The chianti zebrafish mutant provides a model for erythroid-specific disruption of transferrin receptor 1

Iron is a crucial metal for normal development, being required for the production of heme, which is incorporated into cytochromes and hemoglobin. The zebrafish chianti (cia) mutant manifests a hypochromic, microcytic anemia after the onset of embryonic circulation, indicative of a perturbation in red blood cell hemoglobin production. We show that cia encodes tfr1a, which is specifically expressed in the developing blood and requisite only for iron uptake in erythroid precursors. In the process of isolating zebrafish tfr1, we discovered two tfr1-like genes (tfr1a and tfr1b) and a single tfr2 ortholog. Abrogation of tfr1b function using antisense morpholinos revealed that this paralog was dispensable for hemoglobin production in red cells. tfr1b morphants exhibited growth retardation and brain necrosis, similar to the central nervous system defects observed in the Tfr1 null mouse, indicating that tfr1b is probably used by non-erythroid tissues for iron acquisition. Overexpression of mouse Tfr1, mouse Tfr2, and zebrafish tfr1b partially rescued hypochromia in cia embryos, establishing that each of these transferrin receptors are capable of supporting iron uptake for hemoglobin production in vivo. Taken together, these data show that zebrafish tfr1a and tfr1b share biochemical function but have restricted domains of tissue expression, and establish a genetic model to study the specific function of Tfr1 in erythroid cells.     

De novo identification of highly active fluorescent kappa opioid ligands from a rhodamine labeled tetrapeptide positional scanning library

Highly active fluorescent compounds having kappa opioid activity were identified following the screening in a kappa-specific radioligand binding assay of a positional scanning tetrapeptide combinatorial library in which every tetrapeptide was fluorescently labeled. Lissamine rhodamine B sulfonyl chloride was coupled to the N terminal of a mixture-based tetrapeptide positional scanning library made up of over 7.3 million tetrapeptides. Upon determination of the most active mixtures for each position of the library in the kappa binding assay, individual rhodamine labeled tetrapeptides were then synthesized and tested to determine their activities. Eight individual rhodamine labeled peptides were identified that were specific for the kappa opioid receptor, having binding affinities ranging from 5-20 nM. These peptides were poor inhibitors at the mu and delta receptors (K(i)>5,000 nM). Furthermore, neither rhodamine itself nor these same tetrapeptides lacking the N-terminal rhodamine had any significant activity at the kappa receptor, indicating that both the tetrapeptide sequence and the rhodamine moiety are required for receptor binding. This study has demonstrated that novel fluorescent compounds with intrinsic activity can be identified through the use of combinatorial chemistry.     

Requirement for the Rac GTPase in Chlamydia trachomatis invasion of non-phagocytic cells

Chlamydiae are gram-negative obligate intracellular pathogens to which access to an intracellular environment is paramount to their survival and replication. To this end, chlamydiae have evolved extremely efficient means of invading nonphagocytic cells. To elucidate the host cell machinery utilized by Chlamydia trachomatis in invasion, we examined the roles of the Rho GTPase family members in the internalization of chlamydial elementary bodies. Upon binding of elementary bodies on the cell surface, actin is rapidly recruited to the sites of internalization. Members of the Rho GTPase family are frequently involved in localized recruitment of actin. Clostridial Toxin B, which is a known enzymatic inhibitor of Rac, Cdc42 and Rho GTPases, significantly reduced chlamydial invasion of HeLa cells. Expression of dominant negative constructs in HeLa cells revealed that chlamydial uptake was dependent on Rac, but not on Cdc42 or RhoA. Rac but not Cdc42 was found to be activated by chlamydial attachment. The effect of dominant negative Rac expression on chlamydial uptake is manifested through the inhibition of actin recruitment to the sites of chlamydial entry. Studies utilizing Green Fluorescent Protein fusion constructs of Rac, Cdc42 and RhoA, showed Rac to be the sole member of the Rho GTPase family recruited to the site of chlamydial entry.     

Construction, overexpression, and purification of Arthrobacter globiformis amine oxidase-Strep-tag II fusion protein

The copper-containing amine oxidase from Arthrobacter globiformis has been expressed and purified as a fusion protein with a C-terminal Strep-tag II peptide. This tag facilitates the rapid purification of the enzyme on a large scale using the StrepTactin POROS medium. For example, we have demonstrated that 50 mg of protein can be obtained in 2 days from 2 L of Escherichia coli. The purified fusion protein displays turnover and spectroscopic properties that are essentially identical to those of the wild-type enzyme. Given the location of the C-terminus in four amine oxidase crystal structures, this strategy should be quite general for the rapid purification of amine oxidases from multiple sources.     

Evidence for different contributions of archaea and bacteria to the ammonia-oxidizing potential of diverse Oregon soils

A method was developed to determine the contributions of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) to the nitrification potentials (NPs) of soils taken from forest, pasture, cropped, and fallowed (19 years) lands. Soil slurries were exposed to acetylene to irreversibly inactivate ammonia monooxygenase, and upon the removal of acetylene, the recovery of nitrification potential (RNP) was monitored in the presence and absence of bacterial or eukaryotic protein synthesis inhibitors. For unknown reasons, and despite measureable NPs, RNP did not occur consistently in forest soil samples; however, pasture, cropped, and fallowed soil RNPs commenced after lags that ranged from 12 to 30 h after acetylene removal. Cropped soil RNP was completely prevented by the bacterial protein synthesis inhibitor kanamycin (800 μg/ml), whereas a combination of kanamycin plus gentamicin (800 μg/ml each) only partially prevented the RNP (60%) of fallowed soils. Pasture soil RNP was completely insensitive to either kanamycin, gentamicin, or a combination of the two. Unlike cropped soil, pasture and fallowed soil RNPs occurred at both 30°C and 40°C and without supplemental NH(4)(+) (≤ 10 μM NH(4)(+) in solution), and pasture soil RNP demonstrated ～ 50% insensitivity to 100 μM allyl thiourea (ATU). In addition, fallowed and pasture soil RNPs were insensitive to the fungal inhibitors nystatin and azoxystrobin. This combination of properties suggests that neither fungi nor AOB contributed to pasture soil RNP and that AOA were responsible for the RNP of the pasture soils. Both AOA and AOB may contribute to RNP in fallowed soil, while RNP in cropped soils was dominated by AOB.     

Mallard Response to Experimental Walk-In and Shooting Disturbance

ABSTRACT Spatial and temporal closures of anthropogenic activities are a common management strategy to increase waterfowl usage of an area. However, empirical evidence, specifically how individual waterfowl respond to disturbance, is lacking to support their efficacy. We exposed radiomarked mallards (Anas platyrhynchos) to walk-in, shooting, or no disturbance along the South Platte River corridor in Colorado, USA, from September to February during 2006–2007 and 2007–2008. Mallards exposed to shooting disturbance had greater mean flight distance after disturbance (FDAD) during September-November (4.58 km, 95% CI = 3.55–5.62) than December-February (3.04 km, 95% CI = 2.51–3.58) and were 35% and 17% greater than mean FDAD of mallards exposed to walk-in disturbance, respectively. Walk-in and shooting disturbance had a similar effect on return rates, and disturbed mallards had higher (0.09–0.41) movement probabilities away from and lower (0.15–0.20) probabilities of returning to treatment locations than controls. Probability of presence of disturbed mallards was 37% lower than controls during the daytime but was equal at night. Mallards exposed to walk-in (0.38 [95% CI = 0.30–0.46]) and shooting (0.23 [95% CI = 0.17–0.30] disturbance had low return rates the first afternoon after a disturbance compared to controls (0.71 [95% CI = 0.65–0.77]). A high proportion of mallards exposed to walk-in (0.75 [95% CI = 0.67–0.83]) and shooting (0.70 [95% CI = 0.64–0.76]) disturbance returned to treatment locations in ≤1 day. Managers may be able to more effectively manage disturbance regimes by 1) accounting for surrounding lands within <10 km, especially lands within <5 km, 2) being conscientious when establishing regulations that will affect levels of disturbance 1–2 days after a previous disturbance, and 3) considering shooting and walking disturbance equally for refuge design.     

Systemic vasculopathy with altered vasoreactivity in a transgenic mouse model of scleroderma

Introduction  

Vasculopathy, including altered vasoreactivity and abnormal large vessel biomechanics, is a hallmark of systemic sclerosis (SSc). However, the pathogenic link with other aspects of the disease is less clear. To assess the potential role of transforming growth factor beta (TGF-β) overactivity in driving these cardiovascular abnormalities, we studied a novel transgenic mouse model characterized by ligand-dependent activation of TGF-β signaling in fibroblasts.