Heterogeneity of parvalbumin expression in the avian cerebellar cortex and comparisons with zebrin II.

The cerebellar cortex has a fundamental parasagittal organization that is reflected in the physiological responses of Purkinje cells, afferent and efferent connections, and the expression of several molecular markers. The most thoroughly studied of these molecular markers is zebrin II (ZII; a.k.a. aldolase C). ZII is differentially expressed in Purkinje cells, resulting in a pattern of sagittal stripes of high expression interdigitated with stripes of little or no expression. In this study, we examined the expression of the calcium binding protein parvalbumin (PV) in the cerebellum of several avian species (pigeons, hummingbirds, zebra finches) and compared it to the expression of ZII. We found that PV immunoreactivity was distributed across the cerebellar cortex such that there were sagittal stripes of PV immunopositive (PV+) Purkinje cells alternating with PV immunonegative (PV-) Purkinje cells. Although most Purkinje cells in the anterior lobe were PV+, there were several thin (i.e. only a few Purkinje cells wide) PV- stripes spanning the folia. In the posterior lobe, PV+ and PV- stripes were also apparent, but the PV- stripes were much wider than in the anterior lobe. In sections processed for both ZII and PV, the expression was generally complementary: PV+ stripes were ZII-, and vice-versa. This complementary expression was most apparent in folia II-IV and VIII-IXcd. The complementary expression was not, however, absolute; some Purkinje cells co-expressed PV and ZII whereas others lacked both. These novel findings relate to the complex neurochemical organization of the cerebellum, and are likely important to issues regarding cerebellar plasticity.

Congruence of zebrin II expression and functional zones defined by climbing fiber topography in the flocculus.

The cerebellum is organized into parasagittal zones with respect to the topography of climbing fiber (CF) afferents and the expression of molecular markers such as zebrin II. Zebrin is expressed by a subset of Purkinje cells that are distributed as a parasagittal array of immunopositive and immunonegative stripes. Several studies in rodents suggest that, in general, CFs to the zebrin negative stripes convey somatosensory information, whereas CFs to the zebrin positive stripes convey information from visual and other sensory systems. The pigeon flocculus consists of four pairs of zebrin+/- stripes (P4 +/- through P7 +/-), however the CF input consists entirely of visual inputs. Thus, because the correspondence of zebrin expression and CF information must be different from that proposed for rodents, we investigated this relationship in the pigeon flocculus. Floccular Purkinje cells respond to patterns of optic flow resulting from self-rotation about one of two axes: either the vertical axis (zones 0 and 2), or a horizontal axis (zones 1 and 3). Visual CF afferents projecting to the flocculus arise from the medial column of the inferior olive (mcIO). Zones 0 and 2 receive input from the caudal mcIO, whereas zones 1 and 3 receive input from the rostral mcIO. We injected a fluorescent anterograde tracer into the rostral and/or caudal mcIO and visualized zebrin expression. There was a strict concordance between CF organization and zebrin labeling: caudal mcIO injections resulted in CFs in zebrin bands P4 +/- and P6 +/-, whereas rostral mcIO injections resulted in CFs in zebrin bands P5 +/- and P7 +/-. Thus, zebrin stripes P4 +/- and P6 +/- correspond to the vertical axis zones 0 and 2, whereas P5 +/- and P7 +/- correspond to the horizontal axis zones 1 and 3. This is the first explicit demonstration that a series of zebrin stripes corresponds with functional zones in the cerebellum.

A comparison of ventral tegmental neurons projecting to optic flow regions of the inferior olive vs. the hippocampal formation.

The ventral tegmental area (catecholaminergic group A10) is a midbrain region characterized by concentrated dopaminergic immunoreactivity. Previous studies in pigeons show that the ventral tegmental area provides a robust projection to the hippocampal formation and to the medial column of the inferior olive. However, the distribution, morphology, and neurochemical content of the neurons that constitute these projections have not been resolved. In this study, we used a combination of retrograde tracing techniques and immunofluorohistochemistry to address these issues. Retrograde tracers were used to demonstrate that the distribution of ventral tegmental area neurons projecting to the hippocampus and the inferior olive overlap in the caudo-ventral ventral tegmental area. The hippocampus- and inferior olive-projecting ventral tegmental area neurons could not be distinguished based on morphology: most neurons had small- to medium-sized multipolar or fusiform soma. Double-labeling with fluorescent retrograde tracers revealed that the hippocampus- and medial column of the inferior olive-projecting neurons were found intermingled in the ventral tegmental area, but no cells were double labeled; i.e. individual ventral tegmental area neurons do not project to both the hippocampal formation and medial column of the inferior olive. Finally, we found that a minority (8.2%) of ventral tegmental area neurons providing input to the hippocampus were tyrosine hydroxylase-immunoreactive, whereas none of the inferior olive-projecting neurons were tyrosine hydroxylase positive. Combined, our findings show that the projections to the hippocampus and olivocerebellar pathway arise from intermixed subpopulations of ventral tegmental area neurons with indistinguishable morphology but only the hippocampal projection involves dopaminergic neurons. We suggest that equivalent projections from the ventral tegmental area to the hippocampal formation and inferior olive exist in mammals and discuss their potential role in the processing of optic flow and the analysis of self-motion.

Transport characteristics of mammalian Rh and Rh glycoproteins expressed in heterologous systems.

The development and use of heterologous expression systems is critical for deciphering the function of mammalian Rh and Rh-glycoproteins. The studies here use Xenopus oocytes, well known for their ability to readily traffic and express difficult membrane proteins, and S. cerevisiae wild-type strains and mutants that are defective in ammonium transport. Data obtained in both of these expression systems revealed that mammalian Rh-glycoprotein-mediated transport (RhAG, RhBG, and RhCG) is an electroneutral process that is driven by the NH4+ concentration and the transmembrane H+ gradient, effectively exchanging NH4+ for H+ in a process that results in transport of net NH3. Homology modeling and functional studies suggest that the more recently evolved erythrocyte blood group proteins, RhCE and RhD, may not function directly in ammonia transport and may be evolving a new function in the RBC membrane. The relationship of Rh and Rh-glycoproteins to the Amt/Mep ammonium transporters is substantiated with functional transport data and structural modeling.

Responses of neurons in the medial column of the inferior olive in pigeons to translational and rotational optic flowfields.

The responses of neurons in the medial column of the inferior olive to translational and rotational optic flow were recorded from anaesthetized pigeons. Panoramic translational or rotational flowfields were produced by mechanical devices that projected optic flow patterns onto the walls, ceiling and floor of the room. The axis of rotation/translation could be positioned to any orientation in three-dimensional space such that axis tuning could be determined. Each neuron was assigned a vector representing the axis about/along which the animal would rotate/translate to produce the flowfield that elicited maximal modulation. Both translation-sensitive and rotation-sensitive neurons were found. For neurons responsive to translational optic flow, the preferred axis is described with reference to a standard right-handed coordinate system, where +x, +y and +z represent rightward, upward and forward translation of the animal, respectively (assuming that all recordings were from the right side of the brain). t(+y) neurons were maximally excited in response to a translational optic flowfield that results from self-translation upward along the vertical (y) axis. t(-y) neurons also responded best to translational optic flow along the vertical axis but showed the opposite direction preference. The two remaining groups, t(-x+z) and t(-x-z) neurons, responded best to translational optic flow along horizontal axes that were oriented 45 degrees to the midline. There were two types of neurons responsive to rotational optic flow: rVA neurons preferred rotation about the vertical axis, and rH135c neurons preferred rotation about a horizontal axis at 135 degrees contralateral azimuth. The locations of marking lesions indicated a clear topographical organization of the six response types. In summary, our results reinforce that the olivo-cerebellar system dedicated to the analysis of optic flow is organized according to a reference frame consisting of three approximately orthogonal axes: the vertical axis, and two horizontal axes oriented 45 degrees to either side the midline. Previous research has shown that the eye muscles, vestibular semicircular canals and postural control system all share a similar spatial frame of reference.

Empty capsids in column-purified recombinant adenovirus preparations.

Empty capsids from adenovirus, that is, virus particles lacking DNA, are well documented in the published literature. They can be separated from complete virus by CsCl density gradient centrifugation. Here we characterize the presence of empty capsids in recombinant adenovirus preparations purified by column chromatography. The initial purified recombinant adenovirus containing the p53 tumor suppressor gene was produced from 293 cells grown on microcarriers and purified by passage through DEAE-Fractogel and gel-filtration chromatography. Further sequential purification of the column-purified virus by CsCl and glycerol density gradient centrifugations yielded isolated complete virus and empty capsids. The empty capsids were essentially noninfectious and free of DNA. Analysis of empty capsids by SDS-PAGE or RP-HPLC showed the presence of only three major components: hexon, IIIa, and a 31K band. This last protein was identified as the precursor to protein VIII (pVIII) by mass spectrometric analysis. No pVIII was detected from the purified complete virus. Analysis by electron microscopy of the empty capsids showed particles with small defects. The amount of pVIII was used to determine the level of empty capsid contamination. First, the purified empty capsids were used to quantify the relation of pVIII to empty capsid particle concentration (as estimated by either light scattering or hexon content). They were then used as a standard to establish the empty capsid concentration of various recombinant adenovirus preparations. Preliminary research showed changes in empty capsid concentration with variations in the infection conditions. While virus purification on anion-exchange or gel-filtration chromatography has little effect on empty capsid contamination, other chromatographic steps can substantially reduce the final concentration of empty capsids in column-purified adenovirus preparations.

Carboxyalkylated histidine is a pH-dependent product of pegylation with SC-PEG.

PURPOSE: Pegylation of therapeutic protein usually results in a mixture of monopegylated proteins with differing sites of modification. With rh-interferon-alpha2A pegylation, we have found that this heterogeneity includes two classes of pegylation site chemistry, the relative proportions of which can be adjusted by reaction pH. METHODS: The effect of pegylation reaction pH on the relative proportion of three peaks produced was investigated. Products were purified and characterized by peptide mapping, chemical stability to neutral hydroxylamine, and biologic activity. RESULTS: Reactions at basic pH levels produced a mixture of products pegylated at lysine residues as has been observed elsewhere. However, the dominant product of reactions at mildly acidic levels of pH showed distinct chemistry and higher cytopathic effect activity. The primary site of modification at this pH was His34. We developed a quantitative assay using sensitivity to neutral hydroxylamine to measure the proportion of urethane bonds involving carboxyalkylated histidines. This assay showed that histidine was pegylated preferentially at low pH levels with another protein, rh-Interleukin-10. CONCLUSIONS: Reaction pH can be used to select the preferred pegylation site chemistry.

Detection of glass patterns by pigeons and humans: implications for differences in higher-level processing.

Glass patterns have been used to examine mechanisms underlying form perception. The current investigation compared detection of Glass patterns by pigeons and humans and provides evidence for substantial species differences in global form perception. Subjects were required to discriminate, on a simultaneous display, a random dot pattern from a Glass pattern. Four different randomly presented Glass patterns were used (concentric, radial, parallel-vertical, and parallel-horizontal). Detection thresholds were measured by degrading the Glass patterns through the addition of random noise. For both humans and pigeons, discrimination decreased systematically with the addition of noise. Humans showed detection differences among the four patterns, with lowest thresholds to radial and concentric patterns and highest thresholds to the parallel-horiZontal pattern. Pigeons did not show a detection difference across the four patterns. Implications for differences in neural processing of complex forms are discussed.

16Cys encoded by the RHce gene is associated with altered expression of the e antigen and is frequent in the R0 haplotype.

Serological observations have suggested that numerous D, many e (especially in Blacks), several E, and rare c variants exist within the Rh blood group system. The molecular basis for expression of many of these variants has been elucidated. This study describes five unrelated Caucasians whose red blood cells reacted with polyclonal anti-e but did not react with some monoclonal anti-e, which suggested that they carried a variant e antigen. Molecular investigation revealed the presence of a 48G-->C change (encoding cysteine instead of tryptophan at amino acid 16) in their RHce gene. No other differences were found, which suggests that amino acid residues located in the first transmembrane region can affect expression of the e antigen, whose critical residues are on the predicted fourth external loop of the protein. This polymorphism has not previously been observed because polyclonal anti-e does not distinguish this variant from wild type. This position is polymorphic in RHce alleles and the presence of the 48C nucleotide is often found in the R0 (Dce) haplotype.

Fast and slow neurons in the nucleus of the basal optic root in pigeons.

The nucleus of the basal optic root (nBOR) is involved in the generation of the optokinetic response. Previous studies showed that most nBOR neurons exhibit direction-selectivity in response to largefield motion. We investigated the responses of pigeon nBOR neurons to drifting sine wave gratings of varying spatial and temporal frequency (SF,TF). Two groups of neurons were revealed. The first group preferred gratings of low SF (mean, 0.07 cycles per degree (cpd)) and high TF (mean, 0.76 Hz) ('fast' stimuli). The second group preferred gratings of high SF (mean, 0.56 cpd) and lower TF (mean, 0.33 Hz) ('slow' stimuli). Previous studies have demonstrated fast and slow neurons in pretectal nucleus lentiformis mesencephali, which is also involved in the generation of the optokinetic response.

Projections from the nucleus of the basal optic root and nucleus lentiformis mesencephali to the inferior olive in pigeons (Columba livia).

The nucleus of the basal optic root (nBOR) of the accessory optic system (AOS) and the pretectal nucleus lentiformis mesencephali (LM) are involved in the analysis of optic flow and the generation of the optokinetic response. Previous studies have shown that the nBOR projects bilaterally to the medial column (mc) of the inferior olive (IO) and the LM projects to the ipsilateral mc. In the present study the retrograde tracer cholera toxin subunit B was injected into either the caudal or rostral mc. From all injections, retrogradely labeled cells were seen in the ipsilateral pretectum along the border of the medial and lateral subnuclei of the LM. Cells were also seen in bilaterally in the nBOR. On the contralateral side, a discrete group of cells was labeled in the rostral margin of the nBOR. These cells were localized in the dorsal portion of the nBOR proper and some were found in the adjacent nBOR dorsalis. On the ipsilateral side, a diffuse group of cells was seen in the caudal nBOR. Most of these cells were in the nBOR dorsalis and outside the nBOR complex in the area ventralis of Tsai and the reticular formation. From the injections into the caudal mc, a greater proportion of labeled cells was found in the LM, whereas a greater proportion of cells was found in the nBOR from the injections into the rostral mc. This differential projection from LM and nBOR to the caudal and rostral mc is consistent with the optic flow preferences of neurons in the mc, and a similar pattern of connectivity has been found in mammalian species.

Gene targeting in primary fetal fibroblasts from sheep and pig.

Nuclear transfer offers a new cell-based route for introducing precise genetic modifications in a range of animal species. However, significant challenges, such as establishment of somatic gene targeting techniques, must be overcome before the technology can be applied routinely. In this report, we describe targeted deletion at the GGTA1 (alpha 1,3-galactosyl transferase) and PrP (prion protein) loci in primary fibroblasts from livestock. We place particular emphasis on the growth characteristics of the primary cell cultures, since these are key to determining success.

Spatiotemporal properties of fast and slow neurons in the pretectal nucleus lentiformis mesencephali in pigeons.

Neurons in the pretectal nucleus lentiformis mesencephali (LM) are involved in the analysis of optic flow that results from self-motion. Previous studies have shown that LM neurons have large receptive fields in the contralateral eye, are excited in response to largefield stimuli moving in a particular (preferred) direction, and are inhibited in response to motion in the opposite (anti-preferred) direction. We investigated the responses of LM neurons to sine wave gratings of varying spatial and temporal frequency drifting in the preferred and anti-preferred directions. The LM neurons fell into two categories. "Fast" neurons were maximally excited by gratings of low spatial [0.03-0.25 cycles/ degrees (cpd)] and mid-high temporal frequencies (0.5-16 Hz). "Slow" neurons were maximally excited by gratings of high spatial (0.35-2 cpd) and low-mid temporal frequencies (0.125-2 Hz). Of the slow neurons, all but one preferred forward (temporal to nasal) motion. The fast group included neurons that preferred forward, backward, upward, and downward motion. For most cells (81%), the spatial and temporal frequency that elicited maximal excitation to motion in the preferred direction did not coincide with the spatial and temporal frequency that elicited maximal inhibition to gratings moving in the anti-preferred direction. With respect to motion in the anti-preferred direction, a substantial proportion of the LM neurons (32%) showed bi-directional responses. That is, the spatiotemporal plots contained domains of excitation in addition to the region of inhibition. Neurons tuned to stimulus velocity across different spatial frequency were rare (5%), but some neurons (39%) were tuned to temporal frequency. These results are discussed in relation to previous studies of the responses of neurons in the accessory optic system and pretectum to drifting gratings and other largefield stimuli.

Binocular neurons in the nucleus lentiformis mesencephali in pigeons: responses to translational and rotational optic flowfields.

The pretectal nucleus lentiformis mesencephali (LM) receives direct input from the contralateral retina and is dedicated to the analysis of optic flowfields resulting from self-motion. The activity of 126 LM neurons in response to optic flow stimuli was recorded. As with previous studies, it was found that most neurons (approximately 90%) exhibited direction-selectivity to large-field stimuli moving in the contralateral hemifield. However, some neurons (approximately 10%) responded to stimulation of both eyes and had receptive field structures conducive for detection of particular patterns of optic flow resulting from either self-translation or self-rotation. These binocular neurons were maximally responsive to panoramic optic flowfields simulating either translational or rotational optic flow.

Mass spectrometric mapping of disulfide bonds in recombinant human interleukin-13.

Interleukin 13 (IL-13), a member of the a-helical family of cytokines, has approximately 30% primary sequence homology with IL-4 and shares a common receptor component. The biologically active rhIL-13 is monomeric and non-glycosylated, and contains two disulfide bonds as determined by comparative electrospray mass spectrometric (MS) analysis of the protein before and after reduction with dithiothreitol-dithioerythritol. A trypsin-resistant core peptide of rhIL-13 was isolated and analyzed by plasma desorption (PD) MS, identifying a disulfide-linked core peptide. Subsequent digestion of this core peptide by pepsin, followed by PDMS analysis of the resulting cystine-containing peptic fragments, provided rapid determination of the existing disulfide bonds between cysteine residues 28-56 and 44-70. This disulfide arrangement is similar to that observed for the analogous four internal cysteine residues in hIL-4. The conservation of disulfide bond arrangements between hIL-13 and hIL-4, coupled with their alpha-helical structure and sequence homologies, confirms that IL-13 and IL-4 are structural homologues. It is also consistent with their reported similarities in biological function and receptor binding kinetics.

Topographic organization of inferior olive cells projecting to translational zones in the vestibulocerebellum of pigeons.

In the nodulus and ventral uvula of pigeons, there are four parasagittal zones containing Purkinje cells responsive to patterns of optic flow that results from self-translation along a particular axis in three-dimensional space. By using a three-axis system to describe the preferred direction of translational optic flow, where +X, +Y, and +Z represent rightward, upward, and forward self-motion, respectively, the four cell types are: +Y, -Y, -X-Z, and -X+Z (assuming recording from the left side of the head). The -X-Z zone is the most medial, followed in sequence by the -X+Z, -Y zone, and the +Y zones. In this study, we injected the retrograde tracer cholera toxin subunit B into each of the four translational zones to determine the origin of the climbing fiber inputs in the inferior olive. Retrograde labeling in the inferior olive was found in the ventrolateral margin of the medial column from injections into all four translational zones; however, there was a clear functional topography. Retrograde labeling from -Y zone injections was found most rostrally in the medial column, whereas retrogradely labeled cells from -X-Z zone injections were found most caudally in the medial column. Labeling from +Y and -X+Z zone injections were found between the labeling from -Y zones and -X-Z zones, with +Y labeling located slightly caudal to -X+Z labeling.

Evidence supporting the requirement for two proline residues for expression of c.

BACKGROUND: In humans, c antigen expression is associated with a proline residue at amino acid position 103 in the second extracellular loop of the CE protein. Comparison of nonhuman primate Rh proteins suggested that c reactivity might actually involve two proline residues. It has been shown that the RBCs of New World capuchin monkeys (Cebus apella) react with anti-c. To further define the amino acid residues involved in c expression, Rh cDNA from the capuchin was analyzed. STUDY DESIGN AND METHODS: Rh transcripts were amplified by reverse transcription PCR from RNA isolated from the reticulocytes of a capuchin monkey and were cloned and sequenced. RESULTS: Rh transcripts from the capuchin monkey, whose RBCs react with anti-c, were found to encode adjacent proline residues at 102 and 103. CONCLUSION: Sequencing of Rh transcripts from the capuchin monkey supports the hypothesis that the expression of c requires two adjacent proline residues. Proline causes bends or loops in proteins, which, in this case, might form a unique, stable structure resistant to perturbations induced by changes in upstream or downstream residues. This would explain the scarcity in humans of c variants as compared to the other major Rh antigen variants, and the preservation of c reactivity despite 24-percent divergence between the human and capuchin Rh proteins.