Beta-trace protein concentration in cerebrospinal fluid is decreased in patients with bacterial meningitis.

Although meninges represent a major site of biosynthesis, beta-trace protein (beta-trace) has not been studied in the cerebrospinal fluid (CSF) of meningitis patients. We measured beta-trace in lumbar CSF of normal controls (n = 27) and in patients with various neurological diseases (n = 92) by an immunonephelometric assay. The mean concentration of beta-trace in CSF of control patients was 16.6+/-3.6 mg/l. In bacterial meningitis (n = 41), CSF beta-trace was significantly decreased (8.7+/-3.9 mg/l; P< 0.001), whereas in spinal canal stenosis it was elevated (29.2+/-10.3 mg/l; P= 0.002). In viral meningoencephalitis (n = 12), beta-trace CSF concentrations were normal. Beta-trace concentrations remained below the normal range even after curing of bacterial meningitis, and normalisation of CSF leucocytes and blood-CSF barrier function. Beta-trace may be a useful tool for studying the pathophysiology of bacterial meningitis.

An Fe2IVO2 diamond core structure for the key intermediate Q of methane monooxygenase.

A new paradigm for oxygen activation is required for enzymes such as methane monooxygenase (MMO), for which catalysis depends on a nonheme diiron center instead of the more familiar Fe-porphyrin cofactor. On the basis of precedents from synthetic diiron complexes, a high-valent Fe2(micro-O)2 diamond core has been proposed as the key oxidizing species for MMO and other nonheme diiron enzymes such as ribonucleotide reductase and fatty acid desaturase. The presence of a single short Fe-O bond (1.77 angstroms) per Fe atom and an Fe-Fe distance of 2.46 angstroms in MMO reaction intermediate Q, obtained from extended x-ray absorption fine structure and Mössbauer analysis, provides spectroscopic evidence that the diiron center in Q has an Fe2IVO2 diamond core.

Recombinant toluene-4-monooxygenase: catalytic and Mössbauer studies of the purified diiron and rieske components of a four-protein complex.

Expression of the tmoA-F gene cluster from Pseudomonas mendocina KRI in Escherichia coli BL21(DE3) produces a catalytically active form of the toluene-4-monooxygenase (T4MO) complex. Here we report the purification and characterization of four soluble proteins required for the in vitro reconstitution of T4MO catalytic activity. These proteins are a diiron hydroxylase (T4MOH), a Riesketype ferredoxin (T4MOC), an effector protein (T4MOD), and an NADH oxidoreductase (T4MOF). The T4MOH component is composed of the tmoA, tmoB, and tmoE gene products [quaternary structure (alpha beta epsilon)2, Mr approximately 220 kDa]. The T4MOA polypeptide contains two copies of the amino acid sequence motif (D/E)X(28-37)DEXRH; the same motif provides all of the protein-derived ligands to the diiron centers of ribonucleotide reductase, the soluble methane monooxygenase, and the stearoyl-ACP delta 9 desaturase. Mössbauer, optical, and EPR measurements show that the T4MOH contains diiron centers and suggest that the diiron center contains hydroxo bridge(s) in the diferric state, as observed for methane monooxygenase. Mössbauer and EPR measurements also show that the T4MOC contains a Rieske-type iron-sulfur center. This assignment is in accord with the presence of the amino acid sequence motif CPHX(15-17)CX2H, which has also been found in the bacterial, chloroplastic, and mitochondrial Rieske proteins as well as the bacterial NADH-dependent cis-dihydrodiol-forming aromatic dioxygenases. While single-turnover catalytic studies confirm the function of the T4MOH as the hydroxylase, the NADH-dependent multiple-turnover hydroxylation activity is increased by more than 100-fold in the presence of the T4MOC, which mediates highly specific electron transfer between the T4MOF and the T4MOH. The T4MOD can be purified as an 11.6 kDa monomeric protein devoid of cofactors or redox-active metal ions; this component is also detected as a substoichiometric consitutent of the purified T4MOH. The rate of the hydroxylation reaction can be mildly stimulated by the further addition of separately purified T4MOD to the T4MOH, implying the formation of a high affinity, catalytically competent complex between these two components. These characterizations define a novel, four-component oxygenase combining elements from the soluble methane oxidation complex of the methanotrophic bacteria and the aromatic hydroxylation complexes of the soil pseudomonads.

Selection effects on a linked neutral locus.

Selection changes the frequency of alleles at a linked locus as well as at those under selection if the population is not in linkage equilibrium. The magnitude of this frequency change depends on the tightness of the linkage, the selection intensity, and the deviation from linkage equilibrium. Allowing a population to mate randomly without selection brings the population closer to linkage equilibrium. This decreases the effect of selection on allelic frequencies at a linked neutral locus. However, if linkage is very tight it can take many generations to make a large difference in the effect of the linked locus. The loss due to undesirable changes in allelic frequencies at linked loci when the population is not in linkage equilibrium must be weighed against the time and effort saved by beginning intense selection for the primary trait in an early generation. Effects of selection intensity, linkage intensity, and delayed selection on changes in allelic frequency at a neutral linked locus are demonstrated.