Biomedical subjects

K C Leung

Publications and source records attributed to K C Leung.

At least 19 recordsLinked to original sources

PubMed2006

A two-stage refinement approach for the enhancement of excretory production of an exoglucanase from Escherichia coli.

Hyper-expression of a secretory exoglucanase, Exg, encoded by the cex gene of Cellulomonas fimi was previously shown to saturate the SecYEG pathway and result in dramatic cell death of recombinant Escherichia coli (Z.B. Fu, K.L. Ng, T.L. Lam, W.K.R. Wong, Cell death caused by hyper-expression of a secretory exoglucanase in Esherichia coli, Protein Expr. Purif. 42 (2005) 67-77). We propose here that the cell lysate ratio (Pre/Mat RQ) of the unprocessed precursor Exg protein (Pre-Exg) and its processed mature product (Mat-Exg) reflects the capacity of E. coli to secrete Exg. A Pre/Mat RQ of 20/80, designated the "Critical Value," was an important threshold measurement. A rise in the Pre/Mat RQ triggered a mass killing effect. The use of various secretion signal peptides did not improve the viability of cells expressing high levels of Pre-Exg under strong tac promoter control. However, use of the weaker vegG promoter in conjunction with a change in start codon of the spa leader sequence from ATG to TTG in a pM1vegGcexL plasmid construct resulted in a high level (0.9 U ml(-1)) of excreted Exg in shake-flask cultures. This was 50% higher than the best result obtained from plasmid construct lacUV5par8cex, using the lacUV5 promoter and the ompA leader sequence. Variations in the excreted Exg activities were attributable to differences in the Pre/Mat RQ values of the induced cultures harboring pM1vegGcexL and lacUV5par8cex. These values were 18/82 and 10/90, respectively. Employing fed-batch cultivation in two-liter fermentors, an induced JM101(pM1vegGcexL) culture yielded 4.5 U ml(-1) of excreted Exg, which was over six fold greater that previously reported. Our results illustrate the successful application of the Pre/Mat RQ ratio as a guide to the attainment of a maximum level of secreted/excreted Exg.

Cell Death↗

PubMed2003

Estrogen inhibits GH signaling by suppressing GH-induced JAK2 phosphorylation, an effect mediated by SOCS-2.

Oral estrogen administration attenuates the metabolic action of growth hormone (GH) in humans. To investigate the mechanism involved, we studied the effects of estrogen on GH signaling through Janus kinase (JAK)2 and the signal transducers and activators of transcription (STATs) in HEK293 cells stably expressing the GH receptor (293GHR), HuH7 (hepatoma) and T-47D (breast cancer) cells. 293GHR cells were transiently transfected with an estrogen receptor-alpha expression plasmid and luciferase reporters with binding elements for STAT3 and STAT5 or the beta-casein promoter. GH stimulated the reporter activities by four- to sixfold. Cotreatment with 17beta-estradiol (E(2)) resulted in a dose-dependent reduction in the response of all three reporters to GH to a maximum of 49-66% of control at 100 nM (P < 0.05). No reduction was seen when E(2) was added 1-2 h after GH treatment. Similar inhibitory effects were observed in HuH7 and T-47D cells. E(2) suppressed GH-induced JAK2 phosphorylation, an effect attenuated by actinomycin D, suggesting a requirement for gene expression. Next, we investigated the role of the suppressors of cytokine signaling (SOCS) in E(2) inhibition. E(2) increased the mRNA abundance of SOCS-2 but not SOCS-1 and SOCS-3 in HEK293 cells. The inhibitory effect of E(2) was absent in cells lacking SOCS-2 but not in those lacking SOCS-1 and SOCS-3. In conclusion, estrogen inhibits GH signaling, an action mediated by SOCS-2. This paper provides evidence for regulatory interaction between a sex steroid and the GHJAKSTAT pathway, in which SOCS-2 plays a central mechanistic role.

Blotting, Western↗

PubMed2003

Metabolic effects of oestrogens: impact of the route of administration.

To investigate whether oestrogen modulates GH secretion and action in adult life, we studied the impact of oestrogen replacement on circulating GH and IGF-I levels in post-menopausal women. Since the liver is the major source of circulating IGF-I and the oral route of oestrogen delivery causes non-physiologic effects on hepatic proteins, we compared the effects of oral and transdermal route of delivery. Oral ethinyl oestradiol administration resulted in a significant fall in mean IGF-I levels and a 3-fold increase in mean 24h GH. Transdermal administration of 17beta oestradiol resulted in a slight increase in serum IGF-I but no change in mean 24h GH levels. To determine whether differences in oestrogen type rather than in the route of delivery caused the different effects on the GH/IGF-I axis, we compared the effects of three oral oestrogen formulations. Ethinyl oestradiol, conjugated equine oestrogen and oestradiol valerate each induced a fall in IGF-I and a rise of mean 24h GH levels in post-menopausal women. To determine the metabolic significance of oestrogen-induced changes on GH/ IGF-I, we compared the effects of 24 weeks each of oral and transdermal oestrogen on energy metabolism and body composition in 18 post menopausal women in an open-label randomised cross-over study. When compared to the transdermal route, oral oestrogen reduced lipid oxidation, increased fat mass and reduced lean body mass. Oestrogen causes distinct, route dependent effects on the somatotrophic axis. The dissociation of the GH/IGF-I axis by the oral route is likely to arise from impaired hepatic IGF-I production which causes increased GH secretion through reduced feedback inhibition. The route of oestrogen therapy confers divergent effects on substrate oxidation and body composition. The suppression of lipid oxidation during oral oestrogen therapy may increase fat mass while the fall in IGF-I may lead to a loss of lean body mass. The route dependent changes in body composition observed during oestrogen replacement therapy may have important implications for post-menopausal health and oestrogen use in general.

Blood Glucose↗

PubMed2001

The emotional effects of tooth loss in partially dentate people attending prosthodontic clinics in dental schools in England, Scotland and Hong Kong: a preliminary investigation.

AIM: To compare the emotional effects of tooth loss in three partially dentate populations. DESIGN: A questionnaire survey. MATERIALS AND METHODS: A questionnaire was completed by 150 partially dentate subjects undergoing routine prosthodontic care at Guy's, King's and St Thomas's Dental Institute, London; the Dental School, Dundee, Scotland; and the Faculty of Dentistry, University of Hong Kong. Data were analysed using the Chi-square test. RESULTS: Forty nine per cent of all participants reported difficulties in accepting the loss of some of their teeth. People from Dundee were less likely to have difficulties accepting tooth loss (P = 0.001). People from London took longer to come to terms with their tooth loss and were more likely to feel less confident (P < 0.001). Fifty five per cent of all participants restricted their choice of foods and 54 per cent had not enjoyed their food as much as before. Fewer people in Dundee restricted their choice of food (P < 0.001) and were more likely to enjoy their food (P = 0.009). People in Hong Kong were most likely to restrict their choice of food (P = 0.006). Thirty five percent of all subjects felt unprepared for the effects that tooth loss had upon them. People in Hong Kong were more prepared for tooth loss than those in Dundee and London (P = 0.003). In addition, they were less concerned about leaving their dentures out overnight (P = 0.024). CONCLUSIONS: The emotional effects of tooth loss were significant in all groups. People from London took longer to come to terms with their tooth loss.

Attitude to Health↗

PubMed2001

Oral stereognosis in stroke and Parkinson's disease: a comparison of partially dentate and edentulous individuals.

Oral stereognosis was measured in partially dentate and edentulous patients with stroke, Parkinson's disease, and an age and gender-matched control group. Stereognostic tests involving conventional free intra-oral manipulation of test objects were undertaken in the partially dentate and edentulous with and without complete dentures. Comparisons were made using the unpaired t-test and ANOVA. Edentulous stroke patients without dentures had significantly greater error scores and fewer correct identifications compared with partially dentate stroke patients. Stereognostic measures were similar in the partially dentate and edentulous with dentures, within experimental groups. In the partially dentate, there were no differences in stereognostic measures between the three groups. Stereognostic measures were poorer in edentulous stroke patients with and without dentures compared with the edentulous control group. Partially dentate stroke patients are less likely to have impaired oral stereognosis than edentulous stroke patients.

Aged↗

PubMed2001

Measurement of growth hormone, insulin-like growth factor I and their binding proteins: the clinical aspects.

BACKGROUND: Growth hormone (GH) secreted from the pituitary stimulates the production of insulin-like growth factor I (IGF-I) from the liver and extrahepatic tissues, which in turn regulates tissue proliferation and differentiation in an endocrine or autocrine/paracrine manner. Both GH and IGF-I circulates as complexes with specific binding proteins. The GH binding protein (GHBP) corresponds to the extracellular, ligand-binding domain of the GH receptors in tissues and its serum concentration may reflect the status of the tissue receptors. Most serum IGF-I associates with IGF binding protein 3 (IGFBP-3) and another protein, the acid labile subunit (ALS). Like IGF-I, serum concentrations of IGFBP-3 and ALS are tightly regulated by GH. GH secretion (both spontaneous and stimulated), IGF-I, IGFBP-3, and ALS have been assessed as potential biochemical markers for diagnosis of GH-related disorders. CONCLUSIONS: In acromegaly, IGF-I is the most reliable marker. The peak GH response to insulin tolerance test is the diagnostic test of choice, GH deficiency. GHBP has no diagnostic value in acromegaly or GH deficiency. However, it may be a potential biochemical marker for GH insensitivity syndrome as serum GHBP concentrations are undetectable or reduced in >75% of these patients. Other biochemical tests may also prove to be useful in these disorders, but require further validation.

Adult↗

PubMed2001

Binding and functional studies with the growth hormone receptor antagonist, B2036-PEG (pegvisomant), reveal effects of pegylation and evidence that it binds to a receptor dimer.

GH actions are dependent on receptor dimerization. The GH receptor antagonist, B2036-PEG, has been developed for treating acromegaly. B2036 has mutations in site 1 to enhance receptor binding and in site 2 to block receptor dimerization. Pegylation (B2036-PEG) increases half-life and lowers immunogenicity, but high concentrations are required to control insulin-like growth factor-I levels. We examined antagonist structure and function and the impact of pegylation on biological efficacy. Unpegylated B2036 had a 4.5-fold greater affinity for GH binding protein (GHBP) than GH but similar affinity for membrane receptor. Pegylation substantially reduced membrane binding affinity and receptor antagonism, as assessed by a transcription assay, by 39- and 20-fold, respectively. GHBP reduced antagonist activity of unpegylated B2036 but did not effect antagonism by B2036-PEG. B2036 down-regulated receptors, and membrane binding sites doubled in the presence of dimerization-blocking antibodies, suggesting that B2036 binds to a receptor dimer. It is concluded that the high concentration requirement of B2036-PEG for clinical efficacy relates to pegylation, which decreases binding to membrane receptor but has the advantages of reduced clearance, immunogenicity, and interactions with GHBP. Our studies suggest that B2036 binds to a receptor dimer and induces internalization but not signaling.

Carrier Proteins↗

PubMed2001

Prevalence of symptoms associated with temporomandibular disorders in Hong Kong Chinese.

AIMS: To estimate the prevalence of self-reported symptoms associated with temporomandibular disorders (TMD) and treatment-seeking in adult Chinese in Hong Kong. METHODS: A telephone survey technique was used to identify 1,526 randomly selected Cantonese-speaking individuals aged 18 years or over. Standard questions were asked about joint and jaw muscle pain, jaw opening, and joint sounds. In addition, questions on tooth grinding and clenching, sleep patterns, and treatment-seeking behavior were posed. RESULTS: Jaw pain was reported by 33% of the population. Only 5% of them had frequent pain, with two thirds of this subgroup having moderate to severe symptoms. The prevalence of frequent problems with jaw opening and joint clicking was 0.3% and 1.8%, respectively. There were no gender-related differences in the reporting of TMD symptoms or related conditions. CONCLUSION: One percent of the Hong Kong Chinese population had TMD-related jaw pain that was of moderate or severe intensity and occurred frequently; 0.6% of the population had sought treatment for jaw pain, impaired jaw opening, or joint clicking that occurred often in the previous year.

Adolescent↗

PubMed2000

Investigation of a ponding irrigation system to recycle agricultural wastewater.

This article presents the results of natural carrying capacity of ponding irrigation system in Taoyuan agricultural zone, Taiwan. Both the systematic water quality and the ponding effects were examined. The ponding irrigation system included a flow channel and storage ponds. The data showed that most water characteristics deteriorated gradually from upper- to down-stream in the flow channel and the flow channel was not attributed to any self-purification in agricultural returning water practically. On the other hand, the results of storage ponds indicated that they can provide a natural treatment (i.e., the outlet water quality of the ponds is more desirable than that of the inlet). Consequently, the ponding irrigation system offers the natural self-purification in ponds to reuse and recycle the returning agricultural wastewater and to extend the irrigation capacity and efficiency.

Agriculture↗

PubMed2000

Characterization of a low affinity binding protein for growth hormone in rat serum.

GH forms a high Mr complex in rat serum distinct from that with GH-binding protein (GHBP). The present study investigates the nature of this complex. When subjected to AcA44 filtration chromatography, 125I-labeled human GH (hGH) in rat serum eluted in four peaks. Peak 1 eluted at the void volume, whereas peaks 2, 3, and 4 corresponded to the GHBP complex, free hGH, and iodide, respectively. Stripping of GHBP in serum by immunoaffinity chromatography depleted peak 2 but did not affect peak 1. Peak 1 accounted for 11.4 +/- 1.2% of the total radioactivity (mean +/- SEM; n = 6) in stripped serum. Addition of unlabeled hGH (0.9-9 microM) demonstrated the binding of [125I]hGH to be specific, with Scatchard analysis revealing an affinity of 0.88 +/- 0.03 x 10(5) M(-1)(n = 3)and a capacity of 2.46 +/- 0.14 microM. Sepharose CL-6B filtration chromatography showed the complex to be 260 kDa in size. The distribution of GH binding to GHBP and this high Mr serum factor was investigated by incubating [125I]hGH in sera containing a low (5 nM) and a high (35 nM) concentration of GHBP over a range of physiological GH concentrations. In sera containing a low concentration of GHBP, the proportion of GH complexed in peak 1 increased with increasing GH concentrations. In sera with a high concentration of GHBP, GH was complexed mainly in peak 2. Studies with normal rat sera revealed that more GH was complexed in peak 1 in male than in female rats (3.4 +/- 0.4% and 1.4 +/- 0.1%, respectively; P < 0.006), in contrast to that of peak 2 (1.1 +/- 0.2% and 7.6 +/- 0.4%, respectively; P < 0.002). In summary, we provide strong evidence for the existence of a factor in rat serum that binds GH with low affinity and high capacity. It has a Mr of approximately 240 kDa, assuming a 1:1 binding stoichiometry, and is immunologically distinct from GHBP. This factor may provide supplementary capacity for GH binding when binding to GHBP is saturated.

Algorithms↗

PubMed2000

Insulin regulation of human hepatic growth hormone receptors: divergent effects on biosynthesis and surface translocation.

Insulin modulates the biological actions of GH, but little is known about its effect on human hepatic GH receptors (GHRs). Using the human hepatoma cell line HuH7 as a model, we investigated insulin regulation of total, intracellular, and cell surface GHRs and receptor biosynthesis and turnover. Insulin up-regulated total and intracellular GHRs in a concentration-dependent manner. It increased surface GHRs in a biphasic manner, with a peak response at 10 nmol/L, and modulated GH-induced Janus kinase-2 phosphorylation in parallel with expression of surface GHRs. The abundance of GHR messenger ribonucleic acid and protein, as assessed by RT-PCR and Western analysis, respectively, markedly increased with insulin treatment. To examine whether insulin regulates GHRs at the posttranslational level, its effects on receptor surface translocation and internalization were investigated. Insulin suppressed surface translocation in a concentration-dependent manner, whereas internalization was unaffected. Moreover, insulin actions on total GHRs and surface translocation were inhibited by PD98059 and wortmannin, respectively. In conclusion, insulin regulates hepatic GHR biosynthesis and surface translocation in a reciprocal manner, with surface receptor availability the net result of the divergent effects. The divergent actions of insulin appear to be mediated by the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways, respectively.

Androstadienes↗

PubMed2000

Placental growth hormone (GH), GH-binding protein, and insulin-like growth factor axis in normal, growth-retarded, and diabetic pregnancies: correlations with fetal growth.

We previously described significant changes in GH-binding protein (GHBP) in pathological human pregnancy. There was a substantial elevation of GHBP in cases ofnoninsulin-dependent diabetes mellitus and a reduction in insulin-dependent diabetes mellitus. GHBP has the potential to modulate the proportion of free placental GH (PGH) and hence the impact on the maternal GH/insulin-like growth factor I (IGF-I) axis, fetal growth, and maternal glycemic status. The present study was undertaken to investigate the relationship among glycemia, GHBP, and PGH during pregnancy and to assess the impact of GHBP on the concentration of free PGH. We have extended the analysis of specimens to include measurements of GHBP, PGH, IGF-I, IGF-II, IGF-binding protein-1 (IGFBP-1), IGFBP-2, and IGFBP-3 and have related these to maternal characteristics, fetal growth, and glycemia. The simultaneous measurement of GHBP and PGH has for the first time allowed calculation of the free component of PGH and correlation of the free component to indexes of fetal growth and other endocrine markers. PGH, free PGH, IGF-I, and IGF-II were substantially decreased in IUGR at 28-30 weeks gestation (K28) and 36-38 weeks gestation (K36). The mean concentration (+/-SEM) of total PGH increased significantly from K28 to K36 (30.0 +/- 2.2 to 50.7 +/- 6.2 ng/mL; n = 40), as did the concentration of free PGH (23.4 +/- 2.3 to 43.7 +/- 6.0 ng/mL; n = 38). The mean percentage of free PGH was significantly less in IUGR than in normal subjects (67% vs. 79%; P < 0.01). Macrosomia was associated with an increase in these parameters that did not reach statistical significance. Multiple regression analysis revealed that PGH/IGF-I and IGFBP-3 account for 40% of the variance in birth weight. IGFBP-3 showed a significant correlation with IGF-I, IGF-II, and free and total PGH at K28 and K36. Noninsulin-dependent diabetes mellitus patients had a lower mean percentage of free PGH (65%; P < 0.01), and insulin-dependent diabetics had a higher mean percentage of free PGH (87%; P < 0.01) than normal subjects. Mean postprandial glucose at K28 correlated positively with PGH and free PGH (consistent with the hyperglycemic action of GH). GHBP correlated negatively with both postprandial and fasting glucose. Although GHBP correlated negatively with PGH (r = -0.52; P < .001), free PGH and total PGH correlated very closely (r = 0.98). The results are consistent with an inhibitory function for GHBP in vivo and support a critical role for placental GH and IGF-I in driving normal fetal growth.

Adult↗

PubMed2000

Estrogens exert route- and dose-dependent effects on insulin-like growth factor (IGF)-binding protein-3 and the acid-labile subunit of the IGF ternary complex.

We have previously shown that exogenous estrogens exert route-dependent effects on serum GH and insulin-like growth factor I (IGF-I) levels. IGF-I circulates as a ternary complex with IGF-binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS). It is not known whether IGFBP-3 and ALS in blood are regulated by estrogen and, if so, whether this is also route dependent. In the present study we investigate the effects on IGFBP-3 and ALS of oral and transdermal estrogens (study 1), of different oral estrogen formulations (ethinyl estradiol, conjugated estrogen, and estradiol valerate; study 2), of different estrogen dosages (study 3) in normal postmenopausal women, and of oral estrogen in hypogonadal GH-deficient women (study 4). Administration of oral, but not transdermal, estrogen in normal postmenopausal women significantly decreased serum levels of IGFBP-3 and ALS (P < or = 0.005). The suppressive effects were similar with different oral estrogen formulations, and the degree of suppression increased with estrogen dosage. In hypogonadal GH-deficient women, oral estrogen treatment also significantly reduced IGFBP-3 and ALS (P = 0.02). The changes in IGF-I in each of the four studies paralleled the changes in both IGFBP-3 and ALS. In conclusion, exogenous estrogens suppress serum IGFBP-3 and ALS in a route- and dose-dependent manner, which are in parallel with the effects on serum IGF-I. These actions of oral estrogen are independent of endogenous GH status.

Administration, Cutaneous↗

PubMed2000

Distribution and abundance of messenger ribonucleic acid for growth hormone receptor isoforms in human tissues.

Two alternatively spliced exon 9 variants of human GH receptor (GHR) messenger ribonucleic acid (mRNA), GHR-(1-279) and GHR(1-277), were recently identified in liver. They encode receptor proteins lacking most of the intracellular domain and inhibit GH action in a dominant negative manner. Little is known about tissue distribution and abundance of these GHR isoforms. We have developed quantitative RT-PCR assays specific for the full-length and truncated GHRs and investigated their expression in various human tissues and cell lines. The mRNA of full-length GHR and GHR-(1-279) were readily detectable in all tissues investigated, with liver, fat, muscle, and kidney showing high levels of expression. These two receptor isoforms were also detected in a range of human cell lines, with strongest expression in IM9, a lymphoblastoid cell line. In contrast, GHR-(1277) message was expressed at low levels in liver, fat, muscle, kidney, and prostate and in trace amount in IM9 cells. Full-length GHR was the most abundant isoform, accounting for over 90% of total receptor transcripts in liver, fat, and muscle for quantitative RT-PCR. However, liver had 2- to 4-fold more full-length receptor mRNA and 16- to 40-fold more GHR-(1-277) mRNA than fat and muscle, whereas the mRNA levels of GHR-(1-279) were similar in the three tissues. GHR-(1-279) constituted less than 4% in liver and 7-10% in fat and muscle. GHR-(1-277) accounted for 0.5% of total GHR transcripts in liver and less than 0.1% in the other two tissues. These data suggest that the absolute and relative abundance of mRNA of the three GHR isoforms may be tissue specific. The regulation of expression of exon 9 alternatively spliced GHR variants may provide a potential mechanism for modulation of GH sensitivity at the tissue level.

Adipose Tissue↗

PubMed1999

Effect of adhesive drying time on the bond strength of irreversible hydrocolloid to stainless steel.

STATEMENT OF PROBLEM: The efficacy of adhesive material is believed to be affected by the drying time allowed. However, there is no universal consensus on the drying time required for adhesive to achieve optimal bond strength for irreversible hydrocolloid impressions. PURPOSE: This study investigated the effect of drying time on the cleavage bond strength of irreversible hydrocolloid adhesive and determined optimal adhesive drying time. MATERIAL AND METHODS: In the cleavage test, the adhesive bond was stressed at the edge. An offset tensile force was applied to 1 side of the testing plates to split the bond. Cleavage testing plates were designed and machined in stainless steel. Adhesive material (Fix) was applied thinly to the plates and allowed to dry for 1, 3, 5, 7.5, 10, and 20 minutes before loading with an irreversible hydrocolloid (Blueprint). The irreversible hydrocolloid was then allowed to set for 5 minutes before testing. Twenty specimens were tested for each selected drying time. The force at failure was measured by an Instron machine with a crosshead speed of 50 mm/min applied to the edge of the bond. A nonadhesive group was included as a control. RESULTS: The calculated cleavage bond strength at 1-minute drying (32 kPa) improved moderately up to 5-minute drying (37 kPa), but decreased on further drying. When compared with specimens in the nonadhesive group, cleavage bond strength was 3 times greater when the adhesive was applied and dried for 5 minutes. CONCLUSIONS: Adhesive is effective in improving the cleavage bond strength of irreversible hydrocolloid to stainless steel, and drying of adhesive in excess of 5 minutes is not recommended.

2-Propanol↗

PubMed1998

Tensile, shear and cleavage bond strengths of alginate adhesive.

OBJECTIVES: This study determined the effect of alginate adhesive on various bond strengths of alginate to stainless steel. METHODS: Three test assemblies were designed and machined in stainless steel for tension, shear and cleavage tests. Alginate adhesive (Fix) was applied thinly and dried for 5 min. Alginate (Blueprint) was then loaded and allowed to set for 5 min before testing. The force at failure was measured by an Instron machine with a cross-head speed of 50 mm min(-1). RESULTS: Breaking stresses of alginate without adhesive were found to be 65 kPa (tension), 31 kPa (shear) and 10 kPa (cleavage). The bond strengths of Blueprint with Fix were 100 kPa (tension), 42 kPa (shear) and 37 kPa (cleavage) giving improvements of 53%, 37% and 270% respectively (p < 0.05). CONCLUSIONS: Alginate adhesive increases the bond strength of alginate, particularly cleavage, to stainless steel.

Adhesives↗

PubMed1997

Antigenic determination fragments of alpha-momorcharin.

Alpha momorcharin is a protein isolated from the bitter gourd. It has a number of biological activities including induction of abortion, inhibition of tumor growth and anti-HIV. All these activities may be related to the ribosome-inhibiting activity of the protein. Repeated use of alphaMMC can elicit an antigenic response which may neutralize its biological activity. To overcome this problem, we need to know which part of the molecule is the antigenic determinant. In this study, we constructed a random fragment expression library from the alphaMMC cDNA and screened it with three anti-alphaMMC sera. A total of 9 positive clones were picked and sequenced. Based on the sequence information obtained, we were able to deduce three regions at which antibodies raised against native alphaMMC seem to interact. These regions are residues 1-14, residues 71-136 and residues 195-222. Mapping of these regions against a 3D model of alphaMMC indicates that they all are located on the surface of the molecule. As residues 71-136 are found to be in close proximity to the active site involved in ribosome inactivation, treatment with a monoclonal antibody directed to this area was shown to be effective in inactivating the inhibitory effect of alphaMMC on in vitro protein synthesis.

Abortifacient Agents, Nonsteroidal↗

PubMed1997

Insulin and insulin-like growth factor-I acutely inhibit surface translocation of growth hormone receptors in osteoblasts: a novel mechanism of growth hormone receptor regulation.

We previously have demonstrated that insulin and insulin-like growth factor-I (IGF-I) down-regulate growth hormone (GH) binding in osteoblasts by reducing the number of surface GH receptors (GHRs). The present study was undertaken to investigate the mechanism of GHR down-regulation. Treatment with 5 nM insulin or IGF-I for 18 hr significantly decreased surface GH binding to 26.4 +/- 2.9% and 23.0 +/- 2.7% of control (mean +/- SE; P < 0.05), respectively. No corresponding reductions in the mRNA level and total cellular content of GHR were found, nor was the rate of receptor internalization affected. The effects on GHR translocation were assessed by measuring the reappearance of GH binding of whole cells after trypsinization to remove the surface receptors. GH binding of control cultures significantly increased (P < 0.05) over 2 hr after trypsinization, whereas no recovery of binding activity was detected in insulin and IGF-I-treated cultures, indicating that GHR translocation was impaired. Studies on the time course of GHR down-regulation revealed that surface GH binding was reduced significantly by 3-hr treatment (P </= 0.0005), whereas GHR translocation was completely abolished by 75-90 min with insulin and IGF-I. The inhibition of receptor translocation by insulin, but not IGF-I, was attenuated by wortmannin. In conclusion, insulin and IGF-I down-regulated GH binding in osteoblasts by acutely impairing GHR translocation, with their effects exerted through distinct postreceptor signaling pathways.

Androstadienes↗