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PubMed1998

Store-operated calcium entry promotes shape change in pulmonary endothelial cells expressing Trp1.

Activation of Ca2+ entry is known to produce endothelial cell shape change, leading to increased permeability, leukocyte migration, and initiation of angiogenesis in conduit-vessel endothelial cells. The mode of Ca2+ entry regulating cell shape is unknown. We hypothesized that activation of store-operated Ca2+ channels (SOCs) is sufficient to promote cell shape change necessary for these processes. SOC activation in rat pulmonary arterial endothelial cells increased free cytosolic Ca2+ that was dependent on a membrane current having a net inward component of 5.45 +/- 0.90 pA/pF at -80 mV. Changes in endothelial cell shape accompanied SOC activation and were dependent on Ca2+ entry-induced reconfiguration of peripheral (cortical) filamentous actin (F-actin). Because the identity of pulmonary endothelial SOCs is unknown, but mammalian homologues of the Drosophila melanogaster transient receptor potential (trp) gene have been proposed to form Ca2+ entry channels in nonexcitable cells, we performed RT-PCR using Trp oligonucleotide primers in both rat and human pulmonary arterial endothelial cells. Both cell types were found to express Trp1, but neither expressed Trp3 nor Trp6. Our study indicates that 1) Ca2+ entry in pulmonary endothelial cells through SOCs produces cell shape change that is dependent on site-specific rearrangement of the microfilamentous cytoskeleton and 2) Trp1 may be a component of pulmonary endothelial SOCs.

Amino Acid Sequence↗

PubMed1984

Depolarisation-dependent protein phosphorylation in rat cortical synaptosomes is inhibited by fluphenazine at a step after calcium entry.

The sequence of molecular events linking depolarisation-dependent calcium influx to calcium-stimulated protein phosphorylation is unknown. In this study the effect of the neuroleptic drug fluphenazine on depolarisation-dependent protein phosphorylation was investigated using an intact postmitochondrial pellet isolated from rat cerebral cortex. Fluphenazine, in a dose-dependent manner, completely inhibited the increases in protein phosphorylation observed previously. The concentration of fluphenazine required for 50% inhibition varied for different phosphoproteins but for synapsin I was 123 microM. Other neuroleptics produced effects similar to fluphenazine with their order of potency being thioridazine greater than haloperidol greater than trifluoperazine greater than fluphenazine greater than chlorpromazine. Fluphenazine also increased the phosphorylation of proteins in nondepolarised controls at concentrations of 20 and 60 microM. The inhibition of depolarisation-dependent phosphorylation was apparently not due to a loss of synaptosomal integrity or viability, a decrease in calcium uptake, a change in substrate availability, or to a change in protein phosphatase activity. The data are most consistent with an inhibition of protein kinase activity by blockade of calmodulin or phospholipid activation.

Adenosine Triphosphate↗

PubMed1987

Scanning electron microscope studies of sperm incorporation into the zebrafish (Brachydanio) egg.

Morphological studies on the gametes and entry of the spermatozoan into the egg of the zebra danio, Brachydanio rerio, were conducted primarily with scanning electron microscopy. The spermatozoan showed a spherical head, which lacked an acrosome, a midpiece containing several mitochondria, and a flagellum. Observations of the unfertilized egg confirmed and extended prior studies showing a distinct cluster of microvilli on the plasma membrane, identified as the sperm entry site, beneath the inner micropylar aperture (Hart and Donovan, '83). The fertilizing spermatozoan attached to the sperm entry site within 5 seconds of the mixing of a gamete suspension. Binding to the egg microvilli appeared restricted to the equatorial surface of the spermatozoan. Fusion between the plasma membranes of the interacting gametes was followed by the formation of a distinct, nipple-shaped fertilization cone. The sperm head was partially incorporated into the fertilization cone cytoplasm by 60 seconds postinsemination. The incorporation of the entire sperm head, midpiece, and a portion of the flagellum occurred between 1 and 2 minutes. During this time, the fertilization cone shortened and was transformed into a massive, blister-like cytoplasmic swelling. Concurrently, upward movements of the ooplasm resulted in the gradual disappearance of the original depression in the egg surface containing the sperm entry site. The second polar body, fully developed by 10 minutes postinsemination, formed approximately 10-15 microns from the site of sperm penetration. Development of the fertilization cone, formation of the second polar body and exocytosis of cortical granules at the sperm entry site readily occurred in parthenogenetically activated eggs, indicating that these surface rearrangements do not require sperm binding and/or fusion.

Animals↗

PubMed2000

Inhibition by chloral hydrate and trichloroethanol of AMPA-induced Ca(2+) influx in rat cultured cortical neurones.

The effects of chloral hydrate and its main metabolite 2,2, 2-trichloroethanol were investigated on the (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-induced rise of intracellular Ca(2+) concentration ([Ca(2+)](i)) in cultured non-pyramidal cortical neurones of rats by using single-cell fura-2 microfluorimetry. AMPA elicited a concentration-dependent effect that peaked at 300 microM (EC(50), 7. 5 microM). Responses to AMPA (30 microM) were markedly inhibited by superfusion with chloral hydrate (IC(50), 4.5 mM) or trichloroethanol (IC(50), 0.9 mM). By contrast, ethanol (100 mM) caused only slight inhibition. In conclusion, the results demonstrate that chloral hydrate and especially its metabolite trichloroethanol, inhibit the AMPA-induced rise of [Ca(2+)](i) by depressing the entry of Ca(2) into cortical neurones via the AMPA receptor-channel.

Animals↗

PubMed1995

The prospero transcription factor is asymmetrically localized to the cell cortex during neuroblast mitosis in Drosophila.

Both intrinsic and extrinsic factors are known to regulate sibling cell fate. Here we describe a novel mechanism for the asymmetric localization of a transcription factor to one daughter cell at mitosis. The Drosophila CNS develops from asymmetrically dividing neuroblasts, which give rise to a large neuroblast and a smaller ganglion mother cell (GMC). The prospero gene encodes a transcription factor necessary for proper GMC gene expression. We show that the prospero protein is synthesized in the neuroblast where it is localized to the F-actin cell cortex. At mitosis, prospero is asymmetrically localized to the budding GMC and excluded from the neuroblast. After cytokinesis, prospero is translocated from the GMC cortex into the nucleus. Asymmetric cortical localization of prospero in neuroblasts requires entry into mitosis; it does not depend on numb function. prospero is also observed in cortical crescents in dividing precursors of the peripheral nervous system and adult midgut. The asymmetric cortical localization of prospero at mitosis is a mechanism for rapidly establishing distinct sibling cell fates in the CNS and possibly other tissues.

Animals↗

PubMed2005

Store-operated Ca2+ entry: vesicle fusion or reversible trafficking and de novo conformational coupling?

Store-operated Ca2+ entry (SOCE), a mechanism regulated by the filling state of the intracellular Ca2+ stores, is a major pathway for Ca2+ influx. Hypotheses to explain the communication between the Ca2+ stores and plasma membrane (PM) have considered both the existence of small messenger molecules, such as a Ca2+-influx factor (CIF), and both stable and de novo conformational coupling between proteins in the Ca2+ store and PM. Alternatively, a secretion-like coupling model based on vesicle fusion and channel insertion in the PM has been proposed, which shares some properties with the de novo conformational coupling model, such as the role of the actin cytoskeleton and soluble N-ethylmaleimide (NEM)-sensitive-factor attachment proteins receptor (SNARE) proteins. Here we review recent progress made in the characterization of the de novo conformational coupling and the secretion-like coupling models for SOCE. We pay particular attention into the involvement of SNARE proteins and the actin cytoskeleton in both SOCE models. SNAREs are recognized as proteins involved in exocytosis, participating in vesicle transport, membrane docking, and fusion. As with secretion, a role for the cortical actin network in Ca2+ entry has been demonstrated in a number of cell types. In resting cells, the cytoskeleton may prevent the interaction between the Ca2+ stores and the PM, or preventing fusion of vesicles containing Ca2+ channels with the PM. These are processes in which SNARE proteins might play a crucial role upon cell activation by directing a precise interaction between the membrane of the transported organelle and the PM.

Actins↗

PubMed2007

Duration and not strength of activation in temporo-parietal cortex positively correlates with schizotypy.

Impaired self- and own body processing in patients with schizophrenia and individuals along the schizophrenia spectrum have been associated with dysfunctional cortical activation at the temporo-parietal junction. Here we investigated whether strength or duration of temporo-parietal junction activation during an own body processing task correlates with level of abnormal self-processing in healthy subjects as measured by the frequency of spontaneously experienced schizotypal body schema alterations (perceptual aberrations) and dissociative experiences. Participants carried out a mental imagery task with respect to their own body. Behavioral data and high density EEG were measured. EEG data were analyzed using evoked potential mapping and electrical neuroimaging. Participants completed two validated self-report questionnaires, one asking about perceptual aberration and one about dissociative experiences. The own body transformation task activated the right temporo-parietal junction at 310-390 ms. Participants' reaction times and duration of activation at the right temporo-parietal junction, but not its strength, were found to correlate positively with perceptual aberration scores. No relationship was found with dissociative experiences scores. Brain activations proceeding and following activation of the right temporo-parietal junction did not correlate with scores on either scale. The positive correlation between performance and right temporo-parietal activation in an own body transformation task with perceptual aberrations scores in our healthy population suggests that disturbances in self- and body processing in individuals along the schizophrenia spectrum might be due to prolonged, rather than stronger activation of the right temporo-parietal junction. We argue that this might reflect local pathology, pathologies in cortico-cortical connections and/or re-entry of top-down processing.

Adult↗

PubMed1997

Catheterization of the Medullary Cavity of Long Bones.

Vascular catheterization techniques were used in bones to allow access to the entire medullary cavity through a minimal percutaneous approach. The study was carried out on human and animal anatomical specimens and in living sheep. After oblique penetration at an angle of approximately 40û to the surface of the cortical bone, we attempted to pass in the bone shaft using various types of guides and catheters. Different types of instrumentation currently available were compared for ease of advancement in the shaft and quantity of bone marrow that could be aspirated. Once the metaphysis was reached, a variety of techniques were studied to penetrate the cancellous bone and to introduce a catheter. A percutaneous approach to any part of a long bone through a cortical pathway from a remote entry site is feasible in certain diagnostic or therapeutic indications, when direct approach to pathological sites in long bones is dangerous or impossible.

Journal Article↗

PubMed1993

Cortical bone reactions at the interface of external fixation half-pins under different loading conditions.

The pin-bone interface is the weakest link in the mechanical stability of external skeletal fixation. In this investigation, a canine model was used to characterize the nature of cortical bone reactions at the pin-bone interface. Unilateral external fixators were applied to the tibiae of 61 dogs using six tapered cortical half-pins. The pins were inserted after predrilling both cortices, and pin insertion torque was measured. A transverse or oblique osteotomy was performed in each tibia and stabilized under different gap conditions. Unrestricted weight bearing was allowed immediately after surgery. Sequential functional evaluations of the animals and biweekly roentgenograms of the pin tracks were done. Pin removal torques were recorded at the time the animals were killed at different time periods. Pin tracks were analyzed using quantitative tetracycline histomorphometry and microradiography. The results showed that cortical bone undergoes extensive creeping substitution around external fixation half-pins. New bone accounted for approximately 43% of the intracortical space along the pin track, and cortical bone porosity showed a fourfold increase compared with intact bone value. This cortical bone remodeling resulted in a time-related decrease of pin removal torque (p < 0.001). In inherently unstable oblique osteotomies, and less in stable rigidly fixed transverse osteotomies, immediate postoperative weight bearing caused bone thread resorption and adverse cortical bone remodeling at the entry cortex of external fixation half-pins. The unicortical loosening of half-pins that became evident during the first month of fixation obviously represents a consequence of micromotion and local bone yielding failure caused by high dynamic stresses of the pin-bone interface. Effective precautions should be taken to reduce such stresses.

Animals↗

PubMed2005

Seven Lotus japonicus genes required for transcriptional reprogramming of the root during fungal and bacterial symbiosis.

A combined genetic and transcriptome analysis was performed to study the molecular basis of the arbuscular mycorrhiza (AM) symbiosis. By testing the AM phenotype of nodulation-impaired mutants and complementation analysis, we defined seven Lotus japonicus common symbiosis genes (SYMRK, CASTOR, POLLUX, SYM3, SYM6, SYM15, and SYM24) that are required for both fungal and bacterial entry into root epidermal or cortical cells. To describe the phenotype of these mutants at the molecular level, we screened for differentiating transcriptional responses of mutant and wild-type roots by large-scale gene expression profiling using cDNA-amplified fragment length polymorphism. Two percent of root transcripts was found to increase in abundance during AM development, from which a set of AM-regulated marker genes was established. A Ser-protease (SbtS) and a Cys-protease (CysS) were also activated during root nodule development. AM-induced transcriptional activation was abolished in roots carrying mutations in common symbiosis genes, suggesting a central position of these genes in a pathway leading to the transcriptional activation of downstream genes. By contrast, AM fungus-induced gene repression appeared to be unaffected in mutant backgrounds, which indicates the presence of additional independent signaling pathways.

Bacterial Physiological Phenomena↗

PubMed1979

Oocyte maturation: aberrant post-fusion responses of the rabbit primary oocyte to penetrating spermatozoa.

Primary oocytes cannot be fertilized normally; they begin to develop this capacity as meiosis resumes. To elucidate the changes involved in acquisition of their fertilizability, rabbit primary oocytes displaying a germinal vesicle (GV oocytes) were placed in Fallopian tubes inseminated previously with spermatozoa, recovered 2--5 h later and examined by light and electron microscopy. At least 4 aspects of GV oocyte/sperm interaction were abnormal. Although the vestments and oolemma seem normally receptive to spermatozoa, fusion with the oolemma of the primary oocyte did not elicit exocytosis of cortical granules, and consequently multiple entry of spermatozoa into the ooplasm was common. Secondly, the GV oocyte cortex failed to achieve a normal englufment of the anterior part of the sperm head. It sank into the ooplasm capped by only a small rostral vesicle or left the stable inner acrosomal membrane as a patch in the oolemma. Only rarely then was there significant dispersion of the sperm chromatin, and this remained surrounded by nuclear envelope. The persistence of this envelope constitutes a further aberrant feature, for it disappears immediately in secondary oocytes and was absent in primary oocytes in which germinal vesicle breakdown had occurred. The results are discussed with particular reference to current ideas about male pronucleus formation.

Animals↗

PubMed1999

[Nod factors, chemical signal exchange between bacteria and leguminous plants in nitrogen fixing symbiosis].

The early steps of the nitrogen-fixing symbiosis between plant legumes and soil bacteria (rhizobium) are mediated by an exchange of chemical signals between the two partners. Upon gene activation by plant root secretions (flavonoids), bacteria synthesize lipochitooligomers (called Nod Factors, NFs) that induce root hair deformations, cortical cell divisions, allow bacterial entry and produce nodule organogenesis at nano to picomole concentrations. Substitutions occurring on the lipochitooligosaccharide core are essential for recognition and activity. Biosynthesis of these molecules is now fully dissected, by looking at the structural changes in NFs induced by gene mutation or gene transfers. From the biodiversity studies of NFs, it appears that their structures belong with the phylogenetic evolution of plants, rather than that of bacteria, suggesting a coevolution of symbiotic bacteria with their plant receptors. Some preliminary and indirect observations indicate that similar molecules seem to exist in non-legumes plants, in batrachians and fishes beeing possibly involved in their embryogenesis, but they are probably at at a so low concentration that all attempts to detect them directly fail up to now.

Carbohydrate Sequence↗

PubMed1992

Acquisition of signs from American sign language in hearing individuals following left hemisphere damage and aphasia.

Three severely aphasic hearing patients with no prior knowledge of sign language were able to acquire competency in aspects of American Sign Language (ASL) lexicon and finger spelling, in contrast to a near complete inability to speak the English counterparts of these visuo-gestural signs. Two patients with damage in left postero-lateral temporal and inferior parietal cortices mastered production and comprehension of single signs and short meaningful sign sequences, but the one patient with damage to virtually all left temporal cortices was less accurate in single sign processing and was unable to produce sequences of signs at all. These findings suggest that conceptual knowledge is represented independently of the auditory-vocal records for the corresponding lexical entries, and that left anterior temporal cortices outside of traditional "language areas" are part of the neural network which supports the linkage between conceptual knowledge and linguistic signs, especially as they are used in the sequenced activations required for production or comprehension of meaningful sentences.

Adult↗

PubMed1991

Effects of diltiazem on postischaemic renal cortical microcirculation in the pig.

Diltiazem--a calcium entry blocker--was tested in a porcine model under continuous chlormethiazole-pancuronium anaesthesia as protection against renal failure following 60 min of renal ischaemia. Fourteen pigs were randomly allocated to one experimental (diltiazem and ischaemia) and one control group (only ischaemia) (n = 7 in each). Diltiazem was administered as a continuous intravenous infusion started before the ischaemic insult. In two additional animals diltiazem was given but ischemia was not induced. The postischaemic renal cortical microcirculation was simultaneously investigated in four different regions in the left kidney during the first 4 h of reperfusion. Laser Doppler flowmetry (LDF) was performed in two different regions and measurement of tissue oxygenation was done in two other regions. In the two animals treated with diltiazem without ischaemia, only minor variations in central haemodynamic and renal microcirculatory parameters were evident. In the control group (ischaemia), superficial renal cortical blood flow (Qsrc) decreased from 49 +/- 11 (s.d.) arbitrary units at baseline to 24 +/- 4 arb. units 4 h after start of reperfusion (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Kidney Injury↗

PubMed2005

Invasion of Lotus japonicus root hairless 1 by Mesorhizobium loti involves the nodulation factor-dependent induction of root hairs.

In many legumes, including Lotus japonicus and Medicago truncatula, susceptible root hairs are the primary sites for the initial signal perception and physical contact between the host plant and the compatible nitrogen-fixing bacteria that leads to the initiation of root invasion and nodule organogenesis. However, diverse mechanisms of nodulation have been described in a variety of legume species that do not rely on root hairs. To clarify the significance of root hairs during the L. japonicus-Mesorhizobium loti symbiosis, we have isolated and performed a detailed analysis of four independent L. japonicus root hair developmental mutants. We show that although important for the efficient colonization of roots, the presence of wild-type root hairs is not required for the initiation of nodule primordia (NP) organogenesis and the colonization of the nodule structures. In the genetic background of the L. japonicus root hairless 1 mutant, the nodulation factor-dependent formation of NP provides the structural basis for alternative modes of invasion by M. loti. Surprisingly, one mode of root colonization involves nodulation factor-dependent induction of NP-associated cortical root hairs and epidermal root hairs, which, in turn, support bacterial invasion. In addition, entry of M. loti through cracks at the cortical surface of the NP is described. These novel mechanisms of nodule colonization by M. loti explain the fully functional, albeit significantly delayed, nodulation phenotype of the L. japonicus ROOT HAIRLESS mutant.

Base Sequence↗

PubMed2000

A quantitative analysis of L-glutamate-regulated Na+ dynamics in mouse cortical astrocytes: implications for cellular bioenergetics.

The mode of Na+ entry and the dynamics of intracellular Na+ concentration ([Na+]i) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. An elevation of [Na+]i was evoked by glutamate, whose amplitude and initial rate were concentration dependent. The glutamate-evoked Na+ increase was primarily due to Na+-glutamate cotransport, as inhibition of non-NMDA ionotropic receptors by 6-cyano-7-nitroquinoxiline-2,3-dione (CNQX) only weakly diminished the response and D-aspartate, a substrate of the glutamate transporter, produced [Na+]i elevations similar to those evoked by glutamate. Non-NMDA receptor activation could nevertheless be demonstrated by preventing receptor desensitization using cyclothiazide. Thus, in normal conditions non-NMDA receptors do not contribute significantly to the glutamate-evoked Na+ response. The rate of Na+ influx decreased during glutamate application, with kinetics that correlate well with the increase in [Na+]i and which depend on the extracellular concentration of glutamate. A tight coupling between Na+ entry and Na+/K+ ATPase activity was revealed by the massive [Na+]i increase evoked by glutamate when pump activity was inhibited by ouabain. During prolonged glutamate application, [Na+]i remains elevated at a new steady-state where Na+ influx through the transporter matches Na+ extrusion through the Na+/K+ ATPase. A mathematical model of the dynamics of [Na+]i homeostasis is presented which precisely defines the critical role of Na+ influx kinetics in the establishment of the elevated steady state and its consequences on the cellular bioenergetics. Indeed, extracellular glutamate concentrations of 10 microM already markedly increase the energetic demands of the astrocytes.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2004

Presynaptic activity and Ca2+ entry are required for the maintenance of NMDA receptor-independent LTP at visual cortical excitatory synapses.

We have shown that some neural activity is required for the maintenance of long-term potentiation (LTP) at visual cortical inhibitory synapses. We tested whether this was also the case in N-methyl-d-aspartate (NMDA) receptor-independent LTP of excitatory connections in layer 2/3 cells of developing rat visual cortex. This LTP occurred after 2-Hz stimulation was applied for 15 min and always persisted for several hours while test stimulation was continued at 0.1 Hz. When test stimulation was stopped for 1 h after LTP induction, only one-third of the LTP instances disappeared, but most did disappear under a pharmacological suppression of spontaneous firing, indicating that LTP maintenance requires either evoked or spontaneous activities. LTP was totally abolished by a temporary blockade of action potentials with lidocaine or the removal of extracellular Ca(2+) after LTP induction, but it persisted under a voltage clamp of postsynaptic cells or after a temporary blockade of postsynaptic activity with the glutamate receptor antagonist kynurenate, suggesting that LTP maintenance requires presynaptic, but not postsynaptic, firing and Ca(2+) entry. More than one-half of the LTP instances were abolished after a pharmacological blockade of P-type Ca(2+) channels, whereas it persisted after either L-type or Ni(2+)-sensitive Ca(2+) channel blockades. These results show that the maintenance of NMDA receptor-independent excitatory LTP requires presynaptic firing and Ca(2+) channel activation as inhibitory LTP, although the necessary level of firing and Ca(2+) entry seems lower for the former than the latter and the Ca(2+) channel types involved are only partly the same.

Anesthetics, Local↗

PubMed1999

Organisation of Xenopus oocyte and egg cortices.

The division of the Xenopus oocyte cortex into structurally and functionally distinct "animal" and "vegetal" regions during oogenesis provides the basis of the organisation of the early embryo. The vegetal region of the cortex accumulates specific maternal mRNAs that specify the development of the endoderm and mesoderm, as well as functionally-defined "determinants" of dorso-anterior development, and recognisable "germ plasm" determinants that segregate into primary germ cells. These localised elements on the vegetal cortex underlie both the primary animal-vegetal polarity of the egg and the organisation of the developing embryo. The animal cortex meanwhile becomes specialised for the events associated with fertilisation: sperm entry, calcium release into the cytoplasm, cortical granule exocytosis, and polarised cortical contraction. Cortical and subcortical reorganisations associated with meiotic maturation, fertilisation, cortical rotation, and the first mitotic cleavage divisions redistribute the vegetal cortical determinants, contributing to the specification of dorso-anterior axis and segregation of the germ line. In this article we consider what is known about the changing organisation of the oocyte and egg cortex in relation to the mechanisms of determinant localisation, anchorage, and redistribution, and show novel ultrastructural views of cortices isolated at different stages and processed by the rapid-freeze deep-etch method. Cortical organisation involves interactions between the different cytoskeletal filament systems and internal membranes. Associated proteins and cytoplasmic signals probably modulate these interactions in stage-specific ways, leaving much to be understood.

Animals↗