Reduction of High Background Staining by Heating Unfixed Mouse Skeletal Muscle Tissue Sections Allows for Detection of Thermostable Antigens With Murine Monoclonal Antibodies

Antigen detection with indirect immunohistochemical methods is hampered by high background staining if the primary antibody is from the same species as the examined tissue. This high background can be eliminated in unfixed cryostat sections of mouse skeletal muscle by boiling sections in PBS, and several proteins including even the low abundant dystrophin protein can then be easily detected with murine monoclonal antibodies. However, not all antigens withstand the boiling procedure. Immunoreactivity of some of these antigens can be restored by subsequent washing in Triton X-100, whereas immunoreactivity of other proteins is not restored by this detergent treatment. When such thermolabile proteins are labeled with polyclonal primary antibodies followed by dichlorotriazinylaminofluorescein–conjugated secondary antibodies and boiled, the fluorescence signal persists, and sections can then be processed with a monoclonal antibody for double immunostaining of a protein unaffected by boiling. This stability of certain fluorochromes on heating can also be exploited for double immunofluorescence labeling of two different thermostable proteins with murine monoclonal antibodies as well as for combination with Y-chromosome fluorescence in situ hybridization. Our method should extend the range of monoclonal antibodies applicable to tissues derived from the same species as the monoclonal antibodies. (J Histochem Cytochem 56:969–975, 2008)     

An evaluation of CHD‐Specific primer sets for sex typing of birds from feathers

The amplification of the highly conserved chromo‐helicase‐DNA binding region found in both the Z and W chromosome was evaluated with three sets of primers (P8/P2, 1237L/1272H and 2550F/2718R). DNA extracted from feathers through a simple boiling method was used to address its reliability in generating the sex‐linked bands. All the bird samples, including the seven bird families that have not been reported previously, were successfully amplified with the primer set 2550F/2718R. The resulting polymerase chain reaction products showed clearly resolved fragments on a conventional agarose gel electrophoresis with size differences ranging from 80 to 540 bp between the two respective ZW gene copies. Although the P8/P2 primer was not as effective under the same conditions, it was able to produce well‐resolved Z and W bands from bird species under the Antidea family, whereas the 2250F/2718R primer set only produced a single amplified fragment of a different size between the male and the female. Zoo Biol 27:62–69, 2008. © 2007 Wiley‐Liss, Inc.     

mRNA差异显示技术中特异条带回收方法的比较

目的：建立简化的mRNA差异显示技术中特异条带的回收方法。方法：用单个小鼠早期发育的胚胎构建mRNA差异显示技术,用一步法和煮沸法分别从银染的聚丙烯酰胺凝胶中回收差异显示的特异条带,并进行再扩增、回收、克隆及酶切鉴定。结果：两种不同的回收方法经过mRNA差异显示技术程序环节,证明其具有相同的实验效果。结论：应用简化的一步法和煮沸法对单胚构建的mRNA差异显示技术中的特异条带进行回收,具有有效性和可靠性。     

Compositional changes during landfarming of weathered michigan crude oil‐contaminated soii

Laboratory landfarming experiments were conducted to study the bioremediation potential of weathered Michigan crude oil‐contaminated soils. It was found that landfarming was successful in removing up to 90% of the total petroleum hydrocarbons (TPH) in the soil within 22 weeks of treatment. Boiling point analyses of untreated and treated soils indicate a significant removal of TPH compounds independent of molecular weight or carbon number. Up to 85% of heavy petroleum hydrocarbons with carbon numbers above 44 were biode‐graded. In addition, approximately 93% of saturated and 79% of aromatic compounds of the TPH were biodegraded during the 22 week treatment period. The use of polyethylene sheeting as a landfarm cover does not appear to adversely affect biodegradation kinetics under laboratory conditions. Finally, equilibrium leachate concentrations for BTEX and regulated (in Michigan) polynuclear aromatics (PNAs) were below the respective detection limits for each compound. It can be concluded that landfarming of these weathered soils will be highly successful in removing petroleum hydrocarbons while not adversely impacting either ground‐water or surface water quality.     

Changes in M:G ratios of extracted and residual alginate fractions on boiling with water the dried brown alga Kjellmaniella crassifolia (Laminariales,Phaeophyta)

Kjellmaniella crassifolia, the edible macro-brown alga in Japan contained nearly 27% of alginates of which nearly 7% was extractable from the fronds with boiling water for 6 h and the residual alginates in the frond were almost exhaustively extracted with a dilute alkali at 60 °C for 6 h. The alginates dissolved in all these extracts with both boiling water and dilute alkali were purified by fractionation with MgCl₂ and alcohol.The content of MM blocks in the boiling water-soluble alginate sample increased remarkably during heating for 6 h while that of GG blocks from the same sample decreased. In contrast, MM blocks in the alkali-soluble alginate sample decreased during 6 h heating while GG blocks continued to increase. Since the amounts of MG blocks showed slight fluctation, the M:G ratio of alginates extracted with boiling water increased towards the end of extraction whereas the reverse is true for the alkali-soluble alginates.     

三种快速制备含重复序列质粒PCR模板的方法

为了探索含重复序列质粒PCR模板的快速简易制备方法,分别利用高速离心法、TE煮沸法和TE/SDS煮沸法处理过夜培养的含重组质粒大肠杆菌菌体,制备模板后进行PCR扩增发现,均能得到比较清晰的目的条带,可以用于含重复序列质粒的初步鉴定。相比较而言,高速离心5 min、TE煮沸3 min、TE/SDS煮沸2.5 min制备的样品PCR扩增后效果较好。这3种方法快速、简便、费用低、无污染,简化了PCR模板制备的过程,为重组质粒大量筛选鉴定的研究奠定了基础。     

废糟液全循环对自絮凝酵母糖酵解途径关键酶、胁迫相关代谢物及胞内组分的影响

旨在研究废糟液直接全循环对絮凝酵母乙醇发酵、糖酵解关键酶以及细胞组成的影响。在一有效容积1.5 L的搅拌式生物反应器中,使用葡萄糖为220 g/L,添加8 g/L酵母粉和6 g/L蛋白胨的培养基,以0.04 h?1的稀释率进行自絮凝颗粒酵母乙醇连续发酵。每隔3天将收集到的发酵液集中精馏处理,得到的废糟液用于配制发酵培养基。装置运行近20 d,实验结果表明,随着废液循环批次的增加,系统乙醇和生物量浓度明显降低,糖酵解途径3个关键限速酶:己糖激酶、6-磷酸果糖激酶和丙酮酸激酶不同程度受到抑制。为了应对废糟液中高沸点副产物积累导致的环境胁迫,维持细胞正常代谢,甘油和菌体胞内蛋白生物合成加强,碳水化合物积累减弱。这些研究结果对进一步研究高沸点副产物积累对酵母细胞乙醇发酵影响的机理和菌种的代谢工程改造,具有重要意义。     

Alkali Pretreatment of Wheat Straw (Triticum aestivum) at Boiling Temperature for Producing a Bioethanol Precursor

Polyamines have been shown to be necessary for the activity of the extracellular ice–nucleating matter (EIM) from the ice–nucleating bacterium, Erwinia uredovora KUIN-3. When this bacterium was cultured in the presence of methylglyoxal bis(guanylhydrazone), MGBG (2 mM), the ice–nucleating activity of the EIM significantly decreased. Further, the thermal (25–40°C) and pH (alkaline region) stabilities of the activity were stimulated by the addition of spermidine. This phenomenon only occurred in the class A and B structures, and it showed that the hydrophobicities of the class A and B structures in the EIM increased with the addition of spermidine as judged by the freezing difference spectra. We then found by using fluorescent reagents that the physiological roles of spermidine in the EIM controlled the charge, free-amino groups, and hydrophobicities on the surface of the EIM. In conclusion, one could predict that spermidine took part in the charge of the surface, the control of hydrophobicity, and the stability of protein conformation in the class A and B structures in the EIM, and is a critical component in the class A and B nucleating structures.     

A Comparison of Methods for Heat-Mediated Antigen Retrieval for Immunoelectron Microscopy: Demonstration of Cytokeratin No. 18 in Normal and Neoplastic Hepatocytes

Postembedding antigen retrieval is a veil established technique for immnoelectron microscopy; however, many antigens cannot be detected without additional unmasking procedures. This study was undertaken to determine whether microwave oven heating, autoclaving, and pressurized boiling, which are well recognized methods of antigen retrieval for light microscopy, and simple boiling can also be used in electron microscopy. We investigated neoplastic and normal hepatocytes using a commercially available mouse monoclonal antibody against cytokeratin NO. 18 (CK18). The tissue was fixed in paraformaldehyde / gintaraldehyde and embedded in Lowicryl K4M at -40 C. Ultrathin sections in various buffers were exposed to heat using one of four methods or to pronase at 37 C before incubation with the primary antibody. The secondary antibody was gold-labeled goat anti-mouse antibody. Sections that were not heat-treated remained unlabeled, but heat-treated sections showed immu-noreactivity located mainly at the cytoplasmic periphery. Some of the gold particles lay in direct or loose association with intermediate filaments, some were seen in the area of desmosomes, and some did not appear related to any structures. No difference in immunostainlng was found among the four methods of heat treatment. The citrate buffer, pH 6.0, and 10 mM EDTA, pH 8.0, generated the best labeling results.     

熏蒸法测定土壤微生物量碳的改进

自从Ｊｅｎｋｉｎｓｏｎ和Ｐｏｗｌｓｏｎ［１］创造熏蒸培养方法测定土壤微生物量碳，发展到用多种方法来测定土壤微生物量，如土壤ＡＴＰ含量分析方法［２］，基质诱导呼吸法［３］，精氨酸氨化法［４］，熏蒸浸提法［５］，脂肪酸、ＤＮＡ、ＲＮＡ等微生物细胞成分分析...