A simple method of producing low oxygen conditions with oxyrase for cultured cells exposed to radiation and tirapazamine

Several methods of establishing low O(2) conditions have been used in studies on the response of cultured cells to radiation and other agents. These methods, eg, gassing culture vessels with O(2)-free nitrogen with or without carbon dioxide or placing high cell-density suspensions in sealed glass ampoules to consume O(2) in the ampules, can be technically demanding and have experimental limitations. We introduce a simple, versatile, and reliable method of producing low O(2) conditions without special equipment or changes in culture conditions unrelated to hypoxia. The method is based on the ability of Oxyrase (Oxyrase, Inc., Mansfield, OH), membrane fragments prepared from Enterococcus coli, to consume O(2) in solution and is confirmed in the present study by 2 analytical methods. The effects of low O(2) conditions induced by Oxyrase on cellular responses to radiation and treatment with the bioreductive agent tirapazamine (TPZ) were examined with Chinese hamster V79 and human glioma U373 cells. Measured by clonogenic and MTT assays, these cells were less sensitive to radiation but more sensitive to TPZ in treatment media containing native Oxyrase than in media containing heat-inactivated Oxyrase. In addition, Oxyrase treatment increased the basal activity of mitogen-activated protein kinase (ERK1/2) but suppressed its activation induced by radiation. The results suggest that this method might also be useful for other in vitro cancer biologic investigations requiring a low O(2) condition.     

A longitudinal study of the effects of tobacco smoking and other modifiable risk factors on ill health in middle-aged and old Americans: results from the Health and Retirement Study and Asset and Health Dynamics among the Oldest Old survey

BACKGROUND: While the effects of smoking and other modifiable risk factors on mortality and specific diseases are well established, their effects on ill health more generally are less known. Using two national, longitudinal surveys, the objective of this study was to analyze the effect of smoking and other modifiable risk factors on ill health, defined in a multidimensional fashion (i.e., disability, impaired mobility, health care utilization, and self-reported health). METHODS: The analyses were based on the Health and Retirement Study (HRS) (12,652 persons 50-60 years old surveyed in 1992, 1994, 1996, and 1998) and the Asset and Health Dynamics among the Oldest Old survey (8,124 persons 60-70 years old surveyed in 1993, 1996, and 1998). RESULTS: Smoking was strongly related to mortality and to ill health, with similar relative effects in the middle-aged and the elderly. There were consistent adverse dose-response relationships between smoking and ill health in the HRS. Persons who had quit smoking at least 15 years prior to the survey were no more likely than never smokers to experience ill health. A dose-response relationship was found between exercise and ill health. For body mass index and alcohol, there were U-shaped relationships with ill health. CONCLUSIONS: Public health efforts designed to encourage smoking cessation should emphasize improvements in ill health in addition to decreased mortality.     

Dominant negative EGFR-CD533 and inhibition of MAPK modify JNK1 activation and enhance radiation toxicity of human mammary carcinoma cells.

Exposure of MDA-MB-231 human mammary carcinoma cells to an ionizing radiation dose of 2 Gy results in immediate activation and Tyr phosphorylation of the epidermal growth factor receptor (EGFR). Doxycycline induced expression of a dominant negative EGFR-CD533 mutant, lacking the COOH-terminal 533 amino acids, in MDA-TR15-EGFR-CD533 cells was used to characterize intracellular signaling responses following irradiation. Within 10 min, radiation exposure caused an immediate, transient activation of mitogen activated protein kinase (MAPK) which was completely blocked by expression of EGFR-CD533. The same radiation treatment also induced an immediate activation of the c-Jun-NH2-terminal kinase 1 (JNK1) pathway that was followed by an extended rise in kinase activity after 30 min. Expression of EGFR-CD533 did not block the immediate JNK1 response but completely inhibited the later activation. Treatment of MDA-TR15-EGFR-CD533 cells with the MEK1/2 inhibitor, PD98059, resulted in approximately 70% inhibition of radiation-induced MAPK activity, and potentiated the radiation-induced increase of immediate JNK1 activation twofold. Inhibition of Ras farnesylation with a concomitant inhibition of Ras function completely blocked radiation-induced MAPK and JNK1 activation. Modulation of EGFR and MAPK functions also altered overall cellular responses of growth and apoptosis. Induction of EGFR-CD533 or treatment with PD98059 caused a 3-5-fold increase in radiation toxicity in a novel repeated radiation exposure growth assay by interfering with cell proliferation and potentiating apoptosis. In summary, this data demonstrates that both MAPK and JNK1 activation in response to radiation occur through EGFR-dependent and -independent mechanisms, and are mediated by signaling through Ras. Furthermore, we have demonstrated that radiation-induced activation of EGFR results in downstream activation of MAPK which may affect the radiosensitivity of carcinoma cells.     

Ascorbic acid enhances hydroxyl radical formation in iron-fortified infant cereals and infant formulas

Infant cereals and formulas are usually fortified with iron to prevent iron deficiency. To enhance iron bioavailability, supplemental ascorbic acid is recommended. Ascorbic acid is considered to be an antioxidant in vivo, but has pro-oxidant effects when exposed to non-protein-bound iron. We measured formation of free radicals in cereals and infant formulas after addition of ascorbic acid. The production of hydroxyl radicals was assessed by hydroxylation of salicylic acid to 2,5-dihydroxybenzoic acid (2,5-DHBA). Production of 2,5-DHBA increased with increasing ascorbic acid doses added. Addition of 0.8 mM ascorbic acid to breast milk produced less radicals (0.03 ± 0.05 μM) than addition of ascorbic acid to low-iron formula (0.13 ± 0.08 μM, P = 0.019), medium-iron formula (0.34 ± 0.12 μM, P < 0.0001) or high-iron formula (0.44 ± 0.08 μM, P < 0.0001). Even when iron content in breast milk was adjusted to a level comparable with that of formulas, production of 2,5-DHBA was lower. Breast milk seems to contain substances that reduce hydroxyl radical formation. Conclusion Supplemental ascorbic acid causes hydro-xyl radical formation in iron-fortified infant nutrients in vitro. Received: 30 March 1996 / Accepted in revised form: 10 December 1996     

Laparoscopy and ultrasound examination in women with acute pelvic pain

The results of preoperative pelvic examination and eventual ultrasound examination were correlated with the laparoscopic findings in 316 women with acute pelvic pain. The predictive values of normal and abnormal findings at pelvic examination were 46.9 and 82.1%, respectively. 42.1% of the women had ultrasound examination performed. This investigation showed to be helpful especially in patients with normal findings at pelvic examination. If ultrasonic findings were abnormal the results at laparoscopy were also abnormal in 90%. On the contrary, normal findings at ultrasound examination did not exclude abnormal pelvic findings. The predictive value of normal results at ultrasound examination was 50.0%. This discrepancy between ultrasonic and pelvic findings can be explained by the size of the pelvic masses. Ultrasound examination is a valuable tool in the evaluation of patients with acute pelvic pain, but it cannot replace laparoscopy.     

Work-related knee disorders in floor layers and carpenters

Previous studies indicate an increased prevalence of knee disorders in some occupations possibly related to kneeling working positions. The purpose of the present study was to examine the relationship among knee-straining work, self-reported knee-complaints, and physical signs of knee disorders. The duration of knee-straining work was estimated from videotapes of representative work tasks. Floor layers (n = 133), carpenters (n = 506), and compositors (n = 327) aged 26 to 72 years without previous acute knee traumas were examined in a cross-sectional study by questionnaire. A stratified random sample of the questionnaire responders; 67 floor layers, 127 carpenters, and 101 compositors had independent double examinations for physical signs of knee disorders. The videotapes showed that knee-straining work constituted 56% of working time for floor layers, 26% for carpenters, and none for compositors. The prevalences of self-reported knee-complaints were positively associated with the amount of knee-straining work and were significantly different for the three trades. Floor layers and carpenters who were presently working in their trade had a higher prevalence of knee complaints than floor layers and carpenters who had left their trade. Age, seniority, weight, body mass index, smoking, and knee-straining sports activity had no significant effects. The clinical study showed a positive association for knee-straining work, hyperkeratosis, and bursitis. A similar pattern was found for signs of intraarticular knee disorders by one physician but not by another. The reproducibility of these signs was low. More studies are needed to define clinically important knee disorders for epidemiological studies.     

Alpha-1-antitrypsin levels and clinical symptoms in forty-eight children with selective IgA deficiency

Previous studies in adult patients with selective IgA deficiency have pointed towards a possible connection between chombined IgA and alpha-1-antitrypsin deficiency and the development of bronchiectasis. Thus, investigation were conducted of serum alpha-1-antitrypsin levels and clinical symptoms in 48 children with selective IgA deficiency. However, alpha-1-antitrypsin deficiency was not found in this group of patients. Furthermore, radiographic signs of recurrent pneumonia restricted to certain fields of the lungs, suggestive of bronchiectasia, were not observed in the patients. Thus, although a highly selected group of children with selective IgA deficiency was studied, a connection between IgA deficiency and alpha-1-antitrypsin deficiency as not obvious.Abbreviations RAST radio-allergo-sorbent-test - AAT alpha-1-antitrypsin     

Reduction of traumatic, primary anterior shoulder dislocations with local anesthesia

This study evaluates the use of local anesthesia in the reduction of acute shoulder dislocations. Patients with a primary traumatic dislocation of the shoulder were randomly assigned to receive either local anesthesia or intravenous anesthesia. The patients were observed for any complication during and after the procedure, and the methods used were evaluated with a visual analog scale. In the period from November 1991 to September 1993, 81 patients were admitted to our departments, and 68 patients were included in the study. Average age was 48 years (range 15 to 79 years); 29 men and 39 women were studied. Thirty-five patients were randomly assigned to receive intravenous anesthesia; 33 had a successful reduction, and two had a failed reduction. Thirty-three patients received local anesthesia; 32 had a successful reduction, and one had a failed reduction. Ten patients treated with the intravenous method had respiratory depression, and six required an antidote. No systemic or local side effects and no neurovasculor injuries were recorded. We did not observe any superficial or deep infection in the local anesthetic group. No statistical difference was found between the average visual analog value scale in the two groups. Local anesthesia to reduce acute primary anterior dislocation of the shoulder is a simple and safe method.     

Long-term trial of 1-alpha-hydroxycholecalciferol in adults with chronic renal failure

Summary Twentytwo adult patients with chronic renal insufficiency received long-term treatment (mean 15.4 months) with 1-alpha-hydroxycholecalciferol (1(OH)D₃), a synthetic vitamin D analogue. Before treatment 73% of the patients were hypocalcemic, intestinal calcium absorption was reduced in 82%, serum immunoreactive parathyroid hormone (i-PTH) was increased in 86% and serum alkaline phosphatases were increased in 50% of them. Furthermore, 91% of the patients had generalized scintigraphic bone changes indicative of renal osteodystrophy, 59% had reduced bone mineral content (BMC), 32% had radiographic bone changes and 27% had clinical symptoms. During treatment all patients became normocalcemic within one month; intestinal calcium absorption increased to reach normal values; i-PTH declined significantly, but remained elevated in 50% of patients; serum alkaline phosphatases declined significantly and became normal in all except 2 patients; bone scintigraphy and/or radiography showed considerable improvement in 4 patients; the accelerated bone loss in uremia ceased and bone pain disappeared in 5 of the 6 patients who had had this symptom. Nineteen short-lasting acute hypercalcemic episodes were recorded during treatment. In all cases normocalcemia was re-established within 72 hours after discontinuing the drug. No side-effects were observed apart from hypercalcemia. The present data demonstrate the long-term beneficial effects of 1 (OH)D₃ in improving clinical symptoms and reversing several biochemical and skeletal abnormalities in patients with chronic renal failure.     

Endocarditis due toKingella kingae

Four cases of endocarditis due toKingella kingae are described in compromised patients. All had primary heart disease, and two had systemic lupus erythematosis and congenital heart defect respectively, in addition. Confirmation ofKingella kingae was made in one case at autopsy. The literature on 11 cases of endocarditis, 2 bacteremia, 4 osteomyelitis, 5 septic arthritis and 1 intervertebral disc infection, all caused byKingella kingae, is reviewed. Our findings confirm that the organism is of low pathogenicity. Children may be predisposed to infection withKingella kingae.