Modifying fatty acid profiles through a new cytokinin-based plastid transformation system.

The widespread use of herbicides and antibiotics for selection of transgenic plants has not been very successful with regard to commercialization and public acceptance. Hence, alternative selection systems are required. In this study, we describe the use of ipt, the bacterial gene encoding the enzyme isopentenyl transferase from Agrobacterium tumefaciens, as a positive selectable marker for plastid transformation. A comparison between the traditional spectinomycin-based aadA selection system and the ipt selection system demonstrated that selection of transplastomic plants on medium lacking cytokinin was as effective as selection on medium containing spectinomycin. Proof of principle was demonstrated by transformation of the kasIII gene encoding 3-ketoacyl acyl carrier protein synthase III into tobacco plastids. Transplastomic tobacco plants were readily obtained using the ipt selection system, and were phenotypically normal despite over-expression of isopentenyl transferase. Over-expression of KASIII resulted in a significant increase in 16:0 fatty acid levels, and a significant decrease in the levels of 18:0 and 18:1 fatty acids. Our study demonstrates use of a novel positive plastid transformation system that may be used for selection of transplastomic plants without affecting the expression of transgenes within the integrated vector cassette or the resulting activity of the encoded protein. This system has the potential to be applied to monocots, which are typically not amenable to traditional antibiotic-based selection systems, and may be used in combination with a negative selectable marker as part of a two-step selection system to obtain homoplasmic plant lines.

Uridine phosphorylase (-/-) murine embryonic stem cells clarify the key role of this enzyme in the regulation of the pyrimidine salvage pathway and in the activation of fluoropyrimidines.

We have reported the elevation of uridine phosphorylase (UPase) in many solid tumors and the presence of a variant phosphorolytic activity in breast cancer tissues (M. Liu et al., Cancer Res., 58: 5418-5424, 1998). To better understand the biological and pharmacological significance of these findings, we have developed an UPase gene knockout embryonic stem (ES) cell model by specific gene targeting techniques. In this cellular model, we establish the critical role of UPase as an important anabolic enzyme in 5-fluorouracil (5-FU) activation and pyrimidine salvage pathway regulation. It has long been known that UPase regulates the plasma concentration of uridine; however, little is known of the role of UPase in the activation and metabolism of 5-FU and its derivatives, mainly because of the lack of an appropriate model system. The experimental data indicate that the disruption of UPase activity in murine ES cells leads to a 10-fold increase in 5-FU IC(50) and a 2-3-fold reduction in its incorporation into nucleic acids, whereas no differences in toxicity is seen with other pyrimidine nucleoside analogues such as 5-fluorouridine, 2'-deoxy-5-fluorouridine, and 1-beta-D-arabinofuranosylcytosine compared with WT (wild-type) ES cells. Benzylacyclouridine can specifically prevent the WT ES cells from the sensitivity of 5-FU. Our data also shows the effect of UPase on the cytotoxicity of 5'-deoxy-5-fluorouridine (5'DFUR), a 5-FU prodrug. The IC(50) is increased almost 16-fold in the knockout cells compared with the wild type cells, demonstrating the role of UPase in catalyzing the conversion of 5'DFUR to 5-FU. These findings additionally elucidate the tumor-specific selectivity of capecitabine, the oral fluoropyrimidine prodrug approved for the treatment of metastatic breast and colorectal cancers. Not only do the knockout cells present a decreased incorporation of 5-FU into nucleic acids but also an increased reliance on the pyrimidine salvage pathway. The reduced dependence of UPase knockout cells on the pyrimidine de novo synthesis is reflected in the apparent resistance to phosphonacetyl-L-aspartic acid, a specific inhibitor of pyrimidine pathway, with a 5-fold elevation in its IC(50) in UPase-nullified cells compared with WT. In summary, we have successfully generated an UPase gene knockout cell model that presents reduced sensitivity to 5-FU, 5'DFUR, and phosphonacetyl-L-aspartic acid, although it does not affect the basic cellular physiology under normal tissue culture conditions. Considering the role of UPase in 5-FU metabolism and the elevated expression of this protein in cancer cells compared with paired normal tissues, additional investigation should be warranted to firmly establish the clinical role of UPase in the tumor selective activation of 5-FU and capecitabine.

Homeostatic control of uridine and the role of uridine phosphorylase: a biological and clinical update.

Uridine, a pyrimidine nucleoside essential for the synthesis of RNA and bio-membranes, is a crucial element in the regulation of normal physiological processes as well as pathological states. The biological effects of uridine have been associated with the regulation of the cardio-circulatory system, at the reproduction level, with both peripheral and central nervous system modulation and with the functionality of the respiratory system. Furthermore, uridine plays a role at the clinical level in modulating the cytotoxic effects of fluoropyrimidines in both normal and neoplastic tissues. The concentration of uridine in plasma and tissues is tightly regulated by cellular transport mechanisms and by the activity of uridine phosphorylase (UPase), responsible for the reversible phosphorolysis of uridine to uracil. We have recently completed several studies designed to define the mechanisms regulating UPase expression and better characterize the multiple biological effects of uridine. Immunohistochemical analysis and co-purification studies have revealed the association of UPase with the cytoskeleton and the cellular membrane. The characterization of the promoter region of UPase has indicated a direct regulation of its expression by the tumor suppressor gene p53. The evaluation of human surgical specimens has shown elevated UPase activity in tumor tissue compared to paired normal tissue.

Hospital water point-of-use filtration: a complementary strategy to reduce the risk of nosocomial infection.

Cholera, hepatitis and typhoid are well-recognized water-borne illnesses that take the lives of many every year in areas of uncontrollable flood, but far less attention is afforded to the allegedly safe potable water in affluent nations and the presumed healthful quality of water in communities and hospitals. Recent literature, however, points to increasing awareness of serious clinical sequelae particularly experienced by immunocompromised patients at high risk for disease and death from exposure to water-borne microbes in hospitals. This review reflects the literature indicting hospital water as an important source for nosocomial infections, examines patient populations at greatest risk, uncovers examples of failures in remedial water treatment methods and the reasons for them, and introduces point-of-use water filtration as a practical alternative or complementary component of an infection control strategy that may reduce the risk of nosocomial infections.

Contamination control in nursing with filtration. Part 1: filters applied to intravenous fluids and point-of-use hospital water.

Filters often are viewed as screens with openings smaller than the particles intended to be removed by a process technically known as direct interception. However, filter manufacturing embraces far more advanced technological approaches, with an evolution toward selective removal of cells or soluble constituents from complex physiologic solutions. An appreciation of filtration development makes it easy to understand how differently manufactured filters with the same claims may not perform identically. This article focuses on the filtration of intravenous solutions and point-of-use hospital water.

Contamination control in nursing with filtration: part 2: emerging rationale for bedside (final) filtration of prestorage leukocyte-reduced blood products.

The first part of this 2-part series focused on the manufacture of filters and the application of filtration technology to intravenous fluids and point-of-care hospital water. This second part describes an apparent emerging potential for final filtration defined as bedside filtration of blood and component blood products leukocyte-reduced at the blood center prior to storage. Final filtration serves to further reduce the leukocyte burden in a previously leukocyte-reduced blood product. Another target for final filtration includes putative soluble mediators of morbidity.Selected patients may be at greater risk for alloimmunization and refractory to the benefits afforded by transfusion of blood leukocyte reduced to the current established standards. Multiparous patients who subsequently find themselves in need of a transplanted organ are alloimmunized by exposure to fetal proteins and may be further alloimmunized by transfusion. Such effects can put them at risk for increased latency for donor organ availability and organ rejection. Kidney transplant patients find themselves the recipients of transfused blood products particularly during end-stage renal disease and recent data suggest such patients are not benefited by the levels of leukoreduction prescribed by current standards and may need more dramatic leukocyte removal. The process of blood production is described and affords a greater appreciation for the levels of white cells found in component blood products. The development of alloimmunization is reviewed and fosters greater appreciation for a discussion of the potential for therapeutic value of more dramatic leukocyte reduction and blood conditioning accomplished through the removal of soluble mediators of morbidity.

Detection of bacteria in WBC-reduced PLT concentrates using percent oxygen as a marker for bacteria growth.

BACKGROUND: The risk of receiving a PLT concentrate (PC) contaminated with bacteria may be 1000-fold greater than that of pathogenic viral transmission, yet surveillance for this risk is not generally practiced. A novel bacteria detection system (BDS) that overcomes the limitations of current systems is described. The BDS monitors percent oxygen (%O2) in air above aliquots of PCs that have been filtered to remove the confounding effect of respiring PLTs and residual WBCs. STUDY DESIGN AND METHODS: One-day-old WBC-reduced whole-blood-derived PCs (WBPCs) were inoculated with bacteria at 100 to 500 CFU per mL. After 30 minutes, 2- to 3-mL aliquots were processed through a PLT-reducing filter into a sample pouch containing sodium polyanethol sulfonate and entrained air. After incubation at 35 degrees C for at least 24 hours, the %O2 was measured within the pouch. Noninoculated WBC-reduced WBPCs (n = 155), confirmed free of bacteria by routine culture, were tested in a like manner. Results from the latter group of WBC-reduced WBPCs were used to distinguish contaminated from noncontaminated units. RESULTS: After a 24-hour incubation at 35 degrees C, 195 (96.5%) of the 202 sample pouches obtained from inoculated units were detected by the BDS. After an additional 6 hours at room temperature, those that remained and were tested were found positive. None of the noninoculated controls produced a positive reading. CONCLUSION: The BDS is easy to use and provides good levels of sensitivity and specificity.