Experience on postmortem minimally invasive tissue sampling to ascertain the cause of death determination in South African children: A case for implementing as standard of care.

Determining the death burden for prioritising public health interventions necessitates detailed data on the causal pathways to death. Postmortem minimally invasive tissue sampling (MITS), incorporating histology, molecular and microbial culture diagnostics, enhances cause-of-death attribution, particularly for infectious deaths. MITS proves a valid alternative to full diagnostic autopsies, especially in low- and middle-income countries. In Soweto, South Africa (SA), the Child Health and Mortality Prevention Surveillance (CHAMPS) programme has delineated over 1 000 child and stillbirth deaths since 2017. This SA CHAMPS site supports advocating for the use of postmortem MITS as routine practice, for more granular insights into under-5 mortality causes. This knowledge is crucial for SA's pursuit of Sustainable Development Goal 3.2, targeting reduced neonatal and under-5 mortality rates. This commentary explores the public health advantages and ethicolegal considerations surrounding implementing MITS as standard of care for stillbirths, neonatal and paediatric deaths in SA. Furthermore, based on the data from CHAMPS, we present three pragmatic algorithmic approaches to the wide array of testing options for cost-effectiveness and scalability of postmortem MITS in South African state facilities.

Natal homing in a marine fish metapopulation.

Identifying natal origins of marine fishes is challenging because of difficulties in conducting mark-recapture studies in marine systems. We used natural geochemical signatures in otoliths (ear bones) to determine natal sources in weakfish (Cynoscion regalis), an estuarine-spawning marine fish, in eastern North America. Spawning site fidelity ranged from 60 to 81%, comparable to estimates of natal homing in birds and anadromous fishes. These data were in contrast to genetic analyses of population structure in weakfish. Our findings highlight the need for consideration of spatial processes in fisheries models and have implications for the design of marine reserves in coastal regions.

Interstellar carbon in meteorites.

The Murchison and Allende chondrites contain up to 5 parts per million carbon that is enriched in carbon-13 by up to + 1100 per mil (the ratio of carbon-12 to carbon-13 is approximately 42, compared to 88 to 93 for terrestrial carbon). This "heavy" carbon is associated with neon-22 and with anomalous krypton and xenon showing the signature of the s-process (neutron capture on a slow time scale). It apparently represents interstellar grains ejected from late-type stars. A second anomalous xenon component ("CCFXe") is associated with a distinctive, light carbon (depleted in carbon-13 by 38 per mil), which, however, falls within the terrestrial range and hence may be of either local or exotic origin.

Affinity chromatography using biocompatible and reusable biotinylated membranes.

A novel, reusable biotinylated affinity chromatography strategy for the bio-specific binding of bioactive avidin tagged enzymes or polypeptides is reported. Using an avidin coupled peroxidase fusion protein as a test system; non-specific protein shielding and matrix regeneration were also shown. The amphiphilic surfactant Pluronic F108 was used as an affinity linker, by non-covalent binding to membrane chromatographic matrices while the terminal hydroxyl groups of Pluronic were covalently coupled to the biological ligand biotin. Planar nonporous membranes of varying surface chemistry were synthesised to test the matrix dependent affinity binding of biotinylated Pluronic and their respective ability to resist non-specific protein adsorption. Membrane regeneration using sodium dodecyl sulphate (SDS) was capable of displacing both adsorbed proteins and Pluronic. SDS micelles (34 mM) were effective in desorbing membrane bound protein while 5mM SDS removed up to 85% of the bound ligand after 20 h incubation at 20 degrees C. In this study, polyvinylidene membranes had the highest ligand binding capacity of 0.22 mg cm(-2) and specific, competitive affinity binding of avidin-peroxidase was shown in the presence of up to 0.2 mg ml(-1) 'contaminant' proteins. The resultant biocompatible affinity chromatographic system was regenerated and reused with no significant change in performance for up to five cycles.

Sutherlandia frutescens modulates adrenal hormone biosynthesis, acts as a selective glucocorticoid receptor agonist (SEGRA) and displays anti-mineralocorticoid properties.

ETHNOPHARMACOLOGICAL RELEVANCE: Sutherlandia frutescens is a traditional African medicinal plant used in the treatment of stress and anxiety, while also exhibiting anti-inflammatory properties. AIM OF STUDY: The study aimed at linking anti-stress and anti-inflammatory properties of S. frutescens to its influence on glucocorticoid biosynthesis and the inflammatory response via steroid receptor interaction. MATERIALS AND METHODS: The influence of S. frutescens extracts and sutherlandioside B (SUB),10 and 30µM, on key steroidogenic enzymes was assayed in COS-1 cells. Effects were also assayed on basal and stimulated hormone levels in the adrenal H295R cell model. Agonist activity for transactivation and transrepression of the extract and SUB with the glucocorticoid- (GR) and mineralocorticoid receptor (MR) was subsequently investigated. RESULTS: Inhibitory effects of the extract towards progesterone conversion by CYP17A1 and CYP21A2 were significant. SUB inhibited CYP17A1 and 3ß-HSD2, while not affecting CYP21A2. In H295R cells, SUB decreased cortisol and androgen precursors significantly. The extract decreased total steroid production (basal and stimulated) with cortisol and its precursor, deoxycortisol, together with mineralocorticoid metabolites significantly decreased under forskolin stimulated conditions. S. frutescens extracts and SUB repressed NF-κB-driven gene expression without activating GRE-driven gene expression and while neither activated MR mediated gene transcription, both antagonized the effects of aldosterone via the MR. CONCLUSION: Data provide evidence linking anti-stress, anti-inflammatory and anti-hypertensive properties of S. frutescens to inhibition of steroidogenic enzymes and modulation of adrenal hormone biosynthesis. Findings suggesting S. frutescens and SUB exhibit dissociated glucocorticoid characteristics underline potential therapeutic applications in the treatment of inflammation and hypertension.

Preparation and characterization of conjugates of (modified) human serum albumin and liposomes: drug carriers with an intrinsic anti-HIV activity.

Human serum albumin (HSA) derivatized with cis-aconitic anhydride (Aco-HSA) that was earlier shown to inhibit replication of human immunodeficiency virus type 1 (HIV-1), was covalently coupled to conventional liposomes, consisting of phosphatidylcholine, cholesterol and maleimido-4-(p-phenylbutyryl)phosphatidylethanolamine, using the heterobifunctional reagent N-succinimidyl-S-acetylthioacetate (SATA). The amount of HSA that could be coupled to the liposomes depended on derivatization of the HSA and ranged from 64.2 +/- microgram HSA/micromol total lipid for native HSA to 29.5 +/- 2.7 microgram HSA/micromol total lipid for HSA in which 53 of the epsilon amino groups of lysine were derivatized with cis-aconitic anhydride (Aco53-HSA). Incorporation of 3.8 mol% of total lipid of a poly(ethylene glycol) derivative of phosphatidylethanolamine (PEG-PE) in the liposomes resulted in a lower coupling efficiency of Aco-HSA. The elimination and distribution of the liposomal conjugates in rats in vivo was largely dependent on the modification of the HSA coupled to the liposomes. With native HSA-liposomes, more than 70% of the conjugate was still found in the blood plasma 30 min after i.v. injection in rats, while at this time Aco-HSA-liposomes were completely cleared from the circulation. The rapid clearance of conventional Aco-HSA-liposomes was due to a rapid uptake into the liver and could be considerably decreased by incorporating PEG-PE in the liposomal bilayer. After 3 h 60% of Aco-HSA-PEG-liposome conjugates were found in the blood. In an in vitro anti-HIV-1 assay, the 50% inhibitory concentrations (IC50) for Aco39-HSA-liposomes and Aco53-HSA-liposomes expressed as protein weight, were 2.87 microgram/ml and 0.154 microgram/ml, respectively. When PEG-PE was incorporated, the Aco53-HSA-liposomes retained anti HIV-1 activity (IC50:3.13 microgram/ml). The possibility to modulate the residence time in the bloodstream of Aco-HSA-liposomes and the potent anti-HIV-1 activity of these conjugates, may allow the development of an intrinsically active drug carrier system. By incorporating anti HIV-1 drugs such as AZT into such liposomes a drug delivery system can be designed that might act simultaneously on the virus/cell binding by virtue of the coupled Aco-HSA and on the RNA/DNA transcription of the HIV-1 replication cycle through the nucleoside analogue.

Effect of age on the pharmacokinetics of solifenacin in men and women.

OBJECTIVE: The pharmacokinetics of solifenacin succinate (YM905; Vesicare), a new, bladder-selective, muscarinic receptor antagonist for the treatment of overactive bladder in young/middle-aged and elderly subjects were compared. MATERIAL: Solifenacin. METHODS: 47 healthy adults (24 young/middle-aged: mean age 35; and 23 elderly: mean age 68; 12 males in each age group) were enrolled in a single-center, multi-dose, open-label, crossover trial. Solifenacin, 5 or 10 mg, was administered once daily during two 14-day study periods separated by a washout period. Subjects were randomized to one dose in the first period and the other dose in the second period. Primary outcome variables were maximum plasma concentration (C(max)) and area under the curve from time 0 - 24 hours (AUC(0-24)). Secondary parameters included terminal elimination half-life (t1/2), time to C(max) (t(max)), fraction unbound, renal clearance, amount/percent of dose excreted in urine as solifenacin and its metabolites, and trough plasma metabolite concentrations. Adverse events and other safety parameters were also evaluated. RESULTS: Mean C(max) and AUC(0-24) were 16% (90% confidence interval 0.973 - 1.373) and 20% (1.003 - 1.435) higher, respectively, in elderly subjects. Mean t(max) and t1/2 were higher in elderly subjects. In both elderly and younger subjects, increasing the dose from 5 to 10 mg dose proportionally increased C(max), AUC(0- 24), and the amount excreted in urine. As expected, t(max) and t1/2 were not affected. Plasma concentrations and amounts of metabolites excreted in urine also increased dose proportionally. Solifenacin was highly bound to plasma proteins (fraction of the drug unbound in plasma was approximately 0.02), but there was no clear effect of gender or age. Solifenacin 5 or 10 mg once daily for 14 days was well tolerated by all subjects. CONCLUSION: Although C(max) and AUC(0-24) were higher in elderly subjects than in younger subjects and there was a tendency toward longer t(max) and t1/2, these differences were small and not considered clinically relevant. In this study, no consistent safety/tolerability issues were associated with these pharmacokinetic differences and the administration of solifenacin 5 or 10 mg once daily was well tolerated. The number of adverse events in elderly subjects was similar to that in younger subjects, indicating that no age-related dose adjustments are needed with this agent.

A robust approach to studying the adsorption of Pluronic F108 on nonporous membranes.

A method for poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) desorption from synthetic nonporous polymeric membranes, using hexane:isopropanol treatment and subsequent colorimetric quantification, is described. The polymers polysulfone, poly(vinyldiene fluoride), and poly(ether imide) were used to fabricate solid adsorption matrices. The desorbed Pluronic F108 forms a color complex with ammonium ferrothiocyanate (NH4FeSCN) and is based on partitioning of a chromophore present in NH4FeSCN from an aqueous phase to a chloroform phase in the presence of Pluronic. The protocols for Pluronic desorption and detection are simple, sensitive, inexpensive, rapid, and reproducible over a wide range of Pluronic coating concentrations and membrane surface chemistries. A linear response over the concentration range from 3 to 130 microg ml(-1) is obtained. The adsorption isotherms for flat sheet membranes are also described and the Langmuir equation provides the best fit for the adsorption data obtained within the concentration range studied. The absence of any significant interference from certain proteins, vitamins, carbohydrates, plasma, and halogenated derivatives makes the assay equally suitable for the estimation of Pluronic F108 in the attendant Pluronic conjugates or in biomedical applications. Using nonporous hollow fine fibers and capillary membranes as model curved substrates we were also able to correlate an increase in the radius of curvature with a corresponding increase in the surface interfacial adsorption of Pluronic F108.

Relative contribution of P450c17 towards the acute cortisol response: Lessons from sheep and goats.

The rapid release of cortisol from the adrenal cortex upon ACTH receptor activation plays an integral role in the stress response. It has been suggested that the quantitative control over adrenal steroidogenesis (quantity of total steroids produced) depends on the activities of cytochrome P450 side-chain cleavage and steroidogenic acute regulatory protein that supplies pregnenolone precursor to the pathway. The qualitative control (which steroids) then depends on the downstream steroidogenic enzymes, including cytochrome P450 17α-hydroxylase/17,20-lyase (P450c17). In this review we focus on the relative contribution of P450c17 in the qualitative control of cortisol production with data collected from studies on South African Angora and Boer goats, as well as Merino sheep. Unique P450c17 genotypes were identified in these breeds with isoforms differing only with a couple of single amino acid residue substitutions. This review demonstrates how molecular and cellular differences relating to P450c17 activity can affect physiological and behavioural responses.

Salsola tuberculatiformis Botschantzev and an aziridine precursor analog mediate the in vivo increase in free corticosterone and decrease in corticosteroid-binding globulin in female Wistar rats.

Salsola tuberculatiformis Botschantzev causes prolonged gestation in sheep and contraception in rats. An active fraction isolated from the shrub, containing a highly labile hydoxyphenyl aziridine or precursor, and a more stable analog, compound A, inhibits sheep adrenal cytochrome P450c11. In addition, compound A has been shown to bind to and be stabilized by corticosteroid-binding globulin (CBG). Binding may result in concomitant displacement of endogenous steroids, which could contribute to the biological effects of these compounds. The present study was undertaken to establish which mechanism would predominate in female rats. Compound A significantly (P < 0.01) displaced glucocorticoids, but not progesterone, from rat CBG in vitro, whereas in vivo the percentage of free plasma corticosterone in both S. tuberculatiformis (P < 0.05)- and compound A (P < 0.01)-treated rats was also significantly higher due to displacement from CBG. In addition, both ACTH and CBG concentrations were significantly (P < 0.05) lower than control values. The levels of the gonadotropins were also reduced during treatment, but only LH values significantly (P < 0.05) so. These results suggest that binding of the test substances to CBG in female rat plasma and concomitant displacement of endogenous corticosterone could be part of the contraceptive mechanism of S. tuberculatiformis and the aziridine precursor, compound A.

Progesterone 16 alpha-hydroxylase activity is catalyzed by human cytochrome P450 17 alpha-hydroxylase.

Progesterone and pregnenolone are metabolized to 17 alpha-hydroxysteroids by a cytochrome P450-dependent 17 alpha-hydroxylase (P450c17). The same enzyme can also catalyze the removal of the side-chain of these 17 alpha-hydroxylated steroids to yield androstenedione and dehydroepiandrosterone, respectively. We investigated the metabolism of progesterone by monkey kidney tumor (COS 1) cells transfected with a plasmid vector containing the cDNA encoding the complete amino acid sequence for human cytochrome P450c17. Transfected COS 1 cells converted progesterone to 17 alpha-hydroxyprogesterone as well as 16 alpha-hydroxyprogesterone, but no detectable androstenedione was produced. However, pregnenolone was converted to 17 alpha-hydroxypregnenolone and, ultimately, dehydroepiandrosterone. No 16 alpha-hydroxypregnenolone was produced. The kinetics of progesterone metabolism by the enzyme expressed in COS 1 cells indicated that both 17 alpha- and 16 alpha-hydroxylated products were products were produced from a common active site. Microsomes prepared from fetal adrenal and adult testis converted progesterone to 17 alpha-hydroxyprogesterone as well as 16 alpha-hydroxyprogesterone. No detectable androstenedione was produced by these preparations. Antibodies raised against porcine cytochrome P450c17 inhibited the 17 alpha- and 16 alpha-hydroxylation of progesterone to the same extent when using fetal adrenal microsomes, whereas no inhibition of 21-hydroxylation of progesterone was observed. Similar results were obtained with the imidazole antimycotic agent ketoconazole, which is a preferential cytochrome P450c17 inhibitor. From these results we conclude that human cytochrome P450c17 exhibits marked progesterone 16 alpha-hydroxylase activity in addition to its 17 alpha-hydroxylase function when expressed not only in a heterologous cell expression system but also, importantly, in human steroidogenic cells. Furthermore, the human enzyme has extremely low C-17,20-lyase activity toward progesterone, 17 alpha-hydroxyprogesterone, and 16 alpha-hydroxyprogesterone and fails to convert these to corresponding C19 steroids.

Optimization of the reaction conditions for the synthesis of neoglycoprotein-AZT-monophosphate conjugates.

The coupling of the monophosphate derivative of 3-azido-2,3-dideoxythymidine (AZTMP) to glycoproteins by water soluble carbodiimide (1-ethyl-3-[3-(dimethylamino)propyl]-3-ethylcarbodiimide) was greatly improved, relative to a recently reported method, by using also N-hydroxysulfosuccinimide (NHS) in the conjugation reaction. The hydrolysis of the activated AZTMP intermediate, responsible for the low degree of conjugation in the earlier method, could be delayed considerably if the activated phosphate group was converted into an activated ester by addition of NHS. In order to minimize the use of compounds needed for the preparation of AZTMP-protein conjugates, the present study was undertaken to determine if the reaction conditions could be optimized such that a conjugate with 2 AZTMP molecules/mol of neoglycoprotein would result. In addition a low proportion of cross-linked conjugates was desired. Optimization was achieved studying the shape of three-dimensional response surfaces, in which the degree of AZTMP coupling and the percentage of monomeric conjugates were regarded as the relevant responses. It appeared that the optimal conditions for coupling 1-2 mol of AZTMP to 1 mol of glycoprotein were an incubation time of 30 h, an AZTMP amount of 4 mg, an NHS amount between 8 and 15 mg, and a glycoprotein amount of 50 mg.

Plasma lactoferrin levels are decreased in end-stage AIDS patients.

The antimicrobial protein lactoferrin (Lf) is present in plasma and in mucosal secretions. Using ELISA we analysed plasma and saliva of HIV-infected patients, patients with AIDS, and healthy controls for the presence of secreted Lf. The plasma Lf levels of AIDS patients (classification C3) were significantly lower (p < 0.001) as compared to asymptomatic and symptomatic HIV infected patients, or controls. In addition, plasma Lf levels closely correlated with neutrophilic granulocyte counts in the HIV-infected patients. Thus, basal plasma Lf levels are likely the result of Lf release by neutrophilic granulocytes. The Candida titres present in the oral cavity were determined in a part of the HIV-infected patient group. As it appeared, the presence of this opportunistic pathogen always coincided with low levels of salivary Lf levels. We conclude that Lf, as part of the nonspecific immune system, might play an important role in the first line of defense against opportunistic microbial infections in AIDS patients.

Potent inhibition of replication of primary HIV type 1 isolates in peripheral blood lymphocytes by negatively charged human serum albumins.

We previously reported the antiviral capacity of human serum albumin (HSA), which was modified by the introduction of a single (Suc-HSA) or two carboxylic groups (Aco-HSA) per lysine residue, yielding strongly negatively charged polypeptides. Here we report the antiviral effect of these modified HSAs on replication of primary HIV-1 isolates that differed with respect to syncytium-inducing (SI) capacity and cell tropism. Both Suc-HSA and Aco-HSA potently inhibited replication of primary HIV-1 variants, independent of the SI capacity of the HIV-1 variant, with IC50 values in the range of 50 to 187 microg/ml. The inhibition of the formation of syncytia and the absence of proviral DNA products in cells inoculated with HIV-1 in the presence of Suc-HSA or Aco-HSA pointed to interference at an early level in the virus replication cycle. The inhibitory capacity of Suc-HSA and Aco-HSA on primary HIV-1 variants suggests that these agents are potential candidates for use in antiviral therapy in HIV-infected individuals.

Antiviral effects of milk proteins: acylation results in polyanionic compounds with potent activity against human immunodeficiency virus types 1 and 2 in vitro.

A number of native and modified milk proteins from bovine or human sources were analyzed for their inhibitory effects on human immunodeficiency virus type 1 (HIV-1) and HIV-2 in vitro in an MT4 cell test system. The proteins investigated were lactoferrin, alpha-lactalbumin, beta-lactoglobulin A, and beta-lactoglobulin B. By acylation of the amino function of the lysine residues in the proteins, using anhydrides of succinic acid or cis-aconitic acid, protein derivatives were obtained that all showed a strong antiviral activity against human immunodeficiency virus type 1 and/or 2. The in vitro IC50 values of the aconitylated proteins were in the concentration range of 0.3 to 3 nM. Succinylation or aconitylation of alpha-lactalbumin and beta-lactoglobulin A/B also produced strong anti-HIV-2 activity with IC50 values on the order 500 to 3000 nM. All compounds showed virtually no cytotoxicity at the concentration used. Peptide-scanning studies indicated that the native lactoferrin as well as the charged modified proteins strongly bind to the V3 loop of the gp120 envelope protein, with Kd values in the same concentration range as the above-mentioned IC50. Therefore, shielding of this domain, resulting in inhibition of virus-cell fusion and entry of the virus into MT4 cells, may be the likely underlying mechanism of antiviral action.

The in vitro anti-HIV efficacy of negatively charged human serum albumin is antagonized by heparin.

Succinylated human serum albumin (Suc-HSA) was synthesized by treating human serum albumin with succinic anhydride. Among similar proteins and neo(glyco)proteins tested, Suc-HSA exhibits a pronounced net negative charge, a feature that largely contributes to its efficacy against replication of human immunodeficiency virus type 1 (HIV-1). To assess further the antiviral effect of Suc-HSA, the effect on HIV-1 replication was studied in the presence of whole human plasma. Pretreatment of MT2 cells with Suc-HSA was more efficacious than direct Suc-HSA treatment of HIV prior to addition to the cells. No changes in the antiviral effect of Suc-HSA were observed in tissue culture medium, 30% plasma, or whole plasma when CPDA-1 (citrate-phosphate-dextrose-adenine 1) was used as the anticoagulant. However, a dramatic decrease (greater than 99%) in the antiviral activity was observed when these experiments were performed in plasma prepared from blood using heparin as anticoagulant. The antagonistic effect by heparin was observed both in the case that heparin was added prior to or after addition of Suc-HSA to the test system. In the present study we demonstrate that heparin largely reduces Suc-HSA activity on HIV replication in the same concentration in which if affects binding of Suc-HSA to the envelope protein gp120 and in particular its V3 domain. In the same concentration range, heparin reduced binding of Suc-HSA to MT4 cells, another HTLV-I-transformed cell line. It is concluded that heparin can displace Suc-HSA from its binding sites on hybrid lymphoid cells as well as on HIV-1 particles. Therefore, we conclude that both the binding to cells and to virus contribute to the potent anti-HIV-1 effect. The fact that heparin and heparin degradation products antagonize Suc-HSA without having a significant anti-HIV-1 effect indicates that the anticoagulant acts as a relatively weak partial inhibitor.

Resistance of the human immunodeficiency virus to the inhibitory action of negatively charged albumins on virus binding to CD4.

Negatively charged albumins (NCAs) have been identified as potent inhibitors of HIV-1 replication in vitro. Time of addition studies suggest that succinylated and aconitylated human serum albumin (Suc-HSA and Aco-HSA) act at an early stage of the virus life cycle, and surface plasmon resonance (BIAcore) experiments have confirmed a direct interaction of NCAs with HIV-1 gp120. Resistance to Suc-HSA and Aco-HSA was analyzed by characterizing HIV-1 variants that were selected in cell culture after serial passage of the NL4-3 strain in the presence of the compounds. After 24 passages (126 days) we isolated variants that were resistant to Suc-HSA (>27-fold) and Aco-HSA (37-fold), as compared with the wild-type NL4-3 virus. The binding of the NCA-resistant HIV strains to CD4+ MT-4 cells could no longer be inhibited by either Suc- or Aco-HSA. The emergence of mutations in the envelope gp120 of the resistant virus paralleled the emergence of the resistant phenotype. The Suc-HSA-resistant strain was 100-fold cross-resistant to the G quartet-containing oligonucleotide AR177 (Zintevir, an HIV-binding inhibitor), and partially cross-resistant to dextran sulfate, but remained sensitive to the bicyclam AMD3100 and the chemokine SDF-1alpha, which block HIV replication by interaction with the chemokine receptor CXCR4. Furthermore, neither Suc-HSA nor Aco-HSA inhibited the binding of monoclonal antibodies 12G5 and 2D7 (directed to CXCR4 and CCR5, respectively) in SUPT-1 cells or THP-1 cells. These results confirm that NCAs bind primarily to gp120 and do not interact directly with the HIV chemokine receptor but block the binding of the virus particles (through gp120) with CD4+ cells.

Influence of an aziridine precursor on the in vitro binding parameters of rat and ovine corticosteroid-binding globulin (CBG).

Aziridines are highly reactive alkylating compounds used in cancer treatment. Salsola tuberculatiformis Botsch., which causes prolonged gestation in sheep and contraception in rats, contains a very labile hydroxy-phenylaziridine or its precursor. A less labile analogue, 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride (Compound I), was synthesized and has been shown to be contraceptive in rats and to be stabilized by corticosteroid-binding globulin (CBG). The current study compared the binding parameters of rat and ovine CBG and evaluated the effect of the aziridine precursor, Compound I, on these parameters. Kd and Bmax values of 0.646 and 578 nM for corticosterone binding to rat CBG and 0.577 and 19.8 nM for cortisol binding to sheep CBG, respectively, were measured. In competitive binding studies with rat plasma, Ki values of 3.48 nM, 0.856 nM, 22.2 nM, 722 microM, and > 1,000,000 microM for cortisol, corticosterone, progesterone, Compound I, and synephrine (Compound II), respectively, were found, while in sheep plasma the values were 0.409 nM, 1.78 nM, 5.28 nM, 594 microM, and > 1,000,000 microM, respectively. Concentrations of Compound I equivalent to an effective pharmacological dose resulted in a significant (P < 0.01) decrease in CBG bound corticosterone and a significant (P < 0.01) increase in free corticosterone in rat plasma. In sheep, a similar effect was observed with cortisol. Progesterone binding, however, did not appear to be affected significantly by Compound I in either rat or sheep plasma. Compound I was found to be a competitive inhibitor of glucocorticoid binding to CBG. These results suggest that binding of Compound I to CBG with concomitant displacement of endogenous glucocorticoids, but not progesterone, may be part of the mechanism of action of these phenylaziridine compounds.

Inhibition of influenza virus fusion by polyanionic proteins.

Anionic charge-modified human serum albumin (HSA) has previously been shown to exert potent in vitro activity against human immunodeficiency virus type 1 (HIV-1). In these studies, introduction of the additional negative charges was performed by derivatizing the epsilon-amino groups of lysine residues with succinic (Suc-HSA) or cis-aconitic anhydride (Aco-HSA), by which primary amino groups are replaced with carboxylic acids. The anti-HIV-1 activity was related to inhibition of gp41-mediated membrane fusion. Here, we investigated the activity of aconitylated and succinylated proteins on influenza virus membrane fusion, which is mediated by the viral membrane glycoprotein hemagglutinin (HA). Aco-HSA and Suc-HSA markedly inhibited the rates and extents of fusion of fluorescently labeled virosomes bearing influenza HA, with target membranes derived from erythrocytes. The inhibitory activity was dependent on the overall negative-charge density; HSA modified with 36 or less extra negative charges failed to inhibit fusion. The inhibition of fusion showed a certain degree of specificity for the protein carrying the negative charges: polyanionic HSA and beta-lactoglobulin A derivatives had fusion-inhibitory activity, whereas succinylated BSA, lactalbumin, lactoferrin, lysozyme, and transferrin were inactive. Aco60-HSA and Aco-beta-lactoglobulin A inhibited influenza virus membrane fusion in a concentration-dependent manner, IC50 values being about 4 and 10 microg/mL, respectively. HA-mediated membrane fusion is pH dependent. Aco60-HSA did not induce a shift in the pH threshold or in the pH optimum. Fusion with liposomes of another low pH-dependent virus, Semliki Forest virus, was not specifically affected by any of the compounds reported here. In view of some structural and functional similarities between influenza HA and the HIV-1 gp120/gp41 complex, it is tempting to postulate that the current results might have some implications for the anti-HIV-1 mechanism of polyanionic proteins.

Mechanism for the stabilization in vivo of the aziridine precursor --(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride by serum proteins.

Oral and intraperitoneal administration of 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride (Compound A), an analogue of phenyl aziridine precursors that occur in the shrub Salsola tuberculatiformis Botsch, had a contraceptive effect on female Wistar rats with a concomitant decrease in total body, uterus, and every mass and an increase in abronal mass. Compound A elicited a Type II difference spectrum and inhibited the Type I deoxycorticosterone (DOC) induced difference spectrum of sheep adrenal cytochrome P450c11 in a manner similar to that of S2, a biologically active fraction isolated from S. tuberculatiformis. The effects of Compound A on the spectral properties of P450c11 were diminished with time in PBS. Electrospray mass spectrometry (ES-MS) indicated that the rate of cyclization of Compound A to the corresponding aziridine followed a time course similar to the attenuation of cytochrome P450c11 inhibition. It was concluded that the aziridine precursor. Compound A, rather than aziridine itself, was the inhibiting agent of sheep adrenal P450c11. Addition of sheep and rat plasma prevented the attenuation of the effect of Compound A on the spectral properties of cytochrome P450c11. Subsequent ES-MS analysis indicated that Compound A was stabilized in plasma by sex hormone binding globulin and corticosteroid binding globulin. These results suggest a mechanism whereby natural plant products, which are highly reactive and unstable in vitro, can be stabilized by binding to plasma proteins, and so remain biologically active in vivo.