Influence of extended aging on beef quality characteristics and sensory perception of steaks from the biceps femoris and semimembranosus.

The objective was to determine the influence of post-fabrication aging (2, 14, 21, 42, and 63days) on beef quality characteristics and consumer sensory perception of biceps femoris (BF) and semimembranosus (SM) steaks. Lipid oxidation and aerobic plate counts increased (P<0.05) with longer aging periods and retail display times. An aging period by day of retail display interaction (P<0.05) was observed for a* and b* values of the BF and SM. Warner-Bratzler shear force values decreased (P<0.05) with longer aging for the SM, while no difference was observed for the BF. Consumer panel results revealed that longer aging periods increased (P<0.05) acceptability of the SM, tenderness of both muscles, and tended to increase (P=0.07) juiciness of the SM. Our results show that extended aging reduces retail color stability yet has positive effects on consumer perception of tenderness of both muscles and overall acceptability of the SM.

Influence of extended aging on beef quality characteristics and sensory perception of steaks from the gluteus medius and longissimus lumborum.

The objective was to determine the influence of post-fabrication aging (2, 14, 21, 42, and 63 days) on beef quality characteristics and consumer sensory perception of gluteus medius (GM) and longissimus lumborum (LL) steaks. Lipid oxidation and aerobic plate counts increased (P<0.05) with longer aging periods and retail display times. An aging period by day of retail display interaction (P<0.05) was observed for a* and b* values for both muscles and L* values for the LL. Warner-Bratzler shear force values decreased (P<0.05) with longer aging for the LL, while no difference was observed for the GM. Consumer panel results demonstrated that longer aging periods increased (P<0.05) tenderness of both muscles. Our results indicate that extended aging reduces retail color stability yet has positive effects on consumer perception of tenderness of beef loin muscles.

Ligand-selective interactions of ER detected in living cells by fluorescence resonance energy transfer.

Some aspects of ligand-regulated transcription activation by the estrogen receptor (ER) are associated with the estrogen-dependent formation of a hydrophobic cleft on the receptor surface. At least in vitro, this cleft is required for direct interaction of ER with an alpha helix, containing variants of the sequence LXXLL, found in many coactivators. In cells, it is unknown whether ER interactions with the different LXXLL-containing helices are uniformly similar or whether they vary with LXXLL sequence or activating ligand. Using fluorescence resonance energy transfer (FRET), we confirm in the physiological environment a direct interaction between the estradiol (E2)-bound ER and LXXLL peptides expressed in living cells as fusions with spectral variants of the green fluorescent protein. This interaction was blocked by a single amino acid mutation in the hydrophobic cleft. No FRET was detected when cells were incubated with the antiestrogenic ligands tamoxifen and ICI 182,780. E2, diethylstilbestrol, ethyl indenestrol A, and 6,4'-dihydroxyflavone all promoted FRET and activated ER-dependent transcription. Measurement of the level of FRET of ER with different LXXLL-containing peptides suggested that the orientations or affinities of the LXXLL interactions with the hydrophobic cleft were globally similar but slightly different for some activating ligands.

Nanosecond fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell.

Visualizing and quantifying protein-protein interactions is a recent trend in biomedical imaging. The current advances in fluorescence microscopy, coupled with the development of new fluorescent probes such as green fluorescent proteins, allow fluorescence resonance energy transfer (FRET) to be used to study protein interactions in living specimens. Intensity-based FRET microscopy is limited by spectral bleed-through and fluorophore concentration. Fluorescence lifetime imaging (FLIM) microscopy and lifetime measurements are independent of change in fluorophore concentration or excitation intensity, and the combination of FRET and FLIM provides high spatial (nanometre) and temporal (nanoseconds) resolution. Because only the donor fluorophore lifetime is measured, spectral bleed-through is not an issue in FRET-FLIM imaging. In this paper we describe the development of a nanosecond FRET-FLIM microscopy instrumentation to acquire the time-resolved images of donor in the presence and the absence of the acceptor. Software was developed to process the acquired images for single and double exponential decays. Measurement of donor lifetime in two different conditions allowed us to calculate accurately the distance between the interacting proteins. We used this approach to quantify the dimerization of the transcription factor CAATT/enhancer binding protein alpha in living pituitary cells. The one- and two-component analysis of the donor molecule lifetime in the presence of acceptor demonstrates the distance distribution between interacting proteins.

CCAAT/enhancer binding protein alpha assembles essential cooperating factors in common subnuclear domains.

The transcription factor CCAAT/enhancer binding protein alpha (C/EBP alpha) is the DNA binding subunit of a multiprotein complex that regulates the pituitary-specific GH promoter. C/EBP alpha is absent from the GHFT1-5 pituitary progenitor cell line in which ectopic C/EBP alpha expression leads to activation of the otherwise dormant GH promoter. Transcriptional regulatory complexes are commonly envisaged as assembling from components that evenly diffuse throughout the nucleoplasm. We show that C/EBP alpha, expressed in GHFT1-5 cells as a fusion with color variants of the green fluorescent protein (GFP), concentrated specifically at peri-centromeric chromosomal domains. Although we found the CREB-binding protein (CBP) to activate C/EBP alpha-dependent transcription, CBP was absent from the pericentromeric chromatin. C/EBP alpha expression was accompanied by the translocation of endogenous and ectopically expressed CBP to pericentromeric chromatin. The intranuclear recruitment of CBP required the transcriptional activation domains of C/EBP alpha. C/EBP alpha also caused GFP-tagged TATA binding protein (TBP) to relocate to the Hoechst-stained domains. The altered intranuclear distribution of critical coregulatory factors defines complexes formed upon C/EBP alpha expression. It also identifies an organizational activity, which we label "intranuclear marshalling," that may regulate gene expression by determining the cooperative and antagonistic interactions available at specific nuclear sites.

CCAAT/enhancer-binding protein alpha alters histone H3 acetylation at large subnuclear domains.

Transcriptional regulation is commonly associated with local levels of histone acetylation, which controls chromatin structure at specific genes or within contiguous chromosomal domains. Less well understood are the higher order determinants of histone acetylation. The transcription factor, CCAAT/enhancer-binding protein alpha (C/EBPalpha), concentrates at one higher order structure, the peri-centromeric chromatin, and regulates differentiation in many cell types, including pituitary cells. We used quantitative fluorescence microscopy to show that immunostained acetylated histone H3 is relatively absent from peri-centromeric domains visible as large structures in mouse pituitary progenitor GHFT1-5 cells. GHFT1-5 cells do not contain C/EBPalpha. We observed that expression of C/EBPalpha in GHFT1-5 cells leads to an increased level of acetylated histone H3, but not acetylated histone H4, at the peri-centromeric domains. Only transcriptionally active forms of C/EBPalpha altered histone acetylation at the peri-centromeric domain. The altered state of histone acetylation at large intranuclear domains may complement, counteract, or supersede the more gene-local activities of other transcription factors to coordinate C/EBPalpha-induced cellular differentiation.

Fluorescence resonance energy transfer microscopy of localized protein interactions in the living cell nucleus.

Cells respond to environmental cues by modifying protein complexes in the nucleus to produce a change in the pattern of gene expression. In this article, we review techniques that allow us to visualize these protein interactions as they occur in living cells. The cloning of genes from marine organisms that encode fluorescent proteins provides a way to tag and monitor the intracellular behavior of expressed fusion proteins. The genetic engineering of jellyfish green fluorescent protein (GFP) and the recent cloning of a sea anemone red fluorescent protein (RFP) have provided fluorescent tags that emit light at wavelengths ranging from the blue to the red spectrum. Several of these color variants can be readily distinguished by fluorescence microscopy, allowing them to be used in combination to monitor the behavior of two or more independent proteins in the same living cell. We describe the use of this approach to examine where transcription factors are assembled in the nucleus. To demonstrate that these labeled nuclear proteins are interacting, however, requires spatial resolution that exceeds the optical limit of the light microscope. This degree of spatial resolution can be achieved with the conventional light microscope using the technique of fluorescence resonance energy transfer (FRET). The application of FRET microscopy to detect the interactions between proteins labeled with the color variants of GFP and the limitations of the FRET approach are discussed. The use of different-color fluorescent proteins in combination with FRET offers the opportunity to study the complex behavior of key regulatory proteins in their natural environment within the living cell.

Temporally distinct and ligand-specific recruitment of nuclear receptor-interacting peptides and cofactors to subnuclear domains containing the estrogen receptor.

Ligand binding to estrogen receptor (ER) is presumed to regulate the type and timing of ER interactions with different cofactors. Using fluorescence microscopy in living cells, we characterized the recruitment of five different green fluorescent protein (GFP)-labeled ER-interacting peptides to the distinct subnuclear compartment occupied by blue fluorescent protein (BFP)-labeled ER alpha. Different ligands promoted the recruitment of different peptides. One peptide was recruited in response to estradiol (E2), tamoxifen, raloxifene, or ICI 182,780 incubation whereas other peptides were recruited specifically by E2 or tamoxifen. Peptides containing different sequences surrounding the ER-interacting motif LXXLL were recruited with different time courses after E2 addition. Complex temporal kinetics also were observed for recruitment of the full-length, ER cofactor glucocorticoid receptor-interacting protein 1 (GRIP1); rapid, E2-dependent recruitment of GRIP1 was blocked by mutation of the GRIP1 LXXLL motifs to LXXAA whereas slower E2 recruitment persisted for the GRIP1 LXXAA mutant. This suggested the presence of multiple, temporally distinct GRIP 1 recruitment mechanisms. E2 recruitment of GRIP1 and LXXLL peptides was blocked by coincubation with excess ICI 182,780. In contrast, preformed E2/ER/GRIP1 and E2/ER/LXXLL complexes were resistant to subsequent ICI 182,780 addition whereas ICI 182,780 dispersed preformed complexes containing the GRIP1 LXXAA mutant. This suggested that E2-induced LXXLL binding altered subsequent ligand/ER interactions. Thus, alternative, ligand-selective recruitment and dissociation mechanisms with distinct temporal sequences are available for ER alpha action in vivo.

Calcium ions positively modulate follicle-stimulating hormone- and exogenous cyclic 3',5'-adenosine monophosphate-driven transcription of the P450(scc) gene in porcine granulosa cells.

Given the evident modulation of FSH-induced steroidogenesis by Ca2+ in granulosa cells, we here test the hypothesis that Ca2+ controls expression of the enzymatically rate-limiting cytochrome P450(scc) (CYP11A) gene. To test this postulate, we quantitated the ability of Ca2+ to regulate: 1) transcriptional activity of a transiently transfected luciferase reporter gene driven by a 2.32-kb 5'-upstream fragment of the porcine P450(scc) gene promoter region; and 2) accumulation of endogenous P450(scc) transcripts in primary monolayer cultures of porcine granulosa cells. To this end, granulosa cells were stimulated for 4 h with FSH (15 ng/ml, NIDDK-oFSH-20) or 8-Bromo-cAMP (8 Br-cAMP, 1 mM) in serum-free medium containing either 1.8 mM Ca2- or no added Ca2+ with 100 microM EGTA or 100 microM CoCl2. In the presence of extracellular Ca2+, FSH and 8 Br-cAMP stimulated expression of the transfected P450(scc) promoter-reporter fusion construct by 5.6 +/- 1.1 and 3.6 +/- 0.67-fold, respectively over Ca2+-containing unstimulated control (P < or = 0.04, n = 5-6 experiments). The foregoing two agonists augmented 4-h progesterone production by cultured granulosa cells by 1.8 +/- 0.11 and 1.6 +/- 0.16-fold, respectively (P < or = 0.001 for FSH and P < or = 0.01 for 8 Br-cAMP). FSH and 8 Br-cAMP also significantly elevated endogenous P450(scc) transcript levels as measured by homologous solution-hybridization RNase protection assay; i.e. by 3.1 +/- 0.49 and 2.9 +/- 0.45-fold, respectively (P < or = 0.001). In Ca2+-free/EGTA-supplemented medium, basal luciferase reporter-gene activity and endogenous P450(scc) messenger RNA accumulation in granulosa cells declined to 34 +/- 12% and 78 +/- 12%, respectively, of corresponding values in control (unstimulated Ca2+-containing) cultures. Extracellular Ca2+ deprivation inhibited the stimulatory effect of FSH (and 8 Br-cAMP) on P450(scc) promoter-luciferase reporter expression to 58 +/- 30% (and 58 +/- 23%), and restrained endogenous P450(scc) message accumulation to 86 +/- 15% (and 96 +/- 18%) of the value in Ca2+-containing control. Extracellular Ca2+ withdrawal suppressed FSH (and 8 Br-cAMP)-driven progesterone production over 4 h to basal levels but did not alter FSH-stimulated cAMP accumulation by granulosa cells. Ca2+-deprived cells exposed to serum-containing media regained P450(scc) responsiveness to both agonists. Antagonism of cellular uptake of Ca2+ and other divalent cations via administration of cobalt chloride (100 microM) inhibited FSH and 8 Br-cAMP's stimulation of endogenous (but not exogenous promoter-driven) P450(scc) gene expression. In contrast, granulosa-cell concentrations of messenger RNA's encoding sterol-carrier protein-2 (SCP-2) and the low density lipoprotein receptor were not altered by Ca2+ withdrawal. In summary, uptake of extracellular Ca2+ by porcine granulosa cells significantly potentiates transactivation of the endogenously expressed and exogenously transfected P450(scc) gene by FSH and 8 Br-cAMP. The agonistic impact of Ca2+ on P450(scc) promoter activity is requisite downstream of FSH-induced cAMP second-messenger signaling.

Concerted regulation of low density lipoprotein receptor gene expression by follicle-stimulating hormone and insulin-like growth factor I in porcine granulosa cells: promoter activation, messenger ribonucleic acid stability, and sterol feedback.

Insulin-like growth factor I (IGF-I) and the gonadotropin, FSH, can synergize to stimulate progesterone production in primary cultures of maturing granulosa cells. These trophic hormones increase low density lipoprotein (LDL) receptor binding and internalization, and the utilization of LDL-borne cholesterol by granulosa cells. To determine whether and how IGF-I and FSH control the genomic expression of the LDL receptor, we evaluated their individual and concerted effects on LDL receptor messenger RNA (mRNA) accumulation, stability, and gene promoter activity in first passage monolayer (serum-free) cultures of porcine granulosa cells. Ribonuclease protection assays revealed that LDL receptor mRNA accumulation was increased by human recombinant IGF-I (100 ng/ml), FSH (25 ng/ml NIDDK oFSH-20), or their combination by 2.2-, 2.6-, and 4.6-fold, respectively (P < 0.01). Hormonally stimulated LDL receptor mRNA accumulation was suppressed by 54-75% by the concurrent addition of LDL substrate (50 microg/ml). The combination of FSH and IGF-I significantly prolonged the message half-life, even in the presence of LDL. Using a combination of rapid amplification of cDNA 5'-ends, PCR with adapter-ligated genomic DNA, Southern hybridization, and DNA sequencing, we isolated 1076 bp of the porcine LDL receptor gene upstream of the coding region. In transient transfection assays, with a pLDLR1076/luciferase plasmid construct, FSH, FSH plus IGF-I, or 8-bromo-cAMP (1 mM) treatment (but not IGF-I alone) increased luciferase reporter gene activity by 10- to 23-fold in porcine granulosa cells. Over time in serum-free culture, the basal activity of the LDL receptor gene promoter increased and eventually surpassed hormone-stimulated effects, but was suppressed by LDL substrate (by 75%) at 24 h. The foregoing stimulatory hormone effects and sterol repression were localized to a 116-bp region in the porcine promoter between -255 and -139 upstream of the translational start site. We conclude that the combination of FSH and IGF-I can induce accumulation of LDL receptor mRNA in cultured granulosa cells even in the presence of sterol negative feedback and can do so mechanistically by a combination of promoter activation and increased mRNA stability.

Selective inhibition of prolactin gene transcription by the ETS-2 repressor factor.

Regulation of prolactin gene transcription requires cooperative interactions between the pituitary-specific POU domain protein Pit-1 and members of the ETS transcription factor family. We demonstrate here that the ETS-2 repressor factor (ERF) is expressed in pituitary tumor cells and that overexpression of recombinant ERF inhibits prolactin promoter activity, but not the closely related growth hormone promoter. In non-pituitary cell lines, coexpression of ERF disrupts the cooperative interactions between Pit-1 and ETS-1 and blocks the induction of Pit-1-dependent prolactin promoter activity by cAMP. The potential role of ERF in the inhibitory response of the prolactin promoter to dopamine was examined using pituitary tumor cells stably expressing dopamine D2 receptors. The inhibitory responses of the prolactin promoter to ERF and dopamine are additive, suggesting that ERF has a complementary role in this hormonal response. A single Pit-1 DNA-binding element from the prolactin promoter is sufficient to reconstitute the inhibitory response to ERF. DNA binding analysis using either a composite Pit-1/ETS protein-binding site or a Pit-1 element with no known affinity for ETS proteins revealed that ERF interferes with Pit-1 binding. Together, these results demonstrate that ERF is a specific inhibitor of basal and hormone-regulated transcription of the prolactin gene and suggest a new level of complexity for the interaction of ETS factors with Pit-1 target genes.

Dual-function reporter protein for analysis of gene expression in living cells.

The firefly luciferase (Luc) protein and the jellyfish green fluorescent protein (GFP) are two commonly used molecular reporters that can be detected noninvasively in living cells. The properties that make GFP or Luc useful for a particular experimental application are quite distinct. A recombinant protein with both fluorescent and bioluminescent characteristics might take advantage of the strengths of both reporters. An expression vector encoding a chimeric protein in which GFP was tethered to Luc through a 19-amino acid linker was prepared and characterized. Western blotting with antibodies specific for either GFP or Luc showed that a protein of appropriate size was expressed in transfected cells. Fluorescence microscopy revealed bright green fluorescence from transfected cells, indicating proper formation of the GFP chromophore. Luc enzymatic activity in protein extracts from transfected cells showed that Luc was fully functional. The treatment of living cell cultures stably expressing the GFP-Luc fusion protein with the protein translation-inhibitor cycloheximide (Chx) was used to show that the half-life for Luc protein activity was approximately 2 h at 37 degrees C. The utility of this dual-function reporter protein was shown by the identification of single living cells expressing the chimeric protein within a population by fluorescence microscopy, followed by quantification of Luc activity from the same living cells.

Visualization of Pit-1 transcription factor interactions in the living cell nucleus by fluorescence resonance energy transfer microscopy.

The pituitary-specific transcription factor Pit-1 forms dimers when interacting with specific DNA elements and has been shown to associate with several other nuclear proteins. Recently, techniques have become available that allow visualization of protein-protein interactions as they occur in single living cells. In this study, the technique of fluorescence resonance energy transfer (FRET) microscopy was used to visualize the physical interactions of Pit-1 proteins fused to spectral variants of the jellyfish green fluorescent protein (GFP) that emit green or blue light [blue fluorescent protein (BFP)]. An optimized imaging system was used to discriminate fluorescence signals from single cells coexpressing the BFP- and GFP-fusion proteins, and the contribution of spectral overlap to background fluorescence detected in the FRET images was established. Energy transfer signals from living cells expressing a fusion protein in which GFP was tethered to BFP by short protein linker was used to demonstrate acquisition of FRET signals. Genetic vectors encoding GFP- and BFP-Pit-1 proteins were prepared, and biological function of the fusion proteins was confirmed. FRET microscopy of HeLa cells coexpressing the GFP- and BFP-Pit-1 demonstrated energy transfer, which required the two fluorophores to be separated by less than 100 A. Biochemical studies previously demonstrated that Pit-1 physically interacts with both c-Ets-1 and the estrogen receptor. FRET imaging of cells coexpressing BFP-Pit-1 and GFP-Ets-1 demonstrated energy transfer between these fusion proteins, a result consistent with their association in the nucleus of these living cells. In contrast, there was no evidence for energy transfer between the BFP-Pit-1 and an estrogen receptor-GFP fusion proteins. It is likely that the FRET imaging approach described here can be applied to many different protein-partner pairs in a variety of cellular contexts.

Fret imaging of pit-1 protein interactions in living cells.

The combined use of fluorescence resonance energy transfer (FRET) microscopy and expression of genetic vectors encoding protein fusions with green fluorescent protein (GFP) and blue fluorescent protein (BFP) provides an exceptionally sensitive method for detecting the interaction of protein partners in living cells. The acquisition of FRET signals from GFP- and BFP-fusion proteins expressed in living cells was demonstrated using an optimized imaging system and high sensitivity charge coupled device camera. This imaging system was used to detect energy transfer signals from a fusion protein containing GFP physically linked to BFP expressed in living HeLa cells. In contrast, the co-localization of noninteracting GFP- and BFP-fusion proteins was not sufficient for energy transfer. The FRET imaging system was then used to demonstrate dimerization of the pituitary-specific transcription factor Pit-1 within the living cell nucleus. © 1998 Society of Photo-Optical Instrumentation Engineers.