BAX inhibitor-1 is a Ca(2+) channel critically important for immune cell function and survival.

The endoplasmic reticulum (ER) serves as the major intracellular Ca(2+) store and has a role in the synthesis and folding of proteins. BAX (BCL2-associated X protein) inhibitor-1 (BI-1) is a Ca(2+) leak channel also implicated in the response against protein misfolding, thereby connecting the Ca(2+) store and protein-folding functions of the ER. We found that BI-1-deficient mice suffer from leukopenia and erythrocytosis, have an increased number of splenic marginal zone B cells and higher abundance and nuclear translocation of NF-κB (nuclear factor-κ light-chain enhancer of activated B cells) proteins, correlating with increased cytosolic and ER Ca(2+) levels. When put into culture, purified knockout T cells and even more so B cells die spontaneously. This is preceded by increased activity of the mitochondrial initiator caspase-9 and correlated with a significant surge in mitochondrial Ca(2+) levels, suggesting an exhausted mitochondrial Ca(2+) buffer capacity as the underlying cause for cell death in vitro. In vivo, T-cell-dependent experimental autoimmune encephalomyelitis and B-cell-dependent antibody production are attenuated, corroborating the ex vivo results. These results suggest that BI-1 has a major role in the functioning of the adaptive immune system by regulating intracellular Ca(2+) homeostasis in lymphocytes.

Osteoblast differentiation onto different biometals with an endoprosthetic surface topography in vitro.

In this in vitro study, we compared the cytocompatibility and osteoblast promoting potency on human bone marrow cell culture with three different alloys (surgical steel, CoCr, Ti6Al4V) and three different surface structures (polished, sandblasted, porous coated). These biometals were specifically chosen because of their current applications in clinical orthopedic practices. Human mononuclear bone marrow cells were cultivated onto the surface of the different biomaterials and stimulated by dexamethasone, L-ascorbic-acid-2-phoshpate and beta-glycerolphosphate over a 3-week period. Immunofluorescent stainings against several antigens (ALP, RANKL, osteopontin, collagen I), mRNA-expression of collagen (Col) I/II, BSP, osteopontin, osteocalcin, TRAP, light and scanning electron microscopy evaluation were used to evaluate cellular growth and osteoblast differentiation. For surface roughness and energy analysis of the specimen, roughness profile (Ra, Rz) and contact angle (CA) measurements were performed. We found differences between the different biometals and surface structures. Steel showed potential cytotoxic effects whereas CoCr and more Ti6Al4V showed an excellent cytocompatibility. There were no qualitative differences in mRNA expression between each of the tested biomaterials. In terms of antigen expression, a sandblasted Ti6Al4V surface showed enhanced osteoblastic differentiation. A porous-coated surface improved the osteoconductivity of CoCr when compared to a polished surface. In contrast to controls all cells cultivated with biometals induced a RANKL expression. Cells increased the implant roughness with the exception of sandblasted Ti6Al4V. Our data show that surface topography and physicochemical properties of biometals influence osteoblast differentiation in vitro.

Significance of nano- and microtopography for cell-surface interactions in orthopaedic implants.

Cell-surface interactions play a crucial role for biomaterial application in orthopaedics. It is evident that not only the chemical composition of solid substances influence cellular adherence, migration, proliferation and differentiation but also the surface topography of a biomaterial. The progressive application of nanostructured surfaces in medicine has gained increasing interest to improve the cytocompatibility and osteointegration of orthopaedic implants. Therefore, the understanding of cell-surface interactions is of major interest for these substances. In this review, we elucidate the principle mechanisms of nano- and microscale cell-surface interactions in vitro for different cell types onto typical orthopaedic biomaterials such as titanium (Ti), cobalt-chrome-molybdenum (CoCrMo) alloys, stainless steel (SS), as well as synthetic polymers (UHMWPE, XLPE, PEEK, PLLA). In addition, effects of nano- and microscaled particles and their significance in orthopaedics were reviewed. The significance for the cytocompatibility of nanobiomaterials is discussed critically.

CREB binding protein recruitment to the transcription complex requires growth factor-dependent phosphorylation of its GF box.

Growth factors such as epidermal growth factor (EGF) and insulin regulate development and metabolism via genes containing both POU homeodomain (Pit-1) and phorbol ester (AP-1) response elements. Although CREB binding protein (CBP) functions as a coactivator on these elements, the mechanism of transactivation was previously unclear. We now demonstrate that CBP is recruited to these elements only after it is phosphorylated at serine 436 by growth factor-dependent signaling pathways. In contrast, p300, a protein closely related to CBP that lacks this phosphorylation site, binds only weakly to the transcription complex and in a growth factor-independent manner. A small region of CBP (amino acids 312-440), which we term GF box, contains a potent transactivation domain and mediates this effect. Direct phosphorylation represents a novel mechanism controlling coactivator recruitment to the transcription complex.

Structural and mechanical aspects of the skin of Bufo marinus (Anura, Amphibia).

Specific biomechanical characters and some structures possibly related to them were investigated in the skin of the toad Bufo marinus using tensile testing techniques (at constant strain till rupture) as well as morphological methods (histological, immunohistochemical and electronmicroscopical). Mechanical parameters of the native skin varied considerably according to sex, individual variability and/or site of specimen collection. In skin strips of males and females excised from different parts of the body thickness ranged from 0.45 to 0.87 mm, strain (epsilonf) from 96.52 to 211.03, tensile strength (sigmam) from 5.72 to 9.38 MPa, and stiffness (E-modulus) from 5.76 to 6.73. The dermis of B. marinus is provided with a collagenous stratum compactum of considerable thickness, a stratum spongiosum with loosely arranged fibres and a marked calcified layer (substantia amorpha). Collagen appears to be the main determinant of skin mechanics. However, the slope of the J-shaped static stress-strain curves indicates elastin to be responsible for the high values of strain. Contrary to van Gieson and orcein staining, immunostaining with a monoclonal antibody against elastin revealed very few elastic fibers between collagen bundles and in the vertical fiber tracts (perforating bundles), but a considerable amount in the tela subcutanea. This was partly confirmed at the ultrastructural level by tannic acid staining.

cAMP response element-binding protein-binding protein mediates thyrotropin-releasing hormone signaling on thyrotropin subunit genes.

Transcription of pituitary alpha-glycoprotein hormone subunit (alpha-GSU) and thyrotropin beta subunit (TSH-beta) genes is stimulated by thyrotropin-releasing hormone (TRH). Since cAMP response element-binding protein (CREB)-binding protein (CBP) integrates a number of cell signaling pathways, we investigated whether CBP is important for TRH stimulation of the TSH subunit genes. Cotransfection of E1A in GH(3) cells completely blocked TRH stimulation of the TSH subunit genes, suggesting that CBP is a key factor for TRH signaling in the pituitary. CBP and Pit-1 acted synergistically in TRH stimulation of the TSH-beta promoter, and amino acids 1-450 of CBP were sufficient for the TRH effect. In contrast, on the human alpha-GSU promoter, CREB and P-Lim mediated TRH signaling. Intriguingly, CREB was phosphorylated upon TRH stimulation, leading to CBP recruitment to the alpha-GSU promoter. CBP also interacted with P-Lim in a TRH-dependent manner, suggesting that P-Lim is an important factor for non-cAMP response element-mediated TRH stimulation of this promoter. Distinct domains of CBP were required for TRH signaling by CREB and P-Lim on the alpha-GSU promoter, amino acids 450-700 and 1-450, respectively. Thus, the amino terminus of CBP plays a critical role in TRH signaling in the anterior pituitary via both Pit-1-dependent and -independent pathways, yielding differential regulation of pituitary gene products.

Thyroid hormone-independent interaction between the thyroid hormone receptor beta2 amino terminus and coactivators.

Thyroid hormone receptors (TRs) mediate hormone action by binding to DNA response elements (TREs) and either activating or repressing gene expression in the presence of ligand, T(3). Coactivator recruitment to the AF-2 region of TR in the presence of T(3) is central to this process. The different TR isoforms, TR-beta1, TR-beta2, and TR-alpha1, share strong homology in their DNA- and ligand-binding domains but differ in their amino-terminal domains. Because TR-beta2 exhibits greater T(3)-independent activation on TREs than other TR isoforms, we wanted to determine whether coactivators bound to TR-beta2 in the absence of ligand. Our results show that TR-beta2, unlike TR-beta1 or TR-alpha1, is able to bind certain coactivators (CBP, SRC-1, and pCIP) in the absence of T(3) through a domain which maps to the amino-terminal half of its A/B domain. This interaction is specific for certain coactivators, as TR-beta2 does not interact with other co-factors (p120 or the CBP-associated factor (pCAF)) in the absence of T(3). The minimal TR-beta2 domain for coactivator binding is aa 21-50, although aa 1-50 are required for the full functional response. Thus, isoform-specific regulation by TRs may involve T(3)-independent coactivator recruitment to the transcription complex via the AF-1 domain.

CREB-independent regulation by CBP is a novel mechanism of human growth hormone gene expression.

Hypothalamic growth hormone-releasing hormone (GHRH) stimulates growth hormone (GH) gene expression in anterior pituitary somatotrophs by binding to the GHRH receptor, a G-protein-coupled transmembrane receptor, and by mediating a cAMP-mediated protein kinase A (PKA) signal-transduction pathway. Two nonclassical cAMP-response element motifs (CGTCA) are located at nucleotides -187/-183 (distal cAMP-response element; dCRE) and -99/-95 (proximal cAMP-response element; pCRE) of the human GH promoter and are required for cAMP responsiveness, along with the pituitary-specific transcription factor Pit-1 (official nomenclature, POU1F1). Although a role for cAMP-response element binding protein (CREB) in GH stimulation by PKA has been suggested, it is unclear how the effect may be mediated. CREB binding protein (CBP) is a nuclear cofactor named for its ability to bind CREB. However, CBP also binds other nuclear proteins. We determined that CBP interacts with Pit-1 and is a cofactor for Pit-1-dependent activation of the human GH promoter. This pathway appears to be independent of CREB, with CPB being the likely target of phosphorylation by PKA.

Disruption of myoglobin in mice induces multiple compensatory mechanisms.

Myoglobin may serve a variety of functions in muscular oxygen supply, such as O(2) storage, facilitated O(2) diffusion, and myoglobin-mediated oxidative phosphorylation. We studied the functional consequences of a myoglobin deficiency on cardiac function by producing myoglobin-knockout (myo(-/-)) mice. To genetically inactivate the myoglobin gene, exon 2 encoding the heme binding site was deleted in embryonic stem cells via homologous recombination. Myo(-/-) mice are viable, fertile, and without any obvious signs of functional limitations. Hemoglobin concentrations were significantly elevated in myo(-/-) mice. Cardiac function and energetics were analyzed in isolated perfused hearts under resting conditions and during beta-adrenergic stimulation with dobutamine. Myo(-/-) hearts showed no alteration in contractile parameters either under basal conditions or after maximal beta-adrenergic stimulation (200 nM dobutamine). Tissue levels of ATP, phosphocreatine ((31)P-NMR), and myocardial O(2) consumption were not altered. However, coronary flow [6.4 +/- 1.3 ml.min(-1).g(-1) [wild-type (WT)] vs. 8.5 +/- 2.4 ml.min(-1).g(-1) [myo(-/-)] [and coronary reserve [17.1 +/- 2.1 (WT) vs. 20.8 +/- 1.1 (myo(-/-) ml. min(-1).g(-1) were significantly elevated in myo(-/-) hearts. Histological examination revealed that capillary density also was increased in myo(-/-) hearts [3,111 +/- 400 mm(-2) (WT) vs. 4,140 +/- 140 mm(-2) (Myo(-/-)]. These data demonstrate that disruption of myoglobin results in the activation of multiple compensatory mechanisms that steepen the pO(2) gradient and reduce the diffusion path length for O(2) between capillary and the mitochondria; this suggests that myoglobin normally is important for the delivery of oxygen.

Defective retinoic acid regulation of the Pit-1 gene enhancer: a novel mechanism of combined pituitary hormone deficiency.

Pit-1 is a pituitary-specific transcription factor responsible for pituitary development and hormone expression in mammals. Pit-1 contains two protein domains, termed POU-specific and POU-homeo, which are both necessary for DNA binding and activation of the GH and PRL genes and regulation of the PRL, TSH-beta subunit (TSH-beta), and Pit-1 genes. Pit-1 is also necessary for retinoic acid induction of its own gene during development through a Pit-1-dependent enhancer. Combined pituitary hormone deficiency is caused by defective transactivation of target genes in the anterior pituitary. In the present report, we provide in vivo evidence that retinoic acid induction of the Pit-1 gene can be impaired by a Pit-1 gene mutation, suggesting a new molecular mechanism for combined pituitary hormone deficiency in man.

A novel mechanism for cyclic adenosine 3',5'-monophosphate regulation of gene expression by CREB-binding protein.

The pituitary-specific transcription factor, Pit-1, is necessary to mediate protein kinase A (PKA) regulation of the GH, PRL, and TSH-beta subunit genes in the pituitary. Since these target genes lack classical cAMP DNA response elements (CREs), the mechanism of this regulation was previously unknown. We show that CREB binding protein (CBP), through two cysteine-histidine rich domains (C/H1 and C/H3), specifically and constitutively interacts with Pit-1 in pituitary cells. Pit-1 and CBP synergistically activate the PRL gene after PKA stimulation in a mechanism requiring both an intact Pit-1 amino-terminal and DNA-binding domain. A CBP construct containing the C/H3 domain [amino acids (aa) 1678-2441], but not one lacking the C/H3 domain (aa 1891-2441), is sufficient to mediate this response. Neither construct augments PKA regulation of CRE-containing promoters. Fusion of either CBP fragment to the GAL4 DNA-binding domain transferred complete PKA regulation to a heterologous promoter. These findings provide a mechanism for CREB-independent regulation of gene expression by cAMP.

A unique role of the beta-2 thyroid hormone receptor isoform in negative regulation by thyroid hormone. Mapping of a novel amino-terminal domain important for ligand-independent activation.

Negative regulation by thyroid hormone is mediated by nuclear thyroid hormone receptors (TRs) acting on thyroid hormone response elements (TREs). We examine here the role of human TR-beta2, a TR isoform with central nervous system-restricted expression, in the regulation of target genes whose expression are decreased by triiodothyronine (T3). Using transient transfection studies, we found that TR-beta2 achieved significantly greater ligand-independent activation on the thyrotropin-releasing hormone (TRH) and common glycoprotein alpha-subunit genes than either TR-beta1 or TR-alpha1. A chimeric TR-beta isoform containing the TR-beta2 amino terminus linked to the TR-alpha1 DNA- and ligand-binding domains functioned like the TR-beta2 isoform on these promoters, confirming that the amino terminus of TR-beta2 was both necessary and sufficient to mediate this effect. By constructing deletion mutants of the TR-beta2 amino terminus, we demonstrate that amino acids 89-116 mediate this function. This domain, important in ligand-independent activation on negative TREs, is discrete from a previously described activation domain in the amino-terminal portion of TR-beta2. We conclude that the central nervous system-restricted TR-beta2 isoform has a unique effect on negative regulation by T3 that can be mapped to amino acids 89-116 of the amino terminus of the human TR-beta2.

Immunocytochemical localization of lysozyme in the nephrocytes of the harvestman, Leiobunum rotundum.

Nephrocytes are cells involved in the metabolism of hemolymph components and are characterized by peripheral finger-like projections bordering a labyrinthine channel system. The antibacterial protein lysozyme was localized in anterior and pericardial nephrocytes of the harvestman, Leiobunum rotundum, by biochemical and immuno-gold postembedding-labelling techniques. Lysozyme-activity was demonstrated in experimental animals, challenged by Micrococcus luteus Gram positive bacteria. With SDS-electrophoresis, lysozyme was proved to be present in nephrocytes adjoining the heart, before studies at the electron microscopical level were carried out. The opilionid lysozyme was found to have a molecular weight of 14,000 Da, thus belonging to the c-type lysozyme which is represented by hen-egg-white lysozyme. In untreated animals the presence of lysozyme could not be proved by electrophoresis. With immunogold labelling, however, lysozyme could be clearly localized in the vesicles of nephrocytes of untreated animals. Additional control experiments were carried out which revealed that the nephrocytes of L. rotundum in vivo are able to take up native lysozyme from an exterior medium. Therefore, the problem needs to be solved if nephrocyte vesicles transport lysozyme into or out of the cell.

Mechanical properties of the skin of Xenopus laevis (Anura, Amphibia).

The skin of the aquatic pipid frog, Xenopus laevis, was examined for specific biomechanical features: 1) thickness, 2) maximal strain at break (epsilon f), 3) tensile strength (sigma m), 4) modulus of elasticity (E, stiffness), and 5) the area under the stress-strain curve (W) (breaking energy, toughness). Skin freshly removed from dorsal, ventral, and lateral areas of the body was subjected to uniaxial tension. In both sexes, the dorsal skin is thicker than the ventral. The skin of male frogs was consistently thinner in all body regions than that of females. Most biomechanical parameters showed a considerable range of values in both males (epsilon f = 59-63%, sigma m = 15-16.5 MPa, E = 33.5-38.4 MPa, W = 3.8-4.5 MJ/m3) and females (epsilon f = 102-126%, sigma m = 11.5 MPa, E = 10.4-12 MPa, W = 5.2-6.7 MJ/m3). The disparate epsilon f values in males (low) and females (high) might reflect sexual dimorphism. Static stress-strain curves were typically J-shaped; with the exception of a "toe," the curves rose approximately linearly with increasing strain. The skin of X.laevis, although heterogeneous in structure, possesses features similar to those found in tissues with aligned collagen fibers such as tendons or fish skin. However, in anurans, the skin seems to play a more passive mechanical role during locomotion than in fish.

Seven-day administration of the gonadotropin-releasing hormone antagonist Cetrorelix in normal cycling women.

In contrast to gonadotropin-releasing hormone (GnRH) agonists, GnRH antagonists do not show any stimulatory effect on the pituitary but their clinical usage was precluded by severe side effects and high dose requirements. We report here on a 7-day treatment using the potent GnRH antagonist Cetrorelix ([Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]GnRH) on five women 23-33 years old. All women were ovulatory and were studied during three consecutive cycles: a control cycle, a treatment cycle and a post-treatment control cycle. Throughout the control cycles blood samples were obtained daily during cycle days 8-18 and on days 21 and 23 during the remainder of the control cycles. On the eighth day of the treatment cycle women were hospitalized at 07.00 h for 26 h. Repeated blood samples were drawn at 15-min intervals during the entire period. Subjects received 3 mg of Cetrorelix sc for the first time at 09.00 h on the eighth day of the cycle and daily at 08.00 h for the following 6 days. Blood samples were obtained daily over a period of 25 days and every third day throughout the remainder of the treatment cycle. Twenty-four hours after the first application of Cetrorelix, luteinizing hormone (LH) and estradiol were in the subnormal range and remained subnormal until the end of medication. The suppressive effect of Cetrorelix compared to pretreatment values lasted at least 6 days for LH and FSH and 11 days after the last Cetrorelix injection for estradiol.(ABSTRACT TRUNCATED AT 250 WORDS)

Nephrocytes of harvestmen, Leiobunum limbatum and L. rotundum.

Nephrocytes are said to be able to take up substances from the hemolymph. In Opiliones, which were examined electron microscopically three different types of nephrocytes were found. Numerous large nephrocytes lie clustered between the muscles in the anterior region of the body. Smaller nephrocytes occur scattered throughout the opilionid body, often affixed to tracheoles. The third group, pericardial cells, are always attached to the heart wall by connective ligaments. All nephrocytes are surrounded by a thick basement membrane and their plasma membrane forms pedicels. Junctional complexes link the adjacent pedicels to form diaphragm-like slit-membranes, which form the entrance to an extracellular compartment. The cytoplasm of the nephrocytes contains many pinocytotic vesicles and tubular elements. Different types of large electron-dense and electron-lucent vesicles can be distinguished. Occasionally a large bundle of unmyelinated nerve fibers is enclosed by a pericardial cell. Morphological differences between the types of nephrocytes are described and the ultrastructural characteristics of the nephrocytes of harvestmen are compared with those of other arthropods. Functional aspects are discussed with respect to their ultrafiltration structures.