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Biomedical subjects

K J Moore

Publications and source records attributed to K J Moore.

At least 73 records · Page 4Linked to original sources

Oncology and transport. Beware of the presentation and anticipate the clinical course.

These cases demonstrate a few of the presentations that may occur with new onset oncologic problems. While the transport phase of these children's care was not extraordinary, the development and management of the clinical issues might have been influenced by earlier interventions. Would the outcome of Case 1 have been different if the abnormal hematologic parameters demonstrated on the preoperative laboratory results had been further investigated? Would the institution of cerebral resuscitative measures before and during transport have offered this child a better chance of survival? Would the involvement of a specialized pediatric team earlier in the process have addressed some of those issues and would it have made a difference? Should the patient in case two have had cerebral resuscitative measures instituted at the referring hospital or during transport? In hindsight, this clearly would not have been useful or beneficial to the patient. But what if the initial CT interpretation of a brain tumor and increased intracranial pressure with ventricular ablation and midline shift had been correct? Should the transport team have suggested or instituted a different level of therapy with the information that was available at the time of transport? The patient in Case 3 had a dramatic presentation of his ALL. Were there signs and symptoms that should have alerted the referring hospital, transport command physician or transport team to the likely deterioration of that patient? If this patient had presented to a hospital a further distance away, would the impending cardiovascular collapse and respiratory failure have been anticipated or occurred during the transport? Would or should the mode of transport or team configuration have been altered? If this patient had deteriorated during the transport, would the transport team have had the skills to manage this potentially difficult airway? Should the patient in Case 4 have had antihypertensive medication started at the referring hospital or during the transport process? What are the guidelines for antihypertensive intervention in this situation? If antihypertensive therapy were instituted by the transport team, should this have affected time or mode of transport, or was it more prudent not to rock the boat by instituting interventional therapy? Is hypertension a different issue with a liver mass, as suspected at the time of referral, or with a nephrogenic tumor? These cases afford us the ability to review several presentations of oncologic emergencies. The questions above are but a few of the potential areas of discussion that can arise from these cases. We should use these cases as an opportunity to review and refresh our transport teams on the many faces of oncology and potential pitfalls in the care of those patients.

Adolescent↗

A gene transfer system establishes interleukin-6 neither promotes nor suppresses renal injury.

Conflicting reports claim that circulating interleukin (IL)-6 promotes or suppresses renal disease. Although autoimmune MRL-lpr mice have an increase in serum IL-6, and kidneys can produce IL-6, the relevance of systemic and local exposure remains undefined. To investigate the impact of IL-6 on kidney disease, we constructed a gene transfer approach to deliver sustained, stable IL-6 into the kidney and circulation. We infused syngeneic genetically modified tubular epithelial cells (IL-6-TEC) under the renal capsule of autoimmune and nonautoimmune mice. IL-6-TEC did not incite renal injury in any strain. Furthermore, serum IL-6 levels, which were increased three- to fivefold by IL-6-TEC, did not alter the contralateral kidney. Therefore, neither local nor systemic exposure to IL-6 promoted renal injury. As opposed to IL-6, we previously established that granulocyte macrophage (GM)-colony-stimulating factor (CSF) initiates renal injury in autoimmune mice. To determine whether IL-6 could suppress GM-CSF-incited damage, we infused GM-CSF-TEC TEC along with IL-6-TEC. Local production of IL-6 into the kidney did not alter the tempo or severity of GM-CSF-induced injury. Thus neither local nor systemic delivery of IL-6 promotes or suppresses kidney disease.

Animals↗

Homozygosity mapping of the gene for Chediak-Higashi syndrome to chromosome 1q42-q44 in a segment of conserved synteny that includes the mouse beige locus (bg).

Chediak-Higashi syndrome (CHS) is an autosomal recessive disorder characterized by hypopigmentation or oculocutaneous albinism and severe immunologic deficiency with neutropenia and lack of natural killer (NK) cell function. Most patients die in childhood from pyogenic infections or an unusual lymphoma-like condition. A hallmark of the disorder is giant inclusion bodies seen in all granule-containing cells, including granulocytes, lymphocytes, melanocytes, mast cells, and neurons. Similar ultrastructural abnormalities occur in the beige mouse, which thus has been suggested to be homologous to human CHS. High-resolution genetic mapping has indicated that the bg gene region of mouse chromosome 13 is likely homologous to the distal portion of human chromosome 1q. Accordingly, we carried out homozygosity mapping using markers derived from distal human chromosome 1q in four inbred families or probands with CHS. Our results indicate that the human CHS gene maps to an 18.8-cM interval in chromosome segment 1q42-q44 and that human CHS therefore is very likely homologous to mouse bg.

Adult↗

Macrophage growth factors introduced into the kidney initiate renal injury.

BACKGROUND: CSF-1 expression precedes renal injury in autoimmune MRL-lpr mice and is responsible for macrophage (M phi) proliferation and survival in the kidney. By comparison, C3H-lpr mice do not express CSF-1 in the kidney, and despite the lpr mutation, kidneys remain normal. The purpose of this study was to test the capacity of local and systemic expression of M phi growth factor, CSF-1 to initiate renal injury in normal (C3H-(++), MRL-(++) and autoimmune (C3H-lpr, MRL-lpr) mice. MATERIALS AND METHODS: We designed a gene transfer system to deliver cytokines into the kidney by transducing renal tubular epithelial cells (TEC) using retroviral vectors expressing CSF-1 or another M phi growth factor, GM-CSF. We placed transduced syngeneic cytokine-TEC under the renal capsule of normal and autoimmune prone mice prior to renal injury and evaluated renal pathology at 3, 7, 14, 28, and 90 days postimplant. RESULTS: CSF-1-TEC and GM-CSF-TEC, but not uninfected TEC, caused extensive local renal injury in strains with the lpr mutation. At 3-7 days the infiltrating cells were mainly M phi, and by 28 days they were predominantly lymphocytes. By comparison, the kidneys of MRL-(++) and C3H-(++) mice remained normal. Implanted genetically modified TEC caused a sustained increase of CSF-1 or GM-CSF in the circulation which did not modify the contralateral kidney. CONCLUSIONS: Gene transfer of M phi growth factors into the kidney initiates severe local renal injury in autoimmune prone mice with the lpr mutation, but does not compromise the kidney in nonautoimmune hosts. Of note, introduction of M phi growth factors into the kidney of C3H-lpr mice which do not spontaneously develop renal injury incites renal damage. These studies offer a gene transfer approach to explore the impact of local and systemic cytokine production on renal injury.

Animals↗

Identification and expression cloning of a leptin receptor, OB-R.

The ob gene product, leptin, is an important circulating signal for the regulation of body weight. To identify high affinity leptin-binding sites, we generated a series of leptin-alkaline phosphatase (AP) fusion proteins as well as [125I]leptin. After a binding survey of cell lines and tissues, we identified leptin-binding sites in the mouse choroid plexus. A cDNA expression library was prepared from mouse choroid plexus and screened with a leptin-AP fusion protein to identify a leptin receptor (OB-R). OB-R is a single membrane-spanning receptor most related to the gp130 signal-transducing component of the IL-6 receptor, the G-CSF receptor, and the LIF receptor. OB-R mRNA is expressed not only in choroid plexus, but also in several other tissues, including hypothalamus. Genetic mapping of the gene encoding OB-R shows that it is within the 5.1 cM interval of mouse chromosome 4 that contains the db locus.

Amino Acid Sequence↗

Insight into the microphthalmia gene.

The murine microphthalmia gene (mi) is one of the last multi-allelic, classic coat-colour genes to be cloned in the mouse and, similar to many of these genes, encodes an exciting molecule that is is involved in multiple developmental processes. The existence of the numerous alleles has allowed the molecular dissection of the function of the MI bHLH-Zip transcription factor in vivo and offers a unique opportunity to understand the function of a multimeric transcription factor throughout development and in many tissues. It is also the gene mutated in some patients with the human deafness syndrome, Waardenburg's syndrome type II, and hence helps to understand this syndrome.

Alleles↗

Cloning and regional assignment of the human myosin heavy chain 12 (MYH12) gene to chromosome band 15q21.

Sequences encoding 1,235 bp of the human myosin heavy chain 12 (MYH12) gene have been cloned from a human brain cDNA library by PCR amplification. The human sequence is 95.8% identical to the mouse sequence at the amino acid level, indicating that the MYH12 gene has been evolutionarily well conserved. Somatic cell hybrid analysis and in situ hybridization place the MYH12 gene on human chromosome 15, at band q21, and extend distally the known region of chromosome 15 linkage homology on mouse chromosome 9.

Amino Acid Sequence↗

Brown midrib sorghum silage for midlactation dairy cows.

Brown midrib sorghum silage was compared with alfalfa, corn, and normal sorghum silages for its effect on performance, ruminal metabolism, and digestive kinetics of Holstein dairy cows in midlactation. Twelve cows averaging 90 +/- 5 DIM were assigned to one of four diets in replicated 4 x 4 Latin squares with 4-wk periods. Additionally, 3 ruminally fistulated cows (95 +/- 20 DIM) were assigned to the same diets in a 3 x 4 Youden square for measurement of ruminal characteristics. Diets were fed as isonitrogenous TMR that contained 65% silage (DM basis). The DMI was greater for the corn and brown midrib sorghum (4% of BW/d) than for the alfalfa and normal sorghum diets (3.4% of BW/d). The brown midrib sorghum supported FCM production that was similar to that of cows on corn and alfalfa diets (25.8 kg/d), but cows fed normal sorghum produced less milk and fewer milk components. Source of silage had no effect on eating time, but rumination was least for the alfalfa diet. Ruminal pH and ammonia concentrations were similar for all diets. Total VFA concentrations were greatest for the corn and brown midrib sorghum diets. The brown midrib sorghum had greater in situ extent of ruminal NDF digestion than did the normal sorghum, which agreed with in vitro data. The brown midrib sorghum used in this experiment supported FCM production similar to the corn and alfalfa silages commonly fed to dairy cows in midlactation.

Animal Nutritional Physiological Phenomena↗

Helicase-catalyzed DNA unwinding: energy coupling by DNA motor proteins.

DNA helicases catalyze the unwinding of double-stranded (ds) DNA to yield the single-stranded (ss) DNA intermediates required in DNA replication, recombination, and repair. DNA helicases couple the free energy of nucleoside triphosphate (NTP) binding and hydrolysis to separate the two complementary DNA strands while also translocating vectorially along the DNA substrate. As such, helicases are functionally DNA motor proteins. The functional form of helicases generally appears to be oligomeric (usually dimers or hexamers), which provides the helicase with multiple DNA binding sites that are required for translocation and DNA unwinding. The affinity of ss- versus dsDNA for these multiple DNA binding sites is modulated allosterically by NTP binding, hydrolysis, and product release, which is central to helicase-catalyzed DNA unwinding. The mechanistic details of the DNA unwinding, translocation, and NTPase reactions are only starting to emerge. We discuss energy coupling by DNA helicases in general, and by the dimeric E. coli Rep helicase in particular, focusing on the similarities of these enzymes to classical motor proteins.

Adenosine Triphosphatases↗

Kinetic mechanism of adenine nucleotide binding to and hydrolysis by the Escherichia coli Rep monomer. 1. Use of fluorescent nucleotide analogues.

The Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA in a reaction that is coupled to ATP binding and hydrolysis. The Rep protein is a stable monomer in the absence of DNA but dimerizes upon binding either single-stranded or duplex DNA, and the dimer appears to be the functionally active form of the Rep helicase. As a first step toward understanding how ATP binding and hydrolysis are coupled energetically to DNA unwinding, we have investigated the kinetic mechanism of nucleotide binding to the Rep monomer (P) using stopped-flow techniques and the fluorescent ATP analogue, 2'(3')-O-(N-methylanthraniloyl-ATP (mantATP). The fluorescence of mantATP is enhanced upon Rep binding due to energy transfer from tryptophan. The results are consistent with the following two-step mechanism, in which the bimolecular association step is followed by a conformational change in the P-mantATP complex: P + mantATP [formula: see text] P-mantATP [formula: see text] (P-mantATP). The following rate and equilibrium constants were determined at 4 degrees C in 20 mM Tris.HCl (pH 7.5), 6 mM NaCl, 5 mM MgCl2, and 10% (v/v) glycerol: k+1 = (1.1 +/- 0.2) x 10(7) M-1 s-1; k-1 = 3.2 (+/- 0.5) s-1; k+2 = 2.9 (+/- 0.5) s-1; k-2 = 0.04 (+/- 0.005) s-1; K1 = k+1/k-1 = (3.4 +/- 0.8) x 10(6) M-1; K2 = k+2/k-2 = 73 (+/- 10); Koverall = K1K2 = (2.30 +/- 0.6) x 10(8) M-1. Similar rate and equilibrium constants are obtained with mantATP gamma S, whereas the apparent rate constant for mantAMPPNP binding is 15-fold lower than for mantATP and equilibrium binding is weaker (Koverall approximately 10(6) M-1). Rep monomer does bind mantATP in the absence of Mg2+ (Koverall approximately 5 x 10(5) M-1), although the four rate constants in the above reaction increase by at least 8-fold (k-1 and k-2 increase by approximately 100- and approximately 1000-fold, respectively). The affinities of Mg2+ for P-mantATP and (P-mantATP)* are 10- and 1000-fold higher than those for nucleotide-free Rep monomer, indicating that the second step in the reaction is associated with a marked increase in Mg2+ affinity. The bound Mg2+ in a (P-mantATP)*-Mg2+ complex dissociates at a rate that is comparable to the rate of mantATP release.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine Nucleotides↗

Kinetic mechanism of adenine nucleotide binding to and hydrolysis by the Escherichia coli Rep monomer. 2. Application of a kinetic competition approach.

The Escherichia coli Rep protein is a DNA helicase that functions as a homodimer to catalyze the unwinding of duplex DNA during DNA replication in a reaction that is coupled to the binding and hydrolysis of ATP. As a first step toward a molecular understanding of the interactions of Rep with adenine nucleotides, we have investigated the kinetic mechanism of adenine nucleotide binding to the Rep monomer, which is the state of the protein in the absence of DNA. Although ATP binding to Rep does not significantly change the intrinsic tryptophan fluorescence, the binding of the fluorescent nucleotide analogue, 2'(3')-O-(N-methylanthraniloyl)-ATP (mantATP) is associated with a large increase in mant nucleotide fluorescence intensity [lambda ex = 290 nm, lambda em > 420 nm; Moore, K. J. M., & Lohman, T. M. (1994) Biochemistry (preceding article in this issue)]. We have used the fluorescence signal from mantATP binding to monitor the kinetics of nonfluorescent nucleotide binding to Rep by a kinetic competition approach. The simultaneous and parallel binding of a mixture of mantATP and ATP to the Rep monomer is associated with a complex triphasic fluorescence transient during the approach to equilibrium. Global analysis of the fluorescence transients over a range of [ATP] by numerical integration techniques was used to define the kinetic mechanism of ATP binding and to determine the elementary rate constants. Using this approach, the kinetic rate constants for ADP, ATP gamma S, AMPPNP, AMP, adenosine, and inorganic phosphate were also determined at 4 degrees C in 20 mM Tris.HCl (pH 7.5), 6 mM NaCl, 10% (v/v) glycerol, and 5 mM MgCl2. The kinetics of adenine nucleotide binding to the Rep monomer are similar to those observed with the mant nucleotides under identical experimental conditions (Moore & Lohman, 1994). The kinetic competition data are consistent with the following two-step mechanism for the binding of ATP, ADP, and ATP gamma S, where P is the Rep monomer and A is the adenine nucleotide: P+A [formula: see text] P-A [formula: see text] (P-A). In the presence of 5 mM MgCl2, the values of K+1 (approximately 10(7) M-1 s-1) and k+2 (approximately 10 s-1) are comparable for each nucleotide, whereas k+2 > k-1 for ATP and ATP gamma S while for ADP k+2 << k-1; hence, differences in the overall equilibrium binding affinities of these nucleotides are primarily due to changes in k-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine Nucleotides↗

Single-turnover kinetics of helicase-catalyzed DNA unwinding monitored continuously by fluorescence energy transfer.

We describe a fluorescence assay that can be used to monitor helicase-catalyzed unwinding of duplex DNA continuously in real time. The assay is based on the observation that fluorescence resonance energy transfer (FRET) occurs between donor (fluorescein) and acceptor (hexachlorofluorescein) fluorophores that are in close proximity due to their covalent attachment to the 3' and 5' ends of the complementary strands of a duplex oligodeoxynucleotide. FRET results in a reduction in the fluorescence emission intensity of fluorescein in the duplex DNA substrate relative to that observed for fluorescein-labeled single stranded DNA. Therefore, an enhancement of fluorescein fluorescence (lambda ex = 492 nm; lambda em = 520 nm) occurs upon helicase-catalyzed unwinding of the duplex DNA and separation of the complementary strands. The fluorescence assay is extremely sensitive, allowing DNA unwinding reactions to be monitored continuously at DNA concentrations as low as 1 nM in a fluorescence stopped-flow experiment. We demonstrate the use of this DNA substrate in pre-steady state, single turnover studies of duplex DNA unwinding catalyzed by the Escherichia coli Rep helicase, monitored by fluorescence stopped flow. We show that the fluorescence enhancement monitors Rep-catalyzed DNA unwinding by comparisons with identical kinetic studies carried out using rapid chemical quench-flow techniques. Single turnover kinetic studies performed at 1 nM DNA as a function of excess Rep concentration show that Rep-catalyzed unwinding of an 18 base pair duplex containing a 3'-ss-(dT)20 tail is biphasic and can be described by the sum of two exponential terms. The observed rate constant of the first phase is independent of [Rep] (20-300 nM) and measures the rapid single turnover, unwinding of the duplex DNA by Rep dimers bound in productive complexes (1.3 +/- 0.2 s-1; 23 +/- 3 base pairs s-1 at 25.0 degrees C). The observed rate constant for the second phase increases linearly with [Rep], reflecting DNA unwinding that is limited by a Rep binding event occurring with a bimolecular rate constant of (1.8 +/- 0.1) x 10(5) M-1 s-1, which may reflect the rate constant for Rep dimerization on DNA. Kinetic competition studies indicate that both Rep subunits are bound stably to the DNA substrate in the productive complex that is unwound in the fast phase. The results of these kinetic studies are consistent with an active, rolling mechanism for Rep-catalyzed unwinding of DNA [Wong, I., & Lohman, T. M., (1992) Science 256, 350].(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphatases↗

Intracellular infection by Leishmania donovani inhibits macrophage apoptosis.

The phagocytic macrophage plays a critical role in host immune responses to microbial infection, and represents a major source of inflammatory and growth cytokines. Intramacrophage infection by the protozoan parasite Leishmania donovani results in increased viability of the host cell in the absence of exogenous growth factor. We demonstrate that infection of bone marrow-derived macrophages (BMMs) by L. donovani promastigotes or treatment of BMMs with lipophosphoglycan LPG, the major surface molecule of the promastigote, inhibits apoptosis in the macrophage induced by the removal of macrophage (M)-CSF. This effect was also achieved by supernatants collected from L. donovani-infected macrophages, implicating the elaboration of a soluble factor by infected cells as the mediator of this inhibition. To identify candidate factors, reverse transcription PCR was employed to characterize the mRNA cytokine profile of infected macrophages. L. donovani infection of BMMs was found to induce gene expression for granulocyte-macrophage CSF, TNF-alpha, TGF-beta, and IL-6, but not M-CSF or IL-1 beta. Of the cytokines induced by L. donovani, rTNF-alpha and recombinant granulocyte-macrophage CSF were shown to inhibit apoptosis of BMMs induced by the removal of M-CSF. The amount of these cytokines in L. donovani-infected cell supernatants was quantified by ELISA. The mechanism by which L. donovani may inhibit apoptosis is discussed.

Animals↗

Leishmania donovani infection enhances macrophage viability in the absence of exogenous growth factor.

Bone marrow-derived macrophages rapidly die in the absence of macrophage growth factor (M-CSF). However, as demonstrated here, bone marrow-derived macrophages infected with Leishmania donovani exhibit increased viability in the absence of exogenous growth factor. Forty-eight hours after inoculation with promastigotes or amastigotes, infected cell cultures contained 180 and 95% more cells, respectively, than control cultures. This effect was specific to Leishmania infection, as uptake of latex beads or avirulent promastigotes by macrophages did not enhance cell viability. L. donovani-infected macrophages also displayed increased phagocytic capacity, as compared with control macrophages and macrophages grown continuously in M-CSF-containing medium. Supernatants collected from infected cells elaborated a factor(s) that enhanced macrophage viability but did not stimulate macrophage DNA synthesis. This activity of L. donovani-infected cell-conditioned medium could be abrogated by preincubation of macrophages with cycloheximide before inoculation with the parasite, implying that macrophage protein synthesis is required for the elaboration of this factor(s).

Animals↗

Use of pooled time series in the study of naturally occurring clinical events and problem behavior in a foster care setting.

Pooled time series is an underused analytic technique with the potential to increase researchers' ability to exploit clinical data. This article demonstrates the value of pooled time series by analyzing the behavior of youths in a specialized foster care treatment setting in response to a naturally occurring clinical event: changes in the number of youths living together in a treatment foster care setting. Pooled time series moves beyond typical clinical analyses with an increased capability of controlling statistically for complex within-subject effects and with a clinically useful measure of effect size. The complexity of the intrasubject data made it virtually impossible to determine the relevant significance (i.e., clinical meaning) of the clinical event by the use of standard n = 1 visual analysis procedures or standard statistical methods (e.g., chi square). After things such as autocorrelation and individual time trends were statistically controlled, each additional youth increased the number of problematic behaviors by one behavior per youth per day on the Parent Daily Report.

Adolescent↗

Molecular basis of mouse microphthalmia (mi) mutations helps explain their developmental and phenotypic consequences.

Mutations in the mouse microphthalmia (mi) gene affect the development of a number of cell types including melanocytes, osteoclasts and mast cells. Recently, mutations in the human mi gene (MITF) were found in patients with Waardenburg Syndrome type 2 (WS2), a dominantly inherited syndrome associated with hearing loss and pigmentary disturbances. We have characterized the molecular defects associated with eight murine mi mutations, which vary in both their mode of inheritance and in the cell types they affect. These molecular data, combined with the extensive body of genetic data accumulated for murine mi, shed light on the phenotypic and developmental consequences of mi mutations and offer a mouse model for WS2.

Alleles↗

The murine dilute suppressor gene encodes a cell autonomous suppressor.

The murine dilute suppressor gene (dsu) suppresses the coat-color phenotype of three pigment mutations, dilute (d), ashen (ash) and leaden (ln), that each produce adendritic melanocytes. Suppression is due to the ability of dsu to partially restore (ash and ln), or almost completely restore (d), normal melanocyte morphology. While the ash and ln gene products have yet to be identified, the d gene encodes a novel myosin heavy chain (myosin 12), which is speculated to be necessary for the elaboration, maintenance, and/or function of melanocyte cell processes. To begin to discriminate between different models of dsu action, we have produced aggregation chimeras between mice homozygous for dsu and mice homozygous for d to determine if dsu acts cell autonomously or cell nonautonomously. In addition, we have further refined the map location of dsu in order to examine a number of possible dsu candidate genes mapping in the region and to provide a genetic basis for the positional cloning of dsu.

Animals↗