A mutant yeast topoisomerase II (top2G437S) with differential sensitivity to anticancer drugs in the presence and absence of ATP

To further characterize the mechanistic basis for cellular resistance/hypersensitivity to anticancer drugs, a yeast genetic system was used to select a mutant type II topoisomerase that conferred cellular resistance to CP-115,953, amsacrine, etoposide, and ellipticine. The mutant enzyme contained a single point mutation that converted Gly437 --> Ser (top2G437S). Purified top2G437S displayed wild-type enzymatic activity in the absence of drugs but exhibited two properties that were not predicted by the cellular resistance phenotype. First, in the absence of ATP, it was hypersensitive to all of the drugs examined and hypersensitivity correlated with increased drug affinity. Second, in the presence of ATP, top2G437S lost its hypersensitivity and displayed wild-type drug sensitivity. Since the resistance of yeast harboring top2G437S could not be explained by alterations in enzyme-drug interactions, physiological levels of topoisomerase II were determined. The Gly437 --> Ser mutation reduced the stability of topoisomerase II and decreased the cellular concentration of the enzyme. These findings suggest that the physiological drug resistance phenotype conferred by top2G437S results primarily from its decreased stability. This study highlights the need to analyze both the biochemistry and the physiology of topoisomerase II mutants with altered drug sensitivity in order to define the mechanistic bridge that links enzyme function to cellular phenotype.     

DNA and a CpG oligonucleotide derived from Babesia bovis are mitogenic for bovine B cells

DNAs from bacteria and variety of nonvertebrate organisms, including nematodes, mollusks, yeasts, and insects, cause polyclonal activation of murine B lymphocytes. Similar studies have not been reported for bovine B cells, and to date no studies have reported mitogenic properties of protozoal DNA for any species. However, we and others have observed that protozoal parasite antigens can induce the proliferation of lymphocytes from nonexposed donors. Extending these studies, we now show that the mitogenic property of protozoal antigen preparations is in part attributable to parasite DNA and that Babesia bovis DNA is directly mitogenic for bovine B cells. DNase treatment of B. bovis extracts abrogated B. bovis-induced proliferation of peripheral blood mononuclear cells from nonexposed cattle. Like DNAs from other organisms that were mitogenic for murine B cells, B. bovis DNA is largely nonmethylated and induced a dose-dependent proliferation of bovine B cells, which was reduced upon methylation. Furthermore, B. bovis and E. coli DNAs enhanced immunoglobulin secretion by cultured B cells, inducing moderate increases in immunoglobulin G1 and stronger increases in immunoglobulin G2. Because certain nonmethylated CpG motifs present in bacterial DNA are known to stimulate proliferation of murine and human B cells, an 11-kb fragment of B. bovis DNA was analyzed for CG dinucleotide content and for the presence of known immunostimulatory sequences (ISS) centered on a CG motif. The frequency of CG dinucleotides was approximately one-half of the expected frequency, and several CpG hexameric sequences with known activity for murine B cells were identified. An oligodeoxynucleotide containing one of these ISS (AACGTT), which is present within the rhoptry-associated protein-1 (rap-1) open reading frame, was shown to stimulate B-cell proliferation. These ISS may be involved in host immune modulation during protozoal infection and may be useful as vaccine adjuvants.     

Impact of cholesterol reduction on peripheral arterial disease in the Program on the Surgical Control of the Hyperlipidemias (POSCH)

BACKGROUND: Few lipid/atherosclerosis intervention trials have assessed the impact of cholesterol reduction on peripheral arterial disease. The 838 patients evaluated in the Program on the Surgical Control of the Hyperlipidemias (POSCH) trial represent more than the total number of patients in the seven previously reported studies. METHODS: Peripheral arterial disease in POSCH was assessed by progression of clinical disease, serial changes in the systolic blood pressure ankle/brachial index (ABI), and changes on sequential peripheral arteriograms. RESULTS: At the time of formal closure of the POSCH trial on July 19, 1990, claudication or limb-threatening ischemia was exhibited in 72 of 417 control group (CG) patients and in 54 of 421 intervention group (IG) patients (IG relative risk [RR] 0.702, 95% confidence interval [CI] 0.169 to 1.000, p = 0.049). With additional follow-up evaluation to September 30, 1994, clinical peripheral arterial disease was evident in 91 CG patients and 64 IG patients (RR 0.656, 95% CI 0.200 to 0.903, p = 0.009). At the 5-year follow-up evaluation, an ABI of less than 0.95 was present in 41 of 120 CG patients and in 24 of 126 IG patients, all of whom had an ABI of 0.95 or greater at baseline (RR in the IG of 0.557, 95% CI 0.360 to 0.863, p < 0.01). No appreciable differences were noted in the progression or regression of arteriographic peripheral arterial disease between the two groups. CONCLUSIONS: Effective cholesterol reduction in POSCH led to statistically significant differences between the control and the intervention groups in the development of clinically evident peripheral arterial disease and in the ABI values, but not in the peripheral arteriograms. Additional studies need to assess the correlation between peripheral arterial changes and coronary arterial changes and clinical atherosclerosis events. Intervention trials that study peripheral arterial disease have intrinsic value in the evaluation of the impact of risk factor modification on progression of atherosclerotic peripheral arterial disease.     

Treatment of patients admitted to the hospital with congestive heart failure: specialty-related disparities in practice patterns and outcomes

OBJECTIVES: This study sought to define specialty-related differences in the care and outcome of patients admitted to the hospital with congestive heart failure (CHF). BACKGROUND: Congestive heart failure is the leading diagnosis-related group (DRG) discharge diagnosis in the United States and accounts for an estimated annual hospital cost in excess of $7 billion. The clinical impact of aggressive CHF management and the importance of the subspecialist in guiding this care have not been evaluated. METHODS: To define differences in physician practice patterns, we performed a chart review of consecutive patients admitted to a university teaching hospital with a primary DRG discharge diagnosis of CHF. We compared treatment and outcome of patients cared for by a generalist (n = 160) and those whose care was guided by a cardiologist (n = 138) during their index hospital period with CHF and over the next 6 months. RESULTS: At our institution, > 50% of patients admitted to the hospital with CHF cared for by generalists alone had minimal (New York Heart Association functional class I or II) symptoms, compared with < 15% of those cared for by a cardiologist (p < 0.01). Although generalists' patients underwent significantly fewer in-hospital diagnostic tests and had shorter lengths of stay, they had a 1.7-fold increased risk of readmission for CHF within 6 months (p < 0.05). Six-month cardiac and all-cause mortality were not significantly different between the groups. The type of physician caring for the patient and a history of diabetes, previous CHF or myocardial infarction were independent predictors of readmission for CHF. CONCLUSIONS: Involvement of a cardiologist in the care of patients admitted to the hospital with CHF is associated with increased use of diagnostic testing, longer hospital stays and improved clinical outcome. These results substantiate practice guidelines that suggest a role for cardiologists in the care of symptomatic patients with CHF.     

Human biodistribution of an ultrasound contrast agent (Quantison) by radiolabelling and gamma scintigraphy

The biodistribution and kinetics of an air filled human serum albumin microcapsule formulation (Quantison) intended for use as an intravenous ultrasound contrast agent have been examined. 12 healthy subjects were administered with approximately 50 million microcapsules per kilogram body weight, radiolabelled with 50 MBq 123I. Imaging was performed over a period of 58 h using a large field-of-view gamma camera and the amount of labelled material present in the blood, urine and faeces measured. Imaging demonstrated that the liver was the organ with the highest uptake, with a mean uptake of 41.8% (SD 10.4%) of the administered dose 1 h following administration. The maximum uptake of the agent in the lungs was low, mean 4.0% (SD 3.4%). A small amount of uptake was visible in the bone marrow; however, this was not quantifiable. There was also evidence of minimal myocardial activity within 5 min of administration. No adverse events were observed and there were no changes in any of the individual post-study indices. The present study demonstrates the safety of Quantison. Gamma scintigraphy played a useful role in confirming the biodistribution of the agent with little lung uptake, high liver uptake and evidence of myocardial uptake.     

Bovine immunodeficiency virus tat gene: cloning of two distinct cDNAs and identification, characterization, and immunolocalization of the tat gene products

cDNAs encoding the bovine immunodeficiency virus (BIV) transactivator gene (tat) were cloned from virally infected cells and characterized. BIV expresses two distinct tat mRNAs composed of three exons that are derived by alternative splicing. The BIV tat mRNA splice variants encode Tat proteins of 103 (Tat103) and 108 (Tat108) amino acids. The Tat103 coding region is specified only by exon 2, while that of Tat108 is specified by a truncated exon 2 and the first 30 nt of exon 3. Thus, the first 98 amino acids of each Tat are identical, and have amino terminal, cysteine-rich, conserved core, basic, and carboxyl-terminal domains similar to Tats encoded by primate lentiviruses. BIV-infected bovine cells express a 14-kDa phosphorylated Tat protein identical in size to recombinant Tat expressed in bacteria. BIV Tat was shown to localize exclusively in the nucleoli of virally infected and Tat-expressing cells. Reporter gene assays indicated that Tat103 and Tat108 can strongly transactivate the BIV long terminal repeat (LTR) in virally permissive canine Cf2Th and nonpermissive HeLa and mouse NIH 3T3 cells, but not in permissive lapine EREp cells. However, an intact BIV tat gene is required for viral replication in both Cf2Th and EREp cells. Strong LTR activation by BIV Tat requires a TAR (transactivation responsive) element delimited by viral nt +1 to +31 and the Tat basic domain. BIV Tat strongly cross-transactivates the HIV-1 LTR in a TAR-dependent manner in Cf2Th, but not in EREp, HeLa, or NIH 3T3 cells. In contrast, strong, TAR-dependent cross-transactivation of the BIV LTR by HIV-1 Tat could not be demonstrated in any of these cell types. In Cf2Th cells Tat108 effects a moderately stronger transactivation of the BIV LTR than Tat103, indicative of a functional difference in BIV Tat proteins encoded by the mRNA splice variants. The present studies demonstrate that BIV Tat parallels the primate lentiviral Tats in structure and biochemistry but is not interchangeable with the latter.     

Extraction and replacement of permanent pacemaker leads through occluded vessels: use of extraction sheaths as conduits--balloon venoplasty as an adjunct

Patients (pts) may present for lead extraction with symptomatic or asymptomatic subclavian vein or superior vena cava thrombosis. Replacement of permanent pacemaker leads (PPLs) in these pts may be difficult and may require accessing a new site. We examined the utility of replacing PPLs through completely occluded vessels using extraction sheaths as conduits through the total occlusion. Over six years, a total of 210 atrial and/or ventricular PPLs were extracted from 137 pts. Two pts presented with angiographically documented thrombotic occlusion of the subclavian vein. One additional pt. who had presented with a superior vena cava (SVC) syndrome, had a totally occluded innominate vein and SVC occlusion. Balloon venoplasty was used as an adjunct to dilate the SVC. In all pts, after PPLs were removed via a subclavian extraction sheath through the occluded vessel, the retained sheath was used to place a guide wire, then a peel away dilating sheath, to insert new PPLs, in each case on the side of total venous occlusion. Seven PPLs and two lead fragments were extracted, and five new PPLs replaced, ipsilateral to the venous occlusion. These data show that extraction of PPLs through thrombosed veins may be performed successfully and may not require replacing the leads through a new site. This technique spares the pt the need to access the opposite subclavian vein, and it avoids an excessive number of PPLs in the subclavian vein and SVC. The procedure illustrates an efficient means to reintroduce new PPLs with the potential to reduce associated morbidity, since repeat puncture of the subclavian vein is not required. Safety of the procedure as a whole must be considered with regard to the known risks of lead extraction, some complications of which may be substantial using current techniques.     

Prospective study of high grade anal squamous intraepithelial neoplasia in a cohort of homosexual men: influence of HIV infection, immunosuppression and human papillomavirus infection

OBJECTIVE: To determine the risk of developing high grade anal squamous intraepithelial neoplasia (HG-AIN) in relation to HIV infection and immunosuppression, after controlling for the effects of human papillomavirus (HPV) infection. DESIGN: Prospective cohort study of 158 HIV-seropositive and 147 HIV-seronegative homosexual men presenting to a community-based clinic with initially negative anal cytologic and colposcopic findings. METHODS: Subjects completed self-administered questionnaires, underwent cytologic screening, and standardized unaided and colposcopic examination of the proximal anal canal for presence of abnormalities suggestive of AIN. Anal specimens were screened for HPV DNA. RESULTS: HG-AIN developed in eight (5.4%) and 24 (15.2%) HIV-seronegative and -seropositive men, respectively. Risk of HG-AIN among HIV-seronegative men was associated with detection of anal HPV types 16 or 18 by Southern transfer hybridization (STH), detection of HPV 16 or 18 at the lower levels by polymerase chain reaction but not by STH, and with number of positive HPV tests; HG-AIN risk among HIV-seropositive men was associated with detection of HPV 16 or 18 only by STH, detection of HPV types other than 16 or 18, CD4 count < or = 500 x 10(6)/l, and number of positive HPV tests. HIV-induced immunosuppression remained an independent predictor of HG-AIN after adjusting for type and level of detection of HPV; HIV infection predicted HG-AIN risk after adjustment for number of positive HPV tests. CONCLUSIONS: The association of HG-AIN with HIV, independent of HPV type, level of HPV detection and number of positive HPV tests, suggests that this increased risk cannot be entirely explained by an effect of HIV on HPV detection. Future studies focusing on factors more specific to the local microenvironment in the anal canal should help clarify these issues.