Application of synthetic photostable retinoids induces novel limb and facial phenotypes during chick embryogenesis in vivo.

We have recently developed a range of synthetic retinoid analogues which include the compounds EC23 and EC19. They are stable on exposure to light and are predicted to be resistant to the normal metabolic processes involved in the inactivation of retinoids in vivo. Based on the position of the terminal carboxylic acid groups in the compounds we suggest that EC23 is a structural analogue of all-trans retinoic acid (ATRA), and EC19 is an analogue of 13-cis retinoic acid. Their effects on the differentiation of pluripotent stem cells has been previously described in vitro and are consistent with this hypothesis. We present herein the first description of the effects of these molecules in vivo. Retinoids were applied to the anterior limb buds of chicken embryos in ovo via ion-exchange beads. We found that retinoid EC23 produces effects on the wing digits similar to ATRA, but does so at two orders of magnitude lower concentration. When larger quantities of EC23 are applied, a novel phenotype is obtained involving production of multiple digit 1s on the anterior limb. This corresponds to differential effects of ATRA and EC23 on sonic hedgehog (shh) expression in the developing limb bud. With EC23 application we also find digit 1 phenotypes similar to thumb duplications described in the clinical literature. EC23 and ATRA are shown to have effects on the entire proximal-distal axis of the limb, including hitherto undescribed effects on the scapula. This includes suppression of expression of the scapula marker Pax1. EC23 also produces effects similar to those of ATRA on the developing face, producing reductions of the upper beak at concentrations two orders of magnitude lower than ATRA. In contrast, EC19, which is structurally very similar to EC23, has novel, less severe effects on the face and rarely alters limb development. EC19 and ATRA are effective at similar concentrations. These results further demonstrate the ability of retinoids to influence embryonic development. Moreover, EC23 represents a useful new tool to investigate developmental processes and probe the mechanisms underlying congenital abnormalities in vertebrates including man.

Lack of association between beta 2-adrenergic receptor polymorphisms and juvenile idiopathic arthritis.

OBJECTIVE: Juvenile idiopathic arthritis (JIA) is a chronic autoimmune arthropathy. Beta 2-adrenergic receptors are a link between the sympathetic nervous system and the immune system. Associations between variants in the gene encoding the beta 2-adrenergic receptor (ADRB2) and autoimmune disorders such as rheumatoid arthritis (RA) have been demonstrated. We aimed to investigate ADRB2 variants for association with JIA. METHODS: Genotypes and haplotypes of two ADRB2 variants (G16R and Q27E) were determined in 348 children with JIA and 448 healthy controls by direct molecular haplotyping using melting-curve analysis of a fluorescently labelled loci-spanning probe. Case-control analysis was performed to investigate whether ADRB2 variants were associated with JIA. RESULTS: No association was found between JIA and alleles, genotypes, or haplotypes of ADRB2. Specifically, the haplotype that demonstrated a strong association with RA (R16/Q27) was not associated with JIA. None of the variants demonstrated association after stratification by JIA subtypes or gender. CONCLUSIONS: Our results indicate that ADRB2 variants are not associated with JIA or any of the major JIA subtypes. These observations suggest that, although they share several clinical and pathological features, JIA and RA have unique genetic associations.

The role of retinoids in the adult nervous system and their therapeutic potential.

The mode of action of retinoids in relation to their activity in the adult central nervous system and the potential of synthetic retinoid analogues is reviewed. Investigation into the activity of such molecules will further our understanding of the retinoid pathway during nervous system development and in various neurological disease states.

Genetic relatedness between group B streptococci originating from bovine mastitis and a human group B Streptococcus type V cluster displaying an identical pulsed-field gel electrophoresis pattern.

Twenty isolates of group B streptococcus (GBS) were recovered from the milk of cows with bovine mastitis on three farms located in the south and south-east of Brazil between 1987 and 1988. These isolates were characterised by molecular methods and compared with a collection of 103 human GBS isolates from colonised and infected patients in the same region between 1980 and 2003. Some of the bovine isolates shared identical or similar pulsed-field gel electrophoresis (PFGE) patterns with a PFGE clone of human GBS type V. In addition, these bovine and human isolates also possessed the same ribotype. Multilocus sequence typing (MLST) of representative isolates confirmed the genetic relationship between the human and bovine GBS isolates with identical PFGE patterns, which clustered in the same ST-26 clonal complex. These data support the hypothesis that some bovine GBS strains are related closely to human isolates and may infect humans, or vice versa. Further comparative genomic analyses of GBS isolates from bovine and human origins are required to investigate this hypothesis further.

Molecular weights and Stokes radii of soluble elastins.

Soluble elastin was isolated from lathyritic chick aorta using neutral salt solutions in the presence of beta-amino propionitrile. The effect of a carboxy-methylation step in conjunction with proteolytic inhibitors was investigated. Hydrodynamic (Stokes) radii of soluble elastins were measured by gel filtration and the molecular size and weight distribution in purified fractions are reported.

Purification and properties of native titin.

A procedure has been developed for the extraction and purification of the massive myofibrillar protein titin without exposing it to denaturing conditions. The form of the molecule that has been isolated is soluble at high ionic strength and alkaline pH, but precipitates in low salt or at pH values below 7. Sedimentation velocity experiments indicate that titin is a highly asymmetric molecule with a sedimentation coefficient of 13.4 S. This asymmetry is confirmed by electron microscopy of rotary-shadowed specimens, which shows string-like structures of diameter 40 A and lengths up to 8000 A. Significant differences were observed depending on whether the electron microscope specimens were prepared by spraying or by layering of the titin onto a mica substrate; we tentatively attribute these differences to elasticity in the titin, revealed by the high shearing forces that accompany spraying. In accord with this, the circular dichroism spectrum of titin indicates that its secondary structure is largely random coil, a conformation characteristic of elastic proteins such as elastin. Negative staining of titin again shows long string-like structures, but these can now be seen to have an appearance similar to a string of beads, where the spacing between successive beads is about 40 A. Very similar beaded strings have been observed also associated with negatively stained separated native thick filaments; these are found running alongside the cross-bridge regions and in coils near the filament ends. Since the periodicity of the strings is similar to that of end-filaments, recently identified structures at the tips of thick filaments, it is likely that end-filaments are formed from titin. Titin comprises approximately 9% of the myofibrillar mass, which means that it is the third most abundant protein in muscle. The possible role of titin in forming elastic filaments within myofibrils is discussed.

Does titin regulate the length of muscle thick filaments?

The protein titin has been localized by electron microscopy of myofibrils labelled with monoclonal antibodies. The data are consistent with individual titin molecules extending from near the M-line to beyond the ends of thick filaments, a distance of approximately 1 micron. In the A-band, titin appears to be bound to thick filaments, probably to the outside of the filament shaft. Molecules of titin in this configuration provided an obvious mechanism by which the length of thick filaments could be regulated accurately.

The separation and identification by monoclonal antibodies of dog IgG fractions.

Four fractions of IgG from normal dog serum have been successfully isolated by gel filtration followed by protein A and protein G affinity chromatography using the fast protein liquid chromatography (FPLC) system. Protein A chromatography produced three peaks: peak 1 was fallthrough material consisting of components which did not bind to protein A, peak 2 consisted of bound material eluting at pH 6, and peak 3 contained bound material eluting at pH 3.5. The three peaks were then subjected individually to protein G affinity chromatography. Peak 1 from protein A chromatography produced a fallthrough peak followed by a weakly binding component which eluted at pH 8, and was called peak w. Peak 2 from protein A chromatography bound to protein G and eluted as a single peak at pH 3.8, and was called peak x. Peak 3 from protein A chromatography emerged as two separate peaks (y and z) off the protein G column; peak y bound and eluted at pH 4.1, and peak z bound weakly to protein G and emerged as a broad band at pH 8. Peaks w, x, y and z have been named gamma w, gamma x, gamma y and gamma z, respectively, and there purified IgG fractions were used to immunize mice for the preparation of monoclonal antibodies (McAbs). To date, two sets of McAbs have been produced: one which recognizes an epitope present in both gamma w and gamma z fractions and another set of McAbs which recognizes an epitope in the gamma x and gamma y fractions.

Role of the site of protonation in the low-energy decompositions of gas-phase peptide ions.

The dissociation of singly or multiply protonated peptide ions by using low-energy collisional activation (CA) is highly dependent on the sites of protonation. The presence of strongly basic amino acid residues in the peptide primary structure dictates the sites of protonation, which generates a precursor ion population that is largely homogeneous with respect to charge sites. Attempts to dissociate this type of precursor ion population by low-energy CA result in poor fragmentation via few pathways. The work described here represents a systematic investigation of the effects of charge heterogeneity in the precursor ion population of a series of model peptides in low-energy CA experiments. Incorporation of acidic residues in the peptide RLC*IFSC*FR (where C* indicates a cysteic acid residue), for example, balances the charge on the basic arginine residues, which enables the ionizing protons to reside on a number of less basic sites along the peptide backbone. This results in a precursor ion population that is heterogeneous with respect to charge site. Low-energy CA of these ions results in diverse and efficient fragmentation. Molecular modeling has been utilized to demonstrate that energetically preferred conformations incorporate an intraionic interaction between arginine and cysteic acid residues.

Optimal recovery of cytomegalovirus from urine as a function of specimen preparation.

Cytomegalovirus (CMV) is a significant pathogen among immunocompromised patients. We compared supernatant and sediment fractions of centrifuged urine for the optimal recovery of CMV by shell vial culture and polymerase chain reaction (PCR). Of 336 urine specimens, 31 (9.23%) were positive by shell vial culture; of these 29 (93.5%) were identified using the sediment fraction and 17 (54.8%) using the supernatant fraction (p = 0.001, chi2). Of the 29 positive sediment fraction specimens, 24 (82.8%) were identified as CMV positive at 24 h and 5 (17.2%) were identified as positive at 48 h. Two (0.064%) of the total 31 positive specimens were lost to microbial contamination in the sediment inoculated cultures. Of the 17 supernatant fraction specimens, 9 (53.9%) were identified as CMV positive at 24 h and 8 (47.1%) were identified as positive at 48 h. Fourteen (45.2%) of the total 31 positive specimens were lost to either toxicity or microbial contamination in the sediment-inoculated cultures. Thirty-four CMV culture-positive specimens were tested by PCR; 5 of these specimens (14.7%) were PCR negative for both sediment and supernatant fractions; 26 (76.5%) were found to be positive using the sediment fraction and negative using the supernatant; 3 (8.8%) were PCR positive for both the sediment and the supernatant. None of the 34 was identified as positive using the supernatant fraction only (p = 0.001, chi2). These findings demonstrate that the method of specimen preparation can significantly affect the outcome of diagnostic testing for CMV from urine specimens.

Direct evidence for a ruthenium(IV) oxo complex-mediated oxidation of a hydroxamic acid in the presence of phosphine oxide donors.

Ruthenium(II) complexes can be used to oxidise N-Boc hydroxylamine the the presence of tert-butylhydroperoxide to the corresponding nitroso dienophile, which is trapped using cyclohexa-1,3-diene as the hetero-Diels-Alder adduct; direct evidence has been obtained for the intervention of a triphenylphosphine oxide-stabilised ruthenium(IV) oxocomplex as the catalytically active species.

Speed discrimination of stereoscopic (cyclopean) motion.

This study investigated the degree to which speed of stereoscopic translational motion (i.e. moving binocular disparity information) can be discriminated in a display that minimizes position information. Observers viewed dynamic random-element stereograms depicting arrays of randomly positioned stereoscopic dots that moved bidirectionally. Two tasks were performed: a speed discrimination task and a displacement discrimination task. Across a range of conditions, speed could be discriminated under conditions in which displacement could not. Thus, speed of stereoscopic motion can be discriminated when position information is minimal. This result indicates that stereoscopic motion is sensed in a way that cannot be explained by feature tracking or by inferring the motion from memory of position and time.

A novel (6S)-4,6-dimethyldodeca-2E,4E-dienoyl ester of phomalactone and related alpha-pyrone esters from a Phomopsis sp. with cytokine production inhibitory activity.

A series of novel 6-substituted 5,6-dihydro-5-hydroxy-alpha-pyrone esters, 1 approximately 3, isolated from fermentations of a Phomopsis sp. (Xenova culture collection no. X22502) have been identified as inhibitors of lipopolysaccharide (LPS)-induced cytokine production. These include the (6S)-4,6-dimethyldodecadien-2E,4E-dienoyl ester of phomalactone, 1, and two analogues bearing a prop-2E-enoic acid moiety at the 6-position of the alpha-pyrone ring. (6S)-4,6-Dimethyl-2E,4E-dienoic acid, 4, and a hydroxylated analogue, 5, were also isolated and characterised. The most potent cytokine production inhibitor was 1, which inhibited LPS-induced tumour necrosis factor alpha (TNFalpha) production by U937 cells and LPS-induced interleukin 1beta (IL-1beta) production by peripheral blood mononuclear cells (PBMC) with IC50 values of 80 nM and 190 nM respectively. The effect of 1 in PBMC was selective for IL-1beta relative to TNFalpha. The inhibition of IL-1beta production by 1 involved a post-translational mechanism of action at the level of IL-1beta secretion as demonstrated by the lack of an effect on cell-associated IL-1beta production. 1 showed no effect on the activity of caspase 1 in cytosolic extracts from the THP1 monocytic cell line.

The coacervate-sol transition observed with alpha-elastin and its N-formyl O-methyl derivative.

The coacervate phase produced by raising the temperature of solutions of blocked alpha-elastin has water content and fibrillar structure at electron microscope level similar to fibrous elastin (Cox, B.A., Starcher, B.C., Urry, D.W. (1973) Biochim, Biophys. Acta 317, 209-213). The stability ranges of the coacervates under varying conditions of temperature, pH, salt concentration and concentration of added organic solvent have been investigated with results that suggest a marked sensitivity of elastin conformation to solution conditions.

Development of an enzyme-linked immunosorbent assay for the detection of IgG subclasses in the serum of normal and diseased dogs.

The present study describes the development of an enzyme-linked immunosorbent assay capable of quantifying serum antibody of all four canine IgG subclasses. A panel of subclass-restricted and subclass-specific monoclonal antibodies was used to measure IgG subclasses in the serum of healthy dogs, as well as in dogs with a range of clinical diseases. The subclasses have been redefined as IgG1, IgG2, IgG3 and IgG4 based on a comparison with the relative concentration and electrophoretic mobilities of human IgG subclasses. In serum samples from healthy dogs, the concentration of IgG1 (mean, 8.17 +/- 0.95 mg ml-1) and IgG2 (mean, 8.15 +/- 3.16 mg ml-1) were very similar and considerably higher than the levels of IgG3 (mean, 0.36 +/- 0.43 mg ml-1) and IgG4 (mean, 0.95 +/- 0.45 mg ml-1). There was no apparent difference in the level of subclasses between the different breeds comprising this normal population. Sera from dogs with a range of immune-mediated or inflammatory diseases all had markedly elevated levels of IgG2 (more than 13 mg ml-1), but IgG1 decreased (less than 4 mg ml-1) to levels below the normal range.

Preparation of monoclonal antibodies specific for the subclasses of canine IgG.

Canine IgG is composed of four subclasses, which are defined as IgG1, IgG2, IgG3 and IgG4 on the basis of data from fast protein liquid chromatography, and their electrophoretic mobilities and relative concentrations in serum. This paper describes the preparation of mAbs specific for determinants on canine IgG2, IgG3 and IgG4. The mAb specific for IgG2 resulted from a conventional immunisation protocol. The mAb specific for IgG3 was a result of immunisation with IgG3 combined with the suppression of the immune response to IgG1 by passively administered anti-IgG1 antibody. The mAb specific for IgG4 resulted from immunisation with Fab or Fc fragments which were obtained by the cleavage of the IgG4 molecule with papain. The specificity of each mAb was established by using an enzyme-linked immunosorbent assay which showed that all three specific clones recognised a determinant in the Fd region of the canine immunoglobulin molecule.

Degradation of myofibrils from rabbit, chicken and beef by cathepsin l and lysosomal lysates.

The degradation of rabbit, chicken and beef myofibrils by cathepsin L or lysosomal lysates was studied by SDS-polyacrylamide-gel electrophoresis and electron microscopy (EM). Similar degradation patterns were observed for each myofibrillar preparation incubated with cathepsin L, except that myosin heavy chain and tropomyosin of beef were more susceptible than those of rabbit and chicken. Otherwise, troponin T, troponin in I and C-protein were rapidly degraded with slower degradation of titin, nebulin, myosin heavy chain, α-actinin, α-tropomyosin, actin and myosin light chains, LC1 and LC2. However, the component of 30 000 Mr was found to be further degraded to smaller peptides. Degradation at pH 5·5 (approximate post-mortem limit value) was faster than at pH 6·0 but slower than at pH 5·0. A number of new protein bands were identified (130 000, 120 000, 90 000, 85 000, 80 000, 31 000 and 30 000 Mr). The degradation patterns of rabbit myofibrils by rabbit muscle lysosomal lysates were similar to that of myofibrils incubated with purified cathepsin L except for the retention of the 30 000 Mr component and reduced degradation of actin, due presumably to the reduced amount or stability of cathepsin L in the crude enzyme preparations. Electron micrographs revealed that myofibrillar degradation by cathepsin L occurred preferentially at the Z-lines leading to removal of the Z-line proteins and fracturing of the myofibrils at these sites. Catheptic damage was seen to be most rapid in chicken myofibrils and least rapid in beef myofibrils consistent with the more rapid conditioning process in chicken.

Suppression of Myc oncogenic activity by nucleostemin haploinsufficiency.

Nucleostemin (NS), a nucleolar GTPase, is highly expressed in stem/progenitor cells and in most cancer cells. However, little is known about the regulation of its expression. Here, we identify the NS gene as a novel direct transcriptional target of the c-Myc oncoprotein. We show that Myc overexpression enhances NS transcription in cultured cells and in pre-neoplastic B cells from Eµ-myc transgenic mice. Consistent with NS being downstream of Myc, NS expression parallels that of Myc in a large panel of human cancer cell lines. Using chromatin immunoprecipitation we show that c-Myc binds to a well-conserved E-box in the NS promoter. Critically, we show NS haploinsufficiency profoundly delays Myc-induced cancer formation in vivo. NS+/-Eµ-myc transgenic mice have much slower rates of B-cell lymphoma development, with life spans twice that of their wild-type littermates. Moreover, we demonstrate that NS is essential for the proliferation of Myc-overexpressing cells in cultured cells and in vivo: impaired lymphoma development was associated with a drastic decrease of c-Myc-induced proliferation of pre-tumoural B cells. Finally, we provide evidence that in cell culture NS controls cell proliferation independently of p53 and that NS haploinsufficiency significantly delays lymphomagenesis in p53-deficient mice. Together these data indicate that NS functions downstream of Myc as a rate-limiting regulator of cell proliferation and transformation, independently from its putative role within the p53 pathway. Targeting NS is therefore expected to compromise early tumour development irrespectively of the p53 status.