Adjunct effect of the antimicrobial photodynamic therapy to an association of non-surgical and surgical periodontal treatment in modulation of gene expression

Background: This study has evaluated the effect of antimicrobial photodynamic therapy (aPDT) used in conjunction with non-surgical and surgical periodontal treatment (PT) in modulating gene expression during periodontal wound healing. Methods: Fifteen patients with chronic periodontitis, presenting bilaterally lower molars with class III furcation lesions and scheduled for extraction, were selected. In initial therapy, scaling and root planing (SRP) was performed in the Control Group (CG), while SRP + aPDT were performed in the Test Group (TG). 45 days later, flap surgery plus SRP, and flap surgery plus SRP + aPDT were performed in the CG and TG, respectively. At 21 days post-surgery, the newly formed granulation tissue was collected, and Real-time PCR evaluated the expression of the genes: tumor necrosis factor-?, interleukin-1?, interleukin-4, interleukin-10, matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-2 (TIMP-2), osteoprotegerin (OPG), receptor activator of nuclear factor- ?B ligand (RANKL), type I collagen, alkaline phosphatase, osteopontin, osteocalcin, and bone sialoprotein. Results: There were statistically significant differences between the groups in relation to mRNA levels for MMP-2 (TG = 3.26 ± 0.89; CG = 4.23 ± 0.97; p = 0.01), TIMP-2/MMP-2 ratio (TG = 0.91 ± 0.34; CG = 0.73 ± 0.32; p = 0.04), OPG (TG = 0.84 ± 0.45; CG = 0.30 ± 0.26; p = 0.001), and OPG/RANKL ratio (TG = 0.60 ± 0.86; CG = 0.23 ± 0.16; p = 0.04), favoring the TG. Conclusion: The present data suggest that the aPDT associated to nonsurgical and surgical periodontal therapy may modulate the extracellular matrix and bone remodeling by up regulating the TIMP- 2/MMP-2 and OPG/RANKL mRNA ratio, but the clinical relevance needs to be evaluated in further studies.

Differential inflammasome expression and IL-1β secretion in monocyte-derived dendritic cells differentiated with IL-4 or IFN-α

NLRP3-inflammasome activation was evaluated in monocyte-derived dendritic cells (DC) obtained through IL-4 (IL4-DC) or IFN-α (IFN-DC) protocols and pulsed with chemically inactivated HIV-1. Inflammasome' genes expression and IL-1β secretion were compared in DC isolated from 15 healthy subjects (HC) and 10 HIV-1 infected individuals (HIV+). FINDINGS: Whether HIV was able to increased NLRP3-inflammasome genes expression and IL-1β secretion in IL4-DC from HC, the induction of inflammasome appeared significantly reduced in IFN-DC from HC, suggesting a different responsive state of IFN-DC compared to IL4-DC. No inflammasome activation was observed in IL4-DC as well as in IFN-DC derived from HIV + subjects, confirming previous findings on "unresponsive" state of DC derived from HIV + possibly due to chronic inflammatory state of these individuals. CONCLUSIONS: Our results showed that IFN-α differently modulates inflammasome expression during monocytes-DC in vitro differentiation. These findings could be of interest considering the on-going research about DC manipulation and therapeutic strategies for HIV + involving DC-based immune-vaccines.

Manipulation of the periovulatory sex steroidal milieu affects endometrial but not luteal gene expression in early diestrus Nelore cows

In beef cattle, the ability to conceive has been associated positively with size of the preovulatory follicle (POF). Proestrus estradiol and subsequent progesterone concentrations can regulate the endometrium to affect receptivity and fertility. The aim of the present study was to verify the effect of the size of the POF on luteal and endometrial gene expression during subsequent early diestrus in beef cattle. Eighty-three multiparous, nonlactating, presynchronized Nelore cows received a progesterone-releasing device and estradiol benzoate on Day–10 (D 10). Animals received cloprostenol (large follicle-large CL group; LF-LCL; N ¼ 42) or not (small follicle-small CL group; SF-SCL; N ¼ 41) on D 10. Progesterone devices were withdrawn and cloprostenol administered 42 to 60 hours (LF-LCL) or 30 to 36 hours (SF-SCL) before GnRH treatment (D0). Tissues were collected at slaughter on D7. The LF-LCL group had larger (P < 0.0001) POF (13.24 0.33 mm vs. 10.76 0.29 mm), greater (P < 0.0007) estradiol concentrations on D0 (2.94 0.28 pg/mL vs. 1.27 0.20 pg/mL), and greater (P < 0.01) progesterone concentrations on D7 (3.71 0.25 ng/mL vs. 2.62 0.26 ng/mL) compared with the SF-SCL group. Luteal gene expression of vascular endothelial growth factor A, kinase insert domain receptor, fms-related tyrosine kinase 1, steroidogenic acute regulatory protein, cytochrome P450, family 11, subfamily A, polypeptide 1, and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid deltaisomerase 7 was similar between groups. Endometrial gene expression of oxytocin receptor and peptidase inhibitor 3, skin-derived was reduced, and estrogen receptor alpha 2, aldo-keto reductase family 1, member C4, and lipoprotein lipase expression was increased in LF-LCL versus SF-SCL. Results support the hypothesis that the size of the POF alters the periovulatory endocrine milieu (i.e., proestrus estradiol and diestrus progesterone concentrations) and acts on the uterus to alter endometrial gene expression. It is proposed that the uterine environment and receptivity might also be modulated. Additionally, it is suggested that increased progesterone secretion of cows ovulating larger follicles is likely due to increased CL size rather than increased luteal expression of steroidogenic genes.

Genexpressionsanalyse boviner mesenchymaler Stammzellen und deren in-vitro-differenzierten Folgelinien

Mesenchymale Stamzellen (MSC) sind Vertreter der adulten Stammzellen. Sie bergen durch ihre große Plastizität ein immenses Potential für die klinische Nutzung in Form von Stammzelltherapien. Zellen dieses Typs kommen vornehmlich im Knochenmark der großen Röhrenknochen vor und können zu Knochen, Knorpel und Fettzellen differenzieren. MSC leisten einen wichtigen Beitrag im Rahmen regenerativer Prozesse, beispielsweise zur Heilung von Frakturen. Breite Studien demonstrieren bereits jetzt auch bei komplexeren Erkrankungen (z.B. Osteoporose) therapeutisch vielversprechende Einsatzmöglichkeiten. Oft kommen hierbei aus MSC gezielt differenzierte Folgelinien aus Zellkulturen zum Einsatz. Dies bedingt eine kontrollierte Steuerung der Differenzierungsprozesse in vitro. Der Differenzierung einer Stammzelle liegt eine komplexe Veränderung ihrer Genexpression zugrunde. Genexpressionsmuster zur Erhaltung und Proliferation der Stammzellen müssen durch solche, die der linienspezifischen Differenzierung dienen, ersetzt werden. Die mit der Differenzierung einhergehende, transkriptomische Neuausrichtung ist für das Verständnis der Prozesse grundlegend und wurde bislang nur unzureichend untersucht. Ziel der vorliegenden Arbeit ist eine transkriptomweite und vergleichende Genexpressionsanalyse Mesenchymaler Stammzellen und deren in vitro differenzierten Folgelinien mittels Plasmid - DNA Microarrays und Sequenziertechniken der nächsten Generation (RNA-Seq, Illumina Plattform). In dieser Arbeit diente das Hausrind (Bos taurus) als Modellorganismus, da es genetisch betrachtet eine hohe Ähnlichkeit zum Menschen aufweist und Knochenmark als Quelle von MSC gut verfügbar ist. Primärkulturen Mesenchymaler Stammzellen konnten aus dem Knochenmark von Rindern erfolgreich isoliert werden. Es wurden in vitro Zellkultur - Versuche durchgeführt, um die Zellen zu Osteoblasten, Chondrozyten und Adipozyten zu differenzieren. Zur Genexpressionsanalyse wurde RNA aus jungen MSC und einer MSC Langzeitkultur („alte MSC“), sowie aus den differenzierten Zelllinien isoliert und für nachfolgende Experimente wo nötig amplifiziert. Der Erfolg der Differenzierungen konnte anhand der Genexpression von spezifischen Markergenen und mittels histologischer Färbungen belegt werden. Hierbei zeigte sich die Differenzierung zu Osteoblasten und Adipozyten erfolgreich, während die Differenzierung zu Chondrozyten trotz diverser Modifikationen am Protokoll nicht erfolgreich durchgeführt werden konnte. Eine vergleichende Hybridisierung zur Bestimmung differentieller Genexpression (MSC vs. Differenzierung) mittels selbst hergestellter Plasmid - DNA Microarrays ergab für die Osteogenese mit Genen wie destrin und enpp1, für die undifferenzierten MSC mit dem Gen sema3c neue Kandidatengene, deren biologische Funktion aufzuklären in zukünftigen Experimenten vielversprechende Ergebnisse liefern sollte. Die Analyse der transkriptomweiten Genexpression mittels NGS lieferte einen noch umfangreicheren Einblick ins Differenzierungsgeschehen. Es zeigte sich eine hohe Ähnlichkeit im Expressionsprofil von jungen MSC und Adipozyten, sowie zwischen den Profilen der alten MSC (eine Langzeitkultur) und Osteoblasten. Die alten MSC wiesen deutliche Anzeichen für eine spontane Differenzierung in die osteogene Richtung auf. Durch Analyse der 100 am stärksten exprimierten Gene jeder Zelllinie ließen sich für junge MSC und Adipozyten besonders Gene der extrazellulären Matrix (z.B col1a1,6 ; fn1 uvm.) auffinden. Sowohl Osteoblasten, als auch die alten MSC exprimieren hingegen verstärkt Gene mit Bezug zur oxidativen Phosphorylierung, sowie ribosomale Proteine. Eine Betrachtung der differentiellen Genexpression (junge MSC vs. Differenzierung) mit anschließender Pathway Analyse und Genontologie Anreicherungsstatistik unterstützt diese Ergebnisse vor allem bei Osteoblasten, wo nun jedoch zusätzlich auch Gene zur Regulation der Knochenentwicklung und Mineralisierung in den Vordergrund treten. Für Adipozyten konnte mit Genen des „Jak-STAT signaling pathway“, der Fokalen Adhäsion, sowie Genen des „Cytokine-cytokine receptor interaction pathway“ sehr spannende Einsichten in die Biologie dieses Zelltyps erlangt werden, die sicher weiterer Untersuchungen bedürfen. In undifferenzierten MSC konnte durch differentielle Genexpressionsanalyse die Rolle des nicht kanonischen Teils des WNT Signalweges als für die Aufrechterhaltung des Stammzellstatus potentiell äußerst einflussreich ermittelt werden. Die hier diskutierten Ergebnisse zeigen beispielhaft, dass besonders mittels Genexpressionsanalyse im Hochdurchsatzverfahren wertvolle Einblicke in die komplexe Biologie der Stammzelldifferenzierung möglich sind. Als Grundlage für nachfolgende Arbeiten konnten interessante Gene ermittelt und Hypothesen zu deren Einfluss auf Stammzelleigenschaften und Differenzierungsprozesse aufgestellt werden. Um einen besseren Einblick in den Differenzierungsverlauf zu ermöglichen, könnten künftig NGS Analysen zu unterschiedlichen Differenzierungszeitpunkten durchgeführt werden. Zudem wären weitere Anstrengungen zur erfolgreichen Etablierung der chondrogenen Differenzierung zur vollständigen Analyse der Genexpression des trilinearen Differenzierungspotentials von MSC wünschenswert.

Homeodomain-only protein HOP is a novel modulator of late differentiation in keratinocytes

The homeodomain-only protein (HOP) contains an atypical homeodomain which is unable to bind to DNA due to mutations in residues important for DNA binding. Recently, HOP was reported to regulate proliferation/differentiation homeostasis in different cell types. In the present study, we performed transcriptional profiling of cultured primary human keratinocytes and noted a robust induction of HOP upon calcium-induced cell differentiation. Immunohistochemistry of human skin localized HOP to the granular layer in the epidermis. Overexpression of HOP using a lentiviral vector up-regulated FLG and LOR expression during keratinocyte differentiation. Conversely, decreasing HOP expression using small interfering RNA markedly reduced the calcium-induced expression of late markers of differentiation in vitro, with the most prominent effect on profilaggrin (FLG) mRNA. Moreover, mRNA levels of profilaggrin and loricrin were downregulated in the epidermis of HOP knockout mice. Analysis of skin disorders revealed altered HOP expression in lichen planus, psoriasis and squamous cell carcinoma (SCC). Our data indicate that HOP is a novel modulator of late terminal differentiation in keratinocytes.

Life stage differences in mammary gland gene expression profile in non-human primates

Breast cancer (BC) is the most common malignancy of women in the developed world. To better understand its pathogenesis, knowledge of normal breast development is crucial, as BC is the result of disregulation of physiologic processes. The aim of this study was to investigate the impact of reproductive life stages on the transcriptional profile of the mammary gland in a primate model. Comparative transcriptomic analyses were carried out using breast tissues from 28 female cynomolgus macaques (Macaca fascicularis) at the following life stages: prepubertal (n = 5), adolescent (n = 4), adult luteal (n = 5), pregnant (n = 6), lactating (n = 3), and postmenopausal (n = 5). Mammary gland RNA was hybridized to Affymetrix GeneChip(®) Rhesus Macaque Genome Arrays. Differential gene expression was analyzed using ANOVA and cluster analysis. Hierarchical cluster analysis revealed distinct separation of life stage groups. More than 2,225 differentially expressed mRNAs were identified. Gene families or pathways that changed across life stages included those related to estrogen and androgen (ESR1, PGR, TFF1, GREB1, AR, 17HSDB2, 17HSDB7, STS, HSD11B1, AKR1C4), prolactin (PRLR, ELF5, STAT5, CSN1S1), insulin-like growth factor signaling (IGF1, IGFBP1, IGFBP5), extracellular matrix (POSTN, TGFB1, COL5A2, COL12A1, FOXC1, LAMC1, PDGFRA, TGFB2), and differentiation (CD24, CD29, CD44, CD61, ALDH1, BRCA1, FOXA1, POSTN, DICER1, LIG4, KLF4, NOTCH2, RIF1, BMPR1A, TGFB2). Pregnancy and lactation displayed distinct patterns of gene expression. ESR1 and IGF1 were significantly higher in the adolescent compared to the adult animals, whereas differentiation pathways were overrepresented in adult animals and pregnancy-associated life stages. Few individual genes were distinctly different in postmenopausal animals. Our data demonstrate characteristic patterns of gene expression during breast development. Several of the pathways activated during pubertal development have been implicated in cancer development and metastasis, supporting the idea that other developmental markers may have application as biomarkers for BC.

PU.1 is linking the glycolytic enzyme HK3 in neutrophil differentiation and survival of APL cells

The transcription factor PU.1 is a master regulator of myeloid differentiation and function. On the other hand, only scarce information is available on PU.1-regulated genes involved in cell survival. We now identified the glycolytic enzyme hexokinase 3 (HK3), a gene with cytoprotective functions, as transcriptional target of PU.1. Interestingly, HK3 expression is highly associated with the myeloid lineage and was significantly decreased in acute myeloid leukemia patients compared with normal granulocytes. Moreover, HK3 expression was significantly lower in acute promyelocytic leukemia (APL) compared with non-APL patient samples. In line with the observations in primary APL patient samples, we observed significantly higher HK3 expression during neutrophil differentiation of APL cell lines. Moreover, knocking down PU.1 impaired HK3 induction during neutrophil differentiation. In vivo binding of PU.1 and PML-RARA to the HK3 promoter was found, and PML-RARA attenuated PU.1 activation of the HK3 promoter. Next, inhibiting HK3 in APL cell lines resulted in significantly reduced neutrophil differentiation and viability compared with control cells. Our findings strongly suggest that HK3 is: (1) directly activated by PU.1, (2) repressed by PML-RARA, and (3) functionally involved in neutrophil differentiation and cell viability of APL cells.

Inhibition of damage-regulated autophagy modulator-1 (DRAM-1) impairs neutrophil differentiation of NB4 APL cells

The damage-regulator autophagy modulator 1 (DRAM-1) is a lysosomal protein that positively regulates autophagy in a p53-dependent manner. We aimed at analyzing the role of DRAM-1 in granulocytic differentiation of APL cells. We observed a significant increase of DRAM-1 expression during all-trans retinoic acid (ATRA)-induced neutrophil differentiation of NB4 APL cells but not in ATRA-resistant NB4-R2 cells. Next, knocking down DRAM-1 in NB4 APL cells was sufficient to impair neutrophil differentiation. Given that DRAM-1 is a transcriptional target of p53, we tested if DRAM-1 is regulated by the p53 relative p73. Indeed, inhibiting p73 prevented neutrophil differentiation and DRAM-1 induction of NB4 cells. In conclusion, we show for the first time that p73-regulated DRAM-1 is functionally involved in neutrophil differentiation of APL cells.

Ectopic growth hormone-releasing hormone secretion by a metastatic bronchial carcinoid tumor: a case with a non hypophysial intracranial tumor that shrank during long acting octreotide treatment

Ectopic acromegaly represents less than 1% of the reported cases of acromegaly. Although clinical improvement is common after treatment with somatostatin (SMS) analogs, the biochemical response and tumor size of the growth hormone-releasing hormone (GHRH)-producing tumor and its metastases are less predictable. Subject A 36-year-old male was referred because of a 3-year history of acromegaly related symptoms. He had undergone lung surgery in 1987 for a "benign" carcinoid tumor. Endocrine evaluation confirmed acromegaly Plasma IGF-1: 984 ng/ml (63-380), GH: 49.8 ng/ml (<5). MRI showed a large mass in the left cerebellopontine angle and diffuse pituitary hyperplasia. Pulmonary, liver and bone metastases were shown by chest and abdominal CT scans. Ectopic GHRH secretion was suspected. Methods Measurement of circulating GHRH levels by fluorescence immunoassay levels and immunohistochemical study of the primary lung tumor and metastatic tissue with anti-GHRH and anti-somatostatin receptor type 2 (sst2A) antibodies. Results Basal plasma GHRH: 4654 pg/ml (<100). Pathological study of liver and bone biopsy material and lung tissue removed 19 years earlier was consistent with an atypical carcinoid producing GHRH and exhibiting sst2A receptor expression. Treatment with octreotide LAR 20-40 mg q. month resulted in normalization of plasma IGF-1 levels. Circulating GHRH levels decreased dramatically. The size of the left prepontine cistern mass, with SMS receptors shown by a radiolabeled pentetreotide scan, decreased by 80% after 18 months of therapy. Total regression of pituitary enlargement was also observed. No changes were observed in lung and liver metastases. After 24 months of therapy the patient is asymptomatic and living a full and active life.

Comparative quantitative assessment of GnRH- and LH-receptor mRNA expression in the urinary tract of sexually intact and spayed female dogs

Ovariectomy interrupts the regulatory loop in the hypothalamus-pituitary-gonad axis, leading to a several-fold increase in gonadotropin levels. This rise in hormonal secretion may play a causal role in ovariectomy-related urinary incontinence. The purpose of this study was to examine the effect of ovariectomy in bitches on the expression of GnRH- and LH-receptors in the lower urinary tract, and assess the relationship between receptor expression and plasma gonadotropin concentrations. Plasma gonadotropins were measured in 37 client-owned bitches. Biopsies were harvested from the mid-ventral bladder wall in all dogs, and from nine further locations within the lower urinary tract in 17 of the 37 animals. Messenger RNA of the LH and GnRH receptors was quantified using RT-PCR with the TaqMan Universal PCR Master Mix. Gonadotropins were measured with a canine-specific FSH-immunoradiometric assay and LH-radioimmunoassay. The hierarchical mixed ANOVA model using MINITAB, Mann-Whitney U-test, unpaired means comparison and linear regressions using StatView were applied for statistical analyses. Messenger RNA for both receptors was detected in all biopsy samples. Age was negatively correlated to mRNA expression of the LH and the GnRH receptors. A relationship between the mRNA values and the plasma gonadotropin concentrations was not established. Evaluation of results within each of the biopsy locations revealed greater LH-receptor expression in the proximal second quarter of the urethra in spayed bitches than in intact bitches (P=0.0481). Increased mRNA expression of LH receptors in this location could possibly play a role in the decrease in closing pressure of the urethra following ovariectomy.

Immunophenotypic changes of human articular chondrocytes during monolayer culture reflect bona fide dedifferentiation rather than amplification of progenitor cells

In this study, a time-course comparison of human articular chondrocytes (HAC) and bone marrow-derived mesenchymal stem cells (MSC) immunophenotype was performed in order to determine similarities/differences between both cell types during monolayer culture, and to identify HAC surface markers indicative of dedifferentiation. Our results show that dedifferentiated HAC can be distinguished from MSC by combining CD14, CD90, and CD105 expression, with dedifferentiated HAC being CD14+/CD90bright/CD105dim and MSC being CD14-/CD90dim/CD105bright. Surface markers on MSC showed little variation during the culture, whereas HAC showed upregulation of CD90, CD166, CD49c, CD44, CD10, CD26, CD49e, CD151, CD51/61, and CD81, and downregulation of CD49a, CD54, and CD14. Thus, dedifferentiated HAC appear as a bona fide cell population rather than a small population of MSC amplified during monolayer culture. While most of the HAC surface markers showed major changes at the beginning of the culture period (Passage 1-2), CD26 was upregulated and CD49a downregulated at later stages of the culture (Passage 3-4). To correlate changes in HAC surface markers with changes in extracellular matrix gene expression during monolayer culture, CD14 and CD90 mRNA levels were combined into a new differentiation index and compared with the established differentiation indices based on the ratios of mRNA levels of collagen type II to I (COL2/COL1) and of aggrecan to versican (AGG/VER). A correlation of CD14/CD90 ratio at the mRNA and protein level with the AGG/VER ratio during HAC dedifferentiation in monolayer culture validated CD14/CD90 as a new membrane and mRNA based HAC differentiation index.

The vascular ectonucleotidase CD39 is permissive for sinusoidal endothelial cell proliferation during liver regeneration: evidence for synergism between growth factors and extracellular nucleotides

Crosstalk between elements of the sinusoidal vasculature, platelets and hepatic parenchymal cells influences regenerative responses to liver injury and/or resection. Such paracrine interactions include hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), IL-6 and small molecules such as serotonin and nucleotides. CD39 (nucleoside triphosphate diphosphohydrolase-1) is the dominant vascular ectonucleotidase expressed on the luminal surface of endothelial cells and modulates extracellular nucleotide signaling. We have previously shown that integrity of P2-receptors, as maintained by CD39, is required for angiogenesis in Matrigel plugs in vivo and that there is synergism between nucleotide P2-receptor- and growth factor-mediated cell proliferation in vitro. We have now explored effects of CD39 on liver regeneration and vascular endothelial growth factor responses in a standard small animal model of partial hepatectomy. The expression of CD39 on liver sinusoidal endothelial cells (LSEC) is substantially boosted during liver regeneration. This transcriptional upregulation precedes maximal sinusoidal endothelial cell proliferation, noted at day 5-8 in C57BL6 wild type mice. In matched mutant mice null for CD39 (n=14), overall survival is decreased to 71% by day 10. Increased lethality occurs as a consequence of extensive LSEC apoptosis, decreased endothelial proliferation and failure of angiogenesis leading to hepatic infarcts and regenerative failure in mutant mice. This aberrant vascular remodeling is associated with biochemical liver injury, elevated serum levels of VEGF (113.9 vs. 65.5pg/ml, p=0.013), and decreased circulating HGF (0.89 vs. 1.43 ng/ml, p=0.001) in mice null for CD39. In agreement with these observations, wild type LSEC but not CD39 null cultures upregulate HGF expression and secretion in response to exogenous VEGF in vitro. CD39 null LSEC cultures show poor proliferation responses and heightened levels of apoptosis when contrasted to wild type LSEC where agonists of P2Y receptors augment cell proliferation in the presence of growth factors. These observations are associated with features of P2Y-desensitization, normal levels of the receptor tyrosine kinase VEGFR-1 (Flt-1) and decreased expression of VEGFR-2 (FLK/KDR) in CD39 null LSEC cultures. We provide evidence that CD39 and extracellular nucleotides impact upon growth factor responses and tyrosine receptor kinases during LSEC proliferation. We propose that CD39 expression by LSEC might co-ordinate angiogenesis-independent liver protection by facilitating VEGF-induced paracrine release of HGF to promote vascular remodeling in liver regeneration.

Effects of troglitazone on cellular differentiation, insulin signaling, and glucose metabolism in cultured human skeletal muscle cells

To determine the immediate effect of thiazolidinediones on human skeletal muscle, differentiated human myotubes were acutely (1 day) and myoblasts chronically (during the differentiation process) treated with troglitazone (TGZ). Chronic TGZ treatment resulted in loss of the typical multinucleated phenotype. The increase of muscle markers typically observed during differentiation was suppressed, while adipocyte markers increased markedly. Chronic TGZ treatment increased insulin-stimulated phosphatidylinositol (PI) 3-kinase activity and membranous protein kinase B/Akt (PKB/Akt) Ser-473 phosphorylation more than 4-fold. Phosphorylation of p42/44 mitogen-activated protein kinase (42/44 MAPK/ERK) was unaltered. Basal glucose uptake as well as both basal and insulin-stimulated glycogen synthesis increased approximately 1.6- and approximately 2.5-fold after chronic TGZ treatment, respectively. A 2-fold stimulation of PI 3-kinase but no other significant TGZ effect was found after acute TGZ treatment. In conclusion, chronic TGZ treatment inhibited myogenic differentiation of that human muscle while inducing adipocyte-specific gene expression. The effects of chronic TGZ treatment on basal glucose transport may in part be secondary to this transdifferentiation. The enhancing effect on PI 3-kinase and PKB/Akt involved in both differentiation and glycogen synthesis appears to be pivotal in the cellular action of TGZ.

GPI-specific phospholipase D (GPI-PLD) is expressed during mouse development and is localized to the extracellular matrix of the developing mouse skeleton

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in serum and has a well-characterized biochemistry; however, its physiological role is completely unknown. Previous investigations into GPI-PLD have focused on the adult animal or on in vitro systems and a putative role in development has been neither proposed nor investigated. We describe the first evidence of GPI-PLD expression during mouse embryonic ossification. GPI-PLD expression was detected predominantly at sites of skeletal development, increasing during the course of gestation. GPI-PLD was observed during both intramembraneous and endochondral ossification and localized predominantly to the extracellular matrix of chondrocytes and to primary trabeculae of the skeleton. In addition, the mouse chondrocyte cell line ATDC5 expressed GPI-PLD after experimental induction of differentiation. These results implicate GPI-PLD in the process of bone formation during mouse embryogenesis.

Early over-expression of GRP receptors in prostatic carcinogenesis

BACKGROUND The GRP receptor shows high over-expression in prostatic adenocarcinoma and high grade PIN, but low expression in normal prostate glands. This represents the molecular basis for GRP receptor imaging of prostate cancer with radioactive compounds. However, a focal, high density GRP receptor expression can be observed in hitherto uncharacterized prostate glands. METHODS GRP receptors were quantitatively measured with in vitro receptor autoradiography using ¹²⁵I-Tyr⁴ -bombesin in samples from 115 prostates. On successive tissue sections, ¹²⁵I-Tyr⁴ -bombesin autoradiography was compared with H&E staining and MIB-1 and 34βE12 immunohistochemistry. RESULTS On one hand, it was confirmed that GRP receptors were expressed in adenocarcinoma and high grade PIN in high density and high incidence (77% and 73%, respectively), but in normal prostate glands in low density and low frequency (18%). On the other hand, a novel and intriguing observation was the existence of focal non-invasive prostate glands with high GRP receptor density, characterized by low grade nuclear atypia and increased proliferation, compatible with lower grade PIN. There was a significant GRP receptor density gradient (P ≤ 0.005), increasing from normal prostate glands (mean relative optical density, ROD, of ¹²⁵I-Tyr⁴ -bombesin binding: 0.17) over atypical glands without increased MIB-1 labeling (0.28) and atypical glands with increased MIB-1 expression (0.44) to high grade PIN and adenocarcinoma (0.64 and 0.58, respectively). CONCLUSIONS GRP receptor over-expression may be a novel, specific marker of early prostatic neoplastic transformation, arising in low grade PIN, and progressively increasing during malignant progression. This should be considered when interpreting in vivo GRP receptor imaging in males.