D-Alanyl-D-lactate and D-alanyl-D-alanine synthesis by D-alanyl-D-alanine ligase from vancomycin-resistant Leuconostoc mesenteroides. Effects of a phenylalanine 261 to tyrosine mutation

The Gram-positive bacterium Leuconostoc mesenteroides, ATCC 8293, is intrinsically resistant to the antibiotic vancomycin. This phenotype correlates with substitution of D-Ala-D-lactate (D-Ala-D-Lac) termini for D-Ala-D-Ala termini in peptidoglycan intermediates in which the depsipeptide has much lower affinity than the dipeptide for vancomycin binding. Overproduction of the L. mesenteroides D-Ala-D-Ala ligase (LmDdl) 2 in E. coli and its purification to approximately 90% homogeneity allow demonstration that the LmDdl2 does have both depsipeptide and dipeptide ligase activity. Recently, we reported that mutation of an active site tyrosine (Tyr), Tyr216, to phenylalanine (Phe) in the E. coli DdlB leads to gain of D-Ala-D-Lac depsipeptide ligase activity in that enzyme. The vancomycin-resistant LmDdl2 has a Phe at the equivalent site, Phe261. To test the prediction that a Tyr residue predicts dipeptide ligase while an Phe residue predicts both depsipeptide and dipeptide ligase activity, the F261Y mutant protein of LmDdl2 was constructed and purified to approximately 90% purity. F216Y LmDdl2 showed complete loss of the ability to couple D-Lac but retained D-Ala-D-Ala dipeptide ligase activity. The Tyr-->Phe substitution on the active site omega-loop in D-Ala-D-Ala ligases is thus a molecular indicator of both the ability to make D-Ala-D-Lac and intrinsic resistance to the vancomycin class of glycopeptide antibiotics.     

Comparison of calcium phosphate cement mixture and pure calcium hydroxide as direct pulp-capping agents

This review reports the different genetic factors that have been identified either as risk factor for Alzheimer's disease (AD) or directly causing the disease. First are reviewed epidemiological data and biological mechanisms about the apoplipoprotein E gene allele epsilon 4 that is a major risk factor for Alzheimer's disease. The second part describes the mutations responsible for early-onset autosomal dominant AD found in three different genes. The gene located on chromosome 21 encodes the amyloid precusor protein (APP). The presenilin 1 and presenilin 2 genes, located on chromosome 14 and 1 respectively, encode not yet known membrane proteins.     

Necrotizing enterocolitis

Advances in neonatal care have resulted in an enlarging population of vulnerable premature newborns at risk for necrotizing enterocolitis (NEC). This article presents data supporting a unifying hypothesis for the initiation of NEC based on bacteria as the inciting agent(s), and the preterm baby as the vulnerable host. Facts and controversies concerning the pathology, microbiology, clinical presentation, management and outcome of infants afflicted with NEC are presented.     

Correction for photon attenuation in SPECT: analytical framework, average attenuation factors, and a new hybrid approach

We provide a unified framework for the investigation of convergence properties of the iterative algorithms for photon attenuation correction in SPECT, including the iterative Chang method--a commonly used approach in which an average attenuation factor calculated over all projection angles is employed in a pointwise correction scheme. A new average attenuation factor calculated along the projection line is introduced, which can compensate exactly for the attenuation effect in the case of a uniform activity distribution. We propose a new hybrid approach in which we use this new average attenuation factor initially and then shift to the iterative Chang method in later iterations. This hybrid approach was evaluated in a simulation study by use of a computer-generated phantom with both non-uniform activity and non-uniform attenuation distributions. The results demonstrate that this hybrid approach improves the convergence speed of the iterative Chang method and produces reconstructed images of high quality.     

The oxygen and carbon monoxide reactions of heme oxygenase

The O2 and CO reactions with the heme, alpha-hydroxyheme, and verdoheme complexes of heme oxygenase have been studied. The heme complexes of heme oxygenase isoforms-1 and -2 have similar O2 and CO binding properties. The O2 affinities are very high, KO2 = 30-80 microM-1, which is 30-90-fold greater than those of mammalian myoglobins. The O2 association rate constants are similar to those for myoglobins (kO2' = 7-20 microM-1 s-1), whereas the O2 dissociation rates are remarkably slow (kO2 = 0.25 s-1), implying the presence of very favorable interactions between bound O2 and protein residues in the heme pocket. The CO affinities estimated for both isoforms are only 1-6-fold higher than the corresponding O2 affinities. Thus, heme oxygenase discriminates much more strongly against CO binding than either myoglobin or hemoglobin. The CO binding reactions with the ferrous alpha-hydroxyheme complex are similar to those of the protoheme complex, and hydroxylation at the alpha-meso position does not appear to affect the reactivity of the iron atom. In contrast, the CO affinities of the verdoheme complexes are >10,000 times weaker than those of the heme complexes because of a 100-fold slower association rate constant (kCO' approximately 0. 004 microM-1 s-1) and a 300-fold greater dissociation rate constant (kCO approximately 3 s-1) compared with the corresponding rate constants of the protoheme and alpha-hydroxyheme complexes. The positive charge on the verdoporphyrin ring causes a large decrease in reactivity of the iron.     

Rapid, sensitive procedure to determine buspirone levels in rat brains using gas chromatography with nitrogen-phosphorus detection

Reported here is a rapid, sensitive and relatively inexpensive procedure using gas chromatography with nitrogen-phosphorus detection (GC-NPD) to quantify buspirone levels in brains of rats. The analyte was directly extracted from brain homogenate with toluene after basification and then subjected to GC-NPD analysis using a capillary column. The calibration curves were linear over the range of 10 to 320 ng per 2 ml of brain homogenate, with typical r2 values >0.99. The assay was highly reproducible and gave peaks with excellent chromatographic properties.     

Quantitation of intrathecal measles virus IgG antibody synthesis rate: subacute sclerosing panencephalitis and multiple sclerosis

A method for quantitating specific anti-viral antibodies in serum and cerebrospinal fluid (CSF) is established using enzyme-linked immunosorbent assay (ELISA). Quantitated antibody levels are used to determine intrathecal specific IgG synthesis rate for the particular antibody. Measles virus was used as a model for validating this quantitative technique: a mutated form of measles virus is a cause of subacute sclerosing panencephalitis (SSPE) and there is a possibility that measles virus is related to the cause of multiple sclerosis (MS). Matched serum and CSF samples were assayed. Concentration of anti-measles IgG was determined and intrathecal measles-specific IgG synthesis rate was calculated. For the SSPE samples, measles-specific IgG synthesis rate was elevated and comprised > 20% of the total intrathecal IgG synthesis rate; these results are consistent with the literature. The ELISA method can be performed routinely, providing a quick, simple, reproducible means of quantitating specific antibody concentrations, with sensitivity greater than 1 nanogram per milliliter. With this method, quantitation of IgG antibodies to any other viral antigen can be reliably and precisely determined.     

Induced magnetic fields as evidence for subsurface oceans in Europa and Callisto

The Galileo spacecraft has been orbiting Jupiter since 7 December 1995, and encounters one of the four galilean satellites-Io, Europa, Ganymede and Callisto-on each orbit. Initial results from the spacecraft's magnetometer have indicated that neither Europa nor Callisto have an appreciable internal magnetic field, in contrast to Ganymede and possibly Io. Here we report perturbations of the external magnetic fields (associated with Jupiter's inner magnetosphere) in the vicinity of both Europa and Callisto. We interpret these perturbations as arising from induced magnetic fields, generated by the moons in response to the periodically varying plasma environment. Electromagnetic induction requires eddy currents to flow within the moons, and our calculations show that the most probable explanation is that there are layers of significant electrical conductivity just beneath the surfaces of both moons. We argue that these conducting layers may best be explained by the presence of salty liquid-water oceans, for which there is already indirect geological evidence in the case of Europa.     

Interaction of nonylphenol and hepatic CYP1A in rats

Both estradiol and nonylphenol (NP) inhibited hepatic microsomal 7-ethoxyresorufin O-deethylase (EROD) activity of beta-naphthoflavone-treated rats. Enzyme kinetic analyses (Lineweaver-Burk plots) using different estradiol and NP concentrations with graded increases in the concentrations of the substrate, ethoxyresorufin, showed that the inhibition was of a competitive nature at all concentrations of estradiol or NP used. Thus, the mechanism by which NP inhibits EROD activity is similar to that of estradiol. NP, however, was much less potent than estradiol. Young rats treated in vivo with 80 mg/kg body weight of NP demonstrated a slight but significant decrease in their hepatic microsomal EROD activity and CYP1A protein as measured by western blot analysis. In addition, treatment with NP led to a decrease in the steady-state levels of hepatic CYP1A mRNA in rats, suggesting that NP acted at the pre-translational level. The competitive nature of inhibition by NP on hepatic microsomal EROD activity indirectly suggests that this compound is a possible substrate of the CYP1A enzyme. Furthermore, NP had a moderate modulating effect on the expression of CYP1A in rat liver.