Characterization of a macroporous epoxy-polymer based resin for the ion-exchange chromatography of therapeutic proteins.

This study investigated the adsorption capacity and mass transfer properties of a novel macroporous epoxy-polymer-based anion-exchanger, MPR Q, for the efficient separation of therapeutic proteins. MPR Q resin was prepared by phase separation based on spinodal decomposition followed by dextran grafting and ligand conjugation. Under static conditions, MPR Q exhibited a binding capacity of 49.8 mg-IgG/cm3-resin at pH 10, whereas the fastest adsorption was observed among the anion-exchanger resins tested. Inverse size-exclusion chromatography (iSEC) experiments revealed that the apparent pore diameter of MPR Q was approximately 90 nm, which was sufficiently large for the penetration of human IgG and bovine IgM. Moreover, the reduced height equivalent to a theoretical plate, h, of human IgG, determined using the linear gradient elution method was 65.8 and was not significantly changed in the range of linear velocities from 20.37 to 50.93 cm/min. The dynamic binding capacity at 10% breakthrough of MPR Q, determined by frontal analysis, exhibited a capacity of 43.8 mg/cm3 at 5.09 cm/min and 58% of DBC10% was maintained even though the linear velocity was increased to 50.93 cm/min. Furthermore, a resolution for separation of IgG and BSA by MPR Q was 1.06 at 5.09 cm/min, while it was higher than that for the conventional resin at all linear velocities from 5.09 cm/min to 50.93 cm/min. Thus, it was suggested that the MPR Q developed in this study is a promising resin that can efficiently separate large biomacromolecules such as human IgG at higher velocities.

Estimating the extent of subclinical arteriosclerosis of persons with prediabetes and diabetes mellitus among Japanese urban workers and their families: a cross-sectional study.

BACKGROUND: Diabetes mellitus (hereafter called diabetes) is considered to accelerate arteriosclerosis leading to coronary heart disease and stroke. Thus, it is important to quantitatively estimate the extent of subclinical arteriosclerosis. A new method called cardio-ankle vascular index (CAVI) is developed to reflect arterial stiffness independently from blood pressure at the time of measurement. Then, we examined if CAVI scores could discriminate the extent of arteriosclerosis between persons with prediabetes (or borderline diabetes) and with diabetes among Japanese urban workers and their families. METHODS: Subjects were 9881 men and 12033 women of company employees and their families who participated in cardiovascular disease screening in Japan. Persons having diabetes and prediabetes were defined based on the criteria set by American Diabetes Association. CAVI scores were measured by VaSera VS-1000. We applied the established age-sex specific cutoff points of CAVI scores above which were determined to be abnormally high or advanced level of arteriosclerosis. To examine the association of prediabetes and diabetes with CAVI scores, CAVI scores of screening participants were converted to a binary variable: 1 for less than cutoff points and 2 for equal or greater than cutoff points or abnormally high CAVI scores. Logistic regression method was used to examine the association of prediabetes and diabetes with CAVI scores after adjusting for major cardiovascular disease (CVD) risk factors. RESULTS: Prevalence of abnormally high CAVI scores was significantly higher after 40 years of age among persons with diabetes than either among persons with prediabetes or among normal persons in both genders. Significantly elevated odds ratios (ORs) of abnormally high CAVI scores appeared among persons with prediabetes: 1.29 (95 % confidence interval (CI), 1.11-1.48) for men and 1.14 (CI, 1.01-1.28) for women, and among persons with diabetes: 2.41 (CI, 1.97-2.95) for men and 2.52 (CI, 1.94-3.28) for women. CONCLUSIONS: The extent of subclinical arteriosclerosis (including arterial stiffness and atherosclerosis) was moderately enhanced among persons with prediabetes and was further advanced among persons with diabetes. Thus, it is important to introduce earlier interventions for changing lifestyle and diet of persons with prediabetes in order to prevent them from developing diabetes and further advancing arteriosclerosis.

Topographic distribution of cortical projection cells in the rat subiculum.

The topographic distribution of subicular pyramidal cells, which give rise to projections to the entorhinal cortex, presubiculum, parasubiculum, and the retrosplenial granular cortex, was investigated in the rat using retrograde labeling with wheat germ agglutinin-horseradish peroxidase. Using two-dimensional unfolded maps of the entire hippocampal and parahippocampal fields, we found that the cells originating the projections to the above cortical areas were consistently observed throughout the entire septotemporal extent of the subiculum. In the transverse plane, most of the cortical projection cells were vertically located in the middle region of the subicular pyramidal cell layer. The cells giving rise to the projections to the lateral entorhinal cortex were predominantly located in the most proximal (near CA1), superficial region. Few cortical projection cells were located in the deepest (adjacent to the angular bundle) region. The distribution of cortical projection cells showed an oblique tri-laminar pattern, which was similar to the previously reported laminal pattern of subcortical projection cells in the subiculum. These results suggest that cortical projection cells in middle and superficial regions of the subiculum may correspond to layer V of the isocortex and cells in the deepest region corresponding to layer VI.

Zonal distribution of perforant path cells in layer III of the entorhinal area projecting to CA1 and subiculum in the rat.

The distribution pattern of the cells that give rise to perforant path projections, including direct entorhino-CA1 and entorhino-subicular projections, was investigated in layer III of the medial and lateral entorhinal areas in the rat using retrograde labeling with horseradish peroxidase conjugated to wheat germ agglutinin and cholera toxin B subunit. Using two-dimensional unfolded maps of the entire hippocampal and parahippocampal fields, we found that cells projecting to a certain septotemporal level of CA1 and the subiculum were distributed in a band-like zone extending across the medial and lateral entorhinal areas. The transverse axis of these zones was disposed parallel to the rhinal fissure and their longitudinal axis was perpendicular to the boundary between the medial and lateral entorhinal areas. Projections to the septal CA1 originated from the zone near the rhinal fissure, whereas those to the temporal CA1 originated from the zone far from the rhinal fissure. Each zone in both the medial and lateral entorhinal areas involved many neurons projecting to a wide proximodistal range of CA1 and the subiculum. These results suggest that the entorhino-CA1 and entorhino-subicular perforant path projections are generally organized in a band-like zonal fashion with a gradient, rather than a point-to-point topographic arrangement.

New monolithic capillary columns with well-defined macropores based on poly(styrene-co-divinylbenzene).

Macroporous polymer monoliths based on poly(styrene-co-divinylbenzene) with varied styrene/divinylbenzene ratios have been prepared by organotellurium-mediated living radical polymerization. The well-defined cocontinuous macroporous structure can be obtained by polymerization-induced spinodal decomposition, and the pore structures are controlled by adjusting the starting composition. The separation efficiency of small molecules (alkylbenzenes) in the obtained monoliths has been evaluated in the capillary format by high-performance liquid chromatography (HPLC) under the isocratic reversed-phase mode. Baseline separations of these molecules with a low pressure drop (∼2 MPa) have been achieved because of the well-defined macropores and to the less-heterogeneous cross-linked networks.

Establishing baseline criteria of cardio-ankle vascular index as a new indicator of arteriosclerosis: a cross-sectional study.

BACKGROUND: A cardio-ankle vascular index (CAVI) has been developed to represent the extent of arteriosclerosis throughout the aorta, femoral artery and tibial artery independent of blood pressure. To practically use CAVI as a diagnostic tool for determining the extent of arteriosclerosis, our study objectives were (1) to establish the baseline CAVI scores by age and gender among cardiovascular disease (CVD) risk-free persons, (2) to compare CAVI scores between genders to test the hypothesis that the extent of arteriosclerosis in men is greater than in women, and (3) to compare CAVI scores between the CVD risk-free group and the CVD high-risk group in order to test the hypothesis that the extent of arteriosclerosis in the CVD high-risk group is greater than in the CVD risk-free group. METHODS: Study subjects were 32,627 urban residents 20-74 years of age who participated in CVD screening in Japan during 2004-2006. A new device (model VaSera VS-1000) was used to measure CAVI scores. At the time of screening, CVD high-risk persons were defined as those having any clinical abnormalities of CVD, and CVD risk-free persons were defined as those without any clinical abnormalities of CVD. Age-specific average CAVI scores were compared between genders and between the CVD risk-free group and the CVD high-risk group. Student's t-test using two independent samples was applied to a comparison of means between two groups. RESULTS: Average age-specific baseline scores of CAVI in the CVD risk-free group linearly increased in both genders as their age increased. Average age-specific baseline scores of CAVI in the CVD risk-free group were significantly greater among men than among women. Average age-specific baseline scores of CAVI in the CVD risk-free group were significantly smaller than those in the CVD high-risk group in both genders after 40 years of age. CONCLUSIONS: The baseline CAVI scores from the CVD risk-free group are useful for future studies as control values. The CAVI method is a useful tool to screen persons with moderate to advanced levels of arteriosclerosis.

[Longitudinal fiber systems in the hippocampal formation].

Longitudinal fiber systems in the hippocampal formation play an important role in propagating information to the entire septotemporal extent of the hippocampus. Previously, we have elucidated the longitudinal axonal projections of CA3 pyramidal cells (Ishizuka et al., 1990, J Comp Neurol 295: 580-623). Moreover, we have noted the existence of longitudinal fiber systems in the dentate gyrus. In the dentate gyrus, we found 2 types of longitudinal fiber systems originating from the hilar cells in the dentate gyrus-Type I and Type II fiber systems-by using the anterograde labeling technique with Phaseolus vulgaris-leucoagglutinin (PHA-L) and the retrograde labeling method with fluorescent dyes or the cholera toxin-B subunit. We observed that the Type I fiber system distributed its axonal ramifications in the inner molecular layer immediately above the granular cell layer, while the Type II fiber system distributed its terminals in the outer molecular layer. The Type I fiber system extended over almost the entire longitudinal (septotemporal) extent of the dentate gyrus, but in case of injection of the labeling molecule at the extreme temporal level, it extended within the temporal half of the dentate gyrus. In contrast, axonal arborizations of the Type II system were restricted within approximately 1.5 mm from the level of injection in each septotemporal direction. Axonal arborizations of the Type I fiber systems terminated densely in the narrow band of the inner molecular layer, while those of the Type II fiber system were loosely distributed in a slightly wider area of the outer molecular layer. The cells of origin of both Type I and Type II fiber systems were located in the hilar area. It appears likely that the cells of origin of the Type I system distributed their dendrites within the hilar area, since their cell bodies and axons were not labeled when the injection of PHA-L was restricted within the molecular layer. In contrast, the cells of origin of the Type II fiber system seemed to extend their dendrites over the outer molecular layer of the dentate gyrus. It appears likely that these longitudinal fiber systems of the dentate gyrus and the field CA3 function as an important system for analyzing information under physiological conditions, but under pathological conditions such as epileptic seizure abnormal neuronal bursting might expand to the entire extent of the hippocampus via these longitudinal systems.

Organization of connectivity of the rat presubiculum: II. Associational and commissural connections.

The regional, laminar, and longitudinal organization of intrinsic projections in the presubiculum was examined in the rat with the retrograde tracer horseradish peroxidase conjugated to wheat germ agglutinin and the anterograde tracer Phaseolus vulgaris-leucoagglutinin. Cells of origin of intrinsic projections in the presubiculum were distributed in layers II and V, with almost none in layers III and VI. Projections from layer II cells were bilateral and confined to layers II and V and also to the deep portion of layer I, whereas projections from layer V cells were ipsilateral and confined largely to layer V, with fewer projections to layer II. Septotemporal and proximodistal differences in both the projection and the distribution of layer II cells were found: layer II cells in the septal and mid presubiculum, especially those located in the distal part, provided long projections to the temporal presubiculum, whereas layer II cells in the temporal presubiculum provided slightly shorter projections almost entirely within the mid and temporal presubiculum. Layer II cells aggregated massively in the distal portions of the septal and mid presubiculum, but very few layer II cells were found in the most proximal part, especially in the temporal presubiculum. On the other hand, in layer V, cells of origin and their terminals were diffusely and equally distributed throughout the entire proximodistal extent of the presubiculum. Layer V cells did not project longitudinally as far as layer II cells. These longitudinal connections, in layers II and V, make it possible to merge information conveyed by parallel pathways in the presubiculum.

[The review of 'Yakovlev' circuit].

The neuronal circuit, the so-called 'Yakovlev', is recognized as the neuronal connections between the orbitofrontal, temporal, amygdaloid, and thalamic regions, possibly relevant to emotional function. This name is well known in Japan, however, it is not clear how this neural circuit has become known and why it was named after Yakovlev. Yakovlev, himself, just emphasized the relationship between emotion and the limbic lobes. It might be Nauta who first clarified the circuitry connections between these structures. Then, it was named the 'basolateral limbic circuit' by Livingston. The reason that the circuit had been called 'Yakovlev' might be related to this reference by Livingston who associated the circuit with the hypothesis of limbic system and emotion by Yakovlev. Recent sophistication in tracing methods has clarified the minimal contribution of the anterior cingulate gyrus to this circuit and that the projection from the amygdala to thalamus is unidirectional It will then be more appropriate to define this circuit as follows: orbital and cingulate gyri<=> temporal tip<=> amygdala=> mediodorsal thalamus<=> orbital and cingulate gyri. Yakovlev contributed greatly to identifying the possible relevance of these structures to emotion, which was substantiated by Nauta with his anatomical tracing between the structures. The name of this circuit, 'Yakovlev', would be better modified to 'Yakovlev-Nauta'.

Enhanced sequence coverage in tryptic fragment analysis by two-dimensional HPLC/MS using a monolithic silica capillary column.

The HPLC/MS system, in which a monolithic silica capillary column is directly connected to an electronspray-ionization mass spectrometer, showed superior performance at high mobile phase linear velocity. A two-dimensional (2D) HPLC/MS system was established, using an ion-exchange particle-packed capillary column at the first dimension and a monolithic silica capillary column at the second dimension. In an analysis of tryptic fragments from bovine serum albumin, an 81% sequence coverage, obtained by the 2D-HPLC/MS system, increased by 23% as compared to a 1D-HPLC/MS system. This 2D-HPLC/MS system using a monolithic silica capillary column should be useful for enhancing sequence coverage of tryptic fragments in proteomics.

Preparation of glycerol dimethacrylate-based polymer monolith with unusual porous properties achieved via viscoelastic phase separation induced by monodisperse ultra high molecular weight poly(styrene) as a porogen.

The preparation of polymer-based monolith capillary was examined by the use of glycerol dimethacrylate (GDMA) as monomer and monodisperse standard polystyrene (PS) solution in chlorobenzene as porogen. Poly-GDMA monoliths were prepared in situ in test tubes with standard PS having the variety of molecular weight (defined as Mw hereafter) from 50,000 to 3,840,000, and their morphology was compared to that of poly-GDMA monolith prepared in situ with a poor porogenic solvent of GDMA. According to scanning electron micrograph (SEM) observation, the structure of poly-GDMA monolith prepared in situ with toluene as a poor porogenic solvent showed a typical agglomerated globular structure, whereas the morphology of poly-GDMA monolith prepared in situ with the polymer (PS) porogenic solution was transformed from the aggregated globule form to three dimensionally (3D) continuous skeletal structure with the increase of Mw of standard PS utilized. Along with this morphological transformation or change, in the case of poly-GDMA monolith prepared in situ with ultra high Mw standard PS porogenic solution, the pore size distribution showed a sharp bimodal distribution, with one peak being located around 4 nm in the mesopore range (2-50 nm) and the other peak located around 1-2 microm in the macropore range (>50 nm), respectively. The poly-GDMA capillaries were prepared in situ with toluene, low Mw (50,000, 600,000) PS solution in chlorobenzene and the above mentioned ultra high Mw PS solution in chlorobenzene as a porogen, respectively, and measured by mu-HPLC with benzene and n-alkyl phenyl ketone as solutes for the evaluation in aqueous methanol (MeOH/H(2)O = 50/50-80/20, v/v). The permeability of capillaries prepared in situ with ultra high Mw standard PS polymer porogenic solution was much larger, compared to those of the capillaries prepared in situ with low Mw standard PS polymer porogenic solution or with toluene as porogen. On the other hand, the column efficiency was better in the case of the capillary prepared in situ with the ultra high Mw PS solution than in the latter capillaries. Those observations indicated that the ultra high Mw standard PS polymer porogenic solution should delay dynamically the phase separation of polymerizing mixture because of its visco-elasticity and should contribute to the creation of three dimensionally continuous skeletal monolith structure better to afford high separation efficiency.

Two-dimensional HPLC on-line analysis of phosphopeptides using titania and monolithic columns.

Conventional and comprehensive two-dimensional (2D) HPLC systems using the combination of titania and monolithic columns were established for the on-line analysis of phosphopeptides. Compared with immobilized metal affinity chromatography of a general method for the analysis of phosphopeptides, the use of titania columns in the analysis permits the specific isolation of phosphopeptides in a higher yield. Using the current 2D HPLC systems, phosphopeptides were specifically isolated from nonphosphorylated peptides by the first-dimension titania column, followed by the high-speed separation of the phosphopeptides by the second-dimension monolithic column. Proteolytic digests of beta-casein were analyzed within 30 min using the comprehensive 2D HPLC system; all phosphopeptides from beta-casein could be efficiently isolated and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The comprehensive 2D HPLC system coupled with mass spectrometry will be useful for high-throughput and on-line phosphoproteome analyses.

Simple 2D-HPLC using a monolithic silica column for peptide separation.

Separation of peptides by fast and simple two-dimensional (2D)-HPLC was studied using a monolithic silica column as a second-dimension (2nd-D) column. Every fraction from the first column, 5 cm long (2.1 mm ID) packed with polymer-based cation exchange beads, was subjected to separation in the 2nd-D using an octadecylsilylated (C18) monolithic sillica column (4.6 mm ID, 2.5 cm). A capillary-type monolithic silica C18column (0.1 mm ID, 10 cm) was also employed as a 2nd-D column with split flow/injection. Effluentof the first dimension (1st-D) was directly loaded into an injector loop of 2nd-D HPLC. UV and MS detection were successfully carried out at high linear velocity of mobile phase at 2nd-D using flow splitting for the 4.6 mm ID 2nd-D column, or with directconnection of the capillary column to the MS interface. Two-minute fractionation inthe 1st-D, 118-second loading, and 2-second injection by the 2nd-D injector, allowed one minute for gradient separation in the 2nd-D, resulting in a maximum peak capacity of about 700 within 40 min. The use of a capillary column in solvent consumption and better MS detectability compared to a larger-sized column. This kind of fast and simple 2D-HPLC utilizing monolithic silica columns will be useful for the separation of complex mixtures in a short time.

Development of a monolithic silica extraction tip for the analysis of proteins.

In proteomics, pre-treatment of sample is the most important procedure to remove the matrix for interfacing with mass spectrometry (MS). Additionally, for the samples with low concentration, the process of pre-concentration is required before MS analysis. We have newly developed solid-phase extraction (SPE) tool with pipette-tip shape for purification of bio-samples of various characteristics, utilizing monolithic silica gel as medium. The monolithic silica surface was modified with a C18 phase or coated with titania phase. A C18-bonded tip and a non-modified tip were used for sample concentration, desaltination and removal of detergents from sample. A titania-coated tip was also applied for purification and concentration of phosphorylated peptides. This novel pre-treatment method using monolithic silica extraction tip is much effective and suitable for protein analysis.

A quantitative analysis of the laminar distribution of synaptic boutons in field CA3 of the rat hippocampus.

We analyzed the laminar distribution of synaptic boutons in field CA3 of the rat hippocampus using a large montage electron micrograph. The size of boutons and synaptic vesicles was measured using a computer-assisted digitizing system. In all, 3353 synaptic boutons were observed in a 15 microm x 100 microm strip. Of these, 86.3% contained spherical vesicles (S-boutons), 12% contained flat vesicles (F-boutons), and 1.7% were mossy terminals (M-boutons). S-boutons were distributed widely in the strata moleculare (st. Mol), radiatum (st. Rad), and oriens (st. Ori), but there were only a few in the strata lucidum (st. Luc) and pyramidale (st. Pyr). The upper portions of both the st. Rad and Ori contained slightly fewer boutons. In terms of the location of synaptic contacts, 83% of all S-boutons were found on the dendritic spines and the rest were on the dendritic shafts. S-boutons on the dendritic shafts were observed more frequently in the st. Mol than in the other strata. According to the morphometry of the size of synaptic vesicles, S-boutons with small vesicles (mean vesicle area <1109 nm(2)) were located exclusively in the st. Mol, S-boutons with medium-sized vesicles (mean vesicle area 1109-1482 nm(2)) were observed in all strata, and S-boutons with large vesicles (mean vesicle area >1482 nm(2)) were distributed in the st. Luc and Ori, but not in the st. Mol. F-boutons were predominantly distributed in the upper half of the st. Mol and in the area around the st. Pyr, although they were observed in all strata. In the st. Mol, all the F-boutons were in contact with dendritic shafts, while near the st. Pyr, F-boutons were found exclusively on somata, the proximal parts of the dendritic shafts, and the initial segments of axons. The average F-bouton was smaller in the st. Mol (0.23 microm(2)) than near the st. Pyr (0.39 microm(2)). In this synapto-architectural study of the hippocampal CA3 region using large montage electron micrographs, we observed (1) an intimate relationship between synapse distribution and the dendritic structure of pyramidal neurons, (2) the distribution of different types of boutons containing vesicles of various size, and (3) two different plausible foci of postsynaptic inhibition where F-boutons were distributed densely, and (4) estimated the input ratios of pyramidal neurons.

Organization of connectivity of the rat presubiculum: I. Efferent projections to the medial entorhinal cortex.

The organization of the laminar and topographical projections from the presubiculum to the entorhinal area was studied in the rat by anterograde labeling with Phaseolus vulgaris leucoagglutinin and retrograde labeling with horseradish peroxidase conjugated to wheat germ agglutinin. We found that the pattern of presubiculo-entorhinal projections differs between the superficial and deep layers of the presubiculum. The superficial layers (layers II and III) of the presubiculum gave rise to bilateral projections to layers I-VI of the medial entorhinal area (MEA). Many terminals were distributed in layer III, fewer in layer II and the deep portion of layer I, and many fewer terminals in the deep layers (layers V and VI) of MEA. In contrast, the deep layers (layers V and VI) of the presubiculum gave rise to ipsilateral projections to the entorhinal area. Many axon terminals were distributed in layers V and VI of MEA and the most superficial portion of layer I of MEA, but very few in layers II and III. In addition, the ramifications in layer I extended to the lateral entorhinal area (LEA). Using two-dimensional unfolded maps of parahippocampal cortices, we elucidated the distinct topographical relationship in the presubiculo-entorhinal projection: 1) The septotemporal or longitudinal axis of the presubiculum corresponded to the axis on the MEA/LEA boundary, where the septal presubiculum projected toward the rhinal fissure and the temporal presubiculum projected away from the fissure. 2) The proximodistal axis of the presubiculum corresponded to the axis from the MEA/LEA boundary to the MEA/parasubiculum boundary that was virtually perpendicular to the MEA/LEA boundary, where the proximal portion of the presubiculum (close to the subiculum) projected to the region near the MEA/LEA boundary.

Microanalysis for MDR1 ATPase by high-performance liquid chromatography with a titanium dioxide column.

MDR1 is clinically important because it is involved in multidrug resistance of cancer cells and affects the pharmacokinetics of various drugs. Because MDR1 harnesses adenosine 5'-triphosphate (ATP) hydrolysis for transporting drugs, examining the effect on ATPase activity is imperative for understanding the interactions between drugs and MDR1. However, conventional assay systems for ATPase activity are not sensitive enough for screening drugs using purified MDR1. Here we report a novel method to measure ATPase activity of MDR1 using high-performance liquid chromatography equipped with a titanium dioxide column. The amount of adenosine 5'-diphosphate (ADP) produced by the ATPase reaction was determined within 2 min with a titanium dioxide column (4.6 mm ID x 100 mm). The relationship between ADP amount and chromatogram peak area was linear from 5 pmol to 10 nmol. This method made it possible to reduce the amount of purified MDR1 required for a reaction to 0.5 ng, about 1/20th of the conventional colorimetric inorganic phosphate detection assay. This method is sensitive enough to detect any subtle changes in ATPase activity of MDR1 induced by drugs and can be applied to measure ATPase activity of any protein.

Simple and comprehensive two-dimensional reversed-phase HPLC using monolithic silica columns.

Simple and comprehensive two-dimensional (2D)-HPLC was studied in a reversed-phase mode using monolithic silica columns for second-dimension (2nd-D) separation. Every fraction from the first column, 15 cm long (4.6-mm i.d.), packed with fluoroalkylsilyl-bonded (FR) silica particles, was subjected to the separation in the 2nd-D using one or two octadecylsilylated (C(18)) monolithic silica columns (4.6-mm i.d., 3 cm). Monolithic silica columns in the 2nd-D were eluted at a flow rate of up to 10 mL/min with separation time of 30 s that meets the fractionation every 15-30 s at the first dimension (1st-D) operated at a flow rate of 0.4-0.8 mL/min. Three cases were studied. (1) In the simplest scheme of 2D-HPLC, effluent of the 1st-D was directly loaded into an injector loop of 2nd-D HPLC for 28 s, and 2 s was allowed for injection. (2) Two six-port valves each having a sample loop were used to hold the effluent of the 1st-D alternately for 30 s for one 2nd-D column to effect comprehensive 2D-HPLC without the loss of 1st-D effluent. (3) Two monolithic silica columns were used for 2nd-D by using a switching valve and two sets of 2nd-D chromatographs separating each fraction of the 1st-D effluent with the two 2nd-D columns alternately. In this case, two columns of the same stationary phase (C(18)) or different phases, C(18) and (pentabromobenzyloxy)propylsilyl-bonded (PBB), could be employed at the 2nd-D, although the latter needed two complementary runs. The systems produced peak capacity of approximately 1000 in approximately 60 min in cases 1 and 2 and in approximately 30 min in case 3. The three stationary phases, FR, C(18), and PBB, showed widely different selectivity from each other, making 2D separations possible. The simple and comprehensive 2D-HPLC utilizes the stability and high efficiency at high linear velocities of monolithic silica columns.

Triple immunofluorolabeling with two rabbit polyclonal antibodies and a mouse monoclonal antibody allowing three-dimensional analysis of cotton wool plaques in Alzheimer disease.

We established a triple-labeling method with two rabbit polyclonal antibodies and a mouse monoclonal antibody and examined autopsied brain tissue with cotton wool plaques (CWPs). One of the polyclonal antibodies was so diluted (anti-Abeta42 or anti-Abeta40/1:30,000 or anti-von Willebrand factor/1:1000) that its visualization was possible only after amplification with the catalyzed reporter deposition (CARD) method. The other polyclonal antibody (anti-Abeta40 or anti-Abeta42/1:1000) was visualized with a fluorochrome conjugated to an anti-rabbit antibody that specifically visualized the latter polyclonal antibody because of its lower sensitivity. A monoclonal antibody, AT8, was superimposed to yield triple immunofluorolabeling. Serial optical sections with an interval of 0.3 micro m were reconstructed to allow three-dimensional (3D) observation of these three epitopes. Abeta40 was localized to core-like structures, mainly in layers I-III, and was sometimes in contact with the vascular wall, both without neuritic reactions. CWPs, present in layers I-VI, were labeled with anti-Abeta42 and were accompanied by neuritic reactions. These differences suggest that mechanisms of Abeta deposition and its relation to neuritic reactions or to blood vessels differ according to the lesion, even in the same microscopic field.