Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation

Article Abstract:

Mitochondria are organelles found within cells that have their own DNA and are involved in energy production. The production of mitochondria is controlled by the cell nucleus and mitochondrial genome, the set of chromosomes for the mitochondria. Formation of mitochondrial DNA (mtDNA) requires genetic information that originates and is encoded in the nucleus; this nuclear genetic information must be imported into the mitochondria. The extent of specific control and the interaction of these two DNA complexes were examined. Specially treated cells were chemically altered so their mitochondrial DNA (mtDNA) was removed. Cells were exposed to low concentrations of ethidium bromide over a long period, which interfered with the functioning of the respiratory chain; the cells became dependent upon the amino acids uridine and pyruvate for growth. This characteristic was then used as a marker to trace the progress of the cells. These cells were repopulated by a complementation process using exogenous mitochondria. The new cells that were produced were found to have a distinct respiratory appearance, or phenotype, derived from both the parent cells and the various mitochondrial donors. Examination of the new cells, the transformants, indicated that genetic information from both the mitochondrial and nuclear genome interacted with each other and were involved in the respiratory function of the cell. Messenger DNA mutations have been associated with mitochondrial neuromuscular diseases and this technique may prove to be useful in further research. Mitochondria extracted from cells of patients with the disease may be combined with cells specifically altered for traceability. This may enable the source of the defect, either the mtDNA or nuclear encoded DNA, to be identified.

Evaluation, Mitochondrial DNA, Cellular control mechanisms, Cell regulation, Tissue respiration

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Mutants of pertussis toxin suitable for vaccine development

Article Abstract:

Pertussis or whooping cough is a childhood disease that affects over 60 million infants and is responsible for as many as one million deaths annually. The vaccine which has been developed to prevent this disease may result in neurological complications and death. Although this occurs infrequently, it has created a reluctance to use the vaccine in some countries, and a safer immunization process is needed. The bacteria Bordetella pertussis produce the pertussis toxin (PTX), which is both the virulent factor in the disease and the component used in the production of the vaccine. Studies in Sweden have demonstrated that PTX can be chemically detoxified and still remain effective for use in providing immunization when incorporated into a vaccine. The techniques that were used for the detoxification are not yet perfected and although immunity was produced, toxic effects from the vaccine were observed. An attempt was made using genetic manipulation to produce a safer molecule for use in vaccinations. Alleles of the pertussis toxin genes were genetically engineered by changing one or two key amino acid sequences in the active site of the gene. These altered genes were then inserted into the chromosomes of three bacteria that cause pertussis. A mutant pertussis molecule was produced which was then injected into mice. When the mice were exposed to the virulent B. pertussis, they exhibited immunity to the disease and no toxic effects were observed. It was concluded that the PTX mutant molecule may be useful in the creation of a safer pertussis vaccine.

Prevention, Whooping-cough, Whooping cough, Pertussis vaccines, Pertussis toxin

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Engineering human prolactin to bind to the human growth hormone receptor

Article Abstract:

Site-directed mutagenesis is a cellular process whereby a DNA segment (bit of genetic material) is deliberately altered so that different proteins will be expressed by the cell. When applied to homologous proteins (proteins with many segments of similar structure), desired properties can be induced that may, for instance, enable cells to bind to each other that do not normally do so. Prolactin (hPRL) and human growth hormone (hGH) are members of the same family and share certain characteristics. In general, hormones work by binding to receptors located on the surfaces of cells. Once bound, they enter the cell and influence various intracellular processes. Growth hormone binds to two types of receptors, lactogenic and somatogenic, thereby causing both lactation (milk production) and bone growth. Prolactin binds only to the lactogenic receptor, stimulating lactation, with no effect on bone. Using the methods of molecular biology, a segment of DNA from hPRL was mutated until it contained certain sequences known to be critical for binding to the hGH receptor. Several repetitions of the mutagenic strategy were necessary before a form of hPRL was obtained that had an affinity for the hGH receptor. The results indicate that a particular receptor characteristic can be selected and given to a different molecule, even when information about the structure of the original hormone-receptor complex itself is lacking. (Consumer Summary produced by Reliance Medical Information, Inc.)

Somatotropin, Cell receptors, Prolactin

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