Terrequinone A biosynthesis through L-tryptophan oxidation, dimerization and bisprenylation

The antitumor fungal metabolite terrequinone A, identified in extracts of Aspergillus sp., is biosynthesized by the five-gene cluster tdiA-tdiE. In this work, we have overproduced all five proteins (TdiA-TdiE) in the bacterial host Escherichia coli, fully reconstituting the biosynthesis of terrequinone A. This pathway involves aminotransferase activity, head-to-tail dimerization and bisprenylation of the scaffold to yield the benzoquinone natural product. We have established that TdiD is a pyridoxal-5'-phosphate-dependent L-tryptophan aminotransferase that generates indolepyruvate for an unusual nonoxidative coupling by the tridomain nonribosomal peptide synthetase TdiA. TdiC, an NADH-dependent quinone reductase, generates the nucleophilic hydroquinone for two distinct rounds of prenylation by the single prenyltransferase TdiB. TdiE is required to shunt the benzoquinone away from an off-pathway monoprenylated species by an as yet unknown mechanism. Overall, we have biochemically characterized the complete biosynthetic pathway to terrequinone A, highlighting the nonoxidative dimerization pathway and the unique asymmetric prenylation involved in its maturation.     

Determination of the processes driving the acquisition of immunity to malaria using a mathematical transmission model

Acquisition of partially protective immunity is a dominant feature of the epidemiology of malaria among exposed individuals. The processes that determine the acquisition of immunity to clinical disease and to asymptomatic carriage of malaria parasites are poorly understood, in part because of a lack of validated immunological markers of protection. Using mathematical models, we seek to better understand the processes that determine observed epidemiological patterns. We have developed an age-structured mathematical model of malaria transmission in which acquired immunity can act in three ways (“immunity functions”): reducing the probability of clinical disease, speeding the clearance of parasites, and increasing tolerance to subpatent infections. Each immunity function was allowed to vary in efficacy depending on both age and malaria transmission intensity. The results were compared to age patterns of parasite prevalence and clinical disease in endemic settings in northeastern Tanzania and The Gambia. Two types of immune function were required to reproduce the epidemiological age-prevalence curves seen in the empirical data; a form of clinical immunity that reduces susceptibility to clinical disease and develops with age and exposure (with half-life of the order of five years or more) and a form of anti-parasite immunity which results in more rapid clearance of parasitaemia, is acquired later in life and is longer lasting (half-life of >20 y). The development of anti-parasite immunity better reproduced observed epidemiological patterns if it was dominated by age-dependent physiological processes rather than by the magnitude of exposure (provided some exposure occurs). Tolerance to subpatent infections was not required to explain the empirical data. The model comprising immunity to clinical disease which develops early in life and is exposure-dependent, and anti-parasite immunity which develops later in life and is not dependent on the magnitude of exposure, appears to best reproduce the pattern of parasite prevalence and clinical disease by age in different malaria transmission settings. Understanding the effector mechanisms underlying these two immune functions will assist in the design of transmission-reducing interventions against malaria.     

A cis-element in the 5' untranslated region of the preproinsulin mRNA (ppIGE) is required for glucose regulation of proinsulin translation

Insulin production in pancreatic beta cells is predominantly regulated through glucose control of proinsulin translation. Previously, this was shown to require sequences within the untranslated regions (UTRs) of the preproinsulin (ppI) mRNA. Here, those sequences were found to be sufficient for specific glucose-regulated proinsulin translation. Furthermore, an element 40-48 bp from the 5' end of the ppI mRNA specifically bound a factor present in islets of Langerhans. Glucose-responsive factor binding to this cis-element exhibited temporal and glucose-concentration-dependent patterns that paralleled proinsulin biosynthesis. Mutating this cis-element abolished the ability of ppI mRNA UTRs to confer glucose regulation upon translation. Like the rat 5'UTR, the human ppI 5'UTR conferred glucose regulation of translation. However alternative splicing of the human 5'UTR that disrupts the cis-element abolished glucose-regulated translation. These data indicate that glucose regulation of cis-element/trans-acting factor interaction is a key component of the mechanism by which glucose regulates insulin production.     

Systemic hypoxia causes cutaneous vasodilation in healthy humans.

Hypoxia and hypercapnia represent special challenges to homeostasis because of their effects on sympathetic outflow and vascular smooth muscle. In the cutaneous vasculature, even small changes in perfusion can shift considerable blood volume to the periphery and thereby impact both blood pressure regulation and thermoregulation. However, little is known about the influence of hypoxia and hypercapnia on this circulation. In the present study, 35 healthy subjects were instrumented with two microdialysis fibers in the ventral forearm. Each site was continuously perfused with saline (control) or bretylium tosylate (10 mM) to prevent sympathetically mediated vasoconstriction. Skin blood flow was assessed at each site (laser-Doppler flowmetry), and cutaneous vascular conductance (CVC) was calculated as red blood cell flux/mean arterial pressure and normalized to baseline. In 13 subjects, isocapnic hypoxia (85 and 80% O(2) saturation) increased CVC to 120 +/- 10 and 126 +/- 7% baseline in the control site (both P < 0.05) and 113 +/- 3 (P = 0.087) and 121 +/- 4% baseline (P < 0.05) in the bretylium site. Adrenergic blockade did not affect the magnitude of this response (P > 0.05). In nine subjects, hyperpnea (matching hypoxic increases in tidal volume) caused no change in CVC in either site (both P > 0.05). In 13 subjects, hypercapnia (+5 and +9 Torr) increased CVC to 111 +/- 4 and 111 +/- 4% baseline, respectively, in the control site (both P < 0.05), whereas the bretylium site remained unchanged (both P > 0.05). Thus both hypoxia and hypercapnia cause modest vasodilation in nonacral skin. Adrenergic vasoconstriction of neural origin does not restrain hypoxic vasodilation, but may be important in hypercapnic vasodilation.     

Engineering the stem cell microenvironment

Multipotent stem cells in the body facilitate tissue regeneration, growth, and wound healing throughout life. The microenvironment in which they reside provides signals that direct these progenitors to proliferate, differentiate, or remain dormant; these factors include soluble molecules, the extracellular matrix, neighboring cells, and physical stimuli. Recent advances in the culture of embryonic stem cells and adult progenitors necessitate an increased understanding of these phenomena. Here, we summarize the interactions between stem cells and their local environment, drawing on in vivo observations and tissue culture studies. In addition, we describe novel methods of characterizing the effects of various environmental factors and review new techniques that enable scientists and engineers to more effectively direct stem cell fate.     

Fatigue and functional performance of human biceps muscle following concentric or eccentric contractions.

A long-lasting fatigue was measured in human biceps muscle, following 40 maximal isokinetic concentric or eccentric contractions of the forearm, as the response to single-shock stimuli every minute for 4 h. This protocol allowed new observations on the early time course of long-lasting fatigue. Concentric contractions induced a novel progressive decline to 30.2% (SE 7.8, n = 7) of control at 23 min with complete recovery by 120 min. Eccentric contractions lead initially to a smaller force reduction of similar time course followed by a slower decline to 40.0% (SE 5.1, n = 7) control at 120 min with recovery less than half complete at 4 h. A 50-Hz test stimuli overcame both fatigues, identifying low-frequency fatigue. EMG recordings from the biceps muscle showed moderate (<20%) changes during the fatigue. A visual-tracking task showed no decrement in performance at the time of maximal fatigue of the single-shock response. Because the eccentric contractions have a similar activation, a larger force, but much smaller metabolic usage than concentric contractions, it is concluded that the initial decline is related to the effects of metabolites, whereas the slower phase after eccentric contractions is associated with higher mechanical stress.     

Early queen infection shapes developmental dynamics and induces long-term disease protection in incipient ant colonies

Infections early in life can have enduring effects on an organism's development and immunity. In this study, we show that this equally applies to developing ‘superorganisms’––incipient social insect colonies. When we exposed newly mated Lasius niger ant queens to a low pathogen dose, their colonies grew more slowly than controls before winter, but reached similar sizes afterwards. Independent of exposure, queen hibernation survival improved when the ratio of pupae to workers was small. Queens that reared fewer pupae before worker emergence exhibited lower pathogen levels, indicating that high brood rearing efforts interfere with the ability of the queen's immune system to suppress pathogen proliferation. Early-life queen pathogen exposure also improved the immunocompetence of her worker offspring, as demonstrated by challenging the workers to the same pathogen a year later. Transgenerational transfer of the queen's pathogen experience to her workforce can hence durably reduce the disease susceptibility of the whole superorganism.     

Population genetics reveals bidirectional fish movement across the Continental Divide via an interbasin water transfer

Interbasin water transfers are becoming an increasingly common tool to satisfy municipal and agricultural water demand, but their impacts on movement and gene flow of aquatic organisms are poorly understood. The Grand Ditch is an interbasin water transfer that diverts water from tributaries of the upper Colorado River on the west side of the Continental Divide to the upper Cache la Poudre River on the east side of the Continental Divide. We used single nucleotide polymorphisms to characterize population genetic structure in cutthroat trout (Oncorhynchus clarkii) and determine if fish utilize the Grand Ditch as a movement corridor. Samples were collected from two sites on the west side and three sites on the east side of the Continental Divide. We identified two or three genetic clusters, and relative migration rates and spatial distributions of admixed individuals indicated that the Grand Ditch facilitated bidirectional fish movement across the Continental Divide, a major biogeographic barrier. Previous studies have demonstrated ecological impacts of interbasin water transfers, but our study is one of the first to use genetics to understand how interbasin water transfers affect connectivity between previously isolated watersheds. We also discuss implications on native trout management and balancing water demand and biodiversity conservation.