PubMed · 4903222
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M Ia Shkol'nik. [Microelements and nucleic acids].. https://pubmed.ncbi.nlm.nih.gov/4903222/
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Gene therapy for HIV (human immunodeficiency virus) involves the introduction of a therapeutic gene into the infected individual for the purposes of reducing viral load and ultimately reconstituting a healthy immune system. Clinical trials for HIV gene therapy have not yet reported therapeutic benefit. In addition to improving the efficiency of gene delivery and the maintenance of gene expression, better therapeutic genes must be designed before this therapy becomes available to patients. A new class of therapeutic genes expressing nucleases may be designed. These nucleases may be classified into three categories based on their mode of action: (i) 'targeted nucleases' for specifically cleaving HIV RNA within the cell, (ii) 'colocalized nucleases' for cleaving HIV genomic DNA or RNA present within the cell or progeny virus, and (iii) 'cytotoxic nucleases' for conferring selective toxicity to HIV-infected cells. The focus of this review is on the design and application of these nucleases for HIV gene therapy.
BACKGROUND: Real-time polymerase chain reaction (PCR) is a powerful new technique in the evolution of quantitative reverse transcription-PCR assays. With the increased sensitivity and resolution of real-time techniques, the requirements for constitutive expression of endogenous controls have become increasingly stringent. METHODS AND RESULTS: We compare the expression of the mitochondrial gene, adenosine triphosphate synthase 6 (ATPsy6), to the expression of other routinely used endogenous control genes (e.g., beta-actin, glyceraldehyde-3-phosphate dehydrogenase [GAPDH], ribosomal RNA 18S [18S rRNA], and cyclophilin). In a diverse assortment of tissues and across a wide range of disease stages, ATPsy6 shows a relative steady state of expression compared with other endogenous controls. ATPsy6 gene expression has been used as an endogenous control in a quantitative real-time PCR assay designed to evaluate the expression of potential cancer diagnostic leads across a diverse tissue panel. CONCLUSION: Mitochondrial ATPsy6 serves as a good endogenous control to measure target gene expression independent of the tissue- or disease-specific variation inherent with many housekeeping genes.
STUDY OBJECTIVE: To investigate the effects of streptokinase and deoxyribonuclease (DNase) on the viscosity of pus to assess whether the DNase in the old preparation of streptokinase-streptodornase used intrapleurally to treat empyema was contributing to easier drainage of pus compared with purified streptokinase. DESIGN: In vitro measurement of pus viscosity. PATIENTS: Pus from three patients with surgically drained soft tissue abscesses and from six patients with empyema thoracis of varying etiology was studied. INTERVENTIONS: Pus samples were incubated with saline solution as control and with streptokinase, streptokinase-streptodornase, human recombinant DNase, and a mixture of streptokinase and DNase in concentrations approximating those achieved in clinical practice. RESULTS: Purified streptokinase had little effect on pus viscosity, with a mean reduction of 11.1% in the surgical specimens and 1.7% in the empyema samples. Streptokinase-streptodornase reduced viscosity by a mean of 52.8% in the surgical samples and 94.8% in the empyema samples. Human recombinant DNase reduced viscosity by a mean of 32. 79% in surgical samples and 93.4% in empyema samples. Adding streptokinase to human recombinant DNase produced no further reduction in viscosity. Final viscosities in samples treated with DNase were very similar whatever the starting viscosity. CONCLUSIONS: DNase significantly reduces pus viscosity, whereas streptokinase has little or no effect, and in empyema may work simply by breaking down loculations. Clinical studies should be undertaken to see if these in vitro changes produce clinical benefits. The simple viscometer devised for these experiments may also prove useful in other contexts.