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PubMedDate not supplied

A gradient molecule in developing rat retina: expression of 9-O-acetyl GD3 in relation to cell type, developmental age, and GD3 ganglioside.

In the embryonic and postnatal rat retina a cell surface antigen that is detected by monoclonal antibody JONES is distributed in a dorsoventral gradient. Biochemical analysis has determined that the antigen is a modified ganglioside, 9-O-acetyl GD3. In the present study, the distributions of 9-O-acetyl GD3 and its possible precursor GD3 in developing rat retina were compared immunocytochemically using specific monoclonal antibodies JONES and R24. On embryonic day 13 (E13) immunoreactivity to JONES was localized to central retina; however, R24 stained throughout the retinal epithelium. By E20, when JONES binding was distributed in a gradient along the dorsoventral axis, R24 again stained dorsal and ventral regions with uniform intensity. Analysis of freshly dissociated retinal cells further revealed that GD3 and 9-O-acetyl GD3 expressions do not necessarily coincide. At E15 and postnatal day 2 (PN2), the majority of cells (78 and 92%, respectively) were immunolabeled by antibody to GD3, while between E15 and PN2 the percentage of cells immunolabeled by antibody to 9-O-acetyl GD3 rose from 19 to 68%. By PN4, labeling decreased for both molecules; however, the rate of decline in 9-O-acetyl GD3 labeling was more pronounced. Regulation of the numbers of JONES-positive cells does not appear to depend on interaction with the extraretinal environment, for in neural retina explanted at E15 the proportions of 9-O-acetyl GD3-bearing cells was found to be similar to the percentage observed in neural retina developing to an equivalent age in vivo. Experiments to identify the retinal cell types bearing 9-O-acetyl GD3 revealed that it is expressed by both neurons and glia growing in monolayer cultures of rat perinatal neural retina. Examination of freshly dissociated retinal cells following simultaneous labeling for some specific cell types and 9-O-acetyl GD3 demonstrated that the latter determinant is present on photoreceptor, amacrine, and ganglion cells. For each neuronal cell type, however, not all of the cells were immunoreactive with JONES. We conclude that the differences in the percentages of JONES- and R24-positive cells, and in particular the different rates at which JONES and R24 staining declined with age, indicate that the expression of the JONES epitope is regulated with some independence from parent ganglioside GD3.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1995

The effect of mannitol on the oral bioavailability of cimetidine.

The aim of the study was to investigate whether mannitol at amounts relevant to pharmaceutical formulations would alter the oral bioavailability of cimetidine, a drug primarily absorbed from the small bowel. Seven healthy male subjects each received four formulations, a chewable tablet or a solution, containing 0.200 g of cimetidine and either 2.264 g of mannitol or sucrose, in a randomized four-way cross-over study. Frequent blood samples were taken over a 24 h period to allow a cimetidine plasma profile to be obtained for each formulation. Transit of the radiolabeled formulations was followed by gamma scintigraphy. Statistically significant reductions in the AUC0-24 and maximum plasma concentration values were observed with the mannitol dosage forms compared to the sucrose controls. The mean small intestinal transit times were shortened after administration of the mannitol solution and tablet; the transit time of the solution was significantly shorter with values 23% of those for the sucrose solution. The implication of the study findings is that excipients cannot always be regarded as "inert" substances that can be incorporated into a formulation without having any deleterious effect on the overall in vivo behaviour of the product.

Administration, Oral↗
PubMed1980

In vitro methylation of bacterial chemotaxis proteins: characterization of protein methyltransferase activity in crude extracts of Salmonella typhimurium.

A specific in vitro assay was developed for the protein carboxyl methyltransferase that is involved in the chemotactic behavior of Salmonella typhimurium. This cytosolic enzyme catalyzes an S-adenosyl-L-methionine-dependent methyl esterification of glutamyl residues on a class of 60,000-dalton inner-membrane proteins. The activity was found to display a pH optimum of 6.5 and be sensitive to the concentration of salts in the assay medium. No detectable activity was found towards a variety of other proteins which serve as substrates for mammalian and other bacterial carboxyl methyltransferases. This assay was used to quantitate the methylation of the 60,000-dalton methyl-accepting proteins in response to chemoeffectors. Small but reproducible concentration-dependent changes in the initial rates of in vitro methylation were observed with chemotactic attractants and repellents. The specific methyltransferase activity was found to be absent in several mutants in flagellar synthesis (fla-), suggesting that the synthesis of this enzyme is coordinately regulated with that of flagellin and basal bodies. The hydrodynamic properties of the enzyme in crude extracts were determined by gel filtration and sucrose velocity gradient centrifugation, and a native molecular weight of 41,000 was calculated from these data.

Bacterial Proteins↗
PubMed2005

Determination of the three-dimensional, solution-phase structure of the macrolide antibiotic oxolide in CD2Cl2 and D2O from NMR constraints.

The three-dimensional structure of the antibiotic oxolide, (9S,11S)-11-amino-9-deoxo-11,12-deoxy-9,12-epoxyerythromycin, was determined in CD2Cl2 through constrained molecular mechanics with constraints derived from proton NMR. The calculations yielded a well-defined global minimum. Data acquired for oxolide in D2O, although not as complete, indicate that the antibiotic adopts the same conformation in water.

Anti-Bacterial Agents↗
PubMed1980

A comparison in germ-free mice of the pathogenesis of Sendai virus and mouse pneumonia virus infections.

The poathogenesis of Sendai virus and pneumonia virus of mice (PVM) was studied using the immunoperoxidase technique on paraffin lung sections. The pathology of Sendai virus corresponds to that of a bronchopneumonia, with virus demonstrated by immunoperoxidase in the bronchial epithelium, and sometimes in macrophages, for a period of 2--9 days post-infection. Pneumonia virus of mice produces an interstitial pneumonia with virus demonstrated in the bronchial epithelium but also in the alveolar walls and alveolar macrophages. This virus can be demonstrated between 2 and 7 days post-infection. This technique was used to demonstrate PVM in the case of a natural outbreak of this disease and may eventually become a routine technique for the screening of lung tissue for respiratory viruses.

Animals↗
PubMed2007

The location and characterisation of the O-linked glycans of the human insulin receptor.

O-linked glycosylation is a post-translational and post-folding event involving exposed S/T residues at beta-turns or in regions with extended conformation. O-linked sites are difficult to predict from sequence analyses compared to N-linked sites. Here we compare the results of chemical analyses of isolated glycopeptides with the prediction using the neural network prediction method NetOGlyc3.1, a procedure that has been reported to correctly predict 76% of O-glycosylated residues in proteins. Using the heavily glycosylated human insulin receptor as the test protein six sites of mucin-type O-glycosylation were found at residues T744, T749, S757, S758, T759, and T763 compared to the three sites (T759 and T763- correctly, T756- incorrectly) predicted by the neural network method. These six sites occur in a 20 residue segment that begins nine residues downstream from the start of the insulin receptor beta-chain. This region which also includes N-linked glycosylation sites at N742 and N755, is predicted to lack secondary structure and is followed by residues 765-770, the known linear epitope for the monoclonal antibody 18-44.

Acetylgalactosamine↗
PubMed1995

Longitudinal models for analysis of respiratory function.

We compare the results of fitting three longitudinal models, two autoregressive models (the serial correlation model and a damped autoregressive model) and a compound symmetry model, to data on a cohort of 1154 adult men in Boston. The serial correlation model assumes that the error terms are autocorrelated with correlation of the form lambda t for visits t years apart while the damped autoregressive assumes that the correlation between error terms of observations t years apart is of the form lambda t theta. The compound symmetry model assumes that the errors are correlated, with the same correlation regardless of how far apart observations are in time. These three models are all related in that the serial correlation and compound symmetry models are particular cases of the damped autoregressive models (that is, theta = 1 corresponds to the serial correlation model and theta = 0 corresponds to the compound symmetry model). For current smokers, the damped autoregressive model provided a significantly better fit than either of the other two models (p < 0.001); for never smokers the damped autoregressive and compound symmetry models were almost identical with both providing a significantly better fit than the serial correlation model (p < 0.001).

Adult↗
PubMed2000

The morphology of heart development in Xenopus laevis.

We have used serial histological sections to document heart formation in Xenopus laevis, from the formation of a linear heart tube to the appearance of morphologically distinct atrial and ventricular chambers. 3D reconstruction techniques have been used to derive accurate models from digital images, revealing the morphological changes that accompany heart differentiation. To demonstrate the utility of this approach in analysing cardiac gene expression, we have reexamined the distribution of Hand1 transcripts in the linear and looped heart tube. Our results demonstrate that prior to looping, an initial asymmetric, left-sided pattern is replaced by more symmetrical localisation of transcripts to the ventral portion of the myocardium. After the onset of looping, Hand1 expression is restricted to the ventral ventricular myocardium and extends along the entire length of the single ventricle.

Animals↗
PubMed2000

Subdivision of the cardiac Nkx2.5 expression domain into myogenic and nonmyogenic compartments.

Nkx2.5 is expressed in the cardiogenic mesoderm of avian, mouse, and amphibian embryos. To understand how various cardiac fates within this domain are apportioned, we fate mapped the mesodermal XNkx2.5 domain of neural tube stage Xenopus embryos. The lateral portions of the XNkx2.5 expression domain in the neural tube stage embryo (stage 22) form the dorsal mesocardium and roof of the pericardial cavity while the intervening ventral region closes to form the myocardial tube. XNkx2.5 expression is maintained throughout the period of heart tube morphogenesis and differentiation of myocardial, mesocardial, and pericardial tissues. A series of microsurgical experiments showed that myocardial differentiation in the lateral portion of the field is suppressed during normal development by signals from the prospective myocardium and by tissues located more dorsally in the embryo, in particular the neural tube. These signals combine to block myogenesis downstream of XNkx2.5 and at or above the level of contractile protein gene expression. We propose that the entire XNkx2.5/heart field is transiently specified as cardiomyogenic. Suppression of this program redirects lateral cells to adopt dorsal mesocardial and dorsal pericardial fates and subdivides the field into distinct myogenic and nonmyogenic compartments.

Animals↗
PubMed2000

Regulation of the tinman homologues in Xenopus embryos.

Vertebrate homologues of the Drosophila tinman transcription factor have been implicated in the processes of specification and differentiation of cardiac mesoderm. In Xenopus three members of this family have been isolated to date. Here we show that the XNkx2-3, Xnkx2-5, and XNkx2-10 genes are expressed in increasingly distinctive patterns in endodermal and mesodermal germ layers through early development, suggesting that their protein products (either individually or in different combinations) perform distinct functions. Using amphibian transgenesis, we find that the expression pattern of one of these genes, XNkx2-5, can be reproduced using transgenes containing only 4.3 kb of promoter sequence. Sequence analysis reveals remarkable conservation between the distalmost 300 bp of the Xenopus promoter and a portion of the AR2 element upstream of the mouse and human Nkx2-5 genes. Interestingly, only the 3' half of this evolutionarily conserved sequence element is required for correct transgene expression in frog embryos. Mutation of conserved GATA sites or a motif resembling the dpp-response element in the Drosophila tinman tinD enhancer dramatically reduces the levels of transgene expression. Finally we show that, despite its activity in Xenopus embryos, in transgenic mice the Xenopus Nkx2-5 promoter is able to drive reporter gene expression only in a limited subset of cells expressing the endogenous gene. This intriguing result suggests that despite evolutionary conservation of some cis-regulatory sequences, the regulatory controls on Nkx2-5 expression have diverged between mammals and amphibians.

Animals↗
PubMed2002

Distinct enhancers regulate skeletal and cardiac muscle-specific expression programs of the cardiac alpha-actin gene in Xenopus embryos.

During vertebrate embryonic development, cardiac and skeletal muscle originates from distinct precursor populations. Despite the profound structural and functional differences in the striated muscle tissue they eventually form, such progenitors share many features such as components of contractile apparatus. In vertebrate embryos, the alpha-cardiac actin gene encodes a major component of the myofibril in both skeletal and cardiac muscle. Here, we show that expression of Xenopus cardiac alpha-actin in the myotomes and developing heart tube of the tadpole requires distinct enhancers within its proximal promoter. Using transgenic embryos, we find that mutations in the promoter-proximal CArG box and 5 bp downstream of it specifically eliminate expression of a GFP transgene within the developing heart, while high levels of expression in somitic muscle are maintained. This sequence is insufficient on its own to limit expression solely to the myocardium, such restriction requiring multiple elements within the proximal promoter. Two additional enhancers are active in skeletal muscle of the embryo, either one of which has to interact with the proximal CArG box for correct expression to be established. Transgenic reporters containing multimerised copies of CArG box 1 faithfully detect most sites of SRF expression in the developing embryo as do equivalent reporters containing the SRF binding site from the c-fos promoter. Significantly, while these motifs possess a different A/T core within the CC(A/T)(6)GG consensus and show no similarity in flanking sequence, each can interact with a myotome-specific distal enhancer of cardiac alpha-actin promoter, to confer appropriate cardiac alpha-actin-specific regulation of transgene expression. Together, these results suggest that the role of CArG box 1 in the cardiac alpha-actin gene promoter is to act solely as a high-affinity SRF binding site.

Actins↗
PubMed1994

Cytokine regulation of nitric oxide synthase in mouse retinal pigment epithelial cells in culture.

Nitric oxide is an intercellular signaling molecule whose numerous functions include regulation of vascular tone, mediation of the cytotoxic effects of macrophages and potentiation of synaptic transmission. For some cellular functions, nitric oxide synthesis is mediated by the inducible form of nitric oxide synthase. We now show that cultured mouse retinal pigment epithelial cells exposed to interferon-gamma and lipopolysaccharide, express inducible nitric oxide synthase. The latter was detected immuno-cytochemically in interferon-gamma-lipopolysaccharide-treated retinal pigment epithelium using rabbit antiserum to a synthetic peptide of mouse nitric oxide synthase. Untreated cultures of retinal pigment epithelium or cultures treated with either interferon-gamma or or lipopolysaccharide alone were not immunoreactive. Induction of iNOS in gamma-interferon-lipopolysaccharide-stimulated retinal pigment epithelial cells was also evidenced by the presence of nitric oxide synthase enzyme activity in lysates of stimulated but not unstimulated retinal pigment epithelial cells. On immunoblots of lysates of stimulated murine retinal pigment epithelial cells, rabbit antiserum to iNOS recognized a 130-kDa protein which comigrated with the inducible nitric oxide synthase of macrophages and which was not detectable in lysates of unstimulated retinal pigment epithelial cells nor in lysates of cells treated with only interferon-gamma or lipopolysaccharide alone. Nitrite, a stable endproduct of NO formation by cells, was detectable in the culture supernatants after 18-24 hr of exposure to interferon-gamma and lipopolysaccharide, and continued to accumulate in a linear fashion for at least 96 hr. Treatment of cultured retinal pigment epithelium with interferon-gamma, lipopolysaccharide and either basic fibroblast growth factor or epidermal growth factor as third signals augmented inducible nitric oxide synthase expression as evidenced by intensified signals on immunoblots, enhanced accumulation of nitrite and increased iNOS enzyme activity. Conversely, when transforming growth factor-beta was present in the culture medium, gamma-interferon-LPS-induced expression of nitric oxide synthase and NO release were reduced. We conclude that interferon-gamma synergizes with lipopolysaccharide to induce synthesis of inducible nitric oxide synthase and production of nitric oxide by murine retinal pigment epithelium and that this induction can be modulated by basic fibroblast growth factor, epidermal growth factor or transforming growth factor-beta.

Amino Acid Oxidoreductases↗
PubMed1997

CD36 expression is altered in retinal pigment epithelial cells of the RCS rat.

The retinal pigment epithelial cell has several important functions, one of which is the phagocytosis of photoreceptor outer segments which are discarded diurnally. We previously provided evidence in human retinal pigment epithelium that CD36, an 88 kDa integral membrane glycoprotein, participates in the phagocytosis of photoreceptor outer segments. Since in the Royal College of Surgeons dystrophic rat, retinal pigment epithelial cells fail to perform this function and as a result the photoreceptor cells degenerate, the expression of CD36 has now been examined by retinal pigment epithelial cells of the dystrophic rat. Consistent with earlier work using human retinal pigment epithelial cells, expression of CD36 by freshly isolated retinal pigment epithelial cells of Long Evans rats was confirmed by immunoblotting and immunocytochemistry with antibody to rat CD36. The protein was also present in lysates of cultured retinal pigment epithelium. Furthermore, with an in vitro phagocytosis assay using 125I-labeled outer segments, it was demonstrated that the binding and ingestion of outer segments by rat retinal pigment epithelial cells was reduced by 64% in the presence of antibodies to rat CD36. In contrast to observations in the Long Evans rat, immunoblotting of retinal pigment epithelial cells isolated from the adult Royal College of Surgeons rat revealed that CD36 protein was not present. This appeared to be a tissue-specific absence since CD36 protein was present in peritoneal macrophages harvested from the adult Royal College of Surgeons rat. A developmental study of CD36 expression also demonstrated an absence of the protein on the day of birth and at 1 and 2 weeks postnatally. By reverse transcriptase-polymerase chain reaction, CD36 mRNA was detected in freshly harvested retinal pigment epithelial cells of the Royal College of Surgeons rat at only PN1, 1 week and 10 days. Significantly, at 2 weeks of age and in the adult Royal College of Surgeons rat. CD36 transcripts were no longer present. Nevertheless, by Northern blot analysis CD36 mRNA was detected in various other tissues shown previously to express CD36. We conclude that in RPE cells of the Royal College of Surgeons rat, CD36 protein is not expressed while CD36 mRNA is present only transiently during postnatal development.

Animals↗
PubMed1995

Defects in the Drosophila myosin rod permit sarcomere assembly but cause flight muscle degeneration.

We have determined the molecular and ultrastructural defects associated with three homozygous-viable myosin heavy chain mutations of Drosophila melanogaster. These mutations cause a dominant flightless phenotype but allow relatively normal assembly of indirect flight muscle myofibrils. As adults age, the contents of the indirect flight muscle myofibers are pulled to one end of the thorax. This apparently results from myofibril "hyper-contraction", and leads to sarcomere rupture and random myofilament orientation. All three mutations cause single amino acid changes in the light meromyosin region of the myosin rod. Two change the same glutamic acid to a lysine residue and the third affects an amino acid five residues away, substituting histidine for arginine. Both affected residues are conserved in muscle myosins, cytoplasmic myosins and paramyosins. The mutations are associated with age-dependent, site-specific degradation of myosin heavy chain and failure to accumulate phosphorylated forms of flightin, an indirect flight muscle-specific protein previously localized to the thick filament. Given the repeating nature of the hydrophobic and charged amino acid residues of the myosin rod and the near-normal assembly of myofibrils in the indirect flight muscle of these mutants, it is remarkable that single amino acid changes in the rod cause such severe defects. It is also interesting that these severe defects are not apparent in other muscles. These phenomena likely arise from the highly organized nature and rigorous performance requirements of indirect flight muscle, and perhaps from the interaction of myosin with flightin, a protein specific to this muscle type.

Age Factors↗
PubMed2000

Drosophila ACT88F indirect flight muscle-specific actin is not N-terminally acetylated: a mutation in N-terminal processing affects actin function.

Many eukaryotic proteins are co and post-translationally modified at their N termini by removal of one or two amino acid residues and N(alpha)-acetylation. Actins show two different forms of N-terminal processing dependent on their N-terminal sequence. In class II actins, which include muscle actins, the common primary sequence of Met-Cys-Asp-actin is processed to acetyl-Asp-actin. The functional significance of this in vivo is unknown. We have studied the indirect flight muscle-specific actin, ACT88F, of Drosophila melanogaster. Our results show that ACT88F is N-terminally processed in vivo as a class II actin by removal of the first two amino acid residues (Met and Cys), but that uniquely the N terminus is not acetylated. In addition we show that ACT88F is methylated, probably at His73. Flies carrying the mod(-) mutation fail to complete post-translational processing of ACT88F. We propose that the mod gene product is normally responsible for removing N-acetyl-cysteine from actin. The biological significance of this process is demonstrated by observations that retention of the N-acetyl-cysteine in ACT88F affects the flight muscle function of mod(-) flies. This suggests that the extreme N terminus affects actomyosin interactions in vivo, a proposal we have examined by in vitro motility assays of ACT88F F-actin from mod(-) flies. The mod(-) actin only moves in the presence of methylcellulose, a viscosity-enhancing agent, where it moves at velocities slightly, but significantly, reduced compared to wild-type. These data confirm that N-acetyl-cysteine at the N terminus affects actomyosin interactions, probably by reducing formation of the initial actomyosin collision complex, a process known to involve the actin N terminus.

Acetylation↗
PubMed1993

The integrity of the epithelium is a major determinant of the responsiveness of the dog bronchial segment to mucosal provocation.

The intact bronchial segment of the dog was used to investigate the role of the epithelium in modulating bronchial responsiveness: firstly, because it retains many of the properties of the airway wall including the separation of the mucosal and adventitial surfaces; and secondly, because airway tissue from dogs has been principally used in advancing evidence for epithelial relaxant substances. The development of tone by the airway smooth muscle was measured as an increase in luminal pressure of the isovolumic segment. Cholinergic excitatory and sympathetic inhibitory responses were obtained to electrical field stimulation. Responses to acetylcholine (ACh), carbachol, depolarizing K+ solution and vanadate (VO3-) applied to the mucosal surface were greatly attenuated both in sensitivity and reactivity compared with their effect on the adventitial surface. Mechanical removal of the epithelium greatly augmented these responses to agents in the lumen (ACh sensitivity increased 100-fold) to equal those by adventitial stimulation. The latter were unaffected by epithelial removal. No functional evidence for epithelial-derived inhibitory factors could be demonstrated. It is concluded that the impermeable nature of the epithelium acts as a barrier to diffusion of hydrophilic and charged molecules thereby producing large concentration gradients across the airway wall. A breach in the integrity of the epithelium enables molecules to gain access to the smooth muscle.

Acetylcholine↗