Behavioral and Neuroanatomical Abnormalities in Pleiotrophin Knockout Mice

Pleiotrophin (PTN) is an extracellular matrix-associated protein with neurotrophic and neuroprotective effects that is involved in a variety of neurodevelopmental processes. Data regarding the cognitive-behavioral and neuroanatomical phenotype of pleiotrophin knockout (KO) mice is limited. The purpose of this study was to more fully characterize this phenotype, with emphasis on the domains of learning and memory, cognitive-behavioral flexibility, exploratory behavior and anxiety, social behavior, and the neuronal and vascular microstructure of the lateral entorhinal cortex (EC). PTN KOs exhibited cognitive rigidity, heightened anxiety, behavioral reticence in novel contexts and novel social interactions suggestive of neophobia, and lamina-specific decreases in neuronal area and increases in neuronal density in the lateral EC. Initial learning of spatial and other associative tasks, as well as vascular density in the lateral EC, was normal in the KOs. These data suggest that the absence of PTN in vivo is associated with disruption of specific cognitive and affective processes, raising the possibility that further study of PTN KOs might have implications for the study of human disorders with similar features.     

Defect in biosynthesis of the linkage unit between peptidoglycan and teichoic acid in a bacteriophage-resistant mutant of Staphylococcus aureus.

The biosynthesis of the linkage region between peptidoglycan and the ribitol teichoic acid was investigated in the bacteriophage-resistant, teichoic acid-less mutant Staphylococcus aureus 52A5 (Chatterjee et al., J. Bacteriol. 100:846--853, 1969). Membrane preparations of this strain were found to be incapable of forming the first intermediate of the biosynthetic pathway, namely, the transfer of N-acetyl-D-glucosamine (GlcNAc) from UDP-GlcNAc to the acceptor molecule, which presumbably is undecaprenol phosphate (R. Bracha and L. Glaser, Biochem. Biophys. Res. Commun. 72:1091--1098, 1976). The addition of heat-inactivated membrane preparations of S. aureus 52A2 (which normally has ribitol teichoic acid) that had been preincubated with UDP-GlcNAc to membranes of strain 52A5 enabled the synthesis of teichoic acid. These data suggest that the mutational defect in the teichoic acid-less organism is in the synthesis of the first compound of the linkage unit, and this is apparently the reason for its absence in the cell walls.     

Effect of heat inactivation and freezing on fatty acid composition of plasma and red blood cells.

The effect of heat inactivation and freezing on fatty acid composition of plasma and red blood cells was investigated. Analysis was completed at baseline; after freezing; after incubation; after incubation and subsequent freezing; after incubation, freezing and a second incubation; and after freezing and subsequent incubation. There were changes in fatty acid levels observed in all groups with the phospholipid fractions showing the greatest changes. Those bloods that had been incubated, frozen and incubated again, and those which had been frozen initially followed by incubation showed the greatest change when compared to baseline samples. Even though there were changes in fatty acid levels seen in all groups, the changes were small except in those two groups. Treatment of blood with either of those two treatment regimens changes the fatty acid values so that they do not accurately reflect the composition of fatty acids in the blood.     

Chondrogenic differentiation of amniotic fluid-derived stem cells

For regenerating damaged articular cartilage, it is necessary to identify an appropriate cell source that is easily accessible, can be expanded to large numbers, and has chondrogenic potential. Amniotic fluid-derived stem (AFS) cells have recently been isolated from human and rodent amniotic fluid and shown to be highly proliferative and broadly pluripotent. The purpose of this study was to investigate the chondrogenic potential of human AFS cells in pellet and alginate hydrogel cultures. Human AFS cells were expanded in various media conditions, and cultured for three weeks with growth factor supplementation. There was increased production of sulfated glycosaminoglycan (sGAG) and type II collagen in response to transforming growth factor-β (TGF-β) supplementation, with TGF-β1 producing greater increases than TGF-β3. Modification of expansion media supplements and addition of insulin-like growth factor-1 during pellet culture further increased sGAG/DNA over TGF-β1 supplementation alone. Compared to bone marrow-derived mesenchymal stem cells, the AFS cells produced less cartilaginous matrix after three weeks of TGF-β1 supplementation in pellet culture. Even so, this study demonstrates that AFS cells have the potential to differentiate along the chondrogenic lineage, thus establishing the feasibility of using these cells for cartilage repair applications.     

Oligonucleotide conjugate GRN163L targeting human telomerase as potential anticancer and antimetastatic agent

Telomerase is one of the key enzymes responsible for the proliferative immortality of the majority of cancer cells. We recently introduced a new telomerase inhibitor, a 13-mer oligonucleotide N3' --> P5'-thio-phosphoramidate lipid conjugate, designated as GRN163L. This compound inhibits telomerase activity in various tumor cell lines with IC(50) values of 3-300 nM without any cellular uptake enhancers. GRN163L demonstrated potent and sequence specific anti-cancer activity in vivo in multiple animal models. This compound was able to significantly affect not only the growth of primary tumors, but also the spread and proliferation of metastases. GRN163L is currently in Phase I and Phase I/II clinical studies in patients with solid tumors and CLL, respectively.     

Click-based synthesis of triazolobithiazole ΔF508-CFTR correctors for cystic fibrosis

Copper catalyzed azide-alkyne cycloaddition (CuAAC) chemistry is reported for the construction of previously unknown 5-(1H-1,2,3-triazol-1-yl)-4,5'-bithiazoles from 2-bromo-1-(thiazol-5-yl)ethanones. These novel triazolobithiazoles are shown to have cystic fibrosis (CF) corrector activity and, compared to the benchmark bithiazole CF corrector corr-4a, improved logP values (4.5 vs 5.96).     

A three-dimensional model of differentiation of immortalized human bronchial epithelial cells

A therapeutic approach being investigated for a variety of pathologies is tissue regeneration using a patient's own cells. Such studies have been hampered due to the difficulty in growing epithelial cells for prolonged periods in culture. Replicative senescence due to short telomeres and p16 induced by culture stress work together to inhibit cell growth. Forced expression of telomerase (hTERT) can prevent replicative senescence, and expression of the cell cycle protein cdk4 can sequester p16, thereby immortalizing epithelial cells in culture. In the present study, we used this method to immortalize human bronchial epithelial cells (HBECs) to determine whether immortalized HBECs retain the ability to differentiate normally. HBECs were plated atop contracted collagen gels containing lung fibroblasts. This three-dimensional (3D) tissue model was cultured initially submerged, then raised to the air/liquid interface for up to 28 days. Normal differentiation was assessed by the presence of ciliated cells, goblet (mucin-producing) cells, and basal epithelial cells. Scanning electron microscopic observations revealed both ciliated and non-ciliated cells in these 3D tissues. Histological examination revealed the presence of mucin-producing cells, and immunohistochemistry using antibodies against p63 and keratin 14 showed the presence of basal cells. These results demonstrate that immortalized HBECs retain the capacity to differentiate into each of three cell types: basal, mucin-producing, and columnar ciliated epithelial cells. Such cells will be useful cellular reagents for research in aging, cancer progression, as well as normal bronchial epithelial differentiation and will help progress the use of engineered cells to enhance tissue regeneration.