Sara Streichman

bcl-2 and immunoglobulin gene rearrangement in patients with hepatitis C virus infection

An association between chronic hepatitis C virus (HCV) infection and clonal proliferation of B cells, including B cell lymphoma, has recently been demonstrated. However, the mechanism of malignant transformation is still unknown. It has been shown that B cells from patients with type II mixed cryoglobulinaemia (MC), strongly express the antiapoptotic bcl‐2 oncogene product. Therefore, we investigated a possible mechanism of lymphomagenesis, the occurrence of bcl‐2 and immunoglobulin gene rearrangement (IgH) in HCV‐infected patients. Three groups of patients were studied: (1) 44 patients with HCV and MC (anti‐HCV and HCV RNA positive); (2) 59 patients with chronic HCV infection without MC; (3) 50 patients with chronic liver disease (CLD) not related to HCV infection. The t(14;18) translocation (MBR bcl‐2–JH) and IgH rearrangement (FR3/JH) were detected by polymerase chain reaction (PCR) in peripheral mononuclear cells. bcl‐2 translocation was detected in 17/44 (39%), 7/59 (12%) and in none of the patients of groups 1, 2 and 3 respectively (P < 0·01). Monoclonal IgH rearrangement was detected in 15/44 (34%), 5/59 (8·5%) and 2/50 (4%) patients of groups 1, 2 and 3 respectively (P < 0·05). HCV‐infected patients had a higher prevalence of monoclonal IgH rearrangement and bcl‐2 translocation than patients with CLD of other aetiologies. These data suggest that HCV may play a role in the multistep mechanism of lymphomagenesis by inducing clonal proliferation of B cells and inhibition of apoptosis.

Cryohemolysis for the detection of hereditary spherocytosis: Correlation studies with osmotic fragility and autohemolysis

Laboratory methods aimed to assess the presence of spheroidal cells such as osmotic fragility, autohemolysis, and glycerol lysis time are very elaborate, time consuming, and often give inconclusive results. We have developed a diagnostic test based on a unique sensitivity of HS cells to hypertonic cryohemolysis and analyzed blood samples of 55 HS patients. The patients were divided into two subgroups, clinically affected probands and their relatives. To get quantitative comparisons with the classic methods, the cryohemolysis results were compared to two parameters of the osmotic fragility test: the salt concentration that causes 50% hemolysis, and the percent lysis at a constant salt concentration. Autohemolysis results were also compared. To evaluate which of these tests has the best analytical power, we calculated the mean results and 2 SDs of each parameter in a control group, and then looked to see which of them was best in identifying the patients. The cryohemolysis test was the single parameter that identified all cases including asymptomatic carriers of the disease. The ability of this test to identify the less severe cases probably reflects the dependency of the cryohemolysis on factors that are more related to the primary membrane molecular defects and less by the surface area to volume ratio. Am. J. Hematol. 58:206–212, 1998.

Moving Boundary Electrophoresis and Sialic Acid Content of Normal and Polycythaemic Red Blood Cells

Summary. Moving boundary electrophoresis has been used for the assessment of the negative charge on erythrocyte membranes. The equipment is easy to construct and simpler to use than the microelectrophoresis technique. There is a direct relationship between the electrophoretic velocity measured by the moving boundary method and the sialic acid content of the red cell membrane. Polycythaemic erythrocytes have a higher electrophoretic mobility and higher sialic acid content than normals.

Modeling surfactant-induced hemolysis by Weibull survival analysis

It is demonstrated that surfactant-induced, slow hemolysis (usually attributed to an osmotic mechanism) is very well described by the two-parameter Weibull distribution. The Weibull shape parameter depends on blood properties. The Weibull scale parameter is found to be determined by the amount of membrane-bound surfactant per cell, which is controlled by the suspending solution composition. For hemolysis that involves also a rapid stage (usually attributed to solubilization), the Weibull distribution fits the experimental data if the hemolysis fraction and time are measured relative to the end point of the rapid stage.

The role of electrostatic forces in the interaction between the membrane and cytoskeleton of human erythrocytes

Evidence is presented that electrostatic forces play a major role in the interaction between the cell membrane and cytoskeleton of human erythrocytes. Experiments were carried out on the effects of ionic strength variation, Ca2+ and Mg2+ ions, dimethonium ion and lipophilic ions on the release of spectrin from the erythrocyte ghost. In addition it was shown that the release of spectrin for fixed Ca2+ or Mg2+ concentration shows a maximum as a function of Na+ concentration. All results are consistent with the existence of a repulsive electrostatic force between membrane and cytoskeleton.

The use of hemolysis kinetics to evaluate erythrocyte-bound surfactant

Abstract The partition of surfactant between erythrocytes and their suspending medium was evaluated using the mass-conservation law and a partition model for the surfactant. Contrary to previous work, it is demonstrated that such an evaluation has to be done using results for the whole time range of hemolysis. The use of a single time-point, for which a certain amount of hemolysis is achieved, may, in some cases, result in misleading conclusions. The present analysis implies that equilibrium is maintained between the membrane-bound surfactant and the bulk surfactant throughout the hemolysis process, in spite of the release of cell interior. This equilibrium may be described by a simple partition model, which is characterized by a constant partition coefficient. A practical approach to the evaluation of the surfactant partition, which is particularly useful when the whole hemolysis curve needs to be studied, is suggested.

A simple spectroscopic method for studying erythrocyte ghost resealing

A novel spectroscopic method is described for following the kinetics of resealing of hemolysed erythrocyte ghosts. The procedure is based on the broadening of the EPR spectrum of nitroxyl radicals by paramagnetic ions. The method is used to study the effect of Ca2+, Mg2+ and dimethonium ion on the kinetics of resealing.

EPR STUDY OF THE HYDROPHOBIC INTERACTION OF SPECTRIN WITH FATTY ACIDS

The hydrophobic interaction between spin-labelled stearic acid and spectrin was studied by electron paramagnetic resonance (EPR) and fluorescence quenching. The results are quantitatively interpreted in terms of two types of binding site on spectrin. A comparison between the results of the EPR and fluorescence experiments show the drawback of the fluorescence method in binding studies.

Difference in 2-thenoyltrifluoroacetone sensitivity of electron transport with and against the redox potential gradient

Abstract The effect of 2-thenoyltrifluoroacetone on electron transport with and against the redox potential gradient, with succinate or ascorbate plus N , N , N ′, N ′-tetramethyl- p -phenylenediamine (TMPD) as electron donor, was studied in rat liver mitochondria. It was found that 2-thenoyltrifluoroacetone inhibited succinate-linked intramitochondrial pyridine nucleotide reduction at low concentrations, which neither affected succinate oxidation in the controlled state nor interfered with intramitochondrial pyridine nucleotide reduction in the ascorbate plus TMPD case. The effect of 2-thenoyltrifluoroacetone on succinate-linked intramitochondrial pyridine nucleotide reduction is not attributable either to blocking of the overall rate of electron flow in the succinate dehydrogenase branch of the respiratory chain or to interference with energy transformation. Transition from the controlled to the active state enhanced the inhibitory effect of 2-thenoyltrifluoroacetone on succinate-linked respiration, and it became as sensitive to 2-thenoyltrifluoroacetone as the succinate-linked intramitochondrial pyridine nucleotide reduction. In the light of the above findings, the possibility is discussed that electrons from succinate enter the main branch of the respiratory chain by different routes, according to whether the flow is with or against the potential gradient.

ASSESSMENT OF RED BLOOD CELL DEFORMABILITY BY CENTRIFUGAL SEDIMENTATION

A centrifugal sedimentation method (CSM) is proposed for the assessment of deformability of red blood cells. The method is based on the premise that a red blood cell (RBC) should deform in a centrifugal field due to the variation of the centrifugal acceleration with the distance from the center of rotation. This change in shape of the RBC leads to a change in the rate of sedimentation in the centrifugal field. The rate of sedimentation, which serves as a measure of deformability, is characterized by an apparent sedimentation coefficient (ASC) and its normalized value (NASC), which is calculated by comparison with a control group of normal RBCs. It has been shown that the NASC is sensitive to the speed of rotation, to treatments with glutaraldehyde, diamide, or chlorpromazine, to heat treatment and to osmotic pressure variations.