Targeted gadolinium-loaded dendrimer nanoparticles for tumor-specific magnetic resonance contrast enhancement

A target-specific MRI contrast agent for tumor cells expressing high affinity folate receptor was synthesized using generation five (G5) ofpolyamidoamine (PAMAM) dendrimer. Surface modified dendrimer was functionalized for targeting with folic acid (FA) and the remaining terminal primary amines of the dendrimer were conjugated with the bifunctional NCS-DOTA chelator that forms stable complexes with gadolinium (Gd III). Dendrimer-DOTA conjugates were then complexed with GdCl3 followed by ICP-OES as well as MRI measurement of their longitudinal relaxivity (T1 s(-1) mM(-1)) of water. In xenograft tumors established in immunodeficient (SCID) mice with KB human epithelial cancer cells expressing folate receptor (FAR), the 3D MRI results showed specific and statistically significant signal enhancement in tumors generated with targeted Gd(III)-DOTA-G5-FA compared with signal generated by non-targeted Gd(III)-DOTA-G5 contrast nanoparticle. The targeted dendrimer contrast nanoparticles infiltrated tumor and were retained in tumor cells up to 48 hours post-injection of targeted contrast nanoparticle. The presence of folic acid on the dendrimer resulted in specific delivery of the nanoparticle to tissues and xenograft tumor cells expressing folate receptor in vivo. We present the specificity of the dendrimer nanoparticles for targeted cancer imaging with the prolonged clearance time compared with the current clinically approved gadodiamide (Omniscan) contrast agent. Potential application of this approach may include determination of the folate receptor status of tumors and monitoring of drug therapy.     

b-D-glucosidase activity in pelargonium and poinsettia leaves infected by <Emphasis Type="Italic">Botrytis cinerea</Emphasis> Pers. ex Fr.

The involvement of β-D-glucosidase activity in grey mould was studied in two ornamental plant species attacked by Botrytis cinerea. β-D-glucosidase activity in the susceptible pelargonium cultivar ‘Shiva’ gradually increased with the disease development in the leaf spots and their surroundings. The endogenic level of the studied hydrolase in the resistant pelargonium ‘Cascade’ was several times higher than in the susceptible cultivar ‘Shiva’ and in principle underwent no changes after inoculation. The postinfection increase in the activity of β-D-glucosidase noted in the leaves of the susceptible poinsettia cultivar ‘Malibu Red’ was evidently weaker in the intensity, but its tendencies were similar to those of the susceptible pelargonium cultivar. In the leaves of the medium-resistant poinsettia ‘Coco White’ the constitutional level of β-D-glucosidase was 2-3-fold higher in that cultivar than in the susceptible cv. ‘Malibu Red’. In attacked leaves of ‘Coco White’, the enzyme activity continued to increase temporarily until the 3^rd h after inoculation. The process of healthy leaf senescence in both species had no significant influence on the change of the studied enzyme activity which was generally low. A high activity of β-D-glucosidase was also observed in the homogenate prepared from mycelium and in the fungal spores.     

Diagnostic and therapeutic guidelines for gastrointestinal neuroendocrine tumors (recommended by the Polish Network of Neuroendocrine Tumors)

We have recently observed an increased interest in gastro-entero-pancreatic neuroendocrine tumors (GEP NET). They are rare cancer types and therefore collaborative effort of specialists in various disciplines of medicine is necessary to work out the diagnostic and therapeutic guidelines. In this publication we present general guidelines of the Polish Network of Neuroendocrine Tumors for the management of patients with GEP NET, developed at the Round Table Conference which took place in Kliczków near Wroc?aw in November 2007. In the subsequent parts of this publication, we present the rules of diagnostic and therapeutic management of: - endocrine tumors of the stomach and duodenum (including gastrinoma); - pancreatic endocrine tumors; - neuroendocrine tumors of the small intestine and the appendix; - neuroendocrine tumors of the colon. We hope that the proposed guidelines by Polish and foreign experts representing various disciplines of medicine, including endocrinology, gastroenterology, surgery, oncology, nuclear medicine and pathomorphology, will become a useful tool in the diagnostics and treatment of these patients.     

Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response

Traditional production of therapeutic glycoproteins relies on mammalian cell culture technology. Glycoproteins produced by mammalian cells invariably display N-glycan heterogeneity resulting in a mixture of glycoforms the composition of which varies from production batch to production batch. However, extent and type of N-glycosylation has a profound impact on the therapeutic properties of many commercially relevant therapeutic proteins making control of N-glycosylation an emerging field of high importance. We have employed a combinatorial library approach to generate glycoengineered Pichia pastoris strains capable of displaying defined human-like N-linked glycans at high uniformity. The availability of these strains allows us to elucidate the relationship between specific N-linked glycans and the function of glycoproteins. The aim of this study was to utilize this novel technology platform and produce two human-like N-linked glycoforms of recombinant human lactoferrin (rhLF), sialylated and non-sialylated, and to evaluate the effects of terminal N-glycan structures on in vitro secondary humoral immune responses. Lactoferrin is considered an important first line defense protein involved in protection against various microbial infections. Here, it is established that glycoengineered P. pastoris strains are bioprocess compatible. Analytical protein and glycan data are presented to demonstrate the capability of glycoengineered P. pastoris to produce fully humanized, active and immunologically compatible rhLF. In addition, the biological activity of the rhLF glycoforms produced was tested in vitro revealing the importance of N-acetylneuraminic (sialic) acid as a terminal sugar in propagation of proper immune responses.     

Attenuation of acridine mutagen ICR-191--DNA interactions and DNA damage by the mutagen interceptor chlorophyllin

We have investigated the ability of chlorophyllin (CHL) to interact with acridine mutagen ICR-191 (2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine) and also its ability to decrease binding of ICR-191 to DNA in a simple three-component competition system: CHL-ICR–DNA. Our data indicate a strong association of ICR-191 with CHL, stronger even than the association of ICR-191 with DNA. Calculations based on the measured affinity data show that a two- to three-fold excess of CHL reduces by about two-fold the concentration of the mutagen-DNA complex. We also exposed human leukemic HL-60 cells to ICR-191 in the absence and presence of CHL and measured the mutagen-induced DNA damage. The extent of DNA damage was assessed by analysis of histone H2AX phosphorylation. While ICR-191 induced significant increase in expression of phosphorylated H2AX (γH2AX), particularly in DNA replicating cells, this increase was totally abolished in the cells treated with ICR-191 in the presence of CHL.     

Genetic variation in CYP17 and endometrial cancer risk

Genetic variation in CYP17 is suspected to be related to endometrial cancer risk based on its role in the regulation of steroid and non-steroid hormone biosynthesis. Reported associations between CYP17 and higher levels of estradiol in some studies suggest that the C allele of a T-to-C single nucleotide polymorphism (SNP) in the 5′UTR of CYP17 (rs743572) may be associated with an increased risk of hormone-related cancers. However, five relatively small epidemiologic studies of endometrial cancer have reported that women with the rs743572 C allele have a decreased risk of endometrial cancer. To examine this association, we genotyped rs743572 and eight other haplotype-tagging SNPs (htSNPs), which are estimated to capture >80% of the variation in CYP17 in a population-based study of 497 endometrial cancer cases and 1,024 controls in Poland. Significant associations were not found for rs743572 (per C allele: OR = 1.12, 95%CI 0.96–1.30; P-trend = 0.15), for individual htSNPs, or for extended haplotypes (global P-value = 0.60). When we pooled data from previously published studies with our own (a total of 1,004 endometrial cases and 1,907 controls), we observed significant study heterogeneity in summary estimates of the association between rs743572 and endometrial cancer, as well as evidence of publication bias. In conclusion, our data are not consistent with a decreased endometrial cancer risk associated with rs743572, as previously reported, or with other haplotype-tagging polymorphisms. Further evaluation in consortia is necessary to confirm potential weak associations between common variation in CYP17 and endometrial cancer risk and to address the concern of publication bias. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Domain organization and functional analysis of type IIS restriction endonuclease Eco31I

Type IIS restriction endonuclease Eco31I harbors a single HNH active site and cleaves both DNA strands close to its recognition sequence, 5'-GGTCTC(1/5). A two-domain organization of Eco31I was determined by limited proteolysis. Analysis of proteolytic fragments revealed that the N-terminal domain of Eco31I is responsible for the specific DNA binding, while the C-terminal domain contains the HNH nuclease-like active site. Gel-shift and gel-filtration experiments revealed that a monomer of the N-terminal domain of Eco31I is able to bind a single copy of cognate DNA. However, in contrast to other studied type IIS enzymes, the isolated catalytic domain of Eco31I was inactive. Steady-state and transient kinetic analysis of Eco31I reactions was inconsistent with dimerization of Eco31I on DNA. Thus, we propose that Eco31I interacts with individual copies of its recognition sequence in its monomeric form and presumably remains a monomer as it cleaves both strands of double-stranded DNA. The domain organization and reaction mechanism established for Eco31I should be common for a group of evolutionary related type IIS restriction endonucleases Alw26I, BsaI, BsmAI, BsmBI and Esp3I that recognize DNA sequences bearing the common pentanucleotide 5'-GTCTC.     

Laccase immobilization on the tailored cellulose-based Granocel carriers

Extracellular laccase produced by Cerrena unicolor was immobilized by adsorption or covalent bonds formation on the cellulose-based carrier Granocel. Immobilization was optimized by changing the anchor groups and the methods of activation/immobilization. On the base of measured activity and stability of immobilized preparations, the covalent method was selected. It was shown that coupling of the enzyme to the carrier via divinyl sulfone or glutaraldehyde yielded an enzyme-carrier preparation of high activity and storage stability. Further optimization of the carrier's superstructure consisted in changing pore diameters and amount of functional groups on the carriers surface. Three-fold higher activity was noted when the enzyme was immobilized on NH2-modified Granocel with the highest size exclusion limit and amino group content. Relatively low products sorption was observed on the carrier surface. The effects of protein concentration and pH-value of the coupling mixture on immobilization efficiency were evaluated also.