PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization

PTX3 is a prototypic long pentraxin that plays a non-redundant role in innate immunity against selected pathogens and in female fertility. Here, we report that the infertility of Ptx3(-/-) mice is associated with severe abnormalities of the cumulus oophorus and failure of in vivo, but not in vitro, oocyte fertilization. PTX3 is produced by mouse cumulus cells during cumulus expansion and localizes in the matrix. PTX3 is expressed in the human cumulus oophorus as well. Cumuli from Ptx3(-/-) mice synthesize normal amounts of hyaluronan (HA), but are unable to organize it in a stable matrix. Exogenous PTX3 restores a normal cumulus phenotype. Incorporation in the matrix of inter-alpha-trypsin inhibitor is normal in Ptx3(-/-) cumuli. PTX3 does not interact directly with HA, but it binds the cumulus matrix hyaladherin tumor necrosis factor alpha-induced protein 6 (TNFAIP6, also known as TSG6) and thereby may form multimolecular complexes that can cross-link HA chains. Thus, PTX3 is a structural constituent of the cumulus oophorus extracellular matrix essential for female fertility.     

Lipid and carbohydrate metabolism in mice with a targeted mutation in the IL-6 gene: absence of development of age-related obesity

Obesity-related insulin resistance may be caused by adipokines such as IL-6, which is known to be elevated with the insulin resistance syndrome. A previous study reported that IL-6 knockout mice (IL-6(-/-)) developed maturity onset obesity, with disturbed carbohydrate and lipid metabolism, and increased leptin levels. Because IL-6 is associated with insulin resistance, one might have expected IL-6(-/-) mice to be more insulin sensitive. We examined body weights of growing and older IL-6(-/-) mice and found them to be similar to wild-type (IL-6(+/+)) mice. Dual-energy X-ray absorptiometry analysis at 3 and 14 mo revealed no differences in body composition. There were no differences in fasting blood insulin and glucose or in triglycerides. To further characterize these mice, we fed 11-mo-old IL-6(-/-) and IL-6(+/+) mice a high- (HF)- or low-fat diet for 14 wk, followed by insulin (ITT) and glucose tolerance tests (GTT). An ITT showed insulin resistance in the HF animals but no difference due to genotype. In the GTT, IL-6(-/-) mice demonstrated elevated postinjection glucose levels by 60% compared with IL-6(+/+) but only in the HF group. Although IL-6(-/-) mice gained weight and white adipose tissue (WAT) with the HF diet, they gained less weight than the IL-6(+/+) mice. Total lipoprotein lipase activity in WAT, muscle, and postheparin plasma was unchanged in the IL-6 (-/-) mice compared with IL-6(+/+) mice. There were no differences in plasma leptin or TNF-alpha due to genotype. Plasma adiponectin was approximately 53% higher (71.7 +/- 14.1 microg/ml) in IL-6(-/-) mice than in IL-6(+/+) mice but only in the HF group. Thus these data show that IL-6(-/-) mice do not demonstrate obesity, fasting hyperglycemia, or abnormal lipid metabolism, although HF IL-6(-/-) mice demonstrate elevated glucose after a GTT.     

The sensitive giant: the role of titin-based stretch sensing complexes in the heart

Every heart beat is not equal. As physiological demands of the cardiovascular system change, cardiac myocytes modulate contractile parameters including the rate and force of contraction. Adaptive responses require the sensing of biomechanical signals involving the interface between the contractile cytoskeleton (myofibrils) and the sarcolemma at specialized cell-cell junctions (intercalated discs) and cell-substrate adhesion complexes (costameres). Recent studies have shed insight into how protein complexes within cardiac myocytes sense biomechanical signals, processes required for normal adaptive or pathological responses. This new evidence suggests that complexes associated with the giant, myofibrillar protein titin sense myocyte stretch. Here, we discuss evidence supporting titin being an ideal biomechanical sensor.     

Novel chimeric immunomodulatory compounds containing short CpG oligodeoxyribonucleotides have differential activities in human cells

Immunostimulatory DNA sequences (ISS) containing CpG motifs induce interferon-α (IFN-α) and interferon-γ (IFN-γ) from human peripheral blood mononuclear cells and stimulate human B cells to proliferate and produce IL-6. We studied the motif and structural requirements for both types of activity using novel chimeric immunomodulatory compounds (CICs), which contain multiple heptameric ISS connected by non-nucleoside spacers in both linear and branched configurations. We found that the optimal motifs and structure for IFN-α production versus B cell activation differed. IFN-α production was optimal for CICs containing the sequences 5′-TCGXCGX and 5′-TCGXTCG, where X is any nucleotide. The presentation of multiple copies of these heptameric ISS with free 5′-ends via long, hydrophilic spacers, such as hexaethylene glycol, significantly enhanced the induction of IFN-α. Conversely, human B cell activity was predominately dependent on ISS motif, with 5′-TCGTXXX and 5′-AACGTTC being the most active sequences. Thus, we found CICs could be ‘programmed’ for IFN-α production or B cell activation as independent variables. Additionally, CICs with separate human- and mouse-specific motifs were synthesized and these were used to confirm in vivo activity in mice. CICs may offer unique advantages over conventional ISS because identification of the optimal motifs, spacers and structures for different biological properties allows for the assembly of CICs exhibiting a defined set of activities tailored for specific clinical applications.     

Ligand screening by exoproteolysis and mass spectrometry in combination with computer modelling

Here, we present a new approach for protein ligand screening based on the use of limited exoproteolysis coupled to MALDI-TOF mass spectrometry, combined with computational modelling and prediction of binding energies. As a test for this combined approach, we have screened a combinatorial library containing 8000 peptides (organized in 60 peptide samples) based on positional scanning format. This library is attached to a poly-Pro framework, and screened against the Abl-SH3 domain. The results obtained demonstrated the validity of the experimental and theoretical approaches in identifying better ligands and in rationalizing the changes in affinity. Exoproteolysis coupled to MALDI-TOF mass spectrometry could be used to screen complex libraries in a fast and efficient way.     

The muscle ankyrin repeat proteins: CARP, ankrd2/Arpp and DARP as a family of titin filament-based stress response molecules

CARP, ankrd-2/Arpp, and DARP, are three members of a conserved gene family, referred to here as MARPs (muscle ankyrin repeat proteins). The expression of MARPs is induced upon injury and hypertrophy (CARP), stretch or denervation (ankrd2/Arpp), and during recovery following starvation (DARP), suggesting that they are involved in muscle stress response pathways. Here, we show that MARP family members contain within their ankyrin repeat region a binding site for the myofibrillar elastic protein titin. Within the myofibril, MARPs, myopalladin, and the calpain protease p94 appear to be components of a titin N2A-based signaling complex. Ultrastructural studies demonstrated that all three endogenous MARP proteins co-localize with I-band titin N2A epitopes in adult heart muscle tissues. In cultured fetal rat cardiac myocytes, passive stretch induced differential distribution patterns of CARP and DARP: staining for both proteins was increased in the nucleus and at the I-band region of myofibrils, while DARP staining also increased at intercalated discs. We speculate that the myofibrillar MARPs are regulated by stretch, and that this links titin-N2A-based myofibrillar stress/strain signals to a MARP-based regulation of muscle gene expression.     

Tethered-function analysis reveals that elF4E can recruit ribosomes independent of its binding to the cap structure

The cap-binding complex elF4F is involved in ribosome recruitment during the initiation phase of translation and is composed of three subunits: elF4E, -4G, and -4A. The m7GpppN cap-binding subunit eIF4E binds the N-terminal region of eIF4G, which in turn contacts eIF4A through its central and C-terminal regions. We have previously shown, through a tethered-function approach in transfected HeLa cells, that the binding of eIF4G to an mRNA is sufficient to drive productive translation (De Gregorio et al., EMBO J, 1999, 18:4865-4874). Here we exploit this approach to assess which of the other subunits of elF4F can exert this function. eIF4AI or mutant forms of eIF4E were fused to the RNA-binding domain of the lambda phage antiterminator protein N to generate the chimeric proteins lambda4A, lambda4E-102 (abolished cap binding), and lambda4E-73-102 (impaired binding to both, the cap and eIF4G). The fusion proteins were directed to a bicistronic reporter mRNA by means of interaction with a specific lambda-N binding site (boxB) in the intercistronic space. We show that lambda4E-102, but neither the double mutant lambda4E-73-102 nor lambda4A, suffices to promote translation of the downstream gene in this assay. Coimmunoprecipitation analyses confirmed that all lambda-fusion proteins are capable of interacting with the appropriate endogenous eIF4F subunits. These results reveal that eIF4E, as well as eIF4G, can drive ribosome recruitment independent of a physical link to the cap structure. In spite of its interaction with endogenous eIF4G, lambda4A does not display this property. eIF4A thus appears to supply an essential auxiliary function to eIF4F that may require its ability to cycle into and out of this complex.     

Homocysteine modifies development of neurulation and dorsal root ganglia in chick embryos

BACKGROUND: The formation of the neural tube (neurulation) involves two mechanisms: primary and secondary neurulation. In chicks, there is also an overlap zone, where both mechanisms work together. Homocysteine (Hcy) may have an important teratogenic role in neural tube defects (NTD) when folic acid levels are considered normal. Recently, Hcy capability to generate NTD and modify neural crest cell migration has been demonstrated in chick embryos. This study was aimed to evaluate the effects of Hcy on neurulation and the development of the dorsal root ganglia (DRG). METHODS: Chick embryos were treated with L-Hcy thiolactone 20 micromol to produce the highest rate of survival with embryos carrying neural tube defect (NTD) in the spine. Embryos at stages (st) 3-10 were treated and harvested at st 18-23. Only externally normal embryos or those carrying spinal NTD embryos were considered. RESULTS: Histological sections of Hcy-treated embryos showed: open spina bifida (39% of embryos), more than one tube forming the spinal cord (26%), disorganized spinal cord (26%), always affecting lumbosacral regions, probably in the overlap zone. Additionally, 32% of embryos had small and continuous DRG, associated with a slimmed roof plate. Three-dimensional reconstruction showed unsegmented DRG until the C8 ganglion level. There was a 75% reduction of C3 DRG cells in treated embryos in comparison to untreated ganglia. CONCLUSION: Hcy teratogenicity in avian embryos affected the neural tube in the overlap zone, secondary neurulation and the cervical DRG.     

The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy

Muscle cells respond to mechanical stretch stimuli by triggering downstream signals for myocyte growth and survival. The molecular components of the muscle stretch sensor are unknown, and their role in muscle disease is unclear. Here, we present biophysical/biochemical studies in muscle LIM protein (MLP) deficient cardiac muscle that support a selective role for this Z disc protein in mechanical stretch sensing. MLP interacts with and colocalizes with telethonin (T-cap), a titin interacting protein. Further, a human MLP mutation (W4R) associated with dilated cardiomyopathy (DCM) results in a marked defect in T-cap interaction/localization. We propose that a Z disc MLP/T-cap complex is a key component of the in vivo cardiomyocyte stretch sensor machinery, and that defects in the complex can lead to human DCM and associated heart failure.