Expression of transmembrane carbonic anhydrase isoenzymes IX and XII in normal human pancreas and pancreatic tumours

Carbonic anhydrase (CA) IX and XII are transmembrane isoenzymes which are expressed in several epithelia and overexpressed in some carcinomas. They have recently been linked to von Hippel-Lindau gene-mediated carcinogenesis in that both isoenzymes are downregulated by the product of the wild-type von Hippel-Lindau tumour suppressor gene. This paper describes the localisation of CA IX and XII in the normal human pancreas and pancreatic tumours. Both isoenzymes showed positive reaction in the basolateral plasma membrane of the normal acinar and ductal epithelia. The hyperplastic ductal epithelium in tumour specimens generally showed an increased staining for CA IX. Of 29 malignant tumours of exocrine pancreas, 10 showed moderate or strong immunoreaction for CA IX. The signal for CA XII remained weak in most malignant lesions. The present results show that both CA IX and XII are unevenly expressed in the ductal and acinar compartments of the human pancreas. The expression of these isoenzymes in a relatively low number of malignant tumour specimens suggests that they have a limited value in diagnostic evaluation of pancreatic carcinoma. However, the increased expression of CA IX in hyperplastic ductal epithelium may contribute to the pancreatic tumourigenesis.     

Laminin alpha1-chain shows a restricted distribution in epithelial basement membranes of fetal and adult human tissues

Two novel monoclonal antibodies were raised and used to study the expression of laminin (Ln) alpha1-chain in developing and adult human tissues. In both fetal and adult kidney, a distinct immunoreactivity was seen in basement membranes (BM) of most proximal tubules but not in the distal tubular or glomerular BM or in the basal laminae of blood vessels. Immunoprecipitation of metabolically labeled cultured human renal proximal tubular cells showed an abundant production and deposition of Ln alpha1-chain to the extracellular matrix, suggestive of an epithelial origin of kidney Ln-1. Quantitative cell adhesion experiments with JAR choriocarcinoma cells showed that purified human Ln-1 is a good substrate for cell adhesion that it is differently recognized by integrin receptors when compared to mouse Ln-1. In fetal and adult testes immunoreactivity was solely confined to BM of the seminiferous epithelium. In the airways BM-confined reaction was only seen in fetal budding bronchial tubules (16-19 weeks) at the pseudoglandular stage of development. In the skin a distinct immunoreactivity was confined to BM of developing hair buds but not in epithelial BMs of adult epidermis or of epidermal appendages. In other adult tissues, immunoreactivity was found in BMs of thyroid, salivary, and mammary glands as well as in BMs of endometrium and endocervix, but not of ectocervix or vagina. No immunoreactivity was found in BMs of most of the digestive tract, including the liver and pancreas, except for BMs of esophageal submucosal glands and duodenal Brunner's glands. In fetal specimens, BMs of the bottoms of the intestinal and gastric glands were positive. Basal laminae of blood vessels were generally negative for Ln alpha1 chain with the exception of specimens of both fetal and adult central nervous system in which immunoreactivity for Ln alpha1 chain was prominently confined to capillary walls. The results suggest that outside the central nervous system, Ln alpha1 chain shows a restricted and developmentally regulated expression in BMs of distinct epithelial tissues.     

Age and gender dependency of baroreflex sensitivity in healthy subjects

Laitinen, Tomi, Juha Hartikainen, Esko Vanninen, LeoNiskanen, Ghislaine Geelen, and Esko Länsimies. Age andgender dependency of baroreflex sensitivity in healthy subjects.J. Appl. Physiol. 84(2): 576-583, 1998.We evaluated the correlates of baroreflex sensitivity (BRS) inhealthy subjects. The study consisted of 117 healthy, normal-weight,nonsmoking male and female subjects aged 23-77 yr. Baroreflexcontrol of heart rate was measured by using the phenylephrinebolus-injection technique. Frequency- and time-domain analysis of heartrate variability and an exercise test were performed. Plasmanorepinephrine, epinephrine, insulin, and arginine vasopressinconcentrations and plasma renin activity were measured. In theunivariate analysis, BRS correlated with age(r = 0.65,P < 0.001), diastolic blood pressure(r = 0.47, P < 0.001), exercise capacity(r = 0.60, P < 0.001), and the high-frequency component of heart rate variability (r = 0.64, P < 0.001). There was also asignificant correlation between BRS and plasma norepinephrine concentration (r = 0.22,P < 0.05) and plasma renin activity (r = 0.32, P < 0.001). According to themultivariate analysis, age and gender were the most importantphysiological correlates of BRS. They accounted for 52% ofinterindividual BRS variation. In addition, diastolic blood pressureand high-frequency component of heart rate variability were significantindependent correlates of BRS. BRS was significantly higher in men thanin women (15.0 ± 1.2 vs. 10.2 ± 1.1 ms/mmHg, respectively;P < 0.01). Twenty-four percent ofwomen >40 yr old and 18% of men >60 yr old had markedly depressedBRS (<3 ms/mmHg). We conclude that physiological factors, particularly age and gender, have significant impact on BRS in healthysubjects. In addition, we demonstrate that BRS values that have beenproposed to be useful in identifying postinfarction patients at highrisk of sudden death are frequently found in healthy subjects.

     

Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering—A Comparison between Wild-Type Protein and a Hinge Mutant

Myelin protein P2 is a fatty acid-binding structural component of the myelin sheath in the peripheral nervous system, and its function is related to its membrane binding capacity. Here, the link between P2 protein dynamics and structure and function was studied using elastic incoherent neutron scattering (EINS). The P38G mutation, at the hinge between the β barrel and the α-helical lid, increased the lipid stacking capacity of human P2 in vitro, and the mutated protein was also functional in cultured cells. The P38G mutation did not change the overall structure of the protein. For a deeper insight into P2 structure-function relationships, information on protein dynamics in the 10 ps to 1 ns time scale was obtained using EINS. Values of mean square displacements mainly from protein H atoms were extracted for wild-type P2 and the P38G mutant and compared. Our results show that at physiological temperatures, the P38G mutant is more dynamic than the wild-type P2 protein, especially on a slow 1-ns time scale. Molecular dynamics simulations confirmed the enhanced dynamics of the mutant variant, especially within the portal region in the presence of bound fatty acid. The increased softness of the hinge mutant of human myelin P2 protein is likely related to an enhanced flexibility of the portal region of this fatty acid-binding protein, as well as to its interactions with the lipid bilayer surface requiring conformational adaptations.     

Avian top predator and the landscape of fear: responses of mammalian mesopredators to risk imposed by the golden eagle

Top predators may induce extensive cascading effects on lower trophic levels, for example, through intraguild predation (IGP). The impacts of both mammalian and avian top predators on species of the same class have been extensively studied, but the effects of the latter upon mammalian mesopredators are not yet as well known. We examined the impact of the predation risk imposed by a large avian predator, the golden eagle (Aquila chrysaetos, L.), on its potential mammalian mesopredator prey, the red fox (Vulpes vulpes, L.), and the pine marten (Martes martes, L.). The study combined 23 years of countrywide data from nesting records of eagles and wildlife track counts of mesopredators in Finland, northern Europe. The predation risk of the golden eagle was modeled as a function of territory density, density of fledglings produced, and distance to nearest active eagle territory, with the expectation that a high predation risk would reduce the abundances of smaller sized pine martens in particular. Red foxes appeared not to suffer from eagle predation, being in fact most numerous close to eagle nests and in areas with more eagle territories. This is likely due to similar prey preferences of the two predators and the larger size of foxes enabling them to escape eagle predation risk. Somewhat contrary to our prediction, the abundance of pine martens increased from low to intermediate territory density and at close proximity to eagle nests, possibly because of similar habitat preferences of martens and eagles. We found a slightly decreasing trend of marten abundance at high territory density, which could indicate that the response in marten populations is dependent on eagle density. However, more research is needed to better establish whether mesopredators are intimidated or predated by golden eagles, and whether such effects could in turn cascade to lower trophic levels, benefitting herbivorous species.     

Extension of dynamic range of sensitive nanoparticle-based immunoassays

Nanoparticles have successfully been employed in immunometric assays that require high sensitivity. Certain analytes, however, require dynamic ranges (DRs) around a predetermined cut-off value. Here, we have studied the effects that antibody orientation and addition of free solid-phase and detection antibodies have on assay sensitivity and DR in traditional sandwich-type immunoassays. D-dimer and cardiac troponin I (cTnI), both routinely used in critical care testing, were applied as model analytes. The assays were performed in microtitration wells with preimmobilized solid-phase antibody. Inherently fluorescent nanoparticles coated with second antibody were used to detect the analyte. The selection of antibody orientation and addition of free solid-phase or detection antibody, with nanoparticles and calibrator, desensitized the assays and extended the DR. With D-dimer the upper limit of the DR was improved from 50 to 10,000 ng/ml, and with cTnI from 25 to 1000 ng/ml. Regression analysis with the Stago STA Liatest D-dimer assay yielded a slope (95% confidence interval) of 0.09 (0.07–0.11) and a y-intercept of −7.79 (−17.87–2.29) ng/L (n = 65, r = 0.906). Thus it is concluded that Europium(III)-chelate-doped nanoparticles can also be employed in immunoassays that require wide DRs around a certain cut-off limit.     

Quantification of lysophosphatidic acids in rat brain tissue by liquid chromatography–electrospray tandem mass spectrometry

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological functions. A highly selective and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed for the determination of LPAs (16:0 LPA, 18:0 LPA, 18:1 LPA, 20:4 LPA) in rat brain cryosections. After partitioning the LPAs from other lipophilic material present in the tissue with a liquid–liquid extraction, a reversed-phase column and ion pair technique was used for separating analytes with a gradient elution. An internal standard (17:0 LPA) was included in the analysis. Detection and quantification of the LPAs were carried out with a triple quadrupole mass spectrometer using negative electrospray ionization (ESI) and multiple reaction monitoring (MRM). The artificial formation of LPAs from lysophosphatidylcholines during the sample preparation procedure and instrumentation was carefully studied during the method development. The method was validated; acceptable selectivity, accuracy, precision, recovery, and stability were obtained for concentrations within the calibration curve range of 0.02–1.0 μM for LPAs. The quantification limit of the assay was 54 fmol injected into column for each LPAs. The method was applied to comparative studies of LPA levels in rat brain cryosections after the various chemical pre-treatments of the sections.     

Physiological and antioxidant responses of two accessions of Arabidopsis thaliana in different light and temperature conditions

During their lifetime, plants need to adapt to a changing environment, including light and temperature. To understand how these factors influence plant growth, we investigated the physiological and antioxidant responses of two Arabidopsis accessions, Shahdara (Sha) from the Shahdara valley (Tajikistan, Central Asia) in a mountainous area and Lovvik‐5 (Lov‐5) from northern Sweden to different light and temperature conditions. These accessions originate from different latitudes and have different life strategies, both of which are known to be influenced by light and temperature. We showed that both accessions grew better in high‐light and at a lower temperature (16°C) than in low light and at 23°C. Interestingly, Sha had a lower chlorophyll content but more efficient non‐photochemical quenching than Lov‐5. Sha, also showed a higher expression of vitamin E biosynthetic genes. We did not observe any difference in the antioxidant prenyllipid level under these conditions. Our results suggest that the mechanisms that keep the plastoquinone (PQ)‐pool in more oxidized state could play a role in the adaptation of these accessions to their local climatic conditions.     

Attenuated expression of tenascin-c in ovalbumin-challenged STAT4-/- mice

Background

Asthma leads to structural changes in the airways, including the modification of extracellular matrix proteins such as tenascin-C. The role of tenascin-C is unclear, but it might act as an early initiator of airway wall remodelling, as its expression is increased in the mouse and human airways during allergic inflammation. In this study, we examined whether Th1 or Th2 cells are important regulators of tenascin-C in experimental allergic asthma utilizing mice with impaired Th1 (STAT4-/-) or Th2 (STAT6-/-) immunity.

Methods

Balb/c wildtype (WT), STAT4-/- and STAT6-/- mice were sensitized with intraperitoneally injected ovalbumin (OVA) followed by OVA or PBS airway challenge. Airway hyperreactivity (AHR) was measured and samples were collected. Real time PCR and immunohistochemistry were used to study cytokines and differences in the expression of tenascin-C. Tenascin-C expression was measured in human fibroblasts after treatment with TNF-α and IFN-γ in vitro.

Results

OVA-challenged WT mice showed allergic inflammation and AHR in the airways along with increased expression of TNF-α, IFN-γ, IL-4 and tenascin-C in the lungs. OVA-challenged STAT4-/- mice exhibited elevated AHR and pulmonary eosinophilia. The mRNA expression of TNF-α and IFN-γ was low, but the expression of IL-4 was significantly elevated in these mice. OVA-challenged STAT6-/- mice had neither AHR nor pulmonary eosinophilia, but had increased expression of mRNA for TNF-α, IFN-γ and IL-4. The expression of tenascin-C in the lungs of OVA-challenged STAT4-/- mice was weaker than in those of OVA-challenged WT and STAT6-/- mice suggesting that TNF-α and IFN-γ may regulate tenascin-C expression in vivo. The stimulation of human fibroblasts with TNF-α and IFN-γ induced the expression of tenascin-C confirming our in vivo findings.

Conclusions

Expression of tenascin-C is significantly attenuated in the airways of STAT4-/- mice, which may be due to the impaired secretion of TNF-α and IFN-γ in these mice.     

Crystal structure of 2-enoyl-CoA hydratase 2 from human peroxisomal multifunctional enzyme type 2

2-Enoyl-CoA hydratase 2 is the middle part of the mammalian peroxisomal multifunctional enzyme type 2 (MFE-2), which is known to be important in the beta-oxidation of very-long-chain and alpha-methyl-branched fatty acids as well as in the synthesis of bile acids. Here, we present the crystal structure of the hydratase 2 from the human MFE-2 to 3A resolution. The three-dimensional structure resembles the recently solved crystal structure of hydratase 2 from the yeast, Candida tropicalis, MFE-2 having a two-domain subunit structure with a C-domain complete hot-dog fold housing the active site, and an N-domain incomplete hot-dog fold housing the cavity for the aliphatic acyl part of the substrate molecule. The ability of human hydratase 2 to utilize such bulky compounds which are not physiological substrates for the fungal ortholog, e.g. CoA esters of C26 fatty acids, pristanic acid and di/trihydroxycholestanoic acids, is explained by a large hydrophobic cavity formed upon the movements of the extremely mobile loops I-III in the N-domain. In the unliganded form of human hydratase 2, however, the loop I blocks the entrance of fatty enoyl-CoAs with chain-length >C8. Therefore, we expect that upon binding of substrates bulkier than C8, the loop I gives way, contemporaneously causing a secondary effect in the CoA-binding pocket and/or active site required for efficient hydration reaction. This structural feature would explain the inactivity of human hydratase 2 towards short-chain substrates. The solved structure is also used as a tool for analyzing the various inactivating mutations, identified among others in MFE-2-deficient patients. Since hydratase 2 is the last functional unit of mammalian MFE-2 whose structure has been solved, the organization of the functional units in the biologically active full-length enzyme is also discussed.