Preparative electrophoresis on agarose submerged gels of two aggregating proteoglycan monomers from articular cartilage

Analytical electrophoresis on polyacrylamide-agarose gels of aggregating proteoglycan monomers from baboon articular cartilage produces two distinct bands, corresponding to two different aggregating monomer populations. A preparative electrophoresis procedure is described for isolating the two monomers. Proteoglycans were extracted from young baboon articular cartilage in 4 M guanidinium chloride containing proteolysis inhibitors and aggregated after hyaluronic acid addition. The aggregates were separated from non-aggregated proteoglycans by isopycnic centrifugation, followed by gel chromatography on Sepharose CL-2B. The monomers of the aggregates were obtained by isopycnic centrifugation under dissociative conditions. Two monomers were separated by preparative electrophoresis on 0.8 % agarose submerged gels. Approximately 60 % of the proteoglycans were recovered from the gel using a freeze-squeeze procedure. Aliquots of the separated monomers gave single bands when submitted to analytical polyacrylamide-agarose gel electrophoresis. Their migration and appearance were similar to that of the two bands present in the non separated preparation of monomers.     

l-Phenylalanyl-l-Glutamate-Stimulated, Chloride-Dependent Glutamate Binding Represents Glutamate Sequestration Mediated by an Exchange System

Stimulation of glutamate binding by the dipeptide L-phenylalanyl-L-glutamate (Phe-Glu) was inhibited by the peptidase inhibitor bestatin, suggesting that the stimulation was caused by glutamate liberated from the dipeptide and not by the dipeptide itself. It further suggests that this form of glutamate binding should be reinterpreted as glutamate sequestration and that stimulation of binding both by dipeptides and after preincubation with high concentrations of glutamate is likely to be due to counterflow accumulation. Several other criteria indicate that most of glutamate binding stimulated by chloride represents glutamate sequestration: Binding is reduced when the osmolarity of the incubation medium is increased, when membranes incubated with [3H]glutamate are lysed before filtration, and when membranes are made permeable by transient exposure to saponin. Moreover, dissociation of bound glutamate after a 100-fold dilution of the incubation medium is accelerated about 50 times by the addition of glutamate to the dilution medium. This result would be anomalous if glutamate were bound to a receptor site; it suggests instead that glutamate is transported in and out of membrane vesicles by a transport system that preferentially mediates exchange between internal and external glutamate. Glutamate binding contains a component of glutamate sequestration even when measured in the absence of chloride. Sequestration is adequately abolished only after treating membranes with detergents; even extensive lysis, sonication, and freezing/thawing may be insufficient.     

DNA unwinding and inhibition of mouse leukemia L1210 DNA topoisomerase I by intercalators.

The DNA unwinding effects of some 9-aminoacridine derivatives were compared under reaction conditions that could be used to study drug-induced topoisomerase II inhibition. An assay was designed to determine drug-induced DNA unwinding by using L1210 topoisomerase I. 9-aminoacridines could be ranked by decreasing unwinding potency: compound C greater than or equal to 9-aminoacridine greater than o-AMSA greater than or equal to compound A greater than compound B greater than m-AMSA. Ethidium bromide was more potent than any of the 9-aminoacridines. This assay is a fast and simple method to compare DNA unwinding effects of intercalators. It led to the definition of a drug intrinsic unwinding constant (k). An additional finding was that all 9-aminoacridines and ethidium bromide inhibited L1210 topoisomerase I. Enzyme inhibition was detectable at low enzyme concentrations (less than or equal to 1 unit) and when the kinetics of topoisomerase I-mediated DNA relaxation was studied. Topoisomerase I inhibition was not associated with DNA swivelling or cleavage.     

A T cell surface molecule different from CD2 is involved in spontaneous rosette formation with erythrocytes

Increasing evidence indicates that rosettes which spontaneously from between human T cells and E might be of physiologic relevance. We show here that another T cell-surface molecule than CD2 is involved in rosette formation. Four mAb have been obtained reacting with human T cells that block rosettes with E from many species, including autologous cells. They react with a molecule, we termed E2, which is actively synthetized by T and monocytic cells. Immunoprecipitation revealed a major 32-kDa band. Immunoblots revealed a major 32-kDa band and a minor 20-kDa band. This molecule was detected on all T cells tested--and present at high densities on corticothymocytes, but at low densities on medullary thymocytes. It was also found on monocytes but not on B cells. However B-CLL cells did carry this molecule. E2 molecules were also detected on nonhematologic cells. Together with the recent evidence that 3 molecules from the erythrocyte surface are also involved for rosettes, intricate molecular interactions would account for adhesion of T cells to autologous E and possibly autologous nucleated cells.     

Application of a stability statistic to international maize yield trials

Summary Genotype x environment (GE) interaction encountered in experiments complicates genotype selection and varietal recommendation. The integration of yield and stability of genotypes into a single parameter may make selection and recommendation easier. Kang developed a rank-sum method that allows selection for both yield and the stability variance statistics ( _i ² or s _i ² ) of Shukla. The objective of this research was to compare the rank-sum selection method to selection based on yield alone in five international maize (Zea mays L.) yield trials. Ranks were assigned for yield (the highest mean yield received a rank of 1) and for _i ² and s _i ² (the lowest value received a rank of 1). The yield and _i ² ranks and/or the yield and s _i ² ranks for each genotype were summed. Each trial contained two reference entries (REs). Yield rank or rank-sum of each genotype was compared to yield rank or rank-sum of the best RE (BRE). GE interaction was significant for all trials. Heterogeneity in the GE interaction due to the linear effect of a covariate (differences in fertility and/or cultural practices) was significant in Trials 1, 2, and 5. Overall, in all trials, 29 genotypes were selected on the basis of yield alone. On the basis of _i ² and yield rank-sum, 32 genotypes were identified, with 11 being lower yielding than the 29 yield-based selections. On the basis of s _i ² and yield rank-sum, 31 genotypes were selected, with 11 being lower yielding than the yield-bases selections. Obviously, yield is sacrificed when the rank-sum method is used in the selection process. However, selection based on yield alone may not be adequate when GE interaction is significant because of testing in diverse environments.     

Specific binding of three neurotoxins with phospholipase A2 activity to synaptosomal membrane preparations from the guinea pig brain

Snake presynaptic toxins such as crotoxin, β-bungarotoxin and taipoxin block neuromuscular transmission through inhibiting the release of acetylcholine by their phospholipase A₂ activities. On the other hand, many other phospholipase A₂s show little neurotoxicity. It is likely that the difference lies in whether high affinity binding to nerve cell membranes exists or not. To test this idea, crotoxin, β-bungarotoxin and taipoxin were first radioactively labeled with Na(¹²⁵I) without loss of their neurotoxicity. Using the radioactive toxins we have found that each of the three showed specific binding to synaptosomal membranes from guinea pig brain. In contrast, we could not detect specific binding of a non-neurotoxic pancreatic phospholipase A₂. Crotoxin and taipoxin, but not β-bungarotoxin, also bound specifically to membrane preparation from other tissues. The binding of each toxin was not greatly affected by the other two toxins. The photoaffinity labeling technique has been used to obtain further information about the components which bind crotoxin. For this purpose, (¹²⁵I) crotoxin was derivatized with N-hydroxysuccinimidyl-4-azidobenzoate. Autoradiographic analysis of the membranes following photoirradiation in the presence of the modified crotoxin revealed that an 85K dalton component was preferentially covalently conjugated with the crotoxin analogue in a specific manner.     

Biological activities of lipopolysaccharide fractionated by preparative acrylamide gel electrophoresis

Lipopolysaccharide from a smooth strain of Salmonella minnesota was fractionated into two major fractions and one intermediate fraction by using sodium dodecylsulfate-polyacrylamide gel electrophoresis. On the basis of the study by Hitchcock and Brown, it was deduced that the top fraction was mainly long O-side chain LPS and the bottom fraction was O-side chain-less LPS. The middle fraction was a mixture of both short O-side chain LPS and O-side chain-less LPS. The antigenic properties and biological activities were not altered in this fractionation procedure. Comparison of the biological activities of the top fraction with those of the bottom fraction revealed that the bottom fraction had higher activity in polyclonal B-cell activation and spleen-swelling effect and that there was no significant difference in adjuvant activity, ability to render macrophages cytotoxic, induction of colony-stimulating factor and the ability to induce the Schwartzmann reaction. It was suggested that O-side chain makes no contribution to the latter biological activities including adjuvant activity of S. minnesota LPS.     

Comparative antioxidant enzyme study in freshwater fishes. II. Distribution of antioxidant enzymes and lipid peroxidation in omnivorous fish organs

Comparative studies were performed on the activities of superoxide dismutases, catalase and glutathione peroxidase in organ homogenates from three omnivorous fishes, the barbel, crucian carp and common carp. The lipid peroxidation and protein contents of organ homogenates were also compared. These comparative measurements primarily provide control values for subsequent toxicological examinations. The highest total superoxide dismutase activities were found in the liver or roe, kidney, heart and spleen in every cases. The antioxidant enzyme activities and other studied parameters of the organ homogenates partly appear to depend on the feeding mode, but are rather characteristic of the fish variety.     

Nuclear DNA content and separation of Nicotiana sylvestris vegetative and generative nuclei at various stages of male gametogenesis

At all stages of male gametogenesis, generative and vegetative pollen nuclei of Nicotiana sylvestris can be distinguished without ambiguity after Feulgen or ethidium bromide staining. They differ by their morphology and their apparent DNA content, always lower in vegetative nuclei. These differences provide a basis for their separation by sedimentation and fluorometry. After elimination of the another somatic cells and after crushing the pollen, vegetative and generative nuclei are separated by two successive Percoll gradients (purity 80–90%). Analysis of the gradient fractions and final purification can be done with a cell sorter. DNAs of both types are isolated by a cetyltrimethylammonium method, followed by a RNase treatment. Yields are lower for vegetative than for generative nuclei, and decrease with the age of pollen. Molecular weights and digestibility by restriction enzymes are compatible with molecular analyses.