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1.  Kv3.1b and Kv3.3 channel subunit expression in murine spinal dorsal horn GABAergic interneurones 
► Kv3.1b and Kv3.3 expression was studied in dorsal horn GABAergic interneurones. ► Kv3.1b or Kv3.3 was most abundantly expressed in laminae I–III. ► Kv3.1b but not Kv3.3 was associated with GAD65 and GAD67 neurones. ► Capsaicin-induced c-fos expression was localized mainly to GAD65-GFP interneurones.
GABAergic interneurones, including those within spinal dorsal horn, contain one of the two isoforms of the synthesizing enzyme glutamate decarboxylase (GAD), either GAD65 or GAD67. The physiological significance of these two GABAergic phenotypes is unknown but a more detailed anatomical and functional characterization may help resolve this issue. In this study, two transgenic Green Fluorescent Protein (GFP) knock-in murine lines, namely GAD65-GFP and GAD67-GFP (Δneo) mice, were used to profile expression of Shaw-related Kv3.1b and Kv3.3 K+-channel subunits in dorsal horn interneurones. Neuronal expression of these subunits confers specific biophysical characteristic referred to as ‘fast-spiking’. Immuno-labelling for Kv3.1b or Kv3.3 revealed the presence of both of these subunits across the dorsal horn, most abundantly in laminae I–III. Co-localization studies in transgenic mice indicated that Kv3.1b but not Kv3.3 was associated with GAD65-GFP and GAD67-GFP immunopositive neurones. For comparison the distributions of Kv4.2 and Kv4.3 K+-channel subunits which are linked to an excitatory neuronal phenotype were characterized. No co-localization was found between GAD-GFP +ve neurones and Kv4.2 or Kv4.3. In functional studies to evaluate whether either GABAergic population is activated by noxious stimulation, hindpaw intradermal injection of capsaicin followed by c-fos quantification in dorsal horn revealed co-expression c-fos and GAD65-GFP (quantified as 20–30% of GFP +ve population). Co-expression was also detected for GAD67-GFP +ve neurones and capsaicin-induced c-fos but at a much reduced level of 4–5%. These data suggest that whilst both GAD65-GFP and GAD67-GFP +ve neurones express Kv3.1b and therefore may share certain biophysical traits, their responses to peripheral noxious stimulation are distinct.
PMCID: PMC3161392  PMID: 21440618
GAD65; GAD67; Immunohistochemistry; c-fos; Capsaicin; Nociception; Potassium channel
Neuroscience  2007;145(1):66-79.
Excessive glutamate receptor stimulation can produce rapid disruption of dendritic morphology, including dendritic beading. We recently showed that transient NMDA exposure resulted in irreversible loss of synaptic function and loss of MAP2 from apical dendrites. The present study examined the initiation and progression of dendritic injury in slice following this excitotoxic stimulus. NMDA exposure (30μM, 10 min) produced irregularly shaped dendritic swellings, evident first in distal apical dendrite branches, and later (20–90min) involving most proximal dendrites. Over the same timecourse, immunoreactivity for the microtubule associated protein MAP2 was progressively lost from apical dendrites, and increased in CA1 somata. This damage and MAP2 loss was Ca2+-dependent, and was not reversible within the timecourse of these experiments (90 min post-NMDA washout). Formation of regularly-spaced, spherical dendritic varicosities (dendritic beading) was rarely observed, except when NMDA was applied in Ca2+-free ACSF. Under these conditions, beading appeared predominant in interneurons, as assessed from experiments with GAD67-GFP (Δneo) mice. Ca2+-removal was associated with significantly better preservation of dendritic structure (MAP2) following NMDA exposure, and other ionic fluxes (sensitive to Gd3+ and spermine) may contribute to residual damage occurring in Ca2+-free conditions. These results suggest that irregularly shaped dendritic swelling is a Ca2+-dependent degenerative event that may be quite different from Ca2+-independent dendritic beading, and can be a predominant type of injury in CA1 pyramidal neurons in slice.
PMCID: PMC1853289  PMID: 17239543
Beading; excitotoxicity; hippocampus; CA1; dendrite; interneuron
3.  P28-S Digging Deeper and Faster into Proteome by IgY-Immunoaffinity Fractionation 
After separating the highly abundant proteins (HAP) by IgY affinity column, the next layer of abundant protein, moderately abundant proteins (MAP), becomes an obstacle to access the low abundant proteins (LAP), where the majority of biologically relevant and clinically important biomarkers reside. Therefore, isolation of MAP is a new challenge for effective detection and analysis of LAPs. To tackle this challenge, we further developed the IgY-microbead system by immunizing chickens with a flow-through fraction of IgY12 column and constructing the column with affinity-purified IgY antibodies against the flow-through proteins of IgY12 column. The column developed, called SuperMix, was applied for further partitioning of the flow-through fraction of IgY12, which resulted in a bound/eluted fraction (designated as MAP fraction) and the flow-through fraction (designated as LAP fraction). Unfractionated and serial-fractionated samples using IgY12 and SuperMix columns were analyzed by SDS-PAGE and 2DE. Our data demonstrate that SuperMix columns specifically and reproducibly remove the post-IgY12 layer of the abundant proteins. A case study using SDS-PAGE coupled with LC/MS/MS demonstrates that the SuperMix column enabled specific capturing of 207 MAP, with 77 proteins being uniquely identified in high confidence (≥95%). This novel approach enables deeper and more effective access into the population of LAPs. In addition to digging deeper with the SuperMix column, we also have progressed in the direction of digging faster. One of the present challenges of plasma biomarker discovery is sample throughput limitations. In collaboration with PSS Bio Instruments, GenWay Biotech has developed a novel multiplex automated system (SepproTip) for high-throughput plasma sample processing. It permits processing of 12 samples at a time. This Seppro-Tip system can process plasma samples using both IgY12 and SuperMix tips. The turnaround time of 12 samples per 65 min allows a large number of samples to be processed without decrease in sample preparation quality.
PMCID: PMC2291923
4.  P174-T Affinity Protein Purification by Automation Using a Magtration Robotic System 
Affinity purification is a powerful tool for protein enrichment in proteomics studies. We here present a fully automated system for purification of His-tag proteins and IgG using Ni2+/Co2+ and Protein A magnetic beads, respectively. Reagents for His-tag protein or IgG purification are pre-dispensed in a sealed cartridge for automated runs on a Magtration 12GC robot. The automated purification is based on Magtration technology to perform magnetic bead separation similar to a filtration process in a pipette tip. An optimized protocol has been developed for the automated protein purification. High protein purity and yields were obtained using this automated system. His-tag protein human galectin-1 was purified to approx. 1.6 mg with 12 samples processed in parallel within 30 min on the 12GC robot. This system was also used to screen the expression of His-tag water-soluble proteins and inclusion bodies in bacterial cells, even at a very low expression level. Using Protein A magnetic beads and corresponding pre-filled reagent cartridges, various amounts of human serum (15–80 μL) and the magnetic beads (100–200 mg) were tested on the robotic system. With 30 μL serum and 150 mg magnetic beads, we purified IgG with a high yield of 230 μg. A total of approx. 2.8 mg IgG can be obtained within 60 min with 12 samples run in parallel on the robot. The magnetic beads after the affinity purification can be regenerated by automation for repeated use. Magtration robotic system can be extended for purification of GST-tag fusion proteins and Immunoprecipitation by automation. We have provided an automated protein purification system with a Magtration robot and pre-filled reagent cartridges for rapid and multiparallel processing of different proteins.
PMCID: PMC2291955
6.  Matrix metalloproteinases and tissue inhibitors of metalloproteinases in synovial fluids from patients with rheumatoid arthritis or osteoarthritis 
Annals of the Rheumatic Diseases  2000;59(6):455-461.
OBJECTIVE—Matrix metalloproteinases (MMPs) are expressed in joint tissues of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). The objective of this study was to define the steady state levels of seven different MMPs and two tissue inhibitors of metalloproteinases (TIMPs) as well as the potential metalloproteinase activity in the synovial fluid (SF) to provide more insight into the role of MMPs in cartilage destruction in RA and OA.
METHODS—Levels of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13, TIMP-1, and TIMP-2 in SF aspirated from knee joints of 97 patients with RA and 103 patients with OA were measured by the corresponding one step sandwich enzyme immunoassays. Proteolytic activity of MMPs in these SFs was examined in an assay using [3H]carboxymethylated transferrin substrate in the presence of inhibitors of serine and cysteine proteinases after activation with p-aminophenylmercuric acetate (APMA). Destruction of RA knee joints was radiographically evaluated.
RESULTS—Levels of MMP-1, MMP-2, MMP-3, MMP-8, and MMP-9 were significantly higher in RA SF than in OA SF. MMP-7 and MMP-13 were detectable in more than 45% of RA SFs and in less than 20% of OA SFs, respectively. Among the MMPs examined, MMP-3 levels were extremely high compared with those of other MMPs. Direct correlations were seen between the levels of MMP-1 and MMP-3 and between those of MMP-8 and MMP-9 in RA SF. Although the levels of MMP-1 and MMP-3 increased even in the early stage of RA, those of MMP-8 and MMP-9 were low in the early stage and increased with the progression of RA. Molar ratios of the total amounts of the MMPs to those of the TIMPs were 5.2-fold higher in patients with RA than in OA, which was significant. APMA-activated metalloproteinase activity in SF showed a similar result, and a direct correlation was seen between the molar ratios and the activity in RA SF.
CONCLUSIONS—Our results show that high levels of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and TIMP-1 are present in RA SF and suggest that once these MMPs are fully activated, they have an imbalance against TIMPs, which may contribute to the cartilage destruction in RA.

PMCID: PMC1753174  PMID: 10834863
7.  Measurement of carboxypeptidase R by colorimetric assay 
Critical Care  2002;6(Suppl 1):P124.
PMCID: PMC3333538
8.  Brain natriuretic peptide as a novel cardiac hormone in humans. Evidence for an exquisite dual natriuretic peptide system, atrial natriuretic peptide and brain natriuretic peptide. 
Journal of Clinical Investigation  1991;87(4):1402-1412.
Using a specific radioimmunoassay for human brain natriuretic peptide (hBNP) with a monoclonal antibody, we have investigated its synthesis, secretion, and clearance in comparison with those of atrial natriuretic peptide (ANP) in normal subjects and patients with congestive heart failure (CHF). Mean BNP-like immunoreactivity (-LI) levels in normal atrium and ventricle were 250 and 18 pmol/g, respectively. The plasma BNP-LI level in normal subjects was 0.90 +/- 0.07 fmol/ml, which was 16% of the ANP-LI level. In contrast, the plasma BNP-LI level markedly increased in patients with CHF in proportion to its severity, and surpassed the ANP-LI level in severe cases. There was a significant step-up of the plasma BNP-LI level in the coronary sinus (CS) compared with that in the aortic root (Ao) and the difference between these BNP-LI levels, delta(CS-Ao)BNP, also increased with the severity of CHF. In addition, the step-up of the BNP-LI level in the anterior interventricular vein [delta(AIV-Ao)BNP] was comparable to delta(CS-Ao)BNP, indicating that BNP is secreted mainly from the ventricle. Predominant BNP synthesis in the ventricle was also confirmed by Northern blot analysis. Catheterization and pharmacokinetic studies revealed that hBNP is cleared from the circulation more slowly than alpha-hANP; this was in part attributed to lower (about 7%) binding affinity of hBNP to clearance receptors than that of alpha-hANP. A predominant molecular form of BNP-LI in the heart and plasma was a 3-kD form corresponding to hBNP. These results indicate that BNP is a novel cardiac hormone secreted predominantly from the ventricle, and that the synthesis, secretion and clearance of BNP differ from those of ANP, suggesting discrete physiological and pathophysiological roles of BNP in a dual natriuretic peptide system.
PMCID: PMC295184  PMID: 1849149
9.  Augmented expression of atrial natriuretic polypeptide gene in ventricle of human failing heart. 
Journal of Clinical Investigation  1989;83(1):298-305.
To elucidate the expression of the atrial natriuretic polypeptide (ANP) gene in the ventricle of the human failing heart, we have measured ANP and ANP messenger RNA (ANPmRNA) levels in left ventricular aneurysm obtained at operation, biopsy specimens of left ventricles from dilated cardiomyopathy (DCM) and autopsy samples of old myocardial infarction (OMI) and DCM hearts, and compared the levels with those in the normal ventricle. The ANP level (mean +/- SE) was 17.5 +/- 6.9 ng/g in the normal ventricle, and increased to 660.3 +/- 122.2 ng/g in the left ventricular aneurysm tissues and to 3,138.6 +/- 1,642.1 ng/g in the biopsy specimens of the DCM ventricle. These levels were approximately 40 and 200 times higher than in the normal ventricle. The increase of ANP levels was observed in both infarcted and noninfarcted regions of the OMI heart, and in the entire ventricle of the DCM heart. A significant positive correlation was found between the ANP level in aneurysm tissues and pulmonary capillary wedge pressure (r = 0.85). The ANPmRNA level in the left ventricular aneurysm showed about a 10-fold increase compared with that in the normal heart and reached 23% of that in the atrium of the same heart. A similar increase in the ANPmRNA level was observed in the entire ventricle of DCM. These data clearly indicate that the expression of the ANP gene in the ventricle is augmented in the failing heart in accordance with the severity of heart failure. In the atrium of the failing heart, ANP and ANPmRNA levels were only two times higher than those in the normal atrium. Thus, the augmentation in the expression of the ANP gene was more prominent in the ventricle than in the atrium. Taking tissue weight into account, the total content of ANPmRNA in the ventricle of the failing heart is much the same as that in the normal atrium. The ratio of the ANP level to the ANPmRNA level in the ventricle is much smaller than that in the atrium. These results suggest more rapid secretion of ANP after synthesis in the ventricle. These findings demonstrate that the expression of the ANP gene is augmented in the human ventricle of the failing heart and suggest that the ventricle becomes a substantial source of circulating ANP in congestive heart failure.
PMCID: PMC303674  PMID: 2521342
10.  Increased secretion of atrial natriuretic polypeptide from the left ventricle in patients with dilated cardiomyopathy. 
To examine whether atrial natriuretic polypeptide (ANP) is released from the left ventricle in patients with dilated cardiomyopathy (DCM) we measured plasma ANP level in the aortic root (Ao), the anterior interventricular vein (AIV), the great cardiac vein (GCV), and the coronary sinus (CS) in 11 patients with DCM and 18 control subjects. Plasma ANP levels in Ao, AIV, GCV, and CS were 454 +/- 360, 915 +/- 584, 1,308 +/- 926, and 1,884 +/- 1,194 pg/ml, respectively, in the patients with DCM and 108 +/- 42, 127 +/- 55, 461 +/- 224, and 682 +/- 341 pg/ml, respectively, in the control subjects. There was no significant difference in the plasma ANP levels between Ao and AIV in the control subjects. On the contrary, there was a significant (P less than 0.001) step-up in plasma ANP levels between Ao and AIV in patients with DCM. Thus, the difference in ANP levels between Ao and AIV was significantly increased in patients with DCM as compared with the control subjects (461 +/- 248 vs. 19 +/- 59 pg/ml, P less than 0.001). The difference in ANP levels between Ao and CS was also significantly increased in patients with DCM as compared with the control subjects (1,429 +/- 890 vs. 577 +/- 318 pg/ml, P less than 0.001). We conclude that ANP is released in increased amounts into the circulation from the left ventricle as well as from the heart as a whole in patients with DCM.
PMCID: PMC303641  PMID: 2521343
11.  Occupation and bladder cancer in Boston, USA, Manchester, UK, and Nagoya, Japan. 
Relations between occupational history and the development of cancer of the lower urinary tract ("bladder cancer") were evaluated in Boston, Massachusetts, USA, Manchester, UK, and Nagoya, Japan. Population-based series of incident cases and controls were identified and interviewed in each area. The present analysis was limited to men and was based on 430 cases and 397 controls in Boston, 399 cases and 493 controls in Manchester, and 226 cases and 443 controls in Nagoya. In Boston, elevated risk of bladder cancer was associated with employment related to dyes (relative risk = 2 X 1; 90% confidence interval, 0 X 9-5 X 1), leather (1 X 7; 1 X 1-2 X 6), paint (1 X 5; 0 X 9-2 X 4), or organic chemicals (1 X 6; 1 X 1-2 X 5). In Manchester, elevated risk was associated with 0 X 9-3 X 6). No clear association was observed between occupation and risk in Nagoya. Elevations in risk related to occupation generally were stronger in men under 65 years of age compared to older men. Differences from place to place in associations between occupation and risk may be the result of differences in the exposures to hazardous agents that jobs with the same title involve.
PMCID: PMC1052460  PMID: 4086958
12.  Identification of a membrane protein induced concurrently with cell filamentation by cyclic AMP in an Escherichia coli K-12 fic mutant. 
Journal of Bacteriology  1983;155(1):398-401.
A membrane protein with a molecular weight of 40,000 (40K protein) was induced concurrently with cell filamentation by cyclic AMP (cAMP) in a fic mutant. In the crp mutant and the wild-type strain, cell filamentation by cAMP was not observed, and the 40K protein was not induced. Induction of the 40K protein is regulated by the cAMP-cAMP receptor protein complex and is closely related to cell filamentation by cAMP in the fic mutant.
PMCID: PMC217692  PMID: 6305918
13.  Artificial sweeteners and bladder cancer in Manchester, U.K., and Nagoya, Japan. 
British Journal of Cancer  1982;45(3):332-336.
We have evaluated the relation between cancer of the lower urinary tract ("bladder cancer") and the use of artificial sweeteners, by means of case-control studies in Manchester, U.K., and Nagoya, Japan, areas where extensive use occurred 30-40 years ago. In each area, a broadly based series of cases (555 in Manchester, 293 in Nagoya) was interviewed and a series of controls (735 in Manchester, 589 in Nagoya) chosen from the general population. A history of use of sugar substitutes primarily saccharin, was not associated with an elevated risk of bladder cancer in either study area. Risk of bladder cancer did not increase regularly with frequency or duration of use of sugar substitutes. Data on dietetic beverages were not obtained in Nagoya. This exposure was not associated with a greater risk of bladder cancer in Manchester. The results of this study suggest that use of artificial sweeteners confers little or no risk of bladder cancer.
PMCID: PMC2010924  PMID: 7073930

Results 1-13 (13)