Phosphorylation and Palmitoylation of the Human D2L Dopamine Receptor in Sf9 Cells

Abstract: We have expressed and biochemically characterized the human D2_long (D2_L) dopamine receptor isoform using the baculovirus/Sf9 cell system. The expressed receptor bound ligands with a pharmacological profile similar to that reported for neuronal and cloned D2_L receptors expressed in mammalian cell lines. Dopamine binding to D2_L receptor was sensitive to guanine nucleotides, indicating receptor coupling to endogenous G proteins. A D2_L receptor-specific antibody identified two major protein species at ∼44 kDa and at ∼93 kDa in immunoblots, suggesting the presence of D2_L receptor monomers and dimers. Both species were purified by immunoprecipitation from digitonin-solubilized preparation of cells expressing D2_L receptor prelabeled with ³²P_i or [³H]-palmitate. These results constitute the first direct evidence for D2_L receptor phosphorylation and palmitoylation.     

Collagen Peptides in Urine: A New Promising Biomarker for the Detection of Colorectal Liver Metastases

Introduction

For both patients and the outpatient clinic the frequent follow-up visits after a resection of colorectal cancer (CRC) are time consuming and due to large patient numbers expensive. Therefore it is important to develop an effective non-invasive test for the detection of colorectal liver metastasis (CRLM) which could be used outside the hospital. The urine proteome is known to provide detailed information for monitoring changes in the physiology of humans. Urine collection is non-invasive and urine naturally occurring peptides (NOPs) have the advantage of being easily accessible without labour-intensive sample preparation. These advantages make it potentially useful for a quick and reliable application in clinical settings. In this study, we will focus on the identification and validation of urine NOPs to discriminate patients with CRLM from healthy controls.

Materials and Methods

Urine samples were collected from 24 patients with CRLM and 25 healthy controls. In the first part of the study, samples were measured with a nano liquid chromatography (LC) system (Thermo Fisher Scientific, Germaring, Germany) coupled on-line to a hybrid linear ion trap/Orbitrap mass spectrometer (LTQ-Orbitrap-XL, Thermo Fisher Scientific, Bremen, Germany). A discovery set was used to construct the model and consecutively the validation set, being independent from the discovery set, to check the acquired model. From the peptides which were selected, multiple reaction monitoring (MRM''s) were developed on a UPLC-MS/MS system.

Results

Seven peptides were selected and applied in a discriminant analysis a sensitivity of 84.6% and a specificity of 92.3% were established (Canonical correlation:0.797, Eigenvalue:1.744, F:4.49, p:0.005). The peptides AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGA P(-OH)GP and KGNSGEP(-OH)GAPGSKGDTGAKGEP(-OH)GPVG were selected for further quantitative analysis which showed a sensitivity of 88% and a specificity of 88%.

Conclusion

Urine proteomic analysis revealed two very promising peptides, both part from collagen type 1, AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGAP(-OH)GP and KGNSGEP(-OH)GAPGSKGDTGAKGEP(-OH)GPVG which could detect CRLM in a non-invasive manner.     

The role of l-arginine-nitric oxide pathway in bacterial translocation

This study investigated the nitric oxide (NO) role as a mediator of arginine on bacterial translocation (BT) and gut damage in mice after intestinal obstruction (IO). The effects of pretreatment with arginine with or without NO inhibition on the systemic and local immunological response were also assessed. Mice were categorized into four groups. Group ARG received chow containing 2 % arginine, while group ARG + l-NAME received the same diet plus l-NAME (N-nitro-l-arginine methyl ester) by gavage. The IO and Sham groups were fed standard chow. After 7 days, animals were gavaged with radiolabeled Escherichia coli, anesthetized and subjected to IO, except the Sham group. Animals were euthanized after 18 h, and BT was evaluated in the mesenteric lymph nodes, blood, liver, spleen and lungs. In another experiment, the intestinal injury was assessed regarding intestinal permeability and ileum histological analyses. Intestinal secretory immunoglobulin A (sIgA) levels, serum IFN-γ and IL-10 cytokines were assessed. Arginine reduced BT, but NO inhibition enhanced BT compared with the ARG group (p < 0.05). Intestinal permeability in the ARG and ARG + l-NAME groups was similar but decreased when compared with the IO group (p < 0.05). Histological preservation was observed. Arginine treatment increased IL-10 and sIgA levels when compared with the Sham and IO groups (p < 0.05). The cytokines and sIgA concentrations were similar in the ARG + l-NAME and Sham groups. Arginine appeared to reduce BT and its effects on the modulation of cytokines and secretory IgA in mice after IO are mediated by NO production.     

Saccharomyces cerevisiae strain UFMG 905 protects against bacterial translocation, preserves gut barrier integrity and stimulates the immune system in a murine intestinal obstruction model

Probiotic is a preparation containing microorganisms that confers beneficial effect to the host. This work assessed whether oral treatment with viable or heat-killed yeast Saccharomyces cerevisiae strain UFMG 905 prevents bacterial translocation (BT), intestinal barrier integrity, and stimulates the immunity, in a murine intestinal obstruction (IO) model. Four groups of mice were used: mice undergoing only laparotomy (CTL), undergoing intestinal obstruction (IO) and undergoing intestinal obstruction after previous treatment with viable or heat-killed yeast. BT, determined as uptake of ^99mTc-E. coli in blood, mesenteric lymph nodes, liver, spleen and lungs, was significantly higher in IO group than in CTL group. Treatments with both yeasts reduced BT in blood and all organs investigated. The treatment with both yeasts also reduced intestinal permeability as determined by blood uptake of ^99mTc-DTPA. Immunological data demonstrated that both treatments were able to significantly increase IL-10 levels, but only viable yeast had the same effect on sIgA levels. Intestinal lesions were more severe in IO group when compared to CTL and yeasts groups. Concluding, both viable and heat-killed cells of yeast prevent BT, probably by immunomodulation and by maintaining gut barrier integrity. Only the stimulation of IgA production seems to depend on the yeast viability.     

Concentration of <Emphasis Type="Italic">Cupriavidus necator</Emphasis> cells by flocculation and sedimentation

Separation and cells concentration constitute important stages in most biotechnological processes. Particularly, use of flocculation/sedimentation can improve significantly the extraction of biopolymers accumulated by microorganisms and the biodegradation of xenobiotic compounds by cell sludge. In this work the use of tannin and aluminum sulphate (Al₂(SO₄)₃) as flocculating agents for concentration of cells of Cupriavidus necator DSM 545 is evaluated. Cells were grown in broth nutrient medium in Erlenmeyer flasks, submitted to orbital agitation of 160 rpm at 30 °C for 21 h. The optimal concentrations of flocculating agents, as determined with a standard jar test method, were equal to 2,800 mg/L for tannin and 800 mg/L for Al₂(SO₄)₃, allowing for recovery of 95% of the cells in both cases. Obtained flocs presented density and average diameter of 1.03 g/mL ± 0.01 g/mL and 158 μm ± 19 μm for tannin and of 1.05 g/mL ± 0.01 g/mL and 146 μm ± 14 μm for Al₂(SO₄)₃, respectively. Batch settling tests were performed in order to determine the operational capacity of continuous settlers to be used for separation of the investigated flocculent suspensions. Finally, cultivation of cells using flocs as inoculum indicated that the cells remained viable after flocculation with usage of the optimum flocculating agent concentrations.     

Comparison of the cytotoxic response against ovarian cancer by immune effector cells isolated and expanded from normal donors and ovarian cancer patients

Background/AimsThe aim of this study was to compare the cytotoxic response against ovarian cancer (OC) cells elicited by different immune effector cells in combination with the cytokines interleukin (IL)-2 and interferon (IFN) α-2b.MethodsOC cells were co-cultured with peripheral blood mononuclear cells (PBMC) from normal donors or OC patients and IL-2 or IFN α-2b alone or in combination, in order to determine the cytotoxicity. T cells were isolated from healthy donors to determine T cell cytotoxic activity. PBMC from healthy donors and OC patients were expanded in an IL-2/IL-7/IL-12 cocktail with and without anti-CD3 antibody, and the cytotoxic activity measured. Flow cytometry was performed on primary, selected and expanded cells to determine T, B, and natural killer- (NK) cell percentages.ResultsHealthy donor PBMC elicited a significant cytotoxic response (59%) compared with OC patient PBMC (7%). T cells enriched from normal donors elicited a significant cytotoxic response (18%) compared with controls lacking effector cells (1.4%); however, the cytotoxicity observed was significantly less compared with unselected PBMC. Expanded effector cells consisted primarily of T cells (98%) and the fold-expansion was significantly higher in the presence of anti-CD3 (19- versus 132-fold). No significant difference in the expansion (either fold-expansion or cell type) was observed between OC patients and healthy donors. Expanded cells from both healthy donors and OC patients elicited a significant cytotoxic response in the presence of IL-2 (19% and 22%) compared with controls.ConclusionsPBMC from OC patients do not elicit a significant cytotoxic response; however, ex vivo-expanded cells from OC patients are capable of cytotoxic killing similar to unexpanded T cells isolated from normal donors. These data provide the groundwork for further development of cellular therapy against OC.     

Symptomatic differences between the sexes in rural Mexico

This paper addresses the problem of the differential presentation of illness by women and men in two Spiritualist temples and a physician's office situated in rural Mexico. Women's morbidity raises the broader anthropological questions of the interplay between symptomatic expression and women's unequal status in the social structure, their cognitive evaluation of specific life experiences, cultural etiological explanations and Western models of dysphoria. Symptoms presented by patients in different health care delivery sites are compared and case vignettes of patients' illnesses and attributions are presented to demonstrate the ways in which culturally constructed illness attributions and illness expressions comprise a somatic grammar for the articulation of experiential distress. The sick population is compared with a control group of healthy women to highlight the socio-cultural and psychosocial variables that promote illness in women from the same sociocultural strata of rural Mexico. Collective understandings of the role of life events and emotional expression in illness attributions legitimize somatization as a coping style under adverse existential conditions.