Investigation of central mechanism of insulin induced hypoglycemic convulsions in mice

Insulin produces seizures in healthy and diabetic animals. Amongst suggested mechanisms, the role of neuromodulators and neurotransmitters is not clear. The present study explores the mechanisms involved in insulin-induced convulsions. Convulsions were induced in Swiss male albino mice with graded doses of insulin. Blood sugar levels were measured prior to and after the first convulsion. Drugs like 5-HTP (5-HT precursor), pCPA (5-HT depletor), ondansetron (5-HT3 antagonist), ketanserin (5-HT, antagonist), ketamine (NMDA antagonist), 1-dopa (dopamine precursor) and reserpine (amine depletor) were studied for interaction with convulsive behaviour induced by insulin. Insulin in 2 IU/kg dose did not produce convulsions while 4 and 8 IU/kg doses produced convulsions in 50% and 100% of animals respectively. 5-HTP, ondansetron, ketanserin, ketamine and l-dopa significantly protected/inhibited animals from convulsions at all studied doses of insulin. On the contrary, pCPA and reserpine potentiated insulin induced convulsions. Insulin caused mortality in 40 and 100% animals with 4 and 8 IU/kg doses respectively. pCPA and reserpine treatments caused mortality at all doses of insulin, while other drugs did not influence insulin induced mortality. Blood sugar levels were reduced in all groups irrespective of the presence or absence of convulsions. A definitive link of serotonergic, dopaminergic and excitatory amino acid pathways in mediating insulin-induced hypoglycemic convulsions is suggested.     

Adenoviral gene transfer of aspartoacylase ameliorates tonic convulsions of spontaneously epileptic rats

The spontaneously epileptic rat (SER: tm/tm, zi/zi) shows both absence-like seizures and tonic convulsions. Our previous studies have demonstrated that absence-like seizures of the tremor rat (tm/tm), one of the parent strains of SER, were inhibited by adenoviral transfer of the aspartoacylase (ASPA) gene, a deleted gene in the tremor rat. In the present study, we examined whether the adenoviral gene transfer of ASPA inhibited the tonic convulsions of SER. Replication-defective recombinant adenoviral vectors carrying the rat ASPA gene (AxASPA) or Escherichia coli beta-galactosidase gene (AxLacZ), as a control, were constructed. After it was confirmed that AxASPA-infected HeLa cells expressed ASPA in vitro, AxASPA or AxLacZ was administered into the left lateral ventricle of 11-week-old SER. The occurrence and duration of tonic convulsions in SER were evaluated before and after the administration of adenoviral vector. Intracerebroventricular administration of AxASPA (5 x 10(7) plaque forming units) transiently, but significantly, inhibited the occurrence of tonic convulsions in SER without affecting the duration per single convulsion 7 days after the administration. No inhibitory effects were observed 10 and 14 days after AxASPA administration. In contrast, the administration of AxLacZ did not have any effect on tonic convulsions in SER. Survival rates did not differ between AxASPA- and AxLacZ-treated SERs. Adenoviral gene transfer of ASPA, one of the deleted genes of SER, transiently rescued SERs from tonic convulsion, although it did not improve their survival time.     

Role of the Sympathetic Innervation of the Pacinian Corpuscle

An investigation was made into the nature of the role played by the noradrenergic innervation of the pacinian corpuscle. Corpuscles of the cat mesentery and mesocolon were used in all experiments. Blockade of noradrenergic beta receptors by dichloroisoproterenol and interference with norepinephrine release by reserpine are each capable of reversibly blocking mechanoelectric transduction by the pacinian corpuscle. The monoamine oxidase inhibitors iproniazid and phenelzine are capable of protecting the transducer from the blocking effects of reserpine. It is concluded that the presence of norepinephrine, as maintained by sympathetic tonus, is required for the afferent nerve terminal of the pacinian corpuscle to be mechanosensitive.     

The Replicative Capacities of Large E1B-Null Group A and Group C Adenoviruses Are Independent of Host Cell p53 Status

Recent reports suggest that an early region 1B (E1B) 55,000-molecular-weight polypeptide (55K)-null adenovirus type 5 (Ad5) mutant (dl1520) can replicate to the same extent as wild-type (wt) Ad5 in cells either deficient or mutated in p53, implicating p53 in limiting viral replication in vivo. In contrast, we show here that the replicative capacity of Ad5 dl1520 is wholly independent of host cell p53 status, as is the replicative capacity of comparable Ad12 E1B 54K-null adenoviruses (Ad12 dl620 and Ad12 hr703). Furthermore, we show that there is no requirement for complex formation between p53 and Ad5 E1B 55K or Ad12 E1B 54K for a productive infection, such that wt Ad5 and wt Ad12 will both replicate in cells which are null for p53. In addition, we find that these Ad5 and Ad12 mutant viruses induce S phase irrespective of the p53 status of the cell and that, therefore, S-phase induction does not correlate with the replicative capacity of the virus. Interestingly, the replicative capacities of the large E1B-null adenoviruses correlated positively with the ability to express E1B 19K and were related to the ability to repress premature adenovirus-induced apoptosis. Infection of primary human cells indicated that Ad5 dl1520, wt Ad5, and wt Ad12 replicated better in cycling normal human skin fibroblasts (HSFs) than in quiescent HSFs. Thus, the cell cycle status of the host cell, upon infection, also influences viral yield.     

Inhibition of Ornithine Decarboxylase and Growth of the Fungus Helminthosporium maydis

α-dl-Difluoromethylornithine (DFMO), a specific enzyme-activated inhibitor of ornithine decarboxylase, at 0.5 to 2.0 millimolar significantly inhibited mycelial growth and especially sporulation of Helminthosporium maydis in the dark; its inhibitory effect on sporulation was greatly increased under light conditions. Putrescine at 0.25 millimolar fully prevented the inhibitory effects of DFMO; the inhibition caused by the latter could not be prevented by cadaverine or CaCl₂. α-dl-Difluoromethylarginine, a specific enzyme-activated inhibitor of arginine decarboxylase, at 0.1 to 2.0 millimolar had a weak inhibitory effect on the fungus. The effect was not dependent on the inhibitor concentration and there was no detectable arginine decarboxylase activity in the fungus.     

Effect of cytochalasin B and colchicine on the stimulation of -amylase release from rat parotid tissue slices

A role for microfilaments and microtubules in the secretion of α-amylase is indicated since cytochalasin B and colchicine inhibited the stimulation of α-amylase release by epinephrine (30 or 15 μM) but only cytochalasin B inhibited the stimulation by N⁶, O^2′ dibutyryl adenosine 3′,5′monophosphate (1.0 mM). It was necessary to incubate the parotid tissue slices in the presence of cytochalasin B (1 hr.) or colchicine (4 hrs.) before adding the agonist in order to see the inhibitory effects.     

Reserpine-induced reduction in norepinephrine transporter function requires catecholamine storage vesicles

Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5 min decreased [³H]NE uptake capacity, an effect characterized by a robust decrease in the V_max of the transport of [³H]NE. As expected, reserpine did not displace the binding of [³H]nisoxetine from the NET in membrane homogenates. The potency of reserpine for reducing [³H]NE uptake was dramatically lower in SK-N-SH cells that have reduced storage capacity for catecholamines. Reserpine had no effect on [³H]NE uptake in HEK-293 cells transfected with the rat NET (293-hNET), cells that lack catecholamine storage vesicles. NET regulation by reserpine was independent of trafficking of the NET from the cell surface. Pre-exposure of cells to inhibitors of several intracellular signaling cascades known to regulate the NET, including Ca²⁺/Ca²⁺–calmodulin dependent kinase and protein kinases A, C and G, did not affect the ability of reserpine to reduce [³H]NE uptake. Treatment of PC12 cells with the catecholamine depleting agent, α-methyl-p-tyrosine, increased [³H]NE uptake and eliminated the inhibitory effects of reserpine on [³H]NE uptake. Reserpine non-competitively inhibits NET activity through a Ca²⁺-independent process that requires catecholamine storage vesicles, revealing a novel pharmacological method to modify NET function. Further characterization of the molecular nature of reserpine's action could lead to the development of alternative therapeutic strategies for treating disorders known to be benefitted by treatment with traditional competitive NET inhibitors.     

Pharmacological studies on serotonin-mediated behaviour

1. Administration to rats of a monoamine oxidase inhibitor (for example tranylcypromine: Tcp) followed by L-tryptophan increases the rate of synthesis and release of 5-hydroxytryptamine (5-HT) and results in a series of behavioural changes, some of which can be recorded on activity meters or scored. Various putative 5-HT agonists and the releasing drugs, fenfluramine and p-chloroamphetamine, also produce these changes. 2. A supersensitive behavioural response was produced by specific lesioning or p-chlorophenylalanine pretreatment and lesioning and sectioning experiments suggested several of the behaviours to be either hind-brain or spinally mediated. 3. A role for dopamine and GABA in the behaviour was demonstrated, but depletion of brain noradrenaline by specific lesioning or administration of disulfiram did not influence the behavioural changes. 4. The behaviour produced by administration of Tcp/L-tryptophan or 5-methoxy N,N-dimethyl tryptamine was inhibited by the suggested 5-HT antagonists, methysergide, methergoline and (--)-propranolol, but not by cinanserin, mianserin and cyproheptadine, other putative antagonists. In contrast, all the antagonists inhibited the behaviour when it was produced by injection of the agonist, quipazine. 5. The possible reasons for these differences is discussed in the light of the receptor binding characteristics of the drugs and the possible existence of different 5-HT receptor populations.     

Brain serotonin and norepinephrine after convulsions and reserpine

Decreased brain norepinephrine (Schildkraut , 1969) and decreased brain serotonin (Shaw , Camp and Eccleston , 1967; Bourne et al., 1968) have been implicated in current hypotheses about the psychiatric syndrome, depression. Reserpine, which causes depression in some people, depletes brain norepinephrine and serotonin and, possibly, replicates in laboratory animals some of the biochemical changes in brains of depressed patients. Electrically-induced convulsions are an effective treatment of depression. There have been numerous studies of the effects of convulsive electrical stimulation on norepinephrine and serotonin in brains of laboratory animals. They have employed different schedules of administration of convulsions, short (minutes) or long (hours to days) periods after convulsion before killing the animals, and various methods of extraction and assay of the amines. Results have varied. For example, Kato , Gozsy , Roy and Groh (1967) found that a series of convulsions, administered daily for 11 days elevated whole brain serotonin but not norepinephrine. On the other hand, Hinesley , Norton and Aprison (1968) observed that a series of seven convulsions, one every other day, elevated norepinephrine only in cerebral hemispheres, whereas serotonin was elevated only in midbrain and pons-medulla. Our present report is the first to deal with both norepinephrine and serotonin in animals given both serial injections of reserpine and a series of electrically-induced convulsions.     

MORPHOLOGICAL AND PHARMACOLOGICAL EFFECTS OF RESERPINE, GIVEN ALONE OR AFTER IPRONIAZID, ON THE CATECHOL AMINES OF THE ADRENAL GLANDS OF THE RAT

Adrenomedullary cells, after fixation with OsO₄, are filled with well formed granules which are considered to represent their catechol amine content. The submicroscopic appearance of these cells was studied in reserpine-treated rats during the late phase of catechol amine depletion and during the period of its restoration. At 3 days after the beginning of reserpine treatment, the granules appeared to be emptied of their content and small vesicles containing scattered, dense deposits of, presumably, catechol amines began to be seen. At 9 days after the beginning of treatment, these deposits had already become granules and the cells had attained a completely normal appearance. The submicroscopic structure of the adrenomedullary cells of rats pretreated with iproniazid (before reserpine), in which a complete inhibition of monoamine oxidase activity had thus been obtained, was similar to that seen in non-treated animals. In numerous cases, however, some characteristic features were noted: the sacs which usually contained a dense granule of catechol amines appeared swollen and many fine granules could be seen around them; the latter were dispersed in a way suggesting that they may represent a partial breakdown of the large granules which, under the inhibitory action of iproniazid, do not release the catechol amines contained within them.     

Toxicokinetics of a single intravenous dose of rac-propranolol versus optically pure propranolol in the rat

Conscious male Wistar SPF Riv:TOX rats were dosed intravenously with 2.5, 5, or 10 mg/kg rac-propranolol·HCl, or with 5 mg/kg of either (-)-(S)- or (+)-(R)-propranolol·HCl. Disposition of (-)-(S)- and (+)-(R)-propranolol after dosing of rac-propranolol was linear in the dose range examined. Total plasma clearance was not changed in animals dosed with the individual enantiomers compared to the animals that were dosed with rac-propranolol. However, for (-)-(S)-propranolol both volume of distribution and elimination half-life decreased, whereas for (+)-(R)-propranolol increases were observed for these characteristics, in animals dosed with the individual enantiomers. Our observations suggest that the (+)-(R)-enantiomer competes with (-)-(S)-propranolol for plasma protein binding sites, resulting in lower plasma protein binding of the (-)-(S)-enantiomer when the racemate is administered. From recent toxicological experiments, it was concluded that rac-propranolol is more toxic than the individual enantiomers in the rat, when dosed iv at the same total mass. It is concluded that the observed potentiation of toxic effects of propranolol enantiomers when administered as a racemate can at least partly be explained by a pharmacokinetic interaction. © 1995 Wiley-Liss, Inc.     

Effects of putrescine and inhibitors of putrescine biosynthesis on organogenesis inEuphorbia esula L.

Summary Exogenous putrescine (≤5 mM) had little effect on root or shoot formation in aseptically isolated hypocotyl segments of leafy spurge (Euphorbia esula L.) grown on full-strength B5 medium. Unexpectedly, putrescine inhibited root and shoot formation in hypocotyl segments grown on B5 medium diluted 10-fold. In the full-strength medium, root and shoot formation were inhibited by 0.5 mM concentrations ofdl-α-difluoromethylornithine (DFMO) anddl-α-difluoromethylarginine (DFMA). DFMO and DFMA are inhibitors of the ornithine decarboxylase and arginine decarboxylase pathways, respectively, of putrescine biosynthesis in plants. Exogenous putrescine (0.5 to 5 mM) did not reverse either the DFMO-or DFMA-induced inhibition of shoot formation. However, the DFMA-induced inhibition of root formation was partially reversed by exogenous putrescine. The auxin, indole-3-acetic acid (IAA), reduced the inhibitory effects of DFMO+DFMA (applied together) on both roots and shoots. In the first few days of culture, the endogenous levels of putrescine and spermidine, but not of spermine, increased in the presence of IAA. The levels of putrescine and spermidine in the tissues did not correlate well with either root or shoot production in the later stages of organ formation; especially in tissues treated with IAA. These results show that there were no obvious correlations between polyamine levels and organogenesis in leafy spurge hypocotyl segments, although residual putrescine or spermidine or both in the tissues at the time of excision may be indirectly involved in the early stages of root formation.     

The pyruvate branch point in fish liver mitochondria: Effects of acylcarnitine oxidation on pyruvate dehydrogenase and pyruvate carboxylase activities

Summary Palmitoyldl-carnitine inhibits¹⁴CO₂ production from 1-[¹⁴C]-pyruvate and from 1-[¹⁴C]-alanine by mitochondria from rainbow trout liver. The inhibitory effect occurs in both respiratory states III and IV. Fixation of H¹⁴CO ₃ ^– into acid-stable products by intact mitochondria requires pyruvate and ATP and is inhibited by sodium arsenite. This inhibitory effect is completely abolished by acetyldl-carnitine. It is proposed that under these conditions, oxidation of palmitoyldl-carnitine results in inhibition of pyruvate dehydrogenase while oxidation of acetyldl-carnitine results in activation of pyruvate carboxylase in intact rainbow trout liver mitochondria.     

Immobilization of alpha1-acid glycoprotein for chromatographic studies of drug-protein binding II. correction for errors in association constant measurements

A new method for the immobilization of α₁-acid glycoprotein (AGP) in high-performance liquid chromatography (HPLC) columns was recently described for applications such as drug binding studies. Part of this earlier work used self-competition zonal elution studies to measure association equilibrium constants between immobilized AGP and R- or S-propranolol. It was later found that analysis of these data by a common equation derived for linear elution conditions gave erroneous values for experiments actually conducted under nonlinear conditions. This report discusses the nature of this error and uses frontal analysis to estimate the true binding strength between R- and S-propranolol and HPLC columns containing immobilized AGP.     

The influence of reserpine on nitrogen metabolizing enzymes in chick liver

Reserpine, a Rauwolfia alkaloid, was shown to increase activity of the hepatic nitrogen metabolizing enzymes xanthine dehydrogenase, purine nucleoside phosphorylase, and tyrosine aminotransferase, when administered orally to young chicks. Using immunochemical techniques, this increase in xanthine dehydrogenase was shown to result from an enhanced de novo enzyme synthesis. The response pattern of the three enzymes to reserpine follows the same pattern to induction by high dietary protein suggesting that a common mode of action may be involved in the regulation of these enzymes. α-Adrenergic blockers, phentolamine and phenoxybenzamine, effectively prevented the increased enzyme activities caused by administration of reserpine.     

Identification of flavonolignans from Silybum marianum seeds as allosteric protein tyrosine phosphatase 1B inhibitors

Protein tyrosine phosphatase 1B (PTP1B) is an attractive molecular target for anti-diabetes, anti-obesity, and anti-cancer drug development. From the seeds of Silybum marianum, nine flavonolignans, namely, silybins A, B (1, 2), isosilybins A, B (3, 4), silychristins A, B (5, 6), isosilychristin A (7), dehydrosilychristin A (8), and silydianin (11) were identified as a novel class of natural PTP1B inhibitors (IC₅₀ 1.3 7–23.87?µM). Analysis of structure–activity relationship suggested that the absolute configurations at C-7" and C-8" greatly affected the PTP1B inhibitory activity. Compounds 1–5 were demonstrated to be non-competitive inhibitors of PTP1B based on kinetic analyses. Molecular docking simulations resulted that 1–5 docked into the allosteric site, including α3, α6, and α7 helix of PTP1B. At a concentration inhibiting PTP1B completely, compounds 1–5 moderately inhibited VHR and SHP-2, and weakly inhibited TCPTP and SHP-1. These results suggested the potentiality of these PTP1B inhibitors as lead compounds for further drug developments.     

Amino ketone formation and aminopropanol-dehydrogenase activity in rat-liver preparations

1. Rat tissue homogenates convert dl-1-aminopropan-2-ol into aminoacetone. Liver homogenates have relatively high aminopropanol-dehydrogenase activity compared with kidney, heart, spleen and muscle preparations. 2. Maximum activity of liver homogenates is exhibited at pH9·8. The K_m for aminopropanol is approx. 15mm, calculated for a single enantiomorph, and the maximum activity is approx. 9mμmoles of aminoacetone formed/mg. wet wt. of liver/hr.at 37°. Aminoacetone is also formed from l-threonine, but less rapidly. An unidentified amino ketone is formed from dl-4-amino-3-hydroxybutyrate, the K_m for which is approx. 200mm at pH9·8. 3. Aminopropanol-dehydrogenase activity in homogenates is inhibited non-competitively by dl-3-hydroxybutyrate, the K_i being approx. 200mm. EDTA and other chelating agents are weakly inhibitory, and whereas potassium chloride activates slightly at low concentrations, inhibition occurs at 50–100mm. 4. It is concluded that aminopropanol-dehydrogenase is located in mitochondria, and in contrast with l-threonine dehydrogenase can be readily solubilized from mitochondrial preparations by ultrasonic treatment. 5. Soluble extracts of disintegrated mitochondria exhibit maximum aminopropanol-dehydrogenase activity at pH9·1 At this pH, K_m values for the amino alcohol and NAD⁺ are approx. 200 and 1·3mm respectively. Under optimum conditions the maximum velocity is approx. 70mμmoles of aminoacetone formed/mg. of protein/hr. at 37°. Chelating agents and thiol reagents appear to have little effect on enzyme activity, but potassium chloride inhibits at all concentrations tested up to 80mm. dl-3-Hydroxybutyrate is only slightly inhibitory. 6. Dehydrogenase activities for l-threonine and dl-4-amino-3-hydroxybutyrate appear to be distinct from that for aminopropanol. 7. Intraperitoneal injection of aminopropanol into rats leads to excretion of aminoacetone in the urine. Aminoacetone excretion proportional to the amount of the amino alcohol administered, is complete within 24hr., but represents less than 0·1% of the dose given. 8. The possible metabolic role of amino alcohol dehydrogenases is discussed.     

An Adenovirus Type 5 Mutant with the Preterminal Protein Gene Deleted Efficiently Provides Helper Functions for the Production of Recombinant Adeno-Associated Virus

Production of recombinant adeno-associated virus (rAAV) requires helper functions that have routinely been provided by infection of the producer cells with adenovirus. Complete removal and/or inactivation of progeny adenovirus, present in such rAAV preparations, presents significant difficulty. Here, we report that an adenovirus type 5 (Ad5) mutant with the preterminal protein (pTP) gene deleted can provide helper function for the growth of rAAV. At high multiplicity, Ad5dl308_ΔpTP was as efficient as the phenotypically wild-type Ad5dl309 in permitting growth of rAAV. Use of Ad5dl308_ΔpTP, which is incapable of replication in the absence of complementation for pTP, as a helper avoids the need to remove contaminating adenovirus infectious activity by heat inactivation or by purification. Comparison of the transducing ability of rAAV generated with either Ad5dl308_ΔpTP or Ad5dl309 as a helper demonstrated that the heat inactivation protocol generally used does not remove all of the helper Ad5dl309 function.     

Inhibition of protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase by xanthones from Cratoxylum cochinchinense, and their kinetic characterization

Cratoxylum cochinchinense displayed significant inhibition against protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase, both of which are key target enzymes to attenuate diabetes and obesity. The compounds responsible for both enzymes inhibition were identified as twelve xanthones (1–12) among which compounds 1 and 2 were found to be new ones. All of them simultaneously inhibited PTP1B with IC₅₀s of (2.4–52.5?µM), and α-glucosidase with IC₅₀ values of (1.7–72.7?µM), respectively. Cratoxanthone A (3) and γ-mangostin (7) were estimated to be most active inhibitors against both PTP1B (IC₅₀?=?2.4?µM for 3, 2.8?µM for 7) and α-glucosidase (IC₅₀?=?4.8?µM for 3, 1.7?µM for 7). In kinetic studies, all isolated xanthones emerged to be mixed inhibitors of α-glucosidase, whereas they behaved as competitive inhibitors of PTP1B. In time dependent experiments, compound 3 showed isomerization inhibitory behavior with following kinetic parameters: K_i^app?=?2.4?µM; k₅?=?0.05001?µM^?1?S^?1 and k₆?=?0.02076?µM^?1?S^?1.