The PP2A-Aß gene is regulated by multiple transcriptional factors including Ets-1, SP1/SP3, and RXRα/ß.

Protein phosphatase-2A (PP-2A) is a major serine/threonine phosphatase abundantly expressed in eukaryotes. PP-2A is a heterotrimer that contains a 65 kD scaffold A subunit, a 36 kD catalytic C subunit, and a regulatory B subunit of variable isoforms ranging from 54-130 kDs. The scaffold subunits, PP2A-Aα/ß, act as platforms for both the C and B subunits to bind, and thus are key structural components for PP-2A activity. Mutations in both genes encoding PP2A-Aα and PP2A-Aß lead to carcinogenesis and likely other human diseases. Our previous work showed that the gene coding for PP2A-Aα is positively regulated by multiple transcription factors including Ets-1, CREB, and AP-2α but negatively regulated by SP-1/SP-3. In the present study, we have functionally dissected the promoter of the mouse PP2A-Aß gene. Our results demonstrate that three major cis-elements, including the binding sites for Ets-1, SP1/SP3, and RXRα/ß, are present in the proximal promoter of the mouse PP2A-Aß gene. Gel mobility shifting assays reveal that Ets-1, SP1/SP3, and RXRα/ß all bind to PP2A-Aß gene promoter. In vitro mutagenesis and reporter gene activity assays demonstrate that while Ets-1 displays negative regulation, SP1/SP3 and RXRα/ß positively regulate the promoter of the PP2A-Aß gene. Co-expression of the cDNAs encoding Ets-1, SP1/SP3, or RXRα/ß and the luciferase reporter gene driven by PP2A-Aß promoter further confirm their control over the PP2A-Aß promoter. Finally, ChIP assays demonstrate that Ets-1, SP1/SP3, and RXRα/ß can all bind to the PP2A-Aß gene promoter. Together, our results reveal that multiple transcription factors regulate the PP2A-Aß gene. Moreover, our results provide important information explaining why PP2A-Aα and PP2A-Aß display distinct expression levels.

Physiological and biochemical analysis of L. tredecimguttatus venom collected by electrical stimulation.

The L. tredecimguttatus venom was collected by electrical stimulation and systematically analyzed. Gel electrophoresis and RP-HPLC showed that the venom consisted primarily of proteins with molecular weights above 10 kDa, most of which were high-molecular-mass acidic proteins, with fewer proteins and peptides below 10 kDa. The most abundant proteins in the venom were concentrated at around 100 kDa, which included latrotoxins- the principal toxic components of the venom. Injection of the venom in mice and cockroaches P. americana gave rise to obvious poisoned symptoms, with LD50 values of 0.16 mg/kg and 1.87 microg/g, respectively. Electrophysiological experiments showed that the venom could block the neuromuscular transmission in isolated mouse phrenic nerve-hemidiaphragm and rat vas deferens preparations. The low-molecular-weight fraction (<10 kDa) of the venom had no effect on the transmission. Enzymatic analysis indicated that the venom possess activities of several kinds of hydrolases including hyaluronidase and proteases. These results demonstrated that L. tredecimguttatus venom was basically a large-protein-constituted venom and is one of the most poisonous spider venoms known in the world. The mammalian toxicity of the venom was based on its larger proteins rather than on smaller proteins and peptides, and its hydrolase activities might be involved in the latrodectism. The use of electrical stimulation method to collect the venom has the advantages of avoiding contamination and repeated use of the valuable L. tredecimguttatus venom resources.

Extraction and protein component analysis of venom from the dissected venom glands of Latrodectus tredecimguttatus.

Black widow spiders (genus Latrodectus) have attracted increasing attention due to frequently reported human injuries caused by them and the potential applications of biologically active components in their venoms. Although a number of studies have described the biological properties and structures of several venomous proteins such as latrotoxins, a comprehensive analysis of protein component of the venom from the spider is not available. We used combinative proteomic strategies to assess the protein components of the crude venom collected from Latrodectus tredecimguttatus by extracting the dissected venom glands. The experiments demonstrated that the crude venom of L. tredecimguttatus has a high abundance of acidic proteins with molecular masses greater than 15 kDa, and the content of proteins and peptides of below 15 kDa is low. 86 unique proteins were identified, part of which were contaminations of cellular components during the extraction, determined in comparison with venom obtained by electrostimulation. Except for members of latrotoxin family that were commonly considered as the primary toxic components of the venom, several other special enzymes and proteins were detected such as protease, phosphatase, lysozyme, inhibitory protein, and so on. These protein components, particularly the proteases, were speculated to play important roles in the action of L. tredecimguttatus venom.

[Chromatographic behavior and purification of synthetic huwentoxin-I].

A comparative study was made to investigate the chromatographic behavior and purification of chemically synthesized and native polypeptide neurotoxin huwentoxin-I (HWTX-I) by means of reversed-phase HPLC and ion-exchange chromatography. The results showed that the synthetic HWTX-I in crude product has a longer retention time than the native HWTX-I, but has the same retention time as the denatured HWTX-I, suggesting it being in a state of complete denaturation which was confirmed by the MALDI-TOF MS analysis. It has also been discovered that, before complete purification, the synthetic HWTX-I, after oxidative treatment, showed wider and more asymmetric peaks than the native HWTX-I that was denatured and then renatured under the same experimental conditions, which suggests that there is even greater conformational unhomogeneity in the synthetic HWTX-I. It was inferred that, in addition to the wrong disulfide bonds formation during oxidative folding, the racemization during chemical synthesis resulted in more stereoisomers of synthetic HWTX-I. It was needed for the renatured synthetic peptides to be purified using different methods alternatively.

Superimposition and Analysis of ESI Tandem Mass Spectra.

Methionine enkephalin and four synthetic peptides (a heptapeptide and its modified form, a decapeptide and an icosapeptide) were fragmented using ESI tandem mass spectrometry with certain varying instrumental parameters, such as amplitude of excitation, to produce a series of MS/MS spectra. Using the Bruker DataAnalysis software, some complementary MS/MS spectra were chosen to form a superimposed MS/MS spectrum with consecutive triplets (as well as a few doublets) consisting of product ions AnBnCn or X n Y n Z n. The amino acid sequences of the peptides were easily and accurately determined from the superimposed MS/MS spectrum, suggesting that the method is easy and suitable for peptide sequencing by tandem mass spectrometry.

The effect of Huwentoxin-I on Ca(2+) channels in differentiated NG108-15 cells, a patch-clamp study.

Huwentoxin-I (HWTX-I), a 3.75 kDa peptide toxin isolated from the venom of the spider Selenocosmia huwena, was found to be a reversible presynaptic inhibitor by our previous work. Using whole-cell patch clamp methods, we found that HWTX-I had no significant effect on the TTX-sensitive Na(+) current or the delayed rectifier K(+) current (K(r)) in low-serum medium cultured NG108-15 cells, but High-Voltage-Activated Ca(2+) channel expressed in prostaglandin E(1) differentiated NG108-15 cells could be potently inhibited by HWTX-I (EC(50) approximately 100 nM), while it hardly affected low-voltage-activated Ca(2+) channel. Among types of high-voltage-activated Ca(2+) channel, HWTX-I selectively inhibited N-type Ca(2+) channel and had only very weak effect on L-type Ca(2+) channel in prostaglandin E(1) differentiated NG108-15 cells.

[Chemical synthesis and characterization of R20A-HWTX-I, a mutant of huwentoxin-I with single residue replacement].

Huwentoxin-I (HWTX-I) is a polypeptide neurotoxin purified from the venom of the spider Selenocosmia huwena. R20A-HWTX-I, a mutant of HWTX-I in which the Arg was replaced by Ala, was synthesized on solid support by using Fmoc chemistry. The synthetic mutant was oxidatively renatured in glutathione-containing buffer, and then isolated by reversed phase and specially designed ion-exchange HPLC. The chemical structure of R20A-HWTX-I was confirmed by amino acid analysis, Edman degradation and MALDI-TOF mass analysis. Physiological experiment showed that the replacement of R20 by a decreased the bioactivity of the HWTX-I by 92%, indicating that R20 is a key residue closely related to the bioactivity of the HWTX-I.

The presynaptic activity of huwentoxin-I, a neurotoxin from the venom of the chinese bird spider Selenocosmia huwena.

Three different types of isolated nerve-synapse preparations, guinea pig ileum, rat vas deferens and toad heart, were used to investigate the physiological activity of Huwentoxin-I, a neurotoxin from the venom of the spider Selenocosmia huwena. The twitch response of isolated guinea pig ileum induced by electrical stimulus can be inhibited by HWTX-I. After blockage, contraction of the ileum can be induced by exogenously applied acetylcholine. HWTX-I caused the inhibition of the twitch response to electrical nerve stimulation in the rat vas deferens. After the twitch was completely inhibited, noradrenaline triggered rhythmic contraction of the vas deferens. The inhibitory effect on heart of toad induced by stimulating sympathetic-vagus nerve can be reversed by HWTX-I, although exogenously applied acetylcholine still acts as an effective inhibitor. All of these results support the conclusion that HWTX-I has the presynaptic activity that effects the release of neurotransmitter from the nerve endings of both the cholinergic synapse and the adrenergic synapse.

Cloning and functional expression of a synthetic gene encoding huwentoxin-I, a neurotoxin from the Chinese bird spider (Selenocosmia huwena).

Cloning and functional expression of a synthetic gene encoding huwentoxin-I, a neurotoxin from the Chinese bird spider Selenocosmia huwena. A gene encoding huwentoxin-I, a peptide neurotoxin consisted of 33 amino acid residues from the venom of the Chinese bird spider Selenocosmia huwena, was designed, synthesized and expressed in Escherichia coli as a hybrid protein fused with glutathione S-transferase at the N-terminal. The fusion protein was purified by GSH-Sepharose 4B affinity column chromatography and cleaved by thrombin to release the toxin peptide. The amino acid sequence of the recombinant toxin was consistent with the designed one by sequence determination and MALDI-TOF mass analysis, suggesting that the recombinant huwentoxin-I produced the same expression product as the native one. After reduction and renaturation, the biological activity of the recombinant toxin was identical with that of the native huwentoxin-I by electrophysiological method.

Purification and characterization of huwentoxin-II, a neurotoxic peptide from the venom of the Chinese bird spider Selenocosmia huwena.

A neurotoxic peptide, huwentoxin-II (HWTX-II), was purified from the venom of the Chinese bird spider Selenocosmia huwena by ion exchange chromatography and reversed phase HPLC. The toxin can reversibly paralyse cockroaches for several hours, with an ED50 of 127 +/- 54 microg/g. HWTX-II blocks neuromuscular transmission in an isolated mouse phrenic nerve diaphragm preparation and acts cooperatively to potentiate the activity of huwentoxin-I. The complete amino sequence of HWTX-II was determined and found to consist of 37 amino acid residues, including six Cys residues. There is microheterogeneity (Ile/Gln) in position 10, and mass spectrometry indicated that the two isoproteins have a tendency to dimerize. It was determined by mass spectrometry that the six Cys residues are involved in three disulphide bonds. The sequence of HWTX-II is highly homologous with ESTX, a toxin from the tarantula Eurypefina californicum.

Effects of clomethiazole on radial-arm maze performance following global forebrain ischemia in gerbils.

The functional and neuroanatomical protective effects of clomethiazole (CMZ) were examined in an animal model of global forebrain ischemia. Gerbils underwent sham-surgery or were rendered ischemic by the application of aneurysm clips to both carotid arteries for 6 min. Three treatment groups received CMZ (50 mg/kg, 100 mg/kg, or 150 mg/kg) 30 min before ischemia, and one group was given 150 mg/kg of CMZ 30 min after ischemia. Following recovery, the gerbils were tested in a radial-arm maze to assess memory functions. Histological evaluation was assessed blindly using a percentile scoring system. The results indicate that pre-ischemic treatment with 100 mg/kg and 150 mg/kg of CMZ reduced brain damage and working memory errors significantly. Treatment dosage of 150 mg/kg of CMZ was the most effective in preventing neuronal damage in the hippocampus and eliminating the working memory deficit typically induced by ischemia.

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.

A lectin-like peptide isolated from the venom of the Chinese bird spider Selenocosmia huwena.

A peptide with haemagglutination activity was isolated from the venom of the Chinese bird spider Selenocosmia huwena by means of ion-exchange and reverse-phase high-performance liquid chromatography. This peptide, named SHLP-I, agglutinates human and mice erythrocytes at a minimum concentration of 125 micrograms/ml and 31 micrograms/ml, respectively. It consists of 32 amino acid residues including 3 Trp and 6 Cys residues, the latter of which form three disulfide bounds. The complete amino acid sequence was determined. The N-terminal and C-terminal residues were Gly and Trp, respectively. SHLP-I shows homology with a fragment of great nettle lectin and with huwentoxin-I from the venom of the same spider.

Properties and amino acid sequence of huwentoxin-I, a neurotoxin purified from the venom of the Chinese bird spider Selenocosmia huwena.

By means of reverse phase and ion-exchange high performance liquid chromatography, a neurotoxic peptide named huwentoxin-I was purified from the venom of the Chinese bird spider Selenocosmia huwena. The intraperitoneal and intracisternal LD50 in mice of the toxin were 0.70 mg/kg and 9.40 micrograms/kg, respectively. This toxin at the concentration of 1 x 10(-5) g/ml can irreversibly block the neuromuscular transmission of the isolated mouse phrenic nerve-diaphragm preparation in 13.4 +/- 1.3 min (mean +/- S.D., n = 5). The isoelectric point is 8.95 determined by isoelectric focusing electrophoresis. It consists of 33 amino acids including 6 Cys and 6 Lys determined by amino acid analysis. The complete amino sequence of huwentoxin-I was determined. The N-terminal and C-terminal residues were Ala and Leu, respectively. The primary structure showed partial homology with that of mu-agatoxins from the funnel-web spider Agelenopsis aperta.

Single-step electroelution of proteins from SDS-polyacrylamide gels and immobilization on diisothiocyanate-glass beads in prepacked capillary columns for solid-phase microsequencing.

A new method for immobilization of proteins purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) prior to sequencing is described. It utilizes a simple apparatus that permits the simultaneous electroelution of proteins from gel slices and attachment to diisothiocyanate-activated glass beads prepacked in capillary tubes [S-P. Liang and R. A. Laursen, Anal. Biochem. 188, 366-373 (1990)]. Transfer/attachment yields of greater than 80% within 90 min were observed for several 125I-labeled proteins with a range of molecular weights using 0.2 M sodium phosphate (pH 8.9) buffer containing 0.1% SDS. The method has the advantage of high capacity, relative simplicity, and insensitivity to the presence of SDS and Coomassie blue stain. The highest transfer yields were obtained when proteins were run on gels which had been aged for at least 12 h. For 100- to 1000-pmol samples, the sequenceable amount of protein, including transfer, was generally 30-60%, with an average repetitive yield of 95%. Factors which influence sample recovery and sequencing yield are discussed.

Covalent immobilization of proteins and peptides for solid-phase sequencing using prepacked capillary columns.

The use of prepacked capillary columns for immobilizing proteins and peptides for solid-phase Edman degradation is described. Capillary tubes with an internal volume of about 30 microliters are filled with glass beads bearing isothiocyanato groups (DITC-glass), aminophenyl groups (AP-glass), or aminoethylaminopropyl groups (AEAP-glass) and are sealed with porous plugs. Proteins or peptides in appropriate buffers are introduced into the columns by capillary action and are covalently coupled to the glass beads, either by reaction of lysine side-chain amino groups with DITC-glass, by carbodi-imide-mediated reaction of carboxyl groups with AP-glass, or by reaction of homoserine lactone groups with AEAP-glass. Optimization of attachment conditions is described. The capillary columns are loaded into the sequencer and, when sequencing has been completed, are discarded. This technique greatly simplifies polypeptide immobilization and is suitable for microsequencing (less than 50-1000 pmol) or macrosequencing (1-50 nmol).

A comparison of the primary structures of lactate dehydrogenase isozymes M4 from giant panda, red panda, black bear and dog.

Lactate dehydrogenase isozymes M4 have been isolated and purified from red panda (Ailurus fulgens), black bear (Selenarctos thibetanus) and dog (Canis familiars) by affinity chromatography and compared with that from giant panda (Ailuropoda melanoleuca). Experimental results have shown that the N-termini, C-termini and the molecular weights of LDH-M subunits of red panda, black bear and dog are the same as those of the LDH-M subunit of giant panda. Analysis and comparison of HPLC peptide maps from the tryptic digests of the isozymes of red panda, black bear and dog have shown that most of their peptide fragments had the same retention time and amino acid composition as the corresponding peptide fragments from giant panda. Fragments with different retention times and/or amino acid compositions were sequenced. Careful examination of those variant amino acid residues demonstrated clearly that the primary structure of giant panda LDH-M subunit is unique and it appears that the giant panda might be classified as an independent family.

The primary structure of the lactate dehydrogenase isozyme M4 from giant panda.

The amino acid sequences of tryptic, chymotryptic and cyanogen bromide cleavage peptides of the lactate dehydrogenase isozyme M4 from giant panda have been determined. Based on the overlapping peptides and by a comparison with the known sequence of porcine lactate dehydrogenase isozyme M subunit, the complete primary structure of the giant panda lactate dehydrogenase isozyme M subunit has been established. The polypeptide chain of giant panda lactate dehydrogenase isozyme M subunit consists of 331 amino acid residues. There is a variance of 17 amino acid residues between the porcine and the giant panda lactate dehydrogenase isozyme M subunits. Most of the variable residues are substitutions by chemically similar amino acids and take place at residues not related to the active center of the enzyme.