Role of class A penicillin-binding proteins in PBP5-mediated beta-lactam resistance in Enterococcus faecalis

Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of beta-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 (ponA, pbpF, and pbpZ). Among mutants with single or double deletions, only JH2-2 DeltaponA DeltapbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of beta-lactams formed a subset of the class A PBPs of E. faecalis, and heterospecific complementation was observed with an ortholog from E. faecium. Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.     

A new polymeric coating for protein microarrays

Despite the increasing interest in arraying proteins in a high-density format, several technical issues still impede the development of protein microarray technology. One of the major problems is the availability of substrates that are able to bind native proteins with high density. In this study, we investigated the suitability of a novel surface as a support for protein microarrays. A polymeric glass coating is obtained by physical adsorption of a N,N-dimethylacrylamide (DMA), N,N-acryloyloxysuccinimide (NAS), and [3-(methacryloyl-oxy)propyl]trimethoxysilyl (MAPS) copolymer. The coating procedure provides a fast and inexpensive method of producing hydrophilic functional surfaces. The slide performance was investigated in a protein-protein interaction experiment and in the assessment of rheumatoid factor (RF) in human serum samples. The results demonstrate that the ligands immobilized on the polymeric surface maintain an active conformation and are easily accessible, providing a detection limit of 54amol/spot. Moreover, in the RF assay, after hybridization with the sera, the slides have a low background, leading to a detection limit of 900amol/spot.     

Soda consumption and overweight status of 2-year-old mexican-american children in california

Objective: The prevalence of overweight in United States children, 2 to 5 years old, has increased 2‐fold since 1975, with the highest prevalence in Mexican Americans. The objective of this study was to determine the association between current soda consumption and overweight in 2‐year‐old Mexican‐American children. Research Methods and Procedures: The Center for the Health Assessment of Mothers and Children of Salinas study is a longitudinal study of the health of low‐income Latino pregnant women and their children living in the Salinas Valley, CA. Six hundred pregnant women were enrolled (October 1999 to October 2000), and their children were followed until 2 years of age. This cross‐sectional analysis includes the 354 children who completed the 2‐year follow‐up interview. Standing height (centimeters) and weight (grams) were measured at 2 years. Overweight was defined as ≥95th percentile of the sex‐specific BMI for each child's age. Results: Fifty‐five (15.5%) children were overweight. Over half (56%) reported consuming any soda in the last week. After covariate adjustment, compared with no soda consumption, <1 soda/d was not related to overweight (adjusted odds ratio, 0.97; 95% confidence interval, 0.47, 1.99), but ≥1 soda/d was significantly associated with overweight (adjusted odds ratio, 3.39; 95% confidence interval, 1.43, 8.07), and the test for trend was significant (p = 0.02). Discussion: At 2 years of age, the prevalence of overweight among the Center for the Health Assessment of Mothers and Children of Salinas cohort is higher than the national prevalence estimate for Mexican‐American 2‐ to 5‐year‐old children and is significantly associated with current soda consumption. Interventions to reduce consumption of soda in young Mexican‐American children should be considered.     

Synthesis of structurally simplified analogues of aplidinone A,a pro-apoptotic marine thiazinoquinone

The synthesis of analogues of aplidinone A (7), a prenylated quinone isolated from the Mediterranean ascidian Aplidium conicum, has been performed. This work not only allowed confirming the structural assignment of aplidinone A, previously made with the support of GIAO shielding calculations, but, above all, made a series of structurally related quinone derivatives (compounds 8–13 and the natural metabolite) available for a screening in vitro for cytotoxic and pro-apoptotic activity and for SAR studies. The study evidenced one of the synthetic analogues (11) as a potent cytotoxic and pro-apoptotic agent against several tumor cell lines which also inhibits the TNFα-induced NF-κB activation in a human leukemia T cell line. This exemplifies the potential of a natural product to qualify as lead structure for medicinal chemistry campaigns, affording simplified analogues with better bioactivity and easier to synthesize.     

The Angiogenic Inhibitor Long Pentraxin PTX3 Forms an Asymmetric Octamer with Two Binding Sites for FGF2

The inflammation-associated long pentraxin PTX3 plays key roles in innate immunity, female fertility, and vascular biology (e.g. it inhibits FGF2 (fibroblast growth factor 2)-mediated angiogenesis). PTX3 is composed of multiple protomers, each composed of distinct N- and C-terminal domains; however, it is not known how these are organized or contribute to its functional properties. Here, biophysical analyses reveal that PTX3 is composed of eight identical protomers, associated through disulfide bonds, forming an elongated and asymmetric, molecule with two differently sized domains interconnected by a stalk. The N-terminal region of the protomer provides the main structural determinant underlying this quaternary organization, supporting formation of a disulfide-linked tetramer and a dimer of dimers (a non-covalent tetramer), giving rise to the asymmetry of the molecule. Furthermore, the PTX3 octamer is shown to contain two FGF2 binding sites, where it is the tetramers that act as the functional units in ligand recognition. Thus, these studies provide a unifying model of the PTX3 oligomer, explaining both its quaternary organization and how this is required for its antiangiogenic function.     

Pressure effect on the stability and the conformational dynamics of the D-Galactose/D-Glucose-binding protein from Escherichia coli

The effect of the pressure on the structure and stability of the D-Galactose/D-Glucose binding protein (GGBP) from Escherichia coli was studied by steady-state and time-resolved fluorescence spectroscopy, and the ability of glucose ligand to stabilize the GGBP structure was also investigated. Steady-state fluorescence experiments showed a marked quenching of fluorescence emission of GGBP in the absence of glucose. Instead, the presence of glucose seems to stabilize the structure of GGBP at low and moderate pressure values. Time-resolved fluorescence measurements showed that the GGBP taumean in the absence of glucose varies significantly up to 600 bar, while in the presence of the ligand it is almost unaffected by pressure increase up to 600 bar. The effect of the pressure on GGBP was also studied by molecular dynamics simulations. The simulation data support the spectroscopic results and confirm that the presence of glucose is able to contrast the negative effects of pressure on the protein structure. Taken together, the spectroscopic and computer simulation studies suggest that at pressure values up to 2000 bar the structure of GGBP in the absence of glucose remains folded, but a significant perturbation of the protein secondary structures can be detected. The binding of glucose reduces the negative effect of pressure on protein structure and confers protection from perturbation especially at moderate pressure values.     

TP53 and p16INK4A, but not H-KI-Ras, are involved in tumorigenesis and progression of pleomorphic adenomas

The putative role of TP53 and p16(INK4A) tumor suppressor genes and Ras oncogenes in the development and progression of salivary gland neoplasias was studied in 28 cases of pleomorphic adenomas (PA), 4 cases of cystic adenocarcinomas, and 1 case of carcinoma ex-PA. Genetic and epigenetic alterations in the above genes were analyzed by Polymerase Chain Reaction/Single Strand Conformational Polymorphism (PCR/SSCP) and sequencing and by Methylation Specific-PCR (MS-PCR). Mutations in TP53 were found in 14% (4/28) of PAs and in 60% (3/5) of carcinomas. Mutations in H-Ras and K-Ras were identified in 4% (1/28) and 7% (2/28) of PAs, respectively. Only 20% (1/5) of carcinomas screened displayed mutations in K-Ras. p16(INK4A) promoter hypermethylation was found in 14% (4/28) of PAs and 100% (5/5) carcinomas. All genetic and epigenetic alterations were detected exclusively in the epithelial and transitional tumor components, and were absent in the mesenchymal parts. Our analysis suggests that TP53 mutations and p16(INK4A) promoter methylation, but not alterations in the H-Ras and K-Ras genes, might be involved in the malignant progression of PA into carcinoma.     

Protein and peptide arrays: recent trends and new directions

Microarrays of proteins and peptides make it possible the screening of thousands of binding events in a parallel and high throughput fashion; therefore they are emerging as a powerful tool for proteomics and clinical assays. The complex nature of Proteome, the wide dynamic range of protein concentration in real samples and the critical role of immobilized protein orientation must be taken into account to maximize the utility of protein microarrays. Immobilization strategy and designing of an ideal local chemical environment on the solid surface are both essential for the success of a protein microarray experiment. This review article will focus on protein and peptide arrays highlighting their technical challenges and presenting new directions by means of a set of selected recent applications.