Synthesis of polymeric hydrogels incorporating chlorhexidine gluconate as antibacterial wound dressings.

Antibacterial hydrogels based on poly(sodium 2-acrylamido-2-methylpropane sulfonate) and gelatin and incorporating chlorhexidine gluconate (CHG) as a drug were fabricated. The work focused on the effects of varying the CHG concentration. The hydrogel containing 0.02% w/v of CHG was chosen as the drug-loaded hydrogel for comparison with the hydrogel with no drug. From the drug release results, it was found that only 2-5% CHG was released, indicating that the CHG strongly interacted with the hydrogel network. To confirm the antibacterial efficiency of the hydrogels, the shake-flask method and scanning electron microscopy were employed. The antibacterial activity of the drug-loaded hydrogels showed a 7-log reduction for S. aureus gram-positive and a 5-6-log reduction for E. coli gram-negative bacteria. In addition, an MTT assay was performed to evaluate their potential cytotoxicity and showed a percentage cell viability after 24 h of more than 70% which classified them as being non-cytotoxic. In conclusion, the hydrogels containing CHG are considered as one of the interesting candidates for potential biomedical use as antibacterial wound dressings. Further in vivo investigations are planned.[Figure: see text].

Single nucleotide polymorphisms minisequencing in hypervariable regions for screening of Thais.

Mitochondrial DNA (mtDNA) analysis has displayed an important role and been considered as a powerful tool in various fields of forensic science applications. Nowadays, single nucleotide polymorphisms (SNPs) on mtDNA have become additional DNA markers when conventional STR typing practically fails. mtDNA sequencing of polymerase chain reaction (PCR) products from the hypervariable region I (HVRI) and II (HVRII) is the standard method of mtDNA analysis. However, mtDNA sequencing is rather expensive, time consuming and technically complex. This study aims to develop the SNPs minisequencing for screening of Thai populations. For this purpose, sixteen SNPs that possess high discriminating power in hypervariable regions were selected. The DNA samples were obtained from 100 buccal swab samples of Thai healthy individuals. All DNA samples were extracted and were subsequently amplified by single duplex PCR technique. The duplex PCR products were genotyped by SNPs minisequencing. Based on 16 SNPs, a total of 63 haplotypes were observed of which 46 haplotypes were unique. The haplotype diversity, discriminating power and random match probability were calculated to be 0.9830, 0.9732 and 0.0268, respectively. The SNPs at 150, 199, 489, 16129, 16189, 16223, and 16304 were highly polymorphic in the studied population. Our results suggested that the SNPs minisequencing can be an alternative method of SNPs genotyping. This method can be used for an exclusion of a large number of mismatch samples and as a presumptive test prior to do confirmatory mtDNA sequencing.

Nucleotide sequence analysis of the hypervariable region III of mitochondrial DNA in Thais.

This study analyzed the nucleotide sequences of the hypervariable region III (HVRIII) of mitochondrial DNA in Thai individuals. Buccal swab samples were randomly obtained from 100 healthy, unrelated, adult (18-60 years old), volunteer donors living in Thailand. Eighteen different haplotypes were found, of which 11 haplotypes were unique. The most frequent haplotypes observed were 522D-523D. Nucleotide transition from Thymine (T) to Cytosine (C) at position 489 (43%) was the most frequent substitution. Nucleotide transversions were also observed at position 433 (Adenine (A) to C, 1%) and position 499 (Guanine (G) to C, 1%). Fifty-three samples presented nucleotide insertion and deletion of C and A (CA) at position 514-523. Insertion of 1AC (3%) and 2AC (2%) were observed. Deletion of 1CA (53%) and 2CA (2%) at position 514-523 were revealed. The deletion of T at position 459 was observed. The haplotype diversity, random match probability, and discrimination power were calculated to be 0.7770, 0.2308, and 0.7692, respectively.

Carbonic anhydrase inhibitors: inhibition of the ß-class enzyme from the pathogenic yeast Candida glabrata with sulfonamides, sulfamates and sulfamides.

The fungal pathogen Candida glabrata encodes for a ß-carbonic anhydrase (CA, EC 4.2.1.1), CgNce103, recently discovered. Only anions have been investigated as CgNce103 inhibitors up until now. Here we report the first sulfonamides inhibition study of this enzyme. Simple sulfonamides showed weak or moderate CgNce103 inhibitory properties, whereas acetazolamide, and a series of 4-substituted ureido-benzene-sulfonamides, sulfamates and sulfamides showed effective CgNce103 inhibitory properties, with KIs in the range of 4.1-115 nM, being also ineffective as human CA II inhibitors. As there is significant resistance of C. glabrata clinical isolates to many classical antifungal agents, inhibition of the ß-CA from this organism may allow an interesting means of controlling the pathogen growth, eventually leading to antifungals with a novel mechanism of action.

Carbonic anhydrase regulation and CO(2) sensing in the fungal pathogen Candida glabrata involves a novel Rca1p ortholog.

Carbon dioxide (CO2) is a ubiquitous gas present at 0.0391% in atmospheric air and 5.5% in human blood. It forms part of numerous carboxylation and decarboxylation reactions carried out in every cell. Carbonic anhydrases (CA) enhance the hydration of CO2 to generate bicarbonate, which is subsequently used in cellular metabolism. In microorganisms, including the yeasts Candida albicans and Saccharomyces cerevisiae, inactivation of CA leads to a growth defect in air, which is complemented in an atmosphere enriched with CO2. In this study we characterize the CA from the fungal pathogen of humans Candida glabrata, CgNce103p, and report a comparable phenotype following its inactivation. Furthermore, we show that expression of the C. glabrata CA is strongly regulated by environmental CO2 at both the protein and transcript level. Similar to what we have previously reported for C. albicans and S. cerevisiae, C. glabrata CA regulation by CO2 is independent from the cAMP-PKA pathway and requires the novel bZIP transcription factor CgRca1p. We show that CgRca1p is an ortholog of the transcription factors Rca1p from C. albicans and Cst6p from S. cerevisiae and prove that CA induction in low CO2 involves the conserved DNA-binding motif TGACGTCA located on this C. glabrata promoter. However, in contrast to what is found in C. albicans CgRca1p expression itself is not affected by CO2. Although our results suggest a high level of similarity between the CO2 sensing pathways from C. glabrata, S. cerevisiae and C. albicans, they also point out significant intrinsic differences.

The bZIP transcription factor Rca1p is a central regulator of a novel CO2 sensing pathway in yeast.

Like many organisms the fungal pathogen Candida albicans senses changes in the environmental CO(2) concentration. This response involves two major proteins: adenylyl cyclase and carbonic anhydrase (CA). Here, we demonstrate that CA expression is tightly controlled by the availability of CO(2) and identify the bZIP transcription factor Rca1p as the first CO(2) regulator of CA expression in yeast. We show that Rca1p upregulates CA expression during contact with mammalian phagocytes and demonstrate that serine 124 is critical for Rca1p signaling, which occurs independently of adenylyl cyclase. ChIP-chip analysis and the identification of Rca1p orthologs in the model yeast Saccharomyces cerevisiae (Cst6p) point to the broad significance of this novel pathway in fungi. By using advanced microscopy we visualize for the first time the impact of CO(2) build-up on gene expression in entire fungal populations with an exceptional level of detail. Our results present the bZIP protein Rca1p as the first fungal regulator of carbonic anhydrase, and reveal the existence of an adenylyl cyclase independent CO(2) sensing pathway in yeast. Rca1p appears to regulate cellular metabolism in response to CO(2) availability in environments as diverse as the phagosome, yeast communities or liquid culture.

Carbonic anhydrase activators: Activation of the beta-carbonic anhydrase from the pathogenic yeast Candida glabrata with amines and amino acids.

The protein encoded by the NCE103 gene of Candida glabrata, a beta-carbonic anhydrase (CA, EC 4.2.1.1) designated as CgCA, was investigated for its activation with amines and amino acids. CgCA was weakly activated by amino acids such as l-/d-His, l-Phe, l-DOPA, and l-Trp and by histamine or dopamine (K(A)s of 21.2-37microM) but more effectively activated by d-Phe, d-DOPA, d-Trp as well as serotonin, pyridyl-alkylamines, aminoethyl-piperazine/morpholine (K(A)s of 10.1-16.7microM). The best activators were l-/d-Tyr, with activation constants of 7.1-9.5microM. This study may bring a better understanding of the catalytic/activation mechanisms of beta-CAs from pathogenic fungi.

Carbonic anhydrase inhibitors. Inhibition of the beta-class enzyme from the pathogenic yeast Candida glabrata with anions.

A beta-carbonic anhydrase (CA, EC 4.2.1.1), the protein encoded by the NCE103 gene of Candida glabrata which also present in Candida albicans and Saccharomycescerevisiae, was cloned, purified, characterized kinetically and investigated for its inhibition by a series simple, inorganic anions such as halogenides, pseudohalogenides, bicarbonate, carbonate, nitrate, nitrite, hydrogen sulfide, bisulfite, perchlorate, sulfate and some isosteric species. The enzyme showed significant CO(2) hydrase activity, with a k(cat) of 3.8 x 10(5)s(-1) and k(cat)/K(M) of 4.8 x 10(7)M(-1)s(-1). The Cà glabrata CA (CgCA) was moderately inhibited by metal poisons (cyanide, azide, cyanate, thiocyanate, K(I)s of 0.60-1.12 mM) but strongly inhibited by bicarbonate, nitrate, nitrite and phenylarsonic acid (K(I)s of 86-98 microM). The other anions investigated showed inhibition constants in the low millimolar range, with the exception of bromide and iodide (K(I)s of 27-42 mM).