ABSTRACT

Abstract. Transforming growth factor-type 13 has been identified as a constituent of bone matrix 261:5693-5695). We used both developing bone and bone-forming cells in vitro to demonstrate the cellular origin of this peptide. TGF-13 mRNA was detected by Northern analysis in both developing bone tissue and fetal bovine bone-forming cells using human cDNA probes. TGF-13 was shown to be synthesized and secreted by metabolically labeled bone cell cultures by immunoprecipitation from the medium. Further, TGF-13 activity was demonstrated in conditioned media from these cultures by competitive radioreceptor and growth promotion assays. Fetal bovine bone cells (FBBC) were found to have relatively few TGF-~ receptors (5,800/ce11) with an extremely low Kd of 2.2 pM (high binding affinity). In contrast to its inhibitory effects on the growth of many cell types including osteosarcoma cell lines, TGF-I3 stimulated the growth of subconfluent cultures of FBBC; it had little effect on the production of collagen by these cells. We conclude that bone-forming cells are a source for the TGF-I~ that is found in bone, and that these cells may be modulated by this factor in an autocrine fashion. TRANSFORMING growth factor-type 13 (TGF-13), ~ discovered and named for its ability to phenotypically transform nonneoplastic fibroblasts in vitro (30), is emerging as the prototype for a family of multifunctional regulatory peptides (38). Many cells, both nonneoplastic and neoplastic, synthesize TGF-13 and most of these cells have receptors for the peptide. The diverse effects of TGF-[~ on cell function have been further illuminated by the recent finding that TGF-[i is similar, if not identical, to cartilageinducing factor-A (36), a protein isolated from demineralized bone that induces the formation of cartilage proteoglycan and type II collagen from undifferentiated mesenchymal cells in vitro (35). Although platelets are the most concentrated source of TGF-13 in the body (2), the high yield of TGF-I~ from bone (14) (,,ol00-fold greater than from soft tissues such as placenta [10] and kidney [32]) suggests that bone has the greatest total amount of TGF-13. Since the level of serum-derived proteins in bone is high (e.g., ¢t2 HSglycoprotein, which is synthesized in the liver, can account for >2 % of the total bone matrix [40]), the origin of TGF-13 found in bone is not entirely clear, and some of it may simply be absorbed to the tissue from serum. Although recent studies of the production of growth factors by fetal rat calvaria have demonstrated secretion of TGF-13 into bone organ culture medium (5), this developing tissue contains both Abbreviations used in thispaper:FBBC, fetal bovine bone cells; TGF-I~, transforming growth factor-type 13; NRK, normal rat kidney fibroblasts. mineralized and nonmineralized regions, with ceils in nonmineralized regions exhibiting distinctly fibroblastic characteristics (i.e., production of types I and III collagen). Consequently, the cellular origin of TGF-I~ in this organ culture system also is unclear. In th...

Lipoplexes formed by the pEGFP-C3 plasmid DNA (pDNA) and lipid mixtures containing cationic gemini surfactant of the 1,2-bis(hexadecyl dimethyl ammonium) alkanes family referred to as C16CnC16, where n=2, 3, 5, or 12, and the zwitterionic helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) have been studied from a wide variety of physical, chemical, and biological standpoints. The study has been carried out using several experimental methods, such as zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), cryo-TEM, gene transfection, cell viability/cytotoxicity, and confocal fluorescence microscopy. As reported recently in a communication (J. Am. Chem. Soc. 2011, 133, 18014), the detailed physicochemical and biological studies confirm that, in the presence of the studied series lipid mixtures, plasmid DNA is compacted with a large number of its associated Na+ counterions. This in turn yields a much lower effective negative charge, qpDNA−, a value that has been experimentally obtained for each mixed lipid mixture. Consequently, the cationic lipid (CL) complexes prepared with pDNA and CL/DOPE mixtures to be used in gene transfection require significantly less amount of CL than the one estimated assuming a value of qDNA−=−2. This drives to a considerably lower cytotoxicity of the gene vector. Depending on the CL molar composition, α, of the lipid mixture, and the effective charge ratio of the lipoplex, ρeff, the reported SAXS data indicate the presence of two or three structures in the same lipoplex, one in the DOPE-rich region, other in the CL-rich region, and another one present at any CL composition. Cryo-TEMand SAXS studies with C16CnC16/DOPE-pDNA lipoplexes indicate that pDNA is localized between the mixed lipid bilayers of lamellar structures within a monolayer of ∼2 nm. This is consistent with a highly compacted supercoiled pDNA conformation compared with that of linear DNA. Transfection studies were carried out with HEK293T, HeLa, CHO, U343, and H460 cells. The α and ρeff values for each lipid mixture were optimized on HEK293T cells for transfection, and using these values, the remaining cells were also transfected in absence (-FBS-FBS) and presence (-FBS+FBS) of serum. The transfection efficiency was higher with the CLs of shorter gemini spacers (n=2 or 3). Each formulation expressed GFP on pDNA transfection and confocal fluorescence microscopy corroborated the results. C16C2C16/DOPE mixtures were the most efficient toward transfection among all the lipid mixtures and, in presence of serum, even better than the Lipofectamine2000, a commercial transfecting agent. Each lipid combination was safe and did not show any significant levels of toxicity. Probably, the presence of two coexisting lamellar structures in lipoplexes synergizes the transfection efficiency of the lipid mixtures which are plentiful in the lipoplexes formed by CLs with short spacer (n=2, 3) than those with the long spacer (n=5, 12).

The most important objective of the present study was to explain why cationic lipid (CL)-mediated delivery of plasmid DNA (pDNA) is better than that of linear DNA in gene therapy, a question that, until now, has remained unanswered. Herein for the first time we experimentally show that for different types of CLs, pDNA, in contrast to linear DNA, is compacted with a large amount of its counterions, yielding a lower effective negative charge. This feature has been confirmed through a number of physicochemical and biochemical investigations. This is significant for both in vitro and in vivo transfection studies. For an effective DNA transfection, the lower the amount of the CL, the lower is the cytotoxicity. The study also points out that it is absolutely necessary to consider both effective charge ratios between CL and pDNA and effective pDNA charges, which can be determined from physicochemical experiments.

Lipoplex-type nanoaggregates prepared from pEGFP-C3 plasmid DNA (pDNA) and mixed liposomes, with a gemini cationic lipid (CL) [1,2-bis(hexadecyl imidazolium) alkanes], referred as (C16Im)2Cn (where Cn is the alkane spacer length, n = 2, 3, 5, or 12, between the imidazolium heads) and DOPE zwitterionic lipid, have been analyzed by zeta potential, gel electrophoresis, SAXS, cryo-TEM, fluorescence anisotropy, transfection efficiency, fluorescence confocal microscopy, and cell viability/cytotoxicity experiments to establish a structure-biological activity relationship. The study, carried out at several mixed liposome compositions, α, and effective charge ratios, ρeff, of the lipoplex, demonstrates that the transfection of pDNA using CLs initially requires the determination of the effective charge of both. The electrochemical study confirms that CLs with a delocalizable positive charge in their headgroups yield an effective positive charge that is 90% of their expected nominal one, while pDNA is compacted yielding an effective negative charge which is only 10-25% than that of the linear DNA. SAXS diffractograms show that lipoplexes formed by CLs with shorter spacer (n = 2, 3, or 5) present three lamellar structures, two of them in coexistence, while those formed by CL with longest spacer (n = 12) present two additional inverted hexagonal structures. Cryo-TEM micrographs show nanoaggregates with two multilamellar structures, a cluster-type (at low α value) and a fingerprint-type, that coexist with the cluster-type at moderate α composition. The optimized transfection efficiency (TE) of pDNA, in HEK293T, HeLa, and H1299 cells was higher using lipoplexes containing gemini CLs with shorter spacers at low α value. Each lipid formulation did not show any significant levels of toxicity, the reported lipoplexes being adequate DNA vectors for gene therapy and considerably better than both Lipofectamine 2000 and CLs of the 1,2-bis(hexadecyl ammnoniun) alkane series, recently reported.

Purpose: Current methods of classification of astrocytoma based on histopathologic methods are often subjective and less accurate. Although patients with glioblastoma have grave prognosis, significant variability in patient outcome is observed. Therefore, the aim of this study was to identify glioblastoma diagnostic and prognostic markers through microarray analysis. Experimental Design: We carried out transcriptome analysis of 25 diffusely infiltrating astrocytoma samples [WHO grade IIödiffuse astrocytoma, grade IIIöanaplastic astrocytoma, and grade IVöglioblastoma (GBM)] using cDNA microarrays containing 18,981 genes. Several of the markers identified were also validated by real-time reverse transcription quantitative PCR and immunohistochemical analysis on an independent set of tumor samples (n = 100). Survival analysis was carried out for two markers on another independent set of retrospective cases (n = 51). Results: We identified several differentially regulated grade-specific genes. Independent validation by real-time reverse transcription quantitative PCR analysis found growth arrest and DNA-damage^inducible a (GADD45a) and follistatin-like 1 (FSTL1) to be up-regulated in most GBMs (both primary and secondary), whereas superoxide dismutase 2 and adipocyte enhancer binding protein 1 were up-regulated in the majority of primary GBM. Further, identification of the grade-specific expression of GADD45a and FSTL1 by immunohistochemical staining reinforced our findings. Analysis of retrospective GBM cases with known survival data revealed that cytoplasmic overexpression of GADD45a conferred better survival while the coexpression of FSTL1with p53 was associated with poor survival. Conclusions: Our study reveals that GADD45a and FSTLI are GBM-specific whereas superoxide dismutase 2 and adipocyte enhancer binding protein 1are primary GBM-specific diagnostic markers. Whereas GADD45a overexpression confers a favorable prognosis, FSTL1 overexpression is a hallmark of poor prognosis in GBM patients.

Two sets of novel multiheaded cationic amphiphiles bearing one, two, and three trimethylammonium headgroups (T1, T2, and T3) and pyridinium headgroups (P1, P2, and P3), have been synthesized and tested for antimicrobial activities against both Gram-positive and Gram-negative bacteria. The multicationic headgroups in these amphiphiles were attached covalently via scissile ester-type linkages. The results were compared with those for known surface-active, nonhydrolyzable compounds cetyltrimethylammonium bromide (CTAB) and cetylpyridinium bromide (CPB). The killing effects of the new single-headed amphiphiles (T1 and P1) were lower than those of CTAB and CPB, but with an increase in the number of headgroups in the amphiphiles, the killing effects increased for both sets of compounds. It was found that amphiphiles with triple headgroups (T3 and P3) were most active among all the amphiphiles, whereas amphiphile P1 had a very poor killing effect on both types of bacteria. The multiheaded pyridinium amphiphiles were more active compared to their trimethylammonium counterparts. The time needed to kill bacteria with multiheaded amphiphiles was significantly less than that of single-headed amphiphiles. Owing to the presence of a cleavable ester moiety, these new amphiphiles are hydrolyzed spontaneously at physiological conditions. This property enables them to be readily metabolized and therefore have the potential to be superior disinfectants and antiseptics for food and body surfaces.

Background: The aim of this study is to identify serum biomarkers with classification and prognosis utility for astrocytoma, in particular glioblastoma (GBM).Methods: Our previous glioma microarray database was mined to identify genes that encode secreted or membrane-localized proteins. Subsequent analysis was done using significant analysis of microarrays, followed by reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemical validation in tumor tissues, ELISA and Western blot validation in sera, and correlation with survival of GBM patients.Results: Significant analysis of microarrays identified 31 upregulated and 3 downregulated genes specifically in GBMs. RT-qPCR validation on an independent set of samples confirmed the GBM-specific differential expression of several genes, including three upregulated (CALU, CXCL9, and TIMP1) and two downregulated (GPX3 and TIMP3) novel genes. With respect to osteopontin (OPN), we show the GBM-specific upregulation by RT-qPCR and immunohistochemical staining of tumor tissues. Elevated serum OPN levels in GBM patients were also shown by ELISA and Western blot. GBM patients with high serum OPN levels had poorer survival than those with low serum OPN levels (median survival 9 versus 22 months respectively; P = 0.0001). Further, we also show high serum TIMP1 levels in GBM patients compared with grade II/III patients by ELISA and downregulation of serum GPX3 and TIMP3 proteins in GBMs compared with normal control by Western blot analysis.Conclusions: Several novel potential serum biomarkers of GBM are identified and validated. High serum OPN level is found as a poor prognostic indicator in GBMs.Impact: Identified serum biomarkers may have potential utility in astrocytoma classification and GBM prognosis. Cancer Epidemiol Biomarkers Prev; 19(6); 1409-22. ©2010 AACR.