Cardioprotective effects of fucoidan against hypoxia-induced apoptosis in H9c2 cardiomyoblast cells.

CONTEXT: Cardiomyocyte apoptosis plays a critical role in the progress of heart diseases. Fucoidan, a complex-sulfated polysaccharide, has been reported to possess potential cardioprotective efficacy in vivo. OBJECTIVE: The present study determines whether fucoidan could provide cardioprotection on hypoxia-induced cardiomyocyte apoptosis. MATERIALS AND METHODS: H9c2 cardiomyoblast cells were incubated with various concentrations (15, 30, and 60 µg/ml) of fucoidan in a humidified incubator at 37 °C with 95% O2 and 5% CO2. After 6 h, hypoxia was processed and the cardioprotective effects of fucoidan were evaluated by applying MTT, ELISA, Hoechst 33258 nucleus staining, and western blot. RESULTS: Following a 6 h exposure of H9c2 to hypoxic condition, significant reduction was found in cell survival (0.57-fold) and superoxide dismutase (SOD) activity (0.56-fold), which were associated with the increase of malondialdehyde (MDA) level (2.58-fold), creatine phosphokinase (CK, 3.57-fold), and lactate dehydrogenase (LDH) activities (2.39-fold). Moreover, hypoxia-induced apoptosis was confirmed by Hoechst 33258 nuclear staining, and these changes were accompanied by the increase of Bcl-2 (1.27-fold) and Bax expression (2.6-fold). However, preincubation of the cells with fucoidan prior to hypoxia exposure elevated the cell viability (30 µg/ml, 1.18-fold; 60 µg/ml, 1.32-fold) and SOD activity (30 µg/ml, 1.12-fold; 60 µg/ml, 1.25-fold), but decreased the MDA level (30 µg/ml, 0.70-fold; 60 µg/ml, 0.80-fold), CK (30 µg/ml, 0.69-fold; 60 µg/ml, 0.76-fold), and LDH (30 µg/ml, 0.67-fold; 60 µg/ml, 0.86-fold) leakages. Hoechst 33258 nuclear staining observations demonstrated the same protective effect of fucoidan on hypoxia-induced myocardial injury. Also, cardioprotective effects of fucoidan were reflected by increasing Bcl-2 (60 µg/ml, 1.84-fold), as well as decreasing Bax (60 µg/ml, 0.6-fold). CONCLUSION: Fucoidan had protective effect against hypoxia-induced cardiomyocytes apoptosis, and the mechanism might involve protections of the cell from oxidative injury.

Comparative genome-wide gene expression analysis of rheumatoid arthritis and osteoarthritis.

Both rheumatoid arthritis (RA) and osteoarthritis (OA) are complex diseases. Studies and treatment of RA and OA have mainly focused on individual factors. However, there is still no clear understanding of their causes and adequate treatment alternatives are still being sought. We applied gene set-enrichment analysis to microarray datasets of RA and OA to look for regulatory mechanisms. We found 32 highly significant pathways, including 18 downregulated and 14 upregulated pathways associated with RA. We also identified 18 highly significant pathways, including 7 downregulated and 11 up-regulated pathways associated with OA. Several such pathways were found in both RA and OA, including an upregulated PPAR signaling pathway and downregulated leukocyte transendothelial migration. Regulatory mechanisms in RA seem to be more complex than in OA. This information could be useful for diagnosis and treatment of these two diseases.

Newly identified respiratory viruses associated with acute lower respiratory tract infections in children in Lanzou, China, from 2006 to 2009.

Nasopharyngeal aspirates were collected from 813 children ≤ 14 years old with acute lower respiratory tract infections in Lanzhou, China, from December 2006 to November 2009. PCR or RT-PCR was used to screen for the presence of 10 respiratory viruses. Viral agents were identified in 73.92% (601/813) of specimens, including RSV in 40.71%, hMPV in 6.15%, IFVA in 7.13%, IFVB in 0.98%, PIV1-3 in 7.87%, HCoV-HKU1 in 2.21%, HCoV-NL63 in 3.81%, HRV in 19.93%, AdV in 7.50% and HBoV in 11.56%. Two or more viruses were detected in 34.44% (280/813) of cases. The newly identified respiratory viruses, HBoV, hMPV, HCoV-HKU1 and HCoV-NL63, accounted for 22.01% of the detected viral pathogens. RSV and HRV were frequently detected in patients with bronchiolitis, and hMPV was frequently associated with pneumonia. HCoV-NL63 was found to be one of the causative agents of acute respiratory wheezing in young children. No seasonal variation was found in the incidence of detection of HCoV-HKU1, HCoV-NL63 or HBoV. This 3-year study demonstrated that viral pathogens play an important role in children with ALRTIs, and more attention should be paid to these newly identified viral agents.

Retraction of articles by T. Liu et al.

A series of 29 papers by Liu et al. are retracted.

Neural stem/progenitor cells survive and differentiate better in PD rats than in normal rats.

To investigate the effects of grafted neural stem/mesencephalic progenitor cells (NSCs/MP) on rotational behavior of Parkinson's disease (PD) rats and the influence of intracerebral environment on NSCs/MP, we observed the survival and differentiation of NSCs/MP transplanted into 6-hydroxydopamine (6-OHDA)-lesioned and intact striatums. NSCs/MP were prepared from E(11-15) rats and proliferated in serum-free medium with bFGF for several weeks. One day after being primed with serum/dbcAMP to differentiate, cell suspensions were grafted into 6-OHDA-lesioned and intact striatums respectively. It had been found that NSCs/MP were able to survive better and differentiate into more tyrosine hydroxylase (TH)-positive neurons in 6-OHDA-lesioned striatums than in intact ones, and apomorphine-induced rotations were obviously attenuated in MP graft models. The data suggested that NSCs/MP tend to survive and differentiate into TH-positive neurons in 6-OHDA-lesioned striatums. The data demonstrated that striatums in which DAergic terminals are destroyed by 6-OHDA undergo some changes and thus provide more appropriate conditions for NSCs/MP to differentiate into mature DAergic neurons. Furthermore, the finding that MP had greater relieving effects on rotational behavior than NSCs suggests that NSCs could not be used in clinical therapy of PD unless being induced into MP in vitro before transplantation.

Mesencephalic progenitors can improve rotational behavior and reconstruct nigrostriatal pathway in PD rats.

The aim of this study was to investigate the possibility of mesencephalic progenitors (MP) in treating Parkinson's disease (PD). MP were prepared from E(11-13) rats and proliferated in serum-free medium with basic fibroblast growth factor (bFGF) for 10 days. Cells were then collected and implanted into the striatum only--single grafts or simultaneously into the substantia nigra (SN) and the striatum--double grafts. Twelve weeks after transplantation, DiI, a fluorescent dye, was microinjected into the ipsilateral striatum. Using this strategy, it was found that MP of double grafts had more potent effects on rotational behavior than that of single grafts. Injection of the retrograde tracer DiI into the striatum resulted in fluorescent-labeled cells within the intranigral grafts in double grafts. These data greatly support that MP transplants could not only improve rotational behavior, but does help to re-establish nigrostriatal connections so that it may become one efficient way in treating PD.

Nod factors and chitooligomers elicit an increase in cytosolic calcium in aequorin-expressing soybean cells.

Rhizobial Nod factors (NFs) function as nodulation signals that trigger symbiotic responses of leguminous host plants. NFs consist of a chitin oligomer backbone carrying a fatty acid at the non-reducing end. Depending on the rhizobial strain, NFs carry additional substituents, which may determine host specificity. Transgenic suspension-cultured soybean (Glycine max [L.] Merr.) cells expressing aequorin have been used to record cytosolic [Ca(2+)] changes upon treatment with purified NFs and chitin fragments. Both compounds elicited an increase of cytosolic [Ca(2+)] at nanomolar concentrations. The shape and amplitude of cytosolic [Ca(2+)] changes was similar to the response elicited by un-derivatized chitin oligomers. Cells challenged first with NFs did not respond to a subsequent treatment with chitin oligomers and vice versa. Dose-response experiments showed that un-derivatized chitin oligomers were more active compared with NFs. The capacity of NFs to elicit the calcium response depended on their structure. The presence of reducing end substituents in methylfucosylated NFs from Rhizobium sp. NGR234 and the O-acetyl group at the non-reducing end in NFs from Sinorhizobium meliloti attenuated the activity to cause the calcium changes. The sulfate group in NFs from Rhizobium tropici did not affect the elicitor activity. Pentameric S. meliloti NFs were more active than tetrameric molecules, whereas trimeric or dimeric degradation products were inactive. Substituents in NFs may have the function to avoid stimulation of defense reactions mediated by the perception system for chitin oligomers.

[Whole-cell recordings of the supraoptic nucleus neurons from rat hypothalamic slices in vitro].

Fifty-two supraoptic nucleus neurons in rat slice preparations were studied using whole cell patch-clamp technique. The mean passive and active membrane properties were measured as follows: resting membrane potential, 59 +/- 8 mV; input resistance, 535 +/- 129 M omega; time constant, 32 +/- 9 ms; amplitude of the action potentials, 99 +/- 11 mV; overshoot, 37 +/- 13 mV (n = 39). Most of these neurons showed a prominent slow after-hyperpolarization potential or current in response to depolarizing pulse. In votage-clamp condition, it was found that virtually all supraoptic neurons (n = 13) were invaded by spontaneous synaptic inputs. Pharmacological experiments showed that the excitatory postsynaptic currents (EPSCs) were mediated by non-NMDA glutamate receptors, whereas inhibitory postsynaptic currents (IPSCs) by GABAA receptors.

Blockade of neuromuscular transmission by huwentoxin-I, purified from the venom of the Chinese bird spider Selenocosmia huwena.

Huwentoxin-I (HWTX-I) is a neurotoxic peptide purified from the venom of the Chinese bird spider Selenocosmia huwena. The effects of HWTX-I on neuromuscular transmission of vertebrate skeletal muscle have been investigated by means of twitch tension and electrophysiological techniques. On isolated mouse phrenic nerve-hemidiaphragm preparations, HWTX-I blocked the twitch responses to indirect, but not to direct, muscle stimulation. The time needed for complete block of the neuromuscular transmission was dose dependent. The transmission could be mostly restored by prolonged repeated washing with Tyrode's solution. If the preparation was pretreated with D-tubocurarine and then immersed in a mixed solution of D-tubocurarine and HWTX-I, the washout time necessary to restore the neuromuscular transmission was significantly decreased. Intracellular recording at the end-plate region of frog sartorius muscle revealed that HWTX-I could synchronously reduce the amplitude of the acetylcholine potential induced by ionophoretic application of acetylcholine as well as the amplitude of the end-plate potential evoked by nerve stimulation. Both of these effects eventually disappeared; however, both could be restored by prolonged washing. Experiments on Xenopus embryonic myocytes indicated that HWTX-I reduced the open probability of acetylcholine-induced channel activity, and finally blocked the channel. All of these results demonstrated that HWTX-I was a peptide neurotoxin and the postsynaptic nicotinic acetylcholine receptor was its site of action.

[Whole cell recording of sodium, potassium and calcium currents of cultured neonatal rat sympathetic neurons].

Na, K and Ca currents and other electrophysiological characteristics of cultured neonatal rat superior cervical sympathetic neurons were studied using whole cell clamp technique. The mean passive and active membrane properties measured are as follows: resting membrane potential, -51 +/- 6 mV; input resistance, 1432 +/- 389 M omega; time constant, 130 +/- 32 ms; amplitude of action potential, 96 +/- 10 mV; overshoot, 42 +/- 6 mV. Na, K and Ca currents were isolated upon pharmacological manipulations. The predominant type of K current was a noninactivating delayed rectifier. Voltage-clamp studies also showed the presence of a high voltage-activated sustained inward Ca current, while low voltage could not elicit any transient Ca current.

Rhizobial Nodulation Factors Stimulate Mycorrhizal Colonization of Nodulating and Nonnodulating Soybeans.

Legumes form tripartite symbiotic associations with noduleinducing rhizobia and vesicular-arbuscular mycorrhizal fungi. Co-inoculation of soybean (Glycine max [L.] Merr.) roots with Bradyrhizobium japonicum 61-A-101 considerably enhanced colonization by the mycorrhizal fungus Glomus mosseae. A similar stimulatory effect on mycorrhizal colonization was also observed in nonnodulating soybean mutants when inoculated with Bradyrhizobium japonicum and in wild-type soybean plants when inoculated with ineffective rhizobial strains, indicating that a functional rhizobial symbiosis is not necessary for enhanced mycorrhiza formation. Inoculation with the mutant Rhizobium sp. NGR[delta]nodABC, unable to produce nodulation (Nod) factors, did not show any effect on mycorrhiza. Highly purified Nod factors also increased the degree of mycorrhizal colonization. Nod factors from Rhizobium sp. NGR234 differed in their potential to promote fungal colonization. The acetylated factor NodNGR-V (MeFuc, Ac), added at concentrations as low as 10-9 M, was active, whereas the sulfated factor, NodNGR-V (MeFuc, S), was inactive. Several soybean flavonoids known to accumulate in response to the acetylated Nod factor showed a similar promoting effect on mycorrhiza. These results suggest that plant flavonoids mediate the Nod factor-induced stimulation of mycorrhizal colonization in soybean roots.

Effects of muscle electrical activity on the transmission of developing neuromuscular junction.

Miniature endplate potentials (MEPPS) caused by the spontaneous release of ACh from the growth cone of cholinergic neurons, are recorded by the whole-cell patch-clamp technique on a large number of 1-day cultured myoballs which have contact neurites of co-cultured neurons. Both muscle cell and neuron are dissociated from the 1-day-old (about stage 20) Xenopus embryo. Frequency and/or amplitude of MEPPs can obviously increase after the repetitive high-level depolarization caused by the stimuli on muscle cells. No detectable changes of single ACh receptor channel property are observed by using the single-channel recording technique. These results suggest that the mechanism of the increase of MEPPs after electrical activity of postsynaptic muscle cells probably involve some alteration of presynaptic membrane.

The effects of glutamate and GABA (gamma-amino-butyric-acid) on spontaneous acetylcholine release at the neuromuscular junction in Xenopus laevis embryo cell cultures.

The miniature endplate currents (MEPC's) were recorded at the neuromuscular junction of Xenopus laevis embryo neuron-muscle co-cultured cells. These MEPC's were due to the spontaneous release of acetylcholine from the nerve terminal. After perfusion with glutamate (10 mumol/L), both frequency and amplitude of the MEPC's increased. After washing away of glutamate, this effect persisted. We named this phenomena "Long-Term Facilitation". GABA (20 mumol/L) on the other hand had an inhibitory effect on both frequency and amplitude of the MEPC's. After washing away of GABA, the MEPC frequency and amplitude increased. We named this effect "Post-Potentiation". Local perfusion experiments furthermore indicated that the effect of glutamate was restricted to the neuromuscular junction, the effect of GABA was restricted to the soma.

[Blocking effect of anisodamine on acetylcholine receptor channels].

Anisodamine, an analog of atropine, was isolated first in China. Patch-clamp technique was used to study the inhibitory effect of anisodamine (Ani) on acetylcholine receptor on the membrane of muscle innervated by neuron. Neural tube in embryo of Xenopus laevis were cultured. By whole-cell clamp and outside-out patch, we found that the inhibitory effect of Ani was obvious on both miniature end-plate currents (MEPC) and single channel. This effect was reversible and the minimal concentration for complete inhibition was 60 mumol.L-1, vs 1 and 0.5 mmol.L-1 for atropine and scopolamine, respectively. Our results indicate that Ani blocks both M-ACh and N-ACh receptors.

Antibodies to synaptophysin interfere with transmitter secretion at neuromuscular synapses.

The involvement of synaptophysin, a synaptic vesicle-specific protein, in transmitter release at neuromuscular synapses was studied by intracellular application of synaptophysin antibodies into presynaptic neurons. Polyclonal antibodies or their Fab fragments were loaded into spinal neurons by injection into one of the early blastomeres of Xenopus embryos 1 day prior to culturing or, alternatively, directly through a whole-cell recording pipette at the soma of cultured neurons. At synapses made by antibody-loaded neurons in culture, the spontaneous synaptic currents showed marked reduction in frequency without significant change in their mean amplitude. The impulse-evoked synaptic currents showed reduced amplitude and increased failure rate. These results suggest that interference with synaptophysin function by antibody binding inhibits transmitter secretion.

[Two types of nondepolarizing-activated calcium channels on the spinal cholinergic neurons from embryonic Xenopus laevis].

Single calcium channel current was studied on the identified spinal cholinergic neurons from embryonic Xenopus laevis with patch clamp method. The results indicate that some calcium channels show opening activity at resting membrane potential. According to the characteristics of conductance and kinetics of such channels, they could be divided into two types: a stretch sensitive Type-NS with a slope conductance of 7.5 pS (mean open time 0.58 ms at resting membrane potential) and a Type-NL with a slope conductance of 16.7 pS (and two opening times of 2 ms and 19.3 ms). Both types of channel are predominantly active at resting potential and negative membrane phases. It is suggested that they may be involved in the calcium-dependent neuronal events at resting state.

Surgical treatment of myasthenia gravis and evaluation of its efficacy.

This paper reports 30 cases of myasthenia gravis (MG) treated by thymectomy from 1965 to 1990 in our hospital. Of all the 30 cases of MG, peripheral blood lymphocyte subpopulation was determined in 10, and anti-acetylcholine receptor antibody titer in 6, before and after operation. The results demonstrated the efficacy of thymectomy against MG. We considered that with application of hormone before and after operation to regulate immune function of the body, and/or with plasma exchange to remiss symptoms, all the patients with generalized MG may be indicated for treatment by thymectomy. Indication of treatment does not depend on age, sex and the course of the disease. But radical operation, proper anesthesia and appropriate use of antibiotics may ensure safety of the operation and its curative effect.

Studies of nerve-muscle interactions in Xenopus cell culture: analysis of early synaptic currents.

We have studied the spontaneous and nerve-evoked synaptic currents during the initial period of nerve-muscle contact in Xenopus cell cultures. The precise timing of the contact was achieved by physically manipulating embryonic muscle cells into contact with co-cultured spinal neurons. Previous studies have shown that physical contact of the muscle membrane induces pulsatile release of acetylcholine (ACh) from the growth cone of these neurons, resulting in spontaneous synaptic currents (SSCs) in the muscle cell within seconds following the contact. In the present work, we first showed that these SSCs at the manipulated nerve-muscle contacts are similar to those observed at naturally occurring synapses. We then examined the possible cellular mechanisms responsible for the marked variation in SSC amplitude and showed that it most likely results from differences in either the amount of ACh contained in each release event or the extent of close membrane apposition near the release sites. During the first 20 min following the nerve-muscle contact, there was an increase in the frequency and mean amplitude of the SSCs. During a similar period, the evoked synaptic currents (ESCs), which were induced by suprathreshold electrical stimulation of the neuronal soma, also showed an increase in the mean amplitude and a reduction in the delay of onset following the stimulus. These postcontact changes in the efficacy of synaptic transmission may be related to an increase in the total area of close membrane apposition between the nerve and muscle cells. This was suggested by the finding that neurite-muscle adhesion increases over a similar postcontact period. The transition from low- to high-efficacy transmission during the early phase of contact may reflect the process of selective adhesion between the cells, and thus signify the formation of specific synapse. Analysis of the fluctuation in the ESC amplitude at the early nerve-muscle contact suggests that evoked release of ACh occurs as multiples of a quantal unit. However, this unit is apparently related to only a small subpopulation of SSCs of relatively high amplitudes.(ABSTRACT TRUNCATED AT 400 WORDS)

Whole-cell clamp study of Xenopus embryonic cholinergic neurons.

Whole-cell clamped myoballs are placed into direct visible contact with the growth cones of isolated neurons in embryonic Xenopus culture to serve as probe of acetylcholine (AcCHo) release in order to determine whether these neurons are cholinergic or not. Using a G omega -seal, whole-cell recording method, the electrophysiological properties of these identified cholinergic neurons are studied. It is found that these embryonic neurons, like adult frog motor neurons, exhibit repetitive firings in a certain embryonic developing stage. A development of repetitive firings is observed simultaneously. Tracing the development of one neuron, we find that the development of repetitive firing is completed at the 48th h after fertilization. Tetrodotoxin (TTX) which blocks Na+ channels can abolish all firings; and tetraethyl ammonium chloride (TEA), the blocker of K+ channels, reverses this development, i.e. it makes the repetitive firings disappear again. These data show that the nature of the development of repetitive firings is the development of K+ channels.

Initial events in the formation of neuromuscular synapse: rapid induction of acetylcholine release from embryonic neuron.

We have studied the electrical events during the initial phase of nerve-muscle contact in embryonic Xenopus culture. Using a G omega-seal, whole-cell recording method, we monitored the membrane current of a muscle cell continuously while it was manipulated into close proximity of the growth cone of a cocultured spinal neuron. We found a rapid appearance of pulsatile inward currents at the muscle cell after the neurite-muscle contact. These currents were abolished by d-tubocurarine and alpha-bungarotoxin but were unaffected by tetrodotoxin. Both the drug sensitivity and the time course of these currents are similar to that of the spontaneous miniature end-plate currents (MEPCs) resulting from spontaneous release of pulses of acetylcholine (AcCho) from the nerve terminal. Unlike the MEPCs at the mature neuromuscular synapse, these early MEPCs varied greatly in their amplitudes, and there was a gradual increase in the frequency of the MEPCs of larger amplitudes during the first 20 min after the contact. Independent measurement of AcCho concentration near the growth cone by an excised patch of AcCho-sensitive muscle membrane showed that very little AcCho is released from the isolated growth cone, and marked release can be triggered by the contact with a muscle cell or with the excised membrane itself. The induction of release is relatively specific: contact with a neuron or the tip of a clean glass pipette was capable of inducing only a transient release, while persistent release was induced by contacts made with muscle membrane. This contact-dependent AcCho release may be responsible for an early induction of muscle activity and serve as a signal for the establishment of synaptic contact.