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At least 19 recordsLinked to original sources
PubMed2026

E2F7 promotes lung adenocarcinoma progression by affecting phosphorylation and stabilization of β-catenin.

BACKGROUND: E2F transcription factor 7 (E2F7) has been implicated in the tumorigenesis and progression of multiple cancer types; however, the molecular mechanisms through which E2F7 regulates malignant phenotypes in cancer cells remain largely undefined. In this study, we investigated the biological functions and underlying mechanisms of E2F7 in lung adenocarcinoma (LUAD). METHODS: E2F7 expression in LUAD was analyzed using The Cancer Genome Atlas (TCGA) datasets and further validated in clinical specimens via quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry. The effects of E2F7 on cancer cell self‑renewal and epithelial-mesenchymal transition (EMT) were assessed using sphere formation and Transwell assays, respectively. In vivo tumorigenicity and metastasis were evaluated using xenograft models combined with extreme limiting dilution analysis to assess tumor-initiating capacity. Wnt/β‑catenin pathway activity was measured using T-cell factor optimal promoter luciferase reporter plasmid/far-from optimal promoter luciferase reporter plasmid (TOP/FOP) flash reporter assays. β‑Catenin expression, stability, and ubiquitination were examined via western blotting, cycloheximide chase assays, and ubiquitination assays. Protein-protein interactions among E2F7, β‑catenin, and glycogen synthase kinase 3 beta (GSK3β) were verified through co‑immunoprecipitation (Co‑IP), glutathione S‑transferase (GST) pull‑down, and immunofluorescence assays. Truncated mutants were generated to map the functional binding domains of E2F7. In vitro immunoprecipitation and kinase assays were further performed to confirm that E2F7 regulates GSK3β autophosphorylation and β‑catenin phosphorylation. RESULTS: Bioinformatic analyses revealed that E2F7 was significantly upregulated in LUAD tissues, and elevated E2F7 expression correlated with poor patient prognosis. Functional assays demonstrated that E2F7 promoted LUAD cell self‑renewal and EMT. Mechanistically, cytoplasmic E2F7 directly associated with β‑catenin through its DNA‑binding domain (DBD) and PHA03247 domain. E2F7 modulated β‑catenin phosphorylation at Ser675 and Ser33/37/T41, thereby inhibiting ubiquitin‑mediated degradation and enhancing β‑catenin protein stability. Furthermore, E2F7 interacted with GSK3β and suppressed its autophosphorylation at Tyr216, concomitant with reduced β-catenin phosphorylation at Ser33/37/T41 and its accumulation. CONCLUSION: Collectively, these findings indicate that E2F7 drives LUAD malignant progression through regulation of the GSK3β/β‑catenin signaling axis and stabilization of β‑catenin. This study unveils a novel oncogenic mechanism of E2F7 in LUAD and identifies E2F7 as a promising therapeutic target for clinical intervention in LUAD.

E2F7↗
PubMed1991

Optimizing the promoter and ribosome binding sequence for expression of human single chain urokinase-like plasminogen activator in Escherichia coli and stabilization of the product by avoiding heat shock response.

The expression of recombinant single-chain urokinase-like plasminogen activator (rscuPA) in Escherichia coli was optimized by fusing the puk gene to different promoters and ribosome binding sequences. Comparison of the tac, trp and lambda PL promoters showed that expression was maximal under tac control. Variation in the ribosome binding sequence and its distance to the AUG start codon yielded a further slight improvement of expression. The largest increase in rscuPA expression was achieved by variations in the host strain and growth conditions. In E. coli DG75 grown at 37 degrees C maximal expression was achieved 30 min after induction and decreased gradually until 240 min after induction. Growth at 30 degrees C yielded maximal expression 60 min after induction and resulted in reduced activity at longer times. Western blot analysis of the products showed that degradation of rscuPA was much larger at 37 degrees C than at 30 degrees C. Using E. coli CAG630 carrying the htpR mutation, which avoids heat shock response, for expression of rscuPA eliminated the instability of the product at both temperatures. Expression in this strain was even more efficient than in E. coli JM101 carrying the lon mutation. It is concluded that induction of the general heat-shock response in E. coli must be avoided to obtain stabilization of rscuPA. This drastically improves the overall yield of rscuPA from recombinant E. coli strains.

Base Sequence↗
PubMed2025

Codon Composition in Human Oocytes Reveals Age-Associated Defects in mRNA Decay.

Oocytes from women of advanced reproductive age exhibit diminished developmental potential, but the underlying mechanisms remain incompletely defined. Oocyte maturation depends on translational control of maternal mRNA synthesized during growth. We performed a computational analysis on human oocytes from women <30 versus &#x2265;40 years and observed that mRNA GC content correlates negatively with half-life in oocytes from young (<30 yr) but positively with oocytes from aged (>40 yr) women. In young oocytes, longer mRNA half-life is associated with lower protein abundance, whereas in aged oocytes GC content correlates positively with protein abundance. During the GV-to-MII transition, codon composition stratifies stability: codons that support rapid translation (optimal) stabilize mRNA, while slow-translating codons (non-optimal) promote decay. With reproductive aging, GC-containing codons become more optimal and align with increased protein abundance. These findings indicate that reproductive aging remodels codon-optimality-linked, translation-coupled mRNA decay, stabilizing a subset of GC-rich maternal mRNA that may be prone to excess translation during maturation. Our analysis is explicitly within human reproductive aging; it does not revisit cross-species stability rules. Instead, it shows that sequence-stability relations are reprogrammed with age within human oocytes, including an inversion of the GC-stability association during GV-to-MII transition. Disruption of the normal mRNA clearance program in aged oocytes may compromise oocyte competence and alter maternal mRNA dosage, with downstream consequences for early embryonic development.

Humans↗
PubMed1992

Mapping and characterization of the promoter elements of the regulatory nif genes rpoN, nifA1 and nifA2 in Rhodobacter capsulatus.

The promoter elements responsible for the expression of the regulatory nif genes rpoN, nifA1 and nifA2 of Rhodobacter capsulatus were mapped by exonuclease-III-mediated deletions and by primer extension analysis. The rpoN promoter maps 600 bp upstream of rpoN and has the characteristic features of a -24/-12 promoter. The upstream activator sequence (UAS) displays two mismatches with the NIFA consensus sequence and is located 37 bp upstream of a perfect -24/-12 promoter element. The spacing and/or the helical phasing of these two promotor elements was found to be important for promoter function. In addition, an UAS half-site may contribute to optimal promoter function. The rpoN UAS can partially substitute for the UAS of the nifE promoter. An open reading frame with homology to Klebsiella pneumoniae NIFU was identified between the rpoN promoter and rpoN and termed nifU2 since another nifU-like gene (nifU1) is located in a conventional nifUSVW operon in nif region A. Thus, rpoN, encoding an alternative sigma factor for RNA polymerase, is cotranscribed with a nifU analogous gene from an rpoN-dependent promoter. Mapping of the promoter elements involved in the expression of nifA copy 1 and copy 2 identified a novel promoter type. A conserved distal promoter element is likely to represent the binding site of NTRC in R. capsulatus. The DNA region preceding the mapped 5' ends of the nifA transcripts displays much less homology. The distance between the distal and proximal elements is about 100 bp.

Amino Acid Sequence↗
PubMed1992

Protein binding sites within the human thymidine kinase promoter.

DNase I footprint analysis, using total HeLa cell nuclear extract and purified transcription factor Sp1, was carried out to determine the various protein binding sites within the human thymidine kinase promoter. The promoter has two separate CCAAT elements and multiple Sp1-binding sites, as well as at least one undefined protein binding site. Detailed analysis of protein binding to the two CCAAT elements showed that changing the spacing between the two CCAAT elements altered both protein binding to the distal CCAAT element as well as promoter activity. Both CCAAT elements can act as functional transcription elements, but are not oriented for optimal promoter strength in the human tk promoter. Our studies show that a promoter fragment that has been previously shown to be the minimal region to maintain a serum responsive promoter regulation apparently contains only a single Sp1-binding domain and the more distal of the CCAAT elements.

Base Sequence↗
PubMed1979

Enhancement of hexose uptake in human polymorphonuclear leukocytes by activated complement component C5a.

The polymorphonuclear leukocyte (PMNL) depends on glucose as a source of energy for motility, chemotaxis, phagocytosis, and bactericidal activity. Activated complement (C5a) at low concentrations stimulates carrier-mediated carbohydrate transport in PMNLs as measured by the uptake of 2-deoxy-D-[(3)H]glucose. Human PMNLs were preincubated at 37 degrees C for 15 min with zymosan-activated human serum or various purified preparations of human C5a. A concentration-dependent increase in deoxyglucose transport (>700% of control) into PMNLs occurred with all test substances. Reaction was linear for 30 min, and uptake of deoxyglucose followed saturation kinetics. C5a caused a decrease in the K(m) for deoxyglucose, from 0.53 to 0.11 mM, without altering the V(max) (44 nmol/30 min per 5 x 10(6) PMNLs in control and 46.6 with C5a). The optimal concentration of C5a for enhanced carrier-mediated transport of deoxyglucose was similar to that which promoted optimal chemotaxis. Activated serum from C5-deficient mice had little or no effect on deoxyglucose transport whereas that from normal syngeneic mice enhanced deoxyglucose transport. C5a did not enhance deoxyglucose transport into isolated erythrocytes, platelets, or lymphocytes. The deoxyglucose within the cell was primarily in the phosphorylated form, and hexokinase activity was not increased in PMNLs stimulated with C5a, indicating that hexokinase was not rate limiting and that enhanced transport was the mechanism of the C5a activity. Insulin at physiologic concentration (10 ng/ml) had no effect on deoxyglucose transport in PMNL and did not act as a competitive inhibitor of C5a. This insulin-like bioactivity could be detected with the amount of C5a that would be present after activation of 0.1-0.5% of the C5 in 1 ml of serum. This suggests that uptake of [(3)H]deoxyglucose by PMNLs might serve as a highly sensitive test for activation of the fifth component of complement.

Biological Transport↗
PubMed1992

Characterization of the human pleiotrophin gene. Promoter region and chromosomal localization.

The protein (PTN) encoded by the pleiotrophin (PTN) gene belongs to a recently described family of heparin-binding cytokines whose expression is temporally and spatially regulated during development. We have now isolated genomic clones of the human PTN gene, characterized its promoter region, determined its transcription initiation site(s), and established functional activity of the PTN promoter. A fragment -550/+191 that contains a CAAT box, no apparent TATA box, and four consensus sites for the binding of MyoD is sufficient to provide optimal promoter activity. A serum response element is found at -559 to -568. We also have identified the human PTN gene on chromosome 7, band q33 and the mouse Ptn gene on chromosome 6, respectively. The data thus identify and characterize the 5' end of the PTN gene and its promoter region, suggest potential regions that may contribute to the regulation of its transcriptional activity, and localize the PTN gene in human and mouse chromosomes.

3T3 Cells↗
PubMed1992

Functional analysis of the human platelet-derived growth factor A-chain promoter region.

The platelet-derived growth factor (PDGF) A-chain gene is a developmentally regulated gene that is expressed in high levels in a limited number of normal and transformed cell lines and in cells stimulated by cytokines, including PDGF itself. We have now analyzed potential regulatory elements in 3.6 kilobase pairs (kb) of the 5'-flanking sequences of the human PDGF A-chain gene using reporter gene constructs and transient transfection analyses. The region between base pairs (bp) -618 and +392 (relative to the transcription initiation site) is sufficient for optimal promoter activity. A highly G + C region containing three contiguous Sp1 binding sites between bp -150 and -33 contributes over 80% of promotor activity. DNase I footprinting analyses indicates that Sp1 binds to and protects over 57 bp of this G + C region. A functional serum response element is located within bp -477 and -468 and positively regulates induction of PDGF A by PDGF. A negative regulatory (silencer) element is located from -1.9 to -0.9 kb. The results suggest that the major constitutive expression of the PDGF A-chain gene requires a highly G + C-rich region containing three Sp1 binding sites and that induction of the PDGF A-chain gene by PDGF is mediated by a SRE located at bp -477 to -468.

Base Sequence↗
PubMed1992

Polysialic acid is required for optimal growth of axons on a neuronal substrate.

Formation of axonal pathways involves a variety of molecules that influence cell-cell interactions. The polysialic acid (PSA) moiety of the neural cell adhesion molecule (NCAM) is present on neuronal surfaces during process outgrowth. Our studies reveal that the removal of PSA causes a decrease in the rate of elongation of retinal cell processes on a substrate of neuronal membranes derived from chick tectum. This effect was partially reversed by antibodies against the L1 adhesion molecule, but not by antibodies against NCAM, N-cadherin, or beta 1-integrins. This predominant effect of PSA on L1 was also observed in short-term, cell-cell adhesion assays, suggesting that PSA promotes optimal outgrowth on neuronal substrates by limiting the consequences of L1-mediated adhesion.

Animals↗
PubMed1992

Production and secretion of high levels of recombinant human acetylcholinesterase in cultured cell lines: microheterogeneity of the catalytic subunit.

To allow for structural analysis of the human acetylcholinesterase (hAChE) subunit, a series of eukaryotic vectors was designed for efficient expression. Several eukaryotic multicistronic expression vectors were tested in various mammalian cell lines. All expression vectors contained the selectable neo gene under control of a weak promoter, while the hAChE cDNA was under control of the cytomegalovirus (CMV) immediate-early or Rous sarcoma virus long terminal repeat (RSV LTR) or simian virus 40 (SV40) early promoters. Optimal production and secretion of recombinant hAChE (rehAChE) was achieved in the embryonal kidney 293 cell line transfected either with the RSV-hAChE or with CMV-hAChE expression vectors. Clones expressing and secreting as much as 5-25 pg of enzyme per cell per 24 h were obtained without resorting to coamplification techniques or continuous maintenance of cells under selective pressure. The purified (specific activity of 6000 units per mg protein) homodimer and tetramer enzyme molecules displayed typical AChE biochemical properties: a Km value of 120 microM for acetylthiocholine; a kcat value of 3.9 x 10(5)/min, and selective by AChE-specific inhibitors. Catalytic subunit dimers (130 kDa) exhibit differential N-glycosylation patterns, and upon reduction resolve into 67- and 70-kDa monomeric subunits. These two forms appear as a single discrete 62-kDa band following deglycosylation by N-glycanase. The N-terminal amino acid sequence analysis of the purified mature enzyme suggests the existence of two alternative cleavage sites for the removal of the signal peptide, in which the 'mature' position 1 is either Ala31 or Gly33. Both of these positions conform with the consensus signal peptide recognition sequences and demonstrate bidirected processing of signal peptides on a native molecule.

Acetylcholinesterase↗
PubMed1992

Infertility: psychotherapeutic issues.

In supportive therapy with infertility patients, the clinician tries to relieve dysphoria and enhance self-esteem. Dynamically informed supportive interventions are designed to decrease guilt that may relate to past sexual activities, sexually related diseases, or abortions. These interventions should also be empathetic, promote optimism and reality testing, help with problem solving, allow catharsis and ventilation, decrease feelings of isolation and loneliness, educate and clarify, and praise and encourage where appropriate. Mental health clinicians have an important role to play in the treatment of these patients, provided they learn enough about the psychology of the experience of infertility and about the technology utilized in its treatment. As the number of people seeking treatment for infertility grows, the need for skilled therapists for this population will grow at a parallel rate.

Female↗
PubMed1978

Physicians' attitudes toward early intervention.

Fourteen pediatricians serving Boston neighborhood health centers and fourteen pediatric neurologists in the greater Boston area were interviewed in order to determine their attitudes toward early intervention for infants with developmental disorders. The two groups differed significantly over the potential of early treatment for promoting optimal motor, cognitive, and affective development in the disadvantaged infant. Both groups viewed prevention of musculoskeletal deformities as appropriate for physically handicapped children and supported early intervention for the parents. When referral for treatment was delayed or withheld, the prime reasons given were an expression of a maturationist viewpoint, a desire for a definitive diagnosis prior to initiating treatment, the assumption that an adequate family can function without professional involvement, and misinterpretation of professional roles.

Attitude of Health Personnel↗
PubMed1979

Discrepancies between knowledge and use of diagnostic studies in asymptomatic patients.

The value of educational programs designed to promote optimal use of laboratory tests by physicians remains unsolved. To assess the effects of level of training and knowledge of test characteristics in determining laboratory ordering habits, physicians' use of laboratory tests as applied to asymptomatic patients was surveyed and their knowledge of four specific procedures was tested. The survey included 148 physicians both in training and in practice in Rochester, New York. Increased selectivity in the use of tests occurred during residency training, but selectivity correlated poorly with knowledge of test characteristics. These data underline the need for additional studies to determine the role of factors other than knowledge which contribute to the use of laboratory tests. Planners of cost containment programs and educational strategies will need to direct attention to these factors.

Clinical Laboratory Techniques↗
PubMed1992

Efficacy of chromium supplementation in athletes: emphasis on anabolism.

As the biologically active component of glucose tolerance factor (GTF), the essential trace mineral chromium is now being marked to athletes. GTF potentiates insulin activity and is responsible for normal insulin function. Thus, insulin's effects on carbohydrate, fat, and protein metabolism are dependent upon the maintenance of adequate chromium stores. Due to excessive chromium loss and marginal chromium intake, athletes may have an increased requirement for chromium. Therefore, in some circumstances the dietary supplementation of a chromium compound may be efficacious. The restoration and maintenance of chromium stores via supplementation would promote optimal insulin efficiency, necessary for high-level athletic performance. However, potential anabolic effects of enhanced insulin function would likely be marginal, and reports of short-term anabolic increases from the supplementation of an organic chromium compound need to be confirmed.

Animals↗
PubMed1992

Role of spore coats in the germinative response of Bacillus cereus to adenosine and its analogues.

Spores of the strain NCIB 8122 of Bacillus cereus have been depleted of coats by treatment with 0.1% sodium dodecyl sulfate--200 mM 2-mercaptoethanol--0.5 M NaCl (pH 9.6). The coat-depleted spores did not show any decrease in viability, heat resistance, refractility, dipicolinic acid content, or specific activities of several protoplastic enzymes. The germinative response of the coat-depleted spores to adenosine and several analogues thereof was found qualitatively similar to that obtained with intact spores. However, germination kinetics appeared to be affected by coat removal, since germination rate measured as loss of refractility was eight times slower even at inducer concentrations 10-fold higher than those required to promote optimal germination response of intact spores. Loss of heat resistance, on the other hand, was hardly affected by coat removal. These results suggest that, even though spore coats are not essential for the triggering reaction, they are required for a rapid evolution of the later events in the germination process.

Adenosine↗
PubMed1977

Influence of a deficiency of the second component of complement on the bactericidal activity of neutrophils in vitro.

Serum from three patients with a complete, selective deficiency of the second component of complement (C2) did not promote optimal killing of Staphylococcus aureus, 502A by neutrophilic polymorphonuclear leukocytes (PMN) in vitro. The addition of C2 reagent or the presence of heat-stable opsonin in the C2-deficient serum corrected the defective killing of S. aureus that was observed with patient or control PMN. PMN from the patients or control subjects killed bacteria with equal efficiency under conditions of optimal opsonization (normal pooled serum). However, twice-washed control PMN were better than patient PMN in killing S. aureus under circumstances of suboptimal opsonization (C2-deficient serum, heated C2-deficient serum, heated normal pooled serum, or no replacement of serum). The latter finding was due to residual C2 on the surface of twice-washed control cells. As repeated washing of control PMN progressively removed cell-associated C2, the staphylocidal effectiveness of the control RMN decreased to the level of patient PMN. In contrast to the findings with S. aureus, triply-washed PMN from patients or controls killed normal numbers of Escherichia coli, ON2, in C2-deficient serum.

Animals↗
PubMed1975

Dependence and psychotherapy--developmental considerations.

This article illustrates how the assessment of the level and degree of dependency in psychotherapy can be used as a diagnostic and therapeutic tool. Dependency varies with the stage of psychosexual development, with the degree and state of illness, and with the stage of treatment. Appropriate monitoring of this factor promotes optimal growth potential in the patient.

Affective Symptoms↗
PubMed1976

Predictive nursing: the baby and parents.

Predictive nursing as a health care strategy is not entirely new. What is new and emerging are means of sharpening our assessments of the events and conditions that support health, as well as specific models engaging the individual in assessing and altering or maintaining his position on the health continuum. This study has been shared with the readers to provide an encouraging direction toward new modes of health care delivery. We believe it is the most encouraging strategy for assisting families in promoting optimal health of infants from a very early point in the infant's life. The assessment techniques discussed in this paper relate to methods of identifying individual behavior patterns of newborn infants, behavioral interaction patterns of parents and infants during the early months and years of life, and the parents' perception of the infant. All of these measures are now being tested by the Nursing Child Assessment Project staff and will result in a systematic format of screening and assessing infants and families "at risk". With current knowledge, astoundingly correct predictions about cognitive development and achievement at school age of groups of children can be made with information about the mother's levels of education and the events of the perinatal periods. The measures discussed in this article concerning the infants' behavior, parent-child interaction, and the parents' perception of the child are factors which will go beyond our present ability to identify potential problems early. Predictive nursing of the infant and young child is our eventual goal. This means going a step beyond the current level of practice by beginning to systematically identify the proneness of individuals to good or bad health outcomes--specifically, the proneness of children to have developmental delays or problems--and to then provide a meaningful type of intervention before the developmental problem occurs.

Child Behavior↗