Bcl-2 overexpression blocks activation of the death protease CPP32/Yama/apopain

Females who are affected by fragile X syndrome (FXS) can have significant physical, neuropsychological and emotional involvement. This study was designed to explore the relationships between these three domains and to learn how the degree of involvement in each of these phenotypic areas relates to molecular parameters including CGG repeat length and activation ratio (the proportion of normal FMR1 alleles on the active X chromosome). Three groups of females were studied: 35 women who grew up in a fragile X family but do not carry an FMR1 mutation, 92 women with a premutation, and 29 women with a full mutation. Correlations between neurocognitive, physical and emotional traits were calculated for each of the three groups. Within the full mutation group significant correlations were seen between schizotypal traits and full scale IQ. The Lie scale was significantly correlated with the physical findings index. The activation ratio correlated significantly with the measure of executive function (r = .50, P = .01). There was a trend toward correlations of activation ratio with the physical index score, outer ear prominence and IQ. CGG repeat number significantly correlated only with the physical index (r = .44, P = .01). Thus, activation ratio may be the more pertinent molecular parameter in full mutation women in determining the degree of cognitive and physical phenotypic involvement.     

Cloning, expression and sequence analysis of the SphI restriction-modification system

SphI, a type II restriction-modification (R-M) system from the bacterium Streptomyces phaeochromogenes, recognizes the sequence 5'-GCATGC. The SphI methyltransferase (MTase)-encoding gene, sphIM, was cloned into Escherichia coli using MTase selection to isolate the clone. However, none of these clones contained the restriction endonuclease (ENase) gene. Repeated attempts to clone the complete ENase gene along with sphIM in one step failed, presumably due to expression of SphI ENase gene, sphIR, in the presence of inadequate expression of sphIM. The complete sphIR was finally cloned using a two-step process. PCR was used to isolate the 3' end of sphIR from a library. The intact sphIR, reconstructed under control of an inducible promoter, was introduced into an E. coli strain containing a plasmid with the NlaIII MTase-encoding gene (nlaIIIM). The nucleotide sequence of the SphI system was determined, analyzed and compared to previously sequenced R-M systems. The sequence was also examined for features which would help explain why sphIR unlike other actinomycete ENase genes seemed to be expressed in E. coli.     

High-density hybridoma perfusion culture. Limitation vs inhibition

Because our earlier work indicated a strong correlation between specific antibody productivity and cell density in perfusion culture, we conducted experiments to determine the optimum means of increasing cell density while maintaining high antibody productivity. The rates of medium supply and waste removal were varied to determine whether cell density was limited or inhibited, and whether a diffusable substance could be responsible for the correlation between antibody productivity and cell density. Nutrient supply was found to be a stronger determinant of cell density than waste removal; however, the rate of waste removal had a greater effect on cell growth at lower cell densities. Even at noninhibitory levels of ammonia and lactate, cellular metabolism was regulated to minimize their concentrations at lowered rates of waste removal. Separate step changes in glucose and glutamine resulted in increased cell density and antibody concentration. Specific antibody productivity increased following the step in glutamine, but not glucose. Both steps caused changes in cellular metabolism that prevented the levels of lactate and ammonia from reaching toxic levels.     

Small bowel injury in gastroschisis: relation to fetal presentation

The study presented here attempts to assess the cost savings attributable to having an on-site corporate medical clinic. For a period of 4 months, employees using the clinic were surveyed regarding: (1) whether they would have used an outside doctor if the corporate medical department clinic were not available, and (2) how many days per year they estimated that they came to work because there was an on-site medical center when they would otherwise have stayed at home. The results indicated that, on average, employees who used the facility saved 3.3 days of absenteeism. That total cost savings was calculated to be $499,212 per year. In addition, 69% of employees indicated that they would have sought attention elsewhere, suggesting that the presence of an on-site medical center does not induce demand. The findings concerning the hidden savings brought about by an on-site medical center are also discussed.     

Human papillomavirus infection in squamous cell carcinoma of the vulva, in various synchronous epithelial changes and in normal vulvar skin

In patients with inferior vena cava (IVC) injuries, predictors of survival are investigated. From 1987 to 1995, 27 IVC injuries were identified among 514 patients with vascular trauma. The ability of clinical determinants to predict survival were retrospectively assessed. IVC injuries occurred in 7 females and 20 males (mean age, 27.7 +/- 2.5 years) from both blunt (n = 14) and penetrating (n = 13) trauma. The mean revised trauma score was 10.2 +/- 0.6. Injuries were treated by primary repair (n = 22), ligation (n = 4), or prosthetic grafting (n = 1). Thirteen patients died (48%), 10 within 12 hours of admission. Suprahepatic (n = 2), retrohepatic (n = 12), suprarenal (n = 1), and infrarenal (n = 12) injuries were associated with 100, 67, 100, and 20 per cent mortality, respectively. Blood transfusions (16 +/- 4 vs 23 +/- 4 units), coagulation factor replacement (7 +/- 2 vs 7 +/- 2 units), and electrolyte solution use (8.6 +/- 1.4 vs 9.6 +/- 1.4 L) were similar among survivors and nonsurvivors. Four complications [venous hypertension (n = 2), IVC thrombosis (n = 1), and pulmonary embolus (n = 1)] occurred in the 14 survivors (28.6%). Blunt injury, revised trauma score, free perforation, injury location, intraoperative hypotension, and blood loss were predictive of mortality. IVC injuries remain extremely lethal, and improved survival is associated with infrarenal penetrating injuries and a contained hematoma.     

High resolution footprinting of a type I methyltransferase reveals a large structural distortion within the DNA recognition site

The type I DNA methyltransferase M.EcoR124I is a multi-subunit enzyme that binds to the sequence GAAN6RTCG, transferring a methyl group from S-adenosyl methionine to a specific adenine on each DNA strand. We have investigated the protein-DNA interactions in the complex by DNase I and hydroxyl radical footprinting. The DNase I footprint is unusually large: the protein protects the DNA on both strands for at least two complete turns of the helix, indicating that the enzyme completely encloses the DNA in the complex. The higher resolution hydroxyl radical probe shows a smaller, but still extensive, 18 bp footprint encompassing the recognition site. Within this region, however, there is a remarkably hyper-reactive site on each strand. The two sites of enhanced cleavage are co-incident with the two adenines that are the target bases for methylation, showing that the DNA is both accessible and highly distorted at these sites. The hydroxyl radical footprint is unaffected by the presence of the cofactor S-adenosyl methionine, showing that the distorted DNA structure induced by M.EcoR124I is formed during the initial DNA binding reaction and not as a transient intermediate in the reaction pathway.     

Phase I study of a five-day dose schedule of 4-Ipomeanol in patients with non-small cell lung cancer

The mammalian pulmonary toxin 4-ipomeanol (IPO) is activated by the cytochrome P450 system in bronchial Clara cells in animals. The resulting metabolites bind rapidly to macromolecules, producing localized cytotoxicity. IPO has in vitro and in vivo antitumor activity in non-small cell lung cancer (NSCLC) and thus was proposed as a lung cancer-specific antitumor agent. We have completed a directed Phase I trial in patients with NSCLC. Forty-four patients (34 men and 10 women) with NSCLC were treated with IPO. All but two patients had an Eastern Cooperative Oncology Group performance status of 0 or 1. They received 91 courses of therapy with i.v. IPO; 82 courses were administered daily for five days, and 9 were single bolus doses. The dose-limiting toxicity of elevated serum transaminases was observed in three of seven patients at 922 mg/m2/day. The maximum tolerated dose was 693 mg/m2/day on 5 consecutive days every 3 weeks. One patient developed grade 4 pulmonary toxicity at 167 mg/m2/day. There was no significant hematological or renal toxicity. No objective antitumor responses were observed. Pharmacokinetic analysis of 39 patients from day 1 of IPO administration showed biexponential elimination with mean half-lives of 8.6 (alpha half-life) and 76 min (beta half-life). There was a linear relationship between the area under the plasma drug concentration-time curve and the dose of IPO. There was no significant difference between the pharmacokinetic parameters measured on day 1 and day 5. Using a 4-day in vitro cytotoxicity assay, two tumor cell lines established from patients treated at 693 mg/m2/day had IC50s of approximately 6 mM, a concentration more than 75-fold higher than the plasma levels measured in these patients. Thus, although the total amount of drug administered per cycle on a daily times five dose schedule is more than 2.5-fold higher than the recommended single daily dose, IPO is unlikely to be a useful drug for patients with lung cancer.     

Transport of paraquat by a renal epithelial cell line, MDCK

Transport of paraquat (PQ), a herbicidal cation, was previously investigated in a proximal (LLC-PK1), renal epithelial cell line using permeable collagen-coated filters. PQ was actively transported from the basolateral side via a cation transport system by the LLC-PK1 cells. In the present study, the transport of PQ was investigated in a distal renal epithelial cell line, MDCK. PQ was predominantly transported from the basolateral to apical (B to A) side. The basolateral transport of PQ in MDCK cells was not saturable with increasing concentrations and not energy dependent. The flux and uptake of PQ was much lower in the MDCK than LLC-PK1 cells. It is concluded that MDCK, a distal renal tubular cell line, does not have an active transport system for PQ.