A Thionin Stain for Visualizing Bone Cells, Mineralizing Fronts and Cement Lines in Undecalcified Bone Sections

A staining method is described using thionin, for undecalcified deacrylated bone sections. RNA is stained purplish violet, allowing still active osteoblasts to be distinguished from lining cells. Staining intensity of mineralized bone is related to the degree of mineralization. Mineralizing fronts and cement lines are visualized clearly. Lamellae show an alternate pattern. Histomorphometric parameters such as osteon thickness and interstitial bone thickness can be measured without using polarized light. The mineralizing front can be assessed and expressed as a percentage of the osteoblast-covered interface between osteoid and mineralized bone. The stain is also useful for qualitative assessment of metabolic bone disease. Thionin stained sections can be kept for at least one year when stored hi the dark at 7 C.     

Use of flavin photochemistry to probe intraprotein and interprotein electron transfer mechanisms

Photoexcitation of flavin analogs generates the lowest triplet state (via intersystem crossing from the first excited singlet state) in the nanosecond time domain and with high quantum efficiency. The triplet, being a strong oxidant, can abstract a hydrogen atom (or an electron) from a reduced donor in a diffusion-controlled reaction. If the donor is a redox protein, the oxidation process can be used to initiate an electron transfer sequence involving either intramolecular or intermolecular reactions. If the donor is an organic compound such as EDTA, the neutral flavin semiquinone will be produced by H atom abstraction; this is a strong reductant and can subsequently transfer a hydrogen atom (or an electron) to an oxidized redox protein, thereby again initiating a sequence of intramolecular or intermolecular processes. If flavin photoexcitation is accomplished using a pulsed laser light source, the initiation of these protein electron transfer reactions can be made to occur in the nanosecond to microsecond time domain, and the sequence of events can be followed by time-resolved spectrophotometry to obtain rate constants and thus mechanistic information. The present paper describes this technology, and selected examples of its use in the investigation of redox protein mechanisms are given.     

Nitrogen Oxyanion-dependent Dissociation of a Two-component Complex That Regulates Bacterial Nitrate Assimilation

Nitrogen is an essential nutrient for growth and is readily available to microbes in many environments in the form of ammonium and nitrate. Both ions are of environmental significance due to sustained use of inorganic fertilizers on agricultural soils. Diverse species of bacteria that have an assimilatory nitrate/nitrite reductase system (NAS) can use nitrate or nitrite as the sole nitrogen source for growth when ammonium is limited. In Paracoccus denitrificans, the pathway-specific two-component regulator for NAS expression is encoded by the nasT and nasS genes. Here, we show that the putative RNA-binding protein NasT is a positive regulator essential for expression of the nas gene cluster (i.e. nasABGHC). By contrast, a nitrogen oxyanion-binding sensor (NasS) is required for nitrate/nitrite-responsive control of nas gene expression. The NasS and NasT proteins co-purify as a stable heterotetrameric regulatory complex, NasS-NasT. This protein-protein interaction is sensitive to nitrate and nitrite, which cause dissociation of the NasS-NasT complex into monomeric NasS and an oligomeric form of NasT. NasT has been shown to bind the leader RNA for nasA. Thus, upon liberation from the complex, the positive regulator NasT is free to up-regulate nas gene expression.     

CLIX: a search algorithm for finding novel ligands capable of binding proteins of known three-dimensional structure.

A computer algorithm, CLIX, capable of searching a crystallographic data-base of small molecules for candidates which have both steric and chemical likelihood of binding a protein of known three-dimensional structure is presented. The algorithm is a significant advance over previous strategies which consider solely steric or chemical requirements for binding. The algorithm is shown to be capable of predicting the correct binding geometry of sialic acid to a mutant influenza-virus hemagglutinin and of proposing a number of potential new ligands to this protein.     

Top-down and bottom-up regulation of herbivores: Spodoptera frugiperda turns tables on endophyte-mediated plant defence and virulence of an entomopathogenic nematode

Abstract.  1. The fungus Neotyphodium lolii forms a symbiotic relationship with its grass host Lolium perenne (perennial ryegrass). The fungus benefits from access to plant nutrients and photosynthate, whereas the plant benefits from acquired chemical defence against herbivory.
2. This study examined the potential for endophyte-mediated plant defences to influence interactions between fall armyworm Spodoptera frugiperda , and the entomopathogenic nematode Steinernema carpocapsae and clarified biological mechanisms underlying the observations made.
3. In laboratory and greenhouse experiments, S. frugiperda larvae were fed endophytic or non-endophytic L. perenne then exposed to S. carpocapsae or injected with the nematodes' symbiotic bacteria Xenorhabdus nematophila .
4. In all instances, S. frugiperda larvae fed endophyte-infected grass suffered significantly lower mortality than those fed non-endophytic plants. Although larvae fed endophyte-infected grass often had significantly lower biomass than those fed uninfected grass, these differences did not account for altered susceptibility to S. carpocapsae .
5. Endophyte-mediated reductions in herbivore susceptibility to the nematode pathogen represent a herbivore adaptation that effectively turns the tables on both plant and natural enemy by reducing the virulence of the nematodes' symbiotic bacteria while expanding the temporal window of herbivory.