Evolutionary dynamics of autopolyploids in natural populations: the case of Turnera sidoides complex / Dinámica evolutiva de autopoliploides en poblaciones naturales: el caso del complejo Turnera sidoides

ABSTRACT Turnera sidoides (x=7) is one of the few well-studied South American autopolyploid complexes. Since polyploidy has played a prominent role within this complex, ongoing studies in T. sidoides focus on understanding the mechanisms involved in the origin and the establishment of polyploids using integrative approaches. This paper synthesises the results of more than 20 years of research on this topic. Cytogenetics analysis provided evidences for the production of unreduced male and female gametes, supporting the hypothesis of bilateral sexual polyploidization as the mechanism of origin of polyploids in T. sidoides. The finding of viable triploids suggested that unilateral sexual polyploidization could also be an important mechanism for the origin of tetraploids in T. sidoides. The occurrence of plants continuously forming many unreduced gametes would play a key role in the establishment of neopolyploids in natural populations. Also, the higher number of propagules that tetraploids contribute to subsequent generations, the ability to multiply asexually by rhizomes, and the occurrence of occasional cases of self-compatibility and successful illegitimate crosses in polyploids increase the likelihood that a low frequency of neopolyploids can be maintained in natural populations of T. sidoides. In addition, integration of cytogeographic and genetic divergence data together with past niche modelling provided further insights supporting the hypothesis that historical climatic and geomorphological events provided favourable conditions for the establishment of autopolyploids, with the wider distribution of tetraploids of T. sidoides being the result of their range expansion.

RESUMEN Turnera sidoides (x=7) es uno de los pocos complejos autopoliploides sudamericanos bien estudiados. Como la poliploidía ha tenido un papel destacado en el complejo, los estudios en curso en T. sidoides se centraron en la comprensión de los mecanismos implicados en el origen y el establecimiento de los poliploides mediante diferentes enfoques. En este trabajo se sintetizan los resultados de más de 20 años de investigación sobre este tema. El análisis citogenético proporcionó evidencias de la producción de gametos masculinos y femeninos no reducidos, sustentando la hipótesis de la poliploidización sexual bilateral como mecanismo de origen de los poliploides en T. sidoides. Sin embargo, el hallazgo de triploides fértiles sugirió que la poliploidización sexual unilateral también sería un mecanismo importante de origen de tetraploides en T. sidoides. La ocurrencia de plantas que forman continuamente gametos no reducidos desempeñaría un papel clave en el establecimiento de neopoliploides. Además, el mayor número de propágulos que los tetraploides aportan a las siguientes generaciones, la capacidad de multiplicación asexual por rizomas y los casos ocasionales de autocompatibilidad y cruzamientos ilegítimos exitosos aumentarían la probabilidad de que se mantenga una baja frecuencia de neopoliploides en las poblaciones naturales de T. sidoides. Asimismo, la integración de datos citogeográficos y de divergencia genética junto con el modelado de nicho en el pasado aportó información que sustenta la hipótesis de que los eventos climáticos y geomorfológicos históricos proporcionaron las condiciones favorables para el establecimiento y expansión de los tetraploides de T. sidoides.

Modification of the hand-held Vscan ultrasound and verification of its performance for transvaginal applications.

PURPOSE: The purpose of this work was to validate a new clinical obstetrics and gynecology (OB-GYN) application for a hand-held ultrasound (US) device. We modified the smallest hand-held device on the market and tested the system for transvaginal (TV) use. This device was originally conceived for abdominal scanning only. METHODS: The validation involved 80 successive patients examined by the same operator: 25 obstetric and 55 gynecologic cases. US examination was performed transvaginally with two US systems: the hand-held Vscan (General Electrics; GE Vingmed Ultrasound; Norway) for which an intravaginal gadget TTGP-2010® (Troyano transvaginal gadget probe) was designed, and the Voluson 730 Expert (multifrequency transvaginal ultrasound of 3-9MHz; GE Healthcare, Milwaukee, WI, USA). We performed the same measurements with both US systems in order to confirm whether or not their diagnostic capability was similar. Quantitative difference in measurements between the systems was assessed, as well as the overall diagnostic detection rate and suitability for telemedicine. RESULTS: Regarding lesion visibility with Vscan, optimal distance was 8-16cm depending on the examination type, and the total detection rate was 98.7%. The exception was an ovarian endometrioma, diagnosed as a follicular cyst using the hand-held device. Assessment of reproducibility in 180 measurements showed that the measurements obtained with Vscan were 0.3-0.4cm lower than those obtained with the high resolution US device (Voluson 730 Expert). Nevertheless, Pearson's correlation coefficient was high for biparietal diameter (0.72) and gynecological (GYN) (0.99) measurements, and for overall correlation (0.997). Image transport on USB and SD-flash cards proved convenient for telemedicine. CONCLUSIONS: A novel TV application of a hand-held US device is demonstrated for OB-GYN. Heart, abdominal and obstetrics presets of the Vscan together with color-Doppler enable a detection capability comparable to that of a high-definition US device. The lower values of the measurements obtained by the hand-held device (by 0.3-0.4cm) must be taken into account, although they have no effect on its diagnostic capability.

[Toxic shock syndrome associated with nasal packing]. / Shock tóxico estafilocócico asociado a cirugía nasal.

Staphiloccocal Toxic Shock Syndrome is a potentially fatal multisystem disease associated to nasal packing, catheter insertion, retention of foreign materials and uneffective sterile techniques. It is usually developed in the immediate postoperative period (first 48 hours) with hypotension, skin rash, fever, multisystemic failure and shock. We report a case in a 24-year-old man secondary to nasal surgery.

Electron density and gas temperature from line broadening in an argon surface-wave-sustained discharge at atmospheric pressure.

We have used the collisional broadening of neutral argon lines to determine the electron density and gas temperature of a microwave discharge at atmospheric pressure. The gas temperature can be obtained from the Van der Waals broadening, provided that the Stark broadening is negligible. This can be achieved by using lines from low-lying levels (close to the ground state). On the other hand, lines corresponding to transitions from high-lying levels, which are more sensitive to Stark (quadratic) broadening, can be utilized to determine electron density. The electron density values obtained from the quadratic Stark broadening of argon atoms are in reasonable agreement with those derived from the linear Stark broadening of the H(beta) line. The proposed method ensures perturbation-free access to plasma parameters, which is not the case when adding hydrogen to the discharge, even in a small amount, to observe the Balmer series lines.

Neural stem cells and the quest for restorative neurology.

A great deal of interest has attracted the attention of researchers on the potential use of (neural) stem cells in cell replacement or restorative therapies for heretofore incurable CNS pathologies such as brain stroke, spinal cord injury, Parkinson's disease or multiple sclerosis. This short perspective illustrates our view of neural stem cell research with a focus on the stem cell concept, on the in situ identity of neural stem cells and on selected aspects of embryonic and adult neurogenesis. A brief survey of current stem cell-based experimental literature tries to provide a realistic picture of how far we have gone in the quest to establish a restorative neurology.

Virtual sonography through the Internet: volume compression issues.

BACKGROUND: Three-dimensional ultrasound images allow virtual sonography even at a distance. However, the size of final 3-D files limits their transmission through slow networks such as the Internet. OBJECTIVE: To analyze compression techniques that transform ultrasound images into small 3-D volumes that can be transmitted through the Internet without loss of relevant medical information. METHODS: Samples were selected from ultrasound examinations performed during, 1999-2000, in the Obstetrics and Gynecology Department at the University Hospital in La Laguna, Canary Islands, Spain. The conventional ultrasound video output was recorded at 25 fps (frames per second) on a PC, producing 100- to 120-MB files (for from 500 to 550 frames). Processing to obtain 3-D images progressively reduced file size. RESULTS: The original frames passed through different compression stages: selecting the region of interest, rendering techniques, and compression for storage. Final 3-D volumes reached 1:25 compression rates (1.5- to 2-MB files). Those volumes need 7 to 8 minutes to be transmitted through the Internet at a mean data throughput of 6.6 Kbytes per second. At the receiving site, virtual sonography is possible using orthogonal projections or oblique cuts. CONCLUSIONS: Modern volume-rendering techniques allowed distant virtual sonography through the Internet. This is the result of their efficient data compression that maintains its attractiveness as a main criterion for distant diagnosis.

Glutamate N-methyl-D-aspartate receptor blockade prevents induction of GAP-43 after focal ischemia in rats.

Growth associated protein-43 (GAP-43) gene induction may be involved in reactive events that follow cerebral ischemic damage. Antagonists of the N-methyl-D-aspartate (NMDA) subclass of glutamate receptors are thought to ameliorate functional outcome after ischemic injury. To assess whether glutamate NMDA receptor blockade could alter GAP-43 postischemic induction we performed immunocytochemistry in rat brains that had been subjected to middle cerebral artery occlusion. Cortical cells did not constitutively express GAP-43, yet focal ischemia induced its expression, with an intense signal generated in cells over the lesioned area at 6 h, increasing at 24 h postischemia. This signal was effectively decreased by pretreatment with the NMDA receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate (0.1 mg/kg s.c.), but not by the glutamate release blocker riluzole (8 mg/kg i.v.), suggesting that overactivation of NMDA receptor during ischemia is linked to GAP-43 expression.

Whole-mount confocal immunofluorescence of mammalian CNS.

Bright-field wholemount labeling techniques applied to the mammalian central nervous system (CNS) offer advantages over conventional methods based on sections since an immediate and three-dimensional view of the stained components is provided. It thereby becomes possible to survey and count large number of cells and fibers in their natural relationships. The ability of confocal laser scanning microscopy to visualize in one focal plane the fluorescence associated with multiple markers could be most valuable by the availability of reliable wholemount fluorescent techniques. Accordingly, based in our previously published bright-field wholemount protocols [Brain Res. Prot. 2 (1998) 165-173], we have devised an effective immmunofluorescence wholemount procedure. We show that reliable wholemount fluorescent staining can be obtained using isolated complete CNS aged up to rat embryonic day 17, with antibodies penetration in the millimeter range. Examples are shown of preparations in which colocalization can be observed in nerve cells of cytoskeletal and calcium-binding proteins.

Identification of a ubiquitous family of membrane proteins and their expression in mouse brain.

A family of genes encoding membrane proteins with a unique structure has been identified in DNA and cDNA clones of various eukaryotes ranging from yeast to human. The nucleotide sequences of three novel cDNAs from Drosophila melanogaster and mouse were determined. The amino acid sequences of the two mouse proteins have human homologs. The gene (TMS1) encoding the yeast member of this family was disrupted, and the resulting mutant showed no significant phenotype under several stress conditions. The expression of the mouse genes TMS-1 and TMS-2 was examined by in situ hybridization of sections from brain, liver, kidney, heart and testis of an adult mouse as well as in a 1-day-old whole mouse. While the expression of TMS-2 was found to be restricted to the central nervous system, TMS-1 was also expressed in kidney and testis. The expression of TMS-1 and TMS-2 in the brain overlapped and was localized to areas associated with glutamatergic excitatory neurons, such as the hippocampus and cerebral cortex. High-magnification analysis indicated that both mRNAs are expressed in neurons. Semiquantitative analysis of mRNA expression was performed in various parts of the brain. The conservation, unique structure and localization in the mammalian brain of this novel protein family suggest an important biological role.

Why does the central nervous system not regenerate after injury?

Spinal cord injuries in humans and in other mammals are never followed by regrowth. In recent years, considerable progress has been made in analyzing mechanisms that promote and inhibit regeneration. The focus of this review is changes that occur in the transition period in development when the central nervous system (CNS) changes from being able to regenerate to the adult state of failure. In our experiments we have used the neonatal opossum (Monodelphis domestica), which corresponds to a 14-day embryonic rat or mouse. The CNS isolated from an opossum pup and maintained in culture shows dramatic regeneration. Fibers grow through and beyond lesions and reform synaptic connections with their targets. Similarly, anesthetized neonatal pups attached to the mother recover the ability to walk after complete spinal cord transection. Although the CNS isolated from a 9-day-old animal will regenerate in vitro, CNS from a 12-day-old will not. This is the stage at which glial cells in the CNS develop. Present research is devoted toward molecular screening to determine which growth-promoting molecules decrease during development, which inhibitory molecules increase, and which receptors on growing axons become altered. Despite progress in many laboratories, major hurdles must be overcome before patients can hope to be treated. Nevertheless, the picture today is not as discouraging as it was: one can think of strategies for research on spinal cord injury so as to promote regeneration and restore function.

Detection of p75 mRNA in developing marsupial CNS by cross-hybridization with rat oligonucleotide probes.

We analyzed the distribution of mRNAs encoding the low-affinity neurotrophin receptor (p75) in the CNS of adult and neonatal opossum (Monodelphis demestica) by in situ hybridization with oligodeoxynucleotide probes complementary to cloned rat sequences. During the first 2 postnatal weeks high levels of p75 message were present in the mantle zone throughout the neural tube, in basal forebrain neurons, in motoneurons, and in cerebellar cell layers. Transcript expression decreased with age. In adult CNS only a few cells in the basal forebrain expressed high levels of p75 mRNA. Nerve growth factor upregulated p75 mRNA signals in dorsal root ganglia of cultured 7 day old whole-CNS preparations. Our results indicate the usefulness of rat p75 oligodexynucleotide probes to identify homologous species of transcripts in the CNS of a non-eutherian mammal.

Monoamine oxidases: from brain maps to physiology and transgenics to pathophysiology.

The present report reviews recent advances in mapping the cellular sites of synthesis and catalytic activity, as well as age- and disease-related changes of monoamine oxidases A and B in the brain. A transgenic model of oxidative stress is also described. The relevance of these findings for the physiological and pathophysiological roles of monoamine oxidases is briefly discussed.

Three-dimensional visualization of the distribution, growth, and regeneration of monoaminergic neurons in whole mounts of immature mammalian CNS.

At birth, the opossum, Monodelphis domestica, corresponds roughly to a 14-day-old mouse embryo. The aim of these experiments was to compare the distribution of monoaminergic neurons in the two preparations during development and to follow their regeneration after injury. Procedures that allowed antibody staining to be visible in transparent whole mounts of the entire central nervous system (CNS) were devised. Neurons throughout the brain and spinal cord were stained for tyrosine hydroxylase (TH) and for serotonin (5-HT). At birth, patterns of monoaminergic cells in opossum CNS resembled those found in 14-day mouse embryos and other eutherian mammals. By postnatal day 5, immunoreactive cell bodies were clustered in appropriate regions of the midbrain and hindbrain, and numerous axons were already present throughout the spinal cord. Differences found in the opossum were the earlier presence of TH neurons in the olfactory bulb and of 5-HT neuronal perikarya in the spinal cord. Most, if not all, monoaminergic neurons in opossum were already postmitotic at birth. To study regeneration, crushes were made in cervical cords in culture. By 5 days, 8% of all TH-labeled axons and 14% of serotonergic axons had grown beyond lesions. Distal segments of monoaminergic axons degenerated. In CNS preparations from opossums older than 11 days, no regeneration of monoaminergic fibers occurred. Isolated embryonic mouse CNS also showed regeneration across spinal cord lesions, providing the possibility of using knockout and transgenic animals. Our procedures for whole-mount observation of identified cell bodies and their axons obviates the need for serial reconstructions and allows direct comparison of events occurring during development and regeneration.

Procedures for whole-mount immunohistochemistry and in situ hybridization of immature mammalian CNS.

Whole-mount labeling techniques for staining in invertebrates or lower vertebrates cannot simply be applied to the mammalian central nervous system (CNS) because of its large size. Such techniques if possible would offer advantages over conventional methods based on sections since an immediate and 3-dimensional view of the stained components in a transparent CNS is provided. It thereby becomes possible to survey and count large number of cells and fibers in their natural relationships. The aim of our experiments is to follow developing and regenerating expression of proteins and mRNAs in the CNS of mouse embryos and newborn opossums (Monodelphis domestica). Accordingly, we have devised three techniques applicable to whole-mounts: (i) An effective immunohistochemical procedure. This comprises a peroxidase-antiperoxidase method (PAP-WM) based on protocols initially developed for Xenopus embryos and oocytes, including a variation to detect exogenously applied nucleotide analogs such as 5-bromo-2'-deoxyuridine (PAP[BrdU]-WM). For greater resolution we have introduced a novel gold-silver method (IGSS-WM). (ii) An in situ hybridization procedure (ISH[PAP]-WM) which combines PAP-WM with protocols described for Xenopus. (iii) A deconvolution (optical sectioning) procedure which improves resolution for bright-field microscopy. We show that reliable whole-mount staining can be obtained using isolated CNS aged up to mouse embryonic day 17 and newborn opossum up to 15 days. Examples are shown of preparations in which one can directly localize nerve cells containing neurotransmitters, cytoskeletal proteins, nucleotide analogs and growth factor messages.

Detection of MAO-A and MAO-B mRNAs in monkey brainstem by cross-hybridization with human oligonucleotide probes.

The distribution of mRNAs encoding the isoenzymes monoamine oxidase A and B (MAO-A and MAO-B) in monkey locus coeruleus and dorsal raphe nucleus was studied by in situ hybridization histochemistry using 35S-labelled oligodeoxynucleotide probes complementary to cloned human sequences. MAO-A mRNA was highly expressed in noradrenergic neurons of the locus coeruleus while MAO-B mRNA was abundantly and exclusively localized in serotoninergic neurons of the raphe. However, upon emulsion radioautography raphe neurons showed a level of MAO-A mRNA signal noticeably above the background. Our results indicate the utility of human MAO oligodeoxynucleotide probes to identify homologous species of transcripts in the brain of a non-human primate. They also suggest the coexistence of the isoenzymes in raphe neurons as well as the potential role of MAO-A in metabolizing serotonin in vivo.

Distribution and sites of synthesis of NTT4, an orphan member of the Na+/Cl(-)-dependent neurotransmitter transporter family, in the rat CNS.

The distribution and sites of synthesis in rat CNS of NTT4, a novel orphan member of the Na+/Cl(-)-dependent neurotransmitter transporter family, were determined by immunohistochemistry and hybridization histochemistry. Antibodies raised against recombinant fusion proteins, corresponding to residues of NTT4, and 35S-labelled oligodeoxyribonucleotide probes, were used to delineate the cellular distribution of the transporter at the protein and mRNA levels. High levels of immunoreactivity (mainly in the neuropil) were found in the olfactory bulb, cerebral cortex, striatum, hippocampus, thalamus, substantia nigra, pontine nuclei, cerebellum and spinal cord. The lowest levels were associated with the lateral hypothalamic area and deep mesencephalic nuclei. In situ hybridization signals correlated well with the immunoreactivity, and demonstrated a widespread distribution of NTT4 transcripts exclusively in neurons. NTT4 transcripts appeared widely codistributed with the N-methyl-D-aspartate receptor subunit 1 (1-4b), i.e. spliced variants characterized by a common 5' 63 bp insertion. These results indicate that the transporter was associated with neuronal processes in specific glutamate innervated CNS regions. Although the substrate transported by NTT4 remains unknown, our findings suggest a possible role for this carrier protein in glutamate/glycine neurotransmission.

Cellular expression of mRNAs encoding monoamine oxidases A and B in the rat central nervous system.

Monoamine oxidases A and B (MAO-A and MAO-B) oxidatively deaminate neurotransmitter and xenobiotic amines. The cellular localization of these isoenzymes in the central nervous system (CNS) differs markedly and only partly reflects the distribution of their presumed natural substrates. In the present study, by using in situ hybridization with 35S-labelled oligonucleotide probes, we examined the distribution of mRNAs encoding MAO-A and MAO-B in the rat CNS. Probes for tyrosine hydroxylase, histidine decarboxylase, and tryptophan hydroxylase mRNAs were used to demonstrate the catecholaminergic, histaminergic, or serotoninergic nature of some cell populations in adjacent sections. The radioligands [3H]-Ro 41-1049 and [3H]lazabemide (reversible and selective inhibitors of MAO-A and MAO-B, respectively) were used to reveal the protein distribution by enzyme radioautography. The distribution and abundance of transcripts for both isoenzymes in the tissues investigated differed markedly but, in general, correlated with the protein distribution. MAO-A mRNA and protein were most abundant in noradrenergic neurons. However, moderate levels of transcript expression and protein were also detected in the serotoninergic neurons, and low but significant levels were detected in the dopaminergic neurons. An unexpectedly remarkable degree of hybridization signal was apparent in nonaminergic cell populations, e.g., in the cerebral cortices, the hippocampal formation (CA1-3, dentate gyrus), the cerebellar granule cell layer, and the spinal cord motoneurons. In contrast, MAO-B mRNA and protein were most abundant in serotoninergic and histaminergic neurons, Bergmann glial cells, and circumventricular organs, including the ependyma. MAO-B transcripts were also weakly expressed in nonaminergic cells, e.g., in the hippocampal formation (CA1-2). A further nonneuronal localization of MAO-B transcripts was also resolved, e.g., in the glia limitans, the olfactory nerve layer, and the cerebellar peduncle. These findings reveal further the potential of various cell populations to synthesize the isoenzymes, and homologous (aminergic) and heterologous (nonaminergic) patterns of expression as well as coexpression of MAO mRNAs are described.