Joel B. Sheffield

A solid-phase method for the quantitation of protein in the presence of sodium dodecyl sulfate and other interfering substances☆

We describe a simple assay for small amounts of protein that is insensitive to sodium dodecyl sulfate (SDS) or many common interfering substances including Tris and reducing sugars. For this reason, it is particularly useful in the analysis of protein content of samples prior to SDS electrophoresis. The assay consists of the following steps: (i) absorption of protein to nitrocellulose; (ii) fixation of the bound protein with methanol; (iii) staining of the bound protein with amido black; and (iv) elution and spectrophotometric measurement of the bound dye. The assay is sensitive to as little as 0.5 microgram of protein in 1 microliter of solution. Although SDS does not interfere appreciably with measurement, Nonidet-P40 does.

Isolation and characterization of flat cells, a subpopulation of the embryonic chick retina

When the embryonic neutral retina is dissociated into single cells which are maintained in stationary culture, the neuronal cells associate on the surfaces of a second population which we refer to as flat cells. The flat cells appear in the culture in significant numbers after 2 days and are required for neuronal cell attachment. We have been able to isolate pure flat cells from early cultures of mixed retina cells and have identified several antigens which support the concept that these cells are related to the glia. The cells have been tested by immunofluorescence for glial fibrillary acidic protein and have been found positive. Cell surfaces were labeled by transfer of tritiated galactose from UDP-galactose to endogenous acceptors in the presence of exogenous galactosyl transferase. After SDS-PAGE and fluorography, the surface glycoproteins of flat cells were seen to be significantly different from those of the original retina, and from chick fibroblasts. Immunoelectron microscope studies of detergent-extracted flat cells have demonstrated a complex network of intermediate filaments and actin fibers. We conclude that the flat cells are derived from the glia subpopulation of the retina and have adapted to the tissue culture environment by assuming this configuration. The unique surface properties of flat cells may be related to their role as an intermediate substrate between the neuronal cells and the tissue culture dish.

A Novel Dynamic Neonatal Blood-Brain Barrier on a Chip.

Studies of neonatal neural pathologies and development of appropriate therapeutics are hampered by a lack of relevant in vitro models of neonatal blood-brain barrier (BBB). To establish such a model, we have developed a novel blood-brain barrier on a chip (B3C) that comprises a tissue compartment and vascular channels placed side-by-side mimicking the three-dimensional morphology, size and flow characteristics of microvessels in vivo. Rat brain endothelial cells (RBEC) isolated from neonatal rats were seeded in the vascular channels of B3C and maintained under shear flow conditions, while neonatal rat astrocytes were cultured under static conditions in the tissue compartment of the B3C. RBEC formed continuous endothelial lining with a central lumen along the length of the vascular channels of B3C and exhibited tight junction formation, as measured by the expression of zonula occludens-1 (ZO-1). ZO-1 expression significantly increased with shear flow in the vascular channels and with the presence of astrocyte conditioned medium (ACM) or astrocytes cultured in the tissue compartment. Consistent with in vivo BBB, B3C allowed endfeet-like astrocyte-endothelial cell interactions through a porous interface that separates the tissue compartment containing cultured astrocytes from the cultured RBEC in the vascular channels. The permeability of fluorescent 40 kDa dextran from vascular channel to the tissue compartment significantly decreased when RBEC were cultured in the presence of astrocytes or ACM (from 41.0±0.9 x 10−6 cm/s to 2.9±1.0 x 10−6 cm/s or 1.1±0.4 x 10−6 cm/s, respectively). Measurement of electrical resistance in B3C further supports that the addition of ACM significantly improves the barrier function in neonatal RBEC. Moreover, B3C exhibits significantly improved barrier characteristics compared to the transwell model and B3C permeability was not significantly different from the in vivo BBB permeability in neonatal rats. In summary, we developed a first dynamic in vitro neonatal BBB on a chip (B3C) that closely mimics the in vivo microenvironment, offers the flexibility of real time analysis, and is suitable for studies of BBB function as well as screening of novel therapeutics.

Cloning and characterization of angiocidin, a tumor cell binding protein for thrombospondin‐1

Thrombospondin‐1 (TSP‐1) is a matrix protein that has been implicated in mechanisms of tumor progression. Our laboratory previously showed that the CSVTCG (cys‐ser‐val‐thr‐cys‐gly) sequence of TSP‐1 functioned as a tumor cell adhesion domain and CSVTCG peptides as well as an anti‐peptide antibody possessed anti‐metastatic activity in a murine model of lung metastasis. In a subsequent study, a putative TSP‐1 binding protein from lung carcinoma was isolated by CSVTCG‐peptide affinity chromatography. In this study, we present the full‐length cDNA of this binding protein isolated from a prostate cancer cell (PC3‐NI) cDNA library. The purified recombinant protein, termed angiocidin, is a potent inhibitor of tumor growth of Lewis Lung carcinoma in vivo and tumor invasion and angiogenesis in vitro. In addition, the recombinant protein inhibits tumor and endothelial cell proliferation and induces apoptosis. The activity of angiocidin both in vivo and in vitro is partially dependent on its TSP‐1 binding activity, since an angiocidin deletion mutant missing a high affinity‐binding site for TSP‐1 failed to inhibit tumor growth in vivo and was less active in its anti‐tumor and anti‐angiogenic activities in vitro. These results suggest that the anti‐tumor activity of TSP‐1 reported in many studies may be mediated in part by binding proteins such as angiocidin. Such proteins may function as tumor‐suppressor proteins, which limit the growth of tumors by inhibiting angiogenesis and cell matrix interaction.

Light curable dental composites designed with colloidal crystal reinforcement

OBJECTIVES Methods to prepare dental composites with a periodic filler arrangement were developed following a strategy of colloidal crystallization. The aims of this study were to determine the influence of suspension medium, silane treatment and amine additive on colloidal particle redispersion and subsequent ordering, and to evaluate the effect of filler ordering on mechanical properties of composites. METHODS Dry monodisperse silica particles (spherical, approximately 500-nm diameter) were redispersed in selected solvents and monomers (e.g. triethyleneglycol dimethacrylate, TEGDMA) to form sediments or dispersions with ordered particle arrangements. Ordering was evaluated by microscopy and mechanical properties of the composites were measured using compression tests (n=6). RESULTS A face-centered cubic packed structure could form in both the sediment from silica dispersions in polar solvents and stable dispersions in TEGDMA. Dimethylaminoethyl methacrylate (DMAEMA) was found to disrupt an ordered structure when non-silanized silica particles were used. Silanization with 3-methacryloxypropyl trimethoxysilane (MPS) promoted filler ordering. Standard compression tests on composites containing 60wt% silica in TEGDMA with or without DMAEMA indicated that DMAEMA had a clearly significant effect (p<0.05) on failure strain, compressive strength, and toughness, and a marginally significant effect on modulus (p=0.12). SIGNIFICANCE Significant increases in compressive strength (16%), failure strain (71%), and toughness (135%) were observed for composites with ordered filler compared to non-ordered composites.

A Spiroligomer α-Helix Mimic That Binds HDM2, Penetrates Human Cells and Stabilizes HDM2 in Cell Culture

We demonstrate functionalized spiroligomers that mimic the HDM2-bound conformation of the p53 activation domain. Spiroligomers are stereochemically defined, functionalized, spirocyclic monomers coupled through pairs of amide bonds to create spiro-ladder oligomers [1]. Two series of spiroligomers were synthesized, one of structural analogs and one of stereochemical analogs, from which we identified compound 1, that binds HDM2 with a Kd value of 400 nM. The spiroligomer 1 penetrates human liver cancer cells through passive diffusion and in a dose-dependent and time-dependent manner increases the levels of HDM2 more than 30-fold in Huh7 cells in which the p53/HDM2 negative feed-back loop is inoperative. This is a biological effect that is not seen with the HDM2 ligand nutlin-3a. We propose that compound 1 modulates the levels of HDM2 by stabilizing it to proteolysis, allowing it to accumulate in the absence of a p53/HDM2 feedback loop.

Murine mammary tumor virus deficient in the major glycoprotein: Biochemical and biological studies on virions produced by a lymphoma cell line

Abstract A cell line, designated MLA, was established from a spontaneous lymphoma of a DBA/2 mouse. These cells contained large amounts of intracytoplasmic type-A particles, the presumed precursors of mature MuMTV. Electron microscopy revealed that the MLA cells released MuMTV-like virions into the culture fluid, but these virions were devoid of the characteristic envelope “spikes” of MuMTV. Antigenic analysis of virions purified from the culture fluid of MLA cells showed that, by microimmunodiffusion, the glycoproteins of MuMTV, gp49 and gp36, were not detectable, nor was the glycoprotein gp70 of MuLV detectable. Internal structural proteins of MuMTV, p28 and p12, were however, readily detectable in these virions. By the more sensitive radioimmunoassay technique, it was found that the virions produced by MLA cells contained some gp49-related antigen, but the amount present was about 500-fold lower than in other tissue culture-derived MuMTV. MLA cells synthesized a 70,000-dalton precursor of MuMTV glycoproteins. Culture fluids from which the virions were removed by centrifugation did contain gp49, indicating processing of the precursor polypeptide, but this polypeptide was not incorporated into the virions. Molecular hybridization studies revealed that MLA cells contained a large amount of MuMTV-specific RNA; the amount of MuLV-related RNA was about 100-fold lower. Whereas 150–200 MuMTV particles were released into culture fluid per cell per day, few MuLV particles were released. Purified virions from the MLA cells were injected into BALB/c and C57BL weanling females. This resulted in mammary tumor development in BALB/c females, but not in C57BL. The tumors contained MuMTV with the normal complement of envelope spikes and gp49 was detectable in the virions.

THE EFFECTS OF NICOTINAMIDE AND HYPERBARIC OXYGEN ON SKIN FLAP SURVIVAL

Hyperbaric oxygen has been established as an acceptable treatment for the chronic healing wound. Nicotinamide has been shown to be angiogenic and accelerate the physiologic process following wounding. Therefore both nicotinamide and hyperbaric oxygen were evaluated to enhance flap survival in an island pedicle skin flap model. These two treatment modalities were evaluated alone and in combination to assess if there is an addictive effect to enhance flap survival. Forty Sprague-Dawley male rats (weight 300-350 grams) were treated for 14 days preoperatively 1 day post-operatively with either 400 mg of nicotinamide i.p. or saline i.p. On day 14, a 7 X 7 cm island pedicle skin flap was elevated ligating the left inferior epigastric neurovascular pedicle and were sutured in their normal position. Twenty animals then underwent hyperbaric oxygen treatments. Forty-eight hours post-operatively animals were re-anesthetized and were given a single injection of fluorescein (25 mg/kg) via the tail vein. The % survival of the flap and SEM of the groups are as follows: Saline 45.67 +/- 31.14, nicotinamide 85.30 +/- 9.24, saline-hyperbaric oxygen 76.70 +/- 9.42 and nicotinamide-hyperbaric oxygen 90.86 +/- 3.94 with statistical significance of p less than 0.01. Nicotinamide appears to be another acceptable therapeutic modality in the management of the acceleration of wound healing.

Nicotinamide enhances skin flap survival

The effects of nicotinamide in an abdominal island pedicle skin flap were examined. A 7 x 7 cm island pedicle skin flap ligating the left inferior neurovascular pedicle was created on 50 male Sprague Dawley rats (250-275 grams) that were divided into five groups. Animals received either 0.6 cc of saline or doses of nicotinamide for 16 days (14 days preoperatively and 2 days postoperatively): 25 mg b.i.d., 50 mg b.i.d., 100 mg b.i.d. or 200 mg b.i.d. Forty-eight hours postoperatively each animal received 25 mg of Fluorescein via the tail vein. The area of necrosis was visualized and quantified and is presented as % survival. A one factor Fisher PLSD test was performed with a statistical significance of p less than 0.05 with the results as follows: saline 58.8%, 25 mg 68.6%, 50 mg 82%, 100 mg 80.8%, and 200 mg 86%. From this data it would appear that the angiogenic factor nicotinamide may increase random flap survival.

Membrane alterations which accompany MuMTV maturation: I. Studies by freeze-cleave techniques

Abstract As part of a study of the assembly of viral envelope at the surface of cells producing the mouse mammary tumor virus (MuMTV), freeze-cleave replicas were obtained of virus producing regions of mammary tumors. Free virions in the acinar space appeared identical to separately prepared, purified virus as reported earlier, with A (convex) and B (concave) fracture surfaces exposed. The A face of microvilli in the tumor was covered with intramembranous particles (IMP), while the B face was generally devoid of IMP. The A face of microvilli with virus in them was covered with IMP except in the region of virus maturation. There was no observable difference between the B surfaces of microvilli and adjacent maturing virus. Our results suggest that while new virus proteins and antigens are added to the cell membrane, converting it to a viral envelope, some normal cellular membrane components are displaced or absent from the region of virus maturation. Some possible mechanisms for this process are discussed.