Synthesis of a stable and specific surface plasmon resonance biosensor surface employing covalently immobilized peptide nucleic acids.

Biosensors allow the real-time and label-free observation of biochemical reactions between various ligands including antigen-antibody reactions and nucleic acids hybridizations. In our studies, we used a surface plasmon resonance biosensor to elucidate the hybridization characteristics of a peptide nucleic acid (PNA) ligand immobilized on sensor surfaces either through covalent or streptavidin-biotin coupling. A biotin-labeled PNA was employed in the latter approach whereas the covalent immobilization included the following steps: A maleimide group was attached to the N-terminal of the PNA using N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). To generate free thiol groups for coupling, a carboxylated dextran matrix of the sensor surface was activated with N-hydroxysuccinimide (NHS) and N-ethyl-N'-(dimethylaminopropyl)-carbodiimide (EDC) and thiolated by addition of cystamine dihydrochloride followed by reduction with 1, 4-dithioerythrite (DTE). Finally, the modified PNA was coupled to the sulfhydryl groups of the activated dextran matrix. Repetitive hybridizations of a single-stranded synthetic DNA oligomer to the PNAs demonstrated the superior stability of covalent immobilization compared to noncovalent immobilization. Differentiation of point mutations in the analyte molecule was accomplished at 40 degrees C using guanidine thiocyanate concentrations of 1.5-1.7 M. In further experiments, we showed that a perfectly matched PNA allows the detection of a single-stranded DNA at a sensitivity of less than 1% in a background of single-stranded DNA having a single C to T point mutation in the region complementary to the PNA. Consequently, covalently bound PNAs provide a stable and reproducible environment for the development of mutation-specific DNA analysis assays.

Cyst(e)ine is the transport metabolite of assimilated sulfur from bundle-sheath to mesophyll cells in maize leaves

The intercellular distribution of the enzymes and metabolites of assimilatory sulfate reduction and glutathione synthesis was analyzed in maize (Zea mays L. cv LG 9) leaves. Mesophyll cells and strands of bundle-sheath cells from second leaves of 11-d-old maize seedlings were obtained by two different mechanical-isolation methods. Cross-contamination of cell preparations was determined using ribulose bisphosphate carboxylase (EC 4.1.1.39) and nitrate reductase (EC 1.6.6.1) as marker enzymes for bundle-sheath and mesophyll cells, respectively. ATP sulfurylase (EC 2.7.7.4) and adenosine 5'-phosphosulfate sulfotransferase activities were detected almost exclusively in the bundle-sheath cells, whereas GSH synthetase (EC 6.3.2.3) and cyst(e)ine, gamma-glutamylcysteine, and glutathione were located predominantly in the mesophyll cells. Feeding experiments using [35S]sulfate with intact leaves indicated that cyst(e)ine was the transport metabolite of reduced sulfur from bundle-sheath to mesophyll cells. This result was corroborated by tracer experiments, which showed that isolated bundle-sheath strands fed with [35S]sulfate secreted radioactive cyst(e)ine as the sole thiol into the resuspending medium. The results presented in this paper show that assimilatory sulfate reduction is restricted to the bundle-sheath cells, whereas the formation of glutathione takes place predominantly in the mesophyll cells, with cyst(e)ine functioning as a transport metabolite between the two cell types.

Chest wall rigidity during fentanyl- and midazolam-fentanyl induction: ventilatory and haemodynamic effects.

In a double-blind randomised study, we examined if pretreatment with small doses of midazolam, given before anaesthesia induction with fentanyl, influences the occurrence of fentanyl-induced thoracic rigidity (FITR). At the same time, the effect of rigidity on the cardiovascular and respiratory system was assessed. Sixteen patients undergoing coronary artery bypass surgery were divided into two groups. The midazolam group (M) received 0.075 mg/kg midazolam i.v. and the placebo group (P) NaCl 0.9% 3 min before the start of fentanyl induction. During the induction period, FITR was assessed clinically on a 3-point scale. Haemodynamic and respiratory variables were collected before anaesthesia induction, at the end of the fentanyl infusion and 3 min after intubation. The incidence of FITR was high in both groups: 63% in Group M and 75% in Group P (n.s.); however, its severity was less in Group M. The appearance of rigidity affected the cardiovascular and the respiratory system: central venous and pulmonary capillary wedge pressures showed a sharp increase in patients with FITR accompanied by CO2 retention, due to an inability to ventilate these patients adequately. We conclude that small doses of midazolam do not prevent, but may attenuate, FITR and that the appearance of rigidity causes alterations of haemodynamic and respiratory variables during induction.

[Low-dose midazolam. Effect on the induction doses of fentanyl and on hemodynamics in the coronary patient]. / Midazolam à faible dose. Effet sur les doses d'induction de fentanyl et sur l'hémodynamique chez le malade coronarien.

A study was carried out to see whether the administration of a small dose of midazolam determined a reduction of the dose of fentanyl necessary for induction of anaesthesia. Sixteen patients undergoing coronary artery bypass surgery were randomly allocated to either of two groups. Patients in group M received 0.075 mg.kg-1 midazolam intravenously 3 to 5 min prior to induction with fentanyl (5 micrograms.kg-1.min-1), whereas patients in group P only received placebo. The mean dose of fentanyl administered to obtain complete loss of reaction to a painful stimulus was 20 +/- 3 micrograms.kg-1 in group M and 21.5 +/- 2.5 micrograms.kg-1 in group P (NS). However the small dose of midazolam associated with fentanyl caused a significant drop in blood pressure by 20%. After the administration of pancuronium (0.15 mg.kg-1), the patients in group P showed a significant increase in heart rate (+ 14 b.min-1), accompanied by an increase in cardiac index (+0.45 l.min-1.m-2). Pretreatment with midazolam seemed to protect the patient from this undesirable reaction. It was concluded that induction with a combination of a small dose of midazolam and fentanyl did not lead to a reduction in the dose of fentanyl necessary to obtain profound analgesia. However, it gave rise to a haemodynamic pattern quite distinct from that seen during induction with fentanyl alone.

[Accumulation of eosinophils in the nasal secretion in patients with bronchial asthma]. / Eosinophilenanreicherung im Nasensekret bei Asthma bronchiale.

From October 1977 to September 1979 69 out of 500 asthmatic patients were selected in whom case histories, skin tests and IgE blood-levels formed a sub-group with more or less pure allergic asthma and a sub-group with more or less pure intrinsic asthma. All the patients exhibited a large quantity of granulocytes in the bronchial and nasal secretions. Special attention was paid to the contents of eosinophils in the nasal smear and in the bronchial mucus. The intrinsic subgroup had a (non significantly) greater incidence of 100% eosinophils in the bronchial secretion than the allergic sub-group (p less than 0.1). Unexpectedly, the reverse was found in the nasal secretions: only 33% of the intrinsics (9 cases) and as many as 67% of the allergics (28 cases) exhibited 100% eosinophils in the nasal mucus (p less than 0.025). Thus, when discussing the two forms of asthma, allergic and intrinsic, it is always necessary to bear in mind the possible paradoxical behaviour between nasal and bronchial mucus: pure eosinophilia in the lower respiratory tract may often be found concomitantly with pure neutrophilia in the upper respiratory tract, when some of the criteria for intrinsic asthma are fulfilled.