How Microbes Can Achieve Balanced Growth in a Fluctuating Environment

A microbial colony needs several essential nutrients in order to grow. Moreover, the colony requires these nutrients in fixed combinations, which are dictated by the chemical composition of its biomass. Unfortunately, ambient availabilities of the various nutrients vary all the time. This poses the question of how microbes can achieve balanced growth.The present solution to this problem is novel in that the allocation of molecular building blocks among assimilatory machineries within the cell is regarded as dynamic. This paper shows that allocation can be adapted so as to achieve balanced growth, nearly regardless of environmental conditions. Moreover, it is shown that a feedback mechanism, which monitors internal stores, is able to achieve this allocation.     

Prolonged feeding of mice with conjugated linoleic acid increases hepatic fatty acid synthesis relative to oxidation

Feeding mice conjugated linoleic acid (9 cis,11 trans/9 trans,11 cis-and 10 trans,12 cis-CLA in equal amounts) resulted in triacylglycerol accumulation in the liver. The objective of this study was to examine whether this steatosis is associated with changes in hepatic fatty acid synthesis and oxidation. Therefore, we measured the activities of key enzymes of fatty acid synthesis, i.e., acetyl-CoA carboxylase and fatty acid synthase and of fatty acid oxidation, i.e., 3-hydroxy-acyl-CoA dehydrogenase and citrate synthase in livers of mice fed a diet with 0.5% (w/w) CLA. CLA (a 1:1 mixture of the 10 trans, 12 cis and 9 cis, 11 trans isomers of octadecadenoic acid) was administered for 3 and 12 weeks with high-oleic sunflower oil fed as control. The proportion of body fat was significantly lower on the CLA than on the control diet and this effect was already significant after 3 weeks. The specific activites of 3-hydroxy-acyl-CoA dehydrogenase and citrate synthase were unaffected by CLA both after 3 and 12 weeks. The specific activity of fatty acid synthase was nonsignificantly raised (by 12%) after 3 weeks on the CLA diet but had increased significantly (by 34%) after 12 weeks of feeding. The specific activity of acetyl-CoA carboxylase had also increased both after 3 weeks (by 53%) and 12 weeks (by 23%) on the CLA diet, but this effect did not reach statistical significance. Due to CLA-induced hepatomegaly, the overall capacity for both fatty acid oxidation and synthesis-as evidenced by the total hepatic activities of 3-hydroxy-acyl-CoA dehydrogenase, citrate synthase, acetyl-CoA carboxylase, and fatty acid synthase-was significantly greater in the CLA-fed group after 12 weeks, although the overall capacity for fatty acid synthesis had increased more than that for fatty acid oxidation. Thus, this study indicates that prolonged, but not short-term, feeding mice with CLA increased hepatic fatty acid synthesis relative to oxidation, despite the decrease in body fat and the increase in liver weight seen earlier. It is concluded that the observed CLA-induced changes in hepatic fatty acid synthesis and oxidation are the result, rather than the cause, of the lowering of body fat.     

Involvement of putative SNF2 chromatin remodeling protein DRD1 in RNA-directed DNA methylation

In plants, the mechanism by which RNA can induce de novo cytosine methylation of homologous DNA is poorly understood. Cytosines in all sequence contexts become modified in response to RNA signals. Recent work has implicated the de novo DNA methyltransferases (DMTases), DRM1 and DRM2, in establishing RNA-directed methylation of the constitutive nopaline synthase promoter, as well as the DMTase MET1 and the putative histone deacetylase HDA6 in maintaining or enhancing CpG methylation induced by RNA. Despite the identification of enzymes that catalyze epigenetic modifications in response to RNA signals, it is unclear how RNA targets DNA for methylation. A screen for mutants defective in RNA-directed DNA methylation identified a novel putative chromatin-remodeling protein, DRD1. This protein belongs to a previously undefined, plant-specific subfamily of SWI2/SNF2-like proteins most similar to the RAD54/ATRX subfamily. In drd1 mutants, RNA-induced non-CpG methylation is almost eliminated at a target promoter, resulting in reactivation, whereas methylation of centromeric and rDNA repeats is unaffected. Thus, unlike the SNF2-like proteins DDM1/Lsh1 and ATRX, which regulate methylation of repetitive sequences, DRD1 is not a global regulator of cytosine methylation. DRD1 is the first SNF2-like protein implicated in an RNA-guided, epigenetic modification of the genome.     

Formation of transient oxygen complexes of cytochrome p450 BM3 and nitric oxide synthase under high pressure

The kinetics of formation and transformation of oxygen complexes of two heme-thiolate proteins (the F393H mutant of cytochrome P450 BM3 and the oxygenase domain of endothelial nitric oxide synthase, eNOS) were studied under high pressure. For BM3, oxygen-binding characteristics (rate and activation volume) matched those measured for CO-binding. In contrast, pressure revealed a different CO- and oxygen-binding mechanism for eNOS, suggesting that it is hazardous to take CO-binding as a model for oxygen-binding. With eNOS, a ferric NO complex is formed as an intermediate in the second reaction cycle. Here we report the pressure stability of this compound. Furthermore, in the presence of 4-amino-tetrahydrobiopterin (ABH(4)), an analog to the natural second electron donor tetrahydrobiopterin (BH(4)), biphasic pressure profiles of the oxygen-binding rates were observed, both in the first and the second reaction cycles, indicative of the formation of an additional reaction intermediate. This was confirmed by experiments where ABH(4) was replaced by ABH(2), a cofactor which cannot deliver an electron. Altogether, high pressure appears to be a useful tool to characterize elementary steps in the reaction cycle of heme-thiolate proteins.     

Proteomic characterization of wheat amyloplasts using identification of proteins by tandem mass spectrometry

We describe the initial characterization of the wheat amyloplast proteome, consisting of the identification and classification of 171 proteins. Whole amyloplasts and purified amyloplast membranes were prepared from wheat (Triticum aestivum). Protein extracts were examined by one-dimensional and two-dimensional electrophoresis, followed by high performance liquid chromatography-tandem mass spectrometry of separated proteins. Tandem mass spectrometry data of individual peptides was then searched by SEQUEST, using a database containing known protein sequences from both wheat and other homologous cereal crops. Using this approach we identified 108 proteins from whole amyloplasts and 63 proteins from purified amyloplast membranes. The majority of protein identifications were derived from protein sequences from cereal crops other than wheat, for which relatively little gene sequence data is available. The highest percentage of protein identifications obtained from any individual species was 46% of the total number of proteins identified, using sequence data found in our proprietary rice (Oryza sativa) genome database.     

Automated identification of diploid reference cells in cervical smears using image analysis

BACKGROUND: Acquisition of DNA ploidy histograms by image analysis may yield important information regarding the behavior of premalignant cervical lesions. Accurate selection of nuclei for DNA measurement is an important prerequisite for obtaining reliable data. Traditionally, manual selection of nuclei of diagnostic and reference cells is performed by an experienced cytotechnologist. In the present study, a method for the fully automated identification of nuclei of diploid epithelial reference cells in Feulgen- restained Papanicolaou (PAP) smears is described. METHODS: The automated procedure consists of a decision tree implemented on the measurement device, containing nodes with feature threshold values and multivariate discriminant functions. Nodes were constructed to recognize debris and inflammatory cells, as well as diploid and nondiploid epithelial cells of the uterine cervix. Evaluation of the classifier was performed by comparing resulting diploid integrated optical densities with those from manually selected reference cells. RESULTS AND CONCLUSION: On average, automatically acquired values deviated 2.4% from manually acquired values, indicating that the method described in this paper may be useful in cytometric practice.     

Role of adipocyte lipid-binding protein (ALBP) and acyl-CoA binding protein (ACBP) in PPAR-mediated transactivation

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that are activated by a number of fatty acids and fatty acid derivatives. By contrast, we have recently shown that acyl-CoA esters display PPAR antagonistic properties in vitro. We have also shown that the adipocyte lipid binding protein (ALBP), the keratinocyte lipid binding protein (KLBP) and the acyl-CoA binding protein (ACBP) exhibit a prominent nuclear localization in differentiating 3T3-L1 adipocytes. Similarly, ectopic expression of these proteins in CV-1 cells resulted in a primarily nuclear localization. We therefore speculated that FABPs and ACBP might regulate the availability of PPAR agonists and antagonists by affecting not only their esterification in the cytoplasm but also their transport to and availability in the nucleus. We show here that coexpression of ALBP or ACBP exerts a negative effect on ligand-dependent PPAR transactivation, when tetradecylthioacetic (TTA) is used as ligand but not when the thiazolidinedione BRL49653 is used as ligand. The results presented here do not support the hypothesis that ALBP facilitates the transport of the fatty acid-type ligands to the nucleus, rather ALBP appears to sequester or increase the turn-over of the agonist. Similarly, our results are in keeping with a model in which ACBP increase the metabolism of these ligands.     

Characterization of four lectin-like receptor kinases expressed in roots of Medicago truncatula. Structure, location, regulation of expression, and potential role in the symbiosis with Sinorhizobium meliloti

To study the role of LecRK (lectin-like receptor kinase) genes in the legumerhizobia symbiosis, we have characterized the four Medicago truncatula Gaernt. LecRK genes that are most highly expressed in roots. Three of these genes, MtLecRK7;1, MtLecRK7;2, and MtLecRK7;3, encode proteins most closely related to the Class A LecRKs of Arabidopsis, whereas the protein encoded by the fourth gene, MtLecRK1;1, is most similar to a Class B Arabidopsis LecRK. All four genes show a strongly enhanced root expression, and detailed studies on MtLecRK1;1 and MtLecRK7;2 revealed that the levels of their mRNAs are increased by nitrogen starvation and transiently repressed after either rhizobial inoculation or addition of lipochitooligosaccharidic Nod factors. Studies of the MtLecRK1;1 and MtLecRK7;2 proteins, using green fluorescent protein fusions in transgenic M. truncatula roots, revealed that they are located in the plasma membrane and that their central transmembrane-spanning helix is required for correct sorting. Moreover, their lectin-like domains appear to be highly glycosylated. Of the four proteins, only MtLecRK1;1 shows a high conservation of key residues implicated in monosaccharide binding, and molecular modeling revealed that this protein may be capable of interacting with Nod factors. However, no increase in Nod factor binding was found in roots overexpressing a fusion in which the kinase domain of this protein had been replaced with green fluorescent protein. Roots expressing this fusion protein however showed an increase in nodule number, suggesting that expression of MtLecRK1;1 influences nodulation. The potential role of LecRKs in the legume-rhizobia symbiosis is discussed.     

Decolorization of synthetic dyes and production of manganese-dependent peroxidase by new fungal isolates

Two yeasts, Debaryomyces polymorphus, Candida tropicalis, and two filamentous fungi, Umbelopsis isabellina, Penicillium geastrivorus, could completely decolorize 100 mg Reactive Black 5 (RB 5) l^–1 within 16–48 h. Manganese-dependent peroxidase (MnP) activities between 60 and 424 U l^–1 were detected in culture supernatants of three of these organisms indicating the color removal by enzymatic biodegradation but with P. geastrivorus there was no ligninolytic enzyme activity in its culture and the decolorization was mainly due to biosorption to mycelium. Extensive decolorization by D. polymorphus (69–94%) and C. tropicalis (30–97%) was obtained with five other azo dyes and one anthraquinone dye. Except for Reactive Brilliant Blue KNR and Reactive Yellow M-3R, the four azo dyes, Reactive Red M-3BE, Procion Scharlach H-E3G, Procion Marine H-EXL and Reactive Brilliant Red K-2BP, induced D. polymorphus to produce MnP (105–587 U l^–1). However, MnP activities of 198–329 U l^–1 were only detected in the culture of C. tropicalis containing Reactive Red M-3BE and Reactive Brilliant Red K-2BP, respectively.