The role of protein synthesis and degradation in the post-transcriptional regulation of rat multidrug resistance-associated protein 2 (Mrp2, Abcc2).

Multidrug resistance-associated protein 2 (Mrp2, Abcc2), an organic anion transporter present in the apical membrane of hepatocytes, renal epithelial cells, and enterocytes, is postulated to undergo post-transcriptional regulation. We hypothesized that Mrp2 protein undergoes altered rates of protein synthesis or degradation consistent with different Mrp2 protein expression. We analyzed Mrp2 synthesis, expression, and degradation in control female, 19- and 20-day pregnant, and pregnenolone-16alpha-carbonitrile (PCN)-treated rats using in vivo metabolic-labeling studies with [35S]cysteine/methionine or [14C]NaHCO3, polysomal distribution analyses and ribonuclease protection assays (RPA). Mrp2 protein was significantly increased in rats treated with PCN for 2 days but significantly decreased in 19-day pregnant rats relative to controls; no significant differences were observed in Mrp2 mRNA expression among these groups. The measured half-lives of 14C-labeled Mrp2 in control, pregnant, and PCN-treated rats were 27, 36, and 22 h, respectively, and were not significantly different. The rate of incorporation of 35S into Mrp2 was highest in PCN-treated rats. Polysomal distribution analysis of Mrp2 mRNA was consistent with increased Mrp2 protein synthesis after PCN treatment. The major transcription-initiation site for rat liver determined by RPA was -98 nucleotides (nt), with other start sites observed at -213, -163, -132, and -71 nt; use of transcription sites did not differ among the groups. Differences in the degradation of Mrp2 protein cannot explain the post-transcriptional regulation of Mrp2 in control, pregnant, and PCN-treated rats. Rather, the observed difference in protein synthesis suggests an intrinsic role for the translational regulation of rat Mrp2 protein.

Selection of RNA replicons capable of persistent noncytopathic replication in mammalian cells.

The natural life cycle of alphaviruses, a group of plus-strand RNA viruses, involves transmission to vertebrate hosts via mosquitoes. Chronic infections are established in mosquitoes (and usually in mosquito cell cultures), but infection of susceptible vertebrate cells typically results in rapid shutoff of host mRNA translation and cell death. Using engineered Sindbis virus RNA replicons expressing puromycin acetyltransferase as a dominant selectable marker, we identified mutations allowing persistent, noncytopathic replication in BHK-21 cells. Two of these adaptive mutations involved single-amino-acid substitutions in the C-terminal portion of nsP2, the viral helicase-protease. At one of these loci, nsP2 position 726, numerous substitution mutations were created and characterized in the context of RNA replicons and infectious virus. Our results suggest a direct correlation between the level of viral RNA replication and cytopathogenicity. This work also provides a series of alphavirus replicons for noncytopathic gene expression studies (E. V. Agapov, I. Frolov, B. D. Lindenbach, B. M. Prágai, S. Schlesinger, and C. M. Rice, Proc. Natl. Acad. Sci. USA 95:12989-12994, 1998) and a general strategy for selecting RNA viral mutants adapted to different cellular environments.

Alphavirus-based expression vectors: strategies and applications.

Alphaviruses are positive-strand RNA viruses that can mediate efficient cytoplasmic gene expression in insect and vertebrate cells. Through recombinant DNA technology, the alphavirus RNA replication machinery has been engineered for high-level expression of heterologous RNAs and proteins. Amplification of replication-competent alpha-virus RNAs (replicons) can be initiated by RNA or DNA transfection and a variety of packaging systems have been developed for producing high titers of infectious viral particles. Although normally cytocidal for vertebrate cells, variants with adaptive mutations allowing noncytopathic replication have been isolated from persistently infected cultures or selected using a dominant selectable marker. Such mutations have been mapped and used to create new alphavirus vectors for noncytopathic gene expression in mammalian cells. These vectors allow long-term expression at moderate levels and complement previous vectors designed for short-term high-level expression. Besides their use for a growing number of basic research applications, recombinant alphavirus RNA replicons may also facilitate genetic vaccination and transient gene therapy.

Corynebacterium urealyticum (CDC Group D2) associated with staghorn calculus: treatment by percutaneous debulking and chemolysis.

We report the formation of a staghorn calculus in a transplanted kidney caused by infection with a urea-splitting Corynebacterium group D2 organism. The stone was debulked percutaneously followed by intravenous vancomycin administration and urinary acidification with oral acetohydroxamic acid, leading to clearance of nearly all of the stone.

Autoregulation of endothelin-1 secretion by cultured human keratinocytes via the endothelin B receptor.

We investigated endothelin-1 (ET-1) receptor expression on normal human keratinocytes (HK). We show that HK express the ETB receptor isoform and respond to ET-1 with a 2.7-fold increase in intracellular free calcium. HK did not respond to ET-1 with increased proliferation; however, 30 nM ET-1 caused a 51.7% decrease in ET-1 accumulation in HK-conditioned medium. We propose that HK ET-1 receptors function in autocrine regulation of ET-1 secretion.

Human melanoma cells express functional endothelin-1 receptors.

Current evidence suggests that endothelium-derived factors enhance human melanoma vascular invasion. Therefore, we studied human melanoma cell expression of receptors to the endothelium-derived peptide, endothelin-1 (ET-1), and determined if they respond to ET-1 with proliferation and chemokinesis. Human metastatic melanoma cell lines were found to have specific, saturable, high affinity ET-1 binding. Northern analysis and competitive inhibition studies confirmed that melanoma cells express the ETB receptor isoform. Ten nanomolar ET-1 caused an 8.2 to 25.5-fold increase in intracellular free calcium. ET-1 was found to be a weak mitogen for melanoma cells, however, melanoma cell chemokinesis was significantly increased by ET-1. These data suggest that ET-1 may be involved in providing a chemokinetic and growth factor environment that enhances perivascular proliferation and invasiveness of melanoma cells.

Risk and detection of pulmonary artery catheter-related infection in septic surgical patients.

Specimen cultures were evaluated in 49 catheterized patients who had a known focus of infection (primarily intra-abdominal peritonitis). Bacteria were recovered from 2% of flush solutions, 14% of transducer domes, 18% of diaphragms, and 24% of cardiac output fluids; however, these bacteria were not found in cultures of the pulmonary artery (PA) catheter segments. The rates of positive PA catheter-aspirate cultures were 30.6% on day 1, 20.4% on day 2, and 32.7% on day 3 (not statistically different). PA catheter-aspirate cultures had a sensitivity of 5.7% and a positive predictive value of 30% for catheter-related infection, and 15% sensitivity and 40% positive predictive value for peripheral bacteremia. While 95% (55 of 58) of the catheter-aspirate cultures were false-positives, only 0.5% (3 of 588) were true-positives. Peripheral blood cultures were positive in 10% of the patients, but the catheter segments were sterile or grew different organisms. Arterial line cultures had zero sensitivity and predictive value to detect catheter-related infection, and 15% sensitivity and 40% predictive value to detect peripheral bacteremia. Thus, PA catheter-aspirate cultures, routine peripheral blood cultures, and arterial cultures cannot be recommended to detect PA catheter-related infection. Catheter-related infection confirmed by catheter-segment cultures was 10.2% when the PA catheters were removed after 73 +/- 6.5 (SD) h. Bacteria from catheter-segment cultures corresponded to those from the primary infection site.

Cranial CT in acquired immunodeficiency syndrome: spectrum of diseases and optimal contrast enhancement technique.

A retrospective review of cranial CT scans obtained over a 4 year period in patients with acquired immunodeficiency syndrome (AIDS) and documented central nervous system (CNS) pathology is presented. The spectrum of diseases and the value of CT in detecting new, recurrent, and superimposed disease processes were determined. Fifty-one AIDS patients with confirmed CNS pathology were identified. Six of them had two coexistent diseases. Opportunistic infections predominated, especially Toxoplasma encephalitis and cryptococcal meningitis, while tumor was seen infrequently. Initial CT was positive in 76% of cases. In contrast to meningeal processes, where it was not very effective, CT was very sensitive in detecting most parenchymal disease processes. Characteristic although not pathognomonic CT patterns were found for certain diseases. Improvement or resolution of CT abnormalities in patients on medical therapy for Toxoplasma encephalitis correlated well with clinical improvement. Recurrence of CT abnormalities correlated well with medical noncompliance. The optimal contrast enhancement technique for detecting CNS pathology and for monitoring the effectiveness of medical therapy was also evaluated by a prospective study in which both immediate (IDD) and 1 hr delayed (DDD) double-dose contrast CT scans were compared. The examination found to be diagnostically superior in 30 of the 41 IDD/DDD studies was the delayed scan. It is recommended that CT be used routinely and with the 1 hr DDD scan to evaluate and follow AIDS patients with neurologic symptoms and/or signs.

Toxoplasma encephalitis in recent Haitian entrants.

We evaluated clinical features of five cases of Toxoplasma encephalitis (TE) occurring in recent Haitian entrants into the United States. None of the patients had any underlying malignancy or known immunosuppressive therapy. Histopathologic findings of TE at autopsy were confirmed by peroxidase-antiperoxidase method. Four patients had an antecedent episode of disseminated tuberculosis and all five were receiving antituberculous therapy when neurologic manifestations of lethargy, seizures, and motor weakness first developed. These symptoms progressed into coma and death within 15 days. Peripheral lymphocytopenia was noted in all patients; three were anergic. Parenchymal lesions were identified by CT brain scans and total proteins were elevated in spinal fluid in all cases. TE appears to be a manifestation of the acquired immune deficiency syndrome in Haitians; it should be suspected in those with a febrile illness and multiple focal lesions of the central nervous system.

Toxoplasma encephalitis in Haitian adults with acquired immunodeficiency syndrome: a clinical-pathologic-CT correlation.

The clinical data, histologic findings, and computed tomographic (CT) abnormalities in eight adult Haitians with toxoplasma encephalitis were analyzed retrospectively. Diagnosis was established by identification of Toxoplasma gondii on autopsy in five and brain biopsy in three specimens and subsequently confirmed by the immunoperoxidase method. All these patients, six of whom had been in the United States for 24 months or less, had severe idiopathic immunodeficiency syndrome. All were lymphopenic and six were on treatment for tuberculosis when the toxoplasma encephalitis developed. All patients were studied with CT when they developed an altered mental status and fever associated with seizures and/or focal neurologic deficits. Scans before treatment showed multiple intraparenchymal lesions in seven and a single lesion in the thalamus in one. Ring and/or nodular enhancement of the lesions was found in six and hypodense areas in two. Progression of abnormalities occurred on serial studies. These CT findings that were best shown on axial and coronal thin-section double-dose contrast studies were useful but not diagnostically pathognomonic. In patients with similar clinical presentation CT is recommended to identify focal areas of involvement and to guide brain biopsy or excision so that prompt medical therapy of this often lethal infection can be instituted.

Clinical evaluation of piperacillin with observations on penetrability into cerebrospinal fluid.

Piperacillin, a new semisynthetic penicillin, was evaluated for efficacy and safety in 26 patients, most of whom had pneumonia. Included were four patients with gram-negative meningitis in whom the penetration of piperacillin into cerebrospinal fluid was determined. Cure was achieved in 11 of 17 patients with pneumonia; another 4 were improved. One relapse and one failure occurred among nine patients with gram-negative pneumonia. Cure or improvement occurred in seven of nine patients with gram-negative infection in various extrapulmonary sites. Piperacillin given by continuous infusion in a dosage ranging from 324 to 436 mg/kg of body weight per day to four patients with meningitis resulted in a mean cerebrospinal fluid level of 23 micrograms/ml at 24 h; the mean penetration of piperacillin into the cerebrospinal fluid was 32% at this interval. Levels of piperacillin in cerebrospinal fluid collected later during the course of therapy were also adequate. Adverse effects were noted in six patients, but only one episode of granulocytopenia was serious. Emergence of resistance to piperacillin did not occur, and only one superinfection was noted. Piperacillin appeared to be efficacious in the treatment of pneumonia. It penetrated well into the cerebrospinal fluid of patients with meningitis and may be useful for treatment of selected gram-negative infections in extrapulmonary sites.

Effects of inducible beta-lactamase and antimicrobial resistance upon the activity of newer beta-lactam antibiotics against Pseudomonas aeruginosa.

The activity of carbenicillin, ticarcillin, piperacillin, cefotaxime, moxalactam, and N-formimidoyl thienamycin was evaluated against 262 clinical isolates of Pseudomonas aeruginosa. There were 242 (92%) of the isolates that were susceptible to carbenicillin or ticarcillin by an agar dilution method. Against this population of susceptible isolates, the median MICs were 1.56 microgram/ml of N-formimidoyl thienamycin, 3.13 microgram/ml of piperacillin, 25 microgram/ml of ticarcillin, 25 microgram/ml of cefotaxime, 50 microgram/ml of carbenicillin and 50 microgram/ml of moxalactam. N-Formimidoyl thienamycin was the only beta-lactam antibiotic not affected by an inducible beta-lactamase detected in 24 randomly selected susceptible isolates by a disk approximation assay, while cefotaxime was inactivated to a greater extent than any of the other beta-lactam antibiotics. Resistance to carbenicillin and ticarcillin was noted in 20 isolates (8%); these were susceptible to N-formimidoyl thienamycin, but cross-resistance with piperacillin, cefotaxime, and moxalactam was frequent. Only four of these resistant isolates were found to have a constitutive beta-lactamase. Gentamicin resistance occurred in 51 isolates (19%) and was an independent variable of resistance to the beta-lactam drugs.

Evaluation of a gonococcal serologic test.

The fluorescent gonorrhea test--heated (FGT-H), an indirect immunofluorescent method for detecting the presence of antigonococcal antibodies in serum, was evaluated in 1,000 women referred to a private clinic for the termination of pregnancy, 202 women treated at a municipal venereal disease clinic, and six women hospitalized with gonococcal bacteremia. The test was positive for 17.7% of private outpatients, even though gonococci were cultured from only 1.1% of these women, and another 5.1% had histories of gonococcal infection. Positive test results were found for 68.3% of the women in the venereal disease clinic population, in which the prevalence of gonococcal infection was 36.1% and an additional 25.2% had had gonorrhea previously. The test was positive for all six patients with gonococcemia. The sensitivity of the test in the low-risk population was 72.7%, which was not significantly different from the sensitivity of 84% in the high-risk population. Specificity of the test was 83% in low-risk patients, but only 41% in high-risk patients. These findings indicate that a negative test is a highly reliable means for excluding gonorrhea in populations of low-risk women. The predictive value of a positive test is low, ranging from 4.5% in the low-risk group to 45% in the high-risk group.

Comparative evaluation of piperacillin in vitro.

The minimal inhibitory concentrations of piperacillin and seven other betalactam antibiotics were determined against 407 bacterial isolates. Piperacillin was found to be more active than ampicillin against susceptible gram-negative bacilli and more active than either carbenicillin or ticarcillin against Pseudomonas aeruginosa and streptococci. Although piperacillin was active against Klebsiella pneumoniae, this activity was less than that of the cephalosporins. Piperacillin was not active against penicillin-resistant Staphylococcus aureus and Enterobacteriaceae that were resistant to the other test antibiotics.

Staphylococcal endocarditis in addicts.

We retrospectively reviewed 55 episodes that fulfilled criteria for Staphylococcus aureus endocarditis in 50 drug addicts. The most common presenting symptoms were fever(90%), chest pain(58%), and cough(43%). All patients had evidence of right-sided heart involvement, and a murmur of tricuspid insufficiency was noted in 42%. Evidence of left-sided heart involvement was present in only 5%. The most helpful laboratory aid in facilitating an early clinical diagnosis of endocarditis was the chest x-ray film. Roentgenographic evidence of septic pulmonary emboli was present in 67% of initial chest films and eventually in 87% of all cases. All but five patients completed at least four weeks of intravenous antibiotic therapy. No patients required cardiac surgery and there were no deaths. The apparent predilection of S aureus for the right side of the heart and infrequent left-sided involvement may explain why addicts with endocarditis have a favorable response to antibiotic therapy.

Amikacin therapy of gram-negative bacteremia and meningitis. Treatment in diseases due to multiple resistant bacilli.

The therapeutic efficacy of amikacin was evaluated in patients with serious hospital-acquired infections caused by Gram-negative bacilli susceptible to amikacin, but usually resistant to kanamycin, gentamicin, and tobramycin. The infections for which amikacin was given were Gram-negative bacteremia in 15 patients and Gram-negative meningitis in two patients. Therapy with amikacin resulted in a cure in 13 patients, improvement in 1, and failure in 3. Continuous intravenous infusion of amikacin yielded a high cerebrospinal fluid to serum ratio of amikacin in one case of meningitis and intrathecally administered amikacin yielded high ventricular fluid levels in another case of meningitis. The emergence of resistance to amikacin was noted in one patient treated with amikacin in whom Serratia bacteremia persisted. Treatment with amikacin was usually tolerated well. This study indicates that amikacin is an effective antibiotic in the treatment of serious Gram-negative infections caused by gentamicin-resistant organisms.

Waterborne typhoid fever in Dade County, Florida. Clinical and therapeutic evaluation of 105 bacteremic patients.

An extensive outbreak of waterborne typhoid fever occurred in 1973 at a migrant labor camp in Dade County, Florida. Blood cultures from 105 of the 188 patients with proved or presumptive cases of typhoid fever grew Salmonella typhi. The clinical and laboratory findings in these patients were reviewed. Fever, usually with temperatures above 38.8 degrees C and of the sustained type, was a primary manifestation of disease, although a majority of the patients also complained of headache and gastroenteric symptoms. Hepatic or splenic enlargement was present in 52 per cent and 42 per cent, respectively, whereas rose spots were detected in only 13 per cent. The total leukocyte count was normal in 74 per cent, but serum levels of liver and muscle enzymes were frequently elevated. Gastrointestinal, pulmonary and neurologic complications were infrequent; circulatory failure was not observed. Defervescence in response to antibiotic therapy was variable; however, the median response among 68 patients who received chloramphenicol was two days less than that in 34 patients treated with ampicillin. There was one possible treatment failure with ampicillin. The relapse rate of 10 per cent in chloramphenicol-treated patients was not significantly greater than the 3 per cent rate among those treated with ampicillin. Serologic studies for typhoid fever were of limited diagnostic value since the titer of agglutinins was 1:160 or higher in 49 per cent of the serums obtained before treatment, and a fourfold rise in titer occurred in only 24 per cent of 57 patients studied. The serologic response to chloramphenicol treatment did not differ from that to ampicillin.