Metabolic Relations between Methylxanthines and Methyluric Acids in Coffea L

Metabolism of purine alkaloids in the leaves of Coffea dewevrei De Wild et Durand var excelsa Chev, Coffea liberica Bull ex Hiern and Coffea abeokutae Cramer was studied by analyzing leaf discs collected during vegetative development and by feeding the following radioactive tracers: [¹⁴C]theobromine, [¹⁴C]caffeine, and [¹⁴C]theacrine (1,3,7,9-tetramethyluric acid). Their principal metabolites were quantitatively and qualitatively determined. All three species convert the precursors to the same radioactive products, and proceed through the same four maturity stages characterized by the alkaloid accumulation pattern and by a particular transformation potency: (stage 1) young plant accumulating caffeine, transforms theobromine to caffeine; (stage 2) caffeine is gradually replaced by theacrine, theobromine and caffeine are converted to theacrine; (stage 3) theacrine disappears whereas liberine (O(2), 1,9-thrimethyluric acid) accumulates, theacrine is metabolized to liberine; (stage 4) branched-out plant containing liberine but no theacrine, caffeine is converted rapidly to liberine via theacrine. Methylliberine (O(2),1,7,9-tetramethyluric acid), presumably the direct precursor of liberine, is occasionally found in low concentrations at stage 3 and 4.

The collective term `liberio-excelsoid' introduced by geneticists for the numerous races or species of Pachycoffea is in accordance with the phytochemical equality found in this work.

     

Zur Problematik der Chromosomenautoradiographie

The distribution of silver grains on the chromosomes of group G was studied in 216 mitoses from five patients with Down's syndrome after incubation with tritated thymidine in order to test the replication pattern of these autosomes. The different labelling densities over the chromosomes were determined by comparative estimations of the blackened areas and subdivided into four degrees: very strongly labelled, strongly labelled, weakly labelled and unlabelled. The 56 combinations for five G chromosomes and four labelling densitives were divided into three groups: group A, with three strong and two weak labelling types, group B, with two strong and three weak labelling types, and group C, with the remaining combinations. Thereby 51 (23.6%) mitoses were counted in six combinations of group A, 31 (14.4%) in six combinations of group B, and 134 (62.0%) in 44 combinations of group C. In order to test the possibility of identifying the G chromosomes based on a different DNA replication pattern the findings were subjected to a statistical analysis. The expected values were calculated for the different combinations of labelling degrees and were compared with the observed values. This method being used, the question whether the extra chromosome is replicating late or early compared with the remaining G chromosomes could not be answered with sufficient certainty, because P_A=1, P_B=0 could not be assumed on our figures. We conclude from these findings that the termination phases for the chromosomes 21 as well as 22 lie close together and that apparently the autoradiographic representation of the replication pattern can be influenced by various factors. The influencing factors can only be understood in connection with the problems of quantitative autoradiography. They are subdivided into three groups and discussed in this way: A. Factors due to the physiology of the genetic material: 1. Duration of the DNA-synthesis and the G₂-period. 2. Chromosomal replication pattern. 3. The relation of ³H-Thymidin and unlabelled thymidine in the newly synthesized DNA. B. Factors due to the autoradiographic and cytologic method: 1. Statistics of the radioactive disintegrations. 2. -self-absorption. 3. Coincidence. 4. Effectivity. 5. Back-ground. 6. Fading. 7. Condensation of the chromosomes. 8. Cytologic preparation. 9. Thickness of the emulsion layer. 10. Duration of exposition and developping. C. Factors due to the method of evaluation: 1. Accuracy of counting. 2. Deficient analysis of the results. After considering these factors it seems clear that the autoradiographic technique is not capable to demonstrate subtle differencies of the DNA replication. Thus the autoradiographic identification of chromosomes remains questionable if the termination phases of DNA replication lie close together as this seems the case for the chromosomes 21 and 22.

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Studie im Rahmen der Assoziation Hämatologie EURATOM — GSF.     

Managing gynaecological emergencies with laparoscopy.

A consecutive series of 49 women (50 procedures), whose conditions were haemodynamically stable, presenting with acute lower abdominal pain, pelvic tenderness, and either a urine concentration of greater than 50 U/l beta human chorionic gonadotrophin or a pelvic mass shown by ultrasonography were treated with operative laparoscopy under video monitoring (videopelviscopy) as an alternative to laparotomy. Ectopic pregnancy, ovarian and non-ovarian cysts, pelvic adhesions, endometriosis, and fibroids were found, for which salpingotomy, salpingectomy and salpingo-oophorectomy, cystectomy, adhesiolysis, thermocoagulation, and myomectomy were carried out by laparoscopy. In one patient pelviscopy was repeated because of persistent tubal pregnancy after the fimbria was expressed. Laparotomies were carried out on three patients because treatment was not possible by laparoscopy and on a further patient two days after adhesiolysis had been attempted. These were the only serious complications. For the 46 cases (45 patients) in which operative laparoscopy was successful the mean stay in hospital was 1.9 days after operation, and this group of patients returned to normal activities and to work after an average of 2.3 and 2.6 weeks respectively. Most gynaecological emergencies that are managed by laparotomy can be treated by laparoscopy and benefit both patients and the health service.     

Response of human squamous cell carcinoma xenografts of different sizes to irradiation: relationship of clonogenic cells, cellular radiation sensitivity in vivo, and tumor rescuing units

The relationship of clonogenic cells, cellular radiation sensitivity at tumor control does in vivo, and tumor rescuing units at different tumor sizes was investigated in the human squamous cell carcinoma FaDu growing in NCr/Sed nude mice. The composition of the tumors was determined in single cell suspensions and compared to tumor control data after single-dose irradiation. To avoid the influence of varying oxygen concentrations in the tumors, all irradiations were performed under clamp hypoxia. Nude mice and animals further immunosuppressed by 6-Gy whole-body irradiation were used to assess the immunological effects. The numbers of total cells, cells excluding trypan blue, host cells, and colony-forming cells increased linearly with the weight of FaDu tumors. Comparable results were obtained for cell suspensions prepared from tumors growing in nude of pretreated nude mice. The radiation dose required to control 50% of tumors (TCD50) of different sizes between 36 and 470 mm3 increased from 52.1 to 60.1 Gy when the tumors were maintained in normal nude mice and from 50.8 to 61.3 Gy in whole-body-irradiated mice. The D0 of FaDu cells in vivo was calculated by regression analysis of TCD50 vs the logarithm of the clonogenic cell number, assuming an oxygen enhancement ratio of 3.0. The resultant D0S of 1.1 and 1.2 Gy in vivo correspond well to the radiosensitivity of FaDu cells in vitro determined previously. Assuming the single-hit multitarget model of cell killing and extrapolation numbers between 2 and 20, the mean number of tumor rescuing units would be 10(5) to 10(6) for a 100-mm3 tumor growing in whole-body-irradiated nude mice. Comparison of the number of tumor rescuing units to the estimated number of clonogenic cells does not conflict with the assumption that every surviving clonogenic cell is able to repopulate FaDu tumors after irradiation; however, it seems more likely that more than one clonogenic cells is necessary. The proportion of tumor rescuing units in the clonogenic cell population is independent of tumor size.     

Helical epitope of the group B meningococcal alpha(2-8)-linked sialic acid polysaccharide.

The immunological properties of the group B meningococcal alpha(2-8)-linked sialic acid polysaccharide have been rationalized in terms of a model where the random coil nature of the polymer can be described by the presence of local helices. The conformational versatility of the alpha NeuAc(2-8)alpha NeuAc linkage has been explored by NMR studies at 600 MHz in conjunction with potential energy calculations for colominic acid, an alpha(2-8)NeuAc polymer, and the trisaccharide alpha NeuAc(2-8)alpha NeuAc(2-8)beta NeuAc. Potential energy calculations were used to estimate the energetically favorable conformers and to describe the wide range of helices which the polymer can adopt. No unique conformer was found to satisfy all NMR constraints, and only ensemble averaged nuclear Overhauser enhancements could correctly simulate the experimental data. Conformational differences between the polymer and the trisaccharide could be best explained in terms of slight changes in the relative distribution of conformers in solution. Similar helical parameters for the alpha(2-8)NeuAc polymer and poly(A) were proposed as the basis for their cross-reactivity to a monoclonal antibody IgMNOV. The unusual length dependency for binding of oligosaccharide to group B specific antibodies was postulated to arise from the recognition of a high-order local helix with an extended conformation which was not highly populated in solution.     

Patterns of divergence during evolution of alpha 1-proteinase inhibitors in mammals

alpha 1-Proteinase inhibitor (alpha 1-PI), a member of the serine proteinase inhibitor superfamily, has a primary role in controlling neutrophil elastase activity within the mammalian circulation. Several studies have indicated that the reactive center region of alpha 1-PI, the amino acid sequence of which is critical to recognition of and binding to target proteinases, is highly divergent within and among species. This appears to be a consequence of accelerated rates of evolution that may have been driven by positive Darwinian selection. In order to examine this and other features of alpha 1-PI evolution in more detail, we have isolated and sequenced cDNAs representing alpha 1- PI mRNAs of the mouse species Mus saxicola and Mus minutoides and have compared these with a number of other mammalian alpha 1-PI mRNAs. Relative to other mammalian mRNAs, the extent of nonsynonymous substitution is generally high throughout the alpha 1-PI mRNA molecule, indicating greater overall rates of amino acid substitution. Within and among mouse species, the 5'-half of the mRNA, but not the 3'-half, has been homogenized by concerted evolution. Finally, the reactive center is under diversifying or positive Darwinian selection in murid rodents (rats, mice) and guinea pigs yet is under purifying selection in primates and artiodactyls. The significance of these findings to alpha 1-PI function and the possible selective forces driving evolution of serpins in general are discussed.      

Primary structure and functional expression of a Drosophila cyclic nucleotide-gated channel present in eyes and antennae.

Cyclic nucleotide-gated (CNG) ion channels serve as downstream targets of signalling pathways in vertebrate photoreceptors and olfactory sensory neurons. Whether CNG channels subserve similar functions in invertebrate photoreception and olfaction is unknown. We have cloned genomic DNA and cDNA encoding a cGMP-gated channel from Drosophila. The gene contains at least seven exons. Heterologous expression of cloned cDNA in both Xenopus oocytes and HEK 293 cells gives rise to functional ion channels. The Drosophila CNG channel is approximately 50-fold more sensitive to cGMP than to cAMP. The voltage dependence of blockage by divalent cations is different compared with the CNG channel of rod photoreceptors, and the Ca2+ permeability is much larger. The channel mRNA is expressed in antennae and the visual system of Drosophila. It is proposed that CNG channels are involved in transduction cascades of both invertebrate photoreceptors and olfactory sensillae.     

Immunolocalization of Na,K-ATPase in blowfly photoreceptor cells

The Na,K-ATPase (sodium pump) plays a central role in the physiology of arthropod photoreceptors as it re-establishes gradients for Na⁺ and K⁺ after light stimulation. We have mapped the distribution of the Na,K-ATPase in the photoreceptors of the blowfly (Calliphora erythrocephala) by immunofluorescent and immunogold cytochemistry, and demonstrate that the distribution pattern is more complex than previously presumed. High levels of sodium pumps have been detected consistently in all photoreceptors R1-8 on the nonreceptive surface, but no sodium pumps are found on the microvillar rhabdomere. Within the nonreceptive surface of the cells R1-6, however, the sodium pumps are confined to sites juxtaposed to neighboring photoreceptor or glial cells; no sodium pumps have been detected on the parts of the nonreceptive surface exposed to the intra-ommatidial space. In R7 and R8, the sodium pumps are found over the entire nonreceptive surface. The cytoskeletal protein spectrin colocalizes with the sodium pumps suggesting that linkage of the pump molecules to the spectrin-based submembrane cytoskeleton contributes to the maintenance of the complex pattern of pump distribution.