Purification, characterization cloning, and sequencing of metalloendopeptidase from Streptomyces septatus TH-2

Streptomyces septatus TH-2 secretes a large amount of a protease when cultured on a medium containing K(2)HPO(4) and glucose. The enzyme was purified to homogeneity by a three-step procedure. This enzyme had a molecular mass of approximately 35kDa, and was particularly inhibited by EDTA and phosphoramidon. Its substrate specificity was investigated using novel fluorescence energy transfer combinatorial libraries. The protease was found to prefer Phe and Tyr at the P(1) position, a hydrophobic or basic residue at the P(2) position, and a basic or small residue at the P(3) position. Its gene was cloned and sequenced, and its deduced amino acid sequence contained an HEXXH consensus sequence for zinc binding, confirming that it encodes metalloendopeptidase. The primary structure of the enzyme showed 40 and 69% identities with that of thermolysin from Bacillus thermoproteolyticus and that of a metalloendopeptidase from Streptomyces griseus, respectively.     

Cucumisin-like protease from the latex of Euphorbia supina

A protease has been purified from the latex of Euphorbia supina Rafin by two steps of chromatography. The Mr was estimated by SDS-PAGE to be 80 kDa. Its activity was inhibited strongly by diisopropyl fluorophosphate, but not by EDTA, pepstatin, or cysteine protease inhibitors, indicating that the enzyme is a serine protease. The specificity of the protease is broad, but the preferential cleavage sites were C-terminal sites of hydrophobic amino acid residues. The N-terminal sequence of the first fifteen residues was determined and six of the residues match those in cucumisin [EC 3.4.21.25], a protease from the sarcocarp of melon fruit (Cucumis melo L. var. Prince). The results indicate that the E. supina protease is a cucumisin-like serine protease.     

Identification of a Novel Gene Encoding a PrP-Like Protein Expressed as Chimeric Transcripts Fused to PrP Exon 1/2 in Ataxic Mouse Line with a Disrupted PrP Gene

1. Mouse lines lacking prion protein (PrP(C)) have a puzzling phenotypic discrepancy. Some, but not all, developed late-onset ataxia due to Purkinje cell degeneration. 2. Here, we identified aberrant mRNA species in the brain of Ngsk Prnp0/0 ataxic, but not in nonataxic Zrch Prnp0/0 mouse line. These mRNAs were chimeric between the noncoding exons 1 and 2 of the PrP gene (Prnp) and the novel sequence encoding PrP-like protein (PrPLP), a putative membrane glycoprotein with 23% identity to PrP(C) in the primary amino acid structure. The chimeric mRNAs were generated from the disrupted Prnp locus of Ngsk Prnp0/0 mice lacking a part of the Prnp intron 2 and its splice acceptor signal. 3. In the brain of wild-type and Zrch Prnp0/0 mice, PrPLP mRNA was barely detectable. In contrast, in the brain of Ngsk Prnp0/0 mice, PrP/PrPLP chimeric mRNAs were expressed in neurons, at a particularly high level in hippocampus pyramidal cells and Purkinje cells under the control of the Prnp promoter. 4. In addition to the functional loss of PrP(C), ectopic PrPLP expression from the chimeric mRNAs could also be involved in the Purkinje cell degeneration in Ngsk Prnp0/0 mice.     

Roles of amino acid residues near the chromophore of photoactive yellow protein

To investigate the roles of amino acid residues around the chromophore in photoactive yellow protein (PYP), new mutants, Y42A, E46A, and T50A were prepared. Their spectroscopic properties were compared with those of wild-type, Y42F, E46Q, T50V, R52Q, and E46Q/T50V, which were previously prepared and specified. The absorption maxima of Y42A, E46A, and T50A were observed at 438, 469, and 454 nm, respectively. The results of pH titration for the chromophore demonstrated that the chromophore of PYP mutant, like the wild-type, was protonated and bleached under acidic conditions. The red-shifts of the absorption maxima in mutants tended toward a pK(a) increase. Mutation at Glu46 induced remarkable shifts in the absorption maxima and pK(a). The extinction coefficients were increased in proportion to the absorption maxima, whereas the oscillator strengths were constant. PYP mutants that conserved Tyr42 were in the pH-dependent equilibrium between two states (yellow and colorless forms). However, Y42A and Y42F were in the pH-independent equilibrium between additional intermediate state(s) at around neutral pH, in which yellow form was dominant in Y42F whereas the other was dominant in Y42A. These findings suggest that Tyr42 acts as the hinge of the protein, and the bulk as well as the hydroxyl group of Tyr42 controls the protein conformation. In all mutants, absorbance at 450 nm was decreased upon flash irradiation and afterwards recovered on a millisecond time scale. However, absorbance at 340--370 nm was increased vice versa, indicating that the long-lived near-UV intermediates are formed from mutants, as in the case of wild-type. The lifetime changes with mutation suggest the regulation of proton movement through a hydrogen-bonding network.     

A novel imprinted gene, HYMAI, is located within an imprinted domain on human chromosome 6 containing ZAC

Transient neonatal diabetes mellitus (TNDM) is a rare disease characterized by intrauterine growth retardation, dehydration, and failure to thrive due to a lack of normal insulin secretion. This disease is associated with paternal uniparental disomy or paternal duplication of chromosome 6, suggesting that the causative gene(s) for TNDM is imprinted. Recently, Gardner et al. (1999, J. Med. Genet. 36: 192-196) proposed that a candidate gene for TNDM lies within chromosome 6q24.1-q24.3. To find human imprinted genes, we performed a database search for EST sequences that mapped to this region, followed by RT-PCR analysis using monochromosomal hybrid cells with a human chromosome 6 of defined parental origin. Here we report the identification of a novel imprinted gene, HYMAI. This gene exhibits differential DNA methylation between the two parental alleles at an adjacent CpG island and is expressed only from the paternal chromosome. A previously characterized imprinted gene, ZAC/LOT1, is located 70 kb downstream of HYMAI and is also expressed only from the paternal allele. In the pancreas, both genes are moderately expressed. HYMAI and ZAC/LOT1 are therefore candidate genes involved in TNDM. Furthermore, the human chromosome 6q24 region is syntenic to mouse chromosome 10 and represents a novel imprinted domain.     

Effect of Active Site Pocket Structure Modification of <Emphasis Type="SmallCaps">d</Emphasis>-Stereospecific Amidohydrolase on the Recognition of Stereospecific and Hydrophobic Substrates

d-Stereospecific amidohydrolase (DAH) from Streptomyces sp. 82F2 has potential utility for the synthesis of d/l configuration dipeptides by an aminolysis reaction. Structural comparison of DAH with substrate-bound d-amino acid amidase revealed that three residues located in the active site pocket of DAH (Thr145, Ala267, and Gly271) might be involved in interactions with d-phenylalanine substrate. We substituted Ala267 and Gly271, which are located at the bottom of the hydrophobic pocket of DAH, with Phe and observed changes in the stereoselectivity and specific activity toward the free and acetylated forms of d/l-Phe-methyl esters. In contrast, the mutation of Thr145, which likely supplies negative charge for recognition of the amino group of the substrate, hardly affected the stereoselectivity of the enzyme. A similar effect was observed in an investigation of hydrolysis and aminolysis reactions using the acetylated forms of d/l-Phe-methyl esters and 1,8-diaminooctane as an acyl-donor and acyl-acceptor, respectively. Substrate binding by DAH was disrupted by the mutation of Ala267 to Val or Trp and kinetic analysis showed that the hydrophobicity of the bottom of the active site pocket (Ala267 and Gly271) is important for both stereoselectivity and recognition of hydrophobic substrates.     

Clinical effects of constant rate infusions of medetomidine–propofol combined with sevoflurane anesthesia in Thoroughbred racehorses undergoing arthroscopic surgery

Background

The aim of the present study was to evaluate clinical efficacy of constant rate infusions (CRIs) of medetomidine–propofol combined with sevoflurane anesthesia in Thoroughbred racehorses undergoing arthroscopic surgery. Thirty horses were sedated intravenously (IV) with medetomidine (6.0 μg/kg) and midazolam (0.02 mg/kg) and induced IV with ketamine (1.0 mg/kg) and propofol (1.0 mg/kg). These horses were randomly allocated to three groups and maintained with sevoflurane and CRI of either medetomidine (3.0 μg/kg/h) (Group M; n?=?10); or medetomidine (3.0 μg/kg/h) and propofol (3.0 mg/kg/h) (Group MP3; n?=?10); or medetomidine (3.0 μg/kg/h) and propofol (6.0 mg/kg/h) (Group MP6; n?=?10). End-tidal sevoflurane concentration (ET_SEVO), cardiovascular parameters, plasma propofol concentration, and recovery time and quality were compared among groups. Data were analyzed by using ANOVA with Tukey’s multiple comparison test, considering P?<?0.05 significant.

Results

ET_SEVO (%) was 2.4?±?0.1 in Group M, 1.7?±?0.2 in Group MP3, and 1.4?±?0.2 in Group MP6; ET_SEVO declined significantly in a propofol-dose-dependent manner. The rates of dobutamine infusion (μg/kg/min) required to keep the mean arterial blood pressure over 70 mmHg were significantly lower in Group MP3 (0.20?±?0.10) and Group MP6 (0.15?±?0.06) than in Group M (0.37?±?0.18). Recovery time and quality did not differ among groups. All horses in Group MP3 required only one attempt to stand, and recovery quality was excellent. Plasma propofol concentrations were stable throughout maintenance of anesthesia in Group MP3, whereas those in Group MP6 increased significantly with increasing duration of maintenance.

Conclusions

CRIs of medetomidine–propofol reduced the sevoflurane requirement for surgical anesthesia as the propofol dose increased, compared with a CRI of medetomidine alone. Additionally, the two propofol protocols provided good maintenance of cardiovascular function. CRIs of medetomidine (3.0 μg/kg/h) and propofol (3.0 mg/kg/h) resulted in excellent-quality recovery. This protocol could therefore be an especially useful additive to sevoflurane anesthesia in Thoroughbred racehorses undergoing arthroscopic surgery.

     

Identification of Residues Essential for the Activity and Substrate Affinity of l-Carnitine Dehydrogenase

Recently, two l-carnitine dehydrogenases from soil isolates Rhizobium sp. (Rs-CDH) and Xanthomonas translucens (Xt-CDH) have demonstrated to exhibit mutually differing affinities toward l-carnitine. To identify residues important for affinity to the substrate, we compared the primary structure of Xt-CDH and Rs-CDH with the recognized 3D structure of 3-hydroxyacyl-CoA dehydrogenase (PDB code: 1F0Y). Then, six residues of Xt-CDH (Phe143, Gly188, Ile190, Ala191, Gly223, and Ala224) and the corresponding residues of Rs-CDH (Tyr140, Ala185, Val187, Gly188, Ser220, and Phe221) were selected for further mutagenesis. The residues of Xt-CDH were replaced with that of Rs-CDH at the corresponding position and vice versa. All Rs-CDH mutants exhibited slight effects on substrate affinity, except for the double mutants Rs-V187I/G188A, which was devoid of enzyme activity. All Xt-CDH mutants showed different K _m values. Xt-F143Y caused a higher increase in the K _m value. Furthermore, the kinetic parameters of 10 mutants at Xt-F143 and Rs-Y140 were investigated. All Rs-Y140 mutants, except aromatic residues (Phe, Trp), produced proteins that were almost entirely devoid of enzyme activity and with disrupted affinity to l-carnitine. All Xt-F143 variants showed a marked reduction (P ≤ 0.05) in enzyme activity. Overall, our results suggest that the aromatic rings of Tyr140 in Rs-CDH and Phe143 of Xt-CDH are essential for substrate recognition.