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Biomedical subjects

B King

Publications and source records attributed to B King.

At least 37 records · Page 2Linked to original sources
PubMed2001

Essential role of Drosophila Hdac1 in homeotic gene silencing.

Deacetylation of the N-terminal tails of core histones plays a crucial role in gene silencing. Rpd3 and Hda1 represent two major types of genes encoding trichostatin A-sensitive histone deacetylases. Although they have been widely found, their cellular and developmental roles remain to be elucidated in metazoa. We show that Drosophila Hdac1, an Rpd3-type gene, interacts cooperatively with Polycomb group repressors in silencing the homeotic genes that are essential for axial patterning of body segments. The biochemical copurification and cytological colocalization of HDAC1 and Polycomb group repressors strongly suggest that HDAC1 is a component of the silencing complex for chromatin modification on specific regulatory regions of homeotic genes.

Animals↗
PubMed2001

Functional annotation of a full-length mouse cDNA collection.

The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.

Animals↗
PubMed2001

Purification and characterization of recombinant human lysosomal alpha-mannosidase.

Lysosomal alpha-mannosidase (EC 3.2.1.24) is required in the degradation of the asparagine-linked carbohydrates of glycoproteins. Deficiency of this enzyme leads to the lysosomal storage disorder alpha-mannosidosis. As an initial step toward enzyme replacement therapy for alpha-mannosidosis, the human lysosomal alpha-mannosidase cDNA was cloned into the pcDNA 3.1 vector and expressed in Chinese hamster ovary cells. Dimethyl sulfoxide (DMSO) added to the cell culture media to induce growth arrest led to a 4-fold increase in the enzyme production, with an average yield of 3.2 mg L(-1) day(-1). alpha-Mannosidase was secreted as an active homodimer of a 130-kDa precursor that was proteolyzed into two polypeptides of 55 and 72 kDa during the subsequent purification of the enzyme. N-terminal sequence analysis of the purified enzyme revealed that the proteolysis occurred close to a cleavage site previously identified in the intracellular form of lysosomal alpha-mannosidase. Generation of monoclonal antibodies against the recombinant enzyme made it possible to develop a single-step immunoaffinity purification procedure for alpha-mannosidase. The immunoaffinity-purified enzyme which mainly consisted of the 130-kDa precursor, displayed specific activity and kinetics similar to those of the processed form. Recombinant alpha-mannosidase was taken up by cultured alpha-mannosidosis fibroblasts and was trafficked to the lysosomes via the mannose 6-phosphate pathway where it reduced the amounts of stored mannose-containing oligosaccharides.

Animals↗
PubMed2001

GC-FID as a primary method for establishing the purity of organic CRMs used for drugs in sport analysis.

The National Analytical Reference Laboratory has synthesized and characterized 67 anabolic steroid marker metabolites, both unlabelled and deuterated, and 37 key glucuronide and sulfate steroid conjugate pure substance reference materials. Work is also in process to establish their full traceability so that they can be issued as certified and primary reference materials. Both identity and purity have been rigorously characterized using a number of techniques and a primary method for purity assessment developed, based gas chromatography combined with flame ionization detection for the parent steroids and HPLC with evaporative light scattering detection for non-volatile steroid conjugates. Strategies for establishing traceability and for estimating measurement uncertainty are reported. The strategies described are considered applicable to a wide range of organic pure substance reference materials.

Algorithms↗
PubMed2001

Meeting the measurement uncertainty and traceability requirements of ISO/AEC standard 17025 in chemical analysis.

The new laboratory accreditation standard, ISO/IEC 17025, reflects current thinking on good measurement practice by requiring more explicit and more demanding attention to a number of activities. These include client interactions, method validation, traceability, and measurement uncertainty. Since the publication of the standard in 1999 there has been extensive debate about its interpretation. It is the author's view that if good quality practices are already in place and if the new requirements are introduced in a manner that is fit for purpose, the additional work required to comply with the new requirements can be expected to be modest. The paper argues that the rigour required in addressing the issues should be driven by customer requirements and the factors that need to be considered in this regard are discussed. The issues addressed include the benefits, interim arrangements, specifying the analytical requirement, establishing traceability, evaluating the uncertainty and reporting the information.

Journal Article↗
PubMed2001

Biological monitoring of workers exposed to dimethylformamide in a textile polyurethane unit.

Dimethylformamide (DMF) is a universal solvent noted for its solubility in both aqueous and lipid media. It is hepatotoxic, and there are concerns about its carcinogenicity. Our objective was to determine the correlation between air monitoring measurements and biological measurements of a suitable metabolite of DMF in a cohort of operatives in a polyurethane production unit. This was done with a view to assessing how much the inhalation route contributed to total DMF exposure, mainly for control purposes. We investigated the relationship between personal air sample measurements of DMF and biological measurements of N-methylformamide (NMF) in nine adult subjects, recruited across the shifts, with varying levels of exposure to DMF. Personal exposure monitoring was carried out with a low-flow-rate Model 222-4 SKC pump, while post-shift urine samples were obtained for further analysis. Operatives were asked to abstain from consuming alcohol for 24 h before the designated shift, as advised by the laboratory responsible for the analysis of urine samples. We found a very strong statistical association between air sample measurements of DMF and NMF in the urine of the sample population (R(2) = 0.95, P < 0.0001). This study suggests that airborne exposure contributes significantly to elevated levels of NMF and, therefore, indicates that efforts should be concentrated on engineering controls in reducing workplace DMF exposure.

Adult↗
PubMed2000

NGF-induced motoneuron cell death depends on the genetic background and motoneuron sub-type.

Nerve growth factor (NGF) promotes the survival of several neuronal populations, but recently it has also been shown to induce neuronal cell death. Here we report the effects of NGF on lesioned motoneurons. We have analyzed facial and sciatic motoneurons in newborn and adult BALB/c and C57BL/6 mice, in addition to mice deficient in the low-affinity p75 receptor for the neurotrophins (p75NTR). NGF application did not alter survival of lesioned facial motoneurons in any of the strains examined independent of the age of the animals. Only in the adult C57BL/6 mouse strain where the sciatic nerve had been crushed prior to factor application did NGF induce cell death of axotomized sciatic motoneurons. Our results illustrate the importance of the genetic background and the motoneuron sub-type in studies related to cell death and survival of motoneurons in relation to NGF and p75NTR.

Animals↗
PubMed2000

Localization of concealed norplant implants and implant fragments by a modified mammography technique.

A modified mammography technique for localization of concealed Norplant implants and Norplant fragments was applied to four patients, ages 18, 35, 22, and 25. A dedicated mammography unit and mammography film screen system affording high resolution and high contrast was applied under the same conditions to each patient. 26 kilovoltage peak (KVp) was used, ranging from 60 to 150 milliamperes. Using both the automatic exposure control and manual techniques, specific milliampere range depended on the specific case. The technique proved successful, as concealed Norplant capsules were exquisitely visualized by films obtained with a Siemens Mammomat 3000. Applying the same modified technique, precise location of the Norplant implants was determined using a fenestrated compression plate with an alphanumeric grid in a manner similar to hookwire localization of breast lesions under mammographic guidance. The capsules were then safely retrieved. We conclude that this modified technique may provide practitioners with valuable assistance in the exact localization of the Norplant capsule(s) or fragments, thereby facilitating their removal. Application of a modified mammography technique reveals precise localization of concealed Norplant capsules facilitating their removal.

Adolescent↗
PubMed2000

An orally active anti-apoptotic molecule (CGP 3466B) preserves mitochondria and enhances survival in an animal model of motoneuron disease.

Apoptosis and mitochondrial dysfunction are thought to be involved in the aetiology of neurodegenerative diseases. We have tested an orally active anti-apoptotic molecule (CGP 3466B) that binds to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in an animal model with motoneuron degeneration, i.e. a mouse mutant with progressive motor neuronopathy (pmn). In pmn/pmn mice, CGP 3466B was administered orally (10 - 100 nmol kg(-1)) at the onset of the clinical symptoms (2 weeks after birth). CGP 3466B slowed disease progression as determined by a 57% increase in life-span, preservation of body weight and motor performance. This improvement was accompanied by a decreased loss of motoneurons and motoneuron fibres as well as an increase in retrograde transport. Electron microscopic analysis showed that CGP 3466B protects mitochondria which appear to be selectively disrupted in the motoneurons of pmn/pmn mice. The data support evaluation of CGP 3466B as a potential treatment for motor neuron disease.

Administration, Oral↗
PubMed2000

Soluble TNF receptors partially protect injured motoneurons in the postnatal CNS.

There is accumulating evidence that cytokines are involved in the functioning of the brain and the spinal cord. However, it has been controversial whether they exert a neurotoxic or a neuroprotective effect. To address this question in vivo, we have examined the survival of injured motoneurons in a line of transgenic mice that overexpress the soluble form of tumour necrosis factor receptor-1 (sTNFR1). In these animals, all of the circulating TNF and lymphotoxin-alpha are neutralized by the continuous expression of the soluble receptor. Following axotomy of the facial nerve in 7-day-old control mice, we observed a loss of approximately 90% of the motoneurons at two weeks survival. In the transgenic mice under the same conditions, the percentage of motoneuron survival was increased two-fold (515 vs. 224) and varied as a function of the level of the circulating receptor. These results indicate that neutralization of endogenous TNF and lymphotoxin-alpha by means of overexpression of the soluble receptor can decrease cell death of injured motoneurons and suggest that these cytokines may play an important role in neuronal degeneration in the CNS following a lesion.

Age Factors↗
PubMed2000

Beryllium, an adjuvant that promotes gamma interferon production.

Beryllium is associated with a human pulmonary granulomatosis characterized by an accumulation of CD4(+) T cells in the lungs and a heightened specific lymphocyte proliferative response to beryllium (Be) with gamma interferon (IFN-gamma) release (i.e., a T helper 1 [Th1] response). While an animal model of Be sensitization is not currently available, Be has exhibited adjuvant effects in animals. The effects of Be on BALB/c mice immunized with soluble leishmanial antigens (SLA) were investigated to determine if Be had adjuvant activity for IFN-gamma production, an indicator of the Th1 response. In this strain of Leishmania-susceptible BALB/c mice, a Th2 response is normally observed after in vivo SLA sensitization and in vitro restimulation with SLA. If interleukin-12 (IL-12) is given during in vivo sensitization with SLA, markedly increased IFN-gamma production and decreased IL-4 production are detected. We show here that when beryllium sulfate (BeSO(4)) was added during in vivo sensitization of BALB/c mice with SLA and IL-12, significantly increased IFN-gamma production and decreased IL-4 production from lymph node and spleen cells were detected upon in vitro SLA restimulation. No specific responses were observed to Be alone. Lymph node and spleen cells from all mice proliferated strongly and comparably upon in vitro restimulation with SLA and with SLA plus Be; no differences were noted among groups of mice that received different immunization regimens. In vivo, when Be was added to SLA and IL-12 for sensitization of BALB/c mice, more effective control of Leishmania infection was achieved. This finding has implications for understanding not only the development of granulomatous reactions but also the potential for developing Be as a vaccine adjuvant.

Adjuvants, Immunologic↗
PubMed2000

Gonadal steroids regulate the number and activational state of mast cells in the medial habenula.

While mast cells in connective tissues have long been associated with allergic reactions, it is now clear that they are also present within the central nervous system under normal physiological conditions. The mast cell population increases 10-fold in the medial habenular region of the brain within 2 h after pairing in doves. The first study explored whether this increase was due to exposure to gonadal steroids. Light microscopic immunocytochemistry indicates an increased number of brain MC following exposure to either testosterone (T) or dihydrotestosterone (DHT) in the male, or 17beta estradiol (E) in the female, but not in cholesterol-treated controls. Thus, the increased habenular MC population is produced by gonadal hormones in the absence of sexual behavior, is not sexually dimorphic, and does not require aromatization of androgen. In the next study, MC activational state was determined using electron microscopy. Cells were categorized into five states: (I) resting; (II) initiation of degranulation; (III) fully degranulated; (IV) piecemeal secretion; and (V) resynthesizing. Hormone treatment (T, DHT, or E) resulted in a significant increase in the percent of cells in activated states. MC granules contain a wide range of biologically active molecules. The release of these granule contents into the neuropil of the central nervous system is likely to have wide ranging effects at multiple levels including vascular permeability and neuronal excitability. In that steroid treatment is known to result in such effects, the present demonstration of a hormonally induced shift in MC secretory state is one avenue by which these effects are mediated.

Animals↗
PubMed2000

Corticotropin-releasing hormone gene expression in primary placental cells is modulated by cyclic adenosine 3',5'-monophosphate.

CRH, the principal neuropeptide regulator of pituitary ACTH secretion, is also expressed in placenta. Placental CRH has been linked to the process of human parturition. However, the mechanisms regulating transcription of the CRH gene in placenta remain unclear. cAMP signaling pathways play important roles in regulating the expression of a diverse range of endocrine genes in the placenta. Therefore, we have explored the effect of cAMP on CRH promoter activity in primary cultures of human placental cells. Both forskolin and 8-bromo-cAMP, activators of protein kinase A, can increase CRH promoter activity 5-fold in transiently transfected human primary placental cells, in a manner that parallels the increase in endogenous CRH peptide. Maximal stimulation of CRH promoter activity occurs at 500 micromol/L 8-bromo-cAMP and 10 micromol/L forskolin. Electrophoretic mobility shift assay and mutation analysis combined with transient transfection demonstrate that in placental cells cAMP stimulates CRH gene expression through a cAMP regulatory element in the proximal CRH promoter region and involves a placental nuclear protein interacting specifically with the cAMP regulatory element.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2000

Glucocorticoid stimulation of corticotropin-releasing hormone gene expression requires a cyclic adenosine 3',5'-monophosphate regulatory element in human primary placental cytotrophoblast cells.

Production of placental CRH, which is identical to the peptide synthesized and secreted in the hypothalamus, has been linked to human parturition. Glucocorticoids stimulate placental CRH secretion and messenger ribonucleic acid expression, in contrast to their inhibition of CRH synthesis in the hypothalamus. A positive feedforward loop involving glucocorticoid-CRH-ACTH-glucocorticoid is thought to drive the exponential increase in placental CRH leading to delivery. Tissue-specific effects of glucocorticoids on CRH expression are therefore of interest. Using human primary placental cells, we investigated the mechanism by which glucocorticoids stimulate placental CRH gene expression. Nuclear run-on transcription shows that in human placental cells glucocorticoids up-regulate transcription of human CRH (hCRH). Using transient transfection assays we demonstrate that dexamethasone up-regulates both basal and cAMP-stimulated hCRH promoter activity, correlating well with the increase in endogenous CRH peptide levels. Through mutagenesis and deletion analyses we show that dexamethasone stimulation of hCRH gene transcription requires a functional cAMP regulatory element (CRE); this CRE is adequate to confer dexamethasone stimulation upon a heterologous promoter, and electrophoretic mobility shift assay studies show that a placental nuclear protein specifically binds to the hCRH CRE.

Cell Nucleus↗