CO2 induced acute respiratory acidosis and brain tissue intracellular pH: a 31P NMR study in swine

High concentration carbon dioxide (CO(2)) is used to promote pre-slaughter anaesthesia in swine and poultry, as well as short-lasting surgical anaesthesia and euthanasia in laboratory animals. Questions related to animal welfare have been raised, as CO(2) anaesthesia does not set in momentarily. Carbon dioxide promotes anaesthesia by lowering the intracellular pH in the brain cells, but the dynamics of the changes in response to a high concentration of CO(2) is not known. Based on (31)P NMR spectroscopy, we describe CO(2)-induced changes in intracellular pH in the brains of five pigs inhaling 90% CO(2) in ambient air for a period of 60 s, and compare the results to changes in arterial blood pH, P(CO2), O(2) saturation and HCO(3)(-) concentration. The intracellular pH paralleled the arterial pH and P(CO2) during inhalation of CO(2); and it is suggested that the acute reaction to CO(2) inhalation mainly reflects respiratory acidosis, and not metabolic regulation as for example transmembrane fluxes of H(+)/HCO(3)(-). The intracellular pH decreased to approximately 6.7 within the 60 s inhalation period, and the situation was metabolically reversible after the end of CO(2) inhalation. The fast decrease in intracellular pH supports the conclusion that high concentration CO(2) leads to anaesthesia soon after the start of inhalation.     

Muscle interstitial potassium kinetics during intense exhaustive exercise: effect of previous arm exercise

Interstitial K+ ([K+]i) was measured in human skeletal muscle by microdialysis during exhaustive leg exercise, with (AL) and without (L) previous intense arm exercise. In addition, the reproducibility of the [K+]i determinations was examined. Possible microdialysis-induced rupture of the sarcolemma was assessed by measurement of carnosine in the dialysate, because carnosine is only expected to be found intracellularly. Changes in [K+]i could be reproduced, when exhaustive leg exercise was performed on two different days, with a between-day difference of approximately 0.5 mM at rest and 1.5 mM at exhaustion. The time to exhaustion was shorter in AL than in L (2.7 +/- 0.3 vs. 4.0 +/- 0.3 min; P < 0.05). Furthermore, [K+]i was higher from 0 to 1.5 min of the intense leg exercise period in AL compared with L (9.2 +/- 0.7 vs. 6.4 +/- 0.9 mM; P < 0.001) and at exhaustion (11.9 +/- 0.5 vs. 10.3 +/- 0.6 mM; P < 0.05). The dialysate content of carnosine was elevated by exercise, but low-intensity exercise resulted in higher dialysate carnosine concentrations than subsequent intense exercise. Furthermore, no relationship was found between carnosine concentrations and [K+]i. Thus the present data suggest that microdialysis can be used to determine muscle [K+]i kinetics during intense exercise, when low-intensity exercise is performed before the intense exercise. The high [K+]i levels reached at exhaustion can be expected to cause fatigue, which is supported by the finding that a faster accumulation of interstitial K+, induced by prior arm exercise, was associated with a reduced time to fatigue.     

Heparin binding to the urokinase kringle domain.

The binding of urokinase to immobilized heparin and dextran sulfate was studied using activity assays of the bound urokinase. The markedly higher binding observed with high M(r) urokinase compared to low M(r) urokinase indicated a role for the amino-terminal fragment (ATF). This was confirmed by the use of inactive truncated urokinase and monoclonal antibodies specific for the ATF in competition assays of urokinase binding. Antibody competition assays suggested a site in the kringle domain, and a synthetic decapeptide Arg-52-Trp-62 from the kringle sequence (kringle numbering convention) was competitive in assays of urokinase binding to dextran sulfate and heparin. Heparin binding to the urokinase kringle was unambiguously demonstrated via 1H NMR spectroscopy at 500 MHz. Effective equilibrium association constants (K(a)*) were determined for the interaction of isolated kringle fragment and low M(r) heparin at pH 7.2. The binding was strong in salt-free 2H2O (K(a)* approximately 57 mM-1) and remained significant in 0.15 M NaCl (K(a)* approximately 12 mM-1), supporting a potential physiological role for the interaction. This is the first demonstration of a function for the kringle domain of urokinase, and it suggests that while the classical kringle structure has specificity for lysine binding, there may also exist a class of kringles with affinity for polyanion binding.     

Versatile High Resolution Oligosaccharide Microarrays for Plant Glycobiology and Cell Wall Research

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.     

Large scale experimental effects of three levels of sheep densities on an alpine ecosystem

Plant–herbivore dynamics is a major topic in ecological research, but empirical knowledge on the ecological effects of different densities of large grazers from fully replicated experiments is rare. Previous studies have focused on grazing vs no grazing, and our understanding of the extent to which different levels of grazing alter vegetation composition, and how quickly such effects can be measured, is therefore limited. We performed a fully replicated, short‐term (four‐year) experiment using large enclosures (each ～0.3 km²) with three different sheep densities (no grazing, low grazing and high grazing, respectively) in an alpine environment with summer grazing in southern Norway to address these issues. Sheep grazing mainly affected plant species at high densities of sheep as compared to no sheep after a four‐year treatment; few effects of low sheep densities were detectable. Highly selected herbs, herbs suggested vulnerable to trampling, and woody species decreased, while most graminoids, one ruderal, one prostrate species and two bryophyte taxa increased at high sheep densities. We found contrasting responses within main functional groups highlighting that fine details of plant life histories need to be known for responses to grazing to be successfully predicted. Vascular plant cover and bare soil responded to sheep density after two years of treatment, but only for one of the species was frequency change observed at this stage. Overall, plants in low grazing plots were found to be almost unaffected. Changes in abundance were mainly found at the no grazing and high grazing treatments. Plant species that decreased at high grazing generally increased at no grazing and vice versa, suggesting a response to both cessation of grazing and enhanced grazing respectively. Our study demonstrates, beyond a simple comparison of heavily grazed and non‐grazed sites, that herbivore effects on plants are typically non‐linearly related to herbivore density, and that the speed of plant responses will depend both on the plant property examined and the grazing pressure.     

Elevated p16ink4a Expression in Human Labial Salivary Glands as a Potential Correlate of Cognitive Aging in Late Midlife

Background

The cell-cycle inhibitor and tumor suppressor cyclin dependent kinase inhibitor, p16^ink4a, is one of the two gene products of the ink4a/ARF (cdkn2a) locus on chromosome 9q21. Up-regulation of p16^ink4a has been linked to cellular senescence, and findings from studies on different mammalian tissues suggest that p16^ink4a may be a biomarker of organismal versus chronological age.

Objective

The aim of this study was to examine the immunolocalization pattern of p16^ink4a in human labial salivary gland (LSG) tissue, and to analyze whether its expression level in LSGs is a peripheral correlate of cognitive decline in late midlife.

Methods

The present study was a part of a study of causes and predictors of cognitive decline in middle-aged men in a Danish birth cohort. It is based on data from 181 male participants from the Danish Metropolit birth cohort, born in 1953, who were examined for age-associated alterations in cognition, dental health, and morphological and autonomic innervation characteristics of the LSGs. The participants were allocated to two groups based on the relative change in cognitive performance from young adulthood to late midlife. LSG biopsies were analyzed by qRT-PCR for the expression level of p16^ink4a. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections of LSGs.

Results

p16^ink4a immunoreactivity was observed in LSG ductal, myoepithelial, and stromal cells, but not in acinar cells. The mean relative expression of p16^ink4a in LSGs was higher in the group of participants with decline in cognitive performance. A logistic regression analysis revealed that the relative p16 expression was predictive of the participant’s group assignment. A negative correlation was found between relative p16^ink4a expression and the participant’s standardized regression residuals from early adulthood to late midlife cognitive performance scores.

Conclusions

p16^ink4a expression in human LSGs may constitute a potential peripheral correlate of cognitive decline. Human labial salivary glands seem suitable for studies on organismal as opposed to chronological age.     

Matrix metalloproteinase 13 is induced in fibroblasts in polyomavirus middle T antigen-driven mammary carcinoma without influencing tumor progression

Matrix metalloproteinase (MMP) 13 (collagenase 3) is an extracellular matrix remodeling enzyme that is induced in myofibroblasts during the earliest invasive stages of human breast carcinoma, suggesting that it is involved in tumor progression. During progression of mammary carcinomas in the polyoma virus middle T oncogene mouse model (MMTV-PyMT), Mmp13 mRNA was strongly upregulated concurrently with the transition to invasive and metastatic carcinomas. As in human tumors, Mmp13 mRNA was found in myofibroblasts of invasive grade II and III carcinomas, but not in benign grade I and II mammary intraepithelial neoplasias. To determine if MMP13 plays a role in tumor progression, we crossed MMTV-PyMT mice with Mmp13 deficient mice. The absence of MMP13 did not influence tumor growth, vascularization, progression to more advanced tumor stages, or metastasis to the lungs, and the absence of MMP13 was not compensated for by expression of other MMPs or tissue inhibitor of metalloproteinases. However, an increased fraction of thin collagen fibrils was identified in MMTV-PyMT;Mmp13(-/-) compared to MMTV-PyMT;Mmp13(+/+) tumors, showing that collagen metabolism was altered in the absence of MMP13. We conclude that the expression pattern of Mmp13 mRNA in myofibroblasts of invasive carcinomas in the MMTV-PyMT breast cancer model recapitulates the expression pattern observed in human breast cancer. Our results suggest that MMP13 is a marker of carcinoma-associated myofibroblasts of invasive carcinoma, even though it does not make a major contribution to tumor progression in the MMTV-PyMT breast cancer model.     

Phosphate transport in mitochondria: Past accomplishments,present problems,and future challenges

The requirement of inorganic phosphate (P_i) for oxidative phosphorylation in eukaryotic cells is fulfilled through specific P_i transport systems. The mitochondrial proton/phosphate symporter (P_ic) is a membrane-embedded protein which translocates P_i from the cytosol into the mitochondrial matrix. P_ic is responsible for the very rapid transport of most of the P_i used in ATP synthesis. During the past five years there have been advances on several fronts. Genomic and cDNA clones for yeast, bovine, rat, and human P_ic have been isolated and sequenced. Functional expression of yeast P_ic in yeast strains deficient in P_i transport and expression inEscherichia coli of a chimera protein involving P_ic and ATP synthase subunit have been accomplished. P_ic, in contrast to other members of the family of transporters involved in energy metabolism, was demonstrated to have a presequence, which optimizes the import of the precursor protein into mitochondria. Six transmembrane segments appear to be a structural feature shared between P_ic and other mitochondrial anion carriers, and recent-site directed mutagenesis studies implicate structure-functional relationships to bacteriorhodopsin. These recent advances on P_ic will be assessed in light of a more global interpretation of transport mechanism across the inner mitochondrial membrane.