Biomedical subjects

J Lough

Publications and source records attributed to J Lough.

At least 19 recordsLinked to original sources

PubMed2001

Bisulfite-induced cytosine deamination rates in E. coli SSB:DNA complexes.

E. coli single-stranded binding protein (SSB) has been examined for its ability to modulate bisulfite-induced cytosine deamination rates in single-stranded DNA (ssDNA). We used a lacZ alpha-complementation reversion assay to detect C-->U rates at a single codon in M13mp2 DNA, whether in free ssDNA or in an SSB:ssDNA complex. When incubated at 37 degrees C, the average bisulfite-induced reversion rate constant was four-fold less in SSB:ssDNA complexes than in ssDNA, at a single codon. Across a 250 base pair target and over 23 scorable C-->U sites, the forward rate constant was 4.9-fold less in SSB:ssDNA complexes than in ssDNA alone. After treatment with N-uracil glycosylase, ssDNA incubated with bisulfite had reversion frequencies at the background rate of ssDNA incubated without bisulfite, indicating that virtually all mutations scored were due to C-->U events. The decrease in cytosine deamination rates occurred both in a single codon and over a 250 bp target, indicating that interactions between SSB and ssDNA reduce bisulfite-catalyzed mutations. The structural role of SSB is well recognized in multiple cellular processes; SSB can also function to minimize bisulfite-induced ssDNA mutations.

Cytosine↗

PubMed2001

Prognostic significance of the labeling of Adnab-9 in pancreatic intraductal papillary mucinous neoplasms.

BACKGROUND: Pancreatic intraductal papillary mucinous neoplasms (IPMN), morphologically resembling colonic adenomas, often have an indefinable malignant potential. We used a monoclonal antibody (MAb) raised against colonic adenomas, Adnab-9, to identify patients with a better prognosis. METHODS: We assessed Adnab-9-labeled sections of these neoplasms from 50 patients, 13 pancreatic adenocarcinomas, and 32 colonic adenomas using standard immunohistochemical techniques. RESULTS: 26% of the IPMNs labeled with Adnab-9 as compared to 0% of pancreatic ductal cancers or surrounding benign tissues, (p < 0.001) and 53% of adenomas (p < 0.025). Labeling in IPMNs was usually seen in the noninvasive epithelium suggesting that Adnab-9 is a premalignant marker in these lesions. Labeling of invasive IPMN's identified a group of patients with a superior overall survival (p = 0.027). CONCLUSION: Adnab-9 labeling-characteristics appear similar for both IPMNs and adenomatous polyps, suggesting that they are analogous lesions. Adnab-9 labeling may also be a useful prognostic marker for invasive intraductal papillary mucinous neoplasms.

Adenocarcinoma, Mucinous↗

PubMed2000

Differential expression of cSmad1 and cSmad5 in the primitive streak during chick embryo gastrulation.

Whole mount in situ hybridization was performed to determine the expression patterns of cSmad1 and cSmad5 during gastrulation stages (Hamburger-Hamilton 2-6) of chicken embryogenesis. It was revealed that cSmads 1 and 5 mRNAs are prominently expressed in the developing primitive streak. As determined from cross-sectional analysis of stage 4 embryos, most transcripts were associated with the ingressing mesoderm. However, whereas cSmad5 was expressed throughout the streak at all stages examined, the strongest expression of cSmad1 was confined to the posterior half of the streak.

Animals↗

PubMed2000

Endoderm and heart development.

Since the first half of the 20th century, experimental embryologists have noted a relationship between endoderm cells and the development of cardiac tissue from mesoderm. During the past decade, the accumulation of evidence for an obligatory interaction between endoderm and mesoderm during the specification and terminal differentiation of myocardial, and more recently endocardial, cells has markedly accelerated. Moreover, the endoderm-derived molecules that may regulate these processes are being identified. It now appears that endoderm-derived growth factors regulate the formation of both myocardial and endocardial cells during specification, terminal differentiation, and perhaps morphogenesis of cells in the developing embryonic heart.

Animals↗

PubMed2000

Requirement for BMP and FGF signaling during cardiogenic induction in non-precardiac mesoderm is specific, transient, and cooperative.

We previously reported that combined treatment with bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-4 (FGF-4) induces cardiogenic events culminating in full cardiac differentiation of non-precardiac mesoderm explanted from stage 6 avian embryos (Lough et al. [1996] Dev. Biol. 178:198-202.). To elucidate the respective functions of BMP and FGF in initiating and maintaining the cardiogenic process, we have used these ectopic cells as a cardiac specification model to ascertain requirements for growth factor specificity and extent of application, as well as induction of cardiac transcription factors. The inability of some BMP isoforms to replace the inductive activity of BMPs-2/4 indicated a specific requirement for this signaling pathway; moreover, neither activin-A nor insulin, which support terminal differentiation of precardiac mesoderm, nor leukocyte inhibitory factor (LIF), which promotes hypertrophy in cardiac myocytes, could replace BMP's cardiogenic activity. A similarly specific requirement for FGF-2/4 signaling was revealed since neither FGF-7, activin-A nor insulin could replace this activity. The effect of both factors was concentration-dependent; maximal incidence of explant differentiation for each occurred at 50 ng/ml. Surprisingly, the majority of explants treated with high BMP levels (250 ng/ml) exhibited a non-cardiac phenotype that was characterized by intense expression of alkaline phosphatase, suggesting differentiation toward an alternative mesodermal phenotype. Experiments to assess the duration of exposure to each factor that was required revealed that while exposure to BMP and FGF during only the initial 30 min of a 48-hr culture period was sufficient to induce cardiogenesis in a significant percentage of explants, 100% incidence of explant differentiation was obtained only when FGF treatment was restricted to the first 30 min and BMP was continuously present during the 48-hr culture period. Treatment with both growth factors was required to induce the cardiac transcription factors cNkx-2.5 and SRF; neither mRNA was induced by BMP or FGF alone. These findings indicate that: (1) specific members of the BMP and FGF families are required to induce cardiogenesis in non-precardiac mesoderm; (2) BMPs-2/4 may function as a morphogen; (3) brief application of both factors can induce cardiogenesis in a modest number of explants whereas (4) 100% incidence of explant differentiation can only be attained by brief FGF treatment combined with continuous BMP treatment and (5) both factors are necessary to induce downstream cardiac transcription factors. These findings are interpreted in terms of these factors' possible roles during cardiac specification and differentiation.

Bone Morphogenetic Protein 2↗

PubMed2000

FGF-2-induced imbalance in early embryonic heart cell proliferation: a potential cause of late cardiovascular anomalies.

BACKGROUND: This laboratory previously demonstrated that placement of fibroblast growth factor-2 (FGF-2)-soaked beads adjacent to the developing ventricle at stage 24 caused cardiovascular anomalies by embryonic day 15. We sought to characterize early cellular changes that may suggest mechanisms for the abnormalities observed at day 15. Because levels of both myocyte proliferation and immunohistochemically detectable endogenous FGF-2 begin to decline before stage 24 in untreated embryos, it was of interest to determine whether exogenous FGF-2 might maintain cardiac myocyte proliferation at or near peak levels. METHODS: Chick embryos were incubated to stage 18 (2.8 days), at which time beads soaked in phosphate-buffered saline (PBS) or 100 microg/ml FGF-2 were placed adjacent to the developing ventricle and development was allowed to continue. After 3 days (stage 29), bromodeoxyuridine (BrdU) was applied to mark dividing cells, followed by double fluorescent assessments to detect relative numbers of dividing and nondividing cells. RESULTS: Quantitative image analysis, using Metamorph software, showed that exogenous FGF-2 caused a 62% increase in the overall number of dividing cells (P < 0.01), concomitant with a 25% increase in total cell number (cell density: P < 0.05). Expressed in relative terms, these changes corresponded to a 25% increase in the proliferation labeling index: 30% of all cells were proliferating in FGF-treated hearts, in contrast with only 24% in control hearts. CONCLUSIONS: Taken together, these data suggest that an FGF-induced imbalance in myocardial cell proliferation at early developmental stages of heart development causes cardiovascular anomalies during late embryogenesis.

Abnormalities, Drug-Induced↗

PubMed1999

Evidence that FGF receptor signaling is necessary for endoderm-regulated development of precardiac mesoderm.

Endoderm cells in the heart forming region (HFR endoderm) of stage 6 chicken embryos are required to support the proliferation and terminal differentiation of precardiac mesoderm cells in vitro. The endoderm's effect can be substituted by growth factors, including members of the fibroblast growth factor (FGF) family. However, direct implication of FGFs in this process requires evidence that inhibition of FGF signaling interferes with proliferation and/or terminal differentiation. This report examines the consequences of treating endoderm/precardiac mesoderm co-explants with agents that inactivate FGF receptors. Using sodium chlorate, which prevents FGF ligand-receptor interaction, it was observed that the percentage of S-phase precardiac mesoderm cells was markedly reduced, suggesting that cell proliferation was inhibited. To more specifically affect FGF signaling, the explants were treated with an antibody that recognizes an extracellular domain of FGF receptor-1 (FGFR-1). This treatment similarly inhibited cell proliferation. Although both agents modestly delayed cardiac myocyte differentiation as indicated by the contractile function, expression of alpha-sarcomeric actin was not affected. These findings provide additional evidence that an intact FGF signaling pathway is required during heart development.

Animals↗

PubMed1998

Teratogenic effects of implanting fibroblast growth factor-2-soaked beads in the cardiac region of the stage 24 chick embryo.

The identification of fibroblast growth factor-2 (FGF-2), and other family members, in a variety of embryonic tissue extracts has implicated these growth factors as participants in many embryonic events, including cardiogenesis. The present study was conducted in an attempt to characterize the effects of exogenous FGF-2 on the development of the avian heart. Heparin acrylic beads, each soaked in 100 micrograms/ml FGF-2, were applied to the Hamburger and Hamilton [(1951) J. Morphol. 88:49-92] stage 24 (day 4 of incubation) chick heart, near the primitive ventricle. The embryos were allowed to develop until stage 41 (day 15), at which time they were observed for the presence of gross external and internal anomalies. Crown rump length (CRL), wet weight (WW), and various heart measurements were obtained and compared. The survival rate of the control group was significantly higher (P < 0.05) than that for the experimental group and the anomaly rate for the control group was significantly lower (P < 0.01) than that for the experimental group. The experimental group exhibited a wide range of anomalies, most of which were cardiac in nature; however, anomalies of the ventral thoracoabdominal wall were also noted. Our data suggest that application of exogenous FGF-2 has teratogenic effects on cardiac development as well as on the development of the ventral body wall of the avian embryo.

Abnormalities, Drug-Induced↗

PubMed1998

Expression of retinol binding protein and transthyretin during early embryogenesis.

Previous studies have shown that anterior lateral plate endoderm from stage 6 chicken embryos is necessary and sufficient to enable precardiac mesoderm to complete its cardiogenic program in vitro, culminating in a rhythmically contractile multicellular vesicle (Sugi and Lough [1994] Dev. Dyn. 200:155-162). To identify cardiogenic factors, we have begun to characterize proteins that are secreted by endoderm cell explants. Fluorography of proteins from endoderm-conditioned medium revealed 1-2 dozen bands, the most prominent of which migrated at approximately 17 and 25 kD. The bulk of the 17-kD band, which migrates near FGFs and subunits of the transforming growth factor-beta family, was identified by N-terminal sequencing as transthyretin (TTR). A component of the 25-kD band was identified by Western blotting as retinol binding protein (RBP). RT/PCR analysis revealed that mRNAs for both proteins are in the embryo as early as stage 3. In situ hybridization localized these mRNAs to the extraembryonic endoderm at stage 6, after which they were detected in endoderm overlying the embryo proper, including the developing heart. Later, RBP and TTR mRNA and protein were detected in cells associated with the developing heart. Western blotting of whole embryo proteins revealed the presence of RBP by stage 7, followed by sequential increases to stage 25; by contrast, content of RBP in isolated hearts peaked at stage 14, then declined. Immunohistochemistry revealed the presence of RBP protein in the extracellular matrix subjacent to lateral plate endoderm beginning at stage 8; upon formation of the definitive heart, intense staining was observed in the cardiac "jelly." By contrast TTR was intracellular, first detected as subtle deposits in stage 6 embryonic endoderm, which by stage 8 were prominent in the dorsally invaginated endoderm subjacent to the precardiac splanchnic mesoderm. At stages 11-14, TTR was detected only in myocardial cells. Such localization of RBP and TTR may indicate a role in the transport and distribution of retinol and thyroid hormone, respectively, from yolk to embryo prior to establishment of the circulatory system, and is suggestive of a subsequent role in heart development.

Amino Acid Sequence↗

PubMed1997

Muscle hypertrophy in multifocal motor neuropathy is associated with continuous motor unit activity.

Multifocal motor neuropathy (MMN) is typically associated with distal upper limb weakness and wasting. However, proximal muscle bulk, particularly of biceps brachii, may be well preserved even in the presence of severe proximal weakness. Here we report 3 patients with MMN who had true muscle hypertrophy of severely weakened biceps muscles and positive motor symptoms including cramp and fasciculations in these muscles. Electromyographic studies demonstrated markedly impaired recruitment in the affected muscles and continuous motor unit activity comprising multiple fasciculation potentials at a frequency of up to 30 per minute. We propose that this continuous motor unit activity may have contributed to the hypertrophy in these muscles.

Adult↗

PubMed1997

Hematoxylin and eosin staining of gastric tissue for the detection of Helicobacter pylori.

BACKGROUND: Gold standard methods of detection of Helicobacter pylori are expensive, difficult to perform, or not widely available. The purpose of this study was to assess hematoxylin and eosin staining of antral tissue, a relatively inexpensive and widely available method of H. pylori detection. MATERIALS AND METHODS: The hematoxylin and eosin stain was assessed as a method of H. pylori detection, with 2 x 2 table analysis using culture as the gold standard in 133 gastric biopsy specimens obtained from patients both before and following attempted eradication. RESULTS: Performance of the stain was good among untreated patients as well as among patients having previously undergone an eradication treatment, with an overall accuracy of 92% (95% confidence intervals: 86-96), sensitivity of 93% (87-97), and specificity of 87% (69-96). CONCLUSION: The hematoxylin and eosin stain is an inexpensive method of H. pylori detection but with test performance characteristics inferior to Giemsa, Genta, or silver stains.

Biopsy↗

PubMed1997

Role of ERCP in the diagnosis of intraductal papillary mucinous neoplasms.

Mucinous cystic neoplasms of the pancreas may present in a "ductectatic" form, which parallels a distinct clinical presentation. We describe six patients with this entity termed mucinous ductal ectasia, or intraductal papillary mucinous neoplasm. All six patients presented with typical clinical and endoscopic findings and subsequently, almost all were found to have mucinous ductal cystadenocarcinomas. The endoscopic and pancreatographic findings associated with an intraductal papillary mucinous neoplasm are characteristic, unique, and yield a high diagnostic accuracy. It is important to recognize these features of intraductal papillary mucinous neoplasm since the tumor has a lower malignant potential than adenocarcinoma of the pancreas, and surgical resection is curative in many cases.

Aged↗

PubMed1996

Combined BMP-2 and FGF-4, but neither factor alone, induces cardiogenesis in non-precardiac embryonic mesoderm.

Previous work in this laboratory has shown that endoderm cells in the heart forming region (HFR endoderm) of the chicken embryo induce terminal cardiac differentiation in explanted precardiac mesoderm cells. Immunostaining patterns indicating that HFR endoderm cells express Drosophila decapentaplegic (dpp)-like antigens prompted a degenerate polymerase chain reaction (PCR) screen to identify cDNAs in the dpp subgroup of the transforming growth factor-beta (TGF-beta) family. Among 50 clones of PCR products that have been sequenced, over half have identity with bone morphogenetic protein-2 (BMP-2). No other TGF-beta cDNAs have been detected, suggesting that BMP-2 is the major dpp subgroup protein synthesized by HFR endoderm cells. However, BMP-2 protein did not promote survival of either precardiac or non-precardiac mesoderm cells in culture. Whereas FGF-4 supports cardiogenesis in precardiac mesoderm, it did not induce cardiogenesis in nonprecardiac mesoderm, although explant viability was maintained. In contrast to the isolated effects of these growth factors, treatment of non-precardiac mesoderm with combined BMP-2 and FGF-4 induced cardiogenesis in the majority of explants, as revealed by the formation of a rhythmically contractile multicellular vesicle that expresses sarcomeric alpha-actin. These findings suggest that BMP-2 and FGF-4 possess respective differentiative and proliferative activities, the combination of which specifies cells to the cardiac lineage.

Animals↗

PubMed1996

Avian serum response factor expression restricted primarily to muscle cell lineages is required for alpha-actin gene transcription.

Serum response factor (SRF) gene expression in avian embryonic muscle lineages plays a central role in activating alpha-actin gene activity. In early stage HH 6 avian embryos, SRF mRNA expression showed strong localization to the neural groove, primitive streak, lateral plate mesoderm, and Hensen's node, while distinct SRF expression was seen later in the neural folds and the somites by HH stage 8. SRF transcripts appeared in the precardiac splanchnic mesoderm in stage HH 9 embryos and was detected at higher levels in the myocardium, somites, and lateral mesoderm of HH 11 embryos. SRF antibody staining demonstrated significant SRF protein accumulation in the myocardium of the developing heart and the myotomal portion of somites. During primary myogenesis in culture, SRF transcripts and nuclear SRF protein content increased about 40-fold, as primary myoblasts withdrew from the cell cycle, reaching their highest levels prior to the upregulation of the skeletal alpha-actin gene. A dominant-negative SRF mutant, SRFpm1, which inhibited DNA binding, but not dimerization of monomeric SRF subunits, blocked transcriptional activation of a skeletal alpha-actin promoter-luciferase reporter gene during myogenesis. Transcriptional blockade was reversed by co-transfections of a wild-type SRF expression vector, but was not rescued by the expression of other myogenic factors, such as MyoD and Mef-2C. Thus, SRF displayed an embryonic expression pattern restricted primarily to striated muscle cell lineages, in which increased mass of nuclear SRF was obligatory for alpha-actin gene transcription.

Actins↗

PubMed1996

Expression of alternatively spliced and canonical basic fibroblast growth factor mRNAs in the early embryo and developing heart.

Previous studies in this laboratory have revealed the presence of substantial deposits of basic fibroblast growth factor (bFGF; FGF-2) in the myocardium from the earliest stages of heart development (Parlow et al. [1991] Dev. Biol. 146:139-147) and that an autocrine supply of bFGF is required for myocardial cell proliferation (Sugi et al. [1993] Dev, Biol, 157:28-37). Recently, an alternatively spliced isoform of bFGF, termed alt-bFGF, was described during later stages of embryogenesis, after heart morphogenesis is complete (Borja et al. [1993] Dev. Biol. 157:110-118). Because the antibody and nucleic acid probes used in our previous studies would have recognized canonical as well as alt-bFGF proteins and mRNAs, we have examined the expression of alt-and canonical bFGF mRNAs at early stages of embryogenesis, during which the initial differentiative and morphogenetic phases of heart development occur (Hamburger-Hamilton stages 3-24). Reverse transcription/polymerase chain reaction (RT/PCR) analysis detected the presence of both alt-bFGF and bFGF mRNAs in whole embryos as early as stage 3 and in the developing heart from the time of its initial appearance at stage 9. The presence of alt-bFGF mRNA was corroborated by RNase protection analysis which, in assessing RNA from whole embryos, revealed increasing levels of alt-bFGF mRNA between stages 5-18, suggesting that expression of alt-bFGF is developmentally regulated. Utilization of a probe that simultaneously protects segments of both alt- and canonical bFGF mRNAs indicated that alt-bFGF was the more abundant FGF isoform in the developing embryo until stage 24, when equivalent expression of each isoform was detected. Similar analysis revealed that alt-bFGF was the more abundant isoform in the embryonic heart, but that its relative expression was not decreased at stage 24.

Alternative Splicing↗

PubMed1996

Insulin-like growth factor-II/mannose-6-phosphate receptor expression during early heart development.

Expression of the insulin-like growth factor-II/mannose-6-phosphate (ICF-II/ M6P) receptor was examined during the major stages of heart morphogenesis in the chicken embryo. By using an affinity-purified antibody, Western blot analysis of total embryonic proteins from stages 5-24 revealed little if any IGF-II/M6P receptor protein until stage 7, approximately 8 hours prior to the appearance of the rudimentary myocardial tubes. Thereafter, receptor accumulation increased until stage 14, after which receptor protein levels remained constant, up to 7 days in ovo. Immunohistochemical localization revealed that, among all embryonic tissues at stages 10-24, the predominant site of receptor expression was the developing myocardium. Receptor expression was also immunohistochemically evaluated in a defined in vitro model of cardiogenesis in which explanted precardiac mesoderm is induced to undergo differentiation by co-explanted endoderm. In this system, as in vivo, IGF-II/M6P receptors were only detected after precardiac mesoderm had differentiated into a synchronously contractile multilayer which expressed cardiac alpha-actin. These findings indicate that the IGF-II/M6P receptor has an important role during early heart development.

Animals↗

PubMed1996

Evidence that fibroblast growth factors 1 and 4 participate in regulation of cardiogenesis.

Previous studies in this laboratory have indicated that the early embryonic chick heart depends on fibroblast growth factor-2 (FGF-2; bFGF), sequentially utilized in paracrine and autocrine fashion, for its growth and development (Sugi and Lough, [1995] Dev. Biol. 168-567-574). This view emanated from immunohistochemical detection of FGF-like antigens in endoderm cells at stage 6, and later in the early myocardium at stage 9+ (Parlow et al. [1991] Dev. Biol. 146:139-147). To identify other members of the FGF family that are expressed by these cells, we have used peptide-generated antisera that specifically recognize FGFs 1 and 4. Like FGF-2, FGFs 1 and 4 were exclusively detected in the endoderm at stage 5+ and later in the myocardium, appearing as punctate cytoplasmic deposits. However, whereas FGF-2 is first detected at stage 9+, FGFs 1 and 4 did not appear until stages 11 and 15, respectively. Expression of all FGFs peaked at stages 18-24, decreasing thereafter in parallel with reduced myocardial cell proliferation. To determine these isoproteins' ability to facilitate the completion of terminal cardiac myocyte differentiation, stage 5+ precardiac mesoderm was cultured in defined medium with purified FGFs. Like FGF-2, as little as 5-10 ng/ml FGF-1 or FGF-4 supported the proliferation and differentiation of precardiac myoblasts, resulting in the formation of a vesicle containing an adherent multilayer of synchronously contractile cells. Evidence that this represented FGF receptor-mediated signaling rather than a nonspecific effect of exogenous FGF was indicated by the ability of sodium chlorate to inhibit FGF-mediated cardiogenesis. These findings are consistent with the hypothesis that, like FGF-2, FGFs 1 and 4 participate in the regulation of early heart development via paracrine and autocrine mechanisms.

Animals↗

PubMed1996

Selenium homeostasis in the central nervous system of the rat.

These experiments have investigated selenium movement between blood and CNS in anaesthetised rats. Each animal was surgically anaesthetised and the left femoral blood vessels cannulated for blood withdrawal and solute infusion. Each rat received 75-selenium as sodium selenite infused in normal saline and experiments lasted between 5 minutes and 5 hours during which blood samples were periodically taken. At termination, the CNS was removed, regionally dissected and analysed with the plasma samples for 75-Se radioactivity by gamma-counting. Data were analysed by graphical analysis. Results showed unidirectional uptake of 75-Se into the CNS and regional differences were not found except for the hypothalamus. On average the CNS influx rate constant (Kin) was about 7 +/- 1 x 10(-5) ml/min/g. Data suggest that the 75-Se most likely entered the CNS as a free ionic form.

Animals↗