Biomedical subjects

N Robert

Publications and source records attributed to N Robert.

At least 37 records · Page 2Linked to original sources

PubMed1995

Proteolytic activity of proteasome on myofibrillar structures.

The physiologic function of proteasome remains unclear. Evidence suggests a role in degradation of ubiquitin-protein conjugates, MHC antigen presentation, and some specificity of substrate within certain cell types. To explore further the properties of proteasome we have examined its effect on a well defined structure, the myofibril. We find that despite its large size (20S) proteasome is able to degrade myofibrils and intact, permeabilized muscle fibrils. The proteins degraded showed some specificity because actin, myosin and desmin were degraded faster than alpha-actinin, troponin T and tropomyosin. Changes in ultrastructure were slow and included a general loss of structure with Z and I bands effected before the M band and costameres.

Animals↗

PubMed1995

Activity of the interferon-induced 2',5'-oligoadenylate synthetase in patients with chronic hepatitis C.

The interferon-induced 2',5'-oligoadenylate synthetase (2-5OAS) is responsible, at least in part, for the antiviral state established in cells in response to viral infections. The purpose of this work was to study the relationship between hepatitis C virus (HCV) infection and 2-5OAS in patients with chronic hepatitis C. Peripheral blood mononuclear cells (PBMC) of 27 patients with chronic hepatitis C were investigated, as well as PBMC of 10 control subjects. Then, the patients were treated with 3 mu interferon-alpha 2a three times per week. At month 3 of therapy, PBMC were sampled. Of the total PBMC samples obtained, half were used for determination of in vivo 2-5OAS activity. The remaining cells were cultured for 24 h in either the absence or presence of 500 U/ml of interferon-alpha 2a for the determination of in vitro 2-5OAS activity. The mean basal in vivo 2-5OAS activities were 3.6 +/- 2.8 nmol/10(6) cells in patients versus 1.6 +/- 1.1 nmol/10(6) cells in controls (p < 0.01). Basal in vivo 2-5OAS activity did not correlate with mean HCV viremia, quantified by a "branched DNA"-based assay. Before treatment, interferon-alpha was detected in the serum of 2 patients in 27. After a 24 h culture of PBMC in the presence of interferon, in vitro 2-5OAS activity was significantly induced in the PBMC of both the patients and the controls. However, in vitro induction of 2-5OAS activity was significantly lower in the PBMC of the patients than in the PBMC of the controls (p < 0.01). At month 3 therapy, in vivo 2-5OAS activity was significantly induced (20.5 +/- 17.9; p < 0.0001). In vitro IFN inductions of 2-5OAS activity in PBMC before treatment and at month 3 of therapy were not significantly different. In conclusion, in vivo 2-5OAS activity is significantly induced in patients with chronic hepatitis C, but endogenously produced interferon-alpha does not seem to be involved. Chronic induction of 2-5OAS activity results in a decreased sensitivity of PBMC to exogenous interferon induction. Whether this phenomenon plays a role in the resistance of chronic hepatitis C to interferon therapy remains uncertain.

2',5'-Oligoadenylate Synthetase↗

PubMed1995

Leukemogenic potential of adjuvant chemotherapy for early-stage breast cancer: the Eastern Cooperative Oncology Group experience.

PURPOSE: Since large numbers of patients with early-stage breast cancer now receive adjuvant chemotherapy containing cyclophosphamide, a known leukemogenic agent, it is important to determine the risk of secondary acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Therefore, we identified all cases of AML or MDS developing in patients treated on six clinical adjuvant chemotherapy trials conducted by the Eastern Cooperative Oncology Group (ECOG). PATIENTS AND METHODS: The patients population included 2,638 patients with previously untreated primary operable breast cancer entered onto six clinical trials conducted by the ECOG between 1978 and 1987. There are 19,200 persons-years of follow-up study and a mean follow-up duration of 7.3 years. Clinical data were obtained from flow sheets submitted to the ECOG Data Management Office. RESULTS: Of 2,638 patients at risk with 19,200 person-years of follow-up study, three patients developed MDS (two with a characteristic cytogenetic abnormality). Two patients developed acute leukemia; however, one had adult T-cell leukemia associated with human T-lymphotrophic virus type 1 (HTLV-1) and a second patient developed AML after receiving additional cyclophosphamide for metastatic breast cancer. The estimated incidence rate for MDS is three per 19,200 or 16 per 100,000 person-years of follow-up study with a 95 percent confidence interval of three to 46 per 100,000 person-years. If all five patients (three MDS and two acute leukemia) are included, the estimated incidence rate is five per 19,200 or 26 per 100,000 person-years of follow-up study with a 95 percent confidence interval of eight to 61 per 100,000 person-years. CONCLUSION: These data suggest that the risk of secondary AML or MDS among patients with early breast cancer who receive standard-dose cyclophosphamide-containing adjuvant chemotherapy is not much higher than in the general population. However, physicians must remain alert to the possible long-term consequences of alkylating agent and anthracycline-based chemotherapy.

Acute Disease↗

PubMed1995

Eastern Cooperative Oncology Group studies of paclitaxel and doxorubicin in advanced breast cancer.

Based on their single-agent activity in metastatic breast cancer, paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) and doxorubicin have been alternated and combined in Eastern Cooperative Oncology Group studies to identify a tolerable dose and schedule. A pilot trial in patients who had received no more than one prior chemotherapy regimen alternated paclitaxel 200 mg/m2 and doxorubicin 75 mg/m2 every 3 weeks. Seven of 12 patients had objective (complete plus partial) responses. A second (phase I) trial combined doxorubicin as an intravenous push and paclitaxel as a 24-hour continuous infusion. To evaluate the effect of the schedule on toxicity, the drug administration sequence (doxorubicin-->paclitaxel or paclitaxel-->doxorubicin) was alternated both between and within patients. initial doses were paclitaxel 150 mg/m2 and doxorubicin 50 mg/m2 in six patients, with a subsequent six patients receiving 175 and 60 mg/m2, respectively. Dose-limiting mucositis was seen at the second dose level when paclitaxel preceded doxorubicin, suggesting an important role for the administration sequence in determining toxicity (and possibly efficacy) in this regimen. These results support the sequence of doxorubicin 50 mg/m2 followed by paclitaxel 150 mg/m2 as the maximum tolerated dose. This combination has been incorporated as a treatment arm in an ongoing randomized prospective phase III Intergroup trial. By comparing the combination with both drugs as single agents (with crossover to the opposite agent at disease progression), investigators will attempt to assess response, toxicity, and time to progression as well as the degree of cross-resistance between the two agents. Given the poor prognosis of patients with advanced breast cancer, the three arms also will be evaluated in terms of patients' quality of life.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1994

Characterization of an interferon-induced 48-kD protein immunologically related to the double-stranded RNA-activated protein kinase PKR.

Polyclonal antibodies raised against purified and urea-denatured double-stranded protein kinase (PKR) from human origin cross-reacted by immunoblotting with a 48-kD protein (p48) induced by the three types of interferon (IFN), alpha, beta, and gamma. The induction of p48 is IFN dose dependent and its accumulation occurs a few hours after the addition of IFN. The induction of p48 is blocked by actinomycin D. Analysis by two-dimensional gel isoelectric-focusing, revealed p48 as a single spot with an isoelectric point (pI) of 6.8. In the same experiment the PKR was revealed as several subspecies with pI values in the pH range of 7.4-8.0. Cell fractionation experiments indicated that PKR and p48 have different subcellular localizations: PKR was found to be associated with the microsomal pellet as shown previously whereas p48 was recovered in the microsomal supernatant fraction. In addition to these differences, PKR and p48 were found to be differentially expressed in some human cells treated with the three types of IFN. For example, in HeLa cells, IFN-alpha or IFN-beta induced similarly both PKR and p48 whereas IFN-gamma induced mainly p48. In U937 cells in which PKR was not expressed with or without IFN treatment, p48 was strongly induced by all three types of IFN. These results suggest different mechanisms for the induction of PKR and p48. In view of its presence in different types of human cells and its induction by different types of IFN, it is possible to suggest that p48 might play an important role in mediating some of the action of IFN.

Enzyme Activation↗

PubMed1994

Binary vascular reconstruction from a limited number of cone beam projections.

This paper describes a method to perform reconstruction of vascular cross-sectional images from a limited number of x-ray angiographic cone-beam projections. It is assumed that the projection data can be simplified by identifying blood vessels in each angiogram and removing signals due to other structures. Under these conditions, the x-ray attenuation coefficient, mu, can be modeled as a binary variable having a value mu 0 within the vessel and "0" outside. The reconstruction is performed by minimizing a cost function using the method of simulated annealing. In this paper, we demonstrate that the introduction of a priori information allows one to reconstruct a sphere and a simulated branched vessel from three views with, respectively, 97% and 93% of voxels having correct values. The addition of a continuity constraint for the reconstruction of the branched vessel resulted in further reduction in the percentage of misplaced voxels. Calculations require from one to six hours of CPU time on a Sun SparcStation 2 computer for the cases investigated here. The effect of noise, "cooling" schedule, and number of views on the reconstruction are examined using simulated vessel projections. Modifications to our approach to accelerate the reconstruction are also discussed.

Angiography↗

PubMed1994

Performance of glass fiber antiscatter devices at mammographic energies.

Using fiber optic manufacturing techniques, it is possible to produce a radiographic grid that discriminates against scattered radiation in two dimensions. Such grids consist of septa composed of glass with a high lead content; the interspace material is air, so that approximately 80% of the grid area is open. In this way, effective high ratio grids can be produced with relatively low Bucky factors. The performance of samples of such grid material is characterized in terms of both scatter rejection and dose efficiency for application in digital mammography in both slot-beam and area-beam geometry. For area beams, five- to tenfold improved scatter rejection relative to conventional grids was observed. In slot configurations, such grids could provide improved SNR/dose performance and more effective utilization of the heat loading capability of the x-ray source.

Fiber Optic Technology↗

PubMed1994

Cloning, sequencing, and expression in Escherichia coli of the D-hydantoinase gene from Pseudomonas putida and distribution of homologous genes in other microorganisms.

Pseudomonas putida DSM 84 produces N-carbamyl-D-amino acids from the corresponding D-5-monosubstituted hydantoins. The gene encoding this D-hydantoinase enzyme was cloned and expressed in Escherichia coli. The nucleotide sequence of the 1.8-kb insert of subclone pGES19 was determined. One open reading frame of 1,104 bp was found and was predicted to encode a polypeptide with a molecular size of 40.5 kDa. Local regions of identity between the predicted amino acid sequence and that of other known amidohydrolases (two other D-hydantoinases, allantionase and dihydroorotase) were found. The D-hydantoinase gene was used as a probe to screen DNA isolated from diverse organisms. Within Pseudomonas strains of rRNA group I, the probe was specific. The probe did not detect D-hydantoinase genes in pseudomonads not in rRNA group I, other bacteria, or plants known to express D-hydantoinase activity.

Amidohydrolases↗

PubMed1994

Paclitaxel (Taxol)/doxorubicin combinations in advanced breast cancer: the Eastern Cooperative Oncology Group experience.

Paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ), the first taxane to enter routine clinical practice, has aroused considerable interest due to its novel mechanism of action and its significant activity in metastatic breast cancer. Given this activity, it seemed logical to attempt to combine paclitaxel with doxorubicin, the other most active single agent in metastatic breast cancer. The Eastern Cooperative Oncology Group performed two trials investigating paclitaxel/doxorubicin combinations in patients with advanced breast cancer in an attempt to identify a tolerable dose and schedule of the combination. In the first trial, paclitaxel and doxorubicin were alternated every 3 weeks in doses of 200 mg/m2 and 75 mg/m2, respectively, for patients who had received no more than one prior chemotherapeutic regimen. Therapy was well tolerated in this setting. At these doses, paclitaxel induced more granulocytopenia and less thrombocytopenia than did doxorubicin. Objective responses (complete and partial responses) were seen in seven of 12 patients; two other patients had improved disease (relief of pain in bony metastases). A second limited-institution Eastern Cooperative Oncology Group trial evaluated paclitaxel and doxorubicin given in combination. In this phase I trial, doxorubicin was given as an intravenous push and paclitaxel as a 24-hour continuous infusion. The sequence of drug administration (D-->P or P-->D) was alternated both between and within patients, so that we might evaluate the effect of administration schedule on toxicity. Therapy was begun at an initial paclitaxel dose of 150 mg/m2 and an initial doxorubicin dose of 50 mg/m2 in six patients, with a subsequent six patients receiving 175 and 60 mg/m2, respectively, of paclitaxel and doxorubicin. In addition, patients received granulocyte colony-stimulating factor 5 micrograms/kg/d. While therapy at the initial dose level was well tolerated, dose-limiting mucositis was seen at the second dose level, although only when paclitaxel preceded doxorubicin. This suggests that sequence of drug administration in paclitaxel-based regimens may play an important role as a determinant of toxicity and (perhaps) efficacy, a finding similar to that seen when paclitaxel and cisplatin were combined in patients with ovarian cancer. Based on this study, we identified the sequence of doxorubicin (50 mg/m2) followed by paclitaxel (150 mg/m2) to be the maximum tolerated dose. This combination is currently being compared with paclitaxel alone and doxorubicin alone in patients with advanced breast cancer in an intergroup trial led by the Eastern Cooperative Oncology Group.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1993

Isoforms p69 and p100 of 2',5'-oligoadenylate synthetase induced differentially by interferons in vivo and in vitro.

Four isoforms of 2',5'-oligoadenylate (2-5A) synthetase have been identified (40, 44, 69, and 100 kD). The 40- and 44-kD enzymes are encoded by the same gene, probably different from the genes encoding the larger isoforms. In this study, induction of the 100- and 69-KD (p100, p69) isoforms in different individuals and in different cell types was investigated after treatment with recombinant human interferons (IFN): IFN-alpha 2, IFN-beta ser, or IFN-gamma. The p69 and p100 isoforms were quantitated in cell extracts on Western blots using specific polyclonal antibodies, or their activity was measured after purification of cell extracts on immunoaffinity columns. The p69 and p100 isoforms were differentially induced in Daudi, fibroblast, and colon adenocarcinoma cells treated with IFNs. Considerable individual variations in both basal and induced levels of p69 and p100 were observed in peripheral blood mononuclear cells from normal donors cultured with IFNs in vitro, and from cancer patients treated with IFN-alpha 2 or with IFN-beta ser. These results demonstrate that the p69 and p100 isoforms are present in vivo in peripheral blood mononuclear cells and their level is increased following IFN administration. Furthermore, both the in vivo and in vitro observations indicate that the expression of these enzymes is specific to each cell type and varies among individuals.

2',5'-Oligoadenylate Synthetase↗

PubMed1991

[Cataract surgery and implantation in diabetics. Apropos of 92 eyes].

We examined 92 eyes (63 patients) who underwent extracapsular cataract extraction with implantation of a posterior chamber intraocular lens. Visual acuity improved by two lines or more in 65% of cases. Follow-up examination (6 months and 2 years) showed posterior capsule opacification in 30% of cases. Progression of diabetic retinopathy was mild but cystoid macular edema was present in 70% of cases with non proliferative retinopathy. Proliferative retinopathy was treated postoperative by pan retinal photocoagulation. One neovascular glaucoma occurred, secondary to a ruptured capsule.

Aged↗

PubMed1990

Differential expression and distinct structure of 69- and 100-kDa forms of 2-5A synthetase in human cells treated with interferon.

Using specific monoclonal and polyclonal antibodies, the induction, synthesis and subcellular localization of 69- and 100-kDa forms of 2',5'-oligoadenylate (2-5A) synthetase (p69 and p100) were investigated in alpha-interferon-treated human HeLa and Daudi cells. Although both p69 and p100 were induced by interferon, there were significant differences in the interferon dose-response and the kinetics of synthesis of each protein in these cell lines. Both proteins are localized mainly in the cytoplasm. However, immunoenzymatic staining analysis of interferon-treated cells indicated that a proportion of p69 is concentrated around the nuclei and the rest is distributed in a specific pattern in the cytoplasm whereas p100 is found in a diffuse state in the cytoplasm. In accord with its association to cell membranes, p69 is myristilated. The [35S]cysteine and [3H]myristate-labeled p69 preparations were resolved by two-dimensional gel isoelectric focusing with pI values in the pH range of 7.0-8.0 and 7.0-7.5, respectively. These observations suggested the existence of two forms of p69 which might be complexed together as a dimer. In favor with a dimeric form of p69, in gel filtration experiments the peak of p69 was routinely found in fractions corresponding to a molecular mass of 160 kDa whereas p100 was recovered as a monomer. Taken together, these results indicate that p69 and p100 have distinct characteristics and that their expression is a specific property of each cell type in response to interferon.

2',5'-Oligoadenylate Synthetase↗

PubMed1989

The binding of double-stranded RNA and adenovirus VAI RNA to the interferon-induced protein kinase.

The protein kinase from human cells dependent on double-stranded (ds) RNA is a 68-kDa protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. When activated by low concentrations of dsRNA, the p68 kinase becomes phosphorylated and thereby catalyzes the phosphorylation of the protein-synthesis initiation factor, eIF2. Here, we have purified the p68 kinase to homogeneity using a specific monoclonal antibody to investigate its capacity to bind dsRNA, poly(I).poly(C). Our study suggest that p68 kinase has high- and low-affinity binding sites: the high-affinity binding site is responsible for the activation and the low-affinity binding site for the inhibition of kinase activity. This is in accord with the fact that autophosphorylation of p68 kinase occurs at low concentrations of dsRNA whereas high concentrations of dsRNA inhibit its autophosphorylation. We have also investigated the binding of adenoviral VAI RNA to the purified p68 kinase and have found that the affinity of this binding is lower than that of poly(I).poly(C). We show that VAI RNA can activate or inhibit autophosphorylation of p68 kinase in a dose-dependent manner, i.e. activation at less than or equal to 1 microgram/ml or inhibition at greater than 1 microgram/ml of VAI RNA. In spite of its lower affinity of binding, VAI RNA cannot be displaced by poly(I).poly(C) or reovirus dsRNA. These data confirm our previous results to illustrate that VAI RNA can bind p68 kinase and cause its inactivation irreversably.

Adenoviruses, Human↗

PubMed1988

Interferon- and sarcolectin-dependent cellular regulatory interactions.

Interferons, via specific membrane-bound receptors, induce various cellular functions of which antiviral protection is the most extensively studied. We have previously reported the existence of interferon antagonists (referred to as sarcolectins) in various tissue extracts from placental blood, cartilage, brain, muscle, or from sarcomas. These sarcolectins have been fully characterized and purified to homogeneity. In interferon-treated cells, they restore virus sensitivity 4-6 h after the establishment of antiviral protection. In the present study we investigate the effect of sarcolectins on the steady state levels of two double-stranded RNA dependent enzymes, 2-5A (p chi (A2'p)nA) synthetase and protein kinase. Several authors have previously emphasized the role of these enzymes in the mechanism of interferon's antiviral action. Interferon promotes a 4-8 fold increase in protein kinase and 2-5A synthetase in cells. Addition of sarcolectin 5 h after interferon results in a dramatic reduction in the steady state levels of both these enzymes, as shown by their decreased activity and yield observed in Western blot assays. The degradation of the antiviral response in sarcolectin-treated cells might therefore be at least partially attributed to a reduced synthesis of protein kinase and 2-5A synthetase. Since there are no direct interactions between sarcolectins and interferon or its receptors, it can be postulated that sarcolectins exert their effect through these interferon-dependent proteins. We postulate that the opposing biological effects of interferon and sarcolectins strike a balance which may, however, be modified in one direction or the other, depending on their respective concentrations.

2',5'-Oligoadenylate Synthetase↗

PubMed1988

Defective estrogen receptors in human mammary cancers: their significance in defining hormone dependence.

Neither estrogen dependence nor clinical response to hormone therapy of the tumor is guaranteed by the mere presence of receptors for estrogens (ERs) or progesterones (PRs). Immunohistochemical staining of ER with polyclonal anti-ER antibodies after in vitro transformation has enabled the identification of two types of defective ER among human breast cancers: those that are unable to bind to the nucleus in a hormone-filled state and those that bind to the nucleus as naked ER. Preliminary clinical correlation studies demonstrate that a subclassification of ER(+) tumors based on functional abnormalities of ER may predict refractoriness to hormone therapy.

Antibodies, Monoclonal↗

PubMed1988

Characterization of 69- and 100-kDa forms of 2-5A-synthetase from interferon-treated human cells.

The existence of distinct 69- and 100-kDa forms of 2-5A-synthetase in addition to the smaller (40 and 46 kDa) forms has recently been established. Using specific monoclonal antibodies we investigated the induction, synthesis, and activity of 69- and 100-kDa 2',5'-oligoadenylate (2-5A) synthetases in interferon-treated human Daudi cells. Although induction of these synthetases is detectable in cells treated with as little as 1-5 units/ml of human alpha-interferon, higher concentrations are required for maximum synthesis of the 100 kDa than the 69-kDa protein. At 5 units/ml of interferon, enhanced synthesis of both proteins is detectable at 4 h with maximum synthesis occurring between 8 to 12 and 12 to 16 h for 69- and 100-kDa 2-5A-synthetases, respectively. At 24 h after addition of interferon, synthesis of these synthetases declines due to a decrease of active interferon in the culture medium. The synthesis of both synthetases is blocked by actinomycin D, and the half-life of these proteins is estimated to be 8 h. The activities of immunoaffinity purified 69- and 100-kDa synthetases are dependent on double-stranded (ds)RNA but show different requirements for optimum concentration of dsRNA and pH of the reaction. The apparent Km of 69- and 100-kDa synthetases for ATP is 1.7 X 10(-3) M and 3.6 X 10(-3) M, respectively. At optimum conditions for the activity of these enzymes, the pattern of 2',5'-linked oligoadenylates synthesized are different, the 69-kDa protein synthesizing higher oligomers than the 100-kDa species. Taken together, these results indicate that the 69- and 100-kDa 2-5A-synthetases are distinct proteins each with specific characteristics of induction and enzymatic activity.

2',5'-Oligoadenylate Synthetase↗

PubMed1988

Activation of the major late promoter in adenovirus transformed cells by 5-azacytidine.

The adenovirus major late promoter (MLP) is normally not active in transformed cells. We investigated if it could be activated with 5-azacytidine. Three days of treatment with 10 microM 5-azacytidine induced transient activation of the MLP as shown by hybridization with an L1 r-strand-specific probe. The po/III-transcribed VA-RNAs were not activated. L1 activation was not accompanied by detectable changes in methylation of HpaII sites at the promoter or in the body of the transcript. Stably activated cell clones could be obtained at 20% frequency after long-term drug treatment.

Adenoviridae↗

PubMed1987

Identification of 69-kd and 100-kd forms of 2-5A synthetase in interferon-treated human cells by specific monoclonal antibodies.

Recently, the existence of 40-kd and 46-kd 2-5A synthetases in interferon-treated cells has been confirmed by cloning and characterization of cDNA corresponding to these small size enzymes. By the use of specific monoclonal antibodies, we describe here two forms of high mol. wt 2-5A synthetases of 69 and 100 kd in human cells. The monoclonal antibodies immunoprecipitate either a 69- or a 100-kd 2-5A synthetase. These purified 2-5A synthetases in immune complex preparations are active, i.e. addition of poly(I).poly(C) and ATP results in the synthesis of 2-5A. Both 2-5A synthetases are composed of several subspecies with similar isoelectric points in the range of 7-8 but have different subcellular localizations: 100-kd synthetase is recovered from the microsomal pellet whereas 69-kd synthetase is found to be associated with cell membranes as well as with the microsomal pellet. Different types of interferon-treated human cells express both or either forms of these enzymes. The 69- and 100-kd 2-5A synthetases were also identified by electrophoretic transfer immunoblot analysis using rabbit polyclonal antibodies against a synthetic peptide common on both 46- and 40-kd 2-5A synthetases. These results indicate that small and large size isozymes share a common peptide sequence.

2',5'-Oligoadenylate Synthetase↗