Cyclosaligenyl-2',3'-didehydro-2',3'-dideoxythymidine monophosphate: efficient intracellular delivery of d4TMP.

Cyclosaligenyl-2',3'-didehydro-2', 3'-dideoxythymidine-5'-monophosphate (cycloSal-d4TMP) is a potent and selective inhibitor of human immunodeficiency virus replication in cell culture and differs from other nucleotide prodrug approaches in that it is designed to selectively deliver the nucleotide 5'-monophosphate by a controlled, chemically induced hydrolysis. Its antiviral efficacy in cell culture is at least as good as, if not superior to, that of d4T. CycloSal-d4TMP was found to lead to the efficient intracellular release of d4TMP in a variety of cell lines, including both wild-type CEM and thymidine kinase-deficient CEM/TK(-) cells. Under similar experimental conditions, exposure of CEM/TK(-) cells to d4T failed to result in significant d4TTP levels. The intracellular conversion of cycloSal-d4TMP proved to be both time and dose dependent. The half-life of d4TTP generated intracellularly from d4T- or cycloSal-d4TMP-treated CEM cells was approximately 3.5 h, and the intracellular ratios of d4TTP/d4TMP in cells exposed to cycloSal-d4TMP gradually increased from 1 to 3.4 upon prolonged incubation. Radiolabeled cycloSal-d4TMP could be separated as its two R(p) and S(p) diastereomers on high-performance liquid chromatography. The R(p) diastereomer of cycloSal-d4TMP was 3- to 7-fold more efficient in releasing d4TMP and generating d4TTP than the S(p) cycloSal-d4TMP diastereomer. This correlated well with the 5-fold more pronounced antiviral activity of the R(p) diastereomer versus the S(p) diastereomer. d4TMP is a poor substrate for the cytosolic 5'(3')-deoxyribonucleotidase (V(max)/K(m) for d4TMP: 0.08 of V(max)/K(m) for dTMP) and is only slowly hydrolyzed to d4T. This contributes to the efficient conversion of the prodrug of d4TTP.

A deoxyribonucleotidase in mitochondria: involvement in regulation of dNTP pools and possible link to genetic disease.

Three cytosolic and one plasma membrane-bound 5'-nucleotidases have been cloned and characterized. Their various substrate specificities suggest widely different functions in nucleotide metabolism. We now describe a 5'-nucleotidase in mitochondria. The enzyme, named dNT-2, dephosphorylates specifically the 5'- and 2'(3')-phosphates of uracil and thymine deoxyribonucleotides. The cDNA of human dNT-2 codes for a 25.9-kDa polypeptide with a typical mitochondrial leader peptide, providing the structural basis for two-step processing during import into the mitochondrial matrix. The deduced amino acid sequence is 52% identical to that of a recently described cytosolic deoxyribonucleotidase (dNT-1). The two enzymes share many catalytic properties, but dNT-2 shows a narrower substrate specificity. Mitochondrial localization of dNT-2 was demonstrated by the mitochondrial fluorescence of 293 cells expressing a dNT-2-green fluorescent protein (GFP) fusion protein. 293 cells expressing fusion proteins without leader peptide or with dNT-1 showed a cytosolic fluorescence. During in vitro import into mitochondria, the preprotein lost the leader peptide. We suggest that dNT-2 protects mitochondrial DNA replication from overproduction of dTTP, in particular in resting cells. Mitochondrial toxicity of dTTP can be inferred from a severe inborn error of metabolism in which the loss of thymidine phosphorylase led to dTTP accumulation and aberrant mitochondrial DNA replication. We localized the gene for dNT-2 on chromosome 17p11.2 in the Smith-Magenis syndrome-critical region, raising the possibility that dNT-2 is involved in the etiology of this genetic disease.

Mammalian 5'(3')-deoxyribonucleotidase, cDNA cloning, and overexpression of the enzyme in Escherichia coli and mammalian cells.

5'(3')-Deoxyribonucleotidase is a ubiquitous enzyme in mammalian cells whose physiological function is not known. It was earlier purified to homogeneity from human placenta. We determined the amino acid sequences of several internal peptides and with their aid found an expressed sequence tag clone with the complete cDNA for a murine enzyme of 23.9 kDa. The DNA was cloned into appropriate plasmids and introduced into Escherichia coli and ecdyson-inducible 293 and V79 cells. The recombinant enzyme was purified to homogeneity from transformed E. coli and was found to be identical with the native enzyme. After induction with ponasterone, the transfected mammalian cells showed a gradual increase of enzyme activity. A human expressed sequence tag clone contained a large part of the cDNA of the human enzyme but lacked the 5'-end corresponding to 51 amino acids of the murine enzyme. Several polymerase chain reaction-based approaches to find this sequence met with no success. A mouse/human hybrid cDNA that had substituted the missing human 5'-end with the corresponding mouse sequence coded for a fully active enzyme.

Induction of human high K(M) 5'-nucleotidase in cultured 293 cells.

Human 293 cells were stably transfected with a plasmid introducing a receptor for the ecdysone analog muristerone. The cells were further stably transfected with muristerone-inducible expression vectors carrying either the cDNA for the human high K(M) 5'-nucleotidase or the coding sequence of the nucleotidase linked to the 5'-end of the sequence for the green fluorescent protein. Upon induction, both types of transfectants overproduced nucleotidase activity in a time- and dose-dependent manner. Western blots gave values close to the expected subunit molecular masses of 65 and 92 kDa, respectively, excluding processing of the induced proteins. Cells induced to overexpress the nucleotidase showed a decreased growth rate and contained smaller pools of each of the four common ribonucleoside triphosphates. They showed no increased resistance to the toxicity of 2-chlorodeoxyadenosine.

Human high-Km 5'-nucleotidase effects of overexpression of the cloned cDNA in cultured human cells.

5'-Nucleotidases participate, together with nucleoside kinases, in substrate cycles involved in the regulation of deoxyribonucleotide metabolism. Three major classes of nucleotidases are known, one on the plasma membrane and two in the cytosol. The two cytosolic classes have been named high-Km nucleotidases and 5'(3')-nucleotidases. Starting from two plasmids with partial sequences (Oka, J., Matsumoto, A., Hosokawa, Y. & Inoue, S. (1994) Biochem. Biophys. Res. Commun. 205, 917-922) we cloned the complete cDNA of the human high-Km nucleotidase into vectors suitable for transfection of Escherichia coli or mammalian cells. After transfection, E. coli overproduced large amounts of the enzyme. Most of the enzyme was present in inclusion bodies that also contained many partially degraded products of the protein. Part of the enzyme, corresponding to approximately 2% of the soluble proteins, was in a soluble active form. Stably transfected human 293 cells were obtained with a vector where the 3'-end of the nucleotidase coding sequence is linked to the 5'-end of the green fluorescent protein coding sequence. Several green clones overproduced both mRNA and fusion protein. Two clones with 10-fold higher enzyme activity were analyzed further. The nucleotidase activity of cell extracts showed the same substrate specificity and allosteric regulation as the high-Km enzyme. The growth rate of the two clones did not differ from the controls. The cells were not resistant to deoxyguanosine or deoxyadenosine, and did not show an increased ability to phosphorylate dideoxyinosine. Both ribonucleoside and deoxyribonucleoside triphosphate pools were decreased slightly, suggesting participation of the enzyme in their regulation.

[Evaluation of the degree of client satisfaction with ICHC/PAMB regarding secretarial and medical assistance]. / Avaliação do grau de satisfação dos usuários do ICHC/PAMB em relação ao atendimento de secretárias e de médicos.

This survey was intended to evaluate the degree of satisfaction from clients of Prédio dos Ambulatórios do Instituto Central do Hospital das Clínicas concerning waiting time for appointment, kind of reception and quality of attention by secretaries and doctors. It was also intended to evaluate the interest people have in the video system, that has monitors in the majority of the great waiting-rooms, as well as what people think about Instituto Central do Hospital das Clínicas as an institution.

Cell cycle-dependent metabolism of pyrimidine deoxynucleoside triphosphates in CEM cells.

We incorporated 3H-labeled thymidine, deoxycytidine, or cytidine into dNTPs and DNA of exponentially growing CEM cells. G1 and S phase cells were separated by centrifugal elutriation, and the size and specific activity of dNTP pools were determined to study the cell cycle-dependent regulation of specific dNTP synthesizing enzymes in their metabolic context. With [3H]thymidine, we confirm the earlier demonstrated S phase specificity of thymidine kinase. Incorporation of radioactivity from [5-3H]deoxycytidine into dCTP occurred almost exclusively in G1 cells. During S phase, de novo synthesis by ribonucleotide reductase was switched on, resulting in a 70-fold dilution of [3H]dCTP, confirming that ribonucleotide reductase is an S phase-specific enzyme, whereas deoxycytidine kinase is not. [5-3H]Cytidine appeared in dCTP almost to the same extent in G1 as in S phase, despite the S phase specificity of ribonucleotide reductase. During S phase, DNA replication greatly increased the turnover of dCTP, requiring a corresponding increase in ribonucleotide reductase activity. During G1, the enzyme maintained activity to provide dNTPs for DNA repair and mitochondrial DNA synthesis. The poor incorporation of isotope from deoxycytidine into DNA earlier led to the suggestion that the nucleoside is used only for DNA repair (Xu, Y-Z., Peng, H., and Plunkett, W. (1995) J. Biol. Chem. 270, 631-637). The poor phosphorylation of deoxycytidine in S phase provides a better explanation.

Synergistic antiviral action of ribonucleotide reductase inhibitors and 3'-azido-3'-deoxythymidine on HIV type 1 infection in vitro.

Ribonucleotide reductase inhibitors reduce the cellular supply of DNA precursors(dNTP) by interfering with their de novo synthesis. A secondary effect is the stimulation of the uptake and phosphorylation of extracellular deoxynucleosides, including their analogs, e.g., 3'-azidothymidine (AZT). Both effects are relevant to HIV replication, which requires dNTP and is impaired by the triphosphate of AZT. Earlier we demonstrated that ribonucleotide reductase inhibitors--hydroxyurea, and two deoxycytidine analogs specifically active in lymphoid cells--increased the phosphorylation of AZT in CEM cells by prolonging the S phase of the cell cycle. Here we tested the effects of long-term treatments on HIV proliferation in CEM cells and stimulated human lymphocytes infected with HIV-1IIIB. Treatment with low doses of AZT (0.05-0.1 microM) and either hydroxyurea (25-100 microM) or 2'-azido-2'-deoxycytidine (0.25-4 microM) lasted 2 weeks, during which p24 in the culture medium was monitored. Noninfected CEM cells were treated in parallel to measure the inhibition of cell growth, distribution along the cell cycle, dNTP pool size, and level of tritiated AZT phosphorylation. A clear synergism between AZT and ribonucleotide reductase inhibitors was observed at nontoxic doses that induced only minor changes in the cellular parameters measured. The reductase inhibitors by themselves interfered with replication only at doses that inhibited cell proliferation.

Mutagenic effects at hprt locus and in minisatellite sequences induced in V79 cells by treatments with UV and methyl-nitro-nitroso guanidine.

DNA alterations induced in V79 cells treated with UV light or methyl-nitro-nitrosoguanidine were analyzed by the mutagenicity test at the hprt locus and by DNA fingerprint analysis. Treated and control cells were seeded in the presence or absence of 6-thioguanine to determine mutant frequency and cell survival. From clonal cultures of the same cell populations we isolated a number of clones and grew them up individually to obtain appropriate amounts of DNA. High molecular weight DNA was digested with HinfI or HaeIII and hybridized with 32P-labelled 33.15 multilocus probe. The induction of 6-thioguanine resistant cells depended on the mutagen dose. The highest value of mutant frequency obtained was 7475 x 10(-6) (MNNG, 27 microM), corresponding to 0.7 percent of clonable cells. DNA fingerprint analysis carried on the same treated cells showed that DNA rearrangements occurred at minisatellites much more frequently than in transcribed sequences. UV irradiation produced the highest frequency of variation, modifying minisatellite patterns in about 50 percent of the analyzed clones.

[Degree of satisfaction in the users of the Central Institute of the Clinical Hospital-PAMB for the care received]. / Avaliação do grau de satisfação dos usuários do I.C.H.C.-PAMB com relação ao atendimento prestado.

Through a sample of 1346 ambulatory patients, correspondent to the 3% monthly attendance of the ICHC/PAMB in the first semester of 1992, the authors report the degree of satisfaction of the population assisted. The survey reveals the performance of the multiple professionals as well as the evaluation of the ambulatory facilities regarding signs, hygiene and general organization of services, etc. The parameters, specially those concerning the health professionals, demonstrate that its concept the patients of the ICHC-PAMB is considered good.

[Monovascular coronary disease in the pre-angioplasty era]. / La malattia coronarica monovascolare in epoca pre-angioplastica.

Single-vessel coronary disease has been considered so far a benign condition, for which the medical therapy may represent the optimal treatment. In order to assess the effectiveness of this approach, we studied 323 patients who had come to our attention for ischemic heart disease and resulted affected by single-vessel coronary artery disease. From our data it appears that single-vessel coronary artery disease is frequently associated with myocardial infarction and post-infarction aneurysm. Coronary angiography indicated that left anterior descending coronary artery is the most frequently affected vessel, and that its involvement is often associated with lethal outcome. Forty-two out of the 323 patients underwent coronary artery bypass surgery; the remaining patients were medically treated. Surgical patients showed a better improvement respect to the medical group, while survival was not statistically different. An unexpected result was the relatively high risk in circumflex artery lesion. These data may justify a broader utilization of invasive therapy in recent onset angina, in light of the excellent results recently obtained with percutaneous transluminal coronary angioplasty.

Value and limitations of transesophageal echocardiographic monitoring during percutaneous balloon mitral valvotomy.

To determine the utility of transesophageal echocardiographic monitoring during percutaneous balloon mitral valvotomy, we analyzed data from 40 consecutive patients who had been randomly assigned to undergo balloon mitral valvotomy under transesophageal echocardiographic guidance or without echo. All procedures were carried out under general anaesthesia. The completion rate (100% vs 73%), the procedure time (108 +/- 28 min vs 65 +/- 18 min), the X-ray exposure time (62 +/- 13 vs 33 +/- 12 min), resulted significantly (P less than 0.001) more favorable in the echo-monitored patients. Moreover, a lower rate of major complications (cardiac tamponade, large residual atrial shunting, and severe mitral regurgitation) was noted in the echo-monitored patients. The achieved final area of the mitral valve did not differ significantly between the two groups. From an evaluation of results as a whole, 96% of the echo-monitored procedures were successful, whereas only 40% of the procedures conducted without echocardiographic control achieved a satisfactory final result in absence of major complications. We conclude that transesophageal echocardiography is a safe, effective, and valuable tool to monitor each step of balloon mitral valvotomy in order to shorten the time of the procedure, and to improve the results of this complex interventional catheterization technique.

Successful emergency percutaneous balloon mitral valvotomy in a patient with massive left atrial thrombosis: utility of transesophageal echocardiographic monitoring.

Emergency percutaneous balloon mitral valvotomy was carried out successfully in a 65-year-old woman with severe mitral stenosis and massive left atrial thrombosis. Transesophageal echocardiography was of great value in monitoring each step of the procedure in order to avoid systemic embolization due to improper manipulation of the dilating apparatus within the left atrium.

[Short- and mid-term results of percutaneous aortic valvuloplasty for calcific aortic stenosis in elderly patients]. / Risultati a breve e medio termine della valvuloplastica aortica percutanea nel trattamento della stenosi aortica calcifica dell'anziano.

In order to evaluate the short and mid-term results of percutaneous aortic balloon valvuloplasty, 40 consecutive elderly patients with symptomatic severe calcific aortic stenosis, underwent the procedure consecutively, with follow-up by clinical evaluation and Doppler echocardiography. Over a mean follow-up period of 11.2 months there were 5 deaths, and 12 patients underwent subsequent aortic valve replacement. Doppler echocardiography revealed an increase in aortic valve area from 0.62 +/- 0.20 cm2 to 0.91 +/- 0.23 cm2 after the procedure, but there was a significant trend toward restenosis by 12 months follow-up in 23 of 32 patients (72%). Restenosis was accompanied by symptomatic deterioration in 18 of 23 patients (78%). Although balloon valvuloplasty may often improve haemodynamics and relieve symptoms, these benefits seem to be short-lived in most cases. Restenosis has a high rate of occurrence. Aortic balloon valvuloplasty should be reserved for truly inoperable cases and for haemodynamically-unstable patients, who may later undergo surgery.

Mitral valve disruption following percutaneous balloon valvuloplasty.

Two cases of massive mitral regurgitation due to mitral valve disruption following percutaneous balloon valvuloplasty are reported. This severe complication occurred in two elderly women with recurrent mitral stenosis after previous surgical commissurotomy. Due to their unstable hemodynamic and clinical condition, both patients underwent emergency valve replacement. At surgery, the commissures appeared fused and heavily calcified; the chordae tendineae thickened, shortened, and fused; and the leaflets presented a large tear with sheared edges. Because the technical aspects of both procedures were unremarkable, the anatomic features of the mitral valve seemed to affect the occurrence of severe mitral regurgitation. Percutaneous balloon valvuloplasty should be therefore applied carefully to patients with prior surgical valvotomy, in whom the structural alterations of the mitral apparatus may predispose to severe valvular damage.

[Natural history of dilated cardiomyopathy]. / Storia naturale delle miocardiopatie dilatative.

Seventy-seven patients with dilated cardiomyopathy (50 M and 27 F, aged 38 +/- 16 yrs) were followed up until Dec 31, 1985. Haemodynamic investigation was performed in all cases, and all pressure and left ventricular quantitative angiography parameters were collected. In no case did the coronary angiogram show significant lesions. Mean values of haemodynamic parameters for surviving and deceased patients were compared. Forty-six patients survived and 28 died during the follow-up period. Although all patients had an enlarged left ventricle and depressed contractility, survivors had either greater pressure/end systolic volume ratio or a greater stress/end systolic volume ratio. Survival curves confirm a particularly severe prognosis for patients with Suga index less than 1 or stress to end systolic volume ratio less than 2.5. Mass to volume ratio seems to affect two-year but not late survival. NYHA class does not indicate early survival. In conclusion, left ventricular function is obviously related to prognosis in these patients, but it seems still difficult to assess life expectancy from these parameters alone.