New aspects of signal transduction in the Xenopus laevis melanotrope cell.

Light and temperature stimuli act via various brain centers and neurochemical messengers on the pituitary melanotrope cells of Xenopus laevis to control distinct subcellular activities such as the biosynthesis, processing, and release of alpha-melanophore-stimulating hormone (alphaMSH). The melanotrope signal transduction involves the action of a large repertoire of neurotransmitter and neuropeptide receptors and the second messengers cAMP and Ca(2+). Here we briefly review this signaling mechanism and then present new data on two aspects of this process, viz. the presence of a stimulatory beta-adrenergic receptor acting via cAMP and the egress of cAMP from the melanotrope upon a change of alphaMSH release activity.

Role of the forkhead transcription family member, FKHR, in thymocyte differentiation.

While performing a large-scale analysis of mRNA transcripts in the murine thymus, our attention was drawn to the forkhead family transcription factor FKHR. Here we demonstrate that FKHR is expressed in thymocytes, most prominently in those that are undergoing positive selection. Interestingly, FKHR transcripts show a highly regionalized pattern of expression, concentrated in the innermost areas of the medulla. We define the FKHR binding site as (G/C)(A/C)N(G/a)T(A/c)AA(T/c) A(T/g)(T/g)(G/c), a sequence found in the regulatory elements of many genes, including certain that encode molecules crucial for thymocyte differentiation. To study the function of FKHR, we engineered mice expressing a dominant-negative mutant specifically in T cells in a tetracycline-regulatable fashion. In these animals, T cell differentiation appeared quite normal; however, total thymocyte numbers were decreased, owing to reductions in all four of the CD4/CD8 subsets, and incorporation of the thymidine analogue bromo-deoxyuridine was increased, again in all four subsets. These data suggest that, in thymocytes, FKHR may be involved in cell survival and/or cycling.

Critical size defect in the goat's os ilium. A model to evaluate bone grafts and substitutes.

Bone defects and their treatment are a well known problem in orthopaedic surgery. A critical size defect is a suitable model to study bone replacement materials. This study describes a critical size defect in the goal and the evaluation of three bone fillers (particulate autograft, particulate allograft, and a polyethylene oxide/polybutylene terephthalate copolymer) in this defect. The goat allows for implantation of large implants and has a metabolic rate more comparable with that of humans than small animals. The critical size defect, located in the goat's iliac wing, is easily reproducible and allows qualitative and quantitative evaluation of bone grafts and bone graft substitutes. After 3 months of healing, the unfilled defects showed 13.5% bone in the defect, the autografted defects 36.3%, and the allografted 18.5%. The copolymer gave only 1.5% bone in the defect; this is in contrast to previous reports. The described model allows for the evaluation of bone graft substitutes before introduction into clinical practice.

Comparison of the cytotoxicity of molybdenum as powder and as alloying element in a niobium-molybdenum alloy.

Commercially pure metal niobium (c.p. Nb) as well as niobium-molybdenum (Nb-Mo) alloys were produced following several powder metallurgical routes. In brief, niobium and molybdenum powders were blended and milled in order to form Nb-Mo alloys. The alloy powders and the c.p. Nb were then either pressed and sintered, or cold isostatically pressed followed by hot isostatically pressing. In order to assess the cytotoxicity of the c.p. Nb and c.p. Mo powders, a 72 h minimal essential medium-extraction test was performed according to ISO/EN 10993-5. The cytotoxicity of the c.p. Nb metal and the Nb-Mo alloys was tested in a 72 h direct contact test. Compared to a negative control (UHMWPE), c.p. Nb was non-toxic, but c.p. Mo was moderately toxic. None of the powder metallurgically produced materials were toxic. Neither differences in molybdenum concentration, nor in porosity of the samples, due to different production routes, had any influence on the toxicity of the materials. Rat bone marrow cultures showed that only on c.p. Nb was a mineralized extracellular matrix formed, while on the more porous Nb-Mo alloys, cell growth was observed, but no mineralization. In conclusion, c.p. Mo powder is moderately toxic, however, as an alloying element it is non-toxic. Material porosity seems to influence differentiation of bone tissue in vitro.

Application of porous PEO/PBT copolymers for bone replacement.

A range of polyethylene oxide/polybutylene terephthalate (PEO/PBT) copolymers (70-30% PEO) was investigated for nonloadbearing bone replacement application. Porous PEO/PBT cylinders (d = 5 mm, h = 7 mm) were implanted transcortically in the diaphyseal femur of 10 goats, and the animals were sacrificed at 3, 6, 9, 12, and 26 weeks. Qualitative evaluation was performed using light and fluorescence microscopy, scanning electron microscopy, and back-scatter electron imaging with an attached X-ray microanalysis system. The percentage of bone ingrowth and the percentage of bone contact in the pore region were quantitatively assessed using undecalcified histological sections. The hydrogel properties of the PEO/PBT copolymers provided a rapid closure of the defect upon press-fit implantation, due to postoperative water uptake and subsequent swelling behavior of the materials. Bridging of the defect by bone and the occurrence of bone bonding were observed 6 weeks postoperatively for the material with the highest PEO content (70/30). For the 60/40 and 55/45 PEO/PBT proportions, union of the defect and bone bonding were observed at 9 and 12 weeks, respectively. The stiffer 40/60 and 30/70 PEO/PBT implants showed bridging of the 5-mm gap after 12 weeks, but did not reveal bone bonding up to 26 weeks. Peripheral fragmentation, mainly in the marrow cavity, was found for the 70/30 material at 12 weeks and had increased at 26 weeks. Degradation was not seen for the other materials. The histomorphometrical data confirmed the microscopical observations and demonstrated a direct relation between PEO content in the PEO/PBT co-polymers, the rate of bone ingrowth, and the amount of bone contact. Porous PEO/PBT copolymers are degradable, bone-bonding elastomeric substrates with favorable handling properties and a high percentage of bone ingrowth (69-78 at 26 weeks). It was therefore concluded that PEO/ PBT copolymers are highly promising materials for bone-replacement surgery.

Inhibition of alpha-MSH secretion is associated with increased cyclic-AMP egress from the neurointermediate lobe of Xenopus laevis.

Cyclic-AMP is known to be released from cells and tissues and the amounts released have been reported to reflect intracellular cAMP levels. To measure cAMP release the phosphodiesterase inhibitor IBMX is often used to increase the amount of cAMP to a detectable level. Using this method to follow cAMP dynamics of melanotrope cells in the neurointermediate pituitary lobe of the amphibian Xenopus laevis we show that the alpha-MSH secreto-inhibitors baclofen (GABAB receptor agonist) and dopamine inhibit cAMP release, confirming the idea that these factors inhibit alpha-MSH secretion by reducing adenylyl cyclase activity. Using a sensitive cAMP radioimmunoassay we were able to measure cAMP release from Xenopus neurointermediate lobes in the absence of IBMX. Both baclofen and dopamine appeared to inhibit alpha-MSH secretion but strongly stimulated the release of cAMP. This indicates that the extracellular cAMP level is not a reliable parameter to measure the intracellular cAMP level in the absence of IBMX. The data furthermore suggest that cAMP release is a physiologically regulated process, which might be involved in lowering intracellular cAMP levels associated with a cellular secretory compartment. No apparent differences could be found in the lobe content of cAMP at the termination of secreto-inhibitor treatment, leading to the idea that the cAMP compartment associated with secretion in small relative to the total amount of cAMP present in the lobe.

Bone-bonding behaviour of poly(ethylene oxide)-polybutylene terephthalate copolymer coatings and bulk implants: a comparative study.

A range of poly(ethylene oxide)-polybutylene terephthalate (PEO-PBT) copolymers (70-30% PEO), both as coating on titanium alloy as well as bulk cylinders, was press-fit implanted in the diaphyseal femur of 16 goats. At early survival times (4 wk), a high degree of cortical bone contact was observed for bulk implants using light microscopy and this was confirmed by backscatter electron microscopy. This was attributed to the swelling behaviour of PEO-PBT copolymers. At this stage, bone contact was also revealed for coated implants, but to a lesser extent. At a later stage (12 wk), bone bonding was demonstrated both morphologically and by X-ray microanalysis, at the interface of 70:30 PEO-PBT bulk as well as 70:30 PEO-PBT-coated implants. Bone bonding increased with time (26 and 52 wk) for this PEO-PBT proportion and was also observed for 60:40 and 55:45 implants, although less frequently. For 40:60 and 30:70 PEO-PBT proportions, bone bonding was not shown. Based on these qualitative data, it was not possible to differentiate between coated and bulk implants with respect to bone bonding. This study demonstrated that the application of PEO-PBT elastomers as coatings does not alter the bone-bonding properties. It was therefore concluded that PEO-PBT coatings are beneficial over the bone-bonding but brittle ceramic coatings, due to their flexibility. In addition, the bone-bonding capacities of these PEO-PBT coatings surpass the non-bonding behaviour of currently available flexible coatings.

Crystal structure of the adenovirus DNA binding protein reveals a hook-on model for cooperative DNA binding.

The adenovirus single-stranded DNA binding protein (Ad DBP) is a multifunctional protein required, amongst other things, for DNA replication and transcription control. It binds to single- and double-stranded DNA, as well as to RNA, in a sequence-independent manner. Like other single-stranded DNA binding proteins, it binds ssDNA, cooperatively. We report the crystal structure, at 2.6 A resolution, of the nucleic acid binding domain. This domain is active in DNA replication. The protein contains two zinc atoms in different, novel coordinations. The zinc atoms appear to be required for the stability of the protein fold rather than being involved in direct contacts with the DNA. The crystal structure shows that the protein contains a 17 amino acid C-terminal extension which hooks onto a second molecule, thereby forming a protein chain. Deletion of this C-terminal arm reduces cooperativity in DNA binding, suggesting a hook-on model for cooperativity. Based on this structural work and mutant studies, we propose that DBP forms a protein core around which the single-stranded DNA winds.

Interface reactions to PEO/PBT copolymers (Polyactive) after implantation in cortical bone.

The bone reaction at the interface of a range of PEO/PBT copolymers (Polyactive) after press-fit implantation in the diaphyseal femur of 20 goats was investigated. The animals were sacrificed at 1, 4, 12, 26, and 52 weeks. Undecalcified histologic sections were prepared, evaluated qualitatively, and quantified with the aid of an image analysis system. The percentage of bone contact was determined morphometrically for all PEO/PBT proportions; for the 70/30 ratio, the percentage of calcification of the material surface and of bone-bonding (defined as a continuum at the light microscopic level between calcification within the material surface and bone tissue) were also assessed. PEO/PBT copolymers possess hydrogelic properties, and a direct relation has been established between water-uptake and PEO content. The swelling behavior of all PEO/PBT substrates resulted in a high degree of bone contact (over 95%) at early stages. From the 12-week survival time onward the 70/30 revealed higher percentages of bone contact (over 80%) when compared with the other ratios (60/40, 55/45, 40/60, and 30/70). This difference was statistically confirmed 1 year postoperatively. The materials with the higher PEO contents showed the highest amounts of calcification, which is reflected by the rate of bone-bonding (70% of bone-bonding for the 70/30 at 52 weeks). Calcification and bone-bonding were not observed for 40/60 and 30/70 proportions. The quantitative results confirmed the relation between PEO content, water-uptake, calcification, and bone-bonding. It was furthermore demonstrated that PEO/PBT copolymers initially establish a high degree of bone contact, whereas the bone-bonding properties, especially of the 70/30, accomplish a long-term implant fixation.

Interfacial behavior of PEO/PBT copolymers (Polyactive) in a calvarial system: an in vitro study.

Polyactive, a polyethylene oxide/polybutylene terephthalate (PEO/PBT) copolymer, has been reported to display bone-bonding behavior. Although a detailed description of the in vivo bone/Polyactive interface is available, the underlying bone-bonding mechanism is still largely unknown. In this in vitro study, a calvarial envelope method has been adopted to reproduce the in vivo bone-bonding phenomenon and subsequently to obtain information on the biological effect of varying PEO/PBT segment ratios. The following PEO/PBT ratios were examined: 70/30, 60/40, 55/45, 40/60, and 30/70. Light microscopy (LM) and scanning (SEM), transmission (TEM), and backscatter electron microscopy (BSE), as well as X-ray microanalysis (XRMA), were employed. Within the period of analysis (3 weeks), an intimate contact between mineralized deposition and the 70/30, 60/40, and, to a lesser extent, the 55/45 surface was observed. Calcified areas developed within the surface of these PEO/BPT proportions during the culture period. Needle-shaped crystals from the mineralized tissue compartment and from calcified areas within the materials surface were intermingled at the interface, providing a morphologic continuity. A cellular layer was interposed with the mineralization front and the noncalcified 40/60 and 30/70 substrates. Apparently, the percentage of PEO is important for calcification within the near surface of the polymer. This relation is such that the higher the PEO content in PEO/PBT ratios, the more rapid the calcification. The occurrence of material calcification is considered to be largely responsible for the subsequent interfacial interactions. The calvarial envelope culture method allows not only reproduction of the in vivo bone/Polyactive interface, but also a relatively rapid differentiation within the range of PEO/PBT ratios.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of coexisting dopamine, GABA and NPY on alpha-MSH secretion from melanotrope cells of Xenopus laevis.

The secretion of alpha-MSH from the intermediate lobe of the pituitary gland of the amphibian Xenopus laevis is under complex neural control. Three neurotransmitters, dopamine, GABA and NPY, coexist in nerve terminals that contact the melanotrope cells. All three neurotransmitters inhibit alpha-MSH release. We have investigated the significance of this neurotransmitter coexistence for the regulation of alpha-MSH release, using an in vitro superfusion system. From experiments where lobes were treated with various combinations of receptor agonists we conclude that the transmitters act in an additive way but have clear, differential actions. Inhibition of secretion by either dopamine, isoguvacine (GABAA receptor agonist) or baclofen (GABAB receptor agonist) occurs rapidly and alpha-MSH secretion rapidly returns when treatment is terminated (recovery from baclofen being relatively fast, that from dopamine relatively slow); in contrast, inhibition by NPY and recovery from NPY-induced inhibition occurs only very slowly. Differential effects of the transmitters were also seen in experiments with 8-bromo-cyclic AMP, which strongly stimulates alpha-MSH secretion from isoguvacine- or baclofen-treated lobes, but is relatively ineffective in stimulating secretion from lobes treated with dopamine or NPY. NPY, furthermore, enables a short phasic stimulation of secretion by isoguvacine and attenuates the inhibitory action of dopamine and baclofen. Altogether it is concluded that the coexisting factors differentially affect the secretory process of the melanotrope cells of Xenopus laevis. NPY has a slow, sustained action whereas dopamine and GABA act fast.

The CRF-related peptide sauvagine stimulates and the GABAB receptor agonist baclofen inhibits cyclic-AMP production in melanotrope cells of Xenopus laevis.

Release of alpha-MSH from the pars intermedia melanotrope cells of Xenopus laevis is regulated by various classical neurotransmitters and neuropeptides. We have examined the effect of two of these regulatory substances, the neurotransmitter GABA and the CRF-related peptide sauvagine, on the adenylate cyclase system of the melanotrope cells. Sauvagine treatment, which stimulates alpha-MSH release, lead to an elevation in the level of cyclic-AMP, an effect which was potentiated by cholera toxin. Treatment with baclofen, a GABAB receptor agonist, gave a pertussis toxin-sensitive decrease in the cyclic-AMP level and an inhibition of alpha-MSH release. We conclude that sauvagine stimulates alpha-MSH secretion through activation of adenylate cyclase and that GABAB receptor activation inhibits secretion through inhibition of cyclic-AMP production. Baclofen treatment sensitized melanotrope cells to the stimulatory action of 8-bromo-cyclic-AMP on the secretion of alpha-MSH. This observation supports the conclusion that GABAB receptor activation inhibits cyclic-AMP production.