Activity and mRNA abundance of Delta-5 and Delta-6 fatty acid desaturases in two human cell lines

We analyzed fatty acid biosynthesis in Chang and ZR-75-1 cells. Both cell lines could desaturate and further elongate substrates for Delta-5 desaturase. ZR-75-1 but not Chang cells showed Delta-6 desaturation of 18:2n-6, 18:3n-3, 24:4n-6 and 24:5n-3. In both cell lines, the mRNA abundance can be related to Delta-5 or Delta-6 fatty acid desaturase activities. These results suggest that desaturase genes could have, at least in part, independent control mechanisms and that Delta-6 desaturase impairment is not specific to any particular step of the fatty acid metabolic pathways, which may diminish the rationale for the existence of at least two distinct enzymes.     

A fluorescent probe which allows highly specific thiol labeling at low pH

Determination of the thiol-disulfide status in biological systems is challenging as redox pools are easily perturbed during sample preparation. This is particularly pertinent under neutral to mildly alkaline conditions typically required for alkylation of thiols. Here we describe the synthesis and properties of a thiol-specific reagent, fluorescent cyclic activated disulfide (FCAD), which includes the fluorescein moiety as fluorophore and utilizes a variation of thiol-disulfide exchange chemistry. The leaving-group character of FCAD makes it reactive at pH 3, allowing modification at low pH, limiting thiol-disulfide exchange. Different applications are demonstrated including picomolar thiol detection, determination of redox potentials, and in-gel detection of labeled proteins.     

Low cytomegalovirus-specific T-cell counts at reactivation are associated with progression to high-level viremia or disease in seropositive recipients of hematopoietic cell grafts from seropositive but not seronegative donors

Background aimsIdentifying patients who spontaneously resolve cytomegalovirus (CMV) reactivation could spare these patients from the toxicity of antiviral drugs such as ganciclovir. The role of CMV-specific T cells in clearing CMV viremia in patients who do not receive ganciclovir has not been evaluated. We assessed this in patients with CMV viremia between 50 and 50 000 genome copies/mL, because our threshold for initiating ganciclovir is 50 000 copies/mLMethodsWe enumerated CMV-specific T cells in 39 CMV seropositive hematopoietic cell transplantation (HCT) recipients within 4 days of the first positive CMV polymerase chain reaction (PCR). CMV-specific T cells were defined as cells that upon stimulation with CMV lysate or pp65 overlapping peptides produced interferon (IFN)-γ, tumor necrosis factor (TNF)-α or interleukin (IL)-2, alone or in combinationResultsAmong Donor (D⁺), Recipient (R⁺) patients, unifunctional CMV-specific CD4 T-cells were higher in patients who spontaneously resolved CMV viremia (did not receive ganciclovir) versus those who progressed (received ganciclovir) (median 0.20 versus 0.02/μL lysate-stimulated cells, P < 0.05, and 0.26 versus 0.05/μL pp65 peptide-stimulated cells, P<0.05). Among D^? R⁺ patients, there was no difference between patients with spontaneous resolution or progression; all subsets of CMV-specific T cells measured were barely detectable, in both patients with spontaneous resolution and those with progression.ConclusionsAmong D⁺ R⁺ patients (but not D^? R⁺ patients), high CMV-specific CD4 T-cell counts identify patients who can spontaneously resolve CMV reactivation. In D^? R⁺ patients, immune mechanisms other than T cells may control the progression from reactivation to high-level viremia/disease.     

Establishing a network of specialist Porphyria centres - effects on diagnostic activities and services

Background

The porphyrias are a heterogeneous group of rare metabolic diseases. The full spectrum of porphyria diagnostics is usually performed by specialized porphyria laboratories or centres. The European Porphyria Initiative (EPI), a collaborative network of porphyria centres formed in 2001, evolved in 2007 into the European Porphyria Network (EPNET), where participating centres are required to adhere to agreed quality criteria. The aim of this study was to examine the state and distribution of porphyria diagnostic services in 2009 and to explore potential effects of increased international collaboration in the field of these rare diseases in the period 2006–2009.

Methods

Data on laboratory, diagnostic and clinical activities and services reported to EPI/EPNET in yearly activity reports during 2006 through 2009 were compared between reporting centres, and possible time trends explored.

Results

Thirty-five porphyria centres from 22 countries, five of which were non-European associate EPNET members, filed one or more activity reports to EPI/EPNET during the study period. Large variations between centres were observed in the analytical repertoire offered, numbers of analyses performed and type and number of staff engaged. The proportion of centres fulfilling the minimum criteria set by EPNET to be classified as a specialist porphyria centre increased from 80% to 94% during the study period.

Conclusions

Porphyria services are unevenly distributed, and some areas are probably still lacking in specialized porphyria services altogether. However, improvements in the quality of diagnostic services provided by porphyria centres participating in EPI/EPNET were observed during 2006 through 2009.

     

Nonredundant Regulation of Rice Arbuscular Mycorrhizal Symbiosis by Two Members of the PHOSPHATE TRANSPORTER1 Gene Family

Pi acquisition of crops via arbuscular mycorrhizal (AM) symbiosis is becoming increasingly important due to limited high-grade rock Pi reserves and a demand for environmentally sustainable agriculture. Here, we show that 70% of the overall Pi acquired by rice (Oryza sativa) is delivered via the symbiotic route. To better understand this pathway, we combined genetic, molecular, and physiological approaches to determine the specific functions of two symbiosis-specific members of the PHOSPHATE TRANSPORTER1 (PHT1) gene family from rice, ORYsa;PHT1;11 (PT11) and ORYsa;PHT1;13 (PT13). The PT11 lineage of proteins from mono- and dicotyledons is most closely related to homologs from the ancient moss, indicating an early evolutionary origin. By contrast, PT13 arose in the Poaceae, suggesting that grasses acquired a particular strategy for the acquisition of symbiotic Pi. Surprisingly, mutations in either PT11 or PT13 affected the development of the symbiosis, demonstrating that both genes are important for AM symbiosis. For symbiotic Pi uptake, however, only PT11 is necessary and sufficient. Consequently, our results demonstrate that mycorrhizal rice depends on the AM symbiosis to satisfy its Pi demands, which is mediated by a single functional Pi transporter, PT11.     

The Renal Arterial Resistive Index and Stage of Chronic Kidney Disease in Patients with Renal Allograft

Objective

The study investigated the optimal threshold value of renal arterial resistive index as assessed by Doppler ultrasonography determining chronic kidney disease stage 4 or higher in patients with renal allograft.

Methods

In a cross-sectional study the renal arterial resistive index were obtained in interlobar arteries by Doppler ultrasonography in 78 patients with renal allograft. The stage of chronic kidney disease was determined by the estimated glomerular filtration rate equation.

Results

The median renal arterial resistive index was 0.61 (interquartile range, 0.56 to 0.66). We observed a significant association between renal arterial resistive index above the upper quartile and chronic kidney disease stage 4 or higher (relative risk, 4.64; 95% confidence interval, 1.71 to 12.55; p = 0.003 by Fisher’s exact test). Multivariate logistic regression analysis showed that renal arterial resistive indices (p = 0.02) and time since transplantation (p = 0.04), but not age, gender, or blood pressure were significantly associated with chronic kidney disease stage 4 or higher.

Conclusion

A renal arterial resistive index higher than 0.66 may determine the threshold value of chronic kidney disease stage 4 or higher in patients with renal allograft.     

18F-FDG PET Imaging of Murine Atherosclerosis: Association with Gene Expression of Key Molecular Markers

Aim

To study whether ¹⁸F-FDG can be used for in vivo imaging of atherogenesis by examining the correlation between ¹⁸F-FDG uptake and gene expression of key molecular markers of atherosclerosis in apoE^−/− mice.

Methods

Nine groups of apoE^−/− mice were given normal chow or high-fat diet. At different time-points, ¹⁸F-FDG PET/contrast-enhanced CT scans were performed on dedicated animal scanners. After scans, animals were euthanized, aortas removed, gamma counted, RNA extracted from the tissue, and gene expression of chemo (C-X-C motif) ligand 1 (CXCL-1), monocyte chemoattractant protein (MCP)-1, vascular cell adhesion molecule (VCAM)-1, cluster of differentiation molecule (CD)-68, osteopontin (OPN), lectin-like oxidized LDL-receptor (LOX)-1, hypoxia-inducible factor (HIF)-1α, HIF-2α, vascular endothelial growth factor A (VEGF), and tissue factor (TF) was measured by means of qPCR.

Results

The uptake of ¹⁸F-FDG increased over time in the groups of mice receiving high-fat diet measured by PET and ex vivo gamma counting. The gene expression of all examined markers of atherosclerosis correlated significantly with ¹⁸F-FDG uptake. The strongest correlation was seen with TF and CD68 (p<0.001). A multivariate analysis showed CD68, OPN, TF, and VCAM-1 to be the most important contributors to the uptake of ¹⁸F-FDG. Together they could explain 60% of the ¹⁸F-FDG uptake.

Conclusion

We have demonstrated that ¹⁸F-FDG can be used to follow the progression of atherosclerosis in apoE^−/− mice. The gene expression of ten molecular markers representing different molecular processes important for atherosclerosis was shown to correlate with the uptake of ¹⁸F-FDG. Especially, the gene expressions of CD68, OPN, TF, and VCAM-1 were strong predictors for the uptake.