Removal of Buffy Coat from Stored ACD Blood by Dextran: Agglomeration and Subsequent Filtration

Abstract. A method for preparing buffy coat-poor red cell suspensions from stored ACD blood is described. Stored ACD blood is sedimented in presence of dextran and then passed through nylon fibre niters. A removal of 94% of leukocytes and 97% of platelets is achieved with this procedure. Red cell recovery is over 70%.     

BARD1 induces apoptosis by catalysing phosphorylation of p53 by DNA-damage response kinase

The BRCA1-associated RING domain protein BARD1 acts with BRCA1 in double-strand break repair and ubiquitination. BARD1 plays a role as mediator of apoptosis by binding to and stabilizing p53, and BARD1-repressed cells are resistant to apoptosis. We therefore investigated the mechanism by which BARD1 induces p53 stability and apoptosis. The apoptotic activity of p53 is regulated by phosphorylation. We demonstrate that BARD1 binds to unphosphorylated and serine-15 phosphorylated forms of p53 in several cell types and that the region required for binding comprises the region sufficient for apoptosis induction. In addition, BARD1 binds to Ku-70, the regulatory subunit of DNA-PK, suggesting that the mechanism of p53-induced apoptosis requires BARD1 for the phosphorylation of p53. Upregulation of BARD1 alone is sufficient for stabilization of p53 and phosphorylation on serine-15, as shown in nonmalignant epithelial cells and ovarian cancer cells, NuTu-19, which are defective in apoptosis induction and express aberrant splice variants of BARD1. Stabilization and phosphorylation of p53 in NuTu-19 cells, as well as apoptosis, can be induced by the exogenous expression of wild-type BARD1, suggesting that BARD1, by binding to the kinase and its substrate, catalyses p53 phosphorylation.     

Pharmacokinetics of anthocyanidin-3-glycosides following consumption of Hibiscus sabdariffa L. extract

Pharmacokinetic parameters of several dietary anthocyanins following consumption of Hibiscus sabdariffa L. extract were determined in 6 healthy volunteers. Subjects were given a single oral dose of 150 mL of Hibiscus sabdariffa L. extract yielding 62.6 mg of cyanidin-3-sambubioside, 81.6 mg of delphindin-3-sambubioside, and 147.4 mg of total anthocyanins (calculated as cyanidin equivalents). Within 7 hours, the urinary excretion of cyanidin-3-sambubioside, delphinidin-3-sambubioside, and total anthocyanins (ie, the sum of all quantifiable anthocyanidin glycosides) was 0.016%, 0.021%, and 0.018% of the administered doses, respectively. Maximum excretion rates were determined at 1.5 to 2.0 hours after intake. The dose-normalized plasma area under the curve estimates were 0.076, 0.032, and 0.050 ng x h/mL/mg for cyanidin-3-sambubioside, delphinidin-3-sambubioside, and total anthocyanins, respectively. The dose-normalized C(max) estimates were 0.036, 0.015, and 0.023 ng/mL/mg in the same sequence. They were reached each at 1.5 hours (median) after intake. The geometric means of t1/2 were 2.18, 3.34, and 2.63 hours for cyanidin-3-sambubioside, delphinidin-3-sambubioside, and total anthocyanins, respectively. The urinary excretion of intact anthocyanins was fast and appeared to be monoexponential. To evaluate the contribution of anthocyanins to the health-protecting effects of Hibiscus sabdariffa L. extract, it will be necessary to perform further studies on both the intact glycosides and their in vivo metabolites or conjugates in human plasma and urine.     

The effect of oral anticoagulation on clotting during hemodialysis

BACKGROUND: Between 5% and 10% of hemodialysis patients are treated with oral anticoagulants. It is currently unknown whether additional anticoagulation with heparin or low-molecular-weight heparin (LMWH) is needed to prevent clotting during hemodialysis. METHODS: In this prospective, randomized, cross-over study 10 patients treated with oral anticoagulants (phenprocoumon) received either no additional anticoagulation or low dose dalteparin (bolus of 40 IU/kg body weight) before dialysis. Efficacy of hemodialysis was measured by normalized weekly Kt/V and urea reduction rate (URR). Thrombus formation was evaluated by measurement of D-dimer and inspection of air traps and dialyser. RESULTS: The median international normalized ratio (INR) did not differ between both observation periods (phenprocoumon 2.2(2 to 3) vs. dalteparin 2.1(2 to 2.9). The anti-Xa level in dalteparin patients was 0.33 (0.27 to 0.38) IU/mL after 2 hours and 0.16 (0.03 to 0.23) IU/mL after 4 hours of hemodialysis. The median increase of D-dimer was significantly higher in patients without additional dalteparin therapy during hemodialysis (DeltaD-dimer 0.23 microg/mL vs. 0.03 mug/mL) (P= 0.0004). Complete thrombosis of the dialyser membrane occurred in one patient in the phenprocoumon group but in none with combined treatment. The extent of thrombosis in the arterial and venous air trap and dialyser was significantly less in patients with additional dalteparin therapy (P= 0.0014, P= 0.0002, and P= 0.0005, respectively). Weekly Kt/V and URR was similar in both groups. CONCLUSION: Standard oral anticoagulation with an INR between 2 and 3 is insufficient to prevent clotting during hemodialysis. Additional low dose anticoagulation with a LMWH or heparin is necessary to facilitate treatment.     

Molecular cytogenetic analysis of human spermatocytic seminomas

We performed comparative genomic hybridization (CGH) on 8 formalin-fixed, paraffin wax-embedded primary spermatocytic seminomas (SS) from 7 patients, one of whom developed metastatic disease. In general, this tumour type is not associated with development of metastases. Since there are only few reported cases of metastatic SS in the literature, this study is the first report of chromosomal constitution in a patient with metastatic disease. Chromosomal imbalances were observed in all 8 tumours analysed by CGH. Frequent copy number alterations were enh(9), dim(16 or 16p), enh(20) and enh(X), each in 6 samples, followed by dim(7) in 4, and enh(1), enh(18) and dim(15), each in 3 samples. In addition to the CGH analysis, interphase fluorescence in situ hybridisation (I-FISH) was applied to evaluate the CGH results and to define the size of the aberrant cell population. Interphase cytogenetics showed gain of material on chromosomes 9 and X in all tumours analysed. Overall, the I-FISH results were in agreement with the CGH data. In conclusion, gain of chromosome 9 seems to be restricted to SS and point to an important role for this aberration in the development of this tumour type.     

Crosstalk of the mitotic spindle assembly checkpoint with p53 to prevent polyploidy

Treatment of cells with microtubule inhibitors results in activation of the mitotic spindle assembly checkpoint, leading to mitotic arrest before anaphase. Upon prolonged treatment, however, cells can adapt and exit mitosis aberrantly, resulting in the occurrence of tetraploid cells in G1. Those cells subsequently arrest in postmitotic G1 due to the activation of a p53-dependent G1 checkpoint. Failure of the G1 checkpoint leads to endoreduplication and further polyploidization. Using HCT116 and isogenic p53-deficient or spindle checkpoint compromised derivatives, we show here that not only p53 but also a functional spindle assembly checkpoint is required for postmitotic G1 checkpoint function. During transient mitotic arrest, p53 stabilization and activation is triggered by a pathway independent of ATM/ATR, Chk1 and Chk2. We further show that a prolonged spindle checkpoint-mediated mitotic arrest is required for proper postmitotic G1 checkpoint function. In addition, we demonstrate that polyploid cells are inhibited to re-enter mitosis by an additional checkpoint acting in G2. Thus, during a normal cell cycle, polyploidization and subsequent aneuploidization is prevented by the function of the mitotic spindle checkpoint, a p53-dependent G1 checkpoint and an additional G2 checkpoint.     

Immunocytochemical characterization of rat brainstem neurons with vagal afferent input from the stomach challenged by acid or ammonia

Exposure of the gastric mucosa to backdiffusing acid is signalled to the brainstem via vagal afferents. This study examined whether exposure of the Sprague-Dawley rat stomach to hydrochloric acid (HCl) or ammonium hydroxide (NH4OH), a noxious chemical produced by Helicobacter pylori, activates different vagal afferent pathways as reflected by different circuitries in the medullary brainstem. Two hours after intragastric treatment with HCl or NH4OH the activation of neurons in the nucleus tractus solitarii at the rostrocaudal extension of the area postrema (NTSAP) was visualized by c-Fos immunohistochemistry and their chemical coding characterized by double-labelling immunohistochemistry. Exposure of the rat gastric mucosa to HCl (0.15-0.5 M) or NH4OH (0.1-0.3 M) led to a concentration-dependent expression of c-Fos in the NTSAP. The number and distribution of NTSAP neurons activated by 0.35 M HCl and 0.3 M NH4OH were similar; the highest number of activated neurons occurring in the medial part of the NTSAP. Some 60% of the NTSAP neurons activated by intragastric HCl and NH4OH stained for the high affinity glutamate transporter EAAC1, while some 30% contained calbindin or neuropeptide Y. Glutamate receptors of the N-methyl-D-aspartate type were found on approximately 50% of the c-Fos-positive cells in the NTSAP, whereas tachykinin NK1, NK2 and NK3 receptors were present on 5-10% of the activated neurons. The similar number and distribution of c-Fos-expressing neurons within the NTSAP and their identical chemical coding indicate that exposure of the rat stomach to backdiffusing concentrations of HCl and NH4OH activates the same vagal afferent-NTSAP pathway.