Increased expression of the MBP mRNA binding protein HnRNP A2 during oligodendrocyte differentiation.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor that mediates intracellular trafficking of myelin basic protein (MBP) mRNA to the myelin compartment in oligodendrocytes, is most abundant in the nucleus, but shuttles between the nucleus and cytoplasm. In the cytoplasm, it is associated with granules that transport mRNA from the cell body to the processes of oligodendrocytes. We found that the overall level of hnRNP A2 increased in oligodendrocytes as they differentiated into MBP-positive cells, and that this augmentation was reflected primarily in the cytoplasmic pool of hnRNP A2 present in the form of granules. The extranuclear distribution of hnRNP A2 was also observed in brain during the period of myelination in vivo. Methylation and phosphorylation have been implicated previously in the nuclear to cytoplasmic distribution of hnRNPs, so we used drugs that block methylation and phosphorylation of hnRNPs to assess their effect on hnRNP A2 distribution and mRNA trafficking. Cultures treated with adenosine dialdehyde (AdOx), an inhibitor of S-adenosyl-L-homocysteine hydrolase, or with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a drug that inhibits casein kinase 2 (CK2), maintained the preferential nuclear distribution of hnRNP A2. Treatment with either drug affected the transport of RNA trafficking granules that remained confined to the cell body.

Axonal loss in normal-appearing white matter in a patient with acute MS.

BACKGROUND: Brain imaging studies detect abnormalities in normal-appearing white matter in patients with MS. OBJECTIVE: To investigate the histopathologic basis for these changes in autopsy tissue from a patient with MS with 9 months' disease duration and a terminal brain stem lesion. METHODS: The brain stem and spinal cord were analyzed ultrastructurally and immunocytochemically for axons, myelin, and activated microglia/macrophages. RESULTS: Pathologic findings were consistent with a terminal inflammatory demyelinated lesion at the cervicomedullary junction. The ventral spinal cord column, containing descending tracts, exhibited 22% axonal loss at segment C7, but grossly normal immunostaining for myelin. Confocal and electron microscopy revealed myelin sheaths without axonal content and initial stages of myelin degradation by activated microglia/macrophages among intact myelinated axons. Axonal number and appearance was normal in ascending sensory tracts. CONCLUSIONS: These studies confirm axonal degeneration in the absence of myelin loss as one histopathologic correlate to abnormal MR findings in patients with MS.

Duck plague epizootics in the United States, 1967-1995.

In 1967, the first confirmed diagnosis of duck plague (DP) in the USA was made from pekin ducks (Anas platyrhynchos domesticus) on commercial duck farms on Long Island, New York. Within 10 mo, DP was confirmed as the cause of death in migratory waterfowl on a Long Island bay. This paper reviews 120 DP epizootics reported from 1967 to 1995 that involved waterfowl species native to North America or were reported in areas with free-flying waterfowl at risk. Duck plague epizootics occurred in 21 states with the greatest number reported in Maryland (29), New York (18), California (16), and Pennsylvania (13). The greatest frequency of epizootics (86%) was detected during the months of March to June. At least 40 waterfowl species were affected with the highest frequency of epizootics reported in captive or captive-reared ducks including muscovy ducks (Cairina moschata) (68%), mallard ducks (A. platyrhynchos) (18%) and black ducks (A. rubripes) (14%). The greatest number of waterfowl died in three epizootics that involved primarily migratory birds in 1967 and 1994 in New York (USA) and 1973 in South Dakota (USA). The greatest number of DP epizootics reported since 1967 appear to have involved flocks of non-migratory rather than migratory waterfowl; therefore, in our opinion it remains unknown if DP is enzootic in either non-migratory or migratory waterfowl.

Neurological disability correlates with spinal cord axonal loss and reduced N-acetyl aspartate in chronic multiple sclerosis patients.

Axonal degeneration has been proposed as a cause of irreversible neurological disability in multiple sclerosis (MS) patients. The purpose of this study was to quantify axonal loss in spinal cord lesions from 5 paralyzed (Expanded Disability Status Scale score > or =7.5) MS patients and to determine if axonal number or volume correlated with levels of the neuronal marker N-acetyl aspartate (NAA). Axonal loss in MS lesions ranged from 45 to 84% and averaged 68%. NAA levels were significantly reduced (>50%) in cross sections of spinal cords containing MS lesions. Reduced NAA correlated with reduced axonal numbers within lesion areas. In addition, NAA levels per axonal volume were significantly reduced in demyelinated axons (42%) and in myelinated axons in normal-appearing white matter (30%). The data support axonal loss as a major cause of irreversible neurological disability in paralyzed MS patients and indicate that reduced NAA as measured by magnetic resonance spectroscopy can reflect axonal loss and reduced NAA levels in demyelinated and myelinated axons.

Binding of an RNA trafficking response element to heterogeneous nuclear ribonucleoproteins A1 and A2.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 binds a 21-nucleotide myelin basic protein mRNA response element, the A2RE, and A2RE-like sequences in other localized mRNAs, and is a trans-acting factor in oligodendrocyte cytoplasmic RNA trafficking. Recombinant human hnRNPs A1 and A2 were used in a biosensor to explore interactions with A2RE and the cognate oligodeoxyribonucleotide. Both proteins have a single site that bound oligonucleotides with markedly different sequences but did not bind in the presence of heparin. Both also possess a second, specific site that bound only A2RE and was unaffected by heparin. hnRNP A2 bound A2RE in the latter site with a K(d) near 50 nm, whereas the K(d) for hnRNP A1 was above 10 microm. UV cross-linking assays led to a similar conclusion. Mutant A2RE sequences, that in earlier qualitative studies appeared not to bind hnRNP A2 or support RNA trafficking in oligodendrocytes, had dissociation constants above 5 microm for this protein. The two concatenated RNA recognition motifs (RRMs), but not the individual RRMs, mimicked the binding behavior of hnRNP A2. These data highlight the specificity of the interaction of A2RE with these hnRNPs and suggest that the sequence-specific A2RE-binding site on hnRNP A2 is formed by both RRMs acting in cis.

Evidence for spatial tuning in informational masking using the probe-signal method.

Auditory spatial attention is one mechanism that may contribute to the ability to identify one sound source in a multi-source environment. The role of auditory spatial attention in a multi-source environment was investigated using the probe-signal method. The experiment took place in a quiet room with seven speakers arranged in a semi-circle in front of the listener. The speakers were placed at 30-degree intervals at a distance of 5 ft from the listener. The signal was comprised of eight contiguous, 60-ms pure-tone bursts arranged in either a rising or falling frequency pattern. Masker components were also comprised of eight contiguous pure-tone bursts but with durations that varied randomly from 20 to 100 ms. The six maskers were played with the signal and were constructed in order to result in informational rather than energetic masking. The frequency of each masker component was chosen randomly on each burst from a narrow frequency band, independent from the signal frequency band. The task was 1I-2AFC fixed-level identification with response time measurement. The listener was instructed to focus attention on a specified speaker (expected location) for a block of trials. Accuracy and response time were compared across two conditions: (1) signal presented at the expected location and (2) signal presented at an unexpected location. Results indicate a significant increase in accuracy and faster response time when the signal was presented at the expected location as compared to an unexpected location. These results suggest that auditory spatial attention plays an important role in multi-source listening, especially when the listening environment is complex and uncertain.

Nitric oxide deficiency contributes to large cerebral infarct size.

The purpose of this study was to examine the role played by a deficit in nitric oxide (NO) in contributing to the large cerebral infarcts seen in hypertension. Cerebral infarction was produced in rats by occlusion of the middle cerebral artery (MCA). Studies were performed in Sprague-Dawley (SD) rats subjected to NO synthase blockade (N(G)-nitro-L-arginine [L-NNA], 20 mg x kg(-1) x d(-1) in drinking water) and in spontaneously hypertensive stroke-prone rats (SHRSP). NO released in the brain in response to MCA occlusion was monitored with a porphyrinic microsensor in Wistar-Kyoto rats. The increment in NO released with MCA occlusion was 1.31+/-0.05 micromol/L in L-NNA-treated rats, 1.25+/-0.04 micromol/L in SHRSP, 2. 24+/-0.07 micromol/L in control SD rats, and 2.25+/-0.06 micromol/L in Wistar-Kyoto rats (P<0.0001 for control versus the other groups). Infarct sizes in the L-NNA-treated and control SD rats were 8.50+/-0. 8% and 5.22+/-0.7% of the brain weights, respectively (P<0.05). The basilar arterial wall was significantly thicker in L-NNA-treated rats compared with their controls. We conclude that both the deficit in NO and the greater wall thickness contribute to the larger infarct size resulting from MCA occlusion in SHRSP and in L-NNA-treated rats compared with their respective controls.

Cell death and immunohistochemistry of p53, c-Fos and c-Jun after spermine injection into the rat striatum.

Administration of polyamines into the central nervous system results in tissue damage, possibly through the excitotoxic actions of the NMDA receptor. Direct injection of 100 nmol of spermine into the rat striatum produced a lesion equivalent to approximately 50% of the striatum. Analysis of the DNA in this region revealed the distinct ladder-like pattern of degradation often associated with apoptosis. This DNA fragmentation was confirmed in vivo using terminal deoxynucleotidyl-transferase-mediated biotinylated deoxyuridine triphosphate nick end labelling (TUNEL). The morphology of the TUNEL-positive cells showed marked differences at the needle tract when compared with cells in damaged areas away from the needle tract, suggesting a differential mechanism of cell death in these two regions. The patterns of p53, c-Fos and c-Jun protein expression were determined using immunohistochemistry. The number of p53-immunoreactive cells increased up to 14 h and returned to basal levels by 24 h. c-Fos protein expression transiently increased, peaking at 8 h after injection. c-Jun exhibited a protracted pattern of expression, remaining elevated up to 24 h. p53 protein expression was colocalised with TUNEL staining in areas away from the needle tract, but not in cells at the needle tract, suggesting once again a differential mechanism of cell death. At 14 h, c-Fos and c-Jun were not colocalised with TUNEL staining, suggesting that they are either not involved with the cell death process or that the time course of protein expression and the onset of DNA fragmentation do not overlap. This work represents the first characterisation of processes associated with cell death induced by spermine in vivo.

Schwann cell myelination requires timely and precise targeting of P(0) protein.

This report investigated mechanisms responsible for failed Schwann cell myelination in mice that overexpress P(0) (P(0)(tg)), the major structural protein of PNS myelin. Quantitative ultrastructural immunocytochemistry established that P(0) protein was mistargeted to abaxonal, periaxonal, and mesaxon membranes in P(0)(tg) Schwann cells with arrested myelination. The extracellular leaflets of P(0)-containing mesaxon membranes were closely apposed with periodicities of compact myelin. The myelin-associated glycoprotein was appropriately sorted in the Golgi apparatus and targeted to periaxonal membranes. In adult mice, occasional Schwann cells myelinated axons possibly with the aid of endocytic removal of mistargeted P(0). These results indicate that P(0) gene multiplication causes P(0) mistargeting to mesaxon membranes, and through obligate P(0) homophilic adhesion, renders these dynamic membranes inert and halts myelination.

Mutational analysis of a heterogeneous nuclear ribonucleoprotein A2 response element for RNA trafficking.

Cytoplasmic transport and localization of mRNA has been reported for a range of oocytes and somatic cells. The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 response element (A2RE) is a 21-nucleotide segment of the myelin basic protein mRNA that is necessary and sufficient for cytoplasmic transport of this message in oligodendrocytes. The predominant A2RE-binding protein in rat brain has previously been identified as hnRNP A2. Here we report that an 11-nucleotide subsegment of the A2RE (A2RE11) was as effective as the full-length A2RE in binding hnRNP A2 and mediating transport of heterologous RNA in oligodendrocytes. Point mutations of the A2RE11 that eliminated binding to hnRNP A2 also markedly reduced the ability of these oligoribonucleotides to support RNA transport. Oligodendrocytes treated with antisense oligonucleotides directed against the translation start site of hnRNP A2 had reduced levels of this protein and disrupted transport of microinjected myelin basic protein RNA. Several A2RE-like sequences from localized neuronal RNAs also bound hnRNP A2 and promoted RNA transport in oligodendrocytes. These data demonstrate the specificity of A2RE recognition by hnRNP A2, provide direct evidence for the involvement of hnRNP A2 in cytoplasmic RNA transport, and suggest that this protein may interact with a wide variety of localized messages that possess A2RE-like sequences.

Effect of deviations from temporal expectations on tempo discrimination of isochronous tone sequences.

The effect of deviations from temporal expectations on tempo discrimination was studied in 3 experiments using isochronous auditory sequences. Temporal deviations consisted of advancing or delaying the onset of a comparison pattern relative to an "expected" onset, defined by an extension of the periodicity of a preceding standard pattern. An effect of onset condition was most apparent when responses to faster and slower comparison patterns were analyzed separately and onset conditions were mixed. Under these conditions, early onsets produced more "faster" judgments and lower thresholds for tempo increases, and late onsets produced more "slower" judgments and lower thresholds for tempo decreases. In another experiment, pattern tempo had a similar effect: Fast tempos led to lower thresholds for tempo increases and slow tempos led to lower thresholds for tempo decreases. Findings support oscillator-based approaches to time discrimination.

Release from masking due to spatial separation of sources in the identification of nonspeech auditory patterns.

A nonspeech pattern identification task was used to study the role of spatial separation of sources on auditory masking in multisource listening environments. The six frequency patterns forming the signal set were comprised of sequences of eight 60-ms tone bursts. Bursts of masking sounds were played synchronously with the signals. The main variables in the study were (1) the difference in spatial separation in the horizontal plane between signals and maskers and (2) the nature of the masking produced by the maskers. Spatial separation of signal and masker ranged from 0-180 degrees. The maskers were of two types: (1) a sequence of eight 60-ms bursts of Gaussian noise intended to produce predominantly peripherally based "energetic masking" and (2) a sequence of eight 60-ms bursts of eight-tone complexes intended to produce primarily centrally based "informational masking." The results indicated that identification performance improved with increasing separation of signal and masker. The amount of improvement depended upon the type of masker and the center frequency of the signal patterns. Much larger improvements were found for spatial separation of the signal and informational masker than for the signal and energetic masker. This was particularly apparent when the acoustical advantage of the signal-to-noise ratio in the more favorable of the two ears (the ear nearest the signal) was taken into account. The results were interpreted as evidence for an important role of binaural hearing in reducing sound source or message uncertainty and may contribute toward solving the "cocktail party problem."

hnRNP A2 selectively binds the cytoplasmic transport sequence of myelin basic protein mRNA.

Segregation of mRNAs in the cytoplasm of polar cells has been demonstrated for proteins involved in Xenopus and Drosophila oogenesis, and for some proteins in somatic cells. It is assumed that vectorial transport of the messages is generally responsible for this localization. The mRNA encoding the basic protein of central nervous system myelin is selectively transported to the distal ends of the processes of oligodendrocytes, where it is anchored to the myelin membrane and translated. This transport is dependent on a 21-nucleotide cis-acting segment of the 3'-untranslated region (RTS). Proteins that bind to this cis-acting segment have now been isolated from extracts of rat brain. A group of six 35-42-kDa proteins bind to a 35-base oligoribonucleotide incorporating the RTS, but not to several oligoribonucleotides with the same composition but randomized sequences, thus establishing specificity for the base sequence in the RTS. The most abundant of these proteins has been identified, by Edman sequencing of tryptic peptides and mass spectroscopy, as heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a 36-kDa member of a family of proteins that are primarily, but not solely, intranuclear. This protein was most abundant in samples from rat brain and testis, with lower amounts in other tissues. It was separated from the other polypeptides by using reverse-phase HPLC and shown to retain preferential association with the RTS. In cultured oligodendrocytes, hnRNP A2 was demonstrated by confocal microscopy to be distributed throughout the nucleus, cell soma, and processes.

Myelin proteolipid protein expressed in COS-1 cells is targeted to actin-associated surfaces.

The compact myelin sheath represents one of the largest expanses of membrane-membrane contact in the body and, in the central nervous system, requires the myelin proteolipid protein (PLP) for assembly. To determine whether the molecular properties of PLP promote membrane adhesion and direct its subcellular localization in the absence of oligodendrocyte-specific targeting mechanisms, PLP was expressed in COS-1 fibroblasts. Immunofluorescence staining indicated that PLP was translated effectively, transited the rough endoplasmic reticulum and Golgi apparatus, was delivered to the cell surface, and was endocytosed. In the plasma membrane, the PLP distribution was patchy and only sporadically coincided with sites of membrane-membrane contact between PLP-expressing cells. PLP was not randomly distributed, however, but correlated closely with microfilament locations in leading edge membranes and microvilli, as demonstrated by phalloidin double labeling. Our results indicate that even in non-myelinating cells, PLP can be concentrated in membranes associated with movement and growth, and suggest possible roles for the actin cytoskeleton in PLP localization. As PLP, DM20, and the DM20-like M6 protein all associate with actin-enriched membranes, this may be a common feature of PLP/DM20 gene family members.

Asteroid hyalosis and vision loss after posterior capsulotomy.

We describe the case of a 77-year-old man with asteroid hyalosis who had phacoemulsification and implantation of a plate-haptic intraocular lens (IOL). Intraoperatively, a tear occurred in the anterior capsule, and vision loss occurred 3 months after a neodymium:YAG posterior capsulotomy. Because of the asteroid hyalosis, a posteriorly dislocated IOL, which occurred after the capsulotomy, was difficult to diagnose. Careful retinoscopy established the aphakic condition of the eye, and the B-scan ultrasonography indicated the IOL's location.

Phase independence of pitch produced by narrow-band sounds.

Three listeners matched the pitch of a simple tone to that of narrow-band complex signals having different phases. The pitch matches were independent of the phases; the frequency of the simple tone approximately equaled the center of gravity of the power spectrum of each complex signal. This result is inconsistent with a model that calculates the pitch of a waveform as the average of instantaneous frequency weighted by the envelope of the waveform.

Plus-maze retest profile in mice: importance of initial stages of trail 1 and response to post-trail cholinergic receptor blockade.

Recent research has shown that a single undrugged prior experience of the elevated plus-maze produces significant behavioural changes upon 24-h retest in rats and mice. Typically, when reexposed to the maze, animals display an increased avoidance of the open arms and a corresponding preference for the enclosed sections of the apparatus. Using ethological analyses, the present series of experiments sought to further characterize this phenomenon in mice and to determine whether or not it involves cholinergic receptor mechanisms. Results confirmed that behaviour during Trial 2 is markedly different to that seen on initial exposure, and that such changes are independent of the duration of Trial 1 (2 vs. 5 min). Retest behavioural changes included reduced entry latencies, reduced open arm entries, less time on the open arms and centre platform, lower levels of exploratory head-dipping, and increased entries into and time spent in the closed arms. The importance to the retest phenomenon of the first few minutes of initial exposure was further suggested by min-by-min analyses of the behaviour of animals naive to the maze. Results showed that behaviour during the first min is characterized by high levels of risk assessment from the centre platform and relatively low, but equal, levels of open- and closed-arm exploration. From min 2 onwards, however, behaviour showed a marked change with increasing open arm/centre platform avoidance, increasing closed-arm preference, and decreasing levels of risk assessment and exploratory head-dipping. Thus, it would appear that this within-session aversive learning transfers between sessions to account for behavioural profiles on retest. Irrespective of the duration of Trial 1 (2 or 5 min), posttrial administration of the muscarinic antagonist, scopolamine (0.1-1.0 mg/kg), failed to significantly alter the behavioural changes seen between trials. Data are discussed in relation to the apparent sensitization of fear produced by plus-maze exposure, its possible relation to phobia acquisition, and the need for further research on underlying mechanisms.

Axons regulate the distribution of Schwann cell microtubules.

It is well established that axons regulate Schwann cell phenotype. The purpose of the present study was to determine whether axons influence the arrangement of Schwann cell microtubules (MTs). Using double-labeling immunocytochemistry and confocal microscopy, we show that MTs in undifferentiated Schwann cells are nucleated from and attached to a single MT organizing center (MTOC) that is associated with the centrosome. Physical contact with appropriate axons initiates a myelin-forming phenotype that disperses MT minus ends and induces multiple MT-nucleating sites in Schwann cell perinuclear cytoplasm. The axonal signal that initiates myelin breakdown during Wallerian degeneration induces multiple MTOCs and MT bundles in Schwann cell perinuclear cytoplasm and in cytoplasm between degenerating myelin ovoids. These results establish that axons influence Schwann cell MT distribution by regulating the location and number of MT-nucleation sites.