A novel class of plant-specific zinc-dependent DNA-binding protein that binds to A/T-rich DNA sequences.

Complementary DNA encoding a DNA-binding protein, designated PLATZ1 (plant AT-rich sequence- and zinc-binding protein 1), was isolated from peas. The amino acid sequence of the protein is similar to those of other uncharacterized proteins predicted from the genome sequences of higher plants. However, no paralogous sequences have been found outside the plant kingdom. Multiple alignments among these paralogous proteins show that several cysteine and histidine residues are invariant, suggesting that these proteins are a novel class of zinc-dependent DNA-binding proteins with two distantly located regions, C-x(2)-H-x(11)-C-x(2)-C-x((4-5))-C-x(2)-C-x((3-7))-H-x(2)-H and C-x(2)-C-x((10-11))-C-x(3)-C. In an electrophoretic mobility shift assay, the zinc chelator 1,10-o-phenanthroline inhibited DNA binding, and two distant zinc-binding regions were required for DNA binding. A protein blot with (65)ZnCl(2) showed that both regions are required for zinc-binding activity. The PLATZ1 protein non-specifically binds to A/T-rich sequences, including the upstream region of the pea GTPase pra2 and plastocyanin petE genes. Expression of the PLATZ1 repressed those of the reporter constructs containing the coding sequence of luciferase gene driven by the cauliflower mosaic virus (CaMV) 35S90 promoter fused to the tandem repeat of the A/T-rich sequences. These results indicate that PLATZ1 is a novel class of plant-specific zinc-dependent DNA-binding protein responsible for A/T-rich sequence-mediated transcriptional repression.

Trihelix DNA-binding protein with specificities for two distinct cis-elements: both important for light down-regulated and dark-inducible gene expression in higher plants.

The DE1 sequence is a cis-regulatory element necessary and sufficient for light down-regulated and dark-inducible expression of the pea GTPase pra2 gene. This sequence does not show any sequence similarity to the previously reported ones involved in light-regulated gene expression. A one-hybrid screen isolated a cDNA encoding a DNA-binding protein, named DF1, with specificity for the DE1 sequence 5'-TACAGT. DF1 has domains similar to the trihelix DNA-binding domain found in the GT-1 and GT-2 proteins, which are plant transcription factors. The DE1-binding domain of DF1 is most similar to the carboxyl-terminal trihelix domain of the rice GT-2 protein with specificity for the GT2 sequence 5'-GGTAATT, which is also necessary for dark-inducible expression of the rice phyA gene. An electrophoretic mobility shift assay showed that this DNA-binding domain specifically binds to two types of DNA sequences, DE1 and GT2. Additionally, using DF1/GT-1 chimeras, we show that the second and third helices of the trihelix DNA-binding domain of DF1 are responsible for this dual DNA binding specificity. Our results show that DF1 has specificity for the two distinct cis-regulatory elements, both important for light down-regulated and dark-inducible gene expression in higher plants.

Molecular cloning, overexpression, and purification of a major xylanase from Aspergillus oryzae.

The gene encoding xylanase G2 (xynG2) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji. The structural part of xynG2 was found to be 767 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynG2 was interrupted by a single intron which was 71 bp in size and encoded 232 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynG2 had a signal peptide of 44 amino acids. The predicted amino acid sequence of XynG2 has strong similarity to other family 11 xylanases from fungi. The xynG2 gene was successfully overexpressed in A. oryzae and the overpexpressed XynG2 was purified. The molecular weight of XynG2 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 21,000. This was almost the same as the molecular weight of 20,047 calculated from the deduced amino acid sequence. The purified XynG2 showed an optimum activity at pH 6.0 and 58 degrees C. It had a Km of 5.1 mg/ml and a Vmax of 123 micromol/min/mg when birch wood xylan was used as a substrate.

Evidence that a substance P-like peptide mediates the non-cholinergic excitatory response of the carp intestinal bulb (Cyprinus carpio).

The participation of substance P in the noncholinergic contraction induced by transmural stimulation (TMS) of the carp intestinal bulb was examined. In the presence of atropine, substance P caused the contraction of carp intestinal bulb smooth muscle in a concentration dependent manner (1 nmol/l - 1 mumol/l). The EC50 value was 28 +/- 7 nmol/l (n = 6). Substance P-induced desensitization (1 mumol/l for 15 min), decreased the response to substance P and the atropine-resistant contraction induced by TMS (20 Hz) selectively. In contrast, in the absence of atropine, the contraction induced by TMS (20 Hz) was slightly attenuated with the substance P-induced desensitization. The acid extract obtained from the carp intestinal bulb contained a smooth muscle excitatory material whose pharmacological properties were consistent with those of substance P. The present results indicate that a substance P-like peptide is present in the carp intestinal bulb which is involved in the non-cholinergic contraction induced by TMS.

Tumor necrosis factor-inducing activities of lipid A preparations from Pseudomonas diminuta and Pseudomonas vesicularis.

Tumor necrosis factor (TNF)-inducing activities of lipid A preparations from P. diminuta and P. vesicularis, which contain mainly 2 mol of 2,3-diamino-2,3-dideoxy-D-glucose and 1 mol of nonglycosidic phosphate as the backbone component and have partly different fatty acid compositions, were examined. TNF was induced by injecting various lipid A fractions into mice that had previously been sensitized with Mycobacterium bovis BCG vaccine. A major component of lipid A of both strains, referred to as A3 fraction, exhibited stronger TNF-inducing activity than A2 fraction having incomplete acyl residues. The removal of ester-linked fatty acyl groups by mild hydrazinolysis of the P. diminuta lipid A results in a marked decrease of the activity. These results suggest that the structure of the hydrophobic part, including the amide-linked acyloxyacyl group(s), of the lipid A molecule play an important role in inducing TNF in the sera of mice.

Contractile response to substance P in isolated smooth muscle strips from the intestinal bulb of the carp (Cyprinus carpio).

1. The effect of substance P on the mechanical activity of carp intestinal bulb smooth muscle was investigated in vitro. 2. Bath-applied substance P (1 nM-1 microM) caused concentration-dependent contraction of the smooth muscle. The EC50 value was 20 +/- 3 nM (N = 13). 3. Pretreatment with tetrodotoxin (780 nM) or atropine (500 nM) partially decreased the contractile response to substance P, while methysergide (3 microM) did not decrease the response. 4. The contractile response to substance P was not decreased by [D-Pro2, D-Trp7.9]-substance P or [D-Pro4, D-Trp7.9]-substance P (4-11) pretreatment (10 microM for 5 min). 5. Exposure of the intestinal bulb to substance P (100 nM and 1 microM for 15 min) decreased the response to subsequent application of substance P, physalaemin and eledoisin in a concentration dependent manner, while the contractile response to acetylcholine or methionine-enkephalin was not affected. 6. Exposure of the intestinal bulb to physalaemin and eledoisin (100 nM for 15 min) decreased the response to subsequent application of substance P. 7. The above results indicate that substance P causes the contraction of the carp intestinal bulb smooth muscle through its direct action on the smooth muscle and its indirect action through enteric cholinergic nerves. Long-term exposure to substance P causes desensitization of the preparation to substance P, physalaemin and eledoisin at the receptor level.

Pharmacological properties of the atropine-resistant contraction of the carp (Cyprinus carpio) intestinal bulb induced by transmural stimulation.

1. The pharmacological properties of the atropine-resistant contraction of the carp intestinal bulb induced by transmural stimulation were investigated. 2. In the presence of atropine (1 microM), transmural stimulation caused frequency-dependent (2-50 Hz) contraction which was abolished by tetrodotoxin (780 nM). 3. The atropine-resistant contraction was not decreased by tubocurarine (5 microM), hexamethonium (100 microM), carteolol (5 microM) and phentolamine (5.4 microM). 4. In vitro pretreatment with guanethidine (10 microM for 1 hr) markedly decreased the noradrenaline and adrenaline contents of the carp intestinal bulb. The atropine-resistant contraction was not affected by pretreatment with guanethidine. 5. Diphenhydramine (1 microM), methysergide (3 microM) and naloxone (1 microM) did not decrease the atropine-resistant contraction, indicating that histamine, 5-hydroxytryptamine and opioid peptides were not involved in the atropine-resistant response. 6. These results indicate that a non-cholinergic, non-adrenergic excitatory nerve is present in the carp intestinal bulb. The neurotransmitter mediating the excitatory response could not be identified.

Is there functional cholinergic innervation in the frog duodenum (Rana catesbeiana)?

To determine if functional cholinergic innervation occurs in the frog duodenum or not, the effects of exogenous acetylcholine and electrical transmural stimulation, the contractile activity of an acid extract from the frog duodenum, and the distribution of acetylcholinesterase (AChE) activity in the wall of the frog duodenum were investigated. Acetylcholine caused non-sustained contraction in a dose-dependent manner (100 nM-1 mM). The ED50 value was 17 +/- 2.4 microM. Atropine (500 nM) shifted the dose-response curve for acetylcholine parallel to the right. Transmural stimulation of the frog duodenum caused frequency-dependent (0.5-50 Hz) contraction which was not decreased by atropine (500 nM) at all. The acid extract from the frog duodenum caused contraction of a longitudinal muscle strip of guinea-pig ileum but atropine (500 nM) had no significant effect on the contraction. Only a little AChE activity was found in Auerbach's plexus of the frog duodenum compared with that of the rat ileum. These results suggest that a cholinergic nerve is present in the frog duodenum but its physiological significance is very small.