Isokinetic Relationship in the Oxidation of Cuprous Stellacyanin by Cobalt(III) Complexes

Rate parameters have been obtained for the oxidation of cuprous stellacyanin by cobalt(III) ions of the form cis(N)-[CoN₂O₄]^?, including cis(N)-[Co(NTA)(gly)]^?, cis(N)-[Co(IDA)₂]^?, [Co(en)(ox)₂]^?(μ 0.5 M(phosphate), pH 7.0), and Co(EDTA)^?(μ 0.1 M(NaCl), pH 7.2, 0.001 M phosphate). An excellent isokinetic correlation between the activation parameters ΔH^≠ and ΔS^≠ exists for the reactions of aminopolycarboxylatocobalt(III) ions with reduced stellacyanin (β = 300 ± 12 K; correlation coefficient = 0.995). It is concluded that enthalpy-entropy compensation in these reactions may be understood in terms of differing orientations preferred by the various oxidants in forming precursor complexes with the reduced blue protein. While ΔH^≠ and ΔS^≠ values for electron transfer from stellacyanin to cis(N)-[CoN₂O₄]^? ions vary over ranges of 10.7 kcal/mol and 34 cal/mol-deg, respectively, room temperature rate constants are relatively constant (3.6–34.5 M^?1 sec^?1), as expected from Marcus theory for outer sphere electron transfer.     

Substituent effects on the electron transfer reactivity of hydroquinones with laccase blue copper.

Stopped-flow kinetic studies of the anaerobic reduction of Rhus vernicifera laccase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) type 1 copper by 25 mono- and disubstituted hydroquinones (H2Q-X) have been performed at 25 degrees C and pH 7.0 in 0.5 M phosphate. All of the data are compatible with a mechanism involving rapid enzyme-substrate complex formation followed by rate-limiting intra-complex electron transfer. ES complex formation constants (Qp) for many substrates are strikingly insensitive to the electronic characteristics of the substituent X, falling within the range 5--50 M-1. It is shown that this result may be accounted for if only the singly ionized forms of the substituted hydroquinones are bound by the enzyme. All of the substrates exhibiting exceptionally high Qp values (greater than 50 M-1) have X groups capable of functioning as ligands; substituents with lone pairs of electrons may facilitate enzyme-substrate complex formation by enabling hydroquinone to function as a bidentate bridging ligand between the type 2 and type 3 copper sites. Intra-complex electron transfer rate constants for most substrates are remarkably insensitive to the thermodynamic driving force for the oxidation of H2Q-X to the corresponding semiquinone, the average value for ten substrates being 30 +/- 10 s-1. The electron transfer reactivity of polyphenols with laccase blue copper therefore appears to be controlled largely by protein-dependent activation requirements rather than by the oxidizability of the substrate.     

Purification and characterization of aleurain : a plant thiol protease functionally homologous to Mammalian cathepsin h

Barley (Hordeum vulgare L. cv Himilaya) aleurain is a vacuolar thiol protease originally isolated as a cDNA with 65% derived amino acid sequence identity with cathepsin H (JC Rogers, D Dean, GR Heck [1985] Proc Natl Acad Sci USA 82: 6512-6516). We purified aleurain from barley leaves to homogeneity (>1000-fold) and characterized its activity against a number of substrates. Aleurain is best described as an aminopeptidase; it hydrolyzes three different aminopeptidase substrates with similar catalytic efficiency but is less efficient at hydrolyzing an NH₂-blocked substrate analog and azocasein. Our values for K_m and k_cat for three substrates (arginine 4-methyl-7-coumarylamide, l-arginine β-naphthylamide, and N-α-benzoyl-l-arginine β-naphthylamide) and specific activity with azocasein are all within a threefold range of those previously reported for human cathepsin H for these substrates (WN Schwartz, AJ Barrett [1980] Biochem J 191: 487-497). Aleurain also shows a number of other similarities to cathepsin H including heterogeneity of charge forms, position of the NH₂-terminus of the mature protein, and pH-activity profile. The similar properties of aleurain and cathepsin H suggest that these enzymes have a similar function(s) that is required by both plant and animal cells. The availability of a plant system may permit functional ablation experiments in the future to clarify the role of this enzyme in higher eukaryotes.     

The chloroplast ClpP complex in Chlamydomonas reinhardtii contains an unusual high molecular mass subunit with a large apical domain

The composition of the chloroplast-localized protease complex, ClpP, from the green alga Chlamydomonas reinhardtii was characterized by nondenaturing electrophoresis, immunoblotting and MS. The detected ClpP complex has a native mass of approximately 540 kDa, which is approximately 200 kDa higher than ClpP complexes in higher plant chloroplasts, mitochondria or bacteria. The 540-kDa ClpP complex contains two nuclear-encoded ClpP proteins (ClpP3 and P5) and five ClpR (R1, R2, R3, R4 and R6) proteins, as well two proteins, ClpP1L and ClpP1H, both probably derived from the plastid clpP1 gene. ClpP1H is 59 kDa and contains a approximately 30-kDa insertion sequence (IS1) not found in other ClpP proteins, responsible for the high MW of the complex. Based on comparison with other sequences, IS1 protrudes as an additional domain on the apical surface of the ClpP/R complex, probably preventing interaction with the HSP100 chaperone. ClpP1L is a 25-kDa protein similar in size to other ClpP proteins and could arise by post-translational processing of ClpP1H. Chloramphenicol-chase experiments show that ClpP1L and ClpP1H have a similar half-life, indicating that both are stable components of the complex. The structure of the ClpP complex is further discussed in conjunction with a phylogenetic analysis of the ClpP/R genes. A model is proposed for the evolution of the algal and plant complex from its cyanobacterial ancestor.     

Generation of mutant murine cytomegalovirus strains from overlapping cosmid and plasmid clones

We have developed a cosmid and plasmid system to generate mutant strains of murine cytomegalovirus (MCMV). The system is based on a series of seven overlapping cosmid clones that regenerate MCMV when cotransfected into mouse cells. The unaltered cosmids produce MCMV that is indistinguishable from wild-type MCMV based on restriction enzyme digest patterns of virus DNA and growth rates both in vitro and in vivo. Analysis of viral DNA from plaque-purified recombinant isolates taken from in vitro and in vivo stocks indicated that regeneration did not introduce novel mutations in the recombinant viral genomes. Isolation of specific genes and subsequent generation of specific mutant MCMVs was accomplished by replacement of cosmids with overlapping plasmid subclones. A new vector, PmeSUB, featuring a multiple cloning site and a stringent origin of replication, was constructed to make large subclones for use with smaller subclones containing the gene of interest. The utility of this system was demonstrated by the generation of two different mutant MCMVs from different combinations of overlapping plasmid subclones of one cosmid. The advantages of this system are that (i) target genes are maintained as small clones making them amenable to standard in vitro mutagenesis manipulations and that (ii) no reporter or selection genes are necessary to identify mutants.     

Uncommon Alu-mediated NF1 microdeletion with a breakpoint inside the NF1 gene

Neurofibromatosis type 1 (NF1) microdeletion syndrome is caused by haploinsufficiency of the NF1 gene and of gene(s) located in adjacent flanking regions. Most of the NF1 deletions originate by nonallelic homologous recombination between repeated sequences (REP-P and -M) mapped to 17q11.2, while a few uncommon deletions show unusual breakpoints. We characterized an uncommon 1.5-Mb deletion of an NF1 patient displaying a mild phenotype. We applied high-resolution FISH analysis allowing us to obtain the sequence of the first junction fragment of an uncommon deletion showing the telomeric breakpoint inside the IVS23a of the NF1 gene. Sequence analysis of the centromeric and telomeric boundaries revealed that the breakpoints were present in the AluJb and AluSx regions, respectively, showing 85% homology. The centromeric breakpoint is localized inside a chi-like element; a few copies of this sequence are also located very close to both breakpoints. The in silico analysis of the breakpoint intervals, aimed at identifying consensus sequences of several motifs usually involved in deletions and translocations, suggests that Alu sequences, probably associated with the chi-like element, might be the only recombinogenic motif directly mediating this large deletion.     

TNF-α levels are not increased in inflamed patients carrying the CCR5 deletion 32

Background and aims: Recently we reported on a genetically predisposed protection from C-reactive protein (CRP) related mortality in dialysis patients carrying the functional CC-chemokine receptor 5 deletion 32 allele (CCR5Δ32) mutation. Since CCR5Δ32 is associated with a less pro-inflammatory immune response in mice, we hypothesized that the observed protection is (in part) due to a less pro-inflammatory cytokine profile. Methods: Cross-sectional observational study including 263 incident dialysis patients aged 18–70 years, without clinical signs of infection and/or acute vasculitis. TNF-α, IL-6, IL-10 and hsCRP levels were determined and studied in relation to the CCR5 genotype. Results: In the presence of elevated hsCRP, IL-6 concentration was higher irrespective of the CCR5 genotype. However, in patients with the CCR5 deletion, TNF-α did not differ in the presence/absence of elevated hsCRP and was not correlated with hsCRP levels in carriers of the CCR5Δ32 polymorphism. Conclusions: A possible underlying mechanism of the impact of CCR5Δ32 genotype on inflammation driven mortality in dialysis patients could be a reduced Th1 immune response as represented by decreased TNF-α levels.     

18F-Fluorodeoxyglucose Positron Emission Tomography in Elderly Patients with an Elevated Erythrocyte Sedimentation Rate of Unknown Origin

Patients with an elevated erythrocyte sedimentation rate (ESR) and non-specific symptoms often pose a diagnostic dilemma. PET/CT visualises infection, inflammation and malignancy, all of which may cause elevated ESR. The objective of this study was to determine the contribution of 18F-fluorodeoxglucose positron emission tomography (PET/CT) in the diagnostic work-up of referred patients with an elevated ESR, in whom initial routine evaluation did not reveal a diagnosis. We conducted a combined retrospective (A) and prospective (B) study in elderly (>50 years) patients with a significantly elevated ESR of≥50 mm/h and non-specific complaints. In study A, 30 patients were included. Malignancy (8 patients), auto-inflammatory disease (8 patients, including 5 with large-vessel vasculitis) and infection (3 patients) were suggested by PET/CT. Two scans showed non-specific abnormalities and 9 scans were normal. Of the 21 abnormal PET/CT results, 12 diagnoses were independently confirmed and two alternative diagnosis were made. Two diagnoses were established in patients with a normal scan. In study B, 58 patients in whom a prior protocolised work-up was non-diagnostic, were included. Of these, 25 PET/CT-scans showed suspected auto-inflammatory disease, particularly large-vessel vasculitis (14 cases). Infection and malignancy was suspected in 5 and 3 cases, respectively. Seven scans demonstrated non-specific abnormalities, 20 were normal. Of the 40 abnormal PET/CT results, 22 diagnoses were confirmed, 3 alternative diagnoses were established. Only one diagnosis was established in the 20 patients with a normal scan. In both studies, the final diagnosis was based on histology, clinical follow-up, response to therapy or additional imaging. In conclusion, PET/CT may be of potential value in the diagnostic work-up of patients with elevated ESR if routine evaluation reveals no diagnosis. In particular, large-vessel vasculitis appears to be a common finding. A normal PET/CT scan in these patients suggests that it is safe to follow a wait-and-see policy.     

Mature male parr contribution to the effective size of an anadromous Atlantic salmon (Salmo salar) population over 30 years

We describe temporal changes in the genetic composition of a small anadromous Atlantic salmon (Salmo salar) population from South Newfoundland, an area where salmon populations are considered threatened (COSEWIC 2010). We examined the genetic variability (13 microsatellite loci) in 869 out‐migrating smolt and post‐spawning kelt samples, collected from 1985 to 2011 for a total of 22 annual collections and a 30 year span of assigned cohorts. We estimated the annual effective number of breeders (N_b) and the generational effective population size (N_e) through genetic methods and demographically using the adult sex ratio. Comparisons between genetic and demographic estimates show that the adult spawners inadequately explain the observed N_e estimates, suggesting that mature male parr are significantly increasing N_b and N_e over the study period. Spawning as parr appears to be a viable and important strategy in the near absence of adult males.     

Carotid baroreflex responsiveness is impaired in normotensive African American men

There are important differences in autonomic function and cardiovascular responsiveness between African Americans (AA) and Caucasian Americans (CA). This study tested the hypothesis that carotid baroreflex (CBR) responsiveness is impaired in normotensive AA compared with normotensive CA at rest. CBR control of heart rate (HR) and mean arterial blood pressure (MAP) was assessed in 30 nonhypertensive male subjects (15 AA; 15 CA; age 18-33 yr) with 5-s periods of neck pressure (NP; simulated hypotension) and neck suction (NS; simulated hypertension) ranging from +45 to -80 Torr during rest. Carotid-cardiac stimulus-response curves revealed a significantly lower minimum HR response in the CA compared with AA (40.8 ± 2.4 vs. 49.8 ± 2.9 beats/min, respectively; P < 0.05). In addition, the magnitude of the mean HR response to all trials of NS (-20, -40, -60, and -80 Torr) was attenuated in the AA group (AA, -10.1 ± 1.7 vs. CA, -14.9 ± 2.2 beats/min; P < 0.05), while no significant differences were found in the magnitude of the mean HR response to NP (+15, +30, and +45 Torr) between racial groups. There were no significant differences in the carotid-vasomotor stimulus-response curves between racial groups. Also, while no racial differences were found in the magnitude of the mean MAP response to all trials of NS, the magnitude of the mean MAP response to all trials of NP was attenuated in the AA group (AA, 7.2 ± 1.3 vs. CA, 9.3 ± 1.1 mmHg; P < 0.05). Together, these findings support inherent differences in short-term blood pressure regulation between racial groups that exhibit different relative risk for the development of hypertension.