Western blotting for the diagnosis of congenital toxoplasmosis

Toxoplasmosis is a common congenital infection. It does not usually produce recognizable signs of infection at birth so most infected newborns are not detected by routine clinical examination and remain untreated. Infected children without clinical symptoms should nonetheless be identified and treated as early as possible. Serological diagnosis of congenital toxoplasmosis is quite difficult. The aim of this study was to evaluate the utility of Western blot for the diagnosis of congenital toxoplasmosis. We compared the immunological profiles of mothers and children to differentiate between passively transmitted maternal antibodies and antibodies synthesized by the infants in the first three months of life. The method enabled us to diagnose congenital toxoplasmosis in cases in which the infection had not been detected by classical serology techniques.     

Enhancement of luminol-based immunodot and Western blotting assays by iodophenol

The application of 4-iodophenol as a signal enhancer for luminol-based immunodot and Western blotting assays was investigated. Immunodetection of biotin-bovine serum albumin (BSA) using a rat anti-biotin antibody for immunodot binding and goat anti-biotin peroxidase conjugate for Western blotting assays was employed to demonstrate the signal enhancement attainable with 4-iodophenol. The addition of 4-iodophenol at a final working concentration of 52.2 microM to a light-emitting substrate, luminol, resulted in 3-fold and 10-fold signal enhancements for Western blotting and immunodot binding detection of biotin-BSA, respectively.     

A photodetection device for luminol-based immunodot and Western blotting assays

A photodetection device permitting rapid and direct photographic recording of light emission from luminol-based immunodot and Western blotting assays is described in detail. This device permits contact exposure on Polaroid film of luminescent samples on nitrocellulose strips, and thus obviates the need for darkroom and film processing facilities. Immunodetection of mouse alpha-fetoprotein in brown fat homogenate and immunoglobulin E in nonimmune human sera employing luminol-based Western blotting and immunodot assays were used to demonstrate the versatility and operational simplicity of the detection device. The use of the detection device in combination with luminol-based immunoassays resulted in greater signal intensities and increased sensitivity over that attainable with the commonly used chromogenic substrate, 4-chloro-1-naphthol.     

Immunoreactivity enhancement with chelators for increasing the detection sensitivity of human PrPSc by Western blotting

Prion diseases are neurodegenerative disorders affecting humans as Creutzfeldt-Jakob disease. The host-encoded prion protein (PrP(C)) will be converted into a structurally altered isoform (PrP(Sc)). PrP(Sc) differ in sizes and glycoform patterns and can be identified using molecular typing with Western blotting. The electrophoretic mobility of PrP(Sc) changes on treatment with metal ions or chelators prior to digestion with proteases. The effects of chelators applied to PrP(Sc) after protease digestion had not been examined in detail, we investigated these effects in this study. Application of EDTA, NTA and DTPA, and to a lesser extent EGTA, significantly enhanced PrP(Sc) signals in immunoblots. PrP(Sc) intensities increased two- to three-fold compared with untreated PrP(Sc). Since the immunoblot method is highly specific, sensitivity is the limiting factor. Enhancing sensitivity might be important in the determination of PrP(Sc) at levels close to or just below the limits of detection. It is to be expected that application of chelators to digested protein samples will increase the sensitivity of PrP(Sc) detection using the Western blot technique.     

Determining protein stability in cell lysates by pulse proteolysis and Western blotting

Proteins require proper conformational energetics to fold and to function correctly. Despite the importance of having information on conformational energetics, the investigation of thermodynamic stability has been limited to proteins, which can be easily expressed and purified. Many biologically important proteins are not suitable for conventional biophysical investigation because of the difficulty of expression and purification. As an effort to overcome this limitation, we have developed a method to determine the thermodynamic stability of low abundant proteins in cell lysates. Previously, it was demonstrated that protein stability can be determined quantitatively by measuring the fraction of folded proteins with a pulse of proteolysis (Pulse proteolysis). Here, we show that thermodynamic stability of low abundant proteins can be determined reliably in cell lysates by combining pulse proteolysis with quantitative Western blotting (Pulse and Western). To demonstrate the reliability of this method, we determined the thermodynamic stability of recombinant human H‐ras added to lysates of E. coli and human Jurkat T cells. Comparison with the thermodynamic stability determined with pure H‐ras revealed that Pulse and Western is a reliable way to monitor protein stability in cell lysates and the stability of H‐ras is not affected by other proteins present in cell lysates. This method allows the investigation of conformational energetics of proteins in cell lysates without cloning, purification, or labeling.     

An improved method for mRNA isolation and characterization of in vitro translation products by Western blotting

A method is described for isolation of messenger RNA (mRNA) from a rather intractable tissue source, calf stomach. The use of additional RNase inhibitors, vanadyl ribonucleoside complexes and proteinase K, which are used in conjunction with the guanidine thiocyanate/CsCl ultracentrifugation procedure traditionally employed for isolation of mRNA, is described. These modifications make the procedure universally applicable to a wide variety of tissues and cell types. The validity of the procedure is demonstrated by isolation of biologically active full-length preprochymosin mRNA. The integrity of the mRNA is measured by in vitro translation, Northern blot analysis, Southern blot analysis of preprochymosin cDNA using synthetic oligodeoxynucleotide probes and immunospecific identification of in vitro translation products using a modification of the Western blot which is described in this report.     

An epidemiologic and immunologic study of measles under conditions of massive immunization against that infection]

Massive measles immunization in Riga led to a marked reduction of measles incidence and to a change of the principal regularities of the epidemic process in this infection. Among those who contracted the disease there was an increase in the percentage of schoolchildren; affection with measles of children attending creches and kindergartens and the intensity of the spread of the infection in them diminished. Selective examination of the immunological efficacy of the living measles vaccine prepared of the (see article) and applied in 1967--1972 demonstrated the presence of specific stimulation of the antibody formation in about 90% of the persons vaccinated. The intensity of humoral immunity in the persons vaccinated did not diminish with the advance of time after the vaccination, and 6--7 years after the vaccination over 90% of the vaccinated individuals were reliably protected from measles. The presence of numerous negative results in carrying out the vaccinations in individual institutions is apparently attributed chiefly to disturbances of the storage regimen of transportation and of the use of the vaccine.     

Direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometric identification of proteins on membrane detected by Western blotting and lectin blotting

We have developed new procedures to identify proteins after they are detected by Western blotting or other interactions such as lectin blotting on membranes. Our method is based on the combination of on-membrane MALDI-TOF mass spectrometry with piezoelectric chemical inkjet technology. Using this method the GroEL, FtsZ, DnaK, and GroES proteins were successfully identified from Escherichia coli after separation on two-dimensional gels, immunostaining, and on-membrane digestion. A glycoprotein detected by lectin blotting with concanavalin A was also identified using this technique.     

Chemotherapy for falciparum malaria: the armoury, the problems and the prospects

Peter Winstanley here describes the pharmacology and therapeutics of the main drugs used for falciparum malaria in the tropical setting, rather than in the developed world, as an overview for newcomers to the field. He then examines some of the current major problems and prospects for the future.     

Analysis of stage-specific and shared antigens derived from Rhipicephalus sanguineus by electrophoresis and Western blotting

e carried out an SDS-PAGE analysis of antigens of Rhipicephalus sanguineus using extracts of eggs (EE), larvae (LE), nymphs (NE), male salivary glands (MSGE), male midguts (MME), female salivary glands (FSGE) and female midguts (FME). Under non-reducing conditions a common band of about 205 kDa was observed. EE, LE and NE extracts showed groups of bands between 150 and 75 kDa. A protein pattern was observed in FSGE extract with a group of bands between 75 and 50 kDa and four bands between 15 and 6.5 kDa. In this case an apparently exclusive band of molecular weight about 25 kDa was observed. Under reducing conditions similarities between LE and NE extracts increased, separating from the EE pattern. On the other hand, we have determined the presence of stage-specific and common antigens on EE, LE, NE, MSGE, MME, FSGE and FME extracts of R.sanguineus by means of immunoblots using polyclonal sera of rabbits infested with larvae, nymphs or adults of this tick. EE extract was only recognized by the anti-larva sera. Higher reactivity was observed when the extracts were tested with anti-adult sera. In these experiments a very prominent band of molecular weight about 45 kDa was detected. This band was not observed under reducing conditions. Higher reactivity with anti-adult sera was observed against FSGE extract.     

Determination of copy number of c-Myc protein per cell by quantitative Western blotting

The protooncogene c-Myc plays a key role in growth control, differentiation, and apoptosis. An abnormally high expression of c-myc has been found to be associated with many neoplasms. c-Myc gene expression is usually measured at the mRNA level. Few studies have been published on quantitative Myc protein determination. A major drawback of ELISA (enzyme-linked immunosorbent assay) methods is the uncertainty of the specificity of the antibody reaction. In contrast, antibody specificity can be easily controlled by Western/immunoblotting. Here we describe a method to quantify c-Myc protein in primary human IMR90 lung fibroblasts based on Western blotting. Using a high-resolution polyacrylamide gel, we were able to differentiate the cellular c-Myc protein (64 kDa) from a c-Myc internal standard (65 kDa). We determined both the total c-Myc protein content per cell and its distribution in the cytoplasmic and nuclear fractions. About 4000 c-Myc protein molecules were detected in the cytoplasmic fraction and 29,000 copies in the nuclear fraction for proliferating human lung fibroblasts IMR90. The ratio of nuclear (active) to cytoplasmic (inactive) c-Myc protein changed from 17:1 for proliferating cells to 2.5:1 for confluent cells.     

Toward silencing the burden of malaria: progress and prospects for RNAi-based approaches

The discovery of RNA interference (RNAi) is one of the most significant of recent years, with potential for application beyond the laboratory to the clinic. As a tool for functional genomics, RNAi has permitted the characterization of genes in organisms that had previously remained recalcitrant to targeted gene manipulation. Efforts to understand its mode of action have revealed a central role in gene regulation and host defense. Finally, as a therapeutic tool, it has shown enormous promise in the control of a large array of diseases. Here we examine how RNAi is revolutionizing malaria research in an organism, the Anopheles mosquito, that until recently was essentially resistant to genetic study, and show how its application in both the mosquito vector and the Plasmodium parasite might ultimately lead to new ways of controlling and perhaps even eradicating this devastating disease.     

Veterinary endectocides for malaria control and elimination: prospects and challenges

Residual transmission is the persistence of malaria transmission after scale-up of appropriate vector control tools and is one of the key challenges for malaria elimination today. Although long associated with outdoor biting, other mosquito behaviours such as partly feeding upon animals contribute greatly to sustaining transmission. Peri-domestic livestock can be used as decoy to protect humans from blood-seeking vectors but this approach often leads to an increased malaria risk in a phenomenon known as zoopotentiation. Treating the said livestock with drugs capable of killing intestinal parasites as well as mosquitoes that feed upon them has the potential to tackle malaria through a previously unexplored mechanism. The advantages and challenges associated with this approach are briefly discussed here. Numerous references are purposely provided.This article is part of the theme issue ‘Novel control strategies for mosquito-borne diseases’.     

Analysis of electroblotted proteins by mass spectrometry: protein identification after Western blotting

We describe a new approach for the identification and characterization by mass spectrometry of proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting and Removal of Nitrocellulose (BARN)), proteins can be analyzed either as intact proteins for molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus removing the nitrocellulose which can interfere with MS analysis. This method offers improved protein coverage, especially for membrane proteins, such as uroplakins, because the extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble and membrane proteins after Western blotting, obtaining comparable or better results than with in-gel digestion.     

许昌人遗址研究的新收获及展望

许昌人遗址位于河南省许昌市灵井镇,2005-2017年发掘,揭露面积500余平方米,2007、2014年在9号探方出土包括2颗"许昌人"头骨在内的5个古人类个体,大量的石制品和21种哺乳动物化石。头骨具有东亚古人类、欧洲尼安德特人和早期现代人的镶嵌特征,可能代表一种新型的古老型人类。石制品研究显示,石核类型多样,且以小型双锥形盘状石核为特色;小型工具类型分异明显、加工精细,显示出不同于旧石器时代早期遗址中的工具技术,而与西方旧石器时代中期遗址工具技术上的特点较为一致。此外,遗址出土7件软锤工具,以动物长骨或鹿角为原料,用以修理石质工具。软锤工具的发现,对于认识中国旧石器时代技术的发展有重要意义。通过对遗址形成过程的分析,"许昌人"生活时期的沉积环境经历了三个阶段:下部灰绿色滨湖相粉砂堆积所指示的水流动力相对较弱的沉积环境,中部综红色粉砂堆积所指示的水流动力相对较强的沉积环境,以及上部浅棕红色粉砂堆积所指示的相对冷湿、水动力仍然较强的沉积环境。尽管存在水动力强弱上的相对变化,但水动力总体上并不大,以低能量水流为主,文化遗物属于原地埋藏。今后将开展人类艺术行为能力、古人用软锤和压制法制作石器的技术、动物埋藏学和年代学等方面的深入研究。