Genetic nomenclature for chicken immunoglobulin allotypes: An extensive survey of inbred lines and antisera

Based on a survey of 36 inbred and 8 partially inbred chicken lines and outbred jungle fowl, and with 29 alloantisera generated in different laboratories, 13 7S Ig and 5 IgM allotypes were designated and a new system of nomenclature for chicken Ig polymorphisms was developed. The survey also revealed considerable genetic polymorphism in the structural gene(s) (G-1) responsible for the production of 7S Ig H chains. IgM H chains, encoded by theM-1 locus were less polymorphic. NineG-1 and fourM-1 gene alleles were delineated in highly inbred lines by the formation of unique combinations ofG-1 orM-1 specificities. Five additionalG-1 alleles were found in chicken lines and jungle fowl segregating for allotypes. Thirty-three percent of theG-1M-1 haplotypes theoretically expected, were detected in inbred lines.     

High-level expression and purification of a recombinant hBD-1 fused to LMM protein in Escherichia coli

In this work, we present the production of an active 43 aa recombinant human beta-defensin-1 (rhBD-1(43)) in Escherichia coli AD202 cells using specific pLMM1-rhBD-1 expression system. Unique solubility properties of the C-terminal fragment of light meromyosin (LMM) allowed us to overcome foreseeable problems with isolation procedures and toxicity caused by rhBD-1 to the host organism. As a result, the majority of fusion protein (LMM-rhBD-1(43)) was obtained in the soluble state, isolated by a low salt-high salt treatment of total cell protein. The rhBD-1(43) was cleaved from the fusion with Protease 4 and purified on CM Sepharose Fast Flow column with the yield of approximately 1 mg rhBD-1(43) from 6 g of wet weight cells. Purified rhBD-1(43) showed antimicrobial activity against E. coli ML-35p at a concentration of 129 microM. The procedure of rhBD-1 expression and purification we present can provide a reliable and simple method for production of different cationic peptides for biological studies.     

Competitive reactions of a ruthenium arene anticancer complex with histidine, cytochrome c and an oligonucleotide

The ruthenium arene anticancer complex [(⁶-bip)Ru(en)Cl][PF₆] (1) (bip is biphenyl, en is ethylenediamine) reacted slowly with the amino acid L-histidine (L-His) in aqueous solution at 310 K. Two L-His adducts of 1 were separated by high-performance liquid chromatography and identified by electrospray ionisation mass spectrometry and NMR: an imidazole N-bound complex [(⁶-bip)Ru(en)(N–L-His)]²⁺, and an N-bound complex [(⁶-bip)Ru(en)(N–L-His)]²⁺. At 310 K, after 24 h only about 22% of complex 1 (2 mM) reacted with L-His, and of the unreacted 1, 59% had hydrolysed. In the presence of 100 mM NaCl, approximately 90% of 1 remained unreacted. In aqueous solution or triethylammonium acetate (TEAA) buffer (pH 7.6), ¹⁵N-labelled 1 reacted with cytochrome c to give two monoruthenated protein adducts. The reaction reached equilibrium within 2 h by which time approximately 50% of cytochrome c was ruthenated. On the basis of [¹H, ¹⁵N] NMR data, one adduct may have Ru bound to the N-terminus, and the other to a carboxylate group on the protein. In TEAA buffer and at 310 K, more than 90% of the 14-mer oligonucleotide d(TATGTACCATGTAT) reacted with 2 mol Eq of 1 to give rise to monoruthenated and diruthenated oligonucleotide adducts. The presence of cytochrome c (1 mol Eq) or L-His (4 mol Eq) had little effect on the course of the reaction with the oligonucleotide. In cells, DNA (or RNA) may be a favoured reaction site for this Ru anticancer complex.Electronic supplementary material is available for this article at .

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Nuclear distribution and chromatin association of DNA polymerase α-primase is affected by TEV protease cleavage of Cdc23 (Mcm10) in fission yeast

Background  

Cdc23/Mcm10 is required for the initiation and elongation steps of DNA replication but its biochemical function is unclear. Here, we probe its function using a novel approach in fission yeast, involving Cdc23 cleavage by the TEV protease.     

Modification of pectin with UV-absorbing substitutents and its effect on the structural and hydrodynamic properties of the water-soluble derivatives

Citrus pectin with a low degree of methyl esterification (LMP) and its deesterified form, potassium pectate (KP), were modified with a low amount of UV-absorbing substituents. For this purpose, two different substitution reactions were used (a) alkylation of hydroxyl groups with p-carboxybenzyl bromide in aqueous alkali and (b) alkylation of the carboxylate group with benzyl bromide in the DMSO/TBAI/catalyst system. Chemical and spectroscopic methods reveal a low degree of substitution (DS<0.1) for the derivatives. The hydrodynamic properties were assessed by analytical ultracentrifugion, viscometry, and HPGPC. The results indicate that the introduction of small amounts of p-carboxybenzyl ether groups practically had no effect on the hydrodynamic properties in the case of KP, whereas, it was accompanied with a decrease of the molecular mass for LMP. The degradation was more pronounced during the benzyl esterification of LMP. The results confirmed that LMP is susceptible to chain cleavage due to β-elimination during both modification reactions. However, KP seems to be more tolerant of the reaction conditions.     

Insect feeding deterrent activity of bisabolangelone and of some sesquiterpenes of eremophilane type

The insect feeding deterrent activity of some sesquiterpenes of furanoeremophilane type and of the related eremophilanolide type were tested towards three selected storage pests: Sitophilus granarius, Tribolium confusum and Trogoderma granarium. The results were compared with the biogenetically related compound of bakkenolide type, known as a potent antifeedant. Bisabolangelone, tested on the same insects, exhibited the strongest activity, and may be included in the class of very good insect feeding deterrents. The antifeeding activity of bisabolangelone and bakkenolide A towards larvae of Leptinotarsa decemlineata was also observed.     

The Langhian (Middle Badenian) carbonate production event in the Moravian part of the Carpathian Foredeep (Central Paratethys): a multiproxy record

The carbonate production event in the Moravian part of the Carpathian Foredeep is known as a deposition of a carbonate–siliciclastic complex in the marginal part of the basin, correlating with the time period from the last occurrence of Helicosphaera waltrans (14.36 Ma) to the last occurrence of Sphenolithus heteromorphus (13.34 Ma). Sedimentological and microfacial data, analysis of foraminifera, calcareous nannoplankton, red algae, mollusks, palynology, as well as oxygen and carbon stable isotopes from foraminiferal tests, were used to interpret the specific paleoenvironment of the carbonate production event. The event was accelerated by a decrease of terrigenous input due to a large transgression and, primarily, an increasingly arid climate. Production of carbonate was related to oligotrophic conditions, expansion of sea-grass meadows, summer downwelling circulations and winter stratification of the water column. Autochthonous and semi-autochthonous carbonates were deposited in shallow-water near the fair-weather wave-base; allochthonous carbonates were transported to the outer shelf by gravity flows. Climatic instability and relative sea-level changes, induced mainly by substantial tectonic activity, caused the carbonate bodies to be small with a high ratio of siliciclastic components, indicating only a short-term and spatially restricted environment suitable for carbonate production. Exceptionally, carbonate production persisted longer during the whole sea-level cycle (“Rousínov Ridge”). Siliciclastic intercalations in these larger limestone bodies represent catastrophic rain events that transported a higher amount of terrigenous material into the basin. The specific climatic conditions of the carbonate production event, namely climatic instability and aridification with episodic intensive rain, were associated with the Middle Miocene climatic transition in the study area.     

Ultrastructural Changes Caused by Snf7 RNAi in Larval Enterocytes of Western Corn Rootworm (Diabrotica virgifera virgifera Le Conte)

The high sensitivity to oral RNA interference (RNAi) of western corn rootworm (WCR, Diabrotica virgifera virgifera Le Conte) provides a novel tool for pest control. Previous studies have shown that RNAi of DvSnf7, an essential cellular component of endosomal sorting complex required for transport (ESCRT), caused deficiencies in protein de-ubiquitination and autophagy, leading to WCR death. Here we investigated the detailed mechanism leading to larval death by analyzing the ultrastructural changes in midgut enterocytes of WCR treated with double-stranded RNA (ds-DvSnf7). The progressive phases of pathological symptoms caused by DvSnf7-RNAi in enterocytes include: 1) the appearance of irregularly shaped macroautophagic complexes consisting of relatively large lysosomes and multi-lamellar bodies, indicative of failure in autolysosome formation; 2) cell sloughing and loss of apical microvilli, and eventually, 3) massive loss of cellular contents indicating loss of membrane integrity. These data suggest that the critical functions of Snf7 in insect midgut cells demonstrated by the ultrastructural changes in DvSnf7 larval enterocytes underlies the conserved essential function of the ESCRT pathway in autophagy and membrane stability in other organisms.