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Biomedical subjects

P Hunt

Publications and source records attributed to P Hunt.

At least 19 recordsLinked to original sources
PubMed1992

Evidence that stem cell factor is involved in the rebound thrombocytosis that follows 5-fluorouracil treatment.

The mechanisms responsible for 5-fluorouracil (5FU)-induced rebound thrombocytosis are not completely understood. SI/SI(d) mice, which do not undergo rebound thrombocytosis in response to 5FU, provide a genetic approach to the study of this phenomenon. Recent reports by several groups that the SI locus encodes a protein known variably as stem cell factor (SCF), mast cell growth factor, or kit ligand, suggests the possibility that the lack of wild-type SCF in SI/SI(d) mice is responsible for their defective response to 5FU-induced thrombocytopenia. It is shown in this report that SCF-treated SI/SI(d) mice are as capable as their wild-type littermates in undergoing rebound thrombocytosis. W/Wv mice, mutated at the locus encoding the SCF receptor, also do not undergo rebound thrombocytosis, but are not responsive to SCF treatment. In normal mice, it is shown by RNA solution hybridization that SCF mRNA expression is increased during the 5FU-induced platelet nadir period. It is also shown by autoradiography that maturing megakaryocytes express SCF receptors, and that in vivo administration of SCF significantly raises the numbers of megakaryocytes, as well as circulating platelet counts. Taken together, these data indicate that SCF may be an important regulator of platelet production under both normal and physiologically disturbed situations.

Animals↗
PubMed1992

Determination of the enantiomers of a novel 20,21-dinoreburnamenine derivative in rat plasma and brain by high-performance liquid chromatography using a chiral stationary phase.

A high-performance liquid chromatographic method with solid-phase extraction was developed for the assay of the enantiomers of a novel 20,21-dinoreburnamenine derivative (RU 49041) in rat plasma and brain using a chiral stationary phase (Nucleosil Chiral 2) and ultraviolet detection. The limit of detection was 10 ng/ml (or ng/g) in both tissues and the intra-assay precision was satisfactory (plasma, ca. 5%; brain, ca. 1%). The pharmacokinetic profiles of the two enantiomers were determined following oral administration of the racemate (10 mg/kg). The results show that their pharmacokinetics are very different: whereas both enantiomers appear in the brain, only the 3 alpha,16 beta-enantiomer is detected in plasma.

Animals↗
PubMed1992

Molecular mechanisms of pattern formation in the vertebrate hindbrain.

During early stages of neural development a series of repeated bulges, termed rhombomeres, form in the vertebrate hindbrain. Studies in the chick have shown that rhombomeres are segments that underlie the patterning of nerves in the hindbrain, and this raises the question of the molecular basis of segment development. Several genes have been found with expression patterns consistent with roles in the formation or differentiation of rhombomeres. The zinc finger gene Krox-20 is expressed in two alternating rhombomeres, r3 and r5, in the mouse hindbrain; these stripes of gene expression are established prior to the morphological appearance of segments. Krox-20 is also expressed in this pattern in the chick and Xenopus, suggesting that it has a conserved role, possibly in the formation of rhombomeres. Four members of the Hox-2 homeobox gene cluster have limits of expression at rhombomere boundaries. Three genes, Hox-2.6, -2.7 and -2.8 have progressively more anterior limits of expression at two-segment intervals, whereas expression of Hox-2.9 is restricted to one rhombomere, r4. The Hox-2 genes are expressed in spatially restricted patterns in early neural crest cells. These findings suggest that the Hox genes have roles in specifying the identity of rhombomeres and of neural crest.

Animals↗
PubMed1992

Analysis of the murine Hox-2.7 gene: conserved alternative transcripts with differential distributions in the nervous system and the potential for shared regulatory regions.

In this study we have investigated the organization and regulation of the mouse Hox-2.7 gene. There are several alternative transcripts some of which are conserved between mouse and humans. By Northern and in situ analysis we are able to identify at least three types of transcripts which are different in size and splicing pattern and have distinctly different boundaries of expression in the nervous system. One subset of the endogenous transcripts has a boundary of expression that corresponds to the adjacent Hox-2.8 gene instead of Hox-2.7. In another type of transcript there is an alternative reading frame which predicts a protein that has homology to an enzyme ATPase and suggests that a non-homeobox containing gene may be located in the Hox-2 cluster. A Hox-2.7-lacZ transgene is expressed in a similar pattern to the endogenous gene in that spatially-restricted domains of expression are seen in the branchial arches, neural tube, paraxial mesoderm (somites), cranial ganglia, neural crest and gut. However, the anterior boundaries of transgene expression only correspond to the subset of Hox-2.7 transcripts which map to the Hox-2.8 boundary. The proximity of a Hox-2.7 promoter to regions which regulate the adjacent Hox-2.6 gene and the expression of transgenic and endogenous transcripts in a Hox-2.8 pattern, suggest that regulatory elements may be shared by neighbouring genes to establish the complete expression pattern.

Amino Acid Sequence↗
PubMed1992

Secondary cytokine production by lymphoid cells used in cellular immunotherapy.

Interleukin-2 (IL-2) has been used extensively in cellular immunotherapy trials as a systemic activator of the immune system as well as an ex vivo stimulant for lymphoid cell function. Despite the measurement of several in vitro and in vivo immunologic parameters related to cellular immunotherapy, determinants of successful cellular immunotherapy remain unknown. To further delineate the consequences of exposing peripheral blood lymphocytes to high concentrations of IL-2, we assessed the supernatants of IL-2-activated peripheral blood lymphocytes for production of tumour necrosis factor (TNF) and interferon-gamma (IFN-gamma). Exposure of normal monocyte-depleted peripheral blood mononuclear cells (PBMC) to IL-2 caused a dose-dependent increase in secretion of TNF and IFN-gamma which increased linearly after 48 h in culture. Analysis of positively selected, highly purified PBMC subpopulations exposed to IL-2 revealed that TNF-alpha and TNF-beta were produced by both CD3+ and CD16+ subpopulations but not by CD22+ cells. These studies were extended to supernatants obtained from PBMC cultures used in the adoptive cellular immunotherapy of patients with advanced cancer. Patients treated with lymphokine-activated killer (LAK) cell immunotherapy were classified as responders (N = 14) or non-responders (N = 17) to therapy. We found no significant difference in the production of TNF between responders and nonresponders (22 +/- 9 U ml-1 vs. 20 +/- 6 U ml-1), P > 0.05. However, LAK cell supernatants harvested from non-responders contained a significantly higher level of IFN-gamma (232 +/- 94 U ml-1) compared with responders (42 +/- 14), P < 0.05. Furthermore, the linear association between IFN-gamma and TNF-alpha production was different between these two response groups (rs = -0.19 for non-responders and rs = 0.48 for responders). These results suggest that secondary cytokine production by adoptively transferred lymphocytesmay play an important role in the host response to cellular immunotherapy.

Cells, Cultured↗
PubMed1992

Cruciate thinning of the zona pellucida for more successful enhancement of blastocyst hatching in the mouse.

Implantation rates remain low following human in-vitro fertilization (IVF). Suboptimal culture conditions may limit the ability of embryos to hatch as blastocysts, and artificial opening of the zona pellucida has been proposed as a means to promote subsequent hatching (assisted hatching). Such techniques must have minimal adverse effects on the embryos, while maximizing the potential for an embryo to hatch fully as a blastocyst. In a mouse model, we compared embryonic development after zona drilling, and cruciate thinning of the zona (CTOZ) intended to simulate the natural thinning of the zona pellucida. Using acidic Tyrode's solution both zona drilling and cruciate-thinning were performed on day 3 morulae. On day 4 the rates of complete hatching of blastocysts were 0/165, 24/172 and 72/175 in control, zona drilled and thinned groups respectively (P less than 0.0001). On day 5 the rates of complete hatching in the same groups were 20/165, 54/172 and 120/175 respectively (P less than 0.00001) and by day 6, 66/165, 74/172 and 130/175 respectively (P less than 0.00001). The rate of arrest at the morula stage was 24/172 versus 8/175 in the zona drilled and thinned groups respectively (P less than 0.005, whilst the rate of arrest at the blastocyst stage was 21/172 versus 14/175 respectively (NS). Hence cruciate thinning of the zona appears less detrimental at the morula stage than zona drilling, but eventual rates of arrest at the blastocyst stage were comparable. Both techniques significantly increased the rate of hatching, but zona drilling did not guarantee complete hatching.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Hox codes and positional specification in vertebrate embryonic axes.

We have compared the ways in which vertebrate Hox genes are used in the patterning of three distinct embryonic contexts, the branchial region, the somites, and the limb. We have identified common features of the three systems, but have suggested on the basis of their differences (in both embryological properties and use of Hox genes) that it is better to consider each as an independent system for regional specification. Nevertheless, there are sufficient common features to expect that exploitation of the distinct experimental advantages of each system will provide important insights to the mode of operation of the others.

Animals↗
PubMed1991

A distinct Hox code for the branchial region of the vertebrate head.

The branchial region of the vertebrate head forms through complex interactions involving rhombomeric segments, neural crest and branchial arches. It is though that aspects of their patterning mechanisms are linked and involve Hox-2 genes, whose overlapping and spatially restricted expression domains represent a combinatorial code for generating regional diversity. Vertebrates possess four Hox clusters of Antennapedia class homeobox genes, related to each other by duplication and divergence from a common ancestral complex. In consequence, at equivalent positions in different clusters there are highly related genes known as subfamilies or paralogous groups. As Hox-2 genes cannot fully account for patterning individual rhombomeres, we investigated whether offsets in expression limits of paralogous genes could account for the generation of regional diversity. We report here that, with the exception of the labial subfamily, paralogues show identical expression limits in rhombomeres, cranial ganglia and branchial arches, providing a combinatorial Hox code for the branchial region that seems to be different in organization to that of the trunk.

Alleles↗
PubMed1991

Retinoic acid-binding protein, rhombomeres and the neural crest.

We have investigated by immunocytochemistry the spatial and temporal distribution of cellular retinoic acid-binding protein (CRABP) in the developing nervous system of the chick embryo in order to answer two specific questions: do neural crest cells contain CRABP and where and when do CRABP-positive neuroblasts first arise in the neural tube? With regard to the neural crest, we have compared CRABP staining with HNK-1 staining (a marker of migrating neural crest) and found that they do indeed co-localise, but cephalic and trunk crest behave slightly differently. In the cephalic region in tissues such as the frontonasal mass and branchial arches, HNK-1 immunoreactivity is intense at early stages, but it disappears as CRABP immunoreactivity appears. Thus the two staining patterns do not overlap, but are complementary. In the trunk, HNK-1 and CRABP stain the same cell populations at the same time, such as those migrating through the anterior halves of the somites. In the neural tube, CRABP-positive neuroblasts first appear in the rhombencephalon just after the neural folds close and then a particular pattern of immunoreactivity appears within the rhombomeres of the hindbrain. Labelled cells are present in the future spinal cord, the posterior rhombencephalon up to rhombomere 6 and in rhombomere 4 thus producing a single stripe pattern. This pattern is dynamic and gradually changes as anterior rhombomeres begin to label. The similarity of this initial pattern to the arrangement of certain homeobox genes in the mouse stimulated us to examine the expression of the chicken Hox-2.9 gene. We show that at stage 15 the pattern of expression of this gene is closely related to that of CRABP. The relationship between retinoic acid, CRABP and homeobox genes is discussed.

Animals↗
PubMed1991

Patterning the vertebrate head: murine Hox 2 genes mark distinct subpopulations of premigratory and migrating cranial neural crest.

The structures of the face in vertebrates are largely derived from neural crest. There is some evidence to suggest that the form of the facial pattern is determined by the crest, and that it is specified before migration as to the structures that is is able to form. The neural crest is able to control the form of surrounding, non-neural crest tissues by an instructive interaction. Some of this cranial crest is derived from a region of the hindbrain that expresses Hox 2 homeobox genes in an overlapping and segment-restricted pattern. We have found that neurogenic and mesenchymal neural crest expresses Hox 2 genes from its point of origin beside the neural plate, during migration and after migration has ceased and that rhombomeres 3 and 5 do not have any expressing neural crest beside them. Each branchial arch expresses a different combination or code of Hox genes in a segment-restricted way. The surface ectoderm over the arches initially does not express Hox genes, and later adopts an expression pattern that reflects that of neural crest that has come to underlie it. We suggest that initially the neural plate and neural crest are spatially specified, while the surface ectoderm is unpatterned. Subsequently some positional information could be transferred to the surface ectoderm as a result of an interaction with the neural crest. Given that the role of the homologous genes in insects is position specification, and that neural crest is imprinted before migration, we suggest that Hox 2 genes are providing part of this positional information to the neural crest and hence are involved in patterning the structures of the branchial arches.

Animals↗
PubMed1991

Homeobox genes and models for patterning the hindbrain and branchial arches.

Antennapedia class homeobox genes, which in insects are involved in regional specification of the segmented central regions of the body, have been implicated in a similar role in the vertebrate hindbrain. The development of the hindbrain involves the establishment of compartments which are subsequently made distinct from each other by Hox gene expression, implying that the lineage of neural cells may be an important factor in their development. The hindbrain produces the neural crest that gives rise to the cartilages of the branchial skeleton. Lineage also seems to be important in the neural crest, as experiments have shown that the crest will form cartilages appropriate to its level of origin when grafted to a heterotopic location. We show how the Hox genes could also be involved in patterning the mesenchymal structures of the branchial skeleton. Recently it has been proposed that the rhombomere-restricted expression pattern of Hox 2 genes is the result of a tight spatially localised induction from underlying head mesoderm, in which a prepattern of Hox expression is visible. We find no evidence for this model, our data being consistent with the idea that the spatially localised expression pattern is a result of segmentation processes whose final stages are intrinsic to the neural plate. We suggest the following model for patterning in the branchial region. At first a segment-restricted code of Hox gene expression becomes established in the neuro-epithelium and adjacent presumptive neural crest. This expression is then maintained in the neural crest during migration, resulting in a Hox code in the cranial ganglia and branchial mesenchyme that reflects the crest's rhombomere of origin. The final stage is the establishment of Hox 2 expression in the surface ectoderm which is brought into contact with neural crest-derived branchial mesenchyme. The Hox code of the branchial ectoderm is established later in development than that of the neural plate and crest, and involves the same combination of genes as the underlying crest. Experimental observations suggest the idea of an instructive interaction between branchial crest and its overlying ectoderm, which would be consistent with our observations. The distribution of clusters of Antennapedia class genes within the animal kingdom suggests that the primitive chordates ancestral to vertebrates had at least one Hox cluster. The origin of the vertebrates is thought to have been intimately linked to the appearance of the neural crest, initially in the branchial region.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

The branchial Hox code and its implications for gene regulation, patterning of the nervous system and head evolution.

In this study we have examined the expression of murine Hox homeobox containing genes by in situ hybridisation in the branchial region of the head. Genes from the Hox complexes display segmentally restricted domains of expression in the developing hindbrain, which are correlated with similar restricted domains in the neural crest and surface ectoderm of the branchial arches. Comparison of related genes from the different clusters shows that subfamily members are expressed in identical rhombomeres and branchial arches. These patterns suggest a combinatorial system for specifying regional variation in the head, which we refer to as a Hox code. The Hox genes also display dynamic dorso-ventral (D-V) restrictions in the developing neural tube which mirror the timing and spatial distributions of the birth of major classes of neurons in the CNS. Genes in the Hox-2 cluster all have a similar D-V distribution that differs from that of genes from the other Hox clusters, and suggests that members of a subfamily may be used to specify positional values to different subsets of cells at the same axial level. These results are discussed in terms of a system for patterning the branchial regions of the vertebrate head, and evolution of head structures. We have also examined aspects of the transcriptional regulation of Hox-2 genes in transgenic mice using a lacZ reporter gene. We have been able to reconstruct the major pattern of the Hox-2.6 gene on the basis of identical expression of the transgene and the endogenous gene with respect to timing, spatial restrictions and tissue-specific distributions. Deletion analysis has enabled us to identify three regions involved in generating this pattern. Two of these regions have the properties of enhancers which are capable of imposing spatially-restricted domains of expression on heterologous promoters. We have generated similar Hox-lacZ fusions that reconstruct the highly restricted patterns of the Hox-2.1 and Hox-2.8 genes in the developing nervous system, supporting our in situ analysis and the idea of a Hox code. These transgenic experiments are a useful step in examining regulation in the Hox cascade.

Amino Acid Sequence↗
PubMed1991

Platelet factor 4 mRNA expression in human erythroleukemic cells: regulation by phorbol esters and certain cytokines.

Developing megakaryocytes are distinguished from progenitor cells by the appearance of platelet proteins such as platelet factor 4 (PF 4). The human erythroleukemic cell line HEL can also be induced to produce PF 4 by incubation in phorbol esters. HEL cells were used here as a model system in which to study the phenomenon of inducible PF 4 production at both the mRNA and protein levels. The cytokines interleukin 1 beta (IL-1 beta), interleukin 3 (IL-3), interleukin 6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO), and transforming growth factor-beta (TGF-beta) were also evaluated for their effects on PF 4 mRNA induction in HEL cells.

Antigens, Surface↗
PubMed1991

Dry electrolyte-balanced heparinized syringes evaluated for determining ionized calcium and other electrolytes in whole blood.

By analyzing whole blood containing no anticoagulants (uncoagulated whole blood) immediately after collection, we evaluated the relative changes in the concentrations of ionized calcium and other electrolytes in whole blood collected in dry heparinized syringes and in serum prepared from blood collected in evacuated blood-collection tubes. Using these dry heparinized syringes, we collected and analyzed whole blood that contained either 33 or 13 int. units of lithium heparin or 40 int. units of electrolyte-balanced heparin per milliliter of blood. We evaluated the effects both of these heparins at different concentrations of ionized calcium and of the incomplete filling of the syringes. We conclude that: (a) when analyzed within 2-3 min after collection, uncoagulated whole blood provides ionized calcium results unaffected by anticoagulants or cellular metabolism; (b) the preparation of serum unpredictably changes ionized calcium; (c) the use of dry electrolyte-balanced heparin virtually eliminates the interference in ionized calcium concentrations between 0.9 and 1.6 mmol/L; and (d) incomplete filling of electrolyte-balanced heparinized syringes produces no effect in syringes two-thirds full (60 int. units/mL heparin concentration) and a small effect in syringes one-third full (120 int. units/mL heparin).

Adult↗
PubMed1990

Ontogenetic differences in the association of gustatory and tactile cues with lithium chloride and footshock.

Using a double dissociation design with 15% sucrose solution and exposure to a heat lamp (34 degrees C) as CSs and 0.3 M LiCl and 1-mA footshock as USs, the present study found 15-day-old rats to show selective associations similar to those found in adults. Five-day-old pups, however, expressed similar sucrose aversions when this solution was paired with either LiCl or footshock, whereas they expressed heat aversions after heat-LiCl but not heat-shock pairings. Results are discussed with respect to previous data on selective associations in pups, current theories on selective associations, and ontogenetic differences in stimulus selection.

Age Factors↗