Responses of upper cervical spinal neurons in cats to stimulation of the brain-stem locomotor region at different frequencies

Synaptic responses of neurons in segments C₂ and C₃ to stimulation of locomotor points in the medulla or midbrain were recorded extracellularly in mesencephalic cats. Neurons generating responses with an index of 0.4–0.6 to stimulation with a frequency of 2 Hz maintained this same index at frequencies of 20–60 Hz. The discharge index of many neurons during stimulation at 2 Hz was low, and it increased to 0.4–0.6 when high-frequency stimulation was used. More than half of the cells were excited by stimulation of both ipsilateral and contralateral locomotor points; one-quarter of the neurons responded to stimulation of locomotor points in both medulla and midbrain. The cells studied were located 1.8–4.2 mm from the dorsal surface of the spinal cord. The mean latencies of responses with an index of not less than 0.5 lay within the range 2–30 msec, with a mode of 2–8 msec. Considerable fluctuations of latent period were observed for long-latency responses. The possibility that the neurons studied may participate in the transmission of activity from the locomotor region of the brain stem to stepping generators in the spinal cord is discussed.Institute for Problems of Information Transmission, Academy of Sciences of the USSR, Moscow. M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 15, No. 4, pp. 355–361, July–August, 1983.     

The rhombencephalic "locomotor region" in turtles

Electrical stimulation (10–20 µA, 20–30 Hz) of the rhombencephalon in decerebrate turtles can induce cyclic coordinated limb movemnts. The "locomotor region" is a strip, oriented in the rostro-caudal direction, which coincides in its location with the lateral reticular formation. Both in the medial and in the lateral reticular formation extracellular ipsilateral and contralateral synaptic responses of single neurons evoked by stimulation of the "locomotor region," (10–30 µA, 2 Hz), were recorded. Usually these responses had latent periods of between 3 and 12 msec (mode 5–6 msec). Excitation of the "locomotor region" thus leads to extensive spread of activity in the rhombencephalon. The possible mechanisms of this spread are discussed.Institute of Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 12, No. 4, pp. 382–390, July–August, 1980.     

Convergence between influences from midbrain and bulbar locomotor sites and from an inhibitory site in neurons of the medulla

Synaptic response to regular stimulation of midbrain and bulbar locomotor sites (LS) and a pontine inhibitory site (IS) was recorded in medial and lateral bulbar neurons in cats (mesencephalic decerebellate preparation). Excitatory post-synaptic potentials (PSP) and discharges were usually noted in medial neurons; mixed PSP also occurred when stimulating the IS. Almost 50% of lateral and over 25% of medial neurons showed a change in background firing rate, failing to generate response time-locked to stimulus. Medial neurons producing a response time-locked to the stimulus showed equal sensitivity to stimulation of midbrain and bulbar LT and very little reaction to IS stimulation. Medial neurons with a response not time-locked to stimuli together with lateral neurons were most receptive to input from the bulbar LS, less sensitive to stimulation of the midbrain LS, and least responsive of all to IS stimulation. Convergence between influences from midbrain and bulbar LS was the same in neurons of all populations. The part played by different neuronal populations in initiation and cessation of locomotion is discussed.Institute for Research into Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 23, No. 3, pp. 297–306, May–June, 1991.     

Propagation of activity along the “stepping strip” of the cat spinal cord

Three points located approximately 8 mm apart were identified in a dorsolateral funiculus of the lower thoracic spinal cord in mesencephalic cats, each producing stepping movements on the ipsilateral hindlimb when stimulated. An area 5–17 mm caudal to the caudal stepping point (SP) was scanned for neurons responding synaptically to stimulating the rostral or caudal SP prior and subsequent to electrolytic coagulation of the medial SP. Relative incidence of neurons excited by stimulating the caudal SP did not change following this type of lesioning, although stimulation of the rostral SP at the rate of 4 Hz induced response 5 times less frequently than before. Even stimulation of the rostral SP at the rate of 40–60 Hz, which had considerably increased firing index prior to coagulation, could only produce excitation in tiny numbers of neurons. This indicates that synaptic excitation of neurons becomes considerably more difficult once the stepping strip between stimulation and recording sites has been damaged.Institute for Research into Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 20, No. 6, pp. 763–769, November–December, 1988.     

Endangered wolves cloned from adult somatic cells

Over the world, canine species, including the gray wolf, have been gradually endangered or extinct. Many efforts have been made to recover and conserve these canids. The aim of this study was to produce the endangered gray wolf with somatic cell nuclear transfer (SCNT) for conservation. Adult ear fibroblasts from a female gray wolf (Canis lupus) were isolated and cultured in vitro as donor cells. Because of limitations in obtaining gray wolf matured oocytes, in vivo matured canine oocytes obtained by flushing the oviducts from the isthmus to the infundibulum were used. After removing the cumulus cells, the oocyte was enucleated, microinjected, fused with a donor cell, and activated. The reconstructed cloned wolf embryos were transferred into the oviducts of the naturally synchronized surrogate mothers. Two pregnancies were detected by ultrasonography at 23 days of gestation in recipient dogs. In each surrogate dog, two fetal sacs were confirmed by early pregnancy diagnosis at 23 days, but only two cloned wolves were delivered. The first cloned wolf was delivered by cesarean section on October 18, 2005, 60 days after embryo transfer. The second cloned wolf was delivered on October 26, 2005, at 61 days postembryo transfer. Microsatellite analysis was performed with genomic DNA from the donor wolf, the two cloned wolves, and the two surrogate female recipients to confirm the genetic identity of the cloned wolves. Analysis of 19 microsatellite loci confirmed that the cloned wolves were genetically identical to the donor wolf. In conclusion, we demonstrated live birth of two cloned gray wolves by nuclear transfer of wolf somatic cells into enucleated canine oocyte, indicating that SCNT is a practical approach for conserving endangered canids.     

Furin is an endogenous regulator of alpha-secretase associated APP processing

Constitutive and PKC-regulated alpha-secretase pathways have been reported to produce the secreted form of alpha-secretase-derived APP (sAPPalpha). Here, we examined putative role of furin in the regulation of alpha-secretase activity in vitro and in vivo. Overexpression of the prodomain of furin and infection with a furin-specific inhibitor significantly reduced the levels of sAPPalpha regardless of PKC activity, whereas total APP levels remained unchanged. Furin mRNA levels in the brains of AD patients and Tg2576 mice were significantly lower than those in controls, whereas ADAM10 and TACE mRNA levels were much alike between Tg2576 and littermate mice. Moreover, the injection of furin-adenovirus into Tg2576 mouse brains markedly increased alpha-secretase activity and reduced beta-amyloid protein (Abeta) production in infected brain regions. Our results suggest that furin enhances alpha-secretase activity via the cleavage of ADAM10 and TACE, and that attenuated furin activity is connected to the production of Abeta.     

Structure of Chlorobium tepidum sepiapterin reductase complex reveals the novel substrate binding mode for stereospecific production of L-threo-tetrahydrobiopterin

Sepiapterin reductase (SR) is involved in the last step of tetrahydrobiopterin (BH(4)) biosynthesis by reducing the di-keto group of 6-pyruvoyl tetrahydropterin. Chlorobium tepidum SR (cSR) generates a distinct BH(4) product, L-threo-BH(4) (6R-(1'S,2'S)-5,6,7,8-BH(4)), whereas animal enzymes produce L-erythro-BH(4) (6R-(1'R,2'S)-5,6,7,8-BH(4)) although it has high amino acid sequence similarities to the other animal enzymes. To elucidate the structural basis for the different reaction stereospecificities, we have determined the three-dimensional structures of cSR alone and complexed with NADP and sepiapterin at 2.1 and 1.7 A resolution, respectively. The overall folding of the cSR, the binding site for the cofactor NADP(H), and the positions of active site residues were quite similar to the mouse and the human SR. However, significant differences were found in the substrate binding region of the cSR. In comparison to the mouse SR complex, the sepiapterin in the cSR is rotated about 180 degrees around the active site and bound between two aromatic side chains of Trp-196 and Phe-99 so that its pterin ring is shifted to the opposite side, but its side chain position is not changed. The swiveled sepiapterin binding results in the conversion of the side chain configuration, exposing the opposite face for hydride transfer from NADPH. The different sepiapterin binding mode within the conserved catalytic architecture presents a novel strategy of switching the reaction stereospecificities in the same protein fold.     

Daidzein activates choline acetyltransferase from MC-IXC cells and improves drug-induced amnesia

The choline acetyltransferase (ChAT) activator, which enhances cholinergic transmission via an augmentation of the enzymatic production of acetylcholine (ACh), is an important factor in the treatment of Alzheimer's disease (AD). Methanolic extracts from Pueraria thunbergiana exhibited an activation effect (46%) on ChAT in vitro. Via the sequential isolation of Pueraria thunbergiana, the active component was ultimately identified as daidzein (4',7-dihydroxy-isoflavone). In order to investigate the effects of daidzein from Pueraria thunbergiana on scopolamine-induced impairments of learning and memory, we conducted a series of in vivo tests. Administration of daidzein (4.5 mg/kg body weight) to mice was shown significantly to reverse scopolamine-induced amnesia, according to the results of a Y-maze test. Injections of scopolamine into mice resulted in impaired performance on Y-maze tests (a 37% decreases in alternation behavior). By way of contrast, mice treated with daidzein prior to the scopolamine injections were noticeably protected from this performance impairment (an approximately 12%-21% decrease in alternation behavior). These results indicate that daidzein might play a role in acetylcholine biosynthesis as a ChAT activator, and that it also ameliorates scopolamine-induced amnesia.     

Effect of new rotenoid glycoside from the fruits of Amorpha fruticosa LINNE on the growth of human immune cells

A new compound, rotenoid isoflavone glycoside named, 6′-O-β-d-glucopyranosyl-12a-hydroxydalpanol was isolated from the methanolic (MeOH) fruit extract of Amorpha fruticosa LINNE by means of multi-stage column chromatography. Immuno-modulatory activities of this new glycoside were compared with the partitioned fractions of Amorpha fruticosa LINNE. Both of the fractions and purified single compound showed a 19% relatively low cytotoxicity at a maximum concentration of 1.0 g/L in a cultivated normal human lung cell line (HEL299). The purified single compound showed less cytotoxicity than the crude extracts, possibly because residual toxicants were eliminated during purification processes. Cell growth of human T cells was increased by about 15% by adding 0.5 g/L of the fractions compared to the control. Specific production rates of interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) from T cell were higher as 1.16 × 10⁻⁴ and 1.86 × 10⁻⁴ pg/cell, respectively, in the purified compound, compared to 1.38 × 10⁻⁴ and 2.22 × 10⁻⁴ pg/cell, respectively, by adding 0.5 g/L of the dichloromethane fraction. Natural killer cell-92MI (NK-92MI) growth supplemented with the supernatant of human T cell was up to 19% higher with the dichloromethane fraction compared with a new single compound at a concentration of 0.5 g/L. Overall, the dichloromethane fraction showed relatively higher immuno-modulatory activities compared with a new single compound, probably due to the synergic effect given by other substances existing in the fractions.