Appetite of an epiphyte: Quantitative monitoring of bacterial sugar consumption in the phyllosphere

We report here the construction, characterization, and application of a bacterial bioreporter for fructose and sucrose that was designed to monitor the availability of these sugars to microbial colonizers of the phyllosphere. Plasmid pPfruB-gfp[AAV] carries the Escherichia coli fruB promoter upstream from the gfp[AAV] allele that codes for an unstable variant of green fluorescent protein (GFP). In Erwinia herbicola, this plasmid brings about the accumulation of GFP fluorescence in response to both fructose and sucrose. Cells of E. herbicola (pPfruB-gfp[AAV]) were sprayed onto bean plants, recovered from leaves at various time intervals after inoculation, and analyzed individually for GFP content by quantitative analysis of digital microscope images. We observed a positive correlation between single-cell GFP accumulation and ribosomal content as determined by fluorescence in situ hybridization, indicating that foliar growth of E. herbicola occurred at the expense of fructose and/or sucrose. One hour after inoculation, nearly all bioreporter cells appeared to be actively engaged in fructose consumption. This fraction dropped to approximately 11% after 7 h and to ≈1% a day after inoculation. This pattern suggests a highly heterogeneous availability of fructose to individual E. herbicola cells as they colonize the phyllosphere. We estimated that individual cells were exposed to local initial fructose abundances ranging from less than 0.15 pg fructose to more than 4.6 pg.

Microvascular and tissue oxygen gradients in the rat mesentery

One of the most important functions of the blood circulation is O2 delivery to the tissue. This process occurs primarily in microvessels that also regulate blood flow and are the site of many metabolic processes that require O2. We measured the intraluminal and perivascular pO2 in rat mesenteric arterioles in vivo by using noninvasive phosphorescence quenching microscopy. From these measurements, we calculated the rate at which O2 diffuses out of microvessels from the blood. The rate of O2 efflux and the O2 gradients found in the immediate vicinity of arterioles indicate the presence of a large O2 sink at the interface between blood and tissue, a region that includes smooth muscle and endothelium. Mass balance analyses show that the loss of O2 from the arterioles in this vascular bed primarily is caused by O2 consumption in the microvascular wall. The high metabolic rate of the vessel wall relative to parenchymal tissue in the rat mesentery suggests that in addition to serving as a conduit for the delivery of O2 the microvasculature has other functions that require a significant amount of O2.

Autoradiographic characterization of [3H]-5-HT-moduline binding sites in rodent brain and their relationship to 5-HT1B receptors

5-HT-moduline is an endogenous tetrapeptide [Leu-Ser-Ala-Leu (LSAL)] that was first isolated from bovine brain tissue. To understand the physiological role of this tetrapeptide, we studied the localization of 5-HT-moduline binding sites in rat and mouse brains. Quantitative data obtained with a gaseous detector of β-particles (β-imager) indicated that [3H]-5-HT-moduline bound specifically to rat brain sections with high affinity (Kd = 0.77 nM and Bmax = 0.26 dpm/mm2). Using film autoradiography in parallel, we found that 5-HT-moduline binding sites were expressed in a variety of rat and mouse brain structures. In 5-HT1B receptor knock-out mice, the specific binding of [3H]-5-HT-moduline was not different from background labeling, indicating that 5-HT-moduline targets are exclusively located on the 5-HT1B receptors. Although the distribution of 5-HT-moduline binding sites was similar to that of 5-HT1B receptors, they did not overlap totally. Differences in distribution patterns were found in regions containing either high levels of 5-HT1B receptors such as globus pallidus and subiculum that were poorly labeled or in other regions such as dentate gyrus of hippocampus and cortex where the relative density of 5-HT-moduline binding sites was higher than that of 5-HT1B receptors. In conclusion, our data, based on autoradiographic localization, indicate that 5-HT-moduline targets are located on 5-HT1B receptors present both on 5-HT afferents and postsynaptic neurons. By interacting specifically with 5-HT1B receptors, this tetrapeptide may play a pivotal role in pathological states such as stress that involves the dysfunction of 5-HT neurotransmission.

Regulation of sterol regulatory element binding proteins in livers of fasted and refed mice

Hepatic lipid synthesis is known to be regulated by food consumption. In rodents fasting decreases the synthesis of cholesterol as well as fatty acids. Refeeding a high carbohydrate/low fat diet enhances fatty acid synthesis by 5- to 20-fold above the fed state, whereas cholesterol synthesis returns only to the prefasted level. Sterol regulatory element binding proteins (SREBPs) are transcription factors that regulate genes involved in cholesterol and fatty acid synthesis. Here, we show that fasting markedly reduces the amounts of SREBP-1 and -2 in mouse liver nuclei, with corresponding decreases in the mRNAs for SREBP-activated target genes. Refeeding a high carbohydrate/low fat diet resulted in a 4- to 5-fold increase of nuclear SREBP-1 above nonfasted levels, whereas nuclear SREBP-2 protein returned only to the nonfasted level. The hepatic mRNAs for fatty acid biosynthetic enzymes increased 5- to 10-fold above nonfasted levels, a pattern that paralleled the changes in nuclear SREBP-1. The hepatic mRNAs for enzymes involved in cholesterol synthesis returned to the nonfasted level, closely following the pattern of nuclear SREBP-2 regulation. Transgenic mice that overproduce nuclear SREBP-1c failed to show the normal decrease in hepatic mRNA levels for cholesterol and fatty acid synthetic enzymes upon fasting. We conclude that SREBPs are regulated by food consumption in the mouse liver and that the decline in nuclear SREBP-1c upon fasting may explain in part the decrease in mRNAs encoding enzymes of the fatty acid biosynthetic pathway.

Fusicoccin Counteracts the n-Ethylmaleimide and Silver-Induced Stimulation of Oxygen Uptake in Egeria densa Leaves1

It was previously shown that a number of sulfhydryl [SH] group reagents (N-ethylmaleimide [NEM], iodoacetate, Ag+, HgCl2, etc.) can induce a marked, transitory stimulation of O2 uptake (QO2) in Egeria densa leaves, insensitive to CN− and salicylhydroxamic acid and inhibited by diphenylene iodonium and quinacrine. The phytotoxin fusicoccin (FC) also induces a marked increase in O2 consumption in E. densa leaves, apparently independent of the recognized stimulating action on the H+-ATPase. In this investigation we compared the FC-induced increase in O2 consumption with those induced by NEM and Ag+, and we tested for a possible interaction between FC and the two SH blockers in the activation of QO2. The results show (a) the different nature of the FC- and NEM- or Ag+-induced increases of QO2; (b) that FC counteracts the NEM- (and Ag+)-induced respiratory burst; and (c) that FC strongly reduces the damaging effects on plasma membrane permeability observed in E. densa leaves treated with the two SH reagents. Two alternative models of interpretation of the action of FC, in activating a CN−-sensitive respiratory pathway and in suppressing the SH blocker-induced respiratory burst, are proposed.