Lessons from genetics: interpreting complex phenotypes in RNAi screens

Mammalian cell biology is witnessing a new era in which cellular processes are explained through dynamic networks of interacting cellular components. In this fast-pacing field, where image-based RNAi screening is taking a central role, there is a strong need to improve ways to capture such interactions in space and time. Cell biologists traditionally depict these events by confining themselves to the level of a single cell, or to many population-averaged cells. Similarly, classical geneticists observe and interpret phenotypes in a single organism to delineate signaling processes, but have also described genetic phenomena in populations of organisms. The analogy in the two approaches inspired us to draw parallels with, and take lessons from concepts in classical genetics.     

Gene expression of adrenomedullin in failing myocardium: comparison to atrial natriuretic peptide.

The expression of adrenomedullin (AM) and atrial natriuretic factor (ANF) were investigated in the myocardium of a rat model of chronic ischemic heart failure (CHF) compared with sham-operated controls. In addition, human myocardium of patients with end-stage heart failure due to idiopathic dilated cardiomyopathy compared with myocardium of normal subjects (NF) was studied. In CHF, similar AM levels but increased ANF expression were observed in left ventricular myocardium, as assessed by semiquantitative PCR. Functional experiments with freshly excised papillary muscles showed no influence of AM on myocardial contractility. In NF human myocardium, the expression of AM mRNA was threefold higher in atrial compared with ventricular tissue. In analogy, ANF mRNA was increased by approximately 15-fold in atrial tissue. In dilated cardiomyopathy, the expression of AM was significantly increased in right and left ventricles compared with NF. In parallel, ventricular ANF expression was enhanced.     

Oxygen-dependent coproporphyrinogen III oxidase (HemF) from Escherichia coli is stimulated by manganese

During heme biosynthesis in Escherichia coli two structurally unrelated enzymes, one oxygen-dependent (HemF) and one oxygen-independent (HemN), are able to catalyze the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX. Oxygen-dependent coproporphyrinogen III oxidase was produced by overexpression of the E. coli hemF in E. coli and purified to apparent homogeneity. The dimeric enzyme showed a Km value of 2.6 microm for coproporphyrinogen III with a kcat value of 0.17 min-1 at its optimal pH of 6. HemF does not utilize protoporphyrinogen IX or coproporphyrin III as substrates and is inhibited by protoporphyrin IX. Molecular oxygen is essential for the enzymatic reaction. Single turnover experiments with oxygen-loaded HemF under anaerobic conditions demonstrated electron acceptor function for oxygen during the oxidative decarboxylation reaction with the concomitant formation of H2O2. Metal chelator treatment inactivated E. coli HemF. Only the addition of manganese fully restored coproporphyrinogen III oxidase activity. Evidence for the involvement of four highly conserved histidine residues (His-96, His-106, His-145, and His-175) in manganese coordination was obtained. One catalytically important tryptophan residue was localized in position 274. None of the tested highly conserved cysteine (Cys-167), tyrosine (Tyr-135, Tyr-160, Tyr-170, Tyr-213, Tyr-240, and Tyr-276), and tryptophan residues (Trp-36, Trp-123, Trp-166, and Trp-298) were found important for HemF activity. Moreover, mutation of a potential nucleotide binding motif (GGGXXTP) did not affect HemF activity. Two alternative routes for HemF-mediated catalysis, one metal-dependent, the other metal-independent, are proposed.     

Spatial variability in a plant–pollinator community across a continuous habitat: high heterogeneity in the face of apparent uniformity

Large‐scale spatial variability in plant–pollinator communities (e.g. along geographic gradients, across different landscapes) is relatively well understood. However, we know much less about how these communities vary at small scales within a uniform landscape. Plants are sessile and highly sensitive to microhabitat conditions, whereas pollinators are highly mobile and, for the most part, display generalist feeding habits. Therefore, we expect plants to show greater spatial variability than pollinators. We analysed the spatial heterogeneity of a community of flowering plants and their pollinators in 40 plots across a 40‐km² area within an uninterrupted Mediterranean scrubland. We recorded 3577 pollinator visits to 49 plant species. The pollinator community (170 species) was strongly dominated by honey bees (71.8% of the visits recorded). Flower and pollinator communities showed similar beta‐diversity, indicating that spatial variability was similar in the two groups. We used path analysis to establish the direct and indirect effects of flower community distribution and honey bee visitation rate (a measure of the use of floral resources by this species) on the spatial distribution of the pollinator community. Wild pollinator abundance was positively related to flower abundance. Wild pollinator visitation rate was negatively related to flower abundance, suggesting that floral resources were not limiting. Pollinator and flower richness were positively related. Pollinator species composition was weakly related to flower species composition, reflecting the generalist nature of flower–pollinator interactions and the opportunistic nature of pollinator flower choices. Honey bee visitation rate did not affect the distribution of the wild pollinator community. Overall, we show that, in spite of the apparent physiognomic uniformity, both flowers and pollinators display high levels of heterogeneity, resulting in a mosaic of idiosyncratic local communities. Our results provide a measure of the background of intrinsic heterogeneity within a uniform habitat, with potential consequences on low‐scale ecosystem function and microevolutionary patterns.     

Cytosolic proline is required for basal freezing tolerance in Arabidopsis

The amino acid proline accumulates in many plant species under abiotic stress conditions, and various protective functions have been proposed. During cold stress, however, proline content in Arabidopsis thaliana does not correlate with freezing tolerance. Freezing sensitivity of a starchless plastidic phosphoglucomutase mutant (pgm) indicated that localization of proline in the cytosol might stabilize the plasma membrane during freeze–thaw events. Here, we show that re-allocation of proline from cytosol to vacuole was similar in the pyrroline-5-carboxylate synthase 2–1 (p5cs2–1) mutant and the pgm mutant and caused similar reduction of basal freezing tolerance. In contrast, the starch excess 1–1 mutant (sex1-1) had even lower freezing tolerance than pgm but did not affect sub-cellular localization of proline. Freezing sensitivity of sex1-1 mutants affected primarily the photosynthetic electron transport and was enhanced in a sex1-1::p5cs2–1 double mutant. These findings indicate that several independent factors determine basal freezing tolerance. In a pgm::p5cs2–1 double mutant, freezing sensitivity and proline allocation to the vacuole were the same as in the parental lines, indicating that the lack of cytosolic proline was the common cause of reduced basal freezing tolerance in both mutants. We conclude that cytosolic proline is an important factor in freezing tolerance of non-acclimated plants.     

Positron Emission Tomography Imaging Reveals Auditory and Frontal Cortical Regions Involved with Speech Perception and Loudness Adaptation

Considerable progress has been made in the treatment of hearing loss with auditory implants. However, there are still many implanted patients that experience hearing deficiencies, such as limited speech understanding or vanishing perception with continuous stimulation (i.e., abnormal loudness adaptation). The present study aims to identify specific patterns of cerebral cortex activity involved with such deficiencies. We performed O-15-water positron emission tomography (PET) in patients implanted with electrodes within the cochlea, brainstem, or midbrain to investigate the pattern of cortical activation in response to speech or continuous multi-tone stimuli directly inputted into the implant processor that then delivered electrical patterns through those electrodes. Statistical parametric mapping was performed on a single subject basis. Better speech understanding was correlated with a larger extent of bilateral auditory cortex activation. In contrast to speech, the continuous multi-tone stimulus elicited mainly unilateral auditory cortical activity in which greater loudness adaptation corresponded to weaker activation and even deactivation. Interestingly, greater loudness adaptation was correlated with stronger activity within the ventral prefrontal cortex, which could be up-regulated to suppress the irrelevant or aberrant signals into the auditory cortex. The ability to detect these specific cortical patterns and differences across patients and stimuli demonstrates the potential for using PET to diagnose auditory function or dysfunction in implant patients, which in turn could guide the development of appropriate stimulation strategies for improving hearing rehabilitation. Beyond hearing restoration, our study also reveals a potential role of the frontal cortex in suppressing irrelevant or aberrant activity within the auditory cortex, and thus may be relevant for understanding and treating tinnitus.     

Pyrimidine benzamide-based thrombopoietin receptor agonists

A series of pyrimidine benzamide-based thrombopoietin receptor agonists is described. The lead molecule contains a 2-amino-5-unsubstituted thiazole, a group that has been associated with idiosyncratic toxicity. The potential for metabolic oxidation at C-5 of the thiazole, the likely source of toxic metabolites, was removed by substitution at C-5 or by replacing the thiazole with a thiadiazole. Potency in the series was improved by modifying the substituents on the pyrimidine and/or on the thiazole or thiadiazole pendant aryl ring. In vivo examination revealed that compounds from the series are not highly bioavailable. This is attributed to low solubility and poor permeability.