Biophysical characterization of the proton-coupled oligopeptide transporter YjdL

Proton-coupled oligopeptide transporters (POTs) utilize the electrochemical proton gradient to facilitate uptake of di- or tripeptide molecules. YjdL is one of four POTs found in Escherichia coli. It has shown an extraordinary preference for di- rather than tripeptides, and is therefore significantly different from prototypical POTs such as the human hPepT1. Nonetheless YjdL contains several highly conserved POT residues, which include Glu388 that is located in the putative substrate binding cavity. Here we present biophysical characterization of WT-YjdL and Glu388Gln. Isothermal titration calorimetrical studies exhibit a K_d of 14 μM for binding of Ala-Lys to WT-YjdL. Expectedly, no binding could be detected for the tripeptide Ala-Ala-Lys. Surprisingly however, binding could not be detected for Ala-Gln, although earlier studies indicated inhibitory potencies of Ala-Gln to be comparable to Ala-Lys (IC₅₀ values of 0.6 compared to 0.3 mM). Finally, Ala-Lys binding to Glu388Gln was also undetectable which may support a previously suggested role in interaction with the ligand peptide N-terminus.     

Brain glycogen and its role in supporting glutamate and GABA homeostasis in a type 2 diabetes rat model

The number of people suffering from diabetes is hastily increasing and the condition is associated with altered brain glucose homeostasis. Brain glycogen is located in astrocytes and being a carbohydrate reservoir it contributes to glucose homeostasis. Furthermore, glycogen has been indicated to be important for proper neurotransmission under normal conditions. Previous findings from our laboratory suggested that glucose metabolism was reduced in type 2 diabetes, and thus we wanted to investigate more specifically how brain glycogen metabolism contributes to maintain energy status in the type 2 diabetic state. Also, our objective was to elucidate the contribution of glycogen to support neurotransmitter glutamate and GABA homeostasis. A glycogen phosphorylase (GP) inhibitor was administered to Sprague-Dawley (SprD) and Zucker Diabetic Fatty (ZDF) rats in vivo and after one day of treatment [1-13C]glucose was used to monitor metabolism. Brain levels of 13C labeling in glucose, lactate, alanine, glutamate, GABA, glutamine and aspartate were determined. Our results show that inhibition of brain glycogen metabolism reduced the amounts of glutamate in both the control and type 2 diabetes models. The reduction in glutamate was associated with a decrease in the pyruvate carboxylase/pyruvate dehydrogenase ratio in the control but not the type 2 diabetes model. In the type 2 diabetes model GABA levels were increased suggesting that brain glycogen serves a role in maintaining a proper ratio between excitatory and inhibitory neurotransmitters in type 2 diabetes. Both the control and the type 2 diabetic states had a compensatory increase in glucose-derived 13C processed through the TCA cycle following inhibition of glycogen degradation. Finally, it was indicated that the type 2 diabetes model might have an augmented necessity for compensatory upregulation at the glycolytic level.     

The EPI bioassay identifies natural compounds with estrogenic activity that are potent inhibitors of androgenic pathways in human prostate stromal and epithelial cells

The reactive stromal phenotype is an important factor for prostate cancer progression and may be a new target for treatment and prevention. A new high efficiency preclinical protocol, the EPI bioassay, reflects the interaction of endocrine, paracrine and immune, (EPI) factors on induced androgen metabolism in human prostate reactive stroma. The bioassay is based on co-culturing human primary prostate stromal cells and LAPC-4 prostatic adenocarcinoma cells in a downscaled format of 96-well-plates for testing multiple doses of multiple target compounds. Metabolism of dehydroepiandrosterone (DHEA) with or without TGFβ1-induced stimulation (D+T) of the reactive stroma phenotype was assessed by increased testosterone in the media and PSA production of the epithelial prostate cancer cells. Using the non-metabolizable androgen R1881, effects from direct androgen action were distinguished from stromal androgen production from DHEA. Stromal cell androgenic bioactivity was confirmed using conditioned media from D+T-treated stromal cell monocultures in an androgen-inducible AR screening assay. We further showed that both agonists to estrogen receptor (ER), DPN (ERβ) and PPT (ERα), as well as estrogenic natural compounds including soy isoflavones attenuated D+T-induced PSA production. Studies with the pure ER agonists showed that activating either ERα or ERβ could inhibit both D+T-mediated and R1881-mediated PSA production with the D+T effect being more pronounced. In conclusion, natural compounds with estrogenic activity and pure ER agonists are very potent inhibitors of stromal conversion of DHEA to androgenic metabolites. More studies are needed to characterize the mechanisms involved in estrogenic modulation of the endocrine-immune-paracrine balance of the prostate microenvironment.     

Impairment of the organization of locomotor and exploratory behaviors in bile duct-ligated rats

Hepatic encephalopathy (HE) arises from acute or chronic liver diseases and leads to several problems, including motor impairment. Animal models of chronic liver disease have extensively investigated the mechanisms of this disease. Impairment of locomotor activity has been described in different rat models. However, these studies are controversial and the majority has primarily analyzed activity parameters. Therefore, the aim of the present study was to evaluate locomotor and exploratory behavior in bile duct-ligated (BDL) rats to explore the spatial and temporal structure of behavior. Adult female Wistar rats underwent common bile duct ligation (BDL rats) or the manipulation of common bile duct without ligation (control rats). Six weeks after surgery, control and BDL rats underwent open-field, plus-maze and foot-fault behavioral tasks. The BDL rats developed chronic liver failure and exhibited a decrease in total distance traveled, increased total immobility time, smaller number of rearings, longer periods in the home base area and decreased percentage of time in the center zone of the arena, when compared to the control rats. Moreover, the performance of the BDL rats was not different from the control rats for the elevated plus-maze and foot-fault tasks. Therefore, the BDL rats demonstrated disturbed spontaneous locomotor and exploratory activities as a consequence of altered spatio-temporal organization of behavior.     

Cognitive Functions in Middle Aged Individuals Are Related to Metabolic Disturbances and Aerobic Capacity: A Cross-Sectional Study

Aims

Metabolic disturbances may contribute to cognitive dysfunction in patients with type 2 diabetes. We investigated the relation between cognitive impairment and metabolic deteriorations, low physical fitness, low-grade inflammation and abdominal obesity in middle aged individuals.

Methods

We conducted a cross-sectional study including 40 to 65 year-old patients with type 2 diabetes and limited co morbidity (N = 56), age-matched individuals with impaired glucose tolerance (N = 56) as well as age-matched controls with normal glucose tolerance (N = 72). Specific cognitive functions were assessed with focus on verbal memory, processing speed, executive functions, and a composite overall mean score. Oral glucose tolerance test, VO₂max test, systemic inflammation, DXA scanning and abdominal MRI were measured.

Results

Multiple linear regression analyses adjusting for age, gender and verbal intelligence demonstrated that a low score in processing speed, executive functions and overall cognitive function were related to high fasting C-peptide, as well as low insulin sensitivity, beta-cell function and VO₂max. Measurements of blood glucose, obesity and inflammation were not associated with cognitive function.

Conclusion

Low cognitive scores are seen in middle aged individuals with hyperinsulinemia, low insulin sensitivity, beta-cell function and low aerobic capacity. These findings emphasize the importance of appropriate lifestyle and not only blood glucose control in prevention of cognitive disability.     

Regulation and Gene Expression Profiling of NKG2D Positive Human Cytomegalovirus-Primed CD4(+) T-Cells

NKG2D is a stimulatory receptor expressed by natural killer (NK) cells, CD8(+) T-cells, and γδ T-cells. NKG2D expression is normally absent from CD4(+) T-cells, however recently a subset of NKG2D(+) CD4(+) T-cells has been found, which is specific for human cytomegalovirus (HCMV). This particular subset of HCMV-specific NKG2D(+) CD4(+) T-cells possesses effector-like functions, thus resembling the subsets of NKG2D(+) CD4(+) T-cells found in other chronic inflammations. However, the precise mechanism leading to NKG2D expression on HCMV-specific CD4(+) T-cells is currently not known. In this study we used genome-wide analysis of individual genes and gene set enrichment analysis (GSEA) to investigate the gene expression profile of NKG2D(+) CD4(+) T-cells, generated from HCMV-primed CD4(+) T-cells. We show that the HCMV-primed NKG2D(+) CD4(+) T-cells possess a higher differentiated phenotype than the NKG2D(-) CD4(+) T-cells, both at the gene expression profile and cytokine profile. The ability to express NKG2D at the cell surface was primarily determined by the activation or differentiation status of the CD4(+) T-cells and not by the antigen presenting cells. We observed a correlation between CD94 and NKG2D expression in the CD4(+) T-cells following HCMV stimulation. However, knock-down of CD94 did not affect NKG2D cell surface expression or signaling. In addition, we show that NKG2D is recycled at the cell surface of activated CD4(+) T-cells, whereas it is produced de novo in resting CD4(+) T-cells. These findings provide novel information about the gene expression profile of HCMV-primed NKG2D(+) CD4(+) T-cells, as well as the mechanisms regulating NKG2D cell surface expression.     

End-of-life practices in Danish ICUs: development and validation of a questionnaire

ABSTRACT: BACKGROUND: Practices for withholding or withdrawing therapy vary according to professional, cultural and religious differences. No Danish-validated questionnaire examining withholding and withdrawing practices exists, thus the aim of this study was to develop and validate a questionnaire for surveying the views of intensive care nurses, intensivists, and primary physicians regarding collaboration and other aspects of withholding and withdrawing therapy in the ICU. METHODS: A questionnaire was developed on the basis of literature, focus group interviews with intensive care nurses and intensivists, and individual interviews with primary physicians. The questionnaire was validated in the following 3 phases: a qualitative test with 17 participants; a quantitative pilot test with 60 participants; and a survey with 776 participants. The validation process included tests for face and content validity (by interviewing participants in the qualitative part of the pilot study), reliability (by assessing the distribution of responses within the individual response categories), agreement (by conducting a test-retest, evaluated by paired analyses), known groups' validity (as a surrogate test for responsiveness, by comparing two ICUs with a known difference in end-of-life practices), floor and ceiling effect, and missing data. RESULTS: Face and content validity were assessed as good by the participants in the qualitative pilot test; all considered the questions relevant and none of the participants found areas lacking. Almost all response categories were used by the participants, thus demonstrating the questionnaires ability to distinguish between different respondents, agreement was fair (the average test-retest agreement for the Likert scale responses was 0.54 (weighted kappa; range, 0.25-0.73), and known groups' validity was proved by finding significant differences in level of satisfaction with interdisciplinary collaboration and in experiences of withdrawal decisions being unnecessary postponed. Floor and ceiling effect was in accordance with other questionnaires, and missing data was limited to a range of 0-7% for all questions. CONCLUSIONS: The validation showed good and fair areas of validity of the questionnaire. The questionnaire is considered a useful tool to assess the perceptions of collaboration and other aspects of withholding and withdrawing therapy practices in Danish ICUs amongst nurses, intensivists, and primary physicians.     

Deciphering the proteome of the in vivo diagnostic reagent "purified protein derivative" from Mycobacterium tuberculosis

Purified protein derivative (PPD) has served as a safe and effective diagnostic reagent for 60 years and is the only broadly available material to diagnose latent tuberculosis infections. This reagent is also used as a standard control for a number of in vitro immunological assays. Nevertheless, the molecular composition and specific products that contribute to the extraordinary immunological reactivity of PPD are poorly defined. Here, a proteomic approach was applied to elucidate the gene products in the U.S. Food and Drug Administration (FDA) standard PPD-S2. Many known Mycobacterium tuberculosis T-cell antigens were detected. Of significance, four heat shock proteins (HSPs) (GroES, GroEL2, HspX, and DnaK) dominated the composition of PPD. The chaperone activities and capacity of these proteins to influence immunological responses may explain the exquisite solubility and immunological potency of PPD. Spectral counting analysis of three separate PPD reagents revealed significant quantitative variances. Gross delayed-type hypersensitivity (DTH) responses in M. tuberculosis infected guinea pigs were comparable among these PPD preparations; however, detailed histopathology of the DTH lesions exposed unique differences, which may be explained by the variability observed in the presence and abundance of early secretory system (Esx) proteins. Variability in PPD reagents may explain differences in DTH responses reported among populations.     

Macaque Homologs of EBV and KSHV Show Uniquely Different Associations with Simian AIDS-related Lymphomas

Two gammaherpesviruses, Epstein-Barr virus (EBV) (Lymphocryptovirus genus) and Kaposi''s sarcoma-associated herpesvirus (KSHV) (Rhadinovirus genus) have been implicated in the etiology of AIDS-associated lymphomas. Homologs of these viruses have been identified in macaques and other non-human primates. In order to assess the association of these viruses with non-human primate disease, archived lymphoma samples were screened for the presence of macaque lymphocryptovirus (LCV) homologs of EBV, and macaque rhadinoviruses belonging to the RV1 lineage of KSHV homologs or the more distant RV2 lineage of Old World primate rhadinoviruses. Viral loads were determined by QPCR and infected cells were identified by immunolabeling for different viral proteins. The lymphomas segregated into three groups. The first group (n = 6) was associated with SIV/SHIV infections, contained high levels of LCV (1–25 genomes/cell) and expressed the B-cell antigens CD20 or BLA.36. A strong EBNA-2 signal was detected in the nuclei of the neoplastic cells in one of the LCV-high lymphomas, indicative of a type III latency stage. None of the lymphomas in this group stained for the LCV viral capsid antigen (VCA) lytic marker. The second group (n = 5) was associated with D-type simian retrovirus-2 (SRV-2) infections, contained high levels of RV2 rhadinovirus (9–790 genomes/cell) and expressed the CD3 T-cell marker. The third group (n = 3) was associated with SIV/SHIV infections, contained high levels of RV2 rhadinovirus (2–260 genomes/cell) and was negative for both CD20 and CD3. In both the CD3-positive and CD3/CD20-negative lymphomas, the neoplastic cells stained strongly for markers of RV2 lytic replication. None of the lymphomas had detectable levels of retroperitoneal fibromatosis herpesvirus (RFHV), the macaque RV1 homolog of KSHV. Our data suggest etiological roles for both lymphocryptoviruses and RV2 rhadinoviruses in the development of simian AIDS-associated lymphomas and indicate that the virus-infected neoplastic lymphoid cells are derived from different lymphocyte lineages and differentiation stages.