Comparative analysis of IFN-gamma B7.1 and antisense TGF-beta gene transfer on the tumorigenicity of a poorly immunogenic metastatic mammary carcinoma

Cancer progression is attributed in part to immune evasion strategies that include lack of co-stimulation, down-regulation of cell surface MHC molecules, and secretion of immunosuppressive factors, such as transforming growth factor-beta (TGF-beta). Gene therapy has been employed to counter these mechanisms of immune evasion by transference of B7.1, IFN-gamma or antisense TGF-beta genes into tumor cells, resulting in cell surface expression of B7.1, upregulation of MHC class I and class II molecules, or elimination of tumor-derived TGF-beta, respectively. Although each of these transgenes has been shown to alter tumorigenicity in murine models, a direct comparison of their efficacy has not been performed. In this study, we have employed a very aggressive, poorly immunogenic and highly metastatic mammary model, 4T1, to compare the efficacy of B7.1, IFN-gamma and antisense TGF-beta gene transfer in stimulating an anti-tumor response. We demonstrate that both IFN-gamma and antisense TGF-beta gene expression significantly reduced the tumorigenicity of these cells compared to mock transduced cells, with IFN-gamma having a greater effect. In contrast, B7.1 gene transfer did not affect the tumorigenicity of 4T1 cells. The anti-tumor response directed against antisense TGF-beta-expressing 4T1 tumors was mediated by CD4+ and CD8+ T cells. However, CD8+ T cells, and not CD4+ T cells, appeared to mediate the anti-tumor response against IFN-gamma-expressing tumors. Treatment of tumor-bearing animals with IFN-gamma or antisense TGF-beta gene-modified tumor cell vaccines reduced the number of clonogenic metastases to the lungs and liver compared to treatment with mock-transduced cells. Finally, in a residual disease model in which the primary tumor was excised and mice were vaccinated with irradiated tumor cells, treatment of mice with vaccinations consisting of 4T1 cells expressing both antisense TGF-beta and IFN-gamma genes was the most effective in prolonging survival.     

A comparison of techniques for isolation of the outer membrane proteins of Haemophilus influenzae type b

We compared several rapid techniques used for extraction of outer membrane proteins from gram-negative enteric bacteria to Haemophilus influenzae type b. After lysis of cells with a French press, the inner and outer membranes were separated by isopycnic centrifugation. Each membrane was identified by density, morphology, enzymatic activity, and susceptibility to solid-phase iodination of intact cells. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we identified 10 polypeptides which were enriched in the outer membrane band compared to the inner membrane band. Using these proteins, we compared the polypeptide pattern of outer membranes with that obtained by (1) selective solubilization with sodium dodecyl-beta-D-maltoside, octyl-beta-D-glucopyranoside, Triton X-100, sodium, or cholamidopropyl dimethylaminopropanesulfonate; (2) extraction with chaotropic agents and heat; and (3) differential centrifugation of vesicles shed during transition from log growth phase to stationary growth phase. There were definable differences between the polypeptide pattern of membranes obtained with each rapid technique compared to the polypeptide pattern of isolated outer membranes. The polypeptide pattern of lithium extracts and the Triton X-100 insoluble fractions of total membranes most closely approximated the polypeptide pattern of isopycnically isolated outer membranes. Depending on the outer membrane protein sought, one of these rapid techniques can be utilized when a rapid method of outer membrane protein isolation is required.     

Macro-scale topology optimization for controlling internal shear stress in a porous scaffold bioreactor

Shear stress is an important physical factor that regulates proliferation, migration, and morphogenesis. In particular, the homeostasis of blood vessels is dependent on shear stress. To mimic this process ex vivo, efforts have been made to seed scaffolds with vascular and other cell types in the presence of growth factors and under pulsatile flow conditions. However, the resulting bioreactors lack information on shear stress and flow distributions within the scaffold. Consequently, it is difficult to interpret the effects of shear stress on cell function. Such knowledge would enable researchers to improve upon cell culture protocols. Recent work has focused on optimizing the microstructural parameters of the scaffold to fine tune the shear stress. In this study, we have adopted a different approach whereby flows are redirected throughout the bioreactor along channels patterned in the porous scaffold to yield shear stress distributions that are optimized for uniformity centered on a target value. A topology optimization algorithm coupled to computational fluid dynamics simulations was devised to this end. The channel topology in the porous scaffold was varied using a combination of genetic algorithm and fuzzy logic. The method is validated by experiments using magnetic resonance imaging readouts of the flow field.     

Characterization of a high-molecular-weight form of human acrosin. Comparison with human pancreatic trypsin.

A high-molecular-weight form of acrosin (alpha-acrosin, EC 3.4.21.10) was extracted from spermatozoa obtained from frozen semen and purified over 300-fold. Purification was effected by sequential use of Sephadex G-150, CM-cellulose and DEAE-cellulose chromatography. Properties of human acrosin were compared with those of human pancreatic trypsin. The molecular weight (Mr) of acrosin (70000) was greater than that of trypsin (Mr 21000). Isoelectric points for acrosin (pI = 9.0) and trypsin (pI = 8.2) were also different. alpha-N-Benzoyl-L-arginine ethyl ester was hydrolysed 50% more rapidly by acrosin than by trypsin. Acrosin had similar kcat. values for the hydrolysis of esters with different acylating groups (i.e. benzoyl-L-arginine and p-tosyl-L-arginine esters). In contrast, trypsin had dissimilar kcat. values for the hydrolysis of esters with different acylating groups. Kinetic data argue against deacylation as the rate-limiting step in ester hydrolysis by acrosin. Acrosin was less sensitive than trypsin to inhibition by 7-amino-1-chloro-3-L-tosylamidoheptan-2-one ('TLCK'), di-isopropyl fluorophosphate and soya-bean trypsin inhibitor. D-Fructose and D-arabinose inhibited acrosin, but had no effect on trypsin. The data indicate that definite differences exist between human acrosin and trypsin.     

Chloroflexus-like organisms from marine and hypersaline environments: Distribution and diversity

We report the presence of a diverse number ofChloroflexus-like organisms in intertidal marine and submerged hypersaline microbial mats using light, infrared fluorescence, and electron microscopy. The intertidal organisms appear morphologically very similar to thermophilicC. aurantiacus while the 2 hypersaline strains are larger and have a more complex ultrastructure composed of chlorosome-bearing internal membranes that appear to arise as invaginations of the cell membrane. By comparing spectroradiometry of microbial mat layers with microscopic observations, we have confirmed that theChloroflexus-like organisms are major constituents of the hypersaline microbial mat communities. In situ studies on mat layers dominated byChloroflexus-like organisms showed that sulfide-dependent photoautotrophic activity sustained by near infrared radiation prevailed. Autoradiographic analyses revealed that autotrophy was sustained in the filaments by 750 nm radiation. Three morphologically distinct strains are now maintained in mixed culture. One of these appears to be growing photoautotrophically.     

MSA-35: a protein identified by human autoantibodies that colocalizes with microtubules.

We have identified a putative 35-kilodalton protein that colocalizes with microtubules and displays a unique spatial and temporal distribution during the cell cycle of HeLa cells. This protein has been given the designation MSA-35. MSA-35 first appears in association with microtubules and centrosomes of interphase cells exhibiting centrosome separation as a prelude to cell division. This protein is found in conjunction with kinetochore microtubules throughout their appearance. MSA-35 transiently associates with interpolar microtubules following anaphase and the pattern of MSA-35 reactivity in telophase cells suggests that there are at least seven domains within the intercellular bridge. The distribution of MSA-35 during and following recovery from mitotic arrest with nocodazole suggest that it is also present at low levels in interphase cells, can associate with interphase centrosomes, and colocalizes with nascent microtubules. The complex spatial and temporal distribution of MSA-35 indicates that it may be necessary for a series of events in the mitotic process such as the bundling of microtubules.     

Genetic investigation of equine recurrent uveitis in Appaloosa horses

Equine recurrent uveitis (ERU) is characterized by intraocular inflammation that often leads to blindness in horses. Appaloosas are more likely than any other breed to develop insidious ERU, distinguished by low-grade chronic intraocular inflammation, suggesting a genetic predisposition. Appaloosas are known for their white coat spotting patterns caused by the leopard complex spotting allele (LP) and the modifier PATN1. A marker linked to LP on ECA1 and markers near MHC on ECA20 were previously associated with increased ERU risk. This study aims to further investigate these loci and identify additional genetic risk factors. A GWAS was performed using the Illumina Equine SNP70 BeadChip in 91 horses. Additive mixed model approaches were used to correct for relatedness. Although they do not reach a strict Bonferroni genome-wide significance threshold, two SNPs on ECA1 and one SNP each on ECA12 and ECA29 were among the highest ranking SNPs and thus warranted further analysis (P = 1.20 × 10⁻⁵, P = 5.91 × 10⁻⁶, P = 4.91 × 10⁻⁵, P = 6.46 × 10⁻⁵). In a second cohort (n = 98), only an association with the LP allele on ECA1 was replicated (P = 5.33 × 10⁻⁵). Modeling disease risk with LP, age and additional depigmentation factors (PATN1 genotype and extent of roaning) supports an additive role for LP and suggests an additive role for PATN1. Genotyping for LP and PATN1 may help predict ERU risk (AUC = 0.83). The functional role of LP and PATN1 in ERU development requires further investigation. Testing samples across breeds with leopard complex spotting patterns and a denser set of markers is warranted to further refine the genetic components of ERU.     

NMR studies of the anti-apoptotic protein Bcl-xL in micelles

The Bcl-2 family of proteins play a pivotal role in the regulation of programmed cell death. One of the postulated mechanisms for the function of these proteins involves the formation of ion channels in membranes. As a first step to structurally characterize these proteins in a membrane environment, we investigated the structure of a Bcl-x(L) mutant protein when incorporated into small detergent micelles. This form of Bcl-x(L) lacks the loop (residues 49-88) between helix 1 and helix 2 and the putative C-terminal transmembrane helix (residues 214-237). Below the critical micelle concentration (CMC), Bcl-x(L) binds detergents in the hydrophobic groove that binds to pro-apoptotic proteins. However, above the CMC, Bcl-x(L) undergoes a dramatic conformational change. Using NMR methods, we characterized the secondary structure of Bcl-x(L) in the micelle-bound form. Like Bcl-x(L) in aqueous solution, the structure of the protein when dissolved in dodecylphosphocholine (DPC) micelles consists of several alpha-helices separated by loops. However, the length and position of the individual helices of Bcl-x(L) in micelles differ from those in aqueous solution. The location of Bcl-x(L) within the micelle was examined from the analysis of protein-detergent NOEs and limited proteolysis. In addition, the mobility of the micelle-bound form of Bcl-x(L) was investigated from NMR relaxation measurements. On the basis of these studies, a model is proposed for the structure, dynamics, and location of Bcl-x(L) in micelles. In this model, Bcl-x(L) has a loosely packed, dynamic structure in micelles, with helices 1 and 6 and possibly helix 5 partially buried in the hydrophobic interior of the micelle. Other parts of the protein are located near the surface or on the outside of the micelle.