UDP-N-acetylglucosamine 1-carboxyvinyl-transferase from Acinetobacter calcoaceticus

Abstract We have analyzed the sequence downstream of rpoN from Zcinetobacter calcoaceticus and identified an open reading frame encoding a protein with high similarity to UDP- N -acetylgucosamine 1-carboxyvinyl-transferase (MurZ). Multicopy plasmids encoding this enzyme conferred phosphomycin resistance to A. calcoaceticus . The polar effect of a rpoN mutation on the phosphomycin resistance level suggests that murZ is, in part, cotranscribed with rpoN . These observations confirm that A. calcoaceticus represents the first exceptin from a conserved genetic context of rpoN observed in several other Gram-negative bacteria.     

Analysis of flow and vascular resistance in a model of the circle of Willis

A very simple model of the flow in the circle of Willis is described in this paper. Disregarding pulsatility and vessel wall elasticity, fluxes in all segments of the circle of Willis and its afferent and efferent vessels are calculated by applying the Poiseuille-Hagen formula. Comparison with the fluxes calculated numerically from a more sophisticated mathematical model, including pulsatility, vessel wall elasticity and nonlinear effects, revealed only very slight differences. In short, fluxes in the afferent vessels and the segments of the circle of Willis are influenced by any change of resistance within the network, whereas the fluxes in the efferent segments are dominated by the efferent resistance distribution. However, a great advantage of the present simple model is that it offers the possibility of an analytical approach which yields both an easy sensitivity analysis of parameters and an insight into the mechanisms that govern the flow in a network like the circle of Willis. It can be concluded that these mechanisms are similar to the principles of the Wheatstone bridge, known from electrical circuit theory.     

Saturation mutagenesis of the Tn10-encoded tet operator O1. Identification of base-pairs involved in Tet repressor recognition

Saturation mutagenesis of Tn10-encoded tet operator O1 was performed by chemical synthesis of 30 sequence variants yielding all possible point mutations of an operator half side. Their effect on Tet repressor binding was scored by an in-vivo repressor titration system. Tet repressor affinities of selected operator mutants were further characterized in vitro by dissociation rate measurements. The O1 sequence spans 19 base-pairs. Out of these, all 18 palindromic base-pairs are involved in Tet repressor recognition. The central base-pair does not contribute to sequence-specific binding of Tet repressor. At position 1 a pyrimidine residue is sufficient for maximal affinity to the repressor. At positions 2, 3 and 4, each mutation reduces repressor binding at least tenfold. Mutations at positions 5, 6, 7, 8 and 9 result in less drastic reductions of Tet repressor binding. Differential effects of mutations at a given position are used to deduce the chemical functions contacted by Tet repressor. The T.A to A.T transversion at position 9 increases Tet repressor affinity slightly, while all other mutations decrease repressor binding. The increased affinity of the wild-type tet operator O2 compared to wild-type O1 results from the addition of two favorable transversions at positions +/- 9 and an unfavorable T.A to C.G transition at position -7. Deletion or palindromic doubling of the central base-pair of the O1 palindrome reveals that the wild-type spacing of both operator half sides is crucial for efficient Tet repressor binding.     

The variability of the circle of Willis: univariate and bivariate analysis

The results are presented of a statistical analysis of the variability of the circle of Willis using univariate and bivariate methods. For this purpose 100 circles of Willis were available. From each circle 19 indexes of arterial size were determined, the basilar artery was measured in two places. Half the circumference was measured. This data yielded no evidence of differences between left- and right-sided vessels in the sample. An important source of variation is the general size of all vessels considered. When the data are cleared from this general size variation, correlation coefficients reveal interesting relations between the vessels. The posterior communicating arteries are strongly related to the ipsilateral carotid artery, whereas a strong inverse relationship exists with the basilar artery and the precommunicating part of the ipsilateral posterior cerebral artery. These relationships can be understood from the expected patterns of the blood flow in these vessels. Similar relationships can be found in the anterior part of the circle of Willis and in the vertebro-basilar junction. In a different manner, based on previous haemodynamic studies, the relation between blood flow and vessel size within the circle of Willis can be demonstrated by relating the ratios of the sizes of afferent and efferent arteries to the sizes of the posterior communicating arteries, an "intuitive" model. The supposed correlations of the outcome of this "intuitive" model with the size of the communicating arteries appeared to by highly significant. It is concluded that the variations of the circle of Willis are related to the individual variations of the blood flow in this arterial network.     

The primary structure of bovine brain myelin lipophilin (proteolipid apoprotein)

The amino-acid sequence of bovine myelin lipophilin (proteolipid apoprotein, Folch-protein) has been completed. Lipophilin is a 276 amino acid residues containing, extremely hydrophobic membrane protein with molecular mass 30,000 Da. The sequence determination was based on automated Edman degradation of four tryptophan and four cyanogen bromide fragments and of proteolytic peptides of complete lipophilin as well as the fragments obtained by chemical cleavage. Four additional sequences were determined which led to the completion of the primary structure. Lipophilin is esterified at threonine-198 by long chain fatty acids (palmitic, stearic and oleic acid). The attachment site has been established at the same threonine residue in three different peptides isolated from thermolysinolytic, papainolytic and chymotrypsinolytic hydrolysates. This threonine residue is part of a hydrophilic segment of lipophilin. The covalent fatty acyl bond is being discussed together with important structural and functional properties of this membrane protein which can be derived from sequence information. New separation and purification methods of hydrophobic and hydrophilic polypeptides for this sequence determination (fractional solubilization, silica gel exclusion, high-performance liquid chromatography) had to be elaborated as indispensable tools. They are generally applicable to the structural analysis of hydrophobic membrane proteins. Four long (26, 29, 40 and 36 residues) and one medium long (12 residues) hydrophobic segments are separated by four predominantly positively and one negatively charged hydrophilic segments. On the basis of structural data a model for the membrane integration of lipophilin is proposed.     

Ligand-modulated binding of a gene regulatory protein to DNA. Quantitative analysis of cyclic-AMP induced binding of CRP from Escherichia coli to non-specific and specific DNA targets

This paper describes a generally applicable method for quantitative investigation of ligand-dependent binding of a regulatory protein to its target DNA at equilibrium. It is used here to analyse the coupled binding equilibria of cAMP receptor protein from Escherichia coli K12 (CRP) with DNA and the physiological effector cAMP. In principle, the DNA binding parameters of CRP dimers with either one or two ligands bound are determinable in such an approach. The change of protein fluorescence was used to measure CRP binding to its recognition sequence in the lac control region and to non-specific DNA. Furthermore, the binding of cAMP to preformed CRP-DNA complexes was independently studied by equilibrium dialysis. The data were analysed using a simple interactive model for two intrinsically identical sites and site-site interactions. The intrinsic binding constant K and the co-operativity factor alpha for binding of cAMP to free CRP depend only slightly on salt concentration between 0.01 M and 0.2 M. In contrast, the affinity of cAMP for CRP pre-bound to non-specific DNA increases with the salt concentration and the co-operativity changes from positive to negative. This results from cation rebinding to the DNA lattice upon forming the cAMP-CRP-DNA complex from cAMP and the pre-formed CRP-DNA complex. The CRP-cAMP1 complex shows almost the same affinity for specific and non-specific DNA as the CRP-cAMP2 complex, and both displace the same number of cations. It is concluded that the allosteric activation of CRP is induced upon binding of the first cAMP. These results are used to estimate the occupation of the CRP site in the lac control region in relation to the cAMP concentration in vivo. Under physiological conditions the lac promoter is activated by the CRP dimer complexed with only one cAMP. Furthermore, a model for the differential activation of various genes expressed under catabolite repression is presented and discussed.     

A modified mallory-cason staining procedure for large cryosections.

The classic Mallory-Cason staining procedure has been modified for application to sections "on tape" obtained from large deep frozen tissue specimens. These 20 microns cryosections are collected on tape from a large heavy duty cryomicrotome. The stained sections provide anatomical details that are not revealed by other techniques. The merit of this procedure is found in the support of modern medical modalities, both for research and educational purposes.