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Biomedical subjects

J M Smith

Publications and source records attributed to J M Smith.

At least 307 records · Page 17Linked to original sources
PubMed1989

Preliminary crystallographic investigations of glycinamide ribonucleotide transformylase.

Crystals of glycinamide ribonucleotide transformylase have been grown from 0.4 to 1 M ammonium sulfate, 0.6 to 1 M sodium-potassium phosphate, or 0.65 to 1 M citrate in the pH range 4.5-7.0. The single crystals display variable morphology with varying pH. The crystals belong to the orthorhombic space group C222 with cell dimensions a = 141.4 A, b = 98.2 A, c = 103.5 A. Co-crystals have also been obtained in the presence of the inhibitor 5,8-dideazafolate (KI = 18 microM) under similar crystallization conditions. Crystals of a chemically modified enzyme, iodinated at Cys-21, were grown under similar conditions within the pH range 6.5-7.0. These crystals are isomorphous with the unmodified enzyme. Crystals suitable for high resolution (less than 2.5 A) x-ray diffraction studies have been obtained for each of the above.

Acyltransferases↗
PubMed1989

Formylglycinamide ribonucleotide synthetase from Escherichia coli: cloning, sequencing, overproduction, isolation, and characterization.

The purL gene of Escherichia coli encoding the enzyme formylglycinamidine ribonucleotide (FGAM) synthetase which catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), glutamine, and MgATP to FGAM, glutamate, ADP, and Pi has been cloned and sequenced. The mature protein, as deduced by the structural gene sequence, contains 1628 amino acids and has a calculated Mr of 141,418. Comparison of the purL control region to other pur loci control regions reveals a common region of dyad symmetry which may be the binding site for the "putative" repressor protein. Construction of an overproducing strain permitted purification of the protein to homogeneity. N-Terminal sequence analysis and comparison of glutamine binding domain sequences (Ebbole & Zalkin, 1987) confirm the amino acid sequence deduced from the gene sequence. The purified protein exhibits glutaminase activity of 0.02% the normal turnover, and NH3 can replace glutamine as a nitrogen donor with a Km = 1 M and a turnover of 3 min-1 (2% glutamine turnover). The enzyme forms an isolable (1:1) complex with glutamine: t1/2 is 22 min at 4 degrees C. This isolated complex is not chemically competent to complete turnover when FGAR and ATP are added, demonstrating that ammonia and glutamine are not covalently bound as a thiohemiaminal available to complete the chemical conversion to FGAM. hydroxylamine trapping experiments indicate that glutamine is bound covalently to the enzyme as a thiol ester. Initial velocity and dead-end inhibition kinetic studies on FGAM synthetase are most consistent with a sequential mechanism in which glutamine binds followed by rapid equilibrium binding of MgATP and then FGAR. Incubation of [18O]FGAR with enzyme, ATP, and glutamine results in quantitative transfer of the 18O to Pi.

Amino Acid Sequence↗
PubMed1989

Amino acid sequence of the bacterioferritin (cytochrome b1) of Escherichia coli-K12.

The complete amino acid sequence of bacterioferritin (cytochrome b1) from Escherichia coli-K12 has been derived from the nucleotide sequence of the cloned gene. It comprises 158 amino acid residues giving an Mr of 18,495. The identity of the gene product was confirmed by an 87 residue N-terminal sequence obtained from the purified protein, but it differs significantly from much of the previously published partial amino acid sequence (1). Secondary structure prediction indicates a high alpha-helical content consistent with a 4-helix-bundle conformation. The fully assembled bacterioferritin molecule comprising 24 identical subunits and 12 haem moieties is a tetracosamer with an Mr of approximately 452,000.

Amino Acid Sequence↗
PubMed1989

Molecular size and symmetry of the bacterioferritin of Escherichia coli. X-ray crystallographic characterization of four crystal forms.

X-ray crystallographic data from four crystal forms of Escherichia coli bacterioferritin show that the molecule has a diameter in the range 119 to 128 A. Molecules are composed of 24 subunits arranged in 432 symmetry. In both size and symmetry the molecule resembles ferritin from eukaryotes. The four crystal forms are monoclinic, space group P2(1) with unit cell dimensions a = 118.7 A, b = 211.6 A, c = 123.3 A and beta = 119.1 degrees; orthorhombic, C222(1), a = 128.7 A, b = 197.1 A, c = 202.8 A; tetragonal, P4(2)2(1)2, a = b = 210.6 A, c = 145.0 A and cubic, I432, a = 146.9 A.

Bacterial Proteins↗
PubMed1989

Effect of replacing glucose with lipid on the energy metabolism of newborn infants.

1. Indirect calorimetry and primed constant infusion of [U-13C]glucose were combined in 28 appropriate-for-gestational age newborn, parenterally fed infants, in order to measure glucose utilization and glucose oxidation and to estimate lipogenesis from glucose. 2. The infants were randomly allocated to either a group receiving glucose as the non-protein energy source or a group having one-quarter of the glucose energy replaced by intravenous fat. The energy intake (370 kJ day-1 kg-1) and protein intake (3.4 g day-1 kg-1) were similar in both groups. 3. Energy expenditure (P less than 0.005), non-protein carbon dioxide production (P less than 0.005) and non-protein oxygen consumption (P less than 0.05) were lower in the lipid-supplemented group. 4. The significant excess of glucose utilization over oxidation (P less than 0.001) can be accounted for by lipid synthesis from glucose. 5. Fat synthesis from glucose was higher in the glucose/amino acid group (P less than 0.02), but total fat storage was higher in the lipid-supplemented group (P less than 0.02). Nitrogen balance was similar in both groups. 6. As lipogenesis from glucose is an energy- and oxygen-consuming and a carbon dioxide-producing process, the data suggest that the differences between the glucose-only group and the lipid-supplemented group are due to different rates of lipogenesis from glucose.

Calorimetry↗
PubMed1989

Horizontal transfer of penicillin-binding protein genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae.

Resistance to penicillin in clinical isolates of Streptococcus pneumoniae has occurred by the development of altered penicillin-binding proteins (PBPs) that have greatly decreased affinity for the antibiotic. We have investigated the origins of penicillin-resistant strains by comparing the sequences of the transpeptidase domain of PBP2B from 6 penicillin-sensitive and 14 penicillin-resistant strains. In addition we have sequenced part of the amylomaltase gene from 2 of the sensitive and 6 of the resistant strains. The sequences of the amylomaltase gene of all of the strains and of the PBP2B gene of the penicillin-sensitive strain show that S. pneumoniae is genetically very uniform. In contrast the PBP2B genes of the penicillin-resistant strains show approximately equal to 14% sequence divergence from those of the penicillin-sensitive strains and the development of penicillin resistance has involved the replacement, presumably by transformation, of the original PBP2B gene by a homologous gene from an unknown source. This genetic event has occurred on at least two occasions, involving different sources, to produce the two classes of altered PBP2B genes found in penicillin-resistant strains of S. pneumoniae. There is considerable variation among the PBP2B genes of the resistant strains that may have arisen by secondary transformation events accompanied by mismatch repair subsequent to their original introductions into S. pneumoniae.

Aminoacyltransferases↗
PubMed1989

Expression of transforming growth factor-beta 1 in specific cells and tissues of adult and neonatal mice.

We have used immunohistochemical techniques to detect transforming growth factor-beta 1 (TGF-beta 1) in many tissues of adult and neonatal mice. Each of two antibodies raised to the amino-terminal 30 amino acids of TGF-beta 1 selectively stained this molecule in either intracellular or extracellular locations. Strong intracellular staining was found in adrenal cortex, megakaryocytes and other cells of the bone marrow, cardiac myocytes, chondrocytes, renal distal tubules, ovarian glandular cells, and chorionic cells of the placenta. Marked staining of extracellular matrix was found in cartilage, heart, pancreas, placenta, skin, and uterus. Staining was often particularly intense in specialized cells of a given tissue, suggesting unique roles for TGF-beta within that tissue. Levels of expression of mRNA for TGF-beta 1 and its histochemical staining did not necessarily correlate in a given tissue, as in the spleen. The present data lend further support to the concept that TGF-beta has an important role in controlling interactions between epithelia and surrounding mesenchyme.

Adrenal Glands↗
PubMed1989

Extracorporeal shock wave lithotripsy with the Lithostar lithotriptor.

Early experience of extracorporeal shock wave lithotripsy (ESWL) using the Lithostar lithotriptor is reported; 145 patients underwent 185 treatments for 189 stones. Treatments were performed with either intravenous sedation and analgesia (90%) or general anaesthesia (10%). There were 123 renal calculi, 44 ureteric calculi and 22 staghorn calculi. Stone fragmentation was achieved with 1724 +/- 1035 shocks at 19 kv. The immediate stone fragmentation rate was 90% and the 3-month clearance rate 88%. Secondary treatments were required in 19% of patients and additional procedures in 17%. A comparison between stone size and the number of shock waves required to achieve stone fragmentation revealed a linear relationship. The Lithostar is a very effective second generation lithotriptor which can be used for renal, staghorn and ureteric calculi in situ, without manipulation.

Adolescent↗
PubMed1989

Effect of dopamine, ibopamine, and epinine on alpha- and beta-adrenoceptors in canine pulmonary circulation.

Dopamine has been widely utilized in the treatment of acute congestive heart failure, ibopamine, the diisobutyrate ester of N-methyldopamine (epinine), is a novel inotropic agent that, unlike-dopamine, is orally active. In clinical studies at doses that produce favorable hemodynamic responses, ibopamine and dopamine can evoke a slight and transient increase in pulmonary artery pressure and pulmonary capillary wedge pressure, an effect that is no longer apparent 1 h after administration. We have previously demonstrated in anesthetized dogs that this effect is due to stimulation of alpha-adrenoceptors in the pulmonary circulation by dopamine and ibopamine, as well as by the active form of ibopamine, epinine. The aim of the present investigation was to determine how dopamine, ibopamine, and epinine interact with beta-adrenoceptors in the canine pulmonary circulation, since this activity may serve to offset the alpha-adrenoceptor-mediated pulmonary vasoconstrictor responses. Intraarterial injection of dopamine, ibopamine, and epinine resulted in dose-dependent pulmonary vasoconstrictor responses with a maximum increase of approximately 50-60% above resting pulmonary vascular tone. When animals were pretreated with propranolol (1 mg/kg iv) to block beta-adrenoceptors, pulmonary vasoconstrictor responses to dopamine were unchanged, whereas pulmonary vasopressor responses to ibopamine and epinine were significantly potentiated, especially for epinine. Upon intraduodenal administration of a therapeutically effective dose of ibopamine (i.e. 36 mg/kg) to normal dogs, virtually no pulmonary pressor response was observed. However, administration of this same dose of ibopamine to dogs pretreated with propranolol (1 mg/kg iv) resulted in a marked pulmonary pressor response. These data indicate that epinine, and therefore the parent compound ibopamine, have the capacity to stimulate beta 2-adrenoceptors in the pulmonary circulation to a far greater degree than dopamine, and that this activity serves to offset, at least in part, the alpha-adrenoceptor-mediated pulmonary vasoconstriction that occurs in response to ibopamine.

Animals↗
PubMed1989

High frequency electrocardiography of three orthogonal leads in dogs during a coronary artery occlusion.

The ability to detect cardiac ischemia in dogs was studied using high frequency electrocardiography and signal averaging technique. Ischemia was induced via a balloon occluding cuff placed around the left anterior descending coronary artery. Three surface orthogonal bipolar leads (X, Y, and Z) were recorded prior to inflation of the occluding cuff. The cuff was then inflated, causing a complete occlusion of the coronary artery. The surface electrocardiogram was recorded before and during ligation and during reperfusion. The recorded waveforms were divided into sequential 10-15 second segments representing different stages of the ischemic state and were aligned using cross-correlation scheme, averaged and band pass filtered between 150-250 Hz. Analysis of the filtered high frequency QRS complexes revealed that coronary ligation was accompanied by changes in waveform morphology including the appearance of zones of reduced amplitude. In seven out of ten experiments reduced amplitude zones were absent before the ligation, present during the occlusion and absent following reperfusion. In two experiments, reduced amplitude zones that were present prior to ligation became wider during occlusion. In one experiment no reduced amplitude zone was present during any stage of the experiment. Such zones may represent slow conduction in regions of the heart rendered ischemia during coronary ligation. Therefore, the morphology of the high frequency QRS complex, as determined by analysis of the signal averaged electrocardiogram, may provide useful information regarding the presence of myocardial ischemia.

Animals↗
PubMed1989

Nucleotide sequence analysis of the purEK operon encoding 5'-phosphoribosyl-5-aminoimidazole carboxylase of Escherichia coli K-12.

5'-Phosphoribosyl-5-aminoimidazole (AIR) carboxylase (EC 4.1.1.21) catalyzes step 6, the carboxylation of AIR to 5'-phosphoribosyl-5-aminoimidazole-4-carboxylic acid, in the de novo biosynthesis of purine nucleotides. As deduced from the DNA sequence of restriction fragments encoding AIR carboxylase and supported by maxicell analyses, AIR carboxylase was found to be composed of two nonidentical subunits. In agreement with established complementation data, the catalytic subunit (deduced Mr, 17,782) was encoded by the purE gene, while the CO2-binding subunit (deduced Mr, 39,385) was encoded by the purK gene. These two genes formed an operon in which the termination codon of the purE gene overlapped the initiation codon of the purK gene. The 5' end of the purEK mRNA was determined by mung bean nuclease mapping and was located 41 nucleotides upstream of the proposed initiation codon. The purEK operon is regulated by the purR gene product, and a purR regulatory-protein-binding site related to the sequences found in other pur loci was identified in the purEK operon control region.

Amino Acid Sequence↗