Serum immunoassay of a small cell lung cancer associated ganglioside: development of a sensitive scintillation proximity assay

We here report an enzyme linked immunosorbent assay (ELISA) and a scintillation proximity assay (SPA) for detection of the ganglioside FucGM₁ in sera from small cell lung cancer (SCLC) patients. The SPA was more sensitive and reproducible than the ELISA. In this assay, monoclonal antibodies specific for FucGM₁ were bound to SPA particles and incubated with labelled FucGM₁ and 100 µl test-serum overnight, and counted in a -counter. The sensitivity was 0.2 ng. Seven out of twenty sera from SCLC patients were positive, whereas none of twenty sera from healthy individuals were positive for FucGM₁. The SPA was more sensitive than the previously reported HPTLC as well as a direct ELISA.Abbreviations MAb monoclonal antibody - SPA scintillation proximity assay - HPTLC high performance thin layer chromatography - SCLC small cell lung cancer - FucGM₁ Fuc1-2Gal1-3GalNAc1-4(NeuAc2-3)-Gal1-4Glc1-1Cer - ELISA enzyme linked immunosorbent assay - FCS foetal calf serum - PBS phosphate buffered saline     

In situ hybridization of corticotropin-releasing factor-encoding messenger RNA in the hypothalamus of the white sucker,Catostomus commersoni

Summary In situ hybridization procedure with a ³²P-labelled synthetic oligonucleotide probe was used to detect corticotropin-releasing factor-encoding messenger RNA (CRF mRNA) in the hypothalamus of the white sucker, Catostomus commersoni. Adjacent sections were immunostained by a sucker CRF-specific antiserum. CRF mRNA-containing neurons were identified by autoradiography in the magnocellular and parvocellular subdivisions of the preoptic nucleus (PON). Many of these neurons were also immunostained by sucker antiserum, showing the same distribution patterns. These results confirm the presence of CRF mRNA and CRF peptide in the white sucker hypothalamus and support the view that the magnocellular and parvocellular neurons of the PON may be involved in the control of adrenocorticotropic hormone secretion from the pituitary in the white sucker.     

Mineralization budgets in sediment microcosms: Effect of the infauna and anoxic conditions

Abstract A number of sediment incubations were set up to reproduce some of the conditions used by Kristensen and Blackburn [1] and to make a comparison with their results. There were three types of microcosm: aerobic (OX), anaerobic (AN) and aerobic with Nephtys (NOX). In addition to other measurements, dissolved organic nitrogen (DON) pools and fluxes, were measured. The sediment in this experiment contained more particulate organic matter (POM). Nephtys (NOX) had the same effect as Nereis in increasing the rate of mineralization of POC and PON, compared with the OX-cores (2.1 and 2.6 times, respectively). Again, the AN-cores had a higher mineralization rate (loss of POM) than that of the OX-cores, but in addition, mineralization in NOX-cores was not significantly different from AN-cores. It was thus confirmed that anoxic mineralization could be as high, or higher, than the oxic process. Both the temporal patterns of O₂-and and CO₂-fluxes and their magnitudes were very similar to those reported earlier. This contrasts with the higher loss of POM in the present experiment. However, the loss of C in DOC (associated with the measured DON) can account for the extra POM loss. The pore-water profiles of σCO₂ and NH₄⁺ were similar to those in the earlier report, and the fluxes of σCO₂, O₂, NH₄⁺ and NO₃⁻ followed the same temporal pattern.     

Floral scent in dioeciousSalix (Salicaceae)—a cue determining the pollination system?

Floral scents of male and female inflorescences of three dioeciousSalix species were collected by head-space adsorption, and analysed by GC-MS. InSalix caprea andS. cinerea 1,4-dimethoxy benzene was the main compound, and male and female scents showed a high degree of resemblance. No dominant compound was found inS. repens and malefemale scent similarity was low. Floral scent inSalix is likely a strong orientation cue, guiding pollinators between male and female plants ensuring pollen transfer and pollination. We suggest that a high degree of male-female floral scent resemblance is coupled to a high degree of insect pollination. Floral scent does not promote reproductive isolation betweenS. caprea andS. cinerea.     

Redox cycling of potential antitumor aziridinyl quinones

The formation of reactive oxygen intermediates (ROI) during redox cycling of newly synthesized potential antitumor 2,5-bis (1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied by assaying the production of ROI (superoxide, hydroxyl radical, and hydrogen peroxide) by xanthine oxidase in the presence of BABQ derivatives. At low concentrations (< 10 microM) some BABQ derivatives turned out to inhibit the production of superoxide and hydroxyl radicals by xanthine oxidase, while the effect on the xanthine-oxidase-induced production of hydrogen peroxide was much less pronounced. Induction of DNA strand breaks by reactive oxygen species generated by xanthine oxidase was also inhibited by BABQ derivatives. The DNA damage was comparable to the amount of hydroxyl radicals produced. The inhibiting effect on hydroxyl radical production can be explained as a consequence of the lowered level of superoxide, which disrupts the Haber-Weiss reaction sequence. The inhibitory effect of BABQ derivatives on superoxide formation correlated with their one-electron reduction potentials: BABQ derivatives with a high reduction potential scavenge superoxide anion radicals produced by xanthine oxidase, leading to reduced BABQ species and production of hydrogen peroxide from reoxidation of reduced BABQ. This study, using a unique series of BABQ derivatives with an extended range of reduction potentials, demonstrates that the formation of superoxide and hydroxyl radicals by bioreductively activated antitumor quinones can in principle be uncoupled from alkylating activity.     

Differential detachment from resting hosts of replete larval and nymphal Ixodes ticks

To determine whether replete subadult Ixodes ticks detach more frequently from resting than from active hosts, diverse rodents and lizards were caged in an apparatus designed to record the ticks' sites of detachment relative to the resting site of the host. Replete larval Ixodes ricinus and Ixodes dammini accumulated mainly beneath the resting places of the mice (Apodemus agrarius and Peromyscus leucopus) most frequently parasitized in nature. Although nymphal I. ricinus similarly detached where these mice rested, nymphal I. dammini detached more randomly. When lizards were used as hosts, both subadult stages of I. ricinus tended to detach away from their main resting sites; these ticks detached from squirrels more randomly. Detachment ratios for other rodent hosts, that are abundantly infested by the larvae of these ticks in nature (Apodemus flavicollis and Clethrionomys glareolus), could not be derived because nymphs generally failed to attach. Our observations are consistent with reports that both subadult stages of I. dammini, but not the adult, feed on the same kind of nest-dwelling hosts and that the host range of I. ricinus is less focused. Detachment of mouse-feeding larvae from resting mice promotes subsequent nymphal attachment to conspecific hosts, and the absence of such behavior among nymphs facilitates access of the resulting adults to deer.     

Gene expression during induction of somatic embryogenesis in carrot cell suspensions

We report the identification, via their cDNAs, of genes which are temporarily transcribed during the initiation of somatic embryogenesis in carrot (Daucus carota L.) cells cultured in an auxin-free medium. Their expression is roughly associated with the first morphogenetic, or globular, stage. A cDNA library ( gt 10) was established using poly(A)⁺ -rich RNAs from cells deprived of auxin for 8 d. By differential screening a number of clones corresponding to early-induced embryogenic genes were identified. For several a temporary accumulation of the specific mRNA between 6 and 16 d after induction was observed. With regard to the nucleotide sequence and the respective deduced amino-acid sequence, two glycine-rich proteins and a polypeptide with a proline-rich domain were among the products of genes activated at the onset of somatic embryogenesis.Abbreviations b, bp bases, basepairs - 2,4-D 2,4-dichlorophenoxyacetic acid Sequence data reported here will appear in the EMBL Genbank and DDBJ Nucleotide Sequence Databases under the following accession numbers: X 15436 for clone DC 2.15 (proline-rich protein), X 15706 for clone DC 7.1 (glycine-rich protein, DCGRP) and X 14067 for clone DC 9.1 (glycine-rich protein, DCGRP)This research was supported by the Deutsche Forschungsgemeinschaft. We thank Mrs. I. Liebscher for her competent assistance.     

Plant protoplasts and genetic engineering II

Book Review

Plant protoplasts and genetic engineering IIY.P.S. Bajaj (Ed.), (Biotechnology in Agriculture and Forestry, Vol. 9). Berlin: Springer-Verlag, 1989. 510 pages. DM398.00. ISBN 3-540-50789-2     

Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys329 to glu mutation in the MCAD gene,and expression of inactive mutant enzyme protein in E. coli

Summary A series of experiments has established the molecular defect in the medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) gene in a family with MCAD deficiency. Demonstration of intra-mitochondrial mature MCAD indistinguishable in size (42.5-kDa) from control MCAD, and of mRNA with the correct size of 2.4 kb, indicated a point-mutation in the coding region of the MCAD gene to be disease-causing. Consequently, cloning and DNA sequencing of polymerase chain reaction (PCR) amplified complementary DNA (cDNA) from messenger RNA of fibroblasts from the patient and family members were performed. All clones sequenced from the patient exhibited a single base substitution from adenine (A) to guanine (G) at position 985 in the MCAD cDNA as the only consistent base-variation compared with control cDNA. In contrast, the parents contained cDNA with the normal and the mutated sequence, revealing their obligate carrier status. Allelic homozygosity in the patient and heterozygosity for the mutation in the parents were established by a modified PCR reaction, introducing a cleavage site for the restriction endonuclease NcoI into amplified genomic DNA containing G⁹⁸⁵. The same assay consistently revealed A⁹⁸⁵ in genomic DNA from 26 control individuals. The A to G mutation was introduced into an E. coli expression vector producing mutant MCAD, which was demonstrated to be inactive, probably because of the inability to form active tetrameric MCAD. All the experiments are consistent with the contention that the G⁹⁸⁵ mutation, resulting in a lysine to glutamate shift at position 329 in the MCAD polypeptide chain, is the genetic cause of MCAD deficiency in this family. We found the same mutation in homozygous form in 11 out of 12 other patients with verified MCAD deficiency.