Continuous erythropoietin delivery by muscle-targeted gene transfer using in vivo electroporation.

It has been demonstrated that gene transfer by in vivo electroporation of mouse muscle increases the level of gene expression by more than 100-fold over simple plasmid DNA injection. We tested continuous rat erythropoietin (Epo) delivery by this method in normal rats, using plasmid DNA expressing rat Epo (pCAGGS-Epo) as the vector. A pair of electrodes was inserted into the thigh muscles of rat hind limbs and 100 microg of pCAGGS-Epo was injected between the electrodes. Eight 100-V, 50-msec electric pulses were delivered through the electrodes. Each rat was injected with a total of 400 microg of pCAGGS-Epo, which was delivered to the medial and lateral sides of each thigh. The presence of vector-derived Epo mRNA at the DNA injection site was confirmed by RT-PCR. The serum Epo levels peaked at 122.2 +/- 33.0 mU/ml on day 7 and gradually decreased to 35.9 +/- 18.2 mU/ml on day 32. The hematocrit levels increased continuously, from the preinjection level of 49.5 +/- 1.1 to 67.8 +/- 2.2% on day 32 (p < 0.001). In pCAGGS-Epo treated rats, endogenous Epo secretion was downregulated on day 32. In a control experiment, intramuscular injection of pCAGGS-Epo without subsequent electroporation did not significantly enhance the serum Epo levels. These results demonstrate that muscle-targeted pCAGGS-Epo transfer by in vivo electroporation is a useful procedure for the continuous delivery of Epo.

Phorbol 12,13-diacetate-induced contraction of the canine basilar artery: role of protein kinase C.

The pharmacological and biochemical mechanisms of contractile responses to the protein kinase C (PKC) activator phorbol-12,13-diacetate (PDA) were investigated in canine basilar arteries. In the normal medium, PDA elicited a strong, dose-related, and slow-developing sustained contraction. Among the constrictors examined, including serotonin, prostaglandin F2 alpha, and endothelin, only PDA yielded contractions in a Ca2(+)-free medium. In both media, the PDA-induced contractions were virtually inhibited by either staurosporine, H-7, or quinacrine, while neither neurotransmitter blockades nor R24571 (calmidazolium) exerted significant effects. In addition, it was shown that 8-bromocyclic GMP, but not 8-bromocyclic AMP, markedly curtailed the PDA-induced contractions. Biochemical analysis, furthermore, showed that PDA induced increased phosphorylations of 27- and 96-kDa and proteins other than the myosin light chain (MLC) 20-kDa protein. Thus, the present results open up a novel mechanism of sustained cerebral artery contractions, where PKC activation rather than Ca2+/calmodulin/MLC system plays a key role that is regulated both by phospholipase A2 and by cyclic GMP.

Impaired plasticity of neurons in aging. Biochemical, biophysical, and behavioral studies.

Age-related correlation of impaired plasticity of neurons (biochemical and biophysical aspects) and behavioral alterations were investigated in young (3.5 months) and extremely aged (approximately 40 months) female Wistar rats. Age-dependent significant differences in second messenger (cAMP and Ins (1,4,5)P3) concentration and signal transduction via muscarinic and dopaminergic receptors were found. The results point to the specifically impaired coupling between dopamine D1 receptor and GS protein, which underlies normal brain aging. However, cholinergic neurotransmission may be modulated at another level in extremely aged rats. Thus, it appears that the site of affection in coupling of receptor and G protein and/or G protein-dependent signal transduction in aging cannot be generalized. This indicates that alterations in the coupling of signal transduction depend on diverse neurotransmitter receptors with advanced age. The age-dependent alterations in the cAMP and PI signal pathways could be due to changes in the physical properties of the membranes. To support this hypothesis, age-dependent changes in the physical state and the biochemical composition of synaptosomal membranes from the cortex, cerebellum, and striatum were examined by measuring the steady-state fluorescence amisotropy of the membrane probes 1,6-diphenyl-1,3,5-hexatriene (DPH), trimethylammonium-DPH (TMA-DPH), and trimethylammoniumpropyl-DPH (TMAP-DPH). Significant differences in the physical properties of the synaptosomal membranes existed between young and very aged rats, expressed by a higher anisotropy in the 40-month-old rat brain tissue. The changes in the physical properties of the membranes were in line with the determined age-dependent alterations in the chemical composition, e.g., the increase in cholesterol content of the aged membranes.

Enhancement of neurite outgrowth by the soluble form of human L1 (neural cell adhesion molecule).

L1, a neural cell adhesion molecule, is involved in neurite outgrowth, migration and fasciculation. Although L1 is a membrane glycoprotein expressed on neural cells, the soluble form of L1 is generated in vivo by proteolysis. In the present study, a stable transfectant of Chinese hamster ovary (CHO) cells secreting human L1 without cytoplasmic and membrane spanning domains was generated, and the function of the secreted L1 was examined. Explants from embryonic chick brain stem were cultured on a substrate coated with polyethylenimine (PEI) alone, on substrate-bound L1 or in medium containing soluble L1. The neurites induced by L1, both the substrate-bound form and the soluble form, were 2-3 times longer than those cultured on PEI. The ability of the soluble L1 to induce neurite formation was slightly greater than that of the substrate L1. The present results demonstrated that neurite outgrowth was induced not only by substrate-bound L1 but also by soluble L1. Soluble L1 could be a pharmaceutical candidate for the promotion of nerve regeneration.

Age-related alteration in signal transduction: involvement of the cAMP cascade.

In the present study, we have investigated the involvement of the cAMP signal transduction pathways in young and aged rats. A significantly higher endogenous adenosine 3':5'-cyclic monophosphate (cAMP) level and a significant decline of the adenylate cyclase [AC, ATP pyrophosphate-lyase (cyclizing), EC.4.6.1.1.] activity were observed in striatal tissue from young rats (3 months) in comparison to aged rats (approximately 40 months). In the nucleus accumbens (NA), no age-dependent changes in the cAMP concentration and in the AC basal activity were found. To address the question, whether the interactions of guanine nucleotide-binding protein (G-protein) subunits (G alpha s and Gi) with AC have changed in the aging process, various pharmacological agents that modulate the AC activity (e.g., beta, tau-imidoguanine 5'-triphosphate (GppNHp), sodium fluoride (NaF), forskolin (FSK), and the combinations of GppNHp plus FSK, NaF plus FSK, and NaF plus ethanol (ETOH)) were applied. In addition, a [3H]FSK binding test was carried out. In striatal and NA tissue, the stimulation of the AC activity by FSK was inhibited by GppNHp (via Gi-protein) and was superadditive by the combination of FSK and NaF (via Gs-protein). The absolute AC activity upon stimulation by all agents used was significantly lower in the aged striatum compared to young striatum. In the NA, however, the AC activity showed an age-dependent reduction only upon FSK and upon FSK plus GppNHp stimulation. There was no difference in the specific [3H]FSK binding to the G alpha s protein-coupled catalytic subunit of the AC between young and aged animals both in the striatum and NA.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered dopamine receptor mediated signal transmission in the striatum of aged rats.

Striatal membranes of very old (40 months) as against young (3 months) female Wistar rats were used. Binding saturation experiments with [3H]SCH 23390 at the dopamine (DA) D1 receptor (D1) and [3H]spiperone at the DA D2 receptor (D2) revealed no change in the affinity (Kd) but a significant decrease in the density (Bmax) of D1 (-31%, P < 0.005) and of D2 (-22%, P < 0.05), respectively, in the aged vs. young striata. Displacement of either [3H]SCH 23390 or [3H]spiperone binding by DA displayed biphasic curves. The Hill coefficient (nH) was significantly increased in the senescent compared with the young of D1 (0.72 +/- 0.04 vs. 0.61 +/- 0.03, P < 0.025) but unchanged of D2 (0.49 +/- 0.04 vs. 0.51 +/- 0.02). The proportion of the high-affinity agonist binding state (Rhigh) was significantly decreased (P < 0.025) in the older (20.9 +/- 3.2%) in comparison with the young (30.6 +/- 2.0%) in D1 but increased non-significantly in D2 (47.9 +/- 2.6 vs. 40.5 +/- 5.1%). Calculating the resulting Bmax from Scatchard and displacement analyses of each single aged and young animal revealed a highly significant reduction (P < 0.001) of the high-affinity agonist binding state of D1 (-53%) as well as a non-significant reduction of D2 (-8%) in the older. Simultaneously, a significant 57% decrease (P < 0.01) in the adenylate cyclase (AC) activity stimulated by 10 microM DA in the senescent compared with the young animals was monitored. The DA stimulation of AC was reversed in both cases by the addition of 200 nM of the D1 antagonist SCH 23390.

Oxidized low density lipoprotein caused CNS neuron cell death.

Death induced by oxidized low density lipoproteins (oxLDL) to embryonic CNS neuronal and neuroblastoma cells was investigated. Cell damage and viability were evaluated by LDH leakage and the MTT method, respectively. Dose- and time-dependent degeneration of neurons occurred after oxLDL (1-100 microg/ml) treatment but was absent after native low density lipoproteins (LDL). This degeneration was mediated, in part, by apoptosis because increased TUNEL and Hoechst dye-positive staining was observed. These effects occurred in the absence of microglia. However, DNA degradation was not detected. The cytotoxicity was attenuated by pre-treatment with antioxidants. These results suggest that oxidation by oxLDL may be important in neurocytotoxicity in the brain.

Alterations of inositol phosphate turnover in striatum of aged rats.

Age-related inositol phosphate turnover in the rat central nervous system was investigated. Higher phospholipase-C activity and drastically higher (almost 2.5-fold) inositol 1,4,5-trisphosphate concentration in the corpus striatum (caudate-putamen) of extremely old (approximately 40 months) female Wistar rats in comparison to the young adult (approximately 3.5 months) rats were observed. Dopamine seems to slightly inhibit total inositol phosphate formation and this effect was antagonized by (-)-sulpiride.

BY-1949 elicits vasodilation via preferential elevation of cyclic GMP levels within the cerebral artery: possible involvement of endothelium-mediated mechanisms.

The pharmacological mechanisms by which BY-1949, a novel dibenzoxazepine derivative, increases in regional cerebral blood flow, were investigated using the canine basilar artery in vitro. BY-1949 inhibited contractions elicited by serotonin (5-HT), prostaglandin (PG) F2 alpha, endothelin and phorbol-12,13-diacetate (PDA), respectively, to the same extent. In addition, pretreatment of the artery with methylene blue significantly suppressed the vasodilating effect of BY-1949. BY-1949 also dose dependently suppressed contractions of the basilar artery induced by CaCl2 (Ca2+) in a non-competitive manner. Biochemical studies disclosed that BY-1949 significantly increased cyclic GMP without causing any apparent change in cyclic AMP. These increases in cyclic GMP were virtually abolished after the endothelial cells were removed. These results strongly suggest that the increased regional cerebral blood flow induced by BY-1949 is explicable, at least partly, in terms of a preferential elevation of cyclic GMP within the cerebral vasculature, where the endothelium plays a pivotal role.

The role of cGMP in the anti-aggregating properties of BY-1949, a novel dibenzoxazepine derivative.

The anti-aggregatory activity of a novel agent, BY-1949, 3-methoxy-11-methyldibenz (b,f) (1,4) oxazepine-8-carboxylic acid, was examined using rabbit platelets. Oral administration of BY-1949 (10 or 30 mg/kg) inhibited platelet aggregation induced by ADP, collagen, and arachidonate in a dose-related fashion. In in vitro studies, however, neither BY-1949 nor its major metabolites inhibited platelet aggregation, even at a concentration similar to that attained in plasma in vivo. With regard to the anti-aggregatory action of BY-1949, biochemical analysis revealed that BY-1949 preferentially augmented cyclic GMP (cGMP) formation, via inhibition of phosphodiesterase activity, without altering cyclic AMP (cAMP) formation. Furthermore, the in vitro anti-aggregatory activity was significantly enhanced when the platelets were concomitantly treated with nitric oxide (NO). Based on these results, it is suggested that the in vivo anti-aggregatory effects of BY-1949 are at least partly elicited via platelet/endothelium interactions, in which cGMP plays a pivotal role.

A novel dibenzoxazepine derivative (BY-1949) increases regional cerebral blood flow.

The effects of BY-1949, a novel dibenzoxazepine derivative, on the regional cerebral blood flow were investigated in conscious cats. Oral administration of BY-1949 (10-50 mg/kg) significantly increased in a dose-related manner the regional cerebral blood flow in all brain regions examined. Vinpocetine (20 mg/kg p.p.) had similar effects that were shorter-lasted than those of BY-1949. From these results, it seems likely that amelioration by BY-1949 of cognitive impairment following cerebral ischemia/hypoxia or that occurs on ageing is at least partly explainable in terms of its effects on the cerebral circulation.

Five cases of Diphyllobothrium nihonkaiense infection with discovery of plerocercoids from an infective source, Oncorhynchus masou ishikawae.

Five persons from 2 families residing at Miyama Town, Mie Prefecture, Japan, ingested fresh raw fish Oncorhynchus sp. on 9 May 1999 that was caught at Owase district in Mie. They all expelled diphyllobothriid cestodes 11-37 days after ingesting the fish. The parasites were morphologically identical to Diphyllobothrium nihonkaiense Yamane et al., 1986. Five plerocercoids were detected from a portion of the fish. Nucleotide sequence of a region of the cytochrome c oxidase subunit I gene of mitochondrial DNA from an adult worm was identical with that from the plerocercoid. The fish was identified as Oncorhynchus masou ishikawae according to the nucleotide sequence of the nuclear ribosomal second internal transcribed spacer region II gene. This is the first record of D. nihonkaiense plerocercoids from O. m. ishikawae.

Rhabdomyolysis and myocardial damage induced by palytoxin, a toxin of blue humphead parrotfish.

A 55-year-old man had rhabdomyolysis and myocardial damage induced by palytoxin. Weakness and myalgia of four extremities occurred five hours after eating a fish. Rhabdomyolysis developed and the serum creatine phosphokinase (CK) was elevated to 40,000 IU/l on the 3rd day. Gastric lavage with activated charcoal and forced mannitol-alkaline diuresis therapy were performed. The patient recovered with no complication such as renal failure. In this case, palytoxin was suggested to induce myocardial damage which was demonstrated by an elevation of the myosin light chain level and a change in electrocardiogram.

Signal transduction in aging.

The possible age-related involvement of two different signal transduction pathways in the rat CNS was investigated. In the phosphytidyl inostiol (PI) response, higher phospholipase-C (PL-C) activity and drastically higher (almost 2.5-fold) inositol (1,4,5)trisphosphate (Ins(1,4,5) P(3)) concentration in the corpus striatum (caudate-putamen) of extremely old (approximately 40 months) female Wistar rats in comparison to young adult (approximately 3.5 months) rats were observed. In the adenosine 3':5'-cyclic monophosphate (cAMP) cascade, a significantly higher endogenus cAMP level and a significant decline of the adenylate cyclase (AC, ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1.) activity were observed in striatal tissue from young rats in comparison with aged rats. Binding saturation experiments with [(3)H]SCH 23390 at the dopamine (DA) D(1) receptor (D(1)) and [(3)H]spiperone at the DA D(2) receptor (D(2)) revealed no change in the affinity (K(d)) but a significant decrease in the density (B(max)) of D(1) (-31%, p<0.005) and of D(2) (-22%, p<0.05), respectively, in the aged versus young striata. DA seems to slightly inhibit total inositol phosphate formation and this effect was antagonized by (-)-sulpiride. A significant decrease (p<0.05) in the AC activity stimulated by 10 muM DA in the senescent compared to the young animals was monitored. Apparently, the age-related decline of the AC activity was independent of changes of G(S) and G(i) activity.

Assessment of mitral regurgitation using gated radionuclide ventriculography: analysis of left atrial time activity curve.

The authors analyzed the left atrial (LA) time activity curve (TAC) in 18 normal subjects and 30 patients with mitral regurgitation (MR) to assess the usefulness of radionuclide ventriculography (RNV) in detecting MR. The LA TAC was generated from gated blood pool images using phase and amplitude images. The configuration of normal LA TAC was M shaped. The first peak and last peak of LA TAC were represented as points B and D, respectively. In addition, the trough following B was named point C. The presence and severity of MR was analyzed by use of three methods: (1) analysis of LA TAC, (2) analysis of left ventricular TAC, and (3) measurement of the stroke count ratio of left ventricle to right ventricle. In the diagnosis of MR using RNV, the rapid emptying fraction [REF:(B-C)/(B-A)] of LA TAC was the most sensitive index compared with the other two methods. The sensitivity and specificity of MR with Sellers' II degrees or more were 0.84 and 0.90 in LA TAC but were 0.42 and 0.90 in the count method.