Distinguishing small molecular mass differences of proteins by mass spectrometry

This study was undertaken to determine if acidic or basic fibroblast growth factor (FGF1 or FGF2) or vascular endothelial growth factor (VEGF) alters the radiation response of small bowel after total-body irradiation (TBI). Female C3H mice were treated with various doses of angiogenic growth factor administered intravenously 24 h before or 1 h after TBI. Radiation doses ranged from 7 to 18 Gy. End points measured were the number of crypts in three portions of the small bowel, the frequency of apoptosis of crypt cells at various times after TBI, and the LD50/30 (bone marrow syndrome) and LD50/6 (GI syndrome). Fibroblast growth factors alone, without TBI, decreased the number of crypts per circumference significantly. Among the factors tested, FGF2 caused the greatest decline in baseline crypt number. Despite this decrease in the baseline crypt number, after irradiation the number of surviving crypts was greater in animals treated with growth factor. The greatest radioprotection occurred at intermediate doses of growth factor (6 to 18 pg/mouse). Mice treated with FGF1 and FGF2 had crypt survival curves with a slope that was more shallow than that for saline-treated animals, indicating radiation resistance of crypt stem cells in FGF-treated mice. The LD50/6 was increased by approximately 10% for all treatments with angiogenic growth factors, whether given before or after TBI. Apoptosis of crypt cells was maximum at 4 to 8 h after TBI. The cumulative apoptosis was decreased significantly in animals treated with angiogenic growth factors, and the greatest protection against apoptosis was seen in animals treated with FGF2 prior to TBI. All three angiogenic growth factors tested were radioprotective in small bowel whether given 24 h before or 1 h after irradiation. The mechanism of protection is unlikely to involve proliferation of crypt stem cells, but probably does involve prevention of radiation-induced apoptosis or enhanced repair of DNA damage of crypt cells.     

Inhibition of tumor cell growth by RTP/rit42 and its responsiveness to p53 and DNA damage

Through a differential screening technique, we have identified a cDNA clone with differential expression in normal versus tumor cells. This clone, designated rit42 (reduced in tumor, 42 kDa), was previously isolated as a homocysteine-inducible gene in human endothelial cells (RTP), and the same or a highly related androgen-responsive gene in mouse has also been identified. Both Northern blot analysis and in situ hybridization demonstrated a significantly diminished expression in tumor cells, including those derived from breast and prostate when compared with normal cells. It was shown that RTP/rit42 mRNA cycles with cell division, peaking at G1 and G2-M, with lower expression in S phase. The biphasic expression of RTP/rit42 mRNA was absent in tumor cells. Introduction of rit42 cDNA into human cancer cells reduced cell growth both in vitro and in nude mice. Moreover, analysis of a tetracycline-regulated p53-inducible system in null-p53 cell lines showed that RTP/rit42 mRNA expression increased concomitantly with p53 expression and followed a similar time course. In addition, DNA-damaging agents induced RTP/rit42 expression in a p53-dependent manner but independent of a p53-mediated G1 arrest. Immunofluorescence analysis of a FLAG epitope-tagged RTP/rit42 protein revealed a cytoplasmic localization pattern with redistribution to the nucleus upon DNA damage. We have localized RTP/rit42 to human chromosome 8q24.3. Taken together, these results are consistent with a growth inhibitory role for RTP/rit42, and its down-regulation may contribute to the tumor malignant phenotype.     

Tonsillar carcinoma: analysis of treatment results

A diffusion cell with an artificial membrane and the single-pass perfused rabbit ear were used to evaluate the percutaneous absorption of clonazepam from various 2-hydroxyethyl acetate (HEA) patches. The influence on drug permeation of the various type of enhancers (isopropylmyristate, lauryl alcohol, propylene glycol and water) in the patches was tested. A comparison between the two types of systems of percutaneous absorption of clonazepam has been done. The results showed that HEA patches produce controlled uniform drug release, modulated by the addition of enhancers.     

Formaldehyde in cryoprotectant propanediol and effect on mouse zygotes

Cryopreservation of human zygotes and embryos has been routinely performed by in-vitro fertilization clinics for many years. Karran and Legge (1996) first reported that formaldehyde (FA) present in the cryoprotective solutions can have a deleterious effect on mouse oocytes. FA is a cytotoxic, carcinogenic and mutagenic chemical. The effect of FA on mouse zygotes was investigated. In addition, the concentrations of FA in propanediol (PROH) obtained from various sources were determined. Pooled 1-cell embryos were dispensed into droplets of modified Ham's F10 or human tubal fluid containing various concentrations of FA. Since bovine serum albumin (BSA) may minimize toxicity additional trials were done as above in the absence of BSA. FA concentration in the standard 1.5 M PROH, from different sources in water, was measured in the same assay using a standard curve of 0-100 microM FA. FA in a complex medium had a significant deleterious effect on embryo development and hatching but only at 1 mM concentration (P < 0.000001; see Tables I-III). There was no significant effect of FA at 100 microM. However, in a simple medium even 50 microM FA decreased embryo hatching. FA was present in 1.5 M PROH from different sources (range 1.0-35.3 microM concentration). It appears that FA concentrations do not increase with storage because FA concentrations were low even after opening and storage for 3 years on the shelf. This suggests that FA is a contaminant during the manufacturing process and may vary from manufacturer to manufacturer and batch to batch. Until further studies are done to confirm the lack of toxicity to embryos during cryopreservation (with or without FA scavengers) it may be prudent to screen all batches of cryoprotectants for FA as part of quality control.     

Increased mortality after successful treatment for Hodgkin's disease

PURPOSE: To determine the impact of treatment toxicity on long-term survival in pediatric Hodgkin's disease. PATIENTS AND METHODS: We studied late events in 387 patients treated for pediatric Hodgkin's disease on four consecutive clinical trials at St Jude Children's Research Hospital from 1968 to 1990. Relative risks, actuarial risks, and absolute excess risks for cause-specific deaths were calculated. RESULTS: As of April 1997, 316 (82%) of patients were alive, with a median follow-up of 15.1 (range, 2.9 to 28.6) years. In this cohort, which represented 5,623 person-years of follow-up, 71 fatal events resulted from Hodgkin's disease (n=36), second malignancies (n=14), infections (n=7), accidents (n=7), cardiac disease (n=6), and asphyxiation (n=1). The 5-year estimated event-free survival (EFS) for the entire cohort was 79.6%+/-2.1 %, which declined to 63.1%+/-4.4% by 20 years. Cumulative incidences of cause-specific deaths at 25 years were 9.8%+/-1.6% for Hodgkin's disease, 8.1%+/-2.6% for second malignancy, 4.0%+/-1.8% for cardiac disease, 3.9%+/-1.5% for infection, and 2.1%+/-0.8% for accidents. Standardized incidence ratios showed excess risk for all second malignancies (12; 95% confidence interval [CI], 8 to 17), acute myeloid leukemia (81; 95% CI, 16 to 237), solid tumors (11; 95% CI, 7 to 16), and breast cancer (33; 95% CI, 12 to 72). Standardized mortality ratios also showed excess mortality from cardiac disease (22; 95% CI, 8 to 48) and infection (18; 95% CI, 7 to 38). CONCLUSION: Compared with age- and sex-matched control populations, survivors of pediatric Hodgkin's disease who were treated before 1990 face an increased risk of early mortality related to second cancers, cardiac disease, and infection.     

Laparoscopic cholecystectomy under epidural anesthesia in patients with chronic respiratory disease

BACKGROUND: Laparoscopic cholecystectomy (LC) has become firmly established as a procedure of choice for gallstone disease. The procedure usually necessitates general anaesthesia and endotracheal intubation to prevent aspiration and respiratory embarrassment secondary to the induction of pneumoperitoneum. There is a paucity of data in the literature on the procedure being performed under regional (epidural) anaesthesia, especially in patients with coexisting pulmonary disease and pregnancy, who are deemed high risk for general anaesthesia. We report our preliminary experience with LC using epidural anaesthesia in patients with chronic obstructive pulmonary disease (COPD). METHODS: We performed LC in six patients (one man and five women), with a median age of 56 years (range, 38-74), under epidural anaesthesia over an 8-month period. All patients were ASA grade III/IV and the mean FEB1/FVC was 0.52 (range, 0.4-0.68), due to chronic asthma (two cases) and COPD (four cases). They were admitted a day prior to surgery for pulmonary function tests, nebulisers, and chest physiotherapy. An epidural catheter was introduced at T10/11 intervertebral space, and a bolus of 0.5% Bupivacaine was administered. Depending on the patient's pain threshold and the segmental level of analgesia achieved, incremental doses of 2 ml of 0.5% Bupivacaine along with boluses of intravenous 100 mcg Alfentanil was given to each patient. The patients were breathing spontaneously. No nasogastric tube was inserted, and a low-pressure (10 mmHg) pneumoperitoneum was created. LC was performed according to the standard technique. RESULTS: All the patients tolerated the procedure well and made an uneventful postoperative recovery. Median operating time was 50 min; average length of hospital stay was 2.5 days (range, 2-4). The epidural catheter was removed the morning after the operation. Only one patient required postoperative opioid analgesia. Two patients complained of persistent shoulder tip pain during surgery and required intraoperative analgesia (Alfentanil). There was no change in the patient's cardiorespiratory status, including PO2 and pCO2, and no complications occurred either intra- or postoperatively. CONCLUSIONS: LC can be performed safely under epidural anaesthesia in patients with severe COPD. Intraoperative shoulder tip or abdominal pain does not seem to be a major deterrent and can be effectively controlled with small doses of opioid analgesia.     

CVT-313, a specific and potent inhibitor of CDK2 that prevents neointimal proliferation

The activity of cyclin-dependent kinase 2 (CDK2) is essential for progression of cells from G1 to the S phase of the mammalian cell cycle. CVT-313 is a potent CDK2 inhibitor, which was identified from a purine analog library with an IC50 of 0.5 microM in vitro. Inhibition was competitive with respect to ATP (Ki = 95 nM), and selective CVT-313 had no effect on other, nonrelated ATP-dependent serine/threonine kinases. When added to CDK1 or CDK4, a 8.5- and 430-fold higher concentration of CVT-313 was required for half-maximal inhibition of the enzyme activity. In cells exposed to CVT-313, hyperphosphorylation of the retinoblastoma gene product was inhibited, and progression through the cell cycle was arrested at the G1/S boundary. The growth of mouse, rat, and human cells in culture was also inhibited by CVT-313 with the IC50 for growth arrest ranging from 1.25 to 20 microM. To evaluate the effects of CVT-313 in vivo, we tested this agent in a rat carotid artery model of restenosis. A brief intraluminal exposure of CVT-313 to a denuded rat carotid artery resulted in more than 80% inhibition of neointima formation. These observations suggest that CVT-313 is a promising candidate for evaluation in other disease models related to aberrant cell proliferation.