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Biomedical subjects

B D Bennett

Publications and source records attributed to B D Bennett.

At least 19 recordsLinked to original sources
PubMed1997

Cell-specific expression and regulation of a glucokinase gene locus transgene.

Transgenic mice containing one or more extra copies of the entire glucokinase (GK) gene locus were generated and characterized. The GK transgene, an 83-kilobase pair mouse genomic DNA fragment containing both promoter regions, was expressed and regulated in a cell-specific manner, and rescued GK null lethality when crossed into mice bearing a targeted mutation of the endogenous GK gene. Livers from the transgenic mice had elevated GK mRNA, protein, and activity levels, compared with controls, and the transgene was regulated in liver by dietary manipulations. The amount of GK immunoreactivity in hepatocyte nuclei, where GK binds to the GK regulatory protein, was also increased. Pancreatic islets displayed increased GK immunoreactivity and NAD(P)H responses to glucose, but only when isolated and cultured in 20 mM glucose, as a result of the hypoglycemic phenotype of these mice (Niswender, K. D., Shiota, M., Postic, C., Cherrington, A. D., and Magnuson, M. A. (1997) J. Biol. Chem. 272, 22604-22609). Together, these results indicate that the region of the gene from -55 to +28 kilobase pairs (relative to the liver GK transcription start site) contains all the regulatory sequences necessary for expression of both GK isoforms, thereby placing an upper limit on the size of the GK gene locus.

Animals↗
PubMed1997

Blood glucose determination: point of care testing.

Mechanisms for point of care glucose determinations have changed significantly since first introduced approximately 20 years ago. Such tests are now commonly done in acute and chronic care hospitals, as well as in physicians' offices and patients' homes. Although basically reliable, there are a number of potential problems with glucose determination by these methods that may not be considered by physicians interpreting these tests. This is a brief review of such problems, especially in the acute care setting.

Blood Glucose↗
PubMed1997

Adrenoceptor-mediated elevation of ambient GABA levels activates presynaptic GABA(B) receptors in rat sensorimotor cortex.

At inhibitory synapses in the mature neocortex and hippocampus in vitro, spontaneous action-potential-dependent and -independent release of gamma-aminobutyric acid (GABA) activates postsynaptic GABA(A) receptors but not pre- or postsynaptic GABA(B) receptors. Elevation of synaptic GABA levels with pharmacological agents or electrical stimulation can cause activation of GABA(B) receptors, but the physiological conditions under which such activation occurs need further elucidation. In rodent sensorimotor cortex, epinephrine produced a depression in the amplitude of evoked monosynaptic inhibitory postsynaptic currents (IPSCs) and a concomitant, adrenoceptor-mediated increase in the frequency of spontaneous IPSCs. Blockade of GABA(B) receptors prevented the depression of evoked IPSC amplitude by epinephrine but did not affect the increase in spontaneous IPSC frequency. These data show that adrenoceptor-mediated increases in spontaneous IPSCs can cause activation of presynaptic GABA(B) receptors and indirectly modulate impulse-related GABA release, presumably through elevation of synaptic GABA levels.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1996

A role for leptin and its cognate receptor in hematopoiesis.

BACKGROUND: Hematopoiesis entails the production of multiple blood cell lineages throughout the lifespan of the organism. This is accomplished by the regulated expansion and differentiation of hematopoietic precursors that originate from self-renewing hematopoietic stem cells. Studies of lineage commitment and proliferation have shown that the cytokine family of growth factors plays an important role in hematopoietic differentiation. However, in hematopoiesis, as in most self-renewing biological systems, the molecules that regulate the stem cells directly remain largely unknown. In this study, we have undertaken a search for novel cytokines that may influence the fate of hematopoietic stem cells. RESULTS: We have cloned three splice variants of a novel cytokine receptor from human hematopoietic stem cells expressing the CD34 antigen, one of which is identical to the leptin receptor. Expression analysis revealed that the leptin receptor is expressed in both human and murine hematopoietic stem cell populations, and that leptin is expressed by hematopoietic stroma. We show that leptin provides a proliferative signal in hematopoietic cells. Importantly, we demonstrate that leptin provides a proliferative signal in BAF-3 cells and increases the proliferation of hematopoietic stem cell populations. The proliferative effects of leptin seem to be at the level of a multilineage progenitor, as shown by increased myelopoiesis, erythropoiesis and lymphopoiesis. Analysis of db/db mice, in which the leptin receptor is truncated, revealed that the steady-state levels of peripheral blood B cells and CD4-expressing T cells were dramatically reduced, demonstrating that the leptin pathway plays an essential role in lymphopoiesis. Colony assays performed using marrow from db/db and wild-type mice indicated that db/db marrow has a deficit in lymphopoietic progenitors; furthermore, db/db mice are unable to fully recover the lymphopoietic population following irradiation insult, and although the levels of peripheral blood erythrocytes are normal in db/db mice, spleen erythrocyte production is severely compromized. CONCLUSIONS: We have discovered that leptin and its cognate receptor constitute a novel hematopoietic pathway that is required for normal lymphopoiesis. This pathway seems to act at the level of the hematopoietic stem/progenitor cell, and may well also impact upon erythropoiesis, particularly in anemic states that may require output from the spleen. These findings offer a new perspective on the role of the fat cell in hematopoiesis.

Amino Acid Sequence↗
PubMed1996

Quantitative subcellular imaging of glucose metabolism within intact pancreatic islets.

Studies of dispersed beta cells have been used to infer their behavior in the intact pancreatic islet. When dispersed, beta cells exhibit multiple metabolic glucose-response populations with different insulin secretion properties. This has led to a model for glucose-dependent insulin secretion from the islet based on a step-wise recruitment of individual beta cells. However, previously reported synchronous and uniform Ca2+ activity and electrical responses indicate that beta cell behavior within intact islets is more uniform. Therefore, uncertainty remains whether beta cell metabolic heterogeneity is functionally important in intact islets. We have used two-photon excitation microscopy to measure and compare the glucose-induced NAD(P)H autofluorescence response in dispersed beta cells and within intact islets. Over 90% of beta cells in intact islets responded to glucose with significantly elevated NAD(P)H levels, compared with less than 70% of dispersed beta cells. In addition, all responding beta cells within intact islets exhibited a sigmoidal glucose dose response behavior with inflection points of approximately 8 mm glucose. These results suggest that beta cell heterogeneity may be functionally less important in the intact islet than has been predicted from studies of dispersed beta cells and support the role of glucokinase as the rate-limiting enzyme in the beta cell glucose response.

Animals↗
PubMed1995

Molecular cloning of a ligand for the EPH-related receptor protein-tyrosine kinase Htk.

Htk is a receptor protein-tyrosine kinase that is related to the EPH subfamily of tyrosine kinases. The receptor has a wide tissue distribution including expression in several myeloid hematopoietic cell lines. Using an Htk-Fc fusion protein, a protein ligand for this receptor was expression cloned from the murine kidney mesangial cell line SV40MES 13. The Htk ligand cDNA encodes a transmembrane protein of 336 amino acids. Binding competition experiments demonstrated a Kd of 535 pM for binding of Htk-Fc to the Htk ligand. Incubation of 3T3 cells expressing Htk with COS-7 cells expressing the ligand resulted in tyrosine phosphorylation of Htk. The ligand, like its receptor, is widely expressed and may function in a variety of tissues. However, we localized hematopoietic expression of Htk to the monocytic lineage, suggesting that the ligand may play a role in differentiation and/or proliferation of these cells.

3T3 Cells↗
PubMed1994

Cellular and molecular characterization of the role of the flk-2/flt-3 receptor tyrosine kinase in hematopoietic stem cells.

The flk-2/flt-3 receptor tyrosine kinase was cloned from a hematopoietic stem cell population and is considered to play a potential role in the developmental fate of the stem cell. Using antibodies derived against the extracellular domain of the receptor, we show that stem cells from both murine fetal liver and bone marrow can express flk-2/flt-3. However, in both these tissues, there are stem cell populations that do not express the receptor. Cell cycle analysis shows that stem cells that do not express the receptor have a greater percentage of the population in G0 when compared with the flk-2/flt-3-positive population. Development of agonist antibodies to the receptor shows a proliferative role for the receptor in stem cell populations. Stimulation with an agonist antibody gives rise to an expansion of both myeloid and lymphoid cells and this effect is enhanced by the addition of kit ligand. These studies serve to further illustrate the importance of the flk-2/flt-3 receptor in the regulation of the hematopoietic stem cell.

Animals↗
PubMed1994

Localisation of parvalbumin-immunoreactive structures in primate caudate-putamen.

To investigate the morphology, distribution, and connections of parvalbumin-containing neurones in the caudate-putamen of primates, perfuse-fixed sections were stained to reveal parvalbumin immunoreactivity. In agreement with previous observations, the caudate-putamen was rich in parvalbumin-positive neurones and neuropil. The neuropil staining was uneven such that the dense background staining was interspersed with zones of relatively weak staining. The distribution corresponded to the striosome/matrix system as defined by substance P or met-enkephalin immunostaining in adjacent sections. Because parvalbumin-positive neurones are present in regions known to project to the caudate-putamen and the majority of parvalbumin-positive terminals in the matrix formed asymmetric synapses, it is concluded that the uneven staining is probably due to afferents of the neostriatum. The morphology of the parvalbumin-immunoreactive neurones varied between the striosomes and matrix; those in the matrix were smaller and possessed dendritic arborisations that were relatively uniform, whereas those in the striosomes were generally more extensively stained and possessed a greater variation in their dendritic branching patterns. The dendrites frequently crossed the boundary between the striosomes and matrix. A population of giant parvalbumin-immunoreactive neurones was also observed in the putamen. Electron microscopic analysis revealed that, in addition to terminals forming asymmetric synapses, a smaller population formed symmetric synaptic specialisations and are presumed to be derived from the local parvalbumin-immunoreactive neurones. Terminals of the latter group formed synapses with medium-sized spiny neurones. Because parvalbumin-positive neurones receive input from the cortex, they may transmit cortical information to spiny neurones.

Animals↗
PubMed1994

Synaptic relationships between dopaminergic afferents and cortical or thalamic input in the sensorimotor territory of the striatum in monkey.

The cerebral cortex and the intralaminar thalamic nuclei are the major sources of excitatory glutamatergic afferents to the striatum, whereas the midbrain catecholaminergic neurones provide a dense intrastriatal plexus of dopamine-containing terminals. Evidence from various sources suggests that there is a functional interaction between the glutamate- and dopamine-containing terminals in the striatum. The aim of the present study was to determine the synaptic relationships between cortical or thalamic inputs and the dopaminergic afferents in the sensorimotor territory of the monkey striatum. To address this issue, anterograde tracing in combination with immunocytochemistry for tyrosine hydroxylase (TH) was carried out by light and electron microscopy. Squirrel monkeys received injections of biocytin in the primary motor and somatosensory cortical areas or injections of either Phaseolus vulgaris-leucoagglutinin (PHA-L) or biocytin in the centromedian nucleus (CM) of the thalamus. Sections that included the striatum were processed to visualize the anterograde tracers alone or in combination with TH immunoreactivity. The anterogradely labelled fibres from the cerebral cortex and CM display a band-like pattern and are exclusively confined to the postcommissural region of the putamen, whereas TH-immunoreactive axon terminals are homogeneously distributed throughout the entire extent of the striatum. Electron microscopic analysis revealed that the anterogradely labelled terminals from the cerebral cortex form asymmetric synapses almost exclusively with the heads of dendritic spines. The thalamic terminals also form asymmetric synapses, but in contrast to cortical fibres, predominantly with dendrites (67.4%) and less frequently with spines (32.6%). The TH-immunoreactive boutons are heterogeneous in morphology. The most common type (84% of the total population) forms symmetric synapses; of these the majority is in contact with dendritic shafts (72.1%), less with spines (22.5%) and few with perikarya (5.4%). In sections processed to reveal anterogradely labelled cortical fibres and TH-immunoreactive structures, individual spines of striatal neurones were found to receive convergent synaptic inputs from both cortical and TH-immunoreactive boutons. In contrast, anterogradely labelled thalamic terminals and TH-immunoreactive boutons were never seen to form convergent synaptic contacts on the same postsynaptic structure. These findings suggest that the dopaminergic afferents are located to subserve a more specific modulation of afferent cortical input than afferent thalamic input in the sensorimotor territory of the striatum in primates.

Animals↗
PubMed1994

Production of an atrial natriuretic peptide variant that is specific for type A receptor.

Receptor-specific variants of atrial natriuretic peptide (ANP) were selected from libraries of filamentous phage particles that displayed single copies of random ANP mutants fused to gene III protein. These ANP variants were differentially selected by binding to immobilized natriuretic peptide receptor A (NPR-A) over competing receptor C (NPR-C) in solution. This method also selected ANP variants with improved secretion expression in Escherichia coli. Several of the identified mutations were combined to produce an efficiently expressed ANP analog that was as potent as wild-type ANP in stimulating NPR-A guanylyl cyclase activity but resistant to inactivation mediated by NPR-C. Such NPR-A-selective analogs should be useful for correlating the various activities of ANP to the relevant receptor and may also be more potent therapeutics in the targeting of NPR-A.

Amino Acid Sequence↗
PubMed1994

Cloning and characterization of HTK, a novel transmembrane tyrosine kinase of the EPH subfamily.

Using a polymerase chain reaction based strategy, we identified a novel transmembrane tyrosine kinase in CD34+ human bone marrow cells and a human hepatocellular carcinoma cell line, Hep3B. This protein, hepatoma transmembrane kinase or Htk, shares amino acid similarity with the EPH subfamily of tyrosine kinases. The HTK gene is located on human chromosome 7. The predicted 987-amino acid sequence of Htk includes a transmembrane region and signal sequence. In the predicted extracellular domain, a cysteine-rich region and tandem fibronectin type III repeats are present while a single uninterrupted catalytic domain is present in the intracellular domain. These features are consistent with other members of the Eph subfamily. Antibodies raised against Htk extracellular domain immunoprecipitated a 120-kDa protein from either in vitro translated HTK or Hep3B cells which localized primarily to the Hep3B membrane subcellular fraction. Purified in vitro translated Htk was enzymatically active and autophosphorylated on tyrosine in kinase assays. Furthermore, antibodies against Htk ECD were agonistic, inducing Htk tyrosine phosphorylation in transfected NIH3T3 cells. Northern blot analysis demonstrated a single HTK transcript abundantly present in placenta and in a range of primary tissues and malignant cell lines. HTK appears to be expressed in fetal but not adult brain and in primitive and myeloid but not lymphoid hematopoietic cells. The novel transmembrane protein, Htk, may function as a receptor with an expression pattern suggesting a role in events mediating differentiation and development.

3T3 Cells↗
PubMed1994

Identification and characterization of a novel tyrosine kinase from megakaryocytes.

Protein-tyrosine kinases play pivotal roles in cell signal transduction. We have isolated a cDNA clone encoding a novel human intracytoplasmic tyrosine kinase, termed matk (megakaryocyte-associated tyrosine kinase). Expression of matk mRNA was predominantly found in cells of megakaryocytic lineage. The matk cDNA clone encodes a polypeptide of 527 amino acids and has closest sequence similarity to the csk tyrosine kinase. Sequence comparisons also indicate that matk contains src homology region 2 and 3 domains but lacks the NH2-terminal myristylation signal, the negative regulatory tyrosine (Tyr-527), and the autophosphorylation site (Tyr-416) corresponding to those found in src. Antibodies raised against the NH2 terminus of matk immunoprecipitated a 60-kDa protein from the CMK human megakaryocyte cell line. Expression of matk mRNA was up-regulated in megakaryocytic cells induced to differentiate by the phorbol ester. Based on its restriction in expression and its modulation during in vitro differentiation, it is likely that matk participates in signal transduction during megakaryocytopoiesis.

Base Sequence↗
PubMed1994

Synaptic input and output of parvalbumin-immunoreactive neurons in the neostriatum of the rat.

Previous studies have demonstrated that the calcium-binding protein parvalbumin, is located within a population of GABAergic interneurons in the neostriatum of the rat. Anatomical studies have revealed that these cells receive asymmetrical synaptic input from terminals that are similar to identified cortical terminals and that they innervate neurons with the ultrastructural features of medium spiny cells. Furthermore, electrophysiological studies suggest that some GABAergic interneurons in the neostriatum receive direct excitatory input from the cortex and inhibit medium spiny cells following cortical stimulation. The main objectives of the present study were (i) to determine whether parvalbumin-immunoreactive neurons in the rat receive direct synaptic input from the cortex, (ii) to determine whether parvalbumin-immunopositive axon terminals innervate identified striatal projection neurons and (iii) to chemically characterize this anatomical circuit at the fine structural level. Rats received stereotaxic injections of biocytin in the frontal cortex or injections of neurobiotin in the substantia nigra. Following an appropriate survival time, the animals were perfused and the brains were sectioned and treated to reveal the transported tracers. Sections containing the neostriatum were treated for simultaneous localization of the transported tracer and parvalbumin immunoreactivity. Tracer deposits in the cortex gave rise to massive terminal and fibre labelling in the neostriatum. Parvalbumin-immunoreactive elements located within fields of anterogradely labelled terminals were examined in the electron microscope and corticostriatal terminals were found to form asymmetrical synaptic specializations with all parts of parvalbumin-immunoreactive neurons that were examined. Tracer deposits in the substantia nigra produced retrograde labelling of a subpopulation of striatonigral neurons. Areas of the neostriatum and nucleus accumbens containing retrogradely labelled neurons and parvalbumin-immunoreactive structures were selected for electron microscopy. Parvalbumin-immunopositive axon terminals formed symmetrical synaptic specializations with the perikarya of retrogradely labelled medium spiny projection neurons. Postembedding immunocytochemistry for GABA revealed that parvalbumin-immunoreactive boutons in synaptic contact with medium spiny neurons were GABA-positive. These data demonstrate directly a neural circuit whereby cortical information may be passed to medium spiny cells, via GABAergic interneurons, in the form of inhibition and provide an anatomical substrate for the feed-forward inhibition that has been detected in spiny neurons in electrophysiological experiments.

Afferent Pathways↗
PubMed1994

Subcellular localization of p-boronophenylalanine-delivered boron-10 in the rat 9L gliosarcoma: cryogenic preparation in vitro and in vivo.

A well-characterized in vitro cryogenic preparation for ion microscopic isotope imaging, which minimizes redistribution of diffusible species, was used to determine the distribution of boron in GS-9L gliosarcoma cells incubated with the boron neutron capture therapy agent, p-boronophenylalanine (BPA). At the subcellular level, boron from BPA distributes relatively homogeneously within the glioma cell. Boron from BPA was eliminated rapidly, indicating that most is unbound. Thus a large pool of boron is susceptible to diffusion artifact. Removal of this artifact increases the degree of confidence in microdosimetric results inferred from the homogeneous subcellular distribution. The ion microscopic imaging of boron in subcutaneous tumors cryofixed in situ was achieved in rats treated with BPA. Boron signals from BPA were adequate to image microdistributions at the 1-micron resolution level. As in the in vitro case, boron did not localize discretely at the subcellular level. However, boron heterogeneity was seen at the tissue level. Physiologically valid cellular potassium and sodium levels were seen, which demonstrates minimized redistribution artifact. Future tissue studies designed to correlate ion microscopic boron images to microscopic structure are feasible using cryogenic sample preparation and ion microscopy.

Animals↗
PubMed1993

Two populations of calbindin D28k-immunoreactive neurones in the striatum of the rat.

In order to examine the localisation of calbindin D28k in the normal striatum of the rat, immunocytochemistry using monoclonal antibodies was carried out at both the light and electron microscopic levels. As has previously been shown, many striatal spiny neurones were immunopositive, however, a small population of neurones with smooth dendrites and indented nuclei were also identified. At least some of these cells also displayed NADPH-diaphorase activity. These findings indicate that, in common with the primate striatum and grafted striatal tissue in the rat, the normal rat striatum contains at least two populations of neurones that express calbindin D28k.

Animals↗
PubMed1993

Characterization of calretinin-immunoreactive structures in the striatum of the rat.

Previous observations have shown that the striatum contains a population of neurones that display immunoreactivity for calretinin. In order to morphologically characterize these neurones, sections of the rat striatum were immunostained to reveal calretinin and examined at both light and electron microscopic levels. The striatum contained a small population of calretinin-immunoreactive neurones, which were of medium-size (9-17 microns) and possessed few aspiny, infrequently branching dendrites which tapered to become very thin processes in their most distal portions. Although the calretinin-immunoreactive neurones were homogeneously distributed in the frontal plane, there was a marked rostrocaudal gradient with a much greater density of cells in the rostral than in the caudal parts of the striatum. At the ultrastructural level, calretinin-immunoreactive neurones were seen to possess an indented nucleus and to receive synaptic input from at least three types of boutons. In addition to the calretinin-immunoreactive neurones, the striatum also contained axons and terminal boutons that displayed immunoreactivity for calretinin. At least two types of immunoreactive terminals were identified, those forming symmetrical synaptic specialisations and those forming asymmetrical synaptic specialisations. Approximately 50% formed asymmetrical contacts with spines and 30% formed symmetrical synaptic contact with dendritic shafts. In an attempt to further chemically characterize the calretinin-containing neurones, double pre-embedding immunocytochemistry for calretinin and parvalbumin or choline acetyltransferase was carried out and calretinin immunocytochemistry was combined with histochemistry for NADPH-diaphorase. Analysis of these double-stained sections revealed that the population of calretinin-immunoreactive neurones was distinct from the populations of neurones containing parvalbumin, choline acetyltransferase or NADPH-diaphorase. It is concluded that: (1) on the basis of distribution, morphology, chemistry, ultrastructure and afferent synaptic input, the calretinin-immunoreactive neurones are distinct from the major classes of neurones that have been previously recognised in the striatum; (2) calretinin-immunoreactive terminals are heterogeneous and are probably derived from local calretinin-containing neurones and possibly other sources.

Animals↗