactate dehydrogenase release. Myriocin, but not silymarin, blocked the accumulation of sphinganine in fumonisin B1‐treated cells. Silymarin, unlike myriocin, induced expression of TNFα to an extent similar to fumonisin B1, but pretreatment with silymarin decreased the fumonisin B1‐induced TNFα expression in LLC‐PK1 cells. Results suggest that the mechanisms by which myriocin and silymarin protect renal cells are different, and silymarin potentially prevents fumonisin B1‐induced toxicity by modulating TNFα expression or signals downstream of the inhibition of ceramide synthase.
Diazenecarboxamide UP‐91, a Potential Anticancer Agent, Acts by Reducing Cellular Glutathione Content
Authors: Moskatelo Dubravka , Polanc Slovenko , Košmrlj Janez , Vukovi Lidija , Osmak Maja
Published: March 2003

Abstract: Glutathione (GSH) is a ubiquitous non‐protein thiol essential for cellular homeostasis and protection. Diazenecarboxamides (diazenes) are new compounds that could, according to their biochemical properties, lower the intracellular GSH content, thus inhibiting the growth of tumour cells. In the present study we examined four such compounds: JK‐914, JK‐918, JK‐1013 and UP‐91. Their cytotoxic effect on the growth of eight human tumour cell lines (glioblastoma, cervical and laryngeal carcinoma cells, mammary carcinoma cells and four drug‐resistant sublines) was determined using a modified colorimetric MTT assay. The rate of reaction of thiophenol (as a model thiol) with diazenes leading to diphenyl disulfide was established by chromatography (TLC). Reactivity of diazenes with GSH under quasi‐physiological conditions was determined by NMR spectroscopy. Intracellular GSH content was examined spectrophotometrically by the procedure developed by Tietze (1969). Diazene UP‐91 reduced significantly the cell survival of all eight examined cell lines, including four drug‐resistant cell lines. Other diazenes did not influence the survival of tumour cells. Reaction time for quantitative conversion of thiophenol to diphenyl disulfide was shortest for diazene UP‐91, which is highly consistent with high reactivity of the same diazene with GSH, observed under quasi‐physiological conditions. UP‐91 reduced intracellular GSH level, while other diazenes had no effect on it. Thus, diazenecarboxamides UP‐91 is a potential anticancer agent that may inhibit the growth of tumour cells due to reduction in glutathione level.
Transmission of UVA radiation through epithelium of oral mucosa and skin in rat and man
Authors: Kuusilehto Asko
Published: March 2003

Background/Methods: Transmission of 362 nm ultraviolet A radiation (UVA) through epithelium of oral mucosa and skin epidermis was compared in rat and man. The transmitted irradiance was measured by means of a radiometer.

Results: In man, significantly (P<0.01) greater transmittance (2.8‐fold) was observed in skin compared to buccal mucosa. In rat, no significant differences were found.

Conclusion: In different species and different types of epitheliae, separate studies are needed for the evaluation of UVA transmission.
Marathon with cystic fibrosis and bilateral lung transplant
Authors: Stanghelle J. K. , Koss J. O. , Bjørtuft Ø. , Geiran O.
Published: March 2003

The article presents studies performed before, during and after a marathon run (42 195 m) in a 32‐year‐old man who underwent a bilateral lung transplantation because of end‐stage cystic fibrosis (CF) 15 months prior to the race. Before the run his FEV1 was 81% predicted, compared with 19% predicted before the operation, and his maximal oxygen uptake was 31.9 ml/kg−1/min−1. He completed the New York City Marathon 1998 without major problems in 7 h 8 min 50s. Pulmonary tests, biochemical changes and endocrine responses indicated transient changes, mostly as expected in healthy marathon runners. The case demonstrates that physiological trainability and psychological will power following a successful bilateral lung transplantation can transform a chronically ill CF patient into a robust marathon runner.
Waist circumference and VO2max are associated with metabolic and hemostatic risk in premenopausal nurses
Authors: Elich H. , Riese H. , Geus E. J. C.
Published: March 2003

In 21 nurses (34.4±3.9 yr), VO2max, physical activity, body composition and lifestyle parameters were measured to determine which of these characteristics are related to metabolic and hemostatic risk for cardiovascular disease. Physical activity was assessed with the 7‐day recall interview. VO2max was measured in a progressive and continuous treadmill test to volitional fatigue. Fasting insulin, total cholesterol, HDL‐C, triglycerides, fibrinogen, tPA‐act, tPA‐ag, and PAI‐1‐ag were determined from fasting blood samples. Contrary to our expectation, there was no association of physical activity with any of these risk indicators. High VO2max was associated with lower levels of insulin and fibrinogen. Regression analyses indicated that metabolic and hemostatic risk indicators, as measured in healthy premenopausal nurses, were mainly predicted by waist circumference and oral contraceptive use.
Release of muscle proteins after downhill running in male and female subjects
Authors: Sorichter S. , Mair J. , Koller A. , Calzolari C. , Huonker M. , Pau B. , Puschendorf B.
Published: March 2003

The release of muscle proteins after downhill running, which mainly includes eccentric muscle action, was compared in females (F; n=9) and males (M; n=9). They performed 20 min of downhill treadmill running with 16% decline with a target heart rate of 70% of the individual VO2peak, which was determined two weeks before. Blood samples were drawn before, 6 and 24 h after exercise to measure plasma levels of skeletal troponin I (sTnI), myosin heavy chain fragments (MHC), creatine kinase (CK), and myoglobin (Mb). Baseline levels before exercise were significantly higher in males compared to females for the cytoplasmic proteins CK and Mb, but the difference for MHC and sTnI was not significant. Both groups displayed marked and significant early (6 h) increases (P<0.05) for sTnI (median: F: 8.2 μg/L; M: 22.0 μg/L), Mb (median: F: 86.8 μg/L; M: 407 μg/L), and CK (median: F: 162 U/L; M: 339 U/L). A significant (P<0.05) but delayed (24 h) increase was found for MHC (median: F: 482 μU/L; M: 651 μU/L). The absolute values for all four parameters were significantly (P<0.05) higher in males compared to females; however, no difference was found for the relative increases and the time course of all parameters between females and males. We conclude 1) that there were no significant differences in the basal concentrations of predominantly bound proteins, and 2) that there were no differences in the relative muscle protein release between females and males before and after one bout of high‐intensive eccentric exercise. The higher plasma concentrations of all measured muscle proteins in males are probably caused by the higher muscle mass compared to females.
Range of motion training in brace vs. plaster immobilization after anterior cruciate ligament reconstruction: a prospective randomized comparison with a 2‐year follow‐up
Authors: Henriksson M. , Rockborn P. , Good L.
Published: March 2003

The purpose of this prospective and randomized study was to compare rehabilitation with early range of motion (ROM) training vs immobilization following anterior cruciate ligament (ACL) reconstruction. Fifty patients, undergoing an ACL reconstruction with a bone–patellar tendon–bone graft, were postoperatively allocated randomly to either a plaster cast or a brace for 5 weeks. The brace group had ROM exercises from postoperative day 7. The commencement of ROM exercises was postponed 4 weeks for the plaster group compared to the brace group, but progressed subsequently with equal speed. There was no difference between the groups in the ROM of flexion or extension 20 weeks after the ACL reconstruction and later. Twenty‐four months after surgery, the muscle strength deficit in the hamstring muscles (isokinetic measurements; percent difference, injured vs uninjured) was significantly larger in the brace group (mean ± SD: 5.9 ± 7.8%, P < 0.01) than in the plaster group (− 0.9 ± 11.8%, NS) (brace vs plaster group, P < 0.05). Furthermore, there was also a tendency in the brace group to a larger strength deficit in the quadriceps muscle (brace: 11.1 ± 13.2%, P < 0.001; plaster: 3.8 ± 12.9%, NS) (brace vs plaster group, P= 0.07). There was no difference between the groups in the total sagittal knee laxity, as measured with an arthrometer, or in the subjective knee function or activity level (Lysholm score together with the Tegner activity level) between the groups. It is concluded that the postoperative treatment with early range of motion training after ACL reconstruction gave as good ROM, knee stability, subjective knee function and activity level as the treatment with immobilization. It is hypothesized that the larger strength deficit observed after rehabilitation with early range of motion training is secondary to the more intensive training and physical therapist involvement that was demanded in order to achieve full ROM following immobilization.
Is vascular reactivity in skin predictable?
Authors: Lindén Maria , Andersson Thomas , Wårdell Karin , Anderson Chris
Published: March 2003

Background/aims: Cutaneous microdialysis can be used to follow the pharmacology and metabolism of an inflammatory reaction. It is assumed that an equilibration period compensates for the trauma of catheter insertion.

Methods: In the present paper, the vascular reactivity of the skin to histamine was tested using assessment by laser Doppler perfusion imaging. In a group of six subjects, the provocation was preceded by insertion of a microdialysis catheter. In a second control group, histamine provocation alone was performed (six subjects).

Results: The vascular response to histamine was greater in the microdialysis catheter group than the control group. The histamine provocation caused a greater response than catheter insertion. Further, there was a correlation between the response to catheter insertion and the histamine provocation.

Conclusion: It appears that the insertion and/or presence of a microdialysis catheter increases vascular reactivity to subsequent provocation. The response to catheter insertion may predict skin reactivity in general in the individual subject.
Optical analysis of displacement and strain fields on human skin
Authors: Marcellier H. , Vescovo P. , Varchon D. , Vacher P. , Humbert P.
Published: March 2003

Background/aims: The aim of this article is to propose a new way to measure the mechanical behaviour of skin by using optical analysis software. Some examples on scars and stretch marks show the validity and the prospects of such a method.

Methods: Software is used to compare two states of deformation. The user takes two photos of the skin surface he wants to study, before and after the deformation is applied. The software is used to process the data.

Results: The software gives a mapping of the displacement, and strain fields of the area studied. Investigations can be realised in vitro or in vivo, and it is possible to study, for example, cutaneous lesions like scars and stretch marks.

Conclusions: The method presented gives convincing results, which open many prospects in dermatology, surgery and cosmetology.
Age‐dependent response of energy metabolism of human skin to UVA exposure: anin vivostudy by31P nuclear magnetic resonance spectroscopy
Authors: Declercq Lieve , Perin Fabrice , Vial Francis , Savard Sébastien , Petitcollin Bénédicte , Beau Patrick , Collins Don , Mammone Tom , Maes Daniel
Published: March 2003

PurposeThe aim of our study was to evaluate thein vivoenergy metabolism of human skin as a function of age, in conditions of rest and after a mild stress caused by a suberythemal UVA irradiation.

MethodsThe kinetics of UVA‐induced modifications in high‐energy phosphorylated metabolites of young and old skins were non‐invasively monitored over a period of 24 h using31P nuclear magnetic resonance spectroscopy.In vivo31P spectra were obtained on the ventral aspect of the wrist, using a NMR Imaging Spectrometer equipped with a double‐tuned surface coil. Concentrations of phosphocreatine, inorganic phospate, adenosine tri‐phosphate, phosphomono and phosphodiesters were calculated from the spectra and results were expressed as relative concentrations. A total of 20 subjects were enrolled in this study (n = 10 for the age group below 25 years andn = 10 for the age group above 55 years). A second experiment was then performed on 10 old subjects (mean age 60) who were treated on one wrist, twice a day for one month prior to UVA irradiation, with a product that contained active ingredients to restore barrier function and modulate the inflammatory response, the other wrist being an untreated control.

ResultsBaseline levels of phosphorylated metabolites were similar in young and old skins. A suberythemal dose of UVA (6 J.cm−2) led to a significant decrease in the PCr/Pi ratio (index of energy status) and a significant increase in the PME/PDE ratio (index of cellular turnover rate of lipid‐related metabolites) within 1 h. The observed variations were transient and the recovery was complete at T + 24 h post‐UVA, although recovery was significantly slower in the older group. The disturbances were significantly reduced after treatment of the older skin with a formula that restored barrier function of the stratum corneum and modulated the inflammatory response.

Conclusion(i) baseline levels of energy metabolites in skin do not seem to vary with age; (ii) low dose UVA irradiation induces a rapid response in the energy metabolism of the skin; (iii) the kinetics of the response and recovery after an aggression by UVA suggest that older skin has significantly less energy rebound after a stress situation than younger skin; (iv) the energy reserve in older skin can be protected efficiently against UVA‐induced stress by restoring barrier function and modulating the inflammatory response.
A Novel Pathway for Transport and Metabolism of a Fluorescent Phosphatidic Acid Analog in Yeast
Authors: Trotter Pamela J.
Published: March 2003

Phosphatidic acid is a central intermediate of biosynthetic lipid metabolism as well as an important signaling molecule in the cell. These studies assess the internalization, or retrograde transport, and metabolism of phosphatidic acid in yeast using a fluorescent analog. An analog of phosphatidic acid fluorescently labeled at the sn‐2 position with N‐4‐nitrobenz‐2‐oxa‐1, 3‐diazole‐aminocaproic acid (NBD‐phosphatidic acid) was introduced to yeast cells by spontaneous transfer from phospholipid vesicles. Transport and metabolism of the NBD‐phosphatidic acid were then monitored by fluorescence spectrophotometry, fluorescence microscopy and routine biochemical methods. Primary metabolites of the NBD‐phosphatidic acid in yeast were found to be NBD‐diacylgycerol and NBD‐phosphatidylinositol. Experiments in cells possessing different levels of phosphatidate phosphatase activity suggest that conversion of the NBD‐phosphatidic acid to NBD‐diacylglycerol is not a pre‐requisite for internalization in yeast. Internalization is sensitive to decreased temperature, but neither ATP depletion nor a sec6‐4 mutation, which interrupts endocytosis, has an affect. Thus, internalization of NBD‐phosphatidic acid apparently occurs via a non‐endocytic route. These characteristics of retrograde transport of NBD‐phosphatidic acid in yeast differ significantly from transport of other NBD‐phospholipids in yeast as well as NBD‐phosphatidic acid transport in mammalian fibroblasts.
Transient Surface Delivery of Invariant Chain‐MHC II Complexes Via Endosomes: a Quantitative Study
Authors: Lindner Robert
Published: March 2003

Newly synthesized major histocompatibility complex class II needs to be directed to late endocytic compartments to combine with peptide antigens. Efficient transport requires complexes of major histocompatibility complex class II and invariant chain (αβIi). Since such complexes have been detected on the plasma membrane in human cells, this compartment was proposed as the primary destination for αβIi exiting the trans‐Golgi network. Here, I have used density gradient electrophoresis and selective biotinylation to investigate the trafficking route of αβIi quantitatively. Density gradient electrophoresis analysis showed that αβIi was transported from the trans‐Golgi network to endosomes at ∼ 1.7% min−1. Surface delivery of αβIi was delayed relative to endosome transport by ∼ 10 min and showed slower kinetics (∼ 0.4% min−1), suggesting that αβIi reached the plasma membrane only after arrival in endosomes. A biotinylation assay revealed that 20–40% of endosomal αβIi was delivered to the plasma membrane at steady state, suggesting that surface αβIi was entirely derived from endosomes. Surface αβIi was rapidly re‐internalized and either returned to the cell surface or accessed degradative compartments. Peptide loading commenced ∼ 30 min after delivery to endosomes. Thus αβIi directly traffics from trans‐Golgi network to endosomes and enters an endosome–plasma membrane ‘carousel’ until transport to peptide‐loading compartments ensues.
Constitutive Activation and Endocytosis of the Complement Factor 5a Receptor: Evidence for Multiple Activated Conformations of a G Protein‐Coupled Receptor
Authors: Whistler Jennifer L. , Gerber Basil O. , Meng Elaine C. , Baranski Thomas J. , Von Zastrow Mark , Bourne Henry R.
Published: March 2003

Serpentine receptors relay hormonal or sensory stimuli to heterotrimeric guanine nucleotide‐binding proteins (G proteins). In most G protein‐coupled receptors (GPCRs), binding of the agonist ligand elicits both stimulation of the G protein and endocytosis of the receptor. We have begun to address whether these responses reflect the same sets of conformational changes in the receptor using constitutively active mutants of the human complement factor 5a receptor (C5aR). Two different mutant receptors both constitutively activate G protein‐mediated responses, but one (F251A) is endocytosed only in response to ligand stimulation, while the other (NQ) is constitutively internalized in the absence of ligand. Both the constitutive and ligand‐dependent endocytosis are accompanied by recruitment of beta‐arrestin to the receptor. An inactivating mutation (N296A) complements the NQ mutation, producing a receptor that is activated only upon exposure to agonist; this revertant receptor (NQ/N296A) is nevertheless constitutively endocytosed. Thus one mutant (F251A) requires agonist for triggering endocytosis but not for activation of the downstream G protein signal, while another (NQ/N296A) behaves in the opposite fashion. Dissociation of two responses normally dependent on agonist binding indicates that the corresponding functions of an activated GPCR reflect different sets of changes in the receptor's conformation.
The role of immunity in protection from mucosal SIV infection in macaques
Authors: Bergmeier LA , Wang Y , Lehner T
Published: March 2003

The need for an effective vaccine against HIV has prompted a refocusing of attention on mucosal immunity. More than 75% of all infections are acquired across a mucosal surface. It is therefore a prerequisite for a vaccine to target directly the mucosal tissues or indirectly the regional lymph nodes in order to prevent or control viral replication. Although mucosal immunization has induced responses at the genital or rectal surfaces, immune mechanisms alone have not been shown to be sufficient to contain infections in macaques. A growing body of evidence suggests that a dual mechanism may be required for effective mucosal protection, mediated by specific CD4 and CD8 T cell and antibody responses to the immunizing agents, plus innate antiviral factors and β chemokines that down‐regulate CCR5 coreceptors. Targeted iliac lymph node immunization with SIV gp120 and p27 in alum prevents SIV infection or significantly decreases the viral load when immunized macaques were challenged with SIV by the rectal route. Indeed, in addition to specific immunity, including significant SIgA antibody secreting cells in the iliac lymph nodes, CD8‐suppressor factor and the 3β chemokines (RANTES, MIP‐1α and MIP‐1β) are significantly associated with protection against rectal mucosal SIV infection.
Primary aspergillosis affecting the tongue of a leukemic patient
Authors: Correa MEP , Soares AB , De Souza CA , Cintra ML , Jorge J , Almeida OP , Vargas PA
Published: March 2003

We describe a case of primary aspergillosis involving the tongue of a patient with acute myeloid leukemia. Intraoral aspergillosis is very rare and we found only 23 cases reported in the English literature. Clinically it was a 2‐cm, ulcerated, grayish lesion on the dorsum of the tongue. Microscopically there was invasion of the epithelium, connective tissue and muscle of the tongue by fungal hyphae branching at 45° angle. The large hyphae were easily seen by H & E stain, and were strongly positive for periodic acid‐Schiff and Grocott methenamine. The patient was successfully treated with intravenous amphotericin B. Based on clinical, microscopic and culture data, the diagnosis of primary aspergillosis of the tongue was established. Invasive oral aspergillosis is a potentially lethal disease and it should be considered in immunosuppressed patients.
Host pathogen interaction and the development of oral lesions
Authors: Williams DM , Leigh J , Ramirez‐Amador V , Kaonga K , Russo D , Schiødt M
Published: March 2003

The aetiologies of oral ulceration, disseminated interstitial lymphocytosis syndrome and oral lymphomas have been reviewed, with emphasis on the role of HIV infection in the primary causation or modification of the presentation of these entities. There is a paucity of evidence to explain why oral ulceration is so severe in HIV infection, and why major ulceration affects the oropharynx. A number of mechanisms have been proposed to account for the development of lymphomas in patients with HIV infection, including a genetic predisposition, decreased immunosurveillance due to HIV infection, alteration of endothelial cell function and dysregulation of cytokine networks. From this review, it was concluded that there is a need for a prospective multicentre study, to elucidate the aetiological mechanisms involved in lymphomas of the oral regions in this patient group. It was concluded that, although there is anecdotal evidence implicating tobacco use in the aetiology of the lesions reviewed, this is insufficient to allow definitive statements to be made and further systematic evaluation is indicated.
Histone deacetylases (HDACs): characterization of the classical HDAC family
Authors: RUIJTER Annemieke J.M. de , GENNIP Albert H. van , CARON Huib N. , KEMP Stephan , KUILENBURG André B.P. van
Published: March 2003

Transcriptional regulation in eukaryotes occurs within a chromatin setting, and is strongly influenced by the post-translational modification of histones, the building blocks of chromatin, such as methylation, phosphorylation and acetylation. Acetylation is probably the best understood of these modifications: hyperacetylation leads to an increase in the expression of particular genes, and hypoacetylation has the opposite effect. Many studies have identified several large, multisubunit enzyme complexes that are responsible for the targeted deacetylation of histones. The aim of this review is to give a comprehensive overview of the structure, function and tissue distribution of members of the classical histone deacetylase (HDAC) family, in order to gain insight into the regulation of gene expression through HDAC activity. SAGE (serial analysis of gene expression) data show that HDACs are generally expressed in almost all tissues investigated. Surprisingly, no major differences were observed between the expression pattern in normal and malignant tissues. However, significant variation in HDAC expression was observed within tissue types. HDAC inhibitors have been shown to induce specific changes in gene expression and to influence a variety of other processes, including growth arrest, differentiation, cytotoxicity and induction of apoptosis. This challenging field has generated many fascinating results which will ultimately lead to a better understanding of the mechanism of gene transcription as a whole.
Identification, functional expression and enzymic analysis of two distinct CaaX proteases from Caenorhabditis elegans
Authors: CADIÑANOS Juan , SCHMIDT Walter K. , FUEYO Antonio , VARELA Ignacio , LÓPEZ-OTÍN Carlos , FREIJE José M.P.
Published: March 2003

Post-translational processing of proteins such as the Ras GTPases, which contain a C-terminal CaaX motif (where C stands for cysteine, a for aliphatic and X is one of several amino acids), includes prenylation, proteolytic removal of the C-terminal tripeptide and carboxy-methylation of the isoprenyl-cysteine residue. In the present study, we report the presence of two distinct CaaX-proteolytic activities in membrane extracts from Caenorhabditis elegans, which are sensitive to EDTA and Tos-Phe-CH2Cl (tosylphenylalanylchloromethane; ‘TPCK') respectively. A protein similar to the mammalian and yeast farnesylated-proteins converting enzyme-1 (FACE-1)/Ste24p CaaX metalloprotease, encoded by a hypothetical gene (CeFACE-1/C04F12.10) found in C. elegans chromosome I, probably accounts for the EDTA-sensitive activity. An orthologue of FACE-2/Rce1p, the enzyme responsible for the proteolytic maturation of Ras oncoproteins and other prenylated substrates, probably accounts for the Tos-Phe-CH2Cl-sensitive activity, even though the gene for FACE-2/Rce1 has not been previously identified in this model organism. We have identified a previously overlooked gene in C. elegans chromosome V, which codes for a 266-amino-acid protein (CeFACE-2) with 30% sequence identity to human FACE-2/Rce1. We show that both CeFACE-1 and CeFACE-2 have the ability to promote production of the farnesylated yeast pheromone a-factor in vivo and to cleave a farnesylated peptide in vitro. These results indicate that CeFACE-1 and CeFACE-2 are bona fide CaaX proteases and support the evolutionary conservation of this proteolytic system in eukaryotes.
Empirical evidence of a convection–diffusion model for pH patterns in the rhizospheres of root tips
Authors: Nichol S. A. , Silk W. K.
Published: March 2003

A recently formulated convection–diffusion model predicted that root growth plus diffusion of protons in the neighbouring soil would lead to particular pH patterns around the moving root tip. To test the predictions of this theory, pH was measured at differing radial distances from the root surface after 24 h of growth in a medium with low diffusivity (sandy soil) and after a shorter period (55 min of growth) in a medium with high diffusivity (agar). In agreement with the theory, the growth zone was found to influence the pH of the soil for distances less than 1 mm from the root surface (even after many hours) and the pH of the agar for a distance of at least 5 mm (after only 1 h). The axial pattern of pH along the surface of soil‐grownZea maysL. root tips was found to be the same for roots growing at different rates under different temperatures (2·23  mm h−1at 26 °C or 1·27 mm h−1at 20 °C). Thus, the plant can synchronize proton flux with growth to maintain a particular surface pH pattern within the growth zone. This implies that root tips growing at different rates in response to different temperatures can carry the same microenvironment of pH through a homogeneous soil.
Spatial distribution of leaf water‐use efficiency and carbon isotope discrimination within an isolated tree crown
Authors: Le Roux X. , Bariac T. , Sinoquet H. , Genty B. , Piel C. , Mariotti A. , Girardin C. , Richard P.
Published: March 2003

The spatial variations in the stable carbon isotope composition (δ13C) of air and leaves (total matter and soluble sugars) were quantified within the crown of a well‐watered, 20‐year‐old walnut tree growing in a low‐density orchard. The observed leaf carbon isotope discrimination (Δ) was compared with that computed by a three‐dimensional model simulating the intracanopy distribution of irradiance, transpiration and photosynthesis (previously parameterized and tested for the same tree canopy) coupled to a biophysically based model of carbon isotope discrimination. The importance of discrimination associated with CO2 gradients encountered from the substomatal sites to the carboxylation sites was evaluated. We also assessed by simulation the effect of current irradiance on leaf gas exchange and the effect of long‐term acclimation of photosynthetic capacity and stomatal and internal conductances to light regime on intracanopy gradients in Δ. The main conclusions of this study are: (i) leaf Δ can exhibit important variations (5 and 8‰ in total leaf material and soluble sugars, respectively) along light gradients within the foliage of an isolated tree; (ii) internal conductance must be taken into account to adequately predict leaf Δ, and (iii) the spatial variations in Δ and water‐use efficiency resulted from the short‐term response of leaf gas exchange to variations in local irradiance and, to a much lesser extent, from the long‐term acclimation of leaf characteristics to the local light regime.
Comparative physiology of salt and water stress
Authors: Munns R.
Published: March 2003

Plant responses to salt and water stress have much in common. Salinity reduces the ability of plants to take up water, and this quickly causes reductions in growth rate, along with a suite of metabolic changes identical to those caused by water stress. The initial reduction in shoot growth is probably due to hormonal signals generated by the roots. There may be salt‐specific effects that later have an impact on growth; if excessive amounts of salt enter the plant, salt will eventually rise to toxic levels in the older transpiring leaves, causing premature senescence, and reduce the photosynthetic leaf area of the plant to a level that cannot sustain growth. These effects take time to develop. Salt‐tolerant plants differ from salt‐sensitive ones in having a low rate of Na+and Cl–transport to leaves, and the ability to compartmentalize these ions in vacuoles to prevent their build‐up in cytoplasm or cell walls and thus avoid salt toxicity. In order to understand the processes that give rise to tolerance of salt, as distinct from tolerance of osmotic stress, and to identify genes that control the transport of salt across membranes, it is important to avoid treatments that induce cell plasmolysis, and to design experiments that distinguish between tolerance of salt and tolerance of water stress.
Differential genetic variation in adaptive strategies to a common environmental signal in Arabidopsis accessions: phytochrome‐mediated shade avoidance
Authors: Botto J. F. , Smith H.
Published: March 2003

Shade avoidance is a syndrome of plastic responses to light signals encountered in crowded plant communities and is a crucial component of competitive strategy in higher plants. The responses are mediated via signal perception by specific members of the phytochrome family of photoreceptors, which detect the relative proportions of red (R) and far‐red (FR) radiation within dense communities. We analysed two aspects of shade avoidance, the acceleration of flowering and the enhancement of elongation growth, displayed by more than 100 accessions of Arabidopsis thaliana (Heyn.) in response to FR‐proximity signals. Both traits showed wide variation between accessions, which was unrelated to the latitude of the location of original collection. Flowering acceleration is a major feature of shade avoidance in rosette plants such as Arabidopsis, and most accessions showed dramatic responses, but several were identified as being recalcitrant to the proximity signal. These accessions are likely to be informative in the analysis of quantitative variation in shade avoidance. Hypocotyl elongation, treated here as an indicator of elongation growth responses, also varied widely amongst accessions. The variations in flowering acceleration and elongation were not correlated, indicating that microevolution in the downstream pathways from signal perception has occurred separately.
Several lanthanides activate malate efflux from roots of aluminium‐tolerant wheat
Authors: Kataoka T. , Stekelenburg A. , Nakanishi T. M. , Delhaize E. , Ryan P. R.
Published: March 2003

Many elements of the lanthanide series exist as trivalent cations in solution below pH 6. The present study was carried out to investigate whether lanthanides could stimulate malate efflux from wheat (Triticum aestivum L.) roots, as has been found for trivalent aluminium (Al) cations. Excised root apices treated with 100 µm of each of seven different lanthanide elements (lanthanum, praseodymium, europium, gadolinium, terbium, erbium, and ytterbium) stimulated malate efflux, with five‐ to fifty‐fold more malate being released from an Al‐tolerant wheat line than from a near‐isogenic Al‐sensitive line. As erbium stimulated the greatest malate efflux of the lanthanides tested, this response was characterized further. The characteristics of the erbium‐activated efflux were similar to the Al‐activated efflux described previously suggesting that both of these ions activate the same transport mechanism. The capacity for erbium‐activated malate efflux cosegregated with Al tolerance in wheat seedlings derived from a cross between Al‐sensitive and Al‐tolerant near‐isogenic lines. This is the first study to identify cations, other than Al, which can activate malate release from wheat roots. It also provides additional evidence that malate efflux from root apices is the primary mechanism for Al tolerance in wheat.
Preferential production of interferon‐γ by CD4+ T cells expressing the homing receptor integrin α4/β7
Authors: Abramson Oren , Qiu Shiqiang , Erle David J.
Published: March 2003

Recent studies indicate that T helper type 1 (Th1) and 2 (Th2) lymphocytes differ in their expression of molecules that control T‐cell migration, including adhesion molecules and chemokine receptors. We investigated the relationship between cytokine production and expression of the homing receptor integrin α4/β7 on T cells. We began by analysing cytokine production by human CD4+ CD45RA– memory/effector T cells following brief (4 hr) stimulation with phorbol 12‐myristate 13‐acetate (PMA) and ionomycin. α4/ CD4+ T cells were more likely to produce the Th1 cytokine interferon‐γ (IFN‐γ) than were α4/β7− CD4+ T cells in all six subjects studied. In contrast, production of the Th2 cytokine interleukin‐4 (IL‐4) was similar on α4/ and α4/β7− CD4+ T cells. In addition, we found that human CD4+ CD45RA– T cells that adhered to the α4/β7 ligand mucosal addressin cell adhesion molecule‐1 (MAdCAM‐1) had a greater capacity to produce IFN‐γ than did non‐adherent cells, suggesting that the association between α4/β7 expression and IFN‐γ production has functional significance. These results suggested that primary activation under Th1‐promoting conditions might favour expression of α4/β7. We directly examined this possibility, and found that naïve murine CD4+ T cells activated under Th1‐promoting conditions expressed higher levels of α4/β7 compared to cells activated under Th2‐promoting conditions. The association between α4/β7 expression and IFN‐γ production by CD4+ T cells may help to determine the cytokine balance when MAdCAM‐1 is expressed at sites of inflammation in the intestine or elsewhere.