Purification and Characterization of Two Novel Laccases from Peniophora lycii

Although, currently, more than 100 laccases have been purified from basidiomycete fungi, the majority of these laccases were obtained from fungi of the Polyporales order, and only scarce data are available about the laccases from other fungi. In this article, laccase production by the white-rot basidiomycete fungus Peniophora lycii, belonging to the Russulales order, was investigated. It was shown that, under copper induction, this fungus secreted three different laccase isozymes. Two laccase isozymes—Lac5 and LacA—were purified and their corresponding nucleotide sequences were determined. Both purified laccases were relatively thermostable with periods of half-life at 70 °C of 10 and 8 min for Lac5 and LacA, respectively. The laccases demonstrated the highest activity toward ABTS (97 U·mg−1 for Lac5 and 121 U·mg−1 for LacA at pH 4.5); Lac5 demonstrated the lowest activity toward 2,6-DMP (2.5 U·mg−1 at pH 4.5), while LacA demonstrated this towards gallic acid (1.4 U·mg−1 at pH 4.5). Both Lac5 and LacA were able to efficiently decolorize such dyes as RBBR and Bromcresol Green. Additionally, phylogenetic relationships among laccases of Peniophora spp. were reconstructed, and groups of orthologous genes were determined. Based on these groups, all currently available data about laccases of Peniophora spp. were systematized.

Different detection capabilities by mycological media for Candida isolates from mono- or dual-species cultures

ABSTRACTObjectivesThe aim of this study was to compare the Candida bromcresol green (BCG) medium with the chromogenic (CHROM) Brilliance Candida agar and Sabouraud dextrose agar (SDA) media in regard to their capability of detecting Candida isolates from mono-or dual-species cultures.MethodsWe prepared Candida isolates’ suspensions to obtain mono-species (n = 18) or dual-species (n = 153) culture plates per each medium, and three readers independently observed 513 plates at 24-h, 48-h and 72-h incubation time. We scored reading results as correct, over or under detection compared to the expected species number(s).ResultsBCG showed significantly higher correct-detection and lower under-detection rates for all Candida species when observed by at least one reader. At 24-h reading, 12 mono-species cultures had correct (or over) detections in all media, whereas 106, 60 and 78 dual-species cultures had correct (or over) detections in BCG, CHROM or SDA, respectively.ConclusionBCG provides the basis for an accurate laboratory diagnosis of Candida infections.

METODE BROMCRESOL GREEN (BCG) DAN BROMCRESOL PURPLE (BCP) PADA SIROSIS HATI YANG MENDAPAT INFUS ALBUMIN

Albumin infusion is one of the therapeutic options in hypoalbuminemia patients. Serum albumin can be used to determine the albumininfusion therapy, prognosis and monitoring of liver cirrhosis. The time difference in measurement of serum albumin by bromcresol green(BCG) and bromcresol purple (BCP) methods can give different results. Serum albumin examination was done in 20 sera taken fromcirrhosis patients. Serum albumin was then evaluated before treatment, one (1) hour and 24 hours after the patient received an infusionof albumin and examined by bromcresol green (BCG) and bromcresol purple (BCP) methods. The serum albumin level by BCG methodincreased with a coefficient of 0.12 (p-value=0.022) with BCG method before (1.94±0.32 mg/dL) and after one (1) hour (2.06±0.32mg/dL) receiving intravenous albumin. The coefficient of albumin levels before and after 24 hours (2.12±0.38 mg/dL) was 0.18 (pvalue=0.07), whereas the increased levels of serum albumin after one (1) hour and after 24 hours of intravenous albumin, were notsignificant (p-value=0.467). The BCP method showed that serum albumin before, after one (1) hour and after 24 hours receivingintravenous albumin were 1.68±0.36 mg/dL, 1.87±0.36 mg/dL and 2.12±0.63 mg/dL respectively. The albumin levels showed asignificant increase before and after one (1) hour infusion of albumin (p-value=0.00), both levels shown before and after 24 hours(p-value=0.001), as well as one (1) hour and 24 hours after receiving intravenous albumin (p-value=0.04). The results of this studyshowed that increased serum albumin by BCG method could be detected after 1 (one) hour, whereas by BCP method could only be detectedafter 24 hours receiving intravenous albumin.

ALBUMIN SERUM DALAM SIROSIS HATI

Serum albumin measurement can be used for assessing prognosis, determining initial therapy and treatment efficacy for liver cirrhosis patients. The discrepancy result of albumin measurement using different methods can make bias for the albumin concentration. The measurement of albumin was carried out in 20 sera of liver cirrhosis patients using bromcresol green (BCG), BCG conversion as well as bromcresol purple (BCP) methods including protein electrophoresis. The results showed that BCP method has a lower albumin concentration than the other kind (1.68±0.35), BCG (1.94±0.31), BCG conversion (2.09±0.45) as well as protein electrophoresis (2.14±0.35). BCP method has a good correlation with protein electrophoresis as well as BCG conversion (r=0.935 and r=0.90 respectively). It can be concluded in this study, that albumin measurement with BCP method has a good correlation with protein electrophoresis. It is important to know that the measurement results of BCP method is lower but more accurate in the albumin concentration, because there is no cross reaction with globulin. That means, there is no overestimation of albumin concentration in hypoalbuminemia condition of the patients.

Optimization, Validation and Application of UV-Vis Spectrophotometric-Colorimetric Methods for Determination of Trimethoprim in Different Medicinal Products

Two simple, sensitive, selective, precise, and accurate methods for determination of trimethoprim in different sulfonamide formulations intended for use in human and veterinary medicine were optimized and validated. The methods are based on the trimethoprim reaction with bromcresol green (BCG) and 2,4-dinitro-1-fluorobenzene (DNFB). As extraction solvents we used 10 % N,N-dimethylacetamide in methanol and acetone for both methods, respectively. The colored products are quantified applying visible spectrophotometry at their corresponding absorption maxima. The methods were validated for linearity, sensitivity, accuracy, and precision. We tested the method applicability on four different medicinal products in tablet and powder forms containing sulfametrole and sulfamethoxazole in combination with trimethoprim. The results revealed that both methods are equally accurate with recoveries within the range 95-105 %. The obtained between-day precision for both methods, when applied on four different medicinal products, was within in the range 1.08-3.20 %. By applying the F-statistical test (P<0.05), it was concluded that for three medicinal products tested both methods are applicable with statistically insignificant difference in precision. The optimized and validated BCG and DNFB methods could find application in routine quality control of trimethoprim in various formulation forms, at different concentration levels, and in combination with different sulfonamides.