Characterization of the first beta-class carbonic anhydrase from an arthropod (Drosophila melanogaster) and phylogenetic analysis of beta-class carbonic anhydrases in invertebrates

BACKGROUND: The beta-carbonic anhydrase (CA, EC 4.2.1.1) enzymes have been reported in a variety of organisms, but their existence in animals has been unclear. The purpose of the present study was to perform extensive sequence analysis to show that the beta-CAs are present in invertebrates and to clone and characterize a member of this enzyme family from a representative model organism of the animal kingdom, e.g., Drosophila melanogaster. RESULTS: The novel beta-CA gene, here named DmBCA, was identified from FlyBase, and its orthologs were searched and reconstructed from sequence databases, confirming the presence of beta-CA sequences in 55 metazoan species. The corresponding recombinant enzyme was produced in Sf9 insect cells, purified, kinetically characterized, and its inhibition was investigated with a series of simple, inorganic anions. Holoenzyme molecular mass was defined by dynamic light scattering analysis and gel filtration, and the results suggested that the holoenzyme is a dimer. Double immunostaining confirmed predictions based on sequence analysis and localized DmBCA protein to mitochondria. The enzyme showed high CO2 hydratase activity, with a kcat of 9.5 x 105 s-1 and a kcat/KM of 1.1 x 108 M-1s-1. DmBCA was appreciably inhibited by the clinically-used sulfonamide acetazolamide, with an inhibition constant of 49 nM. It was moderately inhibited by halides, pseudohalides, hydrogen sulfide, bisulfite and sulfate (KI values of 0.67 - 1.36 mM) and more potently by sulfamide (KI of 0.15 mM). Bicarbonate, nitrate, nitrite and phenylarsonic/boronic acids were much weaker inhibitors (KIs of 26.9 - 43.7 mM). CONCLUSIONS: The Drosophila beta-CA represents a highly active mitochondrial enzyme that is a potential model enzyme for anti-parasitic drug development

Renal vascular resistance is increased in patients with kidney transplant

Background Despite improvement in short-term outcome of kidney transplants, the long-term survival of kidney transplants has not changed over past decades. Kidney biopsy is the gold standard of transplant pathology but it's invasive. Quantification of transplant blood flow could provide a novel non-invasive method to evaluate transplant pathology. The aim of this retrospective cross-sectional pilot study was to evaluate positron emission tomography (PET) as a method to measure kidney transplant perfusion and find out if there is correlation between transplant perfusion and histopathology. Methods Renal cortical perfusion of 19 kidney transplantation patients [average time from transplantation 33 (17-54) months; eGFR 55 (47-69) ml/min] and 10 healthy controls were studied by [(15) O]H2O PET. Perfusion and Doppler resistance index (RI) of transplants were compared with histology of one-year protocol transplant biopsy. Results Renal cortical perfusion of healthy control subjects and transplant patients were 2.7 (2.4-4.0) ml min(- 1) g(- 1) and 2.2 (2.0-3.0) ml min(- 1) g(- 1), respectively (p = 0.1). Renal vascular resistance (RVR) of the patients was 47.0 (36.7-51.4) mmHg mL(- 1)min(- 1)g(- 1) and that of the healthy 32.4 (24.6-39.6) mmHg mL(- 1)min(-1)g(-1) (p = 0.01). There was a statistically significant correlation between Doppler RI and perfusion of transplants (r = - 0.51, p = 0.026). Transplant Doppler RI of the group of mild fibrotic changes [0.73 (0.70-0.76)] and the group of no fibrotic changes [0.66 (0.61-0.72)] differed statistically significantly (p = 0.03). No statistically significant correlation was found between cortical perfusion and fibrosis of transplants (p = 0.56). Conclusions [(15) O]H2O PET showed its capability as a method in measuring perfusion of kidney transplants. RVR of transplant patients with stage 2-3 chronic kidney disease was higher than that of the healthy, although kidney perfusion values didn't differ between the groups. Doppler based RI correlated with perfusion and fibrosis of transplants

Advanced Oxidation Process for Degradation of Carbamazepine from Aqueous Solution: Influence of Metal Modified Microporous, Mesoporous Catalysts on the Ozonation Process

Carbamazepine (CBZ), a widely used pharmaceutical compound, is one of the most detected drugs in surface waters. The purpose of this work was to identify an active and durable catalyst, which, in combination with an ozonation process, could be used to remove CBZ and its degradation products. It was found that the CBZ was completely transformed after ozonation within the first minutes of the treatment. However, the resulting degradation products, 1-(2-benzaldehyde)-4-hydro-(1H,3H)-quinazoline-2-one (BQM) and 1-(2-benzaldehyde)-(1H,3H)-quinazoline-2,4-dione (BQD), were more resistant during the ozonation process. The formation and degradation of these products were studied in more detail and a thorough catalytic screening was conducted to reveal the reaction kinetics of both the CBZ and its degradation products. The work was performed by non-catalytic ozonation and with six different heterogeneous catalysts (Pt-MCM-41-IS, Ru-MCM-41-IS, Pd-H-Y-12-EIM, Pt-H-Y-12-EIM, Pd-H-Beta-300-EIM and Cu-MCM-41-A-EIM) operating at two temperatures 20 degrees C and 50 degrees C. The influence of temperature on degradation kinetics of CBZ, BQM and BQD was studied. The results exhibited a notable difference in the catalytic behavior by varying temperature. The higher reactor temperature (50 degrees C) showed a higher activity of the catalysts but a lower concentration of dissolved ozone. Most of the catalysts exhibited higher removal rate for BQM and BQD compared to non-catalytic experiments in both temperatures. The Pd-H-Y-12-EIM catalyst illustrated a higher degradation rate of by-products at 50 degrees C compared to other catalysts

Imaging of αvβ3 integrin expression in experimental myocardial ischemia with [68Ga]NODAGA-RGD positron emission tomography

BACKGROUND: Radiolabeled RGD peptides detect αvβ3 integrin expression associated with angiogenesis and extracellular matrix remodeling after myocardial infarction. We studied whether cardiac positron emission tomography (PET) with [68Ga]NODAGA-RGD detects increased αvβ3 integrin expression after induction of flow-limiting coronary stenosis in pigs, and whether αvβ3 integrin is expressed in viable ischemic or injured myocardium.METHODS: We studied 8 Finnish landrace pigs 13 ± 4 days after percutaneous implantation of a bottleneck stent in the proximal left anterior descending coronary artery. Antithrombotic therapy was used to prevent stent occlusion. Myocardial uptake of [68Ga]NODAGA-RGD (290 ± 31 MBq) was evaluated by a 62 min dynamic PET scan. The ischemic area was defined as the regional perfusion abnormality during adenosine-induced stress by [15O]water PET. Guided by triphenyltetrazolium chloride staining, tissue samples from viable and injured myocardial areas were obtained for autoradiography and histology.RESULTS: Stent implantation resulted in a partly reversible myocardial perfusion abnormality. Compared with remote myocardium, [68Ga]NODAGA-RGD PET showed increased tracer uptake in the ischemic area (ischemic-to-remote ratio 1.3 ± 0.20, p = 0.0034). Tissue samples from the injured areas, but not from the viable ischemic areas, showed higher [68Ga]NODAGA-RGD uptake than the remote non-ischemic myocardium. Uptake of [68Ga]NODAGA-RGD correlated with immunohistochemical detection of αvβ3 integrin that was expressed in the injured myocardial areas.CONCLUSIONS: Cardiac [68Ga]NODAGA-RGD PET demonstrates increased myocardial αvβ3 integrin expression after induction of flow-limiting coronary stenosis in pigs. Localization of [68Ga]NODAGA-RGD uptake indicates that it reflects αvβ3 integrin expression associated with repair of recent myocardial injury.</p

Biochemical and Biophysical Characterization of Carbonic Anhydrase VI from Human Milk and Saliva

Carbonic anhydrases (CA, EC 4.2.1.1) catalyze the hydration of carbon dioxide and take part in many essential physiological processes. In humans, 15 CAs are characterized, including the only secreted isoenzyme CA VI. CA VI has been linked to specific processes in the mouth, namely bitter taste perception, dental caries, and maintenance of enamel pellicle, and implicated in several immunity-related phenomena. However, little is known of the mechanisms of the above. In this study, we characterized human CA VI purified from saliva and milk with biophysical methods and measured their enzyme activities and acetazolamide inhibition. Size-exclusion chromatography showed peaks of salivary and milk CA VI corresponding to hexameric state or larger at pH 7.5. At pH 5.0 the hexamer peaks dominated. SDS- PAGE of milk CA VI protein treated with a bifunctional crosslinker further confirmed that a majority of CA VI is oligomers of similar sizes in solution. Mass spectrometry experiments confirmed that both of the two putative N-glycosylation sites, Asn67 and Asn256, are heterogeneously glycosylated. The attached glycans in milk CA VI were di- and triantennary complex-type glycans, carrying both a core fucose and 1 to 2 additional fucose units, whereas the glycans in salivary CA VI were smaller, seemingly degraded forms of core fucosylated complex- or hybrid-type glycans. Mass spectrometry also verified the predicted signal peptide cleavage site and the terminal residue, Gln 18, being in pyroglutamate form. Thorough characterization of CA VI paves way to better understanding of the biological function of the protein.publishedVersionPeer reviewe

Biochemical and Biophysical Characterization of Carbonic Anhydrase VI from Human Milk and Saliva

Carbonic anhydrases (CA, EC 4.2.1.1) catalyze the hydration of carbon dioxide and take part in many essential physiological processes. In humans, 15 CAs are characterized, including the only secreted isoenzyme CA VI. CA VI has been linked to specific processes in the mouth, namely bitter taste perception, dental caries, and maintenance of enamel pellicle, and implicated in several immunity-related phenomena. However, little is known of the mechanisms of the above. In this study, we characterized human CA VI purified from saliva and milk with biophysical methods and measured their enzyme activities and acetazolamide inhibition. Size-exclusion chromatography showed peaks of salivary and milk CA VI corresponding to hexameric state or larger at pH 7.5. At pH 5.0 the hexamer peaks dominated. SDS- PAGE of milk CA VI protein treated with a bifunctional crosslinker further confirmed that a majority of CA VI is oligomers of similar sizes in solution. Mass spectrometry experiments confirmed that both of the two putative N-glycosylation sites, Asn67 and Asn256, are heterogeneously glycosylated. The attached glycans in milk CA VI were di- and triantennary complex-type glycans, carrying both a core fucose and 1 to 2 additional fucose units, whereas the glycans in salivary CA VI were smaller, seemingly degraded forms of core fucosylated complex- or hybrid-type glycans. Mass spectrometry also verified the predicted signal peptide cleavage site and the terminal residue, Gln 18, being in pyroglutamate form. Thorough characterization of CA VI paves way to better understanding of the biological function of the protein.</p

Radiosynthesis and preclinical evaluation of [68Ga]Ga-NOTA-folate for PET imaging of folate receptor β-positive macrophages

Folate receptor β (FR-β), a marker expressed on macrophages, is a promising target for imaging of inflammation. Here, we report the radiosynthesis and preclinical evaluation of [68Ga]Ga-NOTA-folate (68Ga-FOL). After determining the affinity of 68Ga-FOL using cells expressing FR-β, we studied atherosclerotic mice with 68Ga-FOL and 18F-FDG PET/CT. In addition, we studied tracer distribution and co-localization with macrophages in aorta cryosections using autoradiography, histology, and immunostaining. The specificity of 68Ga-FOL was assessed in a blocking study with folate glucosamine. As a final step, human radiation doses were extrapolated from rat PET data. We were able to produce 68Ga-FOL with high radiochemical purity and moderate molar activity. Cell binding studies revealed that 68Ga-FOL had 5.1 nM affinity for FR-β. Myocardial uptake of 68Ga-FOL was 20-fold lower than that of 18F-FDG. Autoradiography and immunohistochemistry of the aorta revealed that 68Ga-FOL radioactivity co-localized with Mac-3–positive macrophage-rich atherosclerotic plaques. The plaque-to-healthy vessel wall ratio of 68Ga-FOL was significantly higher than that of 18F-FDG. Blocking studies verified that 68Ga-FOL was specific for FR. Based on estimations from rat data, the human effective dose was 0.0105 mSv/MBq. Together, these findings show that 68Ga-FOL represents a promising new FR-β–targeted tracer for imaging macrophage-associated inflammation.</p

Personal stigma and use of mental health services among people with depression in a general population in Finland

Background - A minority of people suffering from depression seek professional help for themselves. Stigmatizing attitudes are assumed to be one of the major barriers to help seeking but there is only limited evidence of this in large general population data sets. The aim of this study was to analyze the associations between mental health attitude statements and depression and their links to actual use of mental health services among those with depression. Methods - We used a large cross-sectional data set from a Finnish population survey (N = 5160). Attitudes were measured by scales which measured the belief that people with depression are responsible for their illness and their recovery and attitudes towards antidepressants. Desire for social distance was measured by a scale and depression with the Composite International Diagnostic Interview Short Form (CIDI-SF) instrument. Use of mental health services was measured by self-report. Results - On the social discrimination scale, people with depression showed more social tolerance towards people with mental problems. They also carried more positive views about antidepressants. Among those with depression, users of mental health services, as compared to non-users, carried less desire for social distance to people with mental health problems and more positive views about the effects of antidepressants. More severe depression predicted more active use of services. Conclusions - Although stronger discriminative intentions can reduce the use of mental health services, this does not necessarily prevent professional service use if depression is serious and views about antidepressant medication are realistic.peerReviewe