NAD+ binding to the Escherichia coli K+-uptake protein TrkA and sequence similarity between TrkA and domains of a family of dehydrogenases suggest a role for NAD+ in bacterial transport

The nucleotide sequence of trkA, a gene encoding a surface component of the constitutive K⁺-uptake systems TrkG and TrkH from Escherichia coli, was determined. The structure of the TrkA protein deduced from the nucleotide sequence accords with the view that TrkA is peripherally bound to the inner side of the cytoplasmic membrane. Analysis by a dot matrix revealed that TrkA is composed of similar halves. The M-terminal part of each TrkA half (residues 1–130 and 234–355, respectively) is similar to the complete NAD⁺-binding domain of NAD⁺-dependent dehydrogenases. The C-terminal part of each TrkA half (residues 131–233 and 357–458, respectively) aligns with the first 100 residues of the catalytic domain of glyceraldehyde-3-phosphate dehydrogenase. Strong u.v. illumination at 252 nm led to cross-linking of NAD⁺ or NADH, but not of ATP to the isolated TrkA protein.     

The MsK family of alfalfa protein kinase genes encodes homologues of shaggy/glycogen synthase kinase-3 and shows differential expression patterns in plant organs and development

This paper reports on the isolation of a novel class of plant serine/threonine protein kinase genes, MsK-1 , MsK-2 and MsK-3 . They belong to the superfamily of cdc2 -like genes, but show highest identity to the Drosophila shaggy and rat GSK-3 proteins (66–70%). All of these kinases share a highly conserved catalytic protein kinase domain. Different amino-terminal extensions distinguish the different proteins. The different plant kinases do not originate from differential processing of the same gene as is found for shaggy , but are encoded by different members of a gene family. Similarly to the shaggy kinases, the plant kinases show different organ-specific and stage-specific developmental expression patterns. Since the shaggy kinases play an important role in intercellular communication in Drosophila development, the MsK kinases are expected to perform a similar function in plants.     

Towards a methodology for taking physical degradation of ecosystems into account in LCA

An analytic procedure has been followed to select adequate methods to express ecosystem degradation in LCA. This procedure consisted of problem definition, identification of relevant issues, of a quantitative expression for ecosystem degradation and of possible nature value indicators and building a framework of criteria for selecting adequate methods. With the selection framework a first screening of methods was performed. For full quantification the following formula is proposed: ED = L (N_r- N_a), with land use I. = A t and nature value change (N_r- N_a) Degradation due to an activity appears difficult to operationalise, but ecosystem suppression by activities can well be assessed. N_r is then the natural background or would-be natural situation. N_a can best be described by the actual state during the activity, unless hard data on restoration is available. N_a and N_r can be expressed in the biomass production indicator NPP - NCR Biodiversity and/or erosion may be added to include irreversible effects.     

Polyphosphate kinase is a component of the Escherichia coli RNA degradosome

Xer site-specific recombination functions in the stable inheritance of circular plasmids and bacterial chromosomes. Two related recombinases, XerC and XerD, mediate this recombination, which 'undoes' the potential damage of homologous recombination. Xer recombination on natural plasmid sites is preferentially intramolecular, converting plasmid multimers to monomers. In contrast, recombination at the Escherichia coli recombination site, dif , occurs both intermolecularly and intramolecularly, at least when dif is inserted into a multicopy plasmid. Here the DNA sequence features of a family of core recombination sites in which the XerC- and XerD-binding sites, which are separated by 6 bp, were analysed in order to ascertain what determines whether recombination will be preferentially intramolecular, or will occur both within and between molecules. Sequence changes in either the XerC- or XerD-binding site can alter the recombination outcome. Preferential intramolecular recombination between a pair of recombination sites requires additional accessory DNA sequences and accessory recombination proteins and is correlated with reduced affinities of recombinase binding to recombination core sites, reduced XerC-mediated cleavage in vitro , and an apparent increased overall bending in recombinase–core-site complexes.     

Betaine and L-carnitine transport by Listeria monocytogenes Scott A in response to osmotic signals.

The naturally occurring compatible solutes betaine and L-carnitine allow the food-borne pathogen Listeria monocytogenes to adjust to environments of high osmotic strength. Previously, it was demonstrated that L. monocytogenes possesses an ATP-dependent L-carnitine transporter (A. Verheul, F. M. Rombouts, R. R. Beumer, and T. Abee, J. Bacteriol. 177:3205-3212, 1995). The present study reveals that betaine and L-carnitine are taken up by separate highly specific transport systems and support a secondary transport mechanism for betaine uptake in L. monocytogenes. The initial uptake rates of betaine and L-carnitine are not influenced by an osmotic upshock, but the duration of transport of both osmolytes is directly related to the osmotic strength of the medium. Regulation of uptake of both betaine and L-carnitine is subject to inhibition by preaccumulated solute. Internal betaine inhibits not only transport of external betaine but also that of L-carnitine and, similarly, internal L-carnitine inhibits transport of both betaine and L-carnitine. The inhibition is alleviated upon osmotic upshock, which suggests that alterations in membrane structure are transmitted to the allosteric binding sites for betaine and L-carnitine of both transporters at the inner surface of the membrane. Upon osmotic downshock, betaine and L-carnitine are rapidly released by L. monocytogenes as a consequence of activation of a channel-like activity. The osmolyte-sensing mechanism described is new and is consistent with various unexplained observations of osmoregulation in other bacteria.     

Distribution and disappearance of the radiolabeled carbon derived from L-arginine and taurine in the mouse

L-arginine and taurine are still in the center of physiological and pharmacological research. Although the fate of nitrogen of both compounds and of the ³⁵S-taurine is well-documented, the fate of the carbon skeleton has not been elucidated yet. We studied the organ distribution of ¹⁴C arginine and ¹⁴C taurine over time in the mouse using whole body autoradiography with densitometric image analysis. We describe different organ distribution patterns. Kidney, heart, lung, the Harderian gland, the central nervous system, intestine and testis showed a comparable pattern of arginine disappearance in contrast to rapid disappearance in the salivary gland and the accumulation pattern in bone and spleen. Data on ¹⁴C taurine of liver, kidneys, lung, testis and Harderian gland resembled the arginine pattern; Accumulation of taurine carbon was found in salivary gland, bone, intestine, heart and brain. Our studies challenge and demand further related studies to obtaining more information on the fate of the carbon skeleton of these amino acids.     

Thermochemiluminescent assay of porcine, rat, and human erythrocytes for antioxidative deficiencies

The thermal induction of chemiluminescence of luminol-horseradish peroxidase-labeled erythrocytes from pigs, rats, and man was studied. The luminescent responses of rat, porcine, and human erythrocytes to heating were linear in respect to logs of counts per minute versus temperature. Landrace-Duroc crossbred pigs with a history of malignant hyperthermia (porcine stress syndrome) and Poland-China-miniature pigs inbred for malignant hyperthermia (MH) yielded erythrocytes with high-level thermochemiluminescence (TCL). Sprague-Dawley rat erythrocytes were intermediate in their production of TCL. Normal human and MH-resistant miniature swine erythrocytes produced low-level TCL. However, pretreatment of human erythrocytes with 1-chloro-2,4-dinitrobenzene (CDNB) resulted in high-level TCL. Furthermore, halothane enhanced the TCL of CDNB-treated human erythrocytes and Landrace-Duroc porcine erythrocytes that were not treated with CDNB. Red blood cells from pigs susceptible to the porcine stress syndrome demonstrated a TCL response very similar to CDNB-treated erythrocytes.     

σ Complexes as biochemical and biophysical probes: 4. Development of a novel reporter group delivery system

A novel reporter group delivery system for the chemical modification of proteins and the investigation of enzyme mechanisms is proposed. The design of this system is based on structural analogy with pyridoxal phosphate and σ-complex adduct formation. Progress is described toward the synthesis of suitable compounds, via three different approaches involving structural modification of the pyridine nucleus. A number of new compounds have been prepared, and other directions for future investigations are indicated.