Active-site residues of the transpeptidase domain of penicillin-binding protein 2 from Escherichia coli: similarity in catalytic mechanism to class A beta-lactamases.

By means of amino acid sequence alignment with class A beta-lactamases, the residues essential for the catalytic activity of the peptidoglycan transpeptidase of penicillin-binding protein 2 (PBP2) have been predicted to be Lys333, Asp447, and Lys544, in addition to the acylation site residue for the acyl-enzyme mechanism, Ser330. Accordingly, these residues were replaced by site-directed mutagenesis, and the resultant mutants were examined as to penicillin-binding activity and genetic complementation, which represent only the acylation step and the total reaction during transpeptidation, respectively. All the mutants at position 333 showed the complete loss of both the binding and complementation activities.

[Quantitative analysis of cerebrospinal fluid dynamics in syringomyelia using cine MRI with pre-saturation].

Some reports show the qualitative analysis of cerebrospinal fluid (CSF) pulsation in the subarachnoid space and the syrinx using cine magnetic resonance imaging (MRI). However, few reports studied the quantitative analysis of CSF pulsation. We report here the results of quantitative analysis of CSF pulsation using the cine MRI with pre-saturation pulse.

Involvement of NH2-terminal pro-sequence in the production of active aqualysin I (a thermophilic serine protease) in Escherichia coli.

Aqualysin I is a heat-stable subtilisin-type protease produced by Thermus aquaticus YT-1. The precursor of aqualysin I consists of four domains: an NH2-terminal signal peptide, an NH2-terminal pro-sequence, a protease domain, and a COOH-terminal pro-sequence. In Escherichia coli cells harboring recombinant plasmid carrying the aqualysin I gene, proteolytic activity is obtained on treatment at 65 degrees C and mature enzyme is detected.

Production and Extracellular Secretion of Aqualysin I (a Thermophilic Subtilisin-Type Protease) in a Host-Vector System for Thermus thermophilus.

Aqualysin I is synthesized as a large precursor, processed, and secreted into the culture medium by Thermus aquaticus YT-1. An expression plasmid for the aqualysin I gene in T. thermophilus HB27 was constructed.

Immunological function of food-restricted germfree and specific pathogen-free mice.

The effects of food restriction on immune function was investigated in germfree (GF) and specific pathogen-free (SPF) mice. They were maintained from five weeks of age under either full-fed or food-restricted conditions to 4.5 grams per day (equivalent to approximately 80% of full-fed intake) of a commercial diet.

Effects of germfree status and food restriction on longevity and growth of mice.

An investigation was undertaken to study the effects of germfree (GF) status and mild food restriction on life span in GF and specific pathogen-free (SPF) male ICR mice either full-fed (ad libitum) or on a restricted diet of 4.5 grams per day (equivalent to approximately 80% of full-fed intake) from five-week-old. The mean life span of the full-fed SPF and GF mice was 75.

Coherence function between sympathetic neurogram and arterial pulse wave in animal with total artificial heart.

To assess the influence of the total artificial heart replacement on autonomic nervous system, sympathetic neurogram was analyzed by power spectrum and coherence function. Two pneumatically driven sac-type ventricular assist devices were implanted as biventricular bypass (BVB) in adult mongrel dogs. After the BVB pumping, natural heart was electrically fibrillated to constitute the BVB type TAH model.

Development of the total artificial heart control system.

Sympathetic neurogram is potentially useful for the real time total artificial heart (TAH) control system. In this study, we used sympathetic tone and hemodynamic derivatives to prospect the following cardiac output in acute animal experiments using adult mongrel dogs. Moving averages of the mean left atrial pressure and mean aortic pressure were used as the parameters of the preload and afterload, respectively.

TH-7B pneumatically driven sac-type ventricular assist device and drive system.

TH-7B pneumatically driven sac-type ventricular assist device and drive system including automatic control system were developed and evaluated in the chronic animal experiments. The blood-contacting surfaces of the blood pump are coated with either Cardiothane or Cardiomat. In the chronic experiments using adult goats, only one of fifteen goats showed sudden thrombus formation after 34 pumping days.

Spectral analysis of the renal sympathetic neurogram during left ventricular assistance.

To assess the effect of left ventricular assistance on cardiac-related sympathetic nerve activity, renal sympathetic nerve activity was analysed using power spectrum and coherence function. Our TH-7B pneumatically driven sac type Ventricular Assist Device (VAD) was used in the acute experiment of 7 adult mongrel dogs. VAD were inserted between the left atrium and the descending aorta as left heart bypass.

Clinical application of proton magnetic resonance spectroscopy to cerebral ischemia.

Proton magnetic resonance spectroscopy (MRS) with the depth resolved surface coil spectroscopy technique and the 1331-2662 water suppression method was used to examine two cerebral ischemia patients and 10 normal volunteers. In all cases, N-acetyl-aspartate, creatine, phosphocreatine, and residual lipid were clearly observed. No lactic acid peak was observed in normal volunteers, but a large lactic acid peak appeared in the early stage of cerebral ischemia.

[Longitudinal change of H-1 spectroscopy in cerebral infarction].

Longitudinal change of the proton spectroscopy was observed in 2 cases of cerebral infarction. Proton spectra was acquired utilizing stimulated echo acquisition mode (STEAM). In acute stage, the increase of lactic acid and decrease of N-acetyl-aspartic acid (NAA) was observed prior to the appearance of abnormality in imagings such as magnetic resonance imaging and computer tomography.

Methionyl-tRNA synthetase gene from an extreme thermophile, Thermus thermophilus HB8. Molecular cloning, primary-structure analysis, expression in Escherichia coli, and site-directed mutagenesis.

The gene for the methionyl-tRNA synthetase (MetRS) from an extreme thermophile, Thermus thermophilus HB8, was cloned and sequenced. By expression of the T. thermophilus MetRS gene in Escherichia coli cells, thermostable MetRS was overproduced and purified to homogeneity by heat treatment and one-step column chromatography.

Site-directed mutants, at position 166, of RTEM-1 beta-lactamase that form a stable acyl-enzyme intermediate with penicillin.

Class A beta-lactamases are known to hydrolyze substrates through a Ser70-linked acyl-enzyme intermediate, although the detailed mechanism remains unknown. On the basis of the tertiary structure of the active site, the role of Glu166 of class A enzymes was investigated by replacing the residue in RTEM-1 beta-lactamase with Ala, Asp, Gln, or Asn. All the mutants, in contrast to the wild-type, accumulated a covalent complex with benzylpenicillin which corresponds to an acyl-enzyme intermediate.

Crystallization of allosteric L-lactate dehydrogenase from Thermus caldophilus and preliminary crystallographic data.

L-Lactate dehydrogenase of Thermus caldophilus GK24 was purified from Escherichia coli containing an overexpression plasmid. The enzyme was crystallized from polyethylene glycol 6000 solutions without ligands by the hanging drop vapor diffusion method. Two forms of crystals were obtained.

The balance between different peptidoglycan precursors determines whether Escherichia coli cells will elongate or divide.

The rodA(Sui) mutation allows cell division to take place at 42 degrees C in ftsI23 mutant cells, which produce a thermolabile penicillin-binding protein 3 (PBP3, the septation-specific peptidoglycan transpeptidase). We show here that the mutation in rodA is a single-base change from a glutamine to a chain termination (amber) codon, and that an amber suppressor (supE) present in the strain restores the ability to produce a reduced level of normal RodA protein. The reduced level of RodA is accompanied by an increase in the levels of two other proteins (PBP2 and PBP5) encoded by genes in the rodA operon.

Molecular cloning and nucleotide sequence of the aminopeptidase T gene of Thermus aquaticus YT-1 and its high-level expression in Escherichia coli.

Aminopeptidase T (AP-T) is a metallo-dependent dimeric enzyme of Thermus aquaticus YT-1, an extremely thermophilic bacterium. We cloned the AP-T gene from T. aquaticus YT-1 into Escherichia coli using a synthetic oligonucleotide as a hybridization probe.

Unique precursor structure of an extracellular protease, aqualysin I, with NH2- and COOH-terminal pro-sequences and its processing in Escherichia coli.

Aqualysin I is a subtilisin-type serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extremely thermophilic Gram-negative bacterium. The nucleotide sequence of the entire gene for aqualysin I was determined, and the deduced amino acid sequence suggests that aqualysin I is produced as a large precursor, consisting of at least three portions, an NH2-terminal pre-pro-sequence (127 amino acid residues), the protease (281 residues), and a COOH-terminal pro-sequence (105 residues). When the cloned gene was expressed in Escherichia coli cells, aqualysin I was not secreted.

Enhancement of the thermostability of subtilisin E by introduction of a disulfide bond engineered on the basis of structural comparison with a thermophilic serine protease.

Sites for Cys substitutions to form a disulfide bond were chosen in subtilisin E from Bacillus subtilis, a cysteine-free bacterial serine protease, based on the structure of aqualysin I of Thermus aquaticus YT-1 (a thermophilic subtilisin-type protease containing two disulfide bonds). Cys residues were introduced at positions 61 (wild-type, Gly) and 98 (Ser) in subtilisin E by site-directed mutagenesis. The Cys-61/Cys-98 mutant subtilisin appeared to form a disulfide bond spontaneously in the expression system used and showed a catalytic efficiency equivalent to that of the wild-type enzyme for hydrolysis of a synthetic peptide substrate.

[MR imaging of cervical disc disease: value GRASS imaging].

Since magnetic resonance imaging (MRI) technology has been greatly improved, MRI for cervical disc disease has become widely used in many facilities. Among non-invasive procedures, MRI is regarded as one of the most useful ones. Conventional myelography, CT myelography, and MRI were performed on 10 patients with cervical disc disease.

Nucleotide sequence and characteristics of the gene for L-lactate dehydrogenase of Thermus aquaticus YT-1 and the deduced amino acid sequence of the enzyme.

The gene for L-lactate dehydrogenase (LDH) from Thermus aquaticus YT-1 was cloned in Escherichia coli, using the Thermus caldophilus LDH gene as a hybridization probe, and its complete nucleotide sequence was determined. The LDH gene comprised 930 base pairs, starting with a GTG initiation codon. Its sequence had high homology (85.

Conformation of NAD+ bound to allosteric L-lactate dehydrogenase activated by chemical modification.

On modification of arginine residues with 2,3-butanedione, the Thermus caldophilus L-lactate dehydrogenase is converted to an activated form that is independent of an allosteric effector, fructose 1,6-bisphosphate (Fru-1,6-P2). The conformation of NAD+ bound to the modified enzyme in the absence of Fru-1,6-P2 was investigated by means of proton NMR, analyzing the time dependence of the transferred nuclear Overhauser effect (TRNOE) and TRNOE action spectra. The inter-proton distances determined on TRNOE analysis indicated that both the nicotinamide riboside moiety and the adenosine moiety of NAD+ were in the anti conformation, the ribose rings being in the C3'-endo form.

Nucleotide sequence of the rodA gene, responsible for the rod shape of Escherichia coli: rodA and the pbpA gene, encoding penicillin-binding protein 2, constitute the rodA operon.

The rodA gene, which is responsible for the rod shape of Escherichia coli, was located 5 nucleotides downstream of another rod-shape-determining gene, pbpA, encoding penicillin-binding protein 2. The coding region for the RodA protein was 1,110 base pairs in length. Two plasmids, carrying a rodA-lacZ gene fusion with and without the pbpA promoter upstream of the gene fusion, were constructed.

Identification of the penicillin-binding active site of penicillin-binding protein 2 of Escherichia coli.

We determined the active site of penicillin-binding protein (PBP) 2 of Escherichia coli. A water-soluble form of PBP 2, which was constructed by site-directed mutagenesis, was purified by affinity chromatography, labeled with dansyl-penicillin, and then digested with a combination of proteases. The amino acid composition of the labeled chymotryptic peptide purified by HPLC was identical with that of the amino acid sequence, Ala-Thr-Gln-Gly-Val-Tyr-Pro-Pro-Ala-Ser330-Thr-Val-Lys-Pro (residues 321-334) of PBP 2, which was deduced from the nucleotide sequence of the pbpA gene encoding PBP 2.

Determination of the positions of the disulfide bonds in aqualysin I (a thermophilic alkaline serine protease) of Thermus aquaticus YT-1.

Aqualysin I is a heat-stable alkaline serine protease produced by Thermus aquaticus YT-1. Aqualysin I comprises 281 amino acid residues and contains four cysteine residues. The cysteine residues seemed to form disulfide bonds in the molecule.