Phospholipase A2 activity in human platelets. Isolation and purification of the enzyme

The activity of phospholipase A2 in blood platelets of healthy donors and IHD patients was examined. The enzyme activity was found to be increased 3-fold in platelets possessing a high level of functional activity (IHD) and by one order of magnitude in patients with myocardial infarction as compared with healthy donors. An enzyme preparation possessing a phospholipase activity was isolated from platelets by using salt extraction (KCl) and sonication. Purification of the enzyme by affinity chromatography resulted in two protein peaks both having a phospholipase A2 activity, the purification and molecular masses of these fractions being 768- and 2200-fold, and 13.5 and 15 kDa, respectively. It was supposed that these proteins are substrate-specific forms of phospholipase A2.     

Accurate determination of mean cell volume by isotope dilution in erythrocyte populations with variable deformability.

Variations in erythrocyte deformability and morphology lead to artifacts in electronic determinations of mean cellular volume (MCV) by the aperture-impedance method. The micropipette-aspiration technique loses accuracy when applied to severely aberrant cells such as dense sickle cells. A new light-scattering technique requires that the cells be capable of undergoing isovolumetric sphering. In contrast, the isotope-dilution (ID) method measures absolute mean volume and is free of artifacts associated with abnormal deformability or morphology. It does not depend on any algorithms or correction factors and does not subject the cells to any stringent processing, not even centrifugation. The ID method can be used to determine the mean volume of red cells in hypo- or hypertonic media or in the presence of pharmacologic agents. It requires no more than a 1-ml aliquot of suspended cells at a hematocrit of at least 30%. The cells can be readily recovered, washed, and reused. Using EDTA labeled with 57Co as an extracellular space marker we have used ID to determine the MCV of fractionated normal human red blood cells (RBC), unfractionated RBC containing SS hemoglobin, and RBC from four other mammalian species. In the case of human RBC obtained from eight normal donors, we obtained mean MCV values (+/- SD) of 83.6 +/- 3.0, 87.5 +/- 3.9, and 76.5 +/- 5.3 fl for unfractionated and top and bottom 10% density fractions, respectively. The value 83.6 is significantly lower than the generally accepted range of 89-91 indicated by electronic analyzers calibrated against spun microhematocrits. The discrepancy of about 7% can account for the difference between mean cell hemoglobin concentration (MCHC) data determined by a calibrated Coulter Counter and corresponding data obtained with paired samples using a cyanmethemoglobin procedure specified in NCCLS Standard H15-A and corrected for trapped plasma.     

Chromatographic methods for large-scale preparation of immunoglobulin G2a monoclonal antibodies Lym-1 and TNT-1 F(ab')2 fragments

Lym-1 and TNT-1 are two murine immunoglobulin G_2a monoclonal antibodies (MAbs) which have been used for clinical trials in cancer patients. This paper describes methods for large-scale preparation of F(ab')₂ fragments from 50 mg to 4 g of MAbs Lym-1 and TNT-1. Digestion of MAbs with pepsin was optimized and performed at pH 3.8, a pepsin/antibody ratio of 1:250, and 3–4 h of incubation at 37°C. The F(ab')₂ fragments were purified by tandem column procedures using fast protein liquid chromatography. Quality control analyses of the products included protein purity, isoelectric point, immunoreactivity, and endotoxin level. The results revealed that the chromatographic procedures are practical, simple, and effective, and can be used to produce gram quantities of clinical-grade F(ab')₂ fragments for the diagnosis of cancer in patients.     

An immune response defect due to low levels of class II cell surface expression. Analysis of antigen presentation and positive selection.

The effects of quantitative differences in class II cell surface expression have been difficult to address in intact animals. This study uses several lines of H-2s/s mice carrying an A beta k transgene that differ significantly in terms of class II cell surface expression. Due to inefficient chain pairing, mice carrying 60 to 65 copies of this transgene express only low levels of A alpha s/A beta k on the cell surface, and cell surface expression of the endogenous A alpha s/A beta s complex (and total Ia) is severely reduced (to 7-15% control levels). The significant decrease in class II cell surface expression in the thymic cortex of these mice did not affect the frequency of peripheral T cells expressing at least 10 distinct TCR V beta chains. However, T cell proliferative responses to the A alpha s/A beta s-restricted peptide MBP 89-101 were abrogated in high copy number A beta k mice. Experiments using bone marrow chimeras demonstrated that both inefficient Ag presentation and failure to positively select appropriate T cells contributed to this lack of response. Inefficient Ag presentation was clearly the dominant defect, and the density of class II cell surface expression required for positive selection appeared to be quite low.     

Interaction of menadione and duroquinone with Q-cycle during DT-diaphorase function]

The interaction of quinones (menadione and duroquinone) with DT-diaphorase and mitochondrial electron transport chain translocators at low (120 mosM) and high (400 mosM) values of the medium tonicity in the quinone concentration range of 6-90 microM was studied. It was shown that with a rise in menadione (K3) concentration the number of electron transport carriers interacting with it increase. At K3 concentration of 6 microM the latter is reduced by DT-diaphorase and fully oxidized via the Q-cycle. At K3 concentration of 15 microM the latter is also reduced by DT-diaphorase via the Q-cycle, but in this case the oxidation is incomplete (about 30% K3H2 is oxidized by the terminal part of the respiratory chain). At 90 microM K3 50% of quinone is reduced by DT-diaphorase and 50% by the respiratory chain NADH dehydrogenase complex enzymes; about 30% of K3H2 is oxidized via the Q-cycle, about 20%--by the terminal part of the respiratory chain and about 50%--by O2 without cytochrome oxidase. Unlike menadione, duroquinone (6-90 microM) is reduced only by DT-diaphorase and is oxidized in all cases by cytochrome oxidase. It was shown that the increase in the mitochondrial matrix volume in low tonicity media decreases the rate of the DT-diaphorase shunt operation.     

Sequence of the cDNA encoding ovine tumor necrosis factor-alpha: problems with cloning by inverse PCR.

We have cloned and sequenced the ovine tumor necrosis factor-alpha (TNF-alpha)-encoding cDNA, using gene amplification by polymerase chain reaction (PCR) technology, to aid studies of assorted diseases in this species. We used primers selected from published TnfA sequences of other species on a cDNA template prepared from lipopolysaccharide-stimulated ovine alveolar macrophages, to generate a product representing the central region of the molecule. We then used a novel method based on 'inverse PCR' to generate a product containing the 5' and 3' ends of the molecule. Here, we present the complete sequence of the ovine TNF-alpha cDNA and compare it with other published TNF sequences. The cloned cDNA has a leader sequence of 156 bp followed by a protein-coding sequence of 702 bp and a 3'-untranslated region of 800 bp. The protein product of the gene is a protein of Mr = 25,586, 79% homologous to human TNF-alpha. An mRNA produced by alveolar macrophages, which hybridises to the cloned gene, is induced greatly, with a peak induction time of approx. 135 min, in response to stimulation by lipopolysaccharide and to plating on plastic. We also discuss the resolution of some artefacts of the inverse PCR technique.     

Identification of a hypervariable region in the long terminal repeat of equine infectious anemia virus.

An avirulent, field-derived isolate of equine infectious anemia virus (EIAV), designated MA-1, was molecularly cloned, and the complete nucleotide sequence was determined for the 3' half of the viral genome. Comparisons between MA-1 and the prototype Wyoming strain of EIAV identified a 66-nucleotide stretch between CAAT (-91) and TATAA (-25) in the U3 region of the long terminal repeat, where sequence divergence was as high as 39.3%. The polymerase chain reaction was used to amplify and clone long terminal repeat sequences from Th-1, the in vivo parental stock of MA-1. Results indicated that the nucleotide sequences of MA-1 and Th-1 clones were less variable than was observed between MA-1 and Wyoming. However, MA-1 and Th-1 markedly differed in the types of enhancer sequences located in the hypervariable region. These results suggest that variation in lentivirus regulatory sequences may be important in EIAV host cell tropism and pathogenesis.