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31.
We present here microwave-based modifications of standard protein assays that dramatically reduce the time required to determine
protein concentrations. Typical protein determinations involve incubation times ranging from 15–60 min. Microwave irradiation
of specimens reduces this time requirement to 10–20 s without compromising accuracy or reliability. The remarkable speed with
which protein determinations may be carried out using microwave enhancement greatly simplifies general laboratory procedures
that depend on the estimation of protein concentrations.
An erratum to this article is available at . 相似文献
32.
33.
Delivery of DNA into mammalian cells by receptor-mediated endocytosis and gene therapy 总被引:8,自引:0,他引:8
The correction of genetically based disorders by the introduction of a therapeutic genetic construct into the appropriate
cell type (“gene therapy”), has become a distinct possibility in recent years. In order for gene therapy to be a practical
alternative to more conventional pharmaceutical approaches to treatment, it must be administrable in vivo. This demands that
a system be developed that can specifically target the DNA to the desired cell type once introduced into the patient. Among
the procedures that are currently being pursued, the delivery of DNA to cells by receptor mediated endocytosis (RME), comes
closest to fulfilling this crucial requirement.
The natural physiological process of RME can be exploited to deliver genetic material to cells. An antibody or ligand to a
cell surface receptor that is known to undergo endocytosis, is complexed with DNA through a covalently linked polycationic
adjunct (e.g., polylysine, protamines). Such complexes retain their binding specificity to the cell surface and are taken
up into the cell where they enter the endosomal compartment via normal endocytotic processes. In addition, steps must be taken
to avoid degradation of the DNA within the endosome-lysosome. Cells can be treated with the lysosomatropic agent chloroquine
during the transfection procedure. Alternatively, the components of viruses that enter cells by endocysis and possess an endosomal
“break out” capacity can be used. Replication defective adenovirus coupled to the ligand-DNA complex gives transfection efficiencies
of virtually 100% on tissue culture cells in vitro. Synthetic peptides that mimic the membrane fusing region of influenza
virus hemagglutinin, have also been successfully used as part of the ligand-DNA complex to bring about endosomal escape.
Preliminary studies have demonstrated the potential of this method to specifically target DNA to the cell type of choice in
vivo. Delivery of genes by receptor-mediated endocytosis offers the greatest hope that gene therapy can be an inexpensive,
easily applicable, widespread technology. 相似文献
34.
Models of the serine protease domain of the human antithrombotic plasma factor activated protein C and its zymogen. 总被引:2,自引:2,他引:0 下载免费PDF全文
C. L. Fisher J. S. Greengard J. H. Griffin 《Protein science : a publication of the Protein Society》1994,3(4):588-599
Three-dimensional structural analysis of physiologically important serine proteases is useful in identifying functional features relevant to the expression of their activities and specificities. The human serine protease anticoagulant protein C is currently the object of many genetic site-directed mutagenesis studies. Analyzing relationships between its structure and function and between naturally occurring mutations and their corresponding clinical phenotypes would be greatly assisted by a 3-dimensional structure of the enzyme. To this end, molecular models of the protease domain of protein C have been produced using computational techniques based on known crystal structures of homologous enzymes and on protein C functional information. The resultant models corresponding to different stages along the processing pathway of protein C were analyzed for structural and electrostatic differences arising during the process of protein C maturation and activation. The most satisfactory models included a calcium ion bound to residues homologous to those that ligate calcium in the trypsin structure. Inspection of the surface features of the models allowed identification of residues putatively involved in specific functional interactions. In particular, analysis of the electrostatic potential surface of the model delineated a positively charged region likely to represent a novel substrate recognition exosite. To assist with future mutational studies, binding of an octapeptide representing a protein C cleavage site of its substrate factor Va to the enzyme's active site region was modeled and analyzed. 相似文献
35.
36.
Gary C. du Moulin Zorina Pitkin Yuan-Jin Shen Evelyn Conti Jean Ko Stewart Carla Charles Dylan Hamilton 《Cytotechnology》1994,15(1-3):365-372
Somatic cell and gene therapy involve the application of biological technologies to an individual patient through the use of living cells which provide a therapeutic benefit (Aliski, 1991). Various forms of cellular and gene therapies are being developed and evaluated in an increasing number of clinical trials for congential and acquired disorders. The potential and progress of these therapeutic applications have resulted in an increasing effort by the Food and Drug Administration (FDA) to develop the regulatory framework under which these therapeutic approaches would insure safety and efficacy, the primary mandate of the FDA.Over five years ago Cellcor began to define the parameters, specifications, and conditions relevant to a Quality Assurance/Quality Control (QA/QC) program that has evolved to insure safety and maximize the efficacy of applications of the company'sex vivo technology, autolymphocyte therapy. Autolymphocyte therapy is an outpatient form of somatic cell immunotherapy based upon the infusion of T cells that have been activatedex vivo using a combination of previously generated autologous cytokines and an anti-CD3 monoclonal antibody.We have been able to demonstrate the feasibility for the safe, controlled, and consistent preparation and delivery of a cellular therapy by application of relevant GMP regulations. This presentation reviews aspects of this program and chronicles our experience which at present amounts to over 4400 infusions for over 700 patients. This program provides a high degree of assurance that a cellular therapy program can be carried out in a multisite mode involving hundreds of patients through the strict adherence to cGMP as set forth in existing regulations. It would be prudent that developers of cellular andex vivo gene therapies establish a similar cell processing and QA/QC infrastructure at an early developmental stage to optimize safety and reproducibility and facilitate regulatory review. 相似文献
37.
38.
Daniel R. Mayer Walter Kosmus Helmut Pogglitsch David mayer Wolfgang Beyer 《Biological trace element research》1993,37(1):27-38
Serum arsenic concentrations of persons suffering from renal failure and undergoing hemodialysis treatment (n=85) and of healthy controls (n=25) were determined by hydride-generation AAS technique after microwave digestion. The results were evaluated by comparing
the values of both groups, considering physiological factors and individual data, as well as comorbid conditions of the hemodialysis
(HD) patients. Serum arsenic levels were diminished in the patient group compared with controls (mean values 8.5±1.8 ng/mL
vs 10.6±1.3 ng/mL). Furthermore, additional diseases within the hemodialysis group, particularly injuries of the central nervous
system (CNS), vascular diseases, and cancer, were correlated to occasionally markedly decreased serum arsenic concentrations.
It was concluded that arsenic homeostasis is disturbed by HD treatment and certain additional diseases. Desirable arsenic
concentrations in the body seem to be reasonable. This consideration results in the conclusion that arsenic could play an
essential role in human health. Thus, reference arsenic concentrations in different human tissues and body fluids should be
established in order to recognize not only arsenic intoxication, but also arsenic deficiency. Perhaps arsenic deficiency contributes
to the increased death risk of HD patients, and therefore, arsenic supplementations for patients with extremely low serum
arsenic concentrations should be taken into account. 相似文献
39.
Bosslet K. Czech J. Seemann G. Monneret C. Hoffmann D. 《Cell biochemistry and biophysics》1994,24(1-3):51-63
A two component system, consisting of a fusion protein and an appropriate prodrug, suited to perform selective tumor therapy
in vivo, is presented. The fusion protein, owing to its humanized carcinoembryonic antigen (CEA)-specific variable region,
specifically binds to CEA-expressing tumors and has an enzymatic activity comparable to human β-glucuronidase. The prodrug
is a nontoxic glucuronide-spacer-derivative of doxorubicin decomposing to doxorubicin by enzymatic deglucuronidation.
In vivo studies in nude mice bearing human CEA-expressing tumor xenografts revealed that 7 d after injection of 20 mg/kg fusion
protein, a high specificity ratio (>100:1) was obtained between tumor and plasma. Injection of 250 mg/kg of prodrug at d 7
resulted in tumor therapeutic effects superior to conventional chemotherapy without any detectable toxicity. These superior
therapeutic effects that were observed using established human tumor xenografts can be explained by the approx 10-fold higher
drug concentrations found in tumors of mice treated with fusion protein and prodrug than in those treated with the maximal
tolerable dose of drug alone. 相似文献