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Biomedical subjects

R Franco

Publications and source records attributed to R Franco.

At least 37 records · Page 2Linked to original sources
PubMed2001

Microvascular coagulopathy and disseminated intravascular coagulation.

OBJECTIVE: To review the dual characteristics of disseminated intravascular coagulation (DIC), as both a contributor to multiple organ failure as well as a symptom of severe underlying disease associated with systemic vascular changes. DATA SOURCES: Published literature data and unpublished results from the authors. DATA SUMMARY: Clinical and experimental studies strongly suggest that DIC contributes to multiple organ failure and death in patients with severe systemic disorders such as sepsis. DIC is evoked by systemic cytokine activity, and the inflammatory response aggravates vascular permeability, inflammation, and cell damage in tissues. In addition to intravascular fibrin formation, thrombin and fibrin generation in tissues is also an important aspect of DIC. An example of DIC at the organ level is adult respiratory distress syndrome, where fibrin in the lung is a characteristic feature. Intravascular fibrin formation and occlusion may elicit a hypoxic response with induction of hypoxia related transcription factors. The resulting ischemic preconditioning may offer protective effects to the involved organ(s). CONCLUSIONS: Overall, the beneficial or harmful effects of activated coagulation and fibrin formation for organ pathology and recovery from DIC remain to be explored. This may be a critical element in the assessment of ischemia-reperfusion effects of specific anticoagulant therapy.

Anticoagulants↗
PubMed2001

DNA ploidy and cyclin D1 expression in basal cell carcinoma of the head and neck.

Basal cell carcinomas (BCCs) may be subdivided into primary with a favorable biologic course (BCC1) and recurrent and/or metastatic (BCC2). No clear association between primary tumor location, histologic subtype, or other clinicopathologic variables and predisposition for BCC2 has been found. Histopathologic criteria are limited for prognostication. To identify prognostic factors useful for planning therapy, we studied cyclin D1 immunohistochemical expression, DNA ploidy, and epiluminescence light microscopic (ELM) patterns in 60 cases of BCC (30 BCC1 and 30 BCC2) in the head and neck region, half of which were hyperpigmented. Cyclin D1 was absent in 27 cases, expressed at low level in 4 cases, and overexpressed in 30 cases. Seven BCCs were euploid, 28 exhibited a mixed cellular population, and 25 were aneuploid. Among aneuploid tumors, hypodiploidy was found in 12. Among the 30 pigmented carcinomas, only 15 showed a typical ELM pattern. No association between pigmentation and more aggressive biologic behavior of BCC was found. These results and follow-up data seem to indicate that an unfavorable outcome can be predicted by hyperexpression of cyclin D1, aneuploidy, and an atypical ELM pattern for pigmented cases. A definite hypodiploid peak was associated with worse prognosis. The analysis of cyclin D1 expression and DNA ploidy may help identify BCC with an aggressive phenotype and a poor clinical outcome.

Basal Cell Carcinoma↗
PubMed2001

P53 and hMSH2 expression in basal cell carcinomas and malignant melanomas from photoexposed areas of head and neck region.

Ultraviolet (UV) radiation plays a pivotal role in skin damage and photocarcinogenesis. The basic mechanism of phototoxicity lies in DNA damage, and involves mutation of tumor suppressor genes, oncogenes and genes directly involved in the control of the stability of genome, such as the mismatch repair (MR) genes. The goal of this study was to evaluate the role of p53 and hMSH2 in the UV-related carcinogenetic process. An immunohistochemical study for p53 and hMSH2 was performed in a series of 43 basal cell carcinomas (BCC) and 60 melanomas (MM) from photoexposed areas of head and neck region, comparing the findings with follow-up. A deregulated p53 expression characterized less differentiated, more aggressive BCC (BCC2) but not the well-differentiated ones (BCC1). The hMSH2 protein was present, though expressed at varying levels, in 18 out of 21 BCC1 cases and in 4 out of 22 BCC2. In the remaining 3 cases of BCC1 and 18 cases of BCC2, a complete absence of hMSH2 expression was found, correlating directly with the presence of recurrence and/or death of the disease in case of melanoma (p<0.05). Overall, the expression of hMSH2 correlated inversely with the p53 overexpression (p<0.01). In MM, p53 was found overexpressed in 81.6% of the cases, and this correlated positively with the level of infiltration and with the presence of relapses (p<0.01) or metastasis (p<0.01) and inversely with the disease-free interval (p<0.05). These results are in agreement with the reported association between p53 deregulation and a more aggressive cancer phenotype. The evaluation of the expression of p53 and hMSH2 could improve the management of patients with BCC and MM, and could have a role also in the evaluation of the early cutaneous photo-inducted damage, contributing to the identification of presymptomatic patients predisposed to the development of UV-related new skin tumors, who could become candidates for chemoprevention trials.

Adult↗
PubMed2001

Adenosine A2B receptors behave as an alternative anchoring protein for cell surface adenosine deaminase in lymphocytes and cultured cells.

Adenosine deaminase (ADA) is an enzyme of the purine metabolism that has been largely considered to be cytosolic. Recently, it has been demonstrated that the enzyme appears on the surface of lymphocytes where it interacts with the T-cell activation antigen CD26. ADA also appears on the surface of nonlymphoid cells anchored to adenosine A1 receptors. Here it is demonstrated that cell surface ADA in ADA+/CD26- T lymphocytes anchors to adenosine receptors of the A2B subtype (A2BR). An interaction between A2BR and cell surface ADA has been demonstrated in transfected Chinese hamster ovary cells and Jurkat J32 T lymphocytes. This has been proved by coimmunoprecipitation, binding of exogenous ADA to A2BR+ cells, and coimmunolocalization. The specificity of the interaction has also been demonstrated by the lack of interaction with other members of the G protein-coupled receptor superfamily. Binding of ADA to A2BR increases the affinity of the agonist 5'-N-ethylcarboxamidoadenosine and cAMP production. This effect occurs even when ADA devoid of enzyme activity is used. Therefore, in lymphocytes, cell surface ADA, apart from degrading extracellular adenosine, regulates those actions of adenosine that are mediated via adenosine receptors of the A2B subtype.

Adenosine Deaminase↗
PubMed2001

Involvement of caveolin in ligand-induced recruitment and internalization of A(1) adenosine receptor and adenosine deaminase in an epithelial cell line.

Chronic exposure of A(1) adenosine receptors (A(1)R) to A(1)R agonists leads to activation, phosphorylation, desensitization, and internalization to intracellular compartments of the receptor. Desensitization and internalization of A(1)R is modulated by adenosine deaminase (ADA), an enzyme that regulates the extracellular concentration of adenosine. ADA interacts with A(1)R on the cell surface of the smooth muscle cell line DDT1 MF-2, and both proteins are internalized following agonist stimulation of the receptor. The mechanism involved in A(1)R and ADA internalization upon agonist exposure is poorly understood in epithelial cells. In this report, we show that A(1)R and ADA interact in LLC-PK(1) epithelial cells. Exposure of LLC-PK(1) cells to A(1)R agonists induces aggregation of A(1)R and ADA on the cell surface and their translocation to intracellular compartments. Biochemical and cell biology assays were used to characterize the intracellular vesicles containing both proteins after agonist treatment. A(1)R and ADA colocalized together with the rafts marker protein caveolin. Filipin, a sterol-binding agent that disrupts rafts (small microdomains of the plasma membrane), was able to inhibit A(1)R internalization. In contrast, acid treatment of the cells, which disrupts internalization via clathrin-coated vesicles, did not inhibit agonist-stimulated A(1)R internalization. We demonstrated that A(1)R agonist N(6)-(R)-phenylisopropyl adenosine promotes the translocation of A(1)R into low-density gradient fractions containing caveolin. Furthermore, a direct interaction of the C-terminal domain of A(1)R with caveolin-1 was demonstrated by pull down experiments. These results indicate that A(1)R and ADA form a stable complex in the cell surface of LLC-PK(1) cells and that agonist-induced internalization of the A(1) adenosine receptor and ADA is mediated by clathrin-independent endocytosis.

Adenosine Deaminase↗
PubMed2001

Valsartan alone and as part of combination therapy in general practice in Brazil.

The objective of this study was to assess the efficacy and tolerability of valsartan (Diovan) when given alone or as part of a combination regimen in normal clinical practice, in general practice patients in Brazil. In an open, multicentre post-marketing surveillance (PMS) study, 7256 hypertensive patients were evaluated. Therapy with valsartan either as monotherapy or in combination with 12.5 mg chlorthalidone was observed for up to six months. Assessments at baseline and study endpoints included analysis of adverse events and measurement of systolic and diastolic blood pressure (BP). A total of 3855 patients (53%) received valsartan as monotherapy (mostly as 80 mg once daily), 1162 (16%) received 80 mg valsartan + 12.5 mg chlorthalidone, and 347 (4.8%) received valsartan together with other antihypertensive agents; 858 patients were not evaluable because of inadequate records. A control group of 1034 patients received other antihypertensives. Overall tolerability was high, with 98.5% of patients receiving valsartan alone, 97.7% of those receiving valsartan + chlorthalidone and 92.4% of those receiving other antihypertensives giving tolerability ratings of 'excellent' or 'good'. In this large PMS study, valsartan alone and in combination with chlorthalidone was associated with excellent safety and tolerability in general practice patients in Brazil with primary hypertension, irrespective of age, sex or concomitant diseases.

Adolescent↗
PubMed2001

Cytokines regulate membrane adenosine deaminase on human activated lymphocytes.

CD26 is a lymphocyte marker that can anchor adenosine deaminase (ADA) on the T cell surface. We found that ADA is regulated by cytokines on the cell surface during T cell activation. By means of flow cytometry, immunofluorescence, and immunoblotting techniques, we found that interleukin (IL)-2 and IL-12 up-regulate ecto-ADA and CD26 expression. In clear contrast, IL-4 led to down-regulation of lymphocyte surface ADA without modifying the level of CD26. Moreover, neither circulating ADA transcription nor mRNA translation was regulated by cytokines. These results, along with absence of total-ADA modulation, the variable amount of ADA found in purified plasma membranes, and the different effect of Brefeldin A on the surface presence of ADA and CD26 indicated that cytokines regulate the translocation of ADA towards the cell surface through a mechanism not involving CD26. Ecto-ADA protected activated lymphocytes from the toxic effects of extracellular adenosine. Therefore, this cell surface ADA control might constitute part of the fine immunoregulatory mechanism of adenosine-mediated signaling through purinergic receptors in leukocytes.

Adenosine Deaminase↗
PubMed2000

Efflux of osmolyte amino acids during isovolumic regulation in hippocampal slices.

The efflux of potassium (K(+)) and amino acids from hippocampal slices was measured after sudden exposure to 10% (270 mOsm), 25% (225 mOsm) or 50% (150 mOsm) hyposmotic solutions or after gradual decrease (-2.5 mOsm/min) in external osmolarity. In slices suddenly exposed to 50% hyposmotic solutions, swelling was followed by partial (74%) cell volume recovery, suggesting regulatory volume decrease (RVD). With gradual hyposmotic changes, no increase in cell water content was observed even when the solution at the end of the experiment was 50% hyposmotic, showing the occurrence of isovolumic regulation (IVR). The gradual decrease in osmolarity elicited the efflux of (3)H-taurine with a threshold at -5 mOsm and D-[(3)H]aspartate (as marker for glutamate) and at -20 mOsm for [(3)H]GABA. The efflux rate of [(3)H]taurine was always notably higher than those of [(3)H]GABA and D-[(3)H]aspartate, with a maximal increase over the isosmotic efflux of about 7-fold for [(3)H]taurine and 3- and 2-fold for [(3)H]GABA and D-[(3)H]aspartate, respectively. The amino acid content in slices exposed to 50% hyposmotic solutions (abrupt change) during 20 min decreased by 50. 6% and 62.6% (gradual change). Taurine and glutamate showed the largest decrease. An enhancement in (86)Rb efflux and a corresponding decrease in K(+) tissue content was seen in association with RVD but not with IVR. These results demonstrate the contribution of amino acids to IVR and indicate their involvement in this mechanism of cell volume control.

Amino Acids↗
PubMed2000

Dopamine D1 and adenosine A1 receptors form functionally interacting heteromeric complexes.

The possible molecular basis for the previously described antagonistic interactions between adenosine A(1) receptors (A(1)R) and dopamine D(1) receptors (D(1)R) in the brain have been studied in mouse fibroblast Ltk(-) cells cotransfected with human A(1)R and D(1)R cDNAs or with human A(1)R and dopamine D(2) receptor (long-form) (D(2)R) cDNAs and in cortical neurons in culture. A(1)R and D(1)R, but not A(1)R and D(2)R, were found to coimmunoprecipitate in cotransfected fibroblasts. This selective A(1)R/D(1)R heteromerization disappeared after pretreatment with the D(1)R agonist, but not after combined pretreatment with D(1)R and A(1)R agonists. A high degree of A(1)R and D(1)R colocalization, demonstrated in double immunofluorescence experiments with confocal laser microscopy, was found in both cotransfected fibroblast cells and cortical neurons in culture. On the other hand, a low degree of A(1)R and D(2)R colocalization was observed in cotransfected fibroblasts. Pretreatment with the A(1)R agonist caused coclustering (coaggregation) of A(1)R and D(1)R, which was blocked by combined pretreatment with the D(1)R and A(1)R agonists in both fibroblast cells and in cortical neurons in culture. Combined pretreatment with D(1)R and A(1)R agonists, but not with either one alone, substantially reduced the D(1)R agonist-induced accumulation of cAMP. The A(1)R/D(1)R heteromerization may be one molecular basis for the demonstrated antagonistic modulation of A(1)R of D(1)R receptor signaling in the brain. The persistence of A(1)R/D(1)R heteromerization seems to be essential for the blockade of A(1)R agonist-induced A(1)R/D(1)R coclustering and for the desensitization of the D(1)R agonist-induced cAMP accumulation seen on combined pretreatment with D(1)R and A(1)R agonists, which indicates a potential role of A(1)R/D(1)R heteromers also in desensitization mechanisms and receptor trafficking.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed2000

The HIV-1 gp120 inhibits the binding of adenosine deaminase to CD26 by a mechanism modulated by CD4 and CXCR4 expression.

HIV-1 external envelope glycoprotein gp120 inhibits adenosine deaminase (ADA) binding to its cell surface receptor in lymphocytes, CD26, by a mechanism that does not require the gp120-CD4 interaction. To further characterize this mechanism, we studied ADA binding to murine clones stably expressing human CD26 and/or human CD4, and transiently expressing human CXCR4. In this heterologous model, we show that both recombinant gp120 and viral particles from the X4 HIV-1 isolate IIIB inhibited the binding of ADA to wild-type or catalytically inactive forms of CD26. In cells lacking human CXCR4 expression, this gp120-mediated inhibition of ADA binding to human CD26 was completely dependent on the expression of human CD4. In contrast, when cells were transfected with human CXCR4 the inhibitory effect of gp120 was significantly enhanced and was not blocked by anti-CD4 antibodies. These data suggest that the interaction of gp120 with CD4 or CXCR4 is required for efficient inhibition of ADA binding to CD26, although in the presence of CXCR4 the interaction of gp120 with CD4 may be dispensable.

Adenosine Deaminase↗
PubMed2000

Porphyrin interactions with wild-type and mutant mouse ferrochelatase.

Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, catalyzes Fe(2+) chelation into protoporphyrin IX. Resonance Raman and UV-vis absorption spectroscopies of wild-type and engineered variants of murine ferrochelatase were used to examine the proposed structural mechanism for iron insertion into porphyrin. The recombinant variants (i.e., H207N and E287Q) are enzymes in which the conserved amino acids histidine-207 and glutamate-287 of murine ferrochelatase were substituted with asparagine and glutamine, respectively. Both of these residues are at the active site of the enzyme as deduced from the Bacillus subtilis ferrochelatase three-dimensional structure. On the basis of changes in the UV-vis absorption spectrum, addition of free-base or metalated porphyrins to wild-type ferrochelatase and H207N variant yields a 1:1 complex, most likely a monomeric protein-bound species at the active site. In contrast, the addition of porphyrin (either free base or metalated) to E287Q is substoichiometric, as this variant retains bound porphyrin in the active site during isolation and purification. The specificity of porphyrin binding is confirmed by the narrowing of the structure-sensitive lines and the vinyl vibrational mode in the resonance Raman spectra. Shifts in the resonance Raman lines of free-base and metalated porphyrins bound to the wild-type ferrochelatase indicate a nonplanar distortion of the porphyrin macrocycle. However, the magnitude of the distortion cannot be determined without first defining the specific type of deformation. Significantly, the extent of the nonplanar distortion varies in the case of H207N- and E287Q-bound porphyrins. In fact, resonance Raman spectral decompositions indicate a homogeneous ruffled deformation for the nickel protoporphyrin bound to the wild-type ferrochelatase, whereas both planar and ruffled conformations are present for the H207N-bound porphyrin. Perhaps more revealing is the unusual resonance Raman spectrum of the endogenous E287Q-bound porphyrin, which has the structure-sensitive lines greatly upshifted relative to those of the free-base protoporphyrin in solution. This could be interpreted as an equilibrium between protein conformers, one of which favors a highly distorted porphyrin macrocycle. Taken together, these findings suggest that distortion occurs in murine ferrochelatase for some porphyrins, even without metal binding, which is apparently required for the yeast ferrochelatase.

Amino Acid Substitution↗
PubMed2000

Mechanisms of CD26/dipeptidyl peptidase IV cytokine-dependent regulation on human activated lymphocytes.

Among the cellular pathways activated by IL-12, we had previously found that both the percentage and intensity of CD26(+)cells in the PHA-stimulated T cells increased when IL-12 was present (independently of its CD4 or CD8 phenotype). Now, we examined the molecular mechanisms of this IL-12-mediated effect. The IL-12 regulation pathway is dependent of de novo protein synthesis and independent of cytokine secretion. Our results show two transcripts for CD26 in PBMC for the first time and no regulation by ILs at this level. Furthermore, secretion of the serum forms of CD26/DPPIV were not affected by IL-12. Interestingly, assays with neutralizing mAbs against TNF-alpha suggest that this cytokine negatively modulates CD26 expression. The fact that translation and probably translocation of CD26 toward the cell surface can be regulated by IL-12 and TNF-alpha reveals new aspects about the control of this T(H1)marker.

Cells, Cultured↗
PubMed2000

PCNA in the testis of the frog, Rana esculenta: a molecular marker of the mitotic testicular epithelium proliferation.

Proliferating cell nuclear antigen (PCNA) plays an essential role in nucleic acid metabolism as a component of the replication and repair machinery. This toroidal-shaped protein encircles DNA and can slide bidirectionally along the duplex. One of the well-established functions for PCNA is its role as the processing factor for DNA polymerase delta and epsilon. It has become apparent that PCNA interacts with proteins involved in the cell cycle. The PCNA interactions with different cellular proteins and the importance of these interactions are also discussed. To examine the different mitotic testicular epithelium proliferation during the annual discontinuous frog (Rana esculenta) spermatogenesis, the temporal and the spatial PCNA expression are described and give a useful endogenous molecular marker.

Animals↗
PubMed2000

Evidence for adenosine/dopamine receptor interactions: indications for heteromerization.

Evidence has been obtained for adenosine/dopamine interactions in the central nervous system. There exists an anatomical basis for the existence of functional interactions between adenosine A(1)R and dopamine D(1)R and between adenosine A(2A) and dopamine D(2) receptors in the same neurons. Selective A(1)R agonists affect negatively the high affinity binding of D(1) receptors. Activation of A(2A) receptors leads to a decrease in receptor affinity for dopamine agonists acting on D(2) receptors, specially of the high-affinity state. These interactions have been reproduced in cell lines and found to be of functional significance. Adenosine/dopamine interactions at the behavioral level probably reflect those found at the level of dopamine receptor binding and transduction. All these findings suggest receptor subtype-specific interactions between adenosine and dopamine receptors that may be achieved by molecular interactions (e.g., receptor heterodimerization). At the molecular level adenosine receptors can serve as a model for homomeric and heteromeric protein-protein interactions. A1R forms homodimers in membranes and also form high-order molecular structures containing also heterotrimeric G-proteins and adenosine deaminase. The occurrence of clustering also clearly suggests that G-protein- coupled receptors form high-order molecular structures, in which multimers of the receptors and probably other interacting proteins form functional complexes. In view of the occurrence of homodimers of adenosine and of dopamine receptors it is speculated that heterodimers between these receptors belonging to two different families of G-protein-coupled receptors can be formed. Evidence that A1/D1 can form heterodimers in cotransfected cells and in primary cultures of neurons has in fact been obtained. In the central nervous system direct and indirect receptor-receptor interactions via adaptor proteins participate in neurotransmission and neuromodulation and, for example, in the establishment of high neural functions such as learning and memory.

Animals↗
PubMed2000

Adenosine A(1) receptor in cultured neurons from rat cerebral cortex: colocalization with adenosine deaminase.

Adenosine A(1) receptors (A(1)Rs) have been characterized in primary cultures of neurons from cerebral cortex. The specific adenosine A(1) antagonist 8-cyclopentyl-1,3-[(3)H]dipropylxanthine bound to both membranes and intact cells. When saturation experiments were performed in membranes, a K(D) value of 0.76 nM and a B(max) of 57 fmol/mg of protein were obtained. Competition assays revealed a pharmacological profile characteristic of A(1)Rs. The presence of this receptor was further confirmed by RT-PCR analysis. The expression of the receptor showed no significant changes during the period of culture studied, up to 12 days in vitro. A(1)R agonist inhibited forskolin-stimulated adenylyl cyclase, showing the functional coupling of these receptors with the effector. alphaG(i1, 2) protein level, detected by immunoblot, presented an increase during the period of culture. This increase correlated with an increase in the mRNA level of alphaG(i1) but not alphaG(i2). By immunochemical assays, it is shown that these receptors are expressed in both the neuronal cell body and the proximal dendrites. Colocalization of A(1)Rs with microtubule-associated protein 2 and cell surface adenosine deaminase was shown by confocal microscopy. The high degree of colocalization observed between A(1)Rs and ectoadenosine deaminase in neurons could suggest an important role of the enzyme in adenosine-mediated neuromodulation.

Adenosine Deaminase↗
PubMed2000

The heat shock cognate protein hsc73 assembles with A(1) adenosine receptors to form functional modules in the cell membrane.

A(1) adenosine receptors (A(1)Rs) are G protein-coupled heptaspanning receptors that interact at the outer face of the plasma membrane with cell surface ecto-adenosine deaminase (ecto-ADA). By affinity chromatography the heat shock cognate protein hsc73 was identified as a cytosolic component able to interact with the third intracellular loop of the receptor. As demonstrated by surface plasmon resonance, purified A(1)Rs interact specifically with hsc73 with a dissociation constant in the nanomolar range (0.5 +/- 0.1 nM). The interaction between hsc73 and A(1)R led to a marked reduction in the binding of the ligands and prevented activation of G proteins, as deduced from (35)S-labeled guanosine-5'-O-(3-thio)triphosphate binding assays. Interestingly this effect was stronger than that exerted by guanine nucleotide analogs, which uncouple receptors from G proteins, and was completely prevented by ADA. As assessed by immunoprecipitation a high percentage of A(1)Rs in cell lysates are coupled to hsc73. A relatively high level of colocalization between A(1)R and hsc73 was detected in DDT(1)MF-2 cells by means of confocal microscopy, and no similar results were obtained for other G protein-coupled receptors. Colocalization between hsc73 and A(1)R was detected in specific regions of rat cerebellum and in the body of cortical neurons but not in dendrites or synapses. Remarkably, agonist-induced receptor internalization leads to the endocytosis of A(1)Rs by two qualitatively different vesicle types, one in which A(1)R and hsc73 colocalize and another in which hsc73 is absent. These results open the interesting possibility that signaling via G protein-coupled receptors may be regulated by heat shock proteins.

Adenosine Deaminase↗