Sensitivity and Specificity Determinations with Isoelectric Focusing Fractions of <i>Blastomyces dermatitidis</i>for Antibody Detection in Serum Specimens from Infected Dogs

Blastomycosis and histoplasmosis manifest as lung and systemic fungal infections in mammals caused by Histoplasma capsulatum, and Blastomyces dermatitidis. These infections exhibit cross reactivity of antibodies which makes a correct diagnosis potentially elusive. The purpose of this study was to gain an understanding of which isoelectric focusing fractions (RotoforTM) of B. dermatitidis were reactive or cross reactive with serum specimens from dogs infected with B. dermatitidis, H. capsulatum, and Cryptococcus neoformans. Three serum specimens from dogs that were infected with B. dermatitidis, two dogs infected with H. capsulatum, and one dog infected with C. neoformans were assayed against the 20 B. dermatitidis RotoforTM fractions. Reactivity was determined using the indirect enzyme linked immunoassay (ELISA). Reactivity with B. dermatitidis was found predominantly in the protein fractions 1 - 6, and cross reactivity with H. capsulatum, and C. neoformans sera was found within the B. dermatitidis protein fractions 15 - 19.

The Isoelectric Point and the Points of Zero Charge of Fe-Al-Mg Hydrotalcite-like Compounds

The relation of the isoelectric point (IEP) and the point of zero net charge (PZNC) of the hydrotalcite-like compounds was discussed. It was found that the IEP does not equal to the PZNC and the IEP is higher than the PZNC. The structural positive charges existing in the HTlc, which cause the difference between the IEP and the PZNC. The effects of the structural positive charges of the HTlc on its IEP and PZNC are the same as the specific adsorption of metal cations.

Direct MALDI-MS Analysis of Proteins Isolated by Liquid-phase Isoelectric Focusing Electrophoresis

A liquid-phase isoelectric focusing electrophoresis system(Rotofor) was used as the prefractionation tool for the sample preparation in the MALDI-MS analysis of a protein mixture. Each fraction collected was then directly subjected to MALDI-TOF-MS analysis. By this approach, we are able to resolve two types of hemoglobins, A and C, which cannot be successfully separated by means of the traditional SDS-PAGE method.

Study on Isoelectric Point of Magnesium Aluminum Hydroxide

Yoon's equation which predicted point of zero charge of complex oxide should predict the zero point of net charge (ZPNC) of mixture of variable and permanent charge rather than the point of zero charge (PZC) of the variable charge component. The calculated values with modified Yoon's equation agreed with the experimental values.

Bio-Control of <i>Alternaria alternata</i>during Banana Storage by Purified AFP Using Isoelectric Focusing Technique

Interestingly, antifungal protein AFP was purified from Aspergillus giganteus supernatants with modified isoelectric focusing procedure after adaptation of the secretion conditions. Subsequently, the antifungal activity as well as the mode of action against Alternaria alternata was tested in vitro. Moreover, different concentrations of AFP were applied to banana fruits for 15 days at 20°C in vivo. Obtained results illustrated that A. giganteus was able to secrete about 39.78 ± 2.39 mg AFP·l-1 Olson medium. The employed ammonium sulfate (AS 75%) precipitation procedure followed by dialysis steps yielded about 16 - 22 mg AFP·l-1 culture supernatant with general mean of 18.67 ± 1.98 mg·l-1. The lost amount of AFP during purification using AS and 3KDa cut-off dialysis membrane is about 50% thus, purification procedure must be further improved. Indeed, concluded results from MIC and hyphal extension inhibition test noticed that AFP was efficiently affected by either growth or hyphae form of A. alternata in vitro. The MIC of AFP against A. alternata was 2μg·ml-1. However,short, thick and highly septated hyphae with damaged constricted apical regions extruding from condensed mycelium aggregates in treated hyphae compared to the untreated culture was remarkably shown. The mode of action of in vitro experiment manifested that AFP was effective to act the fungal cell and permeabilize the cell membrane of A. alternata. Furthermore, the in vivo experiment showed that AFP could reduce post-harvest decay on banana caused by A. alternata. AFP at concentration of 15 and 25 μg&#183;ml-1 exhibit Alternaria decayed reduction by 45.45% and 77.27%, respectively. While no Alternaria decayed area was observed when 50 μg·ml-1 was applied during the storage time. Quantification of DNA by species-specific PCR could exude a positive correlation between the DNA amount and decayed area. In conclusion, AFP can be efficiently used as a bio-preservative agent during storage and handling of banana fruits, and considered as an excellent biological alternative to combat secondary growth of filamentous fungi.

Dual-functional poly(2-methyl-2-oxazoline)/poly(2-(dimethylamine)ethyl methacrylate)mixed brushes with switchable protein adsorption and antibacterial properties

Herein,binary mixed brushes consisting of poly(2-methyl-2-oxazoline)(PMOXA)and poly(2-(dimethylamine)ethyl methacrylate)(PDMAEMA)with different chain lengths were fabricated by successive grafting of NH_(2)-terminated PMOXA and SH-terminated PDMAEMA onto polydopamine-anchored substrates.The mixed-brush coating was characterized by variable-angle spectroscopic ellipsometry,X-ray photoelectron spectroscopy,Fourier transform infrared spectroscopy,zeta potential measurements,water contact angle,and atomic force microscopy.The mixed brushes showed tunable surface charge,wettability,and surface roughness,depending on the degree of PDMAEMA swelling under varying pH and ionic strength(Ⅰ).Then the adsorption behaviors of pepsin,bovine serum albumin(BSA),γ-globulin,and lysozyme,four very different proteins with regard to isoelectric point,on the mixed brushes coating were studied by using fluorescence microscopy and surface plasmon resonance.When the chain length of PDMAEMA was about twice as long as PMOXA,the mixed brushes not only had high adsorption capacity for pepsin,BSA,and y-globulin but also had a desorption efficiency of 86.9%,87.1%,and 93.5%,respectively.It is explained that electrostatic attraction between the protonated PDMAEMA and positively charged acidic proteins(pepsin and BSA,whose isoelectric points were below the pK_(a) of PDMAEMA)would drive the intensive adsorption(at pH 3,I=10^(-3)mol·L^(-1)for pepsin,and pH 5,I=10^(-5)mol·L^(-1)for BSA),while desorption was dominated by the hydrophilic PMOXA when PDMAEMA was shrinking(at pH 7,I=10^(-1)mol·L^(-1)for pepsin,and pH 9,I=10^(-1)mol·L^(-1)for BSA).Furthermore,the isoelectric precipitation led to the adsorption of neutral protein(γ-globulin,whose isoelectric point was near the pK_a of PDMAEMA)at pH 7,I=10^(-5)mol·L^(-1),while electrostatic repulsion and antifouling PMOXA triggered the desorption of y-globulin at pH 3,I-10^(-1)mol·L^(-1).However,alkaline protein(lysozyme,whose isoelectric point was higher than the pK_(a) of PDMAEMA)exhibited slight adsorption on PMOXA/PDMAEMA mixed brushes under test conditions,regardless of whether PMOXA or PDMAEMA occupied the outermost layer.The antibacterial property of the mixed brushes against Escherichia coli was investigated.PMOXA/PDMAEMA mixed brushes showed significant bactericidal activity at pH 3,I=10^(-3)mol·L^(-1),while the rinse of pH 9,I=10^(-1)mol·L^(-1)solution could remove most of the residual bacteria.This work not only enables controlled adsorption of proteins with different isoelectric points but also ensures that the surface of the coating is minimized from bacterial contamination.

33 ku protein associated several polypeptides with nearly the same molecular weight but not the same isoelectric point

The 33 ku protein, prepared from NaCI-treated PSII particles, has shown an single band by SDS-PAGE. After being dialyzed against the low-osmotic medium at 4癈, it has been found that the 33 ku protein degraded into several small fragments. This result suggests that the preparations of 33 ku protein probably contain some latent proteinases. it has also been found, by the 2-D electrophoresis and IEF, that the preparations of 33 ku protein not dialyzed against the low-osmotic medium contain several polypeptides with nearly the same molecular weight but not the same isoelectric point as the 33 ku protein.

Effect of cationic monomer structure on the aggregation behavior of amphoteric acrylic polymer around isoelectric point

Amphoteric polymer can be used as retanning agent in leather manufacture. It is particularly useful in chrome-free tanning systems since it can regulate the charge properties of chrome-free leather and enhance the fixation of anionic post-tanning chemicals in leather. However, the aggregation and precipitation of amphoteric polymer retanning agents around the isoelectric point (pI) hinder their wide application. Herein, we synthesized five amphoteric acrylic polymers (AAPs) by free radical copolymerization with acrylic acid and five different cationic acrylic monomers. The effect of cationic monomer structure on the aggregation behavior of AAPs was investigated. The aggregation of AAPs in aqueous solution showed pH and concentration dependence. Light scattering analysis showed that Poly (AA-co-MAPTAC) and Poly (AA-co-DMAPMA) were in the shape of coiled linear flexible chains with small particle size (Rg 7.6 nm and 14.8 nm, respectively) near the pI. However, Poly (AA-co-DAC), Poly (AA-co-DMC) and Poly (AA-co- DMAEMA) were in the shape of hollow spheres and exhibited serious aggregation. Quantum chemical calculations suggested that the amide groups in the cationic monomers MAPTAC and DMAPMA enhanced the nucleophilicity of AAPs. Thus the corresponding AAPs could carry a large number of cationic charges to slow their aggregation when the pH just climbed over the pI. The results are expected to provide theoretical reference for the synthesis and widespread application of AAPs.

近交系小鼠Hb基因位点等电聚焦分析

等电聚焦(isoelectric focusing,简称IFF)也称等电点分离。自1960年代初,瑞典学者首次合成两性载体(商品名称有Ampholine和Pharmalyte等)并成功地用于蛋白质分离以来,这项技术得以迅速发展和应用。由于其分辨力高,可用于混合物的分离或鉴定,国外已应用此项技术对近交系动物进行生化标志基因监测。我们采用本实验室建立的聚丙烯酰胺凝胶等电聚焦技术对近交系小鼠SMMC/B的Hb基因位点进行遗传关系分析,并与常规聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis,PAGE)作分辨力比较。

A Novel and Optimized Method for Electro-focusing and Moving Neutralization Reaction Boundary Formed by HCl and NaOH

A novel method is developed for electro-focusing and moving neutralization reaction boundary (MNRB) created with HCl and NaOH. The optimized conditions are screened out. By using this method, the experiments are performed on MNRB formed with HCl and NaOH in agarose gel. The experiments are quantitatively in coincidence with the predictions with the theory of moving chemical reaction boundary (MCRB).

Studies on the Thiol Components of Isolated Nuclei

The thiol components of the nonhistone proteins prepared from isolated nuclei from rat liver, regenerating liver and hepatoma 223 cells have been investigated after reaction with radio labelled N-ethylmaleimide and 5-5’-dithiobis-(2-nitrobenzoic acid) (DTNB). The labelled adducts formed were examined by isoelectric focusing analysis in polyacrylamide gel and the distribution of the radiolabels within sliced portions of the gels determined. In the case of the 14C labelled NEM adduct the label was found to be spread amongst numerous protein components within the gel however, in the case of the 35S labelled DTNB adducts, only a small proportion of the label was found in the protein material which was retained in the acidic isoelectric point (pI) region of the gel. The bulk of the 35S labelled adduct (56% - 60%) was found to have migrated into the anode solution (10 mM phosphoric acid). This could be adsorbed onto a hydrophobic resin (XAD2) resin and eluted with methanol. Gel filtration chromatographic analysis of this adduct material on BioGel P2, (exclusion limit 1500 daltons) showed low molecular weight components to be present. Slightly different patterns were obtained for these nuclei, each containing several 35S components with molecular weights greater than the Ellman reagent itself. These 35S labelled thiol components did not contain any protein, peptide or amino acid components indicating strongly that a novel species of thiols could be present in these nuclei bound within the non-histone protein matrices.

On the Distribution of Acidic Proline-rich Proteins Phenotypes in Chinese Han Population in Liaoning Area

The distribution of salivary acidic proline-rich proteins pheno-types was investigated by using the ultra-thin PAGIEF technique in 258 ChineseHan populations in Liaoning area. The gene frequencies were as follows: Pr^10.8101, Pr^20.1899; Db^+0.0416, Db^-0.9584; Pa^+0.1717, Pa^-0.8283; PIF^+0.6647, PIF^-0.3353. The observed numbers of the phenotypes are in good a-greement with the expected numbers under the Hardy-Weinberg equilibrium.The gene frequencies among the Chinese and other populations are compared.

Studies on Y- Potential of Positive Sol of Magnesium Aluminum Hydroxide

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Influence of TAC on Dispersion of Aqueous Nano Alumina Suspension

The stability of aqueous nano alumina powder suspensions with and without dispersant tri-ammonium citrate (TAC) was investigated by measuring zeta potential at different pH values. The isoelectric point (IEP) of alumina powder shifts towards more acidic pH range by adding TAC dispersant. The results illustrate that adsorption of TAC on alumina powder surface existed in the aqueous suspension, which leads to a high stability of the suspension.

Responses of bioenergy sorghum cell wall metabolism to agronomic practices

Maximum lignocellulose yield of biomass sorghum [Sorghum bicolor L. (Moench.)] is hampered by complex biological phenomena related to rotation, nitrogen (N) fertilization, soil tillage, and excessive biomass removal. The biochemical basis of the effects of agronomic practices on sorghum production was studied by the enzymology of the active peroxidase (EC 1.11.1.7) isoenzymes that synthesize lignin. All studied practices altered the peroxidase pI values. Control sorghum without rotation and without N fertilization had the most inhibited peroxidase with very low maximum velocity (Vmax) value (3.10 mmol·min﹣1), and very low lignin (857 kg·ha﹣1) yield, which could decrease soil organic carbon possibly leading to adverse changes in soil chemistry. Corn-sorghum rotations with and without N fertilization increased the Vmax values of peroxidase and lignin and cellulose yields. Rotated sorghum subjected to 50% residue return (the percentage of crop residue was returned to the plot immediately after grinding at harvest) and 280 kg·ha﹣1 N fertilization possessed very active peroxidase (Vmax value 66.4 mmole·min﹣1) and the highest lignin (1387 kg·ha﹣1) yield. The 25% residue return rate without N fertilization induced high lignin (1125 kg·ha﹣1) and cellulose (11,961 kg·ha﹣1) but the 25% residue return rate with 280 kg·ha﹣1 N fertilization induced lower lignin (1046 kg·ha﹣1) yield. Continuously cropped sorghum treated with 336 kg·N·ha﹣1 produced active peroxidase that shared competitive inhibition relationship with the peroxidase of the 84 kg·N·ha﹣1 treatment. Ridge tillage combined with 280 kg·ha﹣1 N fertilization under continuous sorghum resulted in inhibited peroxidase possessing low Vmax value (13.0 μmole·min﹣1). Changing to conventional tillage combined with 280 kg·ha﹣1 N fertilization relieved the inhibition and increased the Vmax value to 23.7 mmol·min﹣1. These biological anomalies of sorghum cell wall related to agronomic practices originated from doubly inhibited sorghum peroxidases. This understanding may guide the choice of sustainable agronomic practices for maximizing lignocellulose yields for the bioenergy industry while protecting the environment.

Interaction of Coal Humic Acids with Fungal Laccase

Humic acids (HA) are one of the main environmental factors controlling the fate and behavior of the compounds released into the environment. In particular, they are universally considered of great importance in determining soil extracellular enzyme activity and stability via association with essential soil enzymes. The objective of this study was to investigate the interaction of coal HA with an extracellular multicopper oxidase laccase (EC 1.10.3.2) that catalyze the oxidation of a wide range of reducing substances in the environment. Using size-exclusion chromatography analysis and monitoring laccase activity, the formation of a stable and an enzymatically active complex between HA and laccase was shown. Basing the data obtained by isoelectric focusing of HA-laccase complex, non-covalent character of laccase association with HA was considered and binding of laccase to HA by weak dispersive forces such as van der Waals, hydrophobic, π-π, CH-π and others was hypothesized.

Comparison of Detection of Antibodies with Yeast Lysate Antigens Prepared from Two Isolates of <i>Blastomyces dermatitidis</i>by Two Different Methods: Sensitivity and Specificity Evaluations

Blastomycosis, a systemic fungal disease, caused by the dimorphic fungus Blastomyces dermatitidis, has continually presented clinicians with concerns with regard to laboratory diagnosis and prevention. For years researchers have strived to develop antigens with a high degree of sensitivity and specificity. The purpose of this study was to gain a bet- ter understanding of how two novel yeast lysate antigens, prepared from two B. dermatitidis isolates by different meth- ods, would be able to detect antibody responses in immunized rabbits in a specific and sensitive manner. The en- zyme-linked immunosorbent assay (ELISA) was used to evaluate antibody in serum specimens with yeast lysate re- agents prepared after allowing yeast cells to lyse for 1 or 7 days. The results indicated that reactivity was greater with the day 7 antigens, with both the B5896 and 597 B. dermatitidis isolates, when compared to the day 1 antigens;in con- trast the day 1 preparations exhibited less cross reactivity when assayed against anti-Histoplasma capsulatum serum specimens.

<i>Pseudomonas aeruginosa</i>BUP2—A Novel Strain Isolated from Malabari Goat Produces Type 2 Pyoverdine

This study focuses on the isolation and characterization of a novel strain of siderophore producing bacterium, i.e., Pseudomonas aeruginosa BUP2 (Pa BUP2) from the rumen of Malabari goat, coupled with qualitative and quantitative analyses of the siderophore produced by it. Pa BUP2—a facultative anaerobe was tuned to be an aerobe by repeatedly growing in Benjamin flask. The new isolate was grown in a specially designed semi-synthetic medium, designated as BUP medium, and the yellowish-green pigment produced was identified as a typical siderophore by spectrophotometry, Chromazurol-S assay, thin layer chromatography and isolectric focusing (IEF). The characteristic orange fluorescence upon UV irradiation on chromatogram and absorption maximum at λ404 confirmed that the characteristic siderophore produced by Pa BUP2 was a typical pyoverdine (PVD). This PVD was further categorized under type 2 by comparing its profile on the IEF gel with that of the representative strains of each PVD types, viz., Pa O1, Pa ATCC 27853 and Pa6. Moreover, the type 2 PVD was purified by XAD-4 Amberlite column chromatography and quantified;maximum yield (11.17 mg/ml) was observed on day 4 of incubation (37°C). Thus, it was confirmed that the bacterium isolated from the rumen content of Malabari goat is a novel strain of Pa capable of producing large quantity of PVD type 2 in specially designed BUP medium under aerobic condition, and that its clinical and industrial implications remain elusive.

Incidence of Sickle Cell Disease and Other Hemoglobinopathies in Burkina Faso: Results of a Five-Year Systematic Neonatal Screening (2015-2019) in Four Urban Hospitals

Hemoglobinopathies, mainly Sickle cell disease (SCD), are the most common monogenic disorders in Africa. In Burkina Faso, data on these diseases are scarce, mainly hospital-based in Ouagadougou and its surroundings. In order to assess the incidence and allelic frequencies of the main hemoglobinopathies in newborns in Burkina Faso, we conducted a cross-sectional study from 2015 to 2019 in four hospitals. The study included babies of both sexes, regardless of ethnic group and parents’ hemoglobin status. It was a newborn screening and hemoglobin variants were detected using isoelectric focusing on cord blood samples and confirmed using hemoglobin electrophoresis by high-performance liquid chromatography. The proportions and cumulative incidences of the different hemoglobinopathies were computed. Hardy-Weinberg equilibrium law was applied to calculate genotypic and allelic frequencies. The significant level was p < 0.05. Out of 11,337 newborns included, 47.8% were males and 60.2% were from Bobo-Dioulasso. Abnormal hemoglobin was found in 27.1%, representing a cumulative incidence of 1:4 newborns. The incidence of SCD was 1.9% (1:53 newborns) with 27.9% of homozygous SS. Homozygous CC and compound heterozygous Cβ-Thalassemia accounted for 1.1%. SCD cases were 1.51 times higher in Bobo-Dioulasso (OR = 1.51;95% CI [1.09 - 2.10]: p = 0.013). The observed genotype frequencies were significantly different from the expected ones (p 0.001). The βS and βC alleles represented 5.1 and 9.9%, respectively. This study showed a high incidence of hemoglobinopathies. Such results raise the question of control strategies for these hemoglobinopathies in our country.