Distribution of transmembrane polypeptides in freeze fracture.

Human erythrocytes have been freeze-fractured, and the polypeptides associated with the separate halves of the membrane bilayer have been analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The transmembrane proteins were differentially separated by the fracture process. Although sialoglycoproteins associated with the outer half of the membrane, the anion transport protein (band 3) mainly remained with the inner half of the membrane. Well-defined fragments of the sialoglycoproteins were produced by the freeze-fracture procedure, indicating that selected covalent bonds of these transmembrane proteins were broken.

Membrane skeletal alterations during in vivo mouse red cell aging. Increase in the band 4.1a:4.1b ratio.

We have examined membrane protein profiles for alterations during red blood cell aging. To obtain populations of in vivo-aged red cells, we maintained mice in a state of continuous erythropoietic suppression for up to 8 wk using serial hypertransfusion. The circulating t1/2 of red cells from mice which had been erythropoietically suppressed for 8 wk was less than 1 d compared with a t1/2 of 15 d for red cells from normal animals. The most obvious alteration in membrane proteins was an increase in the ratio of the membrane skeletal components 4.1a:4.1b from 0.3 for the normal red cell population to greater than 1 for these old cells. The 4.1a:4.1b ratio thus appears to be a useful index of red cell age. Analyses of the density profile of cells aged in the hypertransfused mice disclosed that these old cells had a density range similar to that of controls, suggesting that cell density does not increase significantly with red cell age in the mouse.

Isolation and purification of the cytochrome oxidase of Azotobacter vinelandii.

A membrane-bound cytochrome oxidase for Azobacter vinelandii was purified 20-fold using a detergent-solubilization procedure. Activity was monitored using as ascorbate-TMPD oxidation assay. The oxidase was 'solubilized' from a sonic-type electron-transport particle (R3 fraction) using Triton X-100 and deoxycholate. Low detergent concentrations first solubilized the flavoprotein oxidoreductases, then higher concentrations of Triton X-100 and KCl solubilized the oxidase, which was precipitated at 27-70% (NH4)2SO4. The highly purified cytochrome oxidase has a V of 60-78 microgatom O consumed/min per mg protein. TMPD oxidation by the purified enzyme was inhibited by CO, KCN, NaN3 and NH2OH; NaNO2 (but not NaNO3) also had a potent inhibitory effect. Spectral analyses revealed two major hemoproteins, the c-type cytochrome c4 and cytochrome o; cytochromes a1 and d were not detected. The Azotobacter cytochrome oxidase is an integrated cytochrome c4-o complex, TMPD-dependent cytochrome oxidase activity being highest in preparations having a high c-type cytochrome content. This TMPD-dependent cytochrome oxidase serves as a major oxygen-activation site for the A. vinelandii respiratory chain. It appears functionally analogous to cytochrome a+a3 oxidase of mammalian mitochondria.

Observations on Echinostoma revolutum and Echinostoma trivolvis in single and concurrent infections in domestic chicks.

Studies were done to determine specific differences between adults of Echinostoma revolutum and Echinostoma trivolvis grown singly and concurrently in domestic chicks. Worm recovery at 10-21 days post-infection (p.i.) of E. revolutum and E. trivolvis in singly infected chicks was 21 and 17%, respectively; in concurrent infections, worm recovery for E. revolutum and E. trivolvis was 21 and 16%, respectively. The body area of E. trivolvis was 1.5-2.0 times greater than that of E. revolutum at 10-21 days p.i. Most body organ measurements of the suckers and gonads at 10, 14 and 21 days p.i. were significantly greater in E. trivolvis compared with those of E. revolutum. The mean dry weight/worm at 14 days p.i. was 2.5 mg for E. trivolvis and 1.0 mg for E. revolutum. In single infections, E. revolutum was found only in the rectum and E. trivolvis mainly in the lower ileum, and in concurrent infections worms of both species were found in the rectum. Contrary to a previous report that stated that the body size of adult worms is not a reliable taxonomic indicator between E. revolutum and E. trivolvis, the findings reported here show that body area and organ size can be used to distinguish these closely related species.

Loss of functional hemispheric asymmetry in Alzheimer's dementia assessed with near-infrared spectroscopy.

In a total of 10 patients with dementia of the Alzheimer-type (DAT) and in 10 healthy controls near-infrared spectroscopy (NIRS), a new non-invasive optical method, was used to measure the changes of concentrations of oxy- (O2HB) and deoxyhemoglobin (HHB) in left and right hemispheric prefrontal brain tissue areas during performance of the Verbal Fluency Test (VFT). On a neuropsychological level, the healthy subjects performed better in the VFT than patients with DAT. Statistical analysis of the relative concentrations of O2HB and HHB measured with NIRS during performance of the VFT revealed a significant interaction of the hemispheric effects with the diagnosis. A possible interpretation of this finding is that a good performance in the VFT relies on a predominantly left hemispheric activation observed in controls, whereas a low number of correct responses is associated with a loss of this asymmetric activation in patients with DAT. Although both, patients and controls, performed better in the category version of the VFT, the metabolic effects of this task were significantly less pronounced than in the letter version. This indicates that different energy demands, according to the type of access to the memory stores, may be interpreted as the result of a less energy-demanding access to categorically stored information and adds further evidence to the view that memory departments in humans are organized according to categorical principles.

Age-related changes in the brain electrical correlates of response control.

OBJECTIVES: Previously, a quantification method was validated which, on a single case basis, allows one to assess the NoGo-anteriorisation (NGA) of the positive area of long latency event-related potential (ERP) fields elicited by a cued continuous performance test (CPT). The NGA was shown to be associated with right frontal activity. The present study was conducted to investigate the influence of age and gender on this topographical index of cognitive response control. METHODS: Thirty-seven healthy controls were investigated with 21-channel recordings during the execution of a cued CPT, and ERPs of the Go and NoGo condition were obtained. The location of the positive area centroids in a P300 range and the NGA were calculated and related to age and gender by means of correlation analysis and t tests. RESULTS: The centroid locations of the brain electrical activity during the NoGo- and the Go-condition of the CPT, were both located in more anterior brain regions with increasing age (P<0.01 and P<0.1, respectively); the NGA, however, was not correlated with the subject's age. Latencies and amplitudes of the Go- and NoGo-centroids were not correlated with age. No gender differences were found. CONCLUSIONS: The study showed that age is a source of variance for the positive area centroid locations in this Go-NoGo paradigm. The NGA, on the other hand, was robust to age and gender effects. The result is interpreted as a sign of an increasing contribution of frontal brain areas to cognitive response control during lifespan. The finding is consistent with the age-related topographical changes described in acoustic oddball-paradigms and, therefore, appears to be a general topographical ERP effect.

Test-retest reliability of electrophysiological parameters related to cognitive motor control.

Previously, the continuous performance test was demonstrated to elicit distinct electrophysiological correlates of cognitive response during execution (Go) and inhibition (NoGo) of an anticipated motor response. A robust method for topographical quantification of these brain electrical microstates has been established recently. Test reliability is crucial to allow application in the assessment of neuropsychiatric disorders. The present study evaluates the reliability of the Go and NoGo centroid locations as well as the NoGo anteriorisation (NGA) in 23 healthy individuals. Our results show supreme test-alternate retest reliabilities of Pearson's product moment correlations and intraclass correlation coefficients of r> or =0.63 (P< or =0.001) for these parameters which assert a quality well within the range reported for those of other electrophysiological standard paradigms. Go and NoGo centroid locations as well as the NGA are, therefore, reliable correlates of prefrontal motor control and may contribute to the understanding of disorders with allied impairments.

Decreased EEG microstate duration and anteriorisation of the brain electrical fields in mild and moderate dementia of the Alzheimer type.

Spatially oriented segmentation allows researchers to break down the continuous stream of the ongoing EEG into microstates with stable topography of the brain electrical landscapes. The resulting microstates were shown to be related to conscious mental experience as well as to psychiatric disorders typically associated with thought disorders. In the present study, the microstates of the resting EEG of patients presenting with mild or moderate probable dementia of the Alzheimer type (DAT) were investigated. A significant anteriorisation of the centers of gravity of the microstate fields, an increase of the microstates' optimal window size and a reduced duration of sustained microstates were found. These differences were statistically more robust than the typical changes in the frequency domain (diffuse slowing) and were significantly correlated with the cognitive decline. The adaptive spatial segmentation into microstates is discussed as a method to extract meaningful EEG parameters for the early diagnosis and staging of Alzheimer's disease.

Neurophysiological correlates of mental imagery in different sensory modalities.

Mental Imagery is supposed to be a basic phenomenon of conscious mental operations, which is exerted in any sensory modality. The aim of the present study was to investigate neurophysiological correlates of mental imagery in the visual, the acoustic and tactile modality. For that purpose, word lists were created based on their power to evoke mental imagery in these modalities. Nine young healthy volunteers were included. Words were presented on a computer screen with 3-s intervals. Evoked electrical activity was recorded simultaneously with a 21-channel system. After inspection for artifacts and off-line averaging, further analysis was performed in a time range of 250-450 ms, corresponding to the P300 component. Within this range, six topographically stable segments were found. A repeated measures ANOVA was significant for the left-right directions of the positive and of the negative centroid, but not for the anterior-posterior directions. The posterior centroid was most right-located in the visual sensory modality and most left-located for tactile imagery; in the acoustic modality it had a midline-position. These differences in centroid locations during mental imagery in different sensory modalities can be explained by the underlying activity of different neural generator ensembles, possibly involving modality specific primary cortical areas.

Electrophoretic analysis of proteases in Echinostoma Caproni and Echinostoma trivolvis.

Gelatin substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze proteases in 14 day-old adults of Echinostoma caproni and Echinostoma trivolvis. At pH 8.0, E. caproni adults showed 2 protease bands at 36 kDa and 58 kDa, whereas E. trivolvis adults showed 6 bands at 39, 64, 77, 96, 120, and 168 kDa. Each species also showed distinct protease banding patterns in their excretory/secretory (E/S) products. The E. caproni E/S proteases were at 36 and 58 kDa, whereas those of E. trivolvis were at 120 and 168 kDa. Further characterization of E. caproni adult proteases revealed 2 bands (58 and 66 kDa) with optimal activity at pH 3.0-4.5 and 3 bands (38, 61, and 96 kDa) that were most active at pH 7.0-8.0. Four low molecular weight bands (19, 21, 25, and 30 kDa) appeared when E. caproni worm extracts were incubated in the presence of CaCl2 at pH 8.0 but were inhibited with ethylenediaminetetraacetic acid and 1,10-phenanthroline. Echinostoma caproni protease bands at 58 and 38 kDa in the whole worm samples and the E/S products and the 36-kDa band in the whole worm samples were inhibited with phenylmethylsulfonyl fluoride. By showing protease differences in addition to recent work on nucleotide differences, this study helps distinguish these 2 related allopatric species of 37-collar-spined Echinostoma.

Acquired flatfoot secondary to tibialis posterior dysfunction: biomechanical aspects.

The biomechanical implications of the normally-functioning tibialis posterior muscle and tendon unit, pathomechanics of its dysfunction, and its diagnosis are presented. The interaction of the tibialis posterior tendon with the rearfoot ligamentous system is discussed. An anatomic consideration of the glenoidal ligaments of the talonavicular joint new to the English literature is presented. A plan for staging the disease of tibialis posterior dysfunction and classifying its etiology is considered to be a prerequisite for proper treatment.

A physiological model of binocular rivalry.

This paper presents a modified reciprocal inhibition model for the temporal dynamics of binocular rivalry. The model is based on neurophysiological mechanisms and is derived from human psychophysical data. A simple reciprocal inhibition oscillator may be described with a set of four coupled differential equations with a neurophysiological interpretation. However, such a circuit does not account for some aspects of the temporal behavior of binocular rivalry, including the effects of contrast change on alternation rate and on the magnitudes of changes in duration of the suppressed and dominant phases. To better account for these phenomena, the equations and their stimulation are modified to include three new components: (1) presynaptic inhibition of the reciprocal inhibition by the input, (2) the motor delays that occur when a human observer tracks rivalry and (3) a minimum threshold for each neuron's state variable. The result is a much improved fit to psychophysically-obtained data on the temporal behavior of binocular rivalry. Finally, the model is incorporated into a larger model to suggest how rivalry might occur in a network that usually exhibits binocular fusion.

Detection of a variant of protein 3, the major transmembrane protein of the human erythrocyte.

A variant of the major transmembrane protein of the human erythrocyte has been detected following proteolytic digestion of intact erythrocytes. Pronase digestion of normal erythrocytes gives rise to a 60,000 molecular weight fragment of Protein 3, while digestion of erythrocytes with the variant protein produces two fragments of 60,000 and 63,000 molecular weight when peptides are separated by sodium dodecyl sulfate-acrylamide gel electrophoresis using the discontinuous buffer system of Laemmli (Laemmli, U. K. (1970) Nature 227, 680-685). The two fragments cannot be resolved if electrophoresis is conducted using the continuous phosphate or Tris/acetate buffer systems. This increased molecular weight of the variant fragment does not appear to be due to increased glycosylation, since neither sialic acid residues nor terminal galactose units can be detected. Furthermore, the transmembrane segment of Protein 3 can be detected after proteolytic digestion at both the external and cytoplasmic membrane surfaces. These transmembrane segments of both the normal and the variant peptide have identical molecular weights of 20,000 to 21,000. These results suggest that the increased molecular weight of the variant peptide is due to the incorporation of an additional segment into that region of the molecule which is exposed at the cytoplasmic side of the membrane.

The transmembrane proteins in the plasma membrane of normal human erythrocytes. Evaluation employing lactoperoxidase and proteases.

The molecular architecture of the human erythrocyte membrane has been probed using lactoperoxidase-catalyzed iodination in conjunction with Pronase hydrolysis. Resealed, hemoglobin-free ghosts were labeled at the cytoplasmic surface and the external membrane surface was subsequently digested with Pronase. Changes in size of the components labeled at the cytoplasmic surface were readily detected by sodium dodecyl sulfate gel electrophoresis. The protein 3 molecular weight class labeled at the cytoplasmic surface was extensively hydrolyzed at the external surface to produce a major 65000 molecular weight fragment and a minor 45000 molecular weight fragment. When resealed membranes were labeled on the external surface the same 65000 molecular weight labeled component is produced. These results unequivocally demonstrate that the same polypeptides in the protein 3 molecular weight class that can be labeled by lactoperoxidase at the cytoplasmic membrane surface are digested by Pronase at the external surface and are, therefore, transmembrane components. Where it is possible to label one surface of a membrane with lactoperoxidase and reseal the membrane this procedure represents an alternate method for establishing transmembrane configuration of membrane proteins.

Glycoconnectin (PAS 2), a membrane attachment site for the human erythrocyte cytoskeleton.

The sialoglycoprotein PAS 2 is present in cytoskeletons generated by Triton X-100 extraction of isolated human erythrocyte stroma. However, removal of the peripheral cytoskeletal proteins by elution with 0.1N NaOH prior to Triton extraction renders PAS 2 Triton-soluble. This suggests association of PAS 2 with the cytoskeletal elements lining the inner surface of the erythrocyte membrane. For this reason, we are proposing the name glycoconnectin for PAS 2, since it is a glycoprotein which connects the core of the cytoskeleton to the membrane bilayer. The cytoskeletal proteins bands 4.1a,b also appear to interact directly with the membrane, since all of the other peripheral membrane proteins can be eluted with NaOH, pH 11.5, without releasing bands 4.1a,b from the membrane. Removal of spectrin and actin from the membrane results in the solubilization of both glycoconnectin and bands 4.1a,b by Triton X-100. Glycoconnectin is not present in the cytoskeletons derived from a donor whose membranes are devoid of bands 4.1a,b. These data suggest that glycoconnectin may interact directly with bands 4.1a,b.

On the hemolytic and thrombogenic potential of occluder prosthetic heart valves from in-vitro measurements.

An experimental investigation was conducted to determine the magnitude of shear stresses and areas of stasis of several types of prosthetic occluder heart valves. These experiments were performed in a steady-flow test loop using an axisymmetric aortic-shaped test chamber and an aqueous-glycerine solution. The flow loop produced a low-turbulence intensity and uniform mean velocity profile upstream of the test chamber. Tests were performed on a Kay-Shiley disk, a Bjork-Shiley tilting disk and Starr-Edwards Models 1260 and 2320 ball prostheses at Reynolds numbers between 2000 and 6200. Momentum transfer and turbulence data were obtained both around and distal to the valve occluders using laser Doppler and hot-film anemometry. The region directly surrounding the valve occluders contained the largest stresses measured. Aortic wall shear measurements revealed magnitudes potentially damaging to the vessel lining. Regions of slowly moving separated flow found to exist in these occluder valve flow fields correlated with clinical findings of thrombus formation.

A fresh look at the temporal dynamics of binocular rivalry.

Human observers viewed dichoptic orthogonal sine-wave gratings and indicated when exclusive visibility occurred in either eye. Contrast was held constant in one eye and was increased or decreased in the other eye for a number of alternation cycles (continuous presentation) or for only the duration of a single period of exclusive visibility (synchronous presentation). The synchronous presentation condition allowed us to identify the differing effects of contrast during the suppressed and during the dominant periods. Mixed phases were recorded as distinct from suppressed and dominant phases, and new classifications of compound-dominant and compound-suppressed phases are defined. The results indicate that binocular rivalry responds to stimulus contrast in two ways. 1) The duty-cycle of dominance and suppression is determined by the relative image contrast between the two eyes, with dominance of the higher contrast image being favored, and 2) the overall rate of alternation is driven by monocular image contrast during the suppressed phase (increased monocular contrast increases the alternation rate) and to a lesser extent by monocular contrast during the dominant phase (increased monocular contrast decreases the rate). A model is developed to reflect these ideas. These results support a reciprocal inhibition oscillator as the underlying mechanism of binocular rivalry.

The role of erythropoietin in the production of principal erythrocyte proteins other than hemoglobin during terminal erythroid differentiation.

Erythropoietin (EP) controls the terminal phase of differentiation in which proerythroblasts and their precursors, the colony forming units-erythroid (CFU-e), develop into erythrocytes. Biochemical studies of this hormone-directed terminal differentiation have been hindered by the lack of a homogeneous population of erythroid cells at the developmental stages of CFU-e and proerythroblasts that will synchronously differentiate in response to EP. Such a population of cells can be prepared from the spleens of mice with the acute erythroblastosis resulting from infection with anemia-inducing Friend virus (FVA). Using these FVA-infected erythroid cells, which were induced to differentiate with EP, four proteins other than hemoglobin that have key functions in mature erythrocytes were monitored during the 48-hour period of terminal differentiation. Synthesis of spectrin and membrane band 3 proteins were determined by immunoprecipitation and SDS-polyacrylamide gel electrophoresis; accumulation of the cytoskeletal protein band 4.1 was monitored by immunoblotting; carbonic anhydrase activity was measured electrometrically. Band 3 synthesis and band 4.1 accumulation could be detected only after exposure of the cells to EP. Spectrin synthesis was ongoing prior to culture with EP, but it did increase after exposure to the hormone. Carbonic anhydrase-specific activity changed very little throughout the terminal differentiation process. These results reveal at least three patterns of production of principal erythrocyte proteins during EP-mediated terminal differentiation of FVA-infected erythroid cells. Depending on the specific protein examined, de novo synthesis can be induced by EP, an ongoing production can be enhanced by EP, or the production of a protein can be completed at a developmental stage prior to EP-mediated differentiation in these cells.