A novel packaging system for the generation of helper-free oncolytic MVM vector stocks

MVM-based autonomous parvoviral vectors have been shown to target the expression of heterologous genes in neoplastic cells and are therefore of interest for cancer gene therapy. The traditional method for production of parvoviral vectors requires the cotransfection of vector and helper plasmids into MVM-permissive cell lines, but recombination between the cotransfected plasmids invariably gives rise to vector stocks that are heavily contaminated with wild-type MVM. Therefore, to minimise recombination between the vector and helper genomes we have utilised a cell line in which the MVM helper functions are expressed inducibly from a modified MVM genome that is stably integrated into the host cell chromosome. Using this MVM packaging cell line, we could reproducibly generate MVM vector stocks that contained no detectable helper virus.     

A new system for rapid measurement of ATP

The paper introduces a new type instrument for rapid measuring ATP. The system consists of a micromodule ATP sensor and an instrument for measuring weak light transmitted by optic fiber. The micromodule ATP sensor mainly is composed of enzyme membrane, a probe and a bundle of optic fiber. The instrument measuring weak light consists of photomultiplier, high voltage power, pulse amplifier and counter. The instrument was characterized by simple structure, small size, rapid response time (< 5s), high sensitivity (10(-12) mol/L), stable performance (measuring the same sample for 50 times, CV < 5%), long enzyme storage time (> 3 months).     

A novel ELISA format for the rapid and sensitive detection of staphylococcal enterotoxin A

Staphylococcal food poisoning is one of the leading causes of bacterial food poisoning each year. Detection kits for staphylococcal enterotoxins are commercially available and the assays can require from one and a half to twenty-four hours to complete with detection limits ranging from 0.5 to 2 ng enterotoxin per gram of food. We have successfully demonstrated a microsphere-packed capillary (MPC) ELISA for the detection of staphylococcal enterotoxin A (SEA) and have compared it to two commercially available kits. The MPC assay detected a lower amount of SEA in ham, chicken, cheese, and bean sprouts than either of the two commercially available kits. In addition, the novel MPC assay was completed in less than ten minutes, as compared to three and twenty-four hours for the two commercially available kits. This research also demonstrated that the MPC ELISA can contain integrated positive and negative controls and has the potential to simultaneously detect and identify multiple enterotoxins.     

A standardized protocol for the rapid preparation of bacterial DNA for pulsed-field gel electrophoresis

A rapid method for the preparation of bacterial DNA for pulsed-field gel electrophoresis was developed for Gram-positive and Gram-negative bacteria. This method was accomplished by reducing the time for the cell lysis reaction, restriction endonuclease digestion, and electrophoresis to 1, 1.5, and 18 h, respectively. The whole procedure from the initial bacterial culture plate to the final analysis of restriction fragments can be completed within 24 h. This rapid method was successfully achieved for Staphylococcus aureus, Enterococcus faecalis, Neisseria gonorrhoeae, Salmonella typhimurium, Serratia marcescens, and Stenotrophomonas maltophilia.     

Multiple deletions of the mitochondrial DNA in polymyalgia rheumatica

We analyzed the mitochondrial DNA of patients with polymyalgia rheumatica, a disease frequently associated with mitochondrial myopathy. In an attempt to study the deletions, we have developed a qualitative PCR method using a highly thermostable polymerase in order to amplify multiple mitochondrial DNA large fragments (up to 12 kb). PCR serves to observe both deleted and normal fractions of the mitochondrial DNA. We found multiple deletions of the mitochondrial DNA in all of the patient muscles. Although these muscles harbored many ragged red fibers, we found no point mutations of the tRNA(Leu)(UUR)) and the mutation at nucleotide position 8344 was not present.     

A novel, rapid flat-mounting technique for visualizing antibody labeling in the retina

Complete analysis of retinal tissue is difficult because it consists of a thin neural tissue spread across the back of a hemispheric surface. Conventional sectioning in a plane parallel to a central axis of symmetry produces a large number of samples, each containing only a small amount of the tissue of interest. Consequently, quantitative comparison of any feature of interest typically uses a small fraction of the sections from each retina, because analysis of the entire collection of sections is too time consuming. Such a sampling process can lead to misleading or erroneous conclusions. We present a new method which allows complete analysis of the retina using a small number of samples produced by sectioning flattened retinas. This procedure is straightforward as illustrated using an antibody against proliferating cell nuclear antigen (PCNA) to locate dividing cells in the teleost fish retina. Immunocytochemical staining on flat-sectioned retinas was quantified using a computer-based image analysis system. When the cells of interest are randomly distributed, conventional sampling procedures can seriously under- or over-estimate their number. The new technique presented allows significantly more efficient examination and quantification of the entire retina as compared to conventional techniques.     

A flow cytometric method for rapid selection of novel industrial yeast hybrids

We rapidly produced and isolated novel yeast hybrids by using two-color flow cytometric cell sorting. We labeled one parent strain with a fluorescent green stain and the other parent with a fluorescent orange stain, and hybrids were selected based on their dual orange and green fluorescence. When this technique was applied to the production of hybrids by traditional mating procedures, more than 96% of the isolates were hybrids. When it was applied to rare mating, three hybrids were identified among 50 isolates enriched from a population containing 2 x 10(6) cells. This technology is not dependent on genetic markers and has applications in the development of improved industrial yeast strains.     

Aldol sensors for the rapid generation of tunable fluorescence by antibody catalysis

The synthesis of novel fluorogenic retro-aldol substrates for aldolase antibody 38C2 is described. These substrates are efficiently and specifically processed by antibody aldolases but not by natural cellular enzymes. Together, the fluorogenic substrates and antibody aldolases provide reporter gene systems that are compatible with living cells. The broad scope of the antibody aldolase allows for the processing of a range of substrates that can be designed to allow fluorescence monitoring at a variety of wavelengths. We also have developed the following concept in fluorescent protein tags. beta-Diketones bearing a fluorescent tag are bound covalently by the aldolase antibody and not other proteins. We anticipate that proteins fused with the antibody can be tagged specifically and covalently within living cells with fluorophores of virtually any color, thereby providing an alternative to green fluorescent protein fusions.     

DNA optical sensor: a rapid method for the detection of DNA hybridization

A DNA optical sensor system is proposed based on the combination of sandwich solution hybridization, magnetic bead capture, flow injection and chemiluminescence for rapid detection of DNA hybridization. Bacterial alkaline phosphatase (phoA) gene and Hepatitis B virus (HBV) DNA were used as target DNA. A biotinylated DNA probe was used to capture the target gene onto the streptavidin-coated magnetic beads and a calf intestine alkaline phosphatase (CAP)-labelled DNA probe was used for subsequent enzymatic chemiluminescence detection. The detection cycle was less than 30 min, excluding the DNA hybridization time, which was about 100 min. Both the phoA gene and HBV DNA could be detected at picogramme or femtomole level. No response signal was obtained when target DNA did not exist in the sample. Successive sample detection could be made by removing the magnetic field and a washing step.     

The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes

BACKGROUND:. Modern biological research is highly dependent upon recombinant DNA technology. Conventional cloning methods are time-consuming and lack uniformity. Thus, biological research is in great need of new techniques to rapidly, systematically and uniformly manipulate the large sets of genes currently available from genome projects. RESULTS:. We describe a series of new cloning methods that facilitate the rapid and systematic construction of recombinant DNA molecules. The central cloning method is named the univector plasmid-fusion system (UPS). The UPS uses Cre-lox site-specific recombination to catalyze plasmid fusion between the univector - a plasmid containing the gene of interest - and host vectors containing regulatory information. Fusion events are genetically selected and place the gene under the control of new regulatory elements. A second UPS-related method allows for the precise transfer of coding sequences only from the univector into a host vector. The UPS eliminates the need for restriction enzymes, DNA ligases and many in vitro manipulations required for subcloning, and allows for the rapid construction of multiple constructs for expression in multiple organisms. We demonstrate that UPS can also be used to transfer whole libraries into new vectors. Additional adaptations are described, including directional PCR cloning and the generation of 3' end gene fusions using homologous recombination in Escherichia coli. CONCLUSIONS:. Together, these recombination-based cloning methods constitute a new comprehensive approach for the rapid and efficient generation of recombinant DNA that can be used for parallel processing of large gene sets, a feature that will facilitate future genomic analysis.     

A novel culturing and grafting system for the treatment of leg ulcers

The purpose of this study was to develop and test an efficient culturing and grafting system for the treatment of leg ulcers. The culturing system consisted of a Petriperm culture vessel (20 cm2) aseptically placed in a larger standard Petri dish (60 cm2). Skin cultures were established and cultivated in the Petriperm dish. The cells grew on the bottom of the Petriperm dish, which was made of a gas-permeable 25-micron thick transparent Teflon film. Grafts were produced simply by cutting the film from the bottom of the Petriperm dish with a scalpel. The system was used to produce subconfluent epidermal autografts which were used to heal a 32 cm2 chronic rheumatoid arthritis leg ulcer. The cultured autografts were transferred cell side down on to the cleaned wound bed without an enzymatic digestion. The grafts consisted of autologous keratinocytes, melanocytes and fibroblasts. Caution was taken not to disturb the wound bed for 7-9 days at which time the Teflon film was removed. The wound closed 2 weeks after the last grafting and has remained closed for more than a year post-treatment. The culturing and grafting system presented here will make it possible to develop cellular-based therapies that were previously not possible.     

新型PEG基粘结剂的开发

研究了一种新型PEG基粘结剂,新型PEG基粘结剂由HDPE,PEC,聚合物W,SA,DOP等组成,各组元之间具有一定的相互作用,使粘结剂体系具有较好的部分相容性,新型PEG基粘结剂与Fe-2Ni粉具有较强的相互作用,混合均匀性好,且粉末装载量高,喂料注射成形性好,用新型PEG基粘结剂和Fe-2Ni粉所制备的喂料具有良好的流变性能,是一种假塑性流体,粘结剂各组元用量及粉末装载量影响喂料流变性能,新型PEG基粘结剂用乙醇溶剂脱脂,速度快、缺陷少、维形能力强,且可避免环境污染问题。     

Interference of DNA sequence divergence with precise recombinational DNA repair in mammalian cells

Studies done in prokaryotes and eukaryotes have indicated that DNA sequence divergence decreases the frequency of homologous recombination. To determine which step(s) of homologous recombination is sensitive to DNA sequence divergence in mammalian cells we have used an assay that does not rely on the recovery of functional products. The assay is based on the acquisition by homologous recombination of endogenous LINE-1 sequences by exogenous LINE-1 sequences. In parallel experiments, we introduced into mouse cells two gapped exogenous LINE-1 sequences, one from the mouse, L1Md-A2, and the other from the rat, L1Rn-3. Although L1Rn-3 is on average less than 85% homologous to the LINE-1 elements of the mouse, the frequency of homologous recombination with endogenous LINE-1 elements obtained with L1Rn-3 was the same as the one obtained with L1Md-A2 which is on average 95% homologous to the LINE-1 elements of the mouse. The endogenous LINE-1 sequences rescued by L1Rn-3 were 8-18% divergent from L1Rn-3 sequences, whereas those rescued by L1Md-A2 were 2-5% divergent from L1Md-A2 sequences. The gap which had been introduced into the exogenous LINE-1 sequences had been precisely repaired in 50% of the recombinants obtained with L1Md-A2. None of the L1Rn-3 recombinants showed precise gap repair.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of multiple mitochondrial DNA deletions in inclusion body myositis

Exercise induced pain in the posterior part of the leg is common among runners; the underlying reason for these complaints may be very different. The purpose of the present, controlled study was therefore 1. to confirm a clinically diagnosed deep posterior compartment syndrome by using intramuscular pressure measurements and 2. to evaluate the effect of a surgical release on clinical signs and intracompartment pressure values. Fifteen symptomatic runners with the clinical suspicion of a chronic deep posterior compartment syndrome and nine healthy recreational runners as controls were investigated. Intramuscular pressure was measured both at rest and up to two minutes post-exercise, using a pressure-monitor with a transducer. In symptomatic runners, the average pressure was preoperatively 5.6 mmHg (95%-confidence-interval [CI]: 3.4-7.6) at rest, rising to 18.5 mmHg (CI: 15.4-21.8) post-exercise. Corresponding values in healthy control runners were 5.1 mmHg (CI: 1.9-8.3) at rest, with a decrease induced by exercise to 2.8 mmHg (CI: -0.5-6.1). After fasciotomy of the deep posterior compartment in all fifteen symptomatic runners, average pressure values fell to 2.2 mmHg (CI: 1.0-3.4) at rest, and were further reduced after (now pain-free) exercise to 1.6 mmHg (CI: 0.6-2.6). The decrease between pre-operative and post-operative values was statistically highly significant (p < 0.0001 for values after running, p < 0.005 for values at rest). In conclusion, intracompartment pressure measurement is a useful technique to confirm the clinical diagnosis of deep posterior compartment syndrome prior to recommending surgery. Hereby, an exercise-induced rise in pressure of at least 10 mmHg, corresponding to a two- to threefold increase of values measured at rest, may be a more important diagnostic criterion than absolute levels of pressure measured before or after running.     

Ageing-associated large-scale deletions of mitochondrial DNA in human hair follicles

Hair follicles plucked from the bi-temporal region of the scalp of 433 Chinese subjects of different ages were used for the examination of ageing-associated mutations of human mitochondrial DNA (mtDNA). By use of PCR techniques, we detected the 4,977 bp and 7,436 bp deletions of mtDNA in hair follicles from aged individuals. The frequencies of occurrence of both mtDNA deletions were found to increase with age of the subject. Moreover, we employed a semi-quantitative PCR method to determine the proportion of the 4,977 bp deleted mtDNA (dmtDNA) in hair follicles. The results showed that the average proportion of the 4,977 bp dmtDNA in hair follicles were 0.05% +/- 0.01%, 0.00%, 0.55% +/- 0.05%, 0.52% +/- 0.24%, 0.65% +/- 0.17%, 1.33 +/- 0.25%, and 1.89% +/- 0.81% for the subjects in the age groups of 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, and 81-99, respectively. Furthermore, we screened all the subjects harboring the 4,977 bp and/or 7,436 bp deletions for tandem duplications in the D-loop region of mtDNA by PCR with back-to-back primers. The results showed that none of the previously reported tandem duplications were present in all the hair follicles examined. This indicates that tandem duplications do not predispose to large-scale deletions of mtDNA. However, the data suggest that mtDNA deletions occur and accumulate in hair follicles during human ageing. As hair follicles can be easily and non-invasively obtained from the human, we suggest that the aged-dependent accumulation of dmtDNAs in hair follicles may be used for the monitoring of human ageing process.