A transgenic mouse class-III beta tubulin reporter using yellow fluorescent protein

A yellow fluorescence protein (YFP) reporter construct was cloned downstream of the beta-tubulin III promoter and injected to produce two founder lines of transgenic mice. YFP expression was observed in many regions of the developing peripheral and central nervous system. YFP expression was first observed in the peripheral and central nervous system as early as embryonic day 9.0. There was a dramatic increase in the number of neuronal systems expressing YFP through P0. Then as the animals reached adult age, the expression levels decreased, but many neurons still show YFP expression, notably in regions of the brain undergoing adult neurogenesis, i.e., the rostral migratory stream and subgranular layer of the dentate gyrus. This reporter-based staining was compared with anti-class-III beta-tubulin immunocytochemistry and shown to closely parallel the expression of the endogenous protein. These transgenic lines should provide unique models to study in vivo and in vitro neurodevelopment.     

Estimating population cause-specific mortality fractions from in-hospital mortality: validation of a new method

Background

Cause-of-death data for many developing countries are not available. Information on deaths in hospital by cause is available in many low- and middle-income countries but is not a representative sample of deaths in the population. We propose a method to estimate population cause-specific mortality fractions (CSMFs) using data already collected in many middle-income and some low-income developing nations, yet rarely used: in-hospital death records.

Methods and Findings

For a given cause of death, a community''s hospital deaths are equal to total community deaths multiplied by the proportion of deaths occurring in hospital. If we can estimate the proportion dying in hospital, we can estimate the proportion dying in the population using deaths in hospital. We propose to estimate the proportion of deaths for an age, sex, and cause group that die in hospital from the subset of the population where vital registration systems function or from another population. We evaluated our method using nearly complete vital registration (VR) data from Mexico 1998–2005, which records whether a death occurred in a hospital. In this validation test, we used 45 disease categories. We validated our method in two ways: nationally and between communities. First, we investigated how the method''s accuracy changes as we decrease the amount of Mexican VR used to estimate the proportion of each age, sex, and cause group dying in hospital. Decreasing VR data used for this first step from 100% to 9% produces only a 12% maximum relative error between estimated and true CSMFs. Even if Mexico collected full VR information only in its capital city with 9% of its population, our estimation method would produce an average relative error in CSMFs across the 45 causes of just over 10%. Second, we used VR data for the capital zone (Distrito Federal and Estado de Mexico) and estimated CSMFs for the three lowest-development states. Our estimation method gave an average relative error of 20%, 23%, and 31% for Guerrero, Chiapas, and Oaxaca, respectively.

Conclusions

Where accurate International Classification of Diseases (ICD)-coded cause-of-death data are available for deaths in hospital and for VR covering a subset of the population, we demonstrated that population CSMFs can be estimated with low average error. In addition, we showed in the case of Mexico that this method can substantially reduce error from biased hospital data, even when applied to areas with widely different levels of development. For countries with ICD-coded deaths in hospital, this method potentially allows the use of existing data to inform health policy.     

The Cape element in the Afrotemperate flora: from Cape to Cairo?

The build-up of biodiversity is the result of immigration and in situ speciation. We investigate these two processes for four lineages (Disa, Irideae p.p., the Pentaschistis clade and Restionaceae) that are widespread in the Afrotemperate flora. These four lineages may be representative of the numerous clades which are species rich in the Cape and also occur in the highlands of tropical Africa. It is as yet unclear in which direction the lineages spread. Three hypotheses have been proposed: (i) a tropical origin with a southward migration towards the Cape, (ii) a Cape origin with a northward migration into tropical Africa, and (iii) vicariance. None of these hypotheses has been thoroughly tested. We reconstruct the historical biogeography of the four lineages using likelihood optimization onto molecular phylogenies. We find that tropical taxa are nested within a predominantly Cape clade. There is unidirectional migration from the Cape into the Drakensberg and from there northwards into tropical Africa. The amount of in situ diversification differs between areas and clades. Dating estimates show that the migration into tropical East Africa has occurred in the last 17 Myr, consistent with the Mio-Pliocene formation of the mountains in this area.     

Impacts of feral horses in the Australian Alps and evidence‐based solutions

New evidence of impacts by feral horses in Australia's alpine parks systems confirms they endanger threatened species and extensively damage critically endangered bog communities that could take millennia to recover. These impacts are not confounded by effects of deer and accumulate over time, even when only a small number of feral horses (~100) are present. With protected areas representing only a small proportion of the area of the Australian states of New South Wales (9.3%) and Victoria (17%), allowing feral horses to degrade reserves is not a reasonable management compromise, is contrary to the purpose of the protected area system and conflicts with international obligations. Modelling and decades of management experience indicate that trapping alone does not control feral horse numbers. Trapping and fertility control can work in small populations, but not when there are several thousand horses in remote areas. Aerial culling is needed to cost‐effectively and humanely control feral horse populations. The relatively small amount of suffering feral horses experience during a cull is outweighed by (i) avoiding suffering and death of horses from starvation and thirst, (ii) avoiding the suffering of native animals displaced by horses and (iii) avoiding the ethical concerns of driving threatened species towards extinction. Objections to aerial culling on welfare and cultural grounds are contradicted by evidence. Improving knowledge in the general community about what is at stake is long overdue because without this knowledge, small groups with vested interests and unfounded claims have been able to dominate debate and dictate management actions. As a result of ineffective management, horse populations are now expanding and causing well‐documented damage to Australia's alpine parks, placing at risk almost $10M spent on restoration after livestock grazing ended. The costs of horse control and restoration escalate the longer large horse populations remain in the alpine parks. It is crucial that feral horse numbers are rapidly reduced to levels where ecosystems begin to recover. Aerial culling is needed as part of the toolbox to achieve that reduction.     

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::β-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method^1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, β-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit³, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.Download video file.^{(120M, mp4)}     

Transient expression of human interleukin-2 and interferon-γ genes is regulated by interaction between distinct cell subsets

The level of transient expression of human IL-2 and IFN-γ genes, we show, is regulated by dynamic interaction between two functionally distinct cell populations. One is able to express these genes, while the other, bearing one of several specific surface markers, actively inhibits their expression. Defined cell subsets were isolated from PBMC and tonsil cells using immunomagnetic beads coated with monoclonal antibodies directed against surface markers. Depletion of CD8, CD11a (Leu15), or Leu8 subsets led to a pronounced superinduction of IL-2 and IFN-γ gene expression when the remaining cell population was stimulated with mitogen (PHA) or antigen (SEB). Thus, a 10-fold increase in production of IFN-γ was observed after removal of CD11a (Leu15) cells constituting only a small percentage of the total cell population. By contrast, depletion of cells expressing CD19, a B cell marker, did not yield any superinduction. Conversely, CD8, CD11a (Leu15), or Leu8 cell subsets, but not CD19 cells, each inhibited the induction of IL-2 and IFN-γ gene expression almost completely in depleted or total cell populations from which they were derived. Gene expression occurring within one cell subset could be effectively inhibited by cells from a second subset. Introduction of inhibitory cells (Leu8) into a population that actively expressed IL-2 and IFN-γ mRNA resulted in an immediate cessation of gene expression. This suppression involves a soluble mediator, since the culture medium in which such cells were activated exerted a similarly effective inhibition.     

Do Displays Send Information about Ornament Structure and Male Quality in the Ornate Tree Lizard,Urosaurus ornatus?

Displays can transmit information about ornament or male quality; however, few studies have simultaneously explored the relationship between displays, ornament and male quality within a single species. We quantified ornament morphology (five throat color morphs, throat area, and belly area), male quality (bite force, sprint speed, body condition, and body mass), display behavior [percent time displaying (PTD), number of pushups per display, and display duration], and movement behavior among males in a population of the ornate tree lizard, Urosaurus ornatus. Previous studies have shown that male U. ornatus are polymorphic in throat coloration and that morphs differ in behavioral aggression. Our study shows that blue throat morphs use 1.5 more pushups per display than other male color morphs, which suggests that throat color and display behavior act as backup signals for aggression. However, other data support the multiple messages hypothesis, and overall our data do not provide conclusive evidence for any one hypothesis. In addition, we show that body mass is positively related to both PTD and percent time moving and this relationship is independent of color morph. We also found that throat area, belly area, bite force, sprint speed, and body condition are unrelated to display behavior. This result highlights at least some discordance between display behavior, ornaments, and performance in U. ornatus and suggests that these traits may be evolving independently.     

A single amino acid change is responsible for evolution of acyltransferase specificity in bacterial methionine biosynthesis

Bacteria and yeast rely on either homoserine transsuccinylase (HTS, metA) or homoserine transacetylase (HTA; met2) for the biosynthesis of methionine. Although HTS and HTA catalyze similar chemical reactions, these proteins are typically unrelated in both sequence and three-dimensional structure. Here we present the 2.0 A resolution x-ray crystal structure of the Bacillus cereus metA protein in complex with homoserine, which provides the first view of a ligand bound to either HTA or HTS. Surprisingly, functional analysis of the B. cereus metA protein shows that it does not use succinyl-CoA as a substrate. Instead, the protein catalyzes the transacetylation of homoserine using acetyl-CoA. Therefore, the B. cereus metA protein functions as an HTA despite greater than 50% sequence identity with bona fide HTS proteins. This result emphasizes the need for functional confirmation of annotations of enzyme function based on either sequence or structural comparisons. Kinetic analysis of site-directed mutants reveals that the B. cereus metA protein and the E. coli HTS share a common catalytic mechanism. Structural and functional examination of the B. cereus metA protein reveals that a single amino acid in the active site determines acetyl-CoA (Glu-111) versus succinyl-CoA (Gly-111) specificity in the metA-like of acyltransferases. Switching of this residue provides a mechanism for evolving substrate specificity in bacterial methionine biosynthesis. Within this enzyme family, HTS and HTA activity likely arises from divergent evolution in a common structural scaffold with conserved catalytic machinery and homoserine binding sites.     

High Cu(I) and low proton affinities of the CXXC motif of Bacillus subtilis CopZ

CopZ, an Atx1-like copper chaperone from the bacterium Bacillus subtilis, functions as part of a complex cellular machinery for Cu(I) trafficking and detoxification, in which it interacts specifically with the transmembrane Cu(I)-transporter CopA. Here we demonstrate that the cysteine residues of the MXCXXC Cu(I)-binding motif of CopZ have low proton affinities, with both exhibiting pK(a) values of 6 or below. Chelator competition experiments demonstrated that the protein binds Cu(I) with extremely high affinity, with a small but significant pH-dependence over the range pH 6.5-8.0. From these data, a pH-corrected formation constant, beta(2)= approximately 6 x 10(22) M(-2), was determined. Rapid exchange of Cu(I) between CopZ and the Cu(I)-chelator BCS (bathocuproine disulfonate) indicated that the mechanism of exchange does not involve simple dissociation of Cu(I) from CopZ (or BCS), but instead proceeds via the formation of a transient Cu(I)-mediated protein-chelator complex. Such a mechanism has similarities to the Cu(I)-exchange pathway that occurs between components of copper-trafficking pathways.     

Structure and Cu(I)-binding properties of the N-terminal soluble domains of Bacillus subtilis CopA

CopA, a P-type ATPase from Bacillus subtilis, plays a major role in the resistance of the cell to copper by effecting the export of the metal across the cytoplasmic membrane. The N-terminus of the protein features two soluble domains (a and b), that each contain a Cu(I)-binding motif, MTCAAC. We have generated a stable form of the wild-type two-domain protein, CopAab, and determined its solution structure. This was found to be similar to that reported previously for a higher stability S46V variant, with minor differences mostly confined to the Ser(46)-containing beta3-strand of domain a. Chemical-shift analysis demonstrated that the two Cu(I)-binding motifs, located at different ends of the protein molecule, are both able to participate in Cu(I) binding and that Cu(I) is in rapid exchange between protein molecules. Surprisingly, UV-visible and fluorescence spectroscopy indicate very different modes of Cu(I) binding below and above a level of 1 Cu(I) per protein, consistent with a major structural change occurring above 1 Cu(I) per CopAab. Analytical equilibrium centrifugation and gel filtration results show that this is a result of Cu(I)-mediated dimerization of the protein. The resulting species is highly luminescent, indicating the presence of a solvent-shielded Cu(I) cluster.