Inactivation of Giardia muris cysts by free chlorine.

The chlorine resistance of cysts of the flagellate protozoan Giardia muris was examined. This organism, which is pathogenic to mice, is being considered as a model for the inactivation of the human pathogen Giardia lamblia. Excystation was used as the criterion for cyst viability. Experiments were performed at pH 5, 7, and 9 at 25 degrees C and pH 7 at 5 degrees C. Survival curves were "stepladder"-shaped, but concentration-time data generally conformed to Watson's Law. Chlorine was most effective at neutral pH and was only slightly less so in acidic solutions. Comparison of inactivation data based on equivalent hypochlorous acid concentrations, which corrects for chlorine ionization, showed that the cysts have a pH-dependent resistance to inactivation. Concentration-time (C X t') products for free chlorine obtained at 25 degrees C ranged from a low of 50 mg min/liter at pH 5 to a high of 218 mg min/liter at pH 9 and were as high as 1,000 mg min/liter at 5 degrees C. It appears that G. muris cysts are somewhat more resistant to inactivation than G. lamblia cysts and rank among the microorganisms that are most resistant to inactivation by free chlorine.     

Synergistic effects of purified recombinant human and murine B cell growth factor-1/IL-4 on colony formation in vitro by hematopoietic progenitor cells. Multiple actions

Purified recombinant human B cell growth factor-1/IL-4 was evaluated, alone and in combination, with purified preparations of recombinant human (rhu) CSF or erythropoietin (Epo) for effects on colony formation by human bone marrow CFU-GM progenitor cells (GM) and burst forming unit-E progenitor cells. rhu IL-4 synergized with rhu G-CSF to enhance granulocyte colony formation, but had no effect on CFU-GM colony formation stimulated by rhu GM-CSF, rhu IL-3, or rhu CSF-1. Rhu IL-4 synergized with Epo to enhance BFU-E colony formation equal to that of Epo plus either rhu IL-3, rhu GM-CSF, or rhu G-CSF. Removal of adherent cells and T lymphocytes did not influence the synergistic activities of rhu IL-4. Rmu IL-4, synergized with rhu G-CSF, but not with rmu GM-CSF, rmu IL-3, or natural mu CSF-1, to enhance CFU-GM (mainly granulocyte) colony numbers by a greater than 90% pure preparation of murine CFU-GM. Also, rhu IL-4 at low concentrations enhanced release of CSF and at higher concentrations the release also of suppressor molecules from human monocytes and PHA-stimulated human T lymphocytes. Use of specific CSF antibodies suggested that rhu IL-4 was enhancing the release of G-CSF and CSF-1 from monocytes and the release of GM-CSF and possibly G-CSF from PHA-stimulated T lymphocytes. Use of antibodies for TNF-alpha, IFN-gamma, or TNF-beta as well as measurement of TNF and IFN titers suggested that the suppressor molecule(s) released from monocytes were acting with TNF-alpha and those released from PHA-stimulated T lymphocytes were acting with IFN-gamma. These results implicate B cell growth factor-1/IL-4 as a synergistic activity for hematopoietic progenitors and suggest that the actions can be on both progenitor and accessory cells.     

Production of reactive oxygen species in decoupled, Ca2+-depleted PSII and their use in assigning a function to chloride on both sides of PSII

Extraction of Ca²⁺ from the oxygen-evolving complex of photosystem II (PSII) in the absence of a chelator inhibits O₂ evolution without significant inhibition of the light-dependent reduction of the exogenous electron acceptor, 2,6-dichlorophenolindophenol (DCPIP) on the reducing side of PSII. The phenomenon is known as “the decoupling effect” (Semin et al. Photosynth Res 98:235–249, 2008). Extraction of Cl^? from Ca²⁺-depleted membranes (PSII[–Ca]) suppresses the reduction of DCPIP. In the current study we investigated the nature of the oxidized substrate and the nature of the product(s) of the substrate oxidation. After elimination of all other possible donors, water was identified as the substrate. Generation of reactive oxygen species HO, H₂O₂, and O ₂ ^·? , as possible products of water oxidation in PSII(–Ca) membranes was examined. During the investigation of O ₂ ^·? production in PSII(–Ca) samples, we found that (i) O ₂ ^·? is formed on the acceptor side of PSII due to the reduction of O₂; (ii) depletion of Cl^? does not inhibit water oxidation, but (iii) Cl^? depletion does decrease the efficiency of the reduction of exogenous electron acceptors. In the absence of Cl^? under aerobic conditions, electron transport is diverted from reducing exogenous acceptors to reducing O₂, thereby increasing the rate of O ₂ ^·? generation. From these observations we conclude that the product of water oxidation is H₂O₂ and that Cl^? anions are not involved in the oxidation of water to H₂O₂ in decoupled PSII(–Ca) membranes. These results also indicate that Cl^? anions are not directly involved in water oxidation by the Mn cluster in the native PSII membranes, but possibly provide access for H₂O molecules to the Mn₄CaO₅ cluster and/or facilitate the release of H⁺ ions into the lumenal space.     

The Mouse Age Phenome Knowledgebase and Disease-Specific Inter-Species Age Mapping

Background

Similarities between mice and humans lead to generation of many mouse models of human disease. However, differences between the species often result in mice being unreliable as preclinical models for human disease. One difference that might play a role in lowering the predictivity of mice models to human diseases is age. Despite the important role age plays in medicine, it is too often considered only casually when considering mouse models.

Methods

We developed the mouse-Age Phenotype Knowledgebase, which holds knowledge about age-related phenotypic patterns in mice. The knowledgebase was extensively populated with literature-derived data using text mining techniques. We then mapped between ages in humans and mice by comparing the age distribution pattern for 887 diseases in both species.

Results

The knowledgebase was populated with over 9800 instances generated by a text-mining pipeline. The quality of the data was manually evaluated, and was found to be of high accuracy (estimated precision >86%). Furthermore, grouping together diseases that share similar age patterns in mice resulted in clusters that mirror actual biomedical knowledge. Using these data, we matched age distribution patterns in mice and in humans, allowing for age differences by shifting either of the patterns. High correlation (r²>0.5) was found for 223 diseases. The results clearly indicate a difference in the age mapping between different diseases: age 30 years in human is mapped to 120 days in mice for Leukemia, but to 295 days for Anemia. Based on these results we generated a mice-to-human age map which is publicly available.

Conclusions

We present here the development of the mouse-APK, its population with literature-derived data and its use to map ages in mice and human for 223 diseases. These results present a further step made to bridging the gap between humans and mice in biomedical research.     

In vivo characterization of a vertebrate ultraconserved enhancer

Genomic sequence comparisons among human, mouse, and pufferfish (Takifugu rubripes (Fugu)) have revealed a set of extremely conserved noncoding sequences. While this high degree of sequence conservation suggests severe evolutionary constraint and predicts a lack of tolerance to change to retain in vivo functionality, such elements have been minimally explored experimentally. In this study, we describe the in-depth characterization of an ancient conserved enhancer, Dc2, located near the dachshund gene, which displays a human-Fugu identity of 84% over 424 basepairs (bp). In addition to this large overall conservation, we find that Dc2 is characterized by the presence of a large block of sequence (144 bp) that is completely identical among human, mouse, chicken, zebrafish, and Fugu. Through the testing of reporter vector constructs in transgenic mice, we observed that the 424-bp Dc2-conserved element is necessary and sufficient for brain tissue enhancer activity. In vivo analyses also revealed that the 144-bp 100% conserved sequence is necessary, but not sufficient, to replicate Dc2 enhancer function. However, the introduction of two separate 16-bp insertions into the highly conserved enhancer core did not cause any detectable modification of its in vivo activity. Our observations indicate that the 144-bp 100% conserved element is tolerant of change at least at the resolution of this transgenic mouse assay and suggest that purifying selection on the Dc2 sequence might not be as strong as we predicted or that some unknown property also constrains this highly conserved enhancer sequence.     

Plasma membranes of the rat liver. Isolation and enzymatic characterization of a fraction rich in bile canaliculi

A method is described for the rapid isolation of a plasma membrane fraction containing a high concentration of intact bile canaliculi from the rat liver. Isolated bile canaliculi retain most of the ultrastructural features exhibited in the intact liver cell. The final fraction contains 5''-nucleotidase activity at approximately the same concentration as that in previous preparations of plasma membranes. In the presence of 0.01 M Mg⁺⁺, 5''-nucleotidase exhibits a double pH optimum at pH values of 7.5 and 9.5. The activities of glucose-6-phosphatase and alkaline phosphatase are present in low amounts. Cytochrome P-450 is not detectable. Na⁺-K⁺-activation of ATPase is observed to the extent of 20–36% in about half of the assays. The availability of a method for preparation of intact bile canaliculi should prove useful for studying the biochemical events associated with the transport of bile constituents into canaliculi.     

Evidence for the presence of two different types of protein bodies in wheat endosperm

Storage proteins of wheat grains (Triticum L. em Thell) are deposited in protein bodies inside vacuoles. However, the subcellular sites and mechanisms of their aggregation into protein bodies are not clear. In the present report, we provide evidence for two different types of protein bodies, low- and high-density types that accumulate concurrently and independently in developing wheat endosperm cells. Gliadins were present in both types of protein bodies, whereas the high molecular weight glutenins were localized mainly in the dense ones. Pulse-chase experiments verified that the dense protein bodies were not formed by a gradual increase in density but, presumably, by a distinct, quick process of storage protein aggregation. Subcellular fractionation and electron microscopy studies revealed that the wheat homolog of immunoglobulin heavy-chain-binding protein, an endoplasmic reticulum-resident protein, was present within the dense protein bodies, implying that these were formed by aggregation of storage proteins within the endoplasmic reticulum. The present results suggest that a large part of wheat storage proteins aggregate into protein bodies within the rough endoplasmic reticulum. Because these protein bodies are too large to enter the Golgi, they are likely to be transported directly to vacuoles. This route may operate in concert with the known Golgi-mediated transport to vacuoles in which the storage proteins apparently condense into protein bodies at a postendoplasmic reticulum location. Our results further suggest that although gliadins are transported by either one of these routes, the high molecular weight glutenins use only the Golgi bypass route.     

PHYSIOLOGICAL DEPENDENCE OF BRAIN METHIONINE AND S-ADENOSYLMETHIONINE CONCENTRATIONS ON SERUM AMINO ACID PATTERN

Abstract— Animals maintained on rat chow and water ad libitum in quarters illuminated for 12 h/day show diurnal rhythms in serum methionine and brain S -adenosylmethionine (SAM) concentrations. Brain methionine exhibits no such variation, nor does the ratio of serum methionine to the serum concentrations of six neutral amino acids which are believed to compete with methionine for uptake into brain. Administration of methionine to rats in doses that elevate serum methionine, but keep it within the daily physiological range, significantly increases brain concentrations of both methionine and SAM. The acute feeding of either a protein-free or a 40% casein meal also increases brain methionine and SAM, but does not affect serum methionine; however, both diets also increase the ratio of serum methionine to tyrosine, an amino acid whose postprandial concentration is indicative of the concentrations of the other amino acids that compete with methionine for transport into brain. These findings suggest that brain methionine levels increase physiologically after eating as a result of changes in the serum amino acid pattern. Furthermore, such naturally occurring increases in brain methionine appear to be associated with elevations in brain SAM.     

Induction of polynucleotide-protein cross-linkages by ultraviolet irradiation. Peculiarities of the high-intensity laser pulse irradiation

The efficiency and specificity of RNA-protein cross-linking in the 30S subunit of Escherichia coli ribosomes, induced by low-intensity (10(15) photons cm-2 s-1, 254 nm) and high-intensity [(1.6-6.8) X 10(24) photons cm-2 s-1, 266 nm, pulse duration 10(-8) s] ultraviolet radiation, are studied. Under the former conditions proteins S4, S7 and S9/S11, and under the latter conditions these proteins together with S3, S18 and S20, are cross-linked to 16S RNA. Biphotonic processes operate in the latter case. In the presence of 2-mercaptoethanol cross-linking occurs either directly, via a higher excited state or via activated intermediates with life-times less than 25 ns. Cross-links thus formed are specific, i.e. they are formed between regions of macromolecules which are in contact in the native (non-disturbed) complex prior to excitation. The efficiency of cross-linking (per photon absorbed) is 20-100 times higher upon two-step excitation as compared with single-step excitation and an analysable number of cross-links are produced in a single pulse. Only base U-1239 of 16S RNA is cross-linked to protein S7 by low-intensity radiation, whereas the adjacent base, G-1240 is also involved in laser-induced cross-linking. A transition from the former to the latter conditions allows one to reduce the duration of irradiation from several minutes to several nanoseconds.