Non-native interhelical hydrogen bonds in the cystic fibrosis transmembrane conductance regulator domain modulated by polar mutations

Polar residues comprise about 15% of the transmembrane (TM) domains of proteins, where they can stabilize structure via native side chain-side chain interhelical hydrogen bonds between TM helices. However, non-native H-bonds may be implicated in disease states, through limiting protein dynamics during transport and/or misfolding the protein by inducing non-native rotational positions about TM helical axes. Here we have undertaken an investigation of the presence and strength of H-bond interactions within a series of helix-loop-helix ("hairpin") constructs derived from TM helices 3 and 4 (italic) of the cystic fibrosis transmembrane conductance regulator (CFTR) (prototypic sequence G(194)LALAHFVWIAPLQ(207)VALLMGLIWELLQASAFAGLGFLIV(232)LALFQ(237)AGLG(241)) in which wild-type Q207 in TM3 forms an interhelical H-bond with CF-phenotypic mutant V232D in TM4 [Therien, A. G., Grant, F. E., and Deber, C. M. (2001) Nat. Struct. Biol 8, 597-601]. In the present work, a library of 21 TM3/4 constructs was prepared, where Asp residues were placed individually at TM4 positions 221-241. Using gel shift assays-in which H-bond-linked hairpins (closed conformation) migrate faster than the elongated forms (open conformation)-we found that Q207 in TM3 is able to "capture" all 21 TM4 D mutations into measurable populations of interhelical H-bonds. A similar library of TM4 D mutants-but also containing Q207L-reverted to wild-type migration rates, confirming Q207 as the polar partner for TM4 D residues. In view of the broad capture range of Q207, these results emphasize the potential consequences to folding and dynamics of introducing polar mutations into the TM domains of membrane proteins in the vicinity of a native polar TM residue.     

Genomic profiling by DNA amplification of laser capture microdissected tissues and array CGH

Comparative genomic hybridization by means of BAC microarrays (array CGH) allows high-resolution profiling of copy-number aberrations in tumor DNA. However, specific genetic lesions associated with small but clinically relevant tumor areas may pass undetected due to intra-tumor heterogeneity and/or the presence of contaminating normal cells. Here, we show that the combination of laser capture microdissection, 29 DNA polymerase-mediated isothermal genomic DNA amplification, and array CGH allows genomic profiling of very limited numbers of cells. Moreover, by means of simple statistical models, we were able to bypass the exclusion of amplification distortions and variability prone areas, and to detect tumor-specific chromosomal gains and losses. We applied this new combined experimental and analytical approach to the genomic profiling of colorectal adenomatous polyps and demonstrated our ability to accurately detect single copy gains and losses affecting either whole chromosomes or small genomic regions from as little as 2 ng of DNA or 1000 microdissected cells.     

Monitoring of the bacterial composition of dairy starter cultures by RAPD-PCR

Randomly amplified polymorphic DNA (RAPD)-PCR was used to verify the species composition of commercial dairy starters and to detect possible shifts in strain composition of these cultures. After RAPD-PCR analysis, not all the strains isolated in the years 2001 and 2002 fell within the same dendrogram cluster of the strains isolated in the year 2000 and used as reference strains. Changes in composition of the microbial population and/or voluntary immission of new biotypes with respect to the original strain formulation had occurred in the starters. The microbial composition of modern dairy starters represents a key point because the complex relationships among microorganisms can easily be altered. Little variations in the microbial composition could have unexpected effects on cheese quality.     

Ogataea falcaomoraisii sp. nov., a sporogenous methylotrophic yeast from tree exudates

Thirteen strains of a new ascospore-forming, methanol-assimilating yeast species were isolated from sap exudates of Sclerolobium sp. (carvoeiro) in two forest fragments in the state of Toncantins, Brazil, and from Hymenaea courbaril (guapinol, jatobá) in Guanacaste Province, Costa Rica. Analysis of the sequences of the D1/D2 large-subunit ribosomal DNA showed that the species belongs to the genus Ogataea (syn. Pichia), and it was described as Ogataea falcaomoraisii. The closest relatives are Candida ortonii and C. nemodendra. The type culture is UFMG-T264-1T (= CBS 9814T = NRRL Y-27756).     

Phenotypic and functional changes of circulating monocytes and polymorphonuclear leucocytes from elderly persons

The function and phenotype of monocytes and granulocytes in the elderly is consistently remodelled. Because leucocyte adhesion molecules play important roles in mediating a wide variety of leucocyte functions, age-related changes in their expression on granulocyte and monocyte surfaces could be partially responsible for immune dysfunctions during senescence. Considering the central role of innate immunity in the process of immunosenescence and the involvement of cell adhesion molecules (CAM) in the great majority of leucocyte functions, we studied the expression of CD50 and CD62L adhesion molecules in peripheral blood granulocytes and monocytes from healthy elderly and young subjects. We show here that the percentage of granulocytes and monocytes expressing CD62L is decreased in the elderly, whereas its density expression is unchanged on both cell types. A downregulation of the density expression of CD50 at a per cell level characterizes granulocytes in the elderly, whereas CD50 expression on monocytes from old subjects shows a peculiar attitude: its density expression decreases whereas the number of positive cells is expanded. The downregulation of this receptor on granulocytes from aged people could determine a state of hyperactivation contributing to the proinflammatory status of the elderly, while the lower expression on monocytes could therefore contribute to the impaired antigen presentation in the elderly. On the other hand, the increased number of CD50 positive monocytes in the elderly, despite its decreased density expression at a per cell level, could be interpreted as an attempt to counteract the inability to mount strong immune responses. Both CD50 and CD62L changes in ageing polymorphonuclear (PMN) cells allow recognition as non-self or senescent self to permit macrophages in the liver and spleen to remove them from the circulation. The increased proportion of granulocytes and monocytes lacking CD62L and the downregulation of CD50 intensity expression on both cell types may suggest a state of in vivo activation. Therefore, CD50 and CD62L shedding from the cell surface of activated granulocytes and monocytes could be interpreted as a tentative to counteract the dangerous effects of an excessive chronic inflammation in the elderly. However, the increased proportion of CD62L negative granulocytes in the elderly leads to an impairment in cell adhesion which is the first line of response to acute inflammatory stimuli. This phenomenon likely contributes to the increased susceptibility to acute infections of elderly people.     

Antibody recognition of chiral surfaces. Structural models of antibody complexes with leucine-leucine-tyrosine crystal surfaces

Molecular models are built of the recognition domains of two antibodies, which are raised and selected against crystals of (L)leucine-(L)leucine-(L)tyrosine. The model of one antibody, which is stereo- and enantioselective, reveals astounding chemical and structural complementarity to the recognized crystal surface. The enantioselective binding of this antibody is explained by the significantly fewer chemical interactions arising in the complex, after docking of the antibody to the (D)Leu-(D)Leu-(D)Tyr crystal face, relative to its enantiomer, the (L)Leu-(L)Leu-(L)Tyr crystal face. The modeling and docking of the second antibody, which is poorly stereoselective and is not enantioselective, indicates that binding is based on electrostatic interactions. The docking models of the antibody-crystal complexes provide a rationale for the experimental results while demonstrating the power of modeling techniques to meet the challenge of describing antibody-antigen interactions in detail.     

Goat warble fly infestation by Przhevalskiana silenus (Diptera: Oestridae): immunoepidemiologic survey in the Basilicata region (southern Italy)

The demonstration of serological cross-reactivity between the Hypoderma lineatum antigen and anti-Przhevalskiana silenus antibodies led us to prepare an immunological test (ELISA) for an early diagnosis of goat warble fly infestation. Using the Hypodermosis ELISA-Kit (Vétoquinol Diagnostic, France) produced for the immunodiagnosis of bovine hypodermosis, an epidemiological assay was carried out in Basilicata region where goat breeding is very common and no data are reported with regards to the distribution of goat warble fly infestation. Out of a total of 1,100 flocks and 41,200 goats, 105 randomly extracted flocks proved to be infected and 262 sera out of 1,316 were positive; goat warble fly infestation proved to be present in Basilicata with values similar to those recorded in the surrounding regions (Apulia and Calabria). Statistical evaluation showed highly significant differences between the number of infected flocks in the mountainous areas and hills and those of the mountainous areas and the plain, but no differences between hills and plains. The higher number of positive sera and antibody titres in November-December confirmed that these months are the optimal period for sampling sera in order to perform an early diagnosis. The ELISA test was confirmed to be an easy and economic tool especially when sera sampled within a brucellosis eradication program are used.     

Effect of chemokine receptor CX3CR1 deficiency in a murine model of respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is the most common cause of serious lower respiratory illness in infants and young children worldwide, making it a high priority for development of strategies for prevention and treatment. RSV can cause repeat infections throughout life, with serious complications in elderly and immunocompromised patients. Previous studies indicate that the RSV G protein binds through a CX3C chemokine motif to the host chemokine receptor, CX3CR1, and modulates the inflammatory immune response. In the current study, we examined the contribution of CX3CR1 to the immune response to RSV infection in mice. CX3CR1-deficient mice showed an impaired innate immune response to RSV infection, characterized by substantially decreased NK1.1(+) natural killer, CD11b(+), and RB6-8C5(+) polymorphonuclear cell trafficking to the lung and reduced IFNγ production compared with those in wildtype control mice. Leukocytes from CX3CR1-deficient mice were poorly chemotactic toward RSV G protein and CX3CL1. These results substantiate the importance of the RSV G CX3C-CX3CR1 interaction in the innate immune response to RSV infection.     

Release of tissue-specific proteins into coronary perfusate as a model for biomarker discovery in myocardial ischemia/reperfusion injury

Diagnosis of acute coronary syndromes is based on protein biomarkers, such as the cardiac troponins (cTnI/cTnT) and creatine kinase (CK-MB) that are released into the circulation. Biomarker discovery is focused on identifying very low abundance tissue-derived analytes from within albumin-rich plasma, in which the wide dynamic range of the native protein complement hinders classical proteomic investigations. We employed an ex vivo rabbit model of myocardial ischemia/reperfusion (I/R) injury using Langendorff buffer perfusion. Nonrecirculating perfusate was collected over a temporal profile of 60 min reperfusion following brief, reversible ischemia (15 min; 15I/60R) for comparison with irreversible I/R (60I/60R). Perfusate proteins were separated using two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry (MS), revealing 26 tissue-specific proteins released during reperfusion post-15I. Proteins released during irreversible I/R (60I/60R) were profiled using gel-based (2-DE and one-dimensional gel electrophoresis coupled to liquid chromatography and tandem mass spectrometry; geLC-MS) and gel-free (LC-MS/MS) methods. A total of 192 tissue-specific proteins were identified during reperfusion post-60I. Identified proteins included those previously associated with I/R (myoglobin, CK-MB, cTnI, and cTnT), in addition to examples currently under investigation in large cohort studies (heart-type fatty acid binding protein; FABPH). The postischemic release profile of a novel cardiac-specific protein, cysteine and glycine-rich protein 3 (Csrp3; cardiac LIM domain protein) was validated by Western blot analysis. We also identified Csrp3 in serum from 6 of 8 patients postreperfusion following acute myocardial infarction. These studies indicate that animal modeling of biomarker release using ex vivo buffer perfused tissue to limit the presence of obfuscating plasma proteins may identify candidates for further study in humans.