Metallophthalocyanines as triplet sensitizers for highly efficient photon upconversion based on sensitized triplet-triplet annihilation.

Soluble palladium and platinum phthalocyanines with coumarin moieties were synthesized with Q bands in the red and near-IR regions, in which the molar extinction coefficients were up to 1.01 × 10(5) cm(-1) mol(-1). These metallophthalocyanines were coupled with rubrene and applied in photon upconversion systems based on triplet-triplet annihilation. The highest upconversion efficiency of the palladium phthalocyanine was 5.6%, which is higher than that of the platinum phthalocyanine-rubrene system. The larger molar extinction coefficient resulted in high upconversion capability (>10(5) cm(-1) mol(-1)) and low saturation incident power (<20 mW cm(-2)).

Relative vulnerability of various spinal tracts in C3-4 cervical spondylotic myelopathy: multi-modal spinal cord evoked potentials.

STUDY DESIGN: Retrospective study. OBJECTIVE: To investigate the progression of spinal tract lesions in cervical spondylotic myelopathy (CSM) at C3-4 intervertebral level using spinal cord-evoked potensials (SCEPs). SETTING: This study was conducted at the Department of Orthopaedic Surgery, Yamaguchi University Graduate School of Medicine, Japan. METHODS: A total of 30 patients with CSM were investigated in this study. In all patients, only the C3-4 intervertebral level was symptomatic, as shown by examination of SCEPs. SCEPs were recorded following median nerve stimulation (MN-SCEPs), transcranial electric stimulation (TES-SCEPs) and spinal cord stimulation (spinal-SCEPs). RESULTS: The incidence of abnormalities varied in the order of MN-SCEPs (100%), TES-SCEPs (90%) and spinal-SCEPs (67%). Patients were grouped into three types according to SCEPs: transverse (all SCEPs abnormal), post-erolateral (abnormalities in the MN-SCEPs and TES-SCEPs) and upper limbs sensory (abnormal only for MN-SCEPs). In all, 20 of the 30 patients (67%) were the transverse type, 7 (23%) the post-erolateral type and 3 (10%) the upper limbs sensory type. CONCLUSION: The present study showed the lateral part of the posterior funiculus mediating upper limb sensory function was more vulnerable than the lateral corticospinal tract, which is consistent with numbness tending to appear at an early stage of mild CSM.

Preoperative stellate ganglion blockade prevents tourniquet-induced hypertension during general anesthesia.

BACKGROUND: Prolonged and excessive inflation of pneumatic tourniquets leads to a hyperdynamic circulatory response. Sympathomimetic activity is an important factor in tourniquet-induced hypertension. Stellate ganglion block specifically blunts sympathetic efferent nerves and prevents hypertension induced by sympathomimetic stimulation. The present study was performed to investigate the effects of stellate ganglion block (SGB) on arterial pressure and heart rate during prolonged tourniquet use under general anesthesia. METHODS: Twenty patients scheduled for knee arthroscopy were either treated with 10 ml of 1% lidocaine for SGB (SGB group; n = 10), or intramuscular injection (IM group; n = 10) before tourniquet inflation. Comparisons of systolic and diastolic arterial pressure and heart rate were made before and after the induction of anesthesia, 10 min after the lidocaine treatment, every 5 min during the first 60 min after tourniquet inflation, and immediately before and 5 min following deflation. The maximum values of the circulatory variables were compared. RESULTS: Tourniquet inflation caused increases in the circulatory variables in both groups. Systolic arterial pressure in the SGB group was significantly lower than that in the IM group after 55 min of tourniquet inflation. Diastolic arterial pressure also was significantly lower in the SGB group immediately before the deflation. The maximum values of the three hemodynamic variables were significantly lower in the SGB group. Arterial pressure significantly decreased after tourniquet deflation in the IM group. CONCLUSION: Ipsilateral SGB attenuated the hyperdynamic response mediated by prolonged tourniquet inflation during knee arthroscopy.

A new rabbit model for the study on cervical compressive myelopathy.

Development process and pathology of myelopathy due to chronic spinal cord compression have not been fully elucidated. This study was conducted in order to establish an experimental model which can efficiently produce myelopathy and be useful in the studies on myelopathy due to chronic spinal cord compression. Under electrophysiological monitoring of the spinal cord, anterior compression was produced on C5 using a plastic screw. Two weeks later, a plastic plate was inserted under the C5 arch. For the subsequent 10 months on average, walking pattern and MR images were periodically monitored. Before the sacrifice, electrophysiological test was performed and then histopathological examination was done. Palsy appeared at 5 months on average after the addition of posterior compression. Mean compression ratio of the spinal cord calculated on MR images was 34%. All animals with compression showed a high intramedullary signal intensity, and the mean contrast-to-noise ratio (CNR) in the compressed area was 49%. Electrophysiological test showed a significant decrease in the amplitude of spinal cord evoked potentials (SCEPs) at the given compression level. Histology showed flattening of the anterior horn, disappearance and necrosis of anterior horn cells in the gray matter; and demyelination and axonal degeneration in the white matter. The antero-posterior compression produces the condition of spinal canal stenosis. Repeated antero-posterior compression to the spinal cord is important in establishing myelopathy. The present animal model was evaluated to be useful in the studies on myelopathy.

An efficient refolding method for the preparation of recombinant human prethrombin-2 and characterization of the recombinant-derived alpha-thrombin.

Human recombinant prethrombin-2 was produced in Escherichia coli. The expressed prethrombin-2 formed intracellular inclusion bodies from which the protein was refolded by a simple one-step dilution process in buffer consisting of 50 mM Tris-HCl, containing 20 mM CaCl(2), 500 mM NaCl, 1 mM EDTA, 600 mM arginine, 1 mM cysteine, 0.1 mM cystine, 10% (v/v) glycerol, and 0.2% (w/v) Brij-58 at pH 8.5. After refolding, prethrombin-2 was purified by hirudin-based COOH-terminal peptide affinity chromatography, and then activated with Echis carinatus snake venom prothrombin activator (ecarin). The activated protein, alpha-thrombin, was then tested for several activities including activity toward chromogenic substrate, release of fibrinopeptide A from fibrinogen, activation of protein C, and thrombin-activatable fibrinolysis inhibitor, reactivity with antithrombin, clotting activity, and platelet aggregation. The kinetic data showed no differences in activity between our recombinant alpha-thrombin and plasma-derived alpha-thrombin. The yield of refolded recombinant human prethrombin-2 was about 4-7% of the starting amount of solubilized protein. In addition, the final yield of purified refolded protein was 0.5-1%, and about 1 mg of recombinant prethrombin-2 could be isolated from 1 liter of E. coli cell culture.

Differential recording of upper and lower cervical N13 responses and their contribution to scalp recorded responses in median nerve somatosensory evoked potentials.

To distinguish the different origins of cervical N13 potentials in median nerve somatosensory evoked potentials (SSEPs), cervical N13 potentials were recorded by two different montages. The abnormal patterns of the SSEPs were compared to the abnormal evoked spinal cord responses (ESCPs) recorded from posterior epidural space in 13 patients with various cervical lesions. SSEPs from the posterior cervical surface were recorded from the mid-cervical level with anterior neck reference (Cv5-AN) and from the upper cervical level with inion reference (Cv2-IN). Scalp responses were recorded from the parietal region contralateral to the stimulating side with non-cephalic reference (shoulder contralateral to stimulating side). ESCPs were recorded from the posterior epidural space using catheter electrodes or needle electrodes inserted into the ligamentum flavum. Lower cervical N13 (LC-N13) recorded from the Cv5-AN montage showed similar latency to upper cervical N13 (UC-N13) recorded from the Cv2-IN montage. The latency of the early part of the P13-P14 complex in the scalp montage was similar to that of the UC-N13 and the negative peak latency of the ESCPs recorded at the C2-3 level. Attenuation of the LC-N13 and relatively preserved UC-N13 and P13-P14 were characteristic in patients with cervical syringomyelia and compression cervical myelopathy at the mid-cervical levels. Attenuation of the UC-N13 with normal LC-N13 was characteristic in patients with cervical spondylotic myelopathy who showed conduction blockade of the ESCPs at the C3-4 level. In a patient with schwannoma at the C1-2 level, conduction blockade of the ESCPs was observed at the C1-2 level. P13 was normal but P14 was prolonged. UC-N13 and P13 latencies were similar to the negative peak latency of the ESCPs at the C2-3 level. We demonstrated that two different cervical N13 potentials can be recorded by two different montages and they represent different behavior in various spinal cord lesions. In addition, at least the early part of the P13-P14 complex originates in the upper cervical region. To distinguish two different cervical N13, it is useful to detect not only the cervical pathology but also the symptomatic cervical cord compression level in patients with cervical myelopathy.

A correlation between magnetic resonance imaging and electrophysiological findings in cervical spondylotic myelopathy.

STUDY DESIGN: Correlation between compressed spinal cords on magnetic resonance imaging (MRI) and electrophysiological findings in cervical spondylotic myelopathy patients. OBJECTIVE: To clarify the correlation between spinal-cord-evoked potentials and MRI measurements of compressed spinal cords in patients with cervical spondylotic myelopathy. SUMMARY OF BACKGROUND DATA: Compression of the spinal cord does not always cause clinical symptoms and it is difficult to infer the degree of dysfunction of the spinal cord from MRI findings. METHODS: Seventeen patients with cervical spondylotic myelopathy were examined with MRI and spinal-cord-evoked potentials before surgery. Using abnormality in spinal-cord-evoked potentials as indicators of spinal cord morphology, spinal-cord transverse area and compression ratios (central and 1/4-lateral) were measured on T1-weighted axial imaging. The correlations between these dimensions and electrophysiological findings were investigated. RESULTS: The mean preoperative transverse area of the spinal cord was 47.13 mm2. The mean preoperative central compression ratio of the spinal cord was 34.4%. The mean preoperative 1/4-lateral compression ratio of the spinal cord was 27.5%. A correlation (Spearman r=0.65, P < 0.01) was observed between the 1/4-lateral compression ratio of the spinal cord and the amplitude ratio of spinal-cord-evoked potentials after electric stimulation of the brain (Br(E)-SCEPs). CONCLUSIONS: The preoperative 1/4-lateral compression ratio of the spinal cord was found to reflect the degree of dysfunction of the corticospinal tracts.

Mechanism of prolonged central motor conduction time in compressive cervical myelopathy.

OBJECTIVE: To investigate the mechanism of prolonged central motor conduction time (CMCT) in compressive cervical myelopathy, we compared the calculated CMCT following transcranial magnetic stimulation (TCM) and evoked spinal cord potentials (ESCPs) following transcranial electric stimulation (TCE). METHOD: Motor evoked potentials following TCM were recorded from abductor digiti minimi and abductor hallucis brevis muscles in 16 patients with compressive cervical myelopathy. CMCT was calculated by subtracting peripheral conduction time using peripheral nerve stimulation from MEP latency. ESCPs following TCE were recorded intraoperatively from posterior epidural space. RESULTS: CMCT was prolonged and significant attenuation of the ESCP amplitude following TCE was observed in all patients with cervical myelopathy. In 8 of 16 patients CMCT was significantly prolonged but ESCPs were recorded at the C6-7 level with normal negative peak latency. CONCLUSIONS: Prolonged CMCT may occur with only a minor amount of conduction slowing in the corticospinal tract in compressive cervical myelopathy. Impaired temporal summation of multiple descending potentials following TCM produced delays of motor neuron firing that contribute to the mechanism of prolonged CMCT.

Introduction of a specific binding domain on myoglobin surface by new chemical modification.

A new myoglobin, reconstituted with a modified zinc protoporphyrin, having a total of four ammonium groups at the terminal of the two propionate side chains was constructed to introduce a substrate binding site. The protein with a positively charged patch on the surface formed a stable complex with negatively charged substrates, such as hexacyanoferrate(III) and anthraquinonesulfonate via an electrostatic interaction. The complexation was monitored by fluorescence quenching due to singlet electron transfer from the photoexcited reconstituted zinc myoglobin to the substrates. The binding properties were evaluated by Stern-Volmer plots from the fluorescence quenching of the zinc myoglobin by a quencher. Particularly, anthraquinone-2,7-disulfonic acid showed a high affinity with a binding constant of 1.5 x 10(5) M(-1) in 10 mM phosphate buffer, pH 7.0. In contrast, the plots upon the addition of anthraquinone-2-sulfonic acid at different ionic strengths indicated that the complex was formed not only by an electrostatic interaction but also by a hydrophobic contact. The findings from the fluorescence studies conclude that the present system is a useful model for discussion of electron transfer via non-covalently linked donor-acceptor pairing on the protein surface.

[Sensitive screening of antifungal compounds from acetone extracts of medicinal plants with a Bio-Cell Tracer].

Antifungal activity of medicinal plants against Aspergillus niger was evaluated using Bio-Cell Tracer (BCT). By this system enabled the real time determination of hyphal growth rate could be done in the presence or absence of Chinese herbal extracts. Of the 41 herbal extracts with 60% acetone extracts tested by the present method, 26 were found to contain active components against A. niger hyphal growth. In contrast, by conventional methods, no active component was detected from every herbal extract except 3 herbal extracts. From the extract of Anemarrhena asphodeloides, one of active components was isolated and its structure determined by NMR, UV, and mass spectroscopy. The compound was identified as broussonin A (2-3-(4-hydroxyphenyl)propyl)-5-methoxy-phenol) which was formerly reported as a phytoalexin of Broussonetia papyrifera Vent.

Cortical motor neuron excitability during cutaneous silent period.

OBJECTIVE: To investigate cortical motor neuron excitability during cutaneous silent period (CSP), motor evoked potentials (MEPs) from abductor pollicis brevis following transcranial magnetic stimulation (TCM) were recorded with and without a conditioning of ipsilateral painful digital nerve electric stimulation. METHODS: MEPs following TCM were recorded with and without a conditioning stimulation at an interstimulus interval (ISI) from 0 ms to 100ms in 6 controls and four patients who had reduced pain sensation in unilateral upper limbs associated with cervical syringomyelia. In addition MEPs and evoked spinal cord potentials (ESCPs) from cervical epidural space following TCM with and without a conditioning stimulation were recorded in four patients with thoracic myelopathy. RESULTS: MEP amplitude was clearly attenuated by a conditioning stimulation at an ISI from 40 ms to 80 ms in controls (statistically significant at 60 ms). In patients with cervical syringomyelia, MEP amplitude was attenuated by a conditioning stimulation in asymptomatic hands similarly in controls but that was unchanged by a conditioning stimulation in the symptomatic hand with reduced pain sensation. In patients with thoracic myelopathy MEP amplitude was attenuated by conditioning stimulation similarly in controls, but ESCP amplitude was unchanged. CONCLUSIONS: We demonstrated that noxious cutaneous nerve stimulation suppressed spinal motor neurons but cortical motor neuron excitability was unchanged during CSP. In clinical practice, measurement of MEP suppression after noxious cutaneous nerve stimulation may provide useful information in patients with damaged pain related nerve fibers.

Spatial distribution of corticospinal potentials following transcranial electric and magnetic stimulation in human spinal cord.

To investigate the spatial distribution of the human corticospinal tract in the spinal cord, evoked spinal cord potentials (ESCPs) following transcranial electrical and magnetic stimulation were recorded simultaneously from both the anterior and posterior epidural space in five anesthetized patients. One ESCP component following transcranial electrical stimulation (D-wave) and at least two ESCP components (initially D-wave and later I-wave) following transcranial magnetic stimulation were recorded in all subjects. The negative peak latency of all the potentials recorded from the posterior epidural space was the same as that recorded anteriorly. The amplitude ratio of the ESCP following electrical stimulation (posterior/anterior) was 1.10+/-0.12, while that of ESCPs following magnetic stimulation was 1.08+/-0.12 (N1) and 1.15+/-0.16 (N2). These results suggest that lateral corticospinal tract descending dorsolateral fasciculus in the spinal cord is main corticospinal pathway and spatial distribution of D and I-waves are similar in the human cervical cord.

Amino acid sequence and inhibitory activity of rhesus monkey tissue factor pathway inhibitor (TFPI): comparison with human TFPI.

Rhesus monkey cDNA for tissue factor pathway inhibitor (TFPI) was cloned by means of the reverse transcriptase-polymerase chain reaction, using liver mRNA, and its nucleotide sequence was determined by sequencing five independent clones. Monkey TFPI was found to have a signal peptide of 28 amino acid residues and to be a mature protein of 276 amino acid residues, in which three and seventeen amino acid residue substitutions compared to human TFPI were found, respectively. All the cysteine residues, three putative carbohydrate-linked asparagine residues, and the P1 amino acid residues of each of the three Kunitz inhibitor domains were conserved in the two species. Recombinant monkey TFPI (rTFPI) was isolated from the culture medium of transformed Chinese hamster ovary cells. Amino acid sequence analysis and immunoblotting analysis, using polyclonal and monoclonal antibodies, showed that the carboxyl-terminal basic part of Rhesus monkey rTFPI had been truncated. The inhibitory activity of monkey rTFPI was compared with that of human rTFPI without the carboxyl-terminal basic part. The prothrombin time of human plasma was slightly more prolonged by the addition of monkey rTFPI than by that of human rTFPI. However, no significant differences were found between the potencies of human and monkey rTFPI as to the inhibition of factor Xa and tissue factor-factor VIIa complex.

Efficient production of recombinant human factor VIII by co-expression of the heavy and light chains.

We have developed a high-level expression system for human blood coagulation factor VIII (FVIII) consisting of a 90 kDa heavy (H-)chain and an 80 kDa light (L-)chain. Two expression plasmids were prepared, one expressing the H-chain and the other expressing the L-chain. These recombinant plasmids were designed to produce each chain linked to short additional amino acid residues derived from the FVIII precursor sequence. Furthermore, Kozak's translation initiation consensus sequence was introduced into the start codon for the H-chain. These modifications have dramatically increased the levels of expression of these chains. Chinese hamster ovary (CHO) cells co-transfected with these two recombinant plasmids were subjected to gene amplification and cloning. The final cell line, designated CTC-CF8, secretes 15 IU/day/10(6) cells of active FVIII which is indistinguishable from plasma-derived FVIII in its structure and biochemical properties. This system is suitable for large-scale production of pathogen-free recombinant human FVIII which can be used for the treatment of haemophilia A patients.

Expression of hepatitis B virus middle and large surface antigen genes in Saccharomyces cerevisiae.

The hepatitis B virus genome carries the surface antigen (SAg) gene and an open reading frame that encodes two SAg-related polypeptides: SAg with a 55-amino-acid N-terminal extension polypeptide and SAg with a 174-amino-acid N-terminal extension polypeptide. These are termed middle S and large S, respectively. These polypeptides or their glycosylated derivatives have been detected in Dane particles, but their chemical and biological properties have remained largely unknown because of their limited availability. We attempted to produce these proteins in Saccharomyces cerevisiae by placing the coding regions under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Yeast cells carrying middle S and large S coding sequences produced 33,000- and 42,000-dalton products, respectively, each of which reacted with anti-S antibody and bound to polymerized human serum albumin, in accordance with the known properties of pre-S proteins from particles in human sera (K. H. Heermann, U. Goldmann, W. Schwartz, T. Seyffarth, H. Baumgarten, and W. H. Gerlich, J. Virol. 52:396-402, 1984; A. Machida, S. Kishimoto, H. Ohnuma, K. Baba, Y. Ito, H. Miyamoto, G. Funatsu, K. Oda, S. Usuda, S. Togami, T. Nakamura, Y. Miyakawa, and M. Mayumi, Gastroenterology 86:910-918, 1984). The middle S polypeptide is glycosylated and can be assembled into particles whose size and density are similar to those of SAg. However, this polypeptide was highly susceptible to proteolytic degradation into 29,000- and 26,000-dalton polypeptides, of which only the former retained the binding activity to polymerized albumin. The large S polypeptides are nonglycosylated, relatively stable, and do not seem to assemble into particles by themselves.