bluuebunny/crossref_metadata_2025 huggingface dataset:
Reaction of melatonin with hemoglobin‐derived oxoferryl radicals and inhibition of the hydroperoxide‐induced hemoglobin denaturation in red blood cells
Authors: Tesoriere L. , Allegra M. , D'Arpa D. , Butera D. , Livrea M.A.
Published: March 2003

Melatonin has been shown to act as a radical scavenger in various chemical and biological model systems in vitro. Kinetic evidence is now provided showing that melatonin inhibits the irreversible degradation of hemoglobin (Hb), when incubated with red blood cells exposed to the oxidant activity of cumene hydroperoxide (cumOOH). A decrease of heme loss and accumulation of soluble methemoglobin (met‐Hb) are explained in terms of the interaction of the indoleamine with perferryl Hb (⋅Hb[FeIV=O]), a highly reactive Hb‐derived radical species responsible for the irreversible Hb degradation. A kinetic study, in pure chemical solution, showed that melatonin can effectively reduce the oxoferryl heme group of perferryl‐Hb, thus forming met‐Hb. The reducing activity of melatonin is of the same order as that of Trolox, the water‐soluble vitamin E analog. This novel radical‐scavenging activity of melatonin may contribute to the previously observed protective effects of melatonin in ischemia‐reperfusion injury.
Effect of melatonin on cell growth, metabolic activity, and cell cycle distribution
Authors: Natarajan Mohan , Reiter Russel J. , Meltz Martin L. , Herman Terence S.
Published: March 2003

We have recently demonstrated that the pineal secretory product melatonin inhibits the key transcriptional regulator nuclear factor‐κB (NF‐κB). As the activation of NF‐κB is known to regulate the expression of cellular genes associated with cell cycle progression, cell growth, and differentiation, we investigated the effect of melatonin treatment on several cellular processes. These include cell viability, metabolic activity, and cell cycle phase distribution. Human embryonic kidney (293S) cells were treated with melatonin at concentrations of 0.02, 0.2, or 2 mM. When cell viability was measured 24, 48, and 72 hr after continuous exposure to melatonin using the trypan blue dye exclusion method, no significant cell death was observed. Even after exposure to 2 mM melatonin for 72 hr, cell viability remained at 98%. In contrast, another antioxidant compound, pyrrolidine dithiocarbomate (PDTC), at a 2 mM concentration reduced cell viability to 80.7±2.1% as early as 24 hr compared with untreated controls (P<0.05). When the metabolic activity was determined at 24, 48, and 72 hr using the colorimetric MTT assay, no significant changes in metabolic activity were observed. Even if the cells were treated with 10 mM melatonin for 72 hr, the metabolic activity was similar to that of the control cells. When cell cycle analysis was performed by flow cytometry, no marked difference in cell cycle distribution was observed. Melatonin at a concentration of 2 mM, however, did slightly alter the cell cycle (percentage of S phase cells) at 48 hr. This study revealed that when 293S cells are treated with concentrations of melatonin up to 2 mM, no significant alterations in three important cellular functions occurred. Exogenously added melatonin appeared to have a limited influence on the normal functioning of the cells even when the exposure continued for 72 hr.
Injury risk factors in female European football. A prospective study of 123 players during one season
Authors: Östenberg A. , Roos H.
Published: March 2003

The purpose of this study was to register prospectively the injuries in female soccer and to study their correlation to potential risk factors. A total of 123 senior players from eight teams of different levels were followed during one season. Isokinetic knee muscle strength at 60 and 180°/s, one‐leg‐hop, vertical jump, square‐hop, and continuous multistage fitness test (MFT) were tested at the end of the pre‐season. In addition, Body Mass Index (BMI) and general joint laxity were measured. During the season, April–October, all injuries resulting in absence from one practice/game or more were registered. Forty‐seven of the 123 players sustained altogether 65 injuries. The total injury rate was 14.3 per 1000 game hours and 3.7 per 1000 practice hours. The knee (26%) was the most commonly injured region followed by the foot (12%), ankle (11%), thigh (11%) and back (11%). The risk of sustaining moderate and major injuries increased in the later part of the game or practice. Significant risk factors for injuries were an increased general joint laxity (odds ratio (OR)=5.3, P<0.001), a high performance in the functional test square‐hop (OR=4.3, P=0.002), and an age over 25 years (OR=3.7, P=0.01). The injury rate was not different compared to male soccer, but knee injuries were more common, which is in accordance with previous studies. None of the risk factors identified in this study is easily applicable for future intervention studies in the attempts to reduce the injury rate in female soccer.
Seven years follow‐up after ankle inversion trauma
Authors: Konradsen L. , Bech L. , Ehrenbjerg M. , Nickelsen T.
Published: March 2003

During one year all ankle inversion injuries seen at the acute ward of our institution were divided into grades of severity and classified according to the maximal area of tenderness at the time of clinical examination. Seven years later 648 of the subjects (91%) evaluated their ankle with the help of a questionnaire.

Location of maximal tenderness at the time of injury was: lateral fibular ligaments 61%, lateral midfoot ligaments 24%, base of the fifth metatarsal/peroneal tendons 5% and combined lesions 8%. 39% were considered minor, 46% were moderate, and 15% severe. All cases followed a functional treatment protocol.

Seven years post‐ injury 32% reported chronic complaints of pain, swelling or recurrent sprains. 72% of the subjects with residual disability reported that they were functionally impaired by their ankle – in most cases a question of not performing sports at a desired level. 4% experienced pain at rest and were severely disabled. 19% were bothered by repeated inversion injuries – 43% of these subjects felt that they could compensate by using an external ankle support.

There was no correlation between the severity of the sprain as judged at the time of injury and the frequency of residual disability or between the area of maximal tenderness at the time of injury and the area of maximal pain at the time of follow‐up.
Effect of hyperoxia on metabolic responses and recovery in intermittent exercise
Authors: Nummela A , Hämäläinen I , Rusko H
Published: March 2003

The aim of the present study was to investigate whether the breathing of hyperoxic gas affects hemoglobin oxygen saturation (SaO2) and blood acidosis during intense intermittent exercise and recovery in sprint runners. The hypothesis was that the breathing of hyperoxic gas prevents SaO2 from decreasing, delays blood acidosis during the exercise and improves the rate of heart rate recovery after the exercise. Nine sprinters ran three sets of 300 m at different velocities on a treadmill in normoxia (NOX) and in two hyperoxic conditions (ERHOX and RHOX; FIO2 0.40) in a randomized order. In ERHOX the inspired air was hyperoxic during the entire exercise and recovery and in RHOX the hyperoxic air was only inhaled during recovery periods. Blood pH and SaO2 were measured from fingertip blood samples taken after each set of runs. The mean heart rate for the final 15 s of the last run in each set (HRwork), the mean heart rate for the final 15 s of the first minute of recovery (HRrec) and the difference of HRwork and HRrec (HRdec) were determined. In NOX, SaO2 decreased from 95.0 ± 2.0% to 88.7 ± 2.0% (p < 0.001) but SaO2 did not change in ERHOX (from 95.4 ± 1.3% to 95.9 ± 1.8%). A significant correlation was observed between the SaO2 decrease in NOX and the effect of hyperoxia on blood pH in ERHOX (r = 0.63) and on HRdec in both ERHOX (r = 0.74) and RHOX (r = 0.69). We concluded that hemoglobin oxygen de‐saturation occurred during intensive intermittent exercise in normoxia but hyperoxic gas during the exercise prevents SaO2 from decreasing. Furthermore, the present results suggested that the beneficial effects of hyperoxia on heart rate recovery and blood acidosis during intensive intermittent exercise were related to hemoglobin de‐saturation in normoxia.
Effect of treatment with nifedipine, an L‐type calcium channel blocker, on muscular atrophy induced by hindlimb immobilization
Authors: Wagatsuma A. , Fujimoto K. , Yamada S.
Published: March 2003

The purpose of this study was to investigate whether the prevention of calcium influx through L‐type calcium channels contributed to the attenuation of muscular atrophy induced by hindlimb immobilization (HI) in a shortened position. Mice were divided into four groups (8 mice/group): control; nifedipine; HI; and HI with nifedipine. Mice received nifedipine at a dose of 5 mg/kg one day before and during the 8 days of HI. Quantitative alterations in the amount of myosin heavy chain (MyHC) and actin proteins in the soleus muscle were analyzed using SDS‐PAGE. The weight of the soleus muscle decreased significantly by 40.8% (P<0.05) and 27.0% (P<0.05) after the hindlimb immobilization in the HI and HI with nifedipine groups, respectively, when compared to that of the control or nifedipine groups. Treatment with nifedipine alone appeared to have no effect on muscle mass or the amount of myofibrillar proteins. The level of MyHC proteins decreased significantly by 25.1% (P<0.001) and 17.4% (P<0.001) in the HI and HI with nifedipine groups, respectively. The level of MyHC protein in the HI with nifedipine group was significantly greater than that of the HI group (P<0.05), although there were no significant differences in the amount of actin protein. These findings suggest that nifedipine treatment may have a beneficial effect on muscular atrophy.
Skeletal muscle fatigue in long‐distance runners, sprinters and untrained men after repeated drop jumps performed at maximal intensity
Authors: Skurvydas A. , Dudoniene V. , Kalvėnas A. , Zuoza A.
Published: March 2003

One hundred drop jumps were performed at maximal intensity every 20 s in 12 untrained subjects (UT), 9 sprinters (S) and 10 long‐distance runners (LDR). Muscle contraction force (P20, P50) induced by percutaneous electrical stimulation (20 Hz and 50 Hz, respectively) as well as maximal voluntary contraction force and the height of vertical jumps performed in different ways decreased (P<0.05) and was not restored to the initial value 20 min post exercise. There was a marked increase in low frequency fatigue (LFF) in all the groups studied as substantiated by a significant decrease in the ratio of P20/P50 immediately after exercise as well as 20 min post exercise compared to pre exercise values (P<0.05). However, low frequency fatigue was similar in UT, S and LDR. The jump height of the sprinters during counter‐movement jump and drop jump at 90 ° decreased to a smaller extent compared to jumps performed by LDR and UT. Muscle pain did not differ between UT, S and LDR at 24 h post exercise. The present data indicate that endurance training status as well as prevalence of muscle fibres of the slow type does not decrease muscle resistance to LFF nor accelerate the recovery of muscle contraction force following maximal, intermittent stretch‐shortening cycle exercise.
Effects of training on functional performance in 65, 75 and 85 year‐old women: Experiences deriving from community based studies in Odense, Denmark
Authors: Puggaard L.
Published: March 2003

The aim of the Odense training studies is to elucidate if regular physical training influences the expected decline in physical functional ability in order to assess capacity for postponing dependence in old age. All participants were healthy community‐dwelling women representing three different age‐cohorts of 65, 75 and 85 year‐old subjects. The 65 and 85 year‐old participants of the training group took part in physical class‐based exercises for eight months with one session of 60 min a week, whereas the 75 year‐old women trained twice a week over eight months. The multicomponent training consisted of various exercises typically involving body awareness, rhythm, aerobic performance (walking), muscle strength and muscle endurance, flexibility, reaction and balance exercises. Physical ability was measured as physical performance test (PPT), isometric muscle strength of the trunk, hip and leg, aerobic capacity and walking speed. This shows that regular training can significantly improve physical ability of elderly women with regard to PPT, maximal oxygen uptake and maximal walking speed, suggesting that both young‐old and old‐old women are able to benefit from regular tailored exercise training. Thus, physical training of old community‐dwelling women appears to represent a prophylactic remedy that merits further research aimed at evaluating the preliminary findings of the present studies in larger, less selective groups of participants.
Possible beneficial role of exercise in modulating low‐grade inflammation in the elderly
Authors: Pedersen B. K. , Bruunsgaard H.
Published: March 2003

Aging is associated with increased levels of tumor necrosis factor‐α (TNF) and interleukin (IL)‐6. These two cytokines are tightly linked in that TNF induces production of IL‐6, which again inhibits TNF gene expression. In epidemiological studies, both cytokines have been associated with obesity, insulin resistance and atherosclerosis. However, based on basal studies, we suggest that TNF (and not IL‐6) is the driver behind insulin resistancy. Thus, it is possible that selective enhancement of the IL‐6 level may inhibit TNF—induced insulin resistance. Muscle contractions induce production and release of IL‐6, but not TNF, into the circulation, in both young and elderly humans. We suggest that muscle‐derived IL‐6 contributes to mediate the beneficial metabolic effects of exercise and may contribute to inhibit TNF‐production and thereby insulin resistance.
Optical coherence tomography in dermatology: a review
Authors: Welzel Julia
Published: March 2003

Background/aims: Optical coherence tomography (OCT) is a non‐invasive technique for morphological investigation of tissue. Since its development in the late 1980s it is mainly used as a diagnostic tool in ophthalmology. For examination of a highly scattering tissue like the skin, it was necessary to modify the method. Early studies on the value of OCT for skin diagnosis gave promising results.

Methods: The OCT technique is based on the principle of Michelson interferometry. The light sources used for OCT are low coherent superluminescent diodes operating at a wavelength of about 1300 nm. OCT provides two‐dimensional images with a scan length of a few millimeters (mm), a resolution of about 15 μm and a maximum detection depth of 1.5 mm. The image acquisition can be performed nearly in real time. The measurement is non‐invasive and with no side effects.

Results: The in vivo OCT images of human skin show a strong scattering from tissue with a few layers and some optical inhomogeneities. The resolution enables the visualization of architectural changes, but not of single cells. In palmoplantar skin, the thick stratum corneum is visible as a low‐scattering superficial well defined layer with spiral sweat gland ducts inside. The epidermis can be distinguished from the dermis. Adnexal structures and blood vessels are low‐scattering regions in the upper dermis. Skin tumors show a homogenous signal distribution. In some cases, tumor borders to healthy skin are detectable. Inflammatory skin diseases lead to changes of the OCT image, such as thickening of the epidermis and reduction of the light attenuation in the dermis. A quantification of treatment effects, such as swelling of the horny layer due to application of a moisturizer, is possible. Repeated measurements allow a monitoring of the changes over time.

Conclusion: OCT is a promising new bioengineering method for investigation of skin morphology. In some cases it may be useful for diagnosis of skin diseases. Because of its non‐invasive character, the technique allows monitoring of inflammatory diseases over time. An objective quantification of the efficacy and tolerance of topical treatment is also possible. Due to the high resolution and simple application, OCT is an interesting addition to other morphological techniques in dermatology.
The Visi‐Chroma VC‐100®: a new imaging colorimeter for dermatocosmetic research
Authors: Barel André O. , Clarys Peter , Alewaeters Katrien , Duez Christian , Hubinon Jean‐Louis , Mommaerts Marc
Published: March 2003

Background/aims: It was the aim of this study to carry out a comparative evaluation in vitro on standardized color charts and in vivo on healthy subjects using the Visi‐Chroma VC‐100®, a new imaging tristimulus colorimeter and the Minolta Chromameter CR‐200® as a reference instrument. The Visi‐Chroma combines tristimulus color analysis with full color visualization of the skin area measured. The technical performances of both instruments were compared with the purpose of validating the use of this new imaging colorimeter in dermatocosmetic research.

Methods: In vitro L*a*b* color parameters were taken with both instruments on standardized color charts (Macbeth and RAL charts) in order to evaluate accuracy, sensitivity range and repeatability. These measurements were completed by in vivo studies on different sites of human skin and studies of color changes induced by topical chemical agents on forearm skin. The accuracy, sensitivity range and repeatability of measurements of selected distances and surfaces in the measuring zone considered and specific color determinations of specific skin zones were also determined.

Results: The technical performance of this imaging colorimeter was rather good, with low coefficients of variation for repeatability of in vitro and vivo color measurements. High positive correlations were established in vitro and in vivo over a wide range of color measurements. The imaging colorimeter was able to measure the L*a*b* color parameters of specific chosen parts of the skin area considered and to measure accurately selected distances and surfaces in the same skin site considered.

Conclusion: These comparative measurements show that both instruments have very similar technical performances and that high levels of correlation were obtained in vitro and in vivo using the L*a*b* color parameters. In addition, the Visi‐Chroma presents the following improvements: 1) direct visualization and recording of the skin area considered with concomitant color measurements; 2) determination of the specific color parameters of skin areas chosen in the total measuring area; and 3) accurate determination of selected distances and surfaces in the same skin areas chosen.
Contrast enhanced phototrichogram pinpoints scalp hair changes in androgen sensitive areas of male androgenetic alopecia
Authors: Leroy Thérèse , Van Neste Dominique
Published: March 2003

Background/aim

In male androgenetic alopecia (AGA), global changes of scalp hair observed on many years are the cumulative result of discrete changes. Such changes reflect structural and/or functional modifications occurring at the level of individual hair follicles. The patterning of scalp hair loss is the phenotypic expression of clusters of hormone sensitive follicles located in specific scalp areas.The aim of this study was to evaluate, in 21 untreated male subjects with AGA, the relation between various hair measurements using a new validated photographic method with clinical staging (modified Norwood‐Hamilton scale) as compared with five controls.

Methods

As recently demonstrated by comparison with transverse sectioning of scalp biopsies, dynamic changes occurring at the level of individual hair follicles can be accurately explored with the contrast enhanced phototrichogram technique (CE‐PTG). This is a further improvement of the PTG (combined analysis of two photographs taken at 48 h interval) using contrast enhancement together with the scalp immersion proxigraphy method. Visible hair counts per unit area were first evaluated on photographs without and with CE. Then other scalp hair variables (anagen hair counts and proportion of thin hair (≤ 40 µm thickness)) were established on CE‐PTGs. Thickness of scalp hair sampled during the preparation of these sites was also calibrated against an objective ruler with a light microscope.

Results

We confirm that the CE improves hair detection. Controls showed higher densities in the top of the head sites as compared with the occipital ones. In the presumably less hormone responsive scalp occipital sites, AGA subjects did not differ from controls. The top of the head sites, i.e. highly androgen sensitive areas of AGA subjects showed a reduction of total and anagen hair counts as compared to controls. These changes were observed even in mildly affected subjects. The clinical severity of AGA seems to be related to the reduction of anagen counts and the associated relative increase of thin hair. Because the thinnest hair usually lacks pigmentation, they escape detection by PTG in the absence of contrast enhancement. The CE‐PTG also detects higher proportions of thin hair at early stages of AGA as compared with light microscopy. This probably reflects lack of control during the sampling process, i.e. the clipping– collection‐display sequence required for light microscopy observations.

Conclusion

These results indicate that CE‐PTG is a powerful tool for analysis of hair growth and loss and may cast some doubt on results obtained with other methods. The potential use of the CE‐PTG method for calibration of innovative tools is also alluded to. Indeed encouraging results have been obtained with the scalp coverage scoring (SCS) method. These SCS scores were correlated with shortening of anagen phase and hair miniaturisation, the two processes apparently involved in the clinical development of male AGA.
The gold standard for photographing pigmented skin lesions for diagnostic purposes: contact versus distant imaging
Authors: Carli Paolo , De Giorgi Vincenzo , Salvini Camilla , Mannone Francesca , Chiarugi Alessandra
Published: March 2003

Background: It is mandatory that a new diagnostic method be validated by comparison with a well‐established reference procedure before being introduced for use in clinical practice. In the field of pigmented skin lesions (PSL), clinical examination should be considered the reference procedure for new diagnostic methods, such as dermoscopy. However, it has not yet been established which is the best photographic procedure for obtaining the most informative clinical images to be used in a formal diagnostic setting.

Methods: In this study we investigated the diagnostic information provided by the two most popular methods currently used for clinical photographing of PSL: ‘contact’ images obtained with a Dermaphot (Heine Ltd) at original × 10 magnification without oil application and ‘distant’ images obtained with a macro objective from a distance of 10 cm.

Results: Two experienced dermatologists observed clinical images of a series of 57 PSL (11 melanomas, 31 melanocytic nevi, 10 pigmented basal cell carcinomas, and four other diagnoses). The degree of concordance between the diagnoses based on ‘contact’ and ‘distant’ images (melanoma/non‐melanoma) was very good (k = 0.819). Regarding histology, the degree of concordance was better when the diagnosis was based on ‘contact’ images (k = 0.54) than ‘distant’ images (k = 0.47). In particular, ‘contact’ images were superior to ‘distant’ images for diagnosis of non‐melanoma lesions (specificity of melanoma diagnosis 87.7% vs. 83.6%), but we found no difference in melanoma detection (73% of sensitivity for both methods).

Conclusions: Although the two photographic procedures appear to provide similar levels of diagnostic information, the ‘contact’ method seems to provide a higher specificity for melanoma diagnosis.
Radiation‐induced skin fibrosis after treatment of breast cancer: profilometric analysis
Authors: Bourgeois J. F. , Gourgou S. , Kramar A. , Lagarde J. M. , Gall Y. , Guillot B.
Published: March 2003

Background/aim: Treatment of breast cancer involves an association of conservative surgery and radiotherapy. This implies various cutaneous complications, well known for their clinical and histological aspects. Little data are available concerning modifications of the cutaneous microrelief after radiotherapy. We have done a profilometric analysis of the skin of breast cancer patients treated with surgery and radiotherapy. The results obtained on the irradiated breast have been compared with the ones of the controlateral normal breast.

Methods: Twenty women, 43–55 year old were enrolled in the study 6–16 months after the end of a treatment associating conservative surgery and radiotherapy for breast cancer. Imprints using a silicone rubber material were performed over symmetrical areas of the treated breast and the controlateral one. All measures were performed over the upper‐medial quadrant of each breast. The imprints are then blindly analysed using an image analyser. The following parameters were measured: RA (average roughness of the skin), RZ (average of the furrows depth), RS (residual length), Rn (number of furrows) and AR (space between the furrows).

Results: The comparison of the imprints shows important modifications of the microrelief after irradiation. We observe a slight increase of the skin roughness (RA: 26, 39 ± 2, 58 versus 21, 84 ± 1, 59), a significantly increase of the furrows depth (RZ: 121, 66 ± 10, 46 versus 101, 26 ± 6, 99) along with an increase of the residual length (RS: 580, 60 ± 60, 60 versus 450, 46 ± 48, 43). The number of furrows has not significantly decreased but the space between the furrows has increased on the irradiated breast.

Conclusion: This study shows a breakdown effect of irradiation on the skin in correlation with the fibrosis inducing by ionising radiation. The imprints modifications are clearly different from those usually observed in the ageing process. Our results can be a basis for studying the effects of treatments on cutaneous complications linked to the radiation‐induced fibrosis.
Redirection of Host Vesicle Trafficking Pathways by Intracellular Parasites
Authors: Hackstadt Ted
Published: March 2003

Bacterial and protozooan intracellular parasites have evolved diverse mechanisms for evasion of host cellular defenses associated with adaptations for survival in distinct intracellular compartments. As the reagents identifying discrete steps in vesicle maturation and trafficking have become increasingly available, it has become clear that the vacuoles occupied by intracellular parasites are much more diverse than had been previously appreciated. Many parasites induce selective fusion competence with the vacuoles they occupy, without affecting vesicular trafficking elsewhere in the cell. A likely means of controlling vesicular interactions is modification of the parasitophorous vacuole membrane by the insertion of parasite‐specific proteins. A rapidly expanding class of bacterial proteins that modify the vacuolar membrane are the chlamydial inclusion membrane proteins. Although the functions of most of these proteins remain to be defined, the majority are expressed early in the infectious process, suggesting that modification of the vacuole is critical to the outcome of the host–parasite interaction.
Poisons, Ruffles and Rockets: Bacterial Pathogens and the Host Cell Cytoskeleton
Authors: Steele‐Mortimer Olivia , Knodler Leigh A. , Finlay B. Brett
Published: March 2003

The cytoskeleton of eukaryotic cells is affected by a number of bacterial and viral pathogens. In this review we consider three recurring themes of cytoskeletal involvement in bacterial pathogenesis: 1) the effect of bacterial toxins on actin‐regulating small GTP‐binding proteins; 2) the invasion of non‐phagocytic cells by the bacterial induction of ruffles at the plasma membrane; 3) the formation of actin tails and pedestals by intracellular and extracellular bacteria, respectively. Considerable progress has been made recently in the characterization of these processes. It is becoming clear that bacterial pathogens have developed a variety of sophisticated mechanisms for utilizing the complex cytoskeletal system of host cells. These bacterially‐induced processes are now providing unique insights into the regulation of fundamental eukaryotic mechanisms.
Definition of Distinct Compartments in Polarized Madin–Darby Canine Kidney (MDCK) Cells for Membrane‐Volume Sorting, Polarized Sorting and Apical Recycling
Authors: Brown Paul S. , Wang Exing , Aroeti Benjamin , Chapin Steven J. , Mostov Keith E. , Dunn Kenneth W.
Published: March 2003

Previous studies of fibroblasts have demonstrated that recycling of endocytic receptors occurs through a default mechanism of membrane‐volume sorting. Epithelial cells require an additional level of polar membrane sorting, but there are conflicting models of polar sorting, some suggesting that it occurs in early endosomes, others suggesting it occurs in a specialized apical recycling endosome (ARE). The relationship between endocytic sorting to the lysosomal, recycling and transcytotic pathways in polarized cells was addressed by characterizing the endocytic itineraries of LDL, transferrin (Tf) and IgA, respectively, in polarized Madin–Darby canine kidney (MDCK) cells. Quantitative analyses of 3‐dimensional images of living and fixed polarized cells demonstrate that endocytic sorting occurs sequentially. Initially internalized into lateral sorting endosomes, Tf and IgA are jointly sorted from LDL into apical and medial recycling endosomes, in a manner consistent with default sorting of membrane from volume. While Tf is recycled to the basolateral membrane from recycling endosomes, IgA is sorted to the ARE prior to apical delivery. Quantifications of the efficiency of sorting of IgA from Tf between the recycling endosomes and the ARE match biochemical measurements of transepithelial protein transport, indicating that all polar sorting occurs in this step. Unlike fibroblasts, rab11 is not associated with Tf recycling compartments in either polarized or glass‐grown MDCK cells, rather it is associated with the compartments to which IgA is directed after sorting from Tf. These results complicate a suggested homology between the ARE and the fibroblast perinuclear recycling compartment and provide a framework that justifies previous conflicting models of polarized sorting.
Coatomer Vesicles are not Required for Inhibition of Golgi Transport by G‐Protein Activators
Authors: Happe Scott , Cairns Matthew , Roth Robyn , Heuser John , Weidman Peggy
Published: March 2003

The G‐protein activators guanosine 5′‐O‐(3‐thiodiphosphate) (GTPΓS) and aluminum fluoride (AlF) are thought to inhibit transport between Golgi cisternae by causing the accumulation of nonfunctional coatomer‐coated transport vesicles on the Golgi. Although GTPΓS and AlF inhibit transport in cell‐free intra‐Golgi transport systems, blocking coatomer vesicle formation does not. We therefore determined whether inhibition of in vitro Golgi transport by these agents requires coatomer vesicle formation. Depletion of coatomer was found to completely block coated vesicle formation on Golgi cisternae without affecting inhibition of in vitro transport by either GTPΓS or AlF. Depletion of ADP‐ribosylation factor (ARF) prevented inhibition of transport by GTPΓS, but not by AlF, suggesting that the AlF‐sensitive component in transport may not be a GTP‐binding protein. Surprisingly, depletion of cytosolic ARF did not prevent the GTPΓS‐induced formation of Golgi‐coated vesicles, whereas ARF was required for AlF‐induced vesicle formation. Although ARF or coatomer depletion caused an increase in the fenestration of cisternae, no other utrastructural changes were observed that might explain the inhibition of transport by GTPΓS or AlF. These findings suggest that ARF‐GTPΓS and AlF act by distinct and coatomer‐independent mechanisms to inhibit membrane fusion in cell‐free intra‐Golgi transport.
The Dileucine Motif Within the Tail of MPR46 is Required for Sorting of the Receptor in Endosomes
Authors: Tikkanen Ritva , Obermüller Stefanie , Denzer Kristin , Pungitore Rodolfo , Geuze Hans J. , Von Figura Kurt , Höning Stefan
Published: March 2003

The cytoplasmic tail of MPR46 carries a leucine‐based motif that is required for the sorting of lysosomal enzymes by the receptor. In addition, it is one of three independent, but functionally redundant, internalization signals present in the cytoplasmic tail of MPR46. We have analyzed a mutant of MPR46, in which the dileucine pair was replaced by alanines (MPR46 LL/AA) with respect to its intracellular distribution and trafficking. Ultrastructural analysis of cells expressing the MPR46 LL/AA mutant revealed that the substitution of the dileucine pair causes a shift of the receptor distribution from the TGN, where it is packaged into AP1‐containing vesicles, to vesicular structures distributed throughout the cytoplasm. The vesicles could be identified as early endosomes with internalized BSA‐gold and rab5 as markers. By analyzing the receptor trafficking biochemically, we found that return of the LL/AA mutant receptor from the plasma membrane/endosome pool back to the TGN was impaired, while recycling from endosomes to the plasma membrane was enhanced. In conclusion, our data indicate that the dileucine motif in the MPR46 tail is required for a sorting event in endosomes.
Targeting of HIV‐1 Nef to the Centrosome: Implications for Antigen Processing
Authors: Lacaille Vashti G. , Androlewicz Matthew J.
Published: March 2003

To gain a better understanding of the intracellular sites of antigen processing we have looked at the localization of human immunodeficiency virus (HIV)‐1 Nef protein by confocal microscopic and biochemical means. We found that ubiquitin (Ub)‐Nef fusion proteins were localized to the centrosome in transfected COS‐7 cells, and that the colocalization was inhibited by the microtubule‐disrupting agent, nocodazole. Interestingly, we found that Ub‐Nef trafficking to the centrosome was not dependent upon the metabolic stability of Ub‐Nef nor on the inhibition of proteasome activity. We also analyzed the MHC class I antigen processing of a reporter epitope linked to the Ub‐Nef fusion proteins and found that Ub‐Nef was processed in COS‐7 cells. In addition, we show that this processing was inhibited by nocodazole. We suggest that the centrosome may serve as a site of antigen processing in vivo.
Vacuolar Sorting Determinants Within a Plant Storage Protein Trimer Act Cumulatively
Authors: Holkeri Heidi , Vitale Alessandro
Published: March 2003

The mechanism for vacuolar sorting of seed storage proteins is as yet poorly understood and no receptor has been identified to date. The homotrimeric glycoprotein phaseolin, which is the major storage protein of the common bean, requires a transient tetrapeptide at the C‐terminus for its vacuolar sorting. A mutated construct without the tetrapeptide is secreted. We show here that coexpression of wild‐type phaseolin and the mutated, secreted form in transgenic tobacco results in the formation of mixed trimers and partial vacuolar delivery of the mutated polypeptides and partial secretion of wild‐type polypeptides. This indicates that the sorting signal has a cumulative effect within a phaseolin trimer. The result is discussed in the light of the hypothesized mechanisms for vacuolar sorting of seed storage proteins.
The Biogenesis and Functions of Exosomes
Authors: Stoorvogel Willem , Kleijmeer Monique J. , Geuze Hans J. , Raposo Graça
Published: March 2003

Exosomes are membrane vesicles with a diameter of 40–100 nm that are secreted by many cell types into the extracellular milieu. They correspond to the internal vesicles of an endosomal compartment, the multivesicular body and are released upon exocytic fusion of this organelle with the plasma membrane. Intracellularly, they are formed by inward budding of the endosomal membrane in a process that sequesters particular proteins and lipids. The unique composition of exosomes may confer specific functions on them upon secretion. Although their physiological rolein vivois far from being unraveled, it is apparent that they function in a multitude of processes, including intercellular communication during the immune response. Exosomes may have evolved early in the evolution of multicellular organisms and also seem to be important for tissue developmental processes.
SNAP‐25 and Synaptotagmin 1 Function in Ca2+‐Dependent Reversible Docking of Granules to the Plasma Membrane
Authors: Chieregatti Evelina , Witkin Joan W. , Baldini Giulia
Published: March 2003

In neuroendocrine cells, Ca2+ triggers fusion of granules with the plasma membrane and functions at earlier steps by increasing the size of the readily releasable pool of vesicles. The effect of Ca2+ at early steps of secretion may be due to the recruitment at the plasma membrane of granules localized in the cytoplasm. To study the mechanism of granule docking, a new in vitro assay is designed using membrane fractions from mouse pituitary AtT‐20 cells. By using this assay, it is found that granule docking to the plasma membrane is controlled by Ca2+ concentrations in the micromolar range, is reversible and requires intact SNAP‐25, but not VAMP‐2. In the docking assay, addition of Ca2+ induces the formation of a SNAP‐25‐Synaptotagmin 1 complex. The cytosolic domain C2AB of Synaptotagmin 1 and anti‐Synaptotagmin 1 antibodies block granule docking. These results show that Ca2+ modulates dynamic docking of granules to the plasma membrane and that this process is due to a Ca2+‐dependent interaction between SNAP‐25 and Synaptotagmin 1.
The Golgi Apparatus: Balancing New with Old
Authors: Storrie Brian , Nilsson Tommy
Published: March 2003

Most models put forward to explain cellular processes do not stand the test of time. The ‘lucky’ few that are able to survive extensive experimental tests and peer critique may eventually become dogmas or paradigms. When this happens, the amount of experimental data required to overturn the paradigm is extensive. To some, such inertia may seem prohibitive to scientific progress but rather, in our opinion, this helps to maintain a degree of coherence. It is needed so that experiments and interpretations may be conducted within relatively safe boundaries. In the field of protein transport in the secretory pathway, we have enjoyed a relatively stable and productive period for quite some time (more than 30 years!). It is only very recently that the field has entered into a phase where all bets seem to be off. As in any paradigm shift, the accumulation of experimental observations inconsistent with the old dogma eventually reached a critical point. As we ‘reluctantly’ dispense with the long‐standing paradigm of forward vesicular transport, we face a time that is bound to be trying as well as exciting.
Pallidin is a Component of a Multi‐Protein Complex Involved in the Biogenesis of Lysosome‐related Organelles
Authors: Moriyama Kengo , Bonifacino Juan S.
Published: March 2003

The Hermansky–Pudlak syndrome defines a group of genetic disorders characterized by defective lysosome‐related organelles such as melanosomes and platelet dense bodies. Hermansky–Pudlak syndrome can be caused by mutations of at least four genes in humans and 15 genes in mice. One of these genes is mutated in the pallid mouse strain and encodes a novel protein named pallidin (L. Huang, Y. M. Kuo and J. Gitschier, Nat Genet 1999; 23: 329–332). Pallidin has no homology to any other known protein and no recognizable functional motifs. We have conducted a biochemical characterization of human pallidin using a newly developed polyclonal antibody. We show that pallidin is a ubiquitously expressed ∼ 25 kDa protein found both in the cytosol and peripherally associated to membranes. Sedimentation velocity analyses show that native pallidin has a sedimentation coefficient of ∼ 5.1 S, much larger than expected from the molecular mass of the pallidin polypeptide. In line with this observation, cosedimentation and coprecipitation analyses reveal that pallidin is part of a hetero‐oligomeric complex. One of the subunits of this complex is the product of another Hermansky–Pudlak syndrome gene, muted. Fibroblasts derived from the muted mouse strain exhibit reduced levels of pallidin, suggesting that the absence of the muted protein destabilizes pallidin. These observations indicate that pallidin is a subunit of a novel multi‐protein complex involved in the biogenesis of lysosome‐related organelles.
MHC Class II Compartments in Human Dendritic Cells Undergo Profound Structural Changes Upon Activation
Authors: Barois Nicolas , De Saint‐Vis Blandine , Lebecque Serge , Geuze Hans J. , Kleijmeer Monique J.
Published: March 2003

Immature dendritic cells efficiently capture exogenous antigens in peripheral tissues. In an inflammatory environment, dendritic cells are activated and become highly competent antigen‐presenting cells. Upon activation, they lose their ability for efficient endocytosis and gain capability to migrate to secondary lymphoid organs. In addition, peptide loading of MHC class II molecules is enhanced and MHC class II/peptide complexes are redistributed from an intracellular location to the plasma membrane. Using immuno‐electron microscopy, we show that activation of human monocyte‐derived dendritic cells induced striking modifications of the lysosomal multilaminar MHC class II compartments (MIICs), whereby electron‐dense tubules and vesicles emerged from these compartments. Importantly, we observed that MHC class II expression in these tubules/vesicles transiently increased, while multilaminar MIICs showed a strongly reduced labeling of MHC class II molecules. This suggests that formation of the tubules/vesicles from multilaminar MIICs could be linked to transport of MHC class II from these compartments to the cell surface. Further characterization of endocytic organelles with lysosomal marker proteins, such as the novel dendritic cell‐specific lysosomal protein DC‐LAMP, HLA‐DM and CD68, revealed differential sorting of these markers to the tubules and vesicles.
Solitary fibrous tumour of the mouth: report of two cases involving the tongue and cheek
Authors: Vargas PA , Alves FA , Lopes MA , Siqueira SAC , Menezes LFC , Aldred VL , Almeida OP
Published: March 2003

We describe two additional cases of solitary fibrous tumour (SFT) affecting the mouth. SFT is very uncommon in the oral cavity and we found only 14 cases reported in the literature. Our two cases were well circumscribed, slow growing tumours that, after surgical removal, did not recur. Case 1 was a 3 cm nodule on the right cheek. Hypo and hypercellular adjacent areas were mainly patternless, and the stroma was formed by thin collagen fibrils. Case 2 was a 4.8 cm mass in the anterior portion of the tongue. Microscopically it was formed by spindle cells embedded in a vascularized sclerotic collagen matrix. Some areas were hypercellular with scarce collagen fibrils. The immunohistochemical findings were similar in both cases, with strong immunoreactivity for vimentin, CD34, bcl‐2, focal positivity for Ki‐67 and negativity for other immunomarkers. Based on these clinical, microscopical and immunohistochemical features the final diagnosis of these two cases was SFT. Diagnosis of SFT is difficult and, although uncommon, it should be considered in the differential diagnosis of oral soft tissue tumours.
Immunohistochemical study on expression of α‐defensin and β‐defensin‐2 in human buccal epithelia with candidiasis
Authors: Sawaki K , Mizukawa N , Yamaai T , Fukunaga J , Sugahara T
Published: March 2003

OBJECTIVES AND DESIGN: It has been previously reported that α‐defensin (HNPs) and β‐defensin‐2 (HBD‐2) peptides with antifungal and cytotoxic activities can be detected in oral carcinomas and the saliva of patients with oral carcinomas. The present study investigated the presence of HNPs and HBD‐2 in oral epithelia with candidiasis.

MATERIALS AND METHODS: Tissue sections (4 μm) were prepared from biopsy and surgically removed specimens diagnosed as oral candidiasis (n=10). The sections were examined immunohistochemically with antibodies directed against HNPs and HBD‐2.

RESULTS: Tissue sections of oral candidiasis were immunostained with antidefensin antibodies. Neutrophils in the inflamed lamina propria were positively immunostained with anti‐HNPs antibody. The cytoplasm of cells in the upper spinous layer, in the lower spinous layer and in the parakeratinized layer of buccal epithelia with candidiasis was immunostained intensely with anti‐HBD‐2 antibody. In contrast, the expression of HBD‐2 in the normal spinous layer was much weaker than that in oral candidiasis. No signals of HNPs were found in normal buccal epithelium.

CONCLUSION: Buccal specimens from individuals with oral candidiasis show greater levels of expression of both HNPs and HBD‐2. There might be a dual protection manner by defensins against fungal inflammation in infected buccal epithelia locally. Generally, HBD‐2 signals have been found everywhere in the buccal epithelium; however, in an infected area, the signal intensity of HBD‐2 has increased. HNPs signals have not been found in the normal buccal epithelium; however, HNPs signals have increased when the infection occurred.
Cytokeratins in epithelia of odontogenic neoplasms
Authors: Crivelini MM , De Araújo VC , De Sousa SOM , De Araújo NS
Published: March 2003

Neoplasms and tumours related to the odontogenic apparatus may be composed only of epithelial tissue or epithelial tissue associated with odontogenic ectomesenchyme. The immunohistochemical detection of different cytokeratins (CKs) polypeptides and vimentin has made it easier to explain the histogenesis of many epithelial diseases. The present study aimed to describe the immunohistochemical expression of cytokeratins 7, 8, 10, 13, 14, 18, 19 and vimentin in the epithelial components of the dental germ and of five types of odontogenic tumours. The results were compared and histogenesis discussed.

All cells of the dental germ were positive for CK14, except for the preameloblasts and secreting ameloblasts, in which CK14 was gradually replaced by CK19. CK7 was especially expressed in the cells of the Hertwig root sheath and the stellate reticulum. The dental lamina was the only structure to express CK13. The reduced epithelium of the enamel organ contained CK14 and occasionally CK13. Cells similar to the stellate reticulum, present in the ameloblastoma and in the ameloblastic fibroma, were positive for CK13, which indicates a nature other than that of the stellate reticulum of the normal dental germ. The expression of CK14 and the ultrastructural aspects of the adenomatoid odontogenic tumour probably indicated its origin in the reduced dental epithelium. Calcifying odontogenic epithelial tumour is thought to be composed of primordial cells due to the expression of vimentin. Odontomas exhibited an immunohistochemical profile similar to that of the dental germ. In conclusion, the typical IF of odontogenic epithelium was CK14, while CK8, 10 and 18 were absent. Cytokeratins 13 and 19 labelled squamous differentiation or epithelial cells near the surface epithelium, and CK7 had variable expression.