Biomedical subjects

M D Ganfornina

Publications and source records attributed to M D Ganfornina.

17 recordsLinked to original sources

PubMed2005

Comparative gene expression profile of mouse carotid body and adrenal medulla under physiological hypoxia.

The carotid body (CB) is an arterial chemoreceptor, bearing specialized type I cells that respond to hypoxia by closing specific K+ channels and releasing neurotransmitters to activate sensory axons. Despite having detailed information on the electrical and neurochemical changes triggered by hypoxia in CB, the knowledge of the molecular components involved in the signalling cascade of the hypoxic response is fragmentary. This study analyses the mouse CB transcriptional changes in response to low PO2 by hybridization to oligonucleotide microarrays. The transcripts were obtained from whole CBs after mice were exposed to either normoxia (21% O2), or physiological hypoxia (10% O2) for 24 h. The CB transcriptional profiles obtained under these environmental conditions were subtracted from the profile of control non-chemoreceptor adrenal medulla extracted from the same animals. Given the common developmental origin of these two organs, they share many properties but differ specifically in their response to O2. Our analysis revealed 751 probe sets regulated specifically in CB under hypoxia (388 up-regulated and 363 down-regulated). These results were corroborated by assessing the transcriptional changes of selected genes under physiological hypoxia with quantitative RT-PCR. Our microarray experiments revealed a number of CB-expressed genes (e.g. TH, ferritin and triosephosphate isomerase) that were known to change their expression under hypoxia. However, we also found novel genes that consistently changed their expression under physiological hypoxia. Among them, a group of ion channels show specific regulation in CB: the potassium channels Kir6.1 and Kcnn4 are up-regulated, while the modulatory subunit Kcnab1 is down-regulated by low PO2 levels.

Adrenal Medulla↗

PubMed2000

Lazarillo, a neuronal lipocalin in grasshoppers with a role in axon guidance.

In this report we present a review on the grasshopper lipocalin Lazarillo with special emphasis on how its molecular properties could account for its known function: the guidance of pioneer neurons during nervous system development. The expression and function of Lazarillo in a subset of developing neurons, its heavy glycosylation and its glycosylphosphatidylinositol linkage to the plasma membrane, make Lazarillo a unique member of the lipocalin family. We have built a model of the tertiary structure of Lazarillo in which we have studied the exposed surfaces in search for clues about ligand and protein interactions with Lazarillo. Our hypotheses about how this lipocalin can exert its function are discussed.

Animals↗

PubMed2000

Evolution of the lipocalin family as inferred from a protein sequence phylogeny.

The lipocalins constitute a family of proteins that have been found in eubacteria and a variety of eukaryotic cells, where they play diverse physiological roles. It is the primary goal of this review to examine the patterns of change followed by lipocalins through their complex history, in order to stimulate scientists in the field to experimentally contrast our phylogeny-derived hypotheses. We reexamine our previous work on lipocalin phylogeny and update the phylogenetic analysis of the family. Lipocalins separate into 14 monophyletic clades, some of which are grouped in well supported superclades. The lipocalin tree was rooted with the bacterial lipocalin genes under the assumption that they have evolved from a single common ancestor with the metazoan lipocalins, and not by horizontal transfer. The topology of the rooted tree and the species distribution of lipocalins suggest that the newly arising lipocalins show a higher rate of amino acid sequence divergence, a higher rate of gene duplication, and their internal pocket has evolved towards binding smaller hydrophobic ligands with more efficiency.

Amino Acid Sequence↗

PubMed2000

A phylogenetic analysis of the lipocalin protein family.

The lipocalins are a family of extracellular proteins that bind and transport small hydrophobic molecules. They are found in eubacteria and a great variety of eukaryotic cells, in which they play diverse physiological roles. We report here the detection of two new eukaryotic lipocalins and a phylogenetic analysis of 113 lipocalin family members performed with maximum-likelihood and parsimony methods on their amino acid sequences. Lipocalins segregate into 13 monophyletic clades, some of which are grouped in well-supported superclades. An examination of the G + C content of the bacterial lipocalin genes and the detection of four new conceptual lipocalins in other eubacterial species argue against a recent horizontal transfer as the origin of prokaryotic lipocalins. Therefore, we rooted our lipocalin tree using the clade containing the prokaryotic lipocalins. The topology of the rooted lipocalin tree is in general agreement with the currently accepted view of the organismal phylogeny of arthropods and chordates. The rooted tree allows us to assign polarity to character changes and suggests a plausible scenario for the evolution of important lipocalin properties. More recently evolved lipocalins tend to (1) show greater rates of amino acid substitutions, (2) have more flexible protein structures, (3) bind smaller hydrophobic ligands, and (4) increase the efficiency of their ligand-binding contacts. Finally, we found that the family of fatty-acid-binding proteins originated from the more derived lipocalins and therefore cannot be considered a sister group of the lipocalin family.

Amino Acid Sequence↗

PubMed2000

Characterization of two novel lipocalins expressed in the Drosophila embryonic nervous system.

We have found two novel lipocalins in the fruit fly Drosophila melanogaster that are homologous to the grasshopper Lazarillo, a singular lipocalin within this protein family which functions in axon guidance during nervous system development. Sequence analysis suggests that the two Drosophila proteins are secreted and possess peptide regions unique in the lipocalin family. The mRNAs of DNLaz (for Drosophila neural Lazarillo) and DGLaz (for Drosophila glial Lazarillo) are expressed with different temporal patterns during embryogenesis. They show low levels of larval expression and are highly expressed in pupa and adult flies. DNLaz mRNA is transcribed in a subset of neurons and neuronal precursors in the embryonic CNS. DGLaz mRNA is found in a subset of glial cells of the CNS: the longitudinal glia and the medial cell body glia. Both lipocalins are also expressed outside the nervous system in the developing gut, fat body and amnioserosa. The DNLaz protein is detected in a subset of axons in the developing CNS. Treatment with a secretion blocker enhances the antibody labeling, indicating the DNLaz secreted nature. These findings make the embryonic nervous system expression of lipocalins a feature more widespread than previously thought. We propose that DNLaz and DGLaz may have a role in axonal outgrowth and pathfinding, although other putative functions are also discussed.

Amino Acid Sequence↗

PubMed1999

Developmental expression and molecular characterization of two gap junction channel proteins expressed during embryogenesis in the grasshopper Schistocerca americana.

Gap junctions are membrane channels that directly connect the cytoplasm of neighboring cells, allowing the exchange of ions and small molecules. Two analogous families of proteins, the connexins and innexins, are the channel-forming molecules in vertebrates and invertebrates, respectively. In order to study the role of gap junctions in the embryonic development of the nervous system, we searched for innexins in the grasshopper Schistocerca americana. Here we present the molecular cloning and sequence analysis of two novel innexins, G-Inx(1) and G-Inx(2), expressed during grasshopper embryonic development. The analysis of G-Inx(1) and G-Inx(2) proteins suggests they bear four transmembrane domains, which show strong conservation in members of the innexin family. The study of the phylogenetic relationships between members of the innexin family and the new grasshopper proteins suggests that G-Inx(1) is orthologous to the Drosophila 1(1)-ogre. However, G-Inx(2) seems to be a member of a new group of insect innexins. We used in situ hybridization with the G-Inx(1) and G-Inx(2) cDNA clones, and two polyclonal sera raised against different regions of G-Inx(1) to study the mRNA and protein expression patterns and the subcellular localization of the grasshopper innexins. G-Inx(1) is primarily expressed in the embryonic nervous system, in neural precursors and glial cells. In addition, a restricted stripe of epithelial cells in the developing limb, involved in the guidance of sensory growth cones, expresses G-Inx(1). G-Inx(2) expression is more widespread in the grasshopper embryo, but a restricted expression is found in a subset of neural precursors. The generally different but partially overlapping expression patterns of G-Inx(1) and G-Inx(2) supports the combinatorial character of gap junction formation in invertebrates, an essential property to generate specificity in this form of cell-cell communication.

Amino Acid Sequence↗

PubMed1999

Generation of evolutionary novelty by functional shift.

That biological features may change their function during evolution has long been recognized. Particularly, the acquisition of new functions by molecules involved in developmental pathways is suspected to cause important morphologic novelties. However, the current terminology describing functional changes during evolution (co-option or recruitment) fails to recognize important biologic distinctions between diverse evolutionary routes involving functional shifts. The main goal of our work is to stress the importance of an apparently trivial distinction: Whether or not the element that adopts a new function (anything from a morphologic structure to a protein domain) is a single or a duplicated element. We propose that natural selection must act in a radically different way, depending on the historic succession of co-option and duplication events; that is, co-option may provide the selective pressure for a subsequent gene duplication or could be a stabilizing factor that helps maintain redundancy after gene duplication. We review the evidence available on functional changes, focusing whenever possible on developmental molecules, and we propose a conceptual framework for the study of functional shifts during evolution with a level of resolution appropriate to the power of our current methodologies.

Animals↗

PubMed1998

Molecular characterization and phylogenetic relationships of a protein with potential oxygen-binding capabilities in the grasshopper embryo. A hemocyanin in insects?

Arthropodan hemocyanins, prophenoloxidases (PPOs), and insect hexamerins form a superfamily of hemolymph proteins that we propose to call the AHPH superfamily. The evolutionary and functional relationships of these proteins are illuminated by a new embryonic hemolymph protein (EHP) that is expressed during early stages of development in the grasshopper embryo. EHP is a 78-kDa soluble protein present initially in the yolk sac content, and later in the embryonic hemolymph. Protein purification and peptide sequencing were used to identify an embryonic cDNA clone coding for EHP. In situ hybridization identifies hemocytes as EHP-expressing cells. As deduced from the cDNA clone, EHP is a secreted protein with two potential glycosylation sites. Sequence analysis defines EHP as a member of the AHPH superfamily. Phylogenetic analyses with all the currently available AHPH proteins, including EHP, were performed to ascertain the evolutionary history of this protein superfamily. We used both the entire protein sequence and each of the three domains present in the AHPH proteins. The phylogenies inferred for each of the domains suggest a mosaic evolution of these protein modules. Phylogenetic and multivariate analyses consistently group EHP with crustacean hemocyanins and, less closely, with insect hexamerins, relative to cheliceratan hemocyanins and PPOs. The grasshopper protein rigorously preserves the residues involved in oxygen binding, oligomerization, and allosteric regulation of the oxygen transport proteins. Although insects were thought not to have hemocyanins, we propose that EHP functions as an oxygen transport or storage protein during embryonic development.

Amino Acid Sequence↗

PubMed1996

Embryonic development of the enteric nervous system of the grasshopper Schistocerca americana.

The enteric nervous system (ENS) of the grasshopper Schistocerca americana is organized into four ganglia located in the foregut (the dorsal unpaired frontal and hypocerebral ganglia, and the paired ingluvial ganglia), and two plexuses that innervate the foregut and midgut. A dorsomedial recurrent nerve and two lateral esophageal nerves connect the ganglia. The midgut plexus is arranged in four nerves running along the midgut surface. In this study, we have focused on the embryonic development of the grasshopper ENS; we have studied the proliferation pattern, morphogenesis, and some aspects of neuronal differentiation by using a number of specific molecular markers. The grasshopper ENS develops early in embryogenesis (25-30%) from three neurogenic zones (NZs) located on the roof of the stomodeum. These NZs slightly invaginate from an epithelial placode. The expression pattern of specific cell surface proteins and the analysis of the mitotic activity showed that NZs cells delaminate from the epithelium, become neuronal precursors, divide symmetrically, and then actively migrate to their final position in the enteric ganglia or plexuses. The grasshopper enteric ganglia are composed of mixed populations of cells from different NZs. The foregut and midgut plexuses are formed by the dispersal of cells from the developing hypocerebral and ingluvial ganglia. The main ENS nerves are pioneered by axons extending anteriorly from hypocerebral and ingluvial neurons. The insect ENS exhibits an enormous variation in design. Several features of the grasshopper program of neurogenesis and pattern of cell migration are compared to other insects, and some evolutionary implications are discussed.

Animals↗

PubMed1996

Developmental expression and biochemical analysis of conulin, a protein secreted from a subset of neuronal growth cones.

In this report, we analyze the developmental pattern of expression of a new grasshopper protein, Conulin, using the monoclonal antibody 7D2 on whole-mount embryos and dissociated neurons. We also have examined its biochemical properties by immunoblot analysis. Conulin is a protein expressed by a subset of neurons in the grasshopper embryo. The monoclonal antibody 7D2 recognizes Conulin as an M(r) 190 x 10(3) protein that is found in both the soluble and membrane-bound fractions of embryonic proteins. The membrane association is disrupted by alkaline pH and high ionic strength. Conulin first is expressed and stored in vesicles inside the cell bodies and axons of central and peripheral neurons. Later, Conulin is detected on the cell surface, but exclusively in the central nervous system neuropil. This expression is confined to a subset of nerve growth cones. Conulin is detected on growth cones only after pioneer neurons have outlined the axonal scaffold. Immunocytochemistry on cultured embryonic neurons demonstrates that the neurons have the autonomous ability to target Conulin to the growth cones. The protein is secreted but remains transiently associated with the growth cone plasma membrane. The discovery of Conulin confirms the existence of proteins specific for the nerve growth cone. Its transitory presence during axonogenesis in only a subset of follower growth cones suggests that Conulin is involved in guidance through selective fasciculation with pre-existing axons within the ganglionic neuropil.

Animals↗

PubMed1995

Contributions of an orthopteran to the understanding of neuronal pathfinding.

During the development of the nervous system neurons extend axons through a complex embryonic environment. To find a correct target, often located at a long distance, the neuronal growth cones travel along highly specific and stereotyped pathways. Proper neuronal pathfinding is thought to be accomplished by the specific interaction of receptors on the neuronal surface with molecular cues in the environment. We review the information obtained in an invertebrate model system, the grasshopper embryo, about the specific role of the cell surface in wiring the nervous system.

Animals↗

PubMed1995

Lazarillo, a new GPI-linked surface lipocalin, is restricted to a subset of neurons in the grasshopper embryo.

Lazarillo, a protein recognized by the monoclonal antibody 10E6, is expressed by a subset of neurons in the developing nervous system of the grasshopper. It is a glycoprotein of 45x10(3) M(r) with internal disulfide bonds and linked to the extracellular side of the plasma membrane by a glycosylphosphatidylinositol moiety. Peptide sequences obtained from affinity purified adult protein were used to identify an embryonic cDNA clone, and in situ hybridizations confirmed that the distribution of the Lazarillo mRNA paralleled that of the monoclonal antibody labeling on embryos. Sequence analysis defines Lazarillo as a member of the lipocalin family, extracellular carriers of small hydrophobic ligands, and most related to the porphyrin- and retinol-binding lipocalins. Lazarillo is the first example of a lipocalin anchored to the plasma membrane, highly glycosylated, and restricted to a subset of developing neurons.

Amino Acid Sequence↗

PubMed1995

Developmental expression of the lipocalin Lazarillo and its role in axonal pathfinding in the grasshopper embryo.

This article describes the expression pattern and functional analysis of Lazarillo, a novel cell surface glycoprotein expressed in the embryonic grasshopper nervous system, and a member of the lipocalin family. Lazarillo is expressed by a subset of neuroblasts, ganglion mother cells and neurons of the central nervous system, by all sensory neurons of the peripheral nervous system, and by a subset of neurons of the enteric nervous system. It is also present in a few non neuronal cells associated mainly with the excretory system. A monoclonal antibody raised against Lazarillo perturbs the extent and direction of growth of identified commissural pioneer neurons. We propose that Lazarillo is the receptor for a midline morphogen involved in the outgrowth and guidance of these neurons.

Animals↗

PubMed1992

Potassium channel types in arterial chemoreceptor cells and their selective modulation by oxygen.

Single K+ channel currents were recorded in excised membrane patches from dispersed chemoreceptor cells of the rabbit carotid body under conditions that abolish current flow through Na+ and Ca2+ channels. We have found three classes of voltage-gated K+ channels that differ in their single-channel conductance (gamma), dependence on internal Ca2+ (Ca2+i), and sensitivity to changes in O2 tension (PO2). Ca(2+)-activated K+ channels (KCa channels) with gamma approximately 210 pS in symmetrical K+ solutions were observed when [Ca2+]i was greater than 0.1 microM. Small conductance channels with gamma = 16 pS were not affected by [Ca2+]i and they exhibited slow activation and inactivation time courses. In these two channel types open probability (P(open)) was unaffected when exposed to normoxic (PO2 = 140 mmHg) or hypoxic (PO2 approximately 5-10 mmHg) external solutions. A third channel type (referred to as KO2 channel), having an intermediate gamma(approximately 40 pS), was the most frequently recorded. KO2 channels are steeply voltage dependent and not affected by [Ca2+]i, they inactivate almost completely in less than 500 ms, and their P(open) reversibly decreases upon exposure to low PO2. The effect of low PO2 is voltage dependent, being more pronounced at moderately depolarized voltages. At 0 mV, for example, P(open) diminishes to approximately 40% of the control value. The time course of ensemble current averages of KO2 channels is remarkably similar to that of the O2-sensitive K+ current. In addition, ensemble average and macroscopic K+ currents are affected similarly by low PO2. These observations strongly suggest that KO2 channels are the main contributors to the macroscopic K+ current of glomus cells. The reversible inhibition of KO2 channel activity by low PO2 does not desensitize and is not related to the presence of F-, ATP, and GTP-gamma-S at the internal face of the membrane. These results indicate that KO2 channels confer upon glomus cells their unique chemoreceptor properties and that the O2-K+ channel interaction occurs either directly or through an O2 sensor intrinsic to the plasma membrane closely associated with the channel molecule.

Adenosine Triphosphate↗

PubMed1992

Gating of O2-sensitive K+ channels of arterial chemoreceptor cells and kinetic modifications induced by low PO2.

We have studied the kinetic properties of the O2-sensitive K+ channels (KO2 channels) of dissociated glomus cells from rabbit carotid bodies exposed to variable O2 tension (PO2). Experiments were done using single-channel and whole-cell recording techniques. The major gating properties of KO2 channels in excised membrane patches can be explained by a minimal kinetic scheme that includes several closed states (C0 to C4), an open state (O), and two inactivated states (I0 and I1). At negative membrane potentials most channels are distributed between the left-most closed states (C0 and C1), but membrane depolarization displaces the equilibrium toward the open state. After opening, channels undergo reversible transitions to a short-living closed state (C4). These transitions configure a burst, which terminates by channels either returning to a closed state in the activation pathway (C3) or entering a reversible inactivated conformation (I0). Burst duration increases with membrane depolarization. During a maintained depolarization, KO2 channels make several bursts before ending at a nonreversible, absorbing, inactivated state (I1). On moderate depolarizations, KO2 channels inactivate very often from a closed state. Exposure to low PO2 reversibly induces an increase in the first latency, a decrease in the number of bursts per trace, and a higher occurrence of closed-state inactivation. The open state and the transitions to adjacent closed or inactivated states seem to be unaltered by hypoxia. Thus, at low PO2 the number of channels that open in response to a depolarization decreases, and those channels that follow the activation pathway open more slowly and inactivate faster. At the macroscopic level, these changes are paralleled by a reduction in the peak current amplitude, slowing down of the activation kinetics, and acceleration of the inactivation time course. The effects of low PO2 can be explained by assuming that under this condition the closed state C0 is stabilized and the transitions to the absorbing inactivated state I1 are favored. The fact that hypoxia modifies kinetically defined conformational states of the channels suggests that O2 levels determine the structure of specific domains of the KO2 channel molecule. These results help to understand the molecular mechanisms underlying the enhancement of the excitability of glomus cells in response to hypoxia.

Animals↗

PubMed1991

Single K+ channels in membrane patches of arterial chemoreceptor cells are modulated by O2 tension.

Type I cells of the carotid body are known to participate in the detection of O2 tension in arterial blood but the primary chemotransduction mechanisms are not well understood. Here we report the existence in excised membrane patches of type I cells of a single K+ channel type modulated by changes in PO2. Open probability of the O2-sensitive K+ channel reversibly decreased by at least 50% on exposure to hypoxia but single-channel conductance (approximately 20 pS) was unaltered. In the range between 70 and 150 mmHg (1 mmHg = 133 Pa) the decrease of single-channel open probability was proportional to the PO2 measured in the vicinity of the membrane patch. The inhibition of K+ channel activity by low PO2 was independent of the presence of non-hydrolyzable guanine triphosphate analogues at the internal face of the membrane. The results indicate that the O2 sensor of type I cells is in the plasma membrane and suggest that environmental O2 interacts directly with the K+ channels.

Animals↗

PubMed1988

Periaqueductal gray neurons' activity in a mesencephalic slice preparation.

The electrophysiological characteristics of periaqueductal gray (PAG) neurons were studied using intracellular techniques in guinea pig brainstem slices maintained 'in vitro'. Input resistance and time constant ranged between 60-110 M omega and 11-20 ms respectively. Direct activation elicited action potentials generated by a Na+-conductance and characterized by a remarkable Ca2+-dependent plateau in the falling phase and a long-lasting afterhyperpolarization which is probably due to a Ca2+-dependent K+-conductance. These cells also showed a slow return to the resting membrane potential after hyperpolarizing pulses which was Ca2+-dependent. All PAG neurons had a low resting membrane potential and displayed a tonic spontaneous firing at a frequency around 7 impulses/s.

Action Potentials↗