Removal of small non-enveloped viruses by nanofiltration.

BACKGROUND AND OBJECTIVES: Nanofiltration is one of the most effective virus reduction methods in the manufacturing process of plasma products. However, it is difficult to remove small viruses from high molecular weight protein preparations like immunoglobulin G or factor VIII complex by nanofiltration, because the size of the protein is similar to that of viruses. In order to separate the viruses from these proteins by nanofiltration, it is necessary to change the size of either one. In this study, we report that such non-enveloped viruses as human parvovirus B19 (B19), human encephalomyocarditis virus (EMC) or porcine parvovirus (PPV) aggregate in the presence of certain kinds of amino acids and could be easily removed by nanofiltration. MATERIALS AND METHODS: 0.3 M Glycine (or other amino acid) solution spiked with viruses was subjected to dead-end single filtration with a 35-nm pore-size filter. Virus removal by nanofiltration was either evaluated by PCR or by infectivity assay. RESULTS: B19 in a 0.3 M glycine solution was reduced to 1:10(7.5) (7.5-log) by nanofiltration with a 35-nm pore-sized filter, whereas in PBS it was not reduced. Similarly, B19 was also reduced when suspended in other amino acids solutions. This effect was also confirmed with the other small non-enveloped viruses EMC or PPV. When 5% globulin or 5% albumin was added to a 0.3 M glycine solution, the removal rate was decreased. CONCLUSIONS: These data suggest that viruses in the presence of certain kinds of amino acids could be aggregated and effectively removed by a filter that has a pore size larger than the size of the viruses.

Intrinsic collaterals of layer 6 Meynert cells and functional columns in primate V1.

Meynert cells are a distinct type of large neuron which project to area MT/V5 and to subcortical targets, including the superior colliculus. They have recently been shown to have extensive intrinsic collaterals spreading up to 8.0 mm within layer 6 of area V1 [J Comp Neurol 441 (2001) 134]. Using intrinsic signal imaging combined with tracer injections, this study investigates how Meynert cell collaterals are mapped in relation to the functional architecture of area V1 in macaque monkeys. In particular, we examined whether terminations of individual axon segments are selective for same-eye or opposite-eye domains. Analysis of 39 anterogradely labeled axon segments (from six injection sites in four hemispheres) showed that terminal segments cross over several pairs of ocular dominance columns (ODCs) and contact both left- and right-eye ODCs, with a slight bias for the contralateral eye. This contrasts with the same-eye bias previously reported for intrinsic collaterals of pyramidal neurons in layer 3. The suggestion is that the system of Meynert intrinsic collaterals is involved with binocular interactions over wide sectors of the visual field. This might be related to processes such as optic flow or, especially given the wide-field spread, even contour completion or interpolation.

Novel functional imaging technique from brain surface with optical coherence tomography enabling visualization of depth resolved functional structure in vivo.

Mapping of the activity of brain by optical intrinsic signal imaging (OISI) provides a two-dimensional activation pattern of visual cortical areas at a resolution of a few hundred microns. However, integration of the intrinsic signal over depth results in loss of finer information about functional organization across the depth. Here, we report the first successful implementation of optical coherence tomography (OCT) at around 30 microm depth resolution to investigate cortical functions of a cat brain in vivo. This technique, named functional OCT (fOCT) provided visually evoked changes in the OCT signal. The fOCT signal shows stimulus specificity that correlates well with that of the intrinsic signals and provides depth resolved layer specific functional information.

Complex objects are represented in macaque inferotemporal cortex by the combination of feature columns.

Intrinsic signal imaging from inferotemporal (IT) cortex, a visual area essential for object perception and recognition, revealed that visually presented objects activated patches in a distributed manner. When visual features of these objects were partially removed, the simplified stimuli activated only a subset of the patches elicited by the originals. This result, in conjunction with extracellular recording, suggests that an object is represented by a combination of cortical columns, each of which represents a visual feature (feature column). Simplification of an object occasionally caused the appearance of columns that were not active when viewing the more complex form. Thus, not all the columns related to a particular feature were necessarily activated by the original objects. Taken together, these results suggest that objects may be represented not only by simply combining feature columns but also by using a variety of combinations of active and inactive columns for individual features.

Odor maps in the mammalian olfactory bulb: domain organization and odorant structural features.

Psychophysical studies indicate that structural features of odorants differentially influence their perceived odor. In the olfactory bulb (OB), odorants are represented by ensembles of activated glomeruli. Here we used optical imaging of intrinsic signals to examine how these structural features are represented spatially in the sensory map of the rat OB. We found that the dorsal OB contained two topographically fixed domains; constituent glomeruli in each domain could be activated by odorants with particular functional groups. Within each domain, other structural features such as carbon chain length and branching were represented by local differences in patterns. These results suggest that structural features are categorized into two classes, primary features (functional groups) that characterize each domain, and secondary features that are represented by local positions within each domain. Such hierarchical representations of different structural features correlate well with psychophysical structure-odor relationships.

Optical responses evoked by single-pulse stimulation to the dorsal root in the rat spinal dorsal horn in slice.

Neuronal excitation evoked after dorsal-root (DR) stimulation in the spinal dorsal horn (DH) of rats was visualized with a high-resolution optical-imaging method, and the propagation mechanism was studied. Transverse slices of the spinal cord were obtained from 2-4 week-old rats and stained with the voltage-sensitive dye RH-482. Single-pulse stimulation to the primary-afferent A fibers in the DR attached to the slice evoked a weak, brief (<10 ms) excitatory optical response in the laminae I and III-V. When the stimulus intensity and duration were increased to activate both A and C fibers, an additional, much greater, and longer-lasting (>100 ms) excitatory response was generated in the laminae I-III, most intensely in the lamina II. A treatment with excitatory amino acid (EAA) antagonists, dl-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2, 3-dione, significantly reduced the amplitude and duration of the response in the lamina II. The optical response in the antagonists-containing solution was quite similar to that recorded in a Ca2+-free solution that blocked afferent synaptic transmission. The late component (>10 ms) was, however, slightly greater than that in the Ca2+-free solution. Treatment with the ATP-receptor antagonist, suramin, had a minimal effect on the response in the presence of EAA antagonists. These results suggested that the propagation of the DR-stimulus-elicited excitation was contributed largely by EAA receptors, but also by other receptors to a much lesser extent.

Functional architecture in monkey inferotemporal cortex revealed by in vivo optical imaging.

To investigate the functional organization in the monkey inferotemporal cortex, which is the last exclusively visual area along the ventral visual cortical pathway, optical imaging based on intrinsic signals was carried out. We first conducted single-cell recordings with microelectrodes and determined the features critical for the activation of single cells. For the subsequent optical imaging, each critical feature was presented, which evoked multiple dark spots. Individual spots were approximately 0.5 mm in diameter and one of them covered the site of the electrode penetration at which the particular critical feature had been determined. The degree of stimulus selectivity varied from spot to spot, and from region to region even within a spot. Some regions were activated only by one of 12- 16 stimuli, while others by more than three stimuli. There were spots specifically activated by faces, and the position of activation spot changed gradually along the cortical surface as the stimulus face was rotated in depth. The length of the overall region along the direction of shift of these spots was approximately 1 mm. These results confirm the regional clustering of cells with similar stimulus selectivity and suggest larger units in which some parameters of object features are continuously mapped.

Delayed signal propagation via CA2 in rat hippocampal slices revealed by optical recording.

Signal propagation from mossy fibers to CA1 neurons was investigated in rat hippocampal slices by a combination of electrical and optical recordings. The slices were prepared by oblique sectioning of the middle part of the hippocampus to preserve fiber connections. The mossy fibers were stimulated to induce population spikes (PSs) and excitatory postsynaptic potentials in the middle part of the CA1 region. Latencies of maximal PSs in CA1 varied widely among slices; they ranged from 7 to 13.5 ms, with two maxima at 9 and 11.5 ms. The fastest PSs probably are evoked by the Schaffer collaterals that connect the CA3 and CA1 regions in the well-known trisynaptic circuit. However, the slower PSs suggest the existence of additional delayed inputs. To determine the source of the delayed input, slices were stained with a voltage-sensitive dye, RH482, and the optical signals relevant to membrane potential changes were detected by a high-resolution optical imaging system. Optical recording of responses to mossy fiber stimulation indicated two distinct types of signal propagation from CA3 to CA1. In preparations evincing the fast type of propagation, signals spread to CA1 within 7.2 ms after the mossy fiber stimulation. During such propagation, activity flowed directly from CA3 to the stratum radiatum of CA1. Other preparations illustrated slow signal propagation, in which optical signals were generated in CA2 before spreading to CA1. During such slow signal transmission, activity persisted in CA2 and its surrounding area for 3 ms before propagating to the strata radiatum and oriens in CA1. In such cases, CA1 activity was detected within 10.8 ms of mossy fiber stimulation. In some slices, a mixture of the fast and slow propagation patterns was observed, indicating that these two transmission modes can coexist. Our data reveal that CA2 neurons can transmit delayed excitatory signals to CA1 neurons. We therefore conclude that consideration of electrical signal propagation through the hippocampus should include flow through the CA2 region in addition to the traditional dentate gyrus-CA3-CA1 trisynaptic circuit.

Optical responses evoked by white matter stimulation in rat visual cortical slices and their relation to neural activities.

To characterize optical responses (ORs) evoked by white matter (WM) stimulation in slices of rat visual cortex (VC) stained with voltage sensitive dyes, time course of ORs in each layer was investigated by recording ORs with a linearly aligned photodiode array, and the spatial patterns of the ORs at specified time after stimulation were investigated by a CCD camera in combination with stroboscopic illumination. The ORs recorded by the photodiode array were an increase in absorption at 700 nm and a decrease in the wavelength below 650 nm, suggesting that the ORs were dye related. The ORs were compared with field potentials (FPs) to clarify that neural events were represented by the ORs, and in support of this view, we found that the first order spatial differentials of ORs and that of FPs were in good agreement. We further compared ORs with intracellular responses, and found that the ORs mainly represent postsynaptic potentials (PSPs) of VC neurons except for the deeper part of layer VI, where a component representing action potentials in fibers stimulated directly was observed. The time-lapse imaging of ORs showed that excitation first propagated vertically up to layer I and subsequently in the horizontal direction along layers II-III and V-VI as in previous investigations. Spatio-temporal patterns of ORs under blockade of synaptic transmission were also investigated to reveal activity of fibers evoked by WM stimulation which produced such patterns of propagation.

Optical imaging of functional organization in the monkey inferotemporal cortex.

To investigate the functional organization of object recognition, the technique of optical imaging was applied to the primate inferotemporal cortex, which is thought to be essential for object recognition. The features critical for the activation of single cells were first determined in unit recordings with electrodes. In the subsequent optical imaging, presentation of the critical features activated patchy regions around 0.5 millimeters in diameter, covering the site of the electrode penetration at which the critical feature had been determined. Because signals in optical imaging reflect average neuronal activities in the regions, the result directly indicates the regional clustering of cells responding to similar features.

Optical responses recorded after local stimulation in rat neostriatal slice preparations: effects of GABA and glutamate antagonists, and dopamine agonists.

Effects of GABA and glutamate antagonists as well as dopamine agonists and antagonists on the optical responses of neostriatal (Str) slices to local electrical stimulation were examined using a voltage-sensitive dye and a high-speed image sensor. A single local stimulation applied to the Str slices evoked optical responses lasting for 40-80 ms and propagating in every direction up to about 1.5 mm. Bath application of bicuculline methiodide increased the intensity and duration of optical responses, while their spatial response patterns were unchanged. Bath application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) greatly reduced the late part of responses occurring about 4 ms after stimulation, but the early part of responses was unaffected by CNQX. The early part of the response was eliminated by application of tetrodotoxin. Bath application of N-methyl-D-aspartate antagonists, 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid and 2-amino-5-phosphonovaleric acid resulted in only small changes in the optical responses. Bath application of D1 agonist 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5,-tetrahydro-1H-3-benz aze pine hydrobromide consistently increased the intensity but decreased the speed of propagation and duration of the optical response. Bath application of D2 agonist quinpirole had no effect on the optical response. D1 antagonist SCH 23390 and D2 antagonist sulpiride also failed to change optical responses. These results indicate that the early part of the response is due to direct activation of the neuronal elements by electrical stimulation, while the late part of the response is due mainly to glutamatergic ex-citatory postsynaptic potentials (EPSPs) mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors. This study also suggests that dopamine may modulate AMPA/kainate responses through D1 receptors.

Horizontal propagation of excitation in rat visual cortical slices revealed by optical imaging.

Optical imaging with high spatial and temporal resolution of neural activity in rat cortical slices was used to investigate the dynamics of signal transmission through neural connections in the visual cortex. When inhibition due to gamma-aminobutyric acid was slightly suppressed, horizontal propagation of excitation in both the supra- and infragranular layers became prominent. This propagation was not affected by vertical cuts in either the supra- or infragranular layer, which suggests that excitation is at least partially conveyed horizontally by reciprocal vertical connections between neurons in these layers.

Possible presence of the ATP-sensitive K+ channel in isolated spinal dorsal horn neurons of the rat.

The ATP-sensitive K+ channel (KATP channel) is a K+ channel inhibited by cytoplasmic ATP. It was originally found in cardiac cells and recently in neuronal cells. Here, we present evidence indicating that the KATP channel also exists in spinal dorsal horn neurons: membrane currents were recorded by whole-cell voltage-clamp in spinal dorsal horn neurons isolated from young rats. The outward current was augmented by KATP channel activators nicorandil and minoxidil and reduced by the blocker glibenclamide. This glibenclamide-induced change in the current was augmented when the intracellular ATP was lowered and the reversal potential was shifted according to the external K+ concentration.

Signal propagation from piriform cortex to the endopiriform nucleus in vitro revealed by optical imaging.

Optical signals were recorded from the posterior piriform cortex slices of guinea pigs stained with a voltage-sensitive dye to analyse spatio-temporal spread of neural activity evoked by electrical stimulation of afferent fibers. After propagation of activity along layers II and III, an isolated island of activity appeared deep to the layer III and moved caudally. Histological inspection revealed that the area where the island appeared corresponded well to the endopiriform nucleus. The present results provided an evidence for one of the main outflows of olfactory information from the posterior piriform cortex.

Optical imaging of the in vitro guinea pig piriform cortex activity using a voltage-sensitive dye.

The spatio-temporal patterns of signal processing in guinea pig piriform cortex (PC) slices were analyzed by optical imaging using a voltage-sensitive dye. Slices (400 microns thick) were cut in a plane parallel to the lateral olfactory tract and perpendicular to the cortical surface. In all the anterior PC and the majority of the posterior PC preparations, neural activity elicited by electrical stimulation of layer Ia propagated along the same layer, then it invaded into layers II and III and propagated along them. In addition to the above pattern, invasion of activity into the deeper area than layer III was observed in some posterior PC preparations. Real-time imaging of an active zone evoked by Ia shocks and its spatio-temporal behavior will contribute to resolving olfactory information processing.

Two-color cytofluorometry and cellular properties of the urokinase receptor associated with a human metastatic carcinomatous cell line.

Purified human urokinase was labeled with either fluorescein isothiocyanate or iodine-125 and used as a probe for binding to the human metastatic carcinomatous cell line, Detroit 562. Cytofluorometry showed that the ligand bound preferentially to cells that had been exposed to acidic pH. The binding was competitive and decreased after mild tryptic digestion. The bound ligand could be removed by restoration of the cells to a low pH. Therefore, the cells had specific binding sites. The bound urokinase was involved in the breakdown of fibrin. Two-color cytofluorometric maps were constructed by counterstaining with propidium iodide. Results suggested that there were different cell populations that had different numbers of receptors and amounts of DNA. We cloned cells and found that single clones had homogeneous levels of receptors with different dissociation constants (from 10(-13) to 10(-11) mol/mg protein) for different clones. Cells of one clone, C5, which had high levels of receptor production, moved characteristically on a glass substratum coated with gold particles and reacted with wheat germ agglutinin, but not with concanavalin A. The receptors were found together with adhesion proteins at the sites where the cells adhered to the substrate. These results and the data obtained by zymography of the cellular proteins suggested that the urokinase-type plasminogen activators were bound to the receptors. The membrane-associated activator may stimulate local proteolysis, facilitating the migration of the tumor cell across the substrate.

[A case report of postoperative thyroid crisis accompanied with struma ovarii].

A 39-year-old female without any specific past history was scheduled to receive an operation for myoma uteri and ovarian cyst. She was premedicated with atropine. Anesthesia was induced with thiopental and was maintained with nitrous oxide and enflurane. Tachycardia shortly after premedication with atropine and remarkable sweat during the operation were observed. On the 1st postoperative day an outbreak of thyroid crisis as well tachycardia of 180.min-1 and fever (39.3 degrees C) were observed. Such outbreak of thyroid crisis indicated that the patient had been suffering from Grave disease. Pathological diagnosis of extirpated ovarian cyst was struma ovarii. It is, however, still uncertain whether struma ovarii induced thyroid crisis in this case. It might be concluded that screening of hyperthyroidism at preoperative rounds is essential for prevention of thyroid crisis.

Local function of urokinase receptor at the adhesion contact sites of a metastatic tumor cell.

The local activity of urokinase and its receptor associated with a cloned cell (C5) obtained from the cloning of Detroit 562 was investigated. The cellular binding sites, similar structure to adhesion plaques, were visualized by fluorescein labeled urokinase and the number was determined to be 300 per cell. The binding sites for radioiodinated urokinase were found to be 30 thousand per cell. Thus, about as many as 100 receptor molecules per site was estimated to be associated with the cellular membrane domains. Immunofluorescence studies demonstrated that the receptors were colocalized with a set of adhesion and cytoskeletal proteins such as vinculin, alpha-actinin and actin; localizing at the adhesion sites. These proteins soluble in 9 M urea were able to be reconstituted by dialyzing out the urea against low ionic buffer solution. It was demonstrated that vinculin and actin were co-associated. Since cell bound urokinase revealed fibrinolytic activity, it was suggested that the focal adhesions of the migrating cells would facilitate proteolytic action when cells move across the matrix architectures.

Further characterization of the cation channel of a yeast vacuolar membrane in a planar lipid bilayer.

A voltage-dependent and Ca2(+)-activated cation channel recently found in the vacuolar membrane of the yeast Saccharomyces cerevisiae was incorporated into planar lipid bilayers and further characterized in macroscopic and single channel levels. Single channel conductances for various cations were in the order: NH4+ greater than K+ greater than Rb+ greater than Cs+ greater than Na+ greater than Li+, and were nearly consistent with the order of permeability ratio estimated from reversal potentials determined by macroscopic measurement. Up to 6 mM of Ca2+ added to the cis (cytoplasmic) side opened the channel, but higher concentrations closed the channel without affecting the single channel conductance. Ba2+ closed the channel without affecting the single channel conductance. Ba2+ closed the channel from the cis side. In addition to the above channel, a small cation-selective channel of about 40 pS was found.