Transglutaminase-2 Regulation by Arecoline in Gingival Fibroblasts

Transglutaminase-2 (TGM-2) stabilizes extracellular matrix (ECM) proteins by cross-linking and has been implicated in several fibrotic disorders. Arecoline present in betel quid has been proposed as one of the causative factors for oral submucous fibrosis (OSMF). Hence, we hypothesize that arecoline may regulate TGM-2 and may have a role in the pathogenesis of OSMF. The expression of TGM-2 was studied in OSMF tissues by real-time RT-PCR analysis, and significant overexpression was observed in most OSMF tissues (P = 0.0112) compared with normal tissues. Arecoline induced TGM-2 mRNA and protein expression as well as TGM-2 activity in human gingival fibroblast cells. The addition of methocramine hemihydrate (M-2 muscarinic acetylcholine receptor selective antagonist) or 8'-bromo-cAMP abolished arecoline-mediated TGM-2 induction, suggesting a role for M-2 muscarinic acid receptor and a repressor role for cAMP. Our study provides evidence for TGM-2 overexpression in OSMF and its regulation by arecoline in oral fibroblasts.

Cholinergic Muscarinic Receptors in Human Fetal Brain: Ontogeny of [3H]Quinuclidinyl Benzilate Binding Sites in Corpus Striatum, Brainstem, and Cerebellum

The ontogeny of muscarinic receptors was studied in human fetal striatum, brainstem, and cerebellum to investigate general principles of synaptogenesis as well as the physiological balance between various chemical synapses during development in a given region of the brain. [3H]Quinuclidinyl benzilate ([-'H]QNB) binding was assayed in total particulate fraction (TPF) from various parts of brain. In the corpus striatum, QNB binding sites are present at 16 weeks of gestation (average concentration 180 fmol/mg protein of TPF), slowly increase up to 24 weeks (average concentration 217 fmol/mg protein), and rapidly increase during the third trimester to 480 fmol/mg protein of TPF. In contrast, dopaminergic receptors exist as two subpopulations. one with low affinity and the other with high affinity up to the 24th week of gestation; all of them acquire the highaffinity characteristic during the third trimester. In brainstem, the muscarinic receptors show maximum concentration by 16 weeks of age (360 fmolimg protein of TPF). Subsequently the muscarinic receptor concentration shows a gradual decline in the brainstem. In cerebellum, except for a slight increase at 24 weeks (average concentration 90 fmol/mg protein of TPF), the receptor concentration remained nearly constant at about 60-70 fmolimg protein of TPF throughout fetal life. This study demonstrates that the ontogeny of muscarinic receptors varies among the different regions, and the patterns observed suggest that receptor formation occurs principally in the third trimester. Also noteworthy is the finding that the QNB binding sites decreased in all regions of the human brain during adult life. Key Words: Cholinergic muscarinic receptors-Quinuclidinyl benzilate-Corpus striaturn-Brainstem-Cerebellum. Ravikumar B. V. and Sastry P. S. Cholinergic muscarinic receptors in human fetal brain: Ontogeny of [3H]quinuclidinyl benzilate binding sites in corpus striatum, brainstem, and cerebellum. J. Neurochem. 45, 1948- 1950 (1985).

Phagocytosis Of Mast Cell Granules By Cultured Fibroblasts

Cultured rat embryonic skin fibroblasts phagocytosed rat mast cell granules added to the medium or released from co-cultured mast cells by rabbit anti-rat IgE or Compound 48/80. Electron microscopy of fibroblasts incubated with mast cell granules revealed that granules adjacent to the plasmalemma were engulfed by long, thin cytoplasmic processes.

Liveweight prediction from hip height, condition score, fetal age and breed in tropical female cattle

Hip height, body condition, subcutaneous fat, eye muscle area, percentage Bos taurus, fetal age and diet digestibility data were collected at 17 372 assessments on 2181 Brahman and tropical composite (average 28% Brahman) female cattle aged between 0.5 and 7.5 years of age at five sites across Queensland. The study validated the subtraction of previously published estimates of gravid uterine weight to correct liveweight to the non-pregnant status. Hip height and liveweight were linearly related (Brahman: P<0.001, R-2 = 58%; tropical composite P<0.001, R-2 = 67%). Liveweight varied by 12-14% per body condition score (5-point scale) as cows differed from moderate condition (P<0.01). Parallel effects were also found due to subcutaneous rump fat depth and eye muscle area, which were highly correlated with each other and body condition score (r = 0.7-0.8). Liveweight differed from average by 1.65-1.66% per mm of rump fat depth and 0.71-0.76% per cm(2) of eye muscle area (P<0.01). Estimated dry matter digestibility of pasture consumed had no consistent effect in predicting liveweight and was therefore excluded from final models. A method developed to estimate full liveweight of post-weaning age female beef cattle from the other measures taken predicted liveweight to within 10 and 23% of that recorded for 65 and 95% of cases, respectively. For a 95% chance of predicted group average liveweight (body condition score used) being within 5, 4, 3, 2 and 1% of actual group average liveweight required 23, 36, 62, 137 and 521 females, respectively, if precision and accuracy of measurements matches that used in the research. Non-pregnant Bos taurus female cattle were calculated to be 10-40% heavier than Brahmans at the same hip height and body condition, indicating a substantial conformational difference. The liveweight prediction method was applied to a validation population of 83 unrelated groups of cattle weighed in extensive commercial situations on 119 days over 18 months (20 917 assessments). Liveweight prediction in the validation population exceeded average recorded liveweight for weigh groups by an average of 19 kg (similar to 6%) demonstrating the difficulty of achieving accurate and precise animal measurements under extensive commercial grazing conditions.

Cord blood 25-hydroxyvitamin D and fetal growth in the China-Anhui birth cohort study

We determined the association of cord blood 25-hydroxyvitamin D [25(OH)D] with birth weight and the risk of small for gestational age (SGA). As part of the China-Anhui Birth Cohort (C-ABC) study, we measured cord blood levels of 25(OH)D in 1491 neonates in Hefei, China. The data on maternal sociodemographic characteristics, health status, lifestyle, birth outcomes were prospectively collected. Multiple regression models were used to estimate the association of 25(OH)D levels with birth weight and the risk of SGA. Compared with neonates in the lowest decile of cord blood 25(OH)D levels, neonates in four deciles (the fourth, fifth, sixth and seventh deciles) had significantly increased birth weight and decreased risk of SGA. Multiple linear regression models showed that per 10 nmol/L increase in cord blood 25(OH)D, birth weight increased by 61.0 g (95% CI: 31.9, 89.9) at concentrations less than 40 nmol/L, and then decreased by 68.5 g (95% CI: −110.5, −26.6) at concentrations from 40 to 70 nmol/L. This study provides the first epidemiological evidence that there was an inverted U shaped relationship between neonatal vitamin D status and fetal growth, and the risk of SGA reduced at moderate concentration.

Interactions between Mast Cells, Fibroblasts and Connective Tissue Components

It has long been recognized that mast cells occur throughout connective tissues. Histologic studies have revealed that such cells release their granules into the surrounding environment upon exposure to both immunologic and nonimmunologic stimuli. By microscopy these extracellular granules appeared to be phagocytosed by fibroblasts and by blood-borne phagocytic cells as they entered the site of mast cell degranulation. Such in vivo observations led to the suggestion that mast cells both altered connective tissue components and influenced fibroblast function through these discharged granules. Recent in vitro studies using cultured fibroblasts and isolated mast cells and mast cell granules have confirmed both these hypotheses. In addition, such studies have also documented that fibroblasts degrade ingested mast cell granules. Such studies document that a number of critical interactions may occur between mast cells and connective tissue components.

Regulation of extracellular matrix genes by arecoline in primary gingival fibroblasts requires epithelial factors

Background and Objective: Oral submucous fibrosis, a disease of collagen disorder, has been attributed to arecoline present in the saliva of betel quid chewers. However, the molecular basis of the action of arecoline in the pathogenesis of oral submucous fibrosis is poorly understood. The basic aim of our study was to elucidate the mechanism underlying the action of arecoline on the expression of genes in oral fibroblasts. Material and Methods: Human keratinocytes (HaCaT cells) and primary human gingival fibroblasts were treated with arecoline in combination with various pathway inhibitors, and the expression of transforming growth factor-beta isoform genes and of collagen isoforms was assessed using reverse transcription polymerase chain reaction analysis. Results: We observed the induction of transforming growth factor-beta2 by arecoline in HaCaT cells and this induction was found to be caused by activation of the M-3 muscarinic acid receptor via the induction of calcium and the protein kinase C pathway. Most importantly, we showed that transforming growth factor-beta2 was significantly overexpressed in oral submucous fibrosis tissues (p = 0.008), with a median of 2.13 (n = 21) compared with 0.75 (n = 18) in normal buccal mucosal tissues. Furthermore, arecoline down-regulated the expression of collagens 1A1 and 3A1 in human primary gingival fibroblasts; however these collagens were induced by arecoline in the presence of spent medium of cultured human keratinocytes. Treatment with a transforming growth factor-beta blocker, transforming growth factor-beta1 latency-associated peptide, reversed this up-regulation of collagen, suggesting a role for profibrotic cytokines, such as transforming growth factor-beta, in the induction of collagens. Conclusion: Taken together, our data highlight the importance of arecoline-induced epithelial changes in the pathogenesis of oral submucous fibrosis.

Glycemic Control in Diabetic Pregnancies: Effects on Fetal and Maternal Outcome

Background: Both maternal and fetal complications are increased in diabetic pregnancies. Although hypertensive complications are increased in pregnant women with pregestational diabetes, reports on hypertensive complications in women with gestational diabetes mellitus (GDM) have been contradictory. Congenital malformations and macrosomia are the main fetal complications in Type 1 diabetic pregnancies, whereas fetal macrosomia and birth trauma but not congenital malformations are increased in GDM pregnancies. Aims: To study the frequency of hypertensive disorders in gestational diabetes mellitus. To evaluate the risk of macrosomia and brachial plexus injury (Erb’s palsy) and the ability of the 2-hour glucose tolerance test (OGTT) combined with the 24-hour glucose profile to distinguish between low and high risks of fetal macrosomia among women with GDM. To evaluate the relationship between glycemic control and the risk of fetal malformations in pregnancies complicated by Type 1 diabetes mellitus. To assess the effect of glycemic control on the occurrence of preeclampsia and pregnancy-induced hypertension in Type 1 diabetic pregnancies. Subjects: A total of 986 women with GDM and 203 women with borderline glucose intolerance (one abnormal value in the OGTT) with a singleton pregancy, 488 pregnant women with Type 1 diabetes (691 pregnancies and 709 offspring), and 1154 pregnant non-diabetic women (1181 pregnancies and 1187 offspring) were investigated. Results: In a prospective study on 81 GDM patients the combined frequency of preeclampsia and PIH was higher than in 327 non-diabetic controls (19.8% vs 6.1%, p<0.001). On the other hand, in 203 women with only one abnormal value in the OGTT, the rate of hypertensive complications did not differ from that of the controls. Both GDM women and those with only one abnormal value in the OGTT had higher pre-pregnancy weights and BMIs than the controls. In a retrospective study involving 385 insulin-treated and 520 diet-treated GDM patients, and 805 non-diabetic control pregnant women, fetal macrosomia occurred more often in the insulin-treated GDM pregnancies (18.2%, p<0.001) than in the diet-treated GDM pregnancies (4.4%), or the control pregnancies (2.2%). The rate of Erb’s palsy in vaginally delivered infants was 2.7% in the insulin-treated group of women and 2.4% in the diet-treated women compared with 0.3% in the controls (p<0.001). The cesarean section rate was more than twice as high (42.3% vs 18.6%) in the insulin-treated GDM patients as in the controls. A major fetal malformation was observed in 30 (4.2%) of the 709 newborn infants in Type 1 diabetic pregnancies and in 10 (1.4%) of the 735 controls (RR 3.1, 95% CI 1.6–6.2). Even women whose levels of HbA1c (normal values less than 5.6%) were only slightly increased in early pregnancy (between 5.6 and 6.8%) had a relative risk of fetal malformation of 3.0 (95% CI 1.2–7.5). Only diabetic patients with a normal HbA1c level (<5.6%) in early pregnancy had the same low risk of fetal malformations as the controls. Preeclampsia was diagnosed in 12.8% and PIH in 11.4% of the 616 Type 1 diabetic women without diabetic nephropathy. The corresponding frequencies among the 854 control women were 2.7% (OR 5.2; 95% CI 3.3–8.4) for preeclampsia and 5.6% (OR 2.2, 95% CI 1.5–3.1) for PIH. Multiple logistic regression analysis indicated that glycemic control, nulliparity, diabetic retinopathy and duration of diabetes were statistically significant independent predictors of preeclampsia. The adjusted odds ratios for preeclampsia were 1.6 (95% CI 1.3–2.0) for each 1%-unit increment in the HbA1c value during the first trimester and 0.6 (95% CI 0.5–0.8) for each 1%-unit decrement during the first half of pregnancy. In contrast, changes in glycemic control during the second half of pregnancy did not alter the risk of preeclampsia. Conclusions: In type 1 diabetic pregnancies it is extremely important to achieve optimal glycemic control before pregnancy and maintain it throughout pregnancy in order to decrease the complication rates both in the mother and in her offspring. The rate of fetal macrosomia and birth trauma in GDM pregnancies, especially in the group of insulin-treated women, is still relatively high. New strategies for screening, diagnosing, and treatment of GDM must be developed in order to decrease fetal and neonatal complications.

Japanese Encephalitis Virus Utilizes the Canonical Pathway To Activate NF-kappa B but It Utilizes the Type I Interferon Pathway To Induce Major Histocompatibility Complex Class I Expression in Mouse Embryonic Fibroblasts

Flaviviruses have been shown to induce cell surface expression of major histocompatibility complex class I (MHC-I) through the activation of NF-kappa B. Using IKK1(-/-), IKK2(-/-), NEMO-/-, and IKK1-/- IKK2-/- double mutant as well as p50(-/-) RelA(-/-) cRel(-/-) triple mutant mouse embryonic fibroblasts infected with Japanese encephalitis virus (JEV), we show that this flavivirus utilizes the canonical pathway to activate NF-kappa B in an IKK2- and NEMO-, but not IKK1-, dependent manner. NF-kappa B DNA binding activity induced upon virus infection was shown to be composed of RelA: p50 dimers in these fibroblasts. Type I interferon (IFN) production was significantly decreased but not completely abolished upon virus infection in cells defective in NF-kappa B activation. In contrast, induction of classical MHC-I (class 1a) genes and their cell surface expression remained unaffected in these NF-kappa B-defective cells. However, MHC-I induction was impaired in IFNAR(-/-) cells that lack the alpha/beta IFN receptor, indicating a dominant role of type I IFNs but not NF-kappa B for the induction of MHC-I molecules by Japanese encephalitis virus. Our further analysis revealed that the residual type I IFN signaling in NF-kappa B-deficient cells is sufficient to drive MHC-I gene expression upon virus infection in mouse embryonic fibroblasts. However, NF-kappa B could indirectly regulate MHC-I expression, since JEV-induced type I IFN expression was found to be critically dependent on it.

Molecular background of three lethal fetal syndromes

This study identified the molecular defects underlying three lethal fetal syndromes. Lethal Congenital Contracture Syndrome 1 (LCCS1, MIM 253310) and Lethal Arthrogryposis with Anterior Horn Cell Disease (LAAHD, MIM 611890) are fetal motor neuron diseases. They affect the nerve cells that control voluntary muscle movement, and eventually result in severe atrophy of spinal cord motor neurons and fetal immobility. Both LCCS1 and LAAHD are caused by mutations in the GLE1 gene, which encodes for a multifunctional protein involved in posttranscriptional mRNA processing. LCCS2 and LCCS3, two syndromes that are clinically similar to LCCS1, are caused by defective proteins involved in the synthesis of inositol hexakisphosphate (IP6), an essential cofactor of GLE1. This suggests a common mechanism behind these fetal motor neuron diseases, and along with accumulating evidence from genetic studies of more late-onset motor neuron diseases such as Spinal muscular atrophy (SMA) and Amyotrophic lateral sclerosis (ALS), implicates mRNA processing as a common mechanism in motor neuron disease pathogenesis. We also studied gle1-/- zebrafish in order to investigate whether they would be a good model for studying the pathogenesis of LCCS1 and LAAHD. Mutant zebrafish exhibit cell death in their central nervous system at two days post fertilization, and the distribution of mRNA within the cells of mutant zebrafish differs from controls, encouraging further studies. The third lethal fetal syndrome is described in this study for the first time. Cocoon syndrome (MIM 613630) was discovered in a Finnish family with two affected individuals. Its hallmarks are the encasement of the limbs under the skin, and severe craniofacial abnormalities, including the lack of skull bones. We showed that Cocoon syndrome is caused by a mutation in the gene encoding the conserved helix-loop-helix ubiquitous kinase CHUK, also known as IκB kinase α (IKKα). The mutation results in the complete lack of CHUK protein expression. CHUK is a subunit of the IκB kinase enzyme that inhibits NF-κB transcription factors, but in addition, it has an essential, independent role in controlling keratinocyte differentiation, as well as informing morphogenetic events such as limb and skeletal patterning. CHUK also acts as a tumor suppressor, and is frequently inactivated in cancer. This study has brought significant new information about the molecular background of these three lethal fetal syndromes, as well as provided knowledge about the prerequisites of normal human development.

Maturation of a transient outward potassium current in mouse fetal hypothalamic neurons in culture

The whole-cell voltage clamp technique was used to record potassium currents in mouse fetal hypothalamic neurons developing in culture medium from days 1 to 17. The neurons were derived from fetuses of IOPS/OF1 mice on the 14th day of gestation. The mature neurons (>six days in culture) showed both a transient potassium current and a non-inactivating delayed rectifier potassium current. These were identified pharmacologically by using the potassium channel blockers tetraethyl ammonium chloride and 4-aminopyridine, and on the basis of their kinetics and voltage sensitivities. The delayed rectifier potassium current had a threshold of −20 mV, a slow time-course of activation, and was sustained during the voltage pulse. The 4-aminopyridine-sensitive current was transient, and was activated from a holding potential more negative (−80 mV) than that required for evoking the delayed rectifier potassium current (−40 mV). The delayed rectifier potassium current was detectable from day 1 onwards, while the transient potassium current showed a distinct developmental trend. The time-constant of inactivation became faster with age in culture. The half steady-state inactivation potential showed a shift towards less negative membrane potentials with age, and the relationship was best described by a logarithmic regression equation.The developmental trend of the transient potassium current may relate functionally to the progressive morphological changes, and the appearance of synaptic connections during ontogenesis.

Diperoxovanadate can substitute for H(2)O(2) at much lower concentration in inducing features of premature cellular senescence in mouse fibroblasts (NIH3T3)

Stress induced premature senescence (SIPS) in mammalian cells is an accelerated ageing response and experimentally obtained on treatment of cells with high concentrations of H(2)O(2), albeit at sub-lethal doses, because H(2)O(2) gets depleted by abundant cellular catalase. In the present study diperoxovanadate (DPV) was used as it is known to be stable at physiological pH, to be catalase-resistant and to substitute for H(2)O(2) in its activities at concentrations order of magnitudes lower. On treating NIH3T3 cells with DPV, SIPS-like morphology was observed along with an immediate response of rounding of the cells by disruption of actin cytoskeleton and transient G2/M arrest. DPV could bring about growth arrest and senescence associated features at 25 mu M dose, which were not seen with similar doses of either H(2)O(2) or vanadate. A minimal dose of 150 mu M of H(2)O(2) was required to induce similar affects as 25 mu M DPV. Increase in senescent associated markers such as p21, HMGA2 and PAI-1 was more prominent in DPV treated cells compared to similar dose of H(2)O(2). DPV-treated cells showed marked relocalization of Cyclin D1 from nucleus to cytoplasm. These results indicate that DPV, stable inorganic peroxide, is more efficient in inducing SIPS at lower concentrations compared to H(2)O(2). (C) 2011 Elsevier Ireland Ltd. All rights reserved.

An investigation into the feasibility of fetal lung maturity prediction using statistical textural features

Fetal lung and liver tissues were examined by ultrasound in 240 subjects during 24 to 38 weeks of gestational age in order to investigate the feasibility of predicting the maturity of the lung from the textural features of sonograms. A region of interest of 64 X 64 pixels is used for extracting textural features. Since the histological properties of the liver are claimed to remain constant with respect to gestational age, features obtained from the lung region are compared with those from liver. Though the mean values of some of the features show a specific trend with respect to gestation age, the variance is too high to guarantee definite prediction of the gestational age. Thus, we restricted our purview to an investigation into the feasibility of fetal lung maturity prediction using statistical textural features. Out of 64 features extracted, those features that are correlated with gestation age and less computationally intensive are selected. The results of our study show that the sonographic features hold some promise in determining whether the fetal lung is mature or immature.