FLUID PHASE DESTRUCTION OF C2hu BY C1hu : I. ITS ENHANCEMENT AND INHIBITION BY HOMOLOGOUS AND HETEROLOGOUS C4

The fluid phase inactivation of C2^hu by C1^hu is markedly enhanced by the presence of C4^hu. The enhancement is afforded by C1 inactivated C4^hu, namely C4i^hu, and requires the simultaneous presence of enzymatically active C1. Heterologous C4 of guinea pig origin protects C2^hu from the inactivation by C1^hu. Thus, in both the fluid phase and on the cellular intermediate, C4^hu is essential to the specific action of C1^hu on C2^hu. It is possible that C4i alters C2 so as to present a more suitable substrate to the C1 enzyme or that C4i acts on the C1 to uncover a specificity for native C2.     

THE EFFECT OF HEAT ON FLAGELLAR AND SOMATIC AGGLUTINATION

1. Heat at 70°C. destroys the form of the flagella and their ability to combine with flagellar agglutinins but it does not destroy their antigenic nature since they can still generate flagellar agglutinins in the animal body. 2. Heat at 70°C. and even at 120°C. in the autoclave does not destroy the forms of the bacilli themselves nor their ability to become agglutinated and to absorb agglutinins. 3. Somatic agglutinins are destroyed to a considerable extent by heat at 70°C. and completely destroyed by heat at 75°C. 4. Heat at 70°C. causes little or no destruction of flagellar agglutinins but a temperature of 75°C. changes the agglutinins so that they react more slowly and produce a slightly lower reaction with a zone of inhibition in the stronger dilutions.     

The role of C4-binding protein and {beta }1H in proteolysis of C4b and C3b

Two forms of C4-binding protein (C4-bp) (C4-bp low, C4-bp high), which differ slightly in net charge and apparent molecular weight, as determined by SDS- PAGE, were separated by ion-exchange chromatography and contaminants removed with specific antisera. Both forms of C4-bp served as cofactors for the cleavage of C4b in solution by C3b inactivator, and the resulting fragments of the a’-chain of C4b had identical molecular weights. In addition, similarly to β1H, C4-bp low or high served as cofactors for the cleavage of fluid phase C3b by C3bINA. However, important quantitative differences between the activities of C4-bp and β1H were observed. With regard to C3b in solution, the cofactor activity of β1H was {approximately equal to}20 times greater than that of C4-bp on a weight basis. In relation to cell-bound C3b, the differences in activity were even more marked. Whereas β1H enhanced the effects of C3bINA on the erythrocyte intermediate EC3b, inhibiting the assembly of EC3bBb, C4-bp was without effect even at concentrations {approximately equal to}300 times greater than β1H. Therefore, under physiological conditions, it is likely that β1H is the key protein which controls the function of C3b, and that C4-bp activity is directed mainly toward the cleavage of C4b. We also examined the relation between C4-bp and the C3b-C4bINA cofactor described by Stroud and collaborators (3, 4). By functional, physico-chemical and immunological criteria, they are the same protein.     

Interplay of Mre11 Nuclease with Dna2 plus Sgs1 in Rad51-Dependent Recombinational Repair

The Mre11/Rad50/Xrs2 complex initiates IR repair by binding to the end of a double-strand break, resulting in 5′ to 3′ exonuclease degradation creating a single-stranded 3′ overhang competent for strand invasion into the unbroken chromosome. The nuclease(s) involved are not well understood. Mre11 encodes a nuclease, but it has 3′ to 5′, rather than 5′ to 3′ activity. Furthermore, mutations that inactivate only the nuclease activity of Mre11 but not its other repair functions, mre11-D56N and mre11-H125N, are resistant to IR. This suggests that another nuclease can catalyze 5′ to 3′ degradation. One candidate nuclease that has not been tested to date because it is encoded by an essential gene is the Dna2 helicase/nuclease. We recently reported the ability to suppress the lethality of a dna2Δ with a pif1Δ. The dna2Δ pif1Δ mutant is IR-resistant. We have determined that dna2Δ pif1Δ mre11-D56N and dna2Δ pif1Δ mre11-H125N strains are equally as sensitive to IR as mre11Δ strains, suggesting that in the absence of Dna2, Mre11 nuclease carries out repair. The dna2Δ pif1Δ mre11-D56N triple mutant is complemented by plasmids expressing Mre11, Dna2 or dna2K1080E, a mutant with defective helicase and functional nuclease, demonstrating that the nuclease of Dna2 compensates for the absence of Mre11 nuclease in IR repair, presumably in 5′ to 3′ degradation at DSB ends. We further show that sgs1Δ mre11-H125N, but not sgs1Δ, is very sensitive to IR, implicating the Sgs1 helicase in the Dna2-mediated pathway.     

Lucidone Suppresses Hepatitis C Virus Replication by Nrf2-Mediated Heme Oxygenase-1 Induction

Upon screening of plant-derived natural products against hepatitis C virus (HCV) in the replicon system, we demonstrate that lucidone, a phytocompound, isolated from the fruits of Lindera erythrocarpa Makino, significantly suppressed HCV RNA levels with 50% effective concentrations of 15 ± 0.5 μM and 20 ± 1.1 μM in HCV replicon and JFH-1 infectious assays, respectively. There was no significant cytotoxicity observed at high concentrations, with a 50% cytotoxic concentration of 620 ± 5 μM. In addition, lucidone significantly induced heme oxygenase-1 (HO-1) production and led to the increase of its product biliverdin for inducing antiviral interferon response and inhibiting HCV NS3/4A protease activity. Conversely, the anti-HCV activity of lucidone was abrogated by blocking HO-1 activity or silencing gene expression of HO-1 or NF-E2-related factor 2 (Nrf2) in the presence of lucidone, indicating that the anti-HCV action of lucidone was due to the stimulation of Nrf-2-mediated HO-1 expression. Moreover, the combination of lucidone and alpha interferon, the protease inhibitor telaprevir, the NS5A inhibitor BMS-790052, or the NS5B polymerase inhibitor PSI-7977, synergistically suppressed HCV RNA replication. These findings suggest that lucidone could be a potential lead or supplement for the development of new anti-HCV agent in the future.     

Evaluation of PD 404,182 as an Anti-HIV and Anti-Herpes Simplex Virus Microbicide

PD 404,182 (PD) is a synthetic compound that was found to compromise HIV integrity via interaction with a nonenvelope protein viral structural component (A. M. Chamoun et al., Antimicrob. Agents Chemother. 56:672–681, 2012). The present study evaluates the potential of PD as an anti-HIV microbicide and establishes PD''s virucidal activity toward another pathogen, herpes simplex virus (HSV). We show that the anti-HIV-1 50% inhibitory concentration (IC₅₀) of PD, when diluted in seminal plasma, is ∼1 μM, similar to the IC₅₀ determined in cell culture growth medium, and that PD retains full anti-HIV-1 activity after incubation in cervical fluid at 37°C for at least 24 h. In addition, PD is nontoxic toward vaginal commensal Lactobacillus species (50% cytotoxic concentration [CC₅₀], >300 μM), freshly activated human peripheral blood mononuclear cells (CC₅₀, ∼200 μM), and primary CD4⁺ T cells, macrophages, and dendritic cells (CC₅₀, >300 μM). PD also exhibited high stability in pH-adjusted Dulbecco''s phosphate-buffered saline with little to no activity loss after 8 weeks at pH 4 and 42°C, indicating suitability for formulation for transportation and storage in developing countries. Finally, for the first time, we show that PD inactivates herpes simplex virus 1 (HSV-1) and HSV-2 at submicromolar concentrations. Due to the prevalence of HSV infection, the ability of PD to inactivate HSV may provide an additional incentive for use as a microbicide. The ability of PD to inactivate both HIV-1 and HSV, combined with its low toxicity and high stability, warrants additional studies for the evaluation of PD''s microbicidal candidacy in animals and humans.     

Vitamin C transporter Slc23a1 links renal reabsorption,vitamin C tissue accumulation,and perinatal survival in mice

Levels of the necessary nutrient vitamin C (ascorbate) are tightly regulated by intestinal absorption, tissue accumulation, and renal reabsorption and excretion. Ascorbate levels are controlled in part by regulation of transport through at least 2 sodium-dependent transporters: Slc23a1 and Slc23a2 (also known as Svct1 and Svct2, respectively). Previous work indicates that Slc23a2 is essential for viability in mice, but the roles of Slc23a1 for viability and in adult physiology have not been determined. To investigate the contributions of Slc23a1 to plasma and tissue ascorbate concentrations in vivo, we generated Slc23a1^–/– mice. Compared with wild-type mice, Slc23a1^–/– mice increased ascorbate fractional excretion up to 18-fold. Hepatic portal ascorbate accumulation was nearly abolished, whereas intestinal absorption was marginally affected. Both heterozygous and knockout pups born to Slc23a1^–/– dams exhibited approximately 45% perinatal mortality, and this was associated with lower plasma ascorbate concentrations in dams and pups. Perinatal mortality of Slc23a1^–/– pups born to Slc23a1^–/– dams was prevented by ascorbate supplementation during pregnancy. Taken together, these data indicate that ascorbate provided by the dam influenced perinatal survival. Although Slc23a1^–/– mice lost as much as 70% of their ascorbate body stores in urine daily, we observed an unanticipated compensatory increase in ascorbate synthesis. These findings indicate a key role for Slc23a1 in renal ascorbate absorption and perinatal survival and reveal regulation of vitamin C biosynthesis in mice.     

INFLUENCE OF EXTRANEOUS PROTEIN AND VIRUS CONCENTRATION ON THE INACTIVATION OF THE RABBIT PAPILLOMA VIRUS BY X-RAYS

The pronounced resistance to the x-rays manifested by the papilloma virus in ordinary suspensions is due to the protecting influence of extraneous matter and also in considerable degree to the amount of virus present in the preparation. Two to 4 million r were required to inactivate the virus contained in the crude papilloma extracts prepared for the present work, whereas 100,000 r or less was enough to inactivate comparable concentrations of virus after extraneous matter had been excluded by repeated differential centrifugation. The addition of normal rabbit serum or crystalline egg albumin to purified suspensions of virus was found to increase greatly the amount of irradiation required to inactivate the virus. Furthermore the percentage destruction of virus by a given amount of irradiation increases as the concentration is decreased by dilution with saline or buffer solutions. As little as 3,000 r will inactivate much of the virus in very dilute suspensions. The complement-binding antigen of papilloma virus suspensions is also inactivated by x-rays, but requires a somewhat larger amount of irradiation than necessary to destroy the infectivity of the suspensions. The effects of irradiation on the antiviral antibody present in the blood of animals which have become immune to the virus—an antibody that specifically fixes complement in mixture with the papilloma virus—are also conditioned by extraneous material. 250,000 to 500,000 r had only a slight effect on the antibody in whole serum, while this amount of irradiation completely inactivated comparable amounts of antibody in preparations partially purified by precipitation with ammonium sulfate. As a whole the findings indicate that under certain conditions of purity and concentration most of the radiation does not act by direct hits on virus or antibody particles, but indirectly by ionizing or exciting some other molecules present in the exposed suspension, which then react with the virus or antibody molecules.     

Anidulafungin Is Fungicidal and Exerts a Variety of Postantifungal Effects against Candida albicans,C. glabrata,C. parapsilosis,and C. krusei isolates

Anidulafungin targets the cell walls of Candida species by inhibiting β-1,3-glucan synthase, thereby killing isolates and exerting prolonged postantifungal effects (PAFEs). We performed time-kill and PAFE experiments on Candida albicans (n = 4), C. glabrata (n = 3), C. parapsilosis (n = 3), and C. krusei (n = 2) isolates and characterized the PAFEs in greater detail. MICs were 0.008 to 0.125 μg/ml against C. albicans, C. glabrata, and C. krusei and 1.0 to 2.0 μg/ml against C. parapsilosis. During time-kill experiments, anidulafungin caused significant kills at 16× MIC (range, log 2.68 to 3.89) and 4× MIC (log 1.87 to 3.19), achieving fungicidal levels (≥log 3) against nine isolates. A 1-hour drug exposure during PAFE experiments resulted in kills ranging from log 1.55 to 3.47 and log 1.18 to 2.89 (16× and 4× MIC, respectively), achieving fungicidal levels against four isolates. Regrowth of all 12 isolates was inhibited for ≥12 h after drug washout. Isolates of each species collected 8 h after a 1-hour exposure to anidulafungin (16× and 4× MIC) were hypersusceptible to sodium dodecyl sulfate (0.01 to 0.04%) and calcofluor white (40 μg/ml). Moreover, PAFEs were associated with major cell wall disturbances, as evident in electron micrographs of viable cells, and significant reductions in adherence to buccal epithelial cells (P ≤ 0.01). Finally, three of four PAFE isolates tested were hypersusceptible to killing by J774 macrophages (P ≤ 0.007). Our data suggest that the efficacy of anidulafungin in the treatment of candidiasis might stem from both direct fungicidal activity and indirect PAFEs that lessen the ability of Candida cells to establish invasive disease and to persist within infected hosts.Anidulafungin is an echinocandin agent that disrupts the cell walls of Candida species by inhibiting β-1,3-d-glucan synthase. In recent studies of treatment of invasive candidiasis, the agent was shown to be at least as effective as the frontline azole agent fluconazole (12, 22). Additional clinical trial data demonstrating the efficacy of caspofungin and micafungin in the treatment of diverse types of candidiasis make it clear that the echinocandins are significant additions to the antifungal armamentarium (13, 17).In general, MIC₉₀s of anidulafungin are low against the common pathogens Candida albicans, C. glabrata, C. tropicalis, and C. krusei (0.06 to 0.12 μg/ml), including isolates that are resistant to azole agents (20, 21). MIC₉₀s against C. parapsilosis and C. guilliermondii isolates are higher (2 μg/ml), as also noted for other echinocandins (20, 21). Diminished susceptibility to anidulafungin might reflect changes in the glucan synthase subunit Fks1p (2, 18). Regardless of the mechanism, the clinical significance of elevated anidulafungin MICs remains unclear (22). To date, only a few studies have assessed the anticandidal activity of anidulafungin by time-kill or postantifungal effect (PAFE) methods. Similar to other echinocandins, anidulafungin exhibited concentration-dependent fungicidal activity against C. albicans, C. glabrata, C. tropicalis, and C. krusei isolates during time-kill experiments at concentrations of 4× and 16× MIC (10, 23). In PAFE experiments, a 1-hour exposure to anidulafungin at 4× MIC resulted in prolonged growth inhibition of C. albicans (9). To our knowledge, time-kill or PAFE data have not been published for anidulafungin against C. parapsilosis. Caspofungin, however, is fungicidal and causes prolonged PAFE growth inhibition of C. parapsilosis at concentrations of ≥4× MIC (7).We hypothesized that anidulafungin would demonstrate significant PAFEs against Candida isolates of diverse species, as measured by growth inhibition following brief drug exposure in vitro. In addition, we hypothesized that anidulafungin''s PAFEs would cause changes to the candidal cell wall that would result in decreased cell integrity and adherence to host cells and in increased susceptibility to killing by phagocytes. In this study, we assessed the fungicidal activity of anidulafungin against 12 Candida isolates (4 C. albicans, 3 C. glabrata, 3 C. parapsilosis, and 2 C. krusei isolates) by time-kill and PAFE methods. We then tested PAFE-inhibited cells for susceptibility to cell wall-active drugs and visualized cell walls by electron microscopy. Finally, we assessed adherence to human epithelial cells and killing by macrophages.     

ADSORPTION OF INFLUENZA HEMAGGLUTININS AND VIRUS BY RED BLOOD CELLS

A number of experiments were performed on the adsorption of influenza hemagglutinins on chicken red blood cells, from which the following conclusions were drawn:— 1. When chicken red blood cells and preparations of influenza viruses were mixed together, the influenza hemagglutinins present were rapidly adsorbed onto the cells. After varying lengths of time, dependent on the conditions of the experiment, the adsorbed hemagglutinins began to elute from the cells. With the Lee strain at 23°C. and the PR8 strain at 37°C. almost all of the adsorbed agglutinin was released in 4 to 6 hours. 2. When the number of red cells used for adsorption was increased, the speed and degree of adsorption of the hemagglutinins increased. The time of maximum adsorption of hemagglutinins was the same, regardiess of red cell concentration, and with the larger amounts of red cells the speed and degree of elution was decreased. 3. When adsorption of PR8 virus agglutinins was carried out at 4°C. the adsorption was rapid and nearly complete. When the reaction was carried out at higher temperatures (27° and 37°C.), the adsorption was equally rapid but was progressively less complete with rise in temperature. At 4°C. the maximum adsorption was not reached for 5 hours; at 27°C. it was reached in 25 minutes; and at 37°C. the greatest degree of adsorption was attained between 3 and 5 minutes. The amount of elution observed at 4°C. at 18 hours was negligible, but the degree of elution increased with temperature so that at 37°C. almost all of the adsorbed agglutinin was released in 6 hours'' time. 4. Red cells which had adsorbed and then fully eluted the agglutinin were not capable of adsorbing a detectable amount of fresh agglutinin. In addition, such cells would no longer agglutinate even though exposed to fresh virus suspensions. 5. The hemagglutinin of influenza B virus was capable of being adsorbed on and eluted from several successive lots of chicken red cells without appreciable loss of agglutinating activity. 6. The hemagglutinins of the PR8 and Lee strains were rapidly inactivated at 60°C. The presence of active virus was not necessary for the occurrence of the adsorption-elution reaction on chicken red cells. 7. The activity of the portion of the red cells responsible for the adsorption of the hemagglutinins persisted, though in reduced amount, even after heating for 5 minutes at 100°C. Hemagglutinins were adsorbed and eluted from red cell stroma. 8. The infective agent in influenza virus suspensions was adsorbed by chicken red cells simultaneously with the adsorption of hemagglutinins. 95 per cent of the infective agent was removed from suspension by the red cells after contact for 15 minutes. From then on the infective agent was gradually released from the red cells. After 4 hours the 50 per cent mortality titer of the supernatant fluid was as high as at the beginning of the experiment.     

GT-1a or GT-1b Subtype-Specific Resistance Profiles for Hepatitis C Virus Inhibitors Telaprevir and HCV-796

In vitro, telaprevir selects subtype-specific resistance pathways for hepatitis C virus GT-1a and GT-1b, as described to have occurred in patients. In GT-1a, the HCV-796 resistance mutation C316Y has low replication capacity (7%) that can be compensated for by the emergence of the mutation L392F or M414T, resulting in an increase in replication levels of ≥10-fold.The current standard of care for hepatitis C virus (HCV)-infected patients involves a treatment regimen of pegylated alpha interferon in combination with ribavirin, which results in a sustained viral response of approximately 50% for genotype 1 (GT-1)-infected patients (1, 11). There is a clear medical need for more efficacious therapies, and to this effect, a number of novel specific antiviral compounds are currently in preclinical and clinical development. A majority of these compounds inhibit the enzymatic activity of either the NS3/4A serine protease or the NS5B RNA-dependent RNA polymerase.One factor that may limit the clinical efficacy of specific HCV antiviral drugs is the development of resistance. HCV presents a number of features that make drug resistance likely to occur upon treatment, such as the following: (i) the NS5B polymerase lacks proofreading activity, which results in the introduction of random mutations during the replication of the genomic RNA; (ii) HCV replicates as a genetic population known as a quasispecies that allows quick adaptation of the viral population upon changes in the environment (12); (iii) HCV produces a large number of infectious particles (up to 10¹²) per day, which means that each genetic variant made during RNA replication may be packaged into an infectious viral particle and can quickly spread (15); and (iv) the short half-life of the HCV genome, as estimated for the circulating virus (14) and calculated for the HCV replicon (3), is such that a variant present at low prevalence within the quasispecies can quickly become the dominant sequence if it offers a selective advantage. Resistance to specific HCV inhibitors in vitro has been well characterized through the use of the HCV GT-1b replicon system, and these studies have been predictive of the amino acid substitution(s) selected in HCV-infected patients upon drug treatment (4, 7-10, 13). For example, for the NS3/4A protease inhibitor telaprevir and the nonnucleoside polymerase inhibitor HCV-796, the resistance mutations identified in vitro (NS3 substitutions at residues T54 and A156 for telaprevir and an NS5B substitution at residue C316 for HCV-796) were also identified in GT-1b-treated patients (4, 5, 16).One limitation of the majority of the replicon resistance studies reported to date is that only a single HCV subtype, GT-1b, has been used. HCV subtypes can vary by up to 25% at the nucleotide level, and this variability may lead to subtype-specific differences in the resistance profiles. In fact, subtype-specific resistance profiles for HCV-infected patients treated with telaprevir have been described previously. Substitutions at NS3 residues V36 and R155 were identified only in GT-1a-infected patients treated with telaprevir and not in GT-1b-infected patients (5, 16). As a result, the findings of the in vitro replicon resistance studies of telaprevir, which used a GT-1b replicon, were predictive for the GT-1b-infected patients but did not identify the emergence of substitutions at V36 or R155. Therefore, in this study, we determined if the HCV replicon system could be used to identify subtype-specific resistance mutations. For these experiments, we treated both a GT-1b replicon and a GT-1a replicon with either the protease inhibitor telaprevir (synthesized at Acme Bioscience, Inc.) or the nonnucleoside polymerase inhibitor HCV-796 (synthesized at Roche Palo Alto) at 15 times the 50% effective concentration (EC₅₀), which for both compounds approximates the EC₉₉, and monitored the emergence of resistance mutations in the NS3 protease or NS5B polymerase gene, respectively. Four independent selection experiments were performed for GT-1b, and two were performed for GT-1a. The GT-1b and GT-1a replicons are both bicistronic replicons in which the first open reading frame (driven by the HCV internal ribosome entry site) contains the Renilla luciferase gene fused with the neomycin phosphotransferase II gene and the second open reading frame (driven by the encephalomyocarditis virus internal ribosome entry site) contains the HCV nonstructural genes with engineered cell culture-adaptive mutations (2, 6). We monitored the kinetics of the development of telaprevir resistance in both GT-1b (n = 4) and GT-1a (n = 2) replicon-bearing cells and characterized the telaprevir resistance profiles to determine if the differences in resistance profiles identified in patients infected with either GT-1a or GT-1b HCV would also be observed in the replicon system. The stably transfected replicon cells bearing either a GT-1b or GT-1a replicon (GT-1a replicon encodes 75 amino acid residues of NS3 protease from the GT-1b Con 1 strain, as described by Gu et al. [2]) were incubated for a maximum of 21 days with 15 times the EC₅₀ of telaprevir (Table ​(Table1)1) as described previously (13), with the exception that sampling was performed on days 3, 6, 9, 13, 16, and 21. Consistent with the data in previous reports (9, 10, 13), the incubation of GT-1b replicon cells with telaprevir resulted in the emergence of an amino acid substitution at NS3 position 156, and this substitution was identified as early as 3 days after the beginning of treatment (Table ​(Table2)2) by comparing the NS3 protease sequence from the untreated replicon cells with that from replicon cells taken at the specified treatment time points. The incubation of GT-1a replicon cells with telaprevir resulted in the selection of a mixture of sequences with wild-type (WT) and mutant R155R/K residues on day 3, with additional substitutions present at positions 156 (day 6) and 54 (day 9) (Table ​(Table2).2). The selection of R155K in GT-1a replicon cells, but not in GT-1b replicon cells, is consistent with the resistance profile described for treated patients. We were unable to detect the emergence of a resistance amino acid substitution at NS3 position 36 given the GT-1a/1b chimeric nature of the NS3 protease region used in this study, reinforcing the observation that V36M occurs only in GT-1a HCV.

TABLE 1.

Inhibitory activity and cytotoxicity against stable HCV GT-1b and GT-1a replicon cells

Human Plasma Protein C: ISOLATION, CHARACTERIZATION, AND MECHANISM OF ACTIVATION BY α-THROMBIN

Protein C is a vitamin K-dependent protein, which exists in bovine plasma as a precursor of a serine protease. In this study, protein C was isolated to homogeneity from human plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. Human protein C (M(r) = 62,000) contains 23% carbohydrate and is composed of a light chain (M(r) = 21,000) and a heavy chain (M(r) = 41,000) held together by a disulfide bond(s). The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe-Leu- and the heavy chain has an aminoterminal sequence of Asp-Pro-Glu-Asp-Gln. The residues that are identical to bovine protein C are underlined. Incubation of human protein C with human alpha-thrombin at an enzyme to substrate weight ratio of 1:50 resulted in the formation of activated protein C, an enzyme with serine amidase activity. In the activation reaction, the apparent molecular weight of the heavy chain decreased from 41,000 to 40,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. No apparent change in the molecular weight of the light chain was observed in the activation process. The heavy chain of human activated protein C also contains the active-site serine residue as evidenced by its ability to react with radiolabeled diisopropyl fluorophosphate. Human activated protein C markedly prolongs the kaolin-cephalin clotting time of human plasma, but not that of bovine plasma. The amidolytic and anticoagulant activities of human activated protein C were completely obviated by prior incubation of the enzyme with diisopropyl fluorophosphate. These results indicate that human protein C, like its bovine counterpart, exists in plasma as a zymogen and is converted to a serine protease by limited proteolysis with attendant anticoagulant activity.     

CHARACTERISTICS OF A NON-COMPLEMENT-DEPENDENT C3-REACTIVE COMPLEX FORMED FROM FACTORS IN NEPHRITIC AND NORMAL SERUM

When serum from a patient with membrano-proliferative glomerulonephritis and normal serum are mixed at 37°C, C3 is rapidly broken down to two more rapidly migrating components. In the mixture, a heat-labile pseudoglobulin, designated as the C3 nephritic factor or C3NeF, reacts with a pseudogolbulin in the normal serum, designated as cofactor, to form a C3 inactivator. By analogy with the cobra venom factor, the C3 inactivator is most likely a complex of the nephritic factor and cofactor. The complex has been designated as the C3 lytic nephritic factor or C3LyNeF. The reaction which results in the Formation of C3LyNeF requires the presence of Mg⁺⁺, is highly temperature sensitive but occurs very rapidly at 37°C. In 20 min at 37°C, C3LyNeF can break down over 80% of the C3 in a mixture of normal and nephritic serum. The two-step reaction which leads to C3 breakdown has an optimum pH ranging from 6.0 to 9.0. Experiments employing serum depleted of C4 and C2, as well as certain characteristics of the C3NeF system provide evidence that C3 breakdown with nephritic serum is not dependent on complement-inactivating immune complexes or on the action of convertase (C4, 2). Data relating rate of C3 breakdown to the concentrations of C3NeF, C3, and C3LyNeF in the reaction mixture are similar to those for the reaction of enzyme with substrate. The biological significance of C3LyNeF in the production of glomerular inflammation has not been established.     

DEFICIENCY OF C1r IN HUMAN SERUM : EFFECTS ON THE STRUCTURE AND FUNCTION OF MACROMOLECULAR C1

The experiments presented here utilize a human serum markedly deficient in hemolytic complement activity to show that: (a) The hemolytic deficiency is the result of a selective deficiency in hemolytic C1. (b) The relative absence of hemolytic C1 is due to a profound deficit in C1r function associated with less than normal C1s protein and hemolytic function and normal C1q protein concentration and function. This deficit in C1r in the face of normal C1q suggests that different cell types are responsible for the synthesis of each of these components. (c) Whatever the basis for the deficiency of C1r function, this defect results in an inadequate association of the remaining C1 subcomponents, C1q and C1s, even in the presence of calcium ions, thus suggesting that C1r has an important role in the assembly and/or maintenance of macromolecular C1.     

Susceptibility of Enterobacter to Cefamandole: Evidence for a High Mutation Rate to Resistance

Cefamandole minimum inhibitory concentrations (MICs) of 10 strains of Enterobacter were determined by the ICS agar dilution and broth dilution procedures. Agar dilution MICs ranged from 1 to 8 μg/ml, with an inoculum of 10⁴ organisms/spot. Broth dilution MICs were consistently higher, with an inoculum of approximately 7 × 10⁵ organisms/ml. Seven strains showed MICs of ≥64 μg/ml. There was a marked inoculum effect in broth, and skipped tubes were often observed. Variants resistant to 32 μg/ml or more were isolated by direct selection and were shown to occur at a frequency of approximately 10⁻⁶ to 10⁻⁷. A mutant showing a 16-fold increase in agar dilution MIC was also isolated by indirect selection. These variants and others isolated from broth in the presence of cefamandole were tested for ability to inactivate the antibiotic, using both a biological and a chemical procedure. Two distinct classes of variants were seen. Twelve of 28 were shown by both methods to inactivate the antibiotic, whereas the others, including the indirectly selected mutant, did not. The wild types were also negative by both tests. The higher cefamandole MICs of Enterobacter in broth, thus, appeared to reflect a high frequency of resistant variants that were not detected with the inoculum and end point criteria usually used in agar dilution methods. The ability of some variants to inactivate cefamandole may have resulted from a mutation that extended the activity of Enterobacter cephalosporinase to include this antibiotic.     

Unique C1 inhibitor dysfunction in a kindred without angioedema. II. Identification of an Ala443-->Val substitution and functional analysis of the recombinant mutant protein.

We have determined the cause of an unusual C1 inhibitor abnormality in a large kindred. We previously found that half of serum C1 inhibitor molecules in affected kindred members are normal. The other half complexed with C1s but showed little complex formation with C1r. These molecules also appeared to be relatively resistant to digestion by trypsin. Taken together, the findings suggested that members of this kindred are heterozygous for an unusual C1 inhibitor mutation. Sequencing of genomic DNA from the kindred revealed that thymine has replaced cytosine in the codon for Ala443 (P2 residue) in one C1 inhibitor allele, resulting in substitution with a Val residue. To test the effect of this substitution, a mutant C1 inhibitor containing Ala443-->Val was constructed by site-directed mutagenesis and expressed in COS-1 cells. Both the Ala443-->Val mutant and the wild-type C1 inhibitor complexed completely with C1s, kallikrein, and coagulation Factor XIIa after incubation at 37 degrees C for 60 min. In contrast, the mutant inhibitor failed to complex completely with C1r under the same conditions. Time course analysis showed that the ability of the mutant to complex with C1s is also impaired: although it complexed completely in 60 min, the rate of complex formation during a 0-60-min incubation was decreased compared with wild-type C1 inhibitor. The mutant inhibitor also formed a complex with trypsin, a serine protease that cleaves, and is not inhibited by, wild-type C1 inhibitor. The Ala443-->Val mutation therefore converts C1 inhibitor from a substrate to an inhibitor of trypsin. These studies emphasize the role of the P2 residue in the determination of target protease specificity.     

THE ANTIGENIC AND MOLECULAR ALTERATIONS OF C3 IN THE FLUID PHASE DURING AN IMMUNE REACTION IN NORMAL HUMAN SERUM : DEMONSTRATION OF A NEW CONVERSION PRODUCT, C3X

During the reaction of an immune precipitate with fresh human serum, C3 undergoes a number of molecular alterations with the formation of conversion products differing from those obtained when purified components react. Those products which remain in the fluid phase, the subject of the present paper, have been identified by their reaction with monospecific antisera to the three antigenic determinants of C3, A, B, and D, after electrophoresis in agar or polyacrylamide gel. When purified C3 reacts with EAC1,4,2, C3i is found in the fluid phase. C3i, a loose complex of C3a and C3b, is in a conformational state whereby only the A and D antigens, present on its C3b portion, will consume antibody. The B antigen, present on the C3a portion of C3i, is unavailable for combination with antibody until C3i dissociates. In the fluid phase of the reaction of an immune precipitate with whole serum, C3i, C3a, and C3b, formed when purified components react, cannot be found. Instead the end products of the reaction appear to be C3c, which contains the A antigen, and C3d, which contains the D antigen. C3c and C3d are similar to the β1A and α2D produced by the aging of serum but differ in their mobilities in acrylamide gel and in agar. The C3c and C3d generated by an immune precipitate also differ slightly from the C3c and C3d produced by the reaction of trypsin with C3 in whole human serum. As human serum reacts with an immune complex, native C3 appears to undergo a primary alteration before conversion. This alteration results in a molecular species of C3 which is labile at 56°C for 30 min, fails to expose additional A and D antigenic sites upon aging, and which forms β1A and C3d rather than β1A and α2D during aging. In addition to this altered form of native C3, a new conversion product, C3x, is formed as whole serum reacts with an immune complex. C3x is not found in systems utilizing pure complement components. C3x is like C3 in that it bears all three antigenic determinants but differs in that it has a slightly faster mobility in polyacrylamide gel than does native C3. C3x is not only found in the fluid phase but is also bound to the immune precipitate. Finally, the fluid-phase kinetics of each of the antigens of C3 have been determined as normal human serum reacts with an immune precipitate. These illustrate that nearly the entire population of native C3 molecules undergoes conversion rapidly as manifested by the disappearance of the B antigen from the fluid phase. Moreover, the kinetics of the fluid-phase A and D antigens reflect that the conversion of C3 in serum is quantitatively not the same as when purified C3 reacts with C4,2.     

Acetylsalicylic acid regulates MMP-2 activity and inhibits colorectal invasion of murine B16F0 melanoma cells in C57BL/6J mice: Effects of prostaglandin F2α

Epidemiological studies indicate that acetylsalicylic acid may reduce the risk of mortality due to colon cancers. Metastasis is the major cause of cancer death. Matrix metalloproteinases (MMPs) play important roles in tumor invasion regulation, and prostaglandin F₂α (PGF₂α) is a key stimulator of MMP production. Thus, we investigated whether acetylsalicylic acid regulated MMP activity and the invasion of cancer cells and whether PGF₂α attenuated acetylsalicylic acid-inhibited invasion of cancer cells. Gelatin-based zymography assays showed that acetylsalicylic acid inhibited the MMP-2 activity of B16F0 melanoma cells. Matrigel-based chemoinvasion assays showed that acetylsalicylic acid inhibited the invasion of B16F0 cells. Acetylsalicylic acid can inhibit PGF₂α synthesis and PGF₂α is a key stimulator of MMP-2 production. Our data showed that PGF₂α treatment attenuated the acetylsalicylic acid-inhibited invasion of B16F0 cells. In animal experiments, acetylsalicylic acid reduced colorectal metastasis of B16F0 cells in C57BL/6J mice by 44%. Our results suggest that PGF₂α is a therapeutic target for metastasis inhibition and acetylsalicylic acid may possess anti-metastasis ability.