High intensity training for faster water polo.

AIM: The aim of this study, based on the interaction between two aerobic and anaerobic metabolisms with a parallel production of both aerobic and anaerobic ATP, was to develop a high intensity training programme and increase the aerobic contribution. We examined the applicability of a 16-week training programme with an ergospirometer treadmill and field tests on eight water polo players. METHODS: Tests/retests of repeated exercises to 90V (90% of maximum personal speed over 100 m freestyle) and Speed Endurance Training (SET) after eight weeks were developed. A one-way blocked ANOVA with random blocks was used and each player represented a particular block with two before-after treatments with the aim of reducing error by subtracting both the variance due to the difference between the treatments and that due to the difference between the blocks. RESULTS: A reduction (15.2%) in blood lactate was observed in response to the same absolute workload (before-after). Furthermore the anaerobic contribution to VO2max (ESCAna, Estimated Anaerobic Contribution) after eight weeks of training at 90maxV and the anaerobic contribution to VO2max (ESCAna) after speed endurance training (SET) were very significant (P<0.004) with a reduction in the anaerobic contribution of 16%. The results of the field tests show that there was a very significant reduction (P<0.001) in lactate between 90maxV and maximal aerobic power velocity (MAPv) of 24%. CONCLUSION: With 90maxV and SET, space was gained towards those velocities, which had previously required a considerable anaerobic contribution. In this way match speed was increased.

beta-catenin mediates insulin-like growth factor-I actions to promote cyclin D1 mRNA expression, cell proliferation and survival in oligodendroglial cultures.

By promoting cell proliferation, survival and maturation insulin-like growth factor (IGF)-I is essential to the normal growth and development of the central nervous system. It is clear that IGF-I actions are primarily mediated by the type I IGF receptor (IGF1R), and that phosphoinositide 3 (PI3)-Akt kinases and MAP kinases signal many of IGF-I-IGF1R actions in neural cells, including oligodendrocyte lineage cells. The precise downstream targets of these signaling pathways, however, remain to be defined. We studied oligodendroglial cells to determine whether beta-catenin, a molecule that is a downstream target of glycogen synthase kinase-3beta (GSK3beta) and plays a key role in the Wnt canonical signaling pathway, mediates IGF-I actions. We found that IGF-I increases beta-catenin protein abundance within an hour after IGF-I-induced phosphorylation of Akt and GSK3beta. Inhibiting the PI3-Akt pathway suppressed IGF-I-induced increases in beta-catenin and cyclin D1 mRNA, while suppression of GSK3beta activity simulated IGF-I actions. Knocking-down beta-catenin mRNA by RNA interference suppressed IGF-I-stimulated increases in the abundance of cyclin D1 mRNA, cell proliferation, and cell survival. Our data suggest that beta-catenin is an important downstream molecule in the PI3-Akt-GSK3beta pathway, and as such it mediates IGF-I upregulation of cyclin D1 mRNA and promotion of cell proliferation and survival in oligodendroglial cells.

Histone deacetylase 11 regulates oligodendrocyte-specific gene expression and cell development in OL-1 oligodendroglia cells.

Both in vivo and in vitro studies indicate a correlation between reduced acetylation of histone core proteins and oligodendrocyte development. The nature of these histone modifications and the mechanisms mediating them remain undefined. To address these issues, we utilized OL-1 cells, a rat nontransformed oligodendrocyte cell line, and primary oligodendrocyte cultures. We found that the acetylated histone H3 at lysine 9 and lysine 14 (H3K9/K14ac) is reduced in both the myelin basic protein (MBP) and proteolipid protein (PLP) genes of maturing oligodendroglial OL-1 cells, and furthermore, this temporally correlates with increases in MBP, PLP, and histone deacetylase (HDAC) 11 expression. Disruption of developmentally-regulated histone H3 deacetylation within the MBP and PLP genes by the HDAC inhibitor trichostatin A blunts MBP and PLP expression. With its increased expression, interaction of HDAC 11 with acetylated histone H3 and recruitment of HDAC 11 to the MBP and PLP genes markedly increases in maturing OL-1 cells. Moreover, suppressing HDAC 11 expression with small interfering RNA significantly (1) increases H3K9/K14ac globally and within the MBP and PLP genes, (2) decreases MBP and PLP mRNA expression, and (3) blunts the morphological changes associated with oligodendrocyte development. Our data strongly support a specific role for HDAC 11 in histone deacetylation and in turn the regulation of oligodendrocyte-specific protein gene expression and oligodendrocyte development.

Type 1 insulin-like growth factor receptor signaling is essential for the development of the hippocampal formation and dentate gyrus.

Type 1 insulin-like growth factor receptor (IGF1R) signaling in neuronal development was studied in mutant mice with blunted igf1r gene expression in nestin-expressing neuronal precursors. At birth [postnatal (P) day 0] brain weights were reduced to 37% and 56% of controls in mice homozygous (nes-igf1r(-/-)) and heterozygous (nes-igf1r(-/Wt)) for the null mutation, respectively, and this brain growth retardation persisted postnatally. Stereological analysis demonstrated that the volumes of the hippocampal formation, CA fields 1-3, dentate gyrus (DG), and DG granule cell layer (GCL) were decreased by 44-54% at P0 and further by 65-69% at P90 in nes-igf1r(-/Wt) mice. In nes-igf1r(-/-) mice, volumes were 29-31% of controls at P0 and, in the two mice that survived to P90, 6-19% of controls, although the hilus could not be identified. Neuron density did not differ among the mice at any age studied; therefore, decreased volumes were due to reduced cell number. In postnatal nes-igf1r(-/Wt) mice, the percentage of apoptotic cells, as judged by activated caspase-3 immunostaining, was increased by 3.5-5.3-fold. The total number of proliferating DG progenitors (labeled by BrdU incorporation and Ki67 staining) was reduced by approximately 50%, but the percentage of these cells was similar to the percentages in littermate controls. These findings suggest that 1) the postnatal reduction in DG size is due predominantly to cell death, pointing to the importance of the IGF1R in regulating postnatal apoptosis, 2) surviving DG progenitors remain capable of proliferation despite reduced IGF1R expression, and 3) IGF1R signaling is necessary for normal embryonic brain development.

Cellular localization of somatomedin (insulin-like growth factor) messenger RNA in the human fetus.

The somatomedins or insulin-like growth factors (IGFs) are synthesized in many organs and tissues, but the specific cells that synthesize them in vivo have not been defined. By in situ hybridization histochemistry, IGF I (somatomedin C) and IGF II messenger RNAs were localized to connective tissues or cells of mesenchymal origin in 14 organs and tissues from human fetuses. IGF messenger RNAs were localized to perisinusoidal cells of liver, to perichondrium of cartilage, to sclera of eye, and to connective tissue layers, sheaths, septa, and capsules of each organ and tissue. All of the hybridizing regions are comprised predominantly of fibroblasts or other cells of mesenchymal origin. Because these cells are widely distributed and anatomically integrated into tissues and organs, they are ideally located for production of IGFs, which may exert paracrine effects on nearby target cells.

Expanding the mind: insulin-like growth factor I and brain development.

Signaling through the type 1 IGF receptor (IGF1R) after interaction with IGF-I is crucial to the normal brain development. Manipulations of the mouse genome leading to changes in the expression of IGF-I or IGF1R significantly alters brain growth, such that IGF-I overexpression leads to brain overgrowth, whereas null mutations in either IGF-I or the IGF1R result in brain growth retardation. IGF-I signaling stimulates the proliferation, survival, and differentiation of each of the major neural lineages, neurons, oligodendrocytes, and astrocytes, as well as possibly influencing neural stem cells. During embryonic life, IGF-I stimulates neuron progenitor proliferation, whereas later it promotes neuron survival, neuritic outgrowth, and synaptogenesis. IGF-I also stimulates oligodendrocyte progenitor proliferation although inhibiting apoptosis in oligodendrocyte lineage cells and stimulating myelin production. These pleiotropic IGF-I activities indicate that other factors provide instructive signals for specific cellular events and that IGF-I acts to facilitate them. Studies of the few humans with IGF-I and/or IGF1R gene mutations indicate that IGF-I serves a similar role in man.

Insulin-like growth factors as intraovarian regulators of granulosa cell growth and function.

A relatively large body of evidence now appears to support the existence of the essential ingredients for novel intraovarian IGF-driven control mechanisms. Indeed, evidence presented in this communication is in keeping with the possibility that the granulosa cell may be the site of IGF production, reception, and action. Although the relevance of IGFs to ovarian cell types other than the granulosa cell is largely unknown, one cannot at the present time exclude the possibility of nongranulosa cell contributions to intraovarian IGF production, reception, and action. Indeed, preliminary affinity cross-linking studies (Adashi, Resnick, Svoboda, Van Wyk and D'Ercole; unpublished data) suggest the existence of type-I and type-II receptors in nongranulosa cell compartments. The above notwithstanding, IGFs of granulosa (and possibly circulatory) origins may interact with granulosa cell autoreceptors either independently or in synergy with other granulosa cell agonists. According to this view, IGFs may act in the autocrine mode to stimulate granulosa cell replication on the one hand and promote granulosa cell differentiation on the other. Although proliferation and terminal differentiation may prove mutually exclusive under some circumstances, coexistence of the two processes is being increasingly recognized. In this context, some studies of porcine granulosa cells support a dual role for IGFs in granulosa cell ontogeny. As such, the IGFs can be added to a growing list of growth factors known to modulate granulosa cell growth and function, including EGF, PDGF, and FGF. Our findings indicate that Sm-C/IGF-I synergizes with FSH in the induction of rat granulosa cell aromatase activity at nanomolar concentrations compatible with its granulosa cell receptor binding affinity (thus far studied only in porcine cells. A role for Sm-C/IGF-I in the regulation of this key granulosa cell function would be in keeping with the possibility that Sm-C/IGF-I may partake in the assertion and maintenance of dominance by the selected follicle(s) or in promoting juvenile and early follicular development. Moreover, the ability of Sm-C/IGF-I to potentiate this and other FSH-driven ovarian functions may also account, at least in part, for the puberty-promoting effect of growth hormone. This permissive action of growth hormone has been initially suggested by observation in growth hormone-deficient rats, mice (dwarf mutants, and humans (sporadic, hereditary or acquired growth hormone deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)

Developmental expression of histone deacetylase 11 in the murine brain.

Recent studies indicate that neural cell development in the central nervous system (CNS) correlates with a reduction in acetylation of histone core proteins. Moreover, histone hypoacetylation is thought to be important to oligodendrocyte lineage development. The mechanisms mediating the reduction in acetylation during postnatal neural development remain to be defined. To begin to understand these mechanisms, we investigated the expression of histone deacetylase 11 (HDAC11), a newly identified HDAC, in mouse brain during postnatal development. We show that HDAC11 was widely expressed in the brain and that this expression gradually increased in a region-specific pattern between birth and 4 weeks of age. At the cellular level HDAC11 protein was predominately localized in the nuclei of mature oligodendrocytes but only minimally in astrocytes. Although dentate gyrus granule neurons abundantly expressed HDAC11, granule neuron precursors in the subgranule layer exhibited little HDAC11 immunoreactivity. Double-immunostaining of the corpus callosum and dentate gyrus demonstrated that HDAC11 and Ki67, a cell-proliferating marker, are rarely colocalized in same cells. Our data show that HDAC11 was expressed in the developing brain in a temporal and spatial pattern that correlates with the maturation of neural cells, including cells of the oligodendrocyte lineage. These findings support a role for HDAC11 in CNS histone deacetylation and the development of oligodendrocytes and neurons during postnatal development.

Photorefractive solitons of arbitrary and controllable linear polarization determined by the local bias field.

We discuss and experimentally demonstrate a scheme to achieve photorefractive solitons of arbitrary linear polarization using the quadratic electro-optic effect and describe the observation of the self-trapping of a set of linear polarized beams in different positions of a paraelectric photorefractive crystal of potassium-lithium-tantalate-niobate (KLTN) biased by the inhomogeneous field produced by two miniaturized top electrodes. The polarization of the single solitons of the set is determined by the local electrostatic configuration and the underlying tunable anisotropy, which is detected through zero-field electro-activation.

IGF-I receptor inhibition combined with rapamycin or temsirolimus inhibits neuroblastoma cell growth.

BACKGROUND: Neuroblastoma is the third most common solid tumor in children. Treatment continues to be challenging. The pathogenesis of neuroblastoma has been related to expression of the type 1 insulin-like growth factor receptor (IGF1R) and to transcription factor MYC-N amplification. Previous studies have shown that MYC-N expression is disrupted by blockade of the IGF1R with a specific monoclonal antibody, alphaIR3. Inhibition of IGF1R signaling can be accomplished by other agents, including rapamycin or temsirolimus, which target mTOR (mammalian target of rapamycin). MATERIALS AND METHODS: BE-2(c) and IMR-32 neuroblastoma cell lines were treated with varying concentrations of alphaIR3, rapamycin and temsirolimus alone or in combination and the viable cells were counted. RESULTS: Blockade of IGF1R signaling significantly inhibited cell growth as compared to untreated controls (p < 0.05), and a combination of agents was more effective than each agent alone. CONCLUSION: The combination of rapamycin or temsirolimus with alphaIR3 blocks the IGF1R signaling pathway and has an antiproliferative effect on neuroblastoma cells warranting further investigations using inhibitors of IGF1R signaling as novel combination therapy for neuroblastoma.

Affinity-labeled plasma somatomedin-C/insulinlike growth factor I binding proteins. Evidence of growth hormone dependence and subunit structure.

By using disuccinimidyl suberate, we have covalently cross-linked 125I-labeled somatomedin-C (Sm-C)/insulinlike growth factor I to specific binding proteins in human plasma. In unfractionated plasma samples from normal and acromegalic donors, 125I-Sm-C binding-protein complexes with relative molecular weights (Mr) of 160,000, 135,000, 110,000, 80,000, 50,000, 43,000-35,000, and 28,000-24,000 were consistently observed. In contrast, the 43,000-35,000-mol wt species were frequently the only specific complexes observed in hypopituitary plasma and were consistently more intensely labeled in such samples. Reduction of samples with beta-mercaptoethanol did not alter the electrophoretic pattern of these 125I-Sm-C binding-protein complexes. All Sm-C binding proteins, with the exception of the 43,000-35,000-mol wt complex, were adsorbed by concanavalin A-Sepharose. When acromegalic or normal plasma was fractionated on a Sephadex G-200 column and affinity labeled, the same complexes that were adsorbed by concanavalin A were found in fractions that eluted near the gamma-globulin peak. On the other hand, the 43,000-35,000-mol wt complex consistently eluted in size-appropriate fractions near the albumin peak. These data suggest that the growth hormone (GH)-dependent Sm-C binding protein, represented by the 160,000-mol wt complex, is in some way composed of smaller species, i.e., the 135,000-, 110,000-, 80,000-, 50,000-, and 28,000-24,000-mol wt complexes. Acid incubation of plasma prior to Sephadex G-200 chromatography results in the elimination of specific 125I-Sm-C binding-protein complexes which elute near gamma-globulin and a concurrent increase in the labeling intensity of the 28,000-24,000-mol wt complexes. We speculate, therefore, that each of the GH-dependent Sm-C binding-protein complexes represents an oligomer composed of 28,000-24,000-mol wt protomers. The 43,000-35,000-mol wt species is not dependent upon GH and appears to represent a different type of Sm-C binding protein.

Delayed fetal pulmonary maturation in a rabbit model of the diabetic pregnancy.

A rabbit model for the diabetic pregnancy was used to investigate the etiology of delayed pulmonary maturation observed in infants of diabetic mothers. Pregnant rabbit does were made glucose intolerant and insulinopenic by injection of alloxan, a pancreatic beta-cell cytotoxin. At 28 d (term approximately 31 d) fetuses of these animals were hyperglycemic, but were not hyperinsulinemic and did not demonstrate tissue overgrowth. Fetal pulmonary maturity was assessed by measurement of pressure-volume relationships on the fetal lungs. Fetuses of glucose-intolerant does demonstrated less retention of air on deflation. Phospholipid components of pulmonary surfactant were assayed on fluid obtained from lavage of the fetal lungs. Levels of disaturated phosphatidylcholine (per-cent total-lavage phospholipids) were diminished in fetuses of glucose-intolerant does compared to control fetuses (20.5 +/- 4.2 vs. 38.0 +/ 4.3%; P less than 0.01). Lecithin/sphingomyelin ratios were similar in both groups and phosphatidylglycerol was not detected in either group. There was a direct correlation between the percentage of alveolar disaturated phosphatidylcholine and retention of air on lung deflation. These findings suggest that in this model pulmonary instability was a result of diminished alveolar disaturated phosphatidylcholine, and this diminution did not result from fetal hyperinsulinemia.

Estimation of somatomedin-C levels in normals and patients with pituitary disease by radioimmunoassay.

The development of a radioimmunoassay for somatomedin-C has for the first time made it possible to discriminate between serum concentrations of a single peptide or closely related group of peptides and the net somatomedin activity measured by less specific bioassay and radioreceptor techniques. Antibodies to human somatomedin-C were raised in rabbits using a somatomedin-C ovalbumin complex as the antigen. A variety of peptide hormones at concentrations up to 1 muM are not recognized by the antibody. Insulin at concentrations >0.1 muM cross reacts in a non-parallel fashion; purified somatomedin-A is only 3% as active as somatomedin-C; and radiolabeled cloned rat liver multiplication stimulating activity does not bind to the antibody. Immunoreactive somatomedin-C can also be quantitated in the sera of a variety of subhuman species. Unusual assay kinetics, which are manifest when reactants are incubated under classic "equilibrium" assay conditions, appear to result from the failure of (125)I-somatomedin-C to readily equilibrate with the somatomedin-C serum binding protein complex. It is, therefore, necessary to use nonequilibrium assay conditions to quantitate somatomedin-C in serum. With this assay it is possible to detect somatomedin-C in normal subjects using as little as 0.25 mul of unextracted serum. Serum somatomedin-C concentrations in normal subjects were lowest in cord blood and rose rapidly during the first 4 yr of life to near adult levels. In 23 normal adult volunteers, the mean serum somatomedin-C concentration was 1.50+/-0.10 U/ml (SEM) when compared to a pooled adult serum standard. 19 children with hypopituitary dwarfism had concentrations below 0.20 U/ml. 17 of these were below 0.1 U/ml, the lower limit of sensitivity of the assay. The mean concentration in 14 adults with active acromegaly was 6.28+/-0.37 U/ml (SEM), five times greater than the normal volunteers. Significant increases in serum somatomedin-C concentrations were observed in 8 of 10 hypopituitary children within 72 h after the parenteral administration of human growth hormone. Three patients with Cushing's disease had elevated serum somatomedin-C concentrations (2.61+/-0.14 U/ml [SEM]). Three patients with hyperprolactinemia had normal concentrations (1.74+/-0.11 U/ml [SEM]).The important new discovery brought to light by quantitation of immunoassayable somatomedin in patient sera is that all previously used assays detect, in addition to somatomedin-C, serum substances that are not under as stringent growth hormone control.

Cultured fibroblast monolayers secrete a protein that alters the cellular binding of somatomedin-C/insulinlike growth factor I.

We studied somatomedin-C/insulinlike growth factor (Sm-C/IGF-I) binding to human fibroblasts in both adherent monolayers and in suspension cultures. The addition of Sm-C/IGF-I in concentrations between 0.5 and 10 ng/ml to monolayers cultures resulted in a paradoxical increase in 125I-Sm-C/IGF-I binding and concentrations between 25 and 300 ng/ml were required to displace the labeled peptide. The addition of unlabeled insulin resulted in no displacement of labeled Sm-C/IGF-I from the adherent cells. When fibroblast suspensions were used Sm-C/IGF-I concentrations between 1 and 10 ng/ml caused displacement, the paradoxical increase in 125I-Sm-C/IGF-I binding was not detected, and insulin displaced 60% of the labeled peptide. Affinity cross-linking to fibroblast monolayers revealed a 43,000-mol wt 125I-Sm-C-binding-protein complex that was not detected after cross-linking to suspended cells. The 43,000-mol wt complex was not detected after cross-linking to smooth muscle cell monolayers, and binding studies showed that 125I-Sm-C/IGF-I was displaced greater than 90% by Sm-C/IGF-I using concentrations between 0.5 and 10 ng/ml. Because fibroblast-conditioned medium contains the 43,000-mol wt complex, smooth muscle cells were incubated with conditioned medium for 24 h prior to initiation of the binding studies. 125I-Sm-C/IGF-I-binding increased 1.6-fold compared to control cultures and after cross-linking the 43,000-mol wt complex could be detected on the smooth muscle cell surface. Human fibroblast monolayers secrete a protein that binds 125I-Sm-C/IGF-I which can be transferred to the smooth muscle cell surface and alters 125I-Sm-C/IGF-I binding.

Insulin-like growth factor-I (IGF-I) inhibits neuronal apoptosis in the developing cerebral cortex in vivo.

Increased expression of insulin-like growth factor-I (IGF-I) in embryonic neural progenitors in vivo has been shown to accelerate neuron proliferation in the neocortex. In the present study, the in vivo actions of (IGF-I) on naturally occurring neuron death in the cerebral cortex were investigated during embryonic and early postnatal development in a line of transgenic (Tg) mice that overexpress IGF-I in the brain, directed by nestin genomic regulatory elements, beginning at least as early as embryonic day (E) 13. The areal density of apoptotic cells (N(A), cells/mm2) at E16 in the telencephalic wall of Tg and littermate control embryos was determined by immunostaining with an antibody specific for activated caspase-3. Stereological analyses were conducted to measure the numerical density (N(V), cells/mm3) and total number of immunoreactive apoptotic cells in the cerebral cortex of nestin/IGF-I Tg and control mice at postnatal days (P) 0 and 5. The volume of cerebral cortex and both the N(V) and total number of all cortical neurons also were determined in both cerebral hemispheres at P0, P5 and P270. Apoptotic cells were rare in the embryonic telencephalic wall at E16. However, the overall N(A) of apoptotic cells was found to be significantly less by 46% in Tg embryos. The volume of the cerebral cortex was significantly greater in Tg mice at P0 (30%), P5 (13%) and P270 (26%). The total number of cortical neurons in Tg mice was significantly increased at P0 (29%), P5 (29%) and P270 (31%), although the N(V) of cortical neurons did not differ significantly between Tg and control mice at any age. Transgenic mice at P0 and P5 exhibited significant decreases in the N(V) of apoptotic cells in the cerebral cortex (31% and 39%, respectively). The vast majority of these apoptotic cells (> 90%) were judged to be neurons by their morphological appearance. Increased expression of IGF-I inhibits naturally occurring (i.e. apoptotic) neuron death during early postnatal development of the cerebral cortex to a degree that sustains a persistent increase in total neuron number even in the adult animal.

A non-transformed oligodendrocyte precursor cell line, OL-1, facilitates studies of insulin-like growth factor-I signaling during oligodendrocyte development.

The process by which oligodendrocyte progenitors differentiate into mature oligodendrocytes is complex and incompletely understood in part because of the paucity of oligodendrocyte precursors cell lines that can be studied in culture. We have developed a non-immortalized rat oligodendrocyte precursor line, called OL-1, which behaves in a fashion consistent with developing oligodendrocytes in vivo. This OL-1 line provides a model for the study of oligodendrocyte development and offers an alternative to the CG-4 cell line. When OL-1 cells are propagated in conditioned growth media, they have morphology consistent with immature oligodendrocytes and exhibit A2B5 antigen positive and myelin basic protein-negative immunoreactivity. Withdrawal of conditioned growth media and culture in serum-free medium results in OL-1 cell maturation, manifested by a shift to myelin basic protein-positive immunoreactivity, A2B5 antigen-negative immunoreactivity, decreased NG2 mRNA expression, increased expression of proteolipid protein mRNA, and increased expression of CNP protein. In addition, the expression of proteolipid protein and its splicing variant DM-20 exhibit a pattern that is similar to brain proteolipid protein expression during development. When OL-1 cells are exposed to Insulin-like growth factor-I, there are significant increases in proteolipid protein mRNA expression (p<0.05), the number of cell processes (p<0.05), and cell number (p<0.05). Treatment with the caspase inhibitors Z-DEVD-FMK and Z-VAD-FMK (inhibitors of caspases 3, 6, 7, 8, 10 and 1, 3, 4, respectively), Insulin-like growth factor-I, or both, results in a similar increase in cell number. Because Insulin-like growth factor-I does not substantially increase the BrdU labeling of OL-1 cells, these data collectively indicate that Insulin-like growth factor-I increases OL-1 cell number predominately by promoting survival, rather than stimulating proliferation. This non-immortalized oligodendrocyte precursor cell line, therefore, exhibits behavior consistent with the in vivo development of oligodendrocytes and provides an excellent model for the study of developing oligodendrocytes.

Cloning and characterization of a novel RNA involved in cellular growth regulation.

During the course of antisense oligodeoxynucleotide (oligo) inhibition experiments investigating the role of insulin-like growth factor I (IGF-I) in the WI-38 cell cycle, we found that a sense-strand oligo (S oligo), used as a control, inhibited DNA synthesis 90 to 95%. S1 nuclease protection assays demonstrated that this S oligo formed intracellular duplexes with WI-38 RNA, and Northern (RNA) hybridization analyses demonstrated specific hybridization of this 32P-labeled S oligo to 1.8-, 2.3-, and 3.2-kb RNAs. We have cloned and sequenced a 2,251-bp cDNA, designated BB1, corresponding to the 2.3-kb RNA. Decoding of the BB1 cDNA sequence reveals several open reading frames arranged in a motif similar to that seen in proteins subject to translational control mechanisms. Homology searches of nucleic acid and protein data bases reveal no significant homology of BB1 with known sequences other than a 234-bp region in the BB1 5' untranslated region that shared 97% homology with a region in the 3' untranslated region of the human cdc42 mRNA. S1 nuclease protection analyses performed with IGF-I gene fragments and computer homology searches demonstrated that the BB1 RNA does not derive from transcription from the opposite strand of the IGF-I gene. Northern hybridization analyses of RNA extracted from serum-starved HeLa S3 cells demonstrated that steady-state BB1 RNA levels increased upon serum growth stimulation, with steady-state levels peaking 4 h after release from the block induced by serum starvation. Antisense oligo inhibition experiments using specific BB1 antisense oligos targeted to the putative open reading frames of the BB1 RNA reduce DNA synthesis of HeLa S3 cells to 15% of control levels, indicating that the BB1 RNA is essential for cell cycle traversal and, as such, encodes a growth-reguLating gene product.

Insulin-like growth factor-I stimulates histone H3 and H4 acetylation in the brain in vivo.

IGF-I is essential to normal brain growth and exerts actions on neural stem cells and each major neural cell lineage. Whereas many studies show that IGF-I regulates gene expression, mechanisms by which it modulates transcription have not been explored. Chromatin modifications, such as histone phosphorylation, acetylation, and methylation, are known to be important initial steps in gene regulation, and acetylation of histone H3 and H4 is associated with gene activation. In this study, we show that IGF-I modulates the acetylation of H3 and H4 histones in the brain of two transgenic mouse lines and that these effects are associated with activation of the phosphoinositide 3-kinase/Akt signaling pathway. This provides evidence that the chromatin architecture modification contributes to the action of IGF-I on gene expression in the mammalian central nerve system.

Mouse NG2+ oligodendrocyte precursors express mRNA for proteolipid protein but not its DM-20 variant: a study of laser microdissection-captured NG2+ cells.

Despite recent advances in our understanding of lineage of oligodendrocytes, detailed molecular characterization of this lineage in vivo is limited, primarily because of our inability to obtain a pure population of cells in situ. To define the molecular characteristics of oligodendrocyte lineage cells during development and their response to injury, we developed a strategy that uses laser capture microdissection (LCM) to isolate cells from sections and reverse transcription-PCR to determine mRNA expression. As a first step, we examined the expression of myelin-specific protein genes in NG2+ cells in cerebral cortex. We demonstrate that NG2+ cells in both developing and adult mice express NG2 mRNA but not mRNA for proteins specific for astrocytes, neurons, or microglia, indicating that a highly pure population of antigen-specific cells of the oligodendrocyte lineage can be obtained using LCM. Furthermore, we show that NG2+ cells express mRNAs for proteolipid protein (PLP), myelin basic protein, and 2',3'-cyclic nucleotide 3'-phosphodiesterase, but they dot not express DM-20 mRNA, a PLP mRNA splicing variant. Our data demonstrate that antigen-specific cells of oligodendrocyte lineage differentially express mRNA for myelin-specific proteins and their variants in vivo, partly define the gene expression in NG2+ cells, and raise questions about the cellular sites of DM-20 expression. This work also shows that LCM is a valuable tool to define and analyze gene expression in the cells of the oligodendrocyte lineage.