HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

A modular treatment of molecular traffic through the active site of cholinesterase

We present a model for the molecular traffic of ligands, substrates, and products through the active site of cholinesterases (ChEs). First, we describe a common treatment of the diffusion to a buried active site of cationic and neutral species. We then explain the specificity of ChEs for cationic ligands and substrates by introducing two additional components to this common treatment. The first module is a surface trap for cationic species at the entrance to the active-site gorge that operates through local, short-range electrostatic interactions and is independent of ionic strength. The second module is an ionic-strength-dependent steering mechanism generated by long-range electrostatic interactions arising from the overall distribution of charges in ChEs. Our calculations show that diffusion of charged ligands relative to neutral isosteric analogs is enhanced approximately 10-fold by the surface trap, while electrostatic steering contributes only a 1.5- to 2-fold rate enhancement at physiological salt concentration. We model clearance of cationic products from the active-site gorge as analogous to the escape of a particle from a one-dimensional well in the presence of a linear electrostatic potential. We evaluate the potential inside the gorge and provide evidence that while contributing to the steering of cationic species toward the active site, it does not appreciably retard their clearance. This optimal fine-tuning of global and local electrostatic interactions endows ChEs with maximum catalytic efficiency and specificity for a positively charged substrate, while at the same time not hindering clearance of the positively charged products.     

A comprehensive analysis of QTL for abdominal fat and breast muscle weights on chicken chromosome 5 using a multivariate approach

Quantitative trait loci (QTL) influencing the weight of abdominal fat (AF) and of breast muscle (BM) were detected on chicken chromosome 5 (GGA5) using two successive F₂ crosses between two divergently selected 'Fat' and 'Lean' INRA broiler lines. Based on these results, the aim of the present study was to identify the number, location and effects of these putative QTL by performing multitrait and multi-QTL analyses of the whole available data set. Data concerned 1186 F₂ offspring produced by 10 F₁ sires and 85 F₁ dams. AF and BM traits were measured on F₂ animals at slaughter, at 8 (first cross) or 9 (second cross) weeks of age. The F₀, F₁ and F₂ birds were genotyped for 11 microsatellite markers evenly spaced along GGA5. Before QTL detection, phenotypes were adjusted for the fixed effects of sex, F₂ design, hatching group within the design, and for body weight as a covariable. Univariate analyses confirmed the QTL segregation for AF and BM on GGA5 in male offspring, but not in female offspring. Analyses of male offspring data using multitrait and linked-QTL models led us to conclude the presence of two QTL on the distal part of GGA5, each controlling one trait. Linked QTL models were applied after correction of phenotypic values for the effects of these distal QTL. Several QTL for AF and BM were then discovered in the central region of GGA5, splitting one large QTL region for AF into several distinct QTL. Neither the 'Fat' nor the 'Lean' line appeared to be fixed for any QTL genotype. These results have important implications for prospective fine mapping studies and for the identification of underlying genes and causal mutations.     

Description and quantification of pteropod shell dissolution: a sensitive bioindicator of ocean acidification

Anthropogenic ocean acidification is likely to have negative effects on marine calcifying organisms, such as shelled pteropods, by promoting dissolution of aragonite shells. Study of shell dissolution requires an accurate and sensitive method for assessing shell damage. Shell dissolution was induced through incubations in CO₂‐enriched seawater for 4 and 14 days. We describe a procedure that allows the level of dissolution to be assessed and classified into three main types: Type I with partial dissolution of the prismatic layer; Type II with exposure of underlying crossed‐lamellar layer, and Type III, where crossed‐lamellar layer shows signs of dissolution. Levels of dissolution showed a good correspondence to the incubation conditions, with the most severe damage found in specimens held for 14 days in undersaturated condition (Ω ~ 0.8). This methodology enables the response of small pelagic calcifiers to acidified conditions to be detected at an early stage, thus making pteropods a valuable bioindicator of future ocean acidification.     

Naive human CD4+ T cells are a major source of lymphotoxin alpha

It is generally accepted that immunologically naive T cells display a very restricted cytokine production profile consisting mainly of IL-2, which is used as an autocrine growth factor. Here we report that activated naive CD4+ T cells, of neonatal or adult origin, express very high levels of soluble lymphotoxin (LT) alpha (LTalpha3), as determined by ELISA, RNase protection assay, and intracytoplasmic staining. Besides LTalpha3 and IL-2, these cells also produce high levels of TNF-alpha together with significant amounts of IFN-gamma and IL-13. Naive cells also express LTbeta mRNA and the membrane form of LTalpha (LTalphabeta). On average, naive CD4+ T cells secrete four times more LTalpha3 than Th1-like cells, twice more than naive CD8+ T cells, and ten times more than B cells. Thus, naive T cells express a large spectrum of cytokines, mainly of the Th1 type, and the very high levels of LTalpha3/TNF-alpha that they release may play an hitherto unsuspected role in the early stage of T cell-dependent immune responses.     

Effect of gender on endothelium-dependent dilation to bradykinin in human adipose microvessels

We examined the influence of gender and climacteric status, two coronary risk factors, on bradykinin (BK)-induced dilation in adipose arterioles from men and women of different ages [premenopausal women (Pre-W), postmenopausal women (Post-W), and similar aged men (Y-M and O-M), respectively]. We examined the responses from both omental (more closely associated with coronary disease) and subcutaneous fat. Tissues were obtained at surgery and cannulated (60 mmHg) for measurement of internal diameter. In vessels from omental tissue, dilation to BK was more sensitive in Pre-W than other groups, whereas in vessels from subcutaneous tissue, sensitivity to BK was greater in both Pre-W and Post-W compared with Y-M and O-M. Maximal dilation was similar among groups. Indomethacin (Indo; 10(-5) M) alone had no effect on dilation to BK in any groups, but Indo and N(omega)-nitro-L-arginine methyl ester (L-NAME; 10(-4) M) reduced dilation to BK in Pre-W more than in Y-M. L-NAME increased dilation to BK in subcutaneous fat from Y-M but had no effect in Post-W and O-M. Indo- and L-NAME-resistant dilation in all vessels was markedly reduced by 30 mM KCl. There was no difference in sodium nitroprusside-induced dilation among groups. We conclude that gender and climacteric state contribute to mechanisms of microvascular regulation in humans. Functional vascular differences in visceral and subcutaneous fat may underlie the proposed differential influence of these tissues on cardiovascular risk.     

Bidirectional negative regulation of human T and dendritic cells by CD47 and its cognate receptor signal-regulator protein-alpha: down-regulation of IL-12 responsiveness and inhibition of dendritic cell activation

Proinflammatory molecules, including IFN-gamma and IL-12, play a crucial role in the elimination of causative agents. To allow healing, potent anti-inflammatory processes are required to down-regulate the inflammatory response. In this study, we first show that CD47/integrin-associated protein, a ubiquitous multispan transmembrane protein highly expressed on T cells, interacts with signal-regulator protein (SIRP)-alpha, an immunoreceptor tyrosine-based inhibition motif-containing molecule selectively expressed on myelomonocytic cells, and next demonstrate that this pair of molecules negatively regulates human T and dendritic cell (DC) function. CD47 ligation by CD47 mAb or L-SIRP-alpha transfectants inhibits IL-12R expression and down-regulates IL-12 responsiveness of activated CD4(+) and CD8(+) adult T cells without affecting their response to IL-2. Human CD47-Fc fusion protein binds SIRP-alpha expressed on immature DC and mature DC. SIRP-alpha engagement by CD47-Fc prevents the phenotypic and functional maturation of immature DC and still inhibits cytokine production by mature DC. Finally, in allogeneic MLR between mDC and naive T cells, CD47-Fc decreases IFN-gamma production after priming and impairs the development of a Th1 response. Therefore, CD47 on T cells and its cognate receptor SIRP-alpha on DC define a novel regulatory pathway that may be involved in the maintenance of homeostasis by preventing the escalation of the inflammatory immune response.     

Quiescin sulfhydryl oxidase 1 promotes invasion of pancreatic tumor cells mediated by matrix metalloproteinases

Quiescin sulfhydryl oxidase 1 (QSOX1) oxidizes sulfhydryl groups to form disulfide bonds in proteins. We previously mapped a peptide in plasma from pancreatic ductal adenocarcinoma (PDA) patients back to an overexpressed QSOX1 parent protein. In addition to overexpression in pancreatic cancer cell lines, 29 of 37 patients diagnosed with PDA expressed QSOX1 protein in tumor cells, but QSOX1 was not detected in normal adjacent tissues or in a transformed, but nontumorigenic cell line. To begin to evaluate the advantage QSOX1 might provide to tumors, we suppressed QSOX1 protein expression using short hairpin (sh) RNA in two pancreatic cancer cell lines. Growth, cell cycle, apoptosis, invasion, and matrix metalloproteinase (MMP) activity were evaluated. QSOX1 shRNA suppressed both short and long isoforms of the protein, showing a significant effect on cell growth, cell cycle, and apoptosis. However, QSOX1 shRNA dramatically inhibited the abilities of BxPC-3 and Panc-1 pancreatic tumor cells to invade through Matrigel in a modified Boyden chamber assay. Mechanistically, gelatin zymography indicated that QSOX1 plays an important role in activation of MMP-2 and MMP-9. Taken together, our results suggest that the mechanism of QSOX1-mediated tumor cell invasion is by activation of MMP-2 and MMP-9.     

Yersinia pestis and plague: an updated view on evolution,virulence determinants,immune subversion,vaccination and diagnostics

Plague is a vector-borne disease caused by Yersinia pestis. Transmitted by fleas from rodent reservoirs, Y. pestis emerged less than 6000 years ago from an enteric bacterial ancestor through events of gene gain and genome reduction. It is a highly remarkable model for the understanding of pathogenic bacteria evolution, and a major concern for public health as highlighted by recent human outbreaks. A complex set of virulence determinants, including the Yersinia outer membrane proteins (Yops), the broad range protease Pla, pathogen-associated molecular patterns (PAMPs) and iron capture systems play critical roles in the molecular strategies that Y. pestis employs to subvert the human immune system, allowing unrestricted bacterial replication in lymph nodes (bubonic plague) and in lungs (pneumonic plague). Some of these immunogenic proteins as well as the capsular antigen F1 are exploited for diagnostic purposes, which are critical in the context of the rapid onset of death in the absence of antibiotic treatment (less than a week for bubonic plague and less than 48 h for pneumonic plague). In here, we review recent research advances on Y. pestis evolution, virulence factors function, bacterial strategies to subvert mammalian innate immune responses, vaccination and problems associated to pneumonic plague diagnosis.