Ginkgo biloba leaf extract (EGb 761®) and its specific acylated flavonol constituents increase dopamine and acetylcholine levels in the rat medial prefrontal cortex: possible implications for the cognitive enhancing properties of EGb 761®.

Experimental and clinical data suggest that the Ginkgo biloba standardized extract EGb 761® exerts beneficial effects in conditions which are associated with impaired cognitive function. However, the neurochemical correlates of these memory enhancing effects are not yet fully clarified. The aim of this study was to examine the effect of repeated oral administration of EGb 761® and some of its characteristic constituents on extracellular levels of dopamine (DA), noradrenaline (NA), serotonin (5-HT), acetylcholine (ACh) and the metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in the medial prefrontal cortex (mPFC) of awake rats by use of in vivo microdialysis technique. Subacute (14 days, once daily), but not acute, oral treatment with EGb 761® (100 and 300 mg/kg) or the flavonoid fraction, which represents about 24% of the whole extract caused a significant and dose-dependent increase in extracellular DA levels in the mPFC. Repeated administration of EGb 761® also caused a modest but significant increase in the NA levels, whereas the concentrations of 5-HT and those of the metabolites DOPAC, HVA and 5-HIAA were not affected. The same treatment regimen was used in a subsequent study with the aim of investigating the effects of two Ginkgo-specific acylated flavonols, 3-O-(2''-O-(6'''-O-(p-hydroxy-trans-cinnamoyl)-ß-D-glucosyl)-α-L-rhamnosyl)quercetin (Q-ag) and 3-O-(2''-O-(6'''-O-(p-hydroxy-trans-cinnamoyl)-ß-D-glucosyl)-α-L-rhamnosyl)kaempferol (K-ag). Both compounds together represent about 4.5% of the whole extract. Repeated oral treatment with Q-ag (10 mg/kg) for 14 days caused a significant increase in extracellular DA levels of 159% and extracellular acetylcholine (ACh) levels of 151% compared to controls. Similarly, administration of K-ag (10 mg/kg) induced a significant rise of DA levels to 142% and ACh levels to 165% of controls, whereas treatment with isorhamnetin, an O-methylated aglycon component of EGb 761® flavonol glycosides had no effect. None of the tested flavonoids had a significant effect on extracellular DOPAC and HVA levels. The present findings provide evidence that the subacute treatment with EGb 761® and its flavonol constituents increases DA and ACh release in the rat mPFC, and suggest that the two Ginkgo-specific acylated flavonol glycosides Q-ag and K-ag are active constituents contributing to these effects. As seen for isorhamnetin, the effect on neurotransmitter levels seems not to be a general effect of flavonols but rather to be a specific action of acylated flavonol glycosides which are present in EGb 761®. The direct involvement of these two flavonol derivatives in the increase of dopaminergic and cholinergic neurotransmission in the prefrontal cortex may be one of the underlying mechanisms behind the reported effects of EGb 761® on the improvement of cognitive function.

Determination of histamine in microdialysis samples from Guinea pig skin by high-performance liquid chromatography with fluorescence detection.

AIM: To develop a sensitive and selective liquid-chromatographic method for the determination of histamine in microdialysis samples from guinea pig skin following allergenic provocation. METHODS: The novel fluorescence derivatization method is based on an intramolecular excimer-forming reaction between 2 amino moieties of histamine and 2 molecules of 4-(1-pyrene)butanoyl chloride (PBC) yielding the corresponding dipyrene-labeled derivative. RESULTS: The PBC derivative of histamine was separated within 20 min, and the detection limit (signal-to-noise ratio = 3) of histamine was 0.6 fmol/20 µl volume injected. The basal extracellular levels of histamine in guinea pig skin microdialysates were 20.6 ± 1.7 fmol/10 µl. Subcutaneous administration of histamine liberator compound 48/80 (3 mg/kg) increased the extracellular histamine levels in the skin dialysates by about 860%, whereas ovalbumin challenge (2 mg/kg i.v.) in the sensitized guinea pigs increased the extracellular histamine levels by about 3,030%. CONCLUSION: The novel technique for histamine determination in microdialysis samples from the guinea pig skin may be utilized in preclinical research of antihistaminergic drugs and evaluation of allergenic properties of various dermal preparations such as transdermal drug delivery systems.

[Aortitis syndrome with repeated restenoses of a drug eluting stent].

The patient was a 49-year-old female who developed acute myocardial infarction of the right coronary artery in August 2005. In a short period of time, the patient had restenosis repeatedly after percutaneous coronary intervention (PCI). Restenosis could not be prevented even with a drug eluting stent (DES), and thus, off-pump coronary artery bypass grafting (OPCAB) was performed. The diagnosis of aortitis syndrome was made due to protracted postoperative inflammation. Aortitis syndrome was determined to be the main cause of repeated restenosis. This case was a middle-aged female who had restenosis in a short period of time, and aortitis syndrome should have been included in the differential diagnosis. Although some positive results have been reported on DES placement for coronary lesions of aortitis syndrome, DES was completely ineffective in our patient. Further studies with more patients are necessary to examine the effectiveness of DES.

[Anomalous origin of the right coronary artery from the ascending aorta].

The patient was a 77-year-old man. In June 2008, he underwent off-pump coronary artery bypass (OPCAB) for unstable angina Intraoperative epiaortic echo showed an anomalous origin of theright coronary artery from the ascending aorta 4 cm above the sinotubular junction (STJ). The right coronary artery traveled through the planned proximal anastomotic site of the saphenous vein graft (SVG). If diagnosis of the anomalous origin of the right coronary artery had not been made, there would have been a high likelihood that the right coronary artery could have been injured. Thus, the usefulness of epiaortic echo was reaffirmed. An anomalous origin of the coronary artery is a rare congenital anomaly and its incidence is approximately 1%. An anomalous origin of the right coronary artery is very rare from the ascending aorta 4 cm above the STJ and only a few cases have been reported. An anomalous origin of the coronary artery can cause serious complications affecting the prognosis after open heart surgery. Thus, such an anomalous origin needs to be considered in preoperative evaluation.

Hemophilia B (factor IXSeattle 2) due to a single nucleotide deletion in the gene for factor IX.

To understand the molecular basis for hemophilia B in patients with little or no circulating Factor IX antigen, a patient who had less than 0.2% circulating Factor IX antigen (Factor IXSeattle 2) was selected for analysis of his Factor IX gene. Genomic DNA fragments from the abnormal gene were cloned into bacteriophage lambda vectors and recombinant phage were identified using radiolabeled genomic probes obtained from the normal Factor IX gene. The exons and flanking regions of the abnormal gene were sequenced by the dideoxy chain-termination method and this sequence was compared with that of the normal gene. Only one significant difference was observed, the deletion of a single adenine nucleotide in exon V. This resulted in a frameshift that converted an aspartic acid at position 85 in the protein to a valine and the formation of a stop signal at position 86. These data indicate that the gene for Factor IXSeattle 2 codes for an 85 residue polypeptide that terminates after the first epidermal growth factor domain. Thus, the putative Factor IXSeattle 2 polypeptide lacks the second epidermal growth factor domain, the activation peptide, and the catalytic domain present in the normal protein. This provides an explanation for the coagulation disorder in this patient and represents the first report of a single nucleotide deletion and frameshift resulting in hemophilia B.

An intragenic deletion of the factor IX gene in a family with hemophilia B.

A family of seven patients severely afflicted with hemophilia B has been studied for their factor IX genes through the use of factor IX cDNA and genomic DNA probes. The patients had detectable (less than 10% of normal) factor IX antigen in urine and no detectable inhibitors in sera to factor IX protein. Based on the DNA hybridization analysis, these patients showed a partial intragenic deletion in their factor IX gene. The deletion included two exons (exons V and VI) coding for the amino acid sequence from number 85 to 195 of the factor IX protein. The deleted portion of the gene contained the entire factor IX activation peptide. The length of the deletion was estimated to be 10 +/- 0.3 kilobase pairs. This specific gene has been named FIXSeattle. In this family both the deletion and a Taq 1 restriction fragment length polymorphism can be used as a useful marker for accurate detection of female carriers of the deficient factor IX gene.

Clinical relevance of quantified fundus autofluorescence in diabetic macular oedema.

PURPOSE: To quantify the signal intensity of fundus autofluorescence (FAF) and evaluate its association with visual function and optical coherence tomography (OCT) findings in diabetic macular oedema (DMO). METHODS: We reviewed 103 eyes of 78 patients with DMO and 30 eyes of 22 patients without DMO. FAF images were acquired using Heidelberg Retina Angiograph 2, and the signal levels of FAF in the individual subfields of the Early Treatment Diabetic Retinopathy Study grid were measured. We evaluated the association between quantified FAF and the logMAR VA and OCT findings. RESULTS: One hundred and three eyes with DMO had lower FAF signal intensity levels in the parafoveal subfields compared with 30 eyes without DMO. The autofluorescence intensity in the parafoveal subfields was associated negatively with logMAR VA and the retinal thickness in the corresponding subfields. The autofluorescence levels in the parafoveal subfield, except the nasal subfield, were lower in eyes with autofluorescent cystoid spaces in the corresponding subfield than in those without autofluorescent cystoid spaces. The autofluorescence level in the central subfield was related to foveal cystoid spaces but not logMAR VA or retinal thickness in the corresponding area. CONCLUSIONS: Quantified FAF in the parafovea has diagnostic significance and is clinically relevant in DMO.

Anti-atherosclerotic effect of E5324, an inhibitor of acyl-CoA:cholesterol acyltransferase, in Watanabe heritable hyperlipidemic rabbits.

E5324, n-butyl-N'-[2-[3-(5-ethyl-4-phenyl-1H-imidazol-1-yl)propoxy]-6- methylphenyl]urea, a novel and potent inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), was evaluated for its anti-atherosclerotic and lipid-lowering effects in Watanabe heritable hyperlipidemic (WHHL) rabbits. At 3 months of age, 40 male WHHL rabbits were divided into 4 groups. The rabbits were fed a standard rabbit chow (control group), or standard rabbit chow containing E5324 (0.1% or 0.02%) or 1% probucol for 16 weeks. Even the high dose of E5324 did not lower the plasma total cholesterol levels throughout the experiment. Probucol slightly reduced the plasma cholesterol levels, and showed anti-atherosclerotic activity, i.e., reductions of atherosclerotic plaque formation and cholesterol content in the aorta. Although E5324 did not lower plasma cholesterol, atherosclerotic plaque formation in the aortic arch and thoracic aorta was reduced (by about 34% and 41%, respectively, at the high dose; P < 0.05). Cholesterol content in the aortic arch and thoracic aorta was also reduced (by about 59% and 62% at the high dose, respectively) compared with the control. These results suggest that E5324 acts directly on the arterial wall through ACAT inhibition, and prevents the progression of atherosclerosis in WHHL rabbits.

A general and mild procedure for the purification of rabbit Fab' antibodies.

A mild procedure for the purification of rabbit Fab' antibodies is described. A small column of normal goat IgG-Sepharose 4B (3 cm x 5mm) was saturated with F(ab')2 prepared from rabbit anti-goat IgG, and anti-goat IgG Fab was eluted from the column by splitting the disulfide bond in the hinge of the F(ab')2 molecule using 10 or 12 mM 2-mercaptoethylamine at pH 7. The recovery of anti-goat IgG Fab' in the eluate was about 35% of anti-goat IgG F(ab')2 bound to the column, and the purity of anti-goat IgG Fab' eluted was more than 90%. This procedure may be applicable to most kinds of rabbit IgG antibodies and useful for various immunoassays.

Activation of 5-HT(1A) autoreceptors enhances the inhibitory effect of galanin on hippocampal 5-HT release in vivo.

The microdialysis technique was used to examine interactions between 5-HT(1A) and galanin receptors in the dorsal raphe nucleus (DRN), by measuring the extracellular levels of 5-HT in the ventral hippocampus of awake rats. The rats were pretreated with the 5-HT(1A) receptor agonist (R,S)-8-OH-DPAT (0.3 mg/kg, s.c.) or saline. 8-OH-DPAT caused a time-dependent reduction of basal 5-HT levels down to 43-48% at 40 min while at 140 min, the hippocampal 5-HT had returned to control values. At that time point, the rats received a second injection of 8-OH-DPAT or galanin (0.15, 0.5 and 1.5 nmol/0.5 microl) infused into the lateral ventricle. The second injection of 8-OH-DPAT caused a significantly smaller reduction of hippocampal 5-HT levels. In contrast, galanin at all three doses in the 8-OH-DPAT-pretreated groups, was significantly more potent in reducing 5-HT levels (maximal reduction to 74%, 52% and 49%, respectively) than it was in saline-pretreated rats (maximal reduction to 96%, 85% and 69%, respectively). The inhibitory effect of galanin (1.5 nmol) on extracellular 5-HT levels in the rat hippocampus was significantly attenuated by co-administration of the 5-HT(1A) receptor antagonists WAY-100635 (0.3 and 0.6 mg/kg s.c.) and, to a lesser extent, with pindolol (20 mg/kg s.c.). These data provide direct in vivo evidence of agonistic 5-HT(1A)-galanin receptor interaction at the presynaptic level. Furthermore, the findings indicate that a down-regulation of the somato-dendritic 5-HT(1A) autoreceptors, following their stimulation with 8-OH-DPAT and possibly also indirectly with 5-HT reuptake inhibitors, may be compensated by a subsequent 'sensitization' of the inhibitory galanin receptors in the DRN. Thus, the enhanced galanin receptor-mediated inhibition of 5-HT neurotransmission may contribute to the pathophysiology of depression or to the reduced and delayed efficacy of antidepressant therapies.

Importance of left anterior descending coronary artery curvature in determining cross-sectional plaque distribution assessed by intravascular ultrasound.

We assessed the relation between the circumferential distribution of coronary atherosclerotic plaques and the structure of the epicardial coronary arteries in patients with coronary artery disease using intravascular ultrasound in vivo. Coronary atherosclerosis preferentially formed at the inner arc of the curved coronary vessels, and greater vessel curvatures were associated with greater distributions of atherosclerotic lesions along the inner coronary artery wall.

Possible differences in the regenerative roles played by thioltransferase and thioredoxin for oxidatively damaged proteins.

A possible involvement of thioltransferase (also known as glutaredoxin) in the regenerative reaction of proteins inactivated by oxidative stress were examined in vitro using the enzyme purified from bovine liver. Thioltransferase at physiological concentrations, together with glutathione, glutathione reductase and NADPH, regenerated the oxidatively damaged proteins with a comparable potency to that of thioredoxin. Experiments performed with protein substrates with their critical cysteine residues oxidized differently, that is, phosphofruktokinase and glyceraldehyde 3-phosphate dehydrogenase with mixed disulfide bonds and glyceraldehyde 3-phosphate dehydrogenase with sulfenyl or sulfinyl groups, indicated that thioltransferase regenerated the proteins inactivated by mixed disulfide formation more efficiently than thioredoxin, whereas thioredoxin preferentially regenerated the proteins inactivated by monothiol oxidation to sulfenic or sulfinic acid. These findings suggested that thioltransferase exerted regenerative effects on oxidatively damaged proteins like its cognate protein, thioredoxin, but with different substrate specificity, and their relative contribution to the regeneration reaction is dependent on the form of the oxidized thiols of the damaged proteins.

Improved procedure for the conjugation of rabbit IgG and Fab' antibodies with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide.

The procedures for the conjugation of rabbit IgG and Fab' antibodies with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide were improved in several respects as compared with the previous methods (Eur. J. Biochem. 62, 285--292, 1976; J. Immunol. 116, 1554--1560, 1976). Maleimide residues were efficiently introduced into antibodies under an atmosphere of nitrogen; the average number of maleimide residues introduced into IgG and Fab' antibodies were 0.78 (0.65--0.86) and 0.86 (0.80--0.95) per molecule, respectively. The conjugation with the enzyme was performed at 4 degrees C at pH 6.5 for 15 or more hours. The conjugates were almost completely separated from unreacted IgG and Fab' by gel filtration. When the recoveries of IgG, Fab', and beta-D-galactosidase in the conjugates were 23-29, 35-44, and 99%, respectively, the average numbers of IgG and Fab' molecules conjugated with the enzyme were 1.5-1.7 and 2.1-2.8 per molecule, respectively. There was no significant impairment of beta-D-galactosidase activity or the activity of anti-human IgG antibody to bind to human IgG upon conjugation. However, the conjugate preparation was heterogeneous, and one-third of each preparation consisted of aggregated conjugates less useful in sandwich enzymoimmunoassay than the remaining material. The conjugate with Fab' antibody gave lower control values in sandwich enzymoimmunoassay with silicone rubber as a solid phase than that with IgG antibody.

Mild and efficient conjugation of rabbit Fab' and horseradish peroxidase using a maleimide compound and its use for enzyme immunoassay.

A mild and efficient procedure for conjugating rabbit Fab' and horseradish peroxidase using a maleimide compound was developed. The enzyme was treated with N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl) maleimide to introduce maleimide groups. Then, the maleimide-enzyme was allowed to react with thiol groups of Fab', and the conjugate formed was separated from unreacted components by gel filtration with Ultrogel AcA 44. In the peak fraction of the separated conjugate, 98% of peroxidase was associated with Fab' and 90% of antibodies was associated with peroxidase. The recoveries in the conjugate of peroxidase and Fab' incubated for conjugation were 65-74%. The conjugate formed appeared to be largely monomeric. Both the enzyme activity and antigen-binding activity of Fab' were fairly well preserved in the conjugate. The cross-link formed was stable at 4 degrees C at least 6 months. Use of the conjugates obtained by this method gave greater sensitivity in sandwich enzyme immunoassay for human ferritin and human thyroid-stimulating hormone than conjugates prepared by the periodate method. The conjugation using N-hydroxysuccinimide ester of m-maleimidobenzoic acid provided a similar monomeric preparation but was less efficient.

Preparation of monomeric Fab'-horseradish peroxidase conjugate using thiol groups in the hinge and its evaluation in enzyme immunoassay and immunohistochemical staining.

Horseradish peroxidase and Fab' were conjugated by using thiol groups in the hinge of Fab'. Maleimide or pyridyl disulfide groups were introduced into peroxidase by treatment with N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate or N-succinimidyl 3-(2-pyridyldithio)propionate and were allowed to react with thiol groups in the hinge of Fab'. The conjugates were obtained in high yields with a minimal polymerization and without impairing the activities of peroxidase and antibodies, and were superior to those prepared using amino groups of Fab' by the glutaraldehyde and periodate methods in performing sandwich enzyme immunoassay and immunohistochemical staining. The conjugate yield was higher in the maleimide method than in the pyridyl disulfide method.

Transient EDTA-dependent pseudothrombocytopenia in a patient with sepsis.

Ethylenediaminetetraacetic acid-dependent pseudothrombocytopenia (EDTA-PTCP) is the phenomenon of a spurious low platelet count due to antiplatelet antibodies that cause platelet clumping in blood anticoagulated with EDTA. We describe a case of EDTA-PTCP that appeared transiently with the development of sepsis. A 50-year-old man underwent Bentall's aortic root replacement for acute aortic dissection with aortic insufficiency. Postoperatively the patient suffered paralytic ileus followed by methicillin-resistant Staphylococcus aureus enteritis and septicemia with endotoxemia. EDTA-PTCP appeared with the development of sepsis, and disappeared with its resolution. To avoid incorrect diagnoses and inappropriate treatment, EDTA-PTCP should always be considered as a possible cause of reported low platelet counts, even in patients with sepsis.

An abnormal protein C (protein C Yonago) with an amino acid substitution of Gly for Arg-15 caused by a single base mutation of C to G in codon 57 (CGG-->GGG). Deteriorated calcium-dependent conformation of the gamma-carboxyglutamic acid domain relevant to a thrombotic tendency.

Protein C Yonago is a dysfunctional protein C characterized by defective anticoagulant activity determined by a coagulation assay and normal amidolytic activity measured on a synthetic substrate S-2366 (Iijima et al., Thromb Res 1991;63:249-257). We have identified a single point mutation of C to G in codon 57 (CGG-->GGG) of the gene for protein C Yonago by genetic analysis utilizing the polymerase chain reaction. The mutation should have resulted in an amino acid substitution of Gly for Arg at position 15 of the light chain of mature protein C. No mutations were found in nucleotides spanning the putative gamma-carboxylase recognition site or gamma-carboxyglutamic acid residues of protein C. Protein C Yonago was non-reactive to monoclonal antibodies JTC-1 and -3 that solely recognized the calcium-dependent conformation of the gamma-carboxyglutamic acid domain. This indicated that the mutation had critically perturbed the highly conserved structure of the gamma-carboxyglutamic acid domain. Thus, the calcium-dependent conformation required for the phospholipids binding to exert the physiological functions of protein C may not have been elicited normally in this abnormal protein C, resulting in defective generation of anticoagulant activities in plasma. Consequently, no anticoagulant activities may have been generated in vivo.

Gene analyses of abnormal fibrinogens with a mutation in the gamma chain.

In four abnormal fibrinogens with a point mutation in the gamma chain, all characterized by impaired fibrin polymerization, we identified single base exchanges in the respective mutant gamma chain genes by polymerase chain reaction followed by DNA sequence analysis. These base exchanges accounted for the amino acid substitutions previously reported from our laboratory. They were exchanges of C to T (CGC for gamma Arg-275 to TGC for Cys) in fibrinogen Osaka II, T to G (AAT for gamma Asn-308 to AAG for Lys) in fibrinogen Kyoto I, T to C (ATG for gamma Met-310 to ACG for Thr) in fibrinogen Asahi, and G to T (GAT for gamma Asp-330 to TAT for Tyr) in fibrinogen Kyoto III. These base exchanges were found to reside in exon VIII of the gamma chain gene. Since many abnormal molecules are associated with polymerization defects, unless associated with the impaired release of fibrinopeptides A and/or B, exon VIII of the gamma chain gene may deserve careful study to define the structural alterations.

Highly sensitive sandwich enzyme immunoassay of human IgE with beta-D-galactosidase from Escherichia coli.

A highly sensitive sandwich enzyme immunoassay of human IgE was developed. Polystyrene balls were coated with goat anti-human IgE immunoglobulin (IgG) by physical adsorption. Goat anti-human IgE Fab' was purified by affinity chromatography and conjugated with beta-D-galactosidase from Escherichia coli. Using thus prepared anti-IgE-coated polystyrene balls and anti-IgE-beta-D-galactosidase conjugate, 0.2 mU (2 amol)--1 U of IgE per assay could be determined. When 0.1 microliter of serum per assay was used, the range of IgE levels in serum that could be determined was 2--10000 U/ml, and even 0.01 U/ml was measurable by using 20 microliters of serum. The regression equation and coefficient for correlation to radioimmunoassay were gamma (RIA) = 0.94 chi (EIA) + 18.2 and 0.96 (n = 81), respectively. The coefficients of within- and between-assay variations ranged from 5.4 to 8.5%. The mean levels of serum IgE determined by the present assay were 103 U/ml in 70 normal children and 1064 U/ml in 38 children with bronchial asthma.

An enzyme immunoassay for the measurement of thyroglobulin in human serum.

An enzyme-linked sandwich immunoassay using silicone rods coated with rabbit (anti-human thyroglobulin) immunoglobulin G and rabbit (anti-human thyroglobulin) monovalent fragment of immunoglobulin F (Fab') conjugated with beta-D-galactosidase was developed for the measurement of thyroglobulin in human serum. The volume of serum needed for the assay was as little as 2 microliters. The sensitivity of the assay was 3.5 ng/ml, which is equal to or rather higher than that of radioimmunoassay. The specificity of the assay was demonstrated by the following observations: (1) The absence of crossreaction of thyroxine and triiodothyronine, (2) non-detectability of thyroglobulin in the sera of patients who underwent total thyroidectomy, (3) parallelism of the standard curve with dilutions of reference serum. The precision of the assay was proven by the demonstration of the sufficient recovery of human thyroglobulin added to sera (92--99%) and coefficients of variance in within and between assays were 6.2--9.3 and 2.5--5.3%, respectively. Furthermore, a highly significant correlation was observed between thyroglobulin concentrations measured by our enzyme immunoassay and those by radioimmunoassay (r = 0.99, p less than 0.001, n = 63). Human thyroglobulin in serum was detectable in 90% of 146 normal subjects, the concentration (mean +/- S.D.) being 13.3 +/- 10.3 ng/ml.