Antigen retrieval in prion protein immunohistochemistry.

Transmissible spongiform encephalopathies are a group of neurodegenerative diseases occurring in both humans and animals and are most likely caused by prions. Neuropathological confirmation of the clinical diagnosis has been a problem because of the difficulty in epitope retrieval from formalin-fixed, paraffin-embedded brain specimens. Many different protocols for the detection of prions in brain tissue have been used. Thus far, picric and/or formic acid, steam autoclaving at 121C of sections, microwave treatment, and 4 M guanidine thiocyanate treatment have been suggested. The objective of our experiment was to obtain the standard pretreatment(s) resulting in optimal immunostaining. In the experiment, successive tissue slides of brain specimens of several Creutzfeldt-Jakob disease and control patients were stained using different combinations of pretreatments. Using densitometric analysis, several well-defined locations per section were examined and prion immunostaining was quantified. The results showed that autoclaving is necessary for antigen retrieval and cannot be substituted by microwave treatment. The best results were obtained when the following combination was used in the specified order: 15 min saturated picric acid, 10 min steam autoclaving at 121C, 5 min 88% formic acid, and 2 hr 4 M guanidine thiocyanate at 4C. (J Histochem Cytochem 47:1465-1470, 1999)     

Camptothecin: current perspectives

This review provides a detailed discussion of recent advances in the medicinal chemistry of camptothecin, a potent antitumor antibiotic. Two camptothecin analogues are presently approved for use in the clinic as antitumor agents and several others are in clinical trials. Camptothecin possesses a novel mechanism of action involving the inhibition of DNA relaxation by DNA topoisomerase I, and more specifically the stabilization of a covalent binary complex formed between topoisomerase I and DNA. This review summarizes the current status of studies of the mechanism of action of camptothecin, including topoisomerase I inhibition and additional cellular responses. Modern synthetic approaches to camptothecin and several of the semi-synthetic methods are also discussed. Finally, a systematic evaluation of novel and important analogues of camptothecin and their contribution to the current structure-activity profile are considered.     

免疫组织化学中的抗原修复技术

概述了免疫组织化学中固定对组织抗原性的影响,综述了抗原修复的方法及其原理。组织块的大小、固定液的种类、浓度及固定时间的长度都影响组织的抗原性。免疫组织化学实践中常用酶消化法、酸水解法、微波金属盐法、高压锅加热法等修复组织的抗原性。     

Extracellular granzymes: current perspectives

Granzyme A (GrA) and granzyme B (GrB) play key roles in the induction of target cell death induced by cytotoxic lymphocytes. Whilst these roles have been extensively studied, it is becoming apparent that both granzymes also possess extracellular activities. Soluble granzymes are found extracellularly in normal plasma and are elevated in a number of diseases, ranging from viral and bacterial infections to autoimmune diseases. Here, we discuss the current knowledge of extracellular granzyme substrates, inhibitors and functions; and the pathological consequences of extracellular granzymes in disease. In addition, we provide new evidence for the role of glycosaminoglycan-binding sites of granzymes in extracellular matrix remodeling.     

Palaeomicrobiology: current issues and perspectives

Palaeomicrobiology is an emerging field that is devoted to the detection, identification and characterization of microorganisms in ancient remains. Data indicate that host-associated microbial DNA can survive for almost 20,000 years, and environmental bacterial DNA preserved in permafrost samples has been dated to 400,000-600,000 years. In addition to frozen and mummified soft tissues, bone and dental pulp can also be used to search for microbial pathogens. Various techniques, including microscopy and immunodetection, can be used in palaeomicrobiology, but most data have been obtained using PCR-based molecular techniques. Infections caused by bacteria, viruses and parasites have all been diagnosed using palaeomicrobiological techniques. Additionally, molecular typing of ancient pathogens could help to reconstruct the epidemiology of past epidemics and could feed into current models of emerging infections, therefore contributing to the development of appropriate preventative measures.     

Microglia in depression: current perspectives

Major depressive disorder(MDD) is a prevalent psychiatric disease that involves malfunctions of different cell types in the brain.Accumulating studies started to reveal that microglia, the primary resident immune cells, play an important role in the development and progression of depression. Microglia respond to stress-triggered neuroinflammation, and through the release of proinflammatory cytokines and their metabolic products, microglia may modulate the function of neurons and astrocytes to regulate depression. In this review, we focused on the role of microglia in the etiology of depression. We discussed the dynamic states of microglia; the correlative and causal evidence of microglial abnormalities in depression; possible mechanisms of how microglia sense depression-related stress and modulate depression state; and how antidepressive therapies affect microglia. Understanding the role of microglia in depression may shed light on developing new treatment strategies to fight against this devastating mental illness.     

Testosterone and sport: current perspectives

Testosterone and other anabolic-androgenic steroids enhance athletic performance in men and women. As a result, exogenous androgen is banned from most competitive sports. However, due to variability in endogenous secretion, and similarities with exogenous testosterone, it has been challenging to establish allowable limits for testosterone in competition. Endogenous androgen production is dynamically regulated by both exercise and winning in competition. Furthermore, testosterone may promote athletic performance, not only through its long-term anabolic actions, but also through rapid effects on behavior. In women, excess production of endogenous testosterone due to inborn disorders of sexual development (DSD) may convey a competitive advantage. For many years, female competitors have been subject to tests of sexual genotype and phenotype known as gender verification. Although gender verification has not identified any normal man competing as a woman, this process has identified women athletes with DSD. As understanding of DSD has expanded in recent years, women with DSD are increasingly able to continue athletic competition.     

Antioxidants: current situation and perspectives

In the overview one can find analysis medico-biological aspects of free radical theory, clinical experience of antioxidants using, perspectives of new active agents and possible directions of theirs practical use.     

Antigen retrieval by heating en bloc for pre-fixed frozen material.

Antigen retrieval (AR) is frequently required for successful immunohistochemistry (IHC) in archival formalin-fixed, paraffin-embedded tissue sections. Although AR by heating is most generally used, the majority of existing methods are useful only for paraffin-embedded sections. This article describes a simple alternative method for AR that can be used for aldehyde-fixed frozen sections. After fixation in paraformaldehyde, tissue blocks were heated in retrieval solutions and then frozen with dry ice. The optimal temperatures for heating were 90C and above, and the optimal retrieval solutions were distilled water and 10 mM sodium citrate, pH 6.0. Sections were cut with a cryostat and mounted on poly-l-lysine-coated glass slides. After the sections dried, routine IHC was performed. Alternatively, free-floating sections were used. This method not only greatly enhanced the immunoreactivity for a wide range of antigens, especially for nuclear proteins, but also effectively lowered the background staining in some cases. I examined the staining of 14 antibodies using sections of mouse brain and rat testis. The heating process was essential for five antibodies, improved immunoreactivity for seven antibodies, and provided no change for two antibodies.     

Antigen retrieval of basement membrane proteins from archival eye tissues.

Antigen retrieval (AR) methods can unmask tissue antigens that have been altered by fixation, processing, storage, or resin interactions. This is particularly important in the study of archival tissues, because primary fixatives and storage times may vary among specimens. We performed an electron microscopic study of basement membrane components of the aqueous humor drainage pathways from archival eye tissue. AR (heated citrate buffer, pH 6.0, LR White resin) increased the amount of label of collagen IV and fibronectin in tissue fixed in four different fixatives, including those containing glutaraldehyde. Labeling density was approximately doubled after AR for most fixatives, with the largest increase for tissues fixed in 4% paraformaldehyde/2% glutaraldehyde. Duration of storage time for archival tissues did not affect AR results. AR did not change the components of the extracellular matrix labeled; no "new" components were labeled after AR. We conclude that AR in citrate buffer can be used on selected extracellular matrix antigens to enhance label that would otherwise be lost due to fixation and storage.     

Biogas production: current state and perspectives

Anaerobic digestion of energy crops, residues, and wastes is of increasing interest in order to reduce the greenhouse gas emissions and to facilitate a sustainable development of energy supply. Production of biogas provides a versatile carrier of renewable energy, as methane can be used for replacement of fossil fuels in both heat and power generation and as a vehicle fuel. For biogas production, various process types are applied which can be classified in wet and dry fermentation systems. Most often applied are wet digester systems using vertical stirred tank digester with different stirrer types dependent on the origin of the feedstock. Biogas is mainly utilized in engine-based combined heat and power plants, whereas microgas turbines and fuel cells are expensive alternatives which need further development work for reducing the costs and increasing their reliability. Gas upgrading and utilization as renewable vehicle fuel or injection into the natural gas grid is of increasing interest because the gas can be used in a more efficient way. The digestate from anaerobic fermentation is a valuable fertilizer due to the increased availability of nitrogen and the better short-term fertilization effect. Anaerobic treatment minimizes the survival of pathogens which is important for using the digested residue as fertilizer. This paper reviews the current state and perspectives of biogas production, including the biochemical parameters and feedstocks which influence the efficiency and reliability of the microbial conversion and gas yield.     

Proteomic profiling of exosomes: current perspectives

Exosomes are 40-100 nm membrane vesicles of endocytic origin secreted by most cell types in vitro. Recent studies have shown that exosomes are also found in vivo in body fluids such as blood, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, and breast milk. While the biological function of exosomes is still unclear, they can mediate communication between cells, facilitating processes such as antigen presentation and in trans signaling to neighboring cells. Exosome-like vesicles identified in Drosophila (referred to as argosomes) may be potential vehicles for the spread of morphogens in epithelia. The advent of current MS-based proteomic technologies has contributed significantly to our understanding of the molecular composition of exosomes. In addition to a common set of membrane and cytosolic proteins, it is becoming increasingly apparent that exosomes harbor distinct subsets of proteins that may be linked to cell-type associated functions. The secretion of exosomes by tumor cells and their implication in the transport and propagation of infectious cargo such as prions and retroviruses such as HIV suggest their participation in pathological situations. Interestingly, the recent observation that exosomes contain both mRNA and microRNA, which can be transferred to another cell, and be functional in that new environment, is an exciting new development in the unraveling exosome saga. The present review aims to summarize the physical properties that define exosomes as specific cell-type secreted membrane vesicles.     

Oncoproteomics: current trends and future perspectives

Oncoproteomics is the application of proteomics technologies in oncology. Functional proteomics is a promising technique for the rational identification of biomarkers and novel therapeutic targets for cancers. Recent progress in proteomics has opened new avenues for tumor-associated biomarker discovery. With the advent of new and improved proteomics technologies, such as the development of quantitative proteomic methods, high-resolution, -speed and -sensitivity mass spectrometry and protein arrays, as well as advanced bioinformatics for data handling and interpretation, it is now possible to discover biomarkers that can reliably and accurately predict outcomes during cancer management and treatment. However, there are several difficulties in the study of proteins/peptides that are not inherent in the study of nucleic acids. New challenges arise in large-scale proteomic profiling when dealing with complex biological mixtures. Nevertheless, oncoproteomics offers great promise for unveiling the complex molecular events of tumorigenesis, as well as those that control clinically important tumor behaviors, such as metastasis, invasion and resistance to therapy. In this review, the development and advancement of oncoproteomics technologies for cancer research in recent years are expounded.     

Tissue engineering: current state and perspectives

Tissue engineering is an interdisciplinary field that involves cell biology, materials science, reactor engineering, and clinical research with the goal of creating new tissues and organs. Significant advances in tissue engineering have been made through improving singular aspects within the overall approach, e.g., materials design, reactor design, or cell source. Increasingly, however, advances are being made by combining several areas to create environments which promote the development of new tissues whose properties more closely match their native counterparts. This approach does not seek to reproduce all the complexities involved in development, but rather seeks to promote an environment which permits the native capacity of cells to integrate, differentiate, and develop new tissues. Progenitors and stem cells will play a critical role in understanding and developing new engineered tissues as part of this approach.     

Super-SILAC: current trends and future perspectives

Stable isotope labeling with amino acids in cell culture (SILAC) has risen as a powerful quantification technique in mass spectrometry (MS)–based proteomics in classical and modified forms. Previously, SILAC was limited to cultured cells because of the requirement of active protein synthesis; however, in recent years, it was expanded to model organisms and tissue samples. Specifically, the super-SILAC technique uses a mixture of SILAC-labeled cells as a spike-in standard for accurate quantification of unlabeled samples, thereby enabling quantification of human tissue samples. Here, we highlight the recent developments in super-SILAC and its application to the study of clinical samples, secretomes, post-translational modifications and organelle proteomes. Finally, we propose super-SILAC as a robust and accurate method that can be commercialized and applied to basic and clinical research.     

Space biotechnology: current and future perspectives

The potential for obtaining enhanced purities and for achieving greater homogeneity of materials in microgravity first attracted biotechnologists to space bioprocessing. This is but one of the benefits of microgravity. This review discusses the unique opportunities of space biotechnology and the diverse means to achieve microgravity conditions.     

Malt-induced premature yeast flocculation: current perspectives

Premature yeast flocculation (PYF) is a sporadic problem for the malting and brewing industries which can have significant financial and logistical implications. The condition is characterised by abnormally heavy (and sometimes early) flocculation of yeast during brewery fermentations. The resulting low suspended yeast cell counts towards the end of the fermentation can result in flavour defects and incomplete attenuation (fermentation of sugars to alcohol). Despite several decades of research into the phenomenon, its precise nature and mechanisms have not been fully elucidated. In part this is because the term PYF has become a 'catch-all' syndrome which can have multiple origins. Furthermore, there are complex interactions in the malting and brewing processes which together mean that the PYF status of a malt sample is hard to predict at a generic level. Whether or not PYF is observed depends not only on barley quality, but on process factors in the maltings and to a substantial extent on the brewing yeast strain concerned. This article highlights the significance of PYF, and reviews current knowledge relating to the origins of this complex phenomenon.