Expression of beta 2 integrin (CD18) in embryonic mouse and chicken heart.

Integrins are heterodimeric receptors composed of alpha and beta transmembrane subunits that mediate attachment of cells to the extracellular matrix and counter-ligands such as ICAM-1 on adjacent cells. beta2 integrin (CD18) associates with four different alpha (CD11) subunits to form an integrin subfamily, which has been reported to be expressed exclusively on leukocytes. However, recent studies indicate that beta2 integrin is also expressed by other types of cells. Since the gene for beta2 integrin is located in the region of human chromosome 21 associated with congenital heart defects, we postulated that it may be expressed in the developing heart. Here, we show the results from several different techniques used to test this hypothesis. PCR analyses indicated that beta2 integrin and the alphaL, alphaM, and alphaX subunits are expressed during heart development. Immunohistochemical studies in both embryonic mouse and chicken hearts, using antibodies directed against the N- or C-terminal of beta2 integrin or against its alpha subunit partners, showed that beta2 integrin, as well as the alphaL, alphaM, and alphaX subunits, are expressed by the endothelial and mesenchymal cells of the atrioventricular canal and in the epicardium and myocardium during cardiogenesis. In situ hybridization studies further confirmed the presence of beta2 integrin in these various locations in the embryonic heart. These results indicate that the beta2 integrin subfamily may have other activities in addition to leukocyte adhesion, such as modulating the migration and differentiation of cells during the morphogenesis of the cardiac valves and myocardial walls of the heart.

Expression of beta 2 integrin (CD18) in embryonic mouse and chicken heart

Integrins are heterodimeric receptors composed of á and â transmembrane subunits that mediate attachment of cells to the extracellular matrix and counter-ligands such as ICAM-1 on adjacent cells. â2 integrin (CD18) associates with four different á (CD11) subunits to form an integrin subfamily, which has been reported to be expressed exclusively on leukocytes. However, recent studies indicate that â2 integrin is also expressed by other types of cells. Since the gene for â2 integrin is located in the region of human chromosome 21 associated with congenital heart defects, we postulated that it may be expressed in the developing heart. Here, we show the results from several different techniques used to test this hypothesis. PCR analyses indicated that â2 integrin and the áL, áM, and áX subunits are expressed during heart development. Immunohistochemical studies in both embryonic mouse and chicken hearts, using antibodies directed against the N- or C-terminal of â2 integrin or against its á subunit partners, showed that â2 integrin, as well as the áL, áM, and áX subunits, are expressed by the endothelial and mesenchymal cells of the atrioventricular canal and in the epicardium and myocardium during cardiogenesis. In situ hybridization studies further confirmed the presence of â2 integrin in these various locations in the embryonic heart. These results indicate that the â2 integrin subfamily may have other activities in addition to leukocyte adhesion, such as modulating the migration and differentiation of cells during the morphogenesis of the cardiac valves and myocardial walls of the heart.

Attention deficit hyperactivity disorder and increased risk of injury.

PURPOSE: This study describes the influence of attention deficit hyperactivity disorder (ADHD) on the incidence rates of selected injuries. METHODS: A retrospective cohort study design was employed using medical claims data from the Deseret Mutual Benefit Administrators (DMBA), a health insurance company for employees of the Church of Jesus Christ of Latter-day Saints (LDS) and their spouses and dependent children. ADHD diagnosis, injury, medication, and demographic data were extracted from claims files during 1998-2005 for all enrollees aged 0-64 years. RESULTS: Incidence rates of ADHD were 1.83 (95% CI 1.68-2.00) times greater in males than females and highest in the age group 5-9 years and income group $80,000 or greater. ADHD increased the risk of selected injuries. The most common injuries involved sprains and strains of joints, then open wounds of the head, neck and trunk, and upper/lower limb, and then fractures of the upper/lower limb. Medication did not significantly protect against injury in ADHD patients. The rate of severe injury (i.e., fracture of skull, neck and trunk; intracranial injury excluding those with skull fracture; and injuries to nerves and spinal cord) was 3.07 (95% CI 2.37-3.98) times more common in ADHD enrollees compared with non-ADHD enrollees. Those with 1, 2, 3, or 4 or more injuries were 1.67 (1.50-1.86), 2.11 (1.75-2.56), 2.63 (1.80-3.84), and 2.94 (1.47-5.87) times more likely to have ADHD, respectively. CONCLUSIONS: ADHD is positively associated with injuries. More severe injuries have a significantly stronger associated with ADHD than less severe injuries.

Evaluation of hypophosphataemia in tenofovir disoproxil fumarate (TDF)-exposed and TDF-unexposed HIV-infected out-patients receiving highly active antiretroviral therapy.

OBJECTIVES: Cases of hypophosphataemia (often coincident with renal dysfunction) have been reported in HIV-infected patients taking tenofovir disoproxil fumarate (TDF), but randomized placebo-controlled trials of HIV-infected persons with normal baseline renal function have found a comparable incidence of hypophosphataemia in the TDF and placebo groups. We assessed the incidence of grade 2 and higher hypophosphataemia in the HIV Outpatient Study (HOPS). METHODS: We analysed a prospective cohort of patients who initiated either a TDF-containing highly active antiretroviral therapy (HAART) regimen [TDF-exposed (TDF+) group; n = 165] or a TDF-sparing HAART regimen [TDF-unexposed (TDF-) group; n = 90], and who had normal baseline phosphate and creatinine values. RESULTS: The TDF+ and TDF- groups had comparable median follow-up times (10.9 vs 8.8 months, respectively; P = 0.18) and number of phosphate measurements (median = 3 for both) and were similar on most clinical and demographic factors. During follow up, 12.7% of TDF+vs 6.7% of TDF-patients developed grade 2 hypophosphataemia (2.0-2.4 mg/dL), and 2.4% of TDF+ patients vs 0% of TDF-patients developed grade 3 hypophosphataemia (1.0-1.9 mg/dL); none developed grade 4 hypophosphataemia (<1.0 mg/dL). The incidence of grade 2 or higher hypophosphataemia was 16.7 per 100 person-years among TDF+ patients vs 8.0 per 100 person-years among TDF-patients (P = 0.11). CONCLUSIONS: The incidence of hypophosphataemia was somewhat elevated in HOPS patients who took TDF-containing HAART compared with those who took TDF-sparing HAART during the first 1 to 2 years of observation, but the difference was not statistically significant. Longer follow-up of a larger population is needed to determine if this trend towards an association achieves statistical significance and to evaluate the clinical consequences of hypophosphataemia.

Catalytic inhibition of human DNA topoisomerase IIalpha by hypericin, a naphthodianthrone from St. John's wort (Hypericum perforatum).

St. John's wort (Hypericum perforatum) is the most widely used herbal medicine for the treatment of depression. However, concerns have arisen about the potential of its interaction with other drugs due to the induction of cytochrome P450 isozymes 1A2 and 3A4 by the components hypericin and hyperforin, respectively. Structurally similar natural products are often employed as antitumor agents due to their action as inhibitors of DNA topoisomerases, nuclear enzymes that modify DNA during cellular proliferation. Preliminary findings that hypericin inhibited the DNA relaxation activity of topoisomerase IIalpha (topo II; EC 5.99.1.3) led us to investigate the mechanism of enzyme inhibition. Rather than stabilizing the enzyme in covalent complexes with DNA (cleavage complexes), hypericin inhibited the enzyme prior to DNA cleavage. In vitro assays indicate that hypericin is a potent antagonist of cleavage complex stabilization by the chemotherapeutics etoposide and amsacrine. This antagonism appears to be due to the ability of hypericin to intercalate or distort DNA structure, thereby precluding topo II binding and/or DNA cleavage. Supporting its non-DNA damaging, catalytic inhibition of topo II, hypericin was shown to be equitoxic to both wild-type and amsacrine-resistant HL-60 leukemia cell lines. Moreover, hypericin was incapable of stimulating DNA damage-responsive gene promoters that are activated by etoposide. As with the in vitro topo II assay, antagonism of DNA damage stimulated by 30 microM etoposide was evident in leukemia cells pretreated with 5 microM hypericin. Since many cancer patients experience clinical depression and concomitantly self-medicate with herbal remedies, extracts of St. John's wort should be investigated further for their potential to antagonize topo II-directed chemotherapy regimens.

Benzene metabolites antagonize etoposide-stabilized cleavable complexes of DNA topoisomerase IIalpha.

Chronic exposure to benzene is associated with hematotoxicity and acute myelogenous leukemia. Inhibition of topoisomerase IIalpha (topo II) has been implicated in the development of benzene-induced cytogenetic aberrations. The purpose of this study was to determine the mechanism of topo II inhibition by benzene metabolites. In a DNA cleavage/relaxation assay, topo II was inhibited by p-benzoquinone and hydroquinone at 10 microM and 10 mM, respectively. On peroxidase activation, inhibition was seen with 4,4'-biphenol, hydroquinone, and catechol at 10 microM, 10 microM, and 30 microM, respectively. But, in no case was cleavable complex stabilization observed and the metabolites appeared to act at an earlier step of the enzyme cycle. In support of this conclusion, several metabolites antagonized etoposide-stabilized cleavable complex formation and inhibited topo II-DNA binding. It is therefore unlikely that benzene-induced acute myelogenous leukemia stems from events invoked for leukemogenic topo II cleavable complex-stabilizing antitumor agents. (Blood. 2001;98:830-833)

Expression of the receptor tyrosine kinase gene EphB3 during early stages of chick embryo development.

The expression pattern of the receptor tyrosine kinase gene EphB3 was examined during the early stages of chick embryogenesis, and is described in this report. In the gastrula, EphB3 is expressed in epiblast cells adjacent to and entering the anterior portion of the primitive streak; expression is extinguished once cells have ingressed. At headfold stages, EphB3 is strongly transcribed in the floor of the foregut and in anterior lateral endoderm, and is expressed in the subjacent cardiogenic mesoderm. EphB3 is transiently expressed in the lateral ectoderm, neural tube, and neural crest during these stages. Later neural expression is localized to the mesencephalon. In the somitic mesoderm, EphB3 is initially expressed in the sclerotome, but later is expressed predominantly in the dermatome. Prominent expression is also detected in the developing heart, liver, posterior ventral limb bud mesenchyme, pharyngeal arches, and head mesenchyme.

Infants' expectations about object label reference.

The aim of this research was to examine whether infants at the early stages of lexical development were sensitive to the word-category linkage. In Experiment 1, 16-to 19-month-old infants were requested to match a target with either a basic-level or a thematic match, with or without a novel label. Stimuli were presented using the preferential looking paradigm. Infants in the Novel Label condition looked significantly longer at the basic-level match than infants in the No Label condition. In Experiment 2, infants were presented with a target, followed by a basic-level match and a superordinate-level match with or without a novel label. Again, infants in the Novel Label condition looked significantly longer at the basic-level match than infants in the No Label condition. Taken together, these findings indicate that infants initially assume that novel words label basic-level categories and thereby do honour the word-category linkage.

A novel cardiac-restricted target for doxorubicin. CARP, a nuclear modulator of gene expression in cardiac progenitor cells and cardiomyocytes.

Doxorubicin (Dox), a cardiotoxic antineoplastic drug, disrupts the cardiac-specific program of gene expression (Kurabayashi, M., Dutta, S., Jeyaseelan, R., and Kedes, L. (1995) Mol. Cell. Biol. 15, 6386-6397; Jeyaseelan, R., Poizat, C., Wu, H. Y., and Kedes, L. (1997) J. Biol. Chem. 272, 5828-5832). To determine whether this drug might interfere with the function of cardiac-specific regulatory pathways, we used a differential display strategy to clone from neonatal rat cardiomyocyte candidate mRNAs that were rapidly sensitive to Dox. We report here the identification of a constitutively expressed, cardiac-restricted, nuclear protein whose mRNA level is exquisitely sensitive to Dox. Hence we have named this protein cardiac adriamycin-responsive protein (CARP). CARP mRNA is present at the earliest stages of cardiac morphogenesis. It was detected by in situ hybridization within the cardiogenic plate of 7. 5-day post coitum (p.c.) embryos, and in 8.5-day p.c. embryos CARP transcripts are present in uniformly high levels in the myocardium. Throughout cardiac development, CARP expression is specific for the myocardium; endocardial cushions and valves exhibit only background levels of signal. Transcript levels persist but gradually decrease in neonatal, 2-week-old, and adult hearts. There were no stages when CARP mRNA could not be detected. The pattern and timing of CARP mRNA expression, including transient expression in the tongue at 14.5 days p.c., coincides with that of Nkx2.5/Csx (a putative homolog of tinman, the Drosophila melanogaster gene responsible for cardiac development). The cloned full-length 1749 nucleotide CARP cDNA encodes a 319-amino acid 40-kDa polypeptide containing five tandem ankyrin repeats. CARP appears to be the rat homolog of a previously reported human single-copy gene (C-193; Chu, W., Burns, D. K., Swerlick, R. A., and Presky, D. H. (1995) J. Biol. Chem. 270, 10236-10245), whose mRNA is inducible by cytokines only in human endothelial cells. CARP appears to function as a negative regulator of cardiac-specific gene expression. Overexpression of CARP in cardiomyocytes suppresses cardiac troponin C and atrial natriuretic factor transcription. Cotransfection experiments in HeLa cells indicate that CARP inhibits Nkx2.5 transactivation of atrial natriuretic factor promoter. When fused to a GAL4 DNA-binding domain, CARP has transcriptional inhibitory properties in noncardiac cells. CARP thus represents the first example of a cardiac-restricted transcriptional regulatory protein that is sensitive to Dox.

In vitro preselection of gene-trapped embryonic stem cell clones for characterizing novel developmentally regulated genes in the mouse.

We have developed an in vitro gene trap screen for novel murine genes that allows one to determine, prior to making chimeric or transgenic animals, if these genes are expressed in one or more specific embryonic tissues. Totipotent embryonic stem (ES) cells are infected with a retroviral gene trap construct encoding a selectable lacZ/neo fusion gene, which is expressed only if the gene trap inserts within an active transcription unit. G418-resistant ES cell clones are induced to differentiate in vitro, and neurons, glia, myocytes, and chondrocytes are screened for expression of beta-galactosidase (beta-gal). cDNAs of the gene trap transcripts are obtained by 5' rapid amplification of cDNA ends and are sequenced to determine if they represent novel genes. In situ hybridization analyses show that trapped genes are expressed in vivo within the cell types that express beta-gal in vitro. Gene traps and their wild-type alleles are characterized in terms of copy number, alternate splicing of their transcripts, and the proportion of endogenous mRNA sequence that is replaced by lacZ/neo in the hybrid gene trap transcript. This approach, which we term "in vitro preselection," is more economical than standard in vivo gene trap screening because tissue-specific expression of probable knockout alleles is verified before transgenic animals are generated. These results also highlight the utility of ES cell differentiation in vitro as a method with which to study the molecular mechanisms regulating the specification and commitment of a variety of cell and tissue types.

Positive and negative control of the Antennapedia promoter P2.

To understand the nature of the regulatory signals impinging on the second promoter of the Antennapedia gene (Antp P2), analysis of its expression in mutants and in inhibitory drug injected embryos has been carried out. The maternally-active gene osk is identified as one of two general repressors of P2 which prevent Antp transcription until division cycle 14. Products of the zygotically-active segmentation genes ftz, hb, Kr, gt and kni then act as activators or repressors of Antp P2 in a combinatorial fashion. The timing of these events, and their positive versus negative nature, is critical for generating the expression patterns normal for Antp.

Synthetic substrate for eukaryotic signal peptidase. Cleavage of a synthetic peptide analog of the precursor region of preproparathyroid hormone.

A synthetic peptide analog of the precursor region of preproparathyroid hormone has been shown to be a specific substrate for hen oviduct signal peptidase. The sequence of the 31-residue peptide is Ser-Ala-Lys-Asp-norleucine (Nle)-Val-Lys-Val-Nle-Ile-Val-Nle-Leu-Ala-Ile-Ala-Phe-Leu-Ala-Arg-Ser-As p-Gly-Lys-Ser-Val-Lys-Lys-Arg-D-Tyr-amide (Caulfield, M. P., Duong, L. T., O'Brien, R., Majzoub, J. A., and Rosenblatt, M. (1988) Mol. Endocrinol. 2, 452-458). This sulfur-free signal peptide analog can be labeled with 125I on the C-terminal D-tyrosine and is cleaved by purified hen oviduct signal peptidase between Gly and Lys, the correct site of cleavage of preproparathyroid hormone in vivo. Amino acid sequence analysis of the cleavage product released 125I at the seventh cycle of Edman degradation, confirming that enzymatic cleavage occurs at the physiological site. Synthetic peptide analogs of the substrate with Lys, Pro, or Asp substituted for Nle-18 were poor substrates for the enzyme and were also poor competitive inhibitors of catalysis, suggesting that modifications at position -18, 12 amino acids from the site of cleavage, directly influence binding by the enzyme. Analysis of the reactivity of signal peptidase with these synthetic peptides provides insight into the cleavage specificity requirements of this eukaryotic signal peptidase.

Purification and characterization of hen oviduct microsomal signal peptidase.

Hen oviduct signal peptidase requires only two proteins for proteolysis of fully synthesized secretory precursor proteins in vitro: one with a molecular mass of 19 kilodaltons (kDa) and one which is a glycoprotein whose mass varies from 22 to 24 kDa depending on the extent of glycosylation. Purified signal peptidase has been analyzed both as part of an active catalytic unit and after electroelution of the individual proteins out of a preparative polyacrylamide gel. The multiple forms of the glycoprotein component of signal peptidase bind to concanavalin A and are shown to be derived from the same polypeptide backbone. Removal of their oligosaccharides by digestion with N-glycanase converts these proteins to a single 19.5-kDa polypeptide. The glycoproteins all exhibit very similar profiles following individual digestion with trypsin and separation of the resulting peptides by reverse-phase high-performance liquid chromatography. In addition, sequence analysis of selected peptides from corresponding regions in chromatograms representing each form of the glycoprotein reveals the same amino acid sequences. The 19-kDa signal peptidase protein does not bind concanavalin A, has a distinct tryptic peptide map from that of the glycoprotein, and appears to share no amino acid sequences in common with the glycoprotein. Its copurification on a concanavalin A-Sepharose column indicates that it must interact directly with the glycoprotein subunit.

Impaired thromboxane synthesis in preactivated human blood platelets: agonist-specific, irreversible desensitization to thrombin.

The causes for reduced platelet thromboxane synthesis in patients with acquired platelet storage pool disease are incompletely understood. The present study was designed to define the nature of the defect(s) underlying diminished thromboxane synthesis in human platelets previously exposed to thrombin in vitro. Platelets pretreated with high concentrations of thrombin were unable to form measurable amounts of thromboxane in response to a second stimulation with thrombin. In contrast, thrombin-pretreated platelets formed additional thromboxane in response to arachidonate, collagen, or A23,187. Thrombin-pretreated platelets did not recover with respect to thrombin-inducible thromboxane synthesis when incubated for two hours in plasma either in the presence or absence of added arachidonic acid. These observations suggest that neither inactivation of cyclooxygenase nor depletion of endogenous arachidonic acid is responsible for the impaired thrombin-induced thromboxane synthesis in thrombin-prestimulated platelets. Impaired thrombin-induced thromboxane synthesis in these platelets may be due to agonist-specific, irreversible receptor uncoupling.

Partial purification of microsomal signal peptidase from hen oviduct.

Signal peptidase has been purified approximately 600-fold from hen oviduct microsomes. Treatment of microsomes with ice-cold sodium carbonate at pH 11.5 removes soluble and extrinsic membrane proteins prior to solubilization of signal peptidase with Nonidet P-40. After dialysis to pH 8.2, the solubilized enzyme is chromatographed on diethylaminoethyl cellulose at pH 8.2. More than 90% of contaminating proteins bind to the column while signal peptidase and endogenous phospholipid are eluted in the column void volume. Enzyme activity subsequently binds to carboxymethyl cellulose at pH 5.8 and is eluted by approximately 100 to 200 mM NaCl during a NaCl gradient. Polypeptides present in partially purified hen oviduct signal peptidase have relative molecular masses ranging from 54 kD to less than 11 kD with major bands at 29, 23, 22, 19, 18 and 13 kD. The purified peptidase requires phospholipid for activity and is maximally active in the presence of 2 mg/ml phosphatidylcholine.

Thrombin pretreatment of human platelets impairs thromboxane A2 synthesis from endogenous precursors in the presence of normal cyclooxygenase activity.

Exposure of horse platelets to thrombin has been reported to cause nearly complete inactivation of cyclooxygenase within 30 sec. This contrasts with the observation that human platelets, depleted of their granule constituents by stimulation with thrombin, still aggregate in response to arachidonic acid, a reaction presumably mediated by thromboxane A2 (TxA2) formation. Because of this conflicting evidence, TxA2 formation was measured by radioimmunoassay in washed human platelets depleted of their alpha- and dense-storage granule constituents by prior stimulation with thrombin. These platelets aggregated in response to adenosine diphosphate (ADP), collagen, arachidonic acid, and thrombin, and formed TxA2. However, collagen- and thrombin-induced TxA2 formation by these platelets was reduced in comparison to control platelets that had not been depleted of their storage granule constituents by prior thrombin stimulation. In contrast, arachidonic acid-induced TxA2 formation was not significantly different in thrombin-depleted and control platelets. These results demonstrate that thrombin can induce degranulation of platelets without concomitant inactivation of cyclooxygenase.

Role of cytoplasmic and releasable ADP in platelet aggregation induced by laminar shear stress.

We examined the extent of lytic and sublytic platelet injury after exposure of platelets to shear stress and the role in shear-induced PAG of ADP liberated from platelets as a result of shear-induced platelet dense body release and/or platelet damage. Platelets in C-PRP or TAS were subjected to well-defined, laminar shear stress in a rotational viscometer, and PAG (loss of single, nonaggregated platelets), 14C-serotonin release, and loss from platelets of LDH and 51Cr were determined. Increased PAG with increasing shear stresses was associated with progressive loss of LDH and 51Cr. Loss of 51Cr was consistently in excess of that of LDH, indicating sublytic platelet injury, which was confirmed by electron microscopy. At the lowest shear stress used (50 dynes/cm2), PAG in C-PRP was observed in the absence of detectable loss of 51Cr or LDH. When platelets in TAS were sheared in the presence of CP/CPK, an enzyme system capable of removing extracellular ADP, PAG was only partially (approximately 40%) inhibited. However, when platelets were preincubated with CP/CPK and ATP (to saturate platelet ADP receptors), shear-induced PAG was almost completely suppressed. Similar results were obtained with PAG induced by collagen in the aggregometer. The findings indicate that (1) shear-induced PAG in this system may occur without measurable lytic or sublytic platelet damage and (2) ADP liberated from platelets as a result of shear-induced release or damage may represent the major if not sole mediator of shear-induced PAG.