Potential rates of fermentation in digesta from the gastrointestinal tract of pigs: effect of feeding fermented liquid feed

Microbial catabolic capacity in digesta from the gastrointestinal tract of pigs fed either dry feed or fermented liquid feed (FLF) was determined with the PhenePlate multisubstrate system. The in vitro technique was modified to analyze the kinetics of substrate catabolism mediated by the standing stock of enzymes (potential rates of fermentation), allowing a quantitative evaluation of the dietary effect on the catabolic capacity of the microbiota. In total, the potential rates of fermentation were significantly reduced in digesta from the large intestine (cecum, P < 0.1; colon, P < 0.01; and rectum, P < 0.0001) of pigs fed FLF compared to pigs fed dry feed. No effect of diet was observed in the stomach (P = 0.71) or the distal part of the small intestine (P = 0.97). The highest rates of fermentation and the most significant effect of diet were observed for readily fermentable carbohydrates like maltose, sucrose, and lactose. Feeding FLF to pigs also led to a reduction in the large intestine of the total counts of anaerobic bacteria in general and lactic acid bacteria specifically, as well as of microbial activity, as determined by the concentration of ATP and short-chain fatty acids. The low-molecular-weight carbohydrates were fermented mainly to lactic acid in the FLF before being fed to the animals. This may have limited microbial nutrient availability in the digesta reaching the large intestine of pigs fed FLF and may have caused the observed reduction in activity and density of the cecal and colonic microbial population. On the other hand, feeding FLF to pigs reduced the viable counts of coliform bacteria (indicator of Escherichia coli and Salmonella spp.) most profoundly in the stomach and the distal part of the small intestine, probably due to the bactericidal effect of lactic acid and low pH. The results presented clearly demonstrate that feeding FLF to pigs had a great impact on the indigenous microbiota, as reflected in bacterial numbers, short-chain fatty acid concentration, and substrate utilization. However, completely different mechanisms may be involved in the proximal and the distal parts of the gastrointestinal tract. The present study illustrates the utility of the PhenePlate system for quantifying the catabolic capacity of the indigenous gastrointestinal tract microbiota.     

Ligand Binding and Polymerization Characteristics of Human Milk Folate Binding Protein Depend on Concentration of Purified Protein and Presence of Amphiphatic Substances

Two folate binding proteins are present in human milk; one of 27 kDa is a cleavage product of the other one (100 kDa) which possesses a hydrophobic membrane anchor. A drastic change of radioligand binding characteristics and appearance of aggregated weak-radioligand affinity forms on gel filtration occurred at low concentrations of both proteins in the absence of Triton X-100 or other amphiphatic substances, e.g. cetyltrimethylammonium and phospholipids. These findings are consistent with a model predicting association between unliganded and liganded monomers resulting in weak-ligand affinity dimers. Amphiphatic substances form micelles and lipid bilayers which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers (monomers become hydrophilic in the liganded state) thereby preventing association between these monomeric forms prevailing at low concentrations of the protein. Bio-Gel P-300 chromatography of the 27 kDa protein revealed a pronounced polymerization tendency, which diminished with decreasing protein concentrations, however, not in the presence of cetyltrimethylammonium. The data could have some bearings on observations indicating that naturally occurring amphiphatic substances, cholesterol and phospholipids, are necessary for the important clustering of membrane folate receptors.     

Inertial shear forces and the use of centrifuges in gravity research. What is the proper control?

Centrifuges are used for 1 x g controls in space flight microgravity experiments and in ground based research. Using centrifugation as a tool to generate an Earth like acceleration introduces unwanted inertial shear forces to the sample. Depending on the centrifuge and the geometry of the experiment hardware used these shear forces contribute significantly to the total force acting on the cells or tissues. The inertial shear force artifact should be dealt with for future experiment hardware development for Shuttle and the International Space Station (ISS) as well as for the interpretation of previous space-flight and on-ground research data.     

The patchwork nature of rolling-circle plasmids: comparison of six plasmids from two distinct Bacillus thuringiensis serotypes

Bacillus thuringiensis, the entomopathogenic bacteria from the Bacillus cereus group, harbors numerous extrachromosomal molecules whose sizes vary from 2 to more than 200kb. Apart from the genes coding for the biopesticide delta-endotoxins located on large plasmids, little information has been obtained on these plasmids and their contribution to the biology of their host. In this paper, we embarked on a detailed comparison of six small rolling-circle replicating (RCR) plasmids originating from two major B. thuringiensis strains. The complete nucleotide sequences of plasmid pGI1, pGI2, pGI3, pTX14-1, pTX14-2, and pTX14-3 have been obtained and compared. Replication functions, comprising, for each plasmid, the gene encoding the Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified and analyzed. Two new families, or homology groups, of RCR plasmids originated from the studies of these plasmids (Group VI based on pGI3 and Group VII based on pTX14-3). On five of the six plasmids, loci involved in conjugative mobilization (Mob-genes and origin of transfer (oriT)) were identified. Plasmids pTX14-1, pTX14-2, and pTX14-3 each harbor an ORF encoding a polypeptide containing a central domain with repetitive elements similar to eukaryotic collagen (Gly-X-Y triplets). These genes were termed bcol for Bacillus-collagen-like genes.     

Effects of emotion-oriented care on elderly people with cognitive impairment and behavioral problems in residential homes

This study investigates the effects of emotion-oriented care on the behavior of elderly people with cognitive impairment and behavioral problems. This approach is mainly based on the validation approach, but uses also insights from other approaches like reminiscence and sensory stimulation. Sixteen homes for the aged with structured day care units were randomly allocated to an intervention or control group. 151 Residents with cognitive impairment and behavioral problems were included in the study. The eight intervention homes received a training program with regard to emotion-oriented care. In the eight control homes usual care was continued. Measurements were performed at baseline and after three, six and twelve months of follow-up (assessment by caregivers and relatives). The primary outcome measure was the change in behavior of the residents. The results of multilevel analyses (overall, subgroup and per protocol) showed no statistically significant, nor clinically relevant effects in favor of the intervention group on the behavioral outcome measures. A possible reason for this is that the intervention did not result in significant contrast between the study groups. There is insufficient scientific evidence yet to justify the implementation of emotion-oriented care in residential homes, for residents with moderate to severe cognitive impairment and behavioral problems, on a large scale. Additional studies are needed in which special attention should be given to the implementation process.     

Tat-conjugated synthetic macromolecules facilitate cytoplasmic drug delivery to human ovarian carcinoma cells

We have synthesized N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-cell penetrating peptide Tat conjugates and evaluated their subcellular distribution in A2780 human ovarian carcinoma cells by confocal fluorescence microscopy and subcellular fractionation. Our data indicate the transport of these conjugates by a single Tat molecule to both the cytoplasm and nucleus via a nonendocytotic and concentration independent process. The uptake was observed to occur within 3 min, as confirmed by live cell microscopy. In contrast, HPMA copolymers lacking the Tat peptide were internalized solely by endocytosis. For the first time, Tat-mediated cytoplasmic delivery of a polymer bound anticancer drug, doxorubicin, was also demonstrated. These findings establish the feasibility of overcoming major cellular and subcellular obstacles to intracellular macromolecular delivery and hold great promise for the development of polymer-based systems for the cytoplasmic delivery of therapeutic molecules.     

The effect of creatine on treadmill running with high-intensity intervals

To determine whether creatine monohydrate supplementation would improve performance during a submaximal treadmill run interspersed with high-intensity intervals, 15 college soccer players (8 women, 7 men) received either creatine or a maltodextrin placebo at 0.3 g.kg body mass per day for 6 days. The speed of the treadmill was constant at 160.8 m.min, and every 2 minutes the grade was elevated to 15%. Each hill segment was 1 minute long. At the end of the 20-minute protocol, the treadmill was again elevated to 15% and held there until volitional exhaustion occurred. There was a significant treatment effect of creatine supplementation on body mass (p < 0.05) in the men; however, no significant differences were observed in the women (p > 0.05). There were no treatment effects (p > 0.05) on time to exhaustion, ratings of perceived exertion, or blood lactate concentration. There was a tendency for blood lactate levels to be lower after short-term creatine supplementation in the women, but this was not statistically significant. Based on these results, it appears that creatine supplementation does not improve performance in submaximal running interspersed with high-intensity intervals.     

Mgm1p,a dynamin-related GTPase,is essential for fusion of the mitochondrial outer membrane

In Saccharomyces cerevisiae, mitochondrial fusion requires at least two outer membrane proteins, Fzo1p and Ugo1p. We provide direct evidence that the dynamin-related Mgm1 protein is also required for mitochondrial fusion. Like fzo1 and ugo1 mutants, cells disrupted for the MGM1 gene contain numerous mitochondrial fragments instead of the few long, tubular organelles seen in wild-type cells. Fragmentation of mitochondria in mgm1 mutants is rescued by disrupting DNM1, a gene required for mitochondrial division. In zygotes formed by mating mgm1 mutants, mitochondria do not fuse and mix their contents. Introducing mutations in the GTPase domain of Mgm1p completely block mitochondrial fusion. Furthermore, we show that mgm1 mutants fail to fuse both their mitochondrial outer and inner membranes. Electron microscopy demonstrates that although mgm1 mutants display aberrant mitochondrial inner membrane cristae, mgm1 dnm1 double mutants restore normal inner membrane structures. However, mgm1 dnm1 mutants remain defective in mitochondrial fusion, indicating that mitochondrial fusion requires Mgm1p regardless of the morphology of mitochondria. Finally, we find that Mgm1p, Fzo1p, and Ugo1p physically interact in the mitochondrial outer membrane. Our results raise the possibility that Mgm1p regulates fusion of the mitochondrial outer membrane through its interactions with Fzo1p and Ugo1p.     

A squirrel monkey model of poststroke motor recovery

Nonhuman primate models of poststroke recovery have become increasingly rare primarily due to high purchase and maintenance costs and limited availability of nonhuman primate species. Despite this obstacle, nonhuman primate models may offer important advantages over rodent models for understanding many of the brain's mechanisms for self-repair due to greater similarity in cortical organization to humans. Since the mid-1990s, surgical, neurophysiological, and neuroanatomical methods have been developed to understand structural and functional remodeling of the cerebral cortex after an ischemic event, such as occurs in stroke. These methods require long surgical procedures and entail constant physiological monitoring. With careful attention to intraoperative and postsurgical monitoring, these procedures can be repeated multiple times in individual monkeys without untoward events. This model provides a statistically powerful approach for tracking brain plasticity in the ensuing weeks and months after a stroke-like injury, reducing the number of animals required for individual experiments. This methodology is described in detail, and many of the resulting findings that are relevant for understanding stroke recovery and the effects of rehabilitative and pharmacotherapeutic interventions are summarized.     

Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis

Rho family GTPases regulate the cytoskeleton and cell migration and are frequently overexpressed in tumours. Here, we identify two modes of tumour-cell motility in 3D matrices that involve different usage of Rho signalling. Rho signalling through ROCK promotes a rounded bleb-associated mode of motility that does not require pericellular proteolysis. This form of motility requires ezrin, which is localized in the direction of cell movement. In contrast, elongated cell motility is associated with Rac-dependent F-actin-rich protrusions and does not require Rho, ROCK or ezrin function. Combined blockade of extracellular proteases and ROCK negates the ability of tumour cells to switch between modes of motility and synergises to prevent tumour cell invasion.