Rethinking Descartes.

Rethinking Descartes is a brief contribution that seeks to encourage medical operators and clinicians to rethink René Descartes' Soul-Body Dualism.

Patient Associations: a driving force for Rare Diseases research. Resilience: a driving force for Patient Associations.

Until a few decades ago, Rare Diseases were relatively unknown. Their low prevalence made them invisible to public opinion, and were of little concern to researchers and pharmaceutical industries. Rare disease sufferers and their loved ones had become victims of the disease as their implications were overlooked. Consequently, some of these individuals formed associations and embarked on ways to change this situation of neglect they had found themselves, in finally having their rights recognized. These associations have over time gained important roles in planning public health and biomedical research, especially after the introduction of the Human Genome Project. Their active participation and awareness activities have been crucial in establishing reliable Rare Diseases Registries and related Biobanks, essential tools in fully utilizing the data and the new omics technologies derived from the Human Genome Project in the field of Rare Diseases. The founders and members of these associations have a high degree and considerable ability to face the difficulties of life, while also maintaining a positive attitude and a confident vision, best defined as resilience. Not everyone, and not always, is endowed with a resilience capability. However resilience can be improved or reinforced through appropriate training and intervention programs. This review points out specific programs centered mainly on mindfulness.

Biophysical and structural characterization of 1H-NMR-detectable mobile lipid domains in NIH-3T3 fibroblasts.

Nature and subcellular localization of 1H-NMR-detectable mobile lipid domains (ML) were investigated by NMR, Nile red fluorescence and electron microscopy, in NIH-3T3 fibroblasts and their H-ras transformants (3T3ras) transfected with a high number of oncogene copies. Substantial ML levels (ratio of (CH2)n/CH3 peak areas R=1. 56+/-0.33) were associated in untransformed fibroblasts with both (a) intramembrane amorphous lipid vesicles, about 60 nm in diameter, distinct from caveolae; and (b) cytoplasmic, osmiophilic lipid bodies surrounded by own membrane, endowed of intramembrane particles. 2D NMR maps demonstrated that ML comprised both mono- and polyunsaturated fatty chains. Lower ML signals were detected in 3T3ras (R=0.76+/-0.37), under various conditions of cell growth. Very few (if any) lipid bodies and vesicles were detected in the cytoplasmic or membrane compartments of 3T3ras cells with R<0.4, while only intramembrane lipid vesicles were associated with moderate R values. Involvement of phosphatidylcholine hydrolysis in ML generation was demonstrated by selective inhibition of endogenous phospholipase C (PC-plc) or by exposure to bacterial PC-plc. This study indicates that: (1) both cytoplasmic lipid bodies and membrane vesicles (possibly in mutual dynamic exchange) may contribute (although to a different extent) to ML signals; and (2) high levels of ras-transfection either inhibit ML formation or facilitate their extrusion from the cell.

Grossly defective nef gene sequences in a human immunodeficiency virus type 1-seropositive long-term nonprogressor.

We have been investigating a long-term nonprogressor who was found to be human immunodeficiency virus type 1 (HIV-1) seropositive in 1985 and has survived with stable CD4+ T-cell counts (>1,000 CD4 cells/microl) without any AIDS-related illness. We have previously reported that repeated attempts to measure HIV-1 RNA in the peripheral mononuclear cells obtained from this subject have invariably failed. In the present study, we have analyzed the molecular nature of the HIV-1 quasispecies infecting this patient by PCR amplification of two proviral regions, the 5' long terminal repeat (5'LTR)/gag leader and the nef gene, directly from fresh uncultured peripheral mononuclear cells, followed by length polymorphism analysis (with 1994, 1995, and 1996 samples) and sequencing (with a 1996 sample). Only proviral forms with nef deletions were revealed by length polymorphism analysis in samples from all three time points. Sequence analysis of the nef gene from the 1996 sample confirmed the presence of similar proviral quasispecies characterized by the presence of several deletions located in the nef-alone and the nef/U3 overlapping regions. Length polymorphism analysis of the 5'LTR/gag leader region suggested the existence of two major quasispecies populations, one characterized by the presence of forms carrying deletions in the U3 region and the other showing a completely intact, full-length 5'LTR. Evidence of the role of nef gene defects in long-term survival of HIV-1-infected patients has been provided so far in two independent investigations involving patients infected with HIV through blood transfusion. Here we show the existence of a similar condition in a subject who acquired HIV-1 seropositivity through the sexual route.

Lower levels of 1H MRS-visible mobile lipids in H-ras transformed tumorigenic fibroblasts with respect to their untransformed parental cells.

High resolution 1H MRS studies report increased mobile neutral lipid (MNL) signals in transformed and malignant as well as in some in vitro cultured embryonic cells. Nature, subcellular localization and biological function of MNL are still under debate. This work was aimed at assessing alterations induced in MNL signals of NIH-3T3 mouse embryo fibroblasts by transformation with human HJ-ras oncogene. Lower MRS-visible MNL levels were unexpectedly detected in ras-transformed, in vivo tumorigenic fibroblasts, with respect to their untransformed and non-tumorigenic parental cells. MRS, gas chromatography and chemical analysis on cells and their lipid extracts indicated that these spectral differences could hardly be attributed to different triacylglycerol, free fatty acids and total cholesterol levels or to changes in the fatty acyl degree of unsaturation and average chain length. Additional, possibly more relevant mechanisms of regulation of MNL mobility may implicate the extensive morphogenetic changes and reorganization of cytoskeleton components (notably actin) associated with ras-transformation.

Stable transfection of provirus of human immunodeficiency virus into a murine packaging cell line.

In order to generate HIV (murine leukemia virus (MuLV)) pseudotypes, HIV genome was transfected into the ecotropic murine packaging cell line (GP+E86) and four of the nine transfected clones were extensively characterized. One clone (801), harbouring a full copy of integrated HIV sequences, exhibited a detectable level of intracellular HIV p24 antigen expression. Northern blot analysis revealed that clone 801 expressed all three classes of HIV mRNAs. Multispliced 2 kb mRNAs were detected in another clone (8.14). Two other clones (1.31 and 1.32) also exhibited a complete HIV provirus, but did not show any viral expression, as evaluated by Northern blot analysis or HIV p24 ELISA. Reverse transcription-polymerase chain reaction (RT-PCR) experiments revealed the presence of full length genomic RNA in four transfected clones, which were extensively characterized. A co-cultivation of clone 801 with human CD4' cells resulted in syncytia formation. By electron microscopy, mature HIV particles were observed after co-cultivation of uninfected C8166 cells with 801 cells. These results demonstrated that the murine clone was stably transfected with the complete HIV genome and was capable of shuttling infectious HIV to human cells. Clone 801 was co-cultivated with murine NIH-3T3 fibroblasts. In several experiments, HIV infection of NIH-3T3 cells was revealed by PCR technique. Thus, 801 cells appear to produce low levels of HIV (MuLV) pseudotypes capable of transferring the HIV genome into mouse cells.

Detection of phosphatidylcholine-specific phospholipase C in NIH-3T3 fibroblasts and their H-ras transformants: NMR and immunochemical studies.

Although evidence supports constitutive activation of phosphatidylcholine specific phospholipase C (PC-plc) in rastransformed fibroblasts, no studies have been devoted to measure the basal activity levels of this enzyme, its molecular characteristics and subcellular localization. This paper reports for the first time measurements of the activity of different enzymes responsible for PC hydrolysis (PC-plc; phospholipases A2 (pla2) and A1 (pla1)) in homogenates of murine NIH-3T3 fibroblasts (3T3) and their transformants obtained by human H-ras transfection (3T3ras). To this end, 31P NMR analyses were carried out on total cell homogenates, incubated in the presence of mixed diheptanoylphosphatidylcholine: sphingomyelin (DHPC:SM) unilamellar vesicles (SLUV), in which DHPC acts as a suitable substrate for water-soluble lipolytic enzymes. The basal PC-plc activity levels (0.66 +/- 0.14 and 0.38 +/- 0.10 nmol/10(6) cells.hour in 3T3 and 3T3ras fibroblasts, respectively),were substantially higher (over 30-50x) than those reported in the literature for normal mammalian cells (dog heart myocytes). Moreover the PC-plc activity was about 15-30 times lower than the overall PC deacylation activity in both clones. The use of high titer polyclonal antibodies, raised in a rabbit against bacterial PC-plc, allowed identification of one cross-reactive mammalian PC-plc component (M(r) 66 kD) in cell lysates of both 3T3 and 3T3ras fibroblasts, and detection, by indirect immunofluorescence, of its subcellular localization. In control 3T3 fibroblasts (in the late log-phase of growth) the enzyme was exclusively located in the cytosol, while in H-ras transformed cells it was massively exposed on the external side of the membrane. This new finding strongly suggests that the oncogenic product p2Iras is able to induce (or mediate) translocation of PC-plc across the plasma membrane of ras transformed cells, with possible implications not only on cell biochemistry (enhancement of PC-plc activity, and consequent production of intra- and extracellular PCho and accumulation of neutral lipids) but also on cell-cell interaction mechanisms which facilitate tumour invasion and metastasis of oncogene-transformed cells.

Growth properties, differentiation capacity and oncogene expression in metastatic and nonmetastatic Friend leukemia cell variants.

The aims of this study were: (1) to characterize the biologic properties of the WGA-resistant (WR) Friend leukemia cells (FLC) as compared to the original nonmetastatic or highly metastatic FLC; (2) to investigate the possible correlations between the expression of some oncogenes (i.e., c-myc, H-ras and K-ras) and the in vitro and in vivo behavior of FLC. The tumorigenic behavior of the different FLC types strongly depended on the site of tumor injection. Both WR FLC and in vitro passaged FLC did not grow as ascites (when injected intraperitoneally) and developed large solid tumors (when injected subcutaneously), without forming any spleen or liver metastasis. In contrast, in vivo passaged FLC rapidly formed hemorrhagic ascites when injected intraperitoneally; the subcutaneous injection of these cells resulted in the development of solid tumors, which were smaller than the other FLC tumors, but capable of metastasizing to the liver and to the spleen. No significant differences were observed in the in vitro growth characteristics and cell cycle parameters among the different FLC types under various experimental conditions (i.e., FCS concentration or cell seeding densities). Similarly to the metastatic in vivo passaged parental cells, WR FLC exhibited a much lower erythroid differentiation after in vitro addition of either dimethyl sulfoxide or hexamethylene bisacetamide than the in vitro passaged FLC. High levels of c-myc oncogene mRNA were expressed in all FLC variants; no major variations in the c-myc expression were observed in FLC cultivated in medium supplemented with different FCS concentrations and/or seeded at various cell densities. In addition, no changes in the expression of H-ras or K-ras were observed between the different FLC types.

Studies on the expression of H-2 antigens in non-metastatic and highly metastatic Friend erythroleukemia cells: correlation with the in vivo behaviour of tumor cells.

The levels of expression of histocompatibility antigens on the cell membrane and their gene expression in non-metastatic and in highly metastatic Friend leukemia cells (FLC) were measured and the levels of expression of these antigens were correlated with the different in vivo behaviour of the tumor cells. Highly metastatic in vivo passaged FLC (either interferon-sensitive 745 or interferon alpha/beta-resistant 3Cl-8 cells) expressed higher levels of class I H-2K and H-2D antigens on their cell membrane with respect to the non-metastatic in vitro passaged counterparts. The increased expression of H-2 class I antigens was associated with an increased transcription of H-2K and H-2D genes. As both in vitro and in vivo passaged FLC have been shown to be resistant in vitro to the natural killer (NK) cell activity, we tried to correlate the levels of expression of histocompatibility antigens with the in vivo clearance of [125I]UDR-labeled FLC. However, no correlation was found between the levels of expression of H-2 antigens and the in vivo clearance of tumor cells. In fact, in vivo passaged FLC (tested either after 1 or after 15 in vitro passages) expressed virtually identical levels of H-2 antigens; however, the freshly explanted in vivo passaged FLC exhibited markedly lower levels of clearance from the lung, spleen and liver (when injected i.v. in DBA/2 mice) with respect to the corresponding FLC cultivated for several passages in vitro. Pretreatment of in vitro passaged 745 FLC with either interferon alpha/beta or interferon gamma resulted in the acquisition of some metastatic potential of FLC to the liver when interferon-treated FLC were subsequently injected i.v. in DBA/2 mice; such in vitro treatments resulted in a 2-3-fold increase in the expression of H-2K antigens versus the control untreated FLC. We suggest that such increases could represent some advantages for the homing properties of tumor cells and/or for the tumor progression, by mechanisms different from the resistance to the NK cells.

Detection of a transforming gene in spontaneous reticulum cell sarcoma of SJL/J mice: genetically linked and host-dependent neoplasia.

Spontaneous reticulum cell sarcoma (RCS) tumor induction occurs in 90% of SJL/J mice of 8-13 months of age. Tumor induction and growth has been shown to be under the influence of both H-2 and non-H-2 genes as well as the presence of an intact host T-cell system. We postulated that cellular oncogenes may play a role in the induction, growth, and characteristics of RCS. DNA-mediated gene transfer protocols were adopted to investigate the presence of transforming genes in DNA from RCS of SJL/J mice. High molecular weight DNA was isolated from these tumors as well as from brains and livers of control tumor-free SJL/J mice and transfected into NIH-3T3 mouse and F2408 rat fibroblast cell lines. Foci of transformed cells with a peculiar round morphology were scored in both rat and mouse cultures given tumor DNA, but not in those receiving DNA from normal tissues. DNA from first-cycle transformants was transfected in further cycles of transfection, giving rise to foci with similar morphological appearances and growth properties. These experiments suggest that a transforming gene, present in RCS spontaneous tumors, is involved in the malignant conversion of the transfected normal fibroblasts. The implication of these results with respect to the induction and growth properties of RCS is discussed.

ras gene Amplification and malignant transformation.

Morphologic transformation of NIH 3T3 mouse cells occurs upon transfection of these cells with large amounts (greater than or equal to 10 micrograms) of recombinant DNA molecules carrying the normal human H-ras-1 proto-oncogene. We provide experimental evidence indicating that transformation of these NIH 3T3 cells results from the combined effect of multiple copies of the H-ras-1 proto-oncogene rather than from spontaneous mutation of one of the transfected H-ras-1 clones (E. Santos, E.P. Reddy, S. Pulciani, R.J. Feldman, and M. Barbacid, Proc. Natl. Acad. Sci. USA 80:4679-4683, 1983). Levels of H-ras-1 RNA and p21 expression are highly elevated in the NIH 3T3 transformants, and in those cases examined, these levels correlate with the malignant properties of these cells. We have also investigated the presence of amplified ras genes in a variety of human carcinomas. In 75 tumor biopsies, we found amplification of the human K-ras-2 locus in one carcinoma of the lung. These results indicate that ras gene amplification is an alternative pathway by which ras genes may participate in the development of human neoplasia.

Characterization of a human transforming gene isolated from T24 bladder carcinoma cells.

A human transforming gene present in T24 bladder carcinoma cells has been molecularly cloned. The transforming sequences have been located within a 4.6 kilo base pair (kbp) DNA fragment that transforms NIH/3T3 cells with a specific activity of 5 X 10(4) focus-forming units/pmol. Homologous sequences present in normal human DNA have also been molecularly cloned. Comparison of the restriction endonuclease maps of the normal and transforming genes did not reveal any significant differences. These results suggest that subtle molecular changes are responsible for the acquisition of malignant properties by this gene in T24 cells. The T24 oncogene was found to be unrelated to transforming genes present in a variety of human tumors other than bladder carcinomas. In contrast, the T24 oncogene is highly related to the onc genes of the BALB and Harvey strains of murine sarcoma viruses ( MSVs ). Preliminary characterization of the transcriptional and translational products of the T24 oncogene suggests that this gene is transcribed into a 1.2-kbp poly(A)-containing RNA whose translation yields a 23,000-dalton protein antigenically related to the transforming gene products of BALB and Harvey MSVs .

A transforming ras gene in tumorigenic guinea pig cell lines initiated by diverse chemical carcinogens.

Fetal guinea pig cells were transformed by treatment with four different chemical carcinogens including nitroso compounds and polycyclic hydrocarbons. As a consequence of this treatment, oncogenes capable of transforming NIH/3T3 cells became activated in each of five independently established clonal guinea pig cell lines. Molecular characterization of representative NIH/3T3 transformants revealed that the same oncogene was present in each of the cell lines tested. Moreover, detection of this transforming gene paralleled the acquisition of tumorigenic properties by these neoplastic cells.

Spontaneous activation of a human proto-oncogene.

It has been recently shown that malignant activation of the c-has/bas proto-oncogene in T24 human bladder carcinoma cells was mediated by a single point mutation. A deoxyguanosine located at position 35 of the first exon of this proto-oncogene was substituted by thymidine. These findings predicted that the resulting oncogene would code for a structurally altered p21 protein containing valine instead of glycine as its 12th amino acid residue. We now report the spontaneous activation of the human c-has/bas proto-oncogene during transfection of NIH/3T3 cells. As in T24 cells, this in vitro activated oncogene also acquired malignant properties by a single point mutation. In this case we have detected a G leads to A transition, which occurred at the same position as the mutation responsible for the activation of the T24 oncogene. These results predict that the p21 protein coded for by the spontaneously activated c-has/bas gene will incorporate aspartic acid as its 12th amino acid residue. Computer analysis of the secondary structure of c-has/bas encoded p21 proteins indicates that substitution of the glycine residue located at position 12, not only by aspartic acid or valine but also by any other amino acid, would result in the same structural alteration. These findings indicate that a specific conformational change is sufficient to confer transforming properties to this p21 protein. Moreover, they predict that any mutation affecting the coding properties of the 12th codon of the c-has/bas proto-oncogene will lead to its malignant activation.

T24 human bladder carcinoma oncogene is an activated form of the normal human homologue of BALB- and Harvey-MSV transforming genes.

A transforming gene isolated from T24 human bladder carcinoma cells is closely related to the BALB murine sarcoma virus (MSV) onc gene (v-bas). This transforming gene is localized to a 4.6 kilobase pair (kbp) region and is expressed as a 1.2-kbp polyadenylated transcript, which contains v-bas related sequences. Moreover, antisera known to detect the immunologically related onc gene products of BALB- and Harvey-MSVs recognized elevated levels of a related protein in T24 cells. The normal human homologue of v-bas was found to be indistinguishable from the T24 oncogene by heteroduplex and restriction enzyme analysis. These results imply that rather subtle genetic alterations have led to the activation of the normal human homologue of v-bas as a human transforming gene.

Oncogenes in human tumor cell lines: molecular cloning of a transforming gene from human bladder carcinoma cells.

The presence of dominant transforming genes in human tumor cell lines has been investigated. High molecular weight DNAs isolated from cell lines established from carcinomas and sarcomas of various organs as well as from a glioblastoma and two melanomas were utilized to transfect NIH/3T3 mouse fibroblasts. The DNAs of T24 and A2182, two cell lines derived from a bladder and a lung carcinoma, respectively, and of HT-1080, a cell line established from a fibrosarcoma, were able to transform recipient NIH/3T3 cells. First-cycle transformants exhibited anchorage-independent growth and were tumorigenic in athymic and immunocompetent mice. Moreover, they contained human DNA sequences and were able to transmit their malignant phenotype in additional cycles of transfection. Southern blot analysis of T24-derived transformants showed that a single fragment of human DNA specifically cosegregated with the malignant phenotype, suggesting that it contained the T24 oncogene. Therefore, these human sequences were molecularly cloned with lambda Charon 9A as the cloning vector. The resulting recombinant DNA molecule, designated lambda T24-15A, was shown to contain a 15-kilobase-pair EcoRI insert of human cellular DNA. lambda T24-15A DNA (either intact or EcoRI digested) transformed NIH/3T3 fibroblasts with a specific activity of 20,000 focus-forming units per pmol of cloned DNA. Our results indicate that we have molecularly cloned a biologically active oncogene present in T24 human bladder carcinoma cells.

Transforming genes in human tumors.

DNAs isolated from a variety of human tumor cell lines as well as from naturally occurring human carcinomas and sarcomas were shown to induce morphologic transformation upon transfection into NIH/3T3 cells. All tested transformants contained human DNA sequences, some of which specifically cosegregated with the malignant phenotype in additional cycles of transfection. Southern blot analysis of second cycle transformants derived from T24 human bladder carcinoma cells showed the presence of a single 15 kbp EcoRI fragment of human DNA. These sequences were molecularly cloned utilizing lambda Charon 9A as the cloning vector. The resulting recombinant DNA molecule, designated lambda T24-15A, was shown to contain an internal 6.6 kbp Bam HI fragment of human DNA that transformed NIH/3T3 fibroblasts with a specific activity of 5 X 10(4) focus forming units per picomole. These results indicate that we have molecularly cloned an oncogene present in T24 bladder carcinoma cells. Comparison of molecular clones containing the T24 oncogene and its normal homologue did not reveal biochemical differences that helped to explain the malignant properties of this oncogene. Finally, we report preliminary results indicating that the T24 bladder carcinoma oncogene is highly related to the transforming gene of BALB-MSV, an acute transforming retrovirus.

Effects of a chromatin low molecular weight peptidic fraction on differentiation markers and virus production in Friend leukemia cells.

Low molecular weight chromatin peptides exert a dose-dependent inhibition of Dimethylsulfoxide (DMSO)-induced erythroid differentiation of murine Friend Leukemia Cells (FLC). This effect correlates with the degree of purification of the peptide fractions. Crot analysis of globin mRNA amounts in DMSO-treated FLC given the peptides showed a 4-5-fold decrease of messenger RNA in the cytoplasma with no nuclear storage of globin transcripts. Spectrin accumulation in "induced" FLC is inhibited as well. The effects of the peptides on erythroid markers are reversible upon removal of the compounds. They also appear to be specific for induced gene expression as (1) no effects are observed on cell growth and RNA synthesis in normal non-differentiating cell lines; and (2) no changes have been detected with regard to the expression of integrated viral genes coding for continuous shedding of viral particles.