Phosphatases as small-molecule targets: inhibiting the endogenous inhibitors of kinases.

Inositols and their phosphorylated derivatives, phosphoinositides, play an important role in diverse cellular functions. They have been recognized as second messengers and are accurately controlled by phosphatases and kinases, such as phosphoinositide 3-kinase and the phosphatidylinositol 3-phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10). Specific inhibitors targeting phosphoinositide 3-kinase and protein phosphatases have been described and characterized, but no small-molecule tools for phosphoinositide phosphatases are currently available. The present mini-review gives an overview of representative phosphatase inhibitors and summarizes the work that has been done recently on molecules that inhibit PTEN.

Primary cultured hepatocytes of the bony fish, Oreochromis mossambicus, the tilapia: a valid tool for physiological studies on IGF-I expression in liver.

In spite of the importance of IGF-I for growth and development, knowledge about regulation of its production in submammalian species is rather limited. In order to create a tool for investigation of direct regulatory effects on the expression of IGF-I in bony fish liver, a primary cell culture of hepatocytes from Oreochromis mossambicus, the tilapia, was established. The cells were viable for up to 3 days and IGF-I mRNA synthesis was detected by northern blot and semiquantitative reverse transcriptase (RT)-PCR. Northern blot analysis of the primary cultured hepatocytes revealed four different IGF-I transcripts, 0.5, 1.9, 3.9 and 6.0 kb in size, which were identical to those in liver tissue. However, the expression rate was weaker than that in liver. The direct effects of recombinant tilapia (rt) growth hormone (GH) and salmon (s) IGF-I on the expression of IGF-I in primary cultured hepatocytes were investigated in time-course and dose-response experiments. In untreated cultures, IGF-I mRNA decreased with time. Hepatocytes treated with 100 nM rtGH resulted in a pronounced stimulation of IGF-I mRNA expression throughout the experiment. Treatment with rtGH in concentrations ranging from 0.1 nM to 1 microM caused a clear dose-dependent increase in the amount of IGF-I mRNA. Significant stimulation was obtained even with 0.1 nM, reaching a plateau with 10 nM. Neither significant inhibitory nor stimulatory effects were detected by adding sIGF-I from 0.1 nM to 1 microM to the hepataocytes. Our results indicate that the established primary cell culture of tilapia hepatocytes is a useful system in which to study direct effects of potential regulators of bony fish liver cell function.

Effect of growth hormone and insulin-like growth factor I (IGF-I) on the expression of IGF-I messenger ribonucleic acid and peptide in rat tibial growth plate and articular chondrocytes in vivo.

Conflicting data exist as to whether insulin-like growth factor I (IGF-I) messenger RNA (mRNA) and peptide are expressed within chondrocytes. This question is pertinent to the mode of GH action on longitudinal bone growth. We have, therefore, investigated this issue in normal rats and in hypophysectomized rats treated for 24 h with GH or IGF-I using in situ hybridization and immunohistochemistry. Serum IGF-I, body weight, and tibial growth plate, but not articular cartilage, height increased with both treatments. Both IGF-I mRNA and IGF-I immunoreactivity occurred in all chondrocyte layers of growth plate and articular cartilage. The percentage of cells with IGF-I mRNA correlated well with IGF-I immunoreactivity under all experimental conditions. In normal rats, IGF-I expression was highest in the upper hypertrophic zone in growth plate (68-71%) and articular cartilage (32-34%). Hypophysectomy, GH, or IGF-I did not significantly affect this percentage. In the stem cell and proliferative and lower hypertrophic zones of growth plate, hypophysectomy dramatically reduced the percentage of labeled chondrocytes, and GH restored it. IGF-I increased IGF-I mRNA and immunoreactivity only in the proliferative zone. In articular cartilage, both remained unchanged under all experimental conditions. Together with our previous finding that GH infusion of hypophysectomized rats enhances chondrocyte maturation at all differentiation stages, the present results are compatible with the idea that IGF-I produced by all chondrocyte layers under the influence of GH mediates chondrocyte maturation and thus longitudinal bone growth in an autocrine/paracrine manner.

Insulin-like growth factor-I and -II in the ovary of a bony fish, Oreochromis mossambicus, the tilapia: in situ hybridisation, immunohistochemical localisation, Northern blot and cDNA sequences.

There is accumulating evidence that insulin-like growth factor (IGF)-I and IGF-II are present in the mammalian ovary but comparable studies on bony fish remain scarce. Thus, the present study aims to analyse several parameters of the IGFs in the ovary of a bony fish, the tilapia, (Oreochromis mossambicus). Molecular biological and morphological techniques were applied. The IGF-I and IGF-II cDNA sequences established from the ovary indicate that the same molecules are present in ovary and liver. Northern blot analysis revealed four IGF-I mRNA transcripts (6.0, 3.9, 1.9, 0.5 kb) and three IGF-II mRNA transcripts (5.0, 4.0, 2.0 kb) in ovary and liver. The amounts of IGF-I and IGF-II mRNA in the ovary were considerably high when compared to those in liver (IGF-I: 80.7%; IGF-II: 63.7%). The expression of IGF-I mRNA and IGF-II mRNA in the ovary were studied by in situ hybridisation and the peptides located by immunohistochemistry. The expression of IGF-I varied between the different developmental stages. Both IGF-I mRNA and IGF-I immunoreactivity were present in small oocytes. Moderate IGF-I expression and immunoreactivity occurred in granulosa cells of follicles at the lipid stage. A high IGF-I expression was observed in the granulosa and theca cells surrounding oocytes at the yolk globule stages and mature oocytes but neither IGF-I mRNA nor IGF-I immunoreactivity occurred in oocytes of the later stages. Thus, the IGF-I production seems to change from the young oocyte to the surrounding follicle cells at the later stages. In contrast, IGF-II mRNA and IGF-II-immunoreactivity occurred only in granulosa cells of the late follicle stages. The results suggest that both IGF-I and IGF-II are involved in the maturation of bony fish oocytes and in follicle development in a paracrine/autocrine manner. IGF-I and IGF-II may exert their effects at different stages of development. Furthermore, the intraovarian IGF-I and IGF-II systems seem to have a long phylogenetic history indicating the importance of the IGFs in reproductive biology.

Tissue engineering of a bioprosthetic heart valve: stimulation of extracellular matrix assessed by hydroxyproline assay.

Creation of an autologous heart valve by tissue engineering offers a promising approach to cardiac surgery. Although we have demonstrated successful formation of native valve analogous tissue in vitro, hemodynamic competence remains a serious problem. The aim of this study was to optimize in vitro formation of collagen as a precondition for mechanical stability of new tissue. Human myofibroblasts were seeded on square sheets of biodegradable scaffolds (control). To stimulate collagen production, one series was cultured with L-ascorbic acid 2-phosphate. In a second series, the seeded scaffolds were subjected to tension by mounting them on a frame. After 4 weeks of culture time, the collagen content of the different series was assessed by hydroxyproline assay. Light and scanning electron microscopy were performed. Hydroxyproline content of the framed scaffolds was 10 times higher than that of the control group (p < 0.05) and 6 times higher than in the unframed scaffolds grown with ascorbic acid (p < 0.05), respectively. Scanning electron microscopy proved extensive formation of solid tissue in the framed samples. These results demonstrate that supplementation of myofibroblast cultures with ascorbic acid, especially if grown on strained scaffolds, significantly increases collagen content, which is crucial for mechanical stability. This concept is a further step toward the creation of a hemodynamically competent autologous heart valve.

Tissue engineering in cardiovascular surgery: MTT, a rapid and reliable quantitative method to assess the optimal human cell seeding on polymeric meshes.

OBJECTIVE: Currently used valve substitutes for valve replacement have certain disadvantages that limit their long-term benefits such as poor durability, risks of infection, thromboembolism or rejection. A tissue engineered autologous valve composed of living tissue is expected to overcome these shortcomings with natural existing biological mechanisms for growth, repair, remodeling and development. The aim of the study was to improve cell seeding methods for developing tissue-engineered valve tissue. METHODS: Human aortic myofibroblasts were seeded on polyglycolic acid (PGA) meshes. Cell attachment and growth of myofibroblasts on the PGA scaffolds with different seeding intervals were compared to determine an optimal seeding interval. In addition, scanning electron microscopy study of the seeded meshes was also performed to document tissue development. RESULTS: There was a direct correlation between cell numbers assessed by direct counting and MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltertra-zoliu m bromide) assay. Both attach rate and cell growth seeded on meshes with long intervals (24 and 36 h) were significantly higher than those seeded with short intervals (2 and 12 h) (P<0.01), there was no significant difference between 24- and 36-h seeding interval. Scanning electron microscopy also documented more cell attachment with long seeding intervals resulting in a more solid tissue like structure. CONCLUSION: It is feasible to use human aortic myofibroblasts to develop a new functional tissue in vitro. Twenty-four hours is an optimal seeding interval for seeding human aortic myofibroblasts on PGA scaffolds and MTT test is a rapid and reliable quantitative method to assess the optimal human cell seeding on polymeric meshes.

Congenital subaortic stenosis by accessory mitral valve tissue, recognition and management.

Accessory mitral valve tissue as the single cause for left ventricular outflow tract obstruction is a very rare cardiac malformation in normally connected hearts. We report a case in which this condition was present as single cause for left ventricular outflow tract obstruction. The surgical technique is described and a review of the literature presented.

Molecular cloning and tissue expression of the insulin-like growth factor II prohormone in the bony fish Cottus scorpius.

The cDNA encoding pro-IGF-II of an advanced teleost fish, Cottus scorpius (Scorpaeniformes), the daddy sculpin, was isolated from liver by RT-PCR and molecular cloning. Like other IGFs, the deduced 168 amino acid peptide contains B-, C-, A-, D-, and E-domains and six cysteine residues (CysB9, CysB21, CysA6, CysA7, CysA11, and CysA20) necessary for the maintenance of tertiary structure. At the amino acid level, the sculpin IGF-II prohormone exhibits 85-92% homology to pro-IGF-II of other bony fish but only 51% homology to human. The mature sculpin IGF-II peptide comprises 70 amino acids. Its A-, B-, and D-domains exhibit homologies as high as 91, 91, and 100%, respectively, when compared with the other bony fish species studied. The high sequence homologies may indicate a particular physiological impact of IGF-II in bony fish. RT-PCR followed by Southern blotting revealed an IGF-II mRNA transcript of the expected size in liver, pyloric and splenic islets, stomach, small and large intestine, kidney, gill, testis, ovary, brain, and heart. The local production of IGF-II in many organs indicates that IGF-II is involved in organ-specific functions in a paracrine/autocrine manner. Furthermore, the results show that all bony fish organs which have been demonstrated to express IGF-I mRNA also express IGF-II mRNA.

IGF-I in the bony fish Cottus scorpius: cDNA, expression and differential localization in brain and islets.

The cDNA encoding prepro-insulin-like growth factor (IGF)-I of a teleost, Cottus scorpius, (Scorpaeniformes) was established from liver by RT-PCR and molecular cloning. Typically, the deduced 184 amino acid protein contains a signal peptide, B-, C-, A-, D- and E-domains and all residues necessary for maintenance of tertiary structure. C. scorpius IGF-I shares only approximately 57% identity with C. scorpius insulin in the A-domain and 7% in the B-domain. RT-PCR followed by Southern blotting revealed a transcript in liver, pancreatic islets, stomach, small and large intestine, kidney, gill, testis, ovary, heart and brain indicating paracrine/autocrine actions of locally produced IGF-I. IGF-I- and insulin-immunoreactivities coexisted in the islets, but did not in other sites such as brain. Thus, in contrast to other bony fish, sculpin insulin cells most probably produce IGF-I. The results also challenge the current model of the IGF/insulin evolution.

Dose relationships of morning bright white light in seasonal affective disorders (SAD).

Bright white full spectrum light (greater than 2500 lux) can improve depressive symptomatology in a selected group of patients with recurrent autumn and winter depression. This crossover study demonstrates that 0.5-h morning white light is not an effective treatment, whereas 2-h is.

Transcendental meditation, altered reality testing, and behavioral change: a case report.

This paper presents the case of a 39-year-old woman who, several weeks following initiation into transcendental meditation (TM), experienced altered reality testing and behavior. We discuss the course of this episode, present evidence for a causal relationship between her practive of TM and altered behavior, and discuss the appropriate treatment of such phenomena.