Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide.

A polyamide nucleic acid (PNA) was designed by detaching the deoxyribose phosphate backbone of DNA in a computer model and replacing it with an achiral polyamide backbone. On the basis of this model, oligomers consisting of thymine-linked aminoethylglycyl units were prepared. These oligomers recognize their complementary target in double-stranded DNA by strand displacement. The displacement is made possible by the extraordinarily high stability of the PNA-DNA hybrids. The results show that the backbone of DNA can be replaced by a polyamide, with the resulting oligomer retaining base-specific hybridization.

An easy method using glutaraldehyde-introduced fluorescence for the microscopic analysis of plant biotrophic interactions.

The method introduced in this article makes use of the glutaraldehyde-induced auto-fluorescence of proteins after cross-linking with glutaraldehyde for the analysis of cellular and sub-cellular structures. Because the interface of biotrophic interactions is rich in proteins, the method presented is particularly suitable for the analysis of such interactions; we have exemplified its usefulness by analyzing (1) the root feeding sites induced in roots from Arabidopsis thaliana by the root-knot nematode Meloidogyne incognita; (2) leaves from Cucurbita pepo infected by powdery mildew and (3) roots from Nicotiana tabacum colonized by the arbuscular mycorrhizal fungus Glomus intraradices. The use of confocal and multi-photon laser scanning microscopy allows three-dimensional reconstructions from optical sections of complex biotrophic interactions. In the case of root-knot nematode feeding sites, our method enabled us to simultaneously study the development of the plant xylem elements (using lignin auto-fluorescence), the nematode feeding site and the nematode itself.

Effect of antitumor diarylsulfonylureas on in vivo and in vitro mitochondrial structure and functions.

Diarylsulfonylureas are novel oncolytic agents shown to have therapeutic activity against both rodent solid tumors and xenografts of human tumors in mice. Previous studies have shown that diarylsulfonylureas localize in mitochondria and cause morphological changes in these organelles. We have investigated the mechanism of action of diarylsulfonylureas, namely, N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea (ISCU) and the N-4-methyl analogue (MPCU), by studying their effect on mitochondrial morphology and uptake of rhodamine 123 in GC3/c1 cells in culture and the oxidative phosphorylation in isolated mitochondria from mouse liver, using pyruvate-malate and succinate as substrates. Morphometric analysis of mitochondria in GC3/c1 cells exposed to ISCU showed that ISCU (165 microM) doubled the mitochondrial size after 24-h exposure in culture. Also, ISCU (100 microM), like 40 microM carbonylcyanide p-trifluoromethoxyphenylhydrazone, significantly reduced the rhodamine 123 uptake by GC3/c1 cells studied by flow cytometry. In isolated mitochondria both ISCU and MPCU uncoupled oxidative phosphorylation at 50 microM, with pyruvate-malate as substrate, as was indicated by a significant increase in the State 4 oxygen consumption. This resulted in the loss of ADP phosphorylation and, therefore, the ADP/oxygen ratio was reduced to zero and the respiratory control ratio to one. The succinate oxidation was also significantly impaired by ISCU, causing some decrease in ADP phosphorylation. On the other hand, MPCU did not exhibit any significant effect on the oxidation of succinate. At concentrations of lower than 50 microM, both of these compounds exhibited a deleterious effect, causing damage to mitochondrial functions in the presence of pyruvate-malate as substrates. These data confirm, through morphometric analysis, our previous qualitative observations of abnormal mitochondrial morphology observed in GC3/c1 cells grown in the presence of high concentrations of ISCU and MPCU and further suggest that diarylsulfonylureas, by uncoupling mitochondrial oxidative phosphorylation, may lower cellular ATP. It is probable that this mechanism contributes, at least partially, to cytotoxicity in GC3/c1 cells exposed to high concentrations of ISCU for relatively brief periods (2 to 4 h) and possibly contributes to cytotoxicity at drug concentrations that can be achieved in rodents.

Peptide nucleic acids and their potential applications in biotechnology.

Peptide nucleic acids (PNAs) are novel DNA mimics in which the sugar-phosphate backbone has been replaced with a backbone based on amino acids. PNAs exhibit sequence-specific binding to DNA and RNA with higher affinities and specificities than unmodified DNA. They are resistant to nuclease and protease attack in serum and cellular extracts and, thus, appear very promising as diagnostic and biomolecular probes, and possibly as antisense and antigene drugs.

Characterisation of the rat and mouse homologues of gC1qBP, a 33 kDa glycoprotein that binds to the globular 'heads' of C1q.

gC1qBP is a 33 kDa glycoprotein that binds to the globular 'heads' of C1q. We have cloned cDNAs encoding the rat and mouse homologues of gC1qBP. Comparison of the cDNA-derived amino acid sequences of gC1qBP reveals that either of the rodent sequences is 89.9% identical to the reported human sequence. Recombinant rat gC1qBP binds avidly to human C1q. gC1qBP mRNA is abundantly expressed in every rat and mouse tissue analysed. Rat mesangial cells synthesise gC1qBP, but do not express gC1qBP on the cell surface. In rat serum, gC1qBP is present at low levels.

Evidence of a myoepithelial cell in tick salivary glands.

Type III acini from feeding female Dermacentor variabilis varied in size during in vitro and in vivo fluid production. As the type III acinus enlarged, its lumen enlarged and the adlumenal cell became thinner. As the acinus contracted, its lumen became smaller while the adlumenal cell became wider. Actin was demonstrated in salivary glands using an immunoblot technique. Actin was localized in the adlumenal cells of type III acini with fluorescent microscopy using rhodamine-phalloidin and with electron microscopy using heavy meromyosin to decorate actin filaments. Pre-treatment of salivary glands with cytochalasin D abolished fluorescence in adlumenal cells subsequently treated with rhodamine-phalloidin. These results support the hypothesis that the adlumenal cell in type III acini functions as a myoepithelial cell.

Pump-induced platelet aggregation in albumin-coated extracorporeal systems.

OBJECTIVE: Coating of extracorporeal systems with heparin does not prevent platelet activation and subsequent bleeding disorders. We investigated whether this could be due to elevated shear stress caused by a roller pump. METHODS: Human or rat blood was made to flow through an uncoated or an albumin-coated medical polyvinyl chloride tube with or without a roller pump. Aggregation of platelets in the tubing was recorded continuously with a photometric device. RESULTS: Although in vitro gravitational flow in uncoated tubes caused immediate platelet aggregation and platelet loss, this remained absent in coated tubes. When the pump was started in experiments with a coated tube strong platelet aggregation was observed and platelet count fell within 5 minutes to 78% +/- 2% and 71% +/- 3% of control values in human and rat blood, respectively. In vivo, no aggregation was observed during spontaneous flow in rats with an albumin-coated tube running from the carotid artery to the femoral artery, but aggregation started as soon as the blood was pumped. Pump-induced platelet aggregation, both in vitro and in vivo, could be prevented with aurintricarboxylic acid, which specifically inhibits shear-induced platelet aggregation as has recently been shown. Pump perfusion of blood in an uncoated tube did not elicit platelet aggregation. CONCLUSIONS: Pump perfusion of blood in coated systems elicits shear-induced platelet aggregation, which may be prevented by administration of substances that block the binding of von Willebrand factor to glycoprotein Ib receptors on the platelets. The effects of pumping on platelets are masked in uncoated circuits because of the dominant influence of blood-material contact.

A new physiological biomarker for nitrate exposure in humans.

After toxicological studies with nitrate/nitrite in rats it was observed with nuclear magnetic resonance that N-methylnicotinamide (NMN), a metabolite of tryptophan was increased. The use of NMN as a biomarker for nitrate/nitrite exposure was investigated further in additional experiments with rats and in a human study with volunteers. Rats have been exposed to 36 mmol KCl, KNO2 or KNO3 per 1 tap water for 13 weeks. In general, the animals receiving KNO2 showed a statistically significant (P < 0.01) 2-fold increase in NMN compared with the KCl group. This increase was observed after a relatively high exposure (about 800 mg/kg body wt./day). It was also noticed that the initial increase in urinary NMN concentrations decreased after prolonged exposure for 12 weeks. To investigate the induction of urinary NMN in humans, an experiment has been performed in which 8 volunteers received a single oral dose of sodium nitrate, corresponding with 10 mg NaNO3/kg body wt./day (2 times the acceptable daily intake for nitrate). A rapid increase of urinary NMN (up to 6-fold) was observed in 4 volunteers. In the other 4 volunteers the urinary NMN concentration did hardly react. When the experiment was repeated with the same volunteers, it was remarkable to see that in this experiment all volunteers showed the same individual response on urinary NMN as in the first experiment. It is concluded that NMN can possibly be a good biomarker for the internal nitrite exposure of humans, but further studies are necessary to assess its value.

Development of a chemiluminescent immunoassay for 19-nortestosterone (nandrolone).

An immunoassay for nortestosterone with detection by chemiluminescence is described, and applied to the urine and drug administration site of slaughtered cattle.

Identification of several stilbene derivatives in bovine urine by means of high performance liquid chromatographic fractionation and immunochemical detection.

Radioimmunochemical detection (RIA) following fractionation of urine extracts via high performance liquid chromatography (HPLC) turned out to be a very specific method for the identification of stilbene derivatives in bovine urine. Combination of the high resolution of the HPLC with a specific RIA is a suitable method to discriminate between the presence of different stilbene derivatives like diethylstilboestrol (DES), dienoestrol (DE) and hexoestrol (HEX) or other unknown compounds interfering in the celite-RIA used in the screening. Using this screening method 8200 samples of bovine urine were investigated on the presence of stilbene derivatives of which 133 were classified as 'positive'. In 106 'positive' urines the presence of DES was shown and in 19 'positive' urines the presence of DE or HEX, using the method described in this report whereas in 8 'positive' urines an unknown immunochemical active compound was detected. During 1.5 year of comparative investigation no qualitative discrepancies occurred between the results of the HPLC-immunogram procedure and the final confirmation by high resolution gas chromatography-mass spectometry (GCMS).

Application of diethylstilbestrol dipropionate in bulls. II. Residues in bile, liver, kidney, muscle tissues and application site.

The second part of an experiment is described in which 20 one year old bulls were injected with diethylstilbestrol (DES) dipropionate containing preparations. Analysis of DES content was performed in several tissues, such as the injection site, diaphragm muscle, psoas muscle, liver, kidney and bile. In the injection site appreciable amounts of DES were found. Measurable amounts of DES were also found in liver and kidney until 4 weeks after injection. In bile, DES concentrations were even higher than those in urine, and were well correlated with DES concentrations in urine. Implications for screening purposes are discussed.

Changing prevalence of asthma in Australian children.

OBJECTIVE: To investigate whether prevalence of asthma in children increased in 10 years. DESIGN: Serial cross sectional studies of two populations of children by means of standard protocol. SETTING: Two towns in New South Wales: Belmont (coastal and humid) and Wagga Wagga (inland and dry). SUBJECTS: Children aged 8-10 years: 718 in Belmont and 769 in Wagga Wagga in 1982; 873 in Belmont and 795 in Wagga Wagga in 1992. MAIN OUTCOME MEASURES: History of respiratory illness recorded by parents in self administered questionnaire; airway hyperresponsiveness by histamine inhalation test; atopy by skin prick tests; counts of house dust mites in domestic dust. RESULTS: Prevalence of wheeze in previous 12 months increased in Belmont, from 10.4% (75/718) in 1982 to 27.6% (240/873) in 1992 (P < 0.001), and in Wagga Wagga, from 15.5% (119/769) to 23.1% (183/795) (P < 0.001). The prevalence of airway hyperresponsiveness increased twofold in Belmont to 19.8% (173/873) (P < 0.001) and 1.4-fold in Wagga Wagga to 18.1% (P < 0.05). The prevalence of airway hyperresponsiveness increased mainly in atopic children only, but the prevalence of atopy was unchanged (about 28.5% in Belmont and about 32.5% in Wagga Wagga). Numbers of house dust mites increased 5.5-fold in Belmont and 4.5-fold in Wagga Wagga. CONCLUSIONS: We suggest that exposure to higher allergen levels has increased airway abnormalities in atopic children or that mechanisms that protected airways of earlier generations of children have been altered by new environmental factors.

Aggregation of TMV CP plays a role in CP functions and in coat-protein-mediated resistance.

Tobacco mosaic virus (TMV) coat protein (CP) in absence of RNA self-assembles into several different structures depending on pH and ionic strength. Transgenic plants that produce self-assembling CP are resistant to TMV infection, a phenomenon referred to as coat-protein-mediated resistance (CP-MR). The mutant CP Thr42Trp (CP(T42W)) produces enhanced CP-MR compared to wild-type CP. To establish the relationship between the formation of 20S CP aggregates and CP-MR, virus-like particles (VLPs) produced by TMV variants that yield high levels of CP-MR were characterized. We demonstrate that non-helical structures are found in VLPs formed in vivo by CP(T42W) but not by wild-type CP and suggest that the mutation shifts the intracellular equilibrium of aggregates from low to higher proportions of non-helical 20S aggregates. A similar shift in equilibrium of aggregates was observed with CP(D77R), another mutant that confers high level of CP-MR. The mutant CP(D50R) confers a level of CP-MR similar to wild-type CP and aggregates in a manner similar to wild-type CP. We conclude that increased CP-MR is correlated with a shift in intracellular equilibrium of CP aggregates, including aggregates that interfere with virus replication.

Evaluation of spectral imaging for plant cell analysis.

Fluorescence imaging at high spectral resolution is now a practical reality and has great promise in plant cell biology. Emission spectral curve data can be used computationally to distinguish spectrally similar fluorophores, or to remove autofluorescence, and to spectrally analyse autofluorescent molecules, which are especially abundant in plant tissues. Examples of these applications in plant cells are given, and a comparison is made between the current offerings in spectral imaging laser scanning confocal microscopes.

High-performance liquid chromatographic investigation of product formation in the horseradish peroxidase-enhanced chemiluminescence of luminol with different enhancers.

A reversed-phase high-performance liquid chromatographic method for the investigation of the chemiluminescence-producing oxidation of luminol by the enzyme horseradish peroxidase is presented. Both the kinetics and the mechanism of product formation can be monitored. Special attention is paid to the mechanism of enhancement of the chemiluminescence by phenolic compounds, such as p-iodophenol, p-hydroxycinnamic acid and 6-hydroxybenzothiazole. The function of the enhancers was elucidated partially by the observation of a higher degradation rate of luminol. In addition, it was concluded that the mechanism of enhancement is probably different for the various enhancers, based on the product formation.

Murine T cell responses to melittin and its analogs.

The 26-residue peptide melittin from bee venom elicits high IgG1 and IgE responses in selected strains of mice. The antibody responses were shown previously to be specific mainly for the region of residue 20-26. The T cell epitope of melittin in H-2d-restricted mice is now found to be primarily in residue 11-19, corresponding to an alpha-helical amphiphilic segment of the molecule. Melittin-specific T cell lines have varying responses to different structural analogs of the melittin T cell epitope, and the results indicate that the antigenicity of T cell epitope peptides depend more on their primary structure than on their secondary structure. Melittin-specific T cell clones are found to be CD4+ and secrete IL-4, and are restricted to presentation on I-Ad or I-Ed. The I-Ad- or I-Ed-restricted clones differ in their responses to different analogs of melittin.

A chemiluminescent immunoassay for 17 alpha-methyltestosterone.

An immunoassay for the xenobiotic anabolic compound methyltestosterone (MT) is presented. The detection is based on chemiluminescence of a MT-isoluminol conjugate. Applications are presented for detection of methyltestosterone and cross-reacting compounds in bovine urine and tissues of application sites isolated from slaughtered cattle, illegally treated with hormonal anabolics.

A specific radioimmunoassay for the detection of 19-nortestosterone in urine.

The antisera used were raised in rabbits against a 19-nortestosterone-7 alpha-carboxyethylthioether conjugate of bovine serum albumin. Tritium-labeled nortestosterone was used as tracer. Cross reactivities with metabolites of nandrolone, other anabolics and endogenous steroids were very low. To achieve additional specificity a clean up and separation procedure was developed, using isocratic high performance liquid chromatography. So far no qualitative discrepancies have been observed between the assay described here and confirmative determinations by combined high performance liquid chromatography-gas chromatography-mass spectrometry.

Peptide nucleic acids (PNAs): potential antisense and anti-gene agents.

The binding of peptide nucleic acids (PNAs) T10-LysNH2, T5CT4-LysNH2 and T2CT2CT4-LysNH2 to double-stranded DNA targets A10, A5GA4 and A2GA2GA4 was studied by nuclease S1 probing. It is found that the PNAs bind preferentially to their complementary targets, weaker to targets containing one mismatch and not to targets containing two mismatches. Using an RNA polymerase T3 in vitro transcription system, it is found that a PNA T10-LysNH2 bound downstream from the promoter causes transcription elongation arrest at the PNA binding site only when the PNA is bound to the template strand. Finally, it is shown that primer extension by Taq DNA polymerase on a single-stranded template is arrested at an occupied PNA T10 binding site. These results are discussed in relation to PNAs as potential anti-sense and anti-gene drugs.