Large Scale Preparation of Myelin Basic Protein from Central Nervous Tissue of Several Mammalian Species

The wide-spread use of and demand for myelin basic protein for immunologic studies has prompted us to re-examine the details of its isolation from CNS tissue of various species. The procedure described in this communication for the isolation and purification of myelin basic protein does not require column chromatography and is therefore suitable for large scale preparation of a reasonably pure product with simple laboratory equipment. If certain precautions are taken, the yield and quality of the product are reproducible. Certain contaminants which may accompany myelin basic protein during purification by procedures currently in use are pointed out, and their possible influence on the immunologic behavior of myelin basic protein is discussed. Suitable electrophoretic techniques for the detection of these contaminants as well as details for their removal from the myelin basic protein are described.     

Studies with Radioactive Tracers in Plant Nutrition: II. THE ESTIMATION OF RADIOACTIVE TRACERS

Part I Tracer methods can yield valid results only if the behaviourof the tracer within the plant is indistinguishable from thatof the normal element. The extent to which radioactive isotopessatisfy this requirement must be considered from the followingviewpoints:

1. their chemical and physical bahaviour before disintegration;
2. the effects and substances formed by their disintegration;
3. the effects of the radiation they emit.

Whereas, chemically and physically, isotopes with the exceptionof H¹ and H³ satisfy this requirement, they may cause significantradiation damage even when supplied in low concentration ifthey are accumulated metabolically. The investigation of possibleradiation effects is therefore an essential preliminary to allphysiological investigations. The value of tracer methods is considered. Part II Equipment and laboratory procedures for the electronic assayof P³² are described. For the assay of samples containing P³²a wet ashing method and the subsequent use of a liquid countingtube is preferred. Improvements of equipment to suit the requirementsof plant nutritional studies are considered. Sources of experimentalerror are examined. On account of the decay of radioactive isotopes,the time factor may be of special importance in tracer work.Its effect on both experimental design and on laboratory proceduresis considered. It is stressed that the scope and replicationof large experiments, particularly if they are of long duration,may be subject to serious limitations. The importance of simplifyingthe equipment used in tracer assay and increasing its reliabilityis emphasized.     

The use of neutron activation analysis in pollination ecology

Neutron activation analysis in pollination ecology was examined with particular regard to external application of indicator elements. Movement of elements between flowers can be detected after external application either to the anthers or, in members of the Asteraceae, to the entire head. This technique is potentially useful in studying pollen flow within and between populations and in detecting flower constancy of pollinators. This technique is advantageous when compared with the use of radioactive tracers because potential health and environmental hazards inherent in the field use of unstable isotopes are eliminated.     

International Society for Analytical Cytology biosafety standard for sorting of unfixed cells.

BACKGROUND: Cell sorting of viable biological specimens has become very prevalent in laboratories involved in basic and clinical research. As these samples can contain infectious agents, precautions to protect instrument operators and the environment from hazards arising from the use of sorters are paramount. To this end the International Society of Analytical Cytology (ISAC) took a lead in establishing biosafety guidelines for sorting of unfixed cells (Schmid et al., Cytometry 1997;28:99-117). During the time period these recommendations have been available, they have become recognized worldwide as the standard practices and safety precautions for laboratories performing viable cell sorting experiments. However, the field of cytometry has progressed since 1997, and the document requires an update. METHODS: Initially, suggestions about the document format and content were discussed among members of the ISAC Biosafety Committee and were incorporated into a draft version that was sent to all committee members for review. Comments were collected, carefully considered, and incorporated as appropriate into a draft document that was posted on the ISAC web site to invite comments from the flow cytometry community at large. The revised document was then submitted to ISAC Council for review. Simultaneously, further comments were sought from newly-appointed ISAC Biosafety committee members. RESULTS: This safety standard for performing viable cell sorting experiments was recently generated. The document contains background information on the biohazard potential of sorting and the hazard classification of infectious agents as well as recommendations on (1) sample handling, (2) operator training and personal protection, (3) laboratory design, (4) cell sorter set-up, maintenance, and decontamination, and (5) testing the instrument for the efficiency of aerosol containment. CONCLUSIONS: This standard constitutes an updated and expanded revision of the 1997 biosafety guideline document. It is intended to provide laboratories involved in cell sorting with safety practices that take into account the enhanced hazard potential of high-speed sorting. Most importantly, it states that droplet-based sorting of infectious or hazardous biological material requires a higher level of containment than the one recommended for the risk group classification of the pathogen. The document also provides information on safety features of novel instrumentation, new options for personal protective equipment, and recently developed methods for testing the efficiency of aerosol containment.     

A Note on The Computer Marking of Objective Tests

A considerable proportion of biology teachers in schools and technical colleges are at present using or contemplating using radioactive isotopes for certain experiments in their courses. These experiments (a general list appears at the end of the article) are easy to perform and usually biologically instructive.

For the teacher the experiment naturally does not stop with performing it; solutions have to be prepared, supervision and suggestions have to be given to students concerning data handling, half-life and self-absorption corrections, etc. An awareness of the pitfalls in both technique and result interpretation has to be gained and this is usually not possible even in an intensive one week course that most teachers have attended.

For this reason this article covers the minimum necessary nuclear fundamentals such as levels of activity, specific activity and the use of carrier materials. The way the isotope is assayed and the corrections that need to be made or taken into account to obtain a valid result is also described in detail. A minimum of statistics necessary for such a valid result is similarly covered. From experience gained in running short radio-isotope courses for teachers some common misconceptions that often arise due to lack of course time have also been included.     

Use of personal protective equipment for respiratory protection

Management of hazards in biomedical research facilities requires the application of the traditional industrial hygiene responsibilities of anticipation, recognition, evaluation, and control to characterize the work environment, evaluate tasks and equipment, identify hazards, define exposure groups, and recommend controls. Generally, the diversity and unique characteristics of hazards faced by laboratory and animal facility employees and the short-term and low-level nature of the exposures factor into the selection of proper exposure control measures in the laboratory. The proper selection of control measures is based on a hierarchy of elimination and minimization by engineering controls, followed last by personal protective equipment when exposures cannot be eliminated. Once it is decided that personal protective equipment is needed, specific regulations and guidelines define safety standards for research facilities, including the elements of a sound respiratory protection program. These elements include respirator selection (including appropriate protection factors), medical evaluation, fit testing, training, inspection, maintenance and care, quality, quantity and flow of breathing air, and routine and emergency use procedures.     

PCR: how to kill unwanted DNA.

Avoidance of contamination in the PCR laboratory requires the use of strict precautions. Among these, chemical decontamination of surfaces and equipment is desirable to prevent inadvertent contamination of samples by the gloved hand and by pipettors. We have investigated the use of sodium hypochloride (Clorox), in comparison to concentrated HCl, for PCR sterilization. Ten percent Clorox was found to eliminate all ethidium bromide-stainable DNA and to prevent PCR amplification of a 600-bp DNA segment within one minute of template treatment. RNA was similarly destroyed. By contrast, even 2.0 N HCl did not destroy DNA detectable by PCR within five minutes. Because of its high efficacy, low cost and relatively low corrosiveness, we recommend the use of ten percent Clorox as a decontaminant for elimination of DNA templates in the PCR laboratory.     

Non-radioactive probes for specific DNA sequences

Advances in the isolation and detection of genes utilizing the great specificity of base pairing in the hybridization of nucleic acid bases have been built upon the use of radioactively labelled nucleotides. These offer sensitivity and the convenience of familiarity but have disadvantages; short lives and the hazards associated with their production, use and disposal. In extending nucleic acid hybridization to unlicensed laboratories or field use in remote areas and eliminating the hazards from handling radioactive materials, other labels have advantages.     

Detection of adeno- and lentiviral (HIV1) contaminations on laboratory surfaces as a tool for the surveillance of biosafety standards

Aims: As a biosafety laboratory, we survey the handling of adenovirus type 5 (Ad5) and HIV1‐derived lentivirus in contained‐use facilities in Switzerland to identify insufficiencies of the safety precautions taken by the laboratories. Methods and Results: In the past 9 years, we took 430 swab samples from various types of surfaces in research laboratories. Samples were examined for Ad5 contaminations by real‐time PCR and infectivity assay or for the presence of lentivirus (HIV1) nucleic acids by real‐time (RT) PCR. Samples collected from centrifuges did not only contain Ad5 DNA more frequently but also exhibited higher numbers of Ad5 and lentiviral (HIV1) DNA copies than swabs from any other area of sampling. Five of ten samples containing infectious Ad5 particles or lentivirus (HIV1) RNA were found in samples taken from centrifuges. Ad5 contamination rates were higher in the tube holder and lower on the inner wall of the rotor chamber in centrifuges that were fitted with aerosol tight covers compared to centrifuges without covers. Conclusions: Our results allowed the comparison of hygiene standards of different laboratories and lead to the identification of centrifuges as hotspots for contaminations. Significance and Impact of the Study: Based on our results, we propose to use the collected data as a tool for rating future swab results. Furthermore, the amount of Ad5 and HIV1‐derived lentivirus DNA could serve as an indicator of the level of good laboratory practice in contained‐use laboratories handling these viral vectors.     

Comparative analysis of S-fatty acylation of gel-separated proteins by stable isotope-coded fatty acid transmethylation and mass spectrometry

Covalent attachment of palmitic acid or other fatty acids to the thiol groups of cysteine residues of proteins through reversible thioester bonds has an important role in the regulation of diverse biological processes. We describe here the development of a mass spectrometry protocol based on stable isotope-coded fatty acid transmethylation (iFAT) for qualitative and comparative analysis of protein S-fatty acylation under different experimental conditions. In this approach, cellular proteins extracted from different cell states are separated by SDS-PAGE and then the gel is stained with either Coomassie blue or Nile red for improved sensitivity. Protein bands are excised and then an in-gel stable iFAT procedure is performed. The fatty acid methyl esters resulting from derivatization with d0- and d3-methanol are identified by mass spectrometry. By measuring the intensities of labeled and unlabeled fragment ion pairs of fatty acid methyl esters, the levels of S-fatty acylation in different cells or tissues can be compared. This approach has been applied to monitor the changes of S-fatty acylation of zebrafish liver proteome in response to environmental dichlorodiphenyltrichloroethane exposure. Compared with the approach using metabolic incorporation of radioactive fatty acid analogs, it is not only simple and effective but also eliminates the hazards of handling radioactive isotopes.     

How to develop a Standard Operating Procedure for sorting unfixed cells

Written Standard Operating Procedures (SOPs) are an important tool to assure that recurring tasks in a laboratory are performed in a consistent manner. When the procedure covered in the SOP involves a high-risk activity such as sorting unfixed cells using a jet-in-air sorter, safety elements are critical components of the document. The details on sort sample handling, sorter set-up, validation, operation, troubleshooting, and maintenance, personal protective equipment (PPE), and operator training, outlined in the SOP are to be based on careful risk assessment of the procedure. This review provides background information on the hazards associated with sorting of unfixed cells and the process used to arrive at the appropriate combination of facility design, instrument placement, safety equipment, and practices to be followed.     

Neurotoxicity of depleted uranium

Depleted uranium (DU) is a byproduct of the enrichment process of uranium for its more radioactive isotopes to be used in nuclear energy. Because DU is pyrophoric and a dense metal with unique features when combined in alloys, it is used by the military in armor and ammunitions. There has been significant public concern regarding the use of DU by such armed forces, and it has been hypothesized to play a role in Gulf War syndrome. In light of experimental evidence from cell cultures, rats, and humans, there is justification for such concern. However, there are limited data on the neurotoxicity of DU. This review reports on uranium uses and its published health effects, with a major focus on in vitro and in vivo studies that escalate concerns that exposure to DU might be associated with neurotoxic health sequelae.     

Review of endovascular brachytherapy physics for prevention of restenosis

Intraluminal irradiation of coronary and peripheral arteries has been shown to reduce neointimal hyperplasia following balloon angioplasty, thereby inhibiting restenosis. Several irradiation techniques are being investigated, including temporary intravascular insertion of high activity gamma- or beta-emitting seeds and wires; inflation of dilatation balloon catheter with radioactive liquid or gas; insertion of miniature x-ray tubes via coronary catheters; permanent implantation of radioactive stents; and postangioplasty fractionated external beam irradiation. Unlike conventional brachytherapy, intravascular treatment of restenosis requires accurate knowledge of dose at distances of 0.5-5 mm from the radioactive source. This requirement presents special problems with regard to source calibration and dose specification, because dose gradients at such close distances from a radioactive source are extremely large. This makes it virtually impossible to define the characteristics of an ideal radiation source without some knowledge of the location and radiosensitivity of the target tissues, plus the radiotolerance of normal tissues. Hence, the current debate over whether beta or gamma sources are to be preferred. Imprecise knowledge of dose-volume effects for coronary arteries, plus uncertainties in the biological time sequencing of restenosis fuel a second debate on whether external beam treatments may be efficacious, and whether or not permanent radioactive stents may prove superior to high dose, single fraction brachytherapy. We review here the dosimetric properties of the various irradiation techniques and isotopes that have been proposed, including aspects of radiation safety, dose homogeneity, and practical aspects of source delivery.     

浅论与实验动物相关的职业健康安全与人畜共患病

道义上的责任或是法律法规都要求公司或单位提供一个安全和健康的工作环境,以保障员工免受不必要的危害。职业健康与安全（OHS）规程的目的就是预防职业伤害和疾病,它不仅需要满足法规的要求,更重要的是控制危害和减少风险。本文描述了实验动物相关OHS规程的重要性及一些关键要素,如管理措施、设施的设计与运行、控制与危险物的接触、教育和培训、职业健康专业化服务、仪器设备的性能、信息管理、应急措施及对规程的评估和完善。本文还简要地介绍了常见的实验非人灵长类动物相关的人畜共患疾病,以及如何安全地从事实验动物相关工作。     

The 4-6-8 method of sequence analysis

A new method to obtain more sequence data from a single gel run is described. This method allows the reading of over 500 bases of sequence data from a single gel by taking advantage of the differential migration of specific sized dideoxy terminated chain lengths in sequencing gels containing variable percentages of acrylamide. The method is easy to use, requires no special equipment and requires no special technical abilities. We feel the methodology described will be useful for the average laboratory doing sequencing work.     

Effects of heavy ions on rabbit tissues: analysis of low levels of DNA damage in retinal photoreceptor cells

When suitable precautions are taken, sedimentation of DNA through reoriented alkaline sucrose gradients in zonal rotors can be used to determine small amounts of DNA damage in mammalian cells without resorting to radioactive precursors. Hence, the method is especially useful for studying the efficacies of DNA repair mechanisms in the neurons of the central nervous system (CNS) and the accumulation of DNA damage in the ageing CNS. Here we describe the technique as it has been used to examine the DNA damage occurring in the photoreceptor cells of the retina of the New Zealand white rabbit during the course of natural ageing or after exposure to heavy ions. This article is an integral part of a series of reports of the latter studies (Lett et al. 1980, Keng and Lett 1981, Cox et al. 1981, 1982, Keng et al. 1982). With the same analytical technique, very low levels of radioactive DNA precursors can be used to advantage in investigations of proliferating cells.     

Model for inactivation and disposal of infectious human immunodeficiency virus and radioactive waste in a BL3 facility.

A method is described for autoclaving low levels of solid infectious, radioactive waste. The method permits steam penetration to inactivate biologic waste, while any volatile radioactive compounds generated during the autoclave process are absorbed. Inactivation of radiolabeled infectious waste has been problematic because the usual sterilization techniques result in unacceptable radiation handling practices. If autoclaved under the usual conditions, there exists a high probability of volatilization or release of radioisotopes from the waste. This results in the radioactive contamination of the autoclave and the laboratory area where steam is released from the autoclave. Our results provide a practical method to inactivate and dispose of infectious radioactive waste. For our research, Bacillus pumilus spore strips and vaccinia virus were used as more heat-resistant surrogates of the human immunodeficiency virus (HIV). These surrogates were used because HIV is difficult to grow under most conditions and is less heat tolerant than the surrogates. In addition, B. pumilus has defined cell death values, whereas such values have not been established for HIV. Both B. pumilus and vaccinia virus are less hazardous to work with. The autoclave method is time efficient and can be performed by laboratory personnel with minimal handling of the waste. Furthermore, waste site handlers are able to visually inspect the solid waste containers and ascertain that inactivation procedures have been implemented.     

Model for inactivation and disposal of infectious human immunodeficiency virus and radioactive waste in a BL3 facility

A method is described for autoclaving low levels of solid infectious, radioactive waste. The method permits steam penetration to inactivate biologic waste, while any volatile radioactive compounds generated during the autoclave process are absorbed. Inactivation of radiolabeled infectious waste has been problematic because the usual sterilization techniques result in unacceptable radiation handling practices. If autoclaved under the usual conditions, there exists a high probability of volatilization or release of radioisotopes from the waste. This results in the radioactive contamination of the autoclave and the laboratory area where steam is released from the autoclave. Our results provide a practical method to inactivate and dispose of infectious radioactive waste. For our research, Bacillus pumilus spore strips and vaccinia virus were used as more heat-resistant surrogates of the human immunodeficiency virus (HIV). These surrogates were used because HIV is difficult to grow under most conditions and is less heat tolerant than the surrogates. In addition, B. pumilus has defined cell death values, whereas such values have not been established for HIV. Both B. pumilus and vaccinia virus are less hazardous to work with. The autoclave method is time efficient and can be performed by laboratory personnel with minimal handling of the waste. Furthermore, waste site handlers are able to visually inspect the solid waste containers and ascertain that inactivation procedures have been implemented.     

Starch Granulomatosis of the Peritoneum

Starch glove powder is used extensively by surgeons in Britain and is generally considered innocuous so that precautions to prevent granuloma formation, previously taken when talc glove powder was in use, are now neglected. Reported here are five cases of starch granulomatosis of the peritoneum occurring over a period of a few months. This condition requires reoperation within a limited time for its diagnosis and may be confused macroscopically with disseminated malignant disease or tuberculosis or may simply cause adhesions. Recognition is dependent on a high degree of suspicion by both surgeon and histopathologist, as special histological techniques may be necessary. Consequently, it is only in a minority of the florid cases that a diagnosis is made, and the condition would appear to be much more common than is generally realized. A plea is made for scrupulous care to avoid starch powder contamination of the operative field.