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We present two patients (case 1: a 78-year-old right-handed woman, case 2: a 66-year-old right-handed woman) with suspected transient global amnesia. Both patients showed sudden onset amnesia that appeared to resolve within 24 hours, and they showed no abnormal findings on electroencephalography or single photon emission tomography. However, the results of the Wechsler Memory Scale-Revised (WMS-R) on the ninth (case2) or tenth day (case1) after the onset indicated memory impairment. And diffusion-weighted magnetic resonance imaging (DWI) revealed a unilateral high-intensity area in the hippocampus, leading to the diagnosis of hippocampal infarction in both patients. Case 1 had a left hippocampal lesion and exhibited impairment of predominantly verbal memory, while case 2 had a right hippocampal lesion and demonstrated impairment of predominantly visual memory. When evaluating a patient with suspected transient global amnesia, imaging of the hippocampus and tests on memory function after an appropriate interval from the onset are often helpful in establishing a correct diagnosis.

Stinky tofu is a kind of fermented tofu with a strong odor. Although stinky tofu is a very popular snack in the Asian region, the community of microbes, and especially lactic acid bacteria (LAB), indigenous to the fermented brine from which it is made remains poorly described. We examined 168 isolates obtained from the original fermented brine (brine A) and two brines in which the hard tofu (brine B) and soft tofu (brine C) had been soaked. Through random amplified polymorphic DNA (RAPD) analysis for typing and 16S rDNA sequencing, 136 representative strains were identified as belonging to 7 genera and 32 species: Enterococcus (2 species), Lactobacillus (14 species), Lactococcus (3 species), Leuconostoc (6 species), Pediococcus (1 species), Streptococcus (2 species), and Weissella (4 species). The LAB composition of brine A was the most diverse: 19 different species were isolated, and 9 of them were classified as Lactobacillus species. The 16S rDNA sequences of 9 strains (6 from brine A and 3 from brine C) showed low values of similarity (below 98%) with currently known species by analysis using the FASTA software. Thus, a wide variety of LAB strains were associated with the fermentation of stinky tofu brines.

Vascular endothelial growth factor A (VEGF-A) has two kinds of isoforms depending on cellular binding domains. VEGF189 is the largest molecule with the strongest cellular binding ability, and is thought to be most potent for vascularization in various cancers. This study aims to clear the clinicopathological characteristics of VEGF189 in the pulmonary adenocarcinoma. We finely and quantitatively examined the expression of VEGF-A isoforms (VEGF121, VEGF165 and VEGF189) by real-time polymerase chain reaction in a total of 100 pulmonary adenocarcinomas resected by surgical operation. The VEGF isoform expression status was analyzed on clinicopathological features including stromal vascularization, vascular involvement, distant metastasis, lymph nodal metastasis, postoperative relapse time and prognosis of long-term observation periods. All the pulmonary adenocarcinomas showed significant expression of VEGF-A. Twenty-two cases with the adenocarcinomas overexpressing VEGF-A significantly showed earlier postoperative relapse and poorer prognosis between 5- to 15-year periods (p = 0.0093 and p = 0.0240, Kaplan Meier, log-rank test). The expression levels of VEGF189 increased in 13% of the pulmonary adenocarcinoma. These 13 cases with increased VEGF189 expression significantly showed higher distant metastases, earlier postoperative relapse, and poorer prognosis (p = 0.0006, Fisher's test; p = 0.0016 and p = 0.0084, Kaplan Meier, log-rank test) than the other 87 cases. The 13 lung cancers with VEGF189 overexpression also showed increased vessel counts, areas (p = 0.0091 and p < 0.0001, Mann-Whitney U test) and enhanced venous involvement (p = 0.0056, Fisher's test). The cellular binding isoform VEGF189 confers pulmonary adenocarcinoma patients with poorer prognosis with distant metastasis via blood flow.

Three lipid-based delivery systems (AmBisome, Amphocil, and Abelcet) for amphotericin B (AmB) have been marketed to overcome the disadvantages associated with the clinical use of AmB. However, to show their efficacy, they need to be administered at higher doses than the conventional dosage form, Fungizone. In this study, we compared LNS-AmB, our new low-dose therapeutic system for AmB using lipid nano-sphere (LNS), with these commercial formulations in terms of their pharmacokinetics and efficacy. The plasma AmB levels yielded by LNS-AmB after intravenous administration to rats were much higher than those yielded by Amphocil or Abelcet, and similar to those yielded by AmBisome, but in dogs LNS-AmB yielded plasma AmB concentrations about three times higher than did AmBisome. In a carrageenin-induced pleurisy model in rats, LNS-AmB yielded AmB levels in the pleural exudate over 20 times those yielded by Amphocil or Abelcet, and similar to those yielded by AmBisome. From these pharmacokinetic results, it is clear that Amphocil and Abelcet are based on a quite distinct drug-delivery concept from LNS-AmB. In a rat model of localized candidiasis, LNS-AmB significantly inhibited the growth of Candida albicans in the pouch, whereas AmBisome did not, even though the AmB concentrations in the pouch were similar. This difference in antifungal activity between LNS-AmB and AmBisome was also found in vitro. That is, the antifungal activity of LNS-AmB against C. albicans was similar to that of Fungizone and dimethyl sulfoxide-solubilized AmB, while AmBisome showed weaker antifungal activity than did other formulations. Based on these results, the release of AmB from AmBisome was judged to be slow and slight. In a mouse model of systemic candidiasis, LNS-AmB (1.0mg/kg) was much more effective than AmBisome (8.0mg/kg) or Fungizone (1.0mg/kg). These results suggest that LNS-AmB maintained the potent activity of AmB against fungal cells even though the AmB was incorporated into LNS particles. We conclude that LNS-AmB may offer an improved therapeutic profile at lower doses than Fungizone and commercial lipid-based formulations.

It is unclear whether a subunit of glutamate cysteine ligase [a modifier subunit (GCLM) and a catalytic subunit (GCLC)] is an effective target for ameliorating cisplatin (CDDP)-resistance. We inhibited each subunit of GCL mRNA using a specific ribozyme (M-Rz and C-Rz) in the pulmonary adenocarcinoma cell line A549. GCL activity was suppressed by the ribozyme. CDDP-resistance was more effectively ameliorated when GCLM rather than GCLC was inhibited. GCLM is a potentially more effective pharmacologic target for ameliorating CDDP-resistance in non-small cell lung cancer than GCLC.

To develop a low-dose therapeutic system for amphotericin B (AmB), the efficacy and toxicity of lipid nano-sphere (LNS) incorporating AmB (LNS-AmB) were evaluated and compared with those of Fungizone, the conventional dosage form of AmB with sodium deoxycholate. LNS-AmB and Fungizone showed nearly equal activity against fungal cells both in vitro and in vivo. In contrast to Fungizone, however, LNS-AmB did not cause significant hemolysis. In addition, the vomiting toxicity of Fungizone was largely avoided by the use of LNS-AmB in dogs, in spite of the higher plasma AmB concentrations achieved by LNS-AmB. Therefore, LNS-AmB may be selective for fungal cells over mammalian cells. In a study of its toxicity and toxicokinetics in a regimen of daily 2-h intravenous infusions for 14 consecutive days, LNS-AmB showed less toxicity to the kidney than did Fungizone in spite of the higher plasma AmB concentrations achieved. LNS-AmB, therefore, allows the treatment of systemic fungal infections at low doses without the severe nephrotoxicity of Fungizone. Size-exclusion chromatography provided evidence that, when LNS-AmB was administered to rats, AmB was retained in the LNS particles in the blood circulation, but that when Fungizone was administered, AmB was transferred to high-density lipoproteins (HDL). AmB retained in LNS particles seemed to be less toxic to the kidney than was AmB associated with HDL. Consequently, LNS-AmB has the potential to become a low-dose therapeutic system for AmB, minimizing most of the severe side effects of AmB by decreasing the total dose required.

Thrombospondin 2 (TSP2) is an extracellular matrix glycoprotein involved in tumor progression and angiogenesis. We evaluated whether overexpression of the TSP2 gene show an alteration of various genes by cDNA arrays in the colon carcinoma cell line SW480. The transformants with the human TSP2 gene overexpression showed a down-regulation of matrix metalloproteinase 2 (MMP2) and MMP9 in comparison to those with vector-control. Protein production of MMP2 and MMP9 decreased in the transformants overexpressing the TSP2 gene. Conversely, the SW480 transformants showed up-regulation of MMP12 and MMP17. These results suggested that the TSP2 gene is a multifunctional modulator of remodeling tissue in which matrix degradation is required.

The 189 amino acid isoform of vascular endothelial growth factor (VEGF189) has been shown to be more strongly associated with the cell membrane than other isoforms of human VEGF (VEGF121, VEGF165). To analyze the biological activities of these VEGF isoforms on tumor growth, we transfected human VEGF121, VEGF165 or VEGF189 cDNA into the human colon cancer cell line SW-480, and established several clones overexpressing these VEGF isoforms. The total amounts of VEGF protein in the culture supernatants of the VEGF189-transfectants were less than those of VEGF121 and VEGF165-transfectants. These transfectants showed no significant differences in growth in culture. Nevertheless, the rate of in vivo tumor growth of VEGF189-transfectants was faster than or equivalent to that of VEGF121-transfectants, while the VEGF165-transfectant showed the greatest enhancement of tumor growth. The protein levels of VEGF were markedly increased only in the VEGF189-transfectants cultured in the presence of heparin. The enhanced in vivo tumor growth of VEGF189-transfectants can be partly explained by the cell-associated features of VEGF189 molecules. The VEGF189 molecule, which is strongly bound to the cell surface, has unique properties and high potential in local angiogenesis and tumor growth in the cancer inductive microenvironment.

The levels of expression of various genes were altered in cellular transformants with manipulation of expression of single genes. Vascular endothelial growth factor A (VEGF-A) is a key molecule for tumor progression, although it is unclear how VEGF-A expression regulates various genes. Multiple gene expression levels were evaluated using cDNA arrays in a human hepatocellular carcinoma cell line (HLF) with suppression of the VEGF-A gene by anti-VEGF-A ribozyme (alphaVRz). The ribozyme-mediated suppression of VEGF-A gene solely up-regulated matrix metalloproteinase 1 (MMP1) gene level in HLF/alphaVRz. Levels of expression of other members of MMP family or tissue inhibitors of MMPs did not show any alteration. These results suggested that intracellular suppression of VEGF-A gene was specifically linked to up-regulation of MMP1 in human hepatocellular carcinoma cells.

This study was performed to characterize human thrombospondin 2 (TSP2). TSP2 has recently attracted attention as an endogenous negative regulator of angiogenesis in tumorigenesis. We cloned and transfected human TSP2 cDNA into the human colon cancer cell line SW-480. Stable transfectants (TSP2-1, TSP2-6) overexpressing TSP2 were established. Growth characteristics of TSP2-transfectants were investigated in vitro and in vivo. TSP2-transfectants showed similar growth properties to vector-transfectants and wild-type SW-480 cells. The overexpression of transfected human TSP2 cDNA did not affect proliferation of SW-480 cells. When the conditioned media of TSP2-transfectants were added to cultures of bovine pulmonary microvascular endothelial cells (BPMEC), the BPMEC proliferation was significantly inhibited. These results suggested that human TSP2 is a potential inhibitor of angiogenesis.

In recent years, a Drug Delivery System (DDS), a preparative approach attracts the attention in the development of new drugs. DDS focuses on the regulation of the in vivo dynamics, such as absorption, distribution, metabolism, and elimination, thereby improving the effectiveness and the safety of the drugs by an applicable use of drug preparation technologies. A conventional intravenous dosage form of Amphotericin B (AmB), Fungizone, is the most effective clinically available for treating fungal infections. However, the clinical efficacy of AmB is limited by its adverse effects. Several lipid formulations, such as Liposomal AmB (L-AmB), AmB lipid complex (ABLC), and AmB colloidal dispersion (ABCD), with reduced side effects have been developed. These formulations are reported to have excellent safety and efficacy. However, comparable efficacy can be achieved only when they are administered at high doses than AmB. One of the problems of using these formulations is that they are easily taken up by the reticuloendothelial system (RES). An artificial lipoprotein-like particles, a novel drug carrier Lipid Nano-Sphere (LNS), which is 25 - 50 nm in size and is composed of phospholipids and simple lipid. LNS show a higher plasma concentration of drugs and lower uptake by RES-tissue different forms other lipid base drug carriers. In vitro and in vivo, LNS incorporating AmB, NS-718, shows reduced toxicity, while maintaining activity against fungi. LNS have a unique characteristic as an effective carrier of AmB for treatment of fungal infection.

Interleukin 10 (IL-10) is an immuno-suppressive cytokine produced by T-lymphocytes, and a regulatory molecule for angiogenesis in various cancers. We examined IL-10 gene expression in 53 colon cancer patients who underwent surgical resection. IL-10 gene expression was correlated with TSP1 and TSP2 gene expression (P=0.0049, P=0.0285). Colon cancer with IL-10 gene expression (19/53) showed significantly decreased venous involvement (P=0.0433). The mean vessel counts in the colon cancers with IL-10 gene expression were significantly lower than those without IL-10 gene expression (P<0.001). These results suggested that IL-10 stimulates angiostatic factor gene expression, and results in suppression of venous involvement.

A series of 7-substituted-6-fluoro-1-fluoromethyl-4-oxo-4H- [1,3]thiazeto[3,2-a]quinoline-3-carboxylic acid derivatives (2a-1) was prepared and evaluated for antibacterial activity. These compounds were obtained by deacylation of 4-benzoyloxy-2-(1-chloro-2-fluoroethyl)thio-6,7- difluoroquinoline-3-carboxylate (10) and subsequent intramolecular cyclization followed by substitution with cyclic amines and then hydrolysis. The intramolecular cyclization reaction of 18, one of the diastereomers (17, 18) revealed that the cyclization reaction proceeded through an inversion to afford (-)-11a in good chemical and optical yield. The enantiomers of 2a were prepared from the enantiomers of 11a, which were obtained by the optical resolution of the racemate using high-performance liquid chromatography (HPLC). Compounds 2a,b showed excellent in vitro and in vivo antibacterial activity against both gram-negative and gram-positive bacteria including quinolone and Methicillin-resistant Staphylococcus aureus.

We evaluated the in vitro and in vivo potencies of a new lipid nanosphere that incorporates amphotericin B (AmB), NS-718, against Aspergillus fumigatus. The in vitro activity of NS-718 (the MIC at which 90% of strains are inhibited [MIC90], 0.25 microgram/ml) against 18 isolates of A. fumigatus was similar to that of deoxycholate AmB (D-AmB; Fungizone; MIC90, 0.25 microgram/ml), but NS-718 was more potent than liposomal AmB (L-AmB; AmBi-some; MIC90, 1.0 microgram/ml). The in vivo efficacy of NS-718 in a rat model of invasive pulmonary aspergillosis was compared with those of D-AmB and L-AmB. A low dose (1 mg/kg of body weight) of L-AmB was ineffective (survival rate, 0%), although equivalent doses of D-AmB and NS-718 were more effective (survival rate, 17%). However, a higher dose of NS-718 (3 mg/kg) was more effective (survival rate, 100%) than equivalent doses of D-AmB and L-AmB (survival rate, 0%). To explain these differences, pharmacokinetic studies showed higher concentrations of AmB in the plasma of rats treated with NS-718 than in the plasma of those treated with D-AmB. Our results suggest that NS-718, a new preparation of AmB, is a promising antifungal agent with activity against pulmonary aspergillosis.

NM394 (6-fluoro-1-methyl-4-oxo-7-(1-piperazinyl)-4H-[1,3]-thiazeto [3,2-a]quinoline-3-carboxylic acid) has potent, broad-spectrum antibacterial activity in vitro, but not in vivo. To increase the bioavailability of NM394, various prodrugs were synthesized and tested. One of them, NM441, an N-(5-methyl-2-oxo-1,3-dioxol-4-yl) derivative, showed potent in vivo antibacterial activity. Using thin-layer chromatography-bioautography, we confirmed that after oral administration, NM441 was readily absorbed and hydrolyzed to NM394. Other prodrugs of NM394 were only partially metabolized to NM394. In pharmacokinetic studies in mice and monkeys, we found that the blood levels of NM394 were 7.8 and 5.9 times greater, respectively, when NM441 rather than NM394 was administered. These findings suggest that NM441 is an effective prodrug of NM394.

The in vitro antibacterial activity of prulifloxacin (CAS 123447-62-1, NM441), a new quinoline prodrug, against clinical isolates from urinary tract infections was investigated. In addition, it was compared with ofloxacin (CAS 82419-36-1), levofloxacin (CAS 100986-85-4), ciprofloxacin urinary tract infections in mice, as well as its pharmacokinetics. 1. The antibacterial activity of NM394 (6-fluoro-1-methyl-4-oxo-7-(1-piperazinyl)-4H-[1,3]thiazeto[3,2- a]quinoline-3-carboxylic acid), an active metabolite of prulifloxacin, against gram-positive clinical isolates was inferior to that of levofloxacin and tosufloxacin, and equal to that of ofloxacin and ciprofloxacin. Against gram-negative clinical isolates, the activity of NM394 was superior to that of the reference drugs. 2. The therapeutic effect of prulifloxacin on experimental urinary tract infection with Escherichia coli in mice was equal to that of tosufloxacin and ciprofloxacin and superior to that of ofloxacin and levofloxacin. Its therapeutic effect on Pseudomonas aeruginosa infection was equal to that of tosufloxacin and ciprofloxacin, and superior to that of ofloxacin. Against urinary tract infection with olfloxacin-resistant Enterobacter cloacae, prulifloxacin was the most effective of all the drugs tested. 3. The maximal serum concentration of prulifloxacin was slightly higher than that of ciprofloxacin, and the area under the curve (AUC) for prulifloxacin was 1/4 that of ofloxacin, levofloxacin and tosufloxacin. The maximal concentration and AUC of prulifloxacin in lung and kidney were slightly higher than the corresponding values for ciprofloxacin but only 1/2 to 1/4 of the values for ofloxacin, levofloxacin and tosufloxacin. In conclusion, prulifloxacin (NM394) showed potent antibacterial activity against clinical isolates and potent therapeutic efficacy against experimental infection in spite of its lower AUCs compared with the reference drugs. These findings suggest that prulifloxacin may be a useful drug in the treatment of urinary tract infections.

In the course of our search for derivatives with potent antibacterial activity and low cytotoxicity, we have studied the relationships among the structure, antibacterial activity and cytotoxicity of thiazoloquinolone and thiazetoquinolone derivatives (the term quinolone as used in this paper includes the quinolone, 1,8-naphthyridine and pyridopyrimidine nuclei). The antibacterial activities and cytotoxicity of these derivatives were compared with those of norfloxacin, ofloxacin, enoxacin and ciprofloxacin. The antibacterial activities of the thiazoloquinolone derivatives were more potent than those of the dihyrothiazoloquinolone derivatives, and comparable to that of ciprofloxacin. All of the thiazoloquinolone derivatives were highly cytotoxic against mammalian cells, but some of the dihyrothiazoloquinolone derivatives were less cytotoxic, being comparable in cytotoxicity to the reference drugs. The thiazetoquinolone derivatives were less cytotoxic than the thiazoloquinolone derivatives, and one of them, 6-fluoro-1-methyl-4-oxo-7-(1-piperazinyl)-4H-[1,3]thiazeto[3,2-a] quinoline-3-carboxylic acid, showed the most potent antibacterial activity of all compounds tested in this study, as well as a very low cytotoxicity. The antibacterial activity and cytotoxicity of this compound were similar to that of ciprofloxacin.

Optically active isomers of 6-fluoro-1-methyl-4-oxo-7-(1-piperazinyl)-4H- [1,3] thiazeto [3,2-alpha] quinoline-3- carboxylic acid (NM394, 3) were prepared through optical resolution of their racemic intermediate ( +/- )-1 by high-performance liquid chromatography (HPLC). The absolute configuration at the C-1 position in the thiazeto-quinolone ring of ( - )-3 was confirmed by X-ray analysis ( - )-4 to be S. The in vitro antibacterial activity of ( - )-3 was 2--8 times that of (+)-3.

A series of 1,8-disubstituted 6-fluoro-4-oxo-7-piperazinyl-4H-[1,3]thiazeto[3,2-a]quinoline-3- carboxylic acid derivatives was prepared and evaluated for antibacterial activity. In the 7-piperazinyl series, addition of a fluorine at C-8, which increased the in vitro activity for the 1-hydrogen and 1-methyl analogues and decreased it for the 1-phenyl analogue, improved the in vivo activity of all the analogues. Introduction of a methoxy group at C-8 of the 1-methyl-7-piperazinyl analogue also improved its in vivo antibacterial activity. The effect of 8-substituents on the in vitro and in vivo antibacterial activity of the 1-methyl-7-(4-methyl-1-piperazinyl) series is also discussed.

A series of [1,3]thiazeto[3,2-a]quinoline-3-carboxylic acids and their esters were prepared and evaluated for antibacterial activity. The derivatives with a hydrogen or methyl group at C-1, fluorine at C-6, and piperazinyl or 4-methyl-1-piperazinyl group at C-7 showed superior in vitro antibacterial activity, and the derivatives with 4-methyl-1-piperazinyl group at C-7 had potent in vivo activity. Compound 29a (NM394) showed excellent in vitro antibacterial activity and low toxicity but poor absorption from the gastrointestinal tract. Compound 29ee (NM441), an N-[(5-methyl-2-oxo-1,3-dioxol-4-yl)methyl] derivative of 29a, was found to possess a favorable pharmacokinetic profile and oral activity superior to that of ciprofloxacin in experimental animals.

To prevent postoperative dysuria, which occurs inevitably after the radical hysterectomy, several surgical procedures have been tried. The principal method is to suture adnexal flaps to the bladder trigone and fundus. Of four procedures so far tried sequentially, the best one was to fix the bladder trigone and to support it with round ligament flaps sutured with the bladder fundus covered by tubal flaps (Type IV). In follow-up studies, Type IV proved more preferable than the other three types or the non-sutured control group in regard to the following aspects: (1) days necessary for the disappearance of residual urine, (2) appearance of urinary sensation, (3) acquirement of urinary sensation, (4) incidence of urinary incontinence, (5) residual urine/bladder capacity ratio, (6) cystometric findings, and others. Effectiveness of our procedures, particularly of Type IV, may be ascribed to the supported bladder trigone and fundus by sutured tissues and to the acquirement of urinary sensation, rather than to the restoration of nervous contact between the bladder and the micturition center in the spinal cord.