Hox genes: Downstream “effectors” of retinoic acid signaling in vertebrate embryogenesis

One of the major regulatory challenges of animal development is to precisely coordinate in space and time the formation, specification, and patterning of cells that underlie elaboration of the basic body plan. How does the vertebrate plan for the nervous and hematopoietic systems, heart, limbs, digestive, and reproductive organs derive from seemingly similar population of cells? These systems are initially established and patterned along the anteroposterior axis (AP) by opposing signaling gradients that lead to the activation of gene regulatory networks involved in axial specification, including the Hox genes. The retinoid signaling pathway is one of the key signaling gradients coupled to the establishment of axial patterning. The nested domains of Hox gene expression, which provide a combinatorial code for axial patterning, arise in part through a differential response to retinoic acid (RA) diffusing from anabolic centers established within the embryo during development. Hence, Hox genes are important direct effectors of retinoid signaling in embryogenesis. This review focuses on describing current knowledge on the complex mechanisms and regulatory processes, which govern the response of Hox genes to RA in several tissue contexts including the nervous system during vertebrate development.     

Analysis of oxygen transport in a diffusion-limited model of engineered heart tissue

Cardiac tissue engineering has made notable progress in recent years with the advent of an experimental model based on neonatal cardiomyocytes entrapped in collage gels and purified basement membrane extract, known as "engineered heart tissues" (EHTs). EHTs are a formidable display of tissue-level contractile function and cellular-level differentiation, although they suffer greatly from mass transport limitations due to the high density of metabolically active cells and the diffusion-limited nature of the hydrogel. In this report, a mathematical model was developed to predict oxygen levels inside a one-dimensional, diffusion-limited model of EHT. These predictions were then compared to values measured in corresponding experiments with a hypoxia-sensitive stain (pimonidazole). EHTs were cast between two plastic discs, which allowed for mass transfer with the culture medium to occur in only the radial direction. EHTs were cultured for up to 36 h in the presence of pimonidazole, after which time they were snap-frozen, histologically sectioned, and stained for bound pimonidazole. Quantitative image analysis was performed to measure the distance from the culture medium at which hypoxia first occurs under various conditions. As tested by variation of simple design parameters, the trends in oxygen profiles predicted by the model are in reasonable agreement with those obtained experimentally, although a number of ambiguities related to the specific model parameters led to a general overprediction of oxygen concentrations. Based on the sensitivity analysis in the present study, it is concluded that diffusion-reaction models may offer relatively precise predictions of oxygen concentrations in diffusion-limited tissue constructs.     

Influence of systematically varied nano-scale topography on cell morphology and adhesion

The types of cell-matrix adhesions and the signals they transduce strongly affect the cell-phenotype. We hypothesized that cells sense and respond to the three-dimensionality of their environment, which could be modulated by nano-structures on silicon surfaces. Human foreskin fibroblasts were cultured on nano-structures with different patterns (nano-post and nano-grate) and heights for 3 days. The presence of integrin alpha(5), beta(1), beta(3), paxillin and phosphorylated FAK (pFAK) were detected by western blot and immunofluorescence. Integrin beta(3) exhibited stronger signals on nano-grates. pFAK and paxillin were observed as small dot-like patterns on the cell-periphery on nano-posts and as elongated and aligned patterns on nano-grates. Collectively, our observations highlighted the presence of focal (integrin beta(1), beta(3), pFAK, paxillin), fibrillar (integrin alpha(5), beta(1)) and 3-D matrix (integrin alpha(5), beta(1), paxillin) adhesions on nano-structures. The presented nano-structures offer interesting opportunities to study the interaction of cells with topographical features comparable to the size of extracellular matrix components.     

The effect of cholesterol in a liposomal Muc1 vaccine

A liposomal Muc1 mucin vaccine for treatment of adenocarcinomas was formulated by incorporating a synthetic Muc1 mucin-based lipopeptide and Lipid A into a DPPC/cholesterol bilayer. Vaccination of mice with the liposomal formulation produced a peptide-specific immune response dependent on the cholesterol content. The response occurred at a threshold of 20-23 mol% cholesterol, and was optimal at cholesterol levels of > or =30 mol%. To understand this cholesterol dependency, we studied the effect of cholesterol on the liposomal bilayer and surface properties. Freeze-fracture electron microscopy showed a unique surface texture that was codependent upon cholesterol (> or =20 mol%) and lipopeptide content. Fluorescence anisotropy measurements exhibited a significant decrease in the rotational motion of 1,6-diphenyl-1,3,5-hexatriene in formulations containing >20 mol% cholesterol and only in the presence of the lipopeptide. At 20 mol% cholesterol and with lipopeptide, DSC showed a significant increase in the main phase transition of the DPPC bilayers, while Raman spectroscopy indicated a more ordered arrangement of DPPC molecules compared to control liposomes containing DPPC/cholesterol alone. Taken together, the data suggest the presence of lipopeptide-rich microdomains at and above a threshold of 20 mol% cholesterol that may play a role in the induction of a peptide-specific immunological response.     

Adaptation of proteins from hyperthermophiles to high pressure and high temperature

Further clarification of the adaptations permitting the persistence of life at temperatures above 100 degrees C depends in part on the analysis of adaptive mechanisms at the protein level. The hyperthermophiles include both Bacteria and Archaea, although the majority of isolates growing at or above 100 degrees C are Archaea. Newly described adaptive features of hyperthermophiles include proteins whose structural integrity persists at temperatures up to 200 degrees C, and under elevated hydrostatic pressure, which in some cases adds significant increments of stability.     

Cross-subtype neutralizing antibodies induced in baboons by a subtype E gp120 immunogen based on an R5 primary human immunodeficiency virus type 1 envelope

Global human immunodeficiency virus type 1 (HIV-1) diversity may require engineering vaccines to express antigens representing strains prevalent in the target population of vaccine testing. The majority (90%) of incident infections in Thailand are genetic subtype E, with a small percentage of subtype B infections in the intravenous drug user populations. We have evaluated and compared the binding and HIV-1 neutralizing properties of serum antibodies induced in baboons by CHO cell-expressed monomeric gp120 derived from a CCR5-using (R5) subtype E primary HIV-1CM235 or a CXCR4-using (X4) subtype B T-cell line-adapted (TCLA) HIV-1SF2 isolate. In contrast to the subtype-specific HIV-1 neutralizing antibodies induced with recombinant HIV-1SF2 gp120 (rgp120SF2), rgp120CM235 immunization induced antibodies capable of neutralizing both subtype E and subtype B TCLA HIV-1 isolates. However, neither immunogen induced antibodies capable of neutralizing primary HIV-1 isolates. Antibody induced by rgp120CM235 preferentially bound natively folded gp120 and retained strong cross-reactivity against multiple gp120 strains within subtype E as well as subtype B. In contrast, antibody responses to rgp120SF2 were directed predominantly to linear epitopes poorly exposed on native gp120 and had more limited cross-recognition of divergent gp120. Fine epitope mapping revealed differences in antibody specificities. While both rgp120CM235 and rgp120SF2 induced antibodies to regions within C1, V1/V2, V3, and C5, unique responses were induced by rgp120CM235 to multiple epitopes within C2 and by rgp120SF2 to multiple epitopes within C3, V4, and C4. These data demonstrate that strain and/or phenotypic differences of HIV-1 subunit gp120 immunogens can substantially alter antibody binding specificities and subsequent HIV-1 neutralizing capacity.