Binding and complement activation by C-reactive protein via the collagen-like region of C1q and inhibition of these reactions by monoclonal antibodies to C-reactive protein and C1q

Ligand-complexed C-reactive protein (CRP), like aggregated or complexed IgG, can react with C1q and activate the classical C pathway. Whereas IgG is known to bind to the globular region and not to the collagen-like region (CLR) of C1q, the site of interaction of C1q with CRP has not been defined. CRP-trimers were prepared by cross-linking and found to bind to C1q and to activate the C system. Heat-aggregated IgG (Agg-IgG) did not block the binding of CRP-trimers to C1q, nor did CRP-trimers block binding of Agg-IgG to C1q, suggesting that CRP and IgG bind at different sites. ELISA and Western blot analysis showed that CRP-trimers bound to the CLR, whereas Agg-IgG bound only to the globular region; similarly, anti-CLR mAb inhibited binding of CRP-trimers to C1q whereas anti-globular region mAb did not. Reactivity with CRP-trimers as well as with Agg-IgG was retained after reduction/alkylation and SDS treatment of C1q. A group of 22 anti-CRP mAb directed against at least six distinct native-CRP epitopes and eight distinct neo-CRP epitopes was tested for ability to inhibit the CRP-CLR interaction; one mAb, anti-native CRP mAb 8D8, with strong inhibitory activity was identified. Fab' of 8D8 blocked binding of CRP-trimers to intact C1q as well as CLR, and also inhibited CRP (CRP-trimers and CRP-protamine complexes) induced C activation, but had no effect on C1q binding or C activation by Agg-IgG. These results indicate that a conformation-determined region on CRP binds to a sequence-determined region on the CLR of C1q in an interaction which leads to C activation. Anti-CRP and anti-C1q mAb that specifically inhibit this interaction are described.     

The role of EGF in testosterone-induced reproductive tract differentiation

EGF is known to modulate a variety of cellular functions including differentiation. The aim of this investigation was to determine the role of EGF in androgen-induced masculine differentiation. Accordingly, a series of experiments were designed and the results are summarized as described below. (1) We found that the specific deprivation of EGF using anti-EGF serum during the period of masculine differentiation in an organ culture bioassay system resulted in the disintegration of the Wolffian system in a dose-dependent manner. (2) Exogenous EGF supplemented in the above experiment corrected the anti-EGF effect, suggesting a specific role of EGF. (3) Anti-EGF serum was also found to disrupt the differentiation even in the presence of exogenous testosterone, suggesting an effect independent of testosterone synthesis. (4) EGF was found to have a direct masculinizing effect both in vivo and in vitro; however, it was not able to mimic all masculinizing effects of testosterone. The mesonephric segment of the Wolffian duct was retained by EGF in the female fetal tract under in vitro conditions, and under in vivo conditions EGF was able to increase anogenital distance and to induce epididymis in some female fetal mice. (5) We were able to detect an EGF-like material in the fetal genital tract during differentiation and found that the level of this material increased with advancement of differentiation. Thus, it appears from the above results that EGF plays a role in testosterone-induced reproductive tract differentiation.     

Reduced susceptibility to HIV-1 infection of ethyl-methanesulfonate-treated CEM subclones correlates with a blockade in their protein kinase C signaling pathway.

We have described the isolation of chemically induced CEM subclones that express CD4 receptors and bind soluble gp120, yet show a markedly reduced susceptibility to infection with HIV-1. Two subclones were found to have an abnormal response to the protein kinase C (PKC) activator PMA. PMA treatment induced CD3 and CD25 (IL-2R) receptors on the parental line and on other ethyl-methanesulfonate-derived subclones, but not on these two mutants. Direct assays of PKC activity were conducted. Total cellular PKC enzymatic activity was found to be normal in these subclones. PMA-induced CD4 down-modulation occurred normally. In addition, activation of c-raf kinase was normal. Since HIV-1 long terminal repeat contains two functional nuclear factor kB (NF-kB) regulatory elements, we studied the ability of PMA to induce NF-kB binding activity by different assays. Chloramphenicol acetyl transferase (CAT) assays using the HIV-1 (-139)long terminal repeat-CAT construct showed no PMA induction of CAT activity in these subclones (unlike the parental line and other subclones). Okadaic acid, an inhibitor of phosphatases 1 and 2A, did not overcome the defect in these subclones. Gel retardation assays, using a 32P-probe containing the HIV-1 NF-kB probe and nuclear extracts from PMA-treated cells, showed significantly reduced induction of nuclear NF-kB binding proteins in these two subclones compared with wild type CEM and a control subclone. Deoxycholate treatment of cytoplasmic extracts from these subclones released much reduced NF-kB binding proteins from their cytoplasmic pools. Thus, reduced levels of PKC-induced nuclear NF-kB activity in two T cell subclones did not affect their normal cell growth, but correlated with a pronounced reduction in their susceptibility to HIV-1 infection.     

Diversity and origins of endophytic fungal symbionts of the North American grass Festuca arizonica

Summary Acremonium spp. endophytes are mutualistic fungal symbionts of many C3 grasses. They are anamorphs of Epichloë typhina (Clavicipitaceae) that have become strictly seedborne, heritable components of symbiotic units (symbiota). In order to test the possibility that endophytes may contribute to the genetic diversity of symbiota, a survey was conducted of plants from nine populations of Festuca arizonica in the southern Rocky Mountains. Sequence analysis of rRNA gene segments distinguished three Acremonium endophyte types. Parsimony analysis indicated at least two distinct evolutionary origins of the Acremonium endophytes from E. typhina. Either or both of these evolutionary lineages may have involved cospeciation with the host.     

Risk for AIDS in multiethnic neighborhoods in San Francisco,California. The population-based AMEN Study.

To examine the actual and potential spread of human immunodeficiency virus (HIV) from an acquired immunodeficiency syndrome (AIDS) epicenter to surrounding neighborhoods, we studied the prevalence of the viral infection and AIDS risk behaviors from 1988 to 1989 in a representative sample of unmarried whites, African Americans, and Hispanics living in San Francisco. We surveyed 1,770 single men and women aged 20 to 44 years (a 64% response rate) in a random household sample drawn from 3 neighborhoods of varying geographic and cultural proximity to the Castro District where the San Francisco epidemic began. Of 1,369 with blood tests, 69 (5%) had HIV antibodies; all but 5 of these reported either homosexual activity (32% HIV-positive; 95% confidence interval [CI] = 23%, 41%), injection drug use (5% HIV-positive; CI = 1%, 14%), or both (59% HIV-positive; CI 42%, 74%). Homosexual activity was more common among white men than among African-American or Hispanic men, but the proportion of those infected was similar in the 3 races. Both the prevalence of homosexually active men and the proportion infected were much lower in the 2 more outlying neighborhoods. Risk behaviors in the past year for acquiring HIV heterosexually--sex with an HIV-infected person or homosexually active man or injection drug user, unprotected sexual intercourse with more than 4 partners, and (as a proxy) having a sexually transmitted disease--were assessed in 1,573 neighborhood residents who were themselves neither homosexually active men nor injection drug users. The prevalence of reporting at least 1 of these risk behaviors was 12% overall, and race-gender estimates ranged from 5% among Hispanic women to 21% among white women. We conclude that in San Francisco, infection with HIV is rare among people who are neither homosexually active nor injection drug users, but the potential for the use spread of infection is substantial, as 12% of this group reported important risk behaviors for acquiring the virus heterosexually.     

Pyrroline-5-carboxylate reductase in soybean nodules: isolation/partial primary structure/evidence for isozymes

Electrophoretic evidence was obtained for two forms of pyrroline-5-carboxylate reductase (P5CR) in soybean nodules. One form was purified over 2300-fold. The apparent sizes of the polypeptides comprising the pyrroline-5-carboxylate reductases from soybean cytosol (29,700) and Escherichia coli (28,000) were consistent with those predicted from the sequences of the genes encoding them (Deutch et al., 1982 Nucleic Acid Res. 10, 7701-7714; Delauney and Verma, 1990 Mol. Gen. Genet. 221, 299-305). Primary structural analysis of the intact soybean P5CR subunit indicated that the amino-terminal residue is blocked. Analyses of a 12-mer and a 21-mer isolated from a cyanogen bromide digest were consistent with the proposition that the soybean P5CR isolated in these studies is very similar, although perhaps not identical, to the polypeptide predicted for the recently cloned soybean reductase (Delauney and Verma, 1990 Mol. Gen. Genet. 221, 299-305).     

Lamellar/inverted cubic (L alpha/QII) phase transition in N-methylated dioleoylphosphatidylethanolamine

Inverted cubic (QII) phases form in hydrated N-methylated dioleoylphosphatidylethanolamine (DOPE-Me). Previous work indicated that QII phases in this and other systems might be metastable structures. Whether or not QII phases are stable has important implications for models of the factors determining the relative stability of bilayer and nonbilayer phases and of the mechanisms of transitions between those phases. Here, using X-ray diffraction and very slow scan rate differential scanning calorimetry (DSC), we show that thermodynamically stable QII phases form slowly during incubation of multilamellar samples of DOPE-Me at constant temperature. The equilibrium L alpha/QII phase transition temperature is 62.2 +/- 1 degree C. The transition enthalpy is 174 +/- 34 cal/mol, about two-thirds of the L alpha/HII transition enthalpy observed at faster scan rates. This implies that the curvature free energy of lipids in QII phases is substantially lower than in L alpha phases and that this reduction is substantial compared to the reduction achieved in the HII phase. The L alpha/QII transition is slow and is not reliably detected with DSC until the temperature scan rate is reduced to ca. 1 degrees C/h. At faster scan rates, the HII phase forms at a reproducible temperature of 66 degrees C. This HII phase is metastable until ca. 72-79 degrees C, where the equilibrium QII/HII transition seems to occur. These results, as well as the induction of QII phases in similar systems by temperature cycling (observed by others), are consistent with a theory of L alpha/QII/HII transition mechanisms proposed earlier (Siegel, 1986c).     

Transit time of epithelial cells in the small intestines of germfree mice and ex-germfree mice associated with indigenous microorganisms.

Germfree mice housed in isolators under controlled environmental and nutritional conditions were associated with an intestinal microflora. These associated animals and germfree mice drawn from the same population were tested for the rate at which the epithelial cells transited from the crypts of Lieberkuhn to the tips of the villi in their small intestines. The method for estimating the rate of transit of epithelial cells involved the use of liquid scintillation counting to determine the amount of radioactivity entering the cells while the animals were being injected with [3H]thymidine and statistical analysis of th data with a computer program developed for the purpose. As estimated by that method, the cells transited from the crypts to the villous tips in germfree mice in about 115 h and in the associated animals in about 53 h. In monoassociated mice, a strain of a Lactobacillus sp. had no effect on the transit time of the epithelial cells. A strain of Torulopsis pintolopesii stimulated uptake of 3[H]thymidine by the small bowel mucosae in mice monoassociated with the organisms for 5 weeks. In animals monoassociated with the yeast fo 3, 4, and 6 weeks, however, the radioactive compound was incorporated into the bowel mucosae to the same extent as the mucosae of germfree mice. Therefore, similarly to the Lactobacillus strain, T. pintolopesii has no obvious influence on the transit rate of small bowel epithelial cells.     

Genetics of lipid deposition in the Japanese quail

Summary The research presented here was designed to investigate the mode of inheritance of fat and lean tissue deposition, and the relationship between them and body weight in Japanese quail. Heterotic effects were found for weight, size, and number of adipocytes in the abdominal fat depots, weight of the sartorial fat depot and percentage carcass fat with means for the hybrids being lower than those for the parental lines. General inferences concerning the importance of nonadditive genetic variation for lean and body weight were precluded due to inconsistencies observed among mating combinations. Thus, although heterosis and recombination effects were general for characteristics associated with fat deposition, the situation for body weight and lean was unique to the populations involved. It may be hypothesized that heterosis in the efficiency of feed utilization is reflected by the heterosis for fat deposition which explains why hybrids utilize feed better than their parental lines.