Human surfactant protein B promoter in transgenic mice: temporal, spatial, and stimulus-responsive regulation

Surfactant protein B (SP-B) is a developmentally and hormonally regulated lung protein that is required for normal surfactant function. We generated transgenic mice carrying the human SP-B promoter (-1,039/+431 bp) linked to chloramphenicol acetyltransferase (CAT). CAT activity was high in lung and immunoreactive protein localized to alveolar type II and bronchiolar epithelial cells. In addition, thyroid, trachea, and intestine demonstrated CAT activity, and each of these tissues also expressed low levels of SP-B mRNA. Developmental expression of CAT activity and SP-B mRNA in fetal lung were similar and both increased during explant culture. SP-B mRNA but not CAT activity decreased during culture of adult lung, and both were reduced by transforming growth factor (TGF)-beta(1). Treatment of adult mice with intratracheal bleomycin caused similar time-dependent decreases in lung SP-B mRNA and CAT activity. These findings indicate that the human SP-B promoter fragment directs tissue- and lung cell-specific transgene expression and contains cis-acting elements involved in regulated expression during development, fetal lung explant culture, and responsiveness to TGF-beta and bleomycin-induced lung injury.     

Characterization of nitrosoalkane binding and activation of soluble guanylate cyclase

Soluble guanylate cyclase (sGC) is the primary receptor for the signaling agent nitric oxide (NO). Electronic absorption and resonance Raman spectroscopy were used to show that nitrosoalkanes bind to the heme of sGC to form six-coordinate, low-spin complexes. In the sGC-nitrosopentane complex, a band assigned to an Fe-N stretching vibration is observed at 543 cm(-)(1) which is similar to values reported for other six-coordinate NO-bound hemoproteins. Nitrosoalkanes activate sGC 2-6-fold and synergize with YC-1, a synthetic benzylindazole derivative, to activate the enzyme 11-47-fold. In addition, the observed off-rates of nitrosoalkanes from sGC were found to be dependent on the alkyl chain length. A linear correlation was found between the observed off-rates and the alkyl chain length which suggests that the sGC heme has a large hydrophobic distal ligand-binding pocket. Together, these data show that nitrosoalkanes are a novel class of heme-based sGC activators and suggest that heme ligation is a general requirement for YC-1 synergism.     

Profiling small RNA reveals multimodal substructural signals in a Boltzmann ensemble

As the biomedical impact of small RNAs grows, so does the need to understand competing structural alternatives for regions of functional interest. Suboptimal structure analysis provides significantly more RNA base pairing information than a single minimum free energy prediction. Yet computational enhancements like Boltzmann sampling have not been fully adopted by experimentalists since identifying meaningful patterns in this data can be challenging. Profiling is a novel approach to mining RNA suboptimal structure data which makes the power of ensemble-based analysis accessible in a stable and reliable way. Balancing abstraction and specificity, profiling identifies significant combinations of base pairs which dominate low-energy RNA secondary structures. By design, critical similarities and differences are highlighted, yielding crucial information for molecular biologists. The code is freely available via http://gtfold.sourceforge.net/profiling.html.     

Inhibition of human breast cancer Matrigel invasion by Streptolysin O activation of the EGF receptor ErbB1

Streptolysin O (SLO) is a protein cytotoxin derived from Group A beta-hemolytic streptococci that associates with membranes and permeabilizes cells. Oxidation inactivates SLO, eliminating the characteristic hemolytic and cytotoxic activities. However, oxidized SLO produces beneficial therapeutic effects in vivo on scleroderma, scar formation and wound healing. Here we report that oxidized SLO also significantly inhibited invasion by human metastatic breast cancer MDA-MB-231 cells through Matrigel in an in vitro model of metastatic disease. This dose-dependent response corresponded to selective SLO activation of epidermal growth factor receptor (EGFR) ErbB1. SLO and EGF were equally selective in activation of EGFR, but EGF elicited larger relative increases in phosphorylation at various sites, especially pronounced for Tyr845. Addition of SLO did not affect either ERK1/2 or Akt kinases and altered the expression of only 10 of 84 metastasis-related genes in MDA-MB-231 cells. Neither SLO nor EGF promoted growth of several human breast cancer cell lines. Knockdown of EGFR by siRNA ablated the inhibitory effect of SLO on cancer cell invasion, showing SLO selectively activated ErbB1 kinase to reduce invasion without increasing cell growth. The results suggest SLO might have promise as a new therapy to inhibit metastasis.     

The distribution of microplankton in the McMurdo Dry Valley Lakes,Antarctica: response to ecosystem legacy or present-day climatic controls?

Plankton abundance and biomass were investigated in five lakes of the McMurdo Dry Valleys, Antarctica: Lakes Bonney, Fryxell, Joyce, Hoare and Miers. Despite plankton communities being dominated by organisms <100 m in length, there were striking differences between the lakes, including large variations in plankton vertical distribution and differences in total plankton biomass. Bacterial biomass was highest in the anoxic monimolimnia of the meromictic lakes, reaching 191 g C l^–1 in Lake Fryxell. Photosynthetic nanoflagellates dominated phytoplankton in the five lakes studied. Highest chlorophyll a concentrations were recorded at the chemocline of Lake Fryxell (21 g chl a l^–1). Heterotrophic nanoflagellate concentrations were low, ranging from 2 cells ml^–1 in Hoare to 237 cells ml^–1 in Bonney. By Antarctic standards, ciliates were relatively successful in terms of biomass and diversity in Lakes Fryxell and Hoare. In contrast, Lake Miers possessed extremely low ciliate abundance (<0.04 cells ml^–1). On both sampling occasions, copepod nauplii were observed in Lake Joyce. This is the first recording of crustacean zooplankton within the McMurdo Dry Valley Lakes. Because the foodwebs of these lakes are structured by bottom-up forces, differences in plankton distributions could be related to the physicochemical characteristics of each lake. The effect of lake evolution (legacy) and present-day climate change on planktonic dynamics is discussed.     

Scaling laws of ambush predator ‘waiting’ behaviour are tuned to a common ecology

The decisions animals make about how long to wait between activities can determine the success of diverse behaviours such as foraging, group formation or risk avoidance. Remarkably, for diverse animal species, including humans, spontaneous patterns of waiting times show random ‘burstiness’ that appears scale-invariant across a broad set of scales. However, a general theory linking this phenomenon across the animal kingdom currently lacks an ecological basis. Here, we demonstrate from tracking the activities of 15 sympatric predator species (cephalopods, sharks, skates and teleosts) under natural and controlled conditions that bursty waiting times are an intrinsic spontaneous behaviour well approximated by heavy-tailed (power-law) models over data ranges up to four orders of magnitude. Scaling exponents quantifying ratios of frequent short to rare very long waits are species-specific, being determined by traits such as foraging mode (active versus ambush predation), body size and prey preference. A stochastic–deterministic decision model reproduced the empirical waiting time scaling and species-specific exponents, indicating that apparently complex scaling can emerge from simple decisions. Results indicate temporal power-law scaling is a behavioural ‘rule of thumb’ that is tuned to species’ ecological traits, implying a common pattern may have naturally evolved that optimizes move–wait decisions in less predictable natural environments.     

The genetic basis for indole-diterpene chemical diversity in filamentous fungi

Indole-diterpenes are a structurally diverse group of secondary metabolites with a common cyclic diterpene backbone derived from geranylgeranyl diphosphate and an indole group derived from indole-3-glycerol phosphate. Different types and patterns of ring substitutions and ring stereochemistry generate this structural diversity. This group of compounds is best known for their neurotoxic effects in mammals, causing syndromes such as ‘ryegrass staggers’ in sheep and cattle. Because many of the fungi that synthesise these compounds form symbiotic relationships with plants, insects, and other fungi, the synthesis of these compounds may confer an ecological advantage to these associations. Considerable recent progress has been made on understanding indole-diterpene biosynthesis in filamentous fungi, principally through the cloning and characterisation of the genes and gene products for paxilline biosynthesis in Penicillium paxilli. Important insights into how the indole-diterpene backbone is synthesised and decorated have been obtained using P. paxilli mutants in this pathway. This review provides an overview of these recent developments.     

Transduction of the mammary epithelium with adenovirus vectors in vivo

Because the mammary parenchyma is accessible from the exterior of an animal through the mammary duct, adenovirus transduction holds promise for the short-term delivery of genes to the mammary epithelium for both research and therapeutic purposes. To optimize the procedure and evaluate its efficacy, an adenovirus vector (human adenovirus type 5) encoding a green fluorescent protein (GFP) reporter and deleted of E1 and E3 was injected intraductally into the mouse mammary gland. We evaluated induction of inflammation (by intraductal injection of [(14)C]sucrose and histological examination), efficiency of transduction, and maintenance of normal function in transduced cells. We found that transduction of the total epithelium in the proximal portion of the third mammary gland varied from 7% to 25% at a dose of 2 x 10(6) PFU of adenovirus injected into day 17 pregnant mice. Transduction was maintained for at least 7 days with minimal inflammatory response; however, significant mastitis was observed 12 days after transduction. Adenovirus transduction could also be used in the virgin animal with little mastitis 3 days after transduction. Transduced mammary epithelial cells maintained normal morphology and function. Our results demonstrate that intraductal injection of adenovirus vectors provides a versatile and noninvasive method of investigating genes of interest in mouse mammary epithelial cells.