Miguel Freire

Gene sequence, developmental expression, and protein phosphorylation of RAB-17 in maize

The ABA-induced MA12 cDNA from maize, which encodes a set of highly phosphorylated embryo proteins, was used to isolate the corresponding genomic clone. This gene, called RAB-17 (responsive to ABA), encodes a basic, glycine-rich protein (mol. wt. 17 164) containing a cluster of 8 serine residues, seven of them contiguous. It is a homologue of the rice RAB-21 gene (Mundy J, Chua NH, EMBO J 7; 2279–2286, 1988). Phosphoamino acid analysis of the isolated protein indicates that only the serine residues are phosphorylated and a putative casein-type kinase phosphorylatable sequence was identified in the protein. The pattern of expression and in vivo phosphorylation of the RAB-17 protein was studied during maize embryo germination and in calli of both meristematic or embryonic origin. ABA treatment induced the synthesis of RAB-17 mRNA and protein in calli, however, the RAB-17 proteins were found to be highly phosphorylated only in embryos.

Cajal's contributions to glia research

In 1906, Santiago Ramón y Cajal was awarded the Nobel Prize in Physiology or Medicine in recognition of his work on the structure of neurons and their connections. What is less well known is that he also had a keen interest in glia and developed specific staining methods for their study. In addition to describing their morphology, he speculated on a role for glia in sleep and wakefulness and even in executive brain functions such as attention. In this article, we focus on Cajals histological research into glial cells; this research includes original drawings of astrocytes, oligodendrocytes, microglia and radial glia, as well as his scientific writings. We aim to show that, concerning glia as well as neurons, Cajal was far ahead of his time.

Functional characteristics of the maize RNA-binding protein MA16.

The maize RNA-binding protein MA16 is a non-ribosomal nucleolar protein widely distributed in different maize tissues. We have previously shown that the MA16 protein binds preferentially to guanosine-and uridine-rich sequences. As a step towards the identification of specific targets with which MA16 interacts within the cell, we investigated the RNA-binding affinities and several other aspects of the protein by using binding assays and immunochemistry. The MA16 protein showed a wide spectrum of RNA-binding activities with lower affinities to several RNAs that was salt and heparin-sensitive indicative of electrostatic interactions, and higher affinities to particular RNAs including rRNA and translatable mRNA sequences. Among the RNAs found associated with MA16 protein was that encoding MA16 itself. This observation raises the possibility that MA16 gene expression could be self-regulated. Immunoprecipitation studies showed that in vivo MA16 was phosphorylated and that MA16 interacts with RNAs through complex association with several proteins. These results suggest that both phosphorylation and interaction with other proteins may be involved in determining RNA-binding specificities of MA16 in the cell.

Updating old ideas and recent advances regarding the Interstitial Cells of Cajal

Since their discovery by Cajal in 1889, the Interstitial Cells of Cajal (ICC) have generated much controversy in the scientific community. Indeed, the nervous, muscle or fibroblastic nature of the ICC has remained under debate for more than a century, as has their possible physiological function. Cajal and his colleagues considered them to be neurons, while contemporary histologists like Kölliker and Dogiel categorized these cells as fibroblasts. More recently, the role of ICC in the origin of slow-wave peristaltism has been elucidated, and several studies have shown that they participate in neurotransmission (intercalation theory). The fact that ICC assemble in the circular muscular layer and that they originate from cells which emerge from the ventral neural tube (VENT cells), a source of neurons, glia and ICC precursors other than the neural crest, suggests a neural origin for this particular subset of ICC. The discovery that ICC express the Kit protein, a type III tyrosine kinase receptor encoded by the proto-oncogene c-kit, has helped better understand their physiological role and implication in pathological conditions. Gleevec, a novel molecule designed to inhibit the mutant activated version of c-Kit receptors, is the drug of choice to treat the so-called gastrointestinal stromal tumours (GIST), the most common non-epithelial neoplasm of the gastrointestinal tract. Here we review Cajals original contributions with the aid of unique images taken from Cajals histological slides (preserved at the Cajal Museum, Cajal Institute, CSIC). In addition, we present a historical review of the concepts associated with this particular cell type, emphasizing current data that has advanced our understanding of the role these intriguing cells fulfil.

Three-Dimensional Reconstruction and Quantitative Study of a Pyramidal Cell of a Cajal Histological Preparation

The year 2006 marks the centenary of the Nobel Prize for Physiology or Medicine awarded to Santiago Ramón y Cajal and Camilo Golgi. We commemorate this centenary with a three-dimensional reconstruction and a quantitative study of a pyramidal cell of a Cajals histological preparation. This preparation is one of the 4529 histological preparations personally made by Ramón y Cajal and preserved in the Museum Cajal. The three-dimensional reconstruction of the neuron allows visualizing one important discovery of Ramón y Cajal that constitutes an active field of research in present-day neuroscience: dendritic spines.

Dendritic Spines and Development: Towards a Unifying Model of Spinogenesis—A Present Day Review of Cajal's Histological Slides and Drawings

Dendritic spines receive the majority of excitatory connections in the central nervous system, and, thus, they are key structures in the regulation of neural activity. Hence, the cellular and molecular mechanisms underlying their generation and plasticity, both during development and in adulthood, are a matter of fundamental and practical interest. Indeed, a better understanding of these mechanisms should provide clues to the development of novel clinical therapies. Here, we present original results obtained from high-quality images of Cajals histological preparations, stored at the Cajal Museum (Instituto Cajal, CSIC), obtained using extended focus imaging, three-dimensional reconstruction, and rendering. Based on the data available in the literature regarding the formation of dendritic spines during development and our results, we propose a unifying model for dendritic spine development.

An inexpensive and interactive microcomputer system for codifying golgi-impregnated neuronal morphology

An interactive microcomputer system has been developed for the quantitative analysis of Golgi-impregnated neuronal morphology (GINM). The system uses commercially available hardware: a 48K RAM microcomputer (Apple II), a TV monitor (NEC), an optical microscope (Reichert Biovar) with a camera lucida and a specially developed wheel attached to the focus adjustment knob and to a digital planimeter (Tamaya), allowing one to obtain the value of the z-coordinate. The x- and y-coordinates are obtained using the movement of a point plotted by the computer on the TV monitor. This point is superimposed upon the GINM point to be codified employing the camera lucida. The x- and y-coordinates are acquired automatically while the variations of the z-coordinate are introduced by the computer keyboard. The three-dimensional coordinates of the selected GINM points and different codes are stored by the computer in a six-dimensional array. The software is written in BASIC. The results demonstrate that the digitization and quantitative analysis of GINM can be achieved with reasonably inexpensive equipment.

The Histological Slides and Drawings of Cajal

Ramón y Cajals studies in the field of neuroscience provoked a radical change in the course of its history. For this reason he is considered as the father of modern neuroscience. Some of his original preparations are housed at the Cajal Museum (Cajal Institute, CSIC, Madrid, Spain). In this article, we catalogue and analyse more than 4,500 of Cajals histological preparations, the same preparations he used during his scientific career. Furthermore, we catalogued Cajals original correspondence, both manuscripts and personal letters, drawings and plates. This is the first time anyone has compiled an account of Cajals enormous scientific production, offering some curious insights into his work and his legacy.

The Growth Cone as Seen Through Cajal's Original Histological Preparations and Publications

During the development of the nervous system, each neuron must contact its appropriate target cell in order to establish its specific connections. More than a century ago, Ramón y Cajal discovered an amoeboid-like structure at the end of the axon of developing nerve cells. He called this structure the growth cone [cono de crecimiento] and he proposed that this structure was guided towards its target tissue by chemical substances secreted by the different cells that line its course. We have reviewed the discovery of the growth cone by Cajal using his original publications, his original scientific drawings, and by studying his histological preparations conserved at the “Instituto Cajal” (Madrid, Spain). 1 We found a very good correlation between the structure of the growth cone in the Golgi-impregnated and reduced silver-nitrate-stained material used by Cajal, and that which is revealed with present-day methods. Finally, Cajals view of the function of the growth cone and his chemotactic hypothesis will also be considered in the light of present-day knowledge. 1The Cajal Museum is situated within the Cajal Institute at Doctor Arce 37, Madrid, where most of the items that Cajal himself produced are conserved, such as: histological preparations; scientific drawings; his photographic collection; scientific manuscripts; scientific correspondence; artistic drawings, and paintings (Freire, 2003). *Both authors have equally contributed to this work.

Stereoscopic and biplanar microphotography of Golgi-impregnated neurons: A correlative study using conventional and real-time, direct-image confocal microscopies

A correlative study of neuronal reconstruction methods was made using both conventional (non-confocal) and real-time confocal microscopies. Simple and sophisticated (totally automated) methods are described for making both biplanar microphotographs using conventional transmitted light, and stereoscopic microphotographs using real-time confocal microscopy of Golgi-impregnated neurons. Confocal microscopy discriminates against out-of-focus layers to produce optical sections which can be summed on photographic film to obtain neuronal reconstructions. Biplanar images are obtained by fusion, using a stereo-viewer, of two adjacent optical sections obtained with a conventional transmitted light microscope. Stereoscopic and biplanar microphotographs of 3-day-old chick neurons are presented.