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Biomedical subjects

J K Phillips

Publications and source records attributed to J K Phillips.

At least 19 recordsLinked to original sources
PubMed2006

The nuclear oncoprotein TLX1/HOX11 associates with pericentromeric satellite 2 DNA in leukemic T-cells.

TLX1/HOX11, a DNA-binding homeodomain protein, was originally identified by virtue of its aberrant expression in T-cell leukemia and subsequently found to be crucial for normal spleen development. The precise mechanism of TLX1 function remains poorly understood, although it is known that it can act as both a transcriptional activator and repressor and can downregulate the Aldh1a1 gene in embryonic mouse spleen. Using a whole-genome PCR approach, we show here that TLX1 protein directly interacts with pericentromeric human satellite 2 DNA sequences. Such DNA is known to localize to heterochromatin, which among other roles has been implicated in gene silencing. The interaction was confirmed in vitro and in vivo by gel retardation and chromatin immunoprecipitation assays involving satellite 2 DNA, which contained sequences resembling TLX1 binding sites. Using immunofluorescence microscopy, TLX1 demonstrated a punctate pattern of staining in the nuclei of leukemic T-cells (ALL-SIL). Double labelling indicated that TLX1 colocalized with the centromeric protein CENP-B, demonstrating that the TLX1 foci corresponded to clusters of centromeric DNA. The novel interaction of TLX1 with constitutive heterochromatin adds an additional level of complexity to the intracellular functions of this transcriptional regulator and may have relevance to its roles in transcriptional repression and T-cell immortalization.

Acute Disease↗
PubMed2005

Phosphorylated extracellular signal-regulated kinase 1/2 immunoreactivity identifies a novel subpopulation of sympathetic preganglionic neurons.

Distinct chemical codes are thought to reflect functional specificity in sympathetic preganglionic neurons (SPN). Although a number of chemical candidates have been identified including neurotransmitter-related, calcium-binding and other proteins, signal transduction proteins have been largely neglected. Not only might these chemicals allow discrimination of functionally unique chemical signatures, but they may also identify activated neurons. Immunoreactivity (ir) to phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was differentially located within the thoracic spinal cord depending upon which of three forms of killing was used: the only exception to this was the intermediolateral cell column (IML) which was consistently, densely labeled. The presence or absence of p-ERK1/2 in SPN (n=17,541) within the IML of the thoraco-lumbar spinal cord was determined in seven rats. SPN were identified on the basis of their location, size and that they contained choline acetyltransferase ir. On average, 58% of SPN contained p-ERK1/2, however, more SPN in both the upper (72%; C8-T4) and lower (78%; T11-L3) thoraco-lumbar spinal cord contained p-ERK1/2-ir than the middle thoracic region (47%; T4-T10). p-ERK1/2-ir was also examined in SPN (n=1895) innervating the adrenal medulla (identified by retrograde tracing using cholera toxin B subunit) combined with localization of neuronal nitric oxide synthase (nNOS) in three rats. On average, 64% of adrenal SPN contain p-ERK1/2-ir, and it was confirmed that all adrenal SPN contain nNOS-ir. It appears that p-ERK1/2-ir SPN, described in this study, have tonically activated receptors that are coupled to intracellular signal transduction pathways that lead to the phosphorylation of ERK1/2.

Animals↗
PubMed2004

Seasonal variation in preeclampsia based on timing of conception.

OBJECTIVE: Studies have suggested that the incidence of preeclampsia may be partially dependent on the month or season of delivery. We sought to evaluate whether preeclampsia occurs seasonally in our population and whether the timing of conception or delivery is more strongly associated with risk. METHODS: Between January 1995 and August 2003, we identified 142 primiparous women with singleton pregnancies who met the American College of Obstetricians and Gynecologists' definition for preeclampsia and compared them with 7,762 primiparous control deliveries. We analyzed rates of preeclampsia by individual month and 3-month seasonal blocks based on conception and delivery. Data were analyzed with the chi2 test, and logistic regression and odds ratios were calculated where appropriate. RESULTS: Preeclampsia occurred in 1.8% of singleton primiparous gestations (142/7,904). Cases were younger than controls (26.5 +/- 5.6 versus 28.0 +/- 0 6.0 years, P < .003), of similar race (97% white versus 96% white, P = .69), and equally likely to have a female child (45% versus 48%, P = .41). We found no significant association of month (logistic regression P = .20) of delivery with the risk of preeclampsia. There was a significant association of month (P = .003) of conception with risk of preeclampsia. Conception during the summer months had the highest risk (incidence 2.3%; odds ratio 1.7; 95% confidence limits 1.06, 2.75) compared with spring (incidence 1.4%). Fall (1.7%) and winter (1.6%) conceptions were associated with intermediate rates of preeclampsia. CONCLUSION: We identified a seasonal variation in preeclampsia that appears to be more strongly related to timing of conception than to the timing of delivery. The highest incidence of preeclampsia was associated with conception in the summer months. LEVEL OF EVIDENCE: II-2.

Female↗
PubMed2003

Effects of supplementary selenium source on the performance and blood measurements in beef cows and their calves.

On December 2, 1999, 120 pregnant cows were weighed, their body condition scored, and then sorted into six groups of 20 stratified by BCS, BW, breed, and age. Groups were assigned randomly to six, 5.1-ha dormant common bermudagrass (Cynodon dactylon [L.] Pers.) pastures for 2 yr to determine the effects of supplemental Se and its source on performance and blood measurements. During the winter, each group of cows had ad libitum access to bermudagrass/dallisgrass (Paspalum dilatatum Poir.) hay plus they were allowed limited access (1 to 4 d/wk) to a 2.4-ha winter-annual paddock planted in half the pasture. Treatments were assigned randomly to pastures (two pastures per treatment), and cows had ad libitum access to one of three free-choice minerals: 1) no supplemental Se, 2) 26 mg of supplemental Se from sodium selenite/kg, and 3) 26 mg of supplemental Se from seleno-yeast/kg (designed intake = 113 g/cow daily). Data were analyzed using a mixed model; year was the random effect and treatment was the fixed effect. Selenium supplementation or its source had no effect (P > or = 0.19) on cow BW, BCS, conception rate, postpartum interval, or hay DMI. Birth date, birth weight, BW, total BW gain, mortality, and ADG of calves were not affected (P > 0.20) by Se or its source. Whole blood Se concentrations and glutathione peroxidase (GSH-Px) activity at the beginning of the trial did not differ (P > or = 0.17) between cows receiving no Se and cows supplemented with Se or between Se sources. At the beginning of the calving and breeding seasons, cows supplemented with Se had greater (P < 0.01) whole blood Se concentrations and GSH-Px activities than cows receiving no supplemental Se; cows fed selenoyeast had greater (P < or = 0.05) whole blood Se concentrations than cows fed sodium selenite, but GSH-Px did not differ (P > or = 0.60) between the two sources. At birth and on May 24 (near peak lactation), calves from cows supplemented with Se had greater (P < or = 0.06) whole blood Se concentrations than calves from cows fed no Se. At birth, calves from cows fed seleno-yeast had greater (P < or = 0.05) whole blood Se concentrations and GSH-Px activities than calves from cows fed sodium selenite. Although no differences were noted in cow and calf performance, significant increases were noted in whole blood Se concentrations and GSH-Px activities in calves at birth as a result of feeding of seleno-yeast compared to no Se or sodium selenite.

Animal Feed↗
PubMed2001

Differential expression of catecholamine synthetic enzymes in the caudal ventral pons.

The analysis of colocalization of multiple catecholamine biosynthetic enzymes within the ventrolateral part of the medulla oblongata of the rat revealed distinct subpopulations of neurons within the C1 region (Phillips et al., J Comp Neurol 2001, 432:20-34). In extending this study to include the caudal pons, it was shown for the first time that the A5 cell group could be distinguished by the presence of immunoreactivity to tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase (AADC), and dopamine beta hydroxylase (DBH). A novel cell group was also identified. The cells within this new group were immunoreactive to DBH but not TH, AADC, or phenylethanolamine N-methyltransferase (PNMT) and will be referred to as the TH-, DBH+ cell group. The TH-, DBH+ neurons were not immunoreactive for either the dopamine or noradrenaline transporters, suggesting that these neurons do not take up these transmitters. A5 neurons were immunoreactive for the noradrenaline transporter but not the dopamine transporter (as previously shown). Retrograde tracing with cholera toxin B revealed that the TH-, DBH+ neurons do not project to the thoracic spinal cord or to the rostral ventrolateral medulla, but A5 neurons do. A calbindin immunoreactive cell group is located in a region overlapping TH-, DBH+ cell group. However, only a few neurons were immunoreactive for both markers. The physiological role of the TH-, DBH+ cell group remains to be determined.

Animals↗
PubMed2001

Modulation of ACh-induced currents in rat adrenal chromaffin cells by ligands of alpha2 adrenergic and imidazoline receptors.

The aim of this study was to investigate the expression of the alpha2-adrenergic receptors in the adrenal medulla, and to examine the mechanism by which clonidine and related drugs inhibit acetylcholine (ACh)-induced whole-cell currents in adrenal chromaffin cells. Reverse transcription-polymerase chain reaction (RT-PCR) performed on punches of rat adrenal medulla demonstrated expression of mRNA for the 2A-, alpha2B- and alpha2C-adrenergic receptors. Similar experiments conducted with tissue punches obtained from the adrenal cortex did not reveal expression of these receptor subtypes. Whole-cell currents were recorded in isolated chromaffin cells using the perforated-patch configuration. ACh (50 microM) evoked inward currents with a peak amplitude of 117.8+/-9.3 pA (n = 45; Vhol = -60 mV). The currents were inhibited in a dose-dependent manner (0.5-50 microM) by clonidine, UK 14,304 and rilmenidine (agonists of alpha2/imidazoline receptors), as well as by SKF 86466 and efaroxan (antagonists). Adrenaline and noradrenaline (50-100 microM) had no significant effect. Thus, although the adrenal medulla expresses mRNA for the alpha2-adrenergic receptors, the lack of agonist-antagonist specificity observed in our whole-cell recordings (in the absence of intracellular dialysis) provides additional evidence against the possibility that these inhibitory effects are mediated by classical alpha2 or imidazoline receptor interactions.

Acetylcholine↗
PubMed2001

Differential expression of catecholamine biosynthetic enzymes in the rat ventrolateral medulla.

Adrenergic (C1) neurons located in the rostral ventrolateral medulla are considered a key component in the control of arterial blood pressure. Classically, C1 cells have been identified by their immunoreactivity for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and/or phenylethanolamine N-methyltransferase (PNMT). However, no studies have simultaneously demonstrated the expression of aromatic L-amino acid decarboxylase (AADC) and dopamine beta-hydroxylase (DBH) in these neurons. We examined the expression and colocalization of all four enzymes in the rat ventrolateral medulla using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Retrograde tracer injected into thoracic spinal segments T2-T4 was used to identify bulbospinal neurons. Using fluorescence and confocal microscopy, most cells of the C1 group were shown to be double or triple labeled with TH, DBH, and PNMT, whereas only 65-78% were immunoreactive for AADC. Cells that lacked detectable immunoreactivity for AADC were located in the rostral C1 region, and approximately 50% were spinally projecting. Some cells in this area lacked DBH immunoreactivity (6.5-8.3%) but were positive for TH and/or PNMT. Small numbers of cells were immunoreactive for only one of the four enzymes. Numerous fibres that were immunoreactive for DBH but not for TH or PNMT were noted in the rostral C1 region. Single-cell RT-PCR analysis conducted on spinally projecting C1 neurons indicated that only 76.5% of cells that contained mRNA for TH, DBH, and PNMT contained detectable message for AADC. These experiments suggest that a proportion of C1 cells may not express all of the enzymes necessary for adrenaline synthesis.

Animals↗
PubMed2001

Heterogeneous control of blood flow amongst different vascular beds.

The control and maintenance of vascular tone is due to a balance between vasoconstrictor and vasodilator pathways. Vasomotor responses to neural, metabolic and physical factors vary between vessels in different vascular beds, as well as along the same bed, particularly as vessels become smaller. These differences result from variation in the composition of neurotransmitters released by perivascular nerves, variation in the array and activation of receptor subtypes expressed in different vascular beds and variation in the signal transduction pathways activated in either the vascular smooth muscle or endothelial cells. As the study of vasomotor responses often requires pre-existing tone, some of the reported heterogeneity in the relative contributions of different vasodilator mechanisms may be compounded by different experimental conditions. Biochemical variations, such as the expression of ion channels, connexin subtypes and other important components of second messenger cascades, have been documented in the smooth muscle and endothelial cells in different parts of the body. Anatomical variations, in the presence and prevalence of gap junctions between smooth muscle cells, between endothelial cells and at myoendothelial gap junctions, between the two cell layers, have also been described. These factors will contribute further to the heterogeneity in local and conducted responses.

Animals↗
PubMed2001

Differential expression of the noradrenaline transporter in adrenergic chromaffin cells, ganglion cells and nerve fibres of the rat adrenal medulla.

Expression of the noradrenaline transporter (NAT) was identified in various cell and fibre populations of the rat adrenal medulla, examined with immunohistochemistry and confocal microscopy. Immunoreactivity for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), aromatic-L-amino-acid decarboxylase (AADC) and dopamine beta-hydroxylase (DBH) was present in all chromaffin cells, while phenylethanolamine N-methyltransferase (PNMT) was used to determine adrenergic chromaffin cell groups. Labelling with NAT antibody was predominantly cytoplasmic and colocalised with PNMT immunoreactivity. Noradrenergic chromaffin cells were not NAT immunoreactive. Additionally, NAT antibody labelling demonstrated clusters of ganglion cells (presumably Type I) and nerve fibres. Expression of TH, AADC, DBH, PNMT and NAT mRNA was examined using reverse transcription-polymerase chain reaction (RT-PCR) from adrenal medulla punches and single chromaffin cells, and results were consistent with those obtained with immunocytochemistry. Chromaffin cells and fibres labelled with antibodies against growth associated protein-43 (GAP-43) were not NAT immunoreactive, while ganglion cells were doubled labelled with the two antibodies. The presence of NAT in adrenergic chromaffin cells, and its absence from noradrenergic cells, suggests that the adrenergic cell type is primarily responsible for uptake of catecholamines in the adrenal medulla.

Adrenal Medulla↗
PubMed2000

Single-cell RT-PCR as a tool to study gene expression in central and peripheral autonomic neurones.

In studies of the central and peripheral autonomic nervous system, it has become increasingly important to be able to investigate mRNA expression patterns within specific neuronal populations. Traditionally, the identification of mRNA species in discrete populations of cells has relied upon in situ hybridization. An alternative, relatively simple procedure is 'multiplex' reverse transcription-polymerase chain reaction (RT-PCR), conducted on single neurons after their in vitro isolation. Multiplex single-cell RT-PCR can be used to examine the expression of multiple genes within individual cells, and can be combined with electrophysiological, pharmacological and anatomical (retrograde labelling) studies. This review focuses on a number of key aspects of this approach, methodology, and both the advantages and the limitations of the technique. We also provide specific examples of work performed in our laboratory, examining the expression of alpha 2-adrenergic receptors in catecholaminergic cells of the rat brainstem and adrenal medulla. The application of single-cell RT-PCR to future studies of the autonomic nervous system will hopefully provide information on how physiological and pathological conditions affect gene expression in autonomic neurones.

Animals↗
PubMed2000

Heterogeneity in mechanisms underlying vasodilatory responses in small arteries of the rat hepatic mesentery.

We have characterised nerve-mediated vasodilations in small arteries of the rat hepatic mesentery. Stimulation of sympathetic nerves (10 Hz, 10 s) produced a vasodilation which was abolished by the beta-adrenoceptor antagonist, propranolol (2 x 10(-6) M), but was unaffected by NG-nitro-L-arginine methyl ester (L-NAME, 10(-5) M). Stimulation of sensory nerves produced a large vasodilation that was abolished by capsaicin (10(-6) M). This vasodilation was unaffected by L-NAME (10(-5) M), but significantly reduced by the calcitonin gene related peptide (CGRP) antagonist, CGRP8-37 (10(-6) M), or inhibition of adenylate cyclase (SQ22356, 2 x 10(-5) M; 2',5'-dideoxyadenosine, 2 x 10(-4) M). Stimulation of cholinergic nerves produced a small vasodilation which was significantly reduced by scopolamine (10(-6) M). Expression of mRNA for CGRP1 receptors, muscarinic m2, m3 and m5 receptors and neurokinin1 (NK1) and NK3, receptors was detected. Perivascular nerves were immunoreactive for CGRP and substance P. No role was found for substance P, neuronal NO, ATP or adenosine in nerve-mediated responses. L-NAME (10(-5) M) potentiated vasoconstrictions following sympathetic nerve stimulation. This effect was reversed by L-arginine (10(-3) M) and cromakalim (10(-6) M) and mimicked by glybenclamide (10(-5) M), thus implicating KATP channels. Vasodilation in response to sensory nerve stimulation was directly proportional to the level of preconstriction, while vasodilation in response to neurogenic or applied acetylcholine was inhibited at high levels of preconstriction. We hypothesize that, under conditions of intensive vasoconstriction, some endothelial-dependent vasodilations may be less important than vasodilations activated directly through the smooth muscle.

Adenylyl Cyclase Inhibitors↗
PubMed1999

Neuroreceptor mRNA expression in the rat mesenteric artery develops independently of innervation.

Studies in autonomic targets have shown that nerves may be required for the development and maintenance of postsynaptic receptor populations. We have examined this relationship in the rat mesenteric artery, assessing mRNA expression levels for a range of neuroreceptors after neonatal sympathectomy, using 6-hydroxydopamine or antisera directed against nerve growth factor, and sensory denervation, using capsaicin. Total RNA was extracted from 28 day old rats and reverse transcription-polymerase chain reaction was performed, using primers specific for the alpha1(A,B,D)- and alpha2(A,B,C)-adrenergic, neurokinin (NKI-NK3), muscarinic (M1-M5) and P2X purinergic (P2x1-7) receptor families. Results showed no decreases in mRNA expression of any of the specific receptor subtypes after either sympathetic or sensory denervation. Small increases in mRNA expression were detected following sensory denervation for some of the receptor subtypes. We conclude that neither sympathetic nor sensory nerves are mandatory for the expression of mRNA of a range of neuroreceptors in the mesenteric vascular bed of the rat.

Aging↗
PubMed1999

Development of peripheral autonomic synapses: neurotransmitter receptors, neuroeffector associations and neural influences.

1. The functional innervation of autonomic target tissues occurs early during development, at a time when both the nerves and post-synaptic target tissues are still differentiating. 2. Physiological responses appear soon after the arrival of the first fibres when uptake and release mechanisms within the nerves are already functional. Initial responses differ from those in the mature animal, both in the form and, frequently, in the subtypes of receptors involved. 3. Results of a number of studies suggest that the initial expression of neurotransmitter receptors during development is largely independent of neural influences. Changes recorded in neurotransmitter receptor expression during development appear to be similarly independent of neural influences. 4. While signal transduction pathways coupling adrenergic neurotransmitter receptors to effector responses appear to develop independently of the nerves, the efficient coupling of muscarinic receptors often requires the action of the neurotransmitter, acetylcholine. 5. During the period of synapse formation, the neural plexus continues to expand. While developing varicosities can release the neurotransmitter, the capacity for neurotransmitter retention appears to be restricted. Developmental changes in the neurotransmitters that produce functional responses, while well known in the sweat glands, may also be seen in more subtle forms in other target tissues. 6. Ultrastructural studies suggest that close physical associations between the membranes of the release sites of the developing nerves and the target cells may form early during development when physiological responses are still immature. These close associations could enable more specific reciprocal interactions between nerves and target cells involving known and novel growth factors, neuropeptides and cytokines important in shaping the mature synaptic characteristics.

Animals↗
PubMed1999

Management of patients with acute respiratory distress syndrome.

Acute respiratory distress syndrome is a complex clinical syndrome of respiratory failure that presents a challenge to every critical care team. Since the first clear description by Ashbaugh et al more than 30 years ago, much has been learned about the pathophysiologic process that occurs within the lungs after they suffer either a direct or indirect injury. Unfortunately, little success has been achieved in improving outcomes; however, hope is on the horizon. Current research evaluating optimal ventilator management, ECMO, the use of inhaled nitric oxide, and other experimental management strategies will hopefully combine to produce improved outcomes.

Critical Care↗
PubMed1998

Receptors involved in nerve-mediated vasoconstriction in small arteries of the rat hepatic mesentery.

1. We have investigated the neurotransmitters and receptor subtypes involved in nerve-mediated vasoconstriction in small arteries of the rat hepatic mesentery. 2. A dense sympathetic innervation was demonstrated using catecholamine histochemistry and antibodies against the synaptic vesicle protein synaptophysin. 3. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated very strong expression of the alpha1A-adrenergic, neuropeptide Y (NPY) Y1, P2X1- and P2X4-purinergic receptors, moderate expression of the alpha2B-adrenergic receptor and the purinergic P2X5- and P2X7-receptors and weak expression of the alpha1B-, alpha1D-, alpha2A- and alpha2C-adrenergic receptors and the P2X2- and P2X3-purinergic receptors. NPY2 and P2X6 receptor expression was absent. 4. Electrical field stimulation (10 Hz, 10 s) produced contractions which were abolished by tetrodotoxin (10(-6) M) and/or guanethidine (GE, 5 x 10(-6) M) and a combination of benextramine (10(-5) M) and alpha,beta-methylene ATP, (alpha,beta-mATP, 3 x 10(-6) M) or PPADS (10(-5) M). Selective alpah1-adrenergic receptor antagonists showed the potency order of prazosin > WB-4101 > 5-methyl-urapidil > BMY 7378. Yohimbine (10(-8) M, 10(-7) M), alpha,beta-mATP (3 x 10(-6) M) and PPADS (10(-5) M) each enhanced the response to nerve stimulation. 5. Some experiments demonstrated a slow neurogenic contraction which was abolished by GE or the selective NPY1 receptor antagonist 1229U91 (6 x 10(-7) M). 6. We conclude that nerve-mediated vasoconstriction results from the activation of postsynaptic alpha,beta-adrenergic and P2X-purinergic receptors and under some conditions, NPY1 receptors. Neurotransmitter release is modulated by presynaptic alpha2-adrenergic receptors and possibly also P2X-purinoceptors. The major postsynaptic subtypes involved were well predicted by mRNA expression as measured by RT-PCR, suggesting that this technique may be a useful adjunct to studies aimed at identifying functional receptor subtypes.

Animals↗
PubMed1997

Variation in mRNA expression of alpha-adrenergic, neurokinin and muscarinic receptors amongst four arteries of the rat.

Different mechanisms mediate constriction and dilation in different vascular beds. We have used reverse transcription-polymerase chain reaction to investigate whether specific patterns of receptor gene expression may underlie these variable responses. Total RNA, from the basilar, pulmonary, mesenteric and tail arteries of anaesthetised adult Wistar rats, was reverse transcribed and amplified using primers specific for the molecular subtypes of the alpha 1(A, B, D)- and alpha 2(A, B, C)-adrenergic, neurokinin (NK1-NK3) and muscarinic (m1-m5), receptors. Results showed that the pattern of gene expression was variable with no two arteries having the same receptor profile. Messenger RNA for the alpha 1A, alpha 1B, alpha 2B, NK1, NK3, m3 and m5 receptor subtypes were detected in all vessels studied while the remaining subtypes showed a variable expression amongst the arteries. This is the first description of mRNA for the m5 muscarinic receptor in peripheral tissue. The NK3 receptor was the major neurokinin receptor expressed in all vessels except the pulmonary artery, in which the NK1 receptor was also strongly expressed. We conclude that each artery expressed a specific receptor array which may permit some unique neural and hormonal controls.

Animals↗