Biomedical subjects

X Lin

Publications and source records attributed to X Lin.

At least 199 records · Page 11Linked to original sources

PubMed1999

Selection and characterization of feather-degrading bacteria from canola meal compost.

Canola meal that contains a high level of protein (40% crude protein) was used as compost material for the isolation of feather-degrading bacteria. After 7 and 14 days, bacteria were isolated from compost amended and unamended with soil. Eighty bacterial isolates from canola meal compost were then grown on milk-agar and isolates that produced proteolytic enzymes were identified by the formation of clear haloes around the colonies. A feather medium was chosen for a secondary selection of feather-degrading isolates. Of the eight isolates that hydrolyzed milk protein, five isolates hydrolyzed feathers. Their keratinolytic activities were subsequently confirmed by an assay using azo-keratin as substrate. Seven of the eight bacteria that hydrolyzed milk protein were Bacillus spp, and all five isolates that hydrolyzed feathers were strains of Bacillus licheniformis. Protease inhibition studies indicated that serine proteases are the predominant proteolytic enzymes produced by these feather-degrading isolates.

Journal Article↗

PubMed1999

Activation and disturbance of blood haemostasis following strenuous physical exercise.

Physical exercise activates blood coagulation and enhances fibrinolytic activity. To investigate whether these activations of blood coagulation and fibrinolysis are balanced post-exercise and during the period of recovery, 11 moderately active young men were examined immediately after a standardised cycle ergometer test and during the 24 h period of recovery. Blood samples were obtained at rest, immediately after exercise, and 2, 6 and 24 h after exercise. All post-exercise values were corrected for any change in plasma volume. Exercise induced a significant increase in factor VIII activity and this occurred with a significant shortening of activated partial thromboplastin time. A concomitant enhancement of tissue plasminogen activity resulted in significant increases in tissue plasminogen activity antigen and total fibrin/fibrinogen degradation products, and a significant decrease in tissue plasminogen activator inhibitor-1 activity. Increases in coagulation and fibrinolytic activity changed in parallel during exercise. However, during recovery, while the increase in factor VIII activity post-exercise persisted 2 and 6 h into recovery, fibrinolytic activity demonstrated a sharp fall. It is concluded that whereas the enhanced fibrinolytic activity during exercise appears to counterbalance the increase in blood coagulability, this haemostatic balance is not maintained during recovery. This perturbed blood haemostasis could constitute an enhanced risk for coronary artery thrombosis and may contribute to exercise-related cardiovascular events.

Adult↗

PubMed1999

The effect of moderate alcohol ingestion on blood coagulation and fibrinolysis at rest and in response to exercise.

The effect of alcohol ingestion before exercise on blood haemostasis is not known. The present study examined the effects of moderate alcohol ingestion on blood haemostatic variables at rest and in response to exercise. Eleven normal healthy individuals randomly performed two tests separated by 7 days. A moderate dose of ethanol (0.5 g.kg-1) was administered before one test, whereas an equal volume of an alcohol-free drink was administered before the other. Forty-five minutes after the ingestion of either drink, the participants cycled at 65% VO2max for 30 min followed by a 5-min all-out performance. Venous blood samples were obtained before and 45 min after the ingestion of both drinks, and also immediately after exercise. Exercise induced a significant increase in tissue-type plasminogen activator activity and antigen, and factor VIII procoagulant activity. The post-exercise data also showed a significant decrease in plasminogen activator inhibitor activity and soluble fibrin, with a significant shortening in prothrombin time and activated partial thromboplastin time, but not thrombin time. No significant changes were observed in antithrombin III. Although no significant differences were found between trials in the haemostatic and fibrinolytic variables at rest, a significant decrease in fibrinogen concentration was observed after exercise in the alcohol trial. This suggests that ingesting a moderate dose of alcohol does not alter blood coagulation and fibrinolysis at rest. Apart from fibrinogen concentration, which was significantly decreased after exercise in the alcohol trial, most of the haemostatic and fibrinolytic variables were not affected by alcohol. The mechanism responsible for the decrease in fibrinogen following exercise in the alcohol trial remains unknown, but might be related to inhibition of fibrinogen synthesis by the liver or an enhanced rate of its catabolism.

Adult↗

PubMed1999

Changes in methadone treatment practices: results from a panel study, 1988-1995.

Results from several studies conducted in the late 1980s and early 1990s showed that the majority of the nation's methadone treatment units did not use effective treatment practices. Since then, however, many efforts have been made to improve critical treatment practices. This paper examines the extent to which key methadone treatment practices (dose levels, treatment duration, client influence in dose decisions) changed from 1988 to 1995 in a panel sample of methadone maintenance units. We also examine factors that may account for variation in methadone treatment practices. We use panel data from a national random sample of 172 units in 1988 (82% response rate), 140 units in 1990 (87% response rate), and 116 units in 1995 (89% response rate). Unit directors and clinical supervisors provided phone survey data on clients influence on doses, upper limits on doses, average dose levels, unit emphasis on decreasing doses, time when clients are encouraged to detoxify, and average length of treatment. Results from random effects regression analyses indicate that treatment practices concerning methadone dose levels, client influence in dose decisions, and treatment duration improved significantly between 1988 and 1995. Several characteristics of clients (race, age) and treatment units (staff, ownership, geographic location) are associated with the use of less-effective treatment practices. Efforts to improve treatment practices appear to be making progress and certainly need to be continued.

Adult↗

PubMed1999

The promotion effect of anorectic drugs on aflatoxin B(1)-induced hepatic preneoplastic foci.

The ability of three extensively used anorectic drugs, namely fenfluramine (FN), fluoxetine (FX) and amphetamine (AM), to alter the development of aflatoxin B(1) (AFB(1))-induced gamma-glutamyl-positive (GGT(+)) preneoplastic liver foci was investigated in 135 male weanling F344 rats. Following AFB(1) administration, 15 rats were killed, while the rest were divided into four groups and fed diets containing either FN, FX, AM or control diet, with half of the animals in each group subsequently being killed at 4 weeks and half at 10 weeks. All three anorectic drugs as expected suppressed initial food intake, growth rate, body weight gain and food efficiency. They also tended to suppress body fat mass and to decrease plasma levels of T(3) and T(4). FN significantly (P < 0.05) increased GGT(+) foci number/cm(2) and number/cm(3), while FX significantly increased GGT(+) foci number/cm(2) and the volume fraction of foci. Histopathological staining also revealed that FN- and FX-treated animals had more serious morphological alterations in their liver tissue. In contrast, foci development was, if anything, suppressed by AM feeding. These results indicate that serotoninergic drugs (FN and FX), as opposed to dopaminergic drugs (AM), may have tumor promoter activity, at least for liver tissue.

Adipose Tissue, Brown↗

PubMed1999

Absence of spontaneous central nervous system remyelination in class II-deficient mice infected with Theiler's virus.

We previously showed that Theiler's murine encephalomyelitis virus (TMEV)-infected major histocompatibility complex (MHC) class II-deficient mice develop both demyelination and neurologic deficits, whereas MHC class I-deficient mice develop demyelination but no neurologic deficits. The absence of neurologic deficits in the class I-deficient mice was associated with preserved sodium channel densities in demyelinated lesions, a relative preservation of axons, and extensive spontaneous remyelination. In this study, we investigated whether TMEV-infected class II-deficient mice, which have an identical genetic background (C57BL/6 x 129) as the class I-deficient mice, have preserved axons and spontaneous myelin repair following chronic TMEV-infection. Both class I- and class II-deficient mice showed similar extents of demyelination of the spinal cord white matter 4 months after TMEV infection. However, the class I-deficient mice demonstrated remyelination by oligodendrocytes, whereas class II-deficient mice showed minimal if any myelin repair. Demyelinated lesions, characterized by inflammatory infiltrates in both mutants, revealed disruption of axons in class II- but not class I-deficient mice. Further characterization revealed that even though class II-deficient mice lacked TMEV-specific IgG, they had virus-specific IgM, which, however, did not neutralize TMEV in vitro. In addition, class II-deficient mice developed TMEV-specific cytotoxic T-lymphocytes in the CNS during the acute (7 days) disease, but these cytotoxic lymphocytes were not present in the chronic stage of disease, despite a high titer of infectious virus throughout the disease. We envision that the presence of demyelination, high virus titer, absence of remyelination, and axonal disruption in chronically infected class II-deficient mice contributes to the development of paralytic disease.

Acute Disease↗

PubMed1999

SIMEX variance component tests in generalized linear mixed measurement error models.

In the analysis of clustered data with covariates measured with error, a problem of common interest is to test for correlation within clusters and heterogeneity across clusters. We examined this problem in the framework of generalized linear mixed measurement error models. We propose using the simulation extrapolation (SIMEX) method to construct a score test for the null hypothesis that all variance components are zero. A key feature of this SIMEX score test is that no assumptions need to be made regarding the distributions of the random effects and the unobserved covariates. We illustrate this test by analyzing Framingham heart disease data and evaluate its performance by simulation. We also propose individual SIMEX score tests for testing the variance components separately. Both tests can be easily implemented using existing statistical software.

Analysis of Variance↗

PubMed1999

Effect of loss of DNA mismatch repair on development of topotecan-, gemcitabine-, and paclitaxel-resistant variants after exposure to cisplatin.

Loss of DNA mismatch repair (MMR) causes genomic instability by markedly increasing the frequency of sporadic mutations in both coding and noncoding sequences. Little is known about how loss of MMR affects sensitivity to the mutagenic effect of chemotherapeutic agents. We wanted to determine how loss of MMR affects the ability of cisplatin, a known mutagen, to generate human tumor cell variants resistant to other drugs with which cisplatin is commonly combined in treatment regimens. We compared the ability of cisplatin to produce variants resistant to topotecan, gemcitabine, and paclitaxel in two pairs of MMR-proficient and -deficient cells that included sublines of the human colon carcinoma cell line HCT-116 and sublines of the human endometrial adenocarcinoma cell line HEC59. Cells were exposed to increasing concentrations of cisplatin for 1 h, and the surviving population was tested for the frequency of variants resistant to these single molecular target drugs 10 days later. The frequency of variants increased linearly with cisplatin concentration for all three drugs. Cisplatin was 2.6 +/- 0.3- (S.D.), 3.6 +/- 0.9-, and 2.3 +/- 0.1-fold more potent at producing topotecan-, gemcitabine-, and paclitaxel-resistant variants in the MMR-deficient than in the MMR-proficient HCT116 cells (P <.05 for all). Cisplatin was 1.4 +/- 0.3- and 1.4 +/- 0.4-fold more potent at generating topotecan- and gemcitabine-resistant variants in MMR-deficient HEC59 cells than in MMR-proficient HEC59+ch2 cells. Cisplatin was not more potent in generating paclitaxel-resistant variants in the MMR-deficient HEC59 cells. Spontaneous rates of generation of cells resistant to these three drugs were also measured in the HCT116 sublines. MMR-deficient HCT116 cells exhibited rates of generation of resistant variants that were 1.94- and 1.51-fold higher (P <.05) than those in the MMR-proficient cells for topotecan and gemcitabine, respectively; loss of MMR had no effect on the rate of generation of variants resistant to paclitaxel. We conclude that the loss of MMR increases the ability of cisplatin to generate variants resistant to topotecan, gemcitabine, and possibly paclitaxel and that MMR also plays a role in controlling the spontaneous rate of generation of variants resistant to topotecan and gemcitabine.

Antineoplastic Agents↗

PubMed1999

Normalization of atropine-induced postprandial dysrhythmias with gastric pacing.

Gastric pacing has received increasing attention recently. However, few studies have systematically assessed the effect of pacing on gastric dysrhythmias. The aims of this study were to investigate the effect of gastric pacing on gastric dysrhythmia and to explore whether the effect of gastric pacing was mediated via cholinergic nerves. Eight hound dogs implanted with three pairs of serosal electrodes were studied. Three study sessions were performed on each dog. The experiment was conducted sequentially as follows: a 30-min myoelectrical recording immediately after a meal, intravenous injection of atropine or saline, and three sequential 20-min myoelectrical recordings with or without gastric pacing during the second 20-min recording. The percentage of regular slow waves (3.5-7. 0 cycles/min) was calculated using spectral analysis. The percentage of the regular slow waves was progressively reduced from 96.7 +/- 1. 7% at baseline to 29.6 +/- 9.0 (P < 0.001), 23.1 +/- 7.1 (P < 0.001), and 27.3 +/- 4.3% (P < 0.001), respectively, during the first, second, and third 20 min after atropine injection. Normalization of the gastric slow wave was achieved with gastric pacing 2.3 +/- 1.0 min after the initiation of pacing. The percentage of regular slow waves was significantly increased both during pacing (93.6 +/- 2.4 vs. 23.1 +/- 7.1%, P < 0.002) and after pacing (70.9 +/- 6.8 vs. 27. 3 +/- 4.3%, P < 0.003) in comparison with the session without pacing. We conclude that 1) atropine induces gastric myoelectric dysrhythmia in the fed state, 2) gastric pacing is able to normalize gastric postprandial dysrhythmia induced by atropine, and 3) the effect of gastric pacing is not mediated by vagal cholinergic mechanism.

Animals↗

PubMed1999

Detection of gastric slow wave propagation from the cutaneous electrogastrogram.

The gastric slow wave is originated in the proximal stomach and propagates distally toward the pylorus. It determines the maximum frequency and propagation of gastric contractions. The aim of this study was to detect the propagation of the gastric slow wave from the surface electrogastrogram (EGG). The study was performed in 11 healthy subjects of normal weight. Gastric myoelectrical activity was recorded for 1 h in the fasting state with the use of a specially designed multichannel recording device that was composed of four identical amplifiers with cutoff frequencies of 1.8 and 16.0 cycles/min. Four active electrodes were placed on the abdomen along the gastric axis and were connected to a common reference electrode placed near the xiphoid process, yielding four-channel bipolar EGG signals. Cross-covariance analysis was performed to compute the time lag among the four channels. There was a time lag in EGG waveform between channels 1 and 4 (9.6 +/- 1.1 s); the average time during which the time lag was observed (>/=3 s) was 89.9 +/- 9.0%. There was a significant difference in the time lag among different adjacent channels (P < 0.04); the time lag observed between channels 3 and 4 was significantly smaller than that between channels 1 and 2 (P < 0.03). No correlation was found between the body mass index and the time lag between channels 1 and 4 (r = -0.31, P = 0.3). It was concluded that, with a multichannel recording device with identical multiamplifiers and an appropriate arrangement of abdominal electrodes, the propagation of the gastric slow wave could be identified from the EGG in healthy subjects. This method may be used to detect the coupling of the gastric slow wave noninvasively.

Adult↗

PubMed1999

Human NDUFB9 gene: genomic organization and a possible candidate gene associated with deafness disorder mapped to chromosome 8q13.

Human NADH dehydrogenase (ubiquinone) 1beta-subcomplex, 9 (NDUFB9) is a nuclear encoded mitochondrial protein with the respiratory electron transport chain. It has been physically mapped to a 1-Mb deletion at chromosome 8q13 which also contains the gene for branchio-oto-renal (BOR) syndrome. BOR syndrome is characterized by branchial and renal abnormalities with hearing impairment. Since several hereditary deafness disorders have been associated with mitochondrial mutations, NDUFB9 was considered a candidate gene for BOR syndrome. Recently, EYA1 gene has been identified in the region which underlies the BOR syndrome but majority of BOR families did not show mutations in the EYA1 gene. Here we have determined the genomic structure of the NDUFB9 gene, including the nucleotide sequence, organization and the boundaries of the four coding exons. PCR primers were designed from the adjacent intron sequences that allow amplification of the four exons that encode the complete open reading frame. To identify whether mutations in NDUFB9 are involved in causing the BOR syndrome, we screened 9 BOR families which did not show mutations in the EYA1 gene by heteroduplex analysis; however, no mutations were found.

Amino Acid Sequence↗

PubMed1999

Sensitivity comparison for detection of respiratory bovine coronaviruses in nasal samples from feedlot cattle by ELISA and isolation with the G clone of HRT-18 cells.

A monoclonal antibody-based capture enzyme-linked immunosorbent assay (ELISA) was developed to detect respiratory bovine coronavirus (RBCV) antigens in nasal swabs collected from cattle showing signs of respiratory tract disease following shipping. These samples had been previously tested for RBCV by inoculation of G clone cultures of human rectal tumor cells (HRT-18G) and for bovine herpes virus 1, parainfluenza virus 3, bovine adenovirus, bovine respiratory syncytial virus, and bovine viral diarrhea virus on other specifically permissive cell cultures. RBCV has not previously been recognized as an important etiological factor in the bovine respiratory disease complex of feedlot cattle. Thirty of 100 samples tested positive for RBCV antigen by capture ELISA in contrast to 38 of 100 samples that yielded RBCV isolates in G clone cells. Samples yielding other bovine respiratory viruses in the absence of RBCV were negative in the capture ELISA, which was based on the use of a single monoclonal antibody that recognizes one RBCV epitope on the S glycoprotein with the broadest reactivity with different strains of RBCV tested. Some RBCV strains may not be detected by this ELISA, which may account for the higher percentage of RBCV-infected cattle detected by RBCV isolation. However, the ELISA was simple to perform, sensitive, and specific and was more rapid than virus isolation. This assay will be useful for processing large numbers of field samples in future epidemiologic and diagnostic studies of RBCV infections of cattle.

Animals↗

PubMed1999

Do market-level hospital and physician resources affect small area variation in hospital use?

This study evaluates the effect of market-level physician and hospital resources on hospital use. It is anticipated that higher hospital discharges are associated with (1) greater hospital and physician resources, (2) more differentiated hospital and physician resources, and (3) higher levels of teaching intensity in the community. Data on 14 modified diagnostically related groups (DRGs) and 58 hospital market communities in Michigan are analyzed during a 7-year period. Findings indicate that physician resources, hospital resources, differentiation of hospital and physician resources, and teaching intensity contribute only modestly to discharges, holding constant the socioeconomic attributes of the community and adjusting for the variation in hospital use over time. With the inclusion of hospital and physician resource variables, socioeconomic factors remain important determinants of the variation across market communities. Findings are discussed in terms of their implications for health care organizations, managed care programs, and cost control efforts in general.

Adult↗

PubMed1999

Toxicity Screening of a Combinatorial Library: Correlation of Cytotoxicity and Gene Induction to Compound Structure.

Combinatorial chemistry has increased the number of compounds available for efficacy and safety assessment by several orders of magnitude and has made high throughput assays essential. To test whether higher throughput toxicity assays could be of utility in screening compounds in early development, a selected set of combinatorial chemistry compounds was screened for induction of 70-Kd heat shock protein (HSP70) and 45-Kd growth arrest and DNA damage protein (GADD45) mRNA levels as well as cytotoxicity, in HepG2 cells, using a 96-well microtiter plate format. Both assays, the branched DNA (Quantigene) assay for mRNA levels and MTT for cytotoxicity, were robust enough to be incorporated into a screening format using a single replicate and a single concentration of compound. Significantly, a structure/toxicity correlation was established with this set of compounds with cytotoxicity and gene induction patterns linked to compound structure. Therefore, this type of early screening may be useful in identifying toxic substituents, enabling the design of libraries with less potential for toxicity. While structure/toxicity correlations were observed, no relationship was observed between GADD45 gene induction and mutagenesis as measured by the Ames bacterial reverse mutation assay.

Journal Article↗

PubMed1999

Transcriptional activation of gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: involvement of multiple signal transduction pathways.

Previous studies have shown that GnRH activates transcriptional activity of its own receptor (GnRHR) gene in part through the cAMP signal transduction pathway. In the present study we explored the possible involvement of multiple signal transduction pathways in GnRH regulation of GnRHR gene transcription; these studies relied upon a luciferase reporter gene vector (GnRHR-pXP2) containing a 1226-bp promoter fragment (-1164 to +62, relative to the major transcription start site) of the mouse GnRHR gene in GGH3 cells (GH3 cells stably expressing rat GnRHR). Activation of protein kinase C (PKC) by phorbol myristic acid significantly stimulated GnRHR-luciferase reporter gene (GnRHR-Luc) activity, but did not potentiate the stimulation of GnRHR-Luc activity by the GnRH agonist, buserelin (GnRH-A). Inhibition of PKC by PKC inhibitor (GF 109203X) or depletion of PKC blocked phorbol myristic acid- or GnRH-A-stimulated GnRHR-Luc activity, but did not affect (Bu)2cAMP-stimulated GnRHR-Luc activity. In addition, GnRH-A-stimulated GnRHR-Luc activity was inhibited by preventing external Ca2+ influx with the external Ca2+ chelator EGTA or the Ca2+ ion channel antagonist, D600. Surprisingly, overexpression of the mitogen-activated protein kinase (MAPK) kinase kinase (Raf-1) inhibited GnRHR-Luc activity and partially blocked GnRH-A-stimulated GnRHR-Luc activity. In contrast, inhibition of MAPK activity by MAPK kinase inhibitor (PD 98059) or by overexpression of kinase-deficient MAPKs activated basal and GnRH-A-stimulated GnRHR-Luc activity. These results suggested that PKC- and Ca2+-dependent signal transduction pathways participate in the GnRH activation of GnRHR promoter activity, and that the MAPK cascade is involved in the negative regulation of basal and GnRH-stimulated GnRHR transcriptional activity conferred by the 1226-bp promoter fragment.

Animals↗

PubMed1999

Molecular cloning and expression of two type one somatostatin receptors in goldfish brain.

Somatostatin (SRIF or SS) exerts diverse inhibitory actions through binding to specific receptors. In this study, two SRIF receptor complementary DNAs (cDNAs) were cloned and sequenced from goldfish brain using PCR and cDNA library screening. The two cDNAs share 92% similarity in nucleotide sequence and 98% similarity in the deduced amino acid sequences and are presumably derived from duplicate genes, as goldfish are tetraploid. Two cDNAs encode two 367-amino acid goldfish type one SRIF receptors (designated as sst1A and sst1B, respectively), with seven putative transmembrane domains (TMD) and YANSCANP motif in the 7th TMD, a signature sequence for mammalian SRIF receptor (sst) family. In addition, the amino acid sequences of two receptors have 76% and 75% similarity to human or rat sst1, respectively, and 39-55% similarities to other mammalian sst subtypes (sst2-5), suggesting that the two receptors could be the goldfish homologs of mammalian sst1. The difference between goldfish and mammalian sst1 is mainly reflected by the extreme divergence in their extracellular N termini. Both SRIF-14 and [Pro2]SRIF-14, two of the native goldfish SRIF forms, significantly inhibited forskolin-stimulated cAMP release in COS-7 cells transiently expressing goldfish sSt1A or sst1B, suggesting functional coupling of the two receptors to adenylate cyclase. Northern blot and RT-PCR showed that messenger RNAs (mRNAs) for both receptors are widely distributed throughout goldfish brain, whereas only one receptor mRNA is expressed in the pituitary. RT-PCR analysis also detected sst1 receptor mRNAs in several peripheral tissues. These findings provide fundamental information for studying the mechanism of SRIF actions in vertebrates and structural analysis of mammalian sst receptors.

Amino Acid Sequence↗

PubMed1999

Expression of three distinct somatostatin messenger ribonucleic acids (mRNAs) in goldfish brain: characterization of the complementary deoxyribonucleic acids, distribution and seasonal variation of the mRNAs, and action of a somatostatin-14 variant.

In this study, three somatostatin (SRIF) complementary DNAs (cDNAs) were characterized from goldfish brain. The cDNAs encode three distinct preprosomatostatins (PSS), designated as PSS-I, PSS-II, and PSS-III. The goldfish PSS-I, PSS-II, and PSS-III contain enzymatic cleavage recognition sites, potentially yielding SRIF-14 with sequence identical to mammalian SRIF-14, SRIF-28 with [Glu1, Tyr7, Gly10]SRIF-14 at its C-terminus, and [Pro2]SRIF-14, respectively. The brain distribution of the three SRIF messenger RNAs (mRNAs) were differential but overlapping in the telencephalon, hypothalamus and optic tectum-thalamus regions. Seasonal variations in the levels of the three mRNAs were observed, with differential patterns between the three mRNAs and differences between the sexes. However, only the seasonal alteration in the levels of the mRNA encoding PSS-I showed close association with the seasonal variation in brain contents of immunoreactive SRIF-14 and inversely correlated with the seasonal variation in serum GH levels described in the previous studies, suggesting that SRIF-14 is involved in the control of the seasonal variation in serum GH levels. The putative SRIF-14 variant, [Pro2]SRIF-14, inhibited basal GH secretion from in vitro perifused goldfish pituitary fragments, with similar potency to SRIF-14; [Pro2]SRIF-14 also inhibited stimulated GH release from the pituitary fragments, supporting that [Pro2] SRIF-14 is a biologically active form of SRIF in goldfish.

Amino Acid Sequence↗

PubMed1999

Simultaneous and independent visualization of the gonadotropin-releasing hormone receptor and its ligand: evidence for independent processing and recycling in living cells.

The first step in GnRH signaling is binding by the peptide to its plasma membrane receptor (GnRHR). The receptor is a member of the seven transmembrane G protein-coupled class but lacks the characteristic C-terminal cytoplasmic tail, making it among the smallest receptors in this superfamily. It has been known since 1980 that agonist occupancy of the GnRHR results in patching, capping, and internalization, although it has not been possible to localize the unoccupied GnRHR, because elaboration of receptor antisera has not been easy to achieve. The recent production of a green fluorescent protein (GFP) conjugate of the GnRHR ("rGnRHR-C-tail-GFP") that is expressed in cells, targeted to the plasma membrane, binds GnRH analogs and couples to G proteins has made it possible to monitor movement of the unoccupied receptor by confocal microscopy. In the present study, we used this probe, along with Texas Red conjugates of a GnRH agonist, to examine simultaneous processing of the receptor and its ligands. The preparation of the GFP GnRHR chimera has been described. A Texas Red conjugate was made from the GnRH agonist D-Lys6-Pro9-des-Gly10EA-GnRH by standard procedures. Bioactivity of this conjugate was confirmed. Confocal fluorescence images of living GGH3 cells showed that the agonist binds the GFP-GnRH receptor construct on the cell membrane and causes the internalization of vesicles delimited by a membrane. Shortly after internalization, the agonist separates from receptor inside the vesicle, although it is still enclosed in membranes containing free receptor. As the vesicles approach the perinuclear space, the separation between receptor and agonist is more pronounced. Free receptor appears at the cell membrane after the internalization of agonist has been completed. The protein synthesis inhibitor, cycloheximide (1 mM) did not inhibit this process, suggesting that the free receptor results from the recycling of previously internalized vesicles rather than from newly synthesized receptor. These studies show visual evidence for recycling of the GnRH receptor in cultured cells.

Animals↗