Probing the modelled structure of wheatwin1 by controlled proteolysis and sequence analysis of unfractionated digestion mixtures.

We set up a method to get rapid information on the three-dimensional structure of peptide and proteins of known sequence. Both native and alkylated polypeptide is hydrolyzed with a number of proteases at different digestion times and the resulting mixtures are compared by HPLC analysis to establish the differences in the hydrolysis pathways of the folded and unfolded molecule. Then, the unfractionated digestion mixtures of the native polypeptide are submitted to automatic sequence analysis to identify the hydrolysis sites. The sequence of each fragment present in the mixtures is reconstructed and its amount determined by quantitative data of the sequence analyses. We used this approach to determine the amino acid surface accessibility of wheatwin1, a pathogenesis-related protein from wheat, and constructed a predictive three-dimensional model based on the knowledge of the tertiary structure of barwin, a highly homologous protein from barley. The procedure allowed us to quickly identify and quantify the hydrolysis at the susceptible bonds which could be classified as exposed, partially hidden, or inaccessible. The results were useful to evidentiate and discuss concordances and differences between experimental and model predicted accessibilities of amino acid residues. Proteins 1999;36:192-204.     

Conformational sampling of bioactive conformers: a low-temperature NMR study of 15N-Leu–enkephalin

Conformational studies of enkephalins are hampered by their high flexibility which leads to mixtures of quasi-isoenergetic conformers in solution and makes NOEs very difficult to detect in NMR spectra. In order to improve the quality of the NMR data, Leu–enkephalin was synthesized with ¹⁵N-labelled uniformly on all amide nitrogens and examined in a viscous solvent medium at low temperature. HMQC NOESY spectra of the labelled Leu–enkephalin in a DMSO_d6/H₂O mixture at 275 K do show numerous NOEs, but these are not consistent with a single conformer and are only sufficient to describe the conformational state as a mixture of several conformers. Here a different approach to the structure–activity relationships of enkephalins is presented: it is possible to analyse the NMR data in terms of limiting canonical structures (i.e. β- and γ-turns) and finally to select only those consistent with the requirements of δ selective agonists and antagonists. This strategy results in the prediction of a family of conformers that may be useful in the design of new δ selective opioid peptides. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Sutural bone frequency in synostotic rabbit crania

This study tests the hypothesis that crania with synostosed sutures will have a significantly higher incidence of calvarial sutural bones than normal crania. Sutural bones were counted in seven calvarial sutures and compared among four groups of adult New Zealand white rabbit skulls: normal in-colony (NI) controls (N = 14), normal out-colony (NO) controls (N = 12), skulls with familial delayed onset (DO) coronal synostosis (N = 25), and skulls with experimentally immobilized coronal sutures (EI) (N = 20). Comparisons among groups were made with a Kruskal-Wallis one-way ANOVA and between groups with a Mann-Whitney U-test, using a Bonferroni correction for multiple comparisons. Significant differences (P > 0.05) were noted only in the coronal and sagittal sutures, with EI crania having the greatest number of coronal sutural bones; between group differences were undetectable for sagittal sutural bones. A post hoc two-sample binomial test for equal proportions showed that the distribution of coronal sutural bones among individuals across groups was even, while the distribution of sagittal sutural bones was significantly higher in EI crania. These results suggest that altered sutural forces of the calvaria contribute to an increased occurrence of sutural bones. However, the influence of inheritance on increased occurrence of sutural bones cannot be discounted, as reflected in the equivalent number of individuals across groups that possessed coronal sutural bones. Am J Phys Anthropol 102:555–563, 1997. © 1997 Wiley-Liss, Inc.     

Temporal strength changes from resistance exercise and albuterol on unloaded muscle

To assess unloaded knee extensor temporal strength changes, healthy subjects without asthma performed 40 continuous days of unilateral limb suspension, whereby their left leg refrained from normal weight-bearing and ambulatory activity. During the 40-day period, subjects performed resistance exercise (REX) with their unloaded leg on an inertial resistance ergometer and, as part of a double-blind design, consumed the maximal oral therapeutic dosage of albuterol (i.e., 16 mg.d) or a placebo (i.e., lactose) with no crossover. Workout data were partitioned into 4 10-day periods that ran consecutively. Dependent strength variables included concentric total work, eccentric total work, concentric average power (CAP), and eccentric average power (EAP). Dependent variables were analyzed with 5 (time) x 2 (group) x 2 (gender) mixed factorial analyses of variance and the Tukey honestly significant difference test. Concentric total work, CAP, and EAP each demonstrated a time-group-gender (p < 0.05) interaction. Female REX-placebo subjects had the greatest percentage of unloaded knee extensor strength loss. However, female REX-albuterol subjects fared best throughout the 40-day period and incurred significant unloaded knee extensor strength gains. Differences in strength changes between male and female REX-albuterol subjects was likely due to the higher relative dosage administered to the latter, as body mass showed a gender (i.e., men > women) effect. Future research may elucidate the ideal dose-response relationship for REX-albuterol treatment for use aboard manned space flights and in other disuse models. Coaches and practitioners should carefully examine their sport-governing bodies' rules on albuterol administration and give the drug only if an athlete's health warrants such treatment.     

Biomonitoring of polybrominated diphenyl ether (PBDE) pollution: a field study

Polybrominated diphenyl ethers (PBDEs) and cytochrome P450 enzyme activities were investigated in European eels (Anguilla anguilla) collected from seven sites in a coastal lagoon in the north-western Mediterranean Sea, Orbetello lagoon (Italy). Twelve PBDE congeners were measured in muscle and two CYP1A enzyme activities, 7-ethoxyresorufin-O-deethylase (EROD) and benzo(a)pyrene monooxygenase (BP(a)PMO), were investigated in liver microsomal fraction in order to obtain insights into the health of the lagoon environment. PBDE muscle levels were low and the most abundant congeners were 2,2',4,4'-tetrabromodiphenylether (BDE-47), 2,2',4,4',5,5'-hexaBDE (BDE-153) and 2,2',4,5'-tetraBDE (BDE-49). EROD and B(a)PMO activities were also low and no differences were observed between eels from different sites. Multivariate analysis (PCA) did not indicate correlations between PBDEs and either P450 activities.     

Studying protein phosphorylation in low MW CSF fractions with capLC-ICPMS and nanoLC-CHIP-ITMS for identification of phosphoproteins

An initial study of protein phosphorylation in human cerebral spinal fluid (CSF) is described. CSF is an important body fluid for study of proteins and metabolites and may lead to the ultimate development of molecular markers to predict neurological diseases or their complications, such as in the case of hemorrhagic stroke. The use of capillary liquid chromatography coupled to inductively coupled plasma mass spectrometry (capLC-ICPMS) for screening using (31)P as the internal elemental tag atom at ultratrace levels, in combination with molecular mass spectrometry using Spectrum Mill and MASCOT database search engines for peptide identification, is a novel approach in its application to CSF relevant phosphopeptides and phosphorylated proteins. CapLC-ICPMS combined with nano liquid chromatography electrospray ionization, ion trap mass spectrometry (nanoLC-CHIP/ITMS), was utilized for initial experiments with CSF. Specific low-level screening for (31)P containing compounds is accomplished, and nanoLC-CHIP/ITMS provided the corresponding peptide information and subsequent protein identifications. The fractions containing (31)P from screening by the capLC-ICPMS were collected offline and analyzed separately with nanoLC-CHIP/ITMS. Synthetic phosphopeptides were used to test the method and to estimate lowest quantifiable limits for phosphorus. Tryptically digested beta-casein was then used to demonstrate the viability of the methodology for the complex CSF matrix from hemorrhagic stroke patients while also analyzing for native phosphopeptides in the CSF.     

A dramatic conformational rearrangement is necessary for the activation of DNR from Pseudomonas aeruginosa. Crystal structure of wild‐type DNR

The opportunistic pathogen Pseudomonas aeruginosa can grow in low oxygen, because it is capable of anaerobic respiration using nitrate as a terminal electron acceptor (denitrification). An intermediate of the denitrification pathway is nitric oxide, a compound that may become cytotoxic at high concentration. The intracellular levels of nitric oxide are tightly controlled by regulating the expression of the enzymes responsible for its synthesis and degradation (nitrite and nitric oxide reductases). In this article, we present the crystallographic structure of the wild‐type dissimilative nitrate respiration regulator (DNR), a master regulator controlling expression of the denitrification machinery and a putative target for new therapeutic strategies. Comparison with other structures among the CRP‐FNR class of regulators reveals that DNR has crystallized in a conformation that has never been observed before. In particular, the sensing domain of DNR has undergone a rotation of more than 50° with respect to the other structures. This suggests that DNR may undergo an unexpected and very large conformational rearrangement on activation. Proteins 2009. © 2009 Wiley‐Liss, Inc.