Molecular detection and characterisation of sapoviruses and noroviruses in outpatient children with diarrhoea in Northwest Ethiopia.

Childhood morbidity and mortality of diarrhoeal diseases are high, particularly in low-income countries and noroviruses and sapoviruses are among the most frequent causes worldwide. Their epidemiology and diversity remain not well studied in many African countries. To assess the positivity rate and the diversity of sapoviruses and noroviruses in Northwest Ethiopia, during November 2015 and April 2016, a total of 450 faecal samples were collected from outpatient children aged <5 years who presented with diarrhoea. Samples were screened for noroviruses and sapoviruses by real-time RT-PCR. Partial VP1 genes were sequenced, genotyped and phylogenetically analysed. Norovirus and sapovirus stool positivity rate was 13.3% and 10.0%, respectively. Noroviruses included GII.4 (35%), GII.6 (20%), GII.17 (13.3%), GII.10 (10%), GII.2 (6.7%), GII.16 (5%), GII.7 (3.3%), GII.9, GII.13, GII.20 and GI.3 (1.7% each) strains. For sapoviruses, GI.1, GII.1 (20.0% each), GII.6 (13.3%), GI.2 (8.9%), GII.2 (11.1%), GV.1 (8.9%), GIV.1 (6.7%), GI.3 and GII.4 (2.2% each) genotypes were detected. This study demonstrates a high genetic diversity of noroviruses and sapoviruses in Northwest Ethiopia. The positivity rate in stool samples from young children with diarrhoea was high for both caliciviruses. Continued monitoring is recommended to identify trends in genetic diversity and seasonal variations.

Magnetic conjugated polymer nanoparticles doped with a europium complex for biomedical imaging.

Self-assembling conjugated polymer nanoparticles containing PVK and PLGA-PEG as a matrix polymer were doped with both a luminescent rare-earth complex and magnetic nanoparticles (SPIONs), giving rise to materials that are both luminescent and magnetic. Nanoparticle sizes ranged from 80-110 nm without SPIONs and showed an increase in size (200-1000 nm) with additional SPION content (11-54%). Quantum yields (QYs) of 24% and 18% were measured for systems without and with 11% SPIONs, respectively. Optical properties were stable and suitable for biological imaging applications.

PAGAT gel dosimetry for everyone: gel production, measurement and evaluation.

Polymer gel (PG) dosimetry is a valuable tool to measure complex dose distributions in 3D with a high spatial resolution. However, due to complex protocols that need to be followed for in-house produced PGs and the high costs of commercially available gels, PG gels are only rarely applied in quality assurance procedures worldwide. In this work, we provide an introduction to perform highly standardized dosimetric PG experiments using PAGAT (PolyAcrylamide Gelatine gel fabricated at ATmospheric conditions) dosimetry gel. PAGAT gel can be produced at atmospheric conditions, at low costs and is evaluated using magnetic resonance imaging (MRI). The conduction of PG experiments is described in great detail including the gel production, treatment planning, irradiation, MRI evaluation and post-processing procedure. Furthermore, a plugin in an open source image processing tool for post-processing is provided free of charge that allows a standardized and reproducible analysis of PG experiments.

End-to-end test for fractionated online adaptive MR-guided radiotherapy using a deformable anthropomorphic pelvis phantom.

Objective.In MR-guided radiotherapy (MRgRT) for prostate cancer treatments inter-fractional anatomy changes such as bladder and rectum fillings may be corrected by an online adaption of the treatment plan. To clinically implement such complex treatment procedures, however, specific end-to-end tests are required that are able to validate the overall accuracy of all treatment steps from pre-treatment imaging to dose delivery.Approach.In this study, an end-to-end test of a fractionated and online adapted MRgRT prostate irradiation was performed using the so-called ADAM-PETer phantom. The phantom was adapted to perform 3D polymer gel (PG) dosimetry in the prostate and rectum. Furthermore, thermoluminescence detectors (TLDs) were placed at the center and on the surface of the prostate for additional dose measurements as well as for an external dose renormalization of the PG. For the end-to-end test, a total of five online adapted irradiations were applied in sequence with different bladder and rectum fillings, respectively.Main results.A good agreement of measured and planned dose was found represented by highγ-index passing rates (3%/3mmcriterion) of the PG evaluation of98.9%in the prostate and93.7%in the rectum. TLDs used for PG renormalization at the center of the prostate showed a deviation of-2.3%.Significance.The presented end-to-end test, which allows for 3D dose verification in the prostate and rectum, demonstrates the feasibility and accuracy of fractionated and online-adapted prostate irradiations in presence of inter-fractional anatomy changes. Such tests are of high clinical importance for the commissioning of new image-guided treatment procedures such as online adaptive MRgRT.

Development of phantom materials with independently adjustable CT- and MR-contrast at 0.35, 1.5 and 3 T.

Quality assurance in magnetic resonance (MR)-guided radiotherapy lacks anthropomorphic phantoms that represent tissue-equivalent imaging contrast in both computed tomography (CT) and MR imaging. In this study, we developed phantom materials with individually adjustable CT value as well as [Formula: see text]- and [Formula: see text]-relaxation times in MR imaging at three different magnetic field strengths. Additionally, their experimental stopping power ratio (SPR) for carbon ions was compared with predictions based on single- and dual-energy CT. Ni-DTPA doped agarose gels were used for individual adjustment of [Formula: see text] and [Formula: see text] at [Formula: see text] and 3.0 T. The CT value was varied by adding potassium chloride (KCl). By multiple linear regression, equations for the determination of agarose, Ni-DTPA and KCl concentrations for given [Formula: see text] [Formula: see text] and CT values were derived and employed to produce nine specific soft tissue samples. Experimental [Formula: see text] [Formula: see text] and CT values of these soft tissue samples were compared with predictions and additionally, carbon ion SPR obtained by range measurements were compared with predictions based on single- and dual-energy CT. The measured CT value, [Formula: see text] and [Formula: see text] of the produced soft tissue samples agreed very well with predictions based on the derived equations with mean deviations of less than [Formula: see text] While single-energy CT overestimates the measured SPR of the soft tissue samples, the dual-energy CT-based predictions showed a mean SPR deviation of only [Formula: see text] To conclude, anthropomorphic phantom materials with independently adjustable CT values as well as [Formula: see text] and [Formula: see text] relaxation times at three different magnetic field strengths were developed. The derived equations describe the material specific relaxation times and the CT value in dependence on agarose, Ni-DTPA and KCl concentrations as well as the chemical composition of the materials based on given [Formula: see text] and CT value. Dual-energy CT allows accurate prediction of the carbon ion range in these materials.

On the feasibility of absolute 3D dosimetry using LiF thermoluminescence detectors and polymer gels on a 0.35T MR-LINAC.

BACKGROUND AND PURPOSE: As shown in our previous study, highly accurate absolute dosimetry in 3D is feasible by combining polymer gels (PG) with thermoluminescence dosimetry (TLD). In this setup, the thermoluminescence (TL)-based point dose information is used to renormalize the PG. This new PG-TLD reference system is now extended to measurements in magnetic fields. MATERIALS AND METHODS: Experiments were carried out on a conventional 6 MV linear accelerator (LINAC) and a 6 MV 0.35 T magnetic resonance (MR)-LINAC. Signal stability of TLD600 and TLD700 was examined without and with magnetic field. Afterwards, the combination of PAGAT PG and TL detectors was employed within a cylindrical phantom in presence of the magnetic field. Two scenarios were tested: (I) an air-filled phantom and (II) a water-filled phantom. For each scenario, two plans were irradiated: (a) opposed beams with a field size of 10 × 10 cm2 and (b) a 3D conformal plan assuring homogeneous target coverage using three equally distributed coplanar beams. RESULTS: Mean relative uncertainty of TL calibration reproducibility for TLD600/TLD700 was 0.49%/0.85% at the MR-LINAC and 0.48%/0.83% for the conventional LINAC. Individual TL calibration coefficients of TLD600 and TLD700 behaved differently in the presence of the magnetic field. An average difference of (3.29 ± 0.89)% occurred for all TLD600, whereas the result for TLD700 is not quite as clear with (1.09 ± 0.89)% after excluding some outliers. Using the TL dose information for PG renormalization, high 3D gamma passing rates were achieved using the 3%/2 mm criteria: 91.0% (Ia), 92.6% (Ib), 94.3% (IIa), 97.4% (IIb). CONCLUSION: This study shows that TL signal reproducibility is not affected by a low magnetic field. Nevertheless, absolute calibration coefficients of the individual detectors indicate a dependency on the magnetic field. Hence, a calibration at the appropriate LINAC type is recommended. Furthermore, the previously established renormalization method for PG was applied to measurements at a MR-LINAC and was verified as suitable for evaluations of homogeneous dose distribution in the target volume.

Feasibility of markerless fluoroscopic real-time tumor detection for adaptive radiotherapy: development and end-to-end testing.

Respiratory-gated radiotherapy treatments of lung tumors reduce the irradiated normal tissue volume and potentially lower the risk of side effects. However, in clinical routine, the gating signal is usually derived from external markers or other surrogate signals and may not always correlate well with the actual tumor position. This study uses the kV-imaging system of a LINAC in combination with a multiple template matching algorithm for markerless real-time detection of the tumor position in a dynamic anthropomorphic porcine lung phantom. The tumor was realized by a small container filled with polymer dosimetry gel, the so-called gel tumor. A full end-to-end test for a gated treatment was performed and the geometric and dosimetric accuracy was validated. The accuracy of the tumor detection algorithm in SI- direction was found to be [Formula: see text] mm and the gel tumor was automatically detected in 98 out of 100 images. The measured 3D dose distribution showed a uniform coverage of the gel tumor and comparison with the treatment plan revealed a high 3D [Formula: see text]-passing rate of [Formula: see text] ([Formula: see text]). The simulated treatment confirmed the employed margin sizes for residual motion within the gating window and serves as an end-to-end test for a gated treatment based on a markerless fluoroscopic real-time tumor detection.

Absolute dosimetry with polymer gels-a TLD reference system.

BACKGROUND AND PURPOSE: Absolute dosimetry in 3D with polymer gels (PG) is generally complicated and usually requires a second independent measurement with conventional detectors. This is why, PG are often used only for relative dosimetry. To overcome this drawback, we combine PG with a 1D thermoluminescence (TL) detector within the same measurement. The TL detector information is then used as additional information for calibration of the gel. MATERIALS AND METHODS: The PAGAT dosimetry gel was used in combination with TLD600 (LiF:Mg,Ti). TL detectors were attached on the surface of the PG container placed inside a cylindrical phantom. To test the usability of this setup, two irradiation geometries were carried out: (a) homogeneous target coverage and (b) small-field irradiation. PG was evaluated with magnetic resonance imaging (MRI) and the TL detectors with a Harshaw 5500 hot gas reader. RESULTS: PG dosimetry alone showed deviations of up to 4% as compared to calculations. Including additionally the dose information of the TL detectors for PG calibration, this deviation was decreased to less than 1% for both irradiation geometries. This is also reflected by the very high [Formula: see text]-passing rates of > 96% (3%/3 mm) and >93% (2%/2 mm), respectively. CONCLUSION: This study presents a novel method combining 3D PG and TL dose measurements for the purpose of absolute 3D dose measurements that can also be applied in complex anthropomorphic phantoms using only a single measurement. The method was validated for two different irradiation geometries including a homogeneous large field as well as a small field irradiation with sharp dose gradients.

Compatibility of 3D printing materials and printing techniques with PAGAT gel dosimetry.

Polymer gel (PG) dosimetry enables three dimensional (3D) measurement of complex dose distributions. However, PGs are strongly reactive with oxygen and other contaminations, limiting their applicability by the need to use specific container materials. We investigate different 3D printing materials and printing techniques for their compatibility with PG. Suitable 3D printing materials may provide the possibility to perform PG dosimetry in complex-shaped phantoms. 3D printed and PG-filled test vials were irradiated homogenously. The signal response was evaluated with respect to homogeneity and compared to the signal in already validated reference vials. In addition, for the printing material VeroClear™ (StrataSys, Eden Prairie, USA) different methods to remove support material, which was required during the printing process, were investigated. We found that the support material should be used only on the outer side of the container wall with no direct contact to the PG. With the VeroClear™ material a homogenous signal response was achieved with a mean deviation of [Formula: see text] relative to the reference vials. In addition, the homogeneous irradiation of an irregularly-shaped gel container designed with the same printing material and technique also lead to a homogenous PG response. Furthermore, a small field irradiation of an additional test-vial showed an accurate representation of steep dose gradients with a deviation of the maximum position of [Formula: see text] relative to the reference vial.

A glassware-free combinatorial synthesis of green quantum dots using bubble wrap.

Here, we describe the use of commercially-available bubble wrap as the basis for the simple, cheap combinatorial exploration of the synthesis of brightly emitting core/shell quantum dots.

Measurement of isocenter alignment accuracy and image distortion of an 0.35 T MR-Linac system.

For hybrid devices combining magnetic resonance (MR) imaging and a linac for radiation treatment, the isocenter accuracy as well as image distortions have to be checked. This study presents a new phantom to investigate MR-Linacs in a single measurement in terms of (i) isocentricity of the irradiation and (ii) alignment of the irradiation and imaging isocenter relative to each other using polymer dosimetry gel as well as (iii) 3-dimensional (3D) geometric MR image distortions. The evaluation of the irradiated gel was performed immediately after irradiation with the imaging component of the 0.35 T MR-Linac using a T2-weighted turbo spin-echo sequence. Eight plastic grid sheets within the phantom allow for measurement of geometric distortions in 3D by comparing the positions of the grid intersections (control points) within the MR-image with their nominal position obtained from a CT-scan. The distance of irradiation and imaging isocenter in 3D was found to be (0.8 ± 0.9) mm for measurements with 32 image acquisitions. The mean distortion over the whole phantom was (0.60 ± 0.28) mm and 99.8% of the evaluated control points had distortions below 1.5 mm. These geometrical uncertainties have to be considered by additional safety margins.

End-to-end test of an online adaptive treatment procedure in MR-guided radiotherapy using a phantom with anthropomorphic structures.

Online adaptive treatment procedures in magnetic resonance (MR)-guided radiotherapy (MRgRT) allow compensating for inter-fractional anatomical variations in the patient. Clinical implementation of these procedures, however, requires specific end-to-end tests to validate the treatment chain including imaging, treatment planning, positioning, treatment plan adaption and accurate dose delivery. For this purpose, a new phantom with reproducibly adjustable anthropomorphic structures has been developed. These structures can be filled either with contrast materials providing anthropomorphic image contrast in MR and CT or with polymer dosimetry gel (PG) allowing for 3D dose measurements. To test an adaptive workflow at a 0.35 T MR-Linac, the phantom was employed in two settings simulating inter-fractional anatomical variations within the patient. The settings included two PG-filled structures representing a tumour and an adjacent organ at risk (OAR) as well as five additional structures. After generating a treatment plan, three irradiation experiments were performed: (i) delivering the treatment plan to the phantom in reference setting, (ii) delivering the treatment plan after changing the phantom to a displaced setting without adaption, and (iii) adapting the treatment plan online to the new setting and delivering it to the phantom. PG measurements revealed a homogeneous tumour coverage and OAR sparing for experiment (i) and a significant under-dosage in the PTV (down to 45% of the prescribed dose) and over-dosage in the OAR (up to 180% relative to the planned dose) in experiment (ii). In experiment (iii), a uniform dose in the PTV and a significantly reduced dose in the OAR was obtained, well-comparable to that of experiment (i) where no adaption of the treatment plan was necessary. PG measurements were well comparable with the corresponding treatment plan in all irradiation experiments. The developed phantom can be used to perform end-to-end tests of online adaptive treatment procedures at MR-Linac devices before introducing them to patients.

Differential regulation of hyaluronic acid synthase isoforms in human saphenous vein smooth muscle cells: possible implications for vein graft stenosis.

Autologous saphenous vein bypass grafts (SVG) are frequently compromised by neointimal thickening and subsequent atherosclerosis eventually leading to graft failure. Hyaluronic acid (HA) generated by smooth muscle cells (SMC) is thought to augment the progression of atherosclerosis. The aim of the present study was (1) to investigate HA accumulation in native and explanted arterialized SVG, (2) to identify factors that regulate HA synthase (HAS) expression and HA synthesis, and (3) to study the function of the HAS2 isoform. In native SVG, expression of all 3 HAS isoforms was detected by RT-PCR. Histochemistry revealed that native and arterialized human saphenous vein segments were characterized by marked deposition of HA in association with SMC. Interestingly, in contrast to native SVG, cyclooxygenase (COX)-2 expression by SMC and macrophages was detected only in arterialized SVG. In vitro in human venous SMC HAS isoforms were found to be differentially regulated. HAS2, HAS1, and HA synthesis were strongly induced by vasodilatory prostaglandins via Gs-coupled prostaglandin receptors. In addition, thrombin induced HAS2 via activation of PAR1 and interleukin 1beta was the only factor that induced HAS3. By small interfering RNA against HAS2, it was shown that HAS2 mediated HA synthesis is critically involved in cell cycle progression through G1/S phase and SMC proliferation. In conclusion, the present study shows that HA-rich extracellular matrix is maintained after arterialization of vein grafts and might contribute to graft failure because of its proproliferative function in venous SMC. Furthermore, COX-2-dependent prostaglandins may play a key role in the regulation of HA synthesis in arterialized vein grafts.

Feasibility of polymer gel-based measurements of radiation isocenter accuracy in magnetic fields.

For conventional irradiation devices, the radiation isocenter accuracy is determined by star shot measurements on films. In magnetic resonance (MR)-guided radiotherapy devices, the results of this test may be altered by the magnetic field and the need to align the radiation and imaging isocenter may require a modification of measurement procedures. Polymer dosimetry gels (PG) may offer a way to perform both, the radiation and imaging isocenter test, however, first it has to be shown that PG reveal results comparable to the conventionally applied films. Therefore, star shot measurements were performed at a linear accelerator using PG as well as radiochromic films. PG were evaluated using MR imaging and the isocircle radius and the distance between the isocircle center and the room isocenter were determined. Two different types of experiments were performed: i) a standard star-shot isocenter test and (ii) a star shot, where the detectors were placed between the pole shoes of an experimental electro magnet operated either at 0 T or 1 T. For the standard star shot, PG evaluation was independent of the time delay after irradiation (1 h, 24 h, 48 h and 216 h) and the results were comparable to those of film measurements. Within the electro magnet, the isocircle radius increased from 0.39 ± 0.01 mm to 1.37 ± 0.01 mm for the film and from 0.44 ± 0.02 mm to 0.97 ± 0.02 mm for the PG-measurements, respectively. The isocenter distance was essentially dependent on the alignment of the magnet to the isocenter and was between 0.12 ± 0.02 mm and 0.82 ± 0.02 mm. The study demonstrates that evaluation of the PG directly after irradiation is feasible, if only geometrical parameters are of interest. This allows using PG for star shot measurements to evaluate the radiation isocenter accuracy with comparable accuracy as with radiochromic films.

Cloning and characterization of KNR4, a yeast gene involved in (1,3)-beta-glucan synthesis.

k9 killer toxin from Hansenula mrakii was used to select a number of resistant mutants from Saccharomyces cerevisiae. Preliminary biochemical and genetic studies showed that some of them acquired structural defects in the cell wall. One of these mutants, the knr4-1 mutant, displays a number of cell wall defects, including osmotic sensitivity; sensitivity to cercosporamide, a known antifungal agent; and resistance to Zymolyase, a (1,3)-beta-glucanase. We report here the isolation and analysis of the KNR4 gene. DNA sequence analysis revealed an uninterrupted open reading frame which contains five potential start codons. The longest coding template encodes a protein of 505 amino acids with a calculated molecular mass of 57,044 Da. A data base search revealed 100% identity with a nuclear protein, SMI1p. Disruption of the KNR4 locus does not result in cell death; however, it leads to reduced levels of both (1,3)-beta-glucan synthase activity and (1,3)-beta-glucan content in the cell wall. The gene was mapped to the right arm of chromosome VII.

3D dosimetric validation of motion compensation concepts in radiotherapy using an anthropomorphic dynamic lung phantom.

In this study, we developed a new setup for the validation of clinical workflows in adaptive radiation therapy, which combines a dynamic ex vivo porcine lung phantom and three-dimensional (3D) polymer gel dosimetry. The phantom consists of an artificial PMMA-thorax and contains a post mortem explanted porcine lung to which arbitrary breathing patterns can be applied. A lung tumor was simulated using the PAGAT (polyacrylamide gelatin gel fabricated at atmospheric conditions) dosimetry gel, which was evaluated in three dimensions by magnetic resonance imaging (MRI). To avoid bias by reaction with oxygen and other materials, the gel was collocated inside a BAREX™ container. For calibration purposes, the same containers with eight gel samples were irradiated with doses from 0 to 7 Gy. To test the technical feasibility of the system, a small spherical dose distribution located completely within the gel volume was planned. Dose delivery was performed under static and dynamic conditions of the phantom with and without motion compensation by beam gating. To verify clinical target definition and motion compensation concepts, the entire gel volume was homogeneously irradiated applying adequate margins in case of the static phantom and an additional internal target volume in case of dynamically operated phantom without and with gated beam delivery. MR-evaluation of the gel samples and comparison of the resulting 3D dose distribution with the planned dose distribution revealed a good agreement for the static phantom. In case of the dynamically operated phantom without motion compensation, agreement was very poor while additional application of motion compensation techniques restored the good agreement between measured and planned dose. From these experiments it was concluded that the set up with the dynamic and anthropomorphic lung phantom together with 3D-gel dosimetry provides a valuable and versatile tool for geometrical and dosimetrical validation of motion compensated treatment concepts in adaptive radiotherapy.

Seemingly diverse activities of beta-alethine.

beta-Alethine (beta-alanyl-cysteamine disulfide) exhibits striking biological activities in diverse systems. At an optimum of about 10 ng/ml, beta-alethine (a) adapts murine liver cells to culture (53 colonies/10(6) cells versus none in controls), (b) delays aging of human IMR-90 fetal lung fibroblasts (102 population doubling levels versus 47 in controls, producing 3 x 10(16) greater biomass), and (c) markedly stimulates antibody-producing plaque-forming cells from murine splenocytes (16,875/10(6) cells versus 55/10(6) cells in controls) or human peripheral blood leukocytes (1826/10(6) cells versus 0/10(6) cells in controls). Early interventions with beta-alethine (1 ng/kg to 100 micrograms/kg) successfully treat NS-1 myeloma in a syngeneic murine tumor model (NS-1 myeloma). Although there are indications in this model that beta-alethine is also effective when intervention is late, beta-alethine is ineffective in an allogeneic murine melanoma model (Cloudman S-91 melanoma). It is inferred that beta-alethine enhances cellular phenotypic expression, function, and vitality in diverse biological systems and may treat certain types of neoplasia. Because atomic spacings between the amide moieties in beta-alethine are the same as in the differentiating agent hexamethylene-bis-acetamide and because the radioprotectors WR 2721 and WR 1065 lack only the carbonyl oxygen of the thiol form (beta-aletheine), biological activities already reported for these compounds are compared with those presented herein for beta-alethine. Although these comparisons have not been made in the same systems, the tentative conclusion is that the amide moieties of beta-alethine may be critical to its potency and lack of obvious toxicity in cell culture and animal models.

Mediation of anorexia by human recombinant tumor necrosis factor through a peripheral action in the rat.

Human recombinant tumor necrosis factor (TNF) produces significant anorexia in the rat which persists for up to 24 h after a single dose (5 micrograms/325 g rat). Dose-response studies indicate similar potencies for TNF following central or peripheral administration. Brain 125I-TNF levels were more than 100-fold greater after intracerebroventricular than i.v. injection, whereas blood levels of radioactivity were quite similar following both routes of administration. Gel filtration chromatography and precipitation by trichloroacetic acid showed that the radioactive label which exited the central nervous system was associated with intact TNF. The rapid effusion of 125I-TNF from the central nervous system resulted in detection of similar levels of the cytokine in a number of important target tissues (skin, muscle, fat) relative to that detected after peripheral administration. After i.v. or intracerebroventricular administration, blood levels of TNF declined rapidly to nearly undetectable levels over 4 h. However, the anorexia induced by TNF was sustained, and feeding remained depressed between 6 and 24 h postadministration. These observations suggest that TNF produces its anorectic effects at peripheral sites, possibly through mediators.

Senescence-induced alteration in cell surface carbohydrates correlated using proton NMR spectroscopy and a lectin-based affinity-binding assay.

Changes in the cell surface oligosaccharides in human fetal lung fibroblasts (IMR-90) are studied as the cells progress to senescence using nuclear magnetic resonance spectroscopy (NMR) and a biochemical assay. A lectin-based affinity-binding technique is used which measures the organization of carbohydrates on the cell surface. Proton NMR studies of the water in samples of frozen cell suspensions of young and old cells provide information on the local dynamics of the cell surface by monitoring the motion of bound water. Changes in the lectin binding density and affinity class distribution correlate with a decrease in the water proton linewidth in frozen cells. These observations reflect alterations in the conformation or structure of the cell surface oligosaccharides and local constituent water.

Membrane oligosaccharides: structure and function during differentiation.

Recent results gathered by normal light microscopy, immunocytochemistry, fluorescent-analog cytochemistry, and electron microscopy have allowed an improved interpretation of ameboid movement and related phenomena. 1. The contractile system responsible in Amoeba proteus for the generation of motive force for protoplasmic streaming and a large variety of dynamic activities is represented mainly by a thin cortical filament layer at the cytoplasmic face of the cell membrane (Fig. 18I). During normal locomotion this layer exhibits a distinct structural and physiological polarity with three different zones: a zone of reformation at the front (A), a zone of contraction in the intermediate cell region (B), and a zone of destruction at the uroid (C). 2. Two types of filaments participate in the formation of the cortical layer: (1) randomly distributed thin (actin) filaments exhibiting a parallel orientation in the anterior (Fc1) and a disordered arrangement in the intermediate and posterior cell region (Fc2; see also Fig. 17b), and (2) thick (myosin) filaments in close association with F-actin and mostly restricted to the intermediate and posterior cell region (Fc2). 3. The internal hydraulic pressure generated by localized active contraction of the cortical layer is transmitted to the endoplasm via the cell membrane and converted into directed streaming by a gel-sol gradient of decreasing viscosity between the uroid and the front. Calcium ions, ATP, and regulative proteins (profilin and a kinase) play an essential role in controlling both the interaction of actin and myosin and the sol-gel state of the cytoplasmic matrix. 4. Any alterations externally induced in the polarity of the cortical filament system by chemical or physical stimulation and inhibition cause immobilization of the amebas (Fig. 18II) with characteristic changes in (1) cell shape (spherulation and cell flattening), (2) membrane dynamics (cytotic and cytokinetic activities), and (3) cytoplasmic organization (hyalogranuloplasmic separation). pseudopodial tip (Fig. 18III, b----c, d----e), (3) destruction of the old layer at the hyalogranuloplasmic border (Fig. 18III, c,e), and (4) alternate solation (Fig. 18III, b and d) and gelation (Fig. 18III, c and e) of the hyaloplasm between the layer and the plasma membrane. The retraction of pseudopodia is accomplished by a local contraction of the cortical layer in conjunction with a simultaneous gel-sol transformation of the ectoplasmic cylinder. 6. The expression of a rather complex cytoskeleton which is composed not only of microfilaments and associated proteins, but also of intermediate- and microtubularlike structures has to be considered in future