Time course of superoxide generation by leukocytes—The MCLA chemiluminescence system

This study was performed to examine the pattern of Superoxide (O ₂ ^– ·) generation from leukocytes using the O ₂ ^– · specific chemiluminescence (CL) method.Cypridina luciferin analog, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one (MCLA) was used as a CL probe. The appropriate conditions of the MCLA method was first determined for the evaluation of the time course of O ₂ ^– · generation by leukocytes. The time course of O ₂ ^– · generation obtained by the MCLA-CL system was compared with that by the luminol-dependent CL, electron spin resonance (ESR)/spin trapping, and cytochromec systems. Following stimulation by three different stimulants (PMA, OZ, FMLP), leukocytes continuously generated O ₂ ^– · for up to 5 h in the MCLA-CL system, irrespective of the kind of stimulation. The curves obtained by generation ceased more rapidly in the luminol-CL, ESR/spin trapping, and cytochromec systems. A 50% activity of the initial value was observed at 70 min in the MCLA-CL system, but 30, 10 and 35 min in the other systems, respectively. The CL or O ₂ ^– · generation value decreased to less than 1% (possible termination) at 300, 90, 120 and 180 min, respectively. With the exception of ESR studies with OZ, the cell viability was not significantly affected in any of the trials. These results indicate that leukocytes can generate O ₂ ^– · much longer than previously estimated and that the MCLA-CL-system is the most suitable system for the measurement of the O ₂ ^– · generation by leukocytes.     

Improved DOP-PCR-based representational whole-genome amplification using quantitative real-time PCR.

In many cases, only a minute amount of partially degraded genomic DNA can be extracted from archived clinical samples. Diverse whole-genome amplification methods are applied to provide sufficient amount of DNA for comparative genome hybridization, single-nucleotide polymorphism, and microsatellite analyses. In these applications, the reliability of the amplification techniques is particularly important. In PCR-based approaches, the plateau effect can seriously alter the original relative copy number of certain chromosomal regions. To eliminate this distorting effect, we improved the standard degenerate oligonucleotide-primed PCR (DOP-PCR) technique by following the amplification status with quantitative real-time PCR (QRT-PCR). With real-time detection of the products, we could eliminate DNA overamplification. Probes were prepared from 10 different tumor samples: primary and metastatic melanoma tissues, epidermoid and bronchioloalveolar lung carcinomas, 2 renal cell carcinomas, 2 colorectal carcinomas, and a Conn and Cushing adenoma. Probes were generated by using nonamplified and amplified genomic DNA with DOP-PCR and DOP-PCR combined with QRT-PCR. To demonstrate the reliability of the QRT-PCR based amplification protocol, altogether 152 relative copy number changes of 44 regions were determined. There was 85.6% concordance in copy number alterations between the QRT-PCR protocol and the nonamplified samples, whereas this value was only 63.8% for the traditional DOP-PCR. Our results demonstrate that our protocol preserves the original copy number of different chromosomal regions in amplified genomic DNA than standard DOP-PCR techniques more accurately.     

gamma-Aminobutyric acid enables synaptogenesis in the intact superior cervical ganglion of the adult rat

Local gamma-aminobutyric acid (GABA) application into the intact superior cervical ganglion (SCG) of the adult rat allows active innervation of a surgically implanted hypoglossal nerve in addition to the normal nerve supply of the ganglion. In GABA-treated SCG of the adult rat, action potentials could be obtained on stimulation of both the preganglionic nerve trunk and the implanted hypoglossal nerve. Both action potentials were reversibly sensitive to hexamethonium bromide indicating new cholinergic synapses established between axons in the hypoglossal nerve and principal sympathetic neurons. If GABA treatment of the ganglion was omitted, the double innervation did not develop after hypoglossal nerve implantation.     

Nitric oxide synthase immunoreactivity of interstitial cells of Cajal in experimental colitis

OBJECTIVE: There is functional and morphological evidence that interstitial cells of Cajal (ICC) may play a role in nitric oxide (NO) dependent signal transduction. However, little is known about the nitric oxide synthase (NOS) containing ICC during inflammation. MATERIALS AND METHODS: Immunocytochemical methods were used for the ultrastructural localization of NOS1-containing ICC in the wall of the colon of rats in experimental colitis. RESULTS: Large numbers of NOS immunoreactive (IR) nerve terminals were found in very close vicinity to smooth muscle cells as well as to blood vessels. IR nerves were found in close relationship with the ICC. The gap between the NOS IR nerve fibers and the membrane of smooth muscle cells and of ICC was 20-250 nm. In experimental colitis the number of NOS IR nerve fibers slightly decreased, however, large numbers (24%) of the ICC became IR for NOS. In the noninflamed area and in the controls, all these cells were immunonegative for NOS. CONCLUSIONS: Our light- and ultrastructural study suggests that some of the ICC can also synthesize NO, at least during inflammation. Therefore the change in the number and structure of ICC could play an important role in the pathogenesis of a variety of motility disorders.     

Light and electron microscopic immunocytochemical localization of vasoactive intestinal polypeptide VIP-like activity in the rat small intestine

Vasoactive intestinal polypeptide nerve processes and cell bodies were identified by electron microscopic immunocytochemistry in the rat small intestine. Labeled nerve processes were numerous in the inner circular smooth muscle coat and mainly in the mucosa, but were absent in the longitudinal muscle layer. Submucosal blood vessels were often surrounded by immunoreactive vasoactive intestinal polypeptide positive nerves, in close associations (distance less than 40 mn) to blood vessel basement membranes and to smooth muscle cells. In the ganglia of the myenteric and submucous plexuses, labeled fibers surrounded unstained neural cell bodies. The synaptic vesicles of vasoactive intestinal polypeptide positive terminals were 35-40 nm in diameter and some dense core vesicles (80-120 nm in diameter) were also observed in the same profiles. These observations suggest that vasoactive intestinal polypeptide nerves may participate in regulating smooth muscle activity and local blood flow in the small intestine.     

A second field metachronous Merkel cell carcinoma of the lip and the palatine tonsil confirmed by microarray-based comparative genomic hybridisation

Merkel cell carcinoma was diagnosed in a 79-year-old Caucasian woman. The tumour was localised to the upper lip and was in stage T2. After successful cryosurgery and a 7-year tumour-free period, a new tumour developed in her palatine tonsil. Histologically and immunohistochemically, this resembled the tumour in the lip. The regional lymph nodes were devoid of metastasis. The paraffin-embedded material of the two tumours and the unaffected lymphatic tissue were analysed with DNA microarrays for comparative genomic hybridisation to assess the genetic relationship of the tumours. In both tumours, regions on 2p and 10p were commonly over-represented, while 41 regions on chromosomes 1–4, 6, 8–9, 11 and 14–22 were commonly under-represented. Chromosomes 1, 3, 4, 16–18 and X were most frequently involved in the DNA losses. In gene copy numbers in the two tumours, 31 chromosome locations were found to be differently affected. The partly similar and partly different molecular patterns indicated a genetic relationship between the tumours and excluded the possibility that the tonsillar tumour was a metastasis. The findings suggest that a genetically altered field was the reason for the development of the tonsillar cancer; thus, it can be regarded pathogenetically as a second field tumour.     

Contributions to the kinetics of the acetylcholine-receptor function

Summary 1. Action-concentration curves have been plotted for the stimulation of the cat's superior cervical ganglion by acetylcholine. The free and the innervated receptors of the postsynaptic membrane, described by the authors, have been studied separately from this point of view.2. In the case of both the free and the innervated receptors, the action-concentration curves were similar in shape to the enzyme-substrate curve of cholinesterase. Analyzing these curves by the method of Lineweaver and Burk, in the case of free receptors, the dissociation constants of acetylcholine turned out to be: with stimulating RS complexes: 5.4·10^–6–2.1·10^–5M, with inhibitory RS ₂ complexes: 3.2·10^–5 – 5.2·10^–5 M. The pertaining values for innervated receptors were 3.45 · 10^–5 and 2.22 · 10^–4 M, respectively.3. The parallelisms and divergences between the cholinesterase and acetylcholine-receptor functions are discussed.With 4 Figures in the Text     

Synthesis and reactivity of debenzorutaecarpine derivatives]

A series of 7,12-dihydropyrimido[1',2';1,2]pyrido[3,4-b]indol-4(6H)-ones was prepared by Fischer indolization of 9-arylhydrazono-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one s. Quantumchemical calculations (ab initio and AM1) indicate that position 3 of 7,12-dihydro-pyrimido[1',2';1,2]pyrido[3,4-b]indol-4(6H)-one can be involved in electrophilic substitutions, while position 2 is sensitive towards nucleophilic attack. Bromination of 6-methyl-7,12-dihydropyrimido[1',2';1,2]pyrido[3,4-b]indol-4(6H)-o ne (16) with bromine afforded 3-bromo derivative (25), which was reacted with cyclic amines to give 2-amino-7,12-dihydro-pyrimido[1',2';1,2]pyrido[3,4-b]indol-4(6H)-ones (26-30) in an addition-elimination reaction. Vielsmeier-Haack formylation of compound (16) give 12-formyl (31) and 3,12-diformyl (32) derivatives (an N-formyl-1-aza derivative of nauclefidine alkaloid (34) at 60 degrees C and 100 degrees C, respectively. 3,12-dformyl compound (32) was oxidized to 3-carboxyl derivative (33). The quaternary salt (35), obtained from compound (16) with dimethyl sulphate, suffered a ring opening on the action of aqueous sodium hydroxide. The new compounds have been characterized by elemental analyses uv, 1H nmr and in some cases by 13C ruler, CD spectra and X-ray investigations.     

The role of oxidative stress, caused by amiodarone, in the side effects of the drug

It was supposed that free radicals are produced during the metabolism of amiodarone and involved in the mechanism of the drug's side effects. In vitro and in vivo experiments were performed in rats to check this hypothesis. We verified that amiodarone generated free radicals in vitro by chemiluminometric method. The light emission induced by amiodarone was inhibited by dihydroquinoline type antioxidants CH 402 and MTDQ--DA in a dose dependent manner. The malondialdehyde content, --one of the end products of lipid peroxidation--was increased by in vivo amiodarone administration in the serum and liver homogenate of rats. Amiodarone treatment increased significantly the NADPH and Fe3+ induced lipid peroxidation in rat liver microsomal fractions. The protective effect of antioxidants, MTDQ--DA and silibinin were ambiguous in these in vivo "short term" experiments.