Size isn't everything: lessons in genetic miniaturisation from nucleomorphs.

Nucleomorphs are the vestigial nuclear genomes of eukaryotic algal cells now existing as endosymbionts within a host cell. Molecular investigation of the endosymbiont genomes has allowed important insights into the process of eukaryote/eukaryote cell endosymbiosis and has also disclosed a plethora of interesting genetic phenomena. Although nucleomorph genomes retain classic eukaryotic traits such as linear chromosomes, telomeres, and introns, they are highly reduced and modified. Nucleomorph chromosomes are extremely small and encode compacted genes which are disrupted by the tiniest spliceosomal introns found in any eukaryote. Mechanisms of gene expression within nucleomorphs have apparently accommodated increasingly parsimonious DNA usage by permitting genes to become co-transcribed or, in select cases, to overlap.

Review: origin of complex algae by secondary endosymbiosis: a journey through time.

Secondary endosymbiosis-the merging of two eukaryotic cells into one photosynthetic cellular unit-led to the evolution of ecologically and medically very important organisms. We review the biology of these organisms, starting from the first proposal of secondary endosymbiosis up to recent phylogenetic models on the origin of secondarily evolved protists. In addition, we discuss the organelle character of the symbionts based on morphological features, gene transfers from the symbiont into the host and re-import of nucleus-encoded plastid proteins. Finally, we hypothesize that secondary endosymbiosis is more than enslaving a eukaryotic, phototrophic cell, but reflects a complex interplay between host and symbiont, leading to the inseparability of the two symbiotic partners generating a cellular entity.

Numerical experiments and field results on the size of steady state plumes.

Contaminated groundwater poses a serious risk for drinking water supplies. Under certain conditions, however, groundwater contamination remains restricted to a tolerable extent because of natural attenuation processes. We present an innovative approach to evaluate the size of these so-called steady-state plumes by 2-D and 1-D modelling in homogeneous aquifers. If longitudinal mixing is negligible, scenarios can be modelled in a simplified way using a 1-D domain vertical to the direction of flow. We analysed the sensitivity of the plume length with respect to biodegradation kinetics, flow velocity, transverse vertical dispersivity alphat, the source and aquifer geometry and reaction stoichiometry. Our findings indicate that for many readily biodegradable compounds transverse-dispersive mixing rather than reaction kinetics is the limiting factor for natural attenuation. Therefore, if alphat, aquifer and source geometry and concentrations of electron acceptors and donors are known, the length of the steady state contaminant plume can be predicted. The approach is validated under field conditions for an ammonium plume at a former landfill site in SW Germany.

Twelve-year experience with two courses of adjuvant single-agent carboplatin therapy for clinical stage I seminoma.

PURPOSE: During the past 30 years, radiation therapy with 28 to 30 Gy for para-aortic and ipsilateral iliac node areas was the standard adjuvant treatment for clinical stage I seminoma after orchiectomy. However, late effects of radiotherapy prompted a search for alternative adjuvant treatment approaches, including surveillance and application of carboplatin. In this retrospective analysis, we evaluated the efficacy and toxicity of two adjuvant single-agent carboplatin courses in 107 patients who were diagnosed with clinical stage I seminoma at our study centers between 1988 and 1999. PATIENTS AND METHODS: All 107 patients (median age, 39 years; range, 24 to 63 years) received two postoperative adjuvant cycles of carboplatin (400 mg/m(2)). The pathologic tumor stage was pT1 in 84 patients, pT2 in 18 patients, and pT3 in five patients. Whole blood count and serum chemistry were evaluated weekly during treatment to assess hematologic and nonhematologic toxicity. RESULTS: Six patients died from tumor-unrelated causes. The remaining 101 patients are currently alive and free of disease after a median follow-up of 74 months (range, 5 to 145 months). A detailed analysis of hematologic toxicity showed only World Health Organization (WHO) grade 1 leukocytopenia in 10.7% of all cycles and WHO grade 2 leukocytopenia in 2.1% of all cycles. CONCLUSION: Regarding the absence of tumor recurrences in our retrospective analysis and the favorable toxicity profile with no episodes of long-term toxicity, we suggest that two adjuvant courses of single-agent carboplatin for clinical stage I seminoma patients might be equivalent to radiotherapy.

Phenotypic and functional characterization of mast cells derived from renal tumor tissues.

Mast cells (MCs) originate from multipotent hematopoietic progenitor cells. However, MCs in various organs are heterogenous in terms of mediator or receptor expression and response to diverse stimuli. We characterized the phenotype and functional properties of human renal mast cells (HRMCs). Tissue was obtained from 17 patients suffering from renal tumors (transitional cell carcinoma, n = 4; renal cell carcinoma, n = 13). HRMCs were isolated by collagenase digestion. Double staining with toluidine blue and immunofluorescence using monoclonal antibodies (mAbs) revealed expression of stem cell factor (SCF)-receptor (c-kit/CD117), CD9, CD29, CD33, CD43, CD44, CD54, and CD63 on HRMCs. In contrast, HRMCs were not recognized by mAbs to CD2, CD3, CD4, CD11b, CD14, CD15, CD16, CDw17, CD19, or CD23. HRMCs were also negative for CD116 (granulocyte-macrophage colony-stimulating factor [GM-CSF] receptor alpha), CD123 (interleukin [IL]-3Ralpha), CD121a (IL-1R type I), CD122 (IL-2Rbeta), and CD127 (IL-7R) and were also found to lack C5aR (CD88). Ligand-induced activation of HRMCs through immunoglobulin (Ig)E-R or SCF-R (c-kit) resulted in histamine secretion (control: <10%; alphaIgE, 1 microg/mL: 50.12 +/-5.18%; rhSCF, 100 ng/mL: 29.24 +/- 22.39), whereas recombinant C5a, erythropoietin (EPO), IL-1 through 10, and GM-CSF exerted no effects. As determined by in situ staining, HRMCs contained tryptase, but only low or undetectable amounts of chymase. Electron microscopy confirmed the presence of MCs in renal tissues and revealed a scroll-rich granule population in HRMCs. Together, HRMCs are tryptase+, C5aR- mast cells exhibiting phenotypic and functional properties similar to those of lung MCs.

Solution structure of a zinc substituted eukaryotic rubredoxin from the cryptomonad alga Guillardia theta.

The rubredoxin from the cryptomonad Guillardia theta is one of the first examples of a rubredoxin encoded in a eukaryotic organism. The structure of a soluble zinc-substituted 70-residue G. theta rubredoxin lacking the membrane anchor and the thylakoid targeting sequence was determined by multidimensional heteronuclear NMR, representing the first three-dimensional (3D) structure of a eukaryotic rubredoxin. For the structure calculation a strategy was applied in which information about hydrogen bonds was directly inferred from a long-range HNCO experiment, and the dynamics of the protein was deduced from heteronuclear nuclear Overhauser effect data and exchange rates of the amide protons. The structure is well defined, exhibiting average root-mean-square deviations of 0.21 A for the backbone heavy atoms and 0.67 A for all heavy atoms of residues 7-56, and an increased flexibility toward the termini. The structure of this core fold is almost identical to that of prokaryotic rubredoxins. There are, however, significant differences with respect to the charge distribution at the protein surface, suggesting that G. theta rubredoxin exerts a different physiological function compared to the structurally characterized prokaryotic rubredoxins. The amino-terminal residues containing the putative signal peptidase recognition/cleavage site show an increased flexibility compared to the core fold, but still adopt a defined 3D orientation, which is mainly stabilized by nonlocal interactions to residues of the carboxy-terminal region. This orientation might reflect the structural elements and charge pattern necessary for correct signal peptidase recognition of the G. theta rubredoxin precursor.

Differential distribution of G-protein beta-subunits in brain: an immunocytochemical analysis.

Heterotrimeric G proteins play central roles in signal transduction of neurons and other cells. The variety of their alpha-, beta-, and gamma-subunits allows numerous combinations thereby confering specificity to receptor-G-protein-effector interactions. Using antisera against individual G-protein beta-subunits we here present a regional and subcellular distribution of Gbeta1, Gbeta2, and Gbeta5 in rat brain. Immunocytochemical specificity of the subtype-specific antisera is revealed in Sf9 cells infected with various G-protein beta-subunits. Since Gbeta-subunits together with a G-protein gamma-subunit affect signal cascades we include a distribution of the neuron-specific Ggamma2- and Ggamma3-subunits in selected brain areas. Gbeta1, Gbeta2, and Gbeta5 are preferentially distributed in the neuropil of hippocampus, cerebellum and spinal cord. Gbeta2 is highly concentrated in the mossy fibres of dentate gyrus neurons ending in the stratum lucidum of hippocampal CA3-area. High amounts of Gbeta2 also occur in interneurons innervating spinal cord alpha-motoneurons. Gbeta5 is differentially distributed in all brain areas studied. It is found in the pyramidal cells of hippocampal CA1-CA3 as well as in the granule cell layer of dentate gyrus and in some interneurons. In the spinal cord Gbeta5 in contrast to Gbeta2 concentrates around alpha-motoneurons. In cultivated mouse hippocampal and hypothalamic neurons Gbeta2 and Gbeta5 are found in different subcellular compartments. Whereas Gbeta5 is restricted to the perikarya, Gbeta2 is also found in processes and synaptic contacts where it partially colocalizes with the synaptic vesicle protein synaptobrevin. An antiserum recognizing Ggamma2 and Ggamma3 reveals that these subunits are less expressed in hippocampus and cerebellum. Presumably this antiserum specifically recognizes Ggamma2 and Ggamma3 in combinations with certain G alphas and/or Gbetas. The widespread but regionally and cellularly rather different distribution of Gbeta- and Ggamma2/3-subunits suggests that region-specific combinations of G-protein subunits mediate signal transduction in the central nervous system. The different subcellular distribution of Gbeta-subunits in cultivated neurons reflects that observed in tissue where Gbeta5 and Gbeta2 associate preferentially with the perikarya and the neuropil, respectively, and suggests an additional association of Gbeta2 with secretory vesicles.

Pyridostigmine kinetics in healthy subjects and patients with myasthenia gravis.

Comparative pyridostigmine kinetics in plasma were measured in 10 healthy subjects given 4 mg iv and 60 mg oral pyridostigmine bromide. As determined from the AUC ratio, oral availability was 11.5% to 18.9% (means = 14.3%). Mean t 1/2 of the plasma level decline after oral dosing was 200 minutes, twice as long as the terminal elimination t1/2 after intravenous infusion (97 minutes). Thus absorption may proceed at a slower rate than elimination. Comparison of intraindividual data revealed strict dependence of the AUC on the infused dose (2, 4, and 8 mg) in one subject and variability in AUC up to a factor of two when two subjects took oral pyridostigmine three times. Patients with myasthenia who were receiving continuous therapy with oral pyridostigmine had AUC values per unit dose corresponding to those in healthy subjects. Storage stability of pyridostigmine in plasma required acidification of samples and storage at -75 degrees C. When native plasma was kept at -20 degrees C, there was appreciable loss of pyridostigmine within 1 to 2 months, the extent of which depended on the initial concentration.

The cryptomonad histone H4-encoding gene: structure and chromosomal localization.

Cryptomonads are unicellular flagellates whose plastids are surrounded by four membranes. A periplastidal compartment, containing eukaryote-type ribosomes, starch grains and a so-called nucleomorph, is located between the inner and outer membrane pairs. The nucleomorph has been shown to be the vestigial nucleus of a eukaryotic endosymbiont. In order to obtain more information about the chromatin structure of the nucleomorph and the host nuclear chromosomes, we studied the distribution of the histone, H4. H4 was not detectable in the nucleomorph by immunolocalization, thus supporting earlier findings by Gibbs [In: Wiesner et al. (Eds.), Experimental Phycology 1, 1990, pp. 145-157]. Likewise, no H4 DNA was demonstrable in the nucleomorph by Southern hybridization. Sequence analysis, and Southern and Northern blot data of a cryptomonad gene, H4, indicate an intermediate position for these genes between animals and plants.

Identification and characterization of a eukaryotically encoded rubredoxin in a cryptomonad alga.

We have identified an open reading frame with homology to prokaryotic rubredoxins (rds) on a nucleomorph chromosome of the cryptomonad alga Guillardia theta. cDNA analysis let us propose that the rd preprotein has an NH(2)-terminal extension that functions as a transit peptide for import into the plastid. Compared to rds found in non-photosynthetic prokaryotes or found in bacteria that exhibit an anoxigenic photosynthesis apparatus, nucleomorph rd has a COOH-terminal extension, which shows high homology exclusively to the COOH-termini of cyanobacterial rds as well as to a hypothetical rd in the Arabidopsis genome. This extension can be divided into a putative membrane anchor and a stretch of about 20 amino acids with unknown function linking the common rd fold to this anchor. Overexpression of nucleomorph rd in Escherichia coli using a T7 RNA polymerase/promotor system resulted in a mixture of iron-containing holorubredoxin and zinc-substituted protein. Preliminary spectroscopic studies of the iron form of nucleomorph rd suggest the existence of a native rd-type iron site. One-dimensional nuclear magnetic resonance spectroscopy of recombinant Zn-rd suggests the presence of a stable tertiary fold similar to that of other rd structures determined previously.

(-)-2C-methyl-D-erythrono-1,4-lactone is formed after application of the terpenoid precursor 1-deoxy-D-xylulose.

Application of [1,2-14C]1-deoxy-D-xylulose, the committed precursor of terpenoids, thiamine and pyridoxol, to a variety of plant species resulted in the labelling of an unknown metabolite. The isolation and purification of this metabolite from Ipomoea purpurea plants fed with 1-deoxy-D-xylulose (DX), followed by NMR analysis, resulted in the identification of its structure as (-)-2C-methyl-D-erythrono-1,4-lactone (MDEL). MDEL has been previously isolated as a stress metabolite of certain plants. A hypothetical biosynthetic scheme is given.

Local intratumoral tumor necrosis factor-alpha and systemic IFN-alpha 2b in patients with locally advanced prostate cancer.

To examine tolerability and activity of local, intratumoral tumor necrosis factor-alpha (TNF-alpha) and systemic interferon-alpha2b (IFN-alpha2b) in locally advanced, hormone-resistant prostate cancer (LA-HRPC), 10 patients with LA-HRPC (T4N x M0, n = 3, T4N x M1, n = 5; T4N1M1, n = 2) were treated with recombinant TNF-alpha injected locally into prostate tumor tissue at 4-week intervals (maximum of four cycles) combined with intermittent subcutaneous (s.c.) administration of 5 x 10(6) IU IFN-alpha2b. Twenty-nine TNF-alpha cycles were administered. Despite significant TNF-alpha leakage into the systemic circulation 2 h after intraprostatic application (from a mean of 9 to a mean of 416 pg/ml; p = 0.0034), TNF-alpha (and IFN-alpha2b) was well tolerated (WHO grade 1-2 toxicity), possibly because of its rapid neutralization by increasing soluble 55-kDa and 75-kDa TNF receptor levels in the serum (mean increase 268% and 91%, respectively) at the same time. TNF-alpha induced prostate tumor cell necrosis in all patients, leading to a significant reduction of prostate volume in 9 of 10 cases (mean 38%; p = 0.0025). The significant short-term increase of prostate-specific antigen (PSA) (mean 65%; p < 0.001), tissue polypeptide-specific antigen (TPS) (mean 85%; p = 0.001), and possibly interleukin-8 (IL-8) (mean 2687%; p < 0.009) serum levels within 4 h after TNF-alpha confirmed the cytotoxic effect in vivo. In the long term, serum PSA levels dropped by 18%-87%, reaching the nadir value 7 weeks after baseline. Objective responses of metastases were not seen. Intraprostatic administration of TNF-alpha is feasible at a tolerable toxicity in patients with LA-HRPC and, thus, may be a new treatment option for these patients.

High incidence of nephrogenic adenoma of the bladder after renal transplantation.

BACKGROUND: Tumors of the bladder termed nephrogenic adenomas in kidney allograft recipients are believed to develop as urothelial metaplastic proliferations in response to mechanical trauma, chemical noxae, irradiation, and bacterial or viral pathogens. We report on the incidence of nephrogenic adenoma of the bladder in patients who received renal transplants during a period of 7 years and 3 months at the University Hospital of Vienna. METHODS: Diagnosis was obtained by cystoscopy and histological analysis. Nephrogenic adenoma was treated by transurethral electroresection and administration of antibiotics in case of urinary tract infections. Follow-up consisted of cytological controls of urine and bladder irrigation fluid as well as of cystoscopy every 3 months. RESULTS: In 7 of 1328 renal allograft recipients, nephrogenic adenoma could be detected after 7 to 60 months following renal transplantation. In five patients, recurrence was detected 9 to 23 months after diagnosis of the initial lesion. No evidence of malignant degeneration was observed in any patient. Nephrogenic adenoma was not related to immunosuppressive therapy, cytomegalovirus disease, or gancyclovir therapy. CONCLUSIONS: We suggest that after successful transurethral electroresection of nephrogenic adenomas, cytological controls are adequate every 3 months. Only in renal transplant patients with recurrence of voiding disturbances, macrohematuria, or urinary tract infection are cystoscopy and biopsy indicated in the routine follow-up regimen.

Distribution of DNA in human Sertoli cell nucleoli.

For better understanding of nucleolar architecture, different techniques have been used to localize DNA within the dense fibrillar component (DF) or within the fibrillar centers (FC) by electron microscopy (EM). Since it still remains controversial which components contain DNA, we investigated the distribution of DNA in human Sertoli cells using various approaches. In situ hybridization (ISH) with human total genomic DNA as probe and the use of anti-DNA antibody were followed by immunogold detection. This allowed statistical evaluation of the signal density over individual components. The Feulgen-like osmium-ammine (OA) technique for the selective visualization of DNA was also applied. The anti-DNA antibodies detected DNA in mitochondria, in chromatin, and in the DF of the nucleolus. ISH using human total genomic DNA showed similar labeling patterns. The OA technique revealed DNA filaments in the FC and focal agglomerates of decondensed DNA within the DF. We conclude that (a) EM staining techniques that utilize colloidal gold appear to be less sensitive for DNA detection than the OA method, (b) the DF consists of different domains with different molecular composition, and (c) decondensed DNA is not necessarily confined to one particular nucleolar component.

Reconstitution of protein transport across the vacuolar membrane in Plasmodium falciparum-infected permeabilized erythrocytes.

The parasite Plasmodium falciparum induces morphological and biochemical alterations of its host erythrocyte. Some of these changes are mediated by parasite proteins that are transported to specific destinations within the erythrocyte or to the erythrocyte plasma membrane. The pathways underlying this transport are still unknown. We anticipate that at least some aspects of these pathways may be biologically unique and therefore potential targets for chemotherapeutic intervention. We have utilized bacterial pore-forming proteins to establish an experimental system that allows selective permeabilization of the erythrocyte plasma membrane, without affecting the integrity of the vacuolar membrane and the parasite plasma membrane, in order to study protein transport from the parasite into the host erythrocyte. Physiological properties of the parasite within permeabilized erythrocytes, such as the ability to synthesize proteins, will be described. The permeabilization of infected erythrocytes has allowed the dissection of individual steps in protein transport from the parasite surface across the vacuolar membrane. Possible pathways involved in the trafficking of parasite proteins within the erythrocyte cytosol, i.e. in a cell that normally has no need to transport proteins, will be discussed.

Andrological findings in young patients under long-term antidepressive therapy with clomipramine.

Serum hormone levels of follicle stimulating hormone (FSH), luteotropic hormone (LH), testosterone (T), prolactin (hPRL), estradiol (E2) and GnRH test were carried out in 11 patients after antidepressive therapy with clomipramine at a dosage of 75 mg/day for 3 months. Nine of these patients agreed with the evaluation of ejaculate parameter. As an age matched control group 15 patients without psychiatric disorders but under urological treatment because of erectile dysfunction were investigated in the same manner, with 11 patients being able to produce an ejaculate for examination. Both groups were closely comparable according to age, percentage of proven fertility and urological status. All spermiograms evaluated in the clomipramine group were pathological, especially with regard to volume, sperm motility and sperm morphology, whereas only 37% in the control group showed pathological findings, approximately the same as that of healthy individuals in this age range. Serum hormone levels as well as the hypothalamic-hypophyseal-gonadal axis proven with the Gn-RH test were in normal range in both groups. According to these results it seems evident that clomipramine does not alter sexual hormone profiles in males or interfere with the hypothalamic-hypophyseal-gonadal axis and has a significant negative effect on ejaculate parameters.

The clinical value of urinary cytology: 12 years of experience with 615 patients.

AIMS: To analyse the diagnostic value of cytological examination compared with histological findings in a large series of patients (n = 615) with tumours of the urinary tract epithelium. METHODS: Cytological examinations (n = 785) after bladder washing and exfoliative cytology were retrospectively compared and correlated with histological findings. In addition, 1527 bladder washings were obtained during follow up of patients after transurethral resection of bladder tumours. RESULTS: Cytology in bladder washings (overall diagnostic accuracy 66%) provides considerably more information that exfoliative cytology (overall accuracy 49%). Cytological examinations (n = 1125) in patients with bladder tumours receiving intravesical cytostatic drugs (for example, mitomycin C) yielded suspicious or positive results in 28% of patients, without being confirmed by endoscopy during follow up. CONCLUSION: Our results illustrate two major drawbacks of urinary cytology. First, a high rate of false positive results in patients on intravesical chemotherapy. Second, a high rate of false negative results in highly differentiated carcinomas, stressing the need for additional diagnostic tests such as staining with monoclonal antibodies directed against tumour antigens or assessment of ploidy.

Long-term observation after intravesical metaphylaxis with mitomycin C in patients with superficial bladder tumors.

After transurethral resection of a superficial bladder tumor 63 patients were treated by chemoprophylaxis with mitomycin C over a period of two years: 33.3 percent experienced recurrent disease under metaphylaxis and 42 patients who remained recurrence-free during instillation therapy were observed further for an average of 26.4 months. During this period 26.2 percent of the patients showed recurrences ranging between two to forty-four months after completing instillation. The majority of the patients in whom recurrent tumors developed also had recurrent tumor processes before the instillation therapy was begun. Of the 42 patients 73.8 percent stayed recurrence-free during the observation period of twelve to fifty-four months after completing instillation therapy. In conclusion, during an average observation period of 50.4 months, 52.4 percent of the patients in this study showed recurrences.

Nephrogenic adenoma in renal transplant recipients: a truly benign lesion?

OBJECTIVES: Nephrogenic adenoma is a benign metaplastic lesion of the urinary bladder, reported to occur as a response to inflammation, trauma, intravesical therapies, and after renal transplantation. The aim of this study was to evaluate on the basis of chromosomal analysis whether nephrogenic adenoma really is benign. METHODS: Twelve renal transplant recipients with histologically verified nephrogenic adenoma were analyzed for numerical aberrations of chromosomes 7, 9, and 17. Results were related to total DNA content, p53 and Ki-67 positivity, and clinical outcome. Ten patients with superficial bladder cancer and 10 healthy renal transplant recipients formed the control groups. RESULTS: All 12 patients with nephrogenic adenoma had monosomy 9 in a mean of 24.3% (range 20% to 30%) of the evaluated cells; 3 patients had an additional trisomy 7 in a mean of 8% (range 6% to 10%) of the counted cells. Chromosome 1 7 was disomic in all patients. DNA histograms were diploid in 11 of the 12 patients and aneuploid in 1 patient. No p53 and Ki-67 positivity was present in this group. All patients with superficial bladder cancer had monosomy 9 in a mean of 79.8% (range 75% to 85%) of the counted cells. Two patients were found to have an additional trisomy 7 in 50% and 65% of the cells, respectively. The latter had an aneuploid histogram; the others had haploid/diploid histograms. p53 was negative in all specimens. Ki-67 positivity was present in 70% of these patients. All healthy transplant recipients had disomic chromosomal patterns according to diploid DNA histograms and negative immunocytochemical results. CONCLUSIONS: Even if in a lower percentage of cells, aberrations of chromosome 7 and 9 were detected in nephrogenic adenoma. It therefore cannot be excluded that nephrogenic adenomas in immunosuppressed renal transplant recipients may develop into malignant lesions.

Biosynthesis of Erythrina alkaloids in Erythrina crista-galli.

A precursor application system was developed to allow the study of Erythrina alkaloid formation in Erythrina crista-galli. Fruit wall tissue of this species was recognized as the major site of alkaloid biosynthesis. The application of radioactively and 13C-labelled potential precursors showed that the hitherto assumed precursor (S)-norprotosinomenine was not incorporated into the Erythrina alkaloids. In contrast, (S)-coclaurine as well as (S)-norreticuline were metabolized to erythraline and erythrinine, respectively, suggesting that a coclaurine-norreticuline pathway is operative in Erythrina alkaloid formation. Feeding of [1-13C]-labelled (S)-norreticuline with subsequent NMR spectroscopy demonstrated that the resulting erythraline was exclusively labelled at position C-10. Therefore, the participation of a symmetrical intermediate of the diphenoquinone type in Erythrina alkaloid biosynthesis can be excluded.