Engineering orthogonal ligand-receptor pairs from "near drugs".

Cell-permeable small molecules are powerful tools for unraveling complex cellular pathways. We demonstrate that nuclear hormone receptors can be engineered through mutagenesis to create orthogonal ligand-receptor pairs to control transcription. Mutated residues in the retinoid X receptor (RXR) were chosen from structural analysis of RXR and the retinoic acid receptor (RAR) ligand binding domains. The potential ligands screened for activation of variant receptors are "near drugs"--compounds synthesized during structure-activity studies that are structurally similar to an approved drug yet inactive on the wild-type receptor. One variant, Q275C;I310M;F313I, is poorly activated by ligands for the wild-type receptor but is activated by a "near drug", fulfilling the criteria of an orthogonal ligand-receptor pair. These experiments demonstrate that nuclear hormone receptors are well suited to supply orthogonal ligand-receptor pairs for experimental biology, biotechnology, and gene therapy. Our findings also demonstrate the general principle that inactive compounds synthesized during drug discovery can be combined with mutant proteins to rapidly create new tools for controlling cellular processes.

Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA.

Locked nucleic acid is an RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2'-oxygen and the 4'-carbon. This conformation restriction increases binding affinity for complementarity sequences and provides an exciting new chemical approach for the control of gene expression and optimization of microarrays.

Synthesis, analysis, purification, and intracellular delivery of peptide nucleic acids.

Peptide nucleic acids (PNAs) are nonionic DNA mimics. Their novel chemical properties may facilitate the development of selective and potent antisense and antigene strategies for regulating intracellular processes. Described herein are procedures for the synthesis, purification, handling, and characterization of PNAs. A simple protocol for the lipid-mediated introduction of PNAs into in vitro cultures of mammalian cells is provided.

Inhibition of gene expression inside cells by peptide nucleic acids: effect of mRNA target sequence, mismatched bases, and PNA length.

Genome sequencing has revealed thousands of novel genes, placing renewed emphasis on chemical approaches for controlling gene expression. Antisense oligomers designed directly from the information generated by sequencing are one option for achieving this control. Here we explore the rules governing the inhibition of gene expression by peptide nucleic acids (PNAs) inside cells. PNAs are a DNA/RNA mimic in which the phosphate deoxyribose backbone has been replaced by uncharged linkages. Binding to complementary sequences is not hindered by electrostatic repulsion and is characterized by high rates of association and elevated affinities. Here we test the hypothesis that the favorable properties of PNAs offer advantages for recognition of mRNA and antisense inhibition of gene expression in vivo. We have targeted 27 PNAs to 18 different sites throughout the 5'-untranslated region (5'-UTR), start site, and coding regions of luciferase mRNA. PNAs were introduced into living cells in culture as PNA-DNA-lipid complexes, providing a convenient high throughput method for cellular delivery. We find that PNAs targeted to the terminus of the 5'-UTR are potent and sequence-specific antisense agents. PNAs fifteen to eighteen bases in length were optimal inhibitors. The introduction of one or two mismatches abolished inhibition, and complementary PNAs targeted to the sense strand were also inactive. In striking contrast to effective inhibition by PNAs directed to the terminal region, PNAs complementary to other sites within the 5'-UTR do not inhibit gene expression. We also observe no inhibition by PNAs complementary to the start site or rest of the coding region, nor do we detect inhibition by PNAs that are highly C/G rich and possess extremely high affinities for their target sequences. Our results suggest that PNAs can block binding of the translation machinery but are less able to block the progress of the ribosome along mRNA. The high specificity of antisense inhibition by PNAs emphasizes both the promise and the challenges for PNAs as antisense agents and provides general guidelines for using PNAs to probe the molecular recognition of biological targets inside cells.

Cell cycle components and their potential impact on the development of continuous in vitro penaeid cell replication.

In vitro prawn cell culture has yet to produce an established cell line. In an effort to establish some understanding of the cellular blockage that prohibits their division in vitro we conducted several studies to characterize the cytoskeletal components of hemocytes and found no cells undergoing mitosis. Following this discovery, a molecular analysis of cell division regulatory proteins was performed. Cell cycle regulatory proteins (cyclins) have been identified as essential components in the progression of all eukaryotic cells through the cell cycle. We report here the identification of cyclin A and cyclin B proteins and their cofactor (p34(cdc2)) in making up the mitosis promoting factor (MPF) in protein extracts from egg and muscle tissues of Penaeus vannamei. Molecular weight analysis confirmed the size of the target proteins to be similar to the same proteins identified in the Atlantic surf clam (Spisula solidissima).

A method to perfuse liver metastases with heated blood (45 degrees C, 45 min).

To heat the livers in ten pigs (20-30 kg) a system of two pumps, both connected to the cannulated A.femoralis and to two radio-wave heating devices, was used. With pump I the arterial vascular system of the liver was perfused for 45 min via the A.gastroduodenalis with a constant volume of 150-200 ml blood/min, heated to 44-45 degrees C. With pump II 50-700 ml/min heated blood was pumped into the portal vein to raise the temperature and oxygen content of the portal flow. During the heating period the A.hepatica was clamped. A temperature of 45 degrees C is a highly toxic for the differentiated liver tissue, but the more simply structured wall of the arteries tolerates this temperature. This difference in heat resistance allows the perfusion of the arterial vascular system of the liver with blood heated to a temperature that is fatal to liver tissues. On the way from the A.gastroduodenalis to the liver periphery the heated blood becomes cooled by the surrounding cooler liver tissues. Finally the supply to the lobules is a mixture of arterial and venous blood, i.e. the highly toxic agent, heat, becomes "detoxified" by cooling before reaching the heat-sensitive liver lobules. Changing flow and temperature in the portal vein allows the temperature of the liver lobules to be kept within a safe range, i.e. below 43 degrees C. The raised oxygen content in the portal flow allows the arterial perfusion to be stopped for 10 min (to subject a heated metastasis to a period of hypoxia). Liver enzymes reached their maximal level 2 days after heating and returned to normal within 1 week. It is supposed that this method allows the temperature in a liver metastasis to be raised to a tumoricidal level.

[Perfusion hyperthermia of the liver in swine--presentation of procedure for precise control of extreme hyperthermia]. / Perfusionshyperthermie der Leber bei Schweinen--Darstellung eines Verfahrens zur präzisen Steuerung der extremen Hyperthermie.

Porcine liver was perfused by heated blood for 45 min. Vascular isolation was not needed as blood was collected from the aorta, heated and infused in the portal vein by a roller pump. Blood temperature was 43 degrees C, 43.2 degrees C, 43.6 degrees C and 44 degrees C. During perfusion, the liver was thermally insulated by a double layer polyethylene sheet. One pig died during perfusion because of a cardiac arrest, another pig after perfusion of 44 degrees C because of an acute liver failure. Serological examinations after perfusion showed a transient increase of liver enzymes. Morphological changes were obvious but seemed to be reversible. These examinations proved that by this method porcine liver can be heated precisely and homogeneously at temperatures required for extreme hyperthermia.

Correlation between shear dependent blood viscosity, electrical resistance and calculated width of the marginal layer in blood perfused capillary tubes.

Viscosity and shear dependent changes in electrical resistance (delta R) were measured simultaneously in blood perfused capillaries (I.D. = 0.58 mm, 1 = 300 mm, TW = 0.04 to 0.5 Pa). It appears that delta R is primarily caused by the development of a low resistant cell free marginal layer. Red cell aggregation strongly supports the development of delta R, while in non-aggregating blood the shear induced change in conductivity is rather small. Theoretical and experimental data become closely correlated if the calculated width of the marginal layer, being 1 to 5% of the capillary radius, is inserted into a modified form of Poiseuille's law. It is concluded that under the conditions of plug flow the non-Newtonian flow behavior of EDTA blood almost exclusively depends upon the width of the marginal layer, being a function of shear stress and hematocrit.

Dependence of capillary flow resistance upon the width of the marginal layer and the viscosity of the axial core.

Capillary blood flow may be considered as the flow of two compartments a.) the low viscous cell free marginal layer, surrounding b.) the higher viscous axial core. If the flow of both compartments is calculated separately according to Poiseuille, the addition of the two calculations allows to adapt Poiseuille's law to the non-Newtonian blood flow in small capillaries. The modified law correlates theoretical and experimental data with R2 = 0.99. (H = 0.3 - 0.7, tau W = 0.1 - 1.2 Pa, blood viscosity 5 to 20 mPa X s, capillary I.D. 0.3 mm). The width of the marginal was calculated by relating the shear dependent change in electrical resistance in blood perfused capillaries to the development of a cell free marginal layer. The viscosity of the core was determined with a bended glass fibre (phi = 10 microns).

The missing negative effect of red cell aggregation upon blood flow in small capillaries at low shear forces.

Flow resistance of EDTA or defibrinized blood was measured in a vertical capillary viscometer. (Capillary I.D. = 0.3 mm, 1 = 475 mm, tau w = 0.27 to 0.036 Pa). With decreasing shear forces the apparent viscosity increased from 12 to 16 mPa X s, i.e. by about 30%. There was no significant difference in flow resistance between red cells suspended in plasma or serum. The apparent viscosity of the same blood sample increased from 14 to 45 mPa X s for RBC in plasma i.e. by about 320% if measured with a rotational viscometer. The attempt is made to explain this difference in flow resistance with the different effect of shear resistant RBC-aggregates upon a parabolic streaming profile in a capillary, or upon the quasi linear profile within the annular gap of a rotational viscometer.

Red cell flexibility, electrical resistance and viscosity of blood flowing through small glass capillaries.

The influence of varying shear forces (4-10 microN.cm-2) generated by a pulsating flow of 4 cycles/min, on the longitudinal electrical resistance (R) of a blood perfused small glass capillary (I.D. = 0.12 mm, l = 30 mm) was determined. Red cells were stiffened by stepwise addition of bile or by sterile incubation during 24-48 h. The shear dependent changes in R were closely related to red cell flexibility and apparent blood viscosity. In normal defibrinized blood R decreased by about 3%, while more rigid cells evoked a shear dependent increase in R of 1-5%. The measurements performed demonstrate that the typical shapes of the electrical signals provide more information of rheological significance of red blood cell flexibility than the results of viscosity determination alone.