Delayed administration of JTP-2942, a novel thyrotropin-releasing hormone analogue, improves cerebral blood flow and metabolism in rat postischaemic brain.

1. The aim of the present study was to examine the central nervous system action of JTP-2942, a novel thyrotropin-releasing hormone (TRH) analogue, from the point of view of cerebral blood flow (CBF) and metabolism in the postischaemic brain. 2. Left middle cerebral artery ischaemia was induced for 90 min followed by reperfusion. 3. Animals were separated into four groups: (i) low-dose (0.003 mg/kg) JTP-2942; (ii) high-dose (0.03 mg/kg) JTP-2942; (iii) cystidine diphosphate choline (500 mg/kg); and (iv) saline. The test drug or saline was administered intravenously 1 week after ischaemia. 4. Local CBF and local cerebral glucose utilization were measured autoradiographically, adjacent sections were stained with haematoxylin-eosin and infarction size was measured. 5. JTP-2942 ameliorated the reduction of local CBF and glucose utilization except in the ischaemic core. In particular, the higher dose (0.03 mg/kg) of JTP-2942 significantly increased local CBF and glucose utilization not only in peri-infarcted areas, but also in distal and contralateral areas. 6. These results suggest that JTP-2942 treatment may be beneficial for improving cerebral circulation and metabolism in the postischaemic brain.

Visualization of the motor activation area using SPECT in neurosurgical patients with lesions near the central sulcus.

UNLABELLED: The purpose of this study was to visualize the motor area related to finger movement and a fist-making task using SPECT in patients with lesions near the central sulcus. METHODS: Eleven patients (9 with a brain tumor, 1 with cerebral infarction, and 1 with an arteriovenous malformation) were investigated. The first intravenous injection of 99mTc-ethyl cysteinate dimer (ECD) for the motor activation SPECT images was administered 2 min after completion of the fist-making task with the hand contralateral to the brain lesion. The movement was stopped 2 min after injection, and activation SPECT was performed. After the scan, the second dose of 99mTc-ECD was injected into resting patients, and a second set of SPECT images was acquired. The first set of images was subtracted from the second set to obtain control images. Regions of interest were set bilaterally on the sensorimotor hand area; the supplementary motor area; the frontal, temporal, and occipital lobes; and the cerebellar hemispheres. The results of activation SPECT were expressed as positive or negative for a high-count area, and the regional percentage change for activation images relative to resting images was calculated. RESULTS: Visual assessment of activation images was positive in 9 patients for the sensorimotor hand area and 7 patients for the supplementary motor area. The regional percentage change between activation and resting images for the high-count areas was 19.7% for the sensorimotor hand area and 18.2% for the supplementary motor area. Both values were significantly higher than those for other areas (P<0.05). CONCLUSION: Motor activation SPECT using a 99mTc-ECD split-dose method is easy to perform and may be helpful for presurgical visualization and identification of the sensorimotor hand area or the supplementary motor area.

A comparative study of Embelia schiperi and Embelia keniensis on blood glucose and triglycerides in normal and epinephrine-induced hyperglycemic mice.

Intraperitoneal administration of the methanol extract of Embelia schiperi (ES) to normal mice caused a significant decrease in blood glucose (p < 0.01) and a significant increase in triglycerides 4 hours after administration at 100 mg/kg (p < 0.01). The toluene fraction of Embelia keniensis methanol extract (TS) showed hypoglycemic and lipid lowering activity 7 hours after intraperitoneal administration at 100 mg/kg. In addition, TS (100 mg/kg) administration significantly decreased blood glucose in epinephrine-induced hyperglycemic mice (p < 0.01). Moreover, ES tended to increase while TS tended to decrease the blood triglycerides in epinephrine-induced hyperglycemic mice. On the other hand, no changes in blood cholesterol were observed after the administration of ES or TS in normal and epinephrine-induced hyperglycemic mice. We found that two species from Embelia, ES and TS, have different activities on blood glucose and triglycerides in normal and epinephrine-induced hyperglycemic mice.

High-efficiency cloning of Arabidopsis full-length cDNA by biotinylated CAP trapper.

Full-length cDNAs are essential for functional analysis of plant genes. We constructed high-content, full-length cDNA libraries from Arabidopsis thaliana plants based on chemical introduction of a biotin group into the diol residue of the CAP structure of eukaryotic mRNA, followed by RNase I treatment, to select full-length cDNA. More than 90% of the total clones obtained were of full length; recombinant clones were obtained with high efficiency (2.2 x 10(6)/9 micrograms starting mRNA). Sequence analysis of 111 randomly picked clones indicated that 32 isolated cDNA groups were derived from novel genes in the A. thaliana genome.

Overexpression, purification and helix-destabilizing properties of Epstein-Barr virus ssDNA-binding protein.

The Epstein-Barr virus (EBV) ssDNA-binding protein (SSB) encoded by the BALF2 gene is one of the essential replication proteins in the lytic phase of EBV DNA replication. In order to obtain the amount of EBV SSB required for characterization, a recombinant baculovirus containing the complete sequence of the BALF2 open reading frame under the control of the baculovirus polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced a protein of 130 kDa, recognized by anti-BALF2 protein-specific polyclonal antibody. The overexpressed EBV SSB was purified homogeneously from the cytosolic fraction of the recombinant virus-infected cells. The purified protein displaced short DNA strands from their complementary sequences in the single-stranded form of M13. The helix-destabilizing activity was neutralized by the anti-BALF2 protein-specific antibody. Maximum unwinding occurred at EBV SSB concentrations exceeding saturation level of the DNA substrate. The DNA unwinding reaction mediated by the EBV SSB was highly cooperative and extremely rapid. The reaction displayed no directionality and required neither ATP nor MgCl2, two essential cofactors for DNA helicase activity. The helix-destabilizing property of the EBV SSB may function to melt out secondary structures on the ssDNA template, thereby facilitating the movement of the EBV DNA polymerase.

Minor Flavanones from Erythrina abyssinica

Four new prenylated flavanones, abyssinone-V 4'-methyl ether (1) and abyssinoflavanones IV (2), V (3), and VI (4), have been isolated as minor flavanones from the African medicinal plant, Erythrina abyssinica, together with a known flavanone, sigmoidin D. The structure elucidation of compounds 1-4 by spectroscopic studies is described.

Antisporulating and respiration-inhibitory effects of essential oils on filamentous fungi.

Sporulation of four species of filamentous fungi, namely Aspergillus fumigatus, Fusarium solani, Penicillium expansum and Rhizopus oryzae, was suppressed by gaseous contact with citron, lavender and thyme oils and, to a lesser extent, by that of perilla and tea tree oils. Lemongrass and cinnamon bark oils were scarcely active. The antisporulating effect of the essential oils was not observed when they were applied as a solution, indicating that their vapours were the active form. Moreover, exposure of fungal cultures to vapours of the active essential oils caused curling of the tips of aerial hyphae (R. oryzae) or incomplete development of conidiophores (A. fumigatus). These antisporulating effects of the vapourizing essential oils seemed to be correlated with their respiration-inhibitory activity, rather than with their growth-inhibitory activity.

Hypoglycemic activity of some triterpenoid glycosides.

Four triterpenoid glycosides isolated from the rhizomes of Polygala senega var. latifolia, senegins II-IV (1-3) and desmethoxysenegin II (4), were tested for hypoglycemic activity in normal and KK-Ay mice. Compounds 1 and 2 reduced the blood glucose of normal mice 4 h after intraperitoneal administration and also significantly lowered the glucose level of KK-Ay mice under similar conditions. Compounds 3 and 4, as well as senegose A (5), an oligosaccharide ester, were inactive when tested against normal mice.

Overexpression of integrin-associated protein (CD47) in rat kidney treated with a renal carcinogen, ferric nitrilotriacetate.

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces renal proximal tubular necrosis, a consequence of free radical-associated damage, that ultimately leads to a polycystic change of the renal cortex and a high incidence of renal cell carcinoma (RCC) in rodents. The differential display technique was used to search for inducible genes in the kidney of male Wistar rats treated with Fe-NTA and in the induced RCCs. Six fragments were selected that showed specific quantitative changes in mRNA. Two of them exhibited similar patterns in northern blots as well. One fragment showed a high homology (89%) to murine integrin-associated protein (IAP; CD47). We thus cloned rat IAP cDNA including the entire coding region for use in further analysis. Rat IAP cDNA showed a 21-amino-acid deletion that was also observed in human, but not in mouse. Northern blots revealed that IAP was consistently overexpressed in non-tumorous parts of the kidney (2.4-fold increase, n = 9, P < 0.0001) as compared with matched controls 1 to 2 years after Fe-NTA treatment. IAP overexpression of more than 2.9-fold was found in 25% (2/8) of RCCs studied, and was limited to cases of a high histological grade and lung metastasis. Unexpectedly, IAP expression was higher in the non-tumorous part of the kidney after Fe-NTA treatment (2.8-fold) than in RCC (1.5-fold) in each case (n = 4, P < 0.05). Abundant expression of IAP mRNA in the renal tubular epithelium after Fe-NTA treatment and RCC cells was observed by in situ hybridization. The results suggest that IAP overexpression may be associated with Fe-NTA-induced renal cortical tubular damage and regeneration that lead to a polycystic state, and with tumor progression and metastasis of the induced RCCs.

High efficiency selection of full-length cDNA by improved biotinylated cap trapper.

We report here an improved protocol for the preparation of full-length cDNA libraries that improves the previously reported method (Carninci, P., Kvam, K., Kitamura, A. et al. 1996, Genomics, 137, 327-336), that allows long cDNAs to be cloned more efficiently. One potential disadvantage of the original biotinylated CAP trapper protocol is the exposure of mRNA to chemical and enzymatic attacks during the biotinylation of the cap structure, before the first-strand cDNA synthesis (and selection of full-length cDNA by biotinylated cap). Here, we show that the biotinylation of the cap structure is very specific and effective even if biotinylation is performed on the mRNA/cDNA hybrid produced by the first-strand cDNA synthesis reaction. Consequently, mRNA remains protected from chemical and enzymatic degradation during the overnight biotinylation step, thus making it possible to select full-length cDNAs of longer average size. We herein report the efficiency and specificity of the new version of the protocol for cap structure biotinylation and capture of full-length cDNA.

Cardiotonic activity of the rhizome of Polygonatum sibiricum in rats.

The cardiotonic effect of the rhizome of Polygonatum sibiricum was investigated in the left atria of rats. The methanol extract of the rhizome of Polygonatum sibiricum (OM) (1-7 mg/ml) concentration-dependently increased the developed tension of the left atrium. It also strongly inhibited cAMP phosphodiesterase. The increase cAMP level correlated the increase in left atrial contraction. On the other hand, OM did not inhibit Na+, K+-ATPase. The cardiotonic effect of OM was strongly inhibited by reserpine, a sympatholytic agent. Furthermore, OM-treated left atria inhibited the tension produced by propranolol, a beta adrenocepter antagonist. These findings suggested that the cardiotonic effect is due to stimulating beta adrenoceptors through activation of sympathetic nerves.

Hypoglycemic effect of the rhizomes of Polygala senega in normal and diabetic mice and its main component, the triterpenoid glycoside senegin-II.

The hypoglycemic effect of the rhizomes of Polygala senega L. var. latifolia Torrey et Gray (Polygalaceae) was investigated in normal and KK-Ay mice, one of the model animals of non-insulin dependent diabetes mellitus (NIDDM). The n-butanol extract of senega rhizomes (SN) (5 mg/kg) reduced the blood glucose of normal mice from 191 +/- 3 to 120 +/- 3 mg/dl 4 hours after intraperitoneal administration (P < 0.001), and also showed a significant decrease in the glucose level of KK-Ay mice from 469 +/- 38 to 244 +/- 14 mg/dl under similar conditions (P < 0.001). But streptozotocin-induced diabetic mice did not experience a change in the blood glucose after administration of SN. We propose that the hypoglycemic effect of SN occurs without altering the insulin concentration. Moreover, SN needs the presence of insulin in order to act. In addition, one of the active components of the hypoglycemic effect was identified as a triterpenoid glycoside, senegin-II.

Intraperitoneal administration of Senegae Radix extract and its main component, senegin-II, affects lipid metabolism in normal and hyperlipidemic mice.

The hypolipidemic effect of Senegae Radix, i.e. "Senega," was investigated in normal and hyperlipidemic mice. The n-butanol fraction of the methanol extract of Senegae Radix (SN) (5 mg/kg) significantly reduced the blood triglyceride level of normal mice 7 h after intraperitoneal administration (p < 0.05) and also significantly reduced the blood triglyceride level of cholesterol-fed mice under similar conditions (p < 0.05). SN (5 mg/kg) also reduced the blood triglyceride and cholesterol levels after repeated administration to cholesterol-fed mice. SN also decreased the blood triglyceride level in Triton-induced hyperlipidemia. It is suggested that this fraction contains one or more hypolipidemic components including the main triterpenoid glycoside, senegin-II, which significantly reduces the levels of blood triglycerides in normal mice.

Hypoglycemic activity and structure-activity relationship of iridoidal glycosides.

Four iridoidal glycosides, deacetylasperulosidic acid methyl ester (DE), scandoside methyl ester (SC), geniposide (GE) and gardenoside (GA) isolated from Gardenia jasminoides Ellis grandiflora Makino leaves, were tested hypoglycemic activity in mice. DE lowered the blood glucose level in normal mice. However, SC, GE and GA did not affect the blood glucose level in normal mice, indicating that the absolute configuration of 6 position hydroxy based (OH) should be essential for the biological activity.

Mitogenic activities in African traditional herbal medicines (Part II).

Mitogenic activities in African traditional herbal medicines were examined on human peripheral blood lymphocytes and mouse spleen cells using protein fractions obtained from their extracts by precipitation with ammonium sulfate. Target specificity for these mitogens was investigated by using isolated T cells and lymphocytes from athymic nude mice. Among 20 plants investigated, potent mitogenic activities for both human and mouse lymphocytes were found in 7 plants: Monanthotaxis sp. (Annonaceae), Uvaria lucida (Annonaceae), Maytenus buchananii (Celastraceae), Lonchocarpus bussei (Leguminosae), Phytolacca dodecandra (Phytolaccaceae), Phytolacca octandra (Phytolaccaceae), and Toddalia asiatica (Rutaceae). The U. lucida stem demonstrated the highest activity among all and induced mitogenesis both in human and mouse isolated T cells, but not in lymphocytes from athymic nude mice.

[Experimental investigation of 201TlCl-scintigraphy in evaluating therapeutic effect of hyperthermia].

We investigated the usefulness of 201Tl-scintigraphy in the evaluation of therapeutic effect of hyperthermia. Satoh's experimental lung cancer tumors implanted in the right thighs of donryu rats were treated with hyperthermia using water bath. Tumors were heated at 44 degrees C or 46 degrees C for 15 min. 201Tl-scintigraphy was obtained before, immediately after and 24 hrs after treatment. Counts ratio of the tumor to the normal muscle (T/N ratio) was measured by gamma camera. We examined blood flow of the tumor, and relative tumor growth rate, and performed autoradiogram and histopathological examination of the tumor after 201Tl-scintigraphy. Immediately after hyperthermia, T/N ratio and blood flow of the tumor significantly decreased (p < 0.05). Autoradiogram showed that the accumulation of 201TlCl in the tumor decreased diffusely. Histopathological finding showed congestion, thrombosis, and swelling of endothelial cells. These results suggest that the decrease in T/N ratio may be caused by the vascular damage due to hyperthermia. The T/N ratio recovered 24 hrs after hyperthermia but was still lower than that for the control. Autoradiogram showed that 201TlCl greatly accumulated in the viable tumor tissue but was hardly seen in the necrotic tumor tissue. These results suggest that the decrease in T/N ratio may be caused by increase of necrotic areas. The T/N ratio 24 hrs after hyperthermia correlated (r = 0.83) with relative tumor growth rate on 7th day after hyperthermia and, therefore, can be used as an indicator of relative tumor growth rate. 201Tl-scintigraphy can be useful for prediction of therapeutic effect of hyperthermia.

Scintigraphic study on the distribution of radiolabeled cis-diamminedichloroplatinum (II) in the tumor-bearing rabbit: a comparison between intra-arterial injection with lipiodol and intravenous injection.

To compare the distribution of cis-diamminedichloroplatinum (II) (CDDP) on scintigraphic images between intra-arterial injection with lipiodol and intravenous injection, we obtained serial scintigraphic images using radiolabeled CDDP (Pt-195m CDDP), which had a high specific activity (7.4 x 10(6) Bq [200 microCi]/mg-CDDP), in the rabbit VX-2 tumor system. A dose of 9.25 x 10(6) Bq (250 microCi) of Pt-195m CDDP was injected at one shot via an ear vein, while 7.4 x 10(6) Bq (200 microCi of Pt-195m CDDP mixed with 1 ml of lipiodol was injected at one shot via a saphenous artery. Mean pixel count, which was corrected for the total dose injected, in the arterially injected tumor always exceeded than that in the intravenously injected tumor. Pt-195m CDDP uptake by the rabbit tumor at 2 hr was 7.8% of the total injected dose with intravenous injection and 16.0% with intra-arterial injection. These data suggest that intra-arterial injection with lipiodol is a more useful method to increase CDDP uptake by tumors than intra-venous injection.

Mitogenic activities in African traditional herbal medicines.

Mitogenic activities in African traditional herbal medicines were examined using protein fractions obtained from their extracts by precipitation with ammonium sulfate. Potent mitogenic activities for human and mouse lymphocytes were found in the three plants: Croton macrostachyus, Croton megalocarpus (Euphorbiaceae), and Phytolacca dodecandra (Phytolaccaceae). All the gel chromatographic patterns of these protein fractions progressed toward the smaller molecule site with pronase treatment, while their mitogenic activities decreased significantly. Protein fractions from these three plants induced mitogenesis both in human and mouse isolated T cells, but not in lymphocytes from athymic nude mice. By testing further fractionated protein fractions with gel filtration chromatography, it was found that all three plants contained several mitogens having different molecule sizes.

[Temperature distribution and geometry of the electrodes in RF interstitial hyperthermia using circular and interstitial electrodes].

To evaluate the feasibility of clinical application of a newly developed interstitial hyperthermia system, which consists of an 8 MHz radiofrequency generator, interstitial needle electrodes, and a superficial circular electrode, we conducted preclinical experiments using an agar phantom and VX-2 carcinoma in the rabbit. In the experiment with an agar phantom, four 4 cm needle electrodes were placed in a square array at intervals of 1.0, 1.5, and 2.0 cm. Thermography demonstrated homogeneous temperature distribution at electrode intervals of 1.0 and 1.5 cm, but hot spots around the electrodes at an interval of 2.0 cm. When electrode deviation was less than 8 degrees from the parallel plane, no temperature deviation was observed. Using two 2 cm electrodes and two 4 cm electrodes in square array, thermography demonstrated a homogeneous temperature distribution in the area surrounded by the electrodes. Even if the electrodes were located at the periphery of the agar phantom, a homogeneous temperature distribution was obtained in the area surrounded by the electrodes. Using four 4 cm electrodes at intervals of 1.5 cm in VX-2 carcinoma in the rabbit, ideal heating was obtained: 42 degrees C at the periphery of the tumor and 43 degrees C at the center. These data suggest that the newly developed interstitial hyperthermia apparatus provides homogeneous heat distribution at electrode intervals of 1.5 cm or less and can be used in a Phase I study for deep-seated or superficial tumors.

In vitro activities of acridone alkaloids against Pneumocystis carinii.

Acridone alkaloids isolated from plants of the family Rutaceae have antiplasmodial activity in rodent models of malaria. Because a variety of antimalarial agents have also been shown to have activity against Pneumocystis carinii, we tested six of these alkaloids in an established culture model for P. carinii. Atalaphillinine and glycobismine A inhibited growth of cultured P. carinii at concentrations of 2.7 and 1.7 microM, respectively. This potency of effect is similar to that of chloroquine (3 microM) but somewhat less than that of primaquine (0.4 microM), which was previously evaluated in the same system.