Abilities of cumulus and granulosa cells to enhance the developmental competence of bovine oocytes during in vitro maturation period are promoted by midkine; a possible implication of its apoptosis suppressing effects.

We previously reported that when midkine (MK), a heparin-binding growth differentiation factor was used in in vitro maturation (IVM) culture of bovine cumulus-enclosed oocytes (CEOs), their developmental competence to the blastocyst stage after in vitro fertilization (IVF) was enhanced and the effect of MK might be mediated by its action upon mural granulosa cells and cumulus cells that closely surround the oocyte. In the present study, when denuded oocytes (DOs) were matured in IVM medium with or without MK (200 ng/ml) in the presence or absence of isolated cumulus cell masses and subjected to IVF, the enhancing effects of MK on the developmental competence of DOs to the blastocyst stage after IVF were exerted only in the presence of cumulus cells. In addition, we prepared the conditioned media of granulosa cells cultured with or without 200 ng MK/ml (CMMK+ or CMMK- respectively) and examined their effects on the IVM of DOs in terms of their developmental competence to the blastocyst stage after IVF. The supplementation of CMMK+ into IVM medium at 40% (v/v) significantly enhanced the blastocyst development compared with the no additive control and the CMMK- supplemented groups. Furthermore, the effects of MK during IVM of bovine CEOs on the cumulus cell apoptosis were investigated. CEOs were cultured up to 24 h in IVM medium without (control) or with 200 ng MK/ml. The genomic DNA was extracted from CEOs at 0, 6, 12, 18 and 24 h of IVM and subjected to ligation-mediated PCR (LM-PCR) to detect the apoptotic internucleosomal DNA fragmentation. DNA fragmentation was scarcely detected at the start of IVM, whereas it increased time-dependently as the IVM culture progressed. The degree of the fragmentation was significantly lower in the MK-treatment group compared with the control group at 18 and 24 h of IVM. The apoptosis-suppressing effect of MK on cumulus cells was further confirmed in situ by using TUNEL on CEOs. In conclusion, data from the present study further confirmed that MK enhances the developmental competence of bovine oocytes via cumulus and granulosa cells. It was also demonstrated that MK suppresses the apoptosis that occurs in cumulus cells during the period of IVM of bovine CEOs. The putative soluble factor(s) from cumulus cells was suggested from the experiment using CMMK+ . MK may promote the production of such factors in part by its anti-apoptotic effects on cumulus cells.

Apoptosis-inducing activity of high molecular weight fractions of tea extracts.

High molecular weight fractions of green tea, black tea, oolong tea, and pu-erh tea were found to induce apoptosis in human monoblastic leukemia U937 cells by examination of their ability to inhibit cell proliferation and to induce apoptotic body formation and DNA ladder formation. These tea fractions were also shown to induce apoptosis in stomach cancer MKN-45 cells. In addition to known antitumor-promoting activity of tea high molecular weight fractions, their apoptosis-inducing activity may contribute to cancer chemopreventive effects of tea.

Apoptosis-inducing activity of a driselase digest fraction of green tea residue.

We enzymatically digested green tea residue with Driselase, a crude preparation containing cellulase, pectinase and proteases, in order to examine the potential usefulness of the residue. A fraction of the digest soluble in 70% ethanol was found to induce the death of U937 human histiocytic lymphoma cells by apoptosis. Other enzyme preparations gave similar products with cell death-inducing activity of varing potency. The green tea residue may therefore be a useful source of potential agents with anti-cancer activity.

Apoptosis-inducing activity of galloyl monosaccharides in human histiocytic lymphoma U937 cells.

Three galloyl monosaccharides contained in medicinal plants were examined for apoptosis-inducing activity in human histiocytic lymphoma U937 cells. Tetragalloyl glucose (TgG) induced apoptosis as found by chromatin condensation, DNA ladder formation, and inhibition by a caspase inhibitor. Digalloyl hamamelose had moderate activity, while monogalloyl glucose was only marginally active. These findings suggest that the number and disposition of their phenolic groups are important for apoptosis induction. TgG induced apoptosis in human colon and stomach cancer cell lines as well, indicating it is potentially useful as an anti-cancer agent.

Tea catechins and related polyphenols as anti-cancer agents.

Epigallocatechin gallate (EGCg) and theaflavins, a major constituent of green tea infusion and the constituents of black tea, respectively, were found to inhibit matrix metalloproteinases (MMPs) which are intimately associated with tumor invasion and metastasis. EGCg and related polyphenols exhibited apoptosis-inducing activity for several cancer cell lines including human stomach and colon cancer cells. Comparison of the activity of these compounds revealed the importance of the number and the steric disposition of hydroxyl groups. A pyrogallol-type structure in a molecule is a minimum requirement for apoptosis induction of catechin compounds and that in the B ring has an important role in the activity. These data would provide useful information for designing anti-cancer agents on the basis of anti-inhibitory activity for MMPs and/or apoptosis-inducing activity.

Hyperproduction of recombinant ferredoxins in escherichia coli by coexpression of the ORF1-ORF2-iscS-iscU-iscA-hscB-hs cA-fdx-ORF3 gene cluster.

Fe-S proteins acquire Fe-S clusters by an unknown post-translational mechanism. To study the in vivo synthesis of the Fe-S clusters, we constructed an experimental system to monitor the expressed ferredoxin (Fd) as a reporter of protein-bound Fe-S clusters assembled in Escherichia coli. Overexpression of five Fds in a T7 polymerase-based system led to the formation of soluble apoFds and mature holoFds, indicating that assembly of the Fe-S cluster into apoFd polypeptides is a rate-limiting step. We examined the coexpression of the E. coli ORF1-ORF2-iscS-iscU-iscA-hscB-hsc A-fdx-ORF3 gene cluster, which has recently been suggested to be involved in the formation or repair of Fe-S protein [Zheng, L., Cash, V.L., Flint, D.H., and Dean, D.R. (1998) J. Biol. Chem. 273, 13264-13272], with reporter Fds using compatible plasmids. The production of all five reporter holoFds examined was dramatically increased by the coexpression of the gene cluster, and apparent specificity to the polypeptides or to the type of Fe-S clusters was not observed. The increase in holoFd production was observed under the coexpression conditions in all culture media examined, with either 2 x YT medium or Terrific broth, and with or without supplemental cysteine or iron. These results indicate that the proteins encoded by the gene cluster are involved in the assembly of the Fe-S clusters in a wide variety of Fe-S proteins.

Inhibitory effects of tetragalloylglucose and digalloylhamamelose on adhesion and in vitro invasion of mouse lung carcinoma cells.

Tetragalloylglucose (TgG) and digalloylhamamelose (DgH) were found to inhibit adhesion to and invasion through Matrigel of mouse Lewis lung carcinoma LL2-Lu3 cells, which are highly metastatic. TgG inhibited matrix metalloproteinases (MMPs) from the tumor cells like (-)-epigallocatechin gallate, whereas DgH did not. These results suggest that TgG and DgH inhibit tumor cell invasion by inhibiting MMPs and/or cell adhesion of the tumor cells.

Apoptosis-inducing activity of polyphenol compounds derived from tea catechins in human histiolytic lymphoma U937 cells.

Polyphenolic compounds derived from tea catechins were examined for apoptosis-inducing activity in human histiolytic lymphoma U937 cells. (-)-Epigallocatechin gallate, theasinensin D, compound OH-5, theaflavin, and theaflavin digallate induced apoptosis as evidenced by DNA ladder formation, its inhibition by a caspase inhibitor, and chromatin condensation. Theasinensin D was the most potent inducer and the data suggest the importance of the number and three dimensional localization of their phenolic groups in this activity. These apoptosis-inducible compounds may be useful as a cancer chemopreventive and chemotherapeutic agent.

Immunohistochemical localization of macrophage migration inhibitory factor (MIF) in tanycytes, subcommissural organ and choroid plexus in the rat brain.

We investigated the immunohistochemical localization of the macrophage migration inhibitory factor (MIF) in the rat brain. In addition to epithelial ependymal cells lining the ventricular wall, tanycytes in the basomedial hypothalamus were heavily immunostained. The immunoreactive processes of tanycytes made contacts to sinusoidal capillaries and reached the pial surface forming an immuno-positive structure at the floor of the hypothalamus. Other immunoreactive cells contained the subcommissural organ in the roof of the third ventricle and the epithelial lamina of the choroid plexus. The localization of MIF in cells which have contact with cerebrospinal fluid and blood vessels suggests that MIF might play a role as a humoral factor in the brain.

Improved retrograde axonal transport and subsequent visualization of tetramethylrhodamine (TMR) -dextran amine by means of an acidic injection vehicle and antibodies against TMR.

We studied the ability of various dextran amines (DA) to retrogradely label cortical neurons to the full extent of their dendritic configurations. Corticothalamic neurons were labeled by pressure injection of DA into the ventrobasal thalamic nuclei of the rat brain. Of fluorescein-, Texas Red-, Cascade Blue- and tetramethylrhodamine (TMR)-DAs of MW 3000 and TMR-DA of MW 10,000, neurons were most efficiently labeled with TMR-DA of MW 3000. The use of acidic vehicles (pH 1-3) for dissolving TMR-DA enhanced the retrograde labeling, as compared with that of a neutral vehicle. The retrograde labeling with TMR-DA was more clearly demonstrated by using anti-TMR antibodies; the indirect immunofluorescence method with a rhodamine-conjugated secondary antibody and immunoperoxidase method with a peroxidase anti-peroxidase (PAP) complex revealed that the dendrites of many corticothalamic neurons were filled with TMR-DA. The Golgi-like retrograde labeling of TMR-DA visualized by the PAP immunoperoxidase method was comparable with that of biotinylated DA by the avidin-biotinylated peroxidase complex method. Similar Golgi-like dendritic staining was observed among corticospinal neurons after injection of TMR-DA into the corticospinal tract of the spinal cord. Most apical dendrites of corticospinal neurons extended into layer I, whereas those of corticothalamic neurons ended in layer IV or the deep part of layer III. The TMR-DA injection under acidic conditions and immunostaining with the anti-TMR antibodies are considered to be a useful method to visualize the dendrite configuration of cortical projection neurons.

Neuronal glucoprivation enhances hypothalamic histamine turnover in rats.

Histamine (HA) turnover in the rat hypothalamus following insufficient energy supply due to glucoprivation was examined after administration of insulin or 2-deoxy-D-glucose (2-DG). HA turnover was assessed by accumulation of tele-methylhistamine (t-MH), a major metabolite of brain HA, following administration of pargyline. Intraperitoneal injection of 1, 2, and 4 U/kg of insulin, which had no influence on steady-state levels of HA and t-MH, increased pargyline-induced accumulation of t-MH. Accumulation of t-MH due to pargyline was inversely related to the concomitant plasma glucose concentration after different doses of insulin. The level of t-MH accumulated by pargyline did not change compared with that of controls, when a euglycemic condition was maintained or insulin at a dose of 6 mU per rat was infused into the third cerebroventricle. Intracerebroventricular infusion of 24 mumol per rat of 2-DG, which had no influence on steady-state levels of HA and t-MH, increased the level of t-MH enhanced by pargyline. The results indicate that an increase in hypothalamic HA turnover in response to glucoprivation may be involved in homeostatic regulation of energy metabolism in the brain.

Molecular cloning and nucleotide sequences of cDNAs encoding subunits I, II, and IX of Euglena gracilis mitochondrial complex III.

cDNA clones encoding subunits I, II, and IX of Euglena gracilis mitochondrial complex III have been isolated from a lambda gt11 cDNA expression library by immunoscreening with an antiserum against the complex of the organism. Determination of the nucleotide sequences and amino-terminal amino acid sequences of purified subunits revealed that subunits II and IX, respectively, consist of 432 and 70 amino acids as their mature forms and possess potential presequences of 42 and 30 amino acids. The amino-terminal parts of the presequences had typical structural features of the mitochondrial targeting signal. Such features were also found at the amino-terminal region of the predicted subunit I protein, which comprises 494 residues. However, the amino terminus of the purified subunit I could not be detected, possibly because of a post-translational modification. Euglena subunits I and II both showed similarities to the members of the protein family which comprises complex III core proteins, mitochondrial processing peptidases (MPP) and processing enhancing proteins (PEP). Namely, the Euglena subunit I could be assigned to core 1 protein and the subunit II to core 2 protein in the family. In contrast, the subunit IX seemed to be peculiar to Euglena complex III. At 5'-untranslated regions, the three cloned cDNAs for subunits I, II, and IX had a common poly(T)CG structure which has also been reported for other Euglena cDNAs of nuclear genes.

Characterization of histamine release from the rat hypothalamus as measured by in vivo microdialysis.

The release of endogenous histamine (HA) from the hypothalamus of anesthetized rats was measured by in vivo microdialysis coupled with HPLC with fluorescence detection. Freshly prepared Ringer's solution was perfused at a rate of 1 microliter/min immediately after insertion of a dialysis probe into the medial hypothalamus, and brain perfusates were collected every 30 min into microtubes containing 0.2 M perchloric acid. The basal HA output was almost constant between 30 min and 7 h after the start of perfusion, with the mean value being 7.1 pg/30 min. Thus, the extracellular HA concentration was assumed to be 7.8 nM, by a calculation from in vitro recovery through the dialysis membrane. Perfusion with a high K+ (100 mM)-containing medium increased the HA output by 170% in the presence of Ca2+. Systemic administration of either thioperamide (5 mg/kg, i.p.), a selective H3 receptor antagonist, or metoprine (10 mg/kg, i.p.), an inhibitor of HA-N-methyltransferase, caused an approximately twofold increase in the HA output 30-60 min after treatment. The combined treatment with thioperamide and metoprine produced a marked increase (650%) in the HA output. The HA output decreased by approximately 70% 4-5 h after treatment with alpha-fluoromethylhistidine (alpha-FMH; 100 mg/kg, i.p.), an inhibitor of histidine decarboxylase. Furthermore, the effect of combined treatment with thioperamide and metoprine was no longer observed in alpha-FMH-treated rats. These results suggest that both HA-N-methyltransferase and H3 autoreceptors are involved in maintaining a constant level of extracellular HA and that their blockade effectively results in a higher activity level of the endogenous histaminergic system in the CNS.

In vivo measurement of noradrenaline and 3,4-dihydroxyphenylethyleneglycol in the rat hypothalamus by microdialysis: effects of various drugs affecting noradrenaline metabolism.

The extracellular concentrations of noradrenaline (NA) and 3,4-dihydroxyphenylethyleneglycol (DOPEG), one of the major metabolites of brain NA, in the hypothalamus of urethane-anesthetized rats were monitored by in vivo microdialysis followed by a sensitive and simultaneous determination of the two substances using high-performance liquid chromatography with electrochemical detection. The effects of various drugs that affect central NA metabolism were also examined. Resting levels of NA and DOPEG were constant during 1 and 6 hr after the start of perfusion, the mean values being 3.8 +/- 0.4 pg/30 min for NA and 107.5 +/- 9.1 pg/30 min for DOPEG (mean +/- S.E.M. of 7 animals). Tetrodotoxin (1 microM), when added to the perfusion medium, reduced the output of NA below the detection limit (0.5 pg) and also decreased the DOPEG output by 60%. Clonidine (0.2 mg/kg i.p.) caused a marked reduction in both the NA and DOPEG outputs, whereas yohimbine (5 mg/kg i.p.) significantly increased both the NA and DOPEG outputs. Desipramine (2 and 5 mg/kg i.p.) produced a dose-dependent increase in the NA output, although it caused a gradual decline of the DOPEG output. The atypical antidepressant mianserin (2 and 5 mg/kg i.p.), which possesses both alpha-2 antagonist and weak NA uptake inhibitory actions, produced a less marked increase in the NA output with no or only a small decrease in the DOPEG output. Therefore, it is suggested that monitoring the extracellular concentrations of both NA and DOPEG enables the discrimination between the action of drugs inhibiting the NA uptake and that of drugs enhancing the NA release, and that this method is useful to obtain detailed information about central NA metabolism in vivo.

Histamine turnover in the rat hypothalamic nuclei estimated from alpha-fluoromethylhistidine-induced histamine decrease.

The turnover rates, rate constants and half-life values of neuronal histamine (HA) in 10 nuclei of the rat hypothalamus were estimated from the depletion of HA induced by alpha-fluoromethylhistidine (alpha-FMH: 100 mg/kg, i.p.), a specific inhibitor of histidine decarboxylase, on the presumption that alpha-FMH depletable HA pools represent neuronal ones. Marked variation in the HA turnover rates were observed among the hypothalamic nuclei, ranging from 5.7 to 19.5 pmole/mg protein/hr.

tele-Methylhistamine levels and histamine turnover in nuclei of the rat hypothalamus and amygdala.

An HPLC method using fluorescence detection for the determination of tele-methylhistamine (t-MH) was improved to a sensitivity level which enabled the detection of 0.05 pmol of tissue t-MH. The t-MH contents and the histamine turnover rates in various nuclei of the rat hypothalamus and amygdala were subsequently measured. The histamine turnover rates were estimated from pargyline-induced t-MH accumulation. Both the t-MH levels and the histamine turnover rates were shown to be relatively high in the nuclei dorsomedialis and premammillaris ventralis of the hypothalamus, and also in the nucleus medialis of the amygdala. The steady-state t-MH levels in various nuclei of the hypothalamus and amygdala correlated well with the histamine turnover rates in these nuclei.

Changes in histamine metabolism in the brains of mice with streptozotocin-induced diabetes.

Histamine (HA) metabolism in the brain of mice with streptozotocin (STZ)-induced diabetes was examined. The levels of tele-methylhistamine (t-MH), a major metabolite of brain HA, significantly increased 3 and 4 weeks after STZ injection. However, the HA turnover rates in the diabetic mice, determined from the accumulation of t-MH after the administration of pargyline, were not different from the control values when the animals were allowed free access to food. When the mice were starved for 15 h 4 weeks after STZ treatment, the brain levels of L-histidine decreased significantly, whereas HA turnover increased significantly. Such changes were not observed in starved control mice. Histidine decarboxylase or HA N-methyltransferase activity did not change after starvation in either diabetic or control mice. These results show that the histaminergic (HAergic) activity in the brains of diabetic mice remains within normal range as long as the animals are allowed free access to food. However, they also indicate that a marked enhancement of HAergic activity accompanied by a decrease in the brain L-histidine level occurs in starved diabetic mice.

[Pharmacological effects of reiousan on experimental hepatic injuries and hepatic functions].

Pharmacological effects of Reiousan, a crude drug preparation consisting of bezoar and ginseng, on experimental hepatic injuries and hepatic functions were studied. After administration of Reiousan, carbon tetrachloride, d-galactosamine and alpha-naphthylisothiocyanate-induced experimental hepatic injuries in rats were inhibited. Facilitation of recovery from increased retention rate of sulfobromophthalein in hepatectomized rats, increase in hepatic blood flow, inhibition of superoxide anions, and decrease in blood ethanol concentration in rats administered ethanol were observed after application of Reiousan. Inhibitory effects of Reiousan on experimental hepatic injuries may result from the increase in hepatic blood flow and inhibition of superoxide anions.

Effect of acute treatment of mice with L-histidine on the brain levels of amino acids.

The brain histidine level in mice dose-dependently increased 1 and 2 hr after an i.p. injection of 0.5-1.5 g/kg of L-histidine. The treatment with 1.0 and 1.5 g/kg but not 0.5 g/kg of L-histidine significantly decreased the brain levels of tyrosine, phenylalanine and some other amino acids 1 hr later. A complete recovery or a rebound rise of amino acid levels was observed 2 hr after treatment. These results indicate that there is a change in the transport of amino acids into the brain after treatment with large doses of L-histidine.