Acceleration of the precession frequency for optically-oriented electron spins in ferromagnetic/semiconductor hybrids.

Time-resolved Kerr rotation measurements were performed in InGaAs/GaAs quantum wells nearby a doped Mn delta layer. Our magneto-optical results show a typical time evolution of the optically-oriented electron spin in the quantum well. Surprisingly, this is strongly affected by the Mn spins, resulting in an increase of the spin precession frequency in time. This increase is attributed to the variation in the effective magnetic field induced by the dynamical relaxation of the Mn spins. Two processes are observed during electron spin precession: a quasi-instantaneous alignment of the Mn spins with photo-excited holes, followed by a slow alignment of Mn spins with the external transverse magnetic field. The first process leads to an equilibrium state imprinted in the initial precession frequency, which depends on pump power, while the second process promotes a linear frequency increase, with acceleration depending on temperature and external magnetic field. This observation yields new information about exchange process dynamics and on the possibility of constructing spin memories, which can rapidly respond to light while retaining information for a longer period.

Efficacy of electrotactile vestibular substitution in patients with peripheral and central vestibular loss.

Vestibular dysfunction of either central or peripheral origin can significantly affect balance, posture, and gait. We conducted a pilot study to test the effectiveness of training with the BrainPort balance device in subjects with a balance dysfunction due to peripheral or central vestibular loss. The BrainPort balance device transmits information about the patient's head position via electrotactile stimulation of the tongue. Head position data is sensed by an accelerometer and displayed on the tongue as a pattern of stimulation. This pattern of stimulation moves forward, backward, and laterally on the tongue in direct response to head movements. Users of the device were trained to use this stimulation to adjust their position in order to maintain their balance. Twenty-eight subjects with peripheral or central vestibular loss were trained with the BrainPort balance device and tested using the following standardized quantitative measurements of the treatment effects: Computerized Dynamic Posturography (CDP) using the Sensory Organization Test (SOT), Dynamic Gait Index (DGI), Activities-specific Balance Confidence Scale (ABC), and Dizziness Handicap Inventory (DHI). All subjects had chronic balance problems and all but one had previously participated in vestibular rehabilitation therapy. The scores on the clinical tests upon entry into the study were compared to their scores following training with the BrainPort balance device. Our results exhibit consistent positive and statistically significant improvements in balance, posture and gait. These results exceed what could normally be achieved in three to five days of traditional balance training alone. Since this was not a controlled study, we are unable to distinguish the degree to which these improvements are attributable to training with the BrainPort balance device versus the balance exercises performed by all subjects as a part of the BrainPort training sessions. Nonetheless, after training with the BrainPort balance device, all subjects demonstrated significant improvements in performance beyond what might be expected from conventional vestibular rehabilitation therapy.

Efficacy of electrotactile vestibular substitution in patients with bilateral vestibular and central balance loss.

Patients with bilateral vestibular loss (BVL) of both central and peripheral origin experience multiple problems with balance and posture control, movement, and abnormal gait.Wicab, Inc. has developed the BrainPort balance device to transmit head position/orientation information normally provided by the vestibular system to the brain through a substitute sensory channel: electrotactile stimulation of the tongue. Head-orientation data (artificially sensed) serves as the input signal for the BrainPort balance device to control the movement of a small pattern of stimulation on the tongue that relates to head position in real-time. With training, the brain learns to appropriately interpret the information from the device and utilize it to function as it would with data from a normal-functioning natural sense. Ina total of 40 subjects trained with the BrainPort, 18 have been tested using standardized quantitative measurements of the treatment effects. A specialized set of exercises, testing, and training procedures has been developed that may serve as the course of intensive physical therapy with the BrainPort balance device. Our results demonstrate consistent positive and statistically significant balance rehabilitation effects independent of aging and etiology of balance deficit.

Functional plasticity in extrastriate visual cortex following neonatal visual cortex damage and monocular enucleation.

Neonatal lesions of primary visual cortex (areas 17, 18 and 19; VC) in cats lead to significant changes in the organization of visual pathways, including severe retrograde degeneration of retinal ganglion cells of the X/beta class. Cells in posteromedial lateral suprasylvian (PMLS) cortex display plasticity in that they develop normal receptive-field properties despite these changes, but they do not acquire the response properties of striate neurons that were damaged (e.g., high spatial-frequency tuning, low contrast threshold). One possibility is that the loss of X-pathway information, which is thought to underlie striate cortical properties in normal animals, precludes the acquisition of these responses by cells in remaining brain areas following neonatal VC damage. Previously, we have shown that monocular enucleation at the time of VC lesion prevents the X-/beta-cell loss in the remaining eye. The purpose of the present study was to determine whether this sparing of retinal X-cells leads to the development of striate-like response properties in PMLS cortex. We recorded the responses of PMLS neurons to visual stimuli to assess spatial-frequency tuning, spatial resolution, and contrast threshold. Results indicated that some PMLS cells in animals with a neonatal VC lesion and monocular enucleation displayed a preference for higher spatial frequencies, had higher spatial resolution, and had lower contrast thresholds than PMLS cells in cats with VC lesion alone. Taken together, these results suggest that preserving X-pathway input during this critical period leads to the addition of some X-like properties to PMLS visual responses.

VEP and PERG acuity in anesthetized young adult rhesus monkeys.

This study used the swept spatial-frequency method to compare retinal and cortical acuity in anesthetized young adult rhesus monkeys. Visual evoked potentials (VEPs) and pattern electroretinographic responses (PERGs) were recorded from 25 monkeys (age range: 4-12 years) anesthetized with a continuous infusion of propofol. The stimuli were temporally countermodulated sine-wave gratings that increased in spatial frequency within a 10.24-s period. All animals were refracted using acuity estimated from the zero micro-volt intercept of the linear regression of evoked potential amplitude on spatial frequency. Average sweep acuities were 23.7 cycles/deg +/- 1.5 S.E.M. and 23.1 cycles/deg +/- 1.8 S.E.M. for the PERG and VEP, respectively. VEP and PERG acuities were within the range expected based on acuities estimated from behavioral studies in macaques. PERG and VEP acuities were highly correlated (r = 0.90) and equally sensitive to spherical blur. On a subset of animals, test-retest reliability of animals, and interocular correlations, were high (r = 0.87 and r = 0.83, respectively). Increasing propofol dosage 8-fold did not degrade PERG or VEP acuity. This study demonstrates that high spatial-frequency acuities can be rapidly obtained from young adult rhesus monkeys under a wide dose range of propofol anesthesia using the swept spatial-frequency method.

Right subscapular artery catheterization for balloon valvuloplasty of critical aortic stenosis in infants.

This study was performed to evaluate the utility and safety of catheterizing the right subscapular artery for balloon valvuloplasty of critical aortic stenosis in infants. Twenty-one patients, age 20 days to 17 months, underwent attempted valvuloplasty through the surgically exposed right subscapular artery. Five or 7Fr catheters with balloon diameters of 7 to 10 mm were used. Valvuloplasty was successfully performed using this approach in 11 patients. In 2 other patients, the subscapular artery would not accommodate the balloon angioplasty catheter (7Fr), and the arteriotomy was extended into the axillary artery. In these 13 patients, the peak systolic pressure gradient across the aortic valve was decreased from 85 +/- 23 to 33 +/- 7 mm Hg. Moderate aortic regurgitation developed in 3 patients. In the remaining 8 patients, valvuloplasty could not be performed through the right subscapular artery. In 2 patients, the right subclavian artery was anomalous and led to the descending aorta. In 6 small patients, no catheter could be advanced across the aortic valve. In 1 of these patients, a guidewire perforated a coronary sinus of Valsalva causing death. Overall, valvuloplasty using the right subscapular arterial approach was successful in 13 of 19 infants (68%) with normal right subclavian arteries, including all 10 such patients weighing > or = 5.5 kg. No clinically significant peripheral vascular complications or brachial plexus injuries occurred. Thus, the right subscapular arterial approach is an alternative route to be considered when planning balloon aortic valvuloplasty in infants.

Are neurons in cat posteromedial lateral suprasylvian visual cortex orientation sensitive? Tests with bars and gratings.

There is controversy in the literature concerning whether or not neurons in the cat's posteromedial lateral suprasylvian (PMLS) visual cortex are orientation selective. Previous studies that have tested cells with simple bar stimuli have found that few, if any, PMLS cells are orientation selective. Conversely, studies that have used repetitive stimuli such as gratings have found that most or all PMLS cells are orientation selective. It is not known whether this difference in results is due to the stimuli used or the laboratories using them. The present experiments were designed to answer this question by testing individual PMLS neurons for orientation sensitivity with both bar and grating stimuli. Using quantitative response measures, we found that most PMLS neurons respond well enough to stationary flashed stimuli to use such stimuli to test for orientation sensitivity. On the basis of these tests, we found that about 85% of the cells with well-defined receptive fields are orientation sensitive to flashed gratings, and a similar percentage are orientation sensitive to flashed bars. About 80% of the cells were orientation sensitive to both types of stimuli. The preferred orientations typically were similar for the two tests, and they were orthogonal to the preferred direction of movement. The strength of the orientation sensitivity (measured as the ratio of discharge to the preferred and nonpreferred orientations) was similar to both types of stimuli. However, the width of the orientation tuning curves was systematically broader to bars than to gratings. Several hypotheses are considered as to why previous studies using bars failed to find evidence for orientation sensitivity. In addition, a mechanism for the difference in orientation tuning to bars and gratings is suggested.

Selective modulation of band 4.1 binding to erythrocyte membranes by protein kinase C.

We have studied the effects of band 4.1 phosphorylation on its association with red cell inside-out vesicles stripped of all peripheral proteins. Band 4.1 bound to these vesicles in a saturable manner, and binding was characterized by a linear Scatchard plot with an apparent Kd of 1-2 x 10(-7) M. Phosphorylation of band 4.1 by purified protein kinase C reduced its ability to bind to membranes, resulting in a reduction in the apparent binding capacity of the membrane by 60-70% but little or no change in the apparent Kd of binding. By contrast, phosphorylation of band 4.1 by cAMP-dependent kinase had no effect on membrane binding. Digestion of the stripped inside-out vesicles with trypsin cleaved 100% of the cytoplasmic domain of band 3 but had little or no effect on glycophorin. Binding of band 4.1 to these digested vesicles was reduced by 70%. Phosphorylation of band 4.1 by protein kinase C had no effect on its binding to the digested vesicles, suggesting that the cytoplasmic domain of band 3 contained the phosphorylation-sensitive binding sites. This was confirmed by direct measurement of band 4.1 binding to the purified cytoplasmic domain of band 3. Phosphorylation of band 4.1 by protein kinase C reduced its binding to the purified 43-kDa domain by as much as 90%, while phosphorylation by cAMP-dependent kinase was without effect. These results show a selective effect of protein kinase C phosphorylation on the binding of band 4.1 to one of its membrane receptors, band 3, and suggest a mechanism whereby one of the key red cell-skeletal membrane associations may be modulated.

Isolation and characterization of Chinese hamster ovary cell variants deficient in the expression of fibronectin receptor.

Chinese hamster ovary cell populations were enriched for cells displaying low surface expression of the 140-kD integrin fibronectin receptor (FnR) by means of fluorescence-activated cell sorting using monoclonal anti-FnR antibodies. Selected cells were cloned by limiting dilution, and the resulting clones were screened for low cell surface FnR expression by ELISA. Two multiply sorted populations gave rise to variant clones possessing approximately 20 or 2% FnR expression, respectively, compared with wild-type cells. Growth rates of the "20%" and "2%" clones on serum-coated plastic dishes were similar to that of wild-type cells. Variant cells expressing 20% FnR could attach and spread on substrata coated with purified fibronectin, although somewhat more slowly than wild-type cells, while cells expressing 2% FnR could not attach or spread. Cells from all variant clones attached normally to vitronectin substrata, but some of the 2% clones displayed altered morphology on this type of substratum. Motility assays in blind well chambers showed a correlation of movement with level of expression of FnR. The number of cells migrating in response to fibronectin was greatly reduced compared with wild-type cells for the 20% FnR variant clones, while variant clones with 2% FnR showed virtually no migratory activity. Surface labeling with 125I and immunoaffinity purification of FnR showed reduced levels of intact FnR on the plasma membranes of variants with 20% FnR, while none was detected in variants expressing 2% FnR. Nevertheless, beta subunits were detected on the surfaces of all variant clones. Immunoblots of cell lysates from wild-type cells and from both types of variant clones showed substantial amounts of FnR beta chain as well as enhanced amounts of a pre-beta moiety in the variants. alpha chain was markedly reduced in the 20% variants and essentially absent in the 2% variants, indicating that failure to assemble intact FnR in these variants was due to deficiencies of alpha chain production. Dot blots of total mRNA from a representative clone expressing 20% FnR showed reduced levels of material hybridizing to an 0.97-kb hamster FnR alpha chain cDNA probe as compared with wild type, while mRNA from a representative clone expressing 2% FnR had no detectable hybridizable RNA; this seems to agree well with the results obtained by immunoblotting. Thus, the defect in the variant clones seems to be due to reduced levels of alpha chain mRNA leading to a deficit of mature FnR and consequent alterations in cell adhesion and motility on fibronectin substrata.

Wheat germ agglutinin but not concanavalin A modulates protein kinase C-mediated phosphorylation of red cell skeletal proteins.

Human red blood cells contain protein kinase C (PKC) which acts exclusively on the membrane skeletal proteins band 4.1, band 4.9 and adducin. PKC activity can be stimulated by the addition of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate to intact cells. Phosphorylation of band 4.1 by PKC in vitro results in a dramatic reduction in band 4.1 binding to spectrin and actin, as well as to the cytoplasmic domain of band 3. Here we show that the lectin wheat germ agglutinin (WGA), which binds to the extracellular domain of glycophorin results in the inhibition of PKC catalyzed phosphorylation of band 4.1, band 4.9 and likely adducin as well. The lectin concanavalin A, which binds to band 3 was without effect. Our results suggest that the binding of WGA to glycophorin results in a major rearrangement of the membrane skeletal network which correlates with reduced phosphorylation of membrane skeletal proteins by PKC.

(Arg-Gly-Asp)n-albumin conjugates as a model substratum for integrin-mediated cell adhesion.

We have prepared protein-peptide conjugates composed of bovine serum albumin (BSA) derivatized with short peptides containing the Arg-Gly-Asp (RGD) sequence derived from the adhesion site of fibronectin. The RGD-BSA conjugates were used to coat tissue culture plastic surfaces which then served as substrata in cell adhesion experiments. Our results indicate that the efficiency of adhesion to RGD-BSA-coated surfaces is highly dependent on the valency of the (RGD)n-BSA conjugates. For example, on surfaces with approximately equal amounts of RGD ligand, CHO cells adhered virtually 100% to the (RGD)n-BSA (n = 20.8) conjugate and not at all to the (RGD)n-BSA (n = 3.5) conjugate. Adhesion on (RGD)n-BSA-coated substrata and on fibronectin- or vitronectin-coated substrata was also examined in terms of the relationship between cell adhesion and the intermolecular distances of adsorbed proteins. It was observed that for substrata coated with relatively compact, symmetric molecules, such as RGD-BSA or vitronectin, adhesion dropped off sharply as intermolecular distances increased; by contrast, for fibronectin, a large asymmetric molecule, adhesion declined more gradually as intermolecular distances increased. Finally, we have examined the role of different cell-surface receptors in the process of adhesion to RGD-BSA substrata. Interestingly, competition and blocking experiments with antibodies and with soluble competing proteins suggest that it is the vitronectin receptor rather than the fibronectin receptor which mediates adhesion to RGD-BSA.

Phorbol ester modulation of integrin-mediated cell adhesion: a postreceptor event.

Chinese hamster ovary (CHO) suspension culture cells adhere readily to substrata coated with extracellular matrix proteins such as fibronectin, vitronectin, or laminin. In the case of fibronectin, it is known that adhesion is mediated by an integrin-type, cell surface fibronectin receptor (FnR). We demonstrate here that treatment of CHO cells with submicromolar concentrations of phorbol ester produces a remarkable increase in the ability of these cells to adhere to fibronectin. Both the rate of adhesion and the efficiency of adhesion are enhanced about four- to fivefold. Further, phorbol ester treatment renders the fibronectin-mediated adhesion process less sensitive to inhibitors, including GRGDSP peptide and PB1, a monoclonal anti-FnR antibody. By contrast, nonspecific adhesion processes, for example cell attachment to substrata coated with polylysine or concanavalin A, are not affected by phorbol ester treatment. Thus integrin-mediated adhesion is modulated by phorbol esters, but nonspecific adhesion is not. Neither the number of cell surface FnRs nor the receptor affinity, as measured by 125I-fibronectin and 125I-anti-FnR antibody binding, is altered by phorbol ester treatment. Thus, the effect of phorbol ester on cell adhesion seems to occur at a step subsequent to initial ligand-receptor binding events. Since phorbol ester is a potent activator of protein kinase C, we examined phosphorylation patterns in control and phorbol-treated cells. In immunoprecipitates of lysates from suspension culture cells, there was no evidence of phorbol ester-stimulated phosphorylation of FnR or of talin, a protein thought to interact with FnR. These results suggest that phorbol ester effects on fibronectin-dependent adhesion are not due to phosphorylation of the FnR itself but rather may be due to postreceptor events, possibly the phosphorylation of cytoskeletal proteins involved in integrin-mediated adhesion.

Modulation of red cell band 4.1 function by cAMP-dependent kinase and protein kinase C phosphorylation.

Human erythrocyte protein 4.1 is phosphorylated in vivo by several protein kinases including protein kinase C and cAMP-dependent kinase. We have used cAMP-dependent kinase purified from red cells and protein kinase C purified from brain to test the effects of phosphorylation on band 4.1 function. In solution, each kinase catalyzed the incorporation of 1-4 mol of PO4/mol of band 4.1. Phosphorylation of band 4.1 by each kinase resulted in a significant (50-80%) reduction in the ability of band 4.1 to promote spectrin binding to F-actin. Direct measurement of spectrin-band 4.1 binding showed that phosphorylation by each kinase also caused dramatic reduction in this association. Phosphorylation of band 4.1 by each kinase for increasing time periods enabled us to demonstrate an approximately linear inverse relationship between PO4 incorporation into band 4.1 and spectrin binding. These results show that phosphorylation of band 4.1 by cAMP-dependent kinase and protein kinase C may be central to the regulation of red cell cytoskeletal organization and membrane mechanical properties.