Catabolism of the anion transport protein in human erythrocytes

We identified the catabolic products of protein 3 in human erythrocytes. Protein 3, the major protein of the erythrocyte membrane, functions in anion transport and reacts covalently with tritiated 4,4'-diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid ([3H]DIDS), a very selective inhibitor of anion transport. In this study, [3H]DIDS was used to label protein 3 in the membranes of normal cells and those from a donor heterozygous for a variant of protein 3, defined by its elongated amino-terminal end. Both types of cells contained [3H]DIDS-labeled peptides other than protein 3. A protein fragment of 60K molecular weight was found in normal cells, whereas both 60K and 63K fragments were identified in cells from the heterozygote. These peptides are identical with those generated by treatment of intact erythrocytes with Pronase or chymotrypsin. A polyclonal rabbit antibody specific for the purified 60K fragment of protein 3 was used to detect this protein and its products in the erythrocyte membrane. Autoradiographs of membrane peptides that were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and allowed to react with the monospecific antibody showed, in addition to protein 3, a 60K fragment and fragments in the 40K region and in the 20-30K region. Cells containing the protein 3 variant yielded two fragments showing a 3K difference in molecular weight in all three regions, demonstrating that degradation of protein 3 is identical in normal erythrocytes and those heterozygous for the variant. This observation also confirms the common derivation of the fragments from protein 3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of phospholipids on the specific binding of [3H]spiroperidol to the cholate extract of rat brain synaptic membranes

Effects of phospholipids including PC, PE, PI, and PS on the specific [3H]SPD binding to the solubilized dopamine receptors were examined in the cholate extracts of the cortical and striatal synaptic membranes (P2M) of the rat brain. PC and PS, but not PE or PI, at 0.4 mM greatly enhanced the specific [3H]SPD binding to the cholate extracts of both cortical and striatal P2M fractions. PC and PS did not enhance the specific [3H]DA binding to the same cholate extracts. The enhancing effects of PC and PS were temperature-dependent and in a dose-response manner peaking at 0.4 mM and 0.2 mM respectively. Such temperature dependence indicated that the PC effects were not due to trapping of [3H]SPD by PC but represented a possible DAR-PC complex formation that allowed higher binding for the ligand. Failure of natural cerebellar P2M in enhancing the [3H]SPD binding to the cholate extract supports the notion that fluidity of the phospholipids is required for the binding or the formation of the DAR-PC (or PS) complex. Scatchard analysis of the [3H]SPD binding to the cholate extract in the absence or presence of PC or PS indicated that the PC or PS enhancement of the ligand binding may be mainly due to an increase in the number of binding sites since both PC and PS significantly increased the Bmax but not the Kd of the binding.     

Constitutively phosphorylated residues in the NS protein of vesicular stomatitis virus

The NS protein of vesicular stomatitis virus is an auxiliary protein in the virus core (nucleocapsid) that plays a role in virus-specific RNA synthesis. NS exhibits a variety of phosphorylated forms, and the degree of phosphorylation correlates with the rate of RNA synthesis. However, chymotryptic peptide mapping has indicated that all forms of NS share a common cluster of phosphorylated residues. To locate these residues in the primary structure of the molecule, we performed a series of residue-specific chemical and enzymatic cleavages and separated radiophosphate-labeled peptides by gel electrophoresis. The data indicate that the constitutively phosphorylated sites in NS molecules reside in the amino-terminal region of the molecule, between residues 35 and 78. The previously reported resistance of the phosphoamino acids in this region to dephosphorylation by exogenous phosphatase suggests that this domain is embedded within the tertiary structure of the molecule or involved in quaternary interactions. In contrast, the amino acid residues that are phosphorylated secondarily, making NS more active in RNA synthesis, reside in more exposed regions of the molecule.     

Preparation and characterization of recombinant DNA-derived human interferon-gamma

An attempt was made to prepare a highly purified, active recombinant DNA-derived human interferon-gamma. When the protein was denatured in urea and refolded, gel filtration and sedimentation velocity experiments indicated the presence of two forms, which are different in size and are not in a rapid reversible equilibrium. The two forms could be chromatographically separated. Far-UV circular dichroic spectra indicated the presence of secondary structures for both forms. Near-UV circular dichroic spectra revealed that the smaller form is folded into a rigid tertiary structure. The antiviral activity of the two forms of interferon-gamma showed a significant difference, i.e. the smaller form was 4-8-fold more active than the larger form. A variety of experiments show that the smaller form is more active, homogeneous, soluble, and stable than the larger form.     

Leukotriene B4 Release and Polymorphonuclear Cell Infiltration in Spinal Cord Injury

Activation of arachidonic acid occurs after spinal cord injury. Leukotriene B4 is a lipoxygenase metabolite of arachidonic acid. In a rat model of experimental spinal cord injury, we found that the leukotriene B4 content was less than the sensitivity of our assay (8 pg/mg of protein) in non-traumatized spinal cord. Leukotriene B4 was detectable in traumatized cord (mean +/- SE, 25 +/- 5 pg/mg of protein; n = 3). Release of leukotriene B4 from spinal cord slices into the incubation medium was also noted after trauma (9 +/- 1 pg/mg of protein; n = 12) and was enhanced by exposure of traumatized spinal cord slices to the calcium ionophore A23187 (375 +/- 43 pg/mg of protein; n = 12). The amount of leukotriene B4 released corresponded to the extent of post-traumatic polymorphonuclear cell infiltration determined by a myeloperoxidase assay. Results from this study suggest that the source of leukotriene B4 in spinal cord injury is infiltrating polymorphonuclear cells.     

Acrylonitrile-induced sister-chromatid exchanges and DNA single-strand breaks in adult human bronchial epithelial cells

The ability of acrylonitrile to induce cytotoxicity, sister-chromatid exchanges and DNA single-strand breaks was studied in cultured human bronchial epithelial cells. The toxic effect as determined by cloning efficiency was observed at a dose of 600 micrograms/ml but not at doses of both 150 and 300 micrograms/ml. The frequency of sister-chromatid exchange in untreated cells was 3.7 +/- 1.3 per cell. In contrast, cells treated with acrylonitrile at 150 and 300 micrograms/ml exhibited 6.6 +/- 1.3 and 10.7 +/- 1.7 sister-chromatid exchanges per metaphase, respectively. DNA single-strand breaks were induced by acrylonitrile at dose levels of 200 and 500 micrograms/ml. The genotoxic effects on human bronchial epithelial cells that were directly exposed to acrylonitrile are of interest in relation to evidence for the higher lung cancer incidence of acrylonitrile workers in epidemiological studies.     

Cytogenetic identification of interspecies cell-line contamination: procedures for non-cytogeneticists

A few simple procedures and cytological characteristics are described for identification of cell-line contamination involving human cells, or cells of several species of common laboratory and domestic animals. These include gross chromosome morphology, C-banding, fluorescence, and the nucleolus organizer regions as revealed by silver straining.     

The effect of sodium selenite toxicity on tissue distribution of zinc,iron, and copper in rats

Male Sprague-Dawley rats were used to determine the effects of suptoxic and toxic concentrations of selenite in the drinking water on tissue distribution of zinc (Zn), iron (Fe), and copper (Cu). Se (as sodium selenite) was provided in drinking water at concentrations of 0, 2, 4, and 8 ppm. At 19 d, half of the rats in 4 and 8 ppm Sesupplemented groups were kept on drinking water alone for additional 13 d. All rats were sacrificed at the end of 32 d of experiment. Heart, liver, and kidney were analyzed for the concentrations of Fe, Zn, and Cu by atomic absorption spectrophotometry and of Se by a fluorometric method. Results indicated that rats receiving 4 and 8 ppm Se in drinking water showed a marked reduction in food intake and a reduced growth rate. These adverse effects were quickly reversed when high Se intake was discontinued. Se toxicity caused minimal change in zinc status, reduced tissue iron concentrations and caused a marked increase in copper contents in heart, liver, and kidney. The latter findings were only partly reversed after removal of Se in drinking water. The accumulation of Cu in the tissues of Se-toxic rats provides the evidence of some interaction between Se and Cu.     

植物分类学中的繁殖生物学问题

繁殖生物学的知识有助于对分类学上复杂性问题的理解,从而大大地有利于我们作出明智的分类估价,但它不一定为我们提供解决问题的钥匙。异体受精增加群体的杂合性和变异性。互交繁育的程度限定了表型变异的式样;互交繁育受阻往往造成分类群表型上的间断。异形不亲和性系统(如花柱异长、雌雄异株等等)给分类学家提供可鉴别的形态差异来划分密切近缘的分类群,但也有由此而造成分类实践上混乱的情况。自体受精增加群体的纯合性;相继世代持续的自体受精使群体变成纯系或同形小种。但对自体受精植物的分类处理并不存在既定的原则,而现在大多数学者不支持把每一个纯系或小种(或约当种)都定为种的做法仅仅反映了当前分类学的时流。但普遍认为,对自体受精类群的分类处理应不同于异体受精类群。无融合生殖是自体受精的一种极端的形式,包括营养生殖和无融合结籽(包括不定胚生殖、无孢子生殖、双倍孢子生殖、假受精)两大类。在任何情况下,胚总是完全由母体组织形成,因此后代在遗传上与它的母体完全相同。由于许多无融合类群都是兼性的,即既能产生无性的种子,又能产生受精的种子,而且又常与杂交和多倍化密切相关,因此具有特别复杂的变异式样。这三套过程同时存在的类群,称为无融合种综,分类学上称为“困难属”,种的划