Low-level resistance to camptothecin in a human small-cell lung cancer cell line without reduction in DNA topoisomerase I or drug-induced cleavable complex formation

To study the evolution of camptothecin (CPT) resistance, we have established two small-cell lung cancer cell lines with low (3.2-fold, NYH/CAM15) and high (18-fold, NYH/CAM50) resistance to CPT by stepwise drug exposure. NYH/CAM50 cells had reduced topoisomerase I (topo I) content and activity, and consequently CPT-induced DNA single strand breaks (SSBs) were reduced, as measured by alkaline elution. In contrast, NYH/CAM15 cells had identical topo I content and activity as compared with wild-type (wt) cells. CPT-mediated SSBs and the rate of their reversal after drug removal were also equal in wt and NYH/CAM15 cells, as were doubling time, the fraction of cells in S-phase and DNA synthesis rate in response to CPT. As the conversion of DNA SSBs to DNA double strand breaks (DSBs) is thought to represent a critical event leading to cell death, we measured DNA DSBs by neutral elution. In contrast to DNA SSBs, CPT induced fewer DNA DSBs in NYH/CAM15 than in wt cells. DNA flow cytometry showed that, in CPT-treated cells, the G1 phase was emptied as cells accumulated in late S- and G2M phase. A Spearman rank correlation showed that depletion of G1 and accumulation in late S and G2M correlated to CPT sensitivity in these three cell lines. In conclusion, acquired resistance to CPT can occur without a reduction in either topo I enzyme or CPT-induced cleavable complex formation, while a decrease in the level of CPT-induced DNA DSBs may be of major importance in the early stages of CPT resistance.     

Mild posttraumatic hypothermia reduces mortality after severe controlled cortical impact in rats

The effect of posttraumatic hypothermia (brain temperature controlled at 32 degrees C for 4 h) on mortality after severe controlled cortical impact (CCI) was studied in rats. Four posttraumatic brain temperatures were compared: 37 degrees C (n = 10), 36 degrees C (n = 4), 32 degrees C (n = 10), and uncontrolled (UC; n = 6). Rats were anesthetized and subjected to severe CCI (4.0-m/s velocity, 3.0-mm depth) to the exposed left parietal cortex. At 10 min posttrauma the rats were cooled or maintained at their target brain temperature, using external cooling or warming. Brain temperature in the UC group was recorded but not regulated, and rectal temperature was maintained at 37 +/- 0.5 degrees C. After 4 h, rats were rewarmed over a 1-h period to 37 degrees C, extubated, and observed for 24 h. In the 37 and 36 degree C groups, 24-h mortality was 50% (37 degrees C = 5/10, 36 degrees C = 2/4). In the 32 degree C group, 24-h mortality was 10% (1/10). In the UC group, brain temperature was 35.4 +/- 0.6 degrees C during the 4-h treatment period and 24-h mortality was 0% (0/6). Mortality was higher in groups with brain temperatures > or = 36 degrees C versus those with brain temperatures < 36 degrees C (50 vs. 6%, respectively; p < 0.05). Additionally, electroencephalograms (EEG) were recorded in subsets of each temperature group and the percentage of time that the EEG was suppressed (isoelectric) was determined. Percentage of EEG suppression was greater in the hypothermic (32 degrees C, n = 6; UC, n = 4) groups than in the normothermic (36 degrees C, n = 3; 37 degrees C, n = 6) groups (23.3 +/- 14.3 vs. 1.2 +/- 3.1%, respectively; p < 0.05). Posttraumatic hypothermia suppressed EEG during treatment and reduced mortality after severe CCI. The threshold for this protective effect appears to be a brain temperature < 36 degrees C. Thus, even mild hypothermia may be beneficial after severe brain trauma.     

Higher order iminodiacetic acid libraries for probing protein-protein interactions

Full details of the preparation of iminodiacetic acid diamide dimer (2040 compounds), trimer (560 compounds), and tetramer (1596 compounds) libraries by multistep convergent solution-phase synthesis for studying protein-protein interactions are provided. The libraries were assembled in a format providing small 8-10 compound mixtures and the deconvolution of many of the small mixtures to identify screening leads by resynthesis of the individual components have been conducted for 320 of the individual compounds to date. A representative example of the subsequent exploration of the structure-activity relationships for an identified receptor binding antagonist (200 additional individual compounds) and steps taken for potential elaboration to a receptor dimerization agonist are defined with preparation of representative linked dimers (70 compounds).     

Alpha-Tocopherol and beta-carotene supplements and lung cancer incidence in the alpha-tocopherol, beta-carotene cancer prevention study: effects of base-line characteristics and study compliance

BACKGROUND: Experimental and epidemiologic investigations suggest that alpha-tocopherol (the most prevalent chemical form of vitamin E found in vegetable oils, seeds, grains, nuts, and other foods) and beta-carotene (a plant pigment and major precursor of vitamin A found in many yellow, orange, and dark-green, leafy vegetables and some fruit) might reduce the risk of cancer, particularly lung cancer. The initial findings of the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (ATBC Study) indicated, however, that lung cancer incidence was increased among participants who received beta-carotene as a supplement. Similar results were recently reported by the Beta-Carotene and Retinol Efficacy Trial (CARET), which tested a combination of beta-carotene and vitamin A. PURPOSE: We examined the effects of alpha-tocopherol and beta-carotene supplementation on the incidence of lung cancer across subgroups of participants in the ATBC Study defined by base-line characteristics (e.g., age, number of cigarettes smoked, dietary or serum vitamin status, and alcohol consumption), by study compliance, and in relation to clinical factors, such as disease stage and histologic type. Our primary purpose was to determine whether the pattern of intervention effects across subgroups could facilitate further interpretation of the main ATBC Study results and shed light on potential mechanisms of action and relevance to other populations. METHODS: A total of 29,133 men aged 50-69 years who smoked five or more cigarettes daily were randomly assigned to receive alpha-tocopherol (50 mg), beta-carotene (20 mg), alpha-tocopherol and beta-carotene, or a placebo daily for 5-8 years (median, 6.1 years). Data regarding smoking and other risk factors for lung cancer and dietary factors were obtained at study entry, along with measurements of serum levels of alpha-tocopherol and beta-carotene. Incident cases of lung cancer (n = 894) were identified through the Finnish Cancer Registry and death certificates. Each lung cancer diagnosis was independently confirmed, and histology or cytology was available for 94% of the cases. Intervention effects were evaluated by use of survival analysis and proportional hazards models. All P values were derived from two-sided statistical tests. RESULTS: No overall effect was observed for lung cancer from alpha-tocopherol supplementation (relative risk [RR] = 0.99; 95% confidence interval [CI] = 0.87-1.13; P = .86, logrank test). beta-Carotene supplementation was associated with increased lung cancer risk (RR = 1.16; 95% CI = 1.02-1.33; P = .02, logrank test). The beta-carotene effect appeared stronger, but not substantially different, in participants who smoked at least 20 cigarettes daily (RR = 1.25; 95% CI = 1.07-1.46) compared with those who smoked five to 19 cigarettes daily (RR = 0.97; 95% CI = 0.76-1.23) and in those with a higher alcohol intake (> or = 11 g of ethanol/day [just under one drink per day]; RR = 1.35; 95% CI = 1.01-1.81) compared with those with a lower intake (RR = 1.03; 95% CI = 0.85-1.24). CONCLUSIONS: Supplementation with alpha-tocopherol or beta-carotene does not prevent lung cancer in older men who smoke. beta-Carotene supplementation at pharmacologic levels may modestly increase lung cancer incidence in cigarette smokers, and this effect may be associated with heavier smoking and higher alcohol intake. IMPLICATIONS: While the most direct way to reduce lung cancer risk is not to smoke tobacco, smokers should avoid high-dose beta-carotene supplementation.     

Geriatric psychiatry subspecialization in Canada: past, present, and future

We evaluated the clinical data in 83 patients with sepsis, which was diagnosed by both Bone's definition of sepsis and positive isolates from blood culture, according to their underlying diseases. This study enrolled a total of 117 septic episodes in 83 patients (57 males and 26 females, mean age: 52.0 years). We classified 3 groups, including hematological malignancies (46 patients, 72 episodes), solid malignant tumors (23 patients, 25 episodes) and non-malignancies (14 patients, 20 episodes), by the underlying diseases. Of the total number of isolates from blood culture, 53.0% were single gram-positive bacteria, 33.3% were single gram-negative bacteria, 7.7% were single fungus and 6.0% were polymicrobial organisms. In addition, coagulase negative staphylococci was isolated most often in patients with hematological malignancies. Sepsis was often caused by infectious focuses of hemorrhoid, stomatitis or intravenous catheter in patients with hematological malignancies, by pneumonia in patients with solid malignant tumors and by urinary tract infection in patients with non-malignancies. Mortality of sepsis in patients with solid malignant tumors (48%) was highest in 3 groups. Septic patients, who were complicated with shock and/or DIC, has poor prognosis in all groups. Serum albumin level was significantly lower in dead patients than patients who survived. These results suggest that clinical features may be different according to the underlying diseases of patients with sepsis.     

Periodontal repair in intrabony defects treated with a calcium sulfate implant and calcium sulfate barrier

THIS RANDOMIZED, CONTROLLED, CLINICAL STUDY was designed to evaluate outcome following surgical implantation of an allogeneic, freeze-dried, demineralized bone matrix-calcium sulfate (DBM+CS) composite with a CS barrier in intrabony periodontal defects. Twenty-six patients contributing 26 deep intrabony defects completed the study. Thirteen patients received the DBM+CS implant. Thirteen patients received gingival flap surgery alone (GFS; control). Clinical outcome was assessed at 6 and 12 months postsurgery. At 12 months postsurgery, probing depth (PD) reduction (mean +/-SD) for the DBM+CS and GFS group was to 4.3+/-0.5 and 3.0+/-1.3 mm; clinical attachment gain was to 2.9+/-0.8 and 1.7+/-1.5 mm; and probing bone level gain was to 2.9+/-1.4 and 1.2+/-1.2 mm, respectively. There were no apparent differences between evaluations at 6 and 12 months postsurgery. Clinical improvements were significantly different from presurgery for both groups at both observation intervals (P < 0.01). There were no significant differences between groups in PD reduction and clinical attachment gain. Probing bone level gain was significantly greater in the DBM+CS group compared to controls (P < 0.05). In summary, surgical implantation of DBM+CS with a CS barrier resulted in reduced PD and improved attachment levels comparable to that achieved by gingival flap surgery alone. However, gain in probing bone levels in deep intrabony periodontal pockets assessed by clinical parameters was greater than that observed by gingival flap surgery alone. These changes were noted at both 6 and 12 months after surgery. This regenerative technique needs further biologic evaluation before being generally accepted.     

Subtype G and multiple forms of A/G intersubtype recombinant human immunodeficiency virus type 1 in Nigeria

Multiple human immunodeficiency virus type 1 (HIV-1) genetic subtypes, intersubtype recombinants, and group O have been found in west central Africa. In Nigeria, where HIV-1 prevalence is rising rapidly, characterization of HIV-1 strains has been limited. Each of three full-length genome sequences acquired to date shows evidence of recombination: two are largely subtype G with subtype A segments in the midgenome accessory region; the third, IbNG, is subtype G with the long terminal repeats and two segments of pol from subtype A. In this study, peripheral blood mononuclear cells obtained in 1994-1995 from 10 patients hospitalized in northeastern Nigeria were evaluated by sequencing of the complete envelope and, from 7 patients, a portion of gag. Four patients harbored subtype G viruses and six patients had recombinant viruses. Two had strains sharing the A/G recombinant structure of IbNG. Two had a previously undescribed recombinant, mostly subtype A, whose carboxyl-terminal gp41 could not be classified. An A/G recombinant different from IbNG but similar to CA1, a Cameroonian strain, was found in one patient. The remaining patient had a strain that was otherwise subtype G but shared an unclassified carboxyl-terminal gp41 segment with the CA1-like strains. Other subtypes and group O were not found.     

Detection of abscesses with Tc-99m HMPAO leukocyte scintigraphy depends on their stage and location

Two abscesses were shown on CT in a 72-year-old man: one in the left hip and one in the left pelvic region that resulted from a motor vehicle collision that occurred 8 months earlier. Bone scintigraphy showed increased uptake in the left hip area. On Tc-99m HMPAO leukocyte imaging, the hip area appeared to be photopenic, and the abscess of the left pelvis was not identified. Incidental uptake in the left lung base appeared to be an active acute inflammatory process as evidenced by an infiltrating lesion in the left lower lung on a chest radiograph. Because leukocyte scintigraphy cannot detect the presence of a chronic inflammatory process in the absence of acute inflammatory cells, the patient's abscesses in the left hip and the left pelvis did not localize Tc-99m HMPAO-labeled neutrophils. The cold lesion in the left hip area was most likely caused by the lesion in the reticuloendothelial system (bone marrow); the unidentifiable pelvic lesion was related to the area(s) outside the reticuloendothelial system. In interpreting a labeled leukocyte image, clinicians should be alert to the stage (chronic or acute) and location (regardless of whether in the reticuloendothelial system) of infectious lesion(s). In this patient, a wide spectrum of manifestations was evident on leukocyte scintigraphy.     

Human pancreatic ribonuclease--deletion of the carboxyl-terminal EDST extension enhances ribonuclease activity and thermostability

Mammalian ribonucleases constitute one of the fastest evolving protein families in nature. The addition of a four-residue carboxyl-terminal tail: Glu-Asp-Ser-Thr (EDST) in human pancreatic ribonuclease (HPR) in comparison with bovine pancreatic RNase (RNase A) could have adaptive significance in humans. We have cloned and expressed human pancreatic ribonuclease in Escherichia coli to probe the influence of the four-residue extension and neighboring C-terminal residues on the biochemical properties of the enzyme. Removal of the C-terminal extension from HPR yielded an enzyme, HPR-(1-124)-peptide, with enhanced ability to cleave poly(C). HPR-(1-124)-peptide also exhibited a steep increase in thermal stability mimicking that known for RNase A. Wild-type HPR had significantly low thermal stability compared to RNase A. The study identifies the C-terminal boundary in the human pancreatic ribonuclease required for efficient catalysis.