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Biomedical subjects

M Pagano

Publications and source records attributed to M Pagano.

At least 127 records · Page 7Linked to original sources
PubMed1995

Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27.

The p27 mammalian cell cycle protein is an inhibitor of cyclin-dependent kinases. Both in vivo and in vitro, p27 was found to be degraded by the ubiquitin-proteasome pathway. The human ubiquitin-conjugating enzymes Ubc2 and Ubc3 were specifically involved in the ubiquitination of p27. Compared with proliferating cells, quiescent cells exhibited a smaller amount of p27 ubiquitinating activity, which accounted for the marked increase of p27 half-life measured in these cells. Thus, the abundance of p27 in cells is regulated by degradation. The specific proteolysis of p27 may represent a mechanism for regulating the activity of cyclin-dependent kinases.

Adenosine Triphosphate↗
PubMed1995

Rescue of defective mitogenic signaling by D-type cyclins.

Three gene products, including Myc and the D- and E-type G1 cyclins, are rate limiting for G1 progression in mammalian fibroblasts. Quiescent mouse NIH 3T3 fibroblasts engineered to express a mutant colony-stimulating factor (CSF-1) receptor (CSF-1R 809F) fail to synthesize c-myc and cyclin D1 mRNAs upon CSF-1 stimulation and remain arrested in early G1 phase. Ectopic expression of c-myc or either of three D-type cyclin genes, but not cyclin E, resensitized these cells to the mitogenic effects of CSF-1, enabling them to proliferate continuously in liquid culture and to form colonies in agar in response to the growth factor. Rescue by cyclin D1 was enhanced by c-myc but not by cyclin E and was reversed by infecting cyclin D1-reconstituted cells with a retroviral vector encoding catalytically inactive cyclin-dependent kinase 4. Induction of cyclin D1 mRNA by CSF-1 was restored in cells forced to express c-myc, and vice versa, suggesting that expression of the two genes is interdependent. Cells reconstituted with c-myc were prevented from entering S phase when microinjected with a monoclonal antibody to cyclin D1, and conversely, those rescued by cyclin D1 were inhibited from forming CSF-1-dependent colonies when challenged with a dominant-negative c-myc mutant. Cyclin D mutants defective in binding to the retinoblastoma protein were impaired in rescuing mitogenic signaling. Therefore, Myc and D-type cyclins collaborate during the mitogenic response to CSF-1, whereas cyclin E functions in a separate pathway.

3T3 Cells↗
PubMed1995

Reconstitution of p53-ubiquitinylation reactions from purified components: the role of human ubiquitin-conjugating enzyme UBC4 and E6-associated protein (E6AP).

The E6 protein of the high-risk human papillomaviruses inactivates the tumor suppressor protein p53 by stimulating its ubiquitinylation and subsequent degradation. Ubiquitinylation is a multistep process involving a ubiquitin-activating enzyme, one of many distinct ubiquitin-conjugating enzymes, and in certain cases, a ubiquitin ligase. In human papillomavirus-infected cells, E6 and the E6-associated protein are thought to act as a ubiquitin-protein ligase in the ubiquitinylation of p53. Here we describe the cloning of a human ubiquitin-conjugating enzyme that specifically ubiquitinylates E6-associated protein. Furthermore, we define the biochemical pathway of p53 ubiquitinylation and demonstrate that in vivo inhibition of various components in the pathway leads to an inhibition of E6-stimulated p53 degradation.

Amino Acid Sequence↗
PubMed1995

The Pediatric Symptom Checklist. Support for a role in a managed care environment.

OBJECTIVES: To gather data based on studies of the Pediatric Symptom Checklist, identify risk factors associated with high levels of dysfunction in primary care pediatric settings, and explore the relationship between common risk factors and psychosocial problems identified by pediatricians. DESIGN: Retrospective review and cross-sectional, case-referent survey. SETTING: Subjects were selected from three primary care pediatric clinics in Massachusetts: a private practice in a predominantly white, middle-class suburb, an urban health maintenance organization clinic, and an inner-city clinic. PARTICIPANTS: Of 423 outpatients aged 6 to 12 years screened for psychosocial problems, 72 children and their families were seen for in-depth structured and clinical interviews (24 from each site). INTERVENTIONS: None. MEASUREMENTS/MAIN RESULTS: Children with a single parent and/or those who were economically disadvantaged were significantly more likely to show psychosocial impairment. The specificity of the Pediatric Symptom Checklist was 100% in samples with a lower socioeconomic status compared with 68% in middle-class samples, and sensitivity was 95% in middle-class samples compared with 80% in lower-class samples. Pediatricians identified psychosocial problems in eight of 15 children with a history of familial mental illness or substance abuse and seven of eight children with a history of physical or sexual abuse, but only six of 17 cases from single-parent families and four of 11 cases from poor families. CONCLUSIONS: Pediatricians should be sensitive to psychosocial dysfunction especially in single-parent and low-income families. Use of the Pediatric Symptom Checklist for psychosocial screening in a managed health care delivery system could target capitated resources efficiently by providing early identification and secondary prevention of psychosocial morbidity.

Affective Symptoms↗
PubMed1995

Placement outcomes of 206 severely maltreated children in the Boston Juvenile Court system: a 7.5-year follow-up study.

This study examines placement outcomes of 206 severely maltreated children 7.5 years after arraignment in Boston Juvenile Court (BJC) on Care and Protection Petitions. Sixty-seven percent (n = 138) of the sample had been permanently removed from their parents and 33% (n = 68) had their cases dismissed in the BJC. At time of this follow-up, 21% of the full sample (n = 44) were still in temporary custody awaiting permanent placement. In addition, 4% (n = 8) of children had "drifted" back to their abusive/neglectful parents despite prior permanent removal. The average time children in this sample spent in probate proceedings (awaiting permanent placement) had increased substantially to 2.1 years since the last overview study of this sample 4 years ago. The rate of court referral for incidences of reabuse (a C&P filing), or delinquency was significantly lower among children who had been permanently placed (p < .003). Rates of court-referral for reabuse charges were the same (16%) for children who were in temporary custody at the time of follow-up and children who had been dismissed back to the parent for whom the original C&P had been filed. Results are discussed in light of the urgent need to restructure time limits in juvenile court proceedings, integrate adequate tracking of child abuse and neglect cases through and across court and agency boundaries, and the use standardized assessments of abused and neglected children as a tool in the adjudication process.

Adolescent↗
PubMed1995

Human cyclin E, a nuclear protein essential for the G1-to-S phase transition.

Cyclin E was first identified by screening human cDNA libraries for genes that would complement G1 cyclin mutations in Saccharomyces cerevisiae and has subsequently been found to have specific biochemical and physiological properties that are consistent with it performing a G1 function in mammalian cells. Most significantly, the cyclin E-Cdk2 complex is maximally active at the G1/S transition, and overexpression of cyclin E decreases the time it takes the cell to complete G1 and enter S phase. We have now found that mammalian cells express two forms of cyclin E protein which differ from each other by the presence or absence of a 15-amino-acid amino-terminal domain. These proteins are encoded by alternatively spliced mRNAs and are localized to the nucleus during late G1 and early S phase. Fibroblasts engineered to constitutively overexpress either form of cyclin E showed elevated cyclin E-dependent kinase activity and a shortened G1 phase of the cell cycle. The overexpressed cyclin E protein was detected in the nucleus during all cell cycle phases, including G0. Although the cyclin E protein could be overexpressed in quiescent cells, the cyclin E-Cdk2 complex was inactive. It was not activated until 6 to 8 h after readdition of serum, 4 h earlier than the endogenous cyclin E-Cdk2. This premature activation of cyclin E-Cdk2 was consistent with the extent of G1 shortening caused by cyclin E overexpression. Microinjection of affinity-purified anti-cyclin E antibodies during G1 inhibited entry into S phase, whereas microinjection performed near the G1/S transition was ineffective. These results demonstrate that cyclin E is necessary for entry into S phase. Moreover, we found that cyclin E, in contrast to cyclin D1, was required for the G1/S transition even in cells lacking retinoblastoma protein function. Therefore, cyclins E and D1 control two different transitions within the human cell cycle.

Alternative Splicing↗
PubMed1995

Vertical Transmission of HIV-1. Correlation with maternal viral load and plasma levels of CD4 binding site anti-gp120 antibodies.

Almost all childhood HIV-1 is now acquired through vertical transmission. Identifying factors that affect the rate of transmission may lead to the initiation of specific preventive strategies. In this study, antibody levels against different neutralizing epitopes on the envelope glycoprotein of HIV-1 (gp120) were measured in HIV-1-infected pregnant women that either transmitted HIV-1 to their infants (18 women) or did not (29 women). Differences in levels of antibodies directed against the monomeric gp120 molecule and against the V3 loop region of gp120 were not significantly different between the two groups studied. However, significant differences were observed in the levels of CD4 binding site antibodies, as determined by the ability of diluted maternal plasma to inhibit binding of the CD4 binding site monoclonal antibody F105 (mAb F105) to monomeric gp120. In addition, more nontransmitting mothers had low viral load as defined by having two or more negative HIV-1 viral cultures during pregnancy compared with transmitters. This pilot study suggests that in addition to higher viral load, low levels of CD4 binding site antibodies correlate with increased risk of HIV-1 vertical transmission. Passive immunotherapy with broadly neutralizing CD4 binding site antibodies should be considered as a strategy to reduce this risk.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Screening for psychosocial dysfunction in pediatric inpatients.

Screening pediatric inpatients for psychosocial dysfunction offers physicians an opportunity to identify emotional and behavioral problems that might otherwise go unrecognized. In this study, the Pediatric Symptom Checklist (PSC), a brief, parent-completed questionnaire, which has been validated in a variety of outpatient settings, was used to screen 98 pediatric inpatients. Results indicated that the PSC can be easily administered in a busy inpatient setting and is well-tolerated by both house staff and patients' parents as a routine part of the admissions process. The percentage of children who screened positive with the PSC in this inpatient setting was similar to the percentages generated by using the PSC in outpatient settings. Routine use of the PSC in inpatient settings serves to heighten house staff awareness of psychosocial concerns and facilitate parent-physician discussion of pediatric mental health issues.

Adolescent↗
PubMed1995

Models of delayed recovery.

After a biologic insult has impaired function of the developing central nervous system, recovery may not become apparent for years. Probability models adopted from the carcinogenesis, developmental neurobiology, learning decay, and stochastic process literatures are presented so that assumptions about apparent delays in the recovery process can be tested with data from longitudinal studies after a temporally circumscribed adverse event/exposure. This process of evaluating multiple models is exemplified with one data set. Nonlinear models of recovery are important because some children with early deficits first show improvement months to years later.

Brain↗
PubMed1994

Differential expression and cell cycle regulation of the cyclin-dependent kinase 4 inhibitor p16Ink4.

p16Ink4 (inhibitor of cyclin-dependent kinase 4) is a cell cycle regulator that specifically binds to and inhibits Cdk4. Recently, the human mts1 (multiple tumor suppressor 1) gene, deleted or mutated in various primary tumors and in a large number of transformed cell lines, was found to be identical to ink4. In this study we have surveyed by immunoblotting the protein levels of p16Ink4 in normal and transformed human cells. We determined that p16Ink4 was differentially expressed in diploid cells derived from different tissues, in contrast to another cell cycle inhibitor, p21Waf1, which is ubiquitously expressed. In some tumor cell lines p16Ink4 protein was not detected, presumably because of a homozygous deletion of its gene. By contrast, it was found to be overexpressed in other cell lines when compared to levels in their normal counterparts. Interestingly, high levels of p16Ink4 protein correlated with functional inactivation of the retinoblastoma gene product. We also found that p16Ink4 protein expression varies during the cell cycle peaking during S phase. These results show a functional relationship between p16Ink4 and the retinoblastoma gene product and indicate that p16Ink4 is required for Cdk4 inhibition only at the G1-S transition at the time when Cdk4 kinase activity is no longer necessary.

Carrier Proteins↗
PubMed1994

Properties of the mineralocorticoid receptor immunopurified from bovine kidney.

The mineralocorticoid receptor (MCR) from bovine kidney was purified on an affinity column containing covalently linked polyclonal IgG raised in the rabbit against rat kidney protein purified in the presence of RU 26752 that is specific to the MCR. The immuno-affinity eluate was excluded as a single peak during gel permeation chromatography and could be resolved as a single band of approximately 98 kDa by western blot and gel electrophoresis. Immunohistochemistry revealed MCR-specific staining in both the cortical and glomerular regions of bovine kidney. Interestingly, the purified MCR could not be activated in the presence of the specific ligand RU 26752 whereas binding to DNA-cellulose increased by 100% when crude cytosol was left at room temperature for 45 min. The binding of calcium to the MCR resulted in an increase in the fluorescence signal that could be partially reversed by EDTA. By a calcium-specific fluorescence dye technique, 1.13 nM of ionized Ca2+ was bound per 0.01 nM MCR. The binding of ATP32 to the immunopurified receptor was observed following chromatography on P-10 columns. The fluorescence signal of etheno-ATP was maximally attenuated by the receptor at 1/1 stoichiometry of the ATP-MCR complex. Asparagine-linked complex chain N-glycosylation of the purified MCR was also observed. Analysis by far-UV circular dichroism spectra showed that MCR contains 33% alpha helices and 30% beta sheets, compatible with a relatively flat conformation of the native protein. These data provide experimental proof for the predicted computer simulation regarding the structural features of the steroid receptor superfamily and suggest crosstalk between several protein families.

Adenosine Triphosphate↗
PubMed1994

Cyclin D1-mediated inhibition of repair and replicative DNA synthesis in human fibroblasts.

Cyclin D1 is a key regulator of the G1 phase of the cell cycle. Inhibition of cyclin D1 function results in cell cycle arrest, whereas unregulated expression of the protein accelerates G1. Cyclin D1 is localized to the nucleus during G1. We found that during repair DNA synthesis, subsequent to UV-induced DNA damage, G1 cells readily lost their cyclin D1 while the proliferating cell nuclear antigen (PCNA) tightly associated with nuclear structures. Microinjection of cyclin D1 antisense accelerated DNA repair, whereas overexpression of cyclin D1 prevented DNA repair and the relocation of PCNA after DNA damage. Coexpression of cyclin D1 with its primary catalytic subunit, Cdk4, or with Cdk2, also prevented repair. In contrast, coexpression of PCNA, which is also a cyclin D1-associated protein, restored the ability of cells to repair their DNA. Acute overexpression of cyclin D1 in fibroblasts prevented them from entering S phase. Again, these effects were abolished by coexpression of cyclin D1 together with PCNA, but not with Cdk4 or Cdk2. Altogether, these results indicate that down-regulation of cyclin D1 is necessary for PCNA relocation and repair DNA synthesis as well as for the start of DNA replication. Cyclin D1 appears to be an essential component of a G1-checkpoint.

CDC2-CDC28 Kinases↗
PubMed1994

Modulation of cyclin gene expression by adenovirus E1A in a cell line with E1A-dependent conditional proliferation.

To investigate how adenovirus E1A controls cell proliferation, we have fused E1A to the hormone-binding domain of the human estrogen receptor (ER) and introduced the E1A-ER chimeric gene together with an activated ras gene into primary rat embryo fibroblasts. Cell lines derived from this transfection proliferate in an estrogen-dependent manner. Estrogen-dependent activation of E1A-ER led to a rapid induction of both cyclin E and cyclin A gene expression. In contrast, levels of cyclin D1 were strongly reduced by activation of E1A-ER. Similar changes in cyclin gene expression were observed when primary human fibroblasts were infected with wild-type adenovirus and when adenovirus E1A was stably expressed in NIH 3T3 cells. Our findings suggest that activation of cyclin A and E, but not D1, gene expression by E1A precedes and may be responsible for E1A-dependent cell proliferation. In contrast, we found that quantitative disruption of complexes between the E2F transcription factor and the retinoblastoma protein is not required for E1A-dependent S-phase entry.

Adenovirus E1A Proteins↗
PubMed1994

Regression analysis of censored and truncated data: estimating reporting-delay distributions and AIDS incidence from surveillance data.

AIDS surveillance provides a vital source of information for health departments to assess the AIDS epidemic and to plan for future health-care needs. However, the use of surveillance data requires proper adjustments for the underreporting of AIDS cases caused by the delay in reporting diagnosed AIDS cases to the surveillance system. The statistical problem of adjusting for this underreporting concerns making inferences about an unobservable random sample of which only a portion is observed in a chronologic time interval defined by the analysis. Most regression methods for making inferences using right-truncated data employ a reverse-time hazard function, which requires that the observed data be transformed so that methods for left-truncated data can be applied. In this paper, we discuss fitting regression models to data that can be truncated and even censored in arbitrary intervals. The proposed methodology was applied to the national AIDS surveillance data provided by the Centers for Disease Control to analyze the trend of delays over chronologic time and variation among different geographic regions as well as across risk groups.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Differential expression and regulation of Cyclin D1 protein in normal and tumor human cells: association with Cdk4 is required for Cyclin D1 function in G1 progression.

In this study we have surveyed by immunoblotting the protein levels of Cyclin D1, D2, D3 and their catalytic partners, Cdk4 and Cdk6 in normal and transformed human cells. We found that all these proteins were differentially expressed in diploid cells derived from different tissues, in contrast to Cyclin E, Cyclin A and Cdk2 which are ubiquitously expressed. D-type Cyclins were never dramatically overexpressed and often very poorly expressed in tumor cell lines when compared to the levels in their normal counterparts. In contrast, Cdk4 was expressed at high levels in several tumor cell lines and Cdk6 was ectopically expressed in two sarcoma lines, suggesting a possible involvement of these two Cdks in oncogenesis. Interestingly, low levels of Cyclin D1 and D3 proteins always correlated with functional inactivation of the retinoblastoma gene product (pRb). In cells displaying active pRb, Cyclin D1 was found associated with Cdk4 regardless of whether the p53 gene was wild-type or mutant. Microinjection during G1 of Cyclin D1 anti-sense cDNA or anti-Cyclin D1 antibody in these cells arrested the cell cycle in G1. In cells lacking pRb function, Cyclin D1 was dissociated from Cdk4. Microinjection during G1 of Cyclin D1 antisense cDNA or anti-Cyclin D1 antibody in these cells did not affect G1 progression. These results show that (i) in the absence of pRb, Cyclin D1 is expressed at low levels, is dissociated from Cdk4 and becomes dispensable in G1; (ii) Cyclin D1 needs to be associated with its catalytic subunit, Cdk4, to function as a positive regulator of G1 progression.

CDC2 Protein Kinase↗
PubMed1994

Cyclin D1 protein oscillates and is essential for cell cycle progression in human tumour cell lines.

Among the key cell cycle regulators, cyclin D1 has been implicated most strongly in oncogenesis. This G1 cyclin is a putative proto-oncogene whose clonal rearrangement and/or amplification and mRNA overexpression occurs in several types of human neoplasias. We have now raised a series of monoclonal antibodies to human cyclin D1 and analysed its regulation at the protein level in 40 human tumour cell lines. We found that 12 cell lines displayed low or undetectable cyclin D1 protein level, while the remaining lines accumulated the protein to a level comparable to, or moderately higher than, that of four normal diploid non-immortalized cell types. The cell cycle-dependent oscillation and subcellular localization of cyclin D1 were similar in both tumour and normal cells. The protein localized to the nucleus of G1 cells, and it was reduced to immunocytochemically undetectable level in DNA-replicating cells. At the functional level, microinjection and electroporation of anti-D1 antibodies revealed that in most tumour cell lines studied, including those with amplification at the cyclin D1 locus, this cyclin is essential for cell cycle progression in G1. Some tumours, however, seem to have evolved mechanism(s) that enable them to bypass the requirement for functional cyclin D1.

Antibodies, Monoclonal↗
PubMed1993

In vitro activation of pro-cathepsin B by three serine proteinases: leucocyte elastase, cathepsin G, and the urokinase-type plasminogen activator.

In vitro activation of pro-cathepsin B purified from ascitic fluid of ovarian carcinomas by serine proteinases was studied. Both elastase and cathepsin G from human leucocytes were found to be activators, on the basis of generation of cathepsin B activity and processing of the precursor. These results represent a new cooperative pathway between cancer cells and host cells. The urokinase-type plasminogen activator activated pro-cathepsin B faster than leucocyte proteinases. A new relationship is emerging between the cysteine proteinases and the plasmin-activation system. Both pathways suggest an important role of cathepsin B in the proteolytic cascade associated with tumour invasion.

Ascites↗