Evolution of Ycf54-independent chlorophyll biosynthesis in cyanobacteria.

Chlorophylls (Chls) are essential cofactors for photosynthesis. One of the least understood steps of Chl biosynthesis is formation of the fifth (E) ring, where the red substrate, magnesium protoporphyrin IX monomethyl ester, is converted to the green product, 3,8-divinyl protochlorophyllide a In oxygenic phototrophs, this reaction is catalyzed by an oxygen-dependent cyclase, consisting of a catalytic subunit (AcsF/CycI) and an auxiliary protein, Ycf54. Deletion of Ycf54 impairs cyclase activity and results in severe Chl deficiency, but its exact role is not clear. Here, we used a Δycf54 mutant of the model cyanobacterium Synechocystis sp. PCC 6803 to generate suppressor mutations that restore normal levels of Chl. Sequencing Δycf54 revertants identified a single D219G amino acid substitution in CycI and frameshifts in slr1916, which encodes a putative esterase. Introduction of these mutations to the original Δycf54 mutant validated the suppressor effect, especially in combination. However, comprehensive analysis of the Δycf54 suppressor strains revealed that the D219G-substituted CycI is only partially active and its accumulation is misregulated, suggesting that Ycf54 controls both the level and activity of CycI. We also show that Slr1916 has Chl dephytylase activity in vitro and its inactivation up-regulates the entire Chl biosynthetic pathway, resulting in improved cyclase activity. Finally, large-scale bioinformatic analysis indicates that our laboratory evolution of Ycf54-independent CycI mimics natural evolution of AcsF in low-light-adapted ecotypes of the oceanic cyanobacteria Prochlorococcus, which lack Ycf54, providing insight into the evolutionary history of the cyclase enzyme.

Short-term manual compression of hemodialysis fistula leads to a rise in cerebral oxygenation.

BACKGROUND: Decreased cerebral perfusion and oxygenation are common in hemodialysis patients. Magnitude of the arteriovenous fistula involvement in this phenomenon is not known. The aim of this study was to investigate the effect that a short-term arteriovenous fistula flow interruption has on cerebral oxygenation and to review and suggest possible explanations. METHODS: In 19 patients, basic laboratory and clinical data were obtained and arteriovenous fistula flow volume was measured by ultrasonography. Baseline regional cerebral oxygen saturation (rSO2) was measured by near-infrared spectroscopy. Manual pressure was then applied on the fistula, resulting in total blood flow interruption. After 1 min of manual compression, rSO2 and blood pressure values were noted again. The compression-related change in rSO2 was assessed, as well as its association with arteriovenous fistula flow volume, blood pressure, and other parameters. RESULTS: Mean cerebral rSO2 increased after arteriovenous fistula compression (from 53.6% ± 11.4% to 55.6% ± 10.8%; p = 0.000001; 95% confidence interval = 1.39-2.56). The rSO2 increase was higher in patients with lower rSO2 at baseline (r = -0.46; p = 0.045). CONCLUSION: A significant rise in cerebral oxygenation was observed following the manual compression of arteriovenous fistula. Therefore, the arteriovenous fistula could have a role in impaired cerebral oxygenation in hemodialysis patients.

Reduction of arteriovenous access blood flow leads to biventricular unloading in haemodialysis patients.

AIMS: Patients on chronic haemodialysis have a wide range of changes in cardiac function and structure, including left ventricular hypertrophy, dilation and diastolic dysfunction or pulmonary hypertension. All these changes were linked to increased mortality in previous studies. High-flow arteriovenous fistulas (AVF) are supposed to be a factor contributing to their development. This study investigated the early effect of surgical AVF blood flow (Qa) reduction on these changes in patients with or without heart failure changes. METHODS AND RESULTS: Forty-two patients in chronic haemodialysis programme with high-flow AVF (Qa over 1500 mL/min), indicated for surgery for ≥1 of the following indications: 1.manifest heart failure; 2.hand ischemia; 3.advanced structural heart changes detected by echocardiography. The patients underwent echocardiography on selection visit, before blood flow reducing surgery and six weeks thereafter. The Qa reduction led to decrease of left ventricular mass (p = 0.02), end-diastolic volume (p = 0.008), end-diastolic diameter (p = 0.003) and left atrial volume (p = 0.0006). Diastolic function improved. Similarly, right ventricular diameter and right atrial volume decreased (p = 0.000001 and 0.00009, respectively) together with the decrease of estimated pulmonary artery systolic pressure. 81% of patients suffered from pulmonary hypertension prior to surgery, only 36% thereafter. CONCLUSION: The surgical restriction of the hyperkinetic circulation leads to several improvements of heart structure and function, which was linked to higher mortality in other studies. The beneficial effect of Qa reduction is present even in patients without symptoms of heart failure. The contribution of AVF must be considered with structural or functional heart changes.

Review of phenotypic markers used in flow cytometric analysis of MGUS and MM, and applicability of flow cytometry in other plasma cell disorders.

Flow cytometric immunophenotyping is considered an indispensable tool for the diagnosis, classification and monitoring of disease in monoclonal gammopathies. The clinical sensitivity of flow cytometry is comparable with advanced molecular methods. Clinical application of flow cytometry in monoclonal gammopathies has various dimensions, such as differential diagnosis of malignant plasma cell disorder from reactive plasmacytosis, identifying the progression risk in monoclonal gammopathy of undetermined significance (MGUS) and asymptomatic multiple myeloma (MM), and minimal residual disease detection. Flow cytometry-based clonality assessment with immunophenotyping encourages and enables the most stringent method of diagnosis and follow-up. The objective of this review is to update the malignant plasma cells phenotypic profile of MGUS and MM. The most comprehensive antigens, such as CD19, CD27, CD28, CD45, CD56 and CD117, play a significant role in the characterization of normal and malignant plasma cells. Several research groups described the putative phenotype of myeloma cell progenitors, but no remarkable suggestion could be made because of disparity. This review also focuses on the association of malignant phenotypic markers and chromosomal aberrations that identify the specific prognostic features in monoclonal gammopathies.

Levels of angiogenic factors in patients with multiple myeloma correlate with treatment response.

Angiogenesis plays a significant role in the pathogenesis of multiple myeloma (MM). We have measured concentrations of angiogenesis activators, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor, and hepatocyte growth factor (HGF), and inhibitors, including endostatin, thrombospondin-1 (TSP-1), and angiostatin in the peripheral and bone marrow blood of MM patients at diagnosis and after high-dose chemotherapy. We have analyzed 96 patients with secretory MM. Serial measurements of angiogenesis factors/inhibitors were analyzed in the plasma by subgroups based on the best treatment response. Concentrations of angiogenic factors were determined in the peripheral blood and bone marrow plasma. There were significant decreases of VEGF and HGF levels and a significant increase in TSP-1 concentrations in the bone marrow plasma of patients who achieved complete or very good partial response in contrast to those who had partial or no response. VEGF and HGF levels decrease but those of TSP-1 increase after successful treatment for MM, indicating a reduction in the rate of angiogenesis.

Decrease in hand and cerebral oxygenation after percutaneous transluminal angioplasty for arteriovenous fistula stenosis in a patient on chronic hemodialysis.

A 79-year-old woman who was on chronic hemodialysis due to diabetic nephropathy and had previously undergo surgery for radiocephalic arteriovenous fistula (AVF) in her right wrist needed percutaneous transluminal angioplasty (PTA) for stenosis at the juxta-anastomotic access site. After successful PTA, the systemic blood pressure decreased from 144/93 mm Hg to 117/67 mm Hg in response to the increase in AVF blood flow. Furthermore, the regional oxygen saturation (rSO2) value in her dorsal hand decreased from 67.9% to 64.9% and, simultaneously, the cerebral rSO2 decreased from 63.6% to 60.1%. Our experience indicates that the PTA procedure may affect the rapid deterioration of systemic oxygenation, including that in the hand and brain, in association with the increase in the AVF blood flow and change in systemic circulation.

Diastolic dysfunction in asymptomatic hemodialysis patients in the light of the current echocardiographic guidelines.

The prevalence of the left ventricular hypertrophy (LVH) is very high in end-stage renal disease treated by hemodialysis. Diastolic dysfunction is a frequent consequence and leads to the development of heart failure with preserved ejection fraction. New American/European echocardiographic guidelines for the assessment of diastolic function simplified the evaluation and were published recently. The aim of this study was to reveal if the new guidelines stratify asymptomatic hemodialysis patients by the levels of brain-natriuretic peptide (BNP). A cohort of 46 patients hemodialyzed in one center with the lack of overt heart failure, systolic dysfunction, arrhythmia or significant valvular disease were examined by echocardiography before and after a single hemodialysis and blood samples for BNP analysis were drawn at both occasions. The LVH was present in 53% of patients, concentric remodeling in another 17%. Higher indexed left ventricular mass was related to higher BNP levels (r = 0.58, p = 0.0001). Before hemodialysis, diastolic dysfunction was present in 61%: grade 1 in 25%, grade 2 in 21% and grade 3 in 8%. The higher grade of diastolic dysfunction was associated with the incremental increase of BNP. The post-dialysis echocardiography did not allow the assessment of diastolic function in as many as 37% of patients. Our study has shown that the application of the current guidelines for the assessment of diastolic function based on simple four criteria differentiate hemodialysis symptomless patients with preserved systolic function according to BNP levels. BNP levels also rose together with the left ventricular mass. The ratio E/e' medial seemed to be a better predictor of increased BNP than E/e' lateral or E/e' averaged.

Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders.

The European Myeloma Network (EMN) organized two flow cytometry workshops. The first aimed to identify specific indications for flow cytometry in patients with monoclonal gammopathies, and consensus technical approaches through a questionnaire-based review of current practice in participating laboratories. The second aimed to resolve outstanding technical issues and develop a consensus approach to analysis of plasma cells. The primary clinical applications identified were: differential diagnosis of neoplastic plasma cell disorders from reactive plasmacytosis; identifying risk of progression in patients with MGUS and detecting minimal residual disease. A range of technical recommendations were identified, including: 1) CD38, CD138 and CD45 should all be included in at least one tube for plasma cell identification and enumeration. The primary gate should be based on CD38 vs. CD138 expression; 2) after treatment, clonality assessment is only likely to be informative when combined with immunophenotype to detect abnormal cells. Flow cytometry is suitable for demonstrating a stringent complete remission; 3) for detection of abnormal plasma cells, a minimal panel should include CD19 and CD56. A preferred panel would also include CD20, CD117, CD28 and CD27; 4) discrepancies between the percentage of plasma cells detected by flow cytometry and morphology are primarily related to sample quality and it is, therefore, important to determine that marrow elements are present in follow-up samples, particularly normal plasma cells in MRD negative cases.

Decrease of muscle strength in vascular access hand due to silent ischaemia.

BACKGROUND:: Creation of vascular access leads to considerable local haemodynamic changes with decreased hand perfusion. Distal limb tissues then represent a model of hand ischaemia effect on muscles. The aim of our study was to investigate how the presence of vascular access influences the hand muscle strength in end-stage renal disease patients. METHODS:: We included 52 chronically haemodialysed patients with upper limb access without clinical signs of hand ischaemia. Muscle strength was evaluated by dynamometry. Finger pressure was measured on the second and fourth fingers and averaged for further analysis. Thenar tissue oxygenation (rSO2) was analysed using near-infrared spectroscopy. All examinations were performed in both the hands. Basic laboratory analysis was added. Data were processed with unpaired t-test and correlation analysis. RESULTS:: Hands with dialysis access had lower values of handgrip strength (54.2 ± 29.1 lbs vs 48.6 ± 23.4 lbs, p = 0.0006), systolic finger pressure (127.1 ± 32.0 mmHg vs 101.4 ± 31.6 mmHg, p < 10-8) and of thenar rSO2 (45.8% ± 12.9% vs 42.5% ± 13.3%, p = 0.002). Muscle strength (handgrip) was directly related to the thenar oxygenation ( r = 0.36; p = 0.014) and to the finger systolic pressure ( r = 0.38; p = 0.007) on the access extremity. On the extremity without dialysis access, handgrip strength was inversely related to patient's age ( r = -0.41, p = 0.003), dialysis vintage ( r = -0.32, p = 0.02) and red cell distribution width ( r = -0.37, p = 0.01). CONCLUSION:: The presence of dialysis access leads to the decrease of finger pressure, oxygenation, and also muscle strength even in the absence of clinically overt hand ischaemia. All these parameters are interrelated. This study underlines the consequences of inadequate muscle perfusion.

Low Cerebral Oxygenation Is Associated with Cognitive Impairment in Chronic Hemodialysis Patients.

BACKGROUND/AIMS: High rates of cognitive impairment (CI) are an alarming problem in patients undergoing chronic hemodialysis (HD). Its pathophysiology remains unclear and there are indications that brain ischemia might be one of the key causes. Cerebral tissue oxygenation, as measured by near-infrared spectroscopy, is known to be decreased in HD patients. However, it is unknown whether CI is associated or not associated with lower cerebral oxygenation in these patients. The primary aim of our study was to probe this possible association. Our secondary aim was to assess other factors possibly related to cerebral ischemia and CI. METHODS: Thirty-nine patients treated by chronic HD were included in this cross-sectional study. All measurements were performed before the initiation of an HD session. The Montreal Cognitive Assessment (MoCA) was administered according to published recommendations. Regional saturation of oxygen (rSO2) of the left frontal lobe was measured using the INVOS 5100C device. Basic medical history and laboratory data were recorded, and handgrip strength was analyzed. We used the unpaired t test to compare the rSO2 and other variables between cognitively normal patients (MoCA score ≥26) and those who displayed CI (MoCA score <26). Multiple linear regression analysis was used to adjust for principal confounders. RESULTS: Cognitively impaired patients had lower brain rSO2 values compared to cognitively normal patients (48 ± 9 vs. 57 ± 10%, p = 0.01). Among other variables, higher red cell distribution width (15.8 ± 1.9 vs. 13.8 ± 1.6%, p = 0.01) and lower hand grip strength (49.2 ± 23.3 vs. 99.3 ± 31.4 lbs, p < 0.001) also displayed a significant association with CI. The relation between rSO2 and MoCA score was significant after adjustment for age and gender (p = 0.007). CONCLUSION: Decreased brain oxygenation is associated with weaker cognitive performance in patients undergoing chronic HD. Further understanding the causes of cerebral ischemia in HD patients could lead to the prevention of cognitive decline in this population.

Retinoic acid enhances differentiation of v-myb-transformed monoblasts induced by okadaic acid.

Differentiation of various leukemic cells can be induced by liganded retinoic acid receptors and protein phosphatase inhibitors. In this study, we explored the effects of okadaic acid (OA), the phosphatase inhibitor, and retinoic acid (RA) in v-myb-transformed monoblasts BM2. OA induced differentiation of BM2 monoblasts into macrophage-like cells, as documented by analyses of cell morphology, cell cycle, phagocytic activity, non-specific esterase activity, production of reactive oxygen species and expression of vimentin and Mo-1. In contrast to many other leukemic cell lines, BM2 cells do not respond to retinoic acid. However, once exposed to OA and RA simultaneously, BM2 cells differentiate along monocyte/macrophage pathway more efficiently. We conclude that RA enhances differentiation of v-myb-transformed monoblasts induced by protein phosphorylation.

Recognition of DNA modified by antitumor cisplatin by "latent" and "active" protein p53.

Tumor suppressor protein p53 possesses two DNA-binding sites. One that is located within its core domain is responsible for sequence-specific DNA binding of the protein, non-specific binding to internal segments of single- or double-stranded DNA, and to certain kinds of non-B DNA structures. The other that is contained in the C-terminus of the protein binds to damaged DNA. Binding of active, latent, and in vitro-activated p53 protein to DNA fragments modified by antitumor cisplatin was studied using electrophoretic mobility shift assay in agarose gels and immunoblotting analysis. We found that both latent and active p53 forms bound to random sequences of DNA globally modified by cisplatin with a higher affinity than to unmodified DNA. Interestingly, the latent form exhibited a more pronounced selectivity for platinated DNA than the active p53. Consistently with this observation, the preference of the latent form for platinated DNA decreased as a consequence of the activation of latent p53 by phosphorylation at the protein kinase C site within its C-terminus or by binding of the monoclonal antibody Bp53-10.1. Competition experiments involving a 20-bp consensus sequence of p53 suggested that the p53 core domain was a primary binding site of the active p53 when it bound to DNA fragments lacking consensus sequence, but modified by cisplatin. In addition, the latent protein was found to selectively interact with DNA modified by cisplatin probably via its C-terminus.

Surprisingly contrasting metal distribution and fractionation patterns in copper smelter-affected tropical soils in forested and grassland areas (Mufulira, Zambian Copperbelt).

Six soil profiles located near Mufulira (Zambian Copperbelt) were studied to evaluate and compare the extent of environmental pollution of Cu-ore mining and smelting in both forested and grassland areas. The highest metal concentrations were detected in the uppermost soil layers with the following maxima: Co 45.8 mg kg(-1), Cu 8,980 mg kg(-1), Pb 41.6 mg kg(-1), and Zn 97.0 mg kg(-1). Numerous anthropogenic metal-bearing particles were detected in the most polluted soil layers. The spherical smelter-derived particles were mainly composed of covellite (CuS) and chalcocite (Cu2S), while the angular mining-derived particles were mostly composed of chalcopyrite (CuFeS2). Additionally, Fe-Cu oxide particles predominantly corresponding to tenorite (CuO) and delafossite (Cu(1+)Fe(3+)O2), along with hydrated Fe-oxides corresponding to secondary weathering products, were detected. In contrast to smelter-affected soils in temperate climates, where forest soils are significantly more enriched in metals than tilled soils due to high canopy interception, our data indicate a higher proportion of metal-bearing anthropogenic particles and higher metal concentrations in soils from unforested sites. This phenomenon is probably related to the more frequent and intense bushfires in forested areas, leading to the mobilization of pollutants contained in the biomass-rich surface soils back into the atmosphere.

In vitro activation of cytotoxic T-lymphocytes by hTERT-pulsed dendritic cells.

Multiple myeloma has been considered a weakly immunogenic malignancy that can cause profound defects in the immune system. An important issue for the immunotherapy of myeloma is the identification of appropriate tumor-associated antigens (TAAs). Recently, hTERT (human telomerase reverse transcriptase) was detected on a majority of human malignancies. In the studies reported here, we studied antigen-specific and HLA-A2-restricted cytotoxic activity against an ARH77 myeloma cell line in vitro. An HLA-A2-specific hTERT-derived nonapeptide ((540)ILAKFLHWL(548)) was used as a TAA. Myeloma-specific cytotoxic activity of hTERT-reactive cytotoxic lymphocytes (CTLs) was established by repeated stimulation of the CTLs via dendritic cells loaded with hTERT-derived nonapeptide. These studies were able to demonstrate that hTERT-reactive T-lymphocytes can be identified and expanded using relatively simple in vitro techniques consisting of antigen-specific stimulation, immunomagnetic sorting, and then induction of rapid expansion.

Dendritic cell-based immunotherapy for the treatment of hematological malignancies.

Dendritic cells (DCs) are professional antigen-presenting cells and are frequently used in current immunotherapy protocols. The administration of DCs loaded with tumor-associated proteins or peptides results in the induction of immune responses against different types of malignant cells. Methods for large-scale generation of DCs in a sufficient quality and quantity have permitted their use in clinical experiments. DC-based vaccines have already shown promise in follicular non-Hodgkin's lymphoma, and to some extent, in other hematological malignancies. Several strategies have been developed to boost their potency as a new and relatively non-toxic treatment modality. Our review focuses on clinical trials using DCs in the treatment of hematologic malignancies and on recent studies of the immunophenotype, development, and maturation of DCs may have an important impact on designing DC-based antitumor vaccines.

Generation of antigen-loaded dendritic cells in a serum-free medium using different cytokine combinations.

Dendritic cells (DCs) are antigen-presenting cells that play a critical role in the induction of cytotoxic T-lymphocytes. An optimal method for the generation of DC for clinical use remains to be established. The aim of our study was to find an optimal cytokine combination for DC generation from peripheral blood stem cells (PBSC) and peripheral blood mononuclear cells (PBMC) in serum-free conditions. Serial immunophenotyping enabled us to observe changes in DC content during the culture as well as the development of maturation and activation markers. As a source for DC culture, we used PBSC from patients with multiple myeloma after stem cell mobilization using cyclophosphamide and G-CSF, or PBMC from healthy donors without mobilization. The cells were cultured in a serum-free medium with different cytokine combinations including GM-CSF, TNF-alpha, Flt-3, CD40L, IFN-gamma, IL-1alpha, IL-6, PGE1, and IL-4. The cell cultures were evaluated by immunophenotyping. For PBMC, interleukin-12 assay was performed. For PBSC, the yield of DC as determined by CD83+ cell count ranged from 0. 6 x 10(5) to 30.1 x 10(4) (mean: 9.4 x 10(4)) of DC generated per 1 x 10(6) of initially plated nucleated cells from apheresis. This yield corresponded to (0.3-19.1) x 10(5) (mean: 4.3 x 10(5)) per 1 x 10(6) of CD34+ cells in the apheresis products. For PBMC, the yield was (0.4-24.8) x 10(4) (mean: 2.4 x 10(4)) of DC generated per 1 x 10(6) of initially plated mononuclear cells from venous blood. The cultured cells expressed the mature immunophenotype. No significant differences in cell yield or immunophenotype were detected when comparing different cytokine combinations.