Complexes prepared from protein A and human serum,IgG, or Fcγ fragments: characterization by immunochemical analysis of ultracentrifugation fractions and studies on their interconversion

Summary Protein A of Staphylococcus aureus is an Fc receptor for IgG that has been used as a therapeutic reagent to treat cancer in humans and experimental animals. We used ultracentrifugation combined with analysis of isolated fractions by radioimmunoprecipitation and competitive radioimmunoassay with chicken antibodies that bind free protein A or protein A in complexes but do bind free immunoglobulin reagents to localize and characterize the types of complexes formed with different molar ratios of ¹²⁵I-protein A and human ¹³¹I-IgG alone or in serum, and ¹³¹1-Fc fragments. This approach offers a distinct advantage over direct counting of radioactivity in the fractions because resolution of complexes and free reagents is much improved. With excess ¹³¹I-IgG or ¹³¹1-Fc, all the ¹²⁵I-protein A is present only in complexes that contained 4 molecules of immunoglobulin reagent and 2 molecules of protein A (4:2 complexes), whereas with excess ¹²⁵I-protein A the stoichiometry of the complexes was 1:1. We have also shown the preformed 4:2 and 1:1 complexes will interconvert in the presence of added excess protein A or IgG, respectively, and that fresh IgG will exchange with IgG or Fc in preformed complexes. Because protein A has been found to elute from an immobilized reagent used in serotherapy of human cancer and is present in a large excess of IgG, the 4:2 complexes may play an active role in the tumoricidal or toxic reactions observed.Abbreviations SpA protein A of Staphyloccus aureus - VBS EDTA gel, 0.0055 M veronal buffered saline containing 0.01 M EDTA and 0.1% gelatin, pH 7.4 - PBS 0.01 M phosphate buffered saline, pH 7.4     

Comparative analysis of caffeine and 3-aminobenzamide as DNA repair inhibitors in Syrian baby hamster kidney cells

The effects of caffeine and 3-aminobenzamide (3-AB) on Syrian baby hamster kidney cells treated with DNA-alkylating agents and ultraviolet-light suggest that two different DNA-repair mechanisms are involved. Both these agents enhanced the cell kill after methyl methanesulfonate (MMS) treatment. However, enhanced lethality was observed only with caffeine post-treatment when cells were exposed to nitrogen mustard (HN2) or ultraviolet light (UV); 3-AB did not appreciably change cell killing by these agents. With MMS-treated cultures, the effect of caffeine was maximal about 16 h later. The effect of 3-AB on the other hand, was exerted during the first 4 h after exposure to MMS. Caffeine's effect on cell survival could be abolished by low concentrations of cycloheximide, whereas 3-AB's effect could not. Furthermore, the G2 block in cell cycle progression, after MMS treatment, was not observed if the cells were post-treated with caffeine. In the presence of 3-AB, MMS-treated cells were arrested in G2 phase at a much earlier time compared to cells not treated with 3-AB. Finally caffeine post-treatment produced a 10-fold increase in nuclear fragmentation in MMS-treated cells. 3-AB did not cause nuclear fragmentation by itself but further enhanced the nuclear fragmenting effect of caffeine when both agents were present during the posttreatment. Therefore, we propose that 3-AB and caffeine each prevent a different repair mechanism from being effective.     

Effects on pregnancy in mice of passive immunization against ovine LH and human chorionic gonadotrophin

Mice given daily i.p. injections of immunoglobulins against ovine LH on Days 3-7 of pregnancy were devoid of implantation sites on Day 8 whereas mice treated with antibodies to hCG had embryos of normal number and appearance on Day 8. These antibody treatments reduced the mean +/- s.d. serum progesterone concentrations from 65.4 +/- 15.3 ng/ml (control globulins) to 8.6 +/- 4.9 ng/ml (anti-LH) and 9.2 +/- 3.1 ng/ml (anti-hCG) on Day 8 and had no differential effect on serum oestrogen levels on Day 4. However, the mice treated with anti-hCG did not litter; resorption of the embryos took place between Days 10 and 14 of pregnancy. Indirect immunofluorescence and quantitative immunoenzymic assays showed the presence of anti-ovine LH and anti-hCG reacting antigens in the mouse feto-placental unit. On Day 6, the values of reacting antigens (mean +/- s.d. absorbance units/10 micron section of embryo) were 0.050 +/- 0.002 with control globulins, 0.059 +/- 0.002 with anti-hCG-Ig and 0.196 +/- 0.018 with anti-LH-Ig; the corresponding values on Day 12 were 0.075 +/- 0.009, 0.402 +/- 0.02 and 0.416 +/- 0.015. The quantitative disposition of the reacting antigens to the two types of anti-gonadotrophins seems to bear a temporal relationship to their respective antifertility action. The pregnancy terminating action of immunoglobulins to ovine LH (Days 6, 7 & 8) and hCG (Days 8, 9 & 10) was counteracted by administration of 2 mg medroxyprogesterone acetate on Days 6, 9 and 12, indicating the importance of progesterone in the maintenance of pregnancy in the mouse.     

Toxicity oft-butylhydroperoxide in hepatocyte monolayers exposed to hypoxia and reoxygenation

Summary Inasmuch as it is known that the toxicity of anesthetic agents is potentiated by hypoxia and that the reductive metabolism of these agents results in the formation of lipid hydroperoxides, we investigated the toxicity of hydroperoxides under low-oxygen concentrations. We found that hypoxia exacerbates the toxicity oft-butyl hydroperoxide, shifting the dose-response curve oft-butyl hydroperoxide vs. lysis of hepatocytes approximately an order of magnitude to the left. Furthermore, although at the end of a 4-h exposure to 0.5% O₂ hepatocyte monolayers seemed normal by three indices (release of⁵¹Cr and serum glutamate transaminase or exclusion of trypan blue), they were completely lysed after an additional 20 h reoxygenation at 20%. O₂. In contrast, monolayers exposed to 2% O₂ for 4 h seemed normal after 20 h reoxygenation. However, cells exposed to both a subtoxic dose of hydroperoxide and 4 h of 2% O₂, although seeming healthy at the end of the hypoxic period, were completely lysed within 20 h after reoxygenation. The study was supported by grant OH 00978 from the National Institutes for Occupational Safety and Health, Atlanta, Georgia.     

Boar sperm surface glycoproteins: Isolation,localization, and temporal expression during spermatogenesis

Boar sperm glycoprotein fractions were isolated by Lens culinaris hemagglutinin affinity chromatography of detergent-solubilized ejaculated spermatozoa, followed by preparative sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. In order to develop methods for further investigations of the sperm proteins, we proceeded with two of the isolated glycoproteins. Antibodies were raised in female rabbits against each of the two sperm glycoproteins. By a combination of immunosorbent chromatography, using the antibodies obtained, and preparative SDS polyacrylamide gel electrophoresis, highly purified sperm proteins were isolated. The sperm proteins were immobilized on Sepharose gel columns and specific immunoglobulin Fab fragments were enriched by affinity chromatography. The specificity of the Fab fragments was ascertained by immunoprecipitation analysis. The Fab fragments were used in indirect immunofluorescence analysis to localize the corresponding antigens on the surface of boar spermatozoa. Both antigens were exclusively confined to the postacrosomal region. Immunohistochemical staining of boar testis sections revealed that both antigens are expressed from the spermatid stage. This technique also revealed that one of the antigens congregated at the Golgi complex-acrosome region during spermatogenesis.     

Late breast pain following reconstruction with polyurethane-covered implants

Two patients of 56 who were reconstructed with polyurethane-coated implants developed breast pain as a late complication and eventually required implant removal for relief. Although the cause of pain was not proven, it may have been due to contracture of the fibrous capsule which formed between the polyurethane and the shell of the implant. The complication of late pain has not been stressed previously in the literature on reconstruction.     

Model for microneurovascular muscle transplantation in the dog

Previous studies have suggested that successful transplantation of skeletal muscle to replace previously lost function depends on the mass of the transplanted tissue. In the present experiment, the possibility that careful microneurovascular surgical technique substantially improves the chances of successful transplantation of large-sized muscle was tested using dog gracilis muscle averaging 75 gm in weight. Gracilis muscles were completely excised ipsilaterally and were implanted into their original location (orthotopic) by reattaching tendons of insertion and origin. In addition, neurorrhaphies of nerve stumps were performed along with repair of the vascular pedicle using microsurgery techniques. After approximately 1 year, orthotopic transplants weighed about 70 percent of contralateral sham-operated gracilis muscles. Although average tension output of transplants declined to about 60 percent of control values, three of the most successfully transplanted muscles produced between 73 and 88 percent of control force. A significant increase in the number of slow-twitch-oxidative fibers was correlated with a slight but significant reduction in the maximal velocity of shortening of transplanted muscles. The ability of transplants to resist fatigue when repetitively stimulated was similar to the endurance capacity of control muscles. These results suggest that microneurovascular surgery may enhance the more complete restoration of function of transplanted skeletal muscles of relatively large size.