Nitrogen-fixation genes and nitrogenase activity in Clostridium acetobutylicum and Clostridium beijerinckii

Several solvent-producing clostridia, including Clostridium acetobutylicum and C. beijerinckii, were previously shown to be nitrogen-fixing organisms based on the incorporation of ¹⁵N₂ into cellular material. The key nitrogen-fixation (nif) genes, including nifH, nifD, and nifK for nitrogenase component proteins as well as nifE, nifN, nifB and nifV for synthesis of the iron–molybdenum cofactor (FeMoco) of nitrogenase, have now been identified in C. acetobutylicum or C. beijerinckii or both. The organization of these genes is similar to the distinctive pattern that was first observed in Clostridium pasteurianum, with the nifN and nifB genes fused into the nifN-B gene and with the nifV gene split into the nifVω and nifVα genes. The corresponding nif genes of these three clostridial species are highly related to each other. However, in the two solvent-producing clostridia, the nifH and nifD genes are interspersed by two glnB-like genes, which are absent in the corresponding region in C. pasteurianum. However, the nifN-B and nifVω genes of C. pasteurianum are interspersed by the putative modA and modB genes (for molybdate transport), which are absent in the corresponding region in C. acetobutylicum. C. acetobutylicum and C. beijerinckii grew well under nitrogen-fixing conditions, and the acetylene-reducing activity of nitrogenase was measured in the two species. Acetone, butanol, and isopropanol production occurred in nitrogen-fixing cultures, but the peak of nitrogen-fixing activity preceded the active solventogenic phase. Journal of Industrial Microbiology & Biotechnology (2001) 27, 281–286. Received 02 September 2000/ Accepted in revised form 22 November 2000     

Isolation and characterization of stem cells from pancreatic islet: pluripotency,differentiation potential and ultrastructural characteristics

Background aimsStem cells (SC) in different locations have individual characteristics. Important questions to be answered include how these specialties are generated, what the mechanism underlying their generation is, and what their biologic and clinical merits are. A basic approach to answering these questions is to make comparisons between the differences and similarities among the various SC types. They may focus on aspects of biologic marker discovery, capacity of proliferation and differentiation, along with other characteristics. The aim of this study was to characterize in detail the SC isolated from pancreatic islet (PI) and compare their properties with bone marrow (BM)-derived mesenchymal stromal cells (MSC) of the rat.MethodsImmunophenotypic characteristics, proliferation capacities, telomerase activities, pluripotent-related gene expressions, ultrastructure and the potential for multilineage differentiation of PI SC and BM MSC were studied.ResultsWe found that PI SC expressed markers of embryonic SC (Oct-4, Sox-2 and Rex-1) and had a high proliferation capacity, proven also by high telomerase activities. Surprisingly, markers belonging to differentiated cells were expressed by these cells in a constitutive manner. PI SC ultrastructure showed more developed and metabolically active cells.ConclusionsThe immunocytochemical identification of both PI SC and BM MSC was demonstrated to be typical MSC. Without stimulation of differentiation markers of adipogenic, chondrogenic, neurogenic, myogenic and osteogenic cells in these SC, the expression of those markers might explain their multilineage differentiation potential. We suggest that, by reason of the respectively high telomerase activity in PI SC, they could be better candidates than BM MSC for cell replacement therapy of type 1 diabetes.     

The histopathologic evaluation of soft tissue changes in rabbit extremity after different dose-fractionation schemes of interstitial high dose rate (HDR) brachytherapy

Background

The use of HDR in the treatment of soft tissue sarcoma had been on the rise. However, there was limited study to evaluate the effect of different fractionation schemes on soft tissue and the optimal HDR scheme.

Aims

We aimed to assess the histopathologic changes on soft tissue after different HDR brachytherapy doses.

Methods

The subjects were divided into three groups. Each group included 10 limbs. Group A had only an applicator without radiation, group B received a total of 24 Gy at 6 Gy per fraction, twice a day, and group C received a total of 13.5 Gy in a single fraction. The histopathologic findings were grouped into soft tissue pathology-1 (edema, inflammation, endothelial proliferation, necrosis) and soft tissue pathology-2 (atrophy, calcification, vascular hyalinization, fibrosis) (STP-1–2).

Results

The highest mean grade values of STP-1 and STP-2 were observed in group C (0.95 and 1.45) in comparison to group A (0.45 and 0.85) and group B (0.65 and 0.9). The difference in STP-1 was found significant only between groups A and C and the difference in STP-2 was found both between groups A and C and groups B and C.

Conclusion

In our experimental study it was shown that the fractionated interstitial HDR had both lower rate and severity of toxicity in comparison to a single high dose fraction. Before using a single fractionated regimen in the clinic, the increased morbidity related to the irreversible early toxicities or progressive late toxicities should be kept in mind.     

Collective dynamics of EcoRI-DNA complex by elastic network model and molecular dynamics simulations

Anisotropic network model (ANM) is used to analyze the collective motions of restriction enzyme EcoRI in free form and in complex with DNA. For comparison, three independent molecular dynamics (MD) simulations, each of 1.5 ns duration, are also performed for the EcoRI-DNA complex in explicit water. Although high mobility (equilibrium fluctuations) of inner and outer loops that surround the DNA is consistent in both methods and experiments, MD runs sample different conformational subspaces from which reliable collective dynamics cannot be extracted. However, ANM employed on different conformations from MD simulations indicates very similar collective motions. The stems of the inner loops are quite immobile even in the free enzyme and form a large, almost fixed, pocket for DNA binding. As a result, the residues that make specific and non-specific interactions with the DNA exhibit very low fluctuations in the free enzyme. The vibrational entropy difference between the EcoRI complex and free protein + unkinked DNA is positive (favorable), which may partially counteract the unfavorable enthalpy difference of DNA kink formation. Dynamic domains in EcoRI complex and cross-correlations between residue fluctuations indicate possible means of communication between the distal active sites.     

Molecular characterization of cDNA encoding resistance gene-like sequences in Buchloe dactyloides

Current knowledge of resistance (R) genes and their use for genetic improvement in buffalograss (Buchloe dactyloides [Nutt.] Engelm.) lag behind most crop plants. This study was conducted to clone and characterize cDNA encoding R gene-like (RGL) sequences in buffalograss. This report is the first to clone and characterize of buffalograss RGLs. Degenerate primers designed from the conserved motifs of known R genes were used to amplify RGLs and fragments of expected size were isolated and cloned. Sequence analysis of cDNA clones and analysis of putative translation products revealed that most encoded amino acid sequences shared the similar conserved motifs found in the cloned plant disease resistance genes PRS2, MLA6, L6, RPMI, and Xa1. These results indicated diversity of the R gene candidate sequences in buffalograss. Analysis of 5′ rapid amplification of cDNA ends (RACE), applied to investigate upstream of RGLs, indicated that regulatory sequences such as TATA box were conserved among the RGLs identified. The cloned RGL in this study will further enhance our knowledge on organization, function, and evolution of R gene family in buffalo grass. With the sequences of the primers and sizes of the markers provided, these RGL markers are readily available for use in a genomics-assisted selection in buffalograss.     

Radiotherapy applications of patients with malignant mesothelioma: A single center experience

Background

In the management of malignant pleural mesothelioma, radiotherapy has been used for the purpose of prophylaxis to reduce the incidence of recurrence at surgical insertion sites or palliate the symptoms.

Aim

The purpose of the study was to evaluate the techniques and effectiveness of radiotherapy in malignant pleural mesothelioma.

Materials and methods

Forty-four (18 female, 26 male) patients diagnosed with malignant pleural mesothelioma were retrospectively evaluated. All patients had surgery or thoracoscopic biopsy for diagnosis, staging or treatment and all received palliative or prophylactic radiotherapy. Fifty-seven percent of the patients received chemotherapy.

Results

Prophylactic radiation was applied to 27 patients with 4–15 MeV electron energies. The median radiotherapy dose was 30 Gy with 3 Gy daily fraction dose. During treatment, 12 patients had grade 1 erythema according to the RTOG scale. In 3 (12%) patients, a local failure at treatment field was observed. Palliative radiotherapy was applied to 17 patients for pain palliation. The median radiation dose was 40 Gy with 2 Gy daily fraction dose by using 6–18 MV photon and/or 4–12 MeV electron energies. Two patients had grade 1 erythema and one patient had grade 2 odynophagy according to the RTOG scale. For 10 (59%) patients, palliation of chest pain was delivered. No late toxicity was observed for all cases.

Conclusion

Our experience showed that prophylactic and palliative radiotherapy are effective and safe therapy modalities in malignant pleural mesothelioma in preventing seeding metastasis at intervention sites or relieving pain. Prospective randomized studies are still needed to determine the benefits of radiotherapy application and to indicate optimum dose schemes.     

Optimization of Ethanol Production From Microfluidized Wheat Straw by Response Surface Methodology

In this study, wheat straw was pretreated with a microfluidizer to improve its enzymatic hydrolysis and ethanol yields. The pretreatment was performed at various pressures (500, 1000, and 1500 bar) and solid loadings (1, 2, and 3%). The microfluidized biomass was then subjected to hydrolysis and simultaneous saccharification and co-fermentation (SSCF) experiments at different enzyme loadings (5, 10, and 15 FPU/g dry wheat straw) using a mutant yeast. The results indicated that the microfluidization method alters the structure of biomass and leads to a reduction in lignin content. The samples pretreated at 1% solid loading contained the minimum lignin concentration and provided the maximum sugar and ethanol yields. These results signified that the microfluidization method is more effective on biomass at low solid loadings. The process conditions were optimized for higher ethanol and sugar yields using response surface methodology (RSM). The optimum pressure and solid and enzyme loadings were found as 1500 bar, 1%, and 15 FPU/g dry wheat straw, respectively. The yields obtained at this condition were 82%, 94%, and 65% for glucose, xylose, and ethanol, respectively. High sugar yields implied that microfluidization is an effective pretreatment method for cellulosic ethanol production. On the other hand, low ethanol yield may indicate that the microorganism was sensitive to inhibitory compounds present in the fermentation medium.     

Inference on the strength of balancing selection for epistatically interacting loci

Existing inference methods for estimating the strength of balancing selection in multi-locus genotypes rely on the assumption that there are no epistatic interactions between loci. Complex systems in which balancing selection is prevalent, such as sets of human immune system genes, are known to contain components that interact epistatically. Therefore, current methods may not produce reliable inference on the strength of selection at these loci. In this paper, we address this problem by presenting statistical methods that can account for epistatic interactions in making inference about balancing selection. A theoretical result due to Fearnhead (2006) is used to build a multi-locus Wright-Fisher model of balancing selection, allowing for epistatic interactions among loci. Antagonistic and synergistic types of interactions are examined. The joint posterior distribution of the selection and mutation parameters is sampled by Markov chain Monte Carlo methods, and the plausibility of models is assessed via Bayes factors. As a component of the inference process, an algorithm to generate multi-locus allele frequencies under balancing selection models with epistasis is also presented. Recent evidence on interactions among a set of human immune system genes is introduced as a motivating biological system for the epistatic model, and data on these genes are used to demonstrate the methods.     

Consumption rate and functional response of the predaceous mite<Emphasis Type="Italic"> Kampimodromus aberrans</Emphasis> to two-spotted spider mite <Emphasis Type="Italic">Tetranychus urticae</Emphasis> in the laboratory

Prey stage preference of female Kampimodromus aberrans (Oudemans) (Phytoseiidae) at constant densities of different stages of Tetranychus urticae Koch (Tetranychidae), functional response types and parameters of the predator females to the varying densities of eggs, larvae, protonymphs and deutonymps of T. urticae were determined in order to establish its potential for the mite biological control. Experiments were conducted at 25 ± 1°C, 65 ± 10% RH and 16:8 (L:D) photoperiod. Our results indicated that the predator consumed significantly more prey larvae than other prey stages. Functional response type of predator was determined by a logistic regression model. The predator exhibited a Type II response on all prey stages. The attack rate (α) and handling time (T _h ) coefficients of a Type II response were estimated by fitting a “random-predator” equation to the data. The lowest estimated value α and the highest value of T _h (including digestion) were obtanined for the predator feeding on deutonmph. The lowest value of T _h were obtained for the predator feeding on prey larvae, but the attack rate value obtained on larva wasn’t different than that obtained on egg and protonymph. According to our results, K. aberrans could be an efficient biological control agent of T. urticae at least at low prey densities. However, further field based studies are needed to draw firm conclusions.