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.

[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.

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.

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.

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).

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.