Vpu increases susceptibility of human immunodeficiency virus type 1-infected cells to fas killing.

The importance of the Fas death pathway in human immunodeficiency virus (HIV) infection has been the subject of many studies. Missing from these studies is direct measurement of infected cell susceptibility to Fas-induced death. To address this question, we investigated whether T cells infected with HIV are more susceptible to Fas-induced death. We found that Fas cross-linking caused a decrease in the number of HIV-infected Jurkat T cells and CD4(+) peripheral blood leukocytes (PBLs). We confirmed this finding by demonstrating that there were more apoptotic infected than uninfected cells after Fas ligation. The increase in sensitivity of HIV-infected cells to Fas killing mapped to vpu, while nef, vif, vpr, and second exon of tat did not appear to contribute. Furthermore, expression of Vpu in Jurkat T cells rendered them more susceptible to Fas-induced death. These results show that HIV-infected cells are more sensitive to Fas-induced death and that the Vpu protein of HIV contributes to this sensitivity. The increased sensitivity of HIV-infected cells to Fas-induced death might help explain why these cells have such a short in vivo half-life.

Rapid noninvasive detection of experimental atherosclerotic lesions with novel 99mTc-labeled diadenosine tetraphosphates.

The development of a noninvasive imaging procedure for identifying atherosclerotic lesions is extremely important for the clinical management of patients with coronary artery and peripheral vascular disease. Although numerous radiopharmaceuticals have been proposed for this purpose, none has demonstrated the diagnostic accuracy required to replace invasive angiography. In this report, we used the radiolabeled purine analog, 99mTc diadenosine tetraphosphate (Ap4A; AppppA, P1,P4-di(adenosine-5')-tetraphosphate) and its analogue 99mTc AppCHClppA for imaging experimental atherosclerotic lesions in New Zealand White rabbits. Serial gamma camera images were obtained after intravenous injection of the radiolabeled dinucleotides. After acquiring the final images, the animals were sacrificed, ex vivo images of the aortas were recorded, and biodistribution was measured. 99mTc-Ap4A and 99mTc AppCHClppA accumulated rapidly in atherosclerotic abdominal aorta, and lesions were clearly visible within 30 min after injection in all animals that were studied. Both radiopharmaceuticals were retained in the lesions for 3 hr, and the peak lesion to normal vessel ratio was 7.4 to 1. Neither of the purine analogs showed significant accumulation in the abdominal aorta of normal (control) rabbits. The excised aortas showed lesion patterns that were highly correlated with the in vivo and ex vivo imaging results. The present study demonstrates that purine receptors are up-regulated in experimental atherosclerotic lesions and 99mTc-labeled purine analogs have potential for rapid noninvasive detection of plaque formation.

Pharmacologic therapies after myocardial infarction.

Despite the availability and use of effective methods for limiting infarct size with thrombolytic agents and primary angioplasty, patients experiencing a myocardial infarction (MI) are at increased risk for a second cardiac event in the post-MI period (e.g., reinfarction, heart failure, and sudden death). For this reason, postinfarction risk management is crucial. An extensive data base has firmly established the efficacy of beta blockers in reducing cardiovascular risk following acute MI. The full advantages of angiotensin-converting enzyme (ACE) inhibitors have only recently begun to emerge as the result of a growing understanding of the mechanisms of adverse outcomes following MI. The importance of lipid-lowering agents, in particular the "statins," should be considered in all post-MI patients, especially since recent studies have conclusively shown improved survival and reduced rates of MI and coronary artery bypass surgery in this population with this therapy. Aspirin is now considered a standard part of the early management of the acute infarct patient as well as for secondary prevention in post-MI patients. At present, chronic anticoagulation with warfarin should be reserved for selected patients. The nondihydropyridine calcium antagonists diltiazem and verapamil can be considered for post-MI use only in patients in whom beta blockers are contraindicated and who have preserved systolic function and/or those without clinical heart failure. In contrast, the dihydropyridine calcium antagonists, particularly nifedipine, have no role in secondary prevention. Although long-term benefits are minimal, nitrates continue to be useful in post-MI patients with residual ischemia (angina or silent ischemia), heart failure (systolic or diastolic), or postinfarction hypertension. Antiarrhythmic agents, except amiodarone, are relatively contraindicated in post-MI patients. Recent data show that vitamin E reduces the rate of nonfatal MI. Its role in cardiovascular death and overall mortality remains to be clarified. Despite their demonstrated value, agents used in secondary prevention generally appear to be underutilized. In addition, when pharmacologic therapies are administered for secondary prevention, they are often prescribed at lower doses than those tested and proved in trials. A greater appreciation for the efficacy and safety profiles of these agents could lead to more widespread use and more pronounced reductions in morbidity and mortality among post-MI patients.

Antiproliferative effect of retinoid compounds on Kaposi's sarcoma cells.

A panel of retinoid compounds (tretinoin, isotretinoin, acitretin, and RO13-1470) were tested for inhibitory activity against Kaposi's sarcoma cell (KSC) cultures in vitro. Tretinoin was found to be the most effective retinoid tested, inhibiting the growth of KSC in vitro while having no effect on the expression of interleukin-6 and basic fibroblast growth factor, two important cytokines involved in KSC growth. Tretinoin also did not appear to downregulate the expression of receptors for these two cytokines. At low concentrations (10(-9) M), acitretin and tretinoin selectively inhibited growth of early passage KSC. At higher concentrations (10(-6)-10(-5) M), retinoid treatment induced a pattern of DNA degradation and morphological changes in KSC characteristic of apoptosis (programmed cell death). The inhibitory activity of tretinoin on KSC growth was decreased if human serum (but not fetal calf serum) was present in the growth medium, and partially restored by removal of serum lipids. These data suggest that retinoids possess potential as therapeutic agents in Kaposi's sarcoma.

Recognition and management of mitral stenosis.

Mitral stenosis may be recognized simply on routine physical examination from abnormalities on auscultation, which are subsequently confirmed by Doppler echocardiographic examination. The diagnosis may also be suspected in patients in whom the history is suggestive of rheumatic fever and in whom physical diagnosis reveals findings indicative of mitral stenosis. Occasional cases of mitral stenosis are picked up in the Doppler echocardiography laboratory when a patient has not been suspected of having mitral stenosis but in whom a routine echocardiogram has been taken, for whatever reason. If there is any doubt about the presence of mitral stenosis, confirming the presence of a diastolic pressure gradient across the mitral valve during cardiac catheterization permits one to make a definitive diagnosis as well as estimate the area of the stenotic valve orifice. Intervention with either surgery or balloon valvotomy is indicated when the mitral valve area falls to < or = 1.2 cm2 in a symptomatic patient.

Early versus late opening of coronary arteries: the effect of timing.

Intuitively, one would assume that the benefit from the use of thrombolytic therapy in reducing mortality in acute myocardial infarction (AMI) is directly related to early recanalization of the thrombosed infarct-related artery. However, a meta-analysis of early clinical trials and the ISIS-II trial results suggests that thrombolysis initiated between 6 and 24 h after the onset of infarction still reduces mortality. Verification of this observation is currently being sought in three ongoing trials of late reperfusion. These are the EMERA trial in South America, using streptokinase; the LATE study involving several European countries, the United States, Canada, and Australia, using tissue plasminogen activator (tPA); and the TAMI-6 trial, in which either tPA or placebo is given 6 to 24 h postinfarction, and patients with closed infarct-related arteries are randomized further to either angioplasty or no angioplasty. There are theoretical reasons that late reperfusion may be beneficial. These reasons include the prevention of infarct expansion and ventricular remodeling leading to ventricular dilatation, the provision of electrophysiologic stability lessening the likelihood of malignant ventricular arrhythmias, and the ability of collaterals emanating from the recanalized artery to perfuse ischemic but still viable myocardium. There are also reasons that late reperfusion might be harmful, including myocardial stunning, microvascular damage, and intramyocardial hemorrhage leading to an increased likelihood of myocardial rupture. It does appear, however, that once recanalization occurs, maintenance of patency improves late outcome, as shown in the Western Washington Intracoronary Streptokinase Trial, the TAMI trial, the TIMI trial, and the Duke study.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein phosphorylation in response to stress in Clostridium acetobutylicum.

The possible involvement of protein phosphorylation in the clostridial stress response was investigated by radioactively labeling growing cells of Clostridium acetobutylicum with 32Pi or cell extracts with [gamma-32P]ATP. Several phosphoproteins were identified; these were not affected by the growth stage of the culture. Although the extent of protein phosphorylation was increased by heat stress, the phosphoproteins did not correspond to known stress proteins seen in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified clostridial DnaK, a stress protein, acted as a kinase catalyzing the phosphorylation of a 50-kilodalton protein. The phosphorylation of this protein was enhanced in extracts prepared from heat-stressed cells. Diadenosine-5',5"'-P1,P4-tetraphosphate had no influence on protein phosphorylation.

Generation of extracellular ATP in blood and its mediated inhibition of host weight loss in tumor-bearing mice.

Intraperitoneal injections of adenosine 5'-monophosphate (AMP) or adenosine 5'-triphosphate (ATP), but not of adenosine, inorganic phosphate or pyrophosphate, were shown to inhibit tumor growth and host weight loss in tumor-bearing murine hosts. The inhibition of tumor growth and host weight loss did not exhibit a cause-effect relationship, though both were mediated through expansion of red blood cell (RBC) ATP pools which were promoted by administered adenine nucleotides. We then demonstrated that expansion of RBC ATP pools was preceded by expansion of liver ATP pools and that the adenosine precursor for this type of enhanced RBC ATP synthesis originated in the turnover of expanded liver ATP pools. Although adenosine, which is the primary catabolic product of ATP in the peritoneal cavity or the systemic circulation, was sufficient to yield an expansion of mouse liver ATP pools in vivo, external phosphate was required for the subsequent expansion of RBC ATP pools, which in turn produced elevated extracellular (blood plasma) ATP levels. The anticancer activities which correlate with the elevated blood plasma ATP concentrations are proposed to be the result of direct action of extracellular ATP on the tumor and host tissues.

Anticancer activities of adenine nucleotides in mice are mediated through expansion of erythrocyte ATP pools.

ATP and AMP exhibit significant anticancer activities against established footpad CT26 colon adenocarcinoma in CB6F1 mice. Adenosine, inorganic phosphate, and inorganic pyrophosphate were without such effects under identical conditions. Daily intraperitoneal injections of adenine nucleotides in large volumes of saline, starting after the tumors became palpable, resulted in inhibition of tumor growth and a few "cures." The treatment was not toxic to the host as determined by changes in body weights. Weight loss observed in animals upon progression of the fast-growing CT26 tumors was slowed markedly in adenine nucleotide-treated mice. The inhibition of weight loss in tumor-bearing mice was shown to be neither the cause nor the effect of the inhibition of tumor growth. Intraperitoneal injections of AMP or ATP but not of adenosine yielded expansions of erythrocyte ATP pools in host animals. The expanded erythrocyte ATP pools are stable over a period of hours, while slowly releasing micromolar amounts of ATP into the blood plasma compartment, leading to several-fold increases in plasma (extracellular) ATP levels. Based on previous studies in which 1-5 microM extracellular ATP effectively inhibited the growth of a variety of tumor cells in several in vitro systems, it is suggested that similar levels of ATP in blood plasma account for the anticancer activities observed in a murine host.

Experimental cancer therapy in mice by adenine nucleotides.

Adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP) and adenosine 5'-triphosphate (ATP), injected intraperitoneally into tumor-bearing (s.c. implanted footpad tumors) mice, exhibited significant anticancer activity. Daily treatments (for 10 days) inhibited the growth of the fast-growing, aggressive CT 26 colon adenocarcinoma in CB6F1 mice. The growth-inhibitory activity of adenine nucleotides was also observed against a human pancreatic adenocarcinoma, CAPAN-1, xenografts in athymic nude mice. With low tumor burdens some 'cures' were obtained in both model systems. No inherent toxicity, as determined by changes in host weight, were observed during and after the period of treatment. Intraperitoneal injections of 1 ml of 50 mM AMP, ADP or ATP in saline, yielded elevated blood and plasma levels of ATP which lasted for several hours in both strains of mice. The growth-inhibitory activities of adenine nucleotides against tumor cells in vitro, have previously been demonstrated.

Metabolism of adenylylated nucleotides in Clostridium acetobutylicum.

In response to the stresses imposed by temperature upshift or addition of butanol, Clostridium acetobutylicum cultures accumulated diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) and adenosine 5'-P1,P4-tetraphospho-5'-guanosine (Ap4G) to high levels. The two adenylylated nucleotides were also accumulated in batch culture in the absence of imposed stresses when the clostridia switched from the acidogenic phase of growth to the solventogenic phase. Most of the adenylylated nucleotides were extracellular. The intracellular concentrations of these compounds were low throughout batch growth and in cells stressed by added butanol. In contrast to other procaryotes, these clostridia did not possess enzymes to degrade the dinucleotides, as shown with both intact cells and cell-free preparations. Our findings are consistent with the hypothesis that endogenously produced solvents are stressful to the cells, stimulating the synthesis of adenylylated nucleotides. The nucleotides accumulate extracellularly because they cannot be degraded and because the cell membranes are permeabilized by the solvents produced.

Aminoacyl-tRNA synthetases catalyze AMP----ADP----ATP exchange reactions, indicating labile covalent enzyme-amino-acid intermediates.

Aminoacyl-tRNA synthetases (amino acid-tRNA ligases, EC 6.1.1.-) catalyze the aminoacylation of specific amino acids onto their cognate tRNAs with extraordinary accuracy. Recent reports, however, indicate that this class of enzymes may play other roles in cellular metabolism. Several aminoacyl-tRNA synthetases are herein shown to catalyze the AMP----ADP and ADP----ATP exchange reactions (in the absence of tRNAs) by utilizing a transfer of the gamma-phosphate of ATP to reactive AMP and ADP intermediates that are probably the mixed anhydrides of the nucleotide and the corresponding amino acid. AMP and ADP produce active intermediates with amino acids by entering the back-reaction of amino acid activation, reacting with labile covalent amino acid-enzyme intermediates. Gramicidin synthetases 1 and 2, which are known to activate certain amino acids through the formation of intermediate thiol-esters of the amino acids and the enzymes, catalyze the same set of reactions with similar characteristics. Several lines of evidence suggest that these activities are an inherent part of the enzymatic reactions catalyzed by the aminoacyl-tRNA synthetases and gramicidin synthetases and are not due to impurities of adenylate kinase, NDP kinase, or low levels of tRNAs bound to the enzymes. The covalent amino acid-enzyme adducts are likely intermediates in the aminoacylation of their cognate tRNAs. The use of gramicidin synthetases has thus helped to illuminate mechanistic details of amino acid activation catalyzed by the aminoacyl-tRNA synthetases.

Retinoic acid alters subcellular compartmentalization of ATP pools in 3T3 cells but not in HeLa cells.

Retinoic acid (RA; beta-all-trans) inhibits the proliferation of both murine 3T3 cells and human HeLa cells. Flow cytometric analyses of exponentially growing cultures show that 3T3 cells are inhibited during the S phase of their cell cycle, while HeLa cells show only a small increase in G1 phase cells. RA (10 microM) causes a 50% increase in total cellular adenosine triphosphate (ATP) pools of 3T3 cells, but not of HeLa cells. We have previously demonstrated that the effects of RA on cellular ATP pools of 3T3 cells are directly related to its inhibition of cellular growth, and now report data which provide a biochemical basis for this process. Established procedures were utilized to investigate the effects of RA on the functional compartmentalization of the nuclear ATP pool which serves as a precursor for RNA synthesis in these cells, and which is shown to be a small pool in comparison with cytoplasmic ATP pools. Expansion of total cellular ATP pools by 1 mM of exogenously supplied unlabeled adenosine is ineffective in reducing the subsequent incorporation of [3H]adenosine into RNA of 3T3 cells. Similar treatment of HeLa cells yields a modest reduction in the incorporation of [3H]adenosine into RNA. RA treatment of HeLa cells does not affect the preferential uptake of exogenous [3H] adenosine into the immediate precursor ATP pool for RNA synthesis. RA treatment of 3T3 cells markedly reduces the incorporation of [3H] adenosine into RNA, indicating a lesser degree of functional compartmentalization of the nuclear ATP pool. Similar conclusions are drawn from correlations of the specific radioactivities of total cellular [3H] ATP pools and the levels of incorporation of radioactive label into cellular RNA. In addition, pulse-chase experiments show that RA-treated 3T3 cells continue to incorporate radioactive label from pools prelabeled with [3H]adenosine despite the presence of a large excess of unlabeled adenosine in the chase medium. Control 3T3 and both control and RA-treated HeLa cells cease to incorporate label immediately upon the start of the chase, suggesting that the functional precursor ATP pool for RNA synthesis is small and readily diluted. These data suggest that RA decreases the degree of functional compartmentalization for 3T3, but not HeLa cell ATP pools, and provides a probable mechanism for expansion of nuclear ATP pools of 3T3 cells. The expanded nuclear ATP pools may provide the biochemical mechanism for the inhibition of DNA synthesis during the S phase of the 3T3 cell cycle.

Imbalance of total cellular nucleotide pools and mechanism of the colchicine-induced cell activation.

Treatment with colchicine or vinblastine, both inhibitors of microtubule assembly, renders quiescent 3T3 cells in an "activated state" as evidenced by induction of DNA synthesis and other criteria. Microtubule disassembly caused by colchicine or vinblastine brings about a dramatic expansion of total cellular UTP pools with a concomitant diminution in total cellular ATP pools, thus resulting in a marked imbalance in total cellular nucleotide pools. Colchicine and vinblastine also stimulate total cellular RNA synthesis without enhancing uridine phosphorylation, suggesting that these drugs affect the G1 phase of the cell cycle at a point beyond the enhancement of uridine phosphorylation that usually accompanies mitogenic stimulation of quiescent mammalian cells. The markedly expanded cellular UTP pools appear to be necessary for initiation of the colchicine-stimulated DNA synthesis because decreasing cellular UTP pools by addition of D-glucosamine results in a selective inhibition of DNA synthesis in the colchicine-stimulated, but not control, cells. Furthermore, D-glucosamine exerts its inhibitory effect only when it is present in the cultures within the first 14 hr after colchicine treatment. When added at 21 hr, D-glucosamine still decreases cellular UTP pools, but it is no longer inhibitory for DNA synthesis, which commences 14-16 hr after colchicine stimulation. Taxol, an antitumor drug, prevents microtubule disassembly and also blocks such events as expansion of total cellular UTP pools and stimulation of RNA and DNA synthesis, indicating that microtubule depolymerization acts as a primary event initiating the process of cell activation induced by colchicine.

99mTc-labeled nucleotides as tumor-seeking radiodiagnostic agents.

Several lines of human tumor cells in monolayer and soft agar cultures allow permeation of low levels of adenine nucleotides through their plasma membranes, while, in general, untransformed cells do not incorporate adenine nucleotides into their cellular pools without prior degradation of the nucleotides to adenosine. This study determined the uptake of 99mTc-radiolabeled chelated forms of adenine nucleotides, 99mTc-Ap4A (diadenosine 5',5"',P1,P4-tetraphosphate) and 99mTc-ATP chelates as radiodiagnostic agents suitable for the in vivo detection of tumors by radionuclide imaging. Biodistribution studies revealed that Ap4A accumulated preferentially in RT-24 tumors implanted in rats and that V2 carcinoma implanted in rabbits could be readily visualized by in vivo imaging. The biodistribution at various time points showed increased tumor-to-muscle ratios after 99mTc-Ap4A or 99mTc-ATP injections when compared with a nonspecific marker of the extracellular fluid space, 99mTc-labeled diethylenetriaminepentaacetic acid and with an agent known to localize in some tumors, 67Ga-labeled citrate. Studies of ectoenzymatic activities of virus-transformed animal cells and their untransformed counterparts in monolayer cultures showed marked decreases in the ectoenzymatic activities that degrade Ap4A in the transformed cells. Incorporation of en bloc [3H, 32P]Ap4A into cellular acid-soluble nucleotide pools of certain transformed cells was observed. Normal untransformed cells incorporated the radioactive label only by prior degradation to [3H]adenosine and 32Pi.

Adenosine(5')tetraphospho(5')adenosine-binding protein of calf thymus.

An adenosine(5')tetraphospho(5')adenosine (Ap4A) binding protein has been purified from calf thymus. The protein is comprised of a single polypeptide of Mr 54000 and is capable of high-affinity (Kd = 13 microM) binding of Ap4A with great substrate specificity. The Ap4A binding protein has been isolated in two forms: a 'free', or non-polymerase-bound, form which predominates, and a similar form which copurifies with DNA polymerase alpha, but which can be resolved from it. The free form of Ap4A binding protein contains associated adenosine(5')tetraphospho(5')adenosine phosphohydrolase (Ap4Aase) activity, while the form resolved from DNA polymerase alpha contains no such activity. The Ap4Aase activity, which catalyzes the phosphohydrolysis of Ap4A to ATP and AMP, is strongly inhibited by low levels (50-100 microM) of Zn2+ without any effect on the Ap4A binding protein activity. This difference in associated Ap4Aase activity between free and polymerase-bound forms of the protein, plus the copurification mentioned above, indicate a specific association between Ap4A binding protein and DNA polymerase alpha.