SPINE workshop on automated X-ray analysis: a progress report.

The Structural Proteomics In Europe (SPINE) consortium contained a workpackage to address the automated X-ray analysis of macromolecules. The aim of this workpackage was to increase the throughput of three-dimensional structures while maintaining the high quality of conventional analyses. SPINE was able to bring together developers of software with users from the partner laboratories. Here, the results of a workshop organized by the consortium to evaluate software developed in the member laboratories against a set of bacterial targets are described. The major emphasis was on molecular-replacement suites, where automation was most advanced. Data processing and analysis, use of experimental phases and model construction were also addressed, albeit at a lower level.

First steps towards effective methods in exploiting high-throughput technologies for the determination of human protein structures of high biomedical value.

The EC 'Structural Proteomics In Europe' contract is aimed specifically at the atomic resolution structure determination of human protein targets closely linked to health, with a focus on cancer (kinesins, kinases, proteins from the ubiquitin pathway), neurological development and neurodegenerative diseases and immune recognition. Despite the challenging nature of the analysis of such targets, approximately 170 structures have been determined to date. Here, the impact of high-throughput technologies, such as parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens or the use of mass spectrometry to assist sample preparation, on the structural biology of mammalian protein targets is illustrated through selected examples.

Small substrate, big surprise: fold, function and phylogeny of dihydroxyacetone kinases.

Dihydroxyacetone (Dha) kinases are a family of sequence-conserved enzymes which utilize either ATP (in animals, plants and eubacteria) or phosphoenolpyruvate (PEP, in eubacteria) as their source of high-energy phosphate. The kinases consist of two domains/subunits: DhaK, which binds Dha covalently in hemiaminal linkage to the Nepsilon2 of a histidine, and DhaL, an eight-helix barrel that contains the nucleotide-binding site. The PEP-dependent kinases comprise a third subunit, DhaM, which rephosphorylates in situ the firmly bound ADP cofactor. DhaM serves as the shuttle for the transfer of phosphate from the bacterial PEP: carbohydrate phosphotransferase system (PTS) to the Dha kinase. The DhaL and DhaK subunits of the PEP-dependent Escherichia coli kinase act as coactivator and corepressor of DhaR, a transcription factor from the AAA(+) family of enhancerbinding proteins. In Gram-positive bacteria genes for homologs of DhaK and DhaL occur in operons for putative transcription factors of the TetR and DeoR families. Proteins with the Dha kinase fold can be classified into three families according to phylogeny and function: Dha kinases, DhaK and DhaL homologs (paralogs) associated with putative transcription regulators of the TetR and DeoR families, and proteins with a circularly permuted domain order that belong to the DegV family.

Examining the paradoxical relation between number of spikes and gaze amplitude in abducens neurons.

During head-unrestrained gaze shifts, the number of spikes in the burst of abducens neurons increases with gaze amplitude, even when corrected for the component of the discharge related to the change in eye position. We examine this paradoxical dissociation between the number of spikes and eye amplitude, which occurs because eye amplitude in the head saturates for larger gaze shifts. First, we show that the extra spikes are unlikely to be due to antagonist muscle loading because the abducens neurons are completely silent during large gaze shifts when the muscle acts as an antagonist. Next, we divide the firing rate profile of abducens neurons into terms that represent signals related to eye position, velocity, and acceleration; a d.c. offset term specifying the firing associated with straight-ahead gaze; and a slide term, which compensates for the zero of the oculomotor plant. Then we examine the contribution of each term to the number of spikes recorded. A comparison of the number of spikes with the integral of the fitted function, combining all of the terms, for the duration of the burst reveals that the simulation captures much of the actual data. However, even a model with a slide term cannot reproduce the nonlinear relationship of the number of spikes with amplitude that characterizes large gaze shifts.

Multimodal signal integration in vestibular neurons of the primate fastigial nucleus.

The rostral fastigial nucleus contains vestibular neurons, which presumably are involved in spinal mechanisms (neck, gait, posture) and which are not modulated with individual eye movements. Single-unit recordings in the alert behaving monkey during natural stimulus conditions reveal that virtually all neurons demonstrate integration of several sensory inputs. This applies not only for canal-canal and canal-otolith interaction, but also for otolith-otolith interaction. There is also some evidence that most neurons receive not only an utriculus but also a sacculus input. Furthermore, most neurons also respond to large-field optokinetic stimulation, reflecting visual-vestibular interaction. Neurons are also affected by the head on trunk position, which would allow these neurons to operate in a body-centered rather than a head-centered reference frame. These complex, multisensory features could permit fastigial nucleus neurons to rather specifically affect spinal motor functions.

Carbohydrate transporters of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS).

The glucose transporter of Escherichia coli couples translocation with phosphorylation of glucose. The IICB(Glc) subunit spans the membrane eight times. Split, circularly permuted and cyclized forms of IICB(Glc) are described. The split variant was 30 times more active when the two proteins were encoded by a dicistronic mRNA than by two genes. The stability and activity of circularly permuted forms was improved when they were expressed as fusion proteins with alkaline phosphatase. Cyclized IICB(Glc) and IIA(Glc) were produced in vivo by RecA intein-mediated trans-splicing. Purified, cyclized IIA(Glc) and IICB(Glc) had 100% and 30% of wild-type glucose phosphotransferase activity, respectively. Cyclized IIA(Glc) displayed increased stability against temperature and GuHCl-induced unfolding.

Canal-otolith interaction in the fastigial nucleus of the alert monkey.

To determine the contribution of the otoliths as well as the horizontal and vertical semicircular canals to the response of "vestibular only" neurons in the rostral fastigial nucleus of the alert monkey, we applied natural sinusoidal vestibular stimuli (0.6 Hz; +/-15 deg) around different axes. During the experiment the monkey sat erect in a primate chair with the head immobile. Semicircular canal responses were investigated during tilted yaw stimulation around an earth vertical axis. The tilt angle was varied by 30 deg and included the optimal plane for horizontal canal stimulation (15 deg nose down from the stereotactic plane). The otoliths and mainly the vertical canals made contributions during stimulation around an earth-fixed horizontal axis (vertical stimulation). Head orientation was also slowly altered (2-3 deg/s) over a range of 180 deg under both stimulus conditions (tilted yaw and vertical stimulation). Neuronal data for each paradigm were fitted by a least squares best-sine function. Computation of the hypothetical contributions made by all three pairs of semicircular canals and the otoliths to these responses showed that 74% of the 46 neurons investigated received an otolith input; in most instances it was combined with a canal input. Neurons most often received input from the horizontal and vertical canals as well as the otoliths. Only a minority of neurons received a purely otolith (13%), vertical canal (13%), or horizontal canal (4%) input. Conventional criteria (head position-related activity, spatiotemporal convergence, STC) failed to detect an otolith contribution in several such instances. Thus, canal-otolith convergence is the general rule at this central stage of vestibular information processing in the fastigial nucleus. The large variety of response types allows these neurons to participate in multiple tasks of vestibulospinal movement control.

Fastigial nucleus activity during different frequencies and orientations of vertical vestibular stimulation in the monkey.

Neurons in the rostral part of the fastigial nucleus (FN) respond to vestibular stimulation but are not related to eye movements. To understand the precise role of these vestibular-only neurons in the central processing of vestibular signals, unit activity in the FN of alert monkeys (Macaca mulatta) was recorded. To induce vestibular stimulation, the monkey was rotated sinusoidally around an earth-fixed horizontal axis at stimulus frequencies between 0.06 (+/-15 degrees) and 1.4 Hz (+/-7.5 degrees). During stimulation head orientation was changed continuously, allowing for roll, pitch, and intermediate planes of orientation. At a frequency of 0.6 Hz, 59% of the neurons had an optimal response orientation (ORO) and a null response (i.e., no modulation) 90 degrees apart. The phase of neuronal response was constant except for a steep shift of 180 degrees around the null response. This group I response is compatible with a semicircular canal input, canal convergence, or a single otolith input. Several other features indicated more complex responses, including spatiotemporal convergence (STC). 1) For 35% of the responses at 0.6 Hz, phase changes were gradual with different orientations. Fifteen percent of these had a null response (group II), and 20% showed only a minimal response but no null response (group III). The remaining responses (6%), classified as group IV, were characterized by a constant sensitivity at different orientations in most instances. 2) For the vast majority of neurons, the stimulus frequency determined the response group, i.e., an individual neuron could show a group I response at one frequency and a group II (III or IV) response at another frequency. 3) ORO changed with frequency by >45 degrees for 44% of the neurons. 4) Although phase changes at different frequencies were close to head velocity (+/-45 degrees ) or head position (+/-45 degrees ) for most neurons, they exceeded 90 degrees for 29% of the neurons between 0.1 and 1.0 Hz. In most cases, this was a phase advance. The change in sensitivity with change in frequency showed a similar pattern for all neurons; the average sensitivity increased from 1.24 imp. s-1. deg-1 at 0.1 Hz to 2.97 imp. s-1. deg-1 at 1.0 Hz. These data demonstrate that only an analysis based on measurements at different frequencies and orientations reveals a number of complex features. They moreover suggest that for the vast majority of neurons several sources of canal and otolith information interact at this central stage of vestibular information processing.

Otolith processing in the deep cerebellar nuclei.

To investigate the otolith contribution to the responses of "vestibular only" neurons in the rostral fastigial nucleus (FN), single-unit activity was recorded in the alert monkey with the head fixed during static and dynamic stimulation (+/- 15 deg, 0.06-1.4 Hz) around an earth-fixed horizontal axis. Head orientation could be altered allowing for roll, pitch, and intermediate planes of orientation. For the vast majority of neurons a response vector orientation (RVO) with an optimal response and a null-response at a head orientation 90 deg apart could be determined. Presumably more than 30% of the vestibular only neurons had an otolith input, as indicated by responses to static tilt, head-position-related activity, large phase changes (> 100 deg) of neuronal activity between 0.06 and 1.4 Hz, changes of the RVO at different frequencies and complex responses (spatio-temporal convergence). Thus, neurons in FN reflecting an otolith or a combined canal-otolith input are much more common than up to now thought. Vestibular-only neurons are most likely involved in vestibulospinal mechanisms. Their precise functional role has yet to be determined.

Short- and long-term consequences of canal plugging on gaze shifts in the rhesus monkey. I. Effects on gaze stabilization.

Short- and long-term consequences of canal plugging on gaze shifts in the rhesus monkey. I. Effects on gaze stabilization. To study the contribution of the vestibular system to the coordinated eye and head movements of a gaze shift, we plugged the lumens of just the horizontal (n = 2) or all six semicircular canals (n = 1) in monkeys trained to make horizontal head-unrestrained gaze shifts to visual targets. After the initial eye saccade of a gaze shift, normal monkeys exhibit a compensatory eye counterrotation that stabilizes gaze as the head movement continues. This counterrotation, which has a gain (eye velocity/head velocity) near one has been attributed to the vestibuloocular reflex (VOR). One day after horizontal canal plugging, the gain of the passive horizontal VOR at frequencies between 0.1 and 1.0 Hz was <0.10 in the horizontal-canal-plugged animals and zero in the all-canal-plugged animal. One day after surgery, counterrotation gain was approximately 0.3 in the animals with horizontal canals plugged and absent in the animal with all canals plugged. As the time after plugging increased, so too did counterrotation gain. In all three animals, counterrotation gain recovered to between 0.56 and 0.75 within 80-100 days. The initial loss of compensatory counterrotation after plugging resulted in a gaze shift that ended long after the eye saccade and just before the end of the head movement. With recovery, the length of time between the end of the eye saccade and the end of the gaze movement decreased. This shortening of the duration of reduced gain counterrotation occurred both because head movements ended sooner and counterrotation gain returned to 1.0 more rapidly relative to the end of the eye saccade. Eye counterrotation was not due to activation of pursuit eye movements as it persisted when gaze shifts were executed to extinguished targets. Also counterrotation was not due simply to activation of neck receptors because counterrotation persisted after head movements were arrested in midflight. We suggest that the neural signal that is used to cause counterrotation in the absence of vestibular input is an internal copy of the intended head movement.

Strategy for membrane protein crystallization exemplified with OmpA and OmpX.

The bacterial outer membrane proteins OmpA and OmpX were modified in such a manner that they yielded bulky crystals diffracting X-rays isotropically beyond 2 A resolution and permitting detailed structural analyses. The procedure involved semi-directed mutagenesis, mass production into inclusion bodies, and (re)naturation therefrom; it should be applicable for a broader range of membrane proteins.

Linear spatio-temporal convergence in vestibular neurons of the primate nucleus fastigii.

Vestibular responses in the primate fastigial nucleus (FN) do often not follow the simple cosine tuning observed in primary vestibular afferents. The present report demonstrates that these more complex patterns can mostly be attributed to simple linear summation of spatially and temporally diverse cosine-tuned input signals (linear spatio-temporal convergence, STC). Analyses following from this elementary finding, however, reveal frequency-dependent properties in many FN neurons, which are difficult to reconcile with existing concepts of possible functions of STC in central vestibular areas. The demonstration that STC linearity holds for FN responses is thus of both theoretical and practical relevance, allowing shortening of future experimental protocols and facilitating comparison of the observed spatio-temporal response dynamics with those at other stages of vestibular signal processing.

Rostral fastigial nucleus activity in the alert monkey during three-dimensional passive head movements.

The fastigial nucleus (FN) receives vestibular information predominantly from Purkinje cells of the vermis. FN in the monkey can be divided in a rostral part, related to spinal mechanisms, and a caudal part with oculomotor functions. To understand the role of FN during movements in space, single-unit activity in alert monkeys was recorded during passive three-dimensional head movements from rostral FN. Seated monkeys were rotated sinusoidally around a horizontal earth-fixed axis (vertical stimulation) at different orientations 15 degrees apart (including roll, pitch, vertical canal plane and intermediate planes). In addition, sinusoidal rotations around an earth-vertical axis (yaw stimulus) included different roll and pitch positions (+/-10 degrees, +/-20 degrees). The latter positions were also used for static stimulation. One hundred fifty-eight neurons in two monkeys were modulated during the sinusoidal vertical search stimulation. The vast majority showed a uniform response pattern: a maximum at a specific head orientation (response vector orientation) and a null response 90 degrees apart. Detailed analysis was obtained from 111 neurons. On the basis of their phase relation during dynamic stimulation and their response to static tilt, these neurons were classified as vertical semicircular canal related (n = 79, 71.2%) or otolith related (n = 25; 22.5%). Only seven neurons did not follow the usual response pattern and were classified as complex neurons. For the vertical canal-related neurons (n = 79) all eight major response vector orientations (ipsilateral or contralateral anterior canal, posterior canal, roll, and nose-down and nose-up pitch) were found in Fn on one side. Neurons with ipsilateral orientations were more numerous and on average more sensitive than those with contralateral orientations. Twenty-eight percent of the vertical canal-related neurons also responded to horizontal canal stimulation. None of the vertical canal-related neurons responded to static tilt. Otolith-related neurons (n = 25) had a phase relation close to head position and were considerably less numerous than canal-related neurons. Except for pitch, all other response vector orientations were found. Seventy percent of these neurons responding during dynamic stimulation also responded during static tilt. The sensitivity during dynamic stimulation was always higher than during static stimulation. Sixty-one percent of the otolith-related neurons responded also to horizontal canal stimulation. These results show that in FN, robust vestibular signals are abundant. Canal-related responses are much more common than otolith-related responses. Although for many canal neurons the responses can be related to single canal planes, convergence between vertical canals but also with horizontal canals is common.

A simplified calibration method for three-dimensional eye movement recordings using search-coils.

An algorithm is presented which allows the calibration of three-dimensional eye movements in two magnetic fields with two search-coils in one eye, whose relative orientation does not have to be known. Prior to recordings a calibration cube is placed in front of the eye to measure real and apparent crosstalk, produced by imprecisely oriented horizontal or vertical magnetic fields, and to obtain a first approximation of offset voltages. For calibration it suffices if one point is fixated and, in addition, spontaneous eye movements for 30-60 sec are performed. Additional fixation points can be used to improve the calibration. Position quaternions are applied for computing eye movement recordings from man and monkey and to determine Listing's plane.

Rapid horizontal gaze movement in the monkey.

1. We studied horizontal eye and head movements in three monkeys that were trained to direct their gaze (eye position in space) toward jumping targets while their heads were both fixed and free to rotate about a vertical axis. We considered all gaze movements that traveled > or = 80% of the distance to the new visual target. 2. The relative contributions and metrics of eye and head movements to the gaze shift varied considerably from animal to animal and even within animals. Head movements could be initiated early or late and could be large or small. The eye movements of some monkeys showed a consistent decrease in velocity as the head accelerated, whereas others did not. Although all gaze shifts were hypometric, they were more hypometric in some monkeys than in others. Nevertheless, certain features of the gaze shift were identifiable in all monkeys. To identify those we analyzed gaze, eye in head position, and head position, and their velocities at three points in time during the gaze shift: 1) when the eye had completed its initial rotation toward the target, 2) when the initial gaze shift had landed, and 3) when the head movement was finished. 3. For small gaze shifts (< 20 degrees) the initial gaze movement consisted entirely of an eye movement because the head did not move. As gaze shifts became larger, the eye movement contribution saturated at approximately 30 degrees and the head movement contributed increasingly to the initial gaze movement. For the largest gaze shifts, the eye usually began counterrolling or remained stable in the orbit before gaze landed. During the interval between eye and gaze end, the head alone carried gaze to completion. Finally, when the head movement landed, it was almost aimed at the target and the eye had returned to within 10 +/- 7 degrees, mean +/- SD, of straight ahead. Between the end of the gaze shift and the end of the head movement, gaze remained stable in space or a small correction saccade occurred. 4. Gaze movements < 20 degrees landed accurately on target whether the head was fixed or free. For larger target movements, both head-free and head-fixed gaze shifts became increasingly hypometric. Head-free gaze shifts were more accurate, on average, but also more variable. This suggests that gaze is controlled in a different way with the head free. For target amplitudes < 60 degrees, head position was hypometric but the error was rather constant at approximately 10 degrees.(ABSTRACT TRUNCATED AT 400 WORDS)

Human perception of horizontal trunk and head rotation in space during vestibular and neck stimulation.

The vestibular signal of head motion in space must be complemented by a neck signal of the trunk-to-head excursion in order to provide the individual with information on trunk motion in space. This consideration led us to study psychophysically the role of vestibular-neck interaction for human self-motion perception. Subjects (Ss) were presented with passive horizontal rotations of their trunk and/or head (sinusoidal rotations, f = 0.025 - 0.4 Hz) in the dark for vestibular and neck stimulation, as well as for combinations of both. Ss' perception was evaluated in terms of gain (veridical perception of stimulus magnitude, G = 1), phase, and detection threshold. (1) Perception of trunk rotation in space. During vestibular stimulation (whole-body rotation) and neck stimulation (trunk rotation with the head kept stationary) the frequency-transfer characteristics underlying this perception were very similar. The gain fell short; it was only about 0.7 at 0.4 and 0.2 Hz stimulus frequency and was further attenuated with decreasing frequency. In contrast, the phase was close to that of actual trunk position. The gain attenuation was found to be a function of the peak angular velocity of the stimulus, a fact, which we related to a 'velocity threshold' of the order of 1 deg/s. During the various vestibular-neck combinations used, Ss' perception was again erroneous, reflecting essentially the sum of its two non-ideal constituents. However, there was one noticeable exception; during the combination 'head rotation on stationary trunk', Ss veridically perceived their trunk as stationary (compatible with the notion that the sum yielded 'zero'). (2) Perception of head rotation in space. During vestibular stimulation, Ss' estimates showed the same non-ideal gain-vs.-frequency characteristics as described above for the trunk. Neck stimulation induced an illusion as if the head had been rotated in space. This neck contribution was such that, when it was combined with its vestibular counterpart during head rotation on stationary trunk, the perception became almost veridical. On closer inspection, however, this neck contribution was found to reflect the sum of two components; one was the non-ideal neck signal contributing to the perception of 'trunk in space', the other was an almost ideal neck signal of head-on-trunk rotation. (3) The results could be described by a simple model. In this model, the erroneous vestibular signal 'head in space' is primarily used to create an internal representation of 'trunk in space'.(ABSTRACT TRUNCATED AT 400 WORDS)

Hepatitis B vaccine and hepatitis B markers: cost effectiveness of screening prehospital personnel.

The purpose of this study was to evaluate the cost effectiveness of screening emergency medical technicians (EMTs) and paramedical personnel prior to administering hepatitis B vaccine. Hepatitis B screening and Heptavax vaccine were offered to 259 basic EMTs and paramedics. Of the 259 individuals, 62 refused screening, and six who had already received hepatitis B vaccine were excluded from the study. The screening was not continued after the results of the first 174 tests returned negative. All 191 participants were vaccinated. No hepatitis B surface antigen carriers and only three individuals positive for hepatitis B surface antibody were found among those screened. This study corroborates the Centers for Disease Control guidelines of cost effectiveness in screening prehospital health care workers.