Seizurogenic effect of perfluorooctane sulfonate in zebrafish larvae.

While the developmental neurotoxicity of perfluorooctane sulfonate (PFOS) has been reported, its seizurogenic potential has not been investigated. Behavior assessment was conducted in zebrafish larvae exposed to PFOS at concentrations of 0, 0.1, 1, 5, 10, and 20 µM. Changes in electrophysiological signals and in the concentration of 20 neurochemicals were measured. Behavior assessment revealed that PFOS altered larval behaviors and significantly increased the counts and duration of bursting (an irregular high-speed movement). Electrophysiological analysis showed that the number of seizure-like events and duration of seizure-like signals were significantly increased, corresponding to results observed using pentylenetetrazol as a positive seizurogenic agent. The outbreak of seizures detected via abnormal electrophysiological signals was confirmed by the increased expression of c-fos and bdnf, which are typical seizure-related genes. Analysis of neurochemicals indicated that PFOS dysregulated overall neurotransmission systems, and aberrant endogenous concentrations of various neurochemicals in the amino acid, cholinergic, dopaminergic, serotonergic and kynurenergic, and GABAergic systems were associated with seizure-like behavior and signals. This study, the first to demonstrate that exposure to PFOS provokes a seizurogenic effect in developing zebrafish larvae, should stimulate further research on the association between PFOS exposure and neurodevelopmental toxicity or neurological disorders.

[Real-world study on the efficacy and prognostic predictive biomarker of patients with metastatic non-small cell lung cancer treated with programmed death-1/programmed death ligand 1 inhibitors].

Objective: To describe the actual efficacy of programmed death-1 (PD-1)/ programmed-death ligand 1 (PD-L1) inhibitors in patients with metastatic non-small cell lung cancer (NSCLC) and explore potential prognostic predictive biomarkers. Methods: Patients with metastatic NSCLC who were treated with PD-1/PD-L1 inhibitors at Cancer Hospital, Chinese Academy of Medical Sciences from January 2016 to December 2019, either as monotherapy or in combination with other agents, were consecutively enrolled into this study. We retrospectively collected the data of demographics, clinical information and pathologic assessment to evaluate the therapeutic efficacy and conduct the survival analysis. Major endpoint of our study is progression-free survival (PFS). Secondary endpoints include objective response rate (ORR), disease control rate (DCR) and overall survival (OS). Results: The ORR of 174 patients who underwent PD-1/PD-L1 inhibitor was 28.7%, and the DCR was 79.3%. Immune-related adverse events (irAEs) occurred in 23 patients (13.2%). Brain metastasis, line of treatment, and treatment patterns were associated with the ORR of metastatic NSCLC patients who underwent immunotherapy (P<0.05). After a median follow-up duration of 18.8 months, the median PFS was 10.5 months (ranged from 1.5 to 40.8 months) while the median OS was not reached. The 2-year survival rate was estimated to be 63.0%. The pathologic type was related with the PFS of metastatic NSCLC patients who underwent immunotherapy (P=0.028). Sex, age, brain metastasis and autoimmune diseases were associated with OS (P<0.05). Analysis of the receptor characteristic curve (ROC) of neutrophil/lymphocyte ratio (NLR) predicting ORR of immunotherapy in metastatic NSCLC showed that the areas under the curve of NLR before immunotherapy (NLR(C0)), NLR after one cycle of immunotherapy (NLR(C1)) and ΔNLR were 0.600, 0.706 and 0.628, respectively. Multivariate logistic regression analysis showed that NLR(C1) was an independent factor of the ORR of metastatic NSCLC patients who underwent immunotherapy (OR=0.161, 95% CI: 0.062-0.422), and the efficacy of combination therapy was better than that of single agent (OR=0.395, 95% CI: 0.174-0.896). The immunotherapy efficacy in patients without brain metastasis was better than those with metastasis (OR=0.291, 95% CI: 0.095-0.887). Multivariate Cox regression analysis showed that NLR(C1) was an independent influencing factor of PFS of metastatic NSCLC patients after immunotherapy (HR=0.480, 95% CI: 0.303-0.759). Sex (HR=0.399, 95% CI: 0.161-0.991, P=0.048), age (HR=0.356, 95% CI: 0.170-0.745, P=0.006) were independent influencing factors of OS of metastatic NSCLC patients after immunotherapy. Conclusions: PD-1/PD-L1 inhibitors are proved to be efficacious and have tolerable toxicities for patients with metastatic NSCLC. Patients at advanced age could still benefit from immunotherapy. Brain metastasis is related to compromised response. Earlier application of immunotherapy in combination with other modalities enhances the efficacy without elevating risk of irAEs. NLR(C1) is an early predictor of clinical outcome. The OS of patients younger than 75 years may be improved when treated with immunotherapy.

Effects of the chorion on the developmental toxicity of organophosphate esters in zebrafish embryos.

Many toxicological studies have utilized zebrafish embryos to investigate the developmental toxicity of organophosphate esters (OPEs). However, in respect of the presence or absence of the chorion, a consistent experimental methodology has yet to be developed. In this study, we used a fixed exposure scheme to compare the developmental toxicities of six major OPEs in chorionated and dechorionated zebrafish embryos. Removal of the chorion increased sensitivity to OPEs: we found higher incidence of mortality and malformation in dechorionated embryos. In a behavioral assay, the locomotive activity of zebrafish larvae was consistently inhibited by OPEs except tris (1-chloropropyl) phosphate regardless of chorion presence. However, at the molecular level, the expression of ZHE1 and mmp9 was affected by the presence of the chorion in zebrafish embryos exposed to tributyl phosphate and triphenyl phosphate (TPHP), respectively. Furthermore, in zebrafish embryos exposed to TPHP, the increased expression of miR-137 and miR-141 was abolished by the presence of the chorion. Our results demonstrate for the first time that the presence of the chorion influences phenotypic morbidity, organismal behavior, and gene expression in zebrafish embryos exposed to chemicals; thus, we suggest that dechorionation is desirable for exploring the toxicity mechanisms that underlie effects of chemical exposure.

miR-137 and miR-141 regulate tail defects in zebrafish embryos caused by triphenyl phosphate (TPHP).

Exposure to triphenyl phosphate (TPHP), an organophosphate flame retardants (OPFRs), caused developmental toxicity in zebrafish embryos. However, the underlying molecular mechanism at the epigenetic level is largely unknown. Based on developmental toxicity (i.e., mortality and malformation), we measured expression levels of mRNA genes and their targeted miRNA in zebrafish embryos exposed to TPHP. As a result, TPHP caused developmental delay beginning at the 17-somite stage linking to detrimental effects in the tail and even embryonic mortality. Abnormal tail development was found to be associated with down-regulation of mmp9 and sox9b in both qRT-PCR and whole in-situ hybridization analysis. Also, we identified two microRNAs (i.e., miR-137 and miR-141) and observed their differential over-expression in TPHP-exposed zebrafish embryos. In the microinjection of miR-137 and miR-141 inhibitors, the reduced expression of mmp9 and sox9b upon TPHP exposure was compensated, indicating that epigenetic deregulation of miRNAs modulated putative genes involved in phenotypic tail defects triggered by TPHP in developing zebrafish embryos. This study provides insight for future mechanistic research using teleost fish on function of miRNAs in environmental toxicology.

Predictive models of cytotoxicity as mediated by exposure to chemicals or drugs.

Predicting cytotoxicity is a challenging task because of the complex biological mechanisms behind it. Cytotoxicity due to toxin - biologically produced poison - is known to play a substantial role in a disease process. Two objectives in this research are to derive robust general predictive cytotoxicity models to minimize unnecessary toxicity. The first objective is to build accurate predictive statistical models for cytotoxicity data based on lymphoblastoid cell lines obtained from in vitro studies. This could be an important step for accomplishing a goal in biomedecial/biophamarceutical research, by obtaining the best medical outcomes by minimizing toxicity in regard to a person's genetic profile. The second objective is to build predictive models to predict population-level cytotoxicity for unknown compounds based on chemical structural features. These two objectives were accomplished by a proposed variable selection process, the random forests, and the least absolute shrinkage and selection operator method. We achieved an excellent prediction result with the random forests algorithm using SNP markers from the proposed approach, having the smallest root mean squared error among the teams which participated in the DREAM Toxicogenetics Challenge. Since chemical compounds for drugs have great influence on human health, the predictive statistical models for these objectives could be helpful to government agencies in relevant decision-making.

A novel differentiation pathway from CD4⁺ T cells to CD4⁻ T cells for maintaining immune system homeostasis.

CD4(+) T lymphocytes are key players in the adaptive immune system and can differentiate into a variety of effector and regulatory T cells. Here, we provide evidence that a novel differentiation pathway of CD4(+) T cells shifts the balance from a destructive T-cell response to one that favors regulation in an immune-mediated liver injury model. Peripheral CD4(-)CD8(-)NK1.1(-) double-negative T cells (DNT) was increased following Concanavalin A administration in mice. Adoptive transfer of DNT led to significant protection from hepatocyte necrosis by direct inhibition on the activation of lymphocytes, a process that occurred primarily through the perforin-granzyme B route. These DNT converted from CD4(+) rather than CD8(+) T cells, a process primarily regulated by OX40. DNT migrated to the liver through the CXCR3-CXCL9/CXCL10 interaction. In conclusion, we elucidated a novel differentiation pathway from activated CD4(+) T cells to regulatory DNT cells for maintaining homeostasis of the immune system in vivo, and provided key evidence that utilizing this novel differentiation pathway has potential application in the prevention and treatment of autoimmune diseases.

Decreasing the Viscosity in CO2 Capture by Amino-Functionalized Ionic Liquids through the Formation of Intramolecular Hydrogen Bond.

A strategy for decreasing the viscosity variation in the process of CO2 capture by amino-functionalized ionic liquids (ILs) through the formation of intramolecular hydrogen bond was reported. Different with the dramatic increase in viscosity during CO2 uptake by traditional amino-functionalized ILs, slight increase or even decrease in viscosity was achieved through introducing a N or O atom as hydrogen acceptor into amino-functionalized anion, which could stabilize the active hydrogen of produced carbamic acid. Quantum chemical calculations and spectroscopic investigations demonstrated that the formation of intramolecular hydrogen bond between introduced hydrogen acceptor and carbamic acid was the key to avoid the dramatic increase in viscosity during the capture of CO2 by these amino-functionalized ILs.

Development of an enzymatic pretargeting strategy for dual-modality imaging.

A pretargeted imaging strategy based on the HaloTag dehalogenase enzyme is described. Here, a HaloTag-Trastuzumab conjugate has been used as the primary agent targeting HER2 expression, and three new radiolabelled HaloTag ligands have been used as secondary agents, two of which offer dual-modality (SPECT/optical) imaging capability.

Efficient and Energy-Saving CO2 Capture through the Entropic Effect Induced by the Intermolecular Hydrogen Bonding in Anion-Functionalized Ionic Liquids.

A strategy for improving the capture of CO2 was developed through the entropic effect by tuning the geometric construction of anion-functionalized ionic liquids. Several kinds of anion-functionalized ionic liquids with the amino group at the para or ortho position were designed and applied for the capture of CO2, which indicates that the former exhibited both higher capacity and lower enthalpy, resulting in the efficient and energy-saving CO2 capture. Viscosity measurements, spectroscopic investigations, and quantum chemical calculations showed that such a unique behavior originated from the entropic effect, which was induced by the intermolecular hydrogen bonding in these ionic liquids. The entropic control for gas separation developed by this work provides an efficient strategy to both increased capacity and reduced enthalpy.

Flying doctor emergency airway registry: a 3-year, prospective, observational study of endotracheal intubation by the Queensland section of the Royal Flying Doctor Service of Australia.

OBJECTIVE: To describe the profile and success rates of emergency endotracheal intubation conducted by the Queensland Royal Flying Doctor Service aeromedical retrieval team comprising a doctor and flight nurse. METHOD: Each intubator completed a study questionnaire at the time of each intubation for indications, complications, overall success, drugs utilised and deployment of rescue airway devices/adjuncts. RESULTS: 76 patients were intubated; 72 intubations were successful. None required surgical airway and three were managed with laryngeal mask airways; the remaining failure was managed with simple airway positioning for transport. There were two cardiac arrests during intubation. Thiopentone and suxamethonium were the predominant drugs used to facilitate intubation. CONCLUSION: Despite a low rate of endotracheal intubation, the high success rate was similar to other aeromedical organisations' published airway data. This study demonstrates the utility of the laryngeal mask airway device in the retrieval and transport setting, in particular for managing a failed intubation.

Characterization of Baylisascaris schroederi from Qinling subspecies of giant panda in China by the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA.

In the present study, a total of 20 nematode isolates, (including 10 male and 10 female worms) representing Baylisascaris schroederi from 5 Qinling subspecies of giant pandas (Ailuropoda melanoleuca) in Shaanxi Province of China, were characterized and grouped genetically by the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA). The rDNA fragment spanning 3' end of 18S rDNA, complete ITS-1 rDNA, and 5' end of 5.8S rDNA were amplified and sequenced. The sequence variability in ITS-1 rDNA was examined within B. schroederi and among parasites in order Ascaridata available in GenBank™, and their phylogenetic relationships were also reconstructed. The sequences of ITS-1 rDNA for all the B. schroederi isolates were 427 bp in length, with no genetic variation detected among these isolates. Phylogenetic analyses based on the ITS-1 rDNA sequences revealed that all the male and female B. schroederi isolates sequenced in the present study were posited into the clade of genus Baylisascaris, sistered to zoonotic nematodes in genus Ascaris, and the ITS-1 rDNA sequence could distinguish different species in order Ascaridata. These results showed that the ITS-1 rDNA provides a suitable molecular marker for the inter-species phylogenetic analysis and differential identification of nematodes in order Ascaridata.

ARFI-based tissue elasticity quantification and kidney graft dysfunction: first clinical experiences.

BACKGROUND AND PURPOSE: Beyond the medical history, the clinical exam and lab findings, non-invasive ultrasound parameters such as kidney size and Doppler values (e.g. the resistive index) are important tools assisting clinical decision making in the monitoring of renal allografts. The gold standard for the diagnosis of renal allograft dysfunction remains the renal biopsy; while an invasive procedure, the justifiable necessity for this derives from its definitive nature a requirement beyond the synopses of all non-invasive tools. "Acoustic Radiation Force Impulse Imaging"(ARFI)-quantification is a novel ultrasound-based technology measuring tissue elasticity properties. So far experience related to this new method has not been reported in renal transplant follow-up. The purpose of this study was to evaluate changes in ARFI-measurements between clinically stable renal allografts and biopsy-proven transplant dysfunction. METHODS: We employed "Virtual Touch™ tissue quantification" (Siemens Acuson, S2000) for the quantitative measurement of tissue stiffness in the cortex of transplant kidneys. We performed initial baseline and later disease-evaluative ultrasound examinations in 8 renal transplant patients in a prospective study design. Patients were first examined during stable allograft function with a routine post-transplant renal ultrasound protocol. A second follow-up examination was carried out on subsequent presentation with transplant dysfunction prior to allograft biopsy and histological evaluation. All patiens were examined using ARFI-quantification (15 measurements/kidney). Resistive indices (RI) were calculated using pulsed-wave Doppler ultrasound, and transplant kidney size was measured on B-mode ultrasound images. All biopsies were evaluated histologically by a reference nephropathologist unaware of the results of the ultrasound studies. Histopathological diagnoses were based on biopsy results, taking clinical and laboratory findings into account. Finally we calculated the relative changes in ARFI-quantification, resistive indices and the absolute change of kidney size on a percentage basis at these defined assessment times and compared the results with the final pathologic diagnosis. RESULTS: Histological results enumerated five cases of acute T-cell-mediated rejection, one case of calcineurin inhibitor toxicity and two cases of acute tubular necrosis. Calcineurin inhibitor toxicity and acute tubular necrosis were subsumed as "other pathologies". Mean ARFI-values showed an average increase of more than 15% percent in transplants with histologically proven acute rejection whereas no increase was seen in transplants with other pathologies. Mean RI-values showed no increase either in the diagnostic group of acute rejection, nor in the group with other pathologies. Kidney size showed a mean absolute increase of 0.5 centimetres in allografts with acute rejection, whereas a mean decrease of 0.17 centimetres was seen in the group with other pathologies. CONCLUSION: As shown before in other studies, RI values and kidney size are of doubtful utility in the evaluation of kidney allograft dysfunction. ARFI-based elasticity measurement shows promise as a complementary non-invasive parameter in follow-on diagnosis of renal allograft rejection.

ARFI-based tissue elasticity quantification in comparison to histology for the diagnosis of renal transplant fibrosis.

BACKGROUND AND PURPOSE: Until recently clinical diagnosis of chronic renal allograft dysfunction could only be established invasively by renal biopsy. Given the risks of that procedure, a non-invasive, diagnostic test would be very advantageous. Novel ultrasound-based elasticity tools, using "Acoustic Radiation Force Impulse (ARFI)" technology are now available. Previously this technique has been utilised to quantify liver fibrosis. First results of these studies are promising. The purpose of our study was to investigate correlation between stiffness values obtained by ARFI-quantification and histological fibrosis score in renal transplants. METHODS: We employed "Virtual Touch™ tissue quantification" (Siemens Acuson, S2000) to quantitatively measure tissue stiffness in the cortex of transplant kidneys. Eighteen patients were included in this prospective study, recording close temporal ARFI-quantification and fibrosis measurements. All patients undergoing renal transplant biopsy were examined with ARFI-quantification (15 measurements per transplant kidney). Resistive indices were also calculated from pulsed-wave Doppler ultrasound. Transplant biopsies were histologically evaluated by a reference nephropathologist and graded according to the percentage of fibrosis and to the BANFF-score. Due to the non-normal distribution of the data the Spearman-correlation-coefficient (rho) was used to assess the bivariate relationship of ARFI and fibrosis in the transplant kidney. RESULTS: There was a significant positive moderate correlation between mean ARFI-values and the grade of fibrosis (rho = +0.465; p = 0.026). This correlation was also valid for the mean ARFI-values and the BANFF-category (rho = +0.468; p = 0.025). There was no significant correlation between the mean ARFI-values and the resistive indices in the transplant kidney (rho = +0.034; p = 0.904). Nevertheless, a positive correlation between the mean RI-values of the kidney and the grade of fibrosis was established (rho = +0.563; p = 0.015). CONCLUSION: The mean values of ARFI measurements and the resistive indices are potentially independent explanation variables for evaluating the grade of fibrosis in transplant kidneys.

Urodynamic study of bladder function for patients with lumbar spinal stenosis treated by surgical decompression.

Lumbar spinal stenosis usually leads to different degrees of nerve damage, presenting with back and leg pain, and/or neurogenic bladder symptoms. To determine whether lumbar decompression improved urological function, bladder dysfunction was evaluated in this retrospective study of 26 patients with lumbar spinal stenosis who had undergone lumbar decompression surgery. Urodynamic study procedures were performed pre-operatively and 6 months post-operatively. The Japanese Orthopaedic Association score rating system and Oswestry Disability Index were employed for clinical evaluation. Following surgery, post-voiding residual urine, maximum cystometric capacity and maximum flow rate improved significantly. There was no statistically significant improvement in voided volume, bladder compliance, maximum detrusor pressure or upper urinary tract damage. Urodynamic study was important in the diagnosis of neurogenic bladder dysfunction, prevention of renal deterioration and assessment of post-operative effects after surgical decompression for patients with lumbar spinal stenosis.

Microcarrier bioreactor culture system promotes propagation of human intervertebral disc cells.

BACKGROUND: Cell-based tissue engineering has emerged as a potential therapy for intervertebral disc degeneration. However, propagating and maintaining high quantity and quality of the seed cells remains a challenge. AIMS: To investigate the feasibility of culturing human disc cells using a microcarrier bioreactor system. METHODS: Cell counts, growth patterns, cell cycles and cellular viability were examined during the course of cell cultivation and compared between the microcarrier bioreactor culture system and the conventional monolayer culture. RESULTS: Cultures in the microcarrier bioreactor resulted in enhanced disc cell growth and satisfactory cell viability in comparison with the conventional monolayer culture. The cells in the microcarrier bioreactor cultivation exhibited higher S phase ratios, elevated mitotic index and persistent exponential growth. CONCLUSION: The microcarrier bioreactor culture system appears suitable for human disc cell propagation and may provide considerably more seeding cells for the tissue engineering process of intervertebral discs.

Relationship between protein expression of VEGF-C, MMP-2 and lymph node metastasis in papillary thyroid cancer.

Papillary thyroid cancer (PTC) is a common malignancy and has a good prognosis after appropriate treatment. PTC cells spread mainly by lymph node metastasis (LNM), but the mechanism is not well understood. Expression of vascular endothelial growth factor C (VEGF-C) and matrix metalloproteinase 2 (MMP-2) protein was studied immunohistochemically from the archived specimens of 65 PTC patients who initially presented without LNM. In this retrospective study, the frequency of expression differed significantly between thyroid cancer tissue and adjacent normal follicular epithelium (VEGF-C 78.5% and 20.0%, respectively; MMP-2 81.5% and 36.7%, respectively). LNM developed in 35 of the patients during 5 - 15 years of follow-up, by the end of which the frequencies of expression of VEGF-C and MMP-2 protein expression were 91.4% and 94.3%, respectively. Both VEGF-C and MMP-2 protein expression were significantly more frequent in PTC with LNM than without LNM. VEGF-C and MMP-2 protein expression levels were significantly correlated with LNM and it is, therefore, feasible that VEGF-C and MMP-2 may be useful as tumour markers of PTC with cervical LNM.

HBV genes induce cytotoxic T-lymphocyte response upon adeno-associated virus (AAV) vector delivery into dendritic cells.

Hepatitis B virus (HBV) has been an increasing problem throughout the world and remains difficult to treat. But immunotherapeutic approaches offer new, effective treatments. Three recombinant adeno-associated virus (AAV) type 2 vectors, carrying one of the HBV S, C or X gene, were used to load (transduce) professional antigen-presenting dendritic cells (DC) for the purpose of stimulating cytotoxic T lymphocytes (CTL) in vitro. It was found that all three recombinant AAV/HBV antigen virus loaded DC at approximately 90% transduction efficiency. Most importantly, all three AAV-loaded DC stimulated rapid, antigen-specific and major histocompatibility complex (MHC)-restricted CTL. In vitro, these CTL killed (30-50%) synthetic antigen-positive autologous targets as well as HepG2 liver cell targets. In comparing the three antigens, it was found that AAV/HBV-C-derived CTL consistently had the highest killing efficiency. CTL derived from AAV/HBV-C-loaded DC also showed significantly higher killing of targets than that from bacterially generated C-protein-loaded DC. Further studies showed that AAV/HBV-C-derived CTL had higher interferon (IFN)-gamma. These data suggest that AAV/HBV antigen gene-loading of DC may be useful for immunotherapeutic protocols against HBV infection and that the HBV C antigen may be the most useful for this purpose.

Binding of the beta2 adrenergic receptor to N-ethylmaleimide-sensitive factor regulates receptor recycling.

Following agonist stimulation, most G protein-coupled receptors become desensitized and are internalized, either to be degraded or recycled back to the cell surface. What determines the fate of a specific receptor type after it is internalized is poorly understood. Here we show that the rapidly recycling beta2 adrenergic receptor (beta2AR) binds via a determinant including the last three amino acids in its carboxyl-terminal tail to the membrane fusion regulatory protein, N-ethylmaleimide-sensitive factor (NSF). This is documented by in vitro overlay assays and by cellular coimmunoprecipitations. Receptors bearing mutations in any of the last three residues fail to interact with NSF. After stimulation with the agonist isoproterenol, a green fluorescent protein fusion of NSF colocalizes with the wild type beta2AR but not with a tail-mutated beta2AR. The beta2AR-NSF interaction is required for efficient internalization of the receptors and for their recycling to the cell surface. Mutations in the beta2AR tail that ablate NSF binding reduce the efficiency of receptor internalization upon agonist stimulation. Upon subsequent treatment of cells with the antagonist propranolol, wild type receptors return to the cell surface, while tail-mutated receptors remain sequestered. Thus, the direct binding of the beta2AR to NSF demonstrates how, after internalization, the fate of a receptor is reliant on a specific interaction with a component of the cellular membrane-trafficking machinery.

Regulation of membrane targeting of the G protein-coupled receptor kinase 2 by protein kinase A and its anchoring protein AKAP79.

The beta2 adrenergic receptor (beta2AR) undergoes desensitization by a process involving its phosphorylation by both protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs). The protein kinase A-anchoring protein AKAP79 influences beta2AR phosphorylation by complexing PKA with the receptor at the membrane. Here we show that AKAP79 also regulates the ability of GRK2 to phosphorylate agonist-occupied receptors. In human embryonic kidney 293 cells, overexpression of AKAP79 enhances agonist-induced phosphorylation of both the beta2AR and a mutant of the receptor that cannot be phosphorylated by PKA (beta2AR/PKA-). Mutants of AKAP79 that do not bind PKA or target to the beta2AR markedly inhibit phosphorylation of beta2AR/PKA-. We show that PKA directly phosphorylates GRK2 on serine 685. This modification increases Gbetagamma subunit binding to GRK2 and thus enhances the ability of the kinase to translocate to the membrane and phosphorylate the receptor. Abrogation of the phosphorylation of serine 685 on GRK2 by mutagenesis (S685A) or by expression of a dominant negative AKAP79 mutant reduces GRK2-mediated translocation to beta2AR and phosphorylation of agonist-occupied beta2AR, thus reducing subsequent receptor internalization. Agonist-stimulated PKA-mediated phosphorylation of GRK2 may represent a mechanism for enhancing receptor phosphorylation and desensitization.

[Morphological and ultrastructural observation of Blastocystis hominis].

OBJECTIVE: To observe the morphology and ultrastructure of Blastocystis hominis. METHODS: Morphological observation was made with 4-5 days cultured B. hominis by light microscopy, and similar material fixed with 4% glutaraldehyde was used for transmission electron microscopy. RESULTS: Several forms of B. hominis were observed including vacuolar, granular, amebic, multifission and cystic forms. The multiplication patterns of B. hominis included both binary fission and sporogony. Under transmission electron microscope, the nuclei, mitochondria, rough endoplasmic reticula and lysomes were observed in addition to lipid droplets in its cytoplasm, and glycogen in the central vacuole. CONCLUSION: The central vacuole of vacuolar form may be related to the storage of the excreta. The amebic form of B. hominis might be pathogenic.