Human pluripotent stem cells (PSCs) exist in naive and primed states and provide important models to investigate the earliest stages of human development. Naive cells can be obtained through ...primed-to-naive resetting, but there are no reliable methods to prospectively isolate unmodified naive cells during this process. Here we report comprehensive profiling of cell surface proteins by flow cytometry in naive and primed human PSCs. Several naive-specific, but not primed-specific, proteins were also expressed by pluripotent cells in the human preimplantation embryo. The upregulation of naive-specific cell surface proteins during primed-to-naive resetting enabled the isolation and characterization of live naive cells and intermediate cell populations. This analysis revealed distinct transcriptional and X chromosome inactivation changes associated with the early and late stages of naive cell formation. Thus, identification of state-specific proteins provides a robust set of molecular markers to define the human PSC state and allows new insights into the molecular events leading to naive cell resetting.
Display omitted
•Flow cytometry profiles cell surface proteins in naive and primed human PSCs•The human PSC state can be defined using robust state-specific protein markers•Identified cell surface proteins track the dynamics of naive-primed PSC conversions•Analyses of early-stage naive cells reveal transcription events during conversion
Collier et al. use profiling to identify cell surface proteins that are specific for naive versus primed human pluripotent cells and then use them to isolate and characterize live naive cells arising during primed-to-naive resetting.
Selected flavonoids that are known as inducers and a suppressor of nodulation (nod) genes of the symbiotic bacterium Rhizobium leguminosarum bv. viciae were tested for their effect on symbiosis ...formation with garden pea as the host. A solid substrate was omitted from the hydroponic growing system in order to prevent losses of flavonoids due to adsorption and degradation. The presumed interaction of the tested flavonoids with nod genes has been verified for the genetic background of strain 128C30. A stimulatory effect of a nod gene inducer naringenin on symbiotic nodule number formed per plant 14 d after inoculation was detected at concentrations of 0.1 and 1 μg ml–1 nutrient solution. At 10 μg ml–1, the highest concentration tested, naringenin was already inhibitory. By contrast, nodulation was negatively affected by a nod gene suppressor, quercetin, at concentrations above 1 μg ml–1, as well as by another tested nod gene inducer, hesperetin. The deleterious effect of hesperetin might be due to its toxicity or to the toxicity of its degradation product(s) as indicated by the inhibition of root growth. Both the stimulatory effect of naringenin and the inhibitory effect of quercetin on nodule number were more pronounced at earlier stages of nodule development as revealed with specific staining of initial nodules. The lessening of the flavonoid impact during nodule development was ascribed to the plant autoregulatory mechanisms. Feedback regulation of nodule metabolism might also be responsible for the fact that the naringenin‐conditioned increase in nodule number was not accompanied by any increase in nitrogenase activity. By contrast, the inhibitory action of quercetin and hesperetin on nodule number was associated with decreases in total nitrogenase activity. Naringenin also stimulated root hair curling (RHC) as one of the earliest nodulation responses at concentrations of 1 and 10 μg ml–1, however, the same effect was exerted by the nod gene suppressor, quercetin, suggesting that feedback regulatory mechanisms control RHC in the range of nodulation‐inhibiting high flavonoid concentrations. The comparison of the effect of the tested flavonoids in planta with nod gene activity response showed a two orders of magnitude shift to higher concentrations. This shift is explained by the absorption and degradation of flavonoids by both the symbionts during 3 d intervals between hydroponic solution changes. The losses were 99, 96.4, and 90% of the initial concentration of 10 μg ml–1 for naringenin, hesperetin, and quercetin, respectively.
Protein Complexes of the Escherichia coli Cell Envelope Stenberg, Filippa; Chovanec, Peter; Maslen, Sarah L. ...
Journal of biological chemistry/The Journal of biological chemistry,
10/2005, Letnik:
280, Številka:
41
Journal Article
Recenzirano
Odprti dostop
Protein complexes are an intrinsic aspect of life in the membrane. Knowing which proteins are assembled in these complexes is therefore essential to understanding protein function(s). Unfortunately, ...recent high throughput protein interaction studies have failed to deliver any significant information on proteins embedded in the membrane, and many membrane protein complexes remain ill defined. In this study, we have optimized the blue native-PAGE technique for the study of membrane protein complexes in the inner and outer membranes of Escherichia coli. In combination with second dimension SDS-PAGE and mass spectrometry, we have been able to identify 43 distinct protein complexes. In addition to a number of well characterized complexes, we have identified known and orphan proteins in novel oligomeric states. For two orphan proteins, YhcB and YjdB, our findings enable a tentative functional assignment. We propose that YhcB is a hitherto unidentified additional subunit of the cytochrome bd oxidase and that YjdB, which co-localizes with the ZipA protein, is involved in cell division. Our reference two-dimensional blue native-SDS-polyacrylamide gels will facilitate future studies of the assembly and composition of E. coli membrane protein complexes during different growth conditions and in different mutant backgrounds.
In rhizobial symbiosis with legume plant hosts, the symbiotic tissue in the root nodules of indeterminate type is localized to the basal part of the nodule where the symbiotic zones contain infected ...cells (IC) interspersed with uninfected cells (UC) that are devoid of rhizobia. Although IC are easily distinguished in nodule sections using standard histochemical techniques, their observation in intact nodules is hampered by nodule tissue characteristics. Tagging of
Rhizobium leguminosarum
bv.
viciae
strain 128C30 with a constitutively expressed gene for green fluorescent protein (nonshifted mutant form cycle3) in combination with the advantages of the tiny nodules formed by
Vicia tetrasperma
(L.)
Schreb
. allowed for vital observation of symbiotic tissue using fluorescence microscopy. Separation of a red-shifted background channel and digital image stacking along
z
-axis enabled us to construct a nodule image in a classical fluorescence microscopy of nodules exceeding 1 mm in diameter. In parallel, visualization of nodule bacteria inside the symbiotic tissue by confocal microscopy at the excitation wavelength 488 nm clearly distinguished IC/UC pattern in the nodule virtual sections and revealed red-shifted fluorescence of nonrhizobial origin. This signal was located on the periphery of IC and increased with their degradation, thus suggesting accumulation of secondary metabolites, presumably flavonoids. The simultaneous detection of bacteria and secondary metabolites can be used for monitoring changes to intact nodule physiology in the model legumes. The advantage of
V. tetrasperma
as a suggested laboratory model for pea cross-inoculation group has been demonstrated.
The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon ...dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and Fe–S clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.
A technique was optimized for the in situ detection of nodulation (nod) gene activity in Rhizobium leguminosarum bv. viciae symbiosis with compatible plant hosts Vicia tetrasperma (L.) SCHREB. and ...Pisum sativum L. The transcription of nodABC-lacZ fusion was visualized as beta-galactosidase (beta-Gal) activity after reaction with the chromogenic substrate X-Gal and subsequent light microscopy, while the background of the indigenous beta-Gal activity of rhizobia and the host plant was eliminated by glutaraldehyde treatment. V. tetrasperma was suggested as a suitable model plant for pea cross-inoculation group due to its advantages over the common model of V. hirsuta (L.) S.F. GRAY: compactness of the plant, extremely small seeds, fast development and stable nodulation under laboratory conditions. In the roots of both plants, a certain extent of nod gene activity was detectable in all rhizobia colonizing the rhizoplane. In pea 1 d after inoculation (d.a.i.), the maximum was localized in the region of emerging root hairs (RH) later (3 and 6 d.a.i.) shifting upwards from the root tip. Nodulation genes sustained full expression even in the infection threads inside the RH and the root cortex, independently of their association with nodule primordia. Comparison of two pea symbiotic mutant lines, Risnod25 and Risnod27, with the wild type did not reveal any differences in the RH formation, RH curling response and rhizoplane colonization. Both mutants appeared to be blocked at the infection thread initiation stage and in nodule initiation, consistent with the phenotype caused by other mutant alleles in the pea sym8 locus. Judging from the nod gene expression level and pattern in the rhizoplane, flavonoid response upon inoculation is preserved in both pea mutants, being independent of infection thread and nodule initiation.
A technique was optimized for thein situ detection of nodulation (nod) gene activity inRhizobium leguminosarum bv.viciae symbiosis with compatible plant hostsVicia tetrasperma (L.)Schreb. andPisum ...sativum L. The transcription ofnodABC-lacZ fusion was visualized as β-galactosidase (β-Gal) activity after reaction with the chromogenic substrate X-Gal and subsequent light microscopy, while the back-ground of the indigenous β-Gal activity of rhizobia and the host plant was eliminated by glutaraldehyde treatment.V. tetrasperma was suggested as a suitable model plant for pea cross-inoculation group due to its advantages over the common model ofV. hirsuta (L.) S.F.Gray: compactness of the plant, extremely small seeds, fast development and stable nodulation under laboratory conditions. In the roots of both plants, a certain extent ofnod gene activity was detectable in all rhizobia colonizing the rhizoplane. In pea 1 d after inoculation (d.a.i.), the maximum was localized in the region of emerging root hairs (RH) later (3 and 6 d.a.i.) shifting upwards from the root tip. Nodulation genes sustained full expression even in the infection threads inside the RH and the root cortex, independently of their association with nodule primordia. Comparison of two pea symbiotic mutant lines, Risnod25 and Risnod27, with the wild type did not reveal any differences in the RH formation, RH curling response and rhizoplane colonization. Both mutants appeared to be blocked at the infection thread initiation stage and in nodule initiation, consistent with the phenotype caused by other mutant alleles in the peasym8 locus. Judging from thenod gene expression level and pattern in the rhizoplane, flavonoid response upon inoculation is preserved in both pea mutants, being independent of infection thread and nodule initiation.
The cell envelope of Escherichia coli is an essential structure that modulates exchanges between the cell and the extra-cellular milieu. Previous proteomic analyses have suggested that it contains a ...significant number of proteins with no annotated function. To gain insight into these proteins and the general organization of the cell envelope proteome, we have carried out a systematic analysis of native membrane protein complexes. We have identified 30 membrane protein complexes (6 of which are novel) and present reference maps that can be used for cell envelope profiling. In one instance, we identified a protein with no annotated function (YfgM) in a complex with a well-characterized periplasmic chaperone (PpiD). Using the guilt by association principle, we suggest that YfgM is also part of the periplasmic chaperone network. The approach we present circumvents the need for engineering of tags and protein overexpression. It is applicable for the analysis of membrane protein complexes in any organism and will be particularly useful for less-characterized organisms where conventional strategies that require protein engineering (i.e., 2-hybrid based approaches and TAP-tagging) are not feasible.
The growth and conidiation of the aged Trichoderma viride culture grown in the dark, and after an induction by a light pulse, was examined in the presence of selected mono-, di(tri)saccharides, amino ...acids and alcohols as sole carbon sources. Hexoses and disaccharides, but not pentoses and amino acids, promoted proportionally both growth and conidiation induced by aging or light. All compounds but pentoses promoted the conidiation in aged cultures and photoconidiation in a close correlation. Ethanol, glycerol and ethylene glycol supported both growth and conidiation but these processes were not supported equally. Conidia formation with hexoses and amino acids as sole carbon sources seems to be a function of growth promotion, rather than of growth restriction (starvation, stress, aging). With glucose as sole carbon source the conidiation was not triggered by nutrient limitation, nor by the accumulation of waste metabolites. The aging-induced conidiation can be considered to be triggered by the genetic program of the microorganism rather than by its nutrient status.
PDF
