Time-dependent subpopulation induction in heterogeneous tumors

Time-dependent induction of clonal heterogeneity in the neoplastic micro-environment is analysed within the context of a competitive ecology. A model that describes a constant source for clonal emergence was analysed by Michelsonet al. (1987) as an extension of a model proposed by Jansson and Revesz (1974). The extended model has been termed the JRE Model. This paper extends these analyses to time-dependent emergence rates which may represent induction in the presence of a cytotoxic agent. If the analysis is constrained to the tumor micro-environment, and if the emergent subpopulation is drug resistant, then the model may describe the induction and emergence of drug resistant subclones in a growing neoplasm. Asymptotic closed form solutions are derived for a class of emergence rate functions which decay asymptotically to a constant mutation rate. This underlying mutation rate may represent spontaneous mutation to the resistant phenotype, and has been analysed stochastically (Coldmanet al., 1985). The asymptotic solutions to the time-dependent model approach the steady state solution for the JRE Model which represents the dynamics observed in the presence of a constant, spontaneous mutation rate. The clinical and biological implications of these results are discussed. Research support provided in part by Hungarian National Foundation for Scientific Research Grant No. 6032/6319 and ACS Grant IN45-Z and ACS PDT 243B.     

Cis-analysis of a seed protein gene promoter: the conservative RY repeat CATGCATG within the legumin box is essential for tissue-specific expression of a legumin gene

A 2.4 kb fragment containing the 5'-flanking region and the 5'-noncoding sequence of the Vicia faba legumin gene LeB4 mediates high level seed-specific expression in transgenic tobacco plants. Deleted derivatives of this legumin upstream sequence were fused to the npt-II reporter gene to determine the tissue-specific activity of the chimeric constructs in stably transformed tobacco plants. The results indicate the presence of positive regulatory, enhancer-like cis elements within 566 bp of the upstream sequence. Most importantly, however, these elements are only fully functional in conjunction with the core motif CATGCATG of the legumin box around position -95, since destruction of the motif by a 6 bp deletion in an otherwise intact 2.4 kb upstream sequence drastically reduces expression in seeds. At the same time, low level expression in leaves is observed. The occurrence of similar CATGCATG consensus cis elements with alternating purine and pyrimidine base pairs in front of several other plant genes suggests a functional role of the motif in a wider range of plant promoters.     

Studies on epimerization-free methods for the preparation of aminosuccinyl peptides

Summary We have found that guanidine acetate catalyses the transformation of a -benzyl-aspartyl peptide (Boc-Asp-(OBzl)-Leu-Trp-OMe) to an aminosuccinyl peptide (Boc-Asu-Leu-Trp-OMe). The reaction was accompanied by partial epimerization. However, not even a small amount of epimerization could be detected when the aminosuccinyl peptide was synthesised from Boc-Asp-Leu-Trp-OMe with the addition of DIC, HOPfp and guanidine acetate (as a catalyst). This reaction seems to be suitable for the epimerization-free solid phase synthesis of aminosuccinyl peptides, e.g. Asu⁶-Lamprey-III-GnRH (Glp-His-Trp-Ser-His-Asu-Trp-Lys-Pro-Gly-NH₂).     

Physical mapping of cleavage sites recognized by restriction endonucleases on the genome of bacteriophage T5

The DNA of bacteriophage T5 has been treated with restriction endonucleases EcoRi, HindIII, BamI, SmaI, PstI, SalI, KpnI and the electrophoretic pattern obtained in agarose gel has been analyzed in order to localize the specific cleavage sites on the T5 DNA. The localization of cleavage sites has been deduced from the electrophoretic pattern of double and partial digests, the digests of isolated restriction fragments and the digests of deletion mutant T5st(o) DNA.Four BamI cleavage sites have been found and localized on the physical map of T5 DNA at 0.21, 0.225, 0.685 and 0.725 fractional length. Endonuclease SmaI cleaves at 0.39, 0.59 and 0.69 fractional length. Endonuclease PstI cuts T5 DNA at 11 sites: 0.090, 0.210, 0.320, 0.510, 0.635, 0.670, 0.705, 0.770, 0.815, 0.840, 0.875 fractional length. Six KpnI cleavage sites have been mapped at 0.170, 0.215, 0.525, 0.755, 0.830, 0.850 fractional length. A complete cleavage map of the phage genome is presented for seven restriction enzymes.     

Novel recombinant human lactoferrin: Differential activation of oxidative stress related gene expression

Lactoferrin, an iron-binding protein found in high concentrations in mammalian exocrine secretions, is an important component of the host defense system. It is also a major protein of the secondary granules of neutrophils from which is released upon activation. Due to its potential clinical utility, recombinant human lactoferrin (rhLF) has been produced in various eukaryotic expression systems; however, none of these are fully compatible with humans. Most of the biopharmaceuticals approved by the FDA for use in humans are produced in mammalian expression systems. The Chinese hamster ovary cells (CHO) have become the system of choice for proteins that require post-translational modifications, such as glycoproteins.     

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.     

Elucidation of the effects of lipoperoxidation on the mitochondrial electron transport chain using yeast mitochondria with manipulated fatty acid content

Lipoperoxidative damage to the respiratory chain proteins may account for disruption in mitochondrial electron transport chain (ETC) function and could lead to an augment in the production of reactive oxygen species (ROS). To test this hypothesis, we investigated the effects of lipoperoxidation on ETC function and cytochromes spectra of Saccharomyces cerevisiae mitochondria. We compared the effects of Fe²⁺ treatment on mitochondria isolated from yeast with native (lipoperoxidation-resistant) and modified (lipoperoxidation-sensitive) fatty acid composition. Augmented sensitivity to oxidative stress was observed in the complex III-complex IV segment of the ETC. Lipoperoxidation did not alter the cytochromes content. Under lipoperoxidative conditions, cytochrome c reduction by succinate was almost totally eliminated by superoxide dismutase and stigmatellin. Our results suggest that lipoperoxidation impairs electron transfer mainly at cytochrome b in complex III, which leads to increased resistance to antimycin A and ROS generation due to an electron leak at the level of the Q_O site of complex III.     

Direct amidation of poly(gamma-glutamic acid) with benzylamine in dimethyl sulfoxide

Partially benzylamidated, amphipathic poly(gamma-glutamic acid) (BzPGA) was synthesized from poly(gamma-glutamic acid) (PGA) and benzylamine by direct amidation in dimethyl sulfoxide (DMSO). Benzylamine and PGA were heated in DMSO for 1 to 26 h at temperatures between 110 and 130 degrees C, producing derivatives of various degrees of benzylamidation as a function of the reaction time and temperature. Neither any carboxyl-activating agent nor catalyst is needed for the reaction to proceed. After purification by dialysis, the product was identified by 1H and 13C 1D and 2D NMR in DMSO-d(6). BzPGA prepared by the new direct amidation method was identical to that obtained with a conventional carbodiimide-mediated reaction in water. The one-pot amidation procedure described in the present article can probably be applied to the synthesis of amides from other amines and carboxylic acids.