Karl Wah Keung Tsim

The agrin gene codes for a family of basal lamina proteins that differ in function and distribution.

We isolated two cDNAs that encode isoforms of agrin, the basal lamina protein that mediates the motor neuron-induced aggregation of acetylcholine receptors on muscle fibers at the neuromuscular junction. Both proteins are the result of alternative splicing of the product of the agrin gene, but unlike agrin, they are inactive in standard acetylcholine receptor aggregation assays. They lack one (agrin-related protein 1) or two (agrin-related protein 2) regions in agrin that are required for its activity. Expression studies provide evidence that both proteins are present in the nervous system and muscle and that, in muscle, myofibers and Schwann cells synthesize the agrin-related proteins while the axon terminals of motor neurons are the sole source of agrin.

cDNA that encodes active agrin

Agrin is thought to mediate the motor neuron-induced aggregation of AChRs and AChE on the surface of muscle fibers at neuromuscular junctions. We have isolated a cDNA from a chick brain library that, based on sequence homology and expression experiments, codes for active agrin. Examination of the sequence reveals considerable similarity to homologous cDNAs previously isolated from ray and rat libraries. A conspicuous difference is an insertion of 33 bp in chick agrin cDNA, which endows the encoded protein with AChR/AChE aggregating activity. Homologous transcripts having the 33 bp insertion were detected in the ray CNS, which indicates that an insertion of similar size is conserved in agrin in many, if not all, vertebrate species. Results of in situ hybridization studies and PCR experiments on mRNA isolated from motor neuron-enriched fractions of the spinal cord indicate that, consistent with the agrin hypothesis, motor neurons contain transcripts that code for active agrin.

A polysaccharide isolated from Cordyceps sinensis, a traditional Chinese medicine, protects PC12 cells against hydrogen peroxide-induced injury

Cordyceps sinensis, a well-known traditional Chinese medicine, possesses activities in anti-tumour, anti-oxidation and stimulating the immune system; however, the identity of active component(s) is not determined. By using anti-oxidation activity-guided fractionation, a polysaccharide of molecular weight approximately 210 kDa was isolated from cultured Cordyceps mycelia by ion-exchange and sizing chromatography. The isolated polysaccharide, having strong anti-oxidation activity, contains glucose, mannose and galactose in a ratio of 1 : 0.6 : 0.75. The pre-treatment of isolated polysaccharide on the cultured rat pheochromocytoma PC12 cells shows strong protective effect against hydrogen peroxide (H(2)O(2))-induced insult. Treatment of the cells with the isolated polysaccharide at 100 microg/ml prior to H(2)O(2) exposure significantly elevated the survival of PC12 cells in culture by over 60%. In parallel, the H(2)O(2)-induced production of malondialdehyde in cultured cells was markedly reduced by the polysaccharide treatment. Moreover, the pre-treatment of the isolated polysaccharide significantly attenuated the changes of glutathione peroxidase and superoxide dismutase activities in H(2)O(2)-treated cells in a dose-dependent manner. This is the first report in identifying a polysaccharide from Cordyceps, which protects against the free radical-induced neuronal cell toxicity.

Cdk5 is involved in neuregulin-induced AChR expression at the neuromuscular junction

Here we describe an important involvement of Cdk5/p35 in regulating the gene expression of acetylcholine receptor (AChR) at the neuromuscular synapse. Cdk5 and p35 were prominently expressed in embryonic muscle, and concentrated at the neuromuscular junction in adulthood. Neuregulin increased the p35-associated Cdk5 kinase activity in the membrane fraction of cultured C2C12 myotubes. Co-immunoprecipitation studies revealed the association between Cdk5, p35 and ErbB receptors in muscle and cultured myotubes. Inhibition of Cdk5 activity not only blocked the NRG-induced AChR transcription, but also attenuated ErbB activation in cultured myotubes. In light of our finding that overexpression of p35 alone led to an increase in AChR promoter activity in muscle, Cdk5 activation is sufficient to mediate the up-regulation of AChR gene expression. Taken together, these results reveal the unexpected involvement of Cdk5/p35 in neuregulin signaling at the neuromuscular synapse.

Anti-oxidation activity of different types of natural Cordyceps sinensis and cultured Cordyceps mycelia.

Cordyceps, one of the well-known traditional Chinese medicines, consists of the dried fungus Cordyceps sinensis growing on the larva of the caterpillar. It is commonly used for the replenishment of body health. One of the known pharmacological effects is its anti-oxidation activity. However, there is a great variation of the quality in different sources of Cordyceps. Here, the water extracts of various sources of natural C. sinensis and cultured Cordyceps mycelia were analyzed for their anti-oxidation activity by using three different assay methods such as the xanthine oxidase assay, the induction of hemolysis assay and the lipid peroxidation assay. The results showed that Cordyceps, in general, possesses a strong anti-oxidation activity in all assays tested. However, both natural and cultured Cordyceps showed the lowest inhibition in the lipid peroxidation when compared with the other two assay methods. The cultured Cordyceps mycelia had equally strong anti-oxidation activity as compared to the natural Cordyceps. Besides, the anti-oxidation activities were increased to 10-30 folds in the partially purified polysaccharide fractions from the cultured Cordyceps mycelia, which suggested that the activity could be derived partly from Cordyceps polysaccharides.

Determination of nucleosides in natural Cordyceps sinensis and cultured Cordyceps mycelia by capillary electrophoresis

Cordyceps sinensis is a well‐known traditional Chinese medicine, and some of the active components are nucleosides. The analysis of nucleosides in Cordyceps material has been performed by reversed‐phase high‐performance liquid chromatography (HPLC) with gradient elution or by spectrometry. Here, we have explored the possibility of using capillary electrophoresis to determine the content of three major nucleosides (adenosine, guanosine and uridine) in Cordyceps. Capillary electrophoresis needs no gradients, and it provides a better separation due to its higher efficiency. In order to optimize the resolution, the separation of adenosine, guanosine and uridine was determined in Cordyceps with respect to the variation of buffer concentration, pH, temperature, and voltage. By using the calibrated electrophoresis system, the separation was achieved for the three nucleosides in less than 10 min with a background electrolyte consisting of 0.2 M boric acid‐sodium hydroxide buffer, pH 8.5. The nucleoside contents of various types of natural Cordyceps and cultured Cordyceps mycelia were determined and compared. There was a great variation of nucleoside content in different sources of Cordyceps; the cultured Cordyceps mycelia, however, contains a much higher concentration than the natural Cordyceps.

Baicalin, a Flavone, Induces the Differentiation of Cultured Osteoblasts AN ACTION VIA THE Wnt/beta-CATENIN SIGNALING PATHWAY

Flavonoids, a group of natural compounds found in a variety of vegetables and herbal medicines, have been intensively reported on regarding their estrogen-like activities and particularly their ability to affect bone metabolism. Here, different subclasses of flavonoids were screened for their osteogenic properties by measuring alkaline phosphatase activity in cultured rat osteoblasts. The flavone baicalin derived mainly from the roots of Scutellaria baicalensis showed the strongest induction of alkaline phosphatase activity. In cultured osteoblasts, application of baicalin increased significantly the osteoblastic mineralization and the levels of mRNAs encoding the bone differentiation markers, including osteonectin, osteocalcin, and collagen type 1α1. Interestingly, the osteogenic effect of baicalin was not mediated by its estrogenic activity. In contrast, baicalin promoted osteoblastic differentiation via the activation of the Wnt/β-catenin signaling pathway; the activation resulted in the phosphorylation of glycogen synthase kinase 3β and, subsequently, induced the nuclear accumulation of the β-catenin, leading to the transcription activation of Wnt-targeted genes for osteogenesis. The baicalin-induced osteogenic effects were fully abolished by DKK-1, a blocker of Wnt/β-catenin receptor. Moreover, baicalin also enhanced the mRNA expression of osteoprotegerin, which could regulate indirectly the activation of osteoclasts. Taken together, our results suggested that baicalin could act via Wnt/β-catenin signaling to promote osteoblastic differentiation. The osteogenic flavonoids could be very useful in finding potential drugs, or food supplements, for treating post-menopausal osteoporosis.

The fruiting body and its caterpillar host of Cordyceps sinensis show close resemblance in main constituents and anti-oxidation activity

Cordyceps (summer-grass, winter-worm), one of the most valued traditional Chinese medicines, is used commonly for the replenishment of body health. It consists of the dried fungus Cordyceps sinensis growing on caterpillar larvae. For medication, the fruiting body (fungus) and the worm (caterpillar) are used together. However, the pharmacological efficiency and the main constituents of the individual parts have not been determined. In the present study the water extracts from the fruiting body and worm of natural Cordyceps were analyzed for their content of nucleosides and polysaccharides; the results showed that the worm had chemical composition similar to the fruiting body. In addition, both the fruiting body and worm of Cordyceps showed similar potency in their anti-oxidation activities in the xanthine oxidase assay, the induction of hemolysis assay and the lipid-peroxidation assay. These results suggest that the function of the worm in Cordyceps is to provide a growth medium for the fruiting body, and that eventually, the worm is totally invaded by C. sinensis mycelia.

ATP acts via P2Y1 receptors to stimulate acetylcholinesterase and acetylcholine receptor expression: transduction and transcription control.

At the vertebrate neuromuscular junction ATP is known to stabilize acetylcholine in the synaptic vesicles and to be co-released with it. We have shown previously that a nucleotide receptor, the P2Y1 receptor, is localized at the junction, and we propose that this mediates a trophic role for synaptic ATP there. Evidence in support of this and on its mechanism is given here. With the use of chick or mouse myotubes expressing promoter–reporter constructs from genes of acetylcholinesterase (AChE) or of the acetylcholine receptor subunits, P2Y1 receptor agonists were shown to stimulate the transcription of each of those genes. The pathway to activation of the AChE gene was shown to involve protein kinase C and intracellular Ca 2+ release. Application of dominant-negative or constitutively active mutants, or inhibitors of specific kinases, showed that it further proceeds via some of the known intermediates of extracellular signal-regulated kinase phosphorylation. In both chick and mouse myotubes this culminates in activation of the transcription factor Elk-1, confirmed by gel mobility shift assays and by the nuclear accumulation of phosphorylated Elk-1. All of the aforementioned activations by agonist were amplified when the content of P2Y1 receptors was boosted by transfection, and the activations were blocked by a P2Y1-selective antagonist. Two Elk-1 binding site sequences present in the AChE gene promoter were jointly sufficient to drive ATP-induced reporter gene transcription. Thus ATP regulates postsynaptic gene expression via a pathway to a selective transcription factor activation.

The establishment of a sensitive method in determining different neurotransmitters simultaneously in rat brains by using liquid chromatography-electrospray tandem mass spectrometry

An effective way to determine the amount of different neurotransmitters is vital to the study of brain function. Here, a highly sensitive HPLC-MS/MS method was developed to simultaneously measure γ-aminobutyric acid, dopamine, epinephrine, norepinepherine, glutamate and serotonin in one sample. The quantification of the neurotransmitters was achieved by a tandem mass spectrometer using the selected reaction monitoring scan mode. The method validation included selectivity, linearity, accuracy, precision, stability, recovery and matrix effect. For the six neurotransmitters, the linear regression analysis was calibrated by deuterated internal standards with a R(2) of over 0.991, and the limit of detection (LOD) and the limit of quantification (LOQ) were from 2.5 to 500 pg/mg and 7.5 to 1000 pg/mg, respectively. This method was employed here to reveal different types and amounts of neurotransmitters simultaneously in adult and embryonic rat brains. Here, the change of dopamine concentration in embryonic and adult brain was from 0.071 to 0.760 ng/mg of brain tissue, GABA was from 207.643 to 445.148 ng/mg, glutamate was from 679.535 to 1408.920 ng/mg, serotonin was from 0.058 to 0.485 ng/mg and norepinepherine was from 0.054 to 0.290 ng/mg. For epinephrine, it was only detected in embryonic stage but not in adult, with the concentration at 0.241 ng/mg.