Cytotoxicity and mechanical behavior of chitin-bentonite clay based polyurethane bio-nanocomposites

Chitin based polyurethane bio-nanocomposites (PUBNC) were prepared using chitin, Delite^® HPS bentonite nanoclay enriched in montmorillonite (MMT), 4,4′-diphenylmethane diisocyanate (MDI) and polycaprolactone polyol CAPA 231 (3000 g/mol⁻¹). The prepolymers having different concentration of Delite HPS bentonite nanoclay were extended with 2 moles of chitin. The structures of the resulted polymers were determined by FT-IR technique. The effect of nanoclay contents on mechanical properties and in vitro biocompatibility was investigated. The mechanical properties of the synthesized materials were improved with increase in the Delite HPS^® bentonite nanoclay contents. Optimum mechanical properties were obtained from the PU bio-nanocomposite samples having 4% Delite HPS^® bentonite nanoclay. The results revealed that the final PU bio-nanocomposite having 2% Delite HPS^® bentonite nanoclay contents is ideal contenders for surgical threads with on going investigations into their in vitro biocompatibility, non-toxicity, and mechanical properties.     

Effect of Mineral Fortification on Plasma Biochemical Profile in Rats

This study aimed at investigating the changes in biochemical profile of male rats following 8 weeks administration of different concentration of elemental iron, sodium iron ethylenediaminetetraacetate (NaFeEDTA), zinc sulfate (ZnSO₄), and zinc oxide (ZnO) in whole wheat flour. Eight groups comprising five rats each were fed fortified whole wheat flour in the form of baked pallets, while one group served as control. Concentration of total cholesterol, high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), triglycerides, total proteins, albumin, globulin, plasma glucose, and blood urea nitrogen were assayed. Supplementing mineral-fortified diet to male rats did not indicate any significant (p ≤ 0.05) effect on total cholesterol concentration. Diets containing NaFeEDTA alone increased HDL-C and decreased LDL-C; however, the differences remained non significant. Likewise, plasma triglycerides content of male rats remained unchanged on feeding fortified diets. Diets containing iron as NaFeEDTA and elemental iron exerted little effect on total protein concentration in the plasma of rats. Plasma glucose and blood urea nitrogen levels did not exhibit any significant change as a result of ingesting mineral supplemented diets. The study concludes that the forms of fortificants and the fortification levels used in the current study are undamaging for lipid profile, renal function, and glucose levels in rats, suggesting that these may be safely used in wheat flour to combat iron and zinc deficiency in vulnerable groups.     

Prolylcarboxypeptidase regulates proliferation, autophagy, and resistance to 4-hydroxytamoxifen-induced cytotoxicity in estrogen receptor-positive breast cancer cells

Endocrine therapy with tamoxifen (TAM) significantly improves outcomes for patients with estrogen receptor-positive breast cancer. However, intrinsic (de novo) or acquired resistance to TAM occurs in a significant proportion of treated patients. To identify genes involved in resistance to TAM, we introduced full-length cDNA expression library into estrogen receptor-positive MCF7 cells and exposed them to a cytotoxic dose of 4-hydroxytamoxifen (4OHTAM). Four different library inserts were isolated from surviving clones. Re-introduction of the genes individually into naive MCF7 cells made them resistant to 4OHTAM. Cells overexpressing these genes had an increase in acidic autophagic vacuoles induced by 4OHTAM, suggesting their role in autophagy. One of them, prolylcarboxypeptidase (PRCP), was investigated further. Overexpression of PRCP increased cell proliferation, boosted several established markers of autophagy, including expression of LC3-2, sequestration of monodansylcadaverine, and proteolysis of BSA in an ER-α dependent manner, and increased resistance to 4OHTAM. Conversely, knockdown of endogenous PRCP in MCF7 cells increased cell sensitivity to 4OHTAM and at the same time decreased cell proliferation and expression of LC3-2, sequestration of monodansylcadaverine, and proteolysis of BSA. Inhibition of enzymatic activity of PRCP enhanced 4OHTAM-induced cytotoxicity in MCF7 cells. Cells with acquired resistance to 4OHTAM exhibited increased PRCP activity, although inhibition of PRCP prevented development of 4OHTAM resistance in parental MCF7 cells and restored response to 4OHTAM in MCF7 cells with acquired resistance to 4OHTAM. Thus, we have for the first time identified PRCP as a resistance factor for 4OHTAM resistance in estrogen receptor-positive breast cancer cells.     

High molecular weight kininogen activates B2 receptor signaling pathway in human vascular endothelial cells

The nonenzymatic cofactor high molecular weight kininogen (HK) is a precursor of bradykinin (BK). The production of BK from HK by plasma kallikrein has been implicated in the pathogenesis of inflammation and vascular injury. However, the functional role of HK in the absence of prekallikrein (PK), the proenzyme of plasma kallikrein, on vascular endothelial cells is not fully defined. In addition, no clinical abnormality is seen in PK-deficient patients. Therefore, an investigation into the effect of HK, in the absence of PK, on human pulmonary artery endothelial cell (HPAEC) function was performed. HK caused a marked and dose-dependent increase in the intracellular calcium [Ca(2+)](i) level in HPAEC. Gd(3+) and verapamil potentiated the HK-induced increase in [Ca(2+)](i). HK-induced Ca(2+) increase stimulated endothelial nitric oxide (NO) and prostacyclin (PGI(2)) production. The inhibitors of B(2) receptor-dependent signaling pathway impaired HK-mediated signal transduction in HPAEC. HK had no effect on endothelial permeability at physiological concentration. This study demonstrated that HK regulates endothelial cell function. HK could play an important role in maintaining normal endothelial function and blood flow and serve as a cardioprotective peptide.     

The Equilibrative Nucleoside Transporter (ENT1) can be phosphorylated at multiple sites by PKC and PKA

Equilibrative Nucleoside Transporters (SLC29) are a family of proteins that transport nucleosides, nucleobases and nucleoside analogue drugs across cellular membranes. ENT1 is expressed ubiquitously in mammalian tissues and responsible for a significant portion of nucleoside analog drug uptake in humans. Despite the important clinical role of ENT1, many aspects of the regulation of this protein remain unknown. A major outstanding question in this field is the whether ENT1 is phosphorylated directly. To answer this question, we overexpressed tagged human (h) and mouse (m) ENT1, affinity purified protein using the tag, conducted phosphoamino acid analysis and found that m/hENT1 is predominantly phosphorylated at serine residues. The large intracellular loop of ENT1, between transmembrane domains 6 and 7, has been suggested to be a site of regulation by phosphorylation, therefore we generated His/Ubiquitin tagged peptides of this region and used them for in vitro kinase assays to identify target serines. Our data support a role for PKA and PKC in the phosphorylation of ENT1 within the intracellular loop and show that PKA can phosphorylate multiple sites within this loop while PKC specifically targets serines 279 and 286 and threonine 274. These data demonstrate, for the first time, that ENT1 is a phosphoprotein that can be directly phosphorylated at several sites by more than one kinase. The presence of multiple kinase targets within the loop suggests that ENT1 phosphorylation is considerably more complex than previously thought and thus ENT1 may be subject to phosphorylation by multiple pathways.     

Analysis of naïve and memory CD4 and CD8 T cell populations in breast cancer patients receiving a HER2/<Emphasis Type="Italic">neu</Emphasis> peptide (E75) and GM-CSF vaccine

We are conducting clinical trials of the E75 peptide as a vaccine in breast cancer (BrCa) patients. We assessed T cell subpopulations in BrCa patients before and after E75 vaccination and compared them to healthy controls. We obtained 17 samples of blood from ten healthy individuals and samples from 22 BrCa patients prior to vaccination. We also obtained pre- and post-vaccination samples of blood from seven BrCa patients who received the E75/GM-CSF vaccine. CD4, CD8, CD45RA, CD45RO, and CCR7 antibodies were used to analyze the CD4+ and CD8+ T cells by four-color flow cytometry. Compared to healthy individuals, BrCa patients have significantly more memory and less naïve T cells and more effector-memory CD8+ and less effector CD4+ T cells. Phenotypic differences in defined circulating CD4+ and CD8+ T cell subpopulations suggest remnants of an active immune response to tumor distinguished by a predominant memory T cell response and by untapped recruitment of naïve helper and cytotoxic T cells. E75 vaccination induced recruitment of both CD4+ and CD8+ naïve T cells while memory response remained stable. Additionally, vaccination induced global activation of all T cells, with specific enhancement of effector CD4+ T cells. E75 vaccination causes activation of both memory and naïve CD4+ and CD8+ T cells, while recruiting additional naïve CD4+ and CD8+ T cells to the overall immune response.     

Blind trials evaluating in vitro infectivity of Cryptosporidium oocysts using cell culture immunofluorescence

An optimized cell culture immunofluorescence (IFA) procedure, using the HCT-8 cell line, was evaluated in blind trials to determine the sensitivity and reproducibility of measuring the infectivity of flow-cytometry-prepared inocula of Cryptosporidium parvum oocysts. In separate trials, suspensions consisting of between 0% and 100% viable oocysts were prepared at the US Environmental Protection Agency, shipped to the American Water Laboratory, and analyzed blindly by cell culture IFA. Data indicated the control (100% live) oocyst suspensions yielded statistically similar results to cell culture dose-response curve data developed previously at the American Water Laboratory. For test samples containing oocyst suspensions of unknown infectivity, cell culture IFA analyses indicated a high degree of correlation (r2 = 0.89; n = 26) with the values expected by the US Environmental Protection Agency. Cell culture infectivity correlates well with neonatal mouse infectivity assays, and these blind validation trials provide credibility for the cell culture IFA procedure as a cost-effective and expedient alternative to mouse infectivity assays for determining in vitro infectivity of C. parvum oocysts.     

Radiosynthesis,radiochemical, and biological characterization of 177Lu-labeled diethylenetriamine penta-acetic acid

Diethylenetriamine penta-acetic acid (DTPA), when complexed with a gamma (γ)-emitter radioisotope like ^99mTc, is used for renal function diagnosis and many other diagnostic applications. The main aim of this study was to develop a novel and versatile single-step methodology for the synthesis of a new ¹⁷⁷Lu-labeled radiopharmaceutical with high radiochemical yield, which can be used for diagnostic purposes and therapeutic purposes also. The single and well-defined ¹⁷⁷Lu-DTPA complex was radiochemically characterized by paper chromatography, thin-layer chromatography, high-performance liquid chromatography, and electrophoresis techniques. Dependence of the labeling yield of ¹⁷⁷Lu-DTPA complex on different factors was studied in detail. Biological evaluation was also performed in a normal rabbit by developing images under a γ camera at various time intervals. More than 99% labeling yield was obtained by reacting DTPA with ¹⁷⁷Lu at specific conditions (pH 7.0, 15 minutes reaction time at 100 °C). ¹⁷⁷Lu-DTPA complex showed high stability both at room temperature and in vitro. Biodistribution studies in normal mice indicated the fractional renal uptake of intravenously administered ¹⁷⁷Lu-DTPA complex, which reached in the kidneys within 2-3 minutes. Scintigraphy showed rapid clearance from the body. Based on these results, we propose that ¹⁷⁷Lu-DTPA complex might be used as an ideal candidate for functional evaluation of kidneys and the urinary tract, especially when needed to be transported to long-range consumer sites, because of its suitable half-life.