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PubMed1988

The tRNA Required for in Vitro delta-Aminolevulinic Acid Formation from Glutamate in Synechocystis Extracts : Determination of Activity in a Synechocystis in Vitro Protein Synthesizing System.

RNA is an essential component for the enzymic conversion of glutamate to delta-aminolevulinic acid (ALA), the universal heme and chlorophyll precursor, as carried out in plants, algae, and some bacteria. The RNA required in this process was reported to bear a close structural resemblance to tRNA(Glu(UUC)), and it can be isolated by affinity chromatography directed against the UUC anticodon. Affinity-purified tRNA(Glu(UUC)) from the cyanobacterium Synechocystis sp. PCC 6803 was resolved into two major subfractions by reverse-phase HPLC. Only one of these was effectively charged with glutamate in enzyme extract from Synechocystis, but both were charged in Chlorella vulgaris enzyme extract. When charged with glutamate, the two glutamyl-tRNA(Glu(UUC)) species produced were equally effective in supporting both ALA formation and protein synthesis in vitro, as measured by label transfer from [(3)H]glutamyl-tRNA to ALA and protein. These results indicate that one of the two tRNA(Glu(UUC)) species is used by Synechocystis for both protein biosynthesis and ALA formation. Both of the tRNA(Glu(UUC)) subfractions from Synechocystis supported ALA formation in Chlorella enzyme extract. Escherichia coli tRNA(Glu(UUC)) was charged with glutamate, but did not support ALA formation in Synechocystis enzyme extract. Unfractionated tRNA from Chlorella, pea, and E. coli, having been charged with [(3)H] glutamate by Chlorella enzyme extract and then re-isolated, were all able to transfer label to proteins in the Synechocystis enzyme extract.

Journal Article↗
PubMed2003

Application and limitations of chloromethyl-benzamidodialkylcarbocyanine for tracing cells used in bone Tissue engineering.

Bone tissue engineering has the potential to provide us with an autologous bone substitute. Despite extensive research to optimize the technique, little is known about the survival and function of the cells after implantation. To monitor the cells, in vivo labeling is the method of choice. In this study we investigated the use of the fluorescent membrane marker chloromethyl-benzamidodialkylcarbocyanine (CM-Dil) to label cells used in bone tissue engineering. When applying label concentrations up to 50 microM, cells could be labeled efficiently without negative effects on cell vitality, proliferation, or bone-forming capacity. Porous hydroxyapatite scaffolds were seeded with labeled cells, and up to 6 weeks after implantation in nude mice cells could be traced inside tissue-engineered bone. However, contrary to other reports concerning intramembranous labels, transfer of the label from labeled to unlabeled cells was detected. Transfer occurred both in vitro and in vivo between vital cells and between dead and living cells. To determine when in vivo label transfer happened, devitalized, labeled constructs were implanted for various time periods in nude mice. The presence of vital labeled cells inside these constructs, when evaluated at different implantation periods, indicated transfer of the label. Transfer occurred at 7 days postimplantation when 40 microM label was applied, whereas 10 microM labeled constructs showed transfer 10 days after implantation. These findings indicate that CM-Dil label is useful for in vivo tracing of cells for follow-up periods up to 10 days. This makes the label particularly useful for cell survival studies in tissue-engineered implants.

Bone and Bones↗
PubMed1976

Specific spin-labeling of transfer ribonucleic acid molecules.

The spin labels anhydride (ASL), bromoacetamide (BSL) and carbodiimide (CSL) were used to label selectively tRNAGlu, tRNA fMet and tRNAPhe from E. coli. The preparation and characterization of the sites of labeling of eight new spin-labeled tRNAs are described. The sites of labeling are: s2U using ASL, BSL and CLS and tRNAGlu; s4U using ASL and BSL on tRNAfMet and tRNAPhe; U-37 with CSL on tRNfMet; U-33 with CSL on tRNAPhe. The rare base X at position 47 of tRNAPhe has been acylated with a spin-labeled N-hydroxysuccinimide (HSL). The 3'end of unfractionated tRNA molecules has been chemically modified to a morpholino spin-labeled analogue (MSL). Their respective e.s.r. spectra are reported and discussed.

Binding Sites↗
PubMed1993

Spin label saturation transfer EPR determinations of the stoichiometry and selectivity of lipid-protein interactions in the gel phase.

Lipid-protein interactions with the myelin proteolipid protein incorporated in the gel phase of dimyristoylphosphatidylcholine bilayers have been studied by saturation transfer EPR spectroscopy of spin-labelled phospholipids. The integrated intensities of the saturation transfer EPR spectra from spin-labelled phosphatidylcholine are linearly dependent on the protein/lipid ratio, and correspond to a fixed stoichiometry of approximately 11 lipids per monomer associated with the protein in the gel phase. The normalized saturation transfer intensities of spin-labelled phosphatidic acid, on the other hand, display a non-linear dependence on the protein/lipid ratio that can be described well by a selectivity for interaction with the protein in the gel phase with an average association constant relative to phosphatidylcholine of approx. 5.2. These values for the stoichiometry and selectivity of lipid-protein interaction in the lipid gel phase obtained from saturation transfer EPR spectroscopy are comparable to those found previously in fluid phase lipids by conventional EPR spectroscopy.

Dimyristoylphosphatidylcholine↗
PubMed1983

Role of intercellular communication in the promotion of C3H/10T1/2 cell transformation.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) upon intercellular communication and promotion were studied in cultures of C3H/10T1/2 mouse embryo fibroblasts. Cell-to-cell communication was quantitated by autoradiographic analysis of [3H]uridine transfer from prelabelled donor cells to an excess of unlabelled recipient cells during a 4 h co-cultivation. Extensive transfer of label was observed from donor to recipient cells in contact. Treatment of non-transformed C3H/10T1/2 cultures with 250 ng/ml TPA at co-cultivation of donor and recipient cells markedly inhibited intercellular communication during the 4 h incubation, producing an 80% reduction in uridine exchange relative to solvent-treated control cultures. Concentrations of TPA ranging from 0.25 to 25 ng/ml were also effective in inhibiting [3H]uridine exchange in a dose dependent fashion from 13% to 74%. This inhibition of intercellular communication was transient; cells exposed to 250 ng/ml TPA for 1.5 h prior to co-cultivation with TPA exhibited a 60% inhibition and the exchange of uridine had increased to control values in cultures pretreated for 12-72 h. An examination of label transfer between non-transformed and transformed C3H/10T1/2 cells indicated that both the extent of inhibition by TPA and the kinetics of communication inhibition were similar to that observed for non-transformed cells. Initiation and promotion experiments demonstrated that exposure to 250 ng/ml TPA for 5 weeks, but not to reduced concentrations of 0.25, 2.5 or 25 ng/ml, was capable of promoting morphological transformation. The lack of correlation between the dose responses of TPA for promotion and for reduction of cell-to-cell communication, and the transient nature of intercellular communication inhibition by TPA, suggests that an inhibition of cell-to-cell communication is not a sufficient event for promotion of oncogenic transformation in these cells.

Animals↗
PubMed1993

Cell type-specific proteins which interact with the 5' nontranslated region of hepatitis A virus RNA.

The 5' nontranslated region (5'NTR) of hepatitis A virus (HAV) RNA contains structural elements which facilitate 5' cap-independent initiation of virus translation and are likely to interact with cellular proteins functioning as translation initiation factors. To define these interactions, we characterized the binding of ribosome-associated proteins from several cell types to synthetic RNAs representing segments of the 5'NTR by using a UV cross-linking/label transfer assay. Four major proteins (p30, p39, p57, and p110) were identified. p30 and p39 were present in ribosomal salt washes prepared only from HAV-permissive BS-C-1 and FRhK-4 cells, while p57 was found only in HeLa cells and rabbit reticulocyte lysates. p110 was present in all cell types. Both p30 and p39 bound to multiple sites within the 5'NTR. Efficient transfer of label to p30 occurred with minimal RNA probes representing nucleotides (nt) 96 to 155, 151 to 354, and, to a much lesser extent, 634 to 744, while label transfer to p39 occurred with probes representing nt 96 to 155 and 634 to 744. All of these probes represent regions of the 5'NTR which are rich in pyrimidines. Competitive inhibition studies indicated that both p30 and p39 bound with greater affinity to sites in the 5' half of the NTR (a probe representing nt 1 to 354) than to the more 3' site (nt 634 to 744). Binding of p39 to the probe representing nt 96 to 155 was inhibited in the presence of an equal amount of proteins derived from HeLa cells, suggesting that p39 shares binding site specificity with one or more HeLa cell proteins. A 57-kDa protein in HeLa cell protein extracts reacted with antibody to polypyrimidine tract-binding protein in immunoblots, but no immunoreactive protein was identified in a similar BS-C-1 protein fraction. These results demonstrate that ribosome-associated proteins which bind to the 5'NTR of HAV vary substantially among different mammalian cell types, possibly accounting for differences in the extent to which individual cell types support growth of the virus. Mutations in the 5'NTR which enhance the growth of HAV in certain cell types may reflect specific adaptive responses to these or other proteins.

Animals↗
PubMed1993

The small subunit of the vaccinia virus early transcription factor contacts the transcription promoter DNA.

The vaccinia virus early transcription factor (VETF), in addition to the viral RNA polymerase, is required for efficient transcription of early genes in vitro. VETF is a heterodimeric protein that binds specifically to early gene promoters. In order to localize the VETF DNA binding domain, we have used photoreactive oligonucleotide probes with the sequence of the vaccinia virus growth factor promoter. The probes consisted of double-stranded oligonucleotides incorporating radiolabeled dAMP and 5-bromo-dUMP into sequences of the promoter known to contact VETF. Irradiation of a DNA probe having these nucleotides located upstream of the transcription start site in the presence of VETF resulted in the transfer of label to a polypeptide that comigrated with the small subunit of VETF. The label transfer reaction was shown to occur with the recombinant VETF small subunit in the absence of the large subunit. These results indicate that the small subunit comprises at least part of the VETF DNA binding domain and contacts the promoter in the region upstream of the transcription start site.

Base Sequence↗
PubMed2002

Effects of metallic silver particles on resonance energy transfer in labeled bovine serum albumin.

Resonance energy transfer (RET) is widely used to detect proximity between biomolecules. In transparent solution the maximum donor-to-acceptor distance for RET is about 70 A. We measured the effects of metallic silver island films on RET from the intrinsic tryptophan of a protein to a bound probe as the acceptor. These preliminary experiments revealed a dramatic increase in the apparent Förster distance increasing from 28.6 to 63 A. These results suggest the use of silver island films for detecting long range proximity between biomolecules and for biotechnology applications based on RET.

Anilino Naphthalenesulfonates↗
PubMed2001

Genotyping energy-transfer-cassette-labeled short-tandem-repeat amplicons with capillary array electrophoresis microchannel plates.

BACKGROUND: Genetic analysis of microsatellite DNA is a powerful tool used in linkage analysis, gene mapping, and clinical diagnosis. To address the expanding needs of studies of short tandem repeats (STRs), we demonstrated high-performance STR analysis on a high-throughput microchannel plate-based platform. METHODS: Energy-transfer-cassette-labeled STR amplicons were separated and typed on a microfabricated 96-channel radial capillary array electrophoresis (CAE) microchannel plate system. Four-color detection was accomplished with a laser-excited confocal fluorescence rotary scanner. RESULTS: Multiplex STR analysis with single base-pair resolution was demonstrated on denaturing polyacrylamide gel media. The high-throughput multiplex capabilities of this genetic analysis platform were demonstrated by the simultaneous separation of STR amplicons representing 122 samples in ninety-six 5.5-cm-long channels in <8 min. Sizing values obtained for these amplicons on the CAE microchannel plate were comparable to those measured on a conventional commercial CAE instrument and exhibit <1% sizing variance. CONCLUSIONS: Energy-transfer-cassette labeling and microfabricated CAE microchannel plates allow high-performance multiplex STR analyses.

Cell Line↗
PubMed1981

Number and size of human X chromosome fragments transferred to mouse cells by chromosome-mediated gene transfer.

Labeled probes of unique-sequence human X chromosomal deoxyribonucleic acid, prepared by two different procedures, were used to measure the amount of human X chromosomal deoxyribonucleic acid in 12 mouse cell lines expressing human hypoxanthine phosphoribosyltransferase after chromosome-mediated gene transfer. The amount of X chromosomal deoxyribonucleic acid detected by this procedure ranged from undetectable levels in the three stable transformants and some unstable transformants examined to about 20% of the human X chromosome in two unstable transformants. Reassociation kinetics of the X chromosomal probe with deoxyribonucleic acid from the two unstable transformants containing 15 to 20% of the human X chromosome indicate that a single copy of these sequences is present. In one of these lines, the X chromosomal sequences exist as multiple fragments which were not concordantly segregated when the cells were selected for loss of hprt.

Animals↗
PubMed1984

Calcium-promoted fusion of isolated chromaffin granules detected by resonance energy transfer between labeled lipids embedded in the membrane bilayer.

Chromaffin granules isolated from bovine adrenal medulla were labeled with small unilamellar vesicles (SUV) made from N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and/or N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine as donor and acceptor fluorophores. Labeling of granules could be followed by the relief of self-quenching experienced when the labels were incorporated into the granule membrane. The mechanism of incorporation seemed to be fusion of the SUV with the granule membrane although transfer of label could not be ruled out. Granule-granule fusion could be detected either by resonance energy transfer from donor to acceptor fluorophores or by donor quenching, in either of two different experimental designs: by a decrease in donor fluorescence and an increase in acceptor fluorescence when donor-labeled granules fused with acceptor-labeled granules or by an increase in donor fluorescence and a decrease in acceptor fluorescence when granules containing both probes fused with unlabeled granules. Fusion of freshly prepared granules could be initiated by millimolar concentrations of calcium; magnesium was less effective. Mg-ATP had no effect. Fusion was inhibited by potassium glutamate and a variety of organic and inorganic cations and anions, which also inhibited granule-granule aggregation to a lesser extent. The conditions for promotion and inhibition of granule-granule fusion were quite different from those reported by Knight and Baker [Knight, D. E., & Baker, P.F. (1982) J. Membr. Biol. 68, 107-140] for exocytosis of granule contents from permeabilized chromaffin cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Medulla↗
PubMed2001

The rate of lipid transfer during fusion depends on the structure of fluorescent lipid probes: a new chain-labeled lipid transfer probe pair.

A number of fluorescent probes have been used to follow membrane fusion events, particularly intermixing of lipids. None of them is ideal. The most popular pair of probes is NBD-PE and Rh-PE, in which the fluorescent groups are attached to the lipid headgroups, making them sensitive to changes in the surrounding medium. Here we present a new assay for monitoring lipid transfer during membrane fusion using the acyl chain tagged fluorescent probes BODIPY500-PC and BODIPY530-PE. Like the NBD-PE/Rh-PE assay, this assay is based on fluorescence resonance energy transfer (FRET) between the donor, BODIPY500, and the acceptor, BODIPY530. The magnitude of FRET is sensitive to the probe surface concentration, allowing one to detect movement of probes from labeled to unlabeled vesicles during fusion. The high quantum yield of fluorescence, high efficiency of FRET (R(o) is estimated to be approximately 60 A), photostability, and localization in the central hydrophobic region of a bilayer all make this pair of probes quite promising for detecting fusion. We have compared this and two other lipid mixing assays for their abilities to detect the initial events of poly(ethylene glycol) (PEG)-mediated fusion of small unilamellar vesicles (SUVs). We found that the BODIPY500/530 assay showed lipid transfer rates consistent with those obtained using the DPHpPC self-quenching assay, while lipid mixing rates measured with the NBD-PE/Rh-PE RET assay were significantly slower. We speculate that the bulky labeled headgroups of NBD-PE and especially Rh-PE molecules hamper movement of probes through the stalk between fusing vesicles, and thus reduce the apparent rate of lipid mixing.

Boron Compounds↗
PubMedDate not supplied

Energy-transfer cassette labeling for capillary array electrophoresis short tandem repeat DNA fragment sizing.

Energy-transfer (ET) dye-labeled primers significantly improve fluorescent DNA detection because they permit excitation at a single common wavelength and they produce well separated and intense acceptor dye emission. Recently, a new ET cassette technology was developed [Berti, L. et al. (2001) Anal. Biochem. 292, 188-197] that can be used to label any PCR, sequencing, or other primer of interest. In this report we examine the utility of this ET cassette technology by labeling seven different short tandem repeat (STR) specific primers with each of the four ET cassettes and analyzing the PCR products generated on a MegaBACE-1000 capillary array electrophoresis system. More than 60 amplicons were generated and successfully analyzed with the ET cassette-labeled primers. Both forward and reverse primers were labeled for multiplex PCR amplification and analysis. Single base pair resolution was achieved with all four ET cassettes. This ET cassette-primer labeling procedure is ideally suited for creating four-color fluorescent ET primers for STR and other DNA assays where large numbers of different loci are analyzed including sequencing, genetic identification, gene mapping, loss of heterozygosity testing, and linkage analysis.

DNA↗
PubMed1999

Transfer insensitive labeling technique (TILT): application to multislice functional perfusion imaging.

Cerebral blood flow can be studied in a multislice mode with a recently proposed perfusion sequence using inversion of water spins as an endogenous tracer without magnetization transfer artifacts. The magnetization transfer insensitive labeling technique (TILT) has been used for mapping blood flow changes at a microvascular level under motor activation in a multislice mode. In TILT, perfusion mapping is achieved by subtraction of a perfusion-sensitized image from a control image. Perfusion weighting is accomplished by proximal blood labeling using two 90 degrees radiofrequency excitation pulses. For control preparation the labeling pulses are modified such that they have no net effect on blood water magnetization. The percentage of blood flow change, as well as its spatial extent, has been studied in single and multislice modes with varying delays between labeling and imaging. The average perfusion signal change due to activation was 36.9 +/- 9.1% in the single-slice experiments and 38.1 +/- 7.9% in the multislice experiments. The volume of activated brain areas amounted to 1.51 +/- 0.95 cm3 in the contralateral primary motor (M1) area, 0.90 +/- 0.72 cc in the ipsilateral M1 area, 1.27 +/- 0.39 cm3 in the contralateral and 1.42 +/- 0.75 cm3 in the ipsilateral premotor areas, and 0.71 +/- 0.19 cm3 in the supplementary motor area.

Artifacts↗
PubMed2026

EvoSNR-Prom: Predicting promoters at single-nucleotide resolution with label-aware transfer learning of the pretrained EVO model.

The precise identification of promoters is crucial for understanding gene regulation. Deep learning methods have achieved considerable success in promoter prediction, yet most operate at the sequence level with coarse-grained labels. This means they label an entire DNA segment as either a "promoter" or "non-promoter," which results in a lack of the nucleotide-level resolution in prediction. In this study, we propose EvoSNR-Prom, a model designed for promoter prediction at single-nucleotide resolution. EvoSNR-Prom is built on the Evo foundation model and formulates promoter identification as a token-level sequence labeling problem, analogous to named entity recognition in natural language processing. To address the limited contextual information available in single-nucleotide tokenization, we introduce a lexicon-enhanced embedding strategy that incorporates biologically meaningful DNA lexicons, enriching contextual representations and improving the model's ability to capture complex sequence motifs. Furthermore, to enhance predictive performance on small size datasets, we integrate a label-aware transfer learning framework to leverage knowledge from well-annotated source species to a target organism. The results across various prokaryotic datasets show that EvoSNR-Prom achieves excellent performance. This work provides a valuable computational framework for the high-precision analysis of gene regulatory elements, contributing to the advancement of promoter prediction at single-nucleotide resolution.

Promoter Regions, Genetic↗
PubMed2001

Macrophages transfer [14C]-labelled fatty acids to pancreatic islets in culture.

Macrophages are able to produce, export, and transfer fatty acids to lymphocytes in culture. The purpose of this study was to examine if labelled fatty acids could be transferred from macrophages to pancreatic islets in co-culture. We found that after 3 h of co-culture the transfer of fatty acids to pancreatic islets was: arachidonic >> oleic > linoleic = palmitic. Substantial amounts of the transferred fatty acids were found in the phospholipid fraction; 87.6% for arachidonic, 59.9% for oleic, 53.1% for palmitic, and 36.9% for linoleic acids. The remaining radioactivity was distributed among the other lipid fractions analysed (namely polar lipids, cholesterol, fatty acids, triacylglycerol and cholesterol ester), varying with the fatty acid used. For linoleic acid, a significant proportion (63.1%) was almost equally distributed in these lipid fractions. Also, it was observed that transfer of fatty acids from macrophages to pancreatic islets is time-dependent up to 24 h, being constant and linear with time for palmitic acid and remaining constant after 12 h for oleic acid. These results lead us to postulate that in addition to the serum, circulating monocytes may also be a source of fatty acids to pancreatic islets, mainly arachidonic acid.

Animals↗
PubMed2006

Marrow stromal cells transplanted to the adult brain are rejected by an inflammatory response and transfer donor labels to host neurons and glia.

Abstract The remarkable plasticity of marrow stromal cells (MSCs) after transplantation to models of neurological disease and injury has been described. In this report, we investigated the plasticity and long-term survival of MSCs transplanted into the normal brain. MSCs were isolated from green fluorescent protein (GFP) transgenic rats and double-labeled with 5-bromo-2-deoxyuridine (BrdU) and bis benzamide (BBZ) prior to transplantation into the adult hippocampus or striatum. Surgery elicited an immediate inflammatory response. MSC grafts were massively infiltrated by ED1-positive microglia/macrophages and surrounded by a marked astrogliosis. By 14 days, graft volume had retracted and GFP immunoreactivity was absent, indicating complete donor rejection. Consequently, MSCs did not exhibit plasticity formerly identified in other studies. However, BrdU- and BBZ-labeled cells were detected up to 12 weeks. Control transplants of nonviable MSCs demonstrated the transfer of donor labels to host cells. Unexpectedly, BrdU labeling was colocalized to host phagocytes, astrocytes, and neurons in both regions. Our results indicate that MSCs transplanted to the intact adult brain are rejected by an inflammatory response. Moreover, use of the traditional cell labels BrdU and BBZ may provide a misleading index of donor survival and differentiation after transplantation.

Animals↗