Search

Background: Chronic kidney disease (CKD) is common after heart transplantation (HT). There are scarce studies looking at longitudinal changes in estimated glomerular filtration rate (eGFR) after repeat HT (RT) and risk factors for the presence of CKD3 post-RT., Methods: First-time HT recipients (FT) were matched with RT, based on age at transplant, sex, race, and transplant era. eGFR was derived from CKiD-U25 formula using creatinine. Changes in eGFR were measured within and between patients using a mixed effects model. Logistic regression and survival analysis were performed to identify significant risk factors for CKD3 presence post-RT., Results: The unmatched cohort included 393 HT recipients and 47 RT. 29 patients in both groups with at least 2 years of follow-up data underwent matching. Over 6 years, the mean eGFR for FT and RT did not significantly change overtime. For those with CKD3 or higher prior to RT, 73% improved to CKD2 or lower at 2 years post-RT. Risk factors for CKD3 post-RT included female sex (OR 17.5 [1.7-181.9], p = 0.017) and greater than two acute kidney injuries (AKI) between FT and RT (OR 1.77 [1.06-2.95], p = 0.03). Approximately 50% of females and those with greater than two AKIs maintained or developed CKD3 at 7.5 and 3 years, respectively., Conclusion: This study provides data on longitudinal changes in eGFR over 6 years and finds that female sex and repeat HT recipients with greater 2 AKIs between transplants are at greatest risk for CKD3 presence post-RT., (© 2024 Wiley Periodicals LLC.)

Background: Renal function is reduced in patients undergoing heart transplant due to hemodynamic compromise, cardiorenal syndrome, and nephrotoxin exposure. No current studies evaluate renal function in retransplants., Methods: We reviewed all heart transplants at our center from 1995 to 2021 and matched first-time heart transplants with retransplants, based on age at transplant, sex, and race. Estimated glomerular filtration rate (eGFR) was derived from CKiD-U25 calculator using creatinine and measured prior to transplant, 1-week post-transplant, 1-3, 6, and 12 months post-transplant, and recent follow-up. Changes in eGFR were measured within and between patients using a piecewise linear mixed effect model with matching. Exploratory univariate analysis was performed to evaluate pre-transplant risk factors for decreased eGFR., Results: The unmatched cohort included 393 heart transplant recipients, with 47 being retransplants. Thirty-eight patients in both groups with at least 1 year of follow-up underwent matching. Both retransplants and first-time transplants had an initial decline in eGFR. eGFR rebounded to baseline or above baseline at 1-3 months post-transplant, but eGFR in retransplants remained significantly lower. At 1-year post-transplant, the average eGFR was 67.8 ± 4.3 mL/min/1.73 m 2 versus 104.7 ± 4.3 mL/min/1.73 m 2 (p < .001) in the retransplants and first-time transplants group, respectively., Conclusion: This study provides data on anticipated renal trajectory following retransplantation., (© 2023 Wiley Periodicals LLC.)

Introduction: We present a case series of neonates with anuric ESRD undergoing renal replacement therapy (RRT) and discuss the associated ethical implications of RRT in this population., Methods: We reviewed patients who initiated RRT within 1 week of life due to anuric ESRD from 2009-2019 at a single tertiary center. Primary outcomes were receipt of renal transplant (RT), one-year survival, and overall survival., Results: Five patients met the inclusion criteria. Two patients received an RT. One-year survival was 80%, while overall survival was 60% with a median follow-up of 18 months. In the 2 still-living patients who have not undergone RT, they are ineligible, one due to recent malignancy and the other from acquired cardiovascular comorbidities., Conclusion: Patients with anuric ESRD requiring RRT undergo multiple treatment challenges with low RT and survival rates. These findings should be shared with families considering intervention for cases of severe renal disease diagnosed prenatally., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Objective: The objective is to compare robotic sacral colpopexy (RSC) utilizing autologous fascia lata with RSC with synthetic mesh in the treatment of pelvic organ prolapse (POP). Methods: We performed a prospective nonrandomized case comparison trial at a single institution. We compared RSC utilizing either synthetic mesh or autologous fascia lata in women with symptomatic POP, stages II through IV. The primary outcome was anatomic prolapse recurrence determined by the Pelvic Organ Prolapse Quantification (POP-Q) examination. Secondary outcomes included patient-reported outcomes such as the Urogenital Distress Inventory-6 (UDI-6) and Incontinence Impact Questionnaire-7 (IIQ-7). Complications were also recorded and categorized using the Clavien-Dindo (CD) system. The hypothesis is that autologous fascia lata would provide equivalent anatomic and patient-reported outcomes compared with mesh while eliminating mesh-related complications. Results: Sixty-four women underwent RSC with 19 (29.7%) receiving fascia lata graft. The overall operative time was greater in the fascia lata group with mean fascia lata harvest time of 24.8 ± 7.4 minutes. Intragroup comparisons of the fascia and mesh groups demonstrated significant improvement in pelvic measurements as well as patient-reported outcomes. Intergroup comparison demonstrated equivalent success rates at 12.1 ± 8.7 months follow-up. There was one apical failure in the fascia lata RSC group; however, the difference was not statistically significant ( p  = 0.30). Significant complications in the fascia lata harvest group included two CD-II and one CD-IIIb. In the mesh group there was one mesh erosion requiring surgical excision (CD-IIIb). Conclusion: This is the first comparison between RSC with autologous fascia lata and mesh. Short-term anatomic outcomes were similar with autologous fascia lata use without the risk of mesh erosion. Morbidity from graft harvest site was not trivial. These results emphasize the need for a randomized controlled trial.

Background: Studies have found an association between obesity and an increased risk for peripartum depression, which has also been linked to decreased placental lactogen levels. In addition, women with obesity treated for gestational diabetes with insulin were found to have increased levels of placental lactogen. Treatment options exist for perinatal and postpartum depression however they pose a risk to the developing offspring. Thus, prevention as well as markers for early identification of peripartum depression are needed. Therefore, our study objective is to identify the association between insulin treatment in pregnancy and the risk of postpartum psychological distress (abbreviated here as PPD) among cohorts of women with and without obesity., Methods: Administrative health data (2002/03-2018/19) were used to identify a cohort of women (age 15+ years) who gave birth (N = 250,746) and had no pre-existing mood/anxiety disorders or diabetes (N = 222,863 excluded). Women were then divided into two groups: lean (N = 17,975) and with obesity (N = 9908), which was identified by a recorded maternal weight of > 38 to < 65.6 kg and ≥ 85 to < 186 kg (respectively). The risk of PPD within one year after delivery with and without insulin treatment was assessed by Poisson regression analysis. Models were adjusted for maternal age group (at pregnancy start date) and area-level income (at delivery)., Results: The unadjusted risk of PPD was higher in the obesity group (8.56%; 95% CI 8.00-9.15) than in the lean group (6.93%; 95% CI 6.56-7.33). When no insulin treatment was given during pregnancy, mothers with obesity had a significantly higher risk of PPD than the lean group (aRR 1.27; 95% CI 1.17-1.39; p < 0.0001). However, when women with obesity and insulin treatment were compared to the lean group with no insulin treatment, no significant difference in the risk of PPD was observed between the groups (aRR 1.30; 95% CI 0.83-2.02; p = 0.248)., Conclusion: This is the first study to demonstrate a positive association between insulin treatment in pregnancy among women with obesity and reduced PPD rates, suggesting insulin as a possible preventative measure. However, the biological mechanism behind the observed positive effect of insulin on PPD rates remains to be investigated.

Dyslipidemia after kidney transplantation is a common complication that has historically been underappreciated, especially in pediatric recipients. It is also a major modifiable risk factor for cardiovascular disease, a top cause of morbidity and mortality of transplant patients. While most knowledge about post-transplant dyslipidemia has been generated in adults, recommendations and treatment strategies also exist for children and are presented in this review. Awareness of these applicable guidelines and approaches is required, but not sufficient, for the reliable management of dyslipidemia in our patients, and additional needs and opportunities for comprehensive care in this area (e.g., quality improvement) are outlined.

The four genes coding for placental members of the human (h) growth hormone (GH) family include two that code independently for placental lactogen (PL), also known as chorionic somatomammotrophin hormone, one that codes for placental growth hormone (PGH) and a pseudogene for which RNA but no protein product is reported. These genes are expressed preferentially in the villus syncytiotrophoblast of the placenta in pregnancy. In higher primates, the placental members, including hPL and PGH, are the result of multiple duplication events of the GH gene. This contrasts with rodents and ruminants, where PLs result from duplication of the prolactin (PRL) gene. Thus, unlike their mouse counterparts, the hPL and PGH hormones bind both lactogenic and somatogenic receptors with varying affinity. Roles influenced by nutrient availability in both metabolic control in pregnancy and maternal behaviour are supported. However, the effect maternal obesity has on the activation of placental members of the hGH gene family, particularly the expression and function of those genes, is poorly understood. Evidence from partially humanised hGH/PL transgenic mice indicates that both the remote upstream hPL locus control region (LCR) and more gene-related regulatory regions are required for placental expression in vivo. Furthermore, a specific pattern of interactions between the LCR and hPL gene promoter regions is detected in term placenta chromatin from women with a normal body mass index (BMI) in the range 18.5-25 kg m -2 by chromosome conformation capture assay. This pattern is disrupted with maternal obesity (class II BMI > 35 kg m -2 ) and associated with a > 40% decrease in term hPL RNA levels, as well as serum hPL but not PRL levels, during pregnancy. The relative importance of the chromosomal architecture and predicted properties for transcription factor participation in terms of hPL production and response to obesity are considered, based on comparison with components required for efficient human pituitary GH gene expression., (© 2020 British Society for Neuroendocrinology.)

Thrombosis after kidney transplantation may result in catastrophic outcomes, including graft loss. Thrombophilia has been implicated in post-transplant thrombosis; data, however, are inconclusive on the impact of acquired and inherited thrombophilia and resultant thrombosis in renal graft recipients. We aimed to evaluate whether identifying children with thrombophilia during the pretransplant evaluation predicted post-transplant outcomes. We reviewed 100 kidney transplants performed in 100 children, aged 1-18 years, in a single-center retrospective study. Routine pretransplant comprehensive thrombophilia evaluation was completed. Thrombophilia was demonstrated in 36% patients (N = 36). TEs occurred in 11 patients before kidney transplant. Low PS and antithrombin were found in 9/86 (10.5%) and 2/89 (2.2%) children, respectively. Heterozygosity for FLV and PGM were found in 5/81 (6.2%) and 1/93(1.1%) children, respectively. A post-transplant thrombotic event occurred in 10 children (10%); six involved the renal transplant. The association between a history of a pretransplant thrombotic event and post-operative renal graft thrombosis approached, but did not reach significance (P = 0.071). There was no association between preoperative screening abnormalities and post-operative TEs. Graft loss due to a thrombotic event occurred in two patients; none had underlying thrombophilia. Our data suggest that the utility of universal, comprehensive preoperative thrombophilia testing is not beneficial in determining risk of post-operative graft thrombosis. Thrombophilia testing may be considered in a select population with a history of pretransplant thrombotic event., (© 2018 Wiley Periodicals, Inc.)

Human (h) pituitary growth hormone (GH) is both physiologically and clinically important. GH reaches its highest circulatory levels in puberty, where it contributes to energy homeostasis and somatogenic growth. GH also helps to maintain tissues and organs and, thus, health and homeostasis. A reduction in the rate of hGH production begins in middle age but if GH insufficiency occurs this may result in tissue degenerative and metabolic diseases. As a consequence, hGH is prescribed under conditions of GH deficiency and, because of its lipolytic activity, stimulation of hGH release has also been used to treat obesity. However, studies of normal GH production and particularly synthesis versus secretion are not feasible in humans as they require sampling normal pituitaries from living subjects. Furthermore, human (or primate) GH structure and, as such, regulation and potential function, is distinct from non-primate rodent GH. As a result, most information about hGH regulation comes from measurements of secreted levels of GH in humans. Thus, partially humanized hGH transgenic mice, generated containing fragments of human chromosome 17 that include the intact hGH gene locus and many thousands of flanking base pairs as well as the endogenous mouse (m) GH gene provide a potentially useful model. Here we review this mouse model in terms of its ability to allow comparison of hGH versus mGH gene expression, and specifically: (i) GH locus structure as well as regulated and rhythmic expression; (ii) their ability to model a clinical assessment of hGH production in response to overeating and hyperinsulinemia as well as a possible effect of exercise, and (iii) their hGH-related immune tolerance and thus potential for testing hGH-related analogue immunogenicity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The human (h) placental lactogenic hormone chorionic somatomammotropin (CS) is highly produced during pregnancy and acts as a metabolic adaptor in response to maternal insulin resistance. Maternal obesity can exacerbate this "resistance", and a >75% decrease in CS RNA levels was observed in term placentas from obese vs. lean women. The genes coding for hCS ( hCS-A and hCS-B) and placental growth hormone ( hGH-V) as well as the hCS-L pseudogene and pituitary growth hormone (GH) gene ( hGH-N) are located at a single locus on chromosome 17. Three remote hypersensitive sites (HS III-V) located >28 kb upstream of hGH-N as well as local hCS gene promoter and enhancer regions are implicated in hCS gene expression. A placenta-specific chromosomal architecture, including interaction between HS III-V and hCS but not hGH gene promoters, was detected in placentas from lean women (BMI <25 kg/m 2 ) by using the chromosome conformation capture assay. This architecture was disrupted by pre-pregnancy maternal obesity (BMI >35 kg/m 2 ), resulting in a predominant interaction between HS III and the hGH-N promoter, which was also observed in nonplacental tissues. This was accompanied by a decrease in hCS levels, which was consistent with reduced RNA polymerase II and CCAAT/enhancer-binding protein-β association with individual hCS promoter and enhancer sequences, respectively. Thus, pre-pregnancy maternal obesity disrupts the placental hGH/CS gene locus chromosomal architecture. However, based on data from obese women who develop GDM, insulin treatment partially recapitulates the chromosomal architecture seen in lean women and positively affects hCS production, presumably facilitating prolactin receptor-related signaling by hCS.

Since first performed in 1954, kidney transplantation has evolved as the preferred long-term treatment of children with end stage renal disease (ESRD). The etiology of chronic kidney disease (CKD) and ESRD in children is broad and can be quite complicated, necessitating a multidisciplinary team to adequately care for these patients and their myriad needs. Precise surgical techniques and modern protocols for immunosuppression provide excellent long-term patient and graft survival. This article reviews the many etiologies of renal failure in the pediatric population focusing on those most commonly leading to the need for kidney transplantation. The processes of evaluation, kidney transplantation, short-term and long-term complications, as well as long-term outcomes are also reviewed., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The genes coding for human (h) chorionic somatomammotropin (CS), hCS-A and hCS-B, and placental growth hormone (GH-V), hGH-V, are located at a single locus on chromosome 17. Efficient expression of these placental genes has been linked to local regulatory (5' P and 3' enhancer) sequences and a remote locus control region (LCR), in part, through gene transfer in placental and nonplacental tumor cells. However, low levels of endogenous hCS/GH-V transcripts are reported in the same cells compared with term placenta, suggesting that chromatin structure, or regulatory region accessibility, versus transcription factor availability contributes to the relatively low levels. To assess individual hCS-A, CS-B, and GH-V gene expression in placental and nonplacental tumor cells and the effect of increasing chromatin accessibility by inhibiting DNA methylation and histone deacetylation using 5-aza-2'-deoxycytidine (azadC) and trichostatin A (TSA). Low levels of hCS-A, CS-B, and GH-V were detected in placental and nonplacental tumor cells compared with term placenta. A significant >5-fold increase in activity was seen in placental, but not nonplacental, cells transfected with hybrid hCS promoter luciferase genes containing 3' enhancer sequences. Pretreatment of placental JEG-3 cells with azadC resulted in a >10-fold increase in hCS-A, CS-B, and GH-V RNA levels with TSA treatment compared with TSA treatment alone. This effect was specific as reversing the treatment regimen did not have the same effect. An assessment of hyperacetylated H3/H4 in JEG-3 cells treated with azadC and TSA versus TSA alone revealed significant increases consistent with a more open chromatin structure, including the hCS 3' enhancer sequences and LCR. These observations suggest that accessibility of remote and local regulatory regions required for efficient placental hGH/CS expression can be restricted by DNA methylation and histone acetylation status. This includes restricting access of the hCS 3' enhancer sequences to available placental enhancer transcription factors.

Background and Objectives: FSGS is the primary cause of childhood nephrotic syndrome leading to ESRD. Permeability factors, including circulating serum soluble urokinase-type plasminogen activator receptor (suPAR), have been postulated as putative causes in adults with primary FSGS. Similar results have yet to be proven in children., Design, Setting, Participants, & Measurements: This cross-sectional single-center study assessed the association of serum suPAR in children with FSGS or other glomerular and nonglomerular kidney diseases., Results: This study examined 110 samples retrieved from 99 individuals (between January 2011 and April 2012), aged 1-21 years; of these individuals, 20 had primary FSGS, 24 had non-FSGS glomerular disease, 26 had nonglomerular kidney disease, and 29 were healthy controls. suPAR levels were not significantly different in children with FSGS, non-FSGS glomerular disease, and healthy controls (P>0.05). However, suPAR levels (median [25%-75%]) were higher in children with nonglomerular kidney disease (3385 pg/ml [2695-4392]) versus FSGS (2487 pg/ml [2191-3351]; P<0.05). Female patients with nephrotic-range proteinuria (U-Pr/Cr >2) had lower suPAR levels than those without proteinuria (2380 pg/ml [2116-2571] versus 3125 pg/ml [2516-4198], respectively; P<0.001). This trend was not seen among male participants; suPAR levels in all female participants were lower than in male participants (P=0.03). Thirty-four patients studied were kidney transplant recipients; transplant status was not associated with suPAR levels in patients with FSGS or non-FSGS diagnoses, independent of proteinuria, race, or sex (P>0.05)., Conclusions: On the basis of these results, circulating suPAR is unlikely the leading cause for childhood idiopathic FSGS.

Aim: Children with steroidresistant (SR) and steroid-dependent (SD) nephrotic syndrome (NS) pose a treatment challenge. Literature on the use of tacrolimus (TAC), a calcineurin inhibitor, for maintenance treatment of NS is sparse. We aimed to evaluate the efficacy and safety of low-dose, long-term TAC for inducing and sustaining remission in children with SD/SR NS., Methods: Data from patients treated at our center from 1999 to 2009 were analyzed., Results: 40 patients with NS were treated with TAC for 3 - 80-month periods (median 25.2 months). Diagnoses included focal segmental glomerulosclerosis (FSGS) (60%), IgM nephropathy (15%), minimal change disease (20%) and membrano-proliferative glomerulonephritis (MPGN) (5%). 58% of patients had been previously treated with alternate agents. After 1, 2, and 3 years on TAC, complete remission was achieved in 26%, 48%, and 29% of patients; complete or partial remission was achieved in 85%, 100%, and 86%, respectively (p < 0.05). Median time to remission was 41 days (range: 10 - 270 days). FSGS and SR diseases were associated with lower likelihood of remission (p < 0.05). Remission was equally likely in both treatment naïve patients and those who had received prior second-line agents., Conclusion: Our results demonstrate that TAC treatment for children with SR/SD NS is associated with high rates of sustained remission, even when prior second-line agents failed.

A number of interesting issues have been addressed on biological networks about their global and local properties. The connection between the topological properties of proteins in Protein-Protein Interaction (PPI) networks and their biological relevance has been investigated focusing on hubs, i.e. proteins with a large number of interacting partners. We will survey the literature trying to answer the following questions: Do hub proteins have special biological properties? Do they tend to be more essential than non-hub proteins? Are they more evolutionarily conserved? Do they play a central role in modular organization of the protein interaction network? Are there structural properties that characterize hub proteins?, (Copyright © 2012 Elsevier Inc. All rights reserved.)

Disruption of the X-chromosome fibroblast growth factor 16 (Fgf-16) gene, a member of the FGF-9 subfamily with FGF-20, was linked with an effect on cardiac development in two independent studies. However, poor trabeculation with lethality by embryonic day (E) 11.5 was associated with only one, involving maintenance in Black Swiss (Bsw) versus C57BL/6 mice. The aim of this study was to examine the potential influence of genetic background through breeding the null mutation onto an alternate (C57BL/6) background. After three generations, 25% of Fgf-16(-/Y) mice survived to adulthood, which could be reversed by reducing the contribution of the C57BL/6 genetic background by back crossing to another strain. There was no significant difference between FGF-9 and FGF-20 RNA levels in Fgf-16 null versus wild-type mice regardless of strain. However, FGF-8 RNA levels were reduced significantly in Bsw but not C57BL/6 mice. FGF-8 is linked to anterior heart development and like the FGF-9 subfamily is reportedly expressed at E10.5. Like FGF-16, neuregulin as well as signaling via ErbB2 and ErbB4 receptors have been linked to trabeculae formation and cardiac development around E10.5. Basal neuregulin, ErbB2, and ErbB4 as well as FGF-8, FGF-9, and FGF-16 RNA levels varied in Bsw versus C57BL/6 mice. These data are consistent with the ability of genetic background to modify the phenotype and affect embryonic survival in Fgf-16 null mice.

MolLoc stands for Molecular Local surface comparison, and is a web server for the structural comparison of molecular surfaces. Given two structures in PDB format, the user can compare their binding sites, cavities or any arbitrary residue selection. Moreover, the web server allows the comparison of a query structure with a list of structures. Each comparison produces a structural alignment that maximizes the extension of the superimposition of the surfaces, and returns the pairs of atoms with similar physicochemical properties that are close in space after the superimposition. Based on this subset of atoms sharing similar physicochemical properties a new rototranslation is derived that best superimposes them. MolLoc approach is both local and surface-oriented, and therefore it can be particularly useful when testing if molecules with different sequences and folds share any local surface similarity. The MolLoc web server is available at http://bcb.dei.unipd.it/MolLoc.

Discovery of a similar region on two protein surfaces can lead to important inference about the functional role or molecular interaction of this region for one of the proteins if such information is available for the other. We propose a new characterization of protein surfaces based on a spin-image representation of the surfaces that facilitates the simultaneous search of the entire surface of each of two proteins for a matching region. For a surface point, we introduce spin-image profiles that are related to the degree of exposure of the point to identify structurally equivalent surface regions in two proteins. Unlike some related methods, we do not assume that a known fixed region of one of the protein surfaces is to be matched on the other protein surface. Rather, we search for the largest similar regions on each of the two surfaces. In spite of the fact that this approach is entirely geometric and no use is made of physicochemical properties of the protein surfaces or fold information, it is effective in identifying similar regions on both surfaces even when the region corresponds to a binding site on one of the proteins. The discovery of similar regions on two or more proteins also has implications for drug design and pharmacophore identification. We present experimental results from datasets of more than 50 protein surfaces.

We present a method for detecting and comparing cavities on protein surfaces that is useful for protein binding site recognition. The method is based on a representation of the protein structures by a collection of spin-images and their associated spin-image profiles. Results of the cavity detection procedure are presented for a large set of non-redundant proteins and compared with SURFNET-ConSurf. Our comparison method is used to find a surface region in one cavity of a protein that is geometrically similar to a surface region in the cavity of another protein. Such a finding would be an indication that the two regions likely bind to the same ligand. Our overall approach for cavity detection and comparison is benchmarked on several pairs of known complexes, obtaining a good coverage of the atoms of the binding sites.

Objective: Added transforming growth factor beta (TGFbeta) inhibits the proliferation of immature cardiomyocytes. We have now examined the hypothesis that suppression of endogenous TGFbeta signaling will boost the proliferative response (DNA synthesis) of cardiac myocytes to serum and/or to the mitogenic factor fibroblast growth factor-2 (FGF-2)., Methods and Results: Overexpression of a kinase-deficient TGFbeta type II receptor (TGFbetaRIIDeltaKD) resulted in a 2.8-fold increase in cardiomyocyte DNA synthesis in serum-rich cultures, an effect requiring active FGFR-1 since it was not observed in the presence of excess kinase-deficient FGFR-1. This finding suggested that endogenous TGFbeta-TGFbetaRII suppressed endogenous FGFR-1-mediated signals that stimulate or are permissive for DNA synthesis. TGFbeta had no effect, however, on the FGF-2-induced acute stimulation of extracellular signal regulated kinase1/2. FGF-2, added in the absence or presence of TGFbeta inhibition, elicited a 3- or a 13-fold stimulation of DNA synthesis, respectively, pointing to a synergistic effect., Conclusion: Inhibition of TGFbetaRII-transduced signaling upregulates the proliferative response of cardiomyocytes to serum, and greatly potentiates the stimulatory effect of FGF-2. A combinatorial strategy including activation of FGF-2 and inhibition of TGFbeta-triggered signal transduction may be required for maximal stimulation of immature cardiomyocyte DNA synthesis.

The problem of characterizing and detecting recurrent sequence patterns such as substrings or motifs and related associations or rules is variously pursued in order to compress data, unveil structure, infer succinct descriptions, extract and classify features, etc. In molecular biology, exceptionally frequent or rare words in bio-sequences have been implicated in various facets of biological function and structure. The discovery, particularly on a massive scale, of such patterns poses interesting methodological and algorithmic problems and often exposes scenarios in which tables and synopses grow faster and bigger than the raw sequences they are meant to encapsulate. In previous study, the ability to succinctly compute, store, and display unusual substrings has been linked to a subtle interplay between the combinatorics of the subword of a word and local monotonicities of some scores used to measure the departure from expectation. In this paper, we carry out an extensive analysis of such monotonicities for a broader variety of scores. This supports the construction of data structures and algorithms capable of performing global detection of unusual substrings in time and space linear in the subject sequences, under various probabilistic models.

Words that are, by some measure, over- or underrepresented in the context of larger sequences have been variously implicated in biological functions and mechanisms. In most approaches to such anomaly detections, the words (up to a certain length) are enumerated more or less exhaustively and are individually checked in terms of observed and expected frequencies, variances, and scores of discrepancy and significance thereof. Here we take the global approach of annotating the suffix tree of a sequence with some such values and scores, having in mind to use it as a collective detector of all unexpected behaviors, or perhaps just as a preliminary filter for words suspicious enough to undergo a more accurate scrutiny. We consider in depth the simple probabilistic model in which sequences are produced by a random source emitting symbols from a known alphabet independently and according to a given distribution. Our main result consists of showing that, within this model, full tree annotations can be carried out in a time-and-space optimal fashion for the mean, variance and some of the adopted measures of significance. This result is achieved by an ad hoc embedding in statistical expressions of the combinatorial structure of the periods of a string. Specifically, we show that the expected value and variance of all substrings in a given sequence of n symbols can be computed and stored in (optimal) O(n2) overall worst-case, O (n log n) expected time and space. The O (n2) time bound constitutes an improvement by a linear factor over direct methods. Moreover, we show that under several accepted measures of deviation from expected frequency, the candidates over- or underrepresented words are restricted to the O(n) words that end at internal nodes of a compact suffix tree, as opposed to the theta(n2) possible substrings. This surprising fact is a consequence of properties in the form that if a word that ends in the middle of an arc is, say, overrepresented, then its extension to the nearest node of the tree is even more so. Based on this, we design global detectors of favored and unfavored words for our probabilistic framework in overall linear time and space, discuss related software implementations and display the results of preliminary experiments.

Diabetes is a common complication encountered during pregnancy. Earlier studies indicated that diabetic placentas bear morphological alterations consistent with modified placental differentiation, including alterations in the villous cellular content, structure, and total surface. Limited data associating the diabetic status with the expression of terminal placental differentiation markers are available. The human growth hormone/chorionic somatomammotropin (hGH/CS) family consists of five genes, one of which (GH-N) is expressed efficiently in pituitary while the other four (CS-A, B, L, and hGH-V) are expressed in placenta and represent ultimate placental differentiation markers. We developed and applied a sensitive RT-PCR method coupled with diagnostic restriction digestion to determine the relative levels of the hGH/CS family in normal pregnancies and examine whether their mRNA expression pattern is altered in pregnancies complicated by diabetes. We show that relative hCS-L content changes during placental development. Specifically, normal term placentas express higher relative levels of hCS-L, lower relative hGH-V levels and a 70-fold lower hGH-V/CS-L mRNA ratio compared to early placentas. Also, many term placentas from diabetic pregnancies express lower relative levels of hCS-L mRNA and a much higher hGH-V/CS-L mRNA ratio compared to normal term placenta, resembling more an early placenta pattern of expression. Thus, our study suggests that the expression of terminal placental differentiation markers, such as the hGH/CS genes, is altered in term placentas from these diabetics reflecting either impaired placental differentiation or post-differentiation impairment of normal placental function.

The transcription factor GHF-1/Pit-1 is essential for the expression of GH and prolactin (PRL) by somatotrophs and lactotrophs respectively. However, PRL is not expressed in mature somatotrophs despite the presence of GHF-1/Pit-1. A possible mechanism is the presence of a somatotroph-specific repressor in the 5'-flanking sequences of the PRL gene. The region -3500/-1750 of the human (h) PRL gene is associated with negative regulatory activity and contains an element, designated D8, that resembles repressor PSF-A sequences which are located in the distal upstream region of placental members of the human GH family. An internal deletion of D8 sequences resulted in a significant stimulation of promoter activity in somatotroph GC (P < 0.005) and somatolactotroph-like GH3 and GH4C1 cells (P < 0.05), but not lactotroph-like 235-1 cells after gene transfer. However, D8 binding was observed by nuclease protection with lactotroph- as well as somatotroph-like cell nuclear protein. Although proteins that bind to the D8 element appear ubiquitous, this element does yield tissue-specific complexes in mobility shift assays. Further, competition studies do not suggest an interaction between GHF-1/Pit-1 and D8 proteins. The hPRL D8 element was inserted upstream of a thymidine kinase promoter and used to transfect pituitary and non-pituitary HeLa cells, to assess intrinsic repressor activity and/or promoter specificity. Although no repression was observed, a significant ninefold increase in expression was observed in HeLa cells (P < 0.001) which was at least twofold greater than observed in any of the pituitary cell lines tested. These results implicate D8 in the somatotroph-specific repression of hPRL; however, they also suggest that D8 can act as a stimulator as well as a repressor, depending on the interaction of a ubiquitous D8 factor forming promoter and cell-specific complexes with other elements/factors.

Positive (HF-1 and HF-2) and negative (HF-3) elements responsible for cardiac-specificity of the rat ventricular myosin light chain-2 (MLC-2v) promoter are contained in a 250 base pair region. The effect of the simian virus 40 (SV40) enhancer or 3 copies of the HF-1, HF-2 and HF-3 elements on MLC-2v promoter activity and specificity was assessed in neonatal rat cardiac myocytes and cardiac nonmuscle cells as well as rat heart myoblast H9c2 and glioma (nonmuscle) C6 cell lines. The SV40 enhancer increased promoter activity by at least 10-fold in both muscle and nonmuscle cell types; however, there was a decrease in cardiac ventricular myocyte-specificity. In contrast, the 3 copies of HF-1, HF-2 and HF-3 elements stimulated MLC-2v promoter activity approximately 3-fold in neonatal ventricular cardiac myocytes alone and, effectively, displayed about a 5-fold increase in specificity over the wild type MLC-2v promoter.

We used reverse transcriptase-polymerase chain reaction (RT-PCR) to clone fibroblast growth factor receptor (FGFR) 1 isoforms from embryonic mouse heart and as a more sensitive method to characterize FGFR1 RNA expression in embryonic and adult mouse hearts. We describe the cloning of both full-length short (2259 base pairs) and long (2526 base pairs) FGFR1 isoform cDNAs which generated 86 and 102 kilodalton proteins, respectively, following in vitro translation. An assessment of FGFR1 RNA indicates that FGFR1-IIIc is the major form in both the embryonic and adult heart but there is an approximately 8.5-fold decrease in RNA levels in the adult. Differential RNA blotting as well as RT-PCR analyses are consistent with a switch in the relative expression of the short versus long FGFR1 isoforms during heart development. The long isoforms are more abundant in the embryo and the short isoforms predominate in the adult. This may be important in the regulation of growth and development of the heart.

Basic (b) fibroblast growth factor (FGF) mediates various biological responses including mitogenesis and angiogenesis by binding to specific cell surface receptors of the tyrosine kinase family. The bFGF receptor-1 FGFR1) exists in short and long isoforms due to alternate RNA splicing. Minor alterations in the amino acid sequence have also led to reports of different FGFR1 isoforms in different tissues even in the same species. In the absence of any sequence for heart FGFR1 and accumulating evidence for a role of bFGF in heart growth and differentiation, we cloned FGFR1 from embryonic mouse hearts. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to generate full-length short (2259 base pairs) and long (2526 base pairs) forms of FGFR1 cDNAs which generated 86 and 102 kDa proteins, respectively, following in vitro translation. Embryonic mouse heart FGFR1 differed by seven amino acids from the reported sequence for mouse neuroepithelial FGFR1 and appeared more similar to human placental FGFR1. A single FGFR1 transcript of approximately 4.3 kb was seen in RNA isolated from embryonic as well as adult mouse hearts. There was a decrease (approximately 8.5-fold) in FGFR1 RNA levels in the adult. The majority of FGFR1 transcripts in the adult as well as embryonic heart contained exon IIIc (FGFR1-IIIc) which is associated with isoforms that display the highest affinity for bFGF. However, the relative ratio of short versus long FGFR1 RNA expression was 0.5 in the embryonic heart compared to 5.9 in the adult heart. These results indicate that: (i) structurally distinct short and long FGFR1 isoform RNAs are expressed in the embryonic and adult heart; (ii) FGFR1-IIIc is the major form of receptor expressed in the embryonic as well as adult heart; (iii) the transition from the embryo to the adult stage is associated with a decrease but not absence of FGFR1 RNA expression; and (iv) long FGFR1-isoforms are more abundant in the embryo while short FGFR1 isoforms predominate in the adult.

Attempts to assess human placental GH variant (hGH-V) and chorionic somatomammotropin (hCS) RNA in choriocarcinoma cell lines have been hampered by low levels of expression and limited sensitivity of RNA blotting analysis. We examined human choriocarcinoma BeWo, JAR, and JEG-3 cell lines as well as samples of complete hydatidiform moles for expression of members of the human GH (hGH) gene family using reverse transcriptase-polymerase chain reaction. A single and common set of primers was designed and used to detect products of the hGH/hCS genes as well as distinguish processed RNA from any contaminating DNA. Transcripts from the hCS genes hCS-A and -B were distinguished from placental hGH variant (hGH-V) and hCS-like (hCS-L) gene RNA by diagnostic restriction digestion of the polymerase chain reaction products. The expected pattern of hGH/hCS RNA expression was detected in term placenta, where hCS and hGH-V/hCS-L transcripts represented approximately 95% and approximately 5% of the total hGH/hCS RNA, respectively. The level of hCS RNA varied from 22-99% of the total hGH/hCS RNA in the neoplastic trophoblast samples, and variable levels of hGH-V and hCS-L RNA were also observed. In choriocarcinoma JAR cells, hGH-V RNA represented approximately 78% of the total hGH/hCS RNA compared to approximately 22% for hCS. Further, although low hCS-L RNA levels (< 1%) were found in term placenta and two of the hydatidiform moles, hCS-L transcripts represented 11% of the total hGH/hCS RNA in a third hydatidiform mole. Finally, in contrast to the detection of variable levels of hCS-L RNA in term placenta and hydatidiform mole samples, no hCS-L transcripts were detected in the three choriocarcinoma cell lines examined. These patterns reflect either deregulated hGH/hCS gene expression in neoplastic trophoblasts or differences that accompany the process of differentiation of trophoblast subpopulations. Regardless, this suggests that the control of hGH-V and hCS-L gene expression is distinct from that of the hCS-A and hCS-B genes and raises questions about the possible involvement of hGH/hCS family members in the pathology of placental abnormalities.

Rat prolactin-like protein A (rPLP-A) is a member of a rapidly expanding family of prolactin-related proteins that are expressed during pregnancy by the rat placenta according to specific developmental patterns. Although the factors involved in the pituitary-specific expression of the prolactin and growth hormone genes themselves have been extensively studied, essentially nothing is known of the factors responsible for the placental expression of these new family members. In this paper we describe the isolation of rPLP-A genomic clones, analyze a portion of the 5' flanking sequence of this gene and use the recently described rat choriocarcinoma cell line, Rcho, in transient transfection studies to show that a 975 base-pair (bp) fragment of 5' flanking sequence is sufficient to specify placental expression of the rPLP-A gene.

Chorionic somatomammotropin (hCS) genes (hCS-A and hCS-B) and the placental growth hormone variant (hGH-V) gene are expressed in the syncytiotrophoblast in vivo, and at low levels in cytotrophoblast-like choriocarcinoma (BeWo) cells. Treatment of choriocarcinoma cells with methotrexate (MTX) will induce a cell type intermediate between a cytotrophoblast and syncytiotrophoblast. After treatment with MTX, hCS/hGH-V mRNA levels were decreased in BeWo cells, and only hGH-V and minor hCS-A related transcripts of 1.6, 2.1 and 4.2 kilobases, termed hCS-A2, hCS-A3 and hCS-A4, respectively, were detected. By contrast, chorionic gonadotropin RNA levels were increased. This pattern of hCS/hGH-V expression resembles that observed when BeWo cells are grown in thyroid hormone (T3)-depleted serum, where hGH-V/hCS RNA increases in response to T3. This increase is blunted by MTX treatment, but is not due to a decrease in number or affinity of T3 receptors. These data indicate that the hGH-V and hCS genes can be differentially regulated by MTX, and are consistent with MTX interfering with T3 responsiveness of these genes. Also, if BeWo cells treated with MTX do represent a transitional state, these data raise the possibility that hGH-V and hCS possess a different temporal pattern of expression in the developing trophoblast.

The location and sequence of androgen responsive elements (AREs) in the 5'-flanking DNA of the androgen-regulated rat probasin (PB) gene were determined. The DNA- and steroid-binding domains of the rat androgen receptor [glutathione-S-transferase (GST)-AR1] and the DNA-binding domain and hinge region alone (GST-AR2) were expressed in Escherichia coli as isopropyl-B-D-thioglactopyranoside-induced fusion proteins with GST and purified using glutathione affinity chromatography. Band shift assays indicated that the AR1 peptide was at least five times more effective than AR2 in binding to PB 5'-flanking DNA (-426 to +28), although both gave qualitatively similar patterns and were displaced by anti-AR antibodies. DNase I footprinting experiments revealed two putative AREs: one between positions -236 and -223 (ARE-1) and the other between -140 and -117 (ARE-2). Hormonal regulation of PB was determined by cotransfecting reporter constructions containing the PB 5'-flanking region (-426 to +28) linked to the bacterial chloramphenicol acetyl transferase (CAT) gene with androgen, glucocorticoid, or progesterone receptor expression vectors into human prostatic carcinoma cells (PC-3). PB-CAT gene expression was more effectively induced by androgens than by glucocorticoids or progestins. Both 5'- and 3'-deletion mapping of the PB 5'-flanking DNA revealed that ARE-1 and ARE-2 were required for androgen regulation. A single base mutation in either ARE resulted in a more than 95% loss of androgen induction of CAT. In comparable transfection experiments, the PB hormone-responsive elements showed a greater induction by androgens than did mouse mammary tumor virus or tyrosine aminotransferase elements. Thus, the preferential androgen regulation of the PB gene involves the participation of two different cis-acting DNA elements that bind AR.

FEG-3 cells are a clonal line of human choriocarcinoma and resemble villous cytotrophoblasts which are the stem cells for the syncytiotrophoblast in the placenta. FEG-3 cells synthesize and secrete the alpha subunit of human chorionic gonadotrophin (hCG). Treatment of FEG-3 cells with the chemotherapeutic drug (1 microM) methotrexate (MTX) results in an increase in nuclear diameter. Cell division is blocked and a decrease in c-myc mRNA levels in observed. The effects on cell growth and c-myc mRNA expression are reversible, and cells treated with MTX for 48 h retain their proliferative potential. Assessment of placental hormone gene expression reveals that a member of the human growth hormone gene family is expressed at extremely low levels and is unaffected by MTX treatment. Alpha and beta chorionic gonadotrophin (hCG) levels are increased by MTX treatment, but levels decrease following removal of MTX. In contrast to hCG in FEG-3 cells, non-trophoblastic or ectopic production of alpha hCG in human cervical carcinoma cells is inhibited by MTX treatment. These data indicate that MTX will induce morphological and biochemical changes in FEG-3 cells. They reveal an inverse relationship between c-myc and hCG RNA expression, and suggest different mechanisms govern trophoblast versus non-trophoblast production of alpha hCG.

Placental chorionic somatomammotropin (hCS-A or B) and growth hormone variant (hGH-V) are members of the human growth hormone family, and are related by structure and function to pituitary growth hormone (hGH-N). However, while the hGH-N gene is expressed specifically in the anterior pituitary, hGH-V and hCS are produced in the placenta. Hybrid hGH-N, hGH-V and hCS-A genes containing 5'-flanking sequences, including the endogenous promoter, are preferentially expressed in rat pituitary tumor (GC) cells, after gene transfer. Since interaction with a pituitary-specific protein (Pit 1) is required for efficient hGH-N as well as rat growth hormone (rGH) gene expression in GC cells, binding of pituitary proteins to the hGH-V and hCS-A promoter sequences was investigated. Rat Pit 1 binds at two locations on the hGH-N gene, a distal (-140/-107) and proximal site (-97/-66), in a similar manner to that observed with the rGH gene. By contrast, efficient Pit 1 binding was seen only to the distal site of the hGH-V gene and the proximal site of the hCS-A gene. Although binding of a protein to the distal hCS-A sequences was observed, the site of interaction was truncated (-140/-116), not pituitary-specific, and was more consistent with the binding of Sp1. These data indicate that rat Pit 1 binds to the placental hGH-V and hCS-A genes and correlates with their promoter activity in GC cells after gene transfer.(ABSTRACT TRUNCATED AT 250 WORDS)