Site-specific conjugation of a lanthanide chelator and its effects on the chemical synthesis and receptor binding affinity of human relaxin-2 hormone

Diethylenetriamine pentaacetic acid (DTPA) is a popular chelator agent for enabling the labeling of peptides for their use in structure-activity relationship study and biodistribution analysis. Solid phase peptide synthesis was employed to couple this commercially available chelator at the N-terminus of either the A-chain or B-chain of H2 relaxin. The coupling of the DTPA chelator at the N-terminus of the B-chain and subsequent loading of a lanthanide (europium) ion into the chelator led to a labeled peptide (Eu-DTPA-(B)-H2) in low yield and having very poor water solubility. On the other hand, coupling of the DTPA and loading of Eu at the N-terminus of the A-chain led to a water-soluble peptide (Eu-DTPA-(A)-H2) with a significantly improved final yield. The conjugation of the DTPA chelator at the N-terminus of the A-chain did not have any impact on the secondary structure of the peptide determined by circular dichroism spectroscopy (CD). On the other hand, it was not possible to determine the secondary structure of Eu-DTPA-(B)-H2 because of its insolubility in phosphate buffer. The B-chain labeled peptide Eu-DTPA-(B)-H2 required solubilization in DMSO prior to carrying out binding assays, and showed lower affinity for binding to H2 relaxin receptor, RXFP1, compared to the water-soluble A-chain labeled peptide Eu-DTPA-(A)-H2. The mono-Eu-DTPA labeled A-chain peptide, Eu-DTPA-(A)-H2, thus can be used as a valuable probe to study ligand-receptor interactions of therapeutically important H2 relaxin analogs. Our results show that it is critical to choose an approriate site for incorporating chelators such as DTPA. Otherwise, the bulky size of the chelator, depending on the site of incorporation, can affect yield, solubility, structure and pharmacological profile of the peptide.     

Livestock disturbances in Mediterranean temporary ponds: A mesocosm experiment with sheep manure and simulated trampling

相似文献   

In vitro cytotoxic activity of isolated acridones alkaloids from Zanthoxylum leprieurii Guill. et Perr

Chemical investigation of the roots and fruits of Zanthoxylum leprieurii Guill. et Perr. led to the isolation of three new alkaloids including two acridone derivatives, 3-hydroxy-1,4-dimethoxy-10-methyl-9-acridone (2) and 3-hydroxy-1,2-dimethoxy-10-methyl-9-acridone (3) named helebelicine A and B, respectively, and one secobenzo[c]phenantridine, 10-O-demethyl-12-O-methylarnottianamide (10), together with thirteen other compounds. The structures of compounds 2, 3 and 10 as well as those of the known compounds were elucidated by using spectroscopic methods and by comparison with reported data. The brine-shrimp (artemia salina) lethality bioassay of the chloroform extract of the fruits showed modest cytotoxicity with LD₅₀ at 13.1 μg/mL. Isolated compounds 1, 4–6 were found to be moderately active against lung carcinoma cells (A549), colorectal adenocarcinoma cells (DLD-1) and normal cells (WS1) with IC₅₀ values ranging from 27 to 77 μM. In contrast to the positive control etoposide used, the cytotoxicity of the most active compound 4 was found to be selective against cancer cells in comparison to normal cells WS1 with IC₅₀ of 51 ± 8 μM and 4.3 ± 0.4 μM, respectively.     

Diversity and phylogenetic relationships of <Emphasis Type="Italic">Glossina</Emphasis> populations in Nigeria and the Cameroonian border region

Background

Tsetse flies are vectors of trypanosomes, parasites that cause devastating disease in humans and livestock. In the course of vector control programmes it is necessary to know about the Glossina species present in the study area, the population dynamics and the genetic exchange between tsetse fly populations.

Results

To achieve an overview of the tsetse fly diversity in Nigeria and at the Nigeria-Cameroon border, tsetse flies were trapped and collected between February and March 2014 and December 2016. Species diversity was determined morphologically and by analysis of Cytochrome C Oxidase SU1 (COI) gene sequences. Internal transcribed spacer-1 (ITS-1) sequences were compared to analyse variations within populations. The most dominant species were G. m. submorsitans, G. tachinoides and G. p. palpalis. In Yankari Game Reserve and Kainji Lake National Park, G. submorsitans and G. tachinoides were most frequent, whereas in Old Oyo National Park and Ijah Gwari G. p. palpalis was the dominant species. Interestingly, four unidentified species were recorded during the survey, for which no information on COI or ITS-1 sequences exists. G. p. palpalis populations showed a segregation in two clusters along the Cameroon-Nigerian border.

Conclusions

The improved understanding of the tsetse populations in Nigeria will support decisions on the scale in which vector control is likely to be more effective. In order to understand in more detail how isolated these populations are, it is recommended that further studies on gene flow be carried out using other markers, including microsatellites.

     

YcfDRM is a thermophilic oxygen-dependent ribosomal protein uL16 oxygenase

Extremophiles - YcfD from Escherichia coli is a homologue of the human ribosomal oxygenases NO66 and MINA53, which catalyse histidyl-hydroxylation of the 60S subunit and affect cellular...     

A new screening technique for salinity resistance in rice (Oryza sativa L.) seedlings using bypass flow

A lack of screening techniques delays progress in research on salinity resistance in rice. In this study, we report our test of the hypothesis that an apoplastic pathway (the so-called bypass flow) causes a difference in salt resistance between rice genotypes and can be used in screening for salinity resistance. Fourteen-day-old seedlings of low- and high-Na(+) -transporting recombinant inbred lines (10 of each) of rice IR55178 were treated with 50 mm NaCl and 0.2 mm trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid (PTS), a bypass flow tracer, for short (4 d) and long (90 d) periods of time. The results showed that the average shoot Na(+) concentration and bypass flow for high-Na(+) -transporting lines were 1.4 and 2.4 times higher than those of low-Na(+) -transporting lines, respectively. There was a positive linear correlation between the percentage of bypass flow and Na(+) concentrations in the shoots, suggesting that the difference in Na(+) transport in rice is a consequence of different degrees of bypass flow. Moreover, a high correlation was found between bypass flow and seedling survival after prolonged salt stress: the lower the magnitude of bypass flow, the greater the seedling survival. We conclude that bypass flow could be used as a new screening technique for salt resistance in rice.     

Mutations in RIPK4 cause the autosomal-recessive form of popliteal pterygium syndrome

The autosomal-recessive form of popliteal pterygium syndrome, also known as Bartsocas-Papas syndrome, is a rare, but frequently lethal disorder characterized by marked popliteal pterygium associated with multiple congenital malformations. Using Affymetrix 250K SNP array genotyping and homozygosity mapping, we mapped this malformation syndrome to chromosomal region 21q22.3. Direct sequencing of RIPK4 (receptor-interacting serine/threonine kinase protein 4) showed a homozygous transversion (c.362T>A) that causes substitution of a conserved isoleucine with asparagine at amino acid position 121 (p.Ile121Asn) in the serine/threonine kinase domain of the protein. Additional pathogenic mutations-a homozygous transition (c.551C>T) that leads to a missense substitution (p.Thr184Ile) at a conserved position and a homozygous one base-pair insertion mutation (c.777_778insA) predicted to lead to a premature stop codon (p.Arg260ThrfsX14) within the kinase domain-were observed in two families. Molecular modeling of the kinase domain showed that both the Ile121 and Thr184 positions are critical for the protein's stability and kinase activity. Luciferase reporter assays also demonstrated that these mutations are critical for the catalytic activity of RIPK4. RIPK4 mediates activation of the nuclear factor-κB (NF-κB) signaling pathway and is required for keratinocyte differentiation and craniofacial and limb development. The phenotype of Ripk4(-/-) mice is consistent with the human phenotype presented herein. Additionally, the spectrum of malformations observed in the presented families is similar, but less severe than the conserved helix-loop-helix ubiquitous kinase (CHUK)-deficient human fetus phenotype; known as Cocoon syndrome; this similarity indicates that RIPK4 and CHUK might function via closely related pathways to promote keratinocyte differentiation and epithelial growth.     

Tolerance and bioaccumulation of copper in Phragmites australis and Zea mays

The effects of copper on the growth, tolerance indices, mineral composition (N, P, K, Fe, Zn and Mn) and metal uptake of reed (Phragmites australis [Cav. Trin. ex Steudel]) and maize (Zea mays L.) were investigated in hydroponic experiments at copper concentrations ranging from 0.5 to 157 M Cu. A reduction in root length was shown to be a good indicator of copper toxicity, concentrations of 15.7 and 78.7 M Cu inhibiting root growth in maize and reed, respectively. The reed was significantly more tolerant of copper than maize and at 7.85 M Cu (external concentration), reed can be described as a Cu tolerant plant, and maize as a Cu non-tolerant species. As a result of Cu toxicity, the concentrations of macronutrients N, P and K decreased in both shoot and root of maize, while the concentrations were hardly affected in reed tissues. Fe concentration increased in shoots and roots of maize and in roots of reed with increasing Cu treatments, leading to highly significant (p<0.01) linear relationships between tissue Fe and Cu concentrations. The bioconcentration factor (BCF) of Cu was higher in roots than in shoots of both plant species, ranging from 612 to 1592 in reed for the Cu treatments tested. In the roots of maize, BCF of Cu increased from 349 to 1931 when increasing Cu in nutrient solution from 7.85 M to 78.5 M. Therefore, reed could be useful in wastewater treatments for the removal of Cu. However, the use of reed in phytoextraction of Cu from contaminated soils is limited by the low accumulation rate in shoots and although reed can be more efficient than maize for Cu phytoextraction, harvesting the full biomass, including roots, may be required.     

Tectona grandis L.f: A comprehensive review on its patents,chemical constituents,and biological activities

Tectona grandis L.f is a timber plant that is commonly referred to as teak. Its wide use as a medicine in the various indigenous systems makes it a plant of importance. A wide gamut of phytoconstituents like alkaloids, phenolic glycosides, steroids, etc. has been reported. A renewed interest in this plant has resulted in scientific investigations by various researchers towards the isolation and identification of active constituents along with scientific proof of its biological activities. The different parts of the plant have been scientifically evaluated for their antioxidant, antipyretic, analgesic, hypoglycemic, wound healing, cytotoxic, and many more biological activities. Documentation of this scientific knowledge is of importance to have consolidated precise information encompassing the various aspects of this plant, which could provide a base for future studies. This review is a compilation of the salient reports on these investigations concerning phytochemistry, the methods used to identify and quantify the constituents, the evaluation methods of the biological activity, toxicological studies, allergies and the patent/patent applications. This will further help researchers to find an area of the gap for future studies.