Cell surface glycoproteins of 13762NF mammary adenocarcinoma clones of differing metastatic potentials

Rat 13762NF mammary adenocarcinoma cell surface glycoproteins from s.c. tumor- or lung metastases-derived cell clones of differing spontaneous metastatic potentials were examined for their relationship to metastasis. After treatment with neuraminidase, lectin-binding assays showed that highly metastatic clone MTLn3 cells express approximately twice the quantity of peanut agglutinin (PNA) binding sites (approximately 2.3 X 10(8) sites/cell) than clones of lower metastatic potential. However, the number of wheat germ agglutinin (WGA)-binding sites on the various cell clones decreased slightly as the metastatic potential of the clones increased. The quantities of concanavalin A (conA)-binding sites were similar (approximately 1.7 X 10(8) sites/cell) in all cell clones and growth conditions. Glycoprotein analysis was performed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS-PAGE) and subsequent staining with 125I-labeled lectins. SDS-PAGE gels stained with 125I-labeled conA revealed mainly one glycoprotein (Mr approximately 150 kD), and the amounts of this glycoprotein did not correlate with metastasis. Differences in WGA-binding glycoproteins were detected between s.c. tumor- and lung metastases-derived cell clones. Several desialylated glycoproteins were detected with 125I-labeled PNA after SDS-PAGE, and the labeling intensity of one (Mr approximately 580 kD) correlated with the metastatic potentials of the various cell clones. This high Mr galactoprotein was further analyzed by [3H]glucosamine metabolic labeling, solubilization, sequential gel filtration, and chondroitinase ABC treatment prior to SDS-PAGE. The 580 kD galactoprotein was expressed in increased amounts on the more highly metastatic clones. Chemical labeling of cell surface sialic acid residues using periodate treatment followed by [3H]borohydride reduction showed an additional change in a major sialoglycoprotein (Mr approximately 80 kD), which decreased in labeling intensity on clones of increasing metastatic potential. The results suggest quantitative changes in cell surface glycoproteins rather than major qualitative alterations are associated with differences in the metastatic behavior of 13762NF tumor cell clones.     

Composition of the Sulfur Particle of Chromatium vinosum Strain D

Sulfur particles were isolated from the purple sulfur photosynthetic bacterium, Chromatium vinosum strain D. The composition of these particles was determined to be 93% sulfur, 5% protein, and 0.6% lipid. Gel electrophoresis indicated the presence of a single protein species with a molecular weight of 13,500 daltons. From these results, the sulfur particle is postulated to be bounded by a membrane consisting entirely of protein.     

Mechanism of resistance to ricin toxin in selected mouse lymphoma cell lines

The role of impaired toxin uptake in conferring cellular resistance to the plant toxin RCA_II (ricin) has been examined using a murine BW5 147 lymphoma line and a toxin-resistant variant (BW5 147 Ric^R.3) selected by repeated exposure to RCA_II. The toxin-resistant variant is 250 times more resistant to RCA_II in long-term growth experiments and 1,000 times more resistant in short-term protein synthesis assays. Experiments with ferritin-conjugated ¹²⁵I-labeled RCA_II (ferritin-¹²⁵I-RCA_II) indicated that toxin binding to sensitive and resistant cells is similar at low toxin concentrations where maximum differential cytotoxicity occurs but that major difference exist with respect to toxin uptake. In sensitive cells toxin is internalized via endocytosis, and as seen previously in other systems subsequent rupture of some of the toxin-containing endocytotic vesicles releases toxin into the cytoplasm, where it inhibits protein synthesis. The process of toxin transfer to the cytoplasm is presumed to account for the one-hour lag before toxin-induced inhibition of protein synthesis can be detected. Endocytotic uptake of toxin is impaired in resistant BW5147Ric^R.3 cells, and they are unaffected by toxin concentrations that inhibit protein synthesis and kill sensitive parental cells. Killing of resistant cells at low toxin concentrations was accomplished by encapsulating RCA_II into lipid vesicles capable of fusing with the plasma membrane. Direct introduction of toxin into resistant cells using lipid vesicles as carriers produced rapid inhibition (< 15 min) of protein synthesis and eliminated the lag in toxin action seen in sensitive cells exposed to free toxin. These findings are discussed in relation to the mechanism of toxin action and proposals that toxin activity requires structural modification of the toxin molecule at the cell surface before transport into the cell.     

Computational modeling of novel inhibitors targeting the Akt pleckstrin homology domain

Computational modeling continues to play an important role in novel therapeutics discovery and development. In this study, we have investigated the use of in silico approaches to develop inhibitors of the pleckstrin homology (PH) domain of AKT (protein kinase B). Various docking/scoring schemes have been evaluated, and the best combination was selected to study the system. Using this strategy, two hits were identified and their binding behaviors were investigated. Robust and predictive QSAR models were built using the k nearest neighbor (kNN) method to study their cellular permeability. Based on our in silico results, long flexible aliphatic tails were proposed to improve the Caco-2 penetration without affecting the binding mode. The modifications enhanced the AKT inhibitory activity of the compounds in cell-based assays, and increased their activity as in vivo antitumor testing.     

A re-evaluation of the role of type IV antifreeze protein

A lipoprotein-like antifreeze protein (type IV AFP) has previously been isolated only from the blood plasma of the longhorn sculpin. However, the plasma antifreeze activity in all individuals of this species tested from Newfoundland and New Brunswick waters ranges from low to undetectable. A close relative of the longhorn sculpin, the shorthorn sculpin, does have appreciable antifreeze activity in its blood but this is virtually all accounted for by the α-helical, alanine-rich type I AFP, other isoforms of which are also present in the skin of both fishes. We have characterized a putative ortholog of type IV AFP in shorthorn sculpin by cDNA cloning. This 12.2-kDa Gln-rich protein is 87% identical to the longhorn sculpin’s type IV AFP. Recombinant versions of both orthologs were produced in bacteria and shown to have antifreeze activity. Immunoblotting with antibodies raised to type IV AFP shows this protein present in longhorn sculpin plasma at levels of less than 100 μg/mL, which are far too low to protect the blood from freezing at the temperature of icy seawater. This confirms the results of direct antifreeze assays on the plasmas. It appears that type IV AFP has the potential to develop as a functional antifreeze in these fishes but may not have been selected for this role because of the presence of type I AFP. Consistent with this hypothesis is the observation that the type IV AFP gene has not been amplified the way functional antifreeze protein genes have in all other species examined.     

Phosphatidylinositol 3-kinase mediates bronchioalveolar stem cell expansion in mouse models of oncogenic K-ras-induced lung cancer

Background

Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death in Western countries. Developing more effective NSCLC therapeutics will require the elucidation of the genetic and biochemical bases for this disease. Bronchioalveolar stem cells (BASCs) are a putative cancer stem cell population in mouse models of oncogenic K-ras-induced lung adenocarcinoma, an histologic subtype of NSCLC. The signals activated by oncogenic K-ras that mediate BASC expansion have not been fully defined.

Methodology/Principal Findings

We used genetic and pharmacologic approaches to modulate the activity of phosphatidylinositol 3-kinase (PI3K), a key mediator of oncogenic K-ras, in two genetic mouse models of lung adenocarcinoma. Oncogenic K-ras-induced BASC accumulation and tumor growth were blocked by treatment with a small molecule PI3K inhibitor and enhanced by inactivation of phosphatase and tensin homologue deleted from chromosome 10, a negative regulator of PI3K.

Conclusions/Significance

We conclude that PI3K is a critical regulator of BASC expansion, supporting treatment strategies to target PI3K in NSCLC patients.     

No evidence of an effect of alterations in dietary fatty acids on fasting adiponectin over 3 weeks

Objective: Little is known about the effects of alterations in fatty acid classes on adiponectin, a hormone secreted by the adipocyte known to be important in the development of diabetes and cardiovascular disease (CVD). Any factor, including diet, that may positively influence adiponectin gene expression or increase circulating levels might be useful for improving such metabolic abnormalities. We investigated the effects of alterations in dietary fatty acid saturation on fasting serum adiponectin and associated peptides. Methods and Procedures: Double‐blind, randomized, crossover, 2 × 3‐week residential intervention trial where 18 mildly hyperlipidemic adult men were provided with a high saturated:unsaturated fat (SFA:USFA) and lower SFA:USFA treatment separated by an uncontrolled 4‐week washout. Only fatty acid profile was altered between treatments. Fasting blood samples were collected on days 0, 1, 7, 14, 21, 22 of each intervention period for the measurement of adiponectin, tumor necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), high‐sensitivity C–reactive protein (hsC‐RP), leptin, and ghrelin. Results: Body weight was kept constant (±1 kg) throughout each treatment. There was no detectable difference in fasting adiponectin at baseline (mean day 0 + day 1) between the treatment groups (mean ± s.d.; high_SFA:USFA = 7.0 ± 1.7 vs. low_SFA:USFA = 6.7 ± 1.4 μg/ml, P > 0.05). There were neither significant between‐treatment effects of fatty acid saturation (diet × time, P > 0.05) on serum adiponectin nor any significant between‐treatment effects on serum TNF‐α, IL‐6, hsC‐RP, leptin, or ghrelin (P > 0.05). Discussion: Fasting serum adiponectin was not detectably affected by alterations in dietary fatty acid profile in mildly hyperlipidemic men. There was no evidence that an increase in SFA content of the diet significantly worsened fasting serum adiponectin over a 3‐week intervention period.     

Strain-specific virulence phenotypes of Streptococcus pneumoniae assessed using the Chinchilla laniger model of otitis media

Background

Streptococcus pneumoniae [Sp] infection is associated with local and systemic disease. Our current understanding of the differential contributions of genetic strain variation, serotype, and host response to disease phenotype is incomplete. Using the chinchilla model of otitis media [OM] we investigated the disease phenotype generated by the laboratory strain TIGR4 and each of thirteen clinical strains (BS68-75, BS290, BS291, BS293, BS436 and BS437); eleven of the thirteen strains have been genomically sequenced.

Methodology/Principal Findings

For each strain 100 colony forming units were injected bilaterally into the tympanic bullae of 6 young adult chinchillas under general anesthesia. All animals were examined daily for local and systemic disease by a blinded observer. Pneumatic otoscopy was used to evaluate local disease, and behavioral assessments served as the measure of systemic disease. Virulence scoring was performed using a 4-point scale to assess four clinical parameters [severity and rapidity of local disease onset; and severity and rapidity of systemic disease onset] during a 10-day evaluation period. Highly significant variation was observed among the strains in their ability to cause disease and moribundity.

Conclusions/Significance

As expected, there was a significant correlation between the rapidity of systemic disease onset and severity of systemic disease; however, there was little correlation between the severity of otoscopic changes and severity of systemic disease. Importantly, it was observed that different strains of the same serotype produced as broad an array of disease phenotypes as did strains of different serotypes. We attribute these phenotypic differences among the strains to the high degree of genomic plasticity that we have previously documented.