Development and utilization of a somatic cell hybrid mapping panel to assign NotI linking probes to the long arm of human chromosome 6

A somatic cell hybrid mapping panel that defines seven regions of the long arm and one region of the short arm of human chromosome 6 has been developed. Utilizing this panel, 17 NotI boundary clones from a NotI linking library were regionally assigned to the long arm of chromosome 6. The majority of these clones (11) were found to localize within band regions 6q24-q27. The nonuniform distribution of NotI sites may indicate a cluster of HTF islands and likely represents a coincidence of coding sequences in this region of chromosome 6. Cross-hybridization of these linking clones to DNA from other species (zoo blots) provides further evidence for transcribed sequences in 7 of the NotI clones. These NotI clones were also used to identify corresponding NotI fragments using pulsed-field gel electrophoresis, facilitating further physical mapping of this region. Finally, regional assignment of five polymorphic probes to the long arm of chromosome 6 is also presented. These hybrids and probes should facilitate the construction of a physical and genetic linkage map to assist in the identification of disease loci along chromosome 6.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30194/1/0000582.pd

Characterization of biomolecular nanoconjugates by high-throughput delivery and spectroscopic difference

Nanoparticle conjugates have the potential for delivering siRNA, splice-shifting oligomers or nucleic acid vaccines, and can be applicable to anticancer therapeutics. This article compares tripartite conjugates with gold nanoparticles or synthetic methoxypoly(ethylene glycol)-block-polyamidoamine dendrimers

Cytotoxicity Effects of Different Surfactant Molecules Conjugated to Carbon Nanotubes on Human Astrocytoma Cells

Phase contrast and epifluorescence microscopy were utilized to monitor morphological changes in human astrocytoma cells during a time-course exposure to single-walled carbon nanotube (SWCNT) conjugates with different surfactants and to investigate sub-cellular distribution of the nanotube conjugates, respectively. Experimental results demonstrate that cytotoxicity of the nanotube/surfactant conjugates is related to the toxicity of surfactant molecules attached on the nanotube surfaces. Both sodium dodecyl sulfate (SDS) and sodium dodecylbenzene sulfonate (SDBS) are toxic to cells. Exposure to CNT/SDS conjugates (0.5 mg/mL) for less than 5 min caused changes in cell morphology resulting in a distinctly spherical shape compared to untreated cells. In contrast, sodium cholate (SC) and CNT/SC did not affect cell morphology, proliferation, or growth. These data indicate that SC is an environmentally friendly surfactant for the purification and dispersion of SWCNTs. Epifluorescence microscopy analysis of CNT/DNA conjugates revealed distribution in the cytoplasm of cells and did not show adverse effects on cell morphology, proliferation, or viability during a 72-h incubation. These observations suggest that the SWCNTs could be used as non-viral vectors for diagnostic and therapeutic molecules across the blood–brain barrier to the brain and the central nervous system

Alterations in integrin expression, distribution and regulation during carcinogenesis.

Metastatic cancer is the most difficult aspect in the treatment of cancer. Cancer cells that have metastasized show altered adhesion compared to normal cells of the same origin. Altered cell adhesion of cancer cells is central to the process of invasion and metastasis. A family of cell adhesion molecules termed integrins have been shown to be the cell adhesion molecules that are altered on cancer cells. These alterations and the mechanisms responsible for altering integrin expression, function and distribution during tumor progression currently are not fully understood. Altered integrin expression was found to affect the tumorigenicity of human prostate carcinoma cells. The integrin profiles on four human prostate cell lines were analyzed using fluorescence activated cell sorting and immunoprecipitation of surface labeled proteins. The tumorigenicity was tested by injection into SCID mice. Different integrin profiles affected tumor formation differently. Increased expression of α5β1 and α6β4 decreased invasion. Altered non-functioning receptors also contributed to the lack of tumorigenicity. These data show that alterations in integrin expression affect the tumorigenic potential of human prostate cells. A mouse model of skin carcinogenesis comprising normal, benign and malignant stages from a common lineage, was analyzed to determine at which stage integrin expression was altered. The malignant carcinoma stage demonstrated a decrease in the surface expression of integrin α6β4 and loss of the normal basal lateral distribution of this integrin. The changes in the α6β4 on the malignant carcinoma cells parallels integrin expression results in situ. This in vitro cell model system provided an opportunity to study the mechanisms of aberrant integrin expression. The results show the β4 subunit expression in this model is altered by down regulation of the mRNA and posttranslational cleavage resulting ultimately in decreased surface expression. The β4 in the malignant cells appears to be susceptible to degradation due to loss of protein-protein interactions involving the 100 kD β4 cytoplasmic domain. These protein interactions localize the α6β4 to the basal surface. Loss of these interactions results in non-polar distribution and potential susceptibility of the α6β4 to proteolytic degradation. These mechanisms contribute to altered surface expression on cancer cells

Cytotoxicity of Single-Walled Carbon Nanotubes Suspended in Various Surfactants

The cytotoxicity of single-walled carbon nanotubes (SWCNTs) suspended in various surfactants was investigated by phase contrast light microscopy characterization in combination with an absorbance spectroscopy cytotoxicity analysis. Our data indicate that individual SWCNTs suspended in the surfactants, sodium dodecyl sulfate (SDS) and sodium dodecylbenzene sulfonate (SDBS), were toxic to 1321N1 human astrocytoma cells due to the toxicity of SDS and SDBS on the nanotube surfaces. This toxicity was observed when cells were exposed to an SDS or SDBS solution having a concentration as low as 0.05 mg ml−1 for 30 min. The proliferation and viability of the cells were not affected by SWCNTs alone or by conjugates of SWCNTs with various concentrations of sodium cholate (SC) or single-stranded DNA. The cells proliferated similarly to untreated cells when surrounded by SWCNTs as they grow, which indicated that the nanotubes did not affect cells adversely. The cytotoxicity of the nanotube–surfactant conjugates was controlled in these experiments by the toxicity of the surfactants. Consequently, when evaluating a surfactant to be used for the dispersion of nanoscale materials in applications such as nanoscale electronics or non-viral biomolecular transporters, the cytotoxicity needs to be evaluated. The methodology proposed in this study can be used to investigate the cytotoxicity of other nanoscale materials suspended in a variety of surfactants

Characterization of integrin subunits, cellular adhesion and tumorgenicity of four human prostate cell lines

Cellular adhesion to extracellular matrix proteins via integrin molecules is a major factor in the process of invasion and metastasis of human tumor cells. Four human prostate cell lines were characterized according to the presence and quantity of integrin subunits, the ability of the cells to attach to extracellular substrates and the capacity of the cells to form tumors in severe combined immunodeficient (SCID) mice. All four human prostate cell lines expressed three to five integrins on their cell surfaces. The DU145, PC3 and 431P cells expressed primarily α3, α5, and α6 integrin at similar levels. These cell lines expressed the subunits β1, β3 and β4 with β1 predominant. The DU145 cells preferred attachment to fibronectin, followed by laminin and vitronectin. Approximately 50%-60% of the binding of DU145 cells to fibronectin and laminin was dependent on the function of α5β1 and α6 respectively. The cell line LNCaP differed in its low expression of the α3 subunit, 95% of cellular adhesion to fobronectin and laminin being integrin-dependent and its inability to attach to vitronectin, in spite of surface expression of αvβ3. All the cell lines except for LNCaP readily formed tumors within SCID mice and the expression of α3, α6, β1, and β4 integrin subunits was preserved in the resulting tumor tissue. The altered adhesion properties of the LNCaP cells may explain their altered tumorigenicity