Biomedical subjects
Publications and source records attributed to M H Lam.
Polyps of the gall bladder are uncommon conditions in children. We present a case report of a 14-year-old girl who had calculous cholecystitis and an adenomatous hyperplastic polyp of the gall bladder. She was treated by laparoscopic cholecystectomy.
OBJECTIVE: To investigate the effectiveness of misoprostol given vaginally for cervical priming before hysteroscopy in postmenopausal women. DESIGN: Double-blind randomised controlled study. SETTING: Regional hospital, Hong Kong. PARTICIPANTS: One hundred women with postmenopausal bleeding scheduled for hysteroscopy from October 1998 to September 2001 were randomly assigned to receive either misoprostol or placebo vaginally before the operation. MAIN OUTCOME MEASURES: The number of women requiring cervical dilatation, outcome of hysteroscopy and side effects of the medication were assessed. RESULTS: Forty-eight women receiving misoprostol and 48 women receiving placebo were compared. The mean degree of endocervical diameter estimated by Hegar's dilator was similar between the treatment group and the control group. A similar number of women in the treatment group and the control group required cervical dilatation. The operative times for both groups were similar. The incidence of side effects was comparable in both groups. The most common side effects for misoprostol were febrile episodes and diarrhoea. There was no cervical tear nor uterine perforation encountered in both groups. The mean duration of hospital stay in both groups were similar. Subanalysis of results were similar in women receiving vaginal medication at least five hours before the operation. CONCLUSION: Vaginal misoprostol was not shown to reduce the need for cervical dilatation in postmenopausal women. It cannot convert diagnostic hysteroscopy from a hospital procedure into an office one in postmenopausal women with tight cervical os.
Specific cellular stresses, including hyperosmotic stress, caused a dramatic but reversible cytoplasmic accumulation of the otherwise nuclear 45-kDa variant of the protein-tyrosine phosphatase TCPTP (TC45). In the cytoplasm, TC45 dephosphorylated the epidermal growth factor receptor and down-regulated the hyperosmotic stress-induced activation of the c-Jun N-terminal kinase. The hyperosmotic stress-induced nuclear exit of TC45 was not inhibited by leptomycin B, indicating that TC45 nuclear exit was independent of the exportin CRM-1. Moreover, hyperosmotic stress did not induce the cytoplasmic accumulation of a green fluorescent protein-TC45 fusion protein that was too large to diffuse across the nuclear pore. Our results indicate that TC45 nuclear exit may occur by passive diffusion and that cellular stress may induce the cytoplasmic accumulation of TC45 by inhibiting nuclear import. Neither p42(Erk2) nor the stress-activated c-Jun N-terminal kinase or p38 mediated the stress-induced redistribution of TC45. We found that only those stresses that stimulated the metabolic stress-sensing enzyme AMP-activated protein kinase (AMPK) induced the redistribution of TC45. In addition, specific pharmacological activation of the AMPK was sufficient to cause the accumulation of TC45 in the cytoplasm. Our studies indicate that specific stress-activated signaling pathways that involve the AMPK can alter the nucleocytoplasmic distribution of TC45 and thus regulate TC45 function in vivo.
Although the specific role of transcription factors (TFs) is nuclear, surprisingly little is known in quantitative terms regarding the pathways by which TFs localize in the nucleus. In this study, we use direct binding assays, native gel electrophoresis, and fluorescence polarization measurements to show for the first time that the cAMP-response element binding protein (CREB) and related AP-1 and jun and fos constituents are recognized by importin beta1 (Impbeta) with nanomolar affinity. We reconstitute the nuclear import of these TFs in vitro, demonstrating dependence on cytosolic factors, and show that this is due to the requirement for Impbeta, since antibodies to Impbeta, but not to importin alpha (Impalpha), inhibit nuclear accumulation significantly. We show that Impbeta is necessary and sufficient for docking of CREB at the nuclear envelope; that Ran is essential for CREB nuclear import is demonstrated by the reduction of nuclear accumulation effected by RanGTPgammaS but not RanGDP, and by dissociation of the Impbeta-CREB-GFP complex by RanGTPgammaS but not RanGDP as demonstrated using fluorescence polarization assays. The results support the existence of an Impbeta1- and Ran-mediated nuclear import pathway for CREB and related constitutively nuclear TFs, which is Impalpha-independent and thus distinct from import pathways utilized by inducible TFs.
Produced by various types of solid tumors, parathyroid hormone-related protein (PTHrP) is the causative agent of humoral hypercalcemia of malignancy. The similarity of PTHrP's amino-terminus to that of parathyroid hormone enables it to share some of the latter's signalling properties, but its carboxy-terminus confers distinct functions including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. PTHrP nuclear import occurs via a novel importin beta1-mediated pathway. The present study uses several different direct binding assays to map the interaction of PTHrP with importin beta using a series of alanine mutated PTHrP peptides and truncated human importin beta1 derivatives. Our results indicate that PTHrP amino acids 83-93 (KTPGKKKKGK) are absolutely essential for importin beta1 recognition with residues 71-82 (TNKVETYKEQPL) additionally required for high affinity binding; residues 380-643 of importin beta1 are required for the interaction. Binding of importin beta1 to PTHrP is reduced in the presence of the GTP-bound but not GDP-bound form of the guanine nucleotide binding protein Ran, consistent with the idea that RanGTP binding to importin beta is involved in the release of PTHrP into the nucleus following translocation across the nuclear envelope. This study represents the first detailed examination of a modular, non-arginine-rich importin beta1-recognized nuclear targeting signal.
Predatory waterbirds, such as ardeids, are susceptible to bioaccumulation of pollutants through the ingestion of contaminated food sources. High body burdens of contaminants, including PCBs, PAHs, cadmium, mercury, lead, copper, zinc and arsenic have been detected in many bird species worldwide. There is a paucity of literature, however, linking contaminant body burden and effects on reproductive success in waterbirds. This review is a synthesis of pertinent literature on this topic, with specific reference to contaminant residues in various tissue types, relationship between body burden and reproductive success, and the use of biomarkers to predict more serious adverse affects. The impetus for this review was the development of a conservation strategy and management plan (commissioned by the Agriculture, Fisheries and Conservation Department of Hong Kong Special Administrative Region (SAR)) for an important wetland in Hong Kong that supports many threatened waterbirds, including ardeids.
Parathyroid hormone-related protein is responsible for hypercalcemia induced by various tumors. The similarity of its N-terminus to that of parathyroid hormone enables parathyroid hormone-related protein to share parathyroid hormone's signaling properties, but the rest of the molecule possesses distinct functions including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. We have previously shown that parathyroid hormone-related protein nuclear import is mediated by importin beta1. Here we use fluorescence recovery after photobleaching for the first time to show that, in living cells, parathyroid hormone-related protein is exported from the nucleus in a leptomycin B-sensitive manner, implicating CRM1 as the parathyroid hormone-related protein nuclear export receptor. Leptomycin B treatment significantly reduced the rate of nuclear export 4 -10-fold, thereby increasing parathyroid hormone-related protein concentration in the nucleus/nucleolus about 2-fold. Intriguingly, this also led to a 2-fold reduced nuclear import rate. Inhibiting the nuclear export of a protein able to shuttle between nucleus and cytoplasm through distinct receptors thus can also affect nuclear import, indicating that the subcellular localization of a protein containing distinct nuclear import and export signals is the product of an integrated system. Although there have been several recent studies examining the dynamics of intranuclear transport using fluorescence recovery after photobleaching, this represents, to our knowledge, the first use of the technique to examine the kinetics of nucleocytoplasmic flux in living cells.
The use of radiation therapy to inhibit vascular proliferative diseases has produced encouraging results in several clinical trials. However, little is known about the possible side effects of radiation on vascular responsiveness. Our goal was to study the in vitro vascular responses of the rabbit aorta to various agonists immediately after several regimens of radiation therapy administered at doses prescribed in clinical protocols and at two different dose rates. High-dose-rate radiation was administered either by brachytherapy, using a gamma source, iridium 192, or an external electron beam producing beta radiation. Low-dose-rate radiation was administered by brachytherapy using a liquid-filled balloon with the beta emitter 32P. Vascular reactivity after the various regimens of irradiation was determined using the organ bath pharmacology assay. Various agonists were applied to the rabbit aorta to produce full cumulative concentration-response curves. Radiation, administered using an external electron beam, did not alter endothelium-dependent relaxation of the aorta induced by acetylcholine. However, the use of a catheter-based system to deliver radiation disrupted the endothelial cell lining of the vessel, causing a lack of relaxation by acetylcholine. Therefore, to compare all modalities of radiation therapy on vascular responsiveness, the agonists used in this study are known to act directly on the smooth muscle. Radiation therapy had no effect on the contractile responses induced by the following agonists: phenylephrine and potassium chloride. Vascular dilatation induced by nitroglycerin, a nitric oxide donor, was unaffected by radiation therapy. The contractile response induced by des-Arg9-bradykinin, a kinin B1 receptor agonist, was significantly increased twofold to threefold by all types of irradiation under study. This enhanced response is attributable to an increase of mRNA levels coding for this receptor. In all cases, radiation therapy did not alter the effective concentration producing 50% of maximal responsiveness (EC50) and did not reduce the vascular responsiveness induced by agonists. Taken together, we conclude that radiation therapy does not hinder endothelium-independent vascular responsiveness and increases the kinin B1 receptor-mediated vasoconstriction.
A solid-phase microextraction (SPME) method was applied to study microcystin (MC) profiles in a natural Microcystis sp. bloom in a freshwater pond in Guangzhou, China. Three dominant MC variants, namely MC-LR, MC-YR, and MC-RR, were quantified. Simultaneous study of their total, extracellular, and intracellular profiles was made possible using SPME coupled to high-performance liquid chromatography. The total and intracellular concentrations of MC-LR in the bloom were 8.67 x 10(-2) microg/ ml and 1.93 mg/g, respectively. The corresponding concentrations of MC-YR were 1.20 x 10(-3) microg/ml and 0.06 mg/g, respectively, and those of MC-RR were 5.57 x 10(-2) microg/ml and 1.49 mg/g, respectively. Only MC-LR was detectable in the extracellular phase (1.49 x 10(-2) microg/ml) of the bloom, and its concentration was 14% of the intracellular content. Mass balance consideration revealed that only 71.1% of total MC-LR, 36.0% of total MC-YR, and 67.4% of total MC-RR within the cyanobacterial cells were released into the aqueous phase immediately after cell lysis.
Recent progress indicates that there are multiple pathways of nucleocytoplasmic transport which involve specific targeting sequences, such as nuclear localization sequences (NLSs), and cytosolic receptor molecules of the importin/karyopherin superfamily which recognise and dock the NLS-containing proteins at the nuclear pore. This first step of nuclear import/export is of central importance, with the affinity of the importin-targeting sequence interaction a critical parameter in determining transport efficiency. Different importins possess distinct NLS-binding specificities, which allows the system to be modulated through differential expression of the importins themselves, as well as through competition between different importins for the same protein, and between different proteins for the same importin. The targeting sequence-importin interaction can also be influenced directly by phosphorylation increasing the affinity of the interaction with importins or by targeting sequence masking through phosphorylation or specific protein binding. Targeting sequence recognition thus appears to represent a key control point in the regulation of nuclear transport. BioEssays 22:532-544, 2000.
Parathyroid hormone-related protein (PTHrP) was first discovered as the factor causing hypercalcaemia produced by solid tumours frequently associated with the head and neck, breast, lung and kidney. The homology of its amino-terminus to parathyroid hormone (PTH; eight of the first 13 residues are identical), enables it to share the same receptor and perform similar biological functions to PTH. The sequences of PTHrP C-terminal to its PTH-like region confer functions such as transplacental calcium transport, renal bicarbonate excretion and in vitro osteoclast inhibition. Recent findings have shown that PTHrP is a nuclear/nucleolar protein in certain tissues and that this localization is cell cycle-regulated, mediated by the middle portion of the molecule, and involves the nuclear import receptor importin beta1. The present review discusses what is known about the pathway by which PTHrP localizes to the nucleus/nucleolus and the putative roles it may have there.
This paper reviews the toxicity and tumor-promoting properties of microcystins. Methods for screening and/or identification of microcystins in environmental samples are discussed and compared. Specific emphasis is placed on newly developed extraction/detection methods, e.g., solid phase microextraction (SPME) technique, and capillary electrophoresis coupled with laser-induced fluorescence detection. The results of a kinetic analysis of the effects of microcystins on phosphorylase-a binding to phosphatase-2A using a surface plasmon resonance biosensor are also presented.
Parathyroid hormone-related protein (PTHrP) is expressed by a wide variety of cells and is considered to act as a secreted factor; however, evidence is accumulating for it to act in an intracrine manner. We have determined that PTHrP localizes to the nucleus at the G1 phase of the cell cycle and is transported to the cytoplasm when cells divide. PTHrP contains a putative nuclear localization sequence (NLS) (residues 61-94) similar to that of SV40 T-antigen, which may be implicated in the nuclear import of the molecule. We identified that Thr85 immediately prior to the NLS of PTHrP was phosphorylated by CDC2-CDK2 and phosphorylation was cell cycle-dependent. Mutation of Thr85 to Ala85 resulted in nuclear accumulation of PTHrP, while mutation to Glu85 to mimic a phosphorylated residue resulted in localization of PTHrP to the cytoplasm. Combined, the data demonstrate that the intracellular localization of PTHrP is phosphorylation- and cell cycle-dependent, and such control further supports a potential intracellular role (10,34,35) for PTHrP.
Parathyroid hormone-related protein (PTHrP), expressed in a range of tumors, has endocrine, autocrine/paracrine, and intracrine actions, some of which relate to its ability to localize in the nucleus. Here we show for the first time that extracellularly added human PTHrP (amino acids 1-108) can be taken up specifically by receptor-expressing UMR106.01 osteogenic sarcoma cells and accumulate to quite high levels in the nucleus and nucleolus within 40 min. Quantitation of recognition by the nuclear localization sequence (NLS)-binding importin subunits indicated that in contrast to proteins containing conventional NLSs, PTHrP is recognized exclusively by importin beta and not by importin alpha. The sequence of PTHrP responsible for binding was mapped to amino acids 66-94, which includes an SV40 large tumor-antigen NLS-like sequence, although sequence determinants amino-terminal to this region were also necessary for high affinity binding (apparent dissociation constant of approximately 2 nM for importin beta). Nuclear import of PTHrP was assessed in vitro using purified components, demonstrating that importin beta, together with the GTP-binding protein Ran, was able to mediate efficient nuclear accumulation in the absence of importin alpha, whereas the addition of nuclear transport factor NTF2 reduced transport. The polypeptide ligand PTHrP thus appears to be accumulated in the nucleus/nucleolus through a novel, NLS-dependent nuclear import pathway independent of importin alpha and perhaps also of NTF2.
We report a direct immersion SPME-GC/mu ECD method for the determination of non-volatile pollutants (Aroclor 1254) in human blood plasma using an enzymatic proteolytic approach to overcome the protein fouling problem. A nonspecific serine protease, Proteinase K, is used to remove the fouling interference. Significant improvement in relative repeatability from 28.9% to 3.4% (n = 10) was obtained. Analyte recovery up to 93% and detection limit of 1.0 ppb (total PCB) were achieved.
Osteoclasts are the cells responsible for bone resorption, and their number and rate of formation are critical in determining bone mass. To identify and quantify osteoclasts, as well as to study their formation in bone and in osteoclastogenic cultures, osteoclast-specific cell markers are required. Only the calcitonin receptor (CTR) expression unambiguously identifies osteoclasts and distinguishes them from macrophage polykaryons. However, present autoradiographic methods for CTR detection are cumbersome and time consuming. We have developed rabbit polyclonal antibodies specific for the C-terminal intracellular domain of the mouse and rat Cla CTR. These antibodies labeled HEK-293 cells stably transfected with CTR (but not untransfected HEK-293 cells). This labeling is abrogated by preabsorbing the antibodies with the recombinant antigen. The antibodies immunostained primary mouse and rat osteoclasts as well as osteoclasts in sections of mouse bone. Osteoclasts (both mononuclear and multinucleated) formed from mouse bone marrow or spleen cells cocultured with osteoblasts in the presence of 1,25 dihydroxyvitamin D3 and prostaglandin E2 were also specifically immunostained by the CTR antibodies. Cocultures incubated under conditions that did not allow osteoclastogenesis (i.e., omission of mediators or osteoblasts, or culture for less than 4 days) were not immunostained by CTR antibodies. Autoradiographic detection of 125I-labeled salmon calcitonin combined with CTR immunohistochemistry showed that both methods labeled the same cells. A CTR polyclonal antibody and monoclonal antibody F4/80 were used in combination to show immunofluorescence labeling of murine osteoclasts and macrophage populations, respectively, in marrow/osteoblast cocultures. These results indicate that simple and rapid CTR antibody-based methods can be used to identify osteoclasts, and can be used to characterize the antigenic profile of osteoclasts by using double immunofluorescence analysis.
The fluorescent cholesterol analog 22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (fluoresterol) was characterized as a tool for exploring the biochemistry and cell biology of intestinal cholesterol absorption. Hamsters absorbed fluoresterol in a concentration- and time-dependent manner, with an efficiency of about 15-30% that of cholesterol. Fluoresterol absorption was blocked by compounds known to inhibit cholesterol absorption, implying that fluoresterol interacts with those elements of the normal pathway for cholesterol absorption on which the inhibitors act. Confocal microscopy of small intestinal tissue demonstrated that fluoresterol was taken up by absorptive epithelial cells and packaged into lipoprotein particles, suggesting a normal route of intracellular trafficking. Uptake of fluoresterol was confirmed by biochemical analysis of intestinal tissue, and a comparison of [(3)H] cholesterol and fluoresterol content in the mucosa suggested that fluoresterol moved through the enterocytes more rapidly than did cholesterol. This interpretation was supported by measurements of fluoresterol esterification in the mucosa. Four hours after hamsters were given fluoresterol and [(3)H]cholesterol orally, 44% of the fluoresterol in the intestinal mucosa was esterified, compared to 8% of the [(3)H]cholesterol. Caco-2 cells took up 2- to 5-fold more [(3)H]cholesterol than fluoresterol from bile acid micelles, and esterified 21-24% of the fluoresterol but only 1-4% of the [(3)H]cholesterol. Thus fluoresterol apparently interacts with the proteins required for cholesterol uptake, trafficking, and processing in the small intestine.
Parathyroid hormone-related protein (PTHrP) is highly expressed in normal skin keratinocytes, and its involvement in growth and differentiation processes in these cells has been implicated by several lines of evidence which include the use of antisense PTHrP (Kaiser et al., 1994, Mol. Endocrinol., 8:139-147). In this study, we have investigated whether PTHrP expression and its subcellular localization is linked to cell cycle progression in a human keratinocyte cell line (HaCat), which constitutively expresses and secretes PTHrP. PTHrP mRNA and immunoreactive PTHrP were assessed in asynchronous dividing cells and in cells blocked at G1 or G2 + M phases of the cell cycle using several different protocols. The response of PTHrP mRNA expression was examined following readdition of serum in the continued presence of cycle blockers, and after release from cell cycle block, or from cell synchronization by serum deprivation. PTHrP expression was greatest in actively dividing cells when cells were in S and G2 + M phases of the cell cycle and were lowest in quiescent G1 cells. Most notable were the high levels of PTHrP mRNA and protein in cells at G2 + M phase of the cell cycle at division. Furthermore, PTHrP was localized to the nucleolus in quiescent cells, but redistributed to the cytoplasm when cells were actively dividing. Taken together, these results support a role for PTHrP in cell division in keratinocytes. In asynchronously growing cells, PTHrP expression fell as cells became confluent at a time when cell growth is inhibited and cells begin to differentiate. Mitogen stimulation of HaCaT cells resulted in a rapid increase in PTHrP mRNA expression, but was dependent upon cells being in the G1 phase of the cell cycle. Cells blocked in G1 responded to mitogen both in the continued presence of aphidicolin or when released from block. Cells blocked at G2 + M with colcemid expressed high levels of PTHrP mRNA and protein, and PTHrP mRNA did not respond further to mitogen in the continued presence of blocker. However, in cells released from block at G2 + M by addition of serum, an increase in PTHrP expression was seen coincident with the progression of cells into G1. In contrast, in a squamous cancer cell line (COLO16), basal PTHrP expression was high and was not altered during the cell cycle or by cell cycle block, consistent with association of its dysregulated expression in malignant cells. The results of this study suggest that PTHrP may have two roles in the cell cycle; one in G1 in response to mitogen, and a second at cell division when its expression is high and it is relocated from the nucleolus to the cytoplasm.