Bacillus Probiotic Supplementations Improve Laying Performance,Egg Quality,Hatching of Laying Hens,and Sperm Quality of Roosters

Probiotics and Antimicrobial Proteins - The study aims at elucidating the effect of bacilli probiotic preparations on the physiology of laying hens and roosters. Probiotic formulations were...     

Vibrational CD (VCD) and atomic force microscopy (AFM) study of DNA interaction with Cr3+ ions: VCD and AFM evidence of DNA condensation

The interaction of natural calf thymus DNA with Cr³⁺ ions was studied at room temperature by means of vibrational CD (VCD) and infrared absorption (ir) spectroscopy, and atomic force microscopy (AFM). Cr³⁺ ion binding mainly to N₇ (G) and to phosphate groups was demonstrated. ψ‐Type VCD spectra resembling electronic CD (ECD) spectra, which appear during ψ‐type DNA condensation, were observed. These spectra are characterized mainly by an anomalous, severalfold increase of VCD intensity. Such anomalous VCD spectra were assigned to DNA condensation with formation of large and dense particles of a size comparable to the wavelength of the probing ir beam and possessing large‐scale helicity. Atomic force microscopy confirmed DNA condensation by Cr³⁺ ions and the formation of tight DNA particles responsible for the ψ‐type VCD spectra. Upon increasing the Cr³⁺ ion concentration the shape of the condensates changed from loose flower‐like structures to highly packed dense spheres. No DNA denaturation was seen even at the highest concentration of Cr³⁺ ions studied. The secondary structure of DNA remained in a B‐form before and after the condensation. VCD and ir as well as AFM proved to be an effective combination for investigating DNA condensation. In addition to the ability of VCD to determine DNA condensation, VCD and ir can in the same experiment provide unambiguous information about the secondary structure of DNA contained in the condensed particles. © 2002 Wiley Periodicals, Inc. Biopolymers 61: 243–260, 2002     

Endothelial cells are activated by angiopoeitin-1 gene transfer and produce coordinated sprouting in vitro and arteriogenesis in vivo

RATIONAL AND OBJECTIVES: Activation of fully differentiated vascular cells using angiogenic genes can lead to phenotypic changes resulting in formation of new blood vessels. We tested whether Ang-1 gene transfer to endothelial cells (EC) activates these cells. METHODS AND RESULTS: EC and SMC were transduced using retroviral or adenoviral vectors to produce Ang-1 or vascular endothelial growth factor (VEGF). EC Tie-2 receptor was phosphorilated by autologous secretion of Ang-1. Transduced EC and SMC sprouting capacity was tested using collagen embedded spheroids assay and capacity to produce arteriogenesis was tested in a hind limb model of ischemia. EC expressing Ang-1 in the presence of SMC expressing VEGF exhibited high levels of sprouting of the two cell types. Flow and numbers of arteries were increased after transduced cells implantation in vivo. CONCLUSIONS: Autologous secretion of Ang-1 by transduced EC resulted in Tie-2 activation and in the presence of SMC expressing VEGF resulted in coordinated sprouting in vitro and increase in flow and number of arteries in vivo.     

Constraints on error rate revealed by computational study of G•U tautomerization in translation

In translation, G•U mismatch in codon-anticodon decoding is an error hotspot likely due to transition of G•U from wobble (wb) to Watson-Crick (WC) geometry, which is governed by keto/enol tautomerization (wb-WC reaction). Yet, effects of the ribosome on the wb-WC reaction and its implications for decoding mechanism remain unclear. Employing quantum-mechanical/molecular-mechanical umbrella sampling simulations using models of the ribosomal decoding site (A site) we determined that the wb-WC reaction is endoergic in the open, but weakly exoergic in the closed A-site state. We extended the classical ‘induced-fit’ model of initial selection by incorporating wb-WC reaction parameters in open and closed states. For predicted parameters, the non-equilibrium exoergic wb-WC reaction is kinetically limited by the decoding rates. The model explains early observations of the WC geometry of G•U from equilibrium structural studies and reveals discrimination capacity for the working ribosome operating at non-equilibrium conditions. The equilibration of the exoergic wb-WC reaction counteracts the equilibration of the open-closed transition of the A site, constraining the decoding accuracy and potentially explaining the persistence of the G•U as an error hotspot. Our results unify structural and mechanistic views of codon-anticodon decoding and generalize the ‘induced-fit’ model for flexible substrates.     

Comparison of cardiac stem cell sheets detached by Versene solution and from thermoresponsive dishes reveals similar properties of constructs

Cell sheets (CS) from c-kit+ cardiac stem cell (CSC) hold a potential for application in regenerative medicine. However, manufacture of CS may require thermoresponsive dishes, which increases cost and puts one in dependence on specific materials. Alternative approaches were established recently and we conducted a short study to compare approaches for detachment of CS from c-kit+ CSC. Our in-house developed method using chelation by Versene solution was compared to UpCell? thermoresponsive plates in terms of CSC proliferation, viability, gap junction formation and engraftment in a model of myocardial infarction. Use of Versene solution instead of thermoresponsive dishes resulted in comparable CS thickness (approximately 100 mcm), cell proliferation rate and no signs of apoptosis detected in both types of constructs. However, we observed a minor reduction of gap junction count in Versene-treated CS. At day 30 after delivery to infarcted myocardium both types of CS retained at the site of transplantation and contained comparable amounts of proliferating cells indicating engraftment. Thus, we may conclude that detachment of CS from c-kit+ CSC using Versene solution followed by mechanical treatment is an alternative to thermoresponsive plates allowing use of routinely available materials to generate constructs for cardiac repair.     

The Phylogenetic Diversity of Aerobic Organotrophic Bacteria from the Dagang High-Temperature Oil Field

The distribution and species diversity of aerobic organotrophic bacteria in the Dagang high-temperature oil field (China), which is exploited with water-flooding, have been studied. Twenty-two strains of the most characteristic thermophilic and mesophilic aerobic organotrophic bacteria have been isolated from the oil stratum. It has been found that, in a laboratory, the mesophilic and thermophilic isolates grow in the temperature, pH, and salinity ranges characteristic of the injection well near-bottom zones or of the oil stratum, respectively, and assimilate a wide range of hydrocarbons, fatty acids, lower alcohols, and crude oil, thus exhibiting adaptation to the environment. Using comparative phylogenetic 16S rRNA analysis, the taxonomic affiliation of the isolates has been established. The aerobic microbial community includes gram-positive bacteria with a high and low G+C content of DNA, and γ and β subclasses of Proteobacteria. The thermophilic bacteria belong to the genera Geobacillus and Thermoactinomyces, and the mesophilic strains belong to the genera Bacillus, Micrococcus, Cellulomonas, Pseudomonas, and Acinetobacter. The microbial community of the oil stratum is dominated by known species of the genus Geobacillus (G. subterraneus, G. stearothermophilus, and G. thermoglucosidasius) and a novel species “Geobacillus jurassicus.” A number of novel thermophilic oil-oxidizing bacilli have been isolated.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 401–409.Original Russian Text Copyright © 2005 by Nazina, Sokolova, Shestakova, Grigoryan, Mikhailova, Babich, Lysenko, Tourova, Poltaraus, Qingxian Feng, Fangtian Ni, Belyaev.     

Human beta-defensin 3 binds to hemagglutinin B (rHagB), a non-fimbrial adhesin from Porphyromonas gingivalis, and attenuates a pro-inflammatory cytokine response

Regulatory mechanisms in mucosal secretions and tissues recognize antigens and attenuate pro-inflammatory cytokine responses. Here, we asked whether human beta-defensin 3 (HBD3) serves as an upstream suppressor of cytokine signaling that binds and attenuates pro-inflammatory cytokine responses to recombinant hemagglutinin B (rHagB), a non-fimbrial adhesin from Porphyromonas gingivalis strain 381. We found that HBD3 binds to immobilized rHagB and produces a significantly higher resonance unit signal in surface plasmon resonance spectroscopic analysis, than HBD2 and HBD1 that are used as control defensins. Furthermore, we found that HBD3 significantly attenuates (P<0.05) the interleukin (IL)-6, IL-10, granulocyte macrophage colony stimulating factor (GM-CSF) and tumor-necrosis factor-alpha (TNF-alpha) responses induced by rHagB in human myeloid dendritic cell culture supernatants and the extracellular signal-regulated kinases (ERK 1/2) response in human myeloid dendritic cell lysates. Thus, HBD3 binds rHagB and this interaction may be an important initial step to attenuate a pro-inflammatory cytokine response and an ERK 1/2 response.     

The effect of solvated ions on the thermal denaturation of human serum albumin in water-dimethylsulfoxide solutions

The effects of solvated ions on the thermal denaturation of human serum albumin (HAS) in water-dimethylsulfoxide (DMSO) solutions were studied by the method of electron absorption spectroscopy. It was shown that depending on the DMSO concentration, electrolytes (LiCl, LiNO₃, LiClO₄, NaCl, and NaNO₃) contained in these solutions were characterized by different anion and cation solvation degrees: unlike cations, anions were only negligibly solvated, which affected HAS thermal denaturation. Electrostatic interactions between anions and positively charged amino acid residues supporting protein denaturation subsided in the line Cl⁻ > NO₃⁻ > ClO₄⁻.     

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

Background

Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone.

Methods

A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection.

Results

Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene.

Conclusion

These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.