Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor

Conventional immunosuppressive drugs have been used effectively to prevent immunologic rejection in organ transplantation. Individuals taking these drugs are at risk, however, for the development and recurrence of cancer. In the present study we show that the new immunosuppressive drug rapamycin (RAPA) may reduce the risk of cancer development while simultaneously providing effective immunosuppression. Experimentally, RAPA inhibited metastatic tumor growth and angiogenesis in in vivo mouse models. In addition, normal immunosuppressive doses of RAPA effectively controlled the growth of established tumors. In contrast, the most widely recognized immunosuppressive drug, cyclosporine, promoted tumor growth. From a mechanistic perspective, RAPA showed antiangiogenic activities linked to a decrease in production of vascular endothelial growth factor (VEGF) and to a markedly inhibited response of vascular endothelial cells to stimulation by VEGF. Thus, the use of RAPA, instead of cyclosporine, may reduce the chance of recurrent or de novo cancer in high-risk transplant patients.     

Bis(3-hydroxy-4-pyridinone)-EDTA derivative as a potential therapeutic Al-chelating agent. Synthesis, solution studies and biological assays

The 3-hydroxy-4-pyridinones are chelating agents of current interest due to their high affinity for hard metal ions and potential clinical applications as metal-decorporation agents. A new bis-(3-hydroxy-4-pyridinone) derivative of EDTA have been developed, and herein we describe the results of solution studies to determine the protonation constants and the partition coefficient. Biodistribution studies, performed with 67Ga-overload mice, showed rapid clearance of the radiotracer from the body, thus indicating that the new ligand should be a quite effective agent for the in vivo aluminium removal.     

Cyclic peptide models of the Ca2+-binding loop of alpha-lactalbumin

A series of cyclic peptides with different linkers were designed and synthesized to model the elbow-type Ca2+-binding loop of alpha-lactalbumin (LA). All amino acids of the Ca2+-binding loop are strikingly well conserved among LAs of different species with the sequence Lys79-Phe-Leu-Asp82-Asp-Asp-Leu-Thr- Asp87-Asp88, where three carboxylates of Asp82, Asp87, and Asp88 and the amide carbonyl oxygen atoms of Lys79 and Asp84 participate in Ca2+ binding. Alanine-containing models were also prepared for monitoring the role of the binding (82, 87-88) and nonbinding Asp residues (83-84) in coordinating the cation. The structural features of synthetic peptides and their Ca2+-binding properties were investigated in solution by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. In water, the CD curves show a strong negative band below 200 nm as a sign of the presence of unfolded conformers. In TFE, all cyclic peptides were found to have a CD spectrum, reflecting the presence of folded (turn) conformers. The effect of Ca2+ was dependent on the structure and concentration of the model and the Ca2+ to peptide ratio (r(cat)). A surprising time dependence of the FTIR spectra of Ca2+ complexes of the Ala-containing peptides was observed. The shape of the broad amide I band showed no more change after approximately 60 min. Contrary to this, the deprotonation of the side chain COOH group(s) and formation of the final coordination sphere of Ca2+ took more time. Infrared spectra showed that in the Ca2+ complex of model comprising the binding Asp residues of LA, the cation is coordinated to the COO- groups of all three Asps, while in the complex of model comprising nonbinding Asp residues of LA, the two neighboring Asp side chains form a bridged Ca2+-binding system.     

Cytologic diagnosis of intraocular lymphoma in vitreous aspirates

OBJECTIVE: To evaluate the cytologic findings of vitreous fluids with atypical, suspicious for malignancy or malignant lymphoid cells to assess cytologic parameters that may help in reaching the diagnosis of intraoclular lymphoma. STUDY DESIGN: Vitreous aspirates with a malignant, suspicious for malignancy or atypical lymphoid population were identified from the files of Barnes-Jewish Hospital during the previous 11 years. Cytologic preparations were reviewed. Pertinent clinical information was obtained from medical records. RESULTS: Thirteen vitreous aspirates from 12 patients were included. The chief complaints included floaters, blurred vision and decreased visual aculity. Bilateral ocular involvement was present in 8 (67%) patients. Three patients had a history of an extraocular lymphoid malignancy. All patients underwent pars plana vitrectomy and collection of the vitreous aspirate. Cytologic diagnoses included: malignant lymphoma (9 of 13), suspicious for malignant lymphoma (3 of 13) and atypical lymphoid population (1 of 13). Most samples had high cellularity (11 of 13) and necrosis (9 of 13). Abnormal lymphoid cells were large (2-4 times the size of a lymphocyte) and had a high nuclear/cytoplasmic ratio, prominent nucleoli, irregular nuclear contours and a fine to coarse chromatin pattern. All cases with malignant cytology had abundant abnormal lymphoid cells; inconclusive cases had few. Immunocytochemistry for CD20 and CD45RO was performed on 9 of 13 samples and was conclusive in 6 of 9. CONCLUSION: Cytologic analysis of vitreous aspirates can be useful in diagnosing intraocular involvement by malignant lymphoma. Sparse cellularity is the main factor leading to inconclusive diagnoses. Immunostaining can be useful in confirming the lymphoid nature of the malignant cells.     

Autophagy occurs upstream or parallel to the apoptosome during histolytic cell death

Histolysis refers to a widespread disintegration of tissues that is morphologically distinct from apoptosis and often associated with the stimulation of autophagy. Here, we establish that a component of the apoptosome, and pivotal regulator of apoptosis, is also required for histolytic cell death. Using in vivo and ex vivo assays, we demonstrate a global apoptogenic requirement for dark, the fly ortholog of Apaf1, and show that a required focus of dark(-) organismal lethality maps to the central nervous system. We further demonstrate that the Dark protein itself is a caspase substrate and find that alterations of this cleavage site produced the first hypermorphic point mutation within the Apaf1/Ced-4 gene family. In a model of ;autophagic cell death', dark was essential for histolysis but dispensable for characteristic features of the autophagic program, indicating that the induction of autophagy occurs upstream or parallel to histolytic cell death. These results demonstrate that stimulation of autophagy per se is not a ;killing event' and, at the same time, establish that common effector pathways, regulated by the apoptosome, can underlie morphologically distinct forms of programmed cell death.     

Quinolinyl- and phenantridinyl-acetamides as bradykinin B1 receptor antagonists

A new series of quinolinyl- and phenantridinyl-acetamides were synthesizer and evaluated against bradykinin B1 receptor. In vitro metabolic stability data were reported for the key compounds.The analgesic effect of compound 20 from the phenantridine series was proved in-vivo.     

Methylation by a Unique α-class N4-Cytosine Methyltransferase Is Required for DNA Transformation of Caldicellulosiruptor bescii DSM6725

Thermophilic microorganisms capable of using complex substrates offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. Members of the Gram-positive bacterial genus Caldicellulosiruptor are anaerobic thermophiles with optimum growth temperatures between 65°C and 78°C and are the most thermophilic cellulolytic organisms known. In fact, they efficiently use biomass non-pretreated as their sole carbon source and in successive rounds of application digest 70% of total switchgrass substrate. The ability to genetically manipulate these organisms is a prerequisite to engineering them for use in conversion of these complex substrates to products of interest as well as identifying gene products critical for their ability to utilize non-pretreated biomass. Here, we report the first example of DNA transformation of a member of this genus, C. bescii. We show that restriction of DNA is a major barrier to transformation (in this case apparently absolute) and that methylation with an endogenous unique α-class N4-Cytosine methyltransferase is required for transformation of DNA isolated from E. coli. The use of modified DNA leads to the development of an efficient and reproducible method for DNA transformation and the combined frequencies of transformation and recombination allow marker replacement between non-replicating plasmids and chromosomal genes providing the basis for rapid and efficient methods of genetic manipulation.     

FcRn overexpression in transgenic mice results in augmented APC activity and robust immune response with increased diversity of induced antibodies

Our previous studies have shown that overexpression of bovine FcRn (bFcRn) in transgenic (Tg) mice leads to an increase in the humoral immune response, characterized by larger numbers of Ag-specific B cells and other immune cells in secondary lymphoid organs and higher levels of circulating Ag-specific antibodies (Abs). To gain additional insights into the mechanisms underlying this increase in humoral immune response, we further characterized the bFcRn Tg mice. Our Western blot analysis showed strong expression of the bFcRn transgene in peritoneal macrophages and bone marrow derived dendritic cells; and a quantitative PCR analysis demonstrated that the expression ratios of the bFcRn to mFcRn were 2.6- and 10-fold in these cells, respectively. We also found that overexpression of bFcRn enhances the phagocytosis of Ag-IgG immune complexes (ICs) by both macrophages and dendritic cells and significantly improves Ag presentation by dendritic cells. Finally, we determined that immunized bFcRn mice produce a much greater diversity of Ag-specific IgM, whereas only the levels, but not the diversity, of IgG is increased by overexpression of bFcRn. We suggest that the increase in diversity of IgG in Tg mice is prevented by a selective bias towards immunodominant epitopes of ovalbumin, which was used in this study as a model antigen. These results are also in line with our previous reports describing a substantial increase in the levels of Ag-specific IgG in FcRn Tg mice immunized with Ags that are weakly immunogenic and, therefore, not affected by immunodominance.