Tumor targeting based on the effect of enhanced permeability and retention (EPR) and the mechanism of receptor-mediated endocytosis (RME).

This review is focused on the macromolecular drug carrier systems by the effect of enhanced permeability and retention (EPR) and the mechanism of receptor-mediated endocytosis (RME). The effect of EPR is thought to be useful for the targeting of the macromolecular drugs to the tumor tissues on a vasculolymphatic level. The RME reveals the selective recognition, high affinity binding, and immediate internalization for the ligand on a cellular level. In the receptor, recognizing transferrin, a level of expression on the tumor cells is higher than that on the normal cells. We have used serum albumin and transferrin as drug carriers to deliver mitomycin C (MMC) to the tumor tissues and into the tumor cells. The properties of the conjugates of MMC to serum albumin and transferrin were examined in vitro and in vivo. We concluded that MMC could be delivered to the tumor tissue and cells by the use of albumin and transferrin as drug carriers.

The effects of danaparoid, dalteparin and heparin on tissue factor-induced experimental disseminated intravascular coagulation and bleeding time in the rat.

Danaparoid and heparin, on the basis of anti-activated factor X (anti-FXa) activity, were equipotent in accelerating the rate of interaction of FXa and antithrombin III. In rat tissue factor-induced disseminated intravascular coagulation (DIC) models, an intravenous administration of danaparoid inhibited the decrease in plasma fibrinogen and platelet counts and the increase in serum fibrinogen degradation products. Expressed on the basis of anti-FXa activity, these effects were comparable with those of dalteparin and heparin. In rat mesenteric small artery and vein, less bleeding was observed after intravenous administration of danaparoid than after dalteparin or heparin. Danaparoid did not affect adenosine diphosphate- or collagen-induced platelet aggregation, and showed weaker inhibitory effects on aggregation induced by thrombin, or collagen + thrombin, than did dalteparin or heparin. These findings suggest that danaparoid may be useful for the prevention of DIC and has less tendency to cause bleeding than dalteparin or heparin, probably as a result of its weaker ability to inhibit platelet aggregation.

Receptor mediated endocytosis and cytotoxicity of transferrin-mitomycin C conjugate in the HepG2 cell and primary cultured rat hepatocyte.

Intracellular disposition and cytotoxicity of macromolecular conjugate of mitomycin C (MMC) with transferrin (TF) were examined in the human hepatoma cell line HepG2 cell and normal cultured rat hepatocyte. The conjugate (TF-MMC) was specifically bound to the HepG2 cell as well as TF. The number of the binding site and the association constant of TF-MMC in the HepG2 cell were 396000+/-31000 molecules/cell and 3.24 x 10(7)+/-0.58 x 10(7) M(-1), respectively. No difference in the binding parameters of TF-MMC and TF can be detected in the HepG2 cell. The association constant for the TF receptor was almost identical between HepG2 cell and hepatocyte, however, the numbers of the binding site of TF-MMC and TF in the HepG2 cell were from 40-times to 50-times greater than those in the hepatocyte. Furthermore, TF-MMC was internalized into the HepG2 cell and the hepatocyte as well as TF. The rates of internalization of TF-MMC and TF into the HepG2 cell were nearly identical to those into the hepatocyte. However, the levels of the internalization into the HepG2 cell were remarkably higher than those into the hepatocyte because the number of receptors in the HepG2 cell was larger than that in the hepatocyte, and the rate of release from the HepG2 cell was slower than that from the hepatocyte. TF-MMC inhibited the growth of the HepG2 cells. The 50% growth inhibition (GI50) of TF-MMC against the HepG2 cell was 0.9 microg MMC/ml, which was a little higher than that of MMC (GI50=0.5 microg/ml). These results indicated that the TF-MMC might be useful for delivery of MMC to the HepG2 cell.

Regional differences in cerebral glucose metabolism in cirrhotic patients with subclinical hepatic encephalopathy using positron emission tomography.

Although it is recognized that abnormalities of glucose metabolism occur substantially throughout the brain in cirrhotic patients with subclinical hepatic encephalopathy (SHE), little attention has been paid to these abnormalities in situ due to technical difficulties. The aim of this study is to clarify the regional differences of cerebral glucose metabolism (CMRglu) in patients with or without SHE using positron emission tomography (PET). Fifteen patients with posthepatitic cirrhosis and 13 control subjects underwent PET imaging of the brain using 2-[18F]fluoro-2-deoxy-D-glucose. Neurological tests included the digit symbol and block design subtests from the Wechsler Adult Intelligence Scale. Auditory brain stem reaction and electroencephalogram were also used in diagnosis of SHE. Regions were defined as frontal, parietal, occipital, basal ganglia, and the white matter. The CMRglu value in the grey matter of SHE is lower than that of control, particularly in the frontal and temporal regions and basal ganglia (P<0.05, respectively). The CMRglu values in the grey matter of non-SHE and control are almost the same, excepting the basal ganglia. The CMRglu value in the basal ganglia of non-SHE is higher than that of control and SHE. The CMRglu value in the grey matter of SHE is depressed as compared to both non-SHE and control. These data suggest that the abnormalities of cerebral glucose metabolism may be a contributing factor in SHE.

Selective 5-hydroxytryptamine3 (5-HT3) receptor blocking activity of KB-R6933, a novel benzimidazole derivative.

5-Hydroxytryptamine3 (5-HT3)-receptor blocking activities of KB-R6933 (6-amino-5-chloro-1-isopropyl-2-(4-methyl-1-piperazinyl)-benzimidazole dimaleate) were assessed in vivo and in vitro. Intravenous administration of KB-R6933, granisetron, ondansetron and azasetron inhibited 5-HT-induced bradycardia (von Bezold-Jarisch reflex) in anesthetized rats, with ED50 values of 0.071, 0.71, 4.0 and 0.82 microg/kg, respectively. The inhibitory effect of KB-R6933 at a dose of 0.3 microg/kg lasted for at least 8 hr, whereas those of granisetron at 30 microg/kg, ondansetron at 100 microg/kg and azasetron at 30 microg/kg nearly disappeared within 2-4 hr. Oral administration of KB-R6933 and granisetron also inhibited the bradycardia, with ED50 values of 0.41 and 76.3 microg/kg, respectively. In guinea pig ileum, KB-R6933 concentration-dependently antagonized 5-HT-evoked contraction and reduced the maximal contraction (pK(B)=8.75). Granisetron, ondansetron and azasetron shifted the dose-response curve for 5-HT to higher concentrations with no reduction of maximal contraction, and their pK(B)s were 7.65, 7.00 and 6.29, respectively. In a radioligand receptor binding study, KB-R6933, granisetron, ondansetron and azasetron displaced [3H]GR65630 binding to rat entorhinal cortex membrane, with Ki values of 0.066, 0.99, 2.70 and 2.5 nM, respectively. On the other hand, KB-R6933 exhibited negligible affinities for other receptors or binding sites tested, except for a weak affinity for the cholinergic M1-receptor, even at concentrations up to 10 microM. These results suggest that KB-R6933 is a potent and selective 5-HT3-receptor antagonist with a longer duration of action than those of existing 5-HT3-receptor antagonists.

The effect of danaparoid sodium (danaparoid) on endotoxin-induced experimental disseminated intravascular coagulation (DIC) in rats.

Danaparoid sodium (danaparoid) is a low molecular weight heparinoid with anticoagulation properties, which mainly consists of heparan sulfate. Compared with heparin sodium (heparin), danaparoid has a much higher anti-Xa/anti-thrombin ratio. We compared the effect of danaparoid on endotoxin-induced experimental disseminated intravascular coagulation (DIC) in rats with heparin. A bolus injection of endotoxin (10 mg/kg) induced gradual decreases in the platelet count, and the plasma fibrinogen, antithrombin III (AT-III) and heparin cofactor II levels, as well as an increase in the fibrinogen/fibrin degradation products level from 1 to 6 hours after the injection, indicating that both coagulation and fibrinolysis were activated. The intravenous administration of danaparoid or heparin 3 hours after the endotoxin injection inhibited the endotoxin-induced decreases in the platelet count and plasma fibrinogen level and also inhibited the endotoxin-induced increase in glomerular fibrin deposition in the kidney. Differences between danaparoid and heparin were observed in their effects on the plasma AT-III level and clotting time. Danaparoid significantly inhibited both the decrease in the plasma AT-III level and the prolongation of the prothrombin time induced by endotoxin, where as heparin showed no effect on those responses. Moreover, danaparoid enhanced the prolongation of the activated partial thromboplast in time induced by endotoxin to a lesser degree than heparin. These findings suggest that the effects of danaparoid on the endotoxin-induced decrease of the plasma AT-III level and the prolongation of the clotting time are more advantageous than those of heparin. The results may have been due to a higher anti-Xa/anti-thrombin ratio of danaparoid than that of heparin, indicating that danaparoid may be useful in the treatment of DIC.

Aortoiliac occlusion associated with the lupus anticoagulant. Report of two cases.

Aortoiliac occlusion due to atherosclerosis is known to occur in elderly people. Two unique cases are documented here in which aortoiliac occlusion was observed in young men. These patients had no identifiable risk factors for developing atherosclerotic thrombosis except for the lupus anticoagulant, an immunoglobulin associated with thromboembolic episodes. Although previously the lupus anticoagulant has been reported to cause thrombosis only in relatively small-sized arteries or veins, it may also be associated with aortoiliac atheromatous occlusion in young men.

Ambulatory blood pressure monitoring in patients with essential hypertension treated with a new calcium antagonist, cilnidipine.

Cilnidipine (FRC-8653), a new dihydropyridine calcium antagonist, was given to 14 hospitalized patients with essential hypertension, and 24-hour ambulatory blood pressure (BP) monitoring was performed. Once-daily administration of cilnidipine (5-20 mg) for 1-3 weeks decreased the 24-hour average BP significantly from 149 +/- 4/88 +/- 2 mmHg to 141 +/- 3/82 +/- 2 mmHg without any change in the pulse rate. The decrease in ambulatory BP by cilnidipine was evident during the daytime (156 +/- 4/93 +/- 2 mmHg to 143 +/- 5/84 +/- 2 mmHg, p < 0.01 for systolic BP and p < 0.01 for diastolic BP), while it was mild during nighttime (141 +/- 4/80 +/- 2 mmHg to 133 +/- 4/76 +/- 3 mmHg, p < 0.05 for systolic and ns for diastolic BP). The decrease in the ambulatory BP over the whole day and during the nighttime was significantly correlated with the basal ambulatory BP levels. When the subjects were divided into the high ambulatory BP (n = 7) and low ambulatory BP (n = 7) groups, the BP reduction by cilnidipine was evident throughout 24 hours in the high ambulatory BP group, while it was mild and significant only during daytime in the low ambulatory BP group. In summary, once-daily cilnidipine exerts a sufficient and prolonged reduction of BP without an increase in the pulse rate in patients with hypertension. The potency of cilnidipine to decrease ambulatory BP may depend on the basal ambulatory BP level. Cilnidipine is thus a useful antihypertensive drug that may not cause an excessive decrease in BP or a reflex tachycardia.

Synthesis and mitogenic activity of nonreducing-sugar subunit analogs of bacterial lipid A composed only of 3-hydroxytetradecanoic acid and its homologs.

Novel analogs of the nonreducing-sugar subunit of bacterial lipid A, which were composed only of 3-hydroxytetradecanoic acid and its homologs, were synthesized. These analogs exhibited significant mitogenic activity.

Five new triterpene glycosides from Wisteria brachybotrys (Leguminosae).

From the vines of Wisteria brachybotrys (Leguminosae), five new oleanene glycosides, called wistariasaponins YC1,2, B3 and A2,3, together with four known ones were isolated. Their structures have been elucidated to be 3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-xylopyranosyl-(1-->2)-beta-D- glucuronopyranosyl yunganogenin C 21-O-beta-D-glucopyranoside (1), 3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->2)-beta -D- glucuronopyranosyl yunganogenin C 21-O-beta-D-glucopyranoside (2), 3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-xylopyranosyl-(1-->2)-beta- -D-glucuronopyranosyl wistariasapogenol B 30-O-beta-D-glucopyranoside (3), 3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-xylopyranosyl-(1-->2)-beta-D- glucuronopyranosyl wistartiasapogenol A 30-O-beta-D-glucopyranoside (4) and 3-O-beta-D-galactopyranosyl-(1-->2)-beta-D-glucuronopyranosyl wistariasapogenol A 30-O-beta-D-glucopyranoside (5), respectively.

Shear bond strength of four commercial bonding systems to cp Ti.

OBJECTIVES: The purpose of this study was to evaluate the bond strength of veneering composite to commercially pure titanium (cp Ti) using several different bonding systems and a post-cure heat treatment. METHODS: Four commercial bonding systems (Cesead, Kuraray; New Metacolor, Sun Medical; Silicaoater MD, Kulzer; Termoresin LC II, GC) were evaluated. Bonding was attempted with the opaque resin provided by each bonding system as well as with the New Metacolor opaque resin. New Metacolor resin composite was used for the veneering composite. Half of the specimens were subjected to a post-cure heat treatment at 100 degrees C for 30 min. The shear bond strengths were tested after aging the specimens in water at 37 degrees C for 1 d and also after thermocycling for 16.5 d (20,000 cycles). RESULTS: Strong bonds, exceeding 20 MPa, were achieved with all of the bonding systems with the exception of Thermoresin LC II, which is designed for noble metals. Bond strengths were only increased by the post-cure heat treatment for the New Metacolor system. Thermocycling caused a significant reduction in bond strength for the New Metacolor adn the Thermoresin LC II systems. The use of the New Metacolor opaque resin produced increased bonding for the Silicoater MD and the opaque resin produced increased bonding for the Silicoater MD and the Cesead systems, but the effect was eliminated after thermocycling. SIGNIFICANCE: Strong, durable bonds can be achieved between composite and sandblasted cp Ti, thus enhancing the usefulness of this metal for esthetic resin-veneered crowns and other fixed prosthetics.

A 10 kb nucleotide sequence at the 5' flanking region (32 degrees) of srfAA of the Bacillus subtilis chromosome.

The nucleotide sequence of approximately 10 kb at the 5' flanking region (32 degrees) of srfAA of the Bacillus subtilis chromosome was determined. Eleven putative ORFs were identified. Three of them (orf6, orf7 and orf8) coincided with known B. subtilis genes (comJ, comI and tlpC) encoding a competence-specific protein, a DNA-entry nuclease and a transducer-like protein, respectively. The products of two other ORFs showed similarity to GlnP of Escherichia coli (orf1) and beta-glucosidase A of B. polymyxa (orf5).

Crystallization and preliminary X-ray diffraction studies on a recombinant isopenicillin N synthase from Cephalosporium acremonium.

Recombinant isopenicillin N synthase from Cephalosporium acremonium was expressed in Escherichia coli and the protein was purified. After nearly 5000 crystallization trials, the apo enzyme was crystallized by the hanging drop vapour diffusion technique, using polyethylene glycol and lithium sulphate as precipitants. Two crystal forms have been obtained with either octahedral or elongated prismatic habits. The larger octahedral crystals (0.1 mm over-all dimensions) belong to space group I4 with unit cell dimensions of a = b = 124.7 A, c = 156.9 A, and diffract X-rays to about 3.5 A resolution at synchrotrons. The crystallographic asymmetric unit contains a dimer.

[The effects of a novel Ca2+ channel blocker, KB-2796, on 5-HT-induced responses].

The effects of KB-2796, a new Ca(2+)-channel blocker, on 5-hydroxytryptamine (5-HT)-induced responses were investigated in comparison with those of other Ca(2+)-channel blockers such as verapamil, flunarizine, diltiazem and nimodipine. In rat cortical membrane, KB-2796 inhibited specific [3H]spiperone binding to 5-HT2 receptors in a competitive manner (Ki = 0.57 microM), but exhibited negligible affinity for radioligand binding to other 5-HT receptor subtypes such as 5-HT1, 5-HT1A, 5-HT1B, 5-HT1C and 5-HT3 at a concentration of 10 or 100 microM. KB-2796 inhibited both 5-HT-stimulated shape change and 5-HT and collagen-stimulated aggregation in rabbit platelet-rich plasma with IC50 values of 13.4 microM and 96.4 microM, respectively. KB-2796 also inhibited the 5-HT-induced increase of [Ca2+]i in washed rabbit platelets with the IC50 value of 25.7 microM. Furthermore, KB-2796 (3-30 mg/kg, p.o.) dose-dependently inhibited the 5-HT-induced paw edema in rats. In these experiments, the inhibitory effects of KB-2796 and other Ca2+ channel blockers were related to their affinities for the 5-HT2 receptor; and the potency of KB-2796 was stronger than those of diltiazem and nimodipine and almost equal to that of flunarizine, although all these inhibitors had weaker potencies than that of verapamil. These findings indicate that KB-2796 may possess antagonistic effect on the 5-HT2 receptor.

[Analysis of handwriting by the dynamic programming matching].

We examined the temporal invariance hypothesis in handwriting, using dynamic programming (DP) matching algorithm. The DP matching algorithm was originally developed for the speech recognition to eliminate fluctuations of time axis caused by different speech rates. This algorithm can determine the optimal point-to-point correspondences (warping function) between the standard velocity pattern and different patterns. We found that this algorithm worked well for various velocity patterns, even when their total writing time was different from the standard pattern. Only 30 to 50 percent of handwriting movements showed temporal invariance. The writing movements which didn't show temporal invariance consisted of several sub-motor units. The borders between the units were not limited near the end of each letter. The results suggest that handwriting of a word does not show the temporal invariance and that it is executed by some discrete sub-motor units.

Spinal cord infarction associated with primary antiphospholipid syndrome in a young child. Case report.

Antiphospholipid antibodies have been reported to occur in ischemic stroke patients, but there have been no previous reports linking these antibodies to spinal cord infarction. A case of spinal cord infarction associated with primary antiphospholipid syndrome in a 6-year-old boy is reported. Magnetic resonance imaging clearly demonstrated marked swelling of the thoracolumbar spinal cord with gadolinium-diethylenetriamine pentaacetic acid enhancement at an acute stage, followed later by cord atrophy. Serological study disclosed positive lupus anticoagulant and immunoglobulin G anticardiolipin antibody. It is suggested that the role of antiphospholipid antibodies as an etiological factor for spinal cord ischemia should be recognized among causes that might have been categorized as either spontaneous spinal cord infarction or myelitis.

X-ray absorption studies of the ferrous active site of isopenicillin N synthase and related model complexes.

Isopenicillin N synthase (IPNS) from Cephalosporium acremonium (M(r) 38,400) is an iron-containing enzyme that aerobically catalyzes the four-electron oxidative ring closure reactions of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV), forming the beta-lactam and thiazolidine rings of isopenicillin N. Here, we report Fe K-edge X-ray absorption studies that provide insight into the iron coordination environment and the effect of substrate and nitric oxide binding. Our analysis reveals an iron(II) coordination environment consisting of two N/O-containing ligands at 2.01 +/- 0.02 A, three N/O ligands at 2.15 +/- 0.02 A, and one C/O scatterer at approximately 2.6-2.7 A. Three His ligands are associated with the 2.15-A shell, while an unsymmetrically chelated carboxylate is associated with a scatterer at 2.01 and at 2.6-2.7 A, a combination which is consistent with the ligand environment deduced from 1H NMR studies [Ming, L.-J., Que, L., Jr., Kriauciunas, A., Frolik, C. A., & Chen, V. J. (1991) Biochemistry 30, 11653-11659]. The remaining scatterer at 2.01 A is assigned to a coordinated solvent molecule, most likely hydroxide, which can act as the proton acceptor for the incoming substrate. ACV binding to Fe(II)IPNS evinces an Fe-S interaction at 2.35 +/- 0.02 A, indicative of the coordination of substrate cysteine thiolate to the metal center. Analysis of the Fe(II)IPNS-ACV-NO data reveals one Fe-N at 1.71 +/- 0.02 A, three Fe-(N,O) at 2.04 +/- 0.02 A, one Fe-S at 2.32 +/- 0.02 A, and one Fe-(C,O) at 2.61 +/- 0.02 A, the short Fe-N bond being derived from the binding of NO. Our EXAFS conclusions, supported by corresponding analysis of relevant model complexes, corroborate and refine the working model for the Fe(II) coordination environment developed from previous spectroscopic studies.