Crystallization and preliminary X-ray analysis of a depressant insect toxin from the scorpion Buthus martensii Karsch.

Depressant insect toxins are a distinct group of scorpion neurotoxins for which no three-dimensional structures are yet available. A depressant insect toxin named BmK dITAP3 from the scorpion Buthus martensii Karsch (BmK) has been purified and crystallized. Single crystals of dITAP3 grew in the presence of the detergent CYMAL-6 using the hanging-drop vapour-diffusion method with ammonium sulfate as precipitant. A set of diffraction data to 2.6 A resolution has been collected. Preliminary analysis of the diffraction data indicated that the crystal belonged to space group R3, with unit-cell parameters a = b = 73.29, c = 68.90 A, alpha = beta = 90, gamma = 120 degrees. Assuming two molecules in the asymmetric unit, the estimated solvent content is 53.4%.

A new insect neurotoxin AngP1 with analgesic effect from the scorpion Buthus martensii Karsch: purification and characterization.

An insect toxin named BmK AngP1 was purified from the venom of the scorpion Buthus martensii Karsch (BmK). It also shows an evident analgesic effect on mice, but is interestingly devoid of mammalian toxicity. Bioassay showed that the CPU value of AngP1 was 0.01 microg/body ( approximately 30 mg) for the excitatory insect toxicity and 43.0% inhibition efficiency for analgesia at a dose of 5 mg/kg. However, even at the dosage of 10 mg/kg no detectable toxicity on mice could be found. The isoelectric point (pI) value for AngP1 was 4.0, and its molecular mass analyzed by MALDI-TOF MS was 8141.0. The first 15 N-terminal residues of AngP1 were determined by Edman degradation and showed high similarity to that of other excitatory scorpion insect toxins. The circular dichroism spectroscopy measured on a JASCO J-720 system showed that there were 10.4% alpha-helix, 46.2% beta-strand and 14.1% turn structure in this peptide. Under two conditions single crystals of AngP1 were obtained.

Crystallization and preliminary X-ray analysis of the thermostable sweet protein mabinlin II.

Mabinlin II is a sweet protein with the highest known thermostablility and is isolated from the seeds of Capparis masaikai Levl. grown in south China. Two crystal forms of mabinlin II were obtained using the hanging-drop vapour-diffusion method. One of them diffracts to 2.8 A resolution and belongs to space group P2, with unit-cell parameters a = 50.16, b = 50.17, c = 76.60 A, beta = 99.6 degrees. There are four molecules per asymmetric unit, with a solvent content of 35.3%.

Crystallization and preliminary X-ray analyses of insect neurotoxins with analgesic effect from the scorpion Buthus martensii Karsch.

Three insect neurotoxins from the scorpion Buthus martensii Karsch, named BmK I1, BmK I4 and BmK I6, have been purified and crystallized. BmK I1 and BmK I4 show strong toxicity to insects, while BmK I6 is relatively weaker. They all exhibit an evident analgesic effect on mice; this is a novel biological function for scorpion insect toxins. Their crystals diffract to at least 3.5 (BmK I1), 2.8 (BmK I4), 2.8 (BmK I6 crystal form I) and 2.2 A (of BmK I6 crystal form II) resolution on an ordinary X-ray source. Crystals of BmK I1 belong to space group P6, with unit-cell parameters a = b = 66.2, c = 176.7 A. BmK I4 crystallized in the tetragonal space group I4, with unit-cell parameters a = b = 134.5, c = 60.6 A. BmK I6 has been crystallized in two forms: form I belongs to space group C2, with unit-cell parameters a = 46.5, b = 85.2, c = 32.6 A, beta = 110.5 degrees; form II belongs to space group R3, with the hexagonal unit-cell parameters a = b = 44.5, c = 164.7 A.

Drg-1 as a differentiation-related, putative metastatic suppressor gene in human colon cancer.

A gene related to cell differentiation was identified by differential display as a candidate suppressor of metastases in colon cancer. This gene, with a full-length cDNA of 3 kb, is expressed in normal colon and primary colon cancer tissues and cell lines but not in their metastatic counterparts. A GenBank search found that it is identical to a recently cloned gene, differentiation-related gene-1 (Drg-1), isolated from differentiated HT-29 colon cancer cells. Stable transfection of the SW620 metastatic colon cancer cell line with Drg-1 cDNA induced morphological changes consistent with differentiation and up-regulated the expression of several colonic epithelial cell differentiation markers (alkaline phosphatase, carcinoembryonic antigen, and E-cadherin). Moreover, the expression of Drg-1 is controlled by several known cell differentiation reagents, such as ligands of peroxisome proliferator-activated receptor gamma (troglitazone and BRL46593) and of retinoid X receptor (LG268), and histone deacetylase inhibitors (trichostatin A, suberoylanilide hydroxamic acid, and tributyrin). A synergistic induction of Drg-1 expression was seen with the combination of tributyrin and a low dose of 5'-aza-2'-dexoycytidine (100 nM), an inhibitor of DNA methylation. Functional studies revealed that overexpression of Drg-1 in metastatic colon cancer cells reduced in vitro invasion through Matrigel and suppressed in vivo liver metastases in nude mice. We propose that Drg-1 suppresses colon cancer metastasis by inducing colon cancer cell differentiation and partially reversing the metastatic phenotype.

Association of K-ras mutations with p16 methylation in human colon cancer.

BACKGROUND & AIMS: K-ras mutations are early genetic changes in colon cancer. p16, a tumor-suppressor gene, is inactivated in neoplasms by mutation, deletion, or methylation. The aims of this study were to determine p16 methylation status and its possible association with K-ras mutations in human colon cancer. METHODS: DNA isolated from 8 colon cancer cell lines and 41 microdissected human colon tissue samples was analyzed. p16 methylation status was determined using two analytical methods. The level of p16 expression was determined by reverse-transcription polymerase chain reaction and Northern blot. K-ras mutations were determined by DNA sequence analysis. The DNA methyltransferase activity was determined by microassay. Parental and K-ras-transformed IEC-18 cells were used to determine the potential association between K-ras mutations and p16 methylation. RESULTS: Methylated p16 was found in 100% of colon cancer cell lines, 55% of colon cancers, 54% of adenomas, and 25% of transitional mucosa but not in normal colonic epithelium. Forty-five percent of cancers and 38% of adenomas showed both K-ras mutations and p16 methylation. Of 11 cancers and adenomas with K-ras mutation, 10 specimens showed methylated p16. In contrast, of 13 adenomas and cancers with wild-type K-ras, only 3 specimens showed methylated p16 (P = 0.001). Stable transformation of IEC-18 cells with K-ras increased the DNA methyltransferase activity, methylated the p16 gene, and suppressed the expression of p16. Treatment with a DNA methylation inhibitor (azadeoxycytidine) resulted in reexpression of p16 in K-ras-transformed IEC-18 cells, suggesting that the expression of p16 was suppressed by DNA methylation. CONCLUSIONS: p16 methylation occurs frequently in human colonic adenomas and cancers and is closely associated with K-ras mutations.

Evolution of transcriptional control from prokaryotic beginnings to eukaryotic complexities.

Mechanisms for regulating gene transcription became increasingly complex as organisms evolved. In prokaryotes the relatively simple mechanism of repression is based on a few proteins that bind to specific DNA sequences in a ligand-dependent fashion. In eukaryotes large complexes that include ligand binding proteins regulate transcription. Lower eukaryotes developed an additional level of control based on protein complexes that include modifying enzymes. The DNA/histone complex, in combination with gene-specific transcriptional factors, is the basis of gene regulation in eukaryotes. Higher eukaryotes took regulation a level further by methylating CpGs in promoter sequences of DNA, thereby allowing binding of histone deacetylases and inhibiting transcription. Finally, long-lasting "superrepression" provides another mechanism for coordinate transcriptional regulation of large blocks of genes.

Increased intestinal Bak expression results in apoptosis.

Cells in the human intestinal epithelium have a life-span of around 5 days before being lost by apoptosis at the luminal surface. We examined changes in expression of the Bcl-2 gene family which may be responsible for epithelial cell loss. In the normal and neoplastic colon, mucosal expression of immunoreactive Bak co-localized with sites of epithelial cell apoptosis. Inducing apoptosis in the human colon cancer cell line HT29 and the rat normal small intestinal cell line IEC 18 in culture was accompanied by increased Bak expression without consistent changes in expression of other Bcl-2 homologous proteins. Bak appears to be the endogenous Bcl-2 family member best correlated with intestinal cell apoptosis.

30 KDa phosphorylated form of Bcl-2 protein in human colon.

Bcl-2 expression was studied in a human colon cell line (HT-29) and in human colonic biopsies by Western and Northern blotting. Polyclonal antibodies raised against the Bcl-2 protein detected the expected 26 KDa protein in human colon. However, although Bcl-2 mRNA was present, the 26 KDa Bcl-2 protein was absent in HT-29 cells. Instead, a 30 KDa protein band strongly reacting with anti-Bcl-2 antibodies was found in HT-29 cells, and also in human colon, tonsil, and some other tissues. Alkaline phosphatase shifted the 30 KDa protein to the 26 KDa position in a time-dependent manner. 32P-labeling of HT-29 cells showed that the 30 KDa protein was phosphorylated. A 27 KDa phosphorylated protein was also immunoprecipitated by anti-Bcl-2 antibody. Phosphopeptide mapping showed that the 27 KDa protein contained a minimum of 3 and the 30 KDa protein at least an additional four phosphorylation sites. Phosphoamino acid analysis revealed that both the 30 KDa and 27 KDa proteins were phosphorylated on serine residues. These findings strongly suggest that the 30 KDa protein is a phosphorylated form of Bcl-2, which is widely distributed in human tissues.

[A study on the mechanism of hyperinsulinemia in essential hypertension].

Blood glucose(BG), insulin(IS), C-peptide(CP), glucagon(GC) and their area under the curve(AUC), the CP:IS molar ratio and IS:GC molar ratio were measured and calculated in 31 hypertensives and 23 weight control normotensives. Compared with the normotensives, the patients showed higher fasting serum IS, CP and the IS:GC ratio, and exhibited increased BG, IS, CP and their AUC and the IS:GC ratio, and a lower CP:IS molar ratio after the oral glucose load. No significant difference was found in the fasting CP:IS molar ratio, BG, GC between normotensive and hypertensive subjects. The results indicate that there are impaired glucose tolerance, hyperinsulinemia and IS resistance in essential hypertensive subjects. The hyperinsulinemia may be caused by a beta-cell hypersecretory response to the defective peripheral action of the hormone and by a decreased hepatic insulin clearance. The study also suggests that IS resistance in essential hypertensive subjects usually involve other abnormalities of metabolism and associate with increased risk factors for coronary artery disease.

Proline-directed protein kinase (p34cdc2/p58cyclin A) phosphorylates bovine neurofilaments.

Proline-directed protein kinase (PDPK), a complex of p34cdc2 and p58cyclin A, phosphorylates bovine neurofilaments (NFs) in vitro. Incubation of intact filaments with PDPK led to strong labeling of the heavy (NF-H) and middle (NF-M) molecular weight NF proteins and weaker labeling of the low molecular weight protein (NF-L). All three proteins were phosphorylated in solution, with the best substrate being NF-H. Proteins that had been dephosphorylated by enzymatic treatment were better substrates than native proteins--as many as 6 mol of phosphate were incorporated per mole of NF-H. Partial proteolytic cleavage experiments combined with two-dimensional peptide mapping indicated that NF-H and NF-M were phosphorylated predominantly in the tail domains, with some phosphate also appearing in the heads. Soluble NF-L is phosphorylated on the head domain peptide L-3, whereas NF-L within intact filaments is phosphorylated only on the tail domain peptide L-1. Phosphorylation does not lead to filament disassembly. A possible role for PDPK in NF phosphorylation in vivo is discussed.

Phosphorylation of bovine neurofilament proteins by protein kinase FA (glycogen synthase kinase 3).

A highly purified preparation of protein kinase FA (where FA is the activating factor for phosphatase 1)/glycogen synthase kinase 3 from rabbit muscle readily phosphorylated bovine neurofilaments. All three neurofilament proteins, the high, middle, and low molecular proteins (NF-H, NF-M, and NF-L), were phosphorylated when intact filaments were incubated with the kinase. Experiments with individual proteins showed that NF-M was the best substrate. At protein concentrations of 0.13 mg/ml, the initial rate of NF-M phosphorylation was 30% of that observed for glycogen synthase. Km values were 0.24 mg/ml (7 x 10(-7) M tetramer) for glycogen synthase and 0.10 mg/ml (5 x 10(-7) M dimer) for NF-M. Vmax values were 0.36 mumol/min/mg for glycogen synthase and 0.035 mumol/min/mg for NF-M. Dephosphorylated NF-M was phosphorylated only half as much as native NF-M; this is consistent with the known substrate specificity of the kinase. The possible involvement of FA/GSK-3 in the phosphorylation of neurofilaments in vivo is discussed.