Biomedical subjects

L C Ng

Publications and source records attributed to L C Ng.

11 recordsLinked to original sources

PubMed2001

The regulatory N-terminal region of the aromatic-responsive transcriptional activator DmpR constrains nucleotide-triggered multimerisation.

The transcriptional promoting activity of DmpR is under the strict control of its aromatic effector ligands that are bound by its regulatory N-terminal domain. The positive control function of DmpR resides within the central C-domain that is highly conserved among activators of sigma(54)-RNA polymerase. The C-domain mediates ATP hydrolysis and interaction with sigma(54)-RNA polymerase that are essential for open-complex formation and thus initiation of transcription. Wild-type and loss-of-function derivatives of DmpR, which are defective in distinct steps in nucleotide catalysis, were used to address the consequences of nucleotide binding and hydrolysis with respect to the multimeric state of DmpR and its ability to promote in vitro transcription. Here, we show that DmpR derivatives deleted of the regulatory N-terminal domain undergo an aromatic-effector independent ATP-binding triggered multimerisation as detected by cross-linking. In the intact protein, however, aromatic effector activation is required before ATP-binding can trigger an apparent dimer-to-hexamer switch in subunit conformation. The data suggest a model in which the N-terminal domain controls the transcriptional promoting property of DmpR by constraining ATP-mediated changes in its oligomeric state. The results are discussed in the light of recent mechanistic insights from the AAA(+) superfamily of ATPases that utilise nucleotide hydrolysis to restructure their substrates.

Adenosine Triphosphatases↗

PubMed2001

Store-operated channels mediate Ca(2+) influx and contraction in rat pulmonary artery.

Cation channels activated by Ca(2+) store depletion have been proposed to mediate Ca(2+) influx in vascular smooth muscle cells. The aim of this study was to determine if store-operated channels have a functional role in pulmonary artery smooth muscle cells (PASMCs). In intact rat pulmonary artery rings, cyclopiazonic acid (CPA) produced a sustained contraction that was resistant to inhibition by nifedipine, but abolished in Ca(2+)-free solution and 50% blocked in the presence of 6 micromol/L Cd(2+), 10 micromol/L Ni(2+), 600 micromol/L La(3+), and 7 micromol/L SKF96365. In freshly isolated PASMCs loaded with fura-2, CPA increased the intracellular Ca(2+) concentration by stimulating dihydropyridine-resistant Ca(2+) influx, which was approximately 50% blocked by 10 micromol/L Ni(2+) and 7 micromol/L SKF96365. In perforated-patch recordings, CPA activated a sustained inward current at negative membrane potentials, which persisted in cells dialyzed with BAPTA, showed a near linear dependence on membrane potential when Cs(+) was the main intracellular cation, and was blocked by Ni(2+), Cd(2+), and SKF96365 at concentrations preventing contraction. The current showed a bimodal dependence on extracellular Ca(2+), being enhanced 2-fold in the absence of Ca(2+) and around 10-fold on reducing Ca from 1.8 to 0.2 mmol/L. RT-PCR revealed the expression of Trp1, Trp3, Trp4, Trp5, and Trp6 mRNA, whereas immunostaining identified Trp1, Trp3, Trp4, and Trp6 channel proteins in isolated PASMCs. At least one of these subunits may contribute to cation channels in PASMCs, which are activated by store depletion to bring about Ca(2+) influx and contraction.

Animals↗

PubMed2000

Comparison between electroglottography and electromagnetic glottography.

Newly developed glottographic sensors, utilizing high-frequency propagating electromagnetic waves, were compared to a well-established electroglottographic device. The comparison was made on four male subjects under different phonation conditions, including three levels of vocal fold adduction (normal, breathy, and pressed), three different registers (falsetto, chest, and fry), and two different pitches. Agreement between the sensors was always found for the glottal closure event, but for the general wave shape the agreement was better for falsetto and breathy voice than for pressed voice and vocal fry. Differences are attributed to the field patterns of the devices. Whereas the electroglottographic device can operate only in a conduction mode, the electromagnetic device can operate in either the forward scattering (diffraction) mode or in the backward scattering (reflection) mode. Results of our tests favor the diffraction mode because a more favorable angle imposed on receiving the scattered (reflected) signal did not improve the signal strength. Several observations are made on the uses of the electromagnetic sensors for operation without skin contact and possibly in an array configuration for improved spatial resolution within the glottis.

Adult↗

PubMed2000

Microalbuminuria measurements by two in-house ELISA methods.

Detection of microalbuminuria is important in the management of diabetic patients since it is predictive of development of proteinuria and nephropathy. Two sensitive and specific in-house ELISAs for microalbuminuria were established and validated. One of the ELISAs was based on antigen coating while the other employed antibody coating. Recovery and linearity experiments gave acceptable results of 100 +/- 10%, while precision results were <10% for intra-assay and <12% for inter-assay coefficients of variation (CVs). The standard curve ranged from 10-625 ug/l, equivalent to 0.2-12.5 mg/l for urine samples diluted 1:20 fold. When the antibody coated ELISA was compared to antigen coated ELISA, a correlation of r=0.996 was obtained. When compared to commercial kits, the in-house ELISAs gave good correlations of r=0.961 versus the Boehringer Mannheim Micral Test strips and r=0.940 versus Ames Microalb Turbidimetry. The normal microalbumin reference ranges determined for 12h, first morning and random urine samples were 0.7-5.3 mg, 0.1-10.2 mg/l and 0.8-26.1 mg/l respectively. The normal albumin excretion rate (AER) was 1.0-7.3 ug/min while untimed urine samples gave results of 0.1-0.9 and 0.2-1.6 mg/mmol after dividing by creatinine concentrations. The ELISAs were used to detect microalbuminuria in 338 random urine samples from diabetic patients. A high percentage 47.9% was found to be positive for microalbuminuria and 18.0% had macroalbuminuria >25 mg/mmol. Thus screening for microalbuminuria together with creatinine measurements using random urine samples can be used for management of diabetic patients.

Adolescent↗

PubMed1998

Aromatic ligand binding and intramolecular signalling of the phenol-responsive sigma54-dependent regulator DmpR.

The Pseudomonas-derived sigma54-dependent regulator DmpR has an amino-terminal A-domain controlling the specificity of activation by aromatic effectors, a central C-domain mediating an ATPase activity essential for transcriptional activation and a carboxy-terminal D-domain involved in DNA binding. In the presence of aromatic effectors, the DmpR protein promotes transcription from the -24, -12 Po promoter controlling the expression of specialized (methyl)phenol catabolic enzymes. Previous analysis of DmpR has led to a model in which the A-domain acts as an interdomain repressor of DmpR's ATPase and transcriptional promoting property until specific aromatic effectors are bound. Here, the autonomous nature of the A-domain in exerting its biological functions has been dissected by expressing portions of DmpR as independent polypeptides. The A-domain of DmpR is shown to be both necessary and sufficient to bind phenol. Analysis of phenol binding suggests one binding site per monomer of DmpR, with a dissociation constant of 16 microM. The A-domain is also shown to have specific affinity for the C-domain and to repress the C-domain mediated ATPase activity in vitro autonomously. However, physical uncoupling of the A-domain from the remainder of the regulator results in a system that does not respond to aromatics by its normal derepression mechanism. The mechanistic implications of aromatic non-responsiveness of autonomously expressed A-domain, despite its demonstrated ability to bind phenol, are discussed.

Adenosine Triphosphatases↗

PubMed1998

Speech articulator measurements using low power EM-wave sensors.

Very low power electromagnetic (EM) wave sensors are being used to measure speech articulator motions as speech is produced. Glottal tissue oscillations, jaw, tongue, soft palate, and other organs have been measured. Previously, microwave imaging (e.g., using radar sensors) appears not to have been considered for such monitoring. Glottal tissue movements detected by radar sensors correlate well with those obtained by established laboratory techniques, and have used to estimate a voiced excitation function for speech processing applications. The noninvasive access, coupled with the small size, low power, and high resolution of these new sensors, permit promising research and development applications in speech production, communication disorders, speech recognition and related topics.

Electromagnetic Phenomena↗

PubMed1996

Genetic evidence for interdomain regulation of the phenol-responsive final sigma54-dependent activator DmpR.

The final sigma54-dependent DmpR activator regulates transcription of the dmp operon that encodes the enzymes for catabolism of (methyl)phenols. DmpR is expressed constitutively, but its transcriptional promoting activity is controlled positively in direct response to the presence of aromatic pathway substrates (effectors). DmpR has a distinct domain structure with the amino-terminal A-domain controlling the specificity of activation of the regulator by aromatic effectors (signal reception), a central C-domain mediating an ATPase activity essential for transcriptional activation, and a carboxyl-terminal D-domain involved in DNA binding. Deletion of the A-domain has been shown previously to result in an effector-independent transcriptional activator with constitutive ATPase activity. These results, in conjunction with the location of mutations within the A- and C-domains which exhibit an effector-independent (semiconstitutive) property, have led to a working model in which the A-domain serves to mask the ATPase and transcriptional promoting activity of the C-domain in the absence of effectors. To investigate the mechanism by which the A-domain exerts its repressive effect, we developed a genetic system to select positively for intramolecular second site revertants of DmpR. The results demonstrate (i) that mutations within the A-domain can suppress the semiconstitutive activity of C-domain located mutations and vice versa; (ii) that the C-domain located mutations do not influence the intrinsic ATPase and transcriptional promoting property of the C-domain in the absence of the A-domain; and (iii) that semiconstitutive mutations of the A- and C-domain have an additive effect. Taken together these results support a model in which the A-domain represses the function(s) of the C-domain by direct interactions between residues of the two domains.

Adenosine Triphosphatases↗

PubMed1996

Insulin treatment normalizes reduced free insulin-like growth factor-I concentrations in diabetic children.

OBJECTIVE: We have recently demonstrated multiple aberrations in the GH-IGF axis in the sera of children with untreated insulin-dependent diabetes mellitus (IDDM) which were restored after insulin replacement. However, the net result of these alterations in the IGF system on the concentrations of free/biologically available IGF-I in the serum have not been examined directly in diabetic children. In the present study, the effect of diabetes and subsequent insulin replacement on the circulating free IGF-I concentrations are assessed. DESIGN: Fasting venous serum samples were obtained longitudinally, before and at various times after the initiation of insulin treatment in untreated diabetic subjects. SUBJECTS: Ten prepubertal, aged (mean +/- SEM) 6.3 +/- 1.0 years, and six adolescent, aged 12.7 +/- 1.1 years, subjects with newly diagnosed and untreated IDDM, and age and pubertal status-matched control children and adolescents were recruited. METHODS: The serum samples were collected before initiating insulin treatment and 12-24 h, 1 week, and 1 month thereafter in subjects with IDDM. Insulin doses ranged from 0.5 to 1.2 U/kg/day. MEASUREMENTS: Free IGF-I concentration was assayed by a recently developed two-site immunoradiometric assay. Total IGF-I was measured by radioimmunoassay after acid-ethanol extraction of binding proteins. Differences in free and total IGF-I concentrations in IDDM subjects before and during insulin treatment were analysed by repeated measures analysis of variance followed by pairwise multiple comparisons test. In seven subjects with IDDM, where serum IGFBP-1 and IGFBP-3 concentrations, and IGFBP-3 protease activity had also been measured in a previous study, the relationship between these variables and circulating free IGF-I concentrations were examined by linear regression analysis. RESULTS: Free IGF-I concentrations in prepubertal subjects with IDDM were 0.9 +/- 0.2, 1.5 +/- 0.3, 1.6 +/- 0.3 and 2.5 +/- 0.4 micrograms/l before, 1 day, 1 week and 1 month after insulin treatment, respectively. Free IGF-I concentrations of control prepubertal children were 2.6 +/- 0.5 micrograms/l. Pubertal subjects had higher free IGF-I concentrations than prepubertal subjects but demonstrated a similar type of pattern; before insulin 2.3 +/- 1.1, 1 day 3.8 +/- 1.3, 1 week 3.7 +/- 0.6, 1 month 6.5 +/- 1.5 vs pubertal controls 7.7 +/- 2.0 micrograms/l. Total IGF-I concentrations were also reduced in untreated diabetic subjects and showed a slower pattern of normalization than free IGF-I concentrations. Free IGF-I concentrations correlated positively with total IGF-I and negatively with IGFBP-1 concentrations. There was no significant correlation between free IGF-I and either serum IGFBP-3 concentrations or IGFBP-3 protease activity. CONCLUSION: Alterations in the IGF system during untreated IDDM lead to a reduction in circulating free IGF-I concentrations which is restored progressively during insulin treatment. An increase in free IGF-I precedes that of total IGF-I suggesting that the former is a more sensitive indicator of the metabolic status. An inverse correlation between free IGF-I and IGFBP-1 supports the hypothesis that IGFBP-1 plays an important role in the acute modulation of free IGF-I levels.

Adolescent↗

PubMed1995

Aromatic effector activation of the NtrC-like transcriptional regulator PhhR limits the catabolic potential of the (methyl)phenol degradative pathway it controls.

Pseudomonas putida P35X (NCIB 9869) metabolizes phenol and monomethylphenols via a chromosomally encoded meta-cleavage pathway. We have recently described a 13.4-kb fragment of the chromosome that codes for the first eight genes of the catabolic pathway and a divergently transcribed positive regulator, phhR. The eight structural genes lie in an operon, the phh operon, downstream of a -24 TGGC, -12 TTGC promoter sequence. Promoters of this class are recognized by RNA polymerase that utilizes the alternative sigma 54 factor encoded by rpoN (ntrA) and are positively regulated by activators of the NtrC family. In this study, we have identified the coding region for the 63-kDa PhhR gene product by nucleotide sequencing of a 2,040-bp region and polypeptide analysis. PhhR was found to have homology with the NtrC family of transcriptional activators, in particular with DmpR, the pVI150-encoded regulator of (methyl)phenol catabolism by Pseudomonas sp. strain CF600. By using a luciferase reporter system, PhhR alone was shown to be sufficient to activate transcription from the phh operon promoter in an RpoN+ background but not an RpoN- background. Luciferase reporter systems were also used to directly compare the aromatic effector profiles of PhhR and DmpR. Evidence that the difference in the growth substrate ranges of strains P35X and CF600 is due to the effector activation specificities of the regulators of these systems rather than the substrate specificities of the catabolic enzymes is presented.

Bacterial Proteins↗

PubMed1994

Cloning and sequences of the first eight genes of the chromosomally encoded (methyl) phenol degradation pathway from Pseudomonas putida P35X.

Pseudomonas putida P35X (NCIB 9869) metabolises phenol and cresols via a chromosomally encoded meta-cleavage pathway. A 13.4-kb fragment of the chromosome involved in encoding phenol catabolism was cloned and characterized. Deletion analysis and nucleotide sequencing of a 6589-bp region, in conjunction with enzyme assays, were used to identify the phhKLMNOP genes encoding the phenol hydroxylase, the phhB gene encoding catechol 2,3-dioxygenase (EC 1.13.11.2) and the phhQ gene that encodes a small ferredoxin-like protein. The genes are organised in an operon-like structure, in the order phhKLMNOPQB, and the deduced amino-acid sequences share high homology (68.3-99.7%) with those of the plasmid-encoded genes dmpKLMNOPQB of Pseudomonas sp. strain CF600. Genetic evidence is presented that the difference in the growth substrate ranges of Pseudomonas P35X and CF600 are due to the effector activation specificities of the regulators of these systems, rather than the substrate specificities of the catabolic enzymes.

Amino Acid Sequence↗

PubMedDate not supplied

Quantitative comparison of prolactin production by decidua compacta and spongiosa from the first trimester elective termination of pregnancy.

Prolactin (PRL) producing capacity was studied in explants of decidua compacta and decidua spongiosa obtained from 41 patients undergoing termination of pregnancy at gestation 6 to 12 weeks. In vitro PRL producing capacity, expressed as mIU/g protein, of the decidua compacta was significantly higher (P < 0.05) than those of decidua spongiosa. Production of PRL increased with gestation from 6 to 12 weeks with a more rapid rate at the later gestation. The pattern of increase fitted significantly (P < 0.0001) to the exponential model for both decidua compacta and decidua spongiosa. The exponential regression equations for decidua compacta and decidua spongiosa were (ln y = 4.25 + 0.19x) and (ln y = 2.80 + 0.31x) respectively. Hence, although both decidua compacta and decidua spongiosa had a similar pattern of increase in PRL production, the rate of increment was significantly greater in decidua spongiosa than in decidua compacta. These findings suggest that separating decidua compacta and decidua spongiosa of the first trimester would reduce the heterogeneity of decidual tissue and offer a new approach to the studies of the synthesis, release and regulation of PRL production by human decidua.

Abortion, Induced↗