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A fluorescence photobleaching study of vesicular stomatitis virus infected BHK cells. Modulation of G protein mobility by M protein.

The mobility of vesicular stomatitis virus (VSV) G protein on the surface of infected BHK cells was studied by using the technique of fluorescence photobleaching recovery. The fraction of surface G protein that was mobile in that time scale of the measurement (minutes) was at least 75%, a relatively high value among cell surface proteins so far observed. For studies of the effect of an internal viral protein (M protein) on G protein mobility, cells infected with wild-type VSV were compared with those infected with temperature-sensitive VSV mutants of complementation group III, which contains lesions in the M protein. At the permissive temperature, a pronounced decrease in the mobile fraction of surface G was observed for each of three mutants studied, while mobility of surface G at the nonpermissive temperature was indistinguishable in mutant and wild-type infected cells. A significantly lower mobile fraction of G protein was also observed in SV40 transformed 3T3 cells infected with wild-type VSV, but not in 3T3 or chick embryo fibroblast cells similarly infected. None of the variables tested had a measurable effect on the lateral diffusion coefficient of the mobile G protein. These results are interpreted as modulation of the mobility of a specific cell surface protein by a specific intracellular protein.

Animals↗

Mobility of the N-terminal segment of rabbit skeletal muscle F-actin detected by 1H and 19F nuclear magnetic resonance spectroscopy.

After polymerization filamentous actin (F-actin) still shows a number of rather narrow 1H NMR signals in its Mg2+ form which are quenched when Mg2+ is replaced by Ca2+. These resonances originate from mobile residues in F-actin. For assignment of these resonances three different strategies were used, the fluorine labeling of Cys-374 by 4-(perfluoro-tert-butyl)phenyliodoacetamide, binding studies with antibodies (Fab) against the seven N-terminal amino acids of actin, and two-dimensional 1H NMR spectroscopy of a highly concentrated F-actin sample. In contrast to the effects detected earlier by 1H NMR spectroscopy, 19F NMR spectroscopy of actin labeled at its C-terminal cysteine shows no significant spectral changes in dependence on the divalent ion present. In its G- (globular) form a strong, narrow 19F resonance can be observed at 15.06 ppm (relative to the external standard trifluoroacetic acid) which is broadened substantially after polymerization of actin. At 283 K the corresponding transverse relaxation time T2 decreases from 16.7 ms to approximately 0.6 ms. These data suggest that the highly mobile residues observed by 1H NMR spectroscopy do not originate from the C-terminus. Binding of Fab directed against the N-terminal amino acids of actin to Mg-F-actin leads to the disappearing of the 1H NMR resonances assigned to a mobile domain in F-actin. This indicates that the mobile region probably comprises the N-terminal amino acids. By homonuclear two-dimensional 1H NMR spectroscopy it was finally possible to sequentially assign the resonances of the mobile domain of F-actin. It turned out that amino acids 1-22 are in a highly mobile state in Mg-F-actin. The nuclear Overhauser effect data indicate that, rather surprisingly, in this high mobility state some of the beta-pleated structure is still conserved. The population of F-actin protomers in the M- (mobile) state can be obtained from the NMR spectra and was determined under different experimental conditions. In the presence of 150 mM KCl approximately half of the protomers in Mg-F-actin are in the M-state. This number is largely independent of the pH in the range studied (pH 7.2-7.8) and of the temperature in range studied (283-310 K). The equilibrium constant KMI for the equilibrium between the I- and M-states is approximately 1.3 under these conditions.

Acetamides↗

Effect of temperature and glassy states on the molecular mobility of solutes in frozen tuna muscle as studied by electron spin resonance spectroscopy with spin probe detection.

The mobility of solutes in frozen food systems (tuna muscle, sarcoplasmic protein fraction of tuna muscle, and carbohydrate-water) has been studied using the temperature dependence of the shape of electron spin resonance (ESR) spectra of the spin probe 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL). The spin probe was incorporated into the tuna meat from an aqueous solution of TEMPOL or by contact with a layer of TEMPOL crystals. The melting/freezing of freeze-concentrated solutes in frozen tuna meat was observed to take place over a range of temperatures from -25 to -10 degrees C. Lower temperatures gave ESR powder spectra due to the decreased mobility of the spin probe, and the temperature dependence of the mobility of the spin probe did not show abrupt changes at the glass transition temperatures of the systems. The mobility of nonglass forming solutes is concluded to be decoupled from the glass forming components. Similar behavior was also observed for TEMPOL in frozen, aqueous carbohydrate systems. The temperature dependence of the mobility of TEMPOL in the frozen systems was analyzed using the Arrhenius equation, and the logarithm of the Arrhenius preexponential factor tau(a) was found to be linearly correlated with the activation energy for all of the tuna and carbohydrate samples, indicating a common molecular mechanism for the observed mobility of TEMPOL in all of the systems. The linear correlation also suggests that the observed mobility of TEMPOL in the frozen aqueous systems is dominated by enthalpy-entropy compensation effects, where the mobility of TEMPOL is thermodynamically strongly coupled to the closest surrounding molecules.

Animals↗

An ESR spin-label study on molecular mobility in the interface between microphases of a diblock copolymer: effects of admixture of homopolymers that are miscible with one of the blocks.

Molecular mobility in the interfacial region of a microphase-separated structure was studied in binary mixtures of AB-type diblock copolymers and homopolymers (miscible with only A) by the spin-label technique. In this study, we prepared (a) binary blends of polystyrene-block-poly(methyl acrylate) (PS-block-PMA) and homopolymer poly(cyclohexyl acrylate)'s having the number-averaged molecular weight (Mn) of 1000 (PCHA-S) and (b) having the Mn of 17300 (PCHA-L), and (c) binary mixtures of the PS-block-PMA and homopolymer PS with the Mn of 900 (PS-1). Emphasis was placed on effects of the molecular weight and the miscibility of added homopolymers on the mobility in the interfacial region of the microphase separation. Selective incorporation of the added PCHA-S, PCHA-L, and PS-1 into the PS phase of the PS-block-PMA was confirmed by modulated-temperature differential scanning calorimetry (MDSC) measurement as a decrease in the glass transition temperature of the PS phase. Moreover, the MDSC and small-angle X-ray scattering (SAXS) measurements suggested that the spatial distributions of the PCHA-S and PS-1 in the PS phase were relatively uniform because of their small Mn. On the other hand, the distribution of the PCHA-L in the PS phase was somewhat heterogeneous because of the large Mn of the PCHA-L. The spin-label at the junction point of the PS-block-PMA allowed us to estimate the mobility in the interfacial region of the microphase separation. Influence of the PCHA-S and PCHA-L on the mobility in the interfacial region was negligible even though the relatively uniform distribution of the PCHA-S in the PS phase was suggested by the SAXS and MDSC. More uniform distribution of the PS-1 than that of the PCHA-S in the PS phase was suggested by the SAXS, and the mobility in the interfacial region was slightly enhanced by the addition of the PS-1. However, the mobility was almost constant against an increase in the PS-1. The PS-1 was considered to be penetrated into the interfacial region and activated the mobility, but the fraction of the PS-1 in the interfacial region was constant irrespective of the blended amount of the PS-1. These results suggest that effects of homopolymers on the mobility in the interface are significantly related to their spatial distribution in the host phase.

Journal Article↗

Epidermal iontophoresis: II. Application of the ionic mobility-pore model to the transport of local anesthetics.

PURPOSE: An in vitro study was carried out to determine the iontophoretic permeability of local anesthetics through human epidermis. The relationship between physicochemical structure and the permeability of these solutes was then examined using an ionic mobility-pore model developed to define quantitative relationships. METHODS: The iontophoretic permeability of both ester-type anesthetics (procaine, butacaine, tetracaine) and amide-type anesthetics (prilocaine, mepivacaine, lidocaine, bupivacaine, etidocaine, cinchocaine) were determined through excised human epidermis over 2 hrs using a constant d.c. current and Ag/AgCl electrodes. Individual ion mobilities were determined from conductivity measurements in aqueous solutions. Multiple stepwise regression was applied to interrelate the iontophoretic permeability of the solutes with their physical properties to examine the appropriateness of the ionic mobility-pore model and to determine the best predictor of iontophoretic permeability of the local anesthetics. RESULTS: The logarithm of the iontophoretic permeability coefficient (log PC(j,iont)) for local anesthetics was directly related to the log ionic mobility and MW for the free volume form of the model when other conditions are held constant. Multiple linear regressions confirmed that log PC(j,iont) was best defined by ionic mobility (and its determinants: conductivity, pKa and MW) and MW. CONCLUSIONS: Our results suggest that of the properties studied, the best predictors of iontophoretic transport of local anesthetics are ionic mobility (or pKa) and molecular size. These predictions are consistent with the ionic mobility pore model determined by the mobility of ions in the aqueous solution, the total current, epidermal permselectivity and other factors as defined by the model.

Anesthetics, Local↗

Dependence of the molecular mobility and protein stability of freeze-dried gamma-globulin formulations on the molecular weight of dextran.

PURPOSE: The effect of the molecular weight of dextran on the molecular mobility and protein stability of freeze-dried serum gamma-globulin (BGG) formulations was studied. The stabilizing effect of higher molecular weight dextran is discussed in relation to the molecular mobility of the formulations. METHODS: The molecular mobility of freeze-dried BGG formulations containing dextrans of various molecular weights was determined based on the free induction decay of dextran and water protons measured by proton NMR. The protein stability of the formulations was determined at temperatures ranging from 20 to 70 degrees C by size exclusion chromatography. RESULTS: Changes in the molecular mobility of freeze-dried formulations that occurred at temperatures below the glass transition temperature could be detected as the molecular mobility-changing temperature (Tmc), at which dextran protons started to exhibit a Lorentzian relaxation decay due to higher mobility in addition to a Gaussian relaxation decay. Tmc increased as the molecular weight of dextran increased. The proportion of dextran protons which exhibited the higher mobility relaxation process (Phm) at temperatures above Tmc decreased as the molecular weight of dextran increased. Protein stability was closely related to molecular mobility. The temperature dependence of the denaturation rate changed at around Tmc, and denaturation in the microscopically liquidized state decreased as Phm decreased with increasing molecular weight of dextran. CONCLUSIONS: The effect of the molecular weight of dextran on the protein stability of freeze-dried BGG formulations could be explained in terms of the parameters obtained by 1H-NMR such as Tmc and Phm. These parameters appear to be useful in preformulation and stability prediction of freeze-dried formulations.

Calorimetry, Differential Scanning↗

Anandamide-induced mobilization of cytosolic Ca2+ in endothelial cells.

1. Experiments were designed to determine whether anandamide affects cytosolic Ca2+ concentrations in endothelial cells and, if so, whether CB1 cannabinoid receptors are involved. To this effect, human umbilical vein-derived EA.hy926 endothelial cells were loaded with fura-2 to monitor changes in cytosolic Ca2+ using conventional fluorescence spectrometry methods. 2. Anandamide induced an increase in Ca2+ in endothelial cells which, in contrast to histamine, developed slowly and was transient. Anandamide caused a concentration-dependent release of Ca2+ from intracellular stores without triggering capacitative Ca2+ entry, contrary to histamine or the endoplasmic reticulum Ca2+ -ATPase inhibitor thapsigargin. 3. Anandamide pretreatment slightly reduced the mobilization of Ca2+ from intracellular stores that was evoked by histamine. The mobilization of Ca2+ from intracellular stores evoked by anandamide was impaired by 10 mM caffeine. 4. Anandamide and histamine each significantly increased NO synthase activity in EA.hy926 cells, as determined by the enhanced conversion of L-[3H]-arginine to L-[3H]-citruline. 5. The CB1 cannabinoid receptor antagonist SR141716A (1 microM) only produced a marginal reduction of the mobilization of Ca2+ produced by 5 microM anandamide. However, at 5 microM SR141716A elicited the release of Ca2+ from intracellular stores. This concentration strongly impaired the mobilization of cytosolic Ca2+ evoked by either anandamide, histamine or thapsigargin. 6. Pretreatment of the cells with either 200 microM phenylmethylsulphonyl fluoride (to inhibit the conversion of anandamide into arachidonic acid) or 400 ng ml(-1) pertussis toxin (to uncouple CB1 cannabinoid receptors from Gi/o proteins) had no significant effect on the mobilization of cytosolic Ca2+ evoked by either anandamide, or histamine. 7. Taken together the results demonstrate that anandamide mobilizes Ca2+ from a caffeine-sensitive intracellular Ca2+ store that functionally overlaps in part with the internal stores mobilized by histamine. However, a classical CB1 cannabinoid receptor-mediated and pertussis toxin-sensitive mechanism does not mediate this novel effect of anandamide in endothelial cells. 8. The mobilization of cytosolic Ca2+ in endothelial cells may account for the endothelium-dependent and NO-mediated vasodilator actions of anandamide. Due to its non-specific inhibition of Ca2+ signalling in endothelial cells, SR141716A may not be used to assess the physiological involvement of endogenous cannabinoids to endothelium-dependent control of vascular smooth muscle tone.

Arachidonic Acids↗

Submucosal microinfusion of endothelin and adrenaline mobilizes ECL-cell histamine in rat stomach, and causes mucosal damage: a microdialysis study.

Rat stomach ECL cells release histamine in response to gastrin. Submucosal microinfusion of endothelin or adrenaline, known to cause vasoconstriction and gastric lesions, mobilized striking amounts of histamine. While the histamine response to gastrin is sustainable for hours, that to endothelin and adrenaline was characteristically short-lasting (1-2 h). The aims of this study were to identify the cellular source of histamine mobilized by endothelin and adrenaline, and examine the differences between the histamine-mobilizing effects of gastrin, and of endothelin and adrenaline. Endothelin, adrenaline or gastrin were administered by submucosal microinfusion. Gastric histamine mobilization was monitored by microdialysis. Local pretreatment with the H1-receptor antagonist mepyramine and the H2-receptor antagonist ranitidine did not prevent endothelin- or adrenaline-induced mucosal damage. Submucosal microinfusion of histamine did not cause damage. Acid blockade by ranitidine or omeprazole prevented the damage, suggesting that acid back diffusion contributes. Gastrin raised histidine decarboxylase (HDC) activity close to the probe, without affecting the histamine concentration. Endothelin and adrenaline lowered histamine by 50-70%, without activating HDC. Histamine mobilization declined upon repeated administration. Endothelin reduced the number of histamine-immunoreactive ECL cells locally, and reduced the number of secretory vesicles. Thus, unlike gastrin, endothelin (and adrenaline) is capable of exhausting ECL-cell histamine. Microinfusion of alpha-fluoromethylhistidine (known to deplete ECL cells but not mast cells of histamine) reduced the histamine-mobilizing effect of endothelin by 80%, while 1-week pretreatment with omeprazole enhanced it, supporting the involvement of ECL cells. Somatostatin or the prostanoid misoprostol inhibited gastrin-, but not endothelin-stimulated histamine release, suggesting that endothelin and gastrin mobilize histamine via different mechanisms. While gastrin effectively mobilized histamine from ECL cells in primary culture, endothelin had no effect, and adrenaline, a modest effect. Hence, the striking effects of endothelin and adrenaline on ECL cells in situ are probably indirect, possibly a consequence of ischemia.

Animals↗

Digital mobile telephones and interference of ophthalmic equipment.

AIM: To assess the effect of mobile telephone electromagnetic interference on electronic ophthalmic equipment. METHODS: Prospective audit with mobile telephones placed at distances of 3 m, 1 m, and 30 cm from, and in contact with, electronic ophthalmic equipment. Any interruption or cessation of the function of the ophthalmic device was assessed with the mobile telephones in standby, and in dialling or receiving modes. Any alterations of displayed digital figures or numbers were also assessed. RESULTS: A total of 23 electronic ophthalmic devices in two hospital ophthalmology outpatient departments were evaluated. All six mobile telephones used, and 22 (95.7%) of the 23 ophthalmic equipment evaluated had the Conformité Européene (CE) mark. No device showed any interruption or cessation of function. There were no alterations of displayed digital figures or numbers. The only effect of any kind was found with four instruments (1 non-CE marked), where there was temporary flickering on the screen, and only occurred when the mobile telephones were dialling or receiving at a distance of 30 cm or less from the instruments. CONCLUSION: This study shows that among the electronic ophthalmic devices tested, none suffered failure or interruption of function, from mobile telephone interference. Although not comprehensive for all ophthalmic equipment, the results question the need for a complete ban of mobile telephones in ophthalmic departments. It highlights the need for a controlled, objectively measured study of the clinically relevant effects of mobile telephones in the ophthalmology outpatient setting.

Cell Phone↗

Antenatal pelvic organ mobility is associated with delivery mode.

OBJECTIVE: Relaxation of pelvic ligaments may facilitate parturition in certain animal species. Biomechanical properties of pelvic connective tissue may also influence progress of labour in the human female. This study was designed to test whether peripheral joint mobility or pelvic organ mobility as measures of connective tissue biomechanical properties are associated with progress in labour and delivery mode. DESIGN: Prospective clinical observational study. SETTING: Tertiary obstetric service. SAMPLE: 200 nulliparous women recruited in antenatal clinic. METHODS: Translabial ultrasound was used to obtain data on third trimester pelvic organ mobility. Upper limb joint mobility was assessed clinically. MAIN OUTCOME MEASURES: Gestational length, length of first and second stage of labour, delivery mode. RESULTS: Pelvic organ mobility was significantly associated with total length of second stage (P = 0.034 to P = 0.002). This was mainly due to the length of passive, not active second stage. There also was a statistically significant association between delivery mode and pelvic organ descent (P = 0.007 to P = 0.001), with the lowest mobility seen in women who required a Caesarean section in second stage. Joint mobility did not correlate with delivery data. CONCLUSION: Third trimester pelvic organ mobility is associated with duration of second stage and delivery mode.

Adult↗

In vitro safety profile of G-CSF-mobilized whole blood after storage for 7 days in an infusable-grade L15 medium.

BACKGROUND: G-CSF-mobilized whole blood (WB) is a cost-reducing and simple alternative for peripheral blood progenitor cell transplantation. Recently, it was demonstrated that mobilized WB supplemented with Leibovitz's L15 medium permitted prolonged preservation of clonogenic cells at ambient temperature. In this study, an infusable-grade L15 medium (IG-L15) was developed, and the safety profile of mobilized WB after 7 days of storage was investigated. STUDY DESIGN AND METHODS: IG-L15 was manufactured in a closed system under good manufacturing practice conditions. Proinflammatory cytokine levels and hemolysis in mobilized WB were determined after 7 days of storage in different containers and were compared with current clinical mobilized WB values after 1 to 3 days of storage at 4 degrees C. RESULTS: IG-L15 and L15 maintained clonogenic cells equally. In the samples of mobilized WB that were returned to the patient, cytokine levels were not elevated in comparison with freshly collected mobilized WB. By using IG-L15 in polystyrene-coated cell culture bags, median (range) levels of 9.4 (2.2-69.8) pg per mL (IL-1beta), 31.6 (6.1-146.5) pg per mL (TNF-alpha), 76.9 (15.5-934.9) pg per mL (IL-6), and 7195 (104-205,600) pg per mL (IL-8) were found after 7 days. Higher cytokine levels were found with L15 and different containers. He- molysis was less than 0.5 g per dL in all cases. CONCLUSION: The storage of mobilized WB for 7 days in IG-L15 at ambient temperature is possible with adequate preservation of clonogenic cells, but cytokine levels may require plasma removal before return.

Blood Preservation↗

The influence of falling, fear of falling, and balance confidence on prosthetic mobility and social activity among individuals with a lower extremity amputation.

OBJECTIVE: To assess in amputee patients the relationship between having fallen in the past 12 months, fear of falling, and balance confidence on mobility capability, mobility performance, and social activity. DESIGN: Population-based survey and chart review. SETTING: Two university-affiliated outpatient amputee programs in southwestern Ontario. PARTICIPANTS: Community-living individuals (n = 435) with a unilateral lower limb amputation. INTERVENTIONS: Patient chart review and a survey questionnaire. MAIN OUTCOME MEASURES: Self-report assessment of prosthetic capability and performance and social activity participation was assessed with the Prosthetic Evaluation Questionnaire mobility subscale, the Houghton Scale, and the Frenchay Activities Index. RESULTS: Falling experiences in the past 12 months were not significantly associated with any outcomes. Fear of falling was important in univariate relationships in all 3 outcomes, but not when balance confidence was included in multivariable modeling. Balance confidence was statistically significant with each of the outcomes and remained significant with inclusion of the covariates. There was statistical interaction (balance confidence x automatism; balance confidence x medication count) in modeling mobility capability and in modeling mobility performance (balance confidence x pain + balance confidence x amputation level). The final models accounted for 70%, 60%, and 55% of the variation in mobility capability, mobility performance, and social activity, respectively. CONCLUSION: Balance confidence was the only factor associated with mobility capability and performance and social activity in the final adjusted models. Clinicians and researchers should consider this variable in the rehabilitation of amputee patients.

Accidental Falls↗

Sentinel: towards an ambient mobility network.

PURPOSE: We are concerned with aiding the mobility of visually impaired travellers around often complex and unfamiliar internal and urban environments. To do this we focus on a users interaction with ambient devices because these device types provide an easy entry point for visually impaired individuals to interact with their surroundings. By augmenting the physical environment with mobility focused ambient devices and making existing devices universally accessible our goal of easy, focused, and confident mobility can be achieved. METHOD: We identify, through paper reviews and studies of empirical and anecdotal evidence, the social and technical problems that have so far barred consistent and cohesive development of an ambient mobility-network. RESULTS: We suggest that multi-model sensory-interaction with objects and assistive devices within an environment is the only way to accomplish easy, focused, and confident mobility for visually impaired travellers. We find that universal access to ambient devices does not exist when the user interface and the device functionality are conjoined. And we also suggest that this universal access will not occur if there are only benefits for one small minority user group. CONCLUSIONS: We conclude that to assist mobility we need to provide universal access to objects and devices within the environment. Also, to support the mobility of visually impaired travellers we must first support all travellers regardless of their mobility needs. And to accomplish this we must separate the user interface from the real purpose (the functionality) of the object/device.

Electronics↗

Phlebotomy-mobilized iron as a surrogate for liver iron content in hemochromatosis patients.

We sought to establish the relationship of quantitative hepatic iron measurements and phlebotomy-mobilized iron in a large sample of HFE C282Y homozygotes with a hemochromatosis phenotype. Thus, we analyzed data from 79 unrelated C282Y homozygotes from treatment centers in Rochester, NY and Birmingham, AL who had undergone liver biopsy with measurement of hepatic iron content and who had achieved iron depletion (serum ferritin <25 ng/l) with quantitative phlebotomy. The sample consisted of 57 men and 22 women; their median age at diagnosis was 47 years (range 23-76 years). Sixty-three of 79 (79.7%) had hepatic iron index (HII; μmol/g dry weight of liver divided by age in years) ≥1.9, a conventional phenotypic definition of hemochromatosis. The mean quantity of phlebotomy-mobilized iron (± 1 sd) was 6.4 g (±4.0 g) in men (range 2.0-18.0 g) and 6.2 g (±5.8) in women (range 0.7-22.5 g). There was a significant positive correlation of liver iron levels with phlebotomy-mobilized iron in this patient sample (Pearson coefficient 0.75; R 2 =55.5%). This relationship was also demonstrable when data from males and females were analyzed separately. We calculated a phlebotomy-mobilized iron index (MII: phlebotomy-mobilized iron in mg divided by age in years) using the corresponding regression equations and evaluated its use as a surrogate for HII. Thus, a phlebotomy-mobilized iron of 3.5 g corresponds to liver iron levels of 80 μmol/g dry weight, and a MII of 80 corresponds to HII of 1.9. Forty-six of 79 subjects met all four phenotypic criteria for hemochromatosis (liver iron levels ≥80 μmol/g, HII≥1.9, phlebotomy-mobilized iron ≥3.5 g and MII≥80). Of the 20 subjects with MII<80, 9 had a HII≥1.9. Conversely, 5 of 16 subjects with HII<1.9 had MII≥80 and 8 had phlebotomy-mobilized iron ≥3.5 g. Most patients with a hemochromatosis phenotype and evidence of moderate or severe iron overload (>80%) are homozygous for the common HFE missense mutation C282Y. Thus, clinicians rely increasingly on HFE mutation analysis to diagnose hemochromatosis and on quantitative phlebotomy to estimate the severity of iron overload in many cases. Liver biopsy is now employed in selected patients to visualize fibrosis or cirrhosis and to identify coincidental hepatic disease. We conclude that the use of the MII permits a retrospective estimation of the age-adjusted severity of iron overload that has a diagnostic value similar to that of the HII in hemochromatosis patients with C282Y homozygosity.

Adult↗

Evidence from lateral mobility studies for dynamic interactions of a mutant influenza hemagglutinin with coated pits.

Replacement of cysteine at position 543 by tyrosine in the influenza virus hemagglutinin (HA) protein enables the endocytosis of the mutant protein (Tyr 543) through coated pits (Lazarovits, J., and M. G. Roth. 1988. Cell. 53:743-752). To investigate the interactions between Tyr 543 and the clathrin coats in the plasma membrane of live cells, we performed fluorescence photobleaching recovery measurements comparing the lateral mobilities of Tyr 543 (which enters coated pits) and wild-type HA (HA wt, which is excluded from coated pits), following their expression in CV-1 cells by SV-40 vectors. While both proteins exhibited the same high mobile fractions, the lateral diffusion rate of Tyr 543 was significantly slower than that of HA wt. Incubation of the cells in a sucrose-containing hypertonic medium, a treatment that disperses the membrane-associated coated pits, resulted in similar lateral mobilities for Tyr 543 and HA wt. These findings indicate that the lateral motion of Tyr 543 (but not of HA wt) is inhibited by transient interactions with coated pits (which are essentially immobile on the time scale of the lateral mobility measurements). Acidification of the cytoplasm by prepulsing the cells with NH4Cl (a treatment that arrests the pinching-off of coated vesicles from the plasma membrane and alters the clathrin lattice morphology) led to immobilization of a significant part of the Tyr 543 molecules, presumably due to their entrapment in coated pits for the entire duration of the lateral mobility measurement. Furthermore, in both untreated and cytosol-acidified cells, the restrictions on Tyr 543 mobility were less pronounced in the cold, suggesting that the mobility-restricting interactions are temperature dependent and become weaker at low temperatures. From these studies we conclude the following. (a) Lateral mobility measurements are capable of detecting interactions of transmembrane proteins with coated pits in intact cells. (b) The interactions of Tyr 543 with coated pits are dynamic, involving multiple entries of Tyr 543 molecules into and out of coated pits. (c) Alterations in the clathrin lattice structure can modulate the above interactions.

Biological Transport↗

Evaluation of a novel automated protocol for the collection of peripheral blood stem cells mobilized with chemotherapy or chemotherapy plus G-CSF using the Fresenius AS104 cell separator.

Forty-seven peripheral blood stem cell (PBSC) collections were carried out on patients mobilized with chemotherapy and 63 on patients mobilized with chemotherapy plus G-CSF (Filgrastim), using the Fresenius AS104 cell separator and a novel automated PBSC collection protocol. As expected, cell yields were significantly higher in the series mobilized using chemotherapy plus G-CSF. The low platelet and red blood cell contamination permitted freezing of the apheresis product without further manipulation, other than plasma removal in both series. In patients mobilized with chemotherapy we obtained a MNC and a hemopoietic progenitor (CFU-GM, BFU-e, and CD34+ cells) collection efficiency comparable or superior to those reported by Bender (1992) with the Baxter CS3000 Plus after mobilization with cyclophosphamide. A significant decrease in MNC, BFU-e, and CD34+ cell collection efficiency was found in patients mobilized with chemotherapy plus G-CSF compared to those obtained in patients mobilized with chemotherapy alone. Ten patients achieved a prompt and stable engraftment after high dose chemotherapy and the infusion of cryopreserved PBSC collected using this protocol. Studies are in progress in order to improve MNC and hemopoietic progenitor collection efficiency in patients mobilized with G-CSF to obtain a graft in no more than one or two procedures.

Adolescent↗

Falls-related self-efficacy is independently associated with balance and mobility in older women with low bone mass.

BACKGROUND: It is currently unknown whether falling is independently associated with measures of balance and mobility in older adults after accounting for relevant physiological functions. This cross-sectional study assessed the independent association of falls-related self-efficacy to balance and mobility after accounting for age, current physical activity, and performances in relevant physiological domains in 98 older women, aged 75-86 years, with low bone mass. METHODS: Falls-related self-efficacy was assessed by the Activities-Specific Balance Confidence Scale (ABC Scale). Measures of balance and mobility included the 13-item Community Balance and Mobility Scale (CB & M Scale) and gait speed under two conditions: normal-paced and fast-paced. Physiological assessment included postural sway, foot reaction time, dominant quadriceps and dorsiflexor strength, proprioception, tactile sensitivity, edge contrast sensitivity, and visual acuity. RESULTS: Falls-related self-efficacy was independently associated with both balance and mobility after accounting for age, current physical activity level, and performances in relevant physiological domains. Based on the standardized beta coefficients, the ABC Scale score was more associated with measures of balance and mobility than measures of physiological function. CONCLUSION: These results highlight the independent association of falls-related self-efficacy with physical performance in older women with low bone mass. Thus, clinicians may need to consider falls-related self-efficacy when assessing and treating balance and mobility in this population, and falls-related self-efficacy may be useful as a screening tool to identify those persons with impaired balance and mobility.

Accidental Falls↗

Evaluating the accuracy of minimum data set bed-mobility ratings against independent performance assessments: systematic error and directions for improvement.

PURPOSE: The Minimum Data Set (MDS) Activities of Daily Living (ADL) bed-mobility item, which rates the staff-assistance level necessary for bed movement, is used to target scheduled repositioning interventions and to identify physical function changes in nursing home residents; however, accuracy of the item is uncertain. The purpose of this study was to evaluate the accuracy of the MDS ADL bed-mobility item as completed by nursing home nurses with independent performance assessments conducted by research staff. DESIGN AND METHODS: A convenience sample of 197 long-stay residents from 26 California nursing homes participating in a larger project was used in this cross-sectional study to compare independent research-staff performance assessments (using graduated assistance protocols of residents' ability to move in bed) and nursing home nurse MDS bed-mobility ratings. Participants also wore movement monitors to verify performance assessments. RESULTS: Poor agreement existed between the nursing home nurse MDS bed-mobility ratings and the research-staff performance assessments across all assistance levels (kappa range, kappa = 0.007, p =.918 to kappa = 0.484, p <.001), with better agreement seen in totally dependent participants and with fewer elapsed days between MDS ratings and performance assessments. The odds of nursing home nurse errors (underestimating or overestimating dependency) on the MDS bed-mobility item were 2.1 times higher for participants judged independent by research staff compared with participants judged as requiring physical assistance by research staff (95% confidence interval, 1.14-4.03) when adjusted for number of days between nurse MDS ratings and research-staff performance assessments. IMPLICATIONS: Nursing home nurses overestimated resident dependency in bed mobility. The systematic inaccuracies in MDS bed-mobility ratings have implications for their use as a basis for targeting residents for repositioning programs and determining changes in residents' physical function. Performance assessments utilizing graduated assistance protocols are recommended as a method of improving the accuracy of MDS bed-mobility ratings.

Activities of Daily Living↗