Penicillin-binding proteins of clostridia

The penicillin-binding protein (PBP) profiles of 33Clostridium perfringens and sixClostridium species isolated from clinically significant infections were analyzed. Three new PBPs—PBPs 2B, 4B, and 5B (84, 70, and 49 kDa respectively)—and a high-molecular-weight PBP 6 (45 kDa) were demonstrated in theC. perfringens isolates. In addition to PBPs 1 and 2, PBPs 2B and 4B were seen to show low binding affinities for penicillin, although further studies are required to determine their possible roles in the development of penicillin resistance. The PBP profiles of theC. perfringens isolates were complex. Variations in apparent molecular weights (M _r s) of all PBPs, with the exception of PBP 5 and the presence or absence of PBPs 2, 3, and 4B, gave rise to nine different PBP patterns. The high-M _rPBPs 5 and 6, which exhibited high-penicillin-binding affinities, were with only one exception consistent within theC. perfringens isolates. These PBPs 5 and 6 of theC. perfringens isolates and independent PBPs found in the otherClostridium species studied indicate that PBP analysis may assist in the differentiation ofClostridium spacies.     

Characterization of a porcine glucosephosphate isomerase-processed pseudogene at Chromosome 1q1.6-1.7

A porcine glucosephosphate isomeraseprocessed pseudogene has been isolated and sequenced. The pseudogene has several base substitutions as well as an insertion and deletions, and is 83% homologous to the corresponding cDNA. It contains an intervening sequence of 565 bp, is truncated at the 3 end, and is flanked by direct repeats of seven nucleotides. Fluorescent in situ hybridization to porcine metaphase chromosomes localized the processed pseudogene to Chromosome (Chr) 1q1.6-1.7. A (GT)₁₄(AT)₁₅ microsatellite was detected close to the processed pseudogene.     

Reflection contrast microscopy of ultrathin sections in immunocytochemical localization studies: a versatile technique bridging electron microscopy with light microscopy

Reflection contrast microscopy (RCM) of ultrathin sections was recently introduced as a sensitive technique for visualization with enhanced definition in immunogold histochemistry. Experience of using RCM as a major tool in immunocytochemical research in different fields is summarized, e.g. oncology, nephrology and embryology. The sensitive visualization of immunocytochemical labels, gold particles or peroxidase-diaminobenzidine deposits in or on ultrathin sections, by RCM instead of electron microscopy is demonstrated. RCM of ultrathin sections is an adequate light microscopical alternative for immunoelectron microscopy, since an overview of both label and tissue is obtained with a high image definition and high contrast of label. In the studies presented, RCM is shown to provide a better gradation in staining intensity and staining pattern than other light microscopical methods. Moreover, a precise localization of multiple labels is obtained with this method. Besides the applications shown, ultrathin section visualization by RCM is very useful for correlative light- and electron microscopical studies of fine structures. Commercially available fluorescence microscopes can be adapted for proper RCM functioning; an adaptation scheme and list of microscopes tested is provided.     

Backbone Cyclization of the C-terminal Part of Substance P. Part 1: The Important Role of the Sulphur in Position 11

Novel backbone-to-side chain and backbone-to-backbone cyclic analogues of substance P (SP) were prepared by solid-phase synthesis and screened for biological activity. An analogue containing a thioether- lactam ring between positions 9 and 11 showed an EC₅₀ value of 20nM toward the neurokinin 1 (NK-1) and was inactive toward the NK-2 and NK-3 receptors. On the other hand, in a multiple backbone cyclic peptide library of similar analogues, in which the sulphur was excluded from the ring, very low activity was detected. The activity was re-evaluated and was found to be even lower (EC₅₀=0.11 mM ) than the previously published data. These results indicate that the thioether moiety has a crucial role in receptor activation. The results also show tolerance of the NK-1 receptor, but not NK-2 or NK-3, to cyclization of the C-terminal portion of the SP_6–11 hexapeptide.     

Growth and nutrient dynamics of beech (Fagus sylvatica L.) seedlings in acid soils

The relative uptake rates of N, P, K, S, Ca, Mg, Fe, Mn, Zn, Cu, and Al were estimated in beech seedlings pot cultured in the field in six acid soils (treatments). The relative uptake rates were compared with the relative growth rates. The relative uptake rates of N, K and Ca agreed well with the growth rates of the seedlings irrespective of widely differing soil conditions (acid sand-clayey till, pH 4–6). The relative uptake rates of P, Fe, and Al differed from that predicted by the growth rate. The uptake rates of Fe and Al were highest at the lowest growth rates, and the P uptake rate was lower than the growth rate in these treatments. Thus the P availability probably limited growth in an eluvial (E) horizon of a podzol, and possibly in the illuvial (B) horizon of a podzol and in an acid clayey till (Dystric Cambisol). Low P uptake was associated with a tendency towards higher relative root growth rates. In terms of the concept of steady state nutrition the high relative root growth rate in some treatments may be interpreted as an acclimation to low P supply. The P limitation seemed to be related to interactions among Fe, Al and organic compounds of the soil solution.FAX no: +4646104423     

Nitrogen mineralization in native cultivated and abandoned fields in shortgrass steppe

We conducted a set of in situ incubations to evaluate patterns of N availability among dominant land uses in the shortgrass steppe region of Colorado, USA, and to assess recovery of soil fertility in abandoned fields. Replicated 30 d incubations were performed in 3 sets of native (never cultivated), abandoned (cultivated until 1937), and currently cultivated, fallow fields. Net N mineralization and the percentage of total N that was mineralized increased in the order: native, abandoned, cultivated. Higher soil water content in fallow fields is the most likely reason for greater mineralization in cultivated fields, while higher total organic C and C/N ratios in native and abandoned fields may explain differences in mineralization between these land uses. Recovery of soil organic matter in abandoned fields appears to involve accumulation of soil C and N under perennial plants, but probable methodological artifacts complicate evaluation of the role of individual plants in recovery of N availability. Higher N mineralization and turnover in cultivated fields may make them more susceptible to N losses; recovery of N cycling in abandoned fields appears to involve a return to slower N turnover and tighter N cycling similar to native shortgrass steppe.     

Crystal structure and spin crossover studies on bis(2,6-bis(benzimidazol-2-yl)pyridine) iron(II) perchlorate

The structure of the [Fe(bzimpy)₂](ClO₄)₂·xH₂O system (x = 0.25) was determined by single crystal X-ray structure analysis. The Fe(II) ion is hexacoordinated by six donor nitrogen atoms. The magnetic properties of the complex were investigated by powder magnetic susceptibility measurements and ESR. The freshly prepared sample does not show any traces of iron(III) impurities but these are formed as a function of time. After 1 year the sample contains 8.2% iron(III) as shown by UV spectroscopy and indicated by g_eff = 4.3 and 2.0 in its ESR spectrum. This explains the recorded ξ versus T behaviour at low temperature: with increasing temperature the ξ value decreases according to the Curie-Weiss law for a S = 5/2 system having an effective g = 4.3. Above 220 K a continuous increase in the ξ value is observed and a spin crossover applies. The spin transition is not complete at room temperature. A pronounced hysteresis is observed upon heating/cooling the sample between 220 and 414 K on the basis of magnetic data and infrared spectra.     

High Mobility Group 1 Protein Is Not Stably Associated with the Chromosomes of Somatic Cells

High mobility group 1 (HMG1) protein is an abundant and conserved component of vertebrate nuclei and has been proposed to play a structural role in chromatin organization, possibly similar to that of histone H1. However, a high abundance of HMG1 had also been reported in the cytoplasm and on the surface of mammalian cells. We conclusively show that HMG1 is a nuclear protein, since several different anti-HMG1 antibodies stain the nucleoplasm of cultured cells, and epitope-tagged HMG1 is localized in the nucleus only. The protein is excluded from nucleoli and is not associated to specific nuclear structures but rather appears to be uniformly distributed. HMG1 can bind in vitro to reconstituted core nucleosomes but is not stably associated to chromatin in live cells. At metaphase, HMG1 is detached from condensed chromosomes, contrary to histone H1. During interphase, HMG1 readily diffuses out of nuclei after permeabilization of the nuclear membranes with detergents, whereas histone H1 remains associated to chromatin. These properties exclude a shared function for HMG1 and H1 in differentiated cells, in spite of their similar biochemical properties. HMG1 may be stably associated only to a very minor population of nucleosomes or may interact transiently with nucleosomes during dynamic processes of chromatin remodeling.