Complement pores in erythrocyte membranes: Analysis of C8/C9 binding required for functional membrane damage

The number of membrane-bound terminal complement proteins (C5b-9) required to generate a functional pore in the human erythrocyte membrane ghost has been determined. Resealed erythrocyte ghost membranes (ghosts) were treated with human complement proteins C5b6, C7, ¹³¹I-C8, and ¹²⁵I-C9 under non-lytic conditions. Following C5b-9 assembly, sucrose-permeant ghosts were separated from C5b-9 ghosts that remained impermeant to sucrose by centrifugation over density barriers formed of 43% (w/v) sucrose. Analysis of ¹³¹I-C8 and ¹²⁵I-C9 bound to sucrose-permeant and sucrose-impermeant subpopulations of C5b-9 ghosts revealed: 1. Sucrose-permeant C5b-9 ghosts show increased uptake of both ¹³¹I-C8 and ¹²⁵I-C9 as compared to ghosts that remain impermeant to sucrose. Ghosts with less than 300 molecules ¹³¹I-C8 bound remain impermeant to sucrose, irrespective of the total C9 input, or, the multiplicity of C9 uptake by membrane C5b-8. 2. In the presence of excess ¹²⁵I-C9, the ratio of ¹²⁵I-C9/¹³¹I-C8 bound to membrane C5b67 is 3.2 ± 0.8 (mean ± 2 S.D.), suggesting an average stoichiometry of 3 C9 per C5b-8. Under these conditions, the ratio of ¹²⁵I-C9/¹³¹I-C8 bound to sucrose-permeant ghosts (3.3 ± 0.7) does not significantly differ from the ratio bound to sucrose-impermeant ghosts (2.9 ± 0.6). 3. With limiting C9 input, the threshold of total C5b-8 uptake required for sucrose permeability increases significantly above 300 per cell when the ratio of bound ¹²⁵I-C9/¹³¹I-C8 is decreased below unity. In the complete absence of C9, 11 700 C5b-8 complexes are bound to sucrose-permeant ghosts. It is concluded that more than 300 C5b-9 complexes must bind to the human erythrocyte to form a sucrose-permeant lesion. Although the binding of one C9 per C5b-8 is critical to the pore-forming activity of these proteins, the binding of additional molecules of C9 to each complex (C9/C8 > 1) does not significantly alter the threshold of total C5b-9 uptake required for lesion formation.     

Chromosome banding in Amphibia

The mitotic and meiotic chromosomes of the marsupial frog Gastrotheca riobambae were analysed with various banding techniques. The karyotype of this species is distinguished by considerable amounts of constitutive heterochromatin and unusual, heteromorphic XY sex chromosomes. The Y chromosome is considerably larger than the X chromosome and almost completely heterochromatic. The analysis of the banding patterns obtained with GC- and AT-base-pair-specific fluorochromes shows that the constitutive heterochromatin in the Y chromosome consists of at least three different structural categories. The only nucleolus organizer region (NOR) of the karyotype is localized in the short arm of the X chromosome. This causes a sex-specific difference in the number of NOR: female animals have two NORs in diploid cells, male animals one. No cytological indications were found for the inactivation of one of the two X chromosomes in the female cells. In male meiosis, the heteromorphic sex chromosomes form a characteristic sex-bivalent by pairing their telomeres in an end-to-end arrangement. The significance of the XY/XX sex chromosomes of G. riobambae for the study of X-linked genes in Amphibia, the evolution of sex chromosomes and their specific DNA sequences, and the significance of the meiotic process of sex chromosomes are discussed.     

Reactions of polycyclic hydrocarbon epoxides with RNA and polyribonucleotides

RNA, poly(G) and poly(A) were reacted with benz[a]anthracene 5,6-oxide or with 7,12-dimethylbenz[a]anthracene 5,6-oxide and hydrolysates of the alkylated polymers examined using a combination of Sephadex LH20 column chromatography and thin-layer chromatography on silica gel. The results show that two RNA products are formed in reactions with benz[a]anthracene 5,6-oxide, one resulting from reaction with guanine and the other from reaction with adenine. With 7,12-dimethylbenz[a]anthracene 5,6-oxide, six RNA products appeared to be formed, two resulting from reactions with guanine and three from alkylation of adenine; the other product has not been identified.     

THE INCORPORATION AND FATE OF H3-TYROSINE IN THE HAIR CORTEX OF RATS OBSERVED BY RADIOAUTOGRAPHY

The incorporation of H³-tyrosine into the protein of the cells in the cortex of rat hair has been investigated by radioautography. In growing hairs, radioactivity is found in the matrix, the upper bulb, and the whole of the keratogenous zone up to the fully keratinized part of the shaft, 10 and 30 minutes after an injection of labelled tyrosine. This is unequivocal evidence of protein synthesis at these sites. There is a very precise relationship between the end of protein synthesis and the hardening of the cortical cells at the top of the keratogenous zone. The way in which the silver grains of the radioautographs are clustered indicates that at 30 minutes after the injection the isotope is distributed more evenly in the matrix and upper bulb than in the top of the keratogenous zone. Possibly this reflects a difference, at these sites, in the cell components engaged in protein synthesis, or in the proteins being synthesized. The fully keratinized and hardened part of the hair was not radioactive at 10 and 30 minutes after the injection of H³-tyrosine. The rate at which the radioactivity moves into this region shows that the hair of rats grows 0.9 mm/24 hours. Comparison of the degree of radioactivity along the growing hair in the 30-minute, 12-hour, and 36-hour materials shows conclusively that protein accumulates in the cortical cells during their keratinization. An injection of a labelled amino acid does not behave as an ideal pulse dose; consequently, the grain density over the hair cortex at 36 hours is 100 per cent larger than would be expected if an ideal pulse dose situation existed.     

Conjugations with glutathione. The enzymic conjugation of some chlorocyclohexenes

1. α-3,4,5,6-Tetrachlorocyclohex-1-ene and γ-2,3,4,5,6-pentachlorocyclohex-1-ene are conjugated with glutathione in vitro by a rat-liver enzyme that is probably glutathione S-aryltransferase. 2. Chlorocyclohexane and the α-, β-, γ- and δ-isomers of hexachlorocyclohexane were not substrates for rat-liver glutathione S-aryltransferase. 3. Glutathione-S-aryltransferase activity was present in tissue preparations of houseflies of insecticide-resistant and -susceptible strains. More activity was found in a dieldrin-resistant strain of houseflies fed on dieldrin than in either a dieldrin-resistant strain not fed on dieldrin or a control strain of dieldrin-susceptible houseflies. 4. Housefly soluble supernatant preparations converted S-(2-chloro-4-nitrophenyl)glutathione into the corresponding cysteine and mercapturic acid derivatives.     

Complement proteins C5b-9 induce secretion of high molecular weight multimers of endothelial von Willebrand factor and translocation of granule membrane protein GMP-140 to the cell surface

The effect of immune activation of the serum complement system on the secretory response of human endothelial cells was examined. Exposure of antibody sensitized cultured umbilical vein endothelial cells to human serum resulted in secretion of very high molecular weight multimers of von Willebrand factor which coincided with new surface expression of the intracellular granule membrane protein GMP-140. This response required complement activation through deposition of C5b-9 and was not observed with cells exposed to antibody plus C8-deficient serum or to membrane C5b-8 (in the absence of C9). This C5b-9-induced secretion was observed with minimal cell lysis, as assessed by the release of lactic dehydrogenase. Delayed addition of C8 and C9 to cells exposed to antibody plus C8-deficient serum revealed a rapid decay of membrane C8 binding sites accompanied by loss of the secretory response, suggesting a process of removal or inactivation of nascent C5b67 complexes deposited on the endothelial surface. Membrane assembly of C5b-9 complexes caused an increase in endothelial cytosolic [Ca2+], due to influx across the plasma membrane. This C5b-9-dependent increase in cytosolic [Ca2+] and concomitant von Willebrand factor secretion were both abolished by removal of external calcium. In addition to being linked to the level of external Ca2+, the C5b-9-induced secretory response was partially inhibited by the protein kinase inhibitor, sphingosine. The capacity of the C5b-9 proteins to stimulate endothelial cells to secrete a platelet adhesive protein provides one mechanism for increased platelet deposition at sites of inflammation, and suggests the potential for other functional changes in endothelium exposed to C5b-9 during intravascular complement activation.     

Riboflavin Production during Growth of Micrococcus luteus on Pyridine

Micrococcus luteus produced 29 μM riboflavin during growth on 6.5 mM pyridine but not during growth on other substrates. On the basis of the results of radiolabelling studies, riboflavin was not directly synthesized from pyridine. Pyridine may interfere with riboflavin biosynthesis or elicit a general stress response in M. luteus.     

Syringolide 1 Triggers Ca2+ Influx,K+ Efflux,and Extracellular Alkalization in Soybean Cells Carrying the Disease-Resistance Gene Rpg4

Alleles of avirulence gene D (avrD) specify the production by bacteria of syringolides that elicit the hypersensitive response in soybean (Glycine max) plants carrying the disease-resistance gene Rpg4, but not rpg4 plants. Syringolide 1 caused extracellular alkalization, K+ efflux, and Ca2+ influx about 30 min after addition to suspension-cultured cells of two Rpg4 cultivars, Harosoy and Flambeau, but not in two rpg4 cultivars, Acme and Merit. All responses were sustained for at least 1.5 h and were inhibited by La3+, which blocks certain Ca2+ channels. These results suggest that syringolide 1 activates a Ca2+ influx-dependent signaling pathway only in Rpg4 soybean cells.