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Biomedical subjects

D J Rossi

Publications and source records attributed to D J Rossi.

15 recordsLinked to original sources
PubMed2001

Vascular abnormalities and deregulation of VEGF in Lkb1-deficient mice.

The LKB1 tumor suppressor gene, mutated in Peutz-Jeghers syndrome, encodes a serine/threonine kinase of unknown function. Here we show that mice with a targeted disruption of Lkb1 die at midgestation, with the embryos showing neural tube defects, mesenchymal cell death, and vascular abnormalities. Extraembryonic development was also severely affected; the mutant placentas exhibited defective labyrinth layer development and the fetal vessels failed to invade the placenta. These phenotypes were associated with tissue-specific deregulation of vascular endothelial growth factor (VEGF) expression, including a marked increase in the amount of VEGF messenger RNA. Moreover, VEGF production in cultured Lkb1(-/-) fibroblasts was elevated in both normoxic and hypoxic conditions. These findings place Lkb1 in the VEGF signaling pathway and suggest that the vascular defects accompanying Lkb1 loss are mediated at least in part by VEGF.

AMP-Activated Protein Kinases↗
PubMed2001

Inability to enter S phase and defective RNA polymerase II CTD phosphorylation in mice lacking Mat1.

The trimeric Cdk7-cyclin H-Mat1 complex comprises the kinase subunit of basal transcription factor TFIIH and has been shown to function as a cyclin-dependent kinase (Cdk)-activating kinase. Herein we report that disruption of the murine Mat1 gene leads to peri-implantation lethality coincident with depletion of maternal Mat1 protein. In culture, Mat1(-/-) blastocysts gave rise to viable post-mitotic trophoblast giant cells while mitotic lineages failed to proliferate and survive. In contrast to wild-type trophoblast giant cells, Mat1(-/-) cells exhibited a rapid arrest in endoreduplication, which was characterized by an inability to enter S phase. Additionally, Mat1(-/-) cells exhibited defects in phosphorylation of the C-terminal domain (CTD) of RNA polymerase II on both Ser5 and Ser2 of the heptapeptide repeat. Despite this, Mat1(-/-) cells demonstrated apparent transcriptional and translational integrity. These data indicate an essential role for Mat1 in progression through the endocycle and suggest that while Mat1 modulates CTD phosphorylation, it does not appear to be essential for RNA polymerase II-mediated transcription.

Animals↗
PubMed2000

Glutamate release in severe brain ischaemia is mainly by reversed uptake.

The release of glutamate during brain anoxia or ischaemia triggers the death of neurons, causing mental or physical handicap. The mechanism of glutamate release is controversial, however. Four release mechanisms have been postulated: vesicular release dependent on external calcium or Ca2+ released from intracellular stores; release through swelling-activated anion channels; an indomethacin-sensitive process in astrocytes; and reversed operation of glutamate transporters. Here we have mimicked severe ischaemia in hippocampal slices and monitored glutamate release as a receptor-gated current in the CA1 pyramidal cells that are killed preferentially in ischaemic hippocampus. Using blockers of the different release mechanisms, we demonstrate that glutamate release is largely by reversed operation of neuronal glutamate transporters, and that it plays a key role in generating the anoxic depolarization that abolishes information processing in the central nervous system a few minutes after the start of ischaemia. A mathematical model incorporating K+ channels, reversible uptake carriers and NMDA (N-methyl-D-aspartate) receptor channels reproduces the main features of the response to ischaemia. Thus, transporter-mediated glutamate homeostasis fails dramatically in ischaemia: instead of removing extracellular glutamate to protect neurons, transporters release glutamate, triggering neuronal death.

ATP-Binding Cassette Transporters↗
PubMed1998

Spillover-mediated transmission at inhibitory synapses promoted by high affinity alpha6 subunit GABA(A) receptors and glomerular geometry.

Divergence and convergence of synaptic connections make a crucial contribution to the information processing capacity of the brain. Until recently, it was thought that transmitter released at a synapse affected only a specific postsynaptic cell. We show here that spillover of inhibitory transmitter at the Golgi to granule cell synapse produces significant cross-talk to non-postsynaptic cells, which is promoted both by the anatomical specialization of this glomerular synapse and by the presence of the high affinity alpha6 subunit-containing GABA(A) receptor in granule cells. Cross-talk is manifested as a novel slow rising and decaying small amplitude inhibitory postsynaptic current (IPSC) that can also contribute a long-lasting component to more typical IPSCs, which is prolonged by inhibition of the neuronal GABA transporter GAT-1. Because of the long duration of IPSCs generated by spillover, the total charge carried is three times that of IPSCs generated by directly connected terminals. GABA spillover within the mossy fiber glomerulus may play an important role in regulating the number of granule cells active in the cerebellar cortex, a regulation that is suggested by theoretical models to optimize cerebellar information processing.

Animals↗
PubMed1997

Abnormal mesoderm patterning in mouse embryos mutant for the SH2 tyrosine phosphatase Shp-2.

Shp-1, Shp-2 and corkscrew comprise a small family of cytoplasmic tyrosine phosphatases that possess two tandem SH2 domains. To investigate the biological functions of Shp-2, a targeted mutation has been introduced into the murine Shp-2 gene, which results in an internal deletion of residues 46-110 in the N-terminal SH2 domain. Shp-2 is required for embryonic development, as mice homozygous for the mutant allele die in utero at mid-gestation. The Shp-2 mutant embryos fail to gastrulate properly as evidenced by defects in the node, notochord and posterior elongation. Biochemical analysis of mutant cells indicates that Shp-2 can function as either a positive or negative regulator of MAP kinase activation, depending on the specific receptor pathway stimulated. In particular, Shp-2 is required for full and sustained activation of the MAP kinase pathway following stimulation with fibroblast growth factor (FGF), raising the possibility that the phenotype of Shp-2 mutant embryos results from a defect in FGF-receptor signalling. Thus, Shp-2 modulates tyrosine kinase signalling in vivo and is crucial for gastrulation during mammalian development.

Animals↗
PubMed1996

Modification of NMDA receptor channels and synaptic transmission by targeted disruption of the NR2C gene.

A novel strain of mutant mouse has been generated with a deletion of the gene encoding the NR2C subunit of the NMDA receptor, which is primarily expressed in cerebellar granule cells. Patch-clamp recordings from granule cells in thin cerebellar slices were used to assess the consequences of the gene deletion. In granule cells of wild-type animals, a wide range of single-channel conductances were observed (19-60 pS). The disruption of the NR2C gene results in the disappearance of low-conductance NMDA receptor channels ( < 37 pS) normally expressed in granule cells during developmental maturation. The NMDA receptor-mediated synaptic current is markedly potentiated in amplitude, but abbreviated in duration (with no net difference in total charge), and the non-NMDA component of the synaptic current was reduced. We conclude that the NR2C subunit contributes to functional heteromeric NMDA receptor-subunit assemblies at the mossy fiber synapse and extrasynaptic sites during maturation, and the conductance level exhibited by a given receptor macromolecule may reflect the stochiometry of subunit composition.

Animals↗
PubMed1996

Generation of a novel Fli-1 protein by gene targeting leads to a defect in thymus development and a delay in Friend virus-induced erythroleukemia.

The proto-oncogene Fli-1 is a member of the ets family of transcription factor genes. Its activation by either chromosomal translocation or proviral insertion leads to Ewing's sarcoma in humans or erythroleukemia in mice, respectively, Fli-1 is preferentially expressed in hematopoietic and endothelial cells. This expression pattern resembled that of c-ets-1, another ets gene closely related and physically linked to Fli-1. We also generated a germ line mutation in Fli-1 by homologous recombination in embryonic stem cells. Homozygous mutant mice exhibit thymic hypocellularity which is not related to a defect in a specific subpopulation of thymocytes or to increased apoptosis, suggesting that Fli-1 is an important regulator of a prethymic T-cell progenitor. This phenotype was corrected by crossing the Fli-1 deficient mice expressing Fli-1 cDNA. Homozygous mutant mice remained susceptible to erythroleukemia induction by Friend murine leukemia virus, although the latency period was significantly increased. Surprisingly, the mutant Fli-1 allele was still a target for Friend murine leukemia virus integration, and leukemic spleens with a rearranged Fli-1 gene expressed a truncated Fli-1 protein that appears to arise from an internal translation initiation site and alternative splicing around the neo cassette used in the gene targeting. The fortuitous discovery of the mutant Fli-1 protein, revealed only as the result of the clonal expansion of leukemic cells harboring a rearranged Fli-1 gene, suggests caution in the interpretation of gene-targeting experiments that result in either no or only a subtle phenotypic alteration.

Animals↗
PubMed1995

Vascular system defects and neuronal apoptosis in mice lacking ras GTPase-activating protein.

The gene encoding p120-rasGAP, a negative regulator of Ras, has been disrupted in mice. This Gap mutation affects the ability of endothelial cells to organize into a highly vascularized network and results in extensive neuronal cell death. Mutati ons in the Gap and Nf1 genes have a synergistic effect, such that embryos homozygous for mutations in both genes show an exacerbated Gap phenotype. Thus rasGAP and neurofibromin act together to regulate Ras activity during embryonic development.

Animals↗
PubMed1995

Properties of transmission at a giant glutamatergic synapse in cerebellum: the mossy fiber-unipolar brush cell synapse.

1. The synaptic activation by mossy fibers (MFs) of unipolar brush cells (UBCs) in the vestibular cerebellum (nodulus and uvula) was examined using patch-clamp recording methods in thin, rat cerebellar slices with Lucifer yellow-filled pipettes for subsequent fluorescence microscopic verification of the cell morphology. 2. UBCs were distinguished from adjacent granule cells in thin cerebellar slices in the uvula and nodulus regions by their larger soma diameters and short dendritic brush, greater whole-cell capacitance, and a prolonged, biphasic excitatory postsynaptic current (EPSC) to stimulation of MFs. 3. Thin-section transmission electron micrographs of the MF-UBC synapse displayed an unusually extensive area of synaptic apposition estimated to measure 12-40 microns2. The majority of UBCs was innervated by a single MF. At high magnification, individual clusters of presynaptic vesicles could be discerned, separated by regions of presynaptic membrane lacking vesicles, but apposed to continuous regions of postsynaptic density. Thus, after release, transmitter diffusion from the synaptic cleft must traverse considerable stretches of postsynaptic membrane before escape into extracellular space. In contrast, MF-granule cell synapses in these cerebellar regions resembled glutamate synapses in other brain regions in that the total synaptic area measured < or = 4 microns2. These synaptic junctions were flanked by short stretches of unspecialized plasma membrane, providing a short (0.5 micron) diffusional path from the site of neurotransmitter release to a branch point of the extracellular space. 4. The MF-evoked EPSC in UBCs was composed of a fast (10-90% rise time: 0.70 ms) and slow (10-90% rise time: 395 ms; 10-90% decay time: 3.1 s) component. The fast component was blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/KA) antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) and displayed linear current-voltage (I-V) relations in the presence or absence of external magnesium. 5. The slow EPSC was also mediated by glutamate receptors, but in most neurons both AMPA/KA and N-methyl-D-aspartate (NMDA) receptors contributed to the slow EPSC, with the contribution of NMDA receptors predominating in the majority of cells. Consequently, although all cells displayed linear I-V relations in Mg(2+)-free saline, cells in which the slow EPSC was predominently mediated by NMDA receptors exhibited voltage-dependent rectification in the presence of external Mg2+ (1 mM). 6. With increasing postnatal age (10-30 d), the contribution made to the slow EPSC by NMDA receptors declined, with a reciprocal increase in the contribution being made by AMPA/KA receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1993

The developmental onset of NMDA receptor-channel activity during neuronal migration.

Patch-clamp recordings of granule cells in thin slices of developing rat cerebellum maintained in vitro displayed spontaneous single-channel activity mediated via activation of N-methyl-D-aspartate (NMDA) receptors. The frequency of tonic single-channel activity was reversibly inhibited by the NMDA receptor/channel antagonists D-2-amino-5-phosphonovalerate (D-AP5), 7-chloro-kynurenate (7-Cl-Kynu) and MgCl2, potentiated by glycine, and unaffected by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or tetrodotoxin (TTX). Tonic channel activity was also reversibly inhibited by enzymatic degradation of endogenous glutamate by glutamate pyruvate transaminase, which did not affect the NMDA sensitivity of granule cells. Both the frequency of spontaneous channel activity and the NMDA sensitivity were low in premigratory cells of the external germinal layer (EGL), with large increases observed in migrating cells in the molecular layer (ML) and in postmigratory cells within the internal granule cell layer (GCL). Tonic channel activity was enhanced by the glutamate uptake inhibitor L-alpha-aminoadipate (L-alpha-AA), the degree of enhancement being greater in the EGL than the GCL. The results demonstrate that a dramatic increase in the tonic NMDA receptor-channel activity occurs during the stages of granule cell differentiation, migration and synaptogenesis, which is driven by endogenous glutamate release and regulated by NMDA receptor density and local glutamate uptake.

Animals↗
PubMed1993

Adenosine agonists reduce conditioned avoidance responding in the rat.

Because adenosine agonists may possess therapeutic potential as antipsychotic agents, we examined the activity of several prototypic agents in vivo in blocking conditioned avoidance responding (CAR) in the rat, a behavioral test predictive of antipsychotic efficacy in humans. Potency in blocking CAR is directly proportional to potency in alleviating schizophrenia. Hence, the adenosine A1-selective agonists [cyclopentyl adenosine (CPA) and (R)-phenylisopropyl adenosine (R-PIA)], A2-selective agonists [CV-1808 and (2-(p-(carboxyethyl)-phenethylamino)-5'-N-ethyl-carboxamido adenosine (CGS 21680)], and a nonselective agonist [5'-N-carboxamido adenosine (NECA)] were examined in this test. Block of CAR was first determined for standard antipsychotic agents [ED50 mg/kg, IP, and 95% confidence level (CL) in parentheses], such as haloperidol [0.23 (0.18, 0.39)], trifluoroperazine [(0.9 (0.7, 1.0)], thioridazine [12.5 (10.5, 15.3)], metoclopramide [7.8 (6.4, 9.2)], and chlorpromazine [4.9 (4.2, 5.9)]. The paradigm consisted of a light- and tone-signaled footshock that could be avoided via a discrete lever press. Affinity for A1 and A2 binding sites in brain tissue from Fischer 344 rats was ascertained to be similar to that seen in other rodent strains. Each adenosine agonist blocked CAR. NECA [ED50 value (95% CL) = 0.07 (0.004, 0.12) mg/kg, IP] was the most potent agent, followed by: R-PIA [0.34 (0.23, 0.44)]; CGS 21680 [1.1 (0.8, 2.0)]; CV-1808 [1.3 (1.0, 1.8)]; and CPA [1.5 (1.3, 1.7)]. Pretreatment with caffeine (25 mg/kg, IP, -10 min) blocked the inhibition of CAR produced by the adenosine agonists, suggesting the event is mediated via purinergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1992

Role of metabotropic glutamate (ACPD) receptors at the parallel fiber-Purkinje cell synapse.

1. The role of metabotropic glutamate receptors at the parallel fiber (PF)-Purkinje cell synapse in cerebellum was studied by examining the actions of the active stereoisomer (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [1S,3R-ACPD (25-50 microM)] on fura-2-loaded, patch-clamped rat Purkinje cells in thin slices. 2. The bath application of 1S,3R-ACPD evoked a direct post-synaptic depolarization that readily desensitized during prolonged (> 1 min) applications of the drug. This depolarizing response to 1S,3R-ACPD differed from the slow depolarization to 1S,3R-ACPD observed in cortical neurons mediated via closure of potassium channels in that it was not associated with an obvious change in membrane conductance and was not blocked by external barium. Similarly, slow inward rectifier currents were not affected during the 1S,3R-ACPD-induced depolarization. 3. The direct depolarization induced by 1S,3R-ACPD was not mediated by N-methyl-D-aspartate (NMDA) or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid kainate (AMPA)-KA excitatory amino acid (EAA) receptor subtypes, because the response was not blocked in the presence of antagonists of these receptors. 4. The EAA antagonist L-2-amino-3-phosphonopropionic acid, which blocks 1S,3R-ACPD-induced inositide synthesis in other cell types, had no effect on the depolarizing response. 5. Fura-2 measurements of somatic [Ca2+]i revealed that [Ca2+]i was not elevated during the 1S,3R-ACPD-induced depolarization unless the cell fired calcium-dependent action potentials. 6. In addition to the direct depolarization induced by 1S,3R-ACPD, the amplitude of PF-evoked excitatory postsynaptic potentials (EPSPs) was profoundly and reversibly reduced. This effect was observed in all cells regardless of whether a direct depolarization was produced by 1S,3R-ACPD. This reduction of the PF EPSP generally preceded the onset of depolarizing responses, did not desensitize during prolonged applications of 1S,3R-ACPD, and was reversible. 7. The reversible reduction of the PF EPSP by 1S,3R-ACPD was not related to a postsynaptic blocking action of the drug, because responses of Purkinje cells to AMPA, an agonist of the EAA receptor subtype mediating the EPSP, were reversibly potentiated in the presence of 1S,3R-ACPD. 8. The nitric oxide synthesis promoter sodium nitroprusside (1-3 nM) had no effect on the amplitude of PF EPSP or the membrane properties of Purkinje cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Afferent Pathways↗
PubMed1992

Correcting bias from the standard linear adjustment of weaning weight to an age-constant basis for beef calves.

Standard linear adjustment of weaning weight to a constant age has been shown to introduce bias in the adjusted weight due to nonlinear growth from birth to weaning of beef calves. Ten years of field records from the five strains of Beefbooster Cattle Alberta Ltd. seed stock herds were used to investigate the use of correction factors to adjust standard 180-d weight (WT180) for this bias. Statistical analyses were performed within strain and followed three steps: 1) the full data set was split into an estimation set (ES) and a validation set (VS), 2) WT180 from the ES was used to develop estimates of correction factors using a model including herd (H), year (YR), age of dam (DA), sex of calf (S), all two and three-way interactions, and any significant linear and quadratic covariates of calf age at weaning deviated from 180 d (DEVCA) and interactions between DEVCA and DA, S or DA x S, and 3) significant DEVCA coefficients were used to correct WT180 from the VS, then WT180 and the corrected weight (WTCOR) from the VS were analyzed with the same model as in Step 2 and significance of DEVCA terms were compared. Two types of data splitting were used. Adjusted R2 was calculated to describe the proportion of total variation of DEVCA terms explained for WT180 from the ES. The DEVCA terms explained .08 to 1.54% of the total variation for the five strains. Linear and quadratic correction factors were both positive and negative. Bias in WT180 from the ES within 180 +/- 35 d of age ranged from 2.8 to 21.7 kg.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1988

Synthesis and biological activity of ketomethylene-containing nonapeptide analogues of snake venom angiotensin converting enzyme inhibitors.

Two ketomethylene-containing nonapeptide analogues were synthesized to determine if ketomethylene analogues of the nonapeptide venom inhibitors of angiotensin converting enzyme (ACE) would have oral ACE inhibition activity. Both ketomethylene-containing nonapeptides 18 and 19 were potent inhibitors of rabbit lung ACE with I50s of 3.4 and 8.0 nM, respectively, compared to 340 nM for their parent nonapeptide and 450 nM for captopril. Peptide 18 was rapidly cleaved by trypsin, but 19 was reasonably stable to all enzyme degradation systems tested with maximum degradation of 50% by pepsin in 3 h. Both 18 and 19 when given iv to normotensive rats were between 3 and 10 times more potent than captopril in inhibiting an angiotensin I induced blood pressure increase. Peptide 19 showed no ACE inhibition activity in unanesthetized normotensive rats when administered orally at doses of 10 or 100 mg/kg. Experiments were conducted to determine whether 19 is adsorbed from the gastrointestinal track following oral administration. These experiments indicated that 19 is adsorbed. It is concluded that the lack of oral activity of 19 is probably due to its rapid excretion, probably into the bile.

Angiotensin-Converting Enzyme Inhibitors↗