Formic Acid and Acetic Acid Induce a Programmed Cell Death in Pathogenic Candida Species

Cutaneous fungal infections are common and widespread. Antifungal agents used for the treatment of these infections often have undesirable side effects. Furthermore, increased resistance of the microorganisms to the antifungal drugs becomes the growing problem. Accordingly, the search for natural antifungal compounds continues to receive attention. Apoptosis is highly regulated programmed cell death. During yeast cell apoptosis, amino acids and peptides are released and can stimulate regeneration of human epithelium cells. Thus, detection of chemical compounds inducing apoptosis in yeast and nontoxic for humans is of great medical relevance. The aim of this study was to detect chemical compound inducing apoptosis in pathogenic Candida species with the lowest toxicity to the mammalian cells. Five chemical compounds—acetic acid, sodium bicarbonate, potassium carbonate, lithium acetate, and formic acid—were tested for evaluation of antifungal activity on C. albicans, C. guilliermondii, and C. lusitaniae. The results showed that acetic acid and formic acid at the lowest concentrations induced yeast cells death. Apoptosis analysis revealed that cells death was accompanied by activation of caspase. Minimal inhibitory concentrations of potassium carbonate and sodium bicarbonate induced Candida cells necrosis. Toxicity test with mammalian cell cultures showed that formic acid has the lowest effect on the growth of Jurkat and NIH 3T3 cells. In conclusion, our results show that a low concentration of formic acid induces apoptosis-like programmed cell death in the Candida yeast and has a minimal effect on the survivability of mammalian cells, suggesting potential applications in the treatment of these infections.     

Cloning of genes encoding heterotetrameric sarcosine oxidase from Arthrobacter sp.

Summary An isolate of Arthrobacter sp. produced the sarcosine oxidase which was purified to homogeneity. SDS-PAGE indicated that the enzyme was composed of four dissimilar subunits with molecular weights of 106, 43, 24, and 15 kDa. The genes encoding the four subunits of sarcosine oxidase were isolated and expressed in E. coli.     

Fatty acid profiles of monofloral clover beebread and pollen and proteomics of red clover (Trifolium pratense) pollen

Fatty acids were identified in monofloral beebread (BB) and bee pollen (BP) loads collected from Trifolium pratense L. A gas chromatography method was used to identify and quantify fatty acids: Thirty-five fatty acids were identified in BB and 42 in BP. A high amount of the healthy n-3 fatty acids was found. The ratio of polyunsaturated fatty acids n-3 to n-6 reached a value of 8.42 and 3.35 in the latter products. The proteomic analysis also was performed on the manually collected T. pratense pollen, and the most abundant protein groups were subjected to mass spectrometry analysis. Proteins identified in T. pratense pollen are involved in the main cellular functions (cell membrane formation, organelles traffic, and mainly metabolic processes). Because of the composition of fatty acids in BB and BP and a variety of proteins present in pollen, these products are considered to be favorable for human nutrition and health.     

Epigenetic regulation of amniotic fluid mesenchymal stem cell differentiation to the mesodermal lineages at normal and fetus-diseased gestation

Human mesenchymal stem cells isolated from amniotic fluid (AF-MSCs) demonstrate the potency for self-renewal and multidifferentiation, and can, therefore, be a potential alternative source of stem cells adapted for therapeutic purposes. The object of this study is to evaluate the efficacy of MSCs from AF when the pregnancy is normal or when the fetus is affected during pregnancy to differentiate into mesodermal lineage tissues and to elucidate epigenetic states responsible for terminal adipogenic and osteogenic differentiation. The morphology of AF-MSCs from two cell sources and the expression of the cell surface-specific (CD44, CD90, and CD105) markers and pluripotency (Oct4, Nanog, Sox2, and Rex1) genes were quite similar and underwent mesodermal lineage differentiation because this is shown by the typical cell morphology and of genes’ expression specific for adipogenic (peroxisome proliferator-activated receptor-ɣ, adiponectin) and osteoblastic (alkaline phosphatase, osteopontin, and osteocalcin) differentiation. Terminal lineage-specific differentiation was related to differential expression of miR-17, miR-21, miR-34a, and miR-146a, decreased levels of acetylated H4 and H3K9, trimethylated H3K4 and H3K9, and the retention of H3K27me3 along with a reduction in the levels of HDAC1, DNMT1, and PRC1/2 proteins (BMI1/SUZ12). No significant distinction could be identified in the levels of expression of all epigenetic or pluripotency markers between undifferentiated MSCs isolated from AF of normal gestation and pregnancy where the fetus was damaged and between those differentiated toward adipocytes or osteoblasts. The expressional changes of those marks and microRNAs that occurred during terminal differentiation to mesodermal tissues indicate subtle epigenetic regulation in AF-MSCs when the condition of the fetus is healthy normal or diseased. More detailed studies of epigenetic mechanisms may offer a better understanding of AF-MSCs differentiation in fetus-diseased conditions and their usage in an autologous therapeutic application and prenatal disease research.     

Pyrrolidinone-bearing methylated and halogenated benzenesulfonamides as inhibitors of carbonic anhydrases

Two series of benzenesulfonamides bearing methyl groups at ortho/ortho or meta/ortho positions and a pyrrolidinone moiety at para position were synthesized and tested as inhibitors of the twelve catalytically active human carbonic anhydrase (CA) isoforms. Observed binding affinities were determined by fluorescent thermal shift assay and intrinsic binding affinities representing the binding of benzenesulfonamide anion to the Zn(II)-bound water form of CA were calculated. Introduction of dimethyl groups into benzenesulfonamide ring decreased the binding affinity to almost all CA isoforms, but gained in selectivity towards one CA isoform. A chloro group at the meta position of 2,6-dimethylbenzenesulfonamide derivatives did not influence the binding to CA I, but it increased the affinity to all other CAs, especially, CA VII and CA XIII (up to 500 fold). The compounds may be used for further development of CA inhibitors with higher selectivity to particular CA isoforms.     

Clarin-1, Encoded by the Usher Syndrome III Causative Gene, Forms a Membranous Microdomain: POSSIBLE ROLE OF CLARIN-1 IN ORGANIZING THE ACTIN CYTOSKELETON*

Clarin-1 is the protein product encoded by the gene mutated in Usher syndrome III. Although the molecular function of clarin-1 is unknown, its primary structure predicts four transmembrane domains similar to a large family of membrane proteins that include tetraspanins. Here we investigated the role of clarin-1 by using heterologous expression and in vivo model systems. When expressed in HEK293 cells, clarin-1 localized to the plasma membrane and concentrated in low density compartments distinct from lipid rafts. Clarin-1 reorganized actin filament structures and induced lamellipodia. This actin-reorganizing function was absent in the modified protein encoded by the most prevalent North American Usher syndrome III mutation, the N48K form of clarin-1 deficient in N-linked glycosylation. Proteomics analyses revealed a number of clarin-1-interacting proteins involved in cell-cell adhesion, focal adhesions, cell migration, tight junctions, and regulation of the actin cytoskeleton. Consistent with the hypothesized role of clarin-1 in actin organization, F-actin-enriched stereocilia of auditory hair cells evidenced structural disorganization in Clrn1^−/− mice. These observations suggest a possible role for clarin-1 in the regulation and homeostasis of actin filaments, and link clarin-1 to the interactive network of Usher syndrome gene products.Usher syndrome is the most common cause of human inherited deafness and blindness, accounting for ∼50% of all cases (1). There are three clinical types of Usher syndrome, types I, II, and III (1–3). Usher type I is characterized by profound congenital deafness and vestibular dysfunction, and Usher type II is characterized by moderate to severe deafness. Usher type III is distinguished from types I and II by progressive (non-congenital) deafness together with variable impairment of vestibular function. All Usher types lead to progressive retinal degeneration with a retinitis pigmentosa-like appearance. Five causative genes have been identified for Usher syndrome type I, and three genes for type II (3). The protein products of Usher type I and II genes are functionally heterogeneous, including an unconventional myosin, scaffold proteins, G-protein-coupled receptor, and cadherins. Adding to this heterogeneity, the Usher syndrome type III gene encodes a novel transmembrane protein named clarin-1 (CLRN1)³ (4–6) with an unknown function. The heterogeneity of genes involved in Usher syndrome makes it extremely challenging to elucidate shared and distinctive disease mechanisms.CLRN1 belongs to a superfamily of four-transmembrane proteins that includes the tetraspanin and claudin families. CLRN1 and its paralogues, CLRN2 and CLRN3, form the Clarin family, which is conserved throughout vertebrate species and shows limited sequence homology to the tetraspanins (4). Tetraspanins are considered to be structural proteins that interact laterally with other membrane proteins such as ion channels, integrins, and other tetraspanins (7, 8) to form tetraspanin-enriched microdomains. Tetraspanin-enriched microdomains embody other proteins to allow localized transmission of signals, cell-cell adhesion/fusion, cell-matrix interactions, and/or formation of diffusion barriers against small molecules. Similar to tetraspanins, CLRN1 retains only limited hydrophilic regions exposed to cytoplasmic or extracellular aqueous phases (Fig. 1A) and, apparently, lacks any functional domains. Although CLRN1 is structurally related and similar to tetraspanins, it is currently unknown whether CLRN1 can form specific microdomains. The question also remains as to what one or more functions CLRN1 microdomains serve if indeed they do exist.Open in a separate windowFIGURE 1.CLRN1 is a plasma membrane protein localized at F-actin-enriched protrusions. A, the topology and transmembrane domains shown were predicted with the HMMTOP transmembrane topology prediction server (55). The possible N-linked glycosylation site is indicated. Also shown (red circle) is the previously predicted motif near the CLRN1 C-terminal tail that may serve as a PDZ-binding site (4). B, immunolocalization of Human WT CLRN1. C, immunolocalization of Na/K ATPase in HEK293 cells stably expressing CLRN1. D, merged image of B and C indicates that CLRN1 and Na/K ATPase co-localize. Images B–D are single optical sections of HEK293 cells. E, cell surface biotinylation was performed to separate cell surface proteins (avidin-bound) (AB) from intracellular proteins (flow-through) (FT). Immunoblots of both fractions reveal that most of the CLRN1 protein localized to the plasma membrane. HEK293 cells alone and HEK293 cells expressing CLRN1 were preincubated for 30 min with Sulfo-NHS-SS-Biotin to label cell surface proteins. After cells were harvested, biotin-labeled CLRN1 protein levels were measured by immunoblotting. F, localization of human WT CLRN1 in HEK293 cells stably expressing CLRN1. G, F-actin in HEK293 cells stably expressing CLRN1. F-actins were labeled with phalloidin-Alexa 488. H, merged image of F and G. CLRN1 localized at both microvilli (arrows) and lamellipodia (arrowheads). I–K, CLRN1 localization studied by immunofluorescence confocal microscopy after disruption of F-actin by cytochalasin D treatment. I, CLRN1 localized diffusely on the plasma membrane. J, F-actin localization is shown. K, merged image of I and J. After disruption of F-actin, CLRN1 and F-actin no longer co-localize. Images F–K were generated from multiple optical sections by a maximum intensity projection. Scale bars, 50 μm.CLRN1 is expressed in sensory hair cells (4) where it may interact with other co-existing Usher gene products or cellular machinery essential for the maintenance of these cells. Increasing evidence suggests that products of Usher type I and II genes form large networks of interacting proteins, and that F-actin plays a major role in organizing these networks (reviewed in Refs. 2, 9). The core of these networks is the Usher type IC gene product, Harmonin, which interacts directly with F-actin in vitro and stabilizes F-actin when it is expressed heterologously in HeLa cells (10). Harmonins retain multiple PDZ domains dedicated to interacting with products of Usher type I and type II genes (reviewed in Refs. 2, 9) and also serve as PDZ domain-based scaffolds to anchor Usher proteins to F-actin. A link between Usher gene products and actin-based organelles also has been established in vivo. In Usher syndrome I and II mouse models, the actin-enriched stereocilia are morphologically and functionally defective (11–14). Because the causative gene for Usher type III was identified more recently than those of Usher types I and II, little is known about the pathogenesis of Usher syndrome III. Epistatic interactions between Usher syndrome type IB and Usher syndrome III may suggest linkage among CLRN1, Myosin VIIa, and F-actin (15). Clinically, patients with the N48K CLRN1 mutation have a rod and cone degenerative phenotype similar to Usher type IIA patients (16), suggesting a common pathological pathway for Usher types IIA and III. Despite the genetic and phenotypic characterization in humans, the molecular function of CLRN1 remains elusive, as well as its relationship and interaction with other Usher gene products. Therefore, identifying possible interactive partners of CLRN1 should improve understanding of the function of CLRN1 and the common pathological pathways of progressive hearing and vision loss in the Usher syndromes.Here we investigated whether CLRN1 can form microdomains similar to the tetraspanin-enriched microdomain, and if so, what the function of such microdomains might be. Our studies indicate that CLRN1 forms membranous cholesterol-rich compartments on plasma membranes and interacts with and regulates the machinery involved in actin filament organization. To understand the pathogenesis of Usher syndrome, we asked whether and how the Usher syndrome III causative mutation, N48K, results in dysfunction of the clarin-1-enriched microdomains involved in organizing actin. To determine whether Clrn1 is involved in the regulation of actin cytoskeleton in vivo, we studied the structure of F-actin-enriched stereocilia bundles in Clrn1^−/− mouse. Because actin provides important scaffolds in Usher interactome, the observations described herein provide a novel molecular link between CLRN1 and the identified gene products of Usher types I and II.     

Indapamide-like benzenesulfonamides as inhibitors of carbonic anhydrases I, II, VII, and XIII

A series of novel 2-chloro-5-[(1-benzimidazolyl- and 2-benzimidazolylsulfanyl)acetyl]benzene-sulfonamides were designed and synthesized. Their binding to recombinant human carbonic anhydrase (hCA) isozymes I, II, VII, and XIII was determined by isothermal titration calorimetry and thermal shift assay. The designed S-alkylated benzimidazole derivatives exhibited stronger binding than the indapamide-like N-alkylated benzimidazoles, with the K(d) reaching about 50-100 nM with drug-targeted hCAs VII and XIII. The cocrystal structures of selected compounds with hCA II were determined by X-ray crystallography, and structural features of the binding event were revealed.     

Purification and characterisation of PQQ-dependent glucose dehydrogenase from Erwinia sp. 34-1

A pyrroloquinoline quinone-dependent glucose dehydrogenase from an isolate of Erwinia sp. has been purified to homogeneity and characterised. SDS-PAGE showed a single band of 88.4 kDa. The enzyme activity was optimal at 47°C and pH 7.5–8.5. The Michaelis constants for d-glucose and PMS were 3.2 mM and 132 M, respectively (50 mM glycine–NaOH, at pH 8.0).