The C19S Substitution Enhances the Stability of Hepcidin While Conserving Its Biological Activity

Hepcidin, the key hormone of iron homeostasis is responsible for lowering the serum iron level through its interaction with iron exporter ferroportin. Thus, hepcidin agonists provide a promising opportunity in the treatment of iron disorders caused by lacking or decreased hepcidin expression. We investigated the importance of each of the eight highly conserved cysteines for the biological activity of hepcidin. Eight cysteine mutants were created with site directed mutagenesis. The binding ability of these hepcidin mutants to the hepcidin receptor ferroportin was determined using bacterial two-hybrid system and WRL68 human hepatic cells. The biological activity of hepcidin mutants was determined by western blot analysis of ferroportin internalization and ferroportin ubiquitination. To investigate the effect of mutant hepcidins on the iron metabolism of the WRL68 cells, total intracellular iron content was measured with a colorimetric assay. The stability of M6 hepcidin mutant was determined using ELISA technique. Our data revealed that serine substitution of the sixth cysteine (M6) yielded a biologically active but significantly more stable peptide than the original hormone. This result may provide a promising hepcidin agonist worth testing in animal models.     

The resolution of acyclic P‐stereogenic phosphine oxides via the formation of diastereomeric complexes: A case study on ethyl‐(2‐methylphenyl)‐phenylphosphine oxide

As an example of acyclic P‐chiral phosphine oxides, the resolution of ethyl‐(2‐methylphenyl)‐phenylphosphine oxide was elaborated with TADDOL derivatives, or with calcium salts of the tartaric acid derivatives. Besides the study on the resolving agents, several purification methods were developed in order to prepare enantiopure ethyl‐(2‐methylphenyl)‐phenylphosphine oxide. It was found that the title phosphine oxide is a racemic crystal‐forming compound, and the recrystallization of the enantiomeric mixtures could be used for the preparation of pure enantiomers. According to our best method, the (R)‐ethyl‐(2‐methylphenyl)‐phenylphosphine oxide could be obtained with an enantiomeric excess of 99% and in a yield of 47%. Complete racemization of the enantiomerically enriched phosphine oxide could be accomplished via the formation of a chlorophosphonium salt. Characterization of the crystal structures of the enantiopure phosphine oxide was complemented with that of the diastereomeric intermediate. X‐ray analysis revealed the main nonbonding interactions responsible for enantiomeric recognition.     

Evaluation of false ultrasonographic pregnancy diagnoses in sows by measuring the concentration of unconjugated estrogens in feces

On Days 26, 28, and 30 after AI, ultrasonographic pregnancy diagnoses were performed on 207 gilts and sows by using a 3.5 MHz linear-array transducer. Fecal samples were taken from the rectum after each ultrasonographic examination, and the concentrations of unconjugated estrogens in selected samples (n = 73) were measured by RIA. Fecal unconjugated estrogen concentration of 11.7 ng/g feces or higher was indicative of pregnancy. The overall sensitivity and specificity of the ultrasonographic test was 99% for farrowing sows and 73.1% for nonfarrowing sows. With one exception, sows with a false negative diagnosis by ultrasonography on Day 26 were correctly diagnosed pregnant by elevated fecal unconjugated estrogens or repeated ultrasonographic examinations on Days 28 or 30. Return to estrus around the sampling period may cause false positive results in the unconjugated estrogen assay, while early embryonic mortality can result in false positive diagnoses in both the ultrasonographic test and estrogen assay. Although there was a positive correlation between the concentrations of unconjugated estrogens in the feces and litter size at farrowing in the selected sows, it seems very unlikely that fecal estrogens can provide an accurate tool for predicting litter size.     

Interference of S-Alkyl Derivatives of Glutathione with Brain Ionotropic Glutamate Receptors

The effects of glutathione, glutathione sulfonate and S-alkyl derivatives of glutathione on the binding of glutamate and selective ligands of ionotropic N-methyl-D-aspartate (NMDA) and non-NMDA receptors were studied with mouse synaptic membranes. The effects of glutathione and its analogues on ⁴⁵Ca²⁺ influx were also estimated in cultured rat cerebellar granule cells. Reduced and oxidized glutathione, glutathione sulfonate, S-methyl-, -ethyl-, -propyl-, -butyl- and -pentylglutathione inhibited the Na⁺-independent binding of L-[³H]glutamate. They strongly inhibited also the binding of (S)-2-amino-3-hydroxy-5-[³H]methyl-4-isoxazolepropionate [³H]AMPA (IC₅₀ values: 0.8–15.9 M). S-Alkylation of glutathione rendered the derivatives unable to inhibit [³H]kainate binding. The NMDA-sensitive binding of L-[³H]glutamate and the binding of 3-[(R)-2-carboxypiperazin-4-yl][1,2-³H]propyl-1-phosphonate ([³H]CPP, a competitive antagonist at NMDA sites) were inhibited by the peptides at micromolar concentrations. The strychnine-insensitive binding of the NMDA coagonist [³H]glycine was attenuated only by oxidized glutathione and glutathione sulfonate. All peptides slightly enhanced the use-dependent binding of [³H]dizocilpine (MK-801) to the NMDA-gated ionophores. This effect was additive with the effect of glycine but not with that of saturating concentrations of glutamate or glutamate plus glycine. The glutamate- and NMDA-evoked influx of ⁴⁵Ca²⁺ into cerebellar granule cells was inhibited by the S-alkyl derivatives of glutathione. We conclude that besides glutathione the endogenous S-methylglutathione and glutathione sulfonate and the synthetic S-alkyl derivatives of glutathione act as ligands of the AMPA and NMDA receptors. In the NMDA receptor-ionophore these glutathione analogues bind preferably to the glutamate recognition site via their -glutamyl moieties.     

Quantitative amino acid compositions at the picomole level using fluorescence scanning

Simple techniques are described for quantitating dansyl amino acids by direct fluoresence scanning and by photo-copying and densitometry. This technique is accurate between 1.0 to 10 × 10^?12 moles with a lower limit of detection around 1 × 10^?14 moles. It is possible to obtain amino acid compositions on very small quantities of peptides and it seems likely that this method will be useful for automatic peptide sequenators which use subtractive dansyl-Edman degradation as a detection procedure.