[The use of ultrasonographic 3D power Doppler to describe an ovarian Sertoli-Leydig cell tumor]. / Description échographique d'une tumeur ovarienne de Sertoli-Leydig à l'aide du doppler énergie tridimensionnel.

We report one case of unilateral ovarian Sertoli-Leydig tumor with intermediate grade and heterologous element, in a young woman with clinical and biological typical presentation. We discuss 2D and 3D ultrasonographic and doppler features.

Conversion of topoisomerase I cleavage complexes on the leading strand of ribosomal DNA into 5'-phosphorylated DNA double-strand breaks by replication runoff.

Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replication-mediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3' DNA ends are extended by DNA polymerase in vivo closely to the 5' ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are produced by replication runoff. We also found that the 5' ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5' kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA.

Genetically modified dendritic cells prime autoreactive T cells through a pathway independent of CD40L and interleukin 12: implications for cancer vaccines.

Genetic immunization through ex vivo transduction of dendritic cells has been suggested as an effective approach to enhance antitumor immunity by activating both CD4+ and CD8+ T cells. Immunizing mice with dendritic cells transduced with an adenovirus expressing the human melanoma antigen glycoprotein 100 (DCAdhgp100) as a cancer vaccine, we demonstrated complete protective immunity and a potent CTL response against melanomas expressing murine glycoprotein 100 in a CD4+ cell-dependent manner. Surprisingly, however, effective tumor rejection was not the result of cooperation between CD4+ and CD8+ T cells. Protective immunity was completely lost when CD4+ cells were depleted immediately before tumor challenge, whereas it was unaffected by removal of CD8+ cells, establishing a principal role for CD4+ cells in the effector phase of tumor rejection. Neither protective immunity nor CTL generation in this model required interleukin 12, in spite of high levels of IFN-gamma secretion by tumor-reactive T cells. Most notably, the DCAdhgp100 vaccine could elicit protective antitumor CD4+ cells in the absence of CD40 ligand, although it does not bypass the need for CD40-mediated signals to generate melanoma-reactive CTLs. Thus, in contrast to the current thinking that the optimal cancer vaccine should include determinants for both CD4+ and CD8+ cells, the potency of the DCAdhgp100 vaccine appears to be a result of its ability to directly prime autoreactive CD4+ cells through a process that does not require interleukin 12 and CD40 signals.

Genes from the cyanobacterium Agmenellum quadruplicatum isolated by complementation: characterization and production of merodiploids.

The isolation of several biosynthetic genes from a cyanobacterium, Agmenellum quadruplicatum, by complementation of auxotrophic mutations in Escherichia coli, and their partial characterization, is described. Although our search for such genes has not been exhaustive, it appears that complementation of E. coli mutations may be of limited utility for the identification and/or isolation of cyanobacterial genes. Despite some overlap in the complementation abilities of these isolated cyanobacterial DNA fragments, the genes that we have studied in some detail are not located in operons. We have used mutagenized versions of these cyanobacterial DNA fragments to produce mutant phenotypes in the cyanobacterium, but clean auxotrophs were not obtained. Complementation of these mutant phenotypes can be obtained when the appropriate wild-type DNA fragment is introduced into the cyanobacterium on a shuttle vector. Recombination between two copies of a cyanobacterial gene occurs at high frequency in the cyanobacterium.

Polyomavirus large T antigen zinc finger is not required for efficient cellular immortalization of primary rat embryo fibroblasts.

The polyomavirus large T antigen contains a zinc finger domain required for the formation of hexamers involved in viral DNA replication. Since mutations within the zinc finger domains of transforming proteins like SV40 large T antigen and human papilloma virus E7 protein generally decrease their overall transforming activity, we have examined the ability of a mutant polyomavirus large T antigen that harbors a deletion in sequences within the zinc finger motif to immortalize primary rat embryo fibroblasts. In contrast to result obtained with SV40 large T antigen and the human papilloma virus E7 protein we show that deletion of the entire zinc finger motif enhances the immortalization efficiency of this mutant T antigen.

Polyomavirus large T-antigen binds the "pRb related' protein p130 through sequences in conserved region 2.

The transforming potential of DNA tumor viruses derives mainly from the ability of their encoded oncogene products to interact with cellular proteins. Many of these viral oncoproteins share regions of sequence similarity, designated conserved region 1 and 2, which have been implicated in complex formation with pRb, the product of the retinoblastoma tumor suppressor gene, and related p107 and p130 species. It has now been shown that the EIA protein of adenovirus is able to bind to all three pRb-related proteins through sequences in conserved region 1 and 2. We have shown previously that polyomavirus large T-antigen also interacts with pRb and p107 in vitro. The pRb and p107 binding domains reside between residues 141, 158 which include conserved region 2. In the present study, we demonstrate that polyomavirus large T-antigen also interacted with p130 in vitro through the same sequences in conserved region 2.

Mutations in the HIV type 1 integrase of patients receiving long-term dideoxynucleoside therapy do not confer resistance to zidovudine.

Metabolites of AZT can inhibit HIV-1 integrase in vitro (Mazumder A, et al., Proc Natl Acad Sci USA 1994;91:5771-5775). To determine if long-term dideoxynucleoside therapy can lead to the emergence of HIV-1 AZT-resistant variants containing mutations in the integrase, we have sequenced the proviral DNA encoding the HIV-1 integrase of nine HIV-1-infected patients at different time points during treatment. Four of the nine patients developed mutations during the course of treatment. Although most mutations occurred at nonconserved amino acids, one patient developed a mutation at codon (R166T), a residue that is conserved among all integrases from known HIV-1 isolates. This mutation was introduced in the recombinant HIV-1 integrase protein to determine if it could confer resistance to AZT in vitro. We show that the R166T integrase mutant is still proficient at carrying 3'-processing and 3' end-joining but that the enzyme is not resistant to AZT-TP. Our results suggest that it is unlikely that integrase inhibition contributes to the antiviral activity of AZT.

Lack of toxic effects of F 12511, a novel potent inhibitor of acyl-coenzyme A: cholesterol O-acyltransferase, on human adrenocortical cells in culture.

Inhibition of acyl-coenzyme A: cholesterol O-acyltransferase (EC 2.3.1.26; ACAT) reduces intracellular cholesteryl esters that are substrates for steroidogenesis in adrenal cells. The adrenal side effects of ACAT inhibitors remain a key point for their development as antiatherosclerotic agents. The aim of this study was to characterize the effects of a novel and powerful ACAT inhibitor, F 12511 (S)-2',3',5'-trimethyl-4'-hydroxy-alpha-dodecylthio-phenylacetanilide, on the NCI-H295R cell line, which has functional properties comparable to those of normal human adrenal cells. F 12511 incubated with cultured cells for 4-72 hr strongly inhibited cholesteryl oleate formation. The concentrations required to produce 50% inhibition (IC50) values) ranged from 20 to 50 nM; in the presence of low-density lipoproteins (LDL), this effect was paralleled by a decrease in cholesteryl ester mass and an increase in intracellular free cholesterol. At concentrations 100-fold larger than the IC(50) value for up to 48 hr, F 12511 reduced neither the basal release of cortisol and aldosterone nor the production of cortisol stimulated by forskolin. F 12511 did not modify the mRNA levels of the steroidogenic enzyme genes cytochrome P450 cholesterol side-chain cleavage (P450scc), cytochrome P450 17alpha-hydroxylase (P450c17), or cytochrome P450 21-hydroxylase (P450c21) or those of the LDL receptor and high-density lipoprotein scavenger receptor class B, type I (SR-BI) genes, either in the presence or absence of adenosine 3',5'-cyclic monophosphate stimulation for 24 hr. Exposure to F 12511 at up to 3 microM for 24 or 48 hr did not result in significant change in morphological and ultrastructural characteristics; the cytoplasm contained large numbers of mitochondria with intact crystae, and the same typical features of secretory activity were observed in NCI-H295R control cells. Exposure to 3 microM of F 12511 for 96 hr also did not affect cell viability. These data demonstrate that reduction of the substrate for steroidogenesis by the ACAT inhibitor F 12511 impairs neither steroid production nor transcription of genes involved in steroidogenesis and lipoprotein uptake in the pluripotent human adrenal cell line NCI-H295R.

Rationale for the development of recombinant human CC10 as a therapeutic for inflammatory and fibrotic disease.

CC10/uteroglobin is a remarkable protein whose physiological roles have only recently been explored in vivo. Both transgenic mice that have been rendered deficient and humans that have been characterized as deficient in this protein exhibit tendencies toward inflammatory, fibrotic, and oncologic disease, demonstrating the potential of the protein as a therapeutic agent. The protein itself is an excellent candidate for clinical development because of its inherent physical properties. It is relatively small, resistant to proteases, stable to extremes of heat and pH, and can be produced by recombinant methods. The physiological roles of this multifunctional protein continue to be uncovered as research progresses in vitro, in animals, and eventually in humans. The pathways through which CC10 mediates its effects, its receptors, and other family members will be a rich source of exciting research, as well as potential diagnostic and therapeutic agents. This paper is an introductory, noncomprehensive review of some of the scientific and medical rationale in support of CC10-based therapies in selected clinical applications.

High-level expression and efficient recovery of ubiquitin fusion proteins from Escherichia coli.

The transition in growth and induction of bacterial cultures expressing recombinant proteins from the laboratory bench to the pilot scale for production has been performed successfully for several ubiquitin fusion-expressing clones. Increased protein turnover and decreased metabolic efficiency of Escherichia coli at high cell densities are often responsible for failures in fermenter cultures. Current data indicate that (1) yields in shaker flask cultures are directly scalable to a 10 L fermenter, (2) higher cell densities actually augment the specific yield of ubiquitin fusion proteins, (3) an in vivo heat shock during fermentation increases the ubiquitin fusion yield, which provides an initial separation step in the fermenter, and (4) the ubiquitin fusion expression clone makes at least 3-fold more total protein, including host proteins, than the parent strain of E. coli. A series of three fermentations was performed, using the model strain, with varied temperature shift protocols. These fermentations showed that a maximal heat shock of 12 degrees C (from 30 to 42 degrees C), initiated simultaneously with induction, gave a maximal specific yield (over 90% of the total soluble protein by densitometry, 709 mg/L by protein assay), in which the recoverable ubiquitin fusion product comprised 16% of the wet weight of the cell paste. These results illustrate the enormous potential of ubiquitin fusion technology for the economical production of peptides, even in a 10 L fermenter.

Ubiquitin fusion technology: bioprocessing of peptides.

Ubiquitin fusion technology represents an emerging method for economically producing peptides and small proteins in the bacterium Escherichia coli. Our focus is on peptide production where the need for cost-effective, scaleable processes has recently been highlighted by Kelley (1996). There are two principal features: (1) the expression system consists of a suitable E. coli host strain paired with a plasmid that encodes the ubiquitin fusion and (2) an ubiquitin-specific protease, UCH-L3, which cleaves only C-terminal extensions from ubiquitin. In this work, multigram yields were obtained of four ubiquitin fusions derived from cell paste generated in single 10-L fermentations. All were expressed intracellularly and remained soluble at extremely high levels of expression. Bacterial freeze--thaw lysates contained over 95% pure ubiquitin fusion protein. All four fusions were efficiently cleaved to ubiquitin and the peptide products. In one case, the final yield of peptide was 1.08 g from 3 L of low cell density bacterial culture. The combination of exceptional overexpression of the ubiquitin--peptide fusion proteins and a robust and specific protease are unique advantages contributing to a cost-effective, scaleable, and generic bioprocess for peptide production.

[Solitary fibrous tumor of the orbit: an unusual cause of unilateral proptosis. Case report with a review of the literature]. / Tumeur fibreuse solitaire de l'orbite: une cause inhabituelle d'exophtalmie unilatérale. A propos d'un cas avec revue de la littérature.

BACKGROUND: The solitary fibrous tumor (SFT) is a spindle-cell tumor that very rarely involves the orbit. We report a new case that we compare to reports in the literature. CASE: A 72-year-old woman presented a conjunctival inflammation of the right eye developing over 5 months with progressive proptosis. Magnetic resonance imaging revealed an extraconal and homogeneous mass, which showed hypointensity on T1-weighted images and hyperintensity on T2-weighted images, without specificity. Histological examination of the lesion removed by anterior orbitotomy confirmed the diagnosis of the SFT of the orbit. The patient was doing well without recurrence after 9 months. DISCUSSION: The diagnosis of SFT is histological. It is a mesenchymal tumor. Immunohistochemically, the tumor cells are strongly positive for CD34 and vimentin. CONCLUSION: The SFT of the orbit is a very rare and generally benign tumor. It must be immunohistochemically differentiated from other spindle-cell tumors of the orbit. The treatment is a complete surgical excision, and long-term follow-up is necessary because recurrence may appear long after treatment.

Health for all by the year 2000-cultural handicaps and possible solutions.

Health conditions, as part-and-parcel of man's project of life, reflect the way people choose to live together as a collective body. Health behavior analysis cannot ignore cultural conditions that foster different ways of life. Health practices are not a matter of mere compliance to a set of norms, but a cultural product. Professional competencies ask for a sufficient curriculum, a full proficiency. Myth and logos should be related and the symbolic form everyday experience assumes should be discussed. Paradigmatic thought should give place to mutual growth and subjective and objective aspects of reality should have an equal and complementary standing. Disciplinarity and multidisciplinarity should evolve into interdisciplinarity, as a paradigm of cooperation, interconnection, equality, and flexibility in order to define curricula and programs. Conceptual ecology of science should be emphasized. New concepts of growth, wealth, work, and power should be discussed as a way of attaining health for all in the year 2000.