Analysis of a new crystal form of procarboxypeptidase B: Further insights into the catalytic mechanism

A new triclinic crystal structure form of porcine pancreatic procarboxypeptidase B (PCPB) was obtained at higher resolution than the previously known tetragonal crystal structure. This new crystal polymorph has allowed for a corrected, accurate assignment of residues along the polypeptide chain based on the currently available gene sequence information and crystallographic data. The present structure shows unbound PCPB in a distinct molecular packing as compared to the previous benzamidine complexed form. Its catalytically important Tyr248 residue is oriented and hydrogen‐bonded to solvent water molecules, and locates the furthest away from the catalytic zinc ion as compared to previous structures. A relatively long stretch of residues flanking Tyr248 and guarding the access to the catalytic zinc ion was found to be sequentially unique to the M14 family of peptidases. Predictions from a normal mode analysis indicated that this stretch of residues belongs to a rigid subdomain in the protein structure. The specific presence of a tyrosyl residue at the most exposed position in this region would allow for a delicate balance between extreme hydrophobicity and hydrophilicity, and affect substrate binding and the kinetic efficiency of the enzyme. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 178–185, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Topological control of the spin coupling in dinuclear copper(II) complexes with meta- and para-phenylenediamine bridging ligands

A novel series of copper(II) complexes of formula [Cu(tren)(mpda)](ClO₄)₂ · 1/2H₂O (1), [Cu₂(tren)₂(mpda)](ClO₄)₄ · 2H₂O (2), and [Cu₂(tren)₂(ppda)](ClO₄)₄ · 2H₂O (3) containing the tetradentate tris(2-aminoethyl)amine (tren) terminal ligand and the potentially bridging 1,n-phenylenediamine [n = 3 (mpda) and 4 (ppda)] ligand have been prepared and spectroscopically characterized. X-ray diffraction on single crystals of 1 and 3 show the presence of mono- (1) and dinuclear (3) copper(II) units where the mpda (1) and ppda (3) ligands adopt terminal monodentate (1) and bridging bis(monodentate) (3) coordination modes toward [Cu(tren)]²⁺ cations with an overall non-planar, orthogonal disposition of the phenylene group and the N-Cu-N threefold axis of the trigonal bipyramid of each copper(II) ion [values of the Cu-N-C-C torsion angle (?) in the range of 50.8(3)-79.2(2) (1) and 80.9(2)-86.5(2)° (3)]. Variable-temperature magnetic susceptibility measurements on the dinuclear complexes 2 and 3 show the occurrence of moderate ferromagnetic (J = +8.3 cm⁻¹, 2) and strong antiferromagnetic (J = −51.4 cm⁻¹, 3) couplings between the two copper(II) ions across the meta- and para-phenylenediamine bridges, leading to S = 1 (2) and S = 0 (3) ground spin states [H = −JS₁ · S₂ with S₁ = S₂ = S_Cu = 1/2]. Density functional theory (DFT) calculations on the triplet (2) and broken-symmetry (BS) singlet (3) ground spin states, support the occurrence of a spin polarization mechanism for the propagation of the exchange interaction through the predominantly π-type orbital pathway of the 1,n-phenylenediamine bridge. Finally, a new magneto-structural correlation between the magnitude of the magnetic coupling (J) and the Cu-N-C-C torsion angle (?) has been found which reveals the role of σ- versus π-type orbital pathways in the modulation of the magnetic coupling for m- and p-phenylenediamine-bridged dicopper(II) complexes.     

A genome-wide survey does not show the genetic distinctiveness of Basques

Basques are a cultural isolate, and, according to mainly allele frequencies of classical polymorphisms, also a genetic isolate. We investigated the differentiation of Spanish Basques from the rest of Iberian populations by means of a dense, genome-wide SNP array. We found that F _ST distances between Spanish Basques and other populations were similar to those between pairs of non-Basque populations. The same result is found in a PCA of individuals, showing a general distinction between Iberians and other South Europeans independently of being Basques. Pathogen-mediated natural selection may be responsible for the high differentiation previously reported for Basques at very specific genes such as ABO, RH, and HLA. Thus, Basques cannot be considered a genetic outlier under a general genome scope and interpretations on their origin may have to be revised.     

Viability determination of Helicobacter pylori using propidium monoazide quantitative PCR

Background: While Helicobacter pylori exists in a bacillary form in both the natural habitat and the human host, detrimental environmental circumstances have been observed to lead to the conversion of H. pylori from the bacillary to the coccoid form. However, the viability or nonviability of coccoid forms remains to be established in H. pylori. The aim of this study was to determine whether the quantitative PCR combined with propidium monoazide could be an alternative and good technique to determine H. pylori viability in environmental samples and, to contribute to understanding of the role of the H. pylori forms. Materials and Methods: Viability, morphological distribution, and the number of live H. pylori cells were determined using a propidium monoazide‐based quantitative PCR method, at various time points. Results: Under adverse environmental conditions was observed the conversion of H. pylori from the bacillary to the coccoid form, and the decrease in amplification signal, in samples that were treated with propidium monoazide, over the time. Conclusions: Incorporation of propidium monoazide indicates that there is an increase in H. pylori cells with the damaged membrane over the study, leading to the manifestation of cellular degeneration and death. Consequently, quantitative PCR combined with propidium monoazide contributes to our understanding of the role of H. pylori cells, under adverse environmental conditions.     

Uridine metabolism in HIV-1-infected patients: effect of infection, of antiretroviral therapy and of HIV-1/ART-associated lipodystrophy syndrome

Background

Uridine has been advocated for the treatment of HIV-1/HAART-associated lipodystrophy (HALS), although its metabolism in HIV-1-infected patients is poorly understood.

Methods

Plasma uridine concentrations were measured in 35 controls and 221 HIV-1-infected patients and fat uridine in 15 controls and 19 patients. The diagnosis of HALS was performed following the criteria of the Lipodystrophy Severity Grading Scale. Uridine was measured by a binary gradient-elution HPLC method. Analysis of genes encoding uridine metabolizing enzymes in fat was performed with TaqMan RT-PCR.

Results

Median plasma uridine concentrations for HIV-1-infected patients were 3.80 µmol/l (interquartile range: 1.60), and for controls 4.60 µmol/l (IQR: 1.8) (P = 0.0009). In fat, they were of 6.0 (3.67), and 2.8 (4.65) nmol/mg of protein, respectively (P = 0.0118). Patients with a mixed HALS form had a median plasma uridine level of 4.0 (IC95%: 3.40–4.80) whereas in those with isolated lipoatrophy it was 3.25 (2.55–4.15) µmol/l/l (P = 0.0066). The expression of uridine cytidine kinase and uridine phosphorylase genes was significantly decreased in all groups of patients with respect to controls. A higher expression of the mRNAs for concentrative nucleoside transporters was found in HIV-1-infected patients with respect to healthy controls.

Conclusions

HIV-1 infection is associated with a decrease in plasma uridine and a shift of uridine to the adipose tissue compartment. Antiretroviral therapy was not associated with plasma uridine concentrations, but pure lipoatrophic HALS was associated with significantly lower plasma uridine concentrations.     

Aberrant Gene Promoter Methylation Associated with Sporadic Multiple Colorectal Cancer

Background

Colorectal cancer (CRC) multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-concept of an underlying epigenetic defect.

Methodology/Principal Findings

We examined a total of 47 synchronous/metachronous primary CRC from 41 patients, and 41 gender, age (5-year intervals) and tumor location-paired patients with solitary tumors. Exclusion criteria were polyposis syndromes, Lynch syndrome and inflammatory bowel disease. DNA methylation at the promoter region of the MGMT, CDKN2A, SFRP1, TMEFF2, HS3ST2 (3OST2), RASSF1A and GATA4 genes was evaluated by quantitative methylation specific PCR in both tumor and corresponding normal appearing colorectal mucosa samples. Overall, patients with multiple lesions exhibited a higher degree of methylation in tumor samples than those with solitary tumors regarding all evaluated genes. After adjusting for age and gender, binomial logistic regression analysis identified methylation of MGMT2 (OR, 1.48; 95% CI, 1.10 to 1.97; p = 0.008) and RASSF1A (OR, 2.04; 95% CI, 1.01 to 4.13; p = 0.047) as variables independently associated with tumor multiplicity, being the risk related to methylation of any of these two genes 4.57 (95% CI, 1.53 to 13.61; p = 0.006). Moreover, in six patients in whom both tumors were available, we found a correlation in the methylation levels of MGMT2 (r = 0.64, p = 0.17), SFRP1 (r = 0.83, 0.06), HPP1 (r = 0.64, p = 0.17), 3OST2 (r = 0.83, p = 0.06) and GATA4 (r = 0.6, p = 0.24). Methylation in normal appearing colorectal mucosa from patients with multiple and solitary CRC showed no relevant difference in any evaluated gene.

Conclusions

These results provide a proof-of-concept that gene promoter methylation is associated with tumor multiplicity. This underlying epigenetic defect may have noteworthy implications in the prevention of patients with sporadic CRC.     

Investigation of Bolivian plant extracts for their radical scavenging activity and antioxidant activity

Fifty-four different extracts of nine Bolivian plants belonging to the family Asteraceae were evaluated for their radical scavenging activity by the DPPH*, NBT/hypoxanthine superoxide, and (*)OH/luminol chemiluminescence methods, and for their antioxidant activity by the beta-carotene bleaching test. The total phenolic content was also determined by the Folin-Ciocalteu method, and the oxidative stability by the Rancimat test. Both remarkably high phenolic content and radical scavenging and antioxidant activities were found mainly in the ethyl acetate fractions among the different plant extracts. Some ethyl acetate and even some defatted crude extracts exhibited activities comparable to those of commercial extracts/compounds, thus making it possible to consider some of the studied plants as a potential source of antioxidants of natural origin.     

Phosphorylation of Hsl1 by Hog1 leads to a G2 arrest essential for cell survival at high osmolarity

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. Exposure of yeast to osmostress leads to activation of the Hog1 SAPK, which controls cell cycle at G1 by the targeting of Sic1. Here, we show that survival to osmostress also requires regulation of G2 progression. Activated Hog1 interacts and directly phosphorylates a residue within the Hsl7-docking site of the Hsl1 checkpoint kinase, which results in delocalization of Hsl7 from the septin ring and leads to Swe1 accumulation. Upon Hog1 activation, cells containing a nonphosphorylatable Hsl1 by Hog1 are unable to promote Hsl7 delocalization, fail to arrest at G2 and become sensitive to osmostress. Together, we present a novel mechanism that regulates the Hsl1-Hsl7 complex to integrate stress signals to mediate cell cycle arrest and, demonstrate that a single MAPK coordinately modulates different cell cycle checkpoints to improve cell survival upon stress.     

Proteomic identification of desmoglein 2 and activated leukocyte cell adhesion molecule as substrates of ADAM17 and ADAM10 by difference gel electrophoresis

In contrast with the early view of metalloproteases as simple extracellular matrix-degrading entities, recent findings show that they are highly specific modulators of different signaling pathways involved, positively or negatively, in tumor development. Thus, before considering a given metalloprotease a therapeutic target, it seems advisable to characterize its function by identifying its repertoire of substrates. Here, we present a proteomic approach to identify ADAM17 substrates by difference gel electrophoresis. We found that the shedding of the extracellular domain of the transferrin receptor and those of two cell-cell adhesion molecules, activated leukocyte cell adhesion molecule (ALCAM) and desmoglein 2 (Dsg-2), is increased in cells overexpressing ADAM17. Genetic evidence shows that while ADAM17 is responsible for the shedding of ALCAM, both ADAM17 and ADAM10 can act on Dsg-2. Activation of the epidermal growth factor receptor leads to the upregulation of the shedding of Dsg-2 and to the concomitant upregulation of ADAM17, but not ADAM10, supporting the ability of overexpressed ADAM17 to shed Dsg-2. These results unveil a role of ADAM10 and ADAM17 in the shedding of cell-cell adhesion molecules. Since loss of cell adhesion is an early event in tumor development, these results suggest that ADAM17 is a useful target in anticancer therapy.     

Serotonin in body fluids: Characterization of human plasmatic and cerebrospinal fluid pools by means of a new HPLC method

A new HPLC technique for the analysis of picomolar amounts of serotonin (5HT) in plasma and cerebrospinal fluid (CSF) is described. Bufotenin is used as internal standard. Detection is achieved electrochemically or fluorimetrically. The detection limit can be estimated as 50 pg 5HT/mL of either fluid (0.3 picomolar). The method is used to characterize a non-particulate pool of 5HT which is clearly distinct of the platelet pool. Administration of parachlorophenylalanine (PCPA) 300 mg/kg to rats leads to a 90% reduction in the plasmatic pool whereas platelet 5HT is only slightly decreased (3rd day after PCPA) or even increased (7th day after PCPA). Human concentration (n=15) of 5HT in plasma is 2.6 ± 0.9 ng/mL (x ± S.D.). The application of the method to CSF of neurological patients reveals 5HT concentrations ranging from 93 to 962 pg/mL.