Detection of Intravascular Hemolysis in Newborn Infants Using Urinary Carbonic Anhydrase I Immunoreactivity.

BACKGROUND: Mild hemolysis occurs physiologically in neonates, but more severe forms can lead to life-threatening anemia. Newborns in developing regions are particularly at-risk due to the higher incidence of triggers (protozoan infections, sepsis, certain genetic traits). In advanced healthcare facilities, hemolysis is monitored indirectly using resource-intensive methods that probe downstream ramifications. These approaches could potentially delay critical decisions in early-life care, and are not suitable for point-of-care testing. Rapid and cost-effective testing could be based on detecting red blood cell (RBC)-specific proteins, such as carbonic anhydrase I (CAI), in accessible fluids (e.g., urine). METHODS: Urine was collected from 26 full-term male neonates and analyzed for CAI using immunoassays (ELISA, western blot) and proteomics (mass spectrometry). The cohort included a range of hemolytic states, including admissions with infection, ABO incompatibility, and receiving phototherapy. Data were paired with hemoglobin, serum bilirubin (SBR), and C-reactive protein (CRP) measurements. RESULTS: Urine from a control cohort (CRP < 20 mg/L, SBR < 125µmol/L) had no detectable CAI, in line with results from healthy adults. CAI excretion was elevated in neonates with raised SBR (>125 µmol/L), including those qualifying for phototherapy. Newborns with low SBR (<125 µmol/L) but elevated CRP (>20 mg/L) produced urine with strong CAI immunoreactivity. Proteomics showed that CAI was the most abundant RBC-specific protein in CAI-immunopositive samples, and did not associate with other RBC-derived peptides, indicating an intravascular hemolytic source followed by CAI-selective excretion. CONCLUSIONS: CAI is a direct biomarker of intravascular hemolysis that can be measured routinely in urine using non-invasive methods under minimal-laboratory conditions.

Secondary metabolites of Helichrysum plicatum DC. subsp. plicatum flowers as strong carbonic anhydrase, cholinesterase and α-glycosidase inhibitors.

Helichrysum plicatum species are used in Turkish folk medicine as lithagogue, diuretic, and nephritic. Research on the methanol (MeOH) extract of flowers of H. plicatum DC. subsp. plicatum resulted in the isolation of eight known compounds (1-8). The chemical structures of the compounds were determined as ß-sitosterol (1), apigenin (2), nonacosanoic acid (3), astragalin (4), ß-sitosterol-3-O-ß-D-glucopyranoside (5), helichrysin A (6), helichrysin B (7), and isosalipurposide (8) by spectroscopic and chromatographic/spectrometric methods, including 1D and 2D nuclear magnetic resonance and liquid chromatography-tandem mass spectrometry. Nonacosanoic acid (3) was isolated for the first time from H. plicatum DC. subsp. plicatum. The MeOH extract and isolated compounds were evaluated for their in vitro human carbonic anhydrase I (hCAI) and II (hCAII), acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α-glycosidase inhibitory activities. The IC50 values of H. plicatum DC. subsp. plicatum MeOH extract for hCAI, hCAII, AChE, BChE, and α-glycosidase were found to be 77.87, 52.90, 115.50, 117.46, and 81.53 mg/mL, respectively. The compounds showed IC50 values of 1.43-4.47, 1.40-4.32, 1.69-2.90, 1.09-3.89, and 1.61-3.80 µM against hCAI, hCAII, AChE, BChE, and α-glycosidase, respectively. In summary, H. plicatum DC. subsp. plicatum secondary metabolites demonstrated strong inhibitory effects especially against hCAI and hCAII, whereas the MeOH extract showed a weak inhibitory effect on all enzymes.

Ellagitannin and flavonoid constituents from Agrimonia pilosa Ledeb. with their protein tyrosine phosphatase and acetylcholinesterase inhibitory activities.

A new ellagitannin, agritannin (1), a new flavone glycoside, agriflavone (2), and another flavone glycoside with spectroscopic data reported for the first time, kaempferol-3-O-[(S)-3-hydroxy-3-methylglutaryl (1→6)]-ß-d-glucoside (3), along with 16 known compounds were isolated from the aerial parts of Agrimonia pilosa Ledeb. These compounds were evaluated for PTP1B inhibitory activity. Among them, compounds 9 and 18 displayed potential inhibitory activity against PTP1B with IC50 values of 7.14±1.75 and 7.73±0.24µM, respectively. In addition, compound 1 showed significant inhibitory effect with an IC50 value of 17.03±0.09µM. Furthermore, these compounds were tested in AChE inhibitory assays. Most of them were found to have moderate inhibitory effects, with IC50 values ranging from 60.20±1.09 to 92.85±1.12µM. Except compounds 3, 8, and 18 were inactive.

Investigation of the inhibitory properties of some phenolic standards and bee products against human carbonic anhydrase I and II.

Polyphenols are important secondary products of plants with the potential to inhibit carbonic anhydrases. The aim of this study was to investigate the inhibition effects of various phenolic standards, honey, propolis, and pollen species on human carbonic anhydrase I and II. The inhibition values (IC50) of the phenolics (gallic acid, protocatechuic acid, quercetin, catechin, tannic acid, and chrysin) ranged from 0.009 to 0.32 µg/mL, tannic acid emerging as the best inhibitor. The inhibition values of three different types of honey, heather, rhododendron, and chestnut ranged between 2.32 and 25.10 µg/mL, the chestnut honeys exhibiting the best inhibition. The ethanolic extracts of pollen and propolis exhibited good inhibitory properties, with IC50 values between 0.486 and 3.320 µg/mL. In order to evaluate the phenolic composition of bee products, phenolic profiles and total phenolic contents (TFC) were also measured. The inhibition ranking among the natural products studied was phenolic standards > propolis > pollen > honeys, and inhibition was related to TFC.

Microwave-assisted extraction, HPLC analysis, and inhibitory effects on carbonic anhydrase I, II, VA, and VII isoforms of 14 blueberry Italian cultivars.

The multi-component fingerprint and the biological evaluation of plant-derived material are indispensable for the pharmaceutical field, in food quality control procedures, and in all plant-based products. We investigated the quantitative content of biologically active compounds (anthocyanins and chlorogenic acid) of microwave-assisted blueberry extracts from 14 different Italian cultivars, using validated high-performance liquid chromatography-photodiode array detector (HPLC-PDA) method and routinely instrument configuration. The carbonic anhydrase (CA, EC 4.2.1.1) inhibition profiles against several pharmacologically relevant CA isoforms of blueberry extracts and some bioactive compounds were also investigated. The various cultivars showed a highly variable content in anthocyanins and chlorogenic acid, and their CA inhibitory effects were also highly variable. Overall these data prove that antioxidant natural products found in blueberries may be useful for designing pharmacological agents in which various CAs are involved, e.g., antiobesity, antitumor, or anticonvulsants agents.

Inhibition studies of bacterial, fungal and protozoan ß-class carbonic anhydrases with Schiff bases incorporating sulfonamide moieties.

A series of new Schiff bases derived from sulfanilamide, 3-fluorosulfanilamide or 4-(2-aminoethyl)-benzenesulfonamide containing either a hydrophobic or a hydrophilic tail, have been investigated as inhibitors of three ß-carbonic anhydrases (CA, EC 4.2.1.1) from three different microorganisms. Their antifungal, antibacterial and antiprotozoan activities have been determined against the pathogenic fungus Cryptococcus neoformans, the bacterial pathogen Brucella suis and the protozoan parasite Leishmania donovani chagasi, responsible for Leishmaniasis. The results of these inhibition studies show that all three enzymes were efficiently inhibited by the Schiff base sulfonamides with KI values in the nanomolar or submicromolar range, depending on the nature of the tail, coming from the aryl/heteroaryl moiety present in the starting aldehyde employed in the synthesis. Furthermore, the compounds hereby investigated revealed high ß-CAs selectivity over the ubiquitous, physiologically relevant and off-target human isoforms (CA I and II) and to be more potent as antifungal and antibacterial than as antiprotozoan potential drugs.

Transgenic mice over-expressing carbonic anhydrase I showed aggravated joint inflammation and tissue destruction.

BACKGROUND: Studies have demonstrated that carbonic anhydrase I (CA1) stimulates calcium salt precipitation and cell calcification, which is an essential step in new bone formation. Our study had reported that CA1 encoding gene has a strong association with rheumatoid arthritis (RA) and ankylosing spondylitis (AS), two rheumatic diseases with abnormal new bone formation and bone resorption in joints. This study investigated the effect of CA1 on joint inflammation and tissue destruction in transgenic mice that over-express CA1 (CA1-Tg). METHODS: CA1-Tg was generated with C57BL/6J mice by conventional methods. CA1-Tg was treated with collagen-II to induce arthritis (CIA). Wild-type mice, CA1-Tg treated with bovine serum albumin (BSA) and transgenic mice over-expressing PADI4 (PADI4-Tg), a gene known to be involved in rheumatoid arthritis, were used as controls. Histochemistry and X-ray radiographic assay were used to examine joint destruction. Western blotting and real time-PCR were used to examine CA1 expression. RESULTS: CIA was observed in 60% of CA1-Tg, 20% of PADI4-Tg and 20% of wild-type mice after collagen injections. No CIA was found in CA1-Tg mice that received injections of BSA. The arthritic score was 5.5 ± 0.84 in the CA1-Tgs but the score was less than 2 in the injected wild-type mice and the PADI4-Tgs. The thickness of the hind paws in the CA1-Tgs was 3.46 ± 0.11 mm, which was thicker than that of PADI4-Tgs (2.23 ± 0.08 mm), wild-type mice (2.08 ± 0.06 mm) and BSA-treated CA1-Tgs (2.04 ± 0.07 mm). Histochemistry showed obvious inflammation, synovial hyperplasia and bone destruction in the joints of CA1-Tg that was not detected in PADI4-Tgs or wild-type mice. X-ray assays showed bone fusion in the paws and spines of CA1-Tg mice. CONCLUSION: Over-expression of CA1 may aggravate joint inflammation and tissue destruction in the transgenic mice.

Identification of differentially expressed proteins in ruminal epithelium in response to a concentrate-supplemented diet.

Ruminal epithelium adapts to dietary change with well-coordinated alterations in metabolism, proliferation, and permeability. To further understand the molecular events controlling diet effects, the aim of this study was to evaluate protein expression patterns of ruminal epithelium in response to various feeding regimes. Sheep were fed with a concentrate-supplemented diet for up to 6 wk. The control group received hay only. Proteome analysis with differential in gel electrophoresis technology revealed that, after 2 days, 60 proteins were significantly modulated in ruminal epithelium in a comparison between hay-fed and concentrate-fed sheep (P < 0.05). Forty proteins were upregulated and 20 proteins were downregulated in response to concentrate diet. After 6 wk of this diet, only 14 proteins were differentially expressed. Among these, 11 proteins were upregulated and 3 downregulated. To identify proteins that were modulated by dietary change, two-dimensional electrophoresis was coupled with liquid chromatography electrospray ionization mass spectrometry. The differential expression of selected proteins, such as esterase D, annexin 5, peroxiredoxin 6, carbonic anhydrase I, and actin-related protein 3, was verified by immunoblotting and/or mRNA analysis. The identified proteins were mainly associated with functions related to cellular stress, metabolism, and differentiation. These results suggest new candidate proteins that may contribute to a better understanding of the signaling pathways and mechanisms that mediate rumen epithelial adaptation to high-concentrate diet.

Use of 3D QSAR models for database screening: a feasibility study.

The applicability and scope of 3D QSAR methods (CoMFA, CoMSIA) to screen databases are examined. A protocol requiring minimal user intervention has been established to align training and test set molecules using FlexS. As model system isozymes of human carbonic anhydrase (hCA) are used, all results are exemplified studying affinity toward hCA II and selectivity between hCA I and II. The predictive power of the obtained models is assessed through prediction of 663 compounds not included in the training set and compared to 2D QSAR models derived from fragment (MACCS) or property (VSA) based descriptors. The predictive power is evaluated with respect to the following criteria: the numerical, concerning the absolute accuracy of prediction, and the categorical, characterizing the ability to assign a compound to the correct activity class.