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At least 19 recordsLinked to original sources
PubMed1990

Altering enzymatic activity: recruitment of carboxypeptidase activity into an RTEM beta-lactamase/penicillin-binding protein 5 chimera.

The D-Ala-D-Ala carboxypeptidases/transpeptidases (penicillin-binding proteins, PBPs) share considerable structural homology with class A beta-lactamases (EC 3.5.2.6), although these beta-lactamases have no observable D-Ala-D-Ala carboxypeptidase activity. With the objective of recruiting such activity into a beta-lactamase background, we have prepared a chimeric protein by inserting a 28-amino acid segment of PBP-5 of Escherichia coli in place of the corresponding region of the RTEM-1 beta-lactamase. The segment thus inserted encompasses two residues conserved in both families: Ser-70, which forms the acyl-enzyme intermediate during beta-lactam hydrolysis, and Lys-73, whose presence has been shown to be necessary for catalysis. This chimera involves changes of 18 residues and gives a protein that differs at 7% of the residues from the parent. Whereas RTEM beta-lactamase has no D-Ala-D-Ala carboxypeptidase activity, that of the chimera is significant and is, in fact, about 1% the activity of PBP-5 on diacetyl-L-Lys-D-Ala-D-Ala; in terms of free energy of activation, the chimera stabilizes the transition state for the reaction to within about 2.7 kcal/mol of the stabilization achieved by PBP-5. Furthermore, the chimera catalyzes hydrolysis exclusively at the carboxyl-terminal amide bond which is the site of cleavage by D-Ala-D-Ala carboxypeptidase. Though containing all those residues that are conserved throughout class A beta-lactamases and are thought to be essential for beta-lactamase activity, the chimera has considerably reduced activity (approximately 10(-5) on penams such as penicillins and ampicillins as substrates. As a catalyst, the chimera shows an induction period of approximately 30 min, reflecting a slow conformational rearrangement from an inactive precursor to the active enzyme.

Alanine↗
PubMed1994

Osteoclast recruiting activity in bone matrix.

An activity that recruits osteoclasts has been identified and partially characterized from bone matrix. Bone-derived osteoclast recruiting activity (BORA) was co-purified with osteogenin, a bone inductive protein. Osteogenin was extracted from bovine bone with 6 M urea and purified by chromatography on hydroxyapatite, heparin-Sepharose and Sephacryl S-200 gel filtration. The biologically active osteoclast formation-stimulating material was further purified by C18 reverse phase HPLC. BORA is obviously distinct from osteogenin and transforming growth factor beta (TGF-beta), since further purified osteogenin and pure TGF-beta did not stimulate the formation of osteoclast-like cells. BORA (0.1-10 micrograms/ml) stimulated the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNC) in a dose-dependent manner. These multinucleated cells resorbed bone when cultured on bovine bone slices. The effect of BORA is primarily directed to differentiate osteoclast precursors, since it did not stimulate osteoclast function in in vitro resorption assay where disaggregated rat osteoclasts were cultured on bovine bone slices. However, after 24 h preincubation with 50 nM PTH in the mouse calvaria assay, BORA at 10 micrograms/ml significantly stimulated bone resorption.

Animals↗
PubMed2004

Active recruitment increased enrollment in a hereditary cancer registry.

BACKGROUND AND OBJECTIVE: The Mid-Atlantic Cancer Genetics Network (MACGN) targets individuals from cancer risk assessment clinics for recruitment into a national hereditary cancer registry. We sought to determine whether different recruitment methods used in a high-risk breast and ovarian cancer clinic yielded differences into enrollment into MACGN. METHODS: Two methods of recruitment were compared over an 8-month period. A passive recruitment technique, used during the first 4 months of recruitment, involved distribution of a brochure. An active recruitment method, used during the second 4-month period, required a MACGN recruiter to approach patients and initiate a brief discussion of the registry. RESULTS: During the first 4-month period, 158 eight patients were seen in the clinic and 142 were seen in the second 4-month period. During passive recruitment, 20% of available patients were approached, compared with 63% during active recruitment. Active recruitment also resulted in fourfold increase over passive recruitment in enrollment (from 15.6% to 67.4%). CONCLUSION: Allocating research staff specifically for recruitment and personal contact with potential participants is effective in achieving increased enrollment into a national hereditary cancer research registry.

Adult↗
PubMed2005

Recruitment index as a measure of patient recruitment activity in clinical trials.

Recruitment of patients for clinical trials is one of the major hurdles on the way to successful completion of any human research study. Direct comparison of recruitment activity in different sites and projects is not easy due to lack of a unified measure of recruitment efficacy. The author introduces a new variable, the Recruitment Index, which represents the number of days required for an average study site in a multicenter study to recruit one analyzable patient. Once established in previous studies, an indication-specific Recruitment Index can be used for a variety of purposes, e.g. evaluation of efficacy of various recruitment strategies, planning duration of recruitment period for a new study, or projecting the number of participating sites required to supply a given number of analyzable patients within a certain period.

Clinical Trials as Topic↗
PubMed1986

Human placental conditioned medium contains monocyte-derived recruiting activity (MRA).

Monocyte-derived recruiting activity (MRA) stimulates the release of granulopoietic colony-stimulating factors (CSF) by endothelial cells. We carried out studies designed to test the hypotheses that human placental conditioned medium (HPCM), a widely utilized source of CSA for in vitro studies, contains both MRA and CSA and that these molecules could be separated on the basis of their isoelectric points. In five separate studies, concentrated samples of HPCM were chromatofocused and fractions tested in bioassays for MRA and CSA. We found that HPCM contains both MRA and CSA, that the majority of the MRA eluted at a pH of from 7.6 to 8.2 and was 2000-fold purified. CSA eluted at pH 5.6 or below. No CSA was detectable in the major MRA peak. We conclude that HPCM contains heterogeneous granulopoietic activities, that the isoelectric points of MRA and CSA are substantially different and that chromatofocusing provides a rapid single-step method for separating these two distinct granulopoietic factors from complex conditioned media.

Colony-Stimulating Factors↗
PubMed1992

N-acetylation is required for the lactotrope recruitment activity of alpha-melanocyte-stimulating hormone and beta-endorphin.

Pituitary neurointermediate lobe (NIL) cells obtained from ovariectomized rats and exposed to 17 beta-estradiol in vitro have been shown to release an activity that induces acute recruitment of additional PRL-secreting cells. We have recently reported that alpha MSH, a major secretory product of the NIL, can substitute for this lactotrope-recruiting factor released by NIL cells in response to 17 beta-estradiol. beta-Endorphin (beta END) was not effective in this regard. Inasmuch as the degree of acetylation is critical to the activities of both of these molecules in other systems, we decided to assess its importance to lactotrope-recruiting activity in the present study. Anterior pituitary cells from ovariectomized rats were cultured overnight, exposed to various treatments for 3 h, and then subjected to a reverse hemolytic plaque assay for PRL. Exposure to mono- and diacetylated alpha MSH (N-ac-alpha MSH and di-ac-alpha MSH, respectively) or N-acetylated beta END (N-ac-beta END) caused a significant increase in the fraction of anterior pituitary cells that released PRL. In contrast, the Des-acetylated variants of both molecules had no lactotrope-recruiting activity. In a dose-response study, maximally effective doses of di-ac-alpha MSH and N-ac-beta END were equally effective with respect to their function as lactotrope-recruiting factors. Furthermore, the two peptides acted cooperatively when simultaneously applied in submaximal concentrations. Taken together, these results demonstrate that 1) N-acetylation is an essential requirement for the lactotrope-recruiting activity of alpha MSH and beta END in vitro; and 2) di-ac-alpha MSH and N-ac-beta END can act in a cooperative fashion to recruit additional cells into the PRL-secreting population.

Acetylation↗
PubMed1992

An activation-recruitment scheme for use in muscle modeling.

The derivation of a new activation-recruitment scheme and the results of a study designed to test its validity are presented. The activation scheme utilizes input data of processed surface EMG signals, muscle composition, muscle architecture, and experimentally determined activation coefficients. In the derivation, the relationship between muscle activation and muscle fiber recruitment was considered. In the experimental study, triceps muscle force was determined for isometric elbow extension tasks varying in intensity from 10 to 100% of a maximum voluntary contraction (MVC) using both a muscle model that incorporates the activation scheme, and inverse dynamics techniques. The forces calculated using the two methods were compared statistically. The modeled triceps force was not significantly different from the experimental results determined using inverse dynamics techniques for average activation levels greater than 25% of MVC, but was significantly different for activation levels less than 25% of MVC. These results lend support for use of the activation-recruitment scheme for moderate to large activation levels, and suggest that factors in addition to fiber recruitment play a role in force regulation at lower activation levels.

Acceleration↗
PubMed2005

The conserved Leu-724 residue is required for both serine phosphorylation and co-activator recruitment for Stat1-mediated transcription activation in response to interferon-gamma.

The signal transducer and activator of transcription (STAT) proteins, a family of latent cytoplasmic transcription factors, become activated in response to extracellular ligand binding to cell surface receptors through tyrosine phosphorylation. Concurrently, a serine phosphorylation event in the transcription activation domain (serine 727 for Stat1) occurs. This serine phosphorylation is essential for the maximal transcription activity of Stat1. Here we show that, in addition to the Ser-727 residue and its phosphorylation, the conserved Leu-724 residue is also essential for gene activation mediated by Stat1. When Leu-724 is mutated to Ala, phosphorylation of Stat1 Ser-727 is defective both in vivo and in vitro. Surprisingly, we found a StatL724I mutant that lacks transcription activity despite normal Ser-727 phosphorylation. Further analyses show that Leu-724, as well as the phospho-Ser-727, are essential for the recruitment of the transcription co-activator CBP/p300 to the promoters of Stat1 target genes. Our results demonstrate that the conserved Leu-724 residue is a key residue that controls the maximal transcription activities of Stat1 in IFN-gamma signaling.

Base Sequence↗
PubMed1993

Des-acetylated variants of alpha-melanocyte-stimulating hormone and beta-endorphin can antagonize the mammotrope-recruiting activity of their acetylated forms.

We have previously reported that hypophysial neurointermediate lobe peptides, di-acetylated alpha-melanocyte-stimulating hormone (di-ac-alpha-MSH) and N-acetylated beta-endorphin (N-ac-beta-END), can acutely increase the relative number of prolactin-secreting cells in anterior pituitary cell cultures from ovariectomized rats. Inasmuch as the des-acetylated forms of these peptides (des-ac-alpha-MSH and beta-END) were not effective in this regard, we concluded that acetylation was an absolute requirement for manifestation of the recruitment response. The aim of the present study was to determine whether these des-acetylated variants could antagonize the mammotrope-recruiting activity of their acetylated congeners. Treatment of anterior pituitary cell cultures with di-ac-alpha-MSH and N-ac-beta-END increased the relative amount of prolactin secretors above control values. Interestingly, des-acetylated variants of alpha-MSH and beta-END blocked the mammotrope-recruitment activity of their respective acetylated forms. In addition, beta-END antagonized the mammotrope-recruitment activity of di-ac-alpha-MSH while des-ac-alpha-MSH did not attenuate the stimulatory effect of N-ac-beta-END. Given that mammotropes maintained in vivo are exposed to all these peptides, it is possible that these acetylated and non-acetylated congeners may act in an opposing manner to regulate dynamic prolactin release.

Acetylation↗
PubMed2004

The activator-recruited cofactor/Mediator coactivator subunit ARC92 is a functionally important target of the VP16 transcriptional activator.

The human activator-recruited cofactor (ARC), a family of large transcriptional coactivator complexes related to the yeast Mediator, was recently identified based on functional association with the activation domains of multiple cellular and viral transcriptional activators, including the herpes simplex viral activator VP16, sterol regulatory element binding protein, and NF-kappaB. Here we describe the biochemical purification and cloning of the 92-kDa ARC/Mediator subunit, ARC92, that is specifically targeted by the activation domain of the VP16 transactivator. Affinity chromatography using the VP16 activation domain followed by peptide microsequencing led to the identification of ARC92 as a specific cellular interaction partner of the VP16 activation domain. ARC92 associates with the VP16 activation domain in vitro and in vivo, and the VP16 binding domain of ARC92 is a strong competitive inhibitor of Gal4-VP16 in vivo. Moreover, small interfering RNA-mediated knockdown of ARC92 in human cells results in selective inhibition of Gal4-VP16 gene activation. Taken together, our results suggest that ARC92 is a direct and specific target of the VP16 transactivator that serves in the context of the ARC/Mediator coactivator as an important transducer of transcription activating signals from the VP16 activation domain to the RNA polymerase II transcriptional machinery.

Base Sequence↗
PubMed2004

1,25-Dihydroxyvitamin D3 stimulates cyclic vitamin D receptor/retinoid X receptor DNA-binding, co-activator recruitment, and histone acetylation in intact osteoblasts.

UNLABELLED: 1,25(OH)2D3 induces gene expression through the VDR. We used chromatin immunoprecipitation techniques to explore this 1,25(OH)2D3-induced process on the 25-hydroxyvitamin D3-24-hydroxylase (Cyp24) and Opn gene promoters in intact osteoblasts. Our studies show that 1,25(OH)2D3-induced transactivation is a dynamic process that involves promoter-specific localization of VDR and RXR, recruitment of histone acetyltransferase complexes, and in the case of the Cyp24 gene, modification of histone 4. INTRODUCTION: The vitamin D receptor (VDR) binds as a retinoid X receptor (RXR) heterodimer to target DNA sequences and facilitates the recruitment of protein complexes that are essential for transcriptional modulation. These complexes include an acetyltransferase component that contains members of the p160 family and p300/CBP as well as human mediator that contains D receptor interacting protein (DRIP205). The objective of this study was to investigate the kinetics of VDR/RXR binding to 25-hydroxyvitamin D3-24-hydroxylase (Cyp24) and osteopontin (Opn) target gene promoters and to explore the recruitment and subsequent activities of co-activator complexes on these target genes in intact cells. MATERIALS AND METHODS: Mouse osteoblastic MC3T3-E1 cells and mouse primary calvarial osteoblasts (MOBs) were cultured in alphaMEM medium supplemented with 10% FBS. Confluent cells were treated with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or the vitamin D antagonist ZK159222, and the ability of these compounds to induce localization of VDR and RXR to specific regions of Cyp24 and Opn target genes was examined using chromatin immunoprecipitation techniques. The ability of both compounds to induce the recruitment of co-activator proteins such as p160 family members, CBP and DRIP205, and to increase the level of histone acetylation on the two gene promoters in MC3T3-E1 cells was also examined. RESULTS: 1,25(OH)2D3 induces rapid association of the VDR and RXR with both the Cyp24 and the Opn gene promoters in both MC3T3-E1 osteoblasts and MOBs, interactions that are both rapid and cyclic in nature. 1,25(OH)2D3 treatment also induces rapid recruitment of co-regulators such as SRC-1, -2, and -3, CBP, and p300 to both promoters, recruitment that leads to acetylation of histone 4 on Cyp24 but not the Opn. DRIP205 is also recruited to the two promoters in response to hormonal stimulation, an appearance that correlates directly with entry of RNA pol II. Studies with the vitamin D antagonist ZK159222 suggest a complex mode of action of this compound in blocking 1,25(OH)2D3-induced transcription. Our studies indicate that 1,25(OH)2D3-induced transactivation in intact osteoblasts is a dynamic process that involves promoter-specific localization of VDR and RXR as well as the recruitment of a number of co-regulators essential to 1,25(OH)2D3-induced transcription. CONCLUSIONS: We conclude that co-regulators essential for the transcriptional activity of the steroid receptor gene family are indeed critical for the actions of 1,25(OH)2D3. Selective use of co-regulators by target genes, however, may provide a mechanism for the unique and perhaps gene-selective responses observed with synthetic analogs such as ZK159222.

Acetylation↗
PubMedDate not supplied

Monocyte/macrophage recruitment, activation and differentiation modulate interleukin-8 production: a paracrine role of tumor-associated macrophages in tumor angiogenesis.

Tumor-associated macrophages (TAM) have been shown to play an important role in tumor angiogenesis. The purpose of this study was to determine whether monocyte recruitment, activation and differentiation mediated by monocyte chemotactic protein-1 (MCP-1) and macrophage colony stimulating factor (M-CSF) modulate the expression of the angiogenic factor, Interleukin (IL)-8. Isolated human peripheral blood monocytes secreted low basal levels of IL-8. Incubation of monocytes with M-CSF or MCP-1 resulted in an up-regulation of IL-8 mRNA and protein expression. The differential expression of IL-8 by monocytes following MCP-1 and M-CSF treatments involved activation of the NFkB transcription factor. Further activation with lipopolysaccharide (LPS) caused an increase in IL-8 secretion in monocytes but not in monocyte-derived macrophages (MDM). MDM-conditioned media significantly up-regulated IL-8 expression in human malignant melanoma cells in vitro. In summary, we demonstrated that MCP-1 and M-CSF, critical for monocyte recruitment, activation and differentiation, differentially regulate IL-8 expression and may play an important role in monocyte/macrophage-mediated tumor angiogenesis.

Cell Differentiation↗
PubMed2000

Homeodomain and winged-helix transcription factors recruit activated Smads to distinct promoter elements via a common Smad interaction motif.

We have investigated the regulation of the activin-inducible distal element (DE) of the Xenopus goosecoid promoter. The results show that paired-like homeodomain transcription factors of the Mix family, Mixer and Milk, but not Mix.1, mediate activin/TGF-beta-induced transcription through the DE by interacting with the effector domain of Smad2, thereby recruiting active Smad2/Smad4 complexes to the Mixer/Milk-binding site. We identify a short motif in the carboxyl termini of Mixer and Milk, which is demonstrated to be both necessary and sufficient for interaction with the effector domain of Smad2 and is required for mediating activin/TGF-beta-induced transcription. This motif is not confined to these homeodomain proteins, but is also present in the Smad2-interacting winged-helix proteins Xenopus Fast-1, human Fast-1, and mouse Fast-2. We demonstrate directly that transcription factors of different DNA-binding specificity recruit activated Smads to distinct promoter elements via a common mechanism. These observations, together with the temporal and spatial expression patterns of Mixer and Milk, lead us to propose a model for mesoendoderm formation in Xenopus in which these homeodomain transcription factor/Smad complexes play a role in initiating and maintaining transcription of target genes in response to endogenous activin-like signals.

Activins↗
PubMed1985

Monocyte-derived recruiting activity: kinetics of production and effects of endotoxin.

Cultured monocytes release a factor, monocyte-derived recruiting activity (MRA), which stimulates fibroblasts, endothelial cells, and T lymphocytes to produce colony-stimulating activity (CSA). We studied the kinetics of MRA production using a technique in which MRA levels were measured in a two stage bioassay. We used umbilical vein endothelial cells as the MRA-responsive (CSA-producing) cells, and normal colony-forming unit granulocyte-macrophage (CFU-GM)-enriched bone marrow cells (T lymphocyte- and monocyte-depleted, low density bone marrow cells) as the CSA-responsive cells. MRA stimulated a 30-fold increase in CSA production by endothelial cells. MRA production was detected in supernatants from as few as 10(3) monocytes per milliliter, required the presence of fetal calf serum, and was inhibited by cycloheximide (10 to 100 micrograms/mL) and puromycin (10 to 50 micrograms/mL). Production was detectable after 24 hours of monocyte incubation, was maintained for three days, and fell to undetectable levels by seven days. With the addition of bacterial endotoxin (lipopolysaccharide [LPS]) (50 micrograms per 10(6) cells), MRA was detectable after only three hours of incubation, and levels peaked at 24 hours. Further, maximum MRA levels in the supernatants of LPS-stimulated monocytes were up to ten times greater than peak levels in the supernatants of unstimulated monocytes. Endotoxin augmented monocyte production of MRA to a greater extent than it did CSA production, indicating that the stimulation of CSA production by endotoxin may be at least partly indirect. The responsiveness of MRA production to endotoxin in vitro is consistent with the notion that MRA may be a biologically relevant regulator of CSA production by cells of the hematopoietic microenvironment.

Colony-Stimulating Factors↗
PubMed2002

Toward synthetic transcription activators: recruitment of transcription factors to DNA by a PNA-peptide chimera.

A PNA-peptide chimera designed to mimic the biochemical function of transcription activators has been synthesized and characterized. The bis-PNA segment binds specifically to a DNA site while the 20-residue peptide is capable of binding to the transcription factors Gal11 and Gal80. The PNA-peptide chimera thus mimics one of the central functions of a native transcription activator, recruitment of transcription factors to a specific DNA site.

DNA↗
PubMed1999

Comparing participants and nonparticipants in a smoking cessation trial: selection factors associated with general practitioner recruitment activity.

We studied the relationship between smokers' sociodemographic characteristics, their smoking habit, health status, and the probability of their having been approached for recruitment in the smoking cessation trial performed in Turin, Italy, with the aim of gathering information on the role of selection criteria adopted by general practitioners (GPs) in offering anti-smoking counseling. The 965 smokers who were offered participation in the trial were matched to a sample of eligible smokers (n = 277), selected from the rosters of the 42 GPs collaborating in the trial, who had not been invited to participate. The probability of being offered enrollment, estimated through a multiple conditional logistic regression model, assuming the GP as the matching variable, was significantly increased for intermediate (10-19 cigarettes per day: odds ratio [OR] = 4.13; 95% confidence interval [CI]: 2.63-6.47) and heavy (20 cigarettes per day or more: OR = 10.12; 95% CI: 6.51-15.75) smokers, for smokers diagnosed with chronic cardiovascular (OR = 2.06; 95% CI: 1.19-3.58), or respiratory (OR = 2.50; 95% CI: 1.40-4.48) diseases, and for smokers mentioning an intermediate number (2-4) of past quit attempts (OR = 3.70; 95% CI: 2.18-6.28). General Practitioners focused their recruitment activity on higer-risk smokers or smokers who had tried to quit, to offer more clues for intervention. Assessing the potential public health benefit of preventive interventions requires a more systematic evaluation of the generalizability of the reported findings.

Adult↗
PubMed1980

Migration and cell recruiting activity of specifically sensitized lymphocytes in sponge matrix allografts.

The migration of indium-III-labeled specifically sensitized lymphocytes (SSLs) and unsensitized spleen lymphocytes (ULs) into sponge matrix allografts was studied in irradiated mice. Intravenously injected SSLs were preferentially recruited to the graft bearing the sensitizing alloantigen. High numbers of these SSLs, however, were attracted regardless of their immunological specificity to a third-party graft, when cells sensitized against the third-party antigen were given simultaneously. When SSLs were injected locally into a specific and third-party graft, i.v. injected ULs also homed preferentially to the specific graft. Prior treatment of SSLs with anti-theta serum and complement abrogated their cell recruiting capacity. The percentage of injected lymphocytes recruited to the specific allograft during the first 36 hr increased with the number of SSLs injected. The duration of augmented lymphocyte recruitment, however, was inversely related to the number of SSLs injected into the graft. Furthermore, there is some evidence that SSLs which have been recruited to an allograft migrate to a second site of antigen challenge and initiate there a high lymphocyte recruitment. These results suggest that increased lymphocyte recruitment to an allograft is initiated by an immunologically specific interaction between SSLs and alloantigen. The cells, however, which are preferentially recruited to an allograft, comprise small unsensitized lymphocytes and SSLs regardless of their immunological specificity. Lymphocytes with cell recruiting activity are T cells and appear to be very mobile in vivo. The mechanism described might have an important amplifying effect on allograft rejection.

Animals↗
PubMed2002

Activator recruitment by the general transcription machinery: X-ray structural analysis of the Oct-1 POU domain/human U1 octamer/SNAP190 peptide ternary complex.

Transcriptional activation of the human U1 snRNA genes is dependent on a noncanonical octamer element contained within an upstream enhancer. The U1 octamer only weakly recruits the Oct-1 POU domain, although recruitment is stimulated by a peptide containing the Oct-1-binding domain of SNAP190. Structural analysis of the Oct-1 POU domain/U1 octamer/SNAP190 peptide complex revealed that SNAP190 makes extensive protein contacts with the Oct-1 POU-specific domain and with the DNA phosphate backbone within the enhancer. Although SNAP190 and OCA-B both interact with the Oct-1 POU domain through the same Oct-1 interface, a single nucleotide within the U1 octamer ablates OCA-B recruitment without compromising activator recruitment by SNAP190.

DNA-Binding Proteins↗