A mechanical model reveals that non-axisymmetric buckling lowers the energy barrier associated with membrane neck constriction.

Membrane neck formation is essential for scission, which, as recent experiments on tubules have demonstrated, can be location dependent. The diversity of biological machinery that can constrict a neck such as dynamin, actin, ESCRTs and BAR proteins, and the range of forces and deflection over which they operate, suggest that the constriction process is functionally mechanical and robust to changes in biological environment. In this study, we used a mechanical model of the lipid bilayer to systematically investigate the influence of location, symmetry constraints, and helical forces on membrane neck constriction. Simulations from our model demonstrated that the energy barriers associated with constriction of a membrane neck are location-dependent. Importantly, if symmetry restrictions are relaxed, then the energy barrier for constriction is dramatically lowered and the membrane buckles at lower values of forcing parameters. Our simulations also show that constriction due to helical proteins further reduces the energy barrier for neck formation when compared to cylindrical proteins. These studies establish that despite different molecular mechanisms of neck formation in cells, the mechanics of constriction naturally leads to a loss of symmetry that can lower the energy barrier to constriction.

Successful third kidney transplantation with intensive immunosuppression in a highly sensitized recipient.

HLA sensitization associated with previous kidney transplantation is a major drawback to retransplantation. Recently we successfully performed a third graft using intensive immunosuppression for a highly sensitized recipient. The patient was a 31-year-old man who had previously undergone a living donor graft from his father at our institute in 1999. His kidney graft function had deteriorated due to chronic allograft nephropathy, returning to hemodialysis therapy in 2005. He received a second graft from a deceased donor in another country on August 14, 2006. It rejected on postoperative day 3 possibly due to acute accelerated rejection. He was offered a third kidney from his brother. Panel-reactive antibody (PRA) tested before the third procedure revealed positive class I (88%) and class II (96%) PRAs. Mycophenolate mofetil (MMF) was started 3 weeks before the third transplantation, and preoperative plasmapheresis performed thrice. He underwent the living donor graft on March 9, 2007. Immunosuppression consisted of tacrolimus, MMF, methylprednisolone, and basiliximab. Immediately afterward there was a sudden decrease in allograft blood flow and urine output, implying hyperacute rejection. Following treatment with plasmapheresis and a single dose of rituximab (200 mg), the kidney allograft function recovered, although the PRA at 3 weeks was still positive. Six months posttransplantation, he is well with a creatinine of 0.9 mg/dL. Our protocol may reduce the risk for graft loss in a highly sensitized transplant recipient.

Characterization of p21Cip1/Waf1 peptide domains required for cyclin E/Cdk2 and PCNA interaction.

The cyclin-dependent kinase inhibitor p21Cip1/Waf1 is responsible for the p53-dependent growth arrest of cells in G1 phase following DNA damage. In the present study we investigated regions of p21 involved in inhibition of the G1/S phase cyclin-dependent kinase, cyclin E/Cdk2, as well as regions of p21 important for binding to this kinase and recombinant PCNA. To perform these studies we synthesized a series of overlapping peptides spanning the entire p21 sequence and used them in in vitro assays with cyclin E/Cdk2-immune complexes and with recombinant p21 and PCNA proteins. One amino-terminal p21 peptide spanning amino acids 15-40, antagonized p21 binding and inhibition of cyclin E/Cdk2 kinase. Antagonism of p21 binding was, however, lost in a similar peptide lacking amino acids 15-20, or in a peptide in which cysteine-18 was substituted for a serine. These results suggest that this peptide region is important for p21 interaction with cyclin E/Cdk2. A second peptide (amino acids 58-77) also antagonized p21-activity, but this peptide did not affect the ability of p21 to interact with cyclin E/Cdk2. A region of p21 larger than 26 amino acids is presumably required for Cdk-inhibition because none of the peptides we tested inhibited cyclin E/Cdk2. We also found that a peptide spanning amino acids 21-45 bound recombinant p21 in ELISA assays, and additional studies revealed a requirement for amino acids 26 through 45 for this interaction. A p21 peptide spanning amino acids 139-164 was found to bind PCNA in a filter binding assay and this peptide suppressed recombinant p21-PCNA interaction. Conformational analysis revealed that peptides spanning amino acids 21-45 and 139-164 tended towards an alpha-helical conformation in trifluoroethanol buffer, indicating that these regions are probably in a coiled conformation in the native protein. Taken together, our results provide an insight into domains of p21 that are involved in cyclin E/Cdk2 and PCNA interaction. Our results also suggest that a potential p21 dimerization domain may lie in the amino-terminus of p21. Continued exploration of these domains could prove useful in assessing p21-mimetic strategies for cancer treatment.

Cloning, sequence analysis, and expression of a mouse cDNA encoding cytochrome c oxidase subunit VIa liver isoform.

A cDNA encoding cytochrome c oxidase (COX) subunit VIa liver isoform (COX6aL) was isolated from a Mus musculus library and sequenced. The protein translated from the nucleotide sequence contains a presequence and is 91% identical to the human cognate sequence over the processed polypeptide region. Northern analysis shows the expression of COX6aL is developmentally regulated in heart, being about equally transcribed with the heart isoform (COX6aH) in 18-day embryos but consisting of less than 25% in adult heart.

Whole-Genome Sequence of a Bordetella pertussis Brazilian Vaccine Strain.

Despite the reduction in incidence after vaccination, pertussis disease is still considered a public health problem worldwide, mainly due to recent and potential new outbreaks. We report here the complete genome of the Bordetella pertussis Butantan strain used in the Brazilian National Immunization Program as a whole-cell pertussis antigen to compose vaccines such as DTwP (diphtheria, tetanus, and whole-cell pertussis).

Quantitative structure-activity relationships of the bitter thresholds of amino acids, peptides, and their derivatives.

Bitter thresholds of a total of 93 amino acids, peptides, and their derivatives were analyzed quantitatively by use of hydrophobicity parameters reported for amino acid side chains and those for the whole molecules estimated from partition coefficients obtained experimentally. We also explored the steric parameters that best explained the variation in the intensity of bitterness attributable to the molecular shape. The results showed that the total length along the zigzag peptide backbone chain of the molecule is an important factor. The bitterness of nonzwitterionic N-acyl and ester derivatives and that of neutral N-acyl ester derivatives were expressed by a single, common equation together with those of zwitterionic amino acids and peptides. Thus the interaction via the charge with the receptor site was probably not an indispensible factor for triggering of the bitter sensation. This study, together with earlier ones, may serve as a prototype of approaches toward unraveling structure-activity relationships of complex molecules like amino acids, peptides, and their derivatives that are of medicinal or agricultural importance.

L-O-(2-malonyl)tyrosine: a new phosphotyrosyl mimetic for the preparation of Src homology 2 domain inhibitory peptides.

Inhibition of Src homology 2 (SH2) domain-binding interactions affords one potential means of modulating protein-tyrosine kinase-dependent signaling. Small phosphotyrosyl (pTyr)-containing peptides are able to bind to SH2 domains and compete with larger pTyr peptides or native pTyr-containing protein ligands. Such pTyr-containing peptides are limited in their utility as SH2 domain inhibitors in vivo due to their hydrolytic lability to protein-tyrosine phosphatases (PTPs) and the poor cellular penetration of the ionized phosphate moiety. An important aspect of SH2 domain inhibitor design is the creation of pTyr mimetics which are stable to PTPs and have reasonable bioavailability. To date, most PTP-resistant pTyr mimetics which bind to SH2 domains are phosphonates such as (phosphonomethyl)phenylalanine (Pmp, 2), [(monofluorophosphono)methyl]phenylalanine (FPmp, 3) or [(difluorophosphono)methyl]-phenylalanine (F2Pmp, 4). Herein we report the incorporation of a new non-phosphorus-containing pTyr mimetic, L-O-(2-malonyl)tyrosine (L-OMT, 5), into SH2 domain inhibitory peptides using the protected analogue L-N alpha-Fmoc-O'-(O",O"-di-tert-butyl-2-malonyl)tyrosine (6) and solid-phase peptide synthesis techniques. Five OMT-containing peptides were prepared against the following SH2 domains: the PI-3 kinase C-terminal p85 SH2 domain (Ac-D-(L-OMT)-V-P-M-L-amide, 10, IC50 = 14.2 microM), the Src SH2 domain (Ac-Q-(L-OMT)-E-E-I-P-amide, 11, IC50 = 25 microM, and Ac-Q-(L-OMT)-(L-OMT)-E-I-P-amide, 14, IC50 = 23 microM), the Grb2 SH2 domain (Ac-N-(L-OMT)-V-N-I-E-amide, 12, IC50 = 120 microM), and the N-terminal SH-PTP2 SH2 domain (Ac-L-N-(L-OMT)-I-D-L-D-L-V-amide, 13, IC50 = 22.0 microM). These results show that peptides 10, 11, 13, and 14 have reasonable affinity for their respective SH2 domains, with the IC50 value for the SH-PTP2 SH2 domain-directed peptide 13 being equivalent to that previously observed for the corresponding F2Pmp-containing peptide. OMT may afford a new structural starting point for the development of novel and useful SH2 domain inhibitors.

4'-O-[2-(2-fluoromalonyl)]-L-tyrosine: a phosphotyrosyl mimic for the preparation of signal transduction inhibitory peptides.

Development of phosphotyrosyl (pTyr) mimetics which are stable to protein-tyrosine phosphatases (PTPs), yet can retain biological potency when incorporated into peptides, is an active area of drug development. Since a majority of pTyr mimetics derive their "phosphofunctionality" from phosphorus-containing moieties, such as phosphonates, evolution of new inhibitors and modes of prodrug derivatization have been restricted to chemistries appropriate for phosphorus-containing moieties. A new, nonphosphorus-containing pTyr mimetic has recently been reported, L-O-(2-malonyl)tyrosine (OMT,5), which can be incorporated into peptides that exhibit good PTP and Src homology 2 (SH2) domain inhibitory potency. For phosphonate-based pTyr mimetics such as phosphonomethyl phenylalanine (Pmp,2) introduction of fluorines alpha to the phosphorus has provided higher affinity pTyr mimetics. This strategy has now been applied to OMT, and herein is reported 4'-O-[2-(2-fluoromalonyl)]-L-tyrosine (FOMT,6) a new fluorine-containing nonphosphorus pTyr mimetic. Incorporation of FOMT into appropriate peptides results in good inhibition of both PTP and SH2 domains. In an assay measuring the inhibition of PTP 1B-mediated dephosphorylation of phosphorylated insulin receptor, the peptide Ac-D-A-D-E-X-L-amide exhibited a 10-fold enhancement in inhibitory potency for X = FOMT (19) (IC(50) = 10 microM) relative to the unfluorinated peptide, X = OMT (18) (IC(50) = 10 microM. Molecular modeling indicated that this increased affinity may be attributable to new hydrogen-bonding interactions between the fluorine and the enzyme catalytic site, and not due to lowering of pKa values. In a competition binding assay using the p85 PI 3-kinase C-terminal SH2 domain GST fusion construct, the inhibitory peptide, Ac-D-X-V-P-M-L-amide, showed no enhancement of inhibitory potency for X = FOMT (22) (IC(50) = 18 microM) relative to the unfluorinated peptide, X = OMT (21) (IC(50) = 14 microM). The use of FOMT would therefore appear to have particular potential for the development of PTP inhibitors.

Angiocentric immunoproliferative lesions of the skin show lobular panniculitis and are mainly disorders of large granular lymphocytes.

Eleven patients with angiocentric immunoproliferative lesion (AIL) of the skin were studied. Histologically, three patients were grouped into AIL grade II (AIL-II), whereas eight showed angiocentric lymphoma (AIL-III). All the patients' specimens exhibited lobular panniculitis. Infiltrating atypical lymphocytes in nine patients possessed electron-dense membrane bound granules in electron microscopy. Phenotypically, the lymphoid cells in the AIL-II patients were positive for CD3 epsilon; two of these showed a positive reaction to CD2, CD7, and CD8, but lacked natural killer-associated (NKa) antigens CD16, CD56, and CD57. In six AIL-III patients, lymphoma cells were positive for CD2 in all patients, CD56 in five, CD3 epsilon in four, CD7 in four, interleukin-2 beta receptor in four, a pore-forming protein in four, and CD30 in three patients. The remaining two AIL-III patients had B-cell lymphoma. By the Southern blot analysis, three patients with AIL-III showed a rearranged T-cell-receptor beta-gene or a deletion of its germline. The preceding results in nine of 11 patients suggest that abnormal or neoplastic large granular lymphocytes with the characteristics of T and NK cells have an important role in producing the angiocentric/angiodestructive features and lobular panniculitis. Clinically, all three patients with AIL-II and four with AIL-III showed liver dysfunction, cytopenia, and abnormal coagulopathy during the clinical course. Five patients with AIL-III died within 8 months. The histological grading of AIL, patients' age, and limited clinical stage of the disease seem to correlate with response to the treatment and prognosis.

CD30-positive anaplastic large cell lymphoma in a human T-cell lymphotropic virus-I endemic area.

Clinicopathological and cytogenetic studies were performed on specimens from 43 patients with CD30-positive anaplastic large cell lymphoma (ALCL). All patients were born and lived in a human T-cell lymphotropic virus (HTLV)-I endemic area. Twenty-one patients (48.8%) had serum anti-HTLV-I antibody suggesting the presence of adult T-cell leukemia/lymphoma (ATL). Seventeen of them showed a clonal integration of complete HTLV-I proviral DNA by the Southern blot hybridization method in materials obtained by biopsy. Their ages ranged from 37 to 81 years (median, 67.0), and they frequently presented with lymphadenopathy (82.4%) and extranodal tumors with (76.5%) as well as with rare leukemic changes (5.9%). Immunohistologically the lymphoma cells in 15 ATL patients showed a T-cell phenotype. In only one patient (5.9%) was there an expression of epithelial membrane antigen (EMA) in most of the lymphoma cells. Cytogenetically aberration of chromosome band 5q35 was not found in seven patients with HTLV-I proviral DNA. The overall median length of survival was 11.9 months for the patients with HTLV-I proviral DNA, indicating a worse prognosis compared with that of the age-matched patients with negative anti-HTLV-I antibody (P < .01). The specimens of the patients with HTLV-I proviral DNA had unique clinicopathological and cytogenetic features. These findings suggested that T-cell ALCL with HTLV-I proviral DNA should be considered to represent the lymphoma type of ATL.

Angiocentric immunoproliferative lesions of the lymph node.

Clinicopathologic features in 14 cases of lymph node-involved angiocentric immunoproliferative lesions (AILs) are reported. They were selected from 900 cases of lymphoproliferative disorders registered at the Department of Pathology, Fukuoka University. Four cases showed a histologic feature of AIL grade II (AIL-II) and 10 had angiocentric lymphoma (AIL-III). Immunohistologically, transformed B cells were mixed with a large number of small T cells in AIL-II. In AIL-III, there were five cases with B-cell lymphoma, and three had peripheral T-cell lymphoma with no expression of natural-killer (NK)-associated antigens. In the remaining two cases, lymphoma cells expressed both T-cell- and NK-associated antigens. These findings indicate that lymph node-involved AILs are rarely occurring (1.6%) and phenotypically different from sinonasal and cutaneous AILs. Furthermore, NK-associated antigen-positive AILs were found to rarely involve the lymph node. For Epstein-Barr virus (EBV) infection, seven cases of AILs showed many atypical lymphocytes that were positive for EBV-encoded RNA (EBER-1) by using the in situ hybridization analysis. Among them, six cases had latent membrane protein (LMP) positive and EBV nuclear antigen 2 (EBNA-2) negative atypical lymphocytes. The pattern of latent EBV infection was similar to that of Hodgkin's disease, but differed from those of sinonasal T-cell lymphoma and other subtypes of non-Hodgkin's lymphoma. Clinically, 12 patients, including all 4 AIL-II, died within 22 months of the onset of the disease, despite intensive therapy, suggesting that lymph node-involved AILs have a poor prognosis.

Analysis of adhesion molecules in Ki-1 anaplastic large-cell lymphoma.

We analysed the expression of adhesion molecules on lymphoma cells in 13 patients with Ki-1 (CD30)-positive anaplastic large-cell lymphoma (Ki-1 ALCL; lymph nodes in 6, extranodal tumours in 6, and both lymph nodes and bone in 1). Very late activation antigen (VLA)-alpha 4 (CD49d) and Hermes lymph node homing receptor (CD44) were constantly expressed in all specimens, and intercellular adhesion molecule-1 (ICAM-1; CD54) was frequently expressed in 10 of the 14 specimens. The expressions of lymphocyte function-associated antigen-1 alpha (LFA-1 alpha; CD11a) and VLA-alpha 5 (CD49e) occurred in 5 of 14 and 4 of 14 specimens, respectively. The expression of VLA-alpha 2 (CD49b), endothelial leukocyte adhesion molecule-1, neural cell adhesion molecule (CD56) and E cadherin were always lacking. VLA-alpha 6 (CD49f) was absent in all but one specimen. The expression of VLA-alpha 5 on Ki-1 ALCL was high in subcutis-cutis but absent in lymph nodes. Furthermore, in one case, LFA-1 alpha was detected in the primary lymph node, but was absent in a metastatic bone lesion. These results suggest that the expression of ICAM-1 is partially responsible for aleukemic behaviour in Ki-1 ALCL and, moreover, that the Ki-1 ALCL cells modify their expression of adhesion molecules at each of the involved organs.

Preparatory inhibition of cortico-spinal excitability: a transcranial magnetic stimulation study in man.

In order to investigate the preparatory modulations of cortico-spinal excitability, reaction time (RT) methods were combined with transcranial magnetic stimulation (TMS) of the motor cortex. We analyzed the variations in the amplitude of motor potentials evoked in a prime mover (flexor digitorum sublimis) by TMS delivered during the foreperiod of a visual choice RT task. In experiment 1 (n = 10), the TMS was delivered either simultaneously with the warning signal or simultaneously with the response signal in two conditions of foreperiod duration: short (500 ms) and long (2500 ms). The peak amplitude of the motor evoked potentials diminished during the short foreperiod but not during the long foreperiod. Since RT was shorter when the foreperiod lasted 500 ms than when it lasted 2500 ms, this result suggests that the excitability of the cortico-spinal structures is minimal when the subject is optimally ready to react. In experiment 2 (n = 10), the time-course of this decrement was further explored. With this aim, only the short foreperiod was used and the TMS was delivered either 500 ms, 333 ms, 167 ms or 0 ms before the response signal. Cortico-spinal excitability decreased during the first 333 ms and then remained stable until the occurrence of the response signal. In light of previous studies, the present results suggest that the decrement of cortico-spinal excitability during the short foreperiod reflects an adaptative mechanism which increases the sensitivity of the motor structures to the forthcoming voluntary command.

Lasting effect of NO on glutamate release in rat striatum revealed by continuous brain dialysis.

The effect of nitric oxide (NO) on glutamate release in the brain of freely moving rats was investigated using a new, high time-resolution microdialysis system. Coperfusion with veratridine (VER) and NO donors increased glutamate release above than that obtained with VER alone. When steady-state levels were regained after co-perfusion, perfusion of VER alone further potentiated glutamate release. The effect depended on the initial level of VER-induced glutamate release, and was maximum for intermediate glutamate levels. These results suggest that NO influences the glutamate release system by affecting the level of neural activity and that its effect lasts and increases when steady-state levels are regained in rat striatum.

Therapeutic trial of intensified conditioning regimen with high-dose cytosine arabinoside, cyclophosphamide and either total body irradiation or busulfan followed by allogeneic bone marrow transplantation for myelodysplastic syndrome in children.

Ten children with myelodysplastic syndrome underwent an allogeneic bone marrow transplantation (BMT) with an intensified conditioning regimen. The median age of the patients was 8 years (range 2-10), and included 6 males and 4 females. The subtype of the disease was refractory anemia (RA) in 4, RA with excess blasts (RAEB) in 4, RAEB in transformation (RAEB-T) in 1, and juvenile chronic myelogenous leukemia (JCML) in 1. All patients were conditioned with high-dose cytosine arabinoside (12000 mg/m2), cyclophosphamide (120 mg/kg) and either total body irradiation (10-13.2 Gy) or busulfan (16 mg/kg or 560 mg/m2). Cyclosporine A and/or methotrexate were used for the prophylaxis of graft-versus-host disease (GVHD). Engraftment was prompt in all but one patient. Severe acute GVHD (grade 3) (n = 1), interstitial pneumonitis (n = 1) and veno-occlusive disease of the liver (n = 1) occurred. The disease relapsed in one patient with RAEB-T. Seven of the 10 patients were alive and disease free 2-74 months after BMT. The disease-free survival rate at 4 years was 69 +/- 15%. All surviving patients were in the full performance status. The examined children with MDS tolerated this intensified conditioning regimen well.

Quantitative analyses of hydrophobicity of di- to pentapeptides having un-ionizable side chains with substituent and structural parameters.

With experimental conditions established recently, we measured the partition ratio (P') of 124 di- to pentapeptides composed of amino acids having un-ionizable side chains in a 1-octanol:pH 7.0 aqueous phosphate buffer system as an approximate zwitterionized "molecular" partition coefficient (P). Empirical equations of good quality, correlating the variations in log P' value of peptides with free energy-related physicochemical parameters for the side chain substituents and substructures, were formulated. The significant side chain parameters were those representing the intrinsic hydrophobicity, the steric effect on the relative solvation of functional groups on the backbone, and the conformational potential index derived from the Chou-Fasman beta-turn potential parameters. For polar side chains, specific indicator variables were required for intramolecular hydrogen-bond formations and the "polar proximity effect" for reductions of hydrophilicity observed when polar groups are crowded together. The proline residue was shown to contribute to the log P' value depending not only on its location on the backbone but also on the number of residues in peptides. On the basis of the analyses, we proposed a new "effective" hydrophobicity index for un-ionizable side chains which could predict the secondary structure of oligopeptides.

Quantitative structure-hydrophobicity and structure-activity relationships of antibacterial gramicidin S analogs.

The structure-hydrophobicity-antibacterial activity relationships of gramicidin S and its analogs retaining a beta-pleated sheet structure were examined quantitatively with physicochemical substituent and molecular parameters using regression analyses. Variations in their apparent hydrophobicity in an octanol/buffer (pH 7) system, log P' (O/W), were analyzed in terms of the "effective" hydrophobicity and steric parameters of side chain substituents of residues at certain positions in the molecule; however, some of the conformational factors have not been fully defined. For the partitioning into liposomes and the growth inhibitory activity against species of Gram-positive bacteria, the log P' (O/W) value simulated the hydrophobic effects of gramicidin S and its analogs better than substituent parameters. The side chain hydrophobicity was assumed to work together with effects attributed to variations in the entire cyclic peptide structure including conformational components undefined in the structure-log P' (O/W) analysis in these activities.

Quantitative analyses of the structure-hydrophobicity relationship for N-acetyl di- and tripeptide amides.

The partition coefficient (P) of neutral species and the apparent partition ratio (P') at pH 7 of the ionized form were measured with the 1-octanol/water system for a number of N-acetyl di- and tripeptide amides having un-ionizable and ionizable side chains. Their log values were studied in terms of free-energy-related substituent and substructural parameters using regression analysis to give correlation equations of high quality physicochemically as well as statistically. The intrinsic hydrophobicity of side-chain substituents and their steric effect on the relative solvation of the backbone CONH groups were significant in determining the log P values of the un-ionizable acetyl peptide amides. For the log P value of peptides with polar side-chain substituents, respective indicator variable terms were required to account for the sum of specific effects of substituents such as intramolecular hydrogen-bond formation and the "polar proximity factor" for augmentation of the hydrophobicity. For the log P'(pH 7) value of basic and acidic peptides, the ability of counterionic species to form ion-pairs, the change in the apparent hydrophobicity of ionizable groups from the intrinsic value for their nonionized forms, the effect of ion-pairing itself, and other effects were additionally considered. From the regression coefficients of the parameter terms in correlation equations an effective hydrophobicity index was defined for each side chain, and the application and its limitation were suggested.

Multineuronal spike classification based on multisite electrode recording, whole-waveform analysis, and hierarchical clustering.

We proposed here a method of multineuronal spike classification based on multisite electrode recording, whole-waveform analysis, and hierarchical clustering for studying correlated activities of adjacent neurons in nervous systems. Multineuronal spikes were recorded with a multisite electrode placed in the hippocampal pyramidal cell layer of anesthetized rats. If the impedance of each electrode site is relatively low and the distance between electrode sites is sufficiently small, a spike generated by a neuron is simultaneously recorded at multielectrode sites with different amplitudes. The covariance between the spike waveform at each electrode site and a template was calculated as a damping factor due to the volume conduction of the spike from the neuron to the electrode site. Calculated damping factors were vectorized and analyzed by hierarchical clustering using a multidimensional statistical test. Since a cluster of damping vectors was shown to correspond to an antidromically identified neuron, spikes of different neurons are classified by referring to the distributions of damping vectors. Errors in damping vector calculation due to partially overlapping spikes were minimized by successively subtracting preceding spikes from raw data. Clustering errors due to complex spike bursts (i.e., spikes with variable amplitudes) were avoided by detecting such bursts and then using only the first spike of a burst for clustering. These special procedures produced better cluster separation than conventional methods, and enabled multiple neuronal spikes to be classified automatically. Waveforms of classified spikes were well superimposed. We concluded that this method is particularly useful for separating the activities of adjacent neurons that fire partially overlapping spikes and/or complex spike bursts.

[Anticancer susceptibility test method using general-purpose dissolved oxygen meter].

Drug susceptibility of cell can be rapidly measured by continuously monitoring its metabolic changes. We focused on respiration volume as a signal of metabolic change, and developed a new dissolved oxygen measuring system which can detect respiration volume of cells. The main feature of the system is the use of a new type bare oxygen electrode which can easily detect the changing rate of dissolved oxygen concentration. In this study, single type electrode was used to evaluate this rapid anticancer drug susceptibility test. The result obtained was almost equivalent to that with MTT method, which is a conventional method for susceptibility test of HL-60 to various kinds of anticancer drugs. We have also developed multi-type electrode plate with oxygen electrodes embedded in the bottom of 96-well plate, with which clinical evaluation of this method can be easily made.