Biomedical subjects

M Wang

Publications and source records attributed to M Wang.

At least 361 records · Page 20Linked to original sources

PubMed2000

[The growth inhibitory effects by transfection of p16 gene on human pancreatic cancer cell line].

OBJECTIVE: To elevate the growth inhibitory effects by transfection of p16 gene on human pancreatic cancer cell line JF305. METHODS: Recombinant eukaryotic expression vector pDOR-p16 containing exogenous human wt-p16 cDNA and vector containing neomycin resistance gene only were introduced by Liposomes-mediated gene transfection into JF305 cell line which did not express endogenous p16. By using PCR amplification, in situ hybridization, and immunocytochemistry, the clones obtained were detected for efficiency of transfection and effect of vector expression and observed for the changes of their biologic characteristics. RESULTS: Exogenous wt-p16 was successfully transferred into JF305 cells and obtained permanent expression. The growth rate of these transfected JF305 cells in regular medium and soft agar was inhibited. The percentage of phage G(1) cells increased and that of phage S cells decreased by analysing cell cycle. The ultrastructural changes of the cells observed under electron-microscope revealed growth retardation. CONCLUSION: p16 is a candidate for cancer gene replacement therapy of human pancreatic cancer.

Cell Division↗

PubMed2000

[Operative treatment of displaced proximal humeral fractures: follow-up and analysis of 31 patients'].

OBJECTIVE: To study the methods and the results of operative treatment of displaced proximal humeral fractures. METHODS: We reviewed thirty-one patients who had been diagnosed as having displaced proximal humeral fractures and had been operated on from July 1989 to December. 1998 in our hospital. The mean follow-up time was 40.5 months (8 - 124 months). Their age ranged from 15 to 62 years (average, 36.8 years); 18 patients were male and 13 female. Neer fracture classification system and rating system were used. In all patients, delto-pectoral approach was adopted. Twelve fractures were fixed with plates, fifteen fractures with screws, and four fractures with Kirschner wire and plaster. RESULTS: Of two-part surgical neck fractures, nine of thirteen patients (69%) were excellent or good with no necrosis and un-union. In three-part or four-part fractures, the rate of satisfaction with open reduction and internal fixation (ORIF) was rather lower. In three-part fractures, the rate of humeral head necrosis was 44%, and in four-part fractures, over 75%. CONCLUSIONS: In two-part and three-part fractures, ORIF is a better treatment, but care should be taken to avoid using plates. As for classic four-part fractures, the rate of satisfaction with ORIF is poor and the rate of necrosis is higher.

Adult↗

PubMed2000

[Does simple posterior dislocation of the elbow necessitate strict immobilization?].

OBJECTIVE: To research for the ideal treatment of isolated posterior dislocation with the elbow after reduction. METHODS: 36 patients of isolated posterior dislocation of the elbow after closed reduction (male 26, female 10; left 14, right 22; dominant extremity 21, undominant extremity 15; average age 22) were examined by varus and valgus stress test and push-draw test. Satisfactory stability from 30 degrees to 130 degrees flexion of the elbow was found in all patients. For the 30 patients below 35 years old, long-arm posterior splint was applied to immobilize the elbow joint at 90 degrees flexion and pronation for one week. For 6 patients over 35 years old, we only immobilized the elbow joint for 3 to 5 days with a neck-wrist sling. Prompt active motion begin after the swelling and pain reduced slightly, but the last 30 extension was not allowed for the first 1 to 2 weeks. Forceful passive motion and stretching of the elbow joint were avoided in all patients. RESULTS: The average follow-up time was 9 months (3 - 15 months). Range of motion and muscle power were regained within 3 - 4 months. 32 patients rehabilitated completely and 4 of the 6 patients decreased extension for 5 degrees - 10 degrees. No unstable symptoms and signs or recurrent dislocation were found. No ectopic calcification or post-traumatic arthritic changes were found on X-ray follow-up. CONCLUSIONS: The ulna-humeral joint is one of the most highly constrained joints in the body with intrinsic stability. Bony structure is stable enough to allow for capsular and ligamental structure healing, even during active motion. For isolated posterior dislocation of the elbow joint, short-term immobilization and early functional exercise are desirable. Elbow instability and recurrent dislocation are rare and do not necessitate long-term strict immobilization.

Adolescent↗

PubMed2000

[Treatment of acute lymphoblastic leukemia by autologous stem cell transplantation: an analysis of 30 cases].

OBJECTIVE: To evaluate the clinical outcome of autologous stem cell transplantation (ASCT) in acute lymphoblastic leukemia (ALL) and the affected factors. METHODS: Data of 30 ALL patients received ASCT in our hospital between July 1987 and December 1997 were retrospectively analyzed. Twenty-four of them were in the first complete remission (CR(1)) and six in the second complete remission (CR(2)) or early relapse (ER). Conditioning regimens were CTX 120 mg/kg + single total body irradiation 9 - 10 Gy (sTBI) or Bu 16 mg/kg of Mel 140 - 180 mg/m(2) + Ara-c 2 - 4 g/m(2). RESULTS: All patients reconstituted hematopoiesis. The median follow-up duration was 504 (18-3043) days. Transplant-related mortality was 10%. The probabilities of 3 year disease-free survival (DFS) for ALL in CR(1) and CR(2) were 67.7% +/- 10.3% and 16.7% +/- 15.2%, respectively (P = 0.00547); the 3 year DFS was significantly better with posttransplant treatment than without it (92.3% +/- 7.4% vs 50.0% +/- 17.7%, P = 0.0130). CONCLUSION: Acute lymphoblastic leukemia patients without HLA-matched related donor are recommended for ASCT in CR(1). To reduce relapse and improve the outcome, adoptive immunotherapy or maintenance chemotherapy should be given after ASCT.

Adolescent↗

PubMed2000

[The development of immune-mediated aplastic anemia in mice can be blocked by cimetidine].

OBJECTIVE: To investigate the effect of H(2) receptor antagonist cimetidine on the development of immune-mediated aplastic anemia (AA) in mice. METHODS: The immune-mediated aplastic anemia mice model was induced by irradiation and half-matched allogeneic lymphocytes transfusion (ALT). The mice were divided into 3 groups: the irradiation alone control group, the irradiation + ALT group (AA group) and the irradiation + ALT + cimetidine group. In cimetidine group, each mouse was intraperitoneally injected cimetidine at different doses once a day for 8 days. On day 14, the bone marrow histology was examined. RESULTS: In 0.5 mg, 1.0 mg and 2.0 mg x (10g)(-1) x d(-1) cimetidine groups, the percentages of hematopoietic tissue volume in bone marrow were (46.58 +/- 20.41)%, (51.88 +/- 24.94)% and (59.12 +/- 32.48)%, respectively, and all much higher than that in AA group [(19.99 +/- 10.98)%, P < 0.01], but were not different from that in irradiated control group [(53.81 +/- 31.72)%, P > 0.05]. CONCLUSION: The development of immune-mediated aplastic anemia in irradiated mice by half-matched allogeneic lymphocytes transfusion was blocked by H(2) receptor antagonist cimetidine.

Anemia, Aplastic↗

PubMed2000

Salt-loading and simulated microgravity on baroreflex responsiveness in rats.

Cardiovascular adaptations observed during exposure to microgravity results in impairment of baroreflex activity partially as a result of fluid and electrolyte shifts. The head-down tilt rat model mimics some of the physiological observations that have been made in astronauts. We examined the effects of salt-loading on baroreflex activity after 7 day simulated microgravity (30 degrees tail-suspension) and the subsequent 6 hr post-suspension in Sprague-Dawley (SD) rats, using low salt (0.3% NaCl) and high salt (8% NaCl) diets. In suspended animals on a low salt diet, the baroreflex response curve was shifted to the left, while the heart rate (HR) range and MAP50 values were reduced compared to their parallel tethered, non-suspended controls. For non-suspended animals, salt-loading shifted the curve to the right with a reduced HR range. In salt-loaded, suspended animals, the curve and its parameters resemble those of non-suspended animals on a low salt diet. In summary, these data have demonstrated that a short-term (seven days) simulated weightlessness may elicit cardiovascular deconditioning in rats after release from the simulation manifested as an altered responsiveness in baroreceptor-heart rate reflex and a lowered blood pressure while the rats are tethered and horizontal. Our results also suggest the counteracting effect of salt loading on cardiovascular deconditioning.

Animals↗

PubMed2000

[Effect of chrysotile on nitric oxide production and anti-oxidasic activity in rabbit alveolar macrophages].

This study aimed to explore the role of nitric oxide (NO) and nitric oxide synthase (NOS) in chrysotile asbestos-caused diseases. The production of NO2-/NO3- (the final product of NO) and the alterations of NOS, SOD and GSH-Px activity were investigated when rabbit alveolar macrophages (AM) were stimulated by UICC chrysotile. The results revealed that with the dosage elevation of UICC chrysotile, the rabbit alveolar macrophages showed: (1) increased mortality, decreased survival activity; (2) increased product of NO2-/NO3-, decreased SOD and GSH-Px activity; (3) increased NOS activity in the lower dosage groups, but decreased NOS activity in the higher dosage groups. There was significantly negative correlation between NO release and SOD (or GSH-Px) activity (r1 = -0.7125, P < 0.05; r2 = -0.8496, P < 0.05 respectively). Moreover, the significantly positive correlation between NO release and NOS activity was found in the lower dosage groups, but significantly negative correlation in the higher dosage groups. These findings suggest that chrysotile could induce the alveolar macrophages to increase NO release and to decrease SOD and GSH-Px activity, which may play a role in the process of asbestos-caused diseases.

Animals↗

PubMed2000

[TMJ radiographic bone alterations in patients of TMJDs].

OBJECTIVE: The characteristics of temporomandibular joint (TMJ) bone changes showed by X-ray films in patients diagnosed as Temporomandibular Joint Disorders (TMJDs) were investigated. METHODS: X-ray films of 792 TMJDs patients who were diagnosised as TMJDs in our hospital from 1988 to 1995 were studied. Among them, 330 cases were male and 462 cases were female. Their ages ranged from 11 to 66 years old, averaged 27.4 years old. Bilateral TMJ, 1584 sides in all, were checked by X-ray. Among them, 210 cases (420 sides) were examined with transcraniall projective technique, 223 cases (446 sides) with tomogram, and 359 cases (718 sides) with both tomogram and transcranial projective techniques, Clinician observed and discussed the films together with the radiographer, and then the final radio-diagnosis was got. RESULTS: 1. 288 (25.31%) out of 1138 condyles examined with transcranial projective technique (temporal articular surface can not be observed accurately in this kind of films) and 82 (7.04%) out of 1164 condyles, 74 (6.36%) out of 1164 temporal articular surfaces checked with tomogram showed osseous changes. With analyzing on 718 sides of 356 cases checked with both methods, it was found that transcranial projective technique was more sensitive to TMJ osseous changes (P < 0.01). 2. The types of osseous changes: within the osseous changes of condyles showed in transcranial projection, 126 sides (43.75%) were mainly hyperplasia, and 140 sides showed absorptive changes. Moreover, both kinds of alterations appeared in 22 sides (7.64%). Among the condyles with osseous changes showed in tomogram, 28 sides were mainly hyperplasia and 54 sides showed absorptive changes, while two kinds of changes were not found in the same tomogram. But for temporal articular surface, 34 sides showed hyperplasia and 6 sides absorptive changes as their chief alterations. And the other 34 sides showed the two kinds of changes. Hyperplasia appeared more frequently in temporal articular surface than in condyles showed in tomogram (P < 0.01). 3. It was found that the osseous alterations were liable to occur in anterior condyle slope (88.19% in the films checked with transcranial projective technique, 81.71% in the films checked with tomography), the top (72.97% in the films checked with tomography) and the posterior slopes (71.62% in the films checked with tomography) of the articular eminence. CONCLUSION: Being considered the characteristics of the osseous changes, the abnormal mechanics around ICP and physiological or pathological remodeling principles of different parts of TMJ might be explained. Further researches on the selection of the two examinational methods of TMJ were suggested.

Adolescent↗

PubMed2000

[Herbological study for the Beimu categorical Chinese medicine on the original plants].

This article reports the herbological study of "Beimu" categorical Chinese medicine on the original plants. The result showed that the medicinal "Beimu" before "Weijin" period is "Jia Beimu" (Bolbostemma paniculatum); "Nenbei" daynasty began to used "Zhe Beimu" (Fritillaria thunbergii), but "Jia Beimu" and "Hubei Beimu" (F. hupehensis) occupied an important position continuously; Last phase of "Ming" dynasty began to used the "Zhe Beimu" and "Chuan Beimu" (F. cirrhosa, F. unibracteato, F. przewalskii and F. delavayi) for "Beimu" greatly. The "Zhe Beimu" from Xiangshan, Zhejiang Province, is named "Xiang Beimu" and others are named "Tu Beimu" and the "Chuan Beimu" included the "Yi Beimu" (F. walujuewii and F. pallidiflora) continuously. So that, authors suggested that the contemporary documents such as Chinese Pharmcopoeia must recover the name "Beimu" to change the name "Tu Beimu" so as to avoid chaos with the "Tu Beimu" of the herbological documents; "Hubei Beimu" may take the place of "Zhe Beimu" but don't substitue for "Chuan Beimu"; "Yi Beimu" may be merged into "Chuan Beimu" to gear to actual circumstances and history; "Ping Beimu" to take the place of "Chuan Beimu" is not foundation from herbological documents.

Drug Contamination↗

PubMed2000

The analysis of pedigree GZ (Guangzhou). 1 with primary open angle glaucoma.

PURPOSE: To analyze the hereditary modality of primary open-angle glaucoma in China. METHODS: The genetic form of Pedigree GZ. 1 was analyzed using Mandalian hereditary rules. RESULTS: Pedigree GZ. 1 had following characteristics: 1) The pedigree had four generations, and there existed POAG patients in each generation; 2) Each patient had a parent with POAG. If the parents didn't suffer from the disease, their children would not. 3) The incidence of POAG in the relatives of the patients was 1/2. In addition, The age of onset, intraocular pressure, fundus and prognosis was different from each other in the patients. CONCLUSIONS: 1) Pedigree GZ. 1 is inherited as an autosomal dominant trait. 2) There exists individual differences of clinical manifestations in POAG patients.

Adolescent↗

PubMed2000

[Trannasal-transsphenoidal endoscopic surgery of the sphenoid sinus and the sella turcica].

OBJECTIVE: The primary objective of this study is reporting our experience with transnasal endoscopic technique to management of the sphenoidal and the sellar lesions. METHOD: Forty one patients were management under endoscope who suffered from pituitary adenomas, craniopharyngioma and sphenoidal lesions, et al. RESULTS: In forty one cases of the sphenoidal and the sellar lesions, thirty two surgeries were successful. One patient died of bleeding, five developed the foramen of nasal septum, the adhesion of nasal cavity was complicated in three patients. CONCLUSION: The endoscope has the advantage of improved visualization, angled view, and a wide panoramic perspective. The transnasal-transspheniodal endoscopic technique is a effective, minimally invasive approach. But there are its disadvantages and occurred severe complication. Surgeons should be understood its feasibility, limits and indication.

Adolescent↗

PubMed2000

[Nickel cation biosorping studies by yeast with dimethylglyoxime spectrophotometry].

The beer yeast as the biosorbent of Ni2+ is studied. Nickel cation was determined by dimethylglyoxime spectrophotometry. The adsorbed quantity is larger at pH = 6. The equilibrium time was 60 minutes. Salinity and Ca2+, Mg2+ have no significant effect on the biomaterial uptake. Higher the equilibrium concentration(c) is, larger the adsorbed quantity(q) of yeast is. In addition, relations between c and q were fitted with followed equations: 1/q = 0.340 + 0.0753 x 1/c, r = 0.995; lgq = 3.989 + 0.789 lgc, r = 0.995. So we concluded that the models of adsorption are fitted with Langmuir and Freundlich isotherm adsorption equation.

Adsorption↗

PubMed1999

Involvement of DNA-dependent protein kinase in UV-induced replication arrest.

Cells exposed to UV irradiation are predominantly arrested at S-phase as well as at the G(1)/S boundary while repair occurs. It is not known how UV irradiation induces S-phase arrest and yet permits DNA repair; however, UV-induced inhibition of replication is efficiently reversed by the addition of replication protein A (RPA), suggesting a role for RPA in this regulatory event. Here, we show evidence that DNA-dependent protein kinase (DNA-PK), plays a role in UV-induced replication arrest. DNA synthesis of M059K (DNA-PK catalytic subunit-positive (DNA-PKcs(+))), as measured by [(3)H]thymidine incorporation, was significantly arrested by 4 h following UV irradiation, whereas M059J (DNA-PKcs(-)) cells were much less affected. Similar results were obtained with the in vitro replication reactions where immediate replication arrest occurred in DNA-PKcs(+) cells following UV irradiation, and only a gradual decrease in replication activity was observed in DNA-PKcs(-) cells. Reversal of replication arrest was observed at 8 h following UV irradiation in DNA-PKcs(+) cells but not in DNA-PKcs(-) cells. Reversal of UV-induced replication arrest was also observed in vitro by the addition of a DNA-PK inhibitor, wortmannin, or by immunodepletion of DNA-PKcs, supporting a positive role for DNA-PK in damage-induced replication arrest. The RPA-containing fraction from UV-irradiated DNA-PKcs(+) cells poorly supported DNA replication, whereas the replication activity of the RPA-containing fraction from DNA-PKcs(-) cells was not affected by UV, suggesting that DNA-PKcs may be involved in UV-induced replication arrest through modulation of RPA activity. Together, our results strongly suggest a role for DNA-PK in S-phase (replication) arrest in response to UV irradiation.

Androstadienes↗

PubMed1999

Zinc finger of replication protein A, a non-DNA binding element, regulates its DNA binding activity through redox.

Eukaryotic replication protein A (RPA) is a single-stranded DNA-binding protein with multiple functions in DNA replication, repair, and genetic recombination. RPA contains an evolutionarily conserved 4-cysteine-type zinc finger motif (X(3)CX(2-4)CX(12-15)CX(2)C) that has a potential role in regulation of DNA replication and repair (Dong, J., Park, J-S., and Lee, S-H. (1999) Biochem. J. 337, 311-317 and Lin, Y.-L., Shivji, M. K. K., Chen, C., Kolodner, R., Wood, R. D., and Dutta, A. (1998) J. Biol. Chem. 273, 1453-1461), even though the zinc finger itself is not essential for its DNA binding activity (Kim, D. K., Stigger, E., and Lee, S.-H. (1996) J. Biol. Chem. 271, 15124-15129). Here, we show that RPA single-stranded DNA (ssDNA) binding activity is regulated by reduction-oxidation (redox) through its zinc finger domain. RPA-ssDNA interaction was stimulated 10-fold by the reducing agent, dithiothreitol (DTT), whereas treatment of RPA with oxidizing agent, diazene dicarboxylic acid bis[N,N-dimethylamide] (diamide), significantly reduced this interaction. The effect of diamide was reversed by the addition of excess DTT, suggesting that RPA ssDNA binding activity is regulated by redox. Redox regulation of RPA-ssDNA interaction was more effective in the presence of 0.2 M NaCl or higher. Cellular redox factor, thioredoxin, was able to replace DTT in stimulation of RPA DNA binding activity, suggesting that redox protein may be involved in RPA modulation in vivo. In contrast to wild-type RPA, zinc finger mutant (cysteine to alanine mutation at amino acid 486) did not require DTT for its ssDNA binding activity and is not affected by redox. Together, these results suggest a novel function for a putative zinc finger in the regulation of RPA DNA binding activity through cellular redox.

Amino Acid Sequence↗

PubMed1999

[The complete nucleotide sequences of A/Goose/Guangdong/2/96(H5N1) virus RNA segment 1-3 and 5].

OBJECTIVE: To determine the nucleotide and amino acid sequences of PB2, PB1, PA and NP genes and compared them with sequences of A/HK/156/97(H5N1) virus for revealing the relationship between A/Googs/Guangdong/2/96(H5N1) and A/HK/156/97(H5N1) viruses. METHODS: Virion RNA was transcribed into cDNA by reverse transcriptase, cDNA amplified by PCR, the productions of PCR were purified. Afterward, RNA sequence analysis was performed by the dideoxynucleotide chain termination method, using synthetic oligodeoxynucleotide primers. RESULTS: The lengths of A/Goose/Guangdong/2/96(H5N1) virus RNA segment 1-3 and 5 contain 2,341, 2,341, 2,233 and 1,565 nucleotides, respectively. They encode for PB2 (759 amino acids), PB1 (757 amino acids), PA (716 amino acids) and NP (498 amino acids) proteins. The homologies of amino acid sequences of PB2, PB1, PA and NP proteins between A/Goose/Guangdong/2/96 (H5N1) and A/HK/156/97 (H5N1) virus are 96.4%, 97.2%, 97.3% and 97.0%, respectively. CONCLUSION: The lengths of RNA segment 1-3 and 5 of Goose strain are 2,341, 2,341, 2,233 and 1,565 nucleotides, respectively. The nucleotide sequences of these genes are distinguish able from those of Hong Kong virus.

Amino Acid Sequence↗

PubMed1999

Crystal structures of two alpha-like scorpion toxins: non-proline cis peptide bonds and implications for new binding site selectivity on the sodium channel.

The crystal structures of two group III alpha-like toxins from the scorpion Buthus martensii Karsch, BmK M1 and BmK M4, were determined at 1.7 A and 1.3 A resolution and refined to R factors of 0.169 and 0.166, respectively. The first high-resolution structures of the alpha-like scorpion toxin show some striking features compared with structures of the "classical" alpha-toxin. Firstly, a non-proline cis peptide bond between residues 9 and 10 unusually occurs in the five-member reverse turn 8-12. Secondly, the cis peptide 9-10 mediates the spatial relationship between the turn 8-12 and the C-terminal stretch 58-64 through a pair of main-chain hydrogen bonds between residues 10 and 64 to form a unique tertiary arrangement which features the special orientation of the terminal residues 62-64. Finally, in consequence of the peculiar orientation of the C-terminal residues, the functional groups of Arg58, which are crucial for the toxin-receptor interaction, are exposed and accessible in BmK M1 and M4 rather than buried as in the classical alpha-toxins. Sequence alignment and characteristics analysis suggested that the above structural features observed in BmK M1 and M4 occur in all group III alpha-like toxins. Recently, some group III alpha-like toxins were demonstrated to occupy a receptor site different from the classical alpha-toxin. Therefore, the distinct structural features of BmK M1 and M4 presented here may provide the structural basis for the newly recognized toxin-receptor binding site selectivity. Besides, the non-proline cis peptide bonds found in these two structures play a role in the formation of the structural characteristics and in keeping accurate positions of the functionally crucial residues. This manifested a way to achieve high levels of molecular specificity and atomic precision through the strained backbone geometry.

Amino Acid Sequence↗

PubMed1999

Reversible exposure of human platelet fibrinogen receptors by antiplatelet tetraspanin monoclonal antibodies via induction of a conformational change in membrane glycoprotein IIb/IIIa complex.

Antihuman platelet tetraspanin (CD9 antigen) monoclonal antibodies, HI117 and SJ9A4, can induce human platelet aggregation and secretion. As platelet aggregation is mediated by fibrinogen binding to its receptors exposed on platelet glycoprotein IIb/IIIa complex, we, therefore, investigated the induction of platelet fibrinogen receptors by HI117 and SJ9A4. It was found that HI117 and SJ9A4 induced specific fibrinogen binding to human platelets, suggesting that the two monoclonal antibodies evoked obvious exposure of fibrinogen receptors on human platelets. But in the absence of fibrinogen, the monoclonal antibody-exposed fibrinogen receptors gradually lost their capacity to bind fibrinogen and closed. Our results also showed that HI117 and SJ9A4, when activating platelets, caused a conformational change in glycoprotein IIb/IIIa complex, which must contribute to the exposure of functional fibrinogen receptors on this integrin. The effect of HI117 and SJ9A4 on glycoprotein IIb/IIIa complex seems, however, to be indirect, because the HI1117 and SJ9A4-induced fibrinogen binding was reduced by pretreatment of platelets with sphingosine, aspirin, apyrase, and/or PGI2. Taken together, we conclude that the antihuman platelet tetraspanin monoclonal antibodies, HI117 and SJ9A4, reversibly expose platelet fibrinogen receptors via inducing a conformational change in glycoprotein IIb/IlIa complex. Three signaling pathways, that is, thromboxane, secreted ADP, and cAMP pathways may be involved in this process, while protein kinase C activation seems to be the final common step of the three pathways.

Antibodies, Monoclonal↗

PubMed1999

Structural requirement of the calcium-channel subunit alpha2delta for gabapentin binding.

Gabapentin [Neurontin, 1-(aminomethyl)cyclohexaneacetic acid] is a novel anticonvulsant drug with a high binding affinity for the Ca(2+)-channel subunit alpha(2)delta. In this study, the gabapentin-binding properties of wild-type and mutated porcine brain alpha(2)delta proteins were investigated. Removal of the disulphide bonds between the alpha(2) and the delta subunits did not result in a significant loss of gabapentin binding, suggesting that the disulphide linkage between the two subunits is not required for binding. Singly expressed alpha(2) protein remained membrane associated. However, alpha(2) alone was unable to bind gabapentin, unless the cells were concurrently transfected with the expression vector for delta, suggesting that both alpha(2) and delta are required for gabapentin binding. Using internal deletion mutagenesis, we mapped two regions [amino acid residues 339-365 (DeltaF) and 875-905 (DeltaJ)] within the alpha(2) subunit that are not required for gabapentin binding. Further, deletion of three other individual regions [amino acid residues 206-222 (DeltaD), 516-537 (DeltaH) and 583-603 (DeltaI)] within the alpha(2) subunit disrupted gabapentin binding, suggesting the structural importance of these regions. Using alanine to replace four to six amino acid residues in each of these regions abolished gabapentin binding. These results demonstrate that region D, between the N-terminal end and the first putative transmembrane domain of alpha(2), and regions H and I, between the putative splicing acceptor sites (Gln(511) and Ser(601)), may play important roles in maintaining the structural integrity for gabapentin binding. Further single amino acid replacement mutagenesis within these regions identified Arg(217) as critical for gabapentin binding.

Acetates↗