Analysis of Chaperone Function and Formation of Hetero-oligomeric Complexes of Hsp18.1 and Hsp17.7, Representing Two Different Cytoplasmic sHSP Classes in Pisum sativum

Small heat shock proteins (sHSPs) are the most abundant stress proteins in plants. Usually not expressed under permissive conditions, they can accumulate to more than 2% of the total cellular protein content during heat stress. At present several points of evidence indicate that these proteins act as molecular chaperones by keeping partially denatured proteins in a folding-competent state. In plants sHSPs are encoded by a multigene family, which can be segregated into several classes according to their subcellular position and/or sequence homology. Curiously, two different classes appear in the cytoplasm. Their specific role during heat shock remains elusive. Here we present some evidence that both classes of sHSPs enhance recovery of reporter protein activity in the presence of HSP70. Applying peptide arrays prepared by SPOT synthesis and in situ analysis by confocal laser scanning microscopy, we could further show that the two classes of sHSP are attached to each other and are able to interact with non-native proteins both in vivo and in vitro. Although both of the sHSPs act similarly as molecular chaperones, immunohistochemistry experiments support the hypothesis that the two have different cellular functions in the development of heat-induced cytoplasmic heat shock granules under elevated temperatures. Daniela Wagner Deceased 24 Feburary 2004.     

Copper(II) PMDTA and copper(II) TMEDA complexes: precursors for the synthesis of dinuclear dicationic copper(II) complexes

Copper(II) cations coordinated with PMDTA (pentamethyldiethylenetriamine) and TMEDA (tetramethylethylenediamine) possess a high synthetic potential. The synthesis of these cations was carried out by metathesis reactions with silver salts. The cationic copper(II) complexes, [Cu(PMDTA)(Me₂CO)Cl]⁺, [Cu(PMDTA)(H₂O)Cl]⁺, [Cu(PMDTA)(DMF)]⁺, [Cu(PMDTA)Cl]⁺, [Cu(PMDTA)OAc]⁺, [Cu(PMDTA)(MeCN)₂]²⁺, [Cu₂(TMEDA)₂Cl₃]⁺ and [Cu(TMEDA)(MeCN)₃]²⁺ were synthesised as PF₆ salts, crystallised and characterised by single-crystal X-ray diffraction.     

Vanadium-dependent iodoperoxidases in Laminaria digitata, a novel biochemical function diverging from brown algal bromoperoxidases

The brown alga Laminaria digitata features a distinct vanadium-dependent iodoperoxidase (vIPO) activity, which has been purified to electrophoretic homogeneity. Steady-state analyses at pH 6.2 are reported for vIPO (K _m ^I– =2.5 mM; k _cat ^I– =462 s^–1) and for the previously characterised vanadium-dependent bromoperoxidase in L. digitata (K _m ^I– =18.1 mM; k _cat ^I– =38 s^–1). Although the vIPO enzyme specifically oxidises iodide, competition experiments with halides indicate that bromide is a competitive inhibitor with respect to the fixation of iodide. A full-length complementary ANA (cDNA) was cloned and shown to be actively transcribed in L. digitata and to encode the vIPO enzyme. Mass spectrometry analyses of tryptic digests of vIPO indicated the presence of at least two very similar proteins, in agreement with Southern analyses showing that vIPOs are encoded by a multigenic family in L. digitata. Phylogenetic analyses indicated that vIPO shares a close common ancestor with brown algal vanadium-dependent bromoperoxidases. Based on a three-dimensional structure model of the vIPO active site and on comparisons with those of other vanadium-dependent haloperoxidases, we propose a hypothesis to explain the evolution of strict specificity for iodide in L. digitata vIPO.The nucleotide sequence reported in this paper has been submitted to the EBI Data Bank with accession no. AJ619804.     

Identification of equine P-selectin glycoprotein ligand-1 (CD162)

P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a dimeric, mucin-like, transmembrane glycoprotein constitutively expressed on leukocytes. A high baseline level of P-selectin expression in circulating equine platelets suggests a primed state toward inflammation and thrombosis via P-selectin/PSGL-1 adhesion. To investigate the potential role of equine P-selectin in these events, we first identified the cDNA sequence of equine PSGL-1 (ePSGL-1) using degenerate PCR and RACE-PCR and then compared the predicted sequence with that of human PSGL-1 (hPSGL-1). ePSGL-1 protein subunit is predicted to be 43 kDa and composed of 420 amino acids with a predicted 18-amino-acid signal sequence showing 78% homology to hPSGL-1. Previously published work has shown that binding of P-selectin requires sulfation of at least one of three tyrosines and O-glycosylation of one threonine in the N-terminus of human PSGL-1. However, the corresponding domain in ePSGL-1, spanning residues 19–43, contains only one tyrosine in the vicinity of two threonines at positions 25 and 41. ePSGL-1 contains 14 threonine/serine-rich decameric repeats as compared to hPSGL-1 which contains 14–16 threonine-rich decameric repeats. The transmembrane and cytoplasmic domains display 91% and 74% homology to corresponding human PSGL-1 domains, respectively. In summary, there is 71% homology in comparing the open reading frame (ORF) of ePSGL-1 with that of hPSGL-1. The greatest homologies between species exist in the transmembrane domain and cytoplasmic tail while substantial differences exist in the extracellular domain.The nucleotide sequence data reported in this article has been submitted to GenBank and assigned the accession number AY298766.     

Improved Prediction of Body Fat by Measuring Skinfold Thickness,Circumferences, and Bone Breadths

Objective: To develop improved predictive regression equations for body fat content derived from common anthropometric measurements. Research Methods and Procedures: 117 healthy German subjects, 46 men and 71 women, 26 to 67 years of age, from two different studies were assigned to a validation and a cross‐validation group. Common anthropometric measurements and body composition by DXA were obtained. Equations using anthropometric measurements predicting body fat mass (BFM) with DXA as a reference method were developed using regression models. Results: The final best predictive sex‐specific equations combining skinfold thicknesses (SF), circumferences, and bone breadth measurements were as follows: BFM_New (kg) for men = ?40.750 + [(0.397 × waist circumference) + [6.568 × (log triceps SF + log subscapular SF + log abdominal SF)]] and BFM_New (kg) for women = ?75.231 + [(0.512 × hip circumference) + [8.889 × (log chin SF + log triceps SF + log subscapular SF)] + (1.905 × knee breadth)]. The estimates of BFM from both validation and cross‐validation had an excellent correlation, showed excellent correspondence to the DXA estimates, and showed a negligible tendency to underestimate percent body fat in subjects with higher BFM compared with equations using a two‐compartment (Durnin and Womersley) or a four‐compartment (Peterson) model as the reference method. Discussion: Combining skinfold thicknesses with circumference and/or bone breadth measures provide a more precise prediction of percent body fat in comparison with established SF equations. Our equations are recommended for use in clinical or epidemiological settings in populations with similar ethnic background.     

Pyruvate Fermentation by Oenococcus oeni and Leuconostoc mesenteroides and Role of Pyruvate Dehydrogenase in Anaerobic Fermentation

The heterofermentative lactic acid bacteria Oenococcus oeni and Leuconostoc mesenteroides are able to grow by fermentation of pyruvate as the carbon source (2 pyruvate → 1 lactate + 1 acetate + 1 CO₂). The growth yields amount to 4.0 and 5.3 g (dry weight)/mol of pyruvate, respectively, suggesting formation of 0.5 mol ATP/mol pyruvate. Pyruvate is oxidatively decarboxylated by pyruvate dehydrogenase to acetyl coenzyme A, which is then converted to acetate, yielding 1 mol of ATP. For NADH reoxidation, one further pyruvate molecule is reduced to lactate. The enzymes of the pathway were present after growth on pyruvate, and genome analysis showed the presence of the corresponding structural genes. The bacteria contain, in addition, pyruvate oxidase activity which is induced under microoxic conditions. Other homo- or heterofermentative lactic acid bacteria showed only low pyruvate fermentation activity.     

Changes in Antimicrobial Susceptibility of Native Enterococcus faecium in Chickens Fed Virginiamycin

The extent of transfer of antimicrobial resistance from agricultural environments to humans is controversial. To assess the potential hazard posed by streptogramin use in food animals, this study evaluated the effect of virginiamycin exposure on antimicrobial resistance in Enterococcus faecium recovered from treated broilers. Four consecutive broiler feeding trials were conducted using animals raised on common litter. In the first three trials, one group of birds was fed virginiamycin continuously in feed at 20 g/ton, and a second group served as the nontreated control. In the fourth trial, antimicrobial-free feed was given to both groups. Fecal samples were cultured 1 day after chickens hatched and then at 1, 3, 5, and 7 weeks of age. Isolates from each time point were tested for susceptibility to a panel of different antimicrobials. Quinupristin/dalfopristin-resistant E. faecium appeared after 5 weeks of treatment in trial 1 and within 7 days of trials 2 to 4. Following removal of virginiamycin in trial 4, no resistant isolates were detected after 5 weeks. PCR failed to detect vat, vgb, or erm(B) in any of the streptogramin-resistant E. faecium isolates, whereas the msr(C) gene was detected in 97% of resistant isolates. In an experimental setting using broiler chickens, continuous virginiamycin exposure was required to maintain a stable streptogramin-resistant population of E. faecium in the animals. The bases of resistance could not be explained by known genetic determinants.