The influence of motor function on processing speed in preterm and term-born children.

This study investigates the relationship between motor function and processing speed in preterm children. Processing speed was compared in 145 adolescents, born 25-41 weeks gestational age, utilizing tasks including differing motor demands. The influence of motor cortex excitability and functional motor skills on task performance was assessed. For tasks with motoric demands, differences in performance between preterm and term-born children were mediated by the relationship between gestational age, corticomotor excitability, and motor function. There were no differences in non-motor processing speed task performance between preterm and term-born children. Measures of processing speed may be confounded by a timed motor component.

Purification and secondary structure determination of simian immunodeficiency virus p27.

We have developed a novel method for the expression and purification of p27, the major core protein of simian immunodeficiency virus. Circular dichroism measurements of purified p27 were used to determine the relative amounts of alpha-helix, beta-sheet and unordered secondary structural elements. These empirically determined values appear to be inconsistent with previously published theoretical models based on homology comparisons.

A novel method for the purification of HIV-1 p24 protein from hybrid Ty virus-like particles (Ty-VLPs).

The self-assembly properties of a protein encoded by the yeast retrotransposon Ty can be exploited to produce large amounts of recombinant, particulate fusion proteins as hybrid Ty virus-like particles (Ty-VLPs). This system has now been adapted to allow the release of the additional protein by incorporation of a protease cleavage site between the yeast carrier protein and the protein of interest. The purification of the additional protein is facilitated by exploiting the ease with which Ty-VLPs can be purified from other yeast cell components due to their particulate nature. We have used this modified system to produce hybrid particles containing the HIV-1 p24 protein downstream of the recognition sequence for the blood coagulation factor Xa. The p24 was released from the particles by proteolytic cleavage and rapidly separated from the residual particulate material using centrifugation and standard chromatography techniques. This procedure has been used to purify milligram quantities of HIV-1 p24 protein that reacts with anti-p24 sera and elicits the production of p24-specific antibodies in experimental animals.

Performance characteristics of a novel immunoassay based on hybrid Ty virus-like particles (Ty-VLPs): rapid differentiation between HIV-1 and HIV-2 infection.

Recombinant antigens containing all or parts of the HIV-1 proteins p24, Nef and gp41 and HIV-2 gp36 have been purified and used to develop a rapid immunoassay to detect and differentiate between HIV-1 and HIV-2 antibodies in a single test. The antigens were produced as particulate fusion proteins by exploiting the ability of a protein encoded by the yeast retrotransposon Ty to assemble into virus-like particles (Ty-VLPs). Hybrid HIV: Ty-VLPs carrying each of the antigens were applied to nitrocellulose strips at specified locations in a slot-blot format and then used to detect antibodies present in human serum and plasma samples of diverse geographical origin. Previously confirmed HIV-1- and HIV-2-positive samples were readily and reliably identified. The assay was used to identify a case of HIV-2 infection in an African woman who had been resident in the Oxford region for the last 3 years and to analyse the prevalence of anti-HIV antibodies in a longitudinal study of seroconverting patients. We also demonstrate that the assay works efficiently with whole blood.

Retroelement particles as purification, presentation and targeting vehicles.

The manipulation of retrotransposon and retroviral particles to carry biologically active molecules is becoming feasible. In addition, recent experiments suggest that it may be possible to target these engineered particles to specific cell types. This has implications for gene therapy, biological drug delivery and vaccine design.

Properties of brain spectrin (fodrin).

Fodrin, a protein from bovine brain, immunologically related to spectrin, is shown, unlike some other proteins of generally similar appearance in the electron microscope, to resemble spectrin closely in its most distinctive structural characteristic, the very high alpha-helix content. Like spectrin, it is also insoluble below pH 5. One of the subunits only is phosphorylated by the cAMP-independent red cell membrane kinase, that phosphorylates the smaller subunit of spectrin. Fodrin also forms a ternary complex with F-actin and the third constituent of the red cell membranes cytoskeleton, protein 4.1. In the presence of 4.1 the interaction between fodrin and F-actin is enhanced. It is surmised that fodrin plays an analogous functional role in neuronal cells to that of spectrin in the red cell.

Hybrid Ty virus-like particles.

Vaccines need to activate antigen presenting cells, overcome genetic restriction in T-cell responses and elicit both T and B memory cells. In order to produce recombinant vaccines which can do this, considerable effort has been put into developing particulate antigen presentation systems to generate polyvalent, high molecular weight antigens which should maximally stimulate the immune system. One such antigen-carrier system is based on the ability of a protein encoded by the yeast retrotransposon, Ty, to self-assemble into virus-like particles (VLPs). Ty-fusion proteins retain this ability to form particles and a range of hybrid VLPs carrying a variety of heterologous antigens have been produced and shown to elicit potent immune responses. Hybrid VLPs carrying human immunodeficiency virus (HIV) antigens stimulate the three main components of the immune system, namely antibody synthesis, T-cell proliferative responses and cytotoxic T-lymphocyte (CTL) responses.

Yeast retrotransposon particles as antigen delivery systems.

The development of technologies to produce recombinant proteins for use in the pharmaceutical industry has made substantial advances, in particular in the area of generating antigens containing multiple copies of important immunological regions. One such antigen-carrier system is based on the ability of a protein encoded by the yeast retrotransposon, Ty, to self-assemble into virus-like particles. Ty-fusion proteins retain this ability to form particles, and a range of hybrid VLPs carrying a variety of heterologous antigens have been produced and shown to induce potent immune responses. In particular, hybrid VLPs carrying the core protein p24 of HIV (p24-VLPs) have been shown to induce antibody and T-cell proliferative responses in both experimental animals and human volunteers, and immunization of rabbits with VLPs carrying the principal neutralizing determinant of HIV (V3-VLPs) resulted in the induction of neutralizing antibody responses and T-cell proliferation. Further studies with V3-VLPs have shown that this particulate antigen stimulates enhanced V3-specific lymphoproliferative responses as compared to whole recombinant gp120 or to V3 peptide conjugated to albumin. The V3-VLPs also induce potent CTL responses following immunization of mice in the absence of adjuvant. These responses are MHC class I restricted and are mediated by CD8-positive cells. These observations therefore demonstrate that hybrid Ty-VLPs induce both humoral and cellular immune responses against HIV and suggest that these immunogens may be important in combatting AIDS and other infections.

Production and purification of hybrid Ty-VLPs.

This article describes how pure Ty-VLPs (virus-like particles) can be prepared from hybrid Ty-VLPs. Many different hybrid Ty-VLPs have been produced and may be easily purified. Since the sedimentation properties of different hybrid Ty-VLPs are similar, a simple purification process can be used for any VLP. This fast, versatile, and easy process allows for the production of a variety of recombinant proteins.

Production and Purification of Hybrid Ty-VLPs.

The self-assembly properties of a protein encoded by the TYA gene of the yeast Ty element can be exploited to produce hybrid Ty-VLPs (virus-like particles) (1,2). There has been developed a series of expression vectors that allow the construction of Ty fusion genes containing protein coding sequences of interest (see Chapter 24 ). Many different hybrid Ty-VLPs have now been produced that carry additional proteins that range in size from 3 to 42 kDa. These include regions from human immunodeficiency virus-1 (HIV-1) env, pol, tat, rev, nef, and vif genes; influenza virus hemagglutinin; human α-interferon, feline leukemia virus env, and bovine papillomavirus El and E2 (1-5 and unpublished data).

The string measure of the ERP: what does it measure?

The event-related brain potential (ERP) has been investigated extensively inan effort to understand the neurophysiological bases of intelligence. Measures derived from the ERP have been used as indices of intelligence, particularly the string measure of the complexity of the ERP. However, the string measure has been criticised for being non-specific and for being dependent on ERP amplitude. These criticisms were tested by investigating relationships between ERP string measure, ERP amplitude measures, and the ERP power spectrum. It was found that the string measure was non-specific in that it indexes both low and high frequency event-related activity; the string measure is also dependent on ERP amplitude. The string measure is therefore not a valid measure of the ERP. It was concluded that the string measure should be abandoned; human intelligence cannot map in a simple way onto gross measures of scalp-recorded electrocortical activity.

Screening for depression and anxiety in spinal cord injury with DASS-21.

STUDY DESIGN: Comparison of two self-report instruments with a structured diagnostic interview. OBJECTIVE: To investigate the properties of the Depression Anxiety Stress Scales-21 (DASS-21) in patients with spinal cord injuries. SETTING: South Australian Spinal Cord Injuries Service, Hampstead Rehabilitation Centre, Northfield, South Australia. METHODS: Forty paraplegic or tetraplegic patients participated. Two self-report measures, DASS-21 and Brief Symptom Inventory (BSI), assessed Depression, Anxiety and Stress. These measures were compared with each other and with diagnoses based on the Mini International Neuropsychiatric Interview. RESULTS: Mean scores on both self-report measures were below clinical threshold levels. Prevalence rates of anxiety and depression were higher on DASS-21 than on BSI. DASS-21 was as sensitive as BSI, but had lower specificity to detect anxiety and depression. CONCLUSION: DASS-21 is a promising screening measure for patients with spinal cord injury in a rehabilitation setting. It has greater sensitivity for identifying those with possible anxiety disorders than it does for those with depressive disorders.

Streaming and bouncing: observations on motion defined objects.

When two identical objects move in opposite directions on the same path and at the same speed, they can appear, after crossing over to continue n their original directions (streaming), or to reverse direction (bouncing). In order to be able to man pulate visibility by adding no se, we used objects defined by contrast, flicker, or motion, and thereby extended previous findings on luminance-defined objects. Two identical rectangles (1.1 x 1.4 degrees) composed of random dot patterns moved toward each other at a speed of 3.5 degrees/s. In experiment I we used backgrounds of a grey field, static random dots, or dynamic noise, and examined the effect of introducing a pause in motion and a visual distractor. In experiment 2 we introduced visual noise at four levels. For all three types of motion display, we found an increase in the proportion of the bouncing percept when either a pause in motion or an attentional distractor was introduced. Experiment 2 showed that neither of these effects depends on the visibility of the moving objects. An increase in the bouncing percept, due to a pause in motion or the distraction of attention, can be observed for all types of object definition, and is not affected by decreas ng the visibility of the motion-defined objects.This finding suggests that the role of attention in determining the perception of bouncing does not lie in the modulation of object visibility.

Determining the visibility of noise in motion signals.

A procedure is described for generating stimuli to study the detection of noise components in motion signals. By using random dots with intensities distributed according to a Gaussian probability function, a temporally and spatially continuous mixture of signal and noise components can be realized in random dot kinematograms. These stimuli were used in a noise detection task, a signal detection task and a direction discrimination task. Signal-to-noise ratio ('coherence') thresholds for the signal detection and direction discrimination tasks were consistent with previous research. Noise can be detected at levels of approximately 0.5-2.5%, depending on the size of the motion stimulus. We argue that the noise in the motion stimulus becomes detectable when it exceeds the noise intrinsic to the various stages of motion processing. Therefore,the method provides a simple procedure for obtaining measures of equivalent input noise and can be used for estimating internal noise levels of motion processing mechanisms.

Interaction of first- and second-order direction in motion-defined motion.

Motion-defined motion can play a special role in the discussion of whether one or two separate systems are required to process first- and second-order information because, in contrast to other second-order stimuli, such as contrast-modulated contours, motion detection cannot be explained by a simple input nonlinearity but requires preprocessing by motion detectors. Furthermore, the perceptual quality that defines an object (motion on the object surface) is identical to that which is attributed to the object as an emergent feature (motion of the object), raising the question of how these two object properties are linked. The interaction of first- and second-order information in such stimuli has been analyzed previously in a direction-discrimination task, revealing some cooperativity. Because any comprehensive integration of these two types of motion information should be reflected in the most fundamental property of a moving object, i.e., the direction in which it moves, we now investigate how motion direction is estimated in motion-defined objects. Observers had to report the direction of moving objects that were defined by luminance contrast or in random-dot kinematograms by differences in the spatiotemporal properties between the object region and the random-noise background. When the dots were moving coherently with the object (Fourier motion), direction sensitivity resembled that for luminance-defined objects, but performance deteriorated when the dots in the object region were static (drift-balanced motion). When the dots on the object surface were moving diagonally relative to the object direction (theta motion), the general level of accuracy declined further, and the perceived direction was intermediate between the veridical object motion direction and the direction of dot motion, indicating that the first- and second-order velocity vectors are somehow pooled. The inability to separate first- and second-order directional information suggests that the two corresponding subsystems of motion processing are not producing independent percepts and provides clues for possible implementations of the two-layer motion-processing network.

Interaction of calmodulin with the red cell and its membrane skeleton and with spectrin.

The binding of calmodulin to red cell membrane cytoskeletons and to purified spectrin from red cells and bovine brain spectrin (fodrin) has been examined. Under physiological solvent conditions binding can be measured by ultracentrifugal pelleting assays. The membrane cytoskeletons contained a single class of binding sites, with a concentration similar to that of spectrin dimers and an association constant of 1.5 X 10(5) M-1. Binding is calcium dependent and is suppressed by the calmodulin inhibitor trifluoperazine. The binding showed a marked dependence on ionic strength, with a maximum at 0.05 M, and a steep dependence on pH, with a maximum at pH 6.5. It was unaffected by 5 mM magnesium. An azidocalmodulin derivative, under the conditions of our experiments, did not label the spectrin-containing complex, although it could be used to demonstrate binding to fodrin. Binding of calmodulin to spectrin tetramers and fodrin in solution could be demonstrated by a pelleting assay after addition of F-actin. Calculations (which are necessarily rough) suggest that at the free calcium concentration prevailing in a normal red cell about 1 in 20 of the calmodulin binding sites in spectrin will be occupied; this proportion will rise rapidly with increasing intracellular calcium. To determine whether inhibition of calmodulin binding to red cell proteins disturbs the control of cell shape, as has been suggested, calcium ions were removed from the cell by addition of an ionophore and of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to the external medium. This did not affect the discoid shape. Trifluoperazine still induced stomatocytosis, exactly as in untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

A case study and use of sedimentation equilibrium analytical ultracentrifugation as a tool for biopharmaceutical development.

Analytical ultracentrifugation (AUC) has reemerged as a powerful technique for protein characterisation. We report the pivotal role sedimentation equilibrium AUC has played in the development of macrophage inflammatory protein-1 alpha (MIP-1 alpha) as a protein therapeutic. MIP-1 alpha has potential clinical applications in cancer but its clinical use is limited, since it associates to form large insoluble aggregates in physiological buffers. Using AUC as a screening technique, we have produced a biologically active variant of MIP-1 alpha, BB-10010, which has a reduced tendency to aggregate in physiological buffers. The aggregation of protein based pharmaceuticals is routinely monitored by size exclusion chromatography (SEC). Comparison of the data acquired by SEC and AUC, demonstrates that owing to the complexity of BB-10010, AUC analysis is required in addition to SEC to provide a rigorous characterisation of molecular association. This work has been extended to include the use of AUC as an analytical tool to monitor the quality of BB-10010 during formulation and stability studies.

Hybrid human immunodeficiency virus Gag particles as an antigen carrier system: induction of cytotoxic T-cell and humoral responses by a Gag:V3 fusion.

In attempts to increase the immunogenicity of recombinant antigens, a number of particulate antigen presentation systems have been developed. In this study, we used human immunodeficiency virus Gag particles as carriers for the human immunodeficiency virus envelope V3 region. Gag:V3 fusion proteins were expressed from baculovirus expression vectors; they migrated to the insect cell membrane and budded from the cells as hybrid particles. An immunization study carried out with rats showed that the particles elicited a strong anti-Gag antibody response and a weak antibody response to the V3 region. A strong anti-V3 cytolytic T-cell response was elicited in immunized mice. These data show that retroviral Gag particles can be used as antigen presentation vehicles.

Analysis of TYA protein regions necessary for formation of the Ty1 virus-like particle structure.

The yeast retrotransposon, Ty1, produces a macromolecular structure known as a virus-like particle (VLP) as an essential part of its replication cycle. The Ty1 Gag-like structural protein TYA, p1-440, alone is capable of directing assembly of the VLP. In order to determine the TYA sequences required for assembly, we have produced a series of truncated and deleted TYA forms and assessed their ability to assemble into particles. Removal of 100 amino acids from the C-terminus renders the TYA protein, p1-340, incapable of particle assembly; however, p1-363 with 77 residues missing from the C-terminus is capable of assembly. Removal of 40 amino acids from the N-terminus (p41-440 and p41-381) does not affect particle formation but more severely N-truncated forms, p71-381 and p100-381, are present as large aggregates within the cells and are therefore either incapable of or unavailable for VLP formation. Analysis of an internally deleted TYA, p1-381 delta 62-114, has identified this as a possible region of the TYA protein important for subunit:subunit interactions during the particle assembly.