Infestin, a thrombin inhibitor presents in Triatoma infestans midgut, a Chagas' disease vector: gene cloning, expression and characterization of the inhibitor.

This work describes the purification, gene cloning and expression of infestin, a thrombin inhibitor from midguts of Triatoma infestans. Infestin is located in the midgut and its purification was performed by anion-exchange and affinity chromatographies. The N-terminal sequence and the sequence of tryptic peptides were determined. Using RT-PCR, total RNA and infestin cDNA information, a DNA fragment was cloned which encodes a multi non-classical Kazal-type serine protease inhibitor. Isolated native infestin has two non-classical Kazal-type domains and shows an apparent molecular mass of 13 kDa, while its gene codes for a protein with four non-classical Kazal-type domains corresponding to an apparent molecular mass of 22 kDa. Two recombinant infestins, r-infestin 1-2 and r-infestin 1-4, were constructed using the vector pVT102U/alpha and expressed in S. cerevisiae. Native and r-infestin 1-2 showed very similar inhibitory activities towards thrombin and trypsin with dissociation constants of 43.5 and 25 pM for thrombin and 2.0 and 3.1 nM for trypsin, respectively. No other serine protease of the blood coagulation cascade was inhibited by the r-infestin 1-2. Surprisingly, r-infestin 1-4 inhibited not only thrombin and trypsin (K(i) of 0.8 and 5.2 nM, respectively), but also factor XIIa, factor Xa and plasmin (K(i) of 78 pM, 59.2 and 1.1 nM, respectively).

A new pyrrolyl-quinoxalinedione series of non-NMDA glutamate receptor antagonists: pharmacological characterization and comparison with NBQX and valproate in the kindling model of epilepsy.

Antagonists at the ionotropic non-NMDA [AMPA (amino-methyl proprionic acid)/kainate] type of glutamate receptors have been suggested to possess several advantages compared to NMDA (N-methyl-D-aspartate) receptor antagonists, particularly in terms of risk/benefit ratio, but the non-NMDA receptor antagonists available so far have not fulfilled this promise. From a large series of pyrrolyl-quinoxalinedione derivatives, we selected six new competitive non-NMDA receptor antagonists. The basis of selection was high potency and selectivity for AMPA and/or kainate receptors, high in vivo potency after systemic administration, and an acceptable ratio between neuroprotective or anticonvulsant effects and adverse effects. Pharmacological characteristics of these novel compounds are described in this study with special emphasis on their effects in the kindling model of temporal lobe epilepsy, the most common type of epilepsy in humans. In most experiments, NBQX and the major antiepileptic drug valproate were used for comparison with the novel compounds. The novel non-NMDA receptor antagonists markedly differed in their AMPA and kainate receptor affinities from NBQX. Thus, while NBQX essentially did not bind to kainate receptors at relevant concentrations, several of the novel compounds exhibited affinity to rat brain kainate receptors or recombinant kainate receptor subtypes in addition to AMPA receptors. One compound, LU 97175, bound to native high affinity kainate receptors and rat GluR5-GluR7 subunits, i.e. low affinity kainate binding sites, with much higher affinities than to AMPA receptors. All compounds potently blocked AMPA-induced cell death in vitro and, except LU 97175, AMPA-induced convulsions in vivo. In the kindling model, compounds with a high affinity for GluR7 (LU 97175) or compounds (LU 115455, LU 136541) which potently bind to AMPA receptors and low affinity kainate receptor subunits were potent anticonvulsants in the kindling model, whereas the AMPA receptor-selective LU 112313 was the least selective compound in this model, indicating that non-NMDA antagonists acting at both AMPA and kainate receptors are more effective in this model than AMPA receptor-selective drugs. Three of the novel compounds, i.e. LU 97175, LU 115455 and LU 136541, exerted potent anticonvulsant effects without inducing motor impairment in the rotarod test. This combination of actions is thought to be a prerequisite for selective anticonvulsant drug action.

A Kazal-type inhibitor with thrombin specificity from Rhodnius prolixus.

A thrombin-specific inhibitor with an apparent molecular mass of 11 kDa has been purified from the insect Rhodnius prolixus. Amino-terminal protein sequence analysis allowed the molecular cloning of the corresponding cDNA. The open reading frame codes for a protein of about 103 amino acid residues and displays an internal sequence homology of residues 6-48 with residues 57-101 indicating a two-domain structure. Based on the amino acid sequence the two domains exhibit high homology to protease inhibitors belonging to the Kazal-type family. Model building suggests that the first domain binds to the active site with residue His10 pointing into the specificity pocket. From gel filtration and tight-binding inhibition experiments the inhibitor appears to form 1:1 complexes with thrombin. Periplasma-directed heterologous expression of the rhodniin cDNA in Escherichia coli yields the intact thrombin inhibitor. Natural and recombinant rhodniin both display inhibition constants of about 2 x 10(-13) M.

Overexpression and purification of the galactose operon enzymes from Escherichia coli.

A convenient new procedure for the purification of galactokinase, galactose-1-phosphate uridyltransferase, and UDP-galactose 4-epimerase overexpressed in Escherichia coli is presented. The procedure is shorter than any other described in the literature and facilitates the purification of the three recombinant enzymes in considerable amounts and at high purity and specific activity. The purified gal operon enzymes were biochemically characterized by gel-filtration column chromatography and isoelectric focusing, and the Km values for their substrates were determined.

The PreA4(695) precursor protein of Alzheimer's disease A4 amyloid is encoded by 16 exons.

Alzheimer's disease (AD) is characterized by the cerebral deposition of fibrillar aggregates of the amyloid A4 protein. Complementary DNA's coding for the precursor of the amyloid A4 protein have been described. In order to identify the structure of the precursor gene relevant clones from several human genomic libraries were isolated. Sequence analysis of the various clones revealed 16 exons to encode the 695 residue precursor protein (PreA4(695] of Alzheimer's disease amyloid A4 protein. The DNA sequence coding for the amyloid A4 protein is interrupted by an intron. This finding supports the idea that amyloid A4 protein arises by incomplete proteolysis of a larger precursor, and not by aberrant splicing.

The promoter of Alzheimer's disease amyloid A4 precursor gene.

The promoter of the gene for the human precursor of Alzheimer's disease A4 amyloid protein (PAD gene) resembles promoters of housekeeping genes. It lacks a typical TATA box and shows a high GC content of 72% in a DNA region that confers promoter activity to a reporter gene in an in vivo assay. Transcription initiates at multiple sites. Sequences homologous to the consensus binding sites of transcription factor AP-1 and the heat shock control element binding protein were found upstream of the RNA start sites. Six copies of a 9-bp-long GC-rich element are located between positions -200 and -100. A protein--DNA interaction could be mapped to this element. The 3.8 kb of the 5' region of the PAD gene include two Alu-type repetitive sequences. These findings suggest that four mechanisms may participate in the regulation of the PAD gene and could be of relevance for the progression of amyloid deposition in Alzheimer's disease.

Identification, transmembrane orientation and biogenesis of the amyloid A4 precursor of Alzheimer's disease.

The precursor of the Alzheimer's disease-specific amyloid A4 protein is an integral, glycosylated membrane protein which spans the bilayer once. The carboxy-terminal domain of 47 residues was located at the cytoplasmic site of the membrane. The three domains following the transient signal sequence of 17 residues face the opposite side of the membrane. The C-terminal 100 residues of the precursor comprising the amyloid A4 part and the cytoplasmic domain have a high tendency to aggregate, and proteinase K treatment results in peptides of the size of amyloid A4. This finding suggests that there is a precursor-product relationship between precursor and amyloid A4 and we conclude that besides proteolytic cleavage other events such as post-translational modification and membrane injury are primary events that precede the release of the small aggregating amyloid A4 subunit.

Localization of the putative precursor of Alzheimer's disease-specific amyloid at nuclear envelopes of adult human muscle.

Cloning and sequence analysis revealed the putative amyloid A4 precursor (pre-A4) of Alzheimer's disease to have characteristics of a membrane-spanning glycoprotein. In addition to brain, pre-A4 mRNA was found in adult human muscle and other tissues. We demonstrate by in situ hybridization that pre-A4 mRNA is present in adult human muscle, in cultured human myoblasts and myotubes. Immunofluorescence with antipeptide antibodies shows the putative pre-A4 protein to be expressed in adult human muscle and associated with some but not all nuclear envelopes. Despite high levels of a single 3.5-kb pre-A4 mRNA species in cultured myoblasts and myotubes, the presence of putative pre-A4 protein could not be detected by immunofluorescence. This suggests that putative pre-A4 protein is stabilized and therefore functioning in the innervated muscle tissue but not in developing, i.e. non-innervated cultured muscle cells. The selective localization of the protein on distinct nuclear envelopes could reflect an interaction with motor endplates.

The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor.

Alzheimer's disease is characterized by a widespread functional disturbance of the human brain. Fibrillar amyloid proteins are deposited inside neurons as neurofibrillary tangles and extracellularly as amyloid plaque cores and in blood vessels. The major protein subunit (A4) of the amyloid fibril of tangles, plaques and blood vessel deposits is an insoluble, highly aggregating small polypeptide of relative molecular mass 4,500. The same polypeptide is also deposited in the brains of aged individuals with trisomy 21 (Down's syndrome). We have argued previously that the A4 protein is of neuronal origin and is the cleavage product of a larger precursor protein. To identify this precursor, we have now isolated and sequenced an apparently full-length complementary DNA clone coding for the A4 polypeptide. The predicted precursor consists of 695 residues and contains features characteristic of glycosylated cell-surface receptors. This sequence, together with the localization of its gene on chromosome 21, suggests that the cerebral amyloid deposited in Alzheimer's disease and aged Down's syndrome is caused by aberrant catabolism of a cell-surface receptor.

Nucleotide sequences of the gal E gene and the gal T gene of E. coli.

The nucleotide sequences of the gal E gene coding for UDP-galactose-4-epimerase and the gal T gene coding for galactose-1-P uridyltransferase of Escherichia coli have been determined. UDP-galactose-4-epimerase and galactose-1-P uridyltransferase are predicted to consist of 338 and 347 residues, respectively, NH2-terminal methionines included.