Biomedical subjects

K W Tsim

Publications and source records attributed to K W Tsim.

54 records · Page 3Linked to original sources

PubMed1995

Truncated form of pro-acetylcholine receptor-inducing activity (ARIA) induces AChR alpha-subunit but not AChE transcripts in cultured chick myotubes.

Acetylcholine receptor-inducing activity (ARIA) is a glycoprotein initially purified from chick brain based on its ability to increase the synthesis of acetylcholine receptor (AChR). We used reverse transcription-polymerase chain reaction (RT-PCR) to obtain a partial pro-ARIA cDNA clone from methonine-1 to serine-358 including the full functional sequence of ARIA. Northern blot analysis of mRNAs from the embryonic chick brain and muscle showed a transcript with a size of approximately 7.5 kb. The cloned cDNA was subcloned into an eukaryotic expression vector and stably transfected into human embryonic kidney 293 cells. The conditioned medium of the transfected cells was found to increase the level of transcript encoding for the alpha-subunit of AChR by approximately 4.4-fold, but not for acetylcholinesterase (AChE), in the cultured chick myotubes.

Acetylcholinesterase↗

PubMed1995

Cloning of the alpha component of the chick ciliary neurotrophic factor receptor: developmental expression and down-regulation in denervated skeletal muscle.

A full-length cDNA clone encoding for the chick CNTFR alpha (alpha component of the ciliary neurotrophic factor receptor) was isolated by screening an embryonic day 13 chick brain cDNA library with a rat CNTFR alpha probe. The isolated cDNA clone contained a approximately 2-kb insert with an open reading frame of 362 amino acids. The identification of this clone as chick CNTFR alpha was based on the homology in amino acid sequence (approximately 70%) with the rat and human CNTFR alpha. Hydropathy analysis revealed that the chick CNTFR alpha contains a hydrophobic region at the amino terminus that is typical of secretory signal peptides, as well as a hydrophobic region at the carboxyl terminus that is characteristic of glycosylphosphatidylinositol-linked proteins. The expression of chick CNTFR alpha was developmentally regulated and was widely distributed in neural tissues, such as brain and spinal cord. In the periphery, chick CNTFR alpha transcript was expressed at high levels in the skeletal muscle and was only barely detectable in the liver. Unexpectedly, the expression of chick CNTFR alpha mRNA in skeletal muscle was decreased by approximately 10-fold at 1.5 days after denervation. This is in sharp contrast to the result previously obtained with CNTFR alpha in denervated rat muscle.

Aging↗

PubMed1992

cDNA that encodes active agrin.

Agrin is thought to mediate the motor neuron-induced aggregation of AChRs and AChE on the surface of muscle fibers at neuromuscular junctions. We have isolated a cDNA from a chick brain library that, based on sequence homology and expression experiments, codes for active agrin. Examination of the sequence reveals considerable similarity to homologous cDNAs previously isolated from ray and rat libraries. A conspicuous difference is an insertion of 33 bp in chick agrin cDNA, which endows the encoded protein with AChR/AChE aggregating activity. Homologous transcripts having the 33 bp insertion were detected in the ray CNS, which indicates that an insertion of similar size is conserved in agrin in many, if not all, vertebrate species. Results of in situ hybridization studies and PCR experiments on mRNA isolated from motor neuron-enriched fractions of the spinal cord indicate that, consistent with the agrin hypothesis, motor neurons contain transcripts that code for active agrin.

Agrin↗

PubMed1992

The agrin gene codes for a family of basal lamina proteins that differ in function and distribution.

We isolated two cDNAs that encode isoforms of agrin, the basal lamina protein that mediates the motor neuron-induced aggregation of acetylcholine receptors on muscle fibers at the neuromuscular junction. Both proteins are the result of alternative splicing of the product of the agrin gene, but unlike agrin, they are inactive in standard acetylcholine receptor aggregation assays. They lack one (agrin-related protein 1) or two (agrin-related protein 2) regions in agrin that are required for its activity. Expression studies provide evidence that both proteins are present in the nervous system and muscle and that, in muscle, myofibers and Schwann cells synthesize the agrin-related proteins while the axon terminals of motor neurons are the sole source of agrin.

Agrin↗

PubMed1992

Transcripts for the acetylcholine receptor and acetylcholine esterase show distribution differences in cultured chick muscle cells.

In situ hybridization of chick cultured muscle cells using exonic DNA probes for both AChR alpha-sub-unit and the catalytic subunit of AChE, revealed major differences in the distribution of label both over nuclei and in their surrounding cytoplasm, although some overlap in these distributions exists. For the AChR alpha-subunit there is a highly skewed distribution of labeled nuclei, with 35% of the nuclei being relatively inactive (less than 0.25 times the mean label) and approximately 10% being very heavily labeled (greater than 2.5 times the mean label). In contrast the nuclei labeled with the exonic probe for the AChE transcripts had a more Gaussian distribution, yet with some slight skewness in the direction of a few heavily labeled nuclei. There was also a difference in the cytoplasmic distribution of the label. The AChR alpha-subunit mRNA was mainly within 4 microns of labeled nuclei while the AChE mRNA was more widely distributed throughout the cytoplasm, possibly within a 10 microns rim around labeled nuclei. An intronic probe for the AChE gave the identical distribution of nuclear label to that of the exonic probe (but without any cytoplasmic label). In addition, calibration of the technique indicated that per myotube the AChE transcript is about sixfold more abundant than the AChR alpha-subunit transcript.

Acetylcholinesterase↗

PubMed1988

Identification of a 17 S asymmetric butyrylcholinesterase in chick muscle by monoclonal antibodies.

A 20 S asymmetric (non-globular) form of acetylcholinesterase (AChE, E.C. 3.1.1.7) has been purified from 1-day chick muscle. This form is a hybrid molecule containing both AChE and butyrylcholinesterase (BuChE, E.C. 3.1.1.8) catalytic subunits, linked through a collagenous tail. However, the 20 S hybrid AChE/BuChE could not account for the total enzyme activities of AChE and BuChE in a high-salt/Triton X-100 extract of 1-day chick muscle. By applying AChE- and BuChE-specific monoclonal antibodies for immunoadsorption, homogeneous asymmetric AChE and BuChE forms were also identified in that extract. The homogeneous BuChE accounts for 20% of the total activity of the asymmetric BuChE present and sediments at 17 S. About 6% of the asymmetric AChE present is, likewise, in a homogeneous, instead of the hybrid, form. The 17 S asymmetric BuChE does not react with monoclonal antibodies specific for the collagenous tail of the hybrid 20 S AChE/BuChE molecule, suggesting that the collagenous subunit differs between these two forms.

Acetylcholinesterase↗

PubMed1988

Synaptic acetylcholinesterase of chicken muscle changes during development from a hybrid to a homogeneous enzyme.

The asymmetric (20S) form of acetylcholinesterase (AChE) in 1-day-old chick muscle is a hybrid enzyme containing both AChE (110 kd) and butyrylcholinesterase (BuChE, 72 kd) catalytic subunits. However, we now report that the asymmetric AChE extracted or immunopurified from older adult chicken muscles, where it is the endplate form, shows a progressive developmental loss of the BuChE subunit and its activities, centred around 4 weeks of age, while the AChE and collagenous subunits remain. In confirmation, using differential labelling and co-sedimentation it was shown that the hybrid 20S AChE/BuChE form of 1-day chick muscle is gradually and completely replaced during muscle maturation by a 21.3S form, also collagen-tailed but otherwise homogeneous in AChE catalytic subunits. Two other changes occur concomitantly. Firstly, the AChE catalytic subunit of the adult form has a lower apparent mol. wt in gel electrophoresis, by 5 kd, than the same subunit in the 1-day hybrid enzyme; this difference does not reside in the carbohydrate attachments. Secondly, the collagen tail changes, in that some conformation-dependent epitopes on it disappear in the same period. Hence, a major reorganization of the asymmetric AChE, involving all three types of subunit, occurs in the course of muscle development.

Acetylcholinesterase↗

PubMed1988

An asymmetric form of muscle acetylcholinesterase contains three subunit types and two enzymic activities in one molecule.

We have purified completely the principal asymmetric ("heavy") form of acetylcholinesterase (Ac-ChoEase; EC 3.1.1.7) from chick muscle (i.e., the synaptic form in the twitch muscle fibers) by using a monoclonal antibody that recognizes AcChoEase but not pseudocholinesterase (ChoEase; cholinesterase, EC 3.1.1.8). The purified protein exhibits catalytic and inhibition properties characteristic of AcChoEase and ChoEase and contains three distinct subunits of apparent sizes 110 kDa, 72 kDa, and 58 kDa in the ratio 2:2:1. The discovery of an AcChoEase/ChoEase hybrid asymmetric form has been further supported by (i) the identification of active site properties of AcChoEase in the 110-kDa subunit and of ChoEase in the 72-kDa subunit, (ii) the purification or precipitation of both activities together by, also, a ChoEase-specific monoclonal antibody, and (iii) evidence that all subunits are bound in the asymmetric forms by disulfide bonds. The 58-kDa subunit is the only one that is sensitive to digestion with purified collagenase; it carries the collagenous "tail" of the asymmetric form. A model is proposed for this form of AcChoEase.

Acetylcholinesterase↗

PubMed1988

Monoclonal antibodies specific for the different subunits of asymmetric acetylcholinesterase from chick muscle.

The asymmetric (20S) acetylcholinesterase (AChE, EC 3.1.1.7) from 1-day-old chick muscle, purified on a column on which was immobilised a monoclonal antibody (mAb) to chick brain AChE, was used to immunise mice. Eight mAbs against the muscle enzyme were hence isolated and characterised. Five antibodies (4A8, 1C1, 10B7, 7G8, and 8H11) recognise a 110-kilodalton (kDa) subunit with AChE catalytic activity, one antibody (7D11) recognises a 72-kDa subunit with pseudocholinesterase or butyrylcholinesterase (BuChE, EC 3.1.1.8) catalytic activity, and two antibodies (6B6 and 7D7) react with the 58-kDa collagenous tail unit. Those three polypeptides can be recognised together in the 20S enzyme used, which is a hybrid AChE/BuChE oligomer. Antibodies 6B6 and 7D7 are specific for asymmetric AChE. Four of the mAbs recognising the 110-kDa subunit were reactive with it in immunoblots. Sucrose density gradient analysis of the antibody-enzyme complexes showed that the anti-110-kDa subunit mAbs cross-link multiple 20S AChE molecules to form large aggregates. In contrast, there is only a 2-3S increase in the sedimentation constant with the mAbs specific for the 72-kDa or for the 58-kDa subunit, suggesting that those subunits are more inaccessible in the structure to intermolecular cross-linking. The 4A8, 10B7, 7D11, and 7D7 mAbs showed cross-reactivity to the corresponding enzyme from quail muscle; however, none of the eight mAbs reacted with either enzyme type from mammalian muscle or from Torpedo electric organ. All eight antibodies showed immunocytochemical localisation of the AChE form at the neuromuscular junctions of chicken twitch muscles.

Acetylcholinesterase↗

PubMed1988

Acetylcholinesterase antibodies and thyroid autoimmunity.

It has been suggested that anti-thyroglobulin antibodies cross-react with acetylcholinesterase (AChE) and that this may explain the pathogenesis of Graves' ophthalmopathy. We have tested this hypothesis using an ELISA. Antibodies to human red blood cell AChE were found in 21% of 47 patients with thyroid autoimmunity. However antibodies to AChE were also detected in one of 25 normal subjects and two of 16 patients with non-organ specific autoimmunity. The anti-AChE antibodies showed no correlation with anti-thyroglobulin antibody levels and they were not associated with the presence of severe ophthalmopathy. Inhibition studies suggested only limited cross reactivity, if any, between anti-Tg and anti-AChE antibodies. Immunoblotting demonstrated antibody binding to at least four human AChE determinants at 130, 55, 32 and 22 kD. Our results demonstrate quite frequent anti-AChE reactivity in sera but no relationship with the development of orbital pathology.

Acetylcholinesterase↗

PubMed1987

Monoclonal antibodies against chicken brain acetylcholinesterase. Their use in immunopurification and immunochemistry to demonstrate allelic variants of the enzyme.

Acetylcholinesterase (AChE) from 1-day chicken brain was enriched over 2000-fold by affinity chromatography using N-methylacridinium-Sepharose. This preparation was used to prepare monoclonal antibodies (mAb) directed against AChE, of which two were extensively characterised for further application. Both mAbs bound to the enzyme from the chicken with high affinity (Kd approximately 8 X 10(-10) M) and one mAb, in addition, recognised AChE from quail brain and muscle. Neither mAb cross-reacted with mammalian or fish AChE. Both mAbs recognised AChE in the endplate region of adult chicken skeletal muscle and bound with equal affinity to the three major oligomeric forms found in early ambryonic muscle. One mAb was used to immunopurify chicken brain AChE to homogeneity (over 12000-fold enrichment), with nearly complete recovery of the enzyme and without detectable proteolytic breakdown. The other mAb recognised AChE after immunoblotting and was used to screen crude brain extracts from individual chickens for allelic variations. Evidence is presented to show that two allelic forms occur, represented in SDS-PAGE by a doublet polypeptide of Mr approximately 110,000, this pattern is maintained after deglycosylation of the N-linked oligosaccharides. This variation was found throughout development and in both the brain and the muscle of individuals. We conclude that the gene encoding the catalytic subunit of chicken AChE is polymorphic with either one or two equally active alleles being expressed.

Acetylcholinesterase↗

PubMed1985

Prolactin receptor in rat liver: sex difference in estrogenic stimulation and imprinting of the responsiveness to estrogen by neonatal androgen in male rats.

Rat hepatic prolactin receptor is regulated by sex steroids. A high level of the receptor was found in female rats but the level was nearly undetectable in males. Gonadectomy reduced the receptor level in females but increased the level in males. Administration of estradiol benzoate (0.05 mumoles/kg on alternate days subcutaneously for 9 days) to adult gonadectomized females increased the receptor level by 473% whereas the same treatment in adult gonadectomized males produced a more modest 276% increase. This sexually dimorphic pattern in the responsiveness to estrogen stimulation in adult rats appeared to be determined neonatally. Neonatal gonadectomy of male rats changed the hepatic response system to a more female pattern in adulthood. Replacement of testosterone (1.45 mumoles at days 1 and 3 after birth) to these neonatally gonadectomized male rats restored the male pattern. Diethylstilbestrol replacement (1.45 mumoles at days 1 and 3 after birth) to the neonatally gonadectomized male rats showed the same effect as neonatally administered testosterone. Scatchard analysis revealed that the observed changes in binding are related to changes in binding capacity but not affinity. Desaturation by 4 M MgCl2 indicated that the amount of endogenously bound hormone was negligible in our membrane preparations.

Animals↗

PubMed1985

The role of neonatal and pubertal gonadal hormones in regulating the sex dependence of the hepatic microsomal testosterone 5-reductase activity in the rat.

Heptic microsomal testosterone 5-reductase activity was approximately fourfold higher in adult female rats than in males. This discrepancy was only partly androgen-dependent since gonadectomy of male rats at 68 days of age resulted in only a partial increase of the enzyme activity. This increase was reversible by the administration of testosterone. Similar treatment, however, produced no effect in the female rat, indicating that there is a sex difference in testosterone responsivity. Castration of newborn male rats resulted in a marked increase in the basal enzyme activity. This increase was not affected by treating the adults with testosterone. Giving testosterone to male rats immediately after neonatal gonadectomy, or to newborn female rats, did not produce the male pattern of both the basal enzyme activity and the testosterone responsivity in adulthood. These results suggest that a brief exposure to neonatal androgen is not critical for the expression of the male type of enzyme activity, but that the continuous presence of the male gonads up to and including the pubertal period is essential. Exposure of pubescent female rats to testosterone during the period from 35 to 50 days of age resulted in a significant increase in testosterone sensitivity when tested at 90 days of age, suggesting that pubertal exposure to androgen is important for the expression of testosterone responsivity in adulthood. The sensitivity was potentiated when the animals were ovariectomized before puberty. Furthermore, the enzyme activity in prepubertally ovariectomized female rats was significantly lower than that in adult gonadectomized animals.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1984

Evidence for the involvement of essential sulphydryl group in rat hepatic lactogenic receptor but not in somatogenic receptor.

Incorporation of p-chloromercuribenzene sulphonate (PCMBS) (1mM) in the assay medium for rat hepatic lactogenic receptor produced complete inhibition of binding of [125I]oPRL to the membrane. However, the presence of the thiol-reactive agent produced no effect on the binding of [125I]bGH to rat hepatic somatogenic receptor. Pretreatment of rat hepatic membrane with PCMBS inhibited the binding of [125I]oPRL but not that of [125I]bGH. The lactogenic receptor binding inhibition by PCMBS pretreatment was both concentration- and time-dependent, with complete inhibition at 0.5 mM for 60 min at 0 degrees C. Scatchard analysis of [125I]oPRL binding to membrane at 50% inhibition by PCMBS (0.11 mM) revealed that the binding capacity was decreased rather than the binding affinity. Furthermore, the inhibition of lactogenic receptor binding by PCMBS could be reversed by dithioerythritol (DTE) treatment. Following 80% inhibition by 0.2 mM PCMBS, full recovery of receptor binding was achieved at 6 mM DTE for 60 min at 0 degrees C. The 'recovered' membrane showed no difference from the control membrane in terms of binding capacity and affinity.

4-Chloromercuribenzenesulfonate↗

PubMed1984

Rat hepatic prolactin receptor: pubertal exposure to sex steroids alters responsivity to testosterone.

Administration of testosterone enanthate to adult female rats lowered the level of hepatic prolactin receptor. Pubertal exposure of intact female rats to testosterone enanthate at the dose used did not affect the level of hepatic prolactin receptor in adults. However, the responsivity of such treated rats to testosterone challenge in adulthood was enhanced. Prepubertal ovariectomy of female rats lowered the level of hepatic prolactin receptor in adulthood. Exposure of the ovariectomized rats during puberty to mestranol (ethynylestradiol 3-methyl ether) at the dose used did not restore the normal adult female rat's level of hepatic prolactin receptor. However, they did become more resistant to subsequent testosterone challenge in adulthood. Exposure of prepubescent ovariectomized rats to testosterone enanthate during puberty reduced the already diminished level of hepatic prolactin receptor further. In addition, these rats were rendered more responsive to testosterone challenge in adulthood.

Aging↗

PubMed1984

Pubertal gonadal hormones in modulating the testosterone dependency of hepatic aryl hydrocarbon hydroxylase in female rats.

The responsiveness of the hepatic microsomal aryl hydrocarbon hydroxylase (AHH) to testosterone enanthate (TE; 2.5 mumol/kg/day for 9 days) was sex-dependent in adult rats, the enzyme being very resistant to TE in normal adult or ovariectomized females. Administration of testosterone propionate or diethylstilbestrol (1.45 mumol) to neonatal female rats at 1 and 3 days of age did not increase the responsivity to TE in adulthood. However, exposure of female rats to TE (5.0 mumol/kg/day) during the peripubertal period (35-50 days old) resulted in increased sensitivity to TE (+55.2%) when tested in adulthood. The responsivity was further potentiated (+109.3%) if the animals were ovariectomized at 28 days of age. Prepubescent ovariectomized females which received corn oil or estradiol benzoate (1.5 mumol/kg on alternate days) during puberty were not able to respond to TE significantly. These results suggest that the refractoriness of the hepatic AHH to testosterone in adult female rats is determined by the absence of testosterone, as well as the presence of estrogens, during puberty.

Animals↗

PubMedDate not supplied

Prenatal cocaine exposure revealed minimal postnatal changes in rat striatal dopamine D2 receptor sites and mRNA levels in the offspring.

It has been reported from this laboratory that prenatal cocaine exposure results in the postnatal transient alterations of rat striatal dopamine uptake sites examined from postnatal 0-32 wk. The present study aims to examine whether this will result in a direct/indirect stimulation of dopamine D2 receptors. Pregnant rats were dosed orally with cocaine hydrochloride (60 mg/kg/d) from gestational day (GD) 7-21. Control animals received an equivalent volume of water. The striatum from the offspring at postnatal 0-32 wk was examined. The radioligand [3H]sulpiride was used for the Scatchard analysis of the D2 receptors, and the changes in the levels of mRNA for the D2 receptor were studied using Northern blot analysis. Results from the present study revealed that in the control group, there was an age-dependent increase in the number of D2 receptor sites (Bmax: 44.00 +/- 2.12 to 178.00 +/- 45.10 fmol/mg protein) and in the levels of D2 mRNA from PN0-32 wk with the most rapid increase occurring during the first 4 wk of postnatal development. Prenatal cocaine exposure resulting in only a significant decrease (p < 0.001) in the number of D2 receptor sites at PN0 wk and in a 10% increase in mRNA levels at PN3, 4, and 12 wk. It was concluded from this study that prenatal cocaine exposure resulted in minimal postnatal changes in the dopamine D2 receptor.

Aging↗

PubMedDate not supplied

Immunoglobulin G1 Fc fragment-tagged human opioid receptor-like receptor retains the ability to inhibit cAMP accumulation.

The human opioid receptor-like (ORL(1)) receptor was tagged with the immunoglobulin G1 Fc fragment at the carboxy-terminus and expressed in human embryonic kidney 293 cells. Expression of the ORL(1)-Fc receptor was confirmed by immunohistochemical staining. The fusion protein was enriched by affinity chromatography and then verified by immunodetection. The function of the ORL(1)-Fc receptor was determined by examining nociceptin/OFQ-induced inhibition of cAMP accumulation. The ORL(1)-Fc receptor inhibited the forskolin-stimulated cAMP accumulation. The inhibitory response was selectively induced by nociceptin/OFQ in a dose-dependent and pertussis toxin-sensitive manner. Our results indicate that the carboxy-terminal Fc-tagged ORL(1) receptor retained the ability to interact with G(i) proteins to inhibit adenylyl cyclase.

Adenylate Cyclase Toxin↗