Polyethyleneimine as a transmembrane carrier of fluorescently labeled proteins and antibodies.

Polyethyleneimine (PEI) has been used previously as a nonviral DNA transfer vector. In this article, we demonstrate its use as a vehicle for transmembrane delivery of proteins in cell culture conditions. Linking proteins to PEI required no other treatment beyond mixing them with PEI. The bond between PEI and protein combined at optimal ratios was maintained in electrophoresis, even in the presence of 2.5% sodium dodecyl sulfate (SDS). The optimal time for delivery of proteins was determined to be 24 h. We have successfully delivered an Alexa 488-labeled avidin protein into human glioblastoma cells. A functional antibody against the nuclear protein lamin was delivered into human fibroblasts and reacted with lamin inside live cells. PEI-based delivery of antibodies and fluorescently labeled proteins can be used for fluorescent detection, tracking, and evaluation of cellular protein function in vivo.

DNA probes using fluorescence resonance energy transfer (FRET): designs and applications.

Fluorescence resonance energy transfer (FRET) is widely used in biomedical research as a reporter method. Oligonucleotides with a DNA backbone and one or several chromophore tags have found multiple applications as FRET probes. They are especially advantageous for the real-time monitoring of biochemical reactions and in vivo studies. This paper reviews the design and applications of various DNA-based probes that use FRET The approaches used in the design of new DNA FRET probes are discussed.

Left ventricular hypertrophy in ascending aortic stenosis mice: anoikis and the progression to early failure.

BACKGROUND: To determine potential mechanisms of the transition from hypertrophy to very early failure, we examined apoptosis in a model of ascending aortic stenosis (AS) in male FVB/n mice. METHODS AND RESULTS: Compared with age-matched controls, 4-week and 7-week AS animals (n=12 to 16 per group) had increased ratios of left ventricular weight to body weight (4.7+/-0.7 versus 3.1+/-0.2 and 5. 7+/-0.4 versus 2.7+/-0.1 mg/g, respectively, P<0.05) with similar body weights. Myocyte width was also increased in 4-week and 7-week AS mice compared with controls (19.0+/-0.8 and 25.2+/-1.8 versus 14. 1+/-0.5 microm, respectively, P<0.01). By 7 weeks, AS myocytes displayed branching with distinct differences in intercalated disk size and staining for beta(1)-integrin on both cell surface and adjacent extracellular matrix. In vivo left ventricular systolic developed pressure per gram as well as endocardial fractional shortening were similar in 4-week AS and controls but depressed in 7-week AS mice. Myocyte apoptosis estimated by in situ nick end-labeling (TUNEL) was extremely rare in 4-week AS and control mice; however, a low prevalence of TUNEL-positive myocytes and DNA laddering were detected in 7-week AS mice. The specificity of TUNEL labeling was confirmed by in situ ligation of hairpin oligonucleotides. CONCLUSIONS: Our findings indicate that myocyte apoptosis develops during the transition from hypertrophy to early failure in mice with chronic biomechanical stress and support the hypothesis that the disruption of normal myocyte anchorage to adjacent extracellular matrix and cells, a process called anoikis, may signal apoptosis.

DNA damage and p21(WAF1/CIP1/SDI1) in experimental injury of the rat adrenal cortex and trauma-associated damage of the human adrenal cortex.

In vivo models are needed to study the reactions of tissues to DNA damage, such as the induction of the cyclin-dependent kinase inhibitor p21, indicating potential repair of the damage, versus apoptosis, indicating the elimination of the damaged cells. Damage to DNA occurs in tissues during shock, sepsis, and other critical medical conditions. Previous studies have found evidence of damage to the cortex of adrenal glands from organ donors who had undergone severe trauma prior to death. The present experiment studied rats under experimental interventions of clinical relevance to patients with conditions that put them at risk for damage to the adrenal glands. These interventions comprised ischaemia and reperfusion injury, sepsis following caecal ligation and puncture, acute pancreatitis, and administration of chemical agents (zymosan and acrylonitrile). All the interventions caused an increase in p21 mRNA as assessed by northern blotting and in situ hybridization. Increased nuclear p21 protein was shown by immunohistochemistry. All the interventions caused damage to DNA, as shown by labelling of available 3' termini of single-strand breaks with terminal transferase. The number of cells undergoing apoptosis, visualized by ligation of a hairpin oligonucleotide probe to double-strand breaks in DNA, was much lower. In rat adrenal glands, apoptotic cells were infrequent under all the conditions studied. They were more abundant in human organ donor adrenal glands that were previously shown to have extensive DNA damage accompanied by induction of p21. The similarity of the effects of a wide variety of surgical interventions and chemical agents suggest a common pathophysiological mechanism which is not specific to the initiating injury. Experimental injury of the rat adrenal cortex provides a model for investigating the role of organ DNA damage and of mediators of the response to DNA damage, such as p21.

Pathophysiologically relevant concentrations of tumor necrosis factor-alpha promote progressive left ventricular dysfunction and remodeling in rats.

BACKGROUND: Although patients with heart failure express elevated circulating levels of tumor necrosis factor-alpha (TNF-alpha) in their peripheral circulation, the structural and functional effects of circulating levels of pathophysiologically relevant concentrations of TNF-alpha on the heart are not known. METHODS AND RESULTS: Osmotic infusion pumps containing either diluent or TNF-alpha were implanted into the peritoneal cavity of rats. The rate of TNF-alpha infusion was titrated to obtain systemic levels of biologically active TNF-alpha comparable to those reported in patients with heart failure (approximately 80 to 100 U/mL), and the animals were examined serially for 15 days. Two-dimensional echocardiography was used to assess changes in left ventricular (LV) structure (remodeling) and LV function. Video edge detection was used to assess isolated cell mechanics, and standard histological techniques were used to assess changes in the volume composition of LV cardiac myocytes and the extracellular matrix. The reversibility of cytokine-induced effects was determined either by removal of the osmotic infusion pumps on day 15 or by treatment of the animals with a soluble TNF-alpha antagonist (TNFR:Fc). The results of this study show that a continuous infusion of TNF-alpha led to a time-dependent depression in LV function, cardiac myocyte shortening, and LV dilation that were at least partially reversible by removal of the osmotic infusion pumps or treatment of the animals with TNFR:Fc. CONCLUSIONS: These studies suggest that pathophysiologically relevant concentrations of TNF-alpha are sufficient to mimic certain aspects of the phenotype observed in experimental and clinical models of heart failure.

Biotin-labeled hairpin oligonucleotides: probes to detect double-strand breaks in DNA in apoptotic cells.

Hairpin oligonucleotides were synthesized with stems ending in a double-stranded structure, which can be ligated to double-strand breaks in DNA, and with loops that contain nucleotides modified by the attachment of biotin. These probes specifically and sensitively detect double-strand breaks in apoptotic cells. Localization of these probes is restricted to areas of chromatin characteristic of apoptosis, whereas much more diffuse labeling was obtained when all available 3' DNA ends were labeled by terminal transferase. In principle, hairpin oligonucleotide probes can be designed with any type of 3' or 5' overhang complementary to double-strand DNA termini being detected.

Presence of double-strand breaks with single-base 3' overhangs in cells undergoing apoptosis but not necrosis.

Apoptotic cells in rat thymus were labeled in situ in paraffin-embedded and frozen tissue sections by ligation of double-stranded DNA fragments containing digoxigenin or Texas red. Two forms of double-stranded DNA fragments were prepared using the polymerase chain reaction: one was synthesized using Taq polymerase, which yields products with single-base 3' overhangs, and one using Pfu polymerase, which produces blunt-ended products. Both types of fragment could be ligated to apoptotic nuclei in thymus, indicating the presence in such nuclei of DNA double-strand breaks with single-base 3' overhangs as well as blunt ends. However, in nuclei with DNA damage resulting from a variety of nonapoptotic processes (necrosis, in vitro autolysis, peroxide damage, and heating) single-base 3' overhangs were either nondetectable or present at much lower concentrations than in apoptotic cells. Blunt DNA ends were present in such tissues, but at lower concentrations than in apoptotic cells. In contrast, in all of these forms of DNA damage, nuclei contained abundant 3'-hydroxyls accessible to labeling with terminal deoxynucleotidyl transferase. Thus, although single-base 3' overhangs and blunt ends are present in apoptotic nuclei, the specificity of the in situ ligation of 3'-overhang fragments to apoptotic nuclei indicates that apoptotic cells labeled in this way can readily be distinguished from cells with nonapoptotic DNA damage. These data are consistent with the involvement of an endonuclease similar to DNase I in apoptosis, which is predicted to leave short 3' overhangs as well as blunt ends in digestion of chromatin.

A quantitative luminescence assay for nonradioactive nucleic acid probes.

In histochemical work with digoxigenin- or biotin-labeled nucleic acid probes, reproducibility of in situ hybridization depends on accurate measurement of the amount of non-radioactive label being used. We describe a rapid and sensitive assay for nonradioactive label incorporated into nucleic acids employing a luminogenic substrate for alkaline phosphatase, CSPD (disodium 3-(4-methoxyspirol¿1,2-dioxetane-3,2'-(5'-chloro)tricyclo [3.3.1.1(3,7)]decan¿-4-yl)phenyl phosphate). An alkaline phosphatase-antibody conjugate was bound to digoxigenin-labeled nucleic acids spotted on nylon membranes. Light emission from the reaction of the bound alkaline phosphatase with CSPD was measured with a luminometer. This method allows an accurate determination of digoxigenin incorporated into nucleic acid probes in the range of 0.5-500 fmol of nonradioactive label.

Expression of p21(WAF1/CIP1/SDI1) and p53 in apoptotic cells in the adrenal cortex and induction by ischemia/reperfusion injury.

p21(WAF1/CIP1/SDI1), an inhibitor of cyclin-dependent kinases, is expressed at varying levels in human adrenal glands removed during surgery or organ recovery. In glands with p21 mRNA, nuclear p21 immunoreactivity, which was occasionally extensive, colocalized with p53 immunoreactivity and DNA damage, as evidenced by in situ end-labeling. Many cells showed morphological features of apoptosis when observed by fluorescent DNA dye staining and electron microscopy. This pattern was also associated with high levels of cytoplasmic heat shock protein 70. To address the question of the origin of p21 expression in some human adrenal glands, rat adrenal glands were subjected to 30 min of ischemia followed by 8 h of reperfusion. Cells with nuclear p21 and p53 appeared in the adrenal cortex together with DNA damage detected by in situ end-labeling. Nuclear p21 immunoreactivity was also produced in adrenal tissue fragments incubated at 37 degrees C in vitro. However, in this case, p21 expression was confined to the cut edge of the tissue. In contrast, p21 in human adrenal glands, as in ischemic rat glands, was within the inner regions of the cortex, supporting an origin of the protein in vivo rather than postmortem. The p53/p21 pathway of reaction to cellular injury, potentially leading to apoptosis, may play a role in tissue damage such as that resulting from ischemia/reperfusion. In the human adrenal cortex this process may be a precursor of adrenal failure.

Increased expression of p21Sdi1 in adrenocortical cells when they are placed in culture.

Expression of the cell cycle inhibitor p21Sdi1/WAF1/Cip1 was investigated in a differentiated cell type, the adrenocortical cell, at different stages of culture, from the preparation of cells from the adrenal gland to senescence after long-term growth. In bovine adrenocortical cells, expression of SDI1 was much higher in culture than in vivo. Elevation of SDI1 mRNA, accompanied by elevation of the level of a protein reacting with anti-p21Sdi1 antibodies, was observed as early as 3 h after the start of the tissue dissociation procedure used to prepare cells for culture. This level of expression was then maintained during plating and subsequent long-term growth in culture. Growth and quiescence in bovine adrenocortical cells can be modulated by inclusion or removal of FGF from the culture medium. In these cells SDI1 mRNA was not increased by long-term mitotic quiescence resulting from FGF deprivation. In cultured fetal human adrenocortical cells, SDI1 mRNA was also detected at all stages of the culture life span, including 2 days after isolation of cells from the adrenal gland and plating in culture. Mid-life-span cells had higher SDI1 mRNA than senescent human fibroblasts. Clones of human adrenocortical cells nearing senescence in culture had somewhat higher SDI1 mRNA than early passage cells. Thus, SDI1 expression in adrenocortical cells is not associated with mitotic quiescence either in vivo or in vitro, yet isolation of the cells and culturing them exerts a powerful inductive influence on its expression.

Biotinylation of DNA on membrane supports: a procedure for preparation and easy control of labeling of nonradioactive single-stranded nucleic acid probes.

We have used M13 single-stranded DNA bound by uv to small pieces of nylon membrane for the synthesis of biotinylated single-stranded DNA probes. The labeling method requires a large fragment of DNA polymerase I and random hexanucleotides. There is no need for previous linearization of the template. The clean probe is removed from the membrane by a single wash step. The synthesized probe is completely free of unincorporated precursors. This makes possible the easy control of the reaction of incorporation of biotinylated analogues into the probe by simple staining on the filter, thus allowing evaluation of the efficiency of labeling. The DNA membrane can be stored for reuse. With the procedure described it is possible to biotinylate many DNA fragments in parallel, simultaneously controlling the efficiency of labeling in a time- and cost-saving manner.

[Opposite effects on antioxidant enzymes of adaptation to continuous and intermittent hypoxia]. / Protivopolozhnoe vliianie adaptatsii k nepreryvnoi i periodicheskoi gipoksii na antioksidantnye fermenty.

Adaptation to continuous hypoxia under mid-mountain conditions (altitude 2100 m) decreases the content of lipid peroxidation products and the activity of superoxide dismutase and catalase in rat heart, liver, and brain, with a concomitant decline in the resistance to reperfusion arrhythmias. On the contrary, adaptation to intermittent hypoxia in the altitude chamber increases the activity of the antioxidant enzymes in the same organs, while the content of peroxidation products remains normal; at the same time, the heart becomes more resistant to reperfusion arrhythmias. The mechanism is discussed that ensures enhanced antioxidant protection in adaptation to intermittent hypoxia.

[Limitation of ischemic necrosis by the use of antioxidant ionol]. / Ogranichenie ishemicheskogo nekroza s pomoshch'iu antioksidanta ionola.

Inhibitory effect of Ionol on lipid peroxidation (LPO) in the infarction zone and out of it after experimental myocardial infarction in the experiments on rats was investigated. The results of measurements, performed by two independent methods: point counting and the computer image analysis were compared. It was shown that LPO activation out of the ischaemic zone was prevented and dimensions of the ischaemic necrosis were limited by Ionol, which did not influence LPO activation in the ischaemic zone. Data obtained by both methods coincide qualitatively, the computer image analysis being more sensitive and effective.