The consequences for human reproduction of a robust inflammatory response

Innate and adaptive immune responsiveness is variable within the population. Since robust immune reactions are critical to the survival of humans, the existence of immune variability in the population suggests the existence of competing, alternative phenotypes. Although women with powerful immune responsiveness may be more likely to survive to reproduce, their reproductive experiences may be less successful than women who are not as responsive. Normal pregnancy elicits a maternal inflammatory reaction. This can be understood on the basis of maternal-fetal conflict theory: inflammation is a component of the maternal attempt to limit excessive fetal demands. However, an overly aggressive inflammatory reaction has been shown to relate to a variety of adverse reproductive outcomes. Reviewed here are several examples, including the fallopian tube damage that results from pelvic inflammatory disease, the upregulated inflammatory response among women who develop preeclampsia, an association between immune hyperresponsiveness and premature delivery, and the relationship between autoimmune diseases and multiple adverse pregnancy outcomes. The hypothesis that immune hyperresponsiveness limits reproductive capacity suggests many avenues for research.     

Target size analysis by radiation inactivation: the use of free radical scavengers

Several model systems were employed to assess indirect effects that occur in the process of using radiation inactivation analysis to determine protein target sizes. In the absence of free radical scavengers, such as mannitol and benzoic acid, protein functional unit sizes can be drastically overestimated. In the case of glutamate dehydrogenase, inclusion of free radical scavengers reduced the apparent target size from that of a hexamer to that of a trimer based on enzyme activity determinations. For glucose-6-phosphate dehydrogenase, the apparent target size was reduced from a dimer to a monomer. The target sizes for both glutamate dehydrogenase and glucose-6-phosphate dehydrogenase in the presence of free radical scavengers corresponded to subunit sizes when determinations of protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or immunoblotting were done rather than enzyme activity. The free radical scavengers appear to compete with proteins for damage by secondary radiation products, since irradiation of these compounds can result in production of inhibitory species. Addition of benzoic acid/mannitol to samples undergoing irradiation was more effective in eliminating secondary damage than were 11 other potential free radical scavenging systems. Addition of a free radical scavenging system enables more accurate functional unit size determinations to be made using radiation inactivation analysis.     

AUTOPOLYPLOIDY IN HEUCHERA MICRANTHA (SAXIFRAGACEAE)

Heuchera micrantha (Saxifragaceae) is a morphologically variable species comprising five varieties: diversifolia, erubescens, hartwegii, micrantha, and pacifica. Both diploids (2n = 14) and tetraploids (2n = 28) occur within the species. The tetraploid cytotype occupies the central portion of the geographic range of the species, whereas diploids occur primarily in the southern and northern portions of the range. Both diploids and tetraploids have been detected within vars. diversifolia, pacifica, and hartwegii. All counts for vars. erubescens and micrantha are diploid and tetraploid, respectively. Several lines of evidence suggest that tetraploid H. micrantha is of autopolyploid origin. The species is distinct morphologically and is also well separated geographically from other closely related species in subsection Micranthae. The two cytotypes are karyologically identical, possess nearly the same suite of allozymes, and have a very high genetic identity (Ī = 0.971). Significantly, an earlier study documented tetrasomic inheritance in the tetraploid cytotype. Following theoretical expectations, the mean number of alleles per locus, proportion of loci that are polymorphic, and observed heterozygosity are significantly higher for the autotetraploid than for the diploid. The occurrence of both cytotypes in three of the varieties suggests that autopolyploidy may have occurred several times independently in H. micrantha. This was further substantiated by discriminant analysis using morphological characters, which provided evidence for a minimum of two separate origins for the autopolyploid cytotype.     

Cultured AIDS-related Kaposi's sarcoma cells retain a proliferative bioenergetic profile but demonstrate reduced cytoprotective capabilities

Features of AIDS-related Kaposi's sarcoma (AIDS-KS), such as the multifocal presentation at mucosal and epidermal sites subjected to trauma, suggest that AIDS-KS is initially a reactive hyperplasia that subsequently progresses to a neoplasia. It is recognized that there is an association between sustained states and the subsequent development of neoplasia (e.g., ulcerative colitis/colonic adenocarcinoma). Furthermore, patients who develop AIDS-KS experience both a constant immune stimulation due to sustained high levelsof virus-induced cytokines and, because of a sparing effect on their phagoctic cells, retention of the phagocytic inflammatory response. A component of phygocytic activation is the initiation of the oxidative brust, resulting in the generation of reactive oxygen species (ROS), which can be mutagenic to host cells if released beyond the phagolysosome and not inactivated. Our results demonstrate that cultured AIDS-KS cells possess drcreased cytoprotective capabilities. Relativeto either dermal fibroblasts, or human microvascular endothelial cells (HMECs), AIDS-KS cells contained significantly lower levels of glutathione, a tripeptide integral in both cytoprotection and maintenance of cellular thiol status. While HMECs increased catalase activity during culture in the cytokine-rich KS milieu (control medium supplement with conditioned medium from MOT, an TLV II-infected cell line), AIDS-KS cells demonstrated reduced catalase function under these conditions. Furthermore, HMEC cultures showed in inherent biochemical responsiveness, by increasing catalase activity following exposure to exaogenous H₂(O₂). In contrast, the catalase activity of AIDS-KS cells decreased following (H₂O₂) challenge. Our results show that an inherent deficiency in cellular cytoprotection is present in AIDS-KS cells and suggest that oxidant stress may function in the development and progression AIDS-KS.     

Isolation and Characterization of a Double-Stranded Ribonucleic Acid from Penicillium chrysogenum

Double-stranded ribonucleic acid has been obtained from cells of the fungus Penicillium chrysogenum. This ribonucleic acid appears to be associated with mycophage and is an efficient inducer of interferon Its. extraction and partial purification are discussed, and evidence for its double-stranded and ribosidal nature is reviewed. The implications of viral nucleic acid in the life processes of fungi are considered.     

Development of a noncompetitive, solid phase, bridged biotin-avidin enzyme immunoassay for measurement of human leukocyte microsomal HMG-CoA reductase protein concentration

Methods were developed for determination of human mononuclear leukocyte HMG-CoA reductase protein concentration by a noncompetitive, solid phase, bridged biotin-avidin enzyme immunoassay procedure. Leukocyte microsomal HMG-CoA reductase, first immobilized onto a nitrocellulose filter, is sequentially reacted with 1) monospecific, polyclonal rabbit anti-rat liver HMG-CoA reductase antiserum, which crossreacts with the human liver and leukocyte enzymes; 2) biotinylated donkey anti-rabbit immunoglobulin; 3) a streptavidin-horseradish peroxidase conjugate; and 4) 4-chloro-1-naphthol and H2O2 to visualize the quantity of horseradish peroxidase bound to the immunocomplex. Color development was proportional to the quantity of either purified liver or leukocyte microsomal HMG-CoA reductase applied to the nitrocellulose. Color development was not observed, however, when HMG-CoA reductase was omitted from the nitrocellulose, when one of the reactant species was omitted from the incubation reactions, or when anti-rat liver HMG-CoA reductase antiserum was pre-absorbed with either rat liver or human leukocyte HMG-CoA reductase. Immunoreactivity of microsomal HMG-CoA reductase was independent of the phosphorylation state of the enzyme, but was inversely related to the concentration of thiol-reducing agents present in the microsomal preparation up to 4 mM. Further increases in thiol-reductant failed to produce changes in immunoreactivity. Freshly isolated mononuclear leukocyte microsomal HMG-CoA reductase protein concentration in leukocytes from 31 healthy, normocholesterolemic subjects was a linear function of HMG-CoA reductase activity (R = 0.65; P less than 0.001). The catalytic efficiency of the freshly isolated mononuclear leukocyte enzyme was 313 +/- 34 pmol of mevalonate formed per min of incubation at 37 degrees C per mg immunoreactive protein. This methodology, in conjunction with that recently developed to measure human leukocyte HMG-CoA reductase activity (1984. J. Lipid Res. 25: 967-978), should prove useful in discriminating between HMG-CoA reductase regulatory mechanisms involving changes in enzyme protein concentration and those resulting from changes in enzyme catalytic efficiency.     

Characteristics of rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

A procedure for the preparation of rat liver microsomal fractions essentially devoid of contaminating lysosomes is described. When this preparation was examined by immunoblotting with a rabbit antiserum to rat 3-hydroxy-3-methylglutaryl-CoA reductase, a single band corresponding to an Mr of 100000 was observed. No evidence was found for glycosylation of rat liver-3-hydroxy-3-methylglutaryl-CoA reductase. Native rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase differs from the purified proteolytically modified species in that it displays allosteric kinetics towards NADPH.