bluuebunny/crossref_metadata_2025 huggingface dataset:
Ability of closed and open kinetic chain tests of muscular strength to assess functional performance
Authors: Augustsson J. , Thomeé R.
Published: March 2003

The purpose of this study was to investigate the ability of closed and open kinetic chain tests of muscular strength to assess functional performance. Sixteen healthy male subjects, with a mean (±SD) age, body mass and height of 27±5 years, 78±9 kg and 183±9 cm, respectively, volunteered to participate in the study. In the closed kinetic chain test (involving muscles working across multiple joints), the subjects performed a 3 repetition maximum (3 RM) barbell squat. The open kinetic chain test (involving muscles working across a single joint) consisted of a concentric isokinetic knee extension at an angular velocity of 60°/s, and was performed using a Kinetic Communicator II dynamometer. The test of functional performance (vertical jump) was performed with the subject standing erect, quickly performing a countermovement jump for maximal height. Moderately strong significant (P<0.05) correlations between the test of functional performance and the closed and open kinetic chain tests of muscular strength were noted, r=0.51 and r=0.57, respectively. It is suggested that the effect of training or rehabilitation interventions should not be based exclusively on tests of muscular strength. Rather, various forms of dynamometry including functional performance tests could be recommended.
In vivo studies of peritendinous tissue in exercise
Authors: Kjær M. , Langberg H. , Skovgaard D. , Olesen J. , Bülow J. , Krogsgaard M. , Boushel R.
Published: March 2003

Soft tissue injury of tendons represents a major problem within sports medicine. Although several animal and cell culture studies have addressed this, human experiments have been limited in their ability to follow changes in specific tissue directly in response to interventions. Recently, methods have allowed for in vivo determination of tissue concentrations and release rates of substances involved in metabolism, inflammation and collagen synthesis, together with the measurement of tissue blood flow and oxygenation in the peritendinous region around the Achilles tendon in humans during exercise. It can be demonstrated that this region experiences an increase in blood flow during both static and dynamic exercise, and that exercise causes increased metabolic activity, accumulation of inflammatory mediators (prostaglandins) and increased formation of collagen type I in response to acute exercise. This coincides with a surprisingly marked drop in tissue pressure during contraction. With regards to both circulation, metabolism and collagen formation, peritendinous tissue represents a dynamic, responsive region that adapts markedly to acute muscular activity.
Use of positron emission tomography in the assessment of skeletal muscle and tendon metabolism and perfusion
Authors: Nuutila P. , Kalliokoski K.
Published: March 2003

Positron emission tomography (PET) represents the most advanced scintigraphic imaging technology in humans, which allows non‐invasive studies of metabolism and perfusion in vivo. PET has been employed for research of skeletal muscle physiology and pathophysiology as well as for studies of effects of training and exercise. PET is a functional research method, being a powerful tool, especially in studies of skeletal muscle perfusion in humans. It also provides a potential tool for studies of tendon physiology. In this review examples of recent studies of skeletal muscle in athletes and during exercise are given and potential applications of PET in the field of tendon studies discussed.
Outcome after anterior cruciate ligament reconstruction – a comparison of patients’ and surgeons’ assessments
Authors: Roos E. M.
Published: March 2003

Visual analog scale ratings (0–100 mm) by patients (n=38, 26 men, mean age 28 years) and surgeons (n=2) of expectations, satisfaction, current knee function and activity level were compared one year after reconstruction of the anterior cruciate ligament. On average, the surgeons rated the outcome with regard to knee function and activity level as significantly better than the patients (P<0.008). The mean differences ranged from 10 to 19 mm. The discrepancies increased as the patients’ ratings got worse. No such differences were seen for expectations or satisfaction. In order to avoid bias, the patient should assess patient‐relevant outcomes, such as function and activity level. When comparing and interpreting the results of clinical studies, it should be taken into account that the use of observer‐administered questionnaires might introduce a positive bias yielding a significantly better outcome.
Exercise capacity in lean versus obese women
Authors: Hulens M. , Vansant G. , Lysens R. , Claessens A. L. , Muls E.
Published: March 2003

The aim of this study was to assess the nature and magnitude of the differences in submaximal and maximal exercise capacity parameters between lean and obese women. A total of 225 healthy obese women 18–65 years (BMI> or=30 kg/m2) and 81 non‐athletic lean women (BMI< or=26 kg/m2) were selected. Anthropometric measurements (weight and height), body composition assessment (bioelectrical impedance method) and a maximal exercise capacity test on a bicycle ergometer were performed. Oxygen uptake (VO2), carbon dioxide production (VCO2), expired ventilation (VE), respiratory quotient (RQ), breathing efficiency (VE/VO2), mechanical efficiency (ME) and anaerobic threshold (AT) were calculated. At a submaximal intensity load of 70 W, VO2 (l/min) was larger in the obese women and was already 78% of their peak VO2, whereas in the non‐obese it was only 69% (P=0.0001). VE (l/min) was larger, VE/VO2 did not differ and ME was lower in obese compared to the lean women. AT occurred at the same percentage of peak VO2 in both lean and obese women. At peak effort, achieved load, terminal VO2 (l min−1 kg−1), VE, heart rate, RQ respiratory exchange ratio and perceived exertion were lower in obese subjects compared to the non‐obese. Obese subjects mentioned significantly more musculoskeletal pain as a reason to end the test, whereas in lean subjects it was leg fatigue. Lean women recovered better as after 2 min they were already at 35% of the peak VO2, whereas in the obese women it was 47% (P=0.0001). Our results confirm that exercise capacity is decreased in obesity, both at submaximal and peak intensity, and during recovery. Moreover, at peak effort musculoskeletal pain was an important reason to end the test and not true leg fatigue. These findings are important when designing exercise programs for obese subjects.
Plantar pressure and EMG activity of simulated and actual ski jumping take‐off
Authors: Virmavirta M. , Komi P. V.
Published: March 2003

Plantar pressures and activation of the four muscles (VL – vastus lateralis, GL – gluteus, TA – tibialis anterior and GA – lat. gastrocnemius) were measured from ten ski jumpers under simulated laboratory conditions with training shoes (Lab TS) and with jumping boots (Lab JB) as well as in actual hill jumping conditions (Hill). The most significant differences between measured conditions were found in muscle activation patterns and plantar pressures prior to take‐off. The centrifugal force due to the curvature of the inrun under actual hill jumping conditions caused extra pressure under the fore and rear parts of the feet (P<0.001) and therefore higher activation in all muscles (P<0.001 for VL, TA and GA and P<0.01 for GL). The actual take‐off was characterized by high pressure under the toes during the early phase and high pressure under the heel during the latter phase of take‐off. However, this should probably he interpreted as a commonly used improper technique where the balance during take‐off is not well maintained to allow effective force production. Activation of GA, especially with regard to anteroposterior pressure distribution under the feet, differed significantly between the measured conditions. The role of GA for explosive force production during ski jumping take‐off is much smaller as compared to that of the knee and hip extensor muscles.
Inversion injury biomechanics in functional ankle instability: a cadaver study of simulated gait
Authors: Konradsen Lars , Voigt Michael
Published: March 2003

The purpose of this study was to test pathogenetic models for the “unprovoked” ankle inversion injuries seen in functional ankle unstable subjects. The consequence of spatial mal‐alignment of the ankle/foot complex on the risk of producing an ankle inversion torque at heel‐strike and during swing‐phase follow through was analyzed in cadaver simulations. Heel‐strike was simulated using a 5° of freedom rig in a material testing machine. A set‐up capable of accelerating lower limb specimens towards a support surface simulated swing‐phase follow through. Joint excursions were monitored with flexible wire goniometers. The unloaded ankle/foot complex was placed in increasing positions of talar and subtalar joint excursions. The consequences of these settings on the behavior of the ankle/foot complex at heel‐strike and when the lateral part of the foot “caught” the ground during swing‐phase follow through were monitored. An inversion torque at heel‐strike was first seen when the unloaded foot was set in positions exceeding 30° of inversion combined with full plantar flexion and 10° of internal tibial rotation. A collision between the lateral border of a 20° inverted, but otherwise neutral ankle/foot complex and the ground surface during swing‐phase follow through forced the foot into the full limit of inversion, plantar flexion and internal tibial rotation measurable in this set‐up.

Clinical consequence: The study showed that the foot/ankle complex exhibits a high degree of intrinsic stability at heel‐strike. The foot will thus stabilize itself and move into normal eversion at the beginning of the stance‐phase even though it is set to the ground in a substantial degree of mal‐alignment. In contrast, the swing‐phase collision model provides a link that can connect the small deficits in inversion angle awareness measured in chronic functional ankle unstable subjects with an increased risk in this group of sustaining ankle inversion injuries.
Patellofemoral pain syndrome: pain, coping strategies and degree of well‐being
Authors: Thomeé P. , Thomeé R. , Karlsson J.
Published: March 2003

The purpose of this study was to evaluate how patients with patellofemoral pain syndrome (PFPS) experience their pain, what coping strategies they use for the pain, and their degree of well‐being. Fifty patients, 15–52 years old, with PFPS were evaluated with multidimensional pain inventory (MPI), coping strategies questionnaire (CSQ) and Spielberger state trait anxiety inventory (STAI). Reliability of the evaluation methods was established for 12 patients.

Good reliability was established for all instruments except for six of the 13 scales of the MPI. Results on MPI, CSQ and STAI are in agreement with the literature on other patient groups with chronic pain. The most frequently used strategies were ‘coping self statements’ and ‘ignoring sensations’. High scores were found for the strategy ‘catastrophizing’ compared with other patient groups having chronic pain. The STAI scores were in general found to be somewhat higher than the scores found in the literature on healthy subjects.

It is concluded that the way patients with PFPS experience their pain, the coping strategies for pain they use and their degree of well‐being, are in agreement with other patient groups who have chronic pain. Some concern is raised in terms of the high scores reported for the coping strategy ‘catastrophizing’.
Dynamic balance in former elite male athletes and in community control subjects
Authors: Räty H. P. , Impivaara O. , Karppi S.‐L.
Published: March 2003

Dynamic balance was studied in 105 former elite athletes (aged 45–68) and in 966 community control subjects (aged 27–67). Subjects walked barefoot along a 5‐m‐long test track, stepping only on two pads which they alternately put down in front of them and picked up from behind while standing on one foot. A better test result (short completion time) was associated with lower body mass index (in both groups), with jumping height and physical activity during the previous year (in former athletes), and with younger age, better general health, and better perceived physical fitness (in control subjects). Test results in former athletes were, on average, comparable to those of 24–30 years younger community control subjects.
The influence of genetic factors on physical functioning and exercise in second half of life
Authors: Frederiksen H. , Christensen K.
Published: March 2003

During the past decades, a number of studies of families and twins in particular have assessed the relative contribution of genetic and environmental factors to traits reflecting various aspects of physical functioning: maximal O2 uptake, muscular endurance, muscular strength, muscle cross sectional area, flexibility, and trainability. Although the estimate of the size of the genetic component differs between the various studies, they point towards a moderate to substantial genetic influence on these phenotypes. Most of the studies have used only young and healthy study subjects, although in recent years phenotypes of particular importance to the elderly and the oldest‐old (e.g., activities‐of‐daily living abilities) have also been shown to have substantial genetic component. Furthermore, behavioural studies have also revealed a genetic contribution to the disposition to level of leisure time physical activity. At present, there is still a few association studies on specific genetic variants, and the results have either been inconsistent or failed to show an association with physical functioning. Therefore, the mechanisms through which the genetic influence is expressed, is still an enigma. Here, we summarise the evidence currently available for a genetic influence on physical functioning and disposition to leisure time physical activity with a focus on recent Danish twin data.
Satellite cells and training in the elderly
Authors: Thornell L.‐E , Lindström M , Renault V , Mouly V , Butler‐Browne G. S.
Published: March 2003

In the present review, we describe the effects of ageing on human muscle fibres, underlining that each human muscle is unique, meaning that the phenotype becomes specifically changed upon ageing in different muscles, and that the satellite cells are key cells in the regeneration and growth of muscle fibres. Satellite cells are closely associated with muscle fibres, located outside the muscle fibre sarcolemma but beneath the basement lamina. They are quiescent cells, which become activated by stimulation, like muscle fibre injury or increased muscle tension, start replicating and are responsible for the repair of injured muscle fibres and the growth of muscle fibres. The degree of replication is governed by the telomeric clock, which is affected upon excessive bouts of degeneration and regeneration as in muscular dystrophies. The telomeric clock, as in dystrophies, does not seem to be a limiting factor in ageing of human muscle. The number of satellite cells, although reduced in number in aged human muscles, has enough number of cell divisions left to ensure repair throughout the human life span. We propose that an active life, with sufficient general muscular activity, should be recommended to reduce the impairment of skeletal muscle function upon ageing.
A bioengineering study on the efficacy of a skin protectant lotion in preventing SLS‐induced dermatitis
Authors: Zhai Hongbo , Brachman Fred , Pelosi Alessandra , Anigbogu Angela , Ramos Maria Belinda , Torralba Maria Cecilia , Maibach Howard I.
Published: March 2003

Background/aims: This study evaluated the efficacy of a dimethicone skin protectant lotion against sodium lauryl sulfate (SLS)‐induced irritant contact dermatitis (ICD) by clinical visual grading and bioengineering techniques in 12 healthy humans.

Methods: The flexor aspects of both forearms of the subjects were used as test sites. Each test was duplicated to diminish the variations of the test sites. In a random order and a double‐blind manner, two test sites were pretreated either with the testing protectant lotion or with its vehicle control prior to contact with SLS. Thirty minutes later, 0.2 ml of 0.5% SLS in a polypropylene chamber was applied to each pretreated site. One additional test site served as a positive control (without lotion), receiving the irritant only. After 24 h of exposure to the irritant, the chambers were removed. The efficacy of protective effect was determined by four parameters: visual scoring (VS), transepidermal water loss (TEWL), skin color (a* value), and cutaneous blood flow volume (BFV). All test sites were assessed with the parameters daily for 5 days.

Results: The VS data showed a significant decrease on the site pretreated with protectant lotion in comparison with the SLS‐only treated site (P<0.01) and with the site pretreated with control vehicle (P<0.05) (overall for 5 days). TEWL value was significantly decreased in comparison with the SLS‐only treated site (P=0.02 at day 2; P=0.008 at day 4; P=0.014 at day 5) and with the site pretreated with the control vehicle (P<0.05) (day 2, 4 and 5). However, the BFV and a* values did not show a statistical difference between protectant lotion, vehicle, and SLS‐only treated sites.

Conclusions: This study demonstrated that appropriate dimethicone skin protection products may provide certain benefits from surfactant ICD. The skin protectant lotion may be used to prevent ICD in home or work environments, where skin irritants may induce dermatitis or eczema.
Tonality of suntan vs sunless tanning with dihydroxyacetone
Authors: Muizzuddin Neelam , Marenus Kenneth D. , Maes Daniel H.
Published: March 2003

Background/aims: Although there is an increasing awareness of the detrimental effects of solar irradiation on skin, a tanned look is still in fashion. To achieve the tanned look without sun exposure various sunless tanning formulations have become available. Most of these contain dihydroxyacetone (DHA) which binds to the proteins of the stratum cornium and imparts a brown color to skin. This color is similar to a suntan but can be somewhat more yellow, making it appear unnatural. The aim of this study was to determine a quantitative method to define a “natural” tan and to study methods to improve the tonality of sunless tanning on skin.

Methods: Tonality of suntan was determined as the marker for a “natural” tan. In order to achieve this, human volunteers were exposed to the sun and the change in skin color was observed after 3 days. Color measurements obtained with the Minolta Chromameter were plotted in two standard graphs labeled as the “natural universe of tan”, which depicted a balance between Chroma and change in reflectance, while the “natural universe of color” determined the balance between changes in yellow and red components of the suntan. Change in skin color after treatment with DHA was then inserted in these graphs to observe how “natural” the tonality of sunless tan appeared relative to suntan.

Results: Results show a good balance between Chroma and change in reflectance for suntanned skin in the “natural universe of tan”. Conversely, the “natural universe of color” exhibited a good balance between increase in yellow and red components of suntan. Sunless tan data inserted in these graphs showed that for several subjects with skin types I‐II the tonality is not within the realm of “natural” but is unnaturally yellow. Addition of antioxidants, especially caffeic acid phenethyl ester (CAPE) in DHA formulation significantly shifted the tonality towards the center of the “natural universe of color”.

Conclusions: The “natural universes of tan and color” exhibit a simple and quantitative assessment of tonality of suntan and sunless tan. Although skin tonality from DHA‐induced sunless tan can often lie outside the realm of the “natural” tan, it is possible to improve this tonality to a more “natural” look by addition of strong antioxidants in the DHA formulations.
In vivo model of the mechanical properties of the human skin under suction
Authors: Diridollou S. , Patat F. , Gens F. , Vaillant L. , Black D. , Lagarde J. M. , Gall Y. , Berson M.
Published: March 2003

Background/aims: A new method for the in vivo characterization of the mechanical properties of skin has been developed. This comprises a suction chamber and an ultrasound device to measure both the vertical displacement of the skin's surface, and the skin's thickness.

Methods: A mathematical model of the mechanical behaviour of a taught elastic membrane is used to obtain a set of parameters intrinsic to the skin, such as Young's modulus (E) and the initial stress (σ0), which reflect the stiffness and the natural tension of the skin, respectively. We also calculated an index of non‐elasticity of the skin (unrestored energy ratio, UER), which takes into account the volume of tissue mobilized. It determines a ratio between the energy input to the skin and the energy it dissipates. These parameters were evaluated from the volar forearm of 10 normal male volunteers.

Results: The results were: 129±88 kPa for E, 13.5±5 kPa for σ0, and 0.42±0.04 for UER; with reproducibilities of 9.5%, 12.4% and 6.4%, respectively.

Conclusions: This new suction device was found useful for the study of the behaviour of the skin, and the device may be used for the evaluation of certain skin diseases and their therapy.
Comparison of surface free energy between reconstructed human epidermis and in situ human skin
Authors: Lerebour G. , Cupferman S. , Cohen C. , Bellon‐Fontaine M. N.
Published: March 2003

Background/aims: The objectives of this study were to characterise the Episkin® model of reconstructed epidermis histologically and energetic, and to compare the results with the data obtained by Mavon (9, 10) on different areas of skin on the human body and on different skin types.

Methods: Histologically, the two skin systems studied were stained using Goldner's trichome method. Surface free energy was determined by the measurement of contact angles, using the posed drop method.

Results: Histologically, the two skin systems demonstrated the same cellular organisation in stratified layers. With regard to surface free energy, Episkin, skin from the forehead, normal and hyper‐seborrheic skins are hydrophilic surfaces, while skin from the forearm or of an oligo‐seborrheic type tends to be hydrophobic. Episkin and skin from the forehead exhibit similar surface free energy profiles. It is impossible to state whether Episkin has characteristics closer to those of normal or hyper‐seborrheic skin, but they differ significantly from those of oligo‐seborrheic skin.

Conclusion: The results obtained during this study justify the use of Episkin in a range of tests where the use of in situ human skin raises problems.
Evaluation of an experimental patch test model for the detection of irritant skin reactions to moisturisers
Authors: Agner Tove , Held Elisabeth , West Will , Gray John
Published: March 2003

Background/aims: Moisturisers are used daily by a large number of people to prevent dryness of the skin. Irritant skin reactions to moisturisers are, however, known to occur. In order to prevent such irritant reactions reliable test methods for irritancy testing of moisturisers are needed. This study was undertaken to evaluate a non‐invasive patch test model for the detection of irritant skin reactions to moisturisers.

Methods: Twenty healthy volunteers were patch tested with three different moisturisers: empty chamber, sodium lauryl sulphate and a moisturizer known to be non‐irritating. Skin reactions were evaluated by visual scoring, measurement of transepidermal water loss (TEWL) by an Evaporimeter, blood flow by laser Doppler flowmetry and electrical capacitance by a Corneometer.

Results: A statistically significant increase in blood flow was found 48 h after application of one of the moisturisers tested, indicating an irritant effect of the product. A statistically significant decrease in skin hydration was found for the same moisturiser after 48 h. No statistically significant differences between the moisturisers were found by visual scoring. None of the products tested had any negative effect on the skin barrier function.

Conclusion: The non‐invasive patch test model was found useful for detecting irritant skin reactions to moisturisers.
Efficacy of corticosteroids in acute experimental irritant contact dermatitis?
Authors: Levin Cheryl , Zhai Hongbo , Bashir Saqib , Chew Ai‐Lean , Anigbogu Angela , Stern Robert , Maibach Howard
Published: March 2003

Background/aims: Topical corticoids are used to treat irritant contact dermatitis (ICD) in humans. However, their clinical efficacy remains sub judice. This study was designed to assess the efficacy of low‐ and medium‐potency corticosteroids on irritant dermatitis.

Methods: We induced an acute ICD via open application of sodium lauryl sulphate (SLS) on the hands of subjects. The dorsal side of hands was irritated with 10% SLS five times in one day. Once on day 1 and twice daily on days 2–5, 1% hydrocortisone, 0.1% betamethasone‐17‐valerate and vehicle cream (petrolatum) were applied subsequently. Visual grading, bioengineering techniques and squamometry were used to quantify skin response.

Results: Corticosteroids were found ineffective in treating the surfactant‐induced irritant dermatitis when compared with the vehicle and with the untreated control.

Conclusion: The counterintuitive result (in a relatively realistic and robust model) should be interpreted with caution until verified with other irritants of varying physicochemical properties.
Anatomy and physiology of subcutaneous adipose tissue by in vivo magnetic resonance imaging and spectroscopy: Relationships with sex and presence of cellulite
Authors: Querleux B. , Cornillon C. , Jolivet O. , Bittoun J.
Published: March 2003

Background Little is still known concerning subcutaneous adipose tissue and cellulite, and controversial questions are still under discussion.

Aims Magnetic resonance imaging and spectroscopy were used to address two unresolved questions relating to the anatomy and physiology of subcutaneous adipose tissue.

Methods Using high spatial resolution magnetic resonance imaging we characterized the topography of the dermo‐ hypodermal junction, and the three‐dimensional architecture of the subcutaneous fibrous septae. Using proton spectroscopy, we measured water and lipid fractions within a fat lobule, and T1 and T2 values of the detected compounds. All these data were analysed according to sex and presence of cellulite.

Results MR imaging quantified deeper indentations of adipose tissue into the dermis, and evidenced for the first time a great increase in the thickness of the inner fat layer in women with cellulite. Moreover, 3D reconstruction of the fibrous septae network showed a higher percentage of septae in a direction perpendicular to the skin surface in women with cellulite; but our study also depicted the tortuous aspect of this network. MR proton spectroscopy could not show any differences related to sex or presence of cellulite concerning T1 and T2 relaxation times of the detected compounds within a fat lobule, neither the unsaturated lipid fraction, the saturated lipid fraction, nor the water fraction.

Conclusions Magnetic resonance imaging showed that the 3D architecture of fibrous septae couldn't be modelled simply as perpendicular planes for women and tilted planes at 45° for men. MR spectroscopy did not confirm the hypothesis of increased water content in the adipose tissue of women with cellulite as suggested by others, except if such water would be located in the connective septae.
Mechanobiology of force transduction in dermal tissue
Authors: Silver Frederick H. , Siperko Lorraine M. , Seehra Gurinder P.
Published: March 2003

Background/aims:The influence of mechanical forces on skin has been examined since 1861 when Langer first reported the existence of lines of tension in cadaver skin. Internal tension in the dermis is not only passively transferred to the epidermis but also gives rise to active cell‐extracellular matrix and cell–cell mechanical interactions that may be an important part of the homeostatic processes that are involved in normal skin metabolism. The purpose of this review is to analyse how internal and external mechanical loads are applied at the macromolecular and cellular levels in the epidermis and dermis.

Methods:A review of the literature suggests that internal and external forces applied to dermal cells appear to be involved in mechanochemical transduction processes involving both cell–cell and cell–extra‐cellular matrix (ECM) interactions. Internal forces present in dermis are the result of passive tension that is incorporated into the collagen fiber network during development. Active tension generated by fibroblasts involves specific interactions between cell membrane integrins and macromolecules found in the ECM, especially collagen fibrils. Forces appear to be transduced at the cell–ECM interface via re‐arrangement of cytoskeletal elements, activation of stretch‐induced changes in ion channels, cell contraction at adherens junctions, activation of cell membrane‐associated secondary messenger pathways and through growth factor‐like activities that influence cellular proliferation and protein synthesis.

Conclusions:Internal and external mechanical loading appears to affect skin biology through mechanochemical transduction processes. Further studies are needed to understand how mechanical forces, energy storage and conversion of mechanical energy into changes in chemical potential of small and large macromolecules may occur and influence the metabolism of dermal cells.
Membrane Dynamics in Endocytosis: Structure–Function Relationship
Authors: Gagescu Raluca , Gruenberg Jean , Smythe Elizabeth
Published: March 2003

A number of years ago a small group of investigators was torn apart by a debate over the existence or fate of somewhat obscure endosomal vesicles not even represented in text books. During the last decade, however, interest has shifted with the fireworks of newly discovered molecules involved in the regulation of protein transport. We are now entering the post‐genomic era, where individual components can be re‐assembled into functional, multiprotein cellular machines. Borders between disciplines in Life Sciences are fading away as our molecular understanding approaches atomic resolution. It is these exciting developments that were captured by the latest edition of the ESF conference series on endocytosis.
Trafficking and Release of Mycobacterial Lipids from Infected Macrophages
Authors: Beatty Wandy L. , Rhoades Elizabeth R. , Ullrich Heinz‐Joachim , Chatterjee Delphi , Heuser John E. , Russell David G.
Published: March 2003

Analysis of infected macrophages revealed that lipid‐containing moieties of the mycobacterial cell wall are actively trafficked out of the mycobacterial vacuole. To facilitate the analysis of vesicular trafficking from mycobacteria‐containing phagosomes, surface‐exposed carbohydrates were labeled with hydrazide‐tagged markers. The distribution of labeled carbohydrate/lipid moieties and subsequent interaction with cellular compartments were analyzed by immunoelectron microscopy and by fluorescence microscopy of live cells. The released mycobacterial constituents were associated with several intracellular organelles and were enriched strikingly in tubular endocytic compartments. Subcellular fractionation of infected macrophages by density gradient electrophoresis showed temporal movement of labeled bacterial constituents through early and late endosomes. Thin layer chromatography analysis of these subcellular fractions confirmed their lipid nature and revealed five dominant bacteria‐derived species. These mycobacterial lipids were also found in extracellular vesicles isolated from the medium and could be observed in un‐infected ‘bystander’ cells. Their transfer to bystander cells could expand the bacteria's sphere of influence beyond the immediate confines of the host cell.
Targeting of an Apical Endosomal Protein to Endosomes in Madin–Darby Canine Kidney Cells Requires Two Sorting Motifs
Authors: Gokay K.E. , Wilson J.M.
Published: March 2003

The efficient sorting and targeting of endocytosed macromolecules is critical for epithelial function. However, the distribution of endosomal compartments in these cells remains controversial. In this study, we show that polarized Madin–Darby canine kidney (MDCK) cells target the apical endosomal protein endotubin into an apical early endosomal compartment that is distinct from the apical recycling endosomes. Furthermore, through a panel of site‐directed mutations we show that signals required for apical endosomal targeting of endotubin are composed of two distinct motifs on the cytoplasmic domain, a hydrophobic motif and a consensus casein kinase II site. Endotubin‐positive endosomes in MDCK cells do not label with basolaterally internalized transferrin or ricin, do not contain the small guanosine triphosphate‐binding protein rab11, and do not tubulate in response to low concentrations of brefeldin‐A (BFA). Nevertheless, high concentrations of BFA reversibly inhibits the sorting of endotubin from transferrin and cause colocalization in tubular endosomes. These results indicate that, in polarized cells, endotubin targets into a distinct subset of apical endosomes, and the targeting information required both for polarity and endosomal targeting is provided by the cytoplasmic portion of the molecule.
Truncated Brush Border Myosin I Affects Membrane Traffic in Polarized Epithelial Cells
Authors: Durrbach A. , Raposo G. , Tenza D. , Louvard D. , Coudrier E.
Published: March 2003

We investigate, in this study, the potential involvement of an acto‐myosin‐driven mechanism in endocytosis of polarized cells. We observed that depolymerization of actin filaments using latrunculin A decreases the rate of transferrin recycling to the basolateral plasma membrane of Caco‐2 cells, and increases its delivery to the apical plasma membrane. To analyze whether a myosin was involved in endocytosis, we produced, in this polarized cell line, truncated, non‐functional, brush border, myosin I proteins (BBMI) that we have previously demonstrated to have a dominant negative effect on endocytosis of unpolarized cells. These non‐functional proteins affect the rate of transferrin recycling and the rate of transepithelial transport of dipeptidyl‐peptidase IV from the basolateral plasma membrane to the apical plasma membrane. They modify the distribution of internalized endocytic tracers in apical multivesicular endosomes that are accessible to fluid phase tracers internalized from apical and basolateral plasma membrane domains. Altogether, these observations suggest that an acto‐myosin‐driven mechanism is involved in the trafficking of basolaterally internalized molecules to the apical plasma membrane.
How Peroxisomes Arise
Authors: Terlecky Stanley R. , Fransen Marc
Published: March 2003

Peroxisomes are formed by the synthesis and assembly of membrane proteins and lipids, the selective import of proteins from the cytosol, and the growth and division of resultant organelles. To date, 23 proteins, called peroxins, are known to participate in these processes. This review summarizes recent progress in peroxin characterization and examines the underlying molecular mechanisms of peroxisome biosynthesis.
SNARE Protein Trafficking in Polarized MDCK Cells
Authors: Steegmaier Martin , Lee Kelly C. , Prekeris Rytis , Scheller Richard H.
Published: March 2003

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains. This polarity is generated and maintained by the continuous sorting of apical and basolateral components in the secretory and endocytic pathways. Soluble N‐ethyl maleimide‐sensitive factor attachment protein receptors (SNARE) proteins of vesicle‐associated membrane protein (VAMP) and syntaxin families have been suggested to play a role in the biosynthetic transport to the apical and basolateral plasma membranes of polarized cells, where they likely mediate membrane fusion. To investigate the involvement of SNARE proteins in membrane trafficking to the apical and basolateral plasma membrane in the endocytic pathway we have monitored the recycling of various VAMP and syntaxin molecules between intracellular compartments and the two plasma membrane domains in Madin–Darby canine kidney (MDCK) cells. Here we show that VAMP8/endobrevin cycles through the apical but not through the basolateral plasma membrane. Furthermore, we found that VAMP8 localizes to apical endosomal membranes in nephric tubule epithelium and in MDCK cells. This asymmetry in localization and cycling behavior suggests that VAMP8/endobrevin may play a role in apical endosomal trafficking in polarized epithelium cells.
Regulation of Lamp2a Levels in the Lysosomal Membrane
Authors: Cuervo A.M. , Dice J.F.
Published: March 2003

The selective degradation of cytosolic proteins in lysosomes by chaperone‐mediated autophagy depends, at least in part, on the levels of a substrate receptor at the lysosomal membrane. We have previously identified this receptor as the lysosome‐associated membrane protein type 2a (lamp2a) and showed that levels of lamp2a at the lysosomal membrane directly correlate with the activity of the proteolytic pathway. Here we show that levels of lamp2a at the lysosomal membrane are mainly controlled by changes in its half‐life and its distribution between the lysosomal membrane and the matrix. The lysosomal degradation of lamp2a requires the combined action of at least two different proteolytic activities at the lysosomal membrane. Lamp2a is released from the membrane by the action of these proteases, and then the truncated lamp2a is rapidly degraded within the lysosomal matrix. Membrane degradation of lamp2a is a regulated process that is inhibited in the presence of substrates for chaperone‐mediated autophagy and under conditions that activate that type of autophagy. Uptake of substrate proteins also results in transport of some intact lamp2a from the lysosomal membrane into the matrix. This fraction of lamp2a can be reinserted back into the lysosomal membrane. The traffic of lamp2a through the lysosomal matrix is not mediated by vesicles, and lamp2a reinsertion requires the lysosomal membrane potential and protein components of the lysosomal membrane. The distribution of lamp2a between the lysosomal membrane and matrix is a dynamic process that contributes to the regulation of lysosomal membrane levels of lamp2a and consequently to the activity of the chaperone‐mediated autophagic pathway.
Rab2 Requires PKCι/Λ to Recruit β‐COP for Vesicle Formation
Authors: Tisdale Ellen J.
Published: March 2003

The small GTPase Rab2 initiates the recruitment of soluble components necessary for protein sorting and recycling from pre‐Golgi intermediates. Our previous studies showed that Rab2 required protein kinase C (PKC) or a PKC‐like protein to recruit β‐COP to membrane (Tisdale EJ, Jackson M. Rab2 protein enhances coatomer recruitment to pre‐Golgi intermediates. J Biol Chem 1998;273: 17269–17277). We investigated the role of PKC in Rab2 function by first determining the active isoform that associates with membranes used in our assay. Western blot analysis detected three isoforms: PKCα, Γ and ι/Λ. A quantitative binding assay was used to measure recruitment of these kinases when incubated with Rab2. Only PKCι/Λ translocated to membrane in a dose‐dependent manner. Microsomes treated with anti‐PKCι/Λ lost the ability to bind β‐COP, suggesting that Rab2 requires PKCι/Λ for β‐COP recruitment. The recruitment of β‐COP to membranes is not regulated by PKCι/Λ kinase activity. However, PKCι/Λ kinase activity was necessary for Rab2‐mediated vesicle budding. We found that the addition of either a kinase‐deficient PKCι/Λ mutant or atypical PKC pseudosubstrate peptide to the binding assay drastically reduced vesicle formation. These data suggest that Rab2 causes translocation of PKCι/Λ to v esicular t ubular c lusters (VTCs), which promotes the recruitment of COPI to generate retrograde‐transport vesicles.
Regulation of Receptor Tyrosine Kinase Signaling by Endocytic Trafficking
Authors: Wiley H. Steven , Burke Patrick M.
Published: March 2003

Activated receptor tyrosine kinase (RTK) receptors are rapidly internalized and eventually delivered to the lysosomes. Although ligand‐induced endocytosis was originally thought to be a mechanism of receptor inactivation, many studies suggest that receptors remain active within endosomes. This review discusses the role that internalized signaling complexes may play in different RTK systems including recent data on how ubiquitination may regulate this process. In general, it appears that some receptor systems have evolved to enhance endosomal signaling, as is the case for TrkA and NGF. In contrast, the insulin receptor system appears to limit the extent of endosomal signaling. The EGFR system is the intermediate example. In this case, some signals are specifically generated from the cell surface while others appear to be generated from within endosomes. This may act as a mechanism to produce ligand‐specific signals. Thus, trafficking could play diverse roles in receptor signaling, depending on the specific cell and tissue type.
Intracellular Distribution and Late Endosomal Effects of the Ocular Albinism Type 1 Gene Product: Consequences of Disease‐Causing Mutations and Implications for Melanosome Biogenesis
Authors: Shen Bin , Rosenberg Benjamin , Orlow Seth J.
Published: March 2003

To investigate the function of ocular albinism type 1 (OA1), the gene responsible for X‐linked ocular albinism, we employed a construct containing murine Oa1 fused to green fluorescent protein (GFP) in a heterologous COS cell expression system. The cellular distribution of wild‐type (WT) Oa1 protein and Oa1 proteins reflecting mutations causing X‐linked ocular albinism were examined. Comparison with different organelle markers revealed that Oa1‐GFP localized to the late endolysosomal compartments. Some Oa1 mutant proteins failed to exit the endoplasmic reticulum (ER) (Class I mutants), while other mutants partially (Class II mutants) or fully (Class III mutants) exited the ER and trafficked to endolysosomal compartments. We observed that expression of WT Oa1‐GFP in COS cells caused an apparent enlargement of late endosomes and a redistribution of the mannose‐6‐phosphate receptor (M6PR). None of the mutants displayed the full range of effects on the redistribution of M6PR exhibited by WT Oa1. The effects of Oa1 on late endosome structure and content are thus likely to reflect an important biological property of Oa1. We propose that OA1 is involved in reorganizing the endolysosomal compartment as a necessary step in ocular melanosome biogenesis.
The Relationship Between Lumenal and Limiting Membranes in Swollen Late Endocytic Compartments Formed After Wortmannin Treatment or Sucrose Accumulation
Authors: Bright Nicholas A. , Lindsay Margaret R. , Stewart Abigail , Luzio J. Paul
Published: March 2003

Immunofluorescence and electron microscopy were used to evaluate the formation of swollen endosomes in NRK cells after treatment with wortmannin or sucrose and to study the relationship between lumenal and limiting membrane. Both treatments resulted in the formation of two populations of swollen late endocytic vacuoles, positive for lysosomal glycoproteins or cation‐independent mannose 6‐phosphate receptors, but those induced by wortmannin were characterised by time‐dependent accumulation of lumenal vesicles, whereas those induced by sucrose uptake did not accumulate lumenal vesicles. In both cases, the distribution of the late endosomal marker, lysobisphosphatidic acid, remained unchanged and was present within the lumen of the swollen vacuoles. Consumption of plasma membrane and peripheral early endosomes, and the appearance of transferrin receptors in swollen late endosomes, indicated that continued membrane influx from early endocytic compartments, together with inhibition of membrane traffic out of the swollen compartments, is sufficient to account for the observed phenotype of cells treated with wortmannin. The accumulation of organelles with the characteristic morphology of endocytic carrier vesicles in cells that have taken up sucrose offers an explanation for the paucity of lumenal vesicles in swollen sucrosomes. Our data suggest that in fibroblast cells the swollen endosome phenotype induced by wortmannin is a consequence of endocytic membrane influx, coupled with the failure to recycle membrane to other cellular destinations, and not the inhibition of multivesicular body biogenesis.
Total Internal Reflection Fluorescence Microscopy in Cell Biology
Authors: Axelrod Daniel
Published: March 2003

Key events in cellular trafficking occur at the cell surface, and it is desirable to visualize these events without interference from other regions deeper within. This review describes a microscopy technique based on total internal reflection fluorescence which is well suited for optical sectioning at cell‐substrate regions with an unusually thin region of fluorescence excitation. The technique has many other applications as well, most notably for studying biochemical kinetics and single biomolecule dynamics at surfaces. A brief summary of these applications is provided, followed by presentations of the physical basis for the technique and the various ways to implement total internal reflection fluorescence in a standard fluorescence microscope.
Phosphatidylinositol 3‐Kinase and the Control of Endosome Dynamics: New Players Defined by Structural Motifs
Authors: Corvera Silvia
Published: March 2003

Phosphatidylinositol (PtdIns) 3‐kinase (PI 3‐kinase) activity has been implicated in fundamental cellular functions such as endosomal trafficking, growth‐factor receptor signal transduction, and cell survival. This multiplicity of actions can be attributed to the existence of three classes of PI 3‐kinases in mammalian cells, which can together lead to the production of fourknown distinct end products: PtdIns(3)P, PtdIns(3,4)P2, PtdIns(3,4,5)P3 and PtdIns(3,5)P2. The challenge of deciphering the connection between PI 3‐kinase activity, the production of specific phosphoinositides and the control of specific cellular events is being met with the discovery of novel structural motifs that interact specifically with distinct PI 3‐kinase products.
Endosomal Sorting of Amyloid Precursor Protein‐P‐Selectin Chimeras Influences Secretase Processing
Authors: Daugherty Brandy L. , Green Samuel A.
Published: March 2003

Amyloid β protein, the major component of the senile plaques in Alzheimer's disease, is generated by secretory and endocytic processing of amyloid precursor protein. Internalized amyloid precursor protein either recycles to the plasma membrane, where α‐secretase resides, or moves to acidic compartment(s) for β‐secretase exposure. While the trans‐Golgi network contains β‐secretase activity, recent examination of the subcellular distribution of this proteinase, called BACE, has led to the suggestion that β‐secretase activity might also reside at the plasma membrane and in endosomes. To examine the role of endocytic compartments in β‐secretase processing of amyloid precursor protein, the wild‐type and endosomal sorting mutant P‐selectin cytoplasmic domains were used to control movement of amyloid precursor protein through endosomes. Amyloid precursor protein/P‐selectin, which is sorted from early to late endosomes, undergoes significantly less α‐secretase cleavage, and more β‐secretase cleavage, than amyloid precursor protein/P‐selectin768A, a mutant that recycles more efficiently to the cell surface. Our results demonstrate that endosomal sorting influences relative exposure of the amyloid precursor protein/P‐selectin chimeras to α‐ and β‐secretase activities, and suggest that, because delivery to late endocytic compartments favors β‐secretase processing of amyloid precursor protein, there is likely limited β‐secretase activity in early endosomes or at the cell surface. We propose that the trans‐Golgi network may be involved in both secretory and endocytic generation of amyloid β protein.