Rumen fungi: morphological types from Georgia cattle and the attack on forage cell walls

Fungal colonies developing in anaerobic media from zoospores in rumen fluid from cows eating Cynodon dactylon or Medicago sativa included types showing monocentric and polycentric growth. High energy supplements added to diets of Sorghum bicolor silage increased fungal numbers in the rumen, but increases were also affected by the history and predisposition of the animal. Mixed fungal types in rumen fluid and pure cultures of isolates showing monocentric and polycentric growth degraded and weakened lignocellulosic tissues and penetrated the cuticle of C. dactylon leaf blades. By weakening or degrading recalcitrant structures in forages, rumen fungi may alter physical parameters of plants that influence utilization of fibre by ruminants.     

Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis

BACKGROUND: The relatively high transfection efficiency of polyethylenimine (PEI) vectors has been hypothesized to be due to their ability to avoid trafficking to degradative lysosomes. According to the proton sponge hypothesis, the buffering capacity of PEI leads to osmotic swelling and rupture of endosomes, resulting in the release of the vector into the cytoplasm. METHODS: The mechanism of PEI-mediated DNA transfer was investigated using quantitative methods to study individual steps in the overall transfection process. In addition to transfection efficiency, the cellular uptake, local pH environment, and stability of vectors were analyzed. N-Quaternized (and therefore non-proton sponge) versions of PEI and specific cell function inhibitors were used to further probe the proton sponge hypothesis. RESULTS: Both N-quaternization and the use of bafilomycin A1 (a vacuolar proton pump inhibitor) reduced the transfection efficiency of PEI by approximately two orders of magnitude. Chloroquine, which buffers lysosomes, enhanced the transfection efficiency of N-quaternized PEIs and polylysine by 2-3-fold. In contrast, chloroquine did not improve the transfection efficiency of PEI. The measured average pH environment of PEI vectors was 6.1, indicating that they successfully avoid trafficking to acidic lysosomes. Significantly lower average pH environments were observed for permethyl-PEI (pH 5.4), perethyl-PEI (pH 5.1), and polylysine (pH 4.6) vectors. Cellular uptake levels of permethyl-PEI and perethyl-PEI vectors were found to be 20 and 90% higher, respectively, than that of parent PEI vectors, indicating that the reduction in transfection activity of the N-quaternized PEIs is due to a barrier downstream of cellular uptake. A polycation/DNA-binding affinity assessment showed that the more charge dense N-quaternized PEIs bind DNA less tightly than PEI, demonstrating that poor vector unpackaging was not responsible for the reduced transfection activity of the N-quaternized PEIs. CONCLUSIONS: The results obtained are consistent with the proton sponge hypothesis and strongly suggest that the transfection activity of PEI vectors is due to their unique ability to avoid acidic lysosomes.     

Does diet variation determine the digestive tract length of Capoeta banarescui Turan,Kottelat, Ekmekci and Imamoglu, 2006?

This study tested the spatial variations in the digestive/intestine tract length of Capoeta banarescui, with regard to their diets in different habitats. Highly varied diets observed in a previous study within the same river system posed the question whether this flexibility is reflected in the digestive tract and intestine length of the species in the Ye?il?rmak River, Turkey. Totals of 382 specimens (standard length 4.6–19.1 cm) were captured by electro‐fishing along the river in September 2012 at 11 locations spanning elevations from 34 to 992 m. The stomach, intestine and total digestive tract lengths were measured, and stomach contents analysed from 196 specimens. For statistical analyses, the stomach, intestine and total digestive tract length were expressed as percentages of total weight and standard length. The data provided evidence that the digestive tract and intestine lengths varied significantly among locations in association with the diet. Fish having dominantly carnivorous diets (e.g. chironomid larvae/invertebrates) in two locations had significantly shorter intestines and digestive tracts than those with diets dominated by benthic algae and other plants. The data indicated that C. banarescui showed broad flexibility in their feeding habits. Feeding heavily on plant materials might lead to the development of longer digestive tracts, increasing the active surface area for digestion; alternatively, there may be less invested in development of the digestive tract when feeding primarily on carnivorous diets where the respective digestive enzymes are readily available. The data suggest that phenotypic plasticity in the digestive tract length of C. banarescui is associated more with the abundant protein‐rich carnivorous food sources in the studied habitats. Whether this digestive tract plasticity has a genetic background remains to be verified in future studies.     

MAGIC Tool: integrated microarray data analysis

Summary: Several programs are now available for analyzing thelarge datasets arising from cDNA microarray experiments. Mostprograms are expensive commercial packages or require expensivethird party software. Some are freely available to academicresearchers, but are limited to one operating system. MicroArrayGenome Imaging and Clustering Tool (MAGIC Tool) is an open sourceprogram that works on all major platforms, and takes users ‘fromtiff to gif’. Several unique features of MAGIC Tool areparticularly useful for research and teaching. Availability: http://www.bio.davidson.edu/MAGIC Contact: laheyer{at}davidson.edu     

Fe(III) Reduction and U(VI) Immobilization by Paenibacillus sp. Strain 300A,Isolated from Hanford 300A Subsurface Sediments

A facultative iron-reducing [Fe(III)-reducing] Paenibacillus sp. strain was isolated from Hanford 300A subsurface sediment biofilms that was capable of reducing soluble Fe(III) complexes [Fe(III)-nitrilotriacetic acid and Fe(III)-citrate] but unable to reduce poorly crystalline ferrihydrite (Fh). However, Paenibacillus sp. 300A was capable of reducing Fh in the presence of low concentrations (2 μM) of either of the electron transfer mediators (ETMs) flavin mononucleotide (FMN) or anthraquinone-2,6-disulfonate (AQDS). Maximum initial Fh reduction rates were observed at catalytic concentrations (<10 μM) of either FMN or AQDS. Higher FMN concentrations inhibited Fh reduction, while increased AQDS concentrations did not. We also found that Paenibacillus sp. 300A could reduce Fh in the presence of natural ETMs from Hanford 300A subsurface sediments. In the absence of ETMs, Paenibacillus sp. 300A was capable of immobilizing U(VI) through both reduction and adsorption. The relative contributions of adsorption and microbial reduction to U(VI) removal from the aqueous phase were ∼7:3 in PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)] and ∼1:4 in bicarbonate buffer. Our study demonstrated that Paenibacillus sp. 300A catalyzes Fe(III) reduction and U(VI) immobilization and that these reactions benefit from externally added or naturally existing ETMs in 300A subsurface sediments.     

Effect of Strontium Ranelate on Hydrogen Peroxide-Induced Apoptosis of CRL-11372 Cells

In vitro and in vivo studies have proven strontium to be an osteoinductive trace element. The effect of strontium ranelate (SR) on H₂O₂-induced apoptosis of CRL-11372 cells and optimization of its anti-apoptotic dose were the aims of this study. After 1 h of pretreatment with SR 1 μM, 50 μM, 100 μM, 500 μM, and 1,000 μM concentrations, CRL-11372 osteoblasts were exposed to 100 μM H₂O₂ for periods of 6–12 h. The same experiments were repeated without H₂O₂. The apoptotic index and viability of cells were assessed quantitatively with a fluorescent dye and qualitatively with agarose gel electrophoresis. Concentrations of 1–100 μM of SR with a 6-h treatment and only 1 μM concentration with a 12-h treatment inhibited the apoptotic effect of H₂O₂ on cultured osteoblasts significantly (P < 0.05). SR was shown to inhibit H₂O₂-induced apoptosis of CRL-11372 cells in a dose-dependent manner.     

Degradation of Bermuda and Orchard Grass by Species of Ruminal Bacteria

Fiber degradation in Bermuda grass and orchard grass was evaluated gravimetrically and by scanning and transmission electron microscopy after incubation with pure cultures of rumen bacteria. Lachnospira multiparus D-32 was unable to degrade plant cell wall components. Butyrivibrio fibrisolvens 49 degraded 6 and 14.9% of the fiber components in Bermuda grass and orchard grass, respectively, and Ruminococcus albus 7 degraded 11.4% orchard grass fiber but none in Bermuda grass. Both B. fibrisolvens and R. albus lacked capsules, did not adhere to fiber, and degraded only portions of the more easily available plant cell walls. R. flavefaciens FD-1 was the most active fiber digester, degrading 8.2 and 55.3% of Bermuda and orchard grass fiber, respectively. The microbe had a distinct capsule and adhered to fiber, especially that which is slowly degraded, but was able to cause erosion and disorganization of the more easily digested cell walls, apparently by extracellular enzymes. Results indicated that more digestible cell walls could be partially degraded by enzymes disassociated from cellulolytic and noncellulolytic bacteria, and data were consistent with the hypothesis that the more slowly degraded plant walls required attachment. Microbial species as well as the cell wall architecture influenced the physical association with and digestion of plant fiber.     

Effect of phenolic monomers on the growth and beta-glucosidase activity of Bacteroides ruminicola and on the carboxymethylcellulase, beta-glucosidase, and xylanase activities of Bacteroides succinogenes

trans-p-Coumaric acid inhibited the growth of Bacteroides ruminicola on both cellobiose and glucose, while trans-ferulic acid and vanillin retarded growth. The phenolic monomers varied in their potential to inhibit the Bacteroides succinogenes beta-glucosidase, carboxymethylcellulase, and xylanase, with p-coumaric acid being the most inhibitory. The B. ruminicola beta-glucosidase was inhibited less than 10% by all three compounds.     

Recurrence of the unfit

Domination among strategies in an evolutionary game implies that the geometric mean of the frequencies of certain strategies—the unfit—approaches zero. However, as we show by example no one strategy need be eliminated in the limit.     

Processing of a newly identified intermediate of human myeloperoxidase in isolated granules occurs at neutral pH

Myeloperoxidase is a major component of specialized lysosomes known as azurophil granules in polymorphonuclear leukocytes or neutrophils. The processing of myeloperoxidase in human HL-60 promyelocytic leukemia cells was studied by pulse-labeling cells in culture with [35S]methionine followed by immunoprecipitation and identification of myeloperoxidase polypeptides from cell fractions after various chase intervals. These studies revealed the presence of a previously unidentified intermediate with Mr 74,000 which kinetically followed the appearance of a larger Mr 81,000 intermediate. Using an in vitro lysosomal preparation the newly identified Mr 74,000 intermediate was directly converted within protected granules to mature forms of myeloperoxidase (Mr 63,000 and 60,000). This conversion occurred optimally at pH 7.5 and was not inhibited by lysosomotropic agents (chloroquine, NH4Cl) or protonophores (monensin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone). Furthermore, the uptake of radiolabeled amines indicated a neutral intragranular environment (pH 7.35-7.67) which remained unchanged in the presence and absence of 1 mM ATP or 2.5 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone. We conclude that, in contrast to other lysosomal pathways, the final proteolytic cleavage of myeloperoxidase does not require an acidic environment.