Biomedical subjects

K W Chan

Publications and source records attributed to K W Chan.

At least 73 records · Page 4Linked to original sources

PubMed2001

Suppression of the tumorigenicity of mutant p53-transformed rat embryo fibroblasts through expression of a newly cloned rat nonmuscle myosin heavy chain-B.

In our previous study, a rat homolog of human nonmuscle myosin heavy chain-B (nmMHC-B) was identified by mRNA differential display comparing of transformed against nontransformed Rat 6 cells overexpressing mutant p53val135 gene. The nmMHC-B was found to be expressed in normal Rat 6 embryo fibroblast cell line, but markedly suppressed in the mutant p53val135-transformed Rat 6 cells. To examine the possible involvement of nmMHC-B in cell transformation, we first cloned and sequenced the full length cDNA of rat nmMHC-B, which was then cloned into an ecdysone-expression vector. The resulting construct was introduced into the T2 cell line, a mutant p53val135-transformed Rat 6 cells lacking the expression of the endogenous nmMHC-B. The clonal transfectants, expressing muristerone A-induced nmMHC-B, displayed a slightly flatter morphology and reached to a lower saturation density compared to the parental transformed cells. Reconstitution of actin filamental bundles was also clearly seen in cells overexpressing the nmMHC-B. In soft agar assays, nmMHC-B transfectants formed fewer and substantially smaller colonies than the parental cells in response to muristerone A induction. Moreover, it was strikingly effective in suppressing the tumorigenicity of the T2 cells when tested in nude mice. Thus, the nmMHC-B, known as a component of the cytoskeletal network, may act as a tumor suppressor gene. Our current finding may reveal a novel role of nmMHC-B in regulating cell growth and cell signaling in nonmuscle cells. Oncogene (2001) 20, 58 - 68.

Actins↗

PubMed2001

Pre-operative grading of intracranial glioma.

AIM: To compare the accuracy of MR-determined cerebral blood volume (CBV) maps with SPECT imaging with thallium-201 in pre-operative grading of intracranial glioma. MATERIAL AND METHODS: Nineteen patients (7 female and 12 male, mean age 46.8 years) with intracranial gliomas were examined with MR perfusion imaging pre-operatively. Sixteen of these patients were also examined with SPECT imaging with thallium-201. The tumour to contralateral white matter NI (negative integral) and tracer uptake ratios were evaluated. The ratios in high-grade and low-grade tumours were compared. RESULTS: The maximum CBV ratios of grades I and II gliomas (2.958+/-2.217) were significantly lower than the maximum CBV ratio of grades III and IV (9.484+/-4.520), p<0.001. There was no statistical difference when CBV ratios of grades I and II (p=0.381), grades II and III (p=0.229) and grades III and IV (p=0.476) gliomas were compared. Thallium SPECT imaging showed no difference in tumour uptake ratio between low-grade and high-grade gliomas (p=0.299). CONCLUSION: MR-determined NI was useful for pre-operative grading of intracranial gliomas but SPECT thallium-201 imaging was not.

Adolescent↗

PubMed2001

A fludarabine-based conditioning regimen for severe aplastic anemia.

Graft rejection is a common problem after alternative donor transplantation for patients with refractory severe aplastic anemia (SAA). Intensification of the conditioning regimen, with the inclusion of irradiation, has often been advocated to combat this problem. With this approach engraftment rate improved, but the incidence of transplant-related complications is also increased, resulting in little change in the overall outcome. We investigated the use of the combination of fludarabine, cyclophosphamide and anti-thymocyte globulin as the conditioning regimen in five multiply-transfused SAA patients. Three patients received an HLA one-antigen disparate related donor transplant, while two patients were given marrow from matched, unrelated donors. The regimen was well tolerated, with only grade I toxicity encountered. With a median follow-up of 9 months, all patients are alive with complete donor chimerism. We conclude that fludarabine may be used in place of irradiation to augment the conditioning regimen of cyclophosphamide and anti-thymocyte globulin for alternative donor transplantation in children with SAA.

Anemia, Aplastic↗

PubMed2001

Factors affecting lymphocyte subset reconstitution after either related or unrelated cord blood transplantation in children -- a Eurocord analysis.

Immune recovery after cord blood transplantation (CBT) is of concern owing to the low number of lymphocytes transferred with the graft and their immaturity. Risk factors influencing lymphocyte subset reconstitution related to disease, patient, donor and transplant were studied in 63 children (< 16 years), given either related (n = 14) or unrelated (n = 49) CBT for malignant (n = 33) or non-malignant diseases (n = 30). Only children with sustained myeloid engraftment were analysed. Absolute numbers of T (CD3(+), CD4(+), CD8(+)), B and natural killer (NK) cells were reported 2--3, 6, 9, 12 and 12--24 months after CBT. Median patient age was 4.0 years (0--15) and median follow-up was 23 months (1.7--61.0). Twenty-six patients received human leucocyte antigen (HLA)-matched CBT and 37 received HLA-mismatched CBT. The median number of nucleated cells (NCs) collected/recipient weight was 6.1 x 10(7)/kg. In this selected population, the estimate 2 year survival was 85%. Lymphocyte reconstitution (defined as the median time to reach the normal value of age-matched healthy children) was 3, 6 and 8 months for NK, B and CD8(+) cells, while it was 11.7 months for both CD3(+) and CD4(+) lymphocytes. In the multivariate analysis, factors favouring T-cell recovery were: related donor (P = 0.002); higher NCs/kg (P = 0.005) and recipient cytomegalovirus (CMV)-positive serology (P = 0.04). Presence of acute graft-versus-host disease (GVHD) delayed T-cell recovery (P = 0.04). To summarize, in children with sustained myeloid engraftment the concern that lymphocyte recovery after CBT could be delayed does not appear to be substantiated by our results.

Adolescent↗

PubMed2001

Development of follicular dendritic cell sarcoma in hyaline-vascular Castleman's disease of the nasopharynx: tracing its evolution by sequential biopsies.

AIMS: Hyaline-vascular Castleman's disease (HVCD) and follicular dendritic cell (FDC) sarcoma occurring in the nasopharynx are both extremely rare. We report the first case of transformation of the former into the latter as documented by sequential biopsies. The steps involved in the transformation were described in detail and the possible role of p53 studied. METHODS AND RESULTS: The patient presented at the age of 23 years with nasopharyngeal HVCD. Hyaline- vascular Castleman's disease with FDC overgrowth was diagnosed in a recurrence 8 years later, and a frank FDC sarcoma developed at the same site 11 years after initial presentation. The patient remained disease-free 3 years after excision and adjuvant chemotherapy. The FDC sarcoma comprised swirling fascicles of spindly cells with indistinct cell borders. The tumour cells expressed the FDC markers CD21, CD35 and CNA.42 and in-situ hybridization for Epstein-Barr virus-encoded RNAs was negative. Over-expression of p53 protein was observed in the FDC sarcoma and an increased number of weakly p53-positive spindly cells could also be demonstrated in the HVCD specimen. This finding suggested a possible role of p53 in the evolution from HVCD to FDC sarcoma. Critical analysis of the literature shows that, among the 13 reported cases of FDC sarcoma associated with Castleman's disease, possible progression from the latter to the former is documented in only two cases. CONCLUSIONS: The sequential changes observed in the current case provide further evidence to strengthen the role of HVCD as a possible precursor of FDC sarcoma. There is a possible role of p53 in the transformation process but confirmation by future studies is needed.

Adult↗

PubMed2001

Treatment of diffuse proliferative lupus glomerulonephritis: a comparison of two cyclophosphamide-containing regimens.

Cyclophosphamide (CYC) has proven beneficial in preserving renal function in patients with lupus with diffuse proliferative glomerulonephritis (DPGN). However, the optimal route of CYC administration is unknown because direct comparative studies are unavailable. In this open study, we compared the renal outcome of two historical cohorts of patients with diffuse proliferative lupus nephritis (World Health Organization classes IVa and IVb) treated with either intravenous (IV) pulse CYC (group A; n = 22) or sequential oral CYC followed by azathioprine (AZA; group B; n = 21) and followed up prospectively. Both groups of patients had similar clinical, biochemical, and renal parameters at baseline. At 24 months posttreatment, significant improvements in proteinuria, creatinine clearance, serum albumin level, and lupus serological results were evident in both groups. Compared with patients in group A, patients in group B had more complete or partial remission (90% versus 73%) and less risk for treatment failure (5% versus 14%), renal flares (5% versus 14%), and doubling of creatinine levels (5% versus 9%), but the difference was not statistically significant. However, patients treated with oral immunosuppression had an insignificant increase in rates of herpes zoster infection (19% versus 9%) and menstrual disturbance (50% versus 29%). We conclude that sequential oral immunosuppression with CYC and AZA tended to have better efficacy than IV pulse CYC in the treatment of lupus DPGN but was associated with more toxicities. Additional randomized trials involving a larger cohort of patients with a longer period of observation are necessary.

Administration, Oral↗

PubMed2001

Unrelated cord blood transplant experience by the pediatric blood and marrow transplant consortium.

Cord blood (CB) has emerged as a potential source of hematopoietic stem cells for patients who are in need of hematopoietic stem cell transplant (HSCT). The authors analyzed the Pediatric Blood and Marrow Transplant Consortium's (PBMTC) data of consecutive unrelated CB transplants performed during the initial 2 years of using placental blood grafts. From January 1995 to December 1996 PBMTC performed a total of 44 unrelated CB transplant for a variety of diseases consisting of acute leukemias (n = 29), congenital conditions (n = 9), and bone marrow failure (n = 6). There were 15 females and 29 males with median age of 5 years (range 0.4-20.6 years) and median weight of 18.2 kg (range 6.3-70 kg). The median volume of CB units was 80 mL (range 44.5-215 mL) and the median cell dose given was 4.3 x 10(7)/kg of recipient weight (range 1.1-23 x 10(7)/kg). Techniques used for human leukocyte antigen (HLA) matching were serologic typing for class I HLA antigens and high-resolution molecular typing for HLA-DRB1 alleles. HLA disparities were as follows: 4 were 6/6 matches, 21 were 5/6, 15 were 4/6, and 4 were 3/6. Twenty-nine (66%) of CB units were DRB1 matched with recipients. Conditioning regimens consisted of either total body irradiation containing (n = 31) or chemotherapy only (n = 11) regimens. All but 3 patients receive cyclosporine as part of graft vs. host disease (GvHD) prophylaxis in combination with either methotrexate (MTX) or methylprednisolone (Pred). The other 3 patients had FK506 and MTX for GvHD prophylaxis. Myeloid engraftment (absolute neutrophil count > or = 500) occurred at a median of 21 days (range 10-43 days) and platelet > or = 50,000/mm3 was noted at a median of 44 days (range 16-102 days). Eight patients died too early (< day + 28) for evaluation of engraftment (5 for infection, 2 for multiorgan failure, 1 for toxic epidermolysis). The probability of having grade II-IV acute GvHD for all patients was 44 +/- 0.7%. The incidence of a GvHD is similar for 4/6 and 5/6 antigen when DRB1 matched, at 47 and 52%, respectively. Chronic GvHD was noted in 28% of patients surviving > 90 days. The Kaplan-Meier estimate of 4-year event-free survival was 43%. A Cox model for analysis of factors associated with survival was DRB1 matching, p = .001; cell dose, p = .009; and younger age, p = .03. In conclusion, CB transplant offers a good alternative to bone marrow transplant Although GvHD occurs, it is usually of low severity despite the high frequency of multiple HLA antigen mismatches. It also appears that a 4/6 is as good as a 5/6 matched antigen CB unit when DRB1 matched especially in the pediatric setting.

Adolescent↗

PubMed2001

Vesicoureteric reflux and renal scarring in Chinese children.

Vesicoureteric reflux (VUR) and renal scarring are commonly found in children with urinary tract infection (UTI). The prevalence of VUR and scarring may vary between racial groups. There are no published data on the prevalence of VUR and scarring in Chinese children with UTI. A retrospective, single-institution study was made of Hong Kong Chinese children aged less than 5 years with a documented UTI investigated by both micturating cystourethrography and dimercaptosuccinic acid scintigraphy. VUR was identified in 39% of 93 Chinese children with UTI. Renal scarring was present in 28% of boys, which is comparable with published data on Western children. Scarring appears to be less common in Chinese girls with UTI (11%) than in Western girls (30-38% from published data), and its severity is poorly related to VUR grade. There is a significant dependency relationship between grade of VUR and degree of scarring in Chinese boys (p < 0.05). In conclusion, renal scarring appears to be relatively uncommon in Chinese girls. The correlation between grade of VUR and degree of scarring in Chinese boys suggests a relationship, but provides no evidence about the direction of causation.

Child, Preschool↗

PubMed2001

Cloning and characterization of G protein-gated inward rectifier K+ channel (GIRK1) isoforms from heart and brain.

A G protein-gated inward rectifier potassium (K+) channel (GIRK1a) has been cloned from different tissues (Kubo et al., 1993b; Dascal et al., 1993). Here we report the cloning of three additional novel isoforms of GIRK1a from rat atria and and one from human brain. These isoform cDNAs code for proteins that have identical N-termini, M1-H5-M2 (predicted transmembrane and pore domains), and post-M2 amino acid regions to GIRK1a (1-501 amino acids), but they have shorter C-termini (GIRK1b (1-309), GIRK1c (1-308), GIRK1d (1-235), and GIRK1e (1-253). These results indicated that isoforms were generated by alternative splicing and partial genomic analysis confirmed the presence of exons and introns in the rat GIRK1 gene. RNase protection analysis and immunoblot analysis indicated that the isoforms were expressed in both rat atria and brain but at lower levels versus GIRK1a. The physiological role that the isoforms may play in atrial and brain physiology remains to be determined.

Alternative Splicing↗

PubMed2001

Incorporating the ABI GeneScan analysis to a RACE-based technique for mapping multiple transcription initiation sites.

Determination of transcription initiation sites has commonly been performed by primer extension and RNase protection assay using radioactively labeled oligonucleotides. Recently, a protocol based on modified 5' rapid amplification of cDNA ends (RACE) with the use of fluorescently labeled primer was developed. Here, we describe the use of RACE-based technique in conjunction with the GeneScan analysis for the determination of transcription initiation sites of genes of interest. The RACE technique is based on the ligation of an adapter to both ends of the cDNAs. The gene of interest was first amplified by PCR using a gene-specific and a 5' adapter primer. Subsequently, nested PCR was performed using an internal gene-specific primer paired with a fluorescently end-labeled adapter primer. The size of the fluorescently labeled PCR products was directly determined by the ABI PRISM 377 GeneScan Analyzer. This novel approach provides an accurate, sensitive, and convenient method for mapping transcription initiation sites, especially for genes with multiple transcription initiation sites, for genes expressed at low levels, and for splice variants that display alternative splicing farther than a few hundred nucleotides downstream from the transcription initiation site. This article describes the application of this new method in the mapping of transcription initiation sites of two splice variants of rat frizzled related protein transcripts.

Alternative Splicing↗

PubMed2001

Androgen induces differentiation of a human papillomavirus 16 E6/E7 immortalized prostate epithelial cell line.

Androgen signaling is crucial for the growth and development, as well as for tumorigenesis of the prostate. However, many of the prostate epithelial cell lines developed previously, either normal or tumorigenic, do not express androgen receptor (AR) or respond to androgen. In order to advance our understanding on how androgen signaling regulates the growth and the differentiation status, and affects tumorigenicity of the epithelial cell, we performed experiments on HPr-1, a prostate cell line recently immortalized from normal human prostate epithelial cells. In the present study, AR was stably transfected into HPr-1 cells by replication-defective retrovirus. Treatment of HPr-1AR cells with androgen resulted in cell differentiation and growth retardation accompanied with up-regulation of cytokeratins K8 and K18, prostate specific antigen, p21 and p27, and down-regulation of c-myc, bcl-2 and telomerase activity. Our results suggest that androgen promotes the process of differentiation in a human papillomavirus 16 E6/E7 immortalized prostate epithelial cell line which may reflect the normal effects of androgen on prostate cells.

Androgens↗

PubMed2001

Human bone marrow transplant rejection is associated with telomere cleavage.

Telomeres that guard chromosomes are shortened with each cell division because of replication-dependent sequence loss at both termini. The gradual erosion of telomeric length has been directly related to the process of aging in vivo. Recently we have reported, in murine and human cancer cells treated with different apoptogens, cleavage and extrusion of telomeric DNA prior to cell death on one hand and an amplification of telomeric DNA in metastatic epithelial malignancies of different histopathologic origin on the other. This study tested our hypothesis that telomere cleavage is linked to transplant rejection in cancer patients receiving stem cells either from bone marrow (BM) or umbilical cord blood transfusion. Telomere integrity and mitotic catastrophe were studied by cytogenetic and molecular fluorescence in situ hybridization (FISH) techniques in two BM samples taken from a male stem cell transplant recipient diagnosed with aplastic anemia. The first BM sample, which was aspirated 27 days after transplant, was mitotically active. Only one of 50 metaphases showed a chromatid break. Every cell karyotyped was of male origin with 46, XY chromosome constitution. The second BM sample aspirated 52 days after transplant gave no metaphases and most interphase cells appeared dead. FISH preparations of the second BM sample showed cleavage and drastic reduction of telomeric DNA at the time the patient was rejecting the transplant. In contrast, the first BM sample had shown no indication of cleavage of the telomeric DNA, although the percentage of telomeric area was smaller than in the control. The replicative stress imposed on the stem cells engrafted may result in an accelerated aging effect, possibly due to the erosion of telomeric DNA. We, therefore, conclude that BM rejection could be directly associated with the cleavage, clustering, and extrusion of telomeric DNA in the donor cells.

Adolescent↗

PubMed2001

Iron deficiency anemia.

Iron deficiency anemia is the most common cause of anemia worldwide and results from inadequate iron supply for erythropoiesis. Iron deficiency is most prevalent during periods of rapid body growth: in infancy and again at puberty. Insufficient intake accounts for most cases. The clinical manifestations of iron deficiency anemia can be subtle, but irreversible delayed psychomotor development may occur if the anemia is severe and prolonged. The optimal approach is prevention and early treatment.

Adolescent↗

PubMed2000

Decreased steroid hormone synthesis from inorganic nitrite and nitrate: studies in vitro and in vivo.

Nitrites and nitrates are consumed nonchalantly in diet. Organic nitrates are also used as vasodilators in angina pectoris, but the therapy is associated with tolerance whose mechanism remains elusive. Previously, we found inorganic nitrate inhibited steroidogenesis in vitro. Because adrenocorticoids regulate water and electrolyte metabolism, tolerance may ensue from steroid deficiency. We have studied the effects of nitrite and nitrate on in vitro synthesis and in vivo blood levels of steroid hormones. In vitro, nitrite was more potent than nitrate in inhibiting human chorionic gonadotropin (hCG)-stimulated androgen synthesis by Mouse Leydig Tumor cells. At concentrations above 42 mM, nitrite completely inhibited androgen synthesis, and, unlike nitrate, the inhibition was irreversible by increasing hCG concentration. The cAMP production remained intact but reduced with both ions. The nitric oxide (NO) scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide (c-PTIO) significantly increased hCG- or cAMP-stimulated androgen synthesis in all buffers, suggesting that NO is a chemical species directly involved in the nitrite/nitrate-induced inhibition. This is further supported by c-PTIO countering the inhibitory action of methylene blue on androgen synthesis. Rats given distilled water containing 50 mg/L NaNO(2) or NaNO(3) for 4 weeks drank significantly less daily. At the end, their blood corticosterone and testosterone levels were significantly decreased. The adrenocortical histology showed bigger lipid droplets, which are pathogonomic of impaired steroidogenesis. Nitrite and nitrate are metabolized to NO, which binds heme in cytochrome P450 enzymes, thereby inhibiting steroidogenesis. Therapeutic nitrates likewise may decrease adrenal (and gonadal) steroidogenesis. Cortisol deficiency would impair water excretion causing volume expansion, and aldosterone deficiency would cause sodium loss and raised renin. Paradoxically, volume expansion without sodium retention and raised renin has all been reported in tolerance.

Androgens↗

PubMed2000

Spontaneous remission of granulocyte colony-stimulating factor-associated leukemia in a child with severe congenital neutropenia.

Leukemia is observed with increased frequency in patients with severe congenital neutropenia (SCN). In the past decade, recombinant human granulocyte colony-stimulating factor (rh G-CSF) has prolonged the survival of patients with SCN increasingly reported to have leukemias. In this communication acute myelogenous leukemia (AML) associated with a mutation of the G-CSF receptor (G-CSF-R) developed in a patient with SCN maintained on long-term G-CSF therapy. The blast count in the blood and bone marrow fell to undetectable levels twice on withholding G-CSF and without chemotherapy administration, but the mutant G-CSF-R was detectable during this period. The patient subsequently underwent successful allogeneic bone marrow transplantation. After transplantation, the patient's neutrophil elastase (ELA-2) mutation and G-CSF-R mutation became undetectable by polymerase chain reaction. This report provides novel insights on leukemia developing in congenital neutropenia.

Bone Marrow Transplantation↗

PubMed2000

Efficacy of mycophenolate mofetil in patients with diffuse proliferative lupus nephritis. Hong Kong-Guangzhou Nephrology Study Group.

BACKGROUND: The combination of cyclophosphamide and prednisolone is effective for the treatment of severe lupus nephritis but has serious adverse effects. Whether mycophenolate mofetil can be substituted for cyclophosphamide is not known. METHODS: In 42 patients with diffuse proliferative lupus nephritis we compared the efficacy and side effects of a regimen of prednisolone and mycophenolate mofetil given for 12 months with those of a regimen of prednisolone and cyclophosphamide given for 6 months, followed by prednisolone and azathioprine for 6 months. Complete remission was defined as a value for urinary protein excretion that was less than 0.3 g per 24 hours, with normal urinary sediment, a normal serum albumin concentration, and values for serum creatinine and creatinine clearance that were no more than 15 percent above the base-line values. Partial remission was defined as a value for urinary protein excretion that was between 0.3 and 2.9 g per 24 hours, with a serum albumin concentration of at least 30 g per liter. RESULTS: Eighty-one percent of the 21 patients treated with mycophenolate mofetil and prednisolone (group 1) had a complete remission, and 14 percent had a partial remission, as compared with 76 percent and 14 percent, respectively, of the 21 patients treated with cyclophosphamide and prednisolone followed by azathioprine and prednisolone (group 2). The improvements in the degree of proteinuria and the serum albumin and creatinine concentrations were similar in the two groups. One patient in each group discontinued treatment because of side effects. Infections were noted in 19 percent of the patients in group 1 and in 33 percent of those in group 2 (P = 0.29). Other adverse effects occurred only in group 2; they included amenorrhea (in 23 percent of the patients), hair loss (19 percent), leukopenia (10 percent), and death (10 percent). The rates of relapse were 15 percent and 11 percent, respectively. CONCLUSIONS: For the treatment of diffuse proliferative lupus nephritis, the combination of mycophenolate mofetil and prednisolone is as effective as a regimen of cyclophosphamide and prednisolone followed by azathioprine and prednisolone but is less toxic.

Adult↗

PubMed2000

Molecular cloning and functional analysis of the promoter region of rat nonmuscle myosin heavy chain-B gene.

Rat nonmuscle myosin heavy chain-B (r-nmMHC-B) mRNA was previously found downregulated in Rat 6 fibroblasts transformed by mutant p53(val135) [J. W. P. Yam, J. Y. Zheng, and W. L. W. Hsiao (1987) Biochem. Biophys. Res. Commun. 266, 472-480]. Overexpression of exogenous r-nmMHC-B could partially reverse the transforming phenotypes both in vitro and in vivo. The downregulation of r-nmMHC-B was also observed in Rat 6 transformed by c-H-ras and v-myc oncogenes. We cloned a 5.2-kb r-nmMHC-B promoter region. Sequence analysis of -1248 to +1 revealed no TATA box, but did show that it contained CAAT boxes, E12/E47, MyoD, MEF, E2F, CREB, and SP1 binding sites. Based on transient reporter assays, the promoter/enhancer activities were unusually extended to the entire 5.2 kb region in normal Rat 6 cultures, but markedly suppressed in p53(val135)-, and c-H-ras-transformed cells. The activity detected by the reporter assay corresponded to levels of mRNA as analyzed previously by Northern blots in each respective cell line. Thus, the switch-off of the r-nmMHC-B in the transformed cells is very likely controlled by upstream transcriptional factors, which might have been altered in the course of neoplastic transformation.

Animals↗

PubMed2000

Glycosylation of GIRK1 at Asn119 and ROMK1 at Asn117 has different consequences in potassium channel function.

GIRK (G protein-gated inward rectifier K(+) channel) proteins play critical functional roles in heart and brain physiology. Using antibodies directed to either GIRK1 or GIRK4, site-directed mutagenesis, and specific glycosidases, we have investigated the effects of glycosylation in the biosynthesis and heteromerization of these proteins expressed in oocytes. Both GIRK1 and GIRK4 have one extracellular consensus N-glycosylation site. Using chimeras between GIRK1 and GIRK4 as well as a GIRK1 N-glycosylation mutant, we report that GIRK1 was glycosylated at Asn(119), whereas GIRK4 was not glycosylated at Asn(132). GIRK1 membrane-spanning domain 1 was required for optimal glycosylation at Asn(119) because a chimera that contained GIRK4 membrane-spanning domain 1 significantly reduced the addition of a carbohydrate structure at this site. This finding may partly account for the reason that GIRK4 is not glycosylated at Asn(132), either as a homomer or when coexpressed with GIRK1. When the GIRK1(N119Q) mutant was coexpressed with GIRK4, the biophysical properties of the heteromeric channel and the magnitude of the agonist-induced currents were similar to those of controls. Thus, N-glycosylation of GIRK1 at Asn(119) does not appear to affect its physical association with GIRK4, the routing of the heteromer to the cell surface, or heteromeric channel function, unlike the dramatic functional effects of N-glycosylation of ROMK1 at Asn(117) (Schwalbe, R. A., Wang, Z., Wible, B. A., and Brown, A. M. (1995) J. Biol. Chem. 270, 15336-15340).

Amidohydrolases↗