Biomedical subjects

C Robinson

Publications and source records attributed to C Robinson.

At least 73 records · Page 4Linked to original sources

PubMed2000

Different lumen-targeting pathways for nuclear-encoded versus cyanobacterial/plastid-encoded Hcf136 proteins.

Lumenal proteins are transported across the thylakoid membrane by two very different pathways: Sec-dependent or twin-arginine translocase (Tat)-dependent, where the substrate protein can be transported in a folded state. We present the first evidence that a given protein can be targeted by different pathways in different organisms. Arabidopsis Hcf136 is targeted exclusively by the Tat pathway in pea chloroplasts and no Sec-dependent transport is evident even when the twin-arginine is replaced by twin-lysine. However, twin-arginine motifs are absent from the presequences of Hcf136 proteins encoded by plastid or cyanobacterial genomes, strongly implying translocation by another pathway (presumably Sec). We suggest that the Hcf136 protein was transferred to the Tat pathway when the gene became incorporated into the nuclear genome, possibly due to the tighter folding associated with the more involved, post-translational targeting pathway.

Adenosine Triphosphatases↗

PubMed2000

Novel Cystic Fibrosis mutation L1093P: functional analysis and possible Native American origin.

A novel mutation was detected using single-strand conformation polymorphism and heteroduplex analysis in a cystic fibrosis subject of mixed ancestry. Mutation 3410T-->C in exon 17b caused the novel missense mutation L1093P; the other chromosome has mutation N1303K. The 31-year-old subject is pancreatic insufficient, had an FEV(1) score that was 33% of normal prior to a heart/lung transplant, and sweat chloride values of 116 and 95 mM when tested at ages 1 and 11. Functional analysis using forskolin-stimulated efflux of (125)I in HEK cells transfected with an ABCC7 construct harboring the L1093P mutation confirmed that cAMP-mediated anion efflux was abnormal, but some function was preserved. Analysis of parental DNA established that N1303K was of English origin, while L1093P was of Greek, Irish or Native American (Cherokee) origin. Given the intensive screening for CF mutations in European populations, we hypothesize that L1093P is of Native American origin. Hum Mutat 15:208, 2000.

Adult↗

PubMed2000

Magnesium and fluoride distribution in human cementum with age.

Sixty-two human teeth, obtained from subjects aged 11 to 80 years, were used to determine the magnesium and fluoride concentration and distribution with age in human cementum. Transverse sections were prepared from the root region of teeth. Samples, each 30 microm thick, were abraded in sequence from the cementum surface and the cemento-dentine junction by an abrasive micro-sampling technique. Magnesium concentrations were lower in the cementum surface, and increased towards the cemento-dentine junction (CDJ), while fluoride concentrations were higher in cementum surfaces and tended to decrease towards CDJ. Fluoride distribution patterns were similar to that reported earlier while average fluoride concentration increased with age, however, either no change or decreasing tendencies were observed with magnesium.

Adolescent↗

PubMed2000

A microbiological survey of Montserrat Island hydrothermal biotopes.

In March 1996, a survey of hydrothermal sites on the island of Montserrat was carried out. Six sites (Galway's Soufrière. Gages Upper and Lower Soufrières, Hot Water Pond, Hot River, and Tar River Soufrière) were mapped and sampled for chemical, ATP, and microbial analyses. The hydrothermal Soufrière sites on the slopes of the active Chances Peak volcano exhibited temperatures up to almost 100 degrees C and were generally either mildly acidic at pH 5-7 or strongly acidic at pH 1.5-3, but with some hot streams and pools of low redox potential at pH 7-8. Hot Water Pond sites, comprising a series of heated pools near the western shoreline of the island. were neutral and saline, consistent with subsurface heating of entrained seawater. Biological activity shown by ATP analyses was greatest in near-neutral pH samples and generally decreased as acidity increased. A variety of heterotrophic and chemolithotrophic thermophilic organisms were isolated or observed in enrichment cultures. Most of the bacteria that were obtained in pure culture were familiar acidophiles and neutrophiles, but novel, iron-oxidizing species of Sulfobacillus were revealed. These species included the first mesophilic iron-oxidizing Sulfobacillus strains to be isolated and a strain with a higher maximum growth temperature (65 degrees C) than the previously described moderately thermophilic Sulfobacillus species.

Adenosine Triphosphate↗

PubMed2000

The chemical composition of tooth enamel in junctional epidermolysis bullosa.

The junctionalis form of epidermolysis bullosa (EBJ) is associated with a number of clinical problems involving tooth enamel, including increased susceptibility to caries. The aim here was to carry out a chemical characterization of the enamel of teeth from EBJ patients compared with that of unaffected controls. The results showed that while protein concentration, amino acid composition and carbonate content were similar in both groups, EBJ enamel contained a significantly reduced mineral per volume content, resulting in enamel hypoplasia. In addition, Western blotting revealed the presence of serum albumin (a known inhibitor of enamel crystal growth) in EBJ enamel. This was not detected in control enamel or in enamel of teeth from patients with the dystrophic form of the disease. It is concluded that EBJ enamel is developmentally compromised and that the enamel defects are commensurate with the reported genetic lesions.

Amelogenesis↗

PubMed2000

Fluoride and magnesium concentrations in human dental calculus obtained from Japanese and Chinese patients.

Calculus deposited on a total of 68 permanent teeth from patients 30-60 years old from Nagoya in Japan and Beijing in China was investigated. An abrasive microsampling method was used to examine the fluoride (F) and magnesium (Mg) distribution, using a fluoride ion-specific electrode and atomic absorption spectrophotometry, respectively. F concentrations decreased from the surface towards the interior of the calculus. Mg concentrations, however, gradually rose towards the innermost surface adjacent to the tooth. In all parts of the depth profiles, the average concentrations of both magnesium and fluoride were higher in the Japanese than in the Chinese calculus. Towards the inner surface of the calculus, F and Mg concentrations were also much higher in the Japanese than in the Chinese group. A greater intake of sea foods and greater use of fluoride dentifrices are possible reasons for the higher F and Mg concentrations in the Japanese individuals.

Adult↗

PubMed2000

Spatially related amelogenin interactions in developing rat enamel as revealed by molecular cross-linking studies.

A cleavable cross-linker (dithiobis[succinimidyl propionate], DTSP) was used to investigate the subunit structure of the developing enamel matrix. Intact matrix was cross-linked under conditions chosen to simulate those found in vivo. The cross-linked complexes were isolated by preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and their subunit composition determined by analytical SDS-PAGE following reductive cleavage of the cross-links. Western blotting using antiamelogenin antibodies was used to confirm the identity of the proteins involved. The results showed that nascent amelogenins tended to be cross-linked to other nascent amelogenins while amelogenin-processing products tended to be cross-linked to other processed molecules at the same stage of processing. The results suggest that nascent amelogenins are in close association after secretion and during extracellular processing, and that processed products are not free to associate with nascent molecules, presumably due to diffusion constraints in the tissue. This conclusion implies that individual amelogenin molecules within supramolecular aggregates (nanospheres) are processed in situ and remain in the same nanosphere while all the individual component amelogenins undergo processing. The biological function of amelogenin processing remains unclear but the fact that amelogenin-amelogenin associations are maintained during processing indicates that matrix stability is an important factor while the enamel layer is being deposited.

Amelogenesis↗

PubMed2000

A case of monosymptomatic hypochondriacal psychosis treated with olanzapine.

OBJECTIVE: To emphasize the availability of safer alternatives to currently standard therapy for monosymptomatic hypochondriacal psychosis (MHP). METHOD: We report a case of treatment of monosymptomatic hypochondriacal psychosis in an elderly woman with the atypical neuroleptic olanzapine. RESULTS: Treatment with rather low doses of olanzapine led to complete resolution of delusional symptoms. CONCLUSIONS: Olanzapine, like other atypical neuroleptic agents, shows promise as a treatment for this syndrome, and it offers considerable safety and side-effect advantages over older agents.

Aged↗

PubMed2000

Tight junction properties of the immortalized human bronchial epithelial cell lines Calu-3 and 16HBE14o-.

Tight junctions (TJs) make a vital contribution to the barrier properties of the airway lining. Opening of TJs, or their frank cleavage, is suspected as a pathophysiological event in the lung, but research into the cellular and molecular mechanisms involved has been impeded by technical limitations of available experimental models. The authors have compared the properties of two epithelial cell lines derived from bronchial epithelium to explore whether these cell lines could constitute appropriate tools for the study of TJ regulation in bronchial epithelium. Investigations of TJs in 16HBE14o- cells and Calu-3 cells were made by fluorescent antibody labelling in conjunction with wide-field, confocal or 2-photon molecular excitation microscopy (2PMEM). The presence of TJ proteins was confirmed by immunoblotting and functional properties of the monolayers were studied by measurements of transepithelial electrical resistance and mannitol permeability. Cells of both lines formed confluent monolayers in which the cells expressed the TJ proteins occludin and ZO-1 in continuous circumferential patterns suggestive of functional TJs. This interpretation was supported by the development of transepithelial electrical resistances and of low paracellular permeability to solutes. Within the limits of resolution offered by 2PMEM, occludin and ZO-1 appeared to colocalize at TJs. These studies suggest that the 16HBE14o- cells and Calu-3 cell lines are potentially useful in vitro models to study how tight junction opening or cleavage changes the functional barrier properties of bronchial epithelium.

Animals↗

PubMed2000

Pretreatment with cationic lipid-mediated transfer of the Na+K+-ATPase pump in a mouse model in vivo augments resolution of high permeability pulmonary oedema.

Resolution of pulmonary oedema is mediated by active absorption of liquid across the alveolar epithelium. A key component of this process is the sodium-potassium ATPase (Na+K+-ATPase) enzyme located on the basolateral surface of epithelial cells and up-regulated during oedema resolution. We hypothesised that lung liquid clearance could be further up-regulated by lipid-mediated transfer and expression of exogenous Na+K+-ATPase cDNA. We demonstrate proof of this principle in a model of high permeability pulmonary oedema induced by intraperitoneal injection of thiourea (2.5 mg/kg) in C57/BL6 mice. Pretreatment of mice (24 h before thiourea) by nasal sniffing of cationic liposome (lipid #67)-DNA complexes encoding the alpha and beta subunits of Na+K+-ATPase (160 microg per mouse), significantly (P<0.01) decreased the wet:dry weight ratios measured 2 h after thiourea injection compared with control animals, pretreated with an equivalent dose of an irrelevant gene. Whole lung Na+K+-ATPase activity was significantly (P<0.05) increased in mice pretreated with Na+K+-ATPase cDNA compared both with untreated control animals as well as animals pretreated with the irrelevant gene. Nested RT-PCR on whole lung homogenates confirmed gene transfer by detection of vector-specific mRNA in three of four mice studied 24 h after gene transfer. This demonstration of a significant reduction in pulmonary oedema following in vivo gene transfer raises the possibility of gene therapy as a novel, localised approach for pulmonary oedema in clinical settings such as ARDS and lung transplantation.

Animals↗

PubMed2000

The 18-kDa form of cat allergen Felis domesticus 1 (Fel d 1) is associated with gelatin- and fibronectin-degrading activity.

BACKGROUND: Fel d 1, an important allergen from domestic cats, is a significant cause of asthma. In addition to directly promoting IgE synthesis, other biological activities of allergens may contribute to either allergic sensitization or the magnitude of allergic effector responses. For example, allergens that degrade proteins have been suggested to facilitate allergen presentation by increasing parallelular permeability of airways epithelium. However, little information exists to indicate whether Fel d 1 has other activities relevant to allergic responses. OBJECTIVE: To study whether Fel d 1 is associated with enzyme activity. METHODS: Fel d 1 was obtained by a rigorous purification strategy and its identity confirmed by laser desorption mass spectrometry, cleavage and sequencing. The ability of Fel d 1 to degrade gelatin, fibronectin and the artificial substrate N-benzoyl-FVR-p-nitroanilide was studied. The effect of Fel d 1 on the morphology of tight junctions in epithelial cell monolayers was also investigated. RESULTS: The 18-kDa form of Fel d 1 caused degradation of denatured collagens (gelatin) and cleaved a 20-kDa fragment from the A chain of plasma fibronectin. Catalytic activity was not altered by inhibitors of cysteine peptidases, matrix metallopeptidases or by removal of divalent cations. In contrast, aprotinin and TLCK were inhibitors of Fel d 1. The absence of a serine peptidase catalytic triad in Fel d 1, together with the stoichiometry of the inhibition of TLCK and aprotinin, suggest that their inhibitory action may be due to noncatalytic site interactions. Alternatively, highly purified Fel d 1 may be associated with an active contaminant, although none were found. CONCLUSION: These results suggest that Fel d 1 is another example of a domestic allergen which is associated with enzyme activity. It remains to be established whether the activity resides in Fel d 1 itself or in an unresolved, and possibly related, protein.

Allergens↗

PubMed2000

Quantitative structural and biochemical analyses of tight junction dynamics following exposure of epithelial cells to house dust mite allergen Der p 1.

BACKGROUND: House dust mite allergen Der p 1 is a cysteine peptidase. Previously, we have suggested that the proteolytic activity of this allergen may contribute to asthma by damaging the barrier formed by the airways epithelium. OBJECTIVE: The present study applied novel techniques to compare changes in permeability with quantitative events in tight junctions (TJs) and desmosomes (DMs) of epithelial cells exposed to Der p 1. METHODS: Confluent monolayers of Madin-Darby canine kidney (MDCK) and 16HBE14o-human bronchial epithelial cells were used as experimental models. Permeability was estimated from mannitol clearance. Digital imaging with quantification of TJs and DMs was achieved by fluorescent antibody staining and 2-photon molecular excitation microscopy (2PMEM). Biochemical changes in TJs were studied by immunoblotting, radiolabelling and immunoprecipitation. RESULTS: Der p 1 caused a time-dependent breakage of TJs and reduction in their content of the protein ZO-1. Reduction in ZO-1 immunofluorescence at TJs occurred with a small increase in the amount of diffuse, cytoplasmic immunoreactive ZO-1 staining. Morpho-logical changes in TJs occurred in synchrony with increases in epithelial permeability. DM puncta increased both in size and intensity of staining. Immunoblotting demonstrated that the disruption of TJ morphology was associated with cleavage of ZO-1 and occludin. Cells recovered from allergen exposure by de novo synthesis of occludin. CONCLUSION: Der p 1 could contribute to sensitization and allergic responses by degrading the function of the airway epithelial barrier.

Animals↗

PubMed2000

Transport of proteins into and across the thylakoid membrane.

The biogenesis of thylakoid proteins is a complex issue that requires the operation of at least four pathways within the chloroplast. Two of the pathways are used for soluble lumenal proteins, where the proteins bear cleavable targeting signals that are recognized by one of two distinct translocases. These pathways differ in fundamental respects. A subset of lumenal proteins are transported in an unfolded state by a typical Sec system, whereas others are transported by a novel class of translocase that appears to function primarily in the transport of fully-folded proteins. Related protein translocases have now been shown to operate in a wide variety of bacterial species, suggesting a widespread requirement for the translocation of folded proteins across biological membranes. Numerous integral membrane proteins are also targeted into the thylakoid membrane, and these too follow at least two distinct routes. Some proteins use a signal recognition particle-dependent pathway that requires GTP and unidentified apparatus in the thylakoid membrane. Others, however, require none of the known targeting factors and may insert spontaneously into the membrane. In this article, the rationale behind this pathway complexity is discussed in relation to the properties of the substrate proteins and the evolutionary origins of the chloroplast.

Amino Acid Sequence↗

PubMed2000

Valganciclovir results in improved oral absorption of ganciclovir in liver transplant recipients.

The pharmacokinetics of an orally administered valine ester of ganciclovir (GCV), valganciclovir (VGC), were studied. These were compared to the pharmacokinetics of oral and intravenous GCV. Twenty-eight liver transplant recipients received, in an open-label random order with a 3- to 7-day washout, each of the following: 1 g of oral GCV three times a day; 450 mg of VGC per os (p.o.) once a day (q.d.); 900 mg of VGC p.o. q.d.; and 5 mg of intravenous (i.v.) GCV per kg of body weight q.d., given over 1 h. GCV and VGC concentrations were measured in blood over 24 h. One-sided equivalence testing was performed to test for noninferiority of 450 mg of VGC relative to oral GCV (two-sided 90% confidence interval [CI] > 80%) and nonsuperiority of 900 mg of VGC relative to i.v. GCV (two-sided 90% CI < 125%). The exposure of 450 mg of VGC (20.56 microg. h/ml) was found to be noninferior to that of oral GCV (20.15 microg. h/ml; 90% CI for relative bioavailability of 95 to 109%), and the exposure of 900 mg of VGC (42.69 microg. h/ml) was found to be nonsuperior to that of i.v. GCV (47.61 microg. h/ml; 90% CI = 83 to 97%). Oral VGC delivers systemic GCV exposure equivalent to that of standard oral GCV (at 450 mg) or i.v. GCV (at 900 mg of VGC). VGC has promise for effective CMV prophylaxis or treatment with once-daily oral dosing in transplant recipients.

Antiviral Agents↗

PubMed2000

Serologic responses in patients with malignant mesothelioma: evidence for both public and private specificities.

Malignant mesothelioma (MM) is a pulmonary malignancy that appears to be immunogenic based on a large number of studies in both animals and humans. This notion is supported by our recent demonstration using Western blot analysis of immunoglobulin G antibodies reactive with a variety of autoantigens in many patients with MM. In view of the enormous potential of such antigens in early diagnosis, immunotherapy, and vaccination of at-risk individuals, it was essential to identify these antigens. We therefore applied the SEREX technique (serologic identification by recombinant expression cloning), using a serum pool from six patients as the probe against an expressed complementary DNA library derived from a cloned MM cell line. We screened over one million recombinants and obtained sequence information on eight antigens that had provoked immunoglobulin heavy chain class switching, presumably as a consequence of T-cell recognition. Six of these antigens were identifiable (U2AF[65], Siah binding protein, topoisomerase IIbeta, ZFM1, mIre1, and pendulin), and of the others, one was found as a single EST from a myotube library (Jemm-1); the other (Jemm-2) was not represented in any EST database even as a weak homolog. Consistent with our previous findings, each of the characterizable antigens would be expected to be associated with the cell nucleus. Each of the autoantibody specificities was uniquely associated with a single patient with the exception of antibodies to TOPIIbeta and U2AF(65). We found 13 of 14 (93%) patients with MM had antibodies to TOPIIbeta and two of 14 (14%) patients had antibodies to U2AF(65). The number of serum reactivities, taken as a measure of the complexity of the immune response, correlates with patient survival and with an index of systemic inflammation. These data suggest that a broader range of serologic reactivities reflects a more active host response to the presence of tumor.

Animals↗

PubMed2000

Architecture of intact natural human plaque biofilms studied by confocal laser scanning microscopy.

Determination of the structure of human plaque will be of great benefit in the prediction of its formation and also the effects of treatment. However, a problem lies in the harvesting of undisturbed intact plaque samples from human volunteers and the viewing of the biofilms in their natural state. In this study, we used an in situ device for the in vivo generation of intact dental plaque biofilms on natural tooth surfaces in human subjects. Two devices were placed in the mouths of each of eight healthy volunteers and left to generate biofilm for 4 days. Immediately upon removal from the mouth, the intact, undisturbed biofilms were imaged by the non-invasive technique of confocal microscopy in both reflected light and fluorescence mode. Depth measurements indicated that the plaque formed in the devices was thicker round the edges at the enamel/nylon junction (range = 75-220 microm) than in the center of the devices (range = 35-215 microm). The reflected-light confocal images showed a heterogeneous structure in all of the plaque biofilms examined; channels and voids were clearly visible. This is in contrast to images generated previously by electron microscopy, suggesting a more compact structure. Staining of the biofilms with fluorescein in conjunction with fluorescence imaging suggested that the voids were fluid-filled. This more open architecture is consistent with recent models of biofilm structure from other habitats and has important implications for the delivery of therapeutics to desired targets within the plaque.

Biofilms↗

PubMed2000

Evidence for charge domains on developing enamel crystal surfaces.

The control of hydroxyapatite crystal initiation and growth during enamel development is thought to be mediated via the proteins of the extracellular matrix. However, the precise nature of these matrix-mineral interactions remains obscure. The aim of the present study was to use a combination of atomic and chemical force microscopy to characterize developing enamel crystal surfaces and to determine their relationship with endogenous enamel matrix protein (amelogenin). The results show regular and discrete domains of various charges or charge densities on the surfaces of hydroxyapatite crystals derived from the maturation stage of enamel development. Binding of amelogenin to individual crystals at physiological pH was seen to be coincident with positively charged surface domains. These domains may therefore provide an instructional template for matrix-mineral interactions. Alternatively, the alternating array of charge on the crystal surfaces may reflect the original relationship with, and influence of, matrix interaction with the crystal surfaces during crystal growth.

Amelogenesis↗

PubMed2000

The chemistry of enamel caries.

The chemical changes which occur during the process of carious destruction of enamel are complex due to a number of factors. First, substituted hydroxyapatite, the main component of dental enamel, can behave in a very complex manner during dissolution. This is due not only to its ability to accept substituent ions but also to the wide range of calcium phosphate species which can form following dissolution. In addition, the composition, i.e., the extent of substitution, changes throughout enamel in the direction of carious attack, i.e., from surface to interior. Both surface and positively birefringent zones of the lesion clearly illustrate that carious destruction is not simple dissolution. Selective dissolution of soluble minerals occurs, and there is the probability of reprecipitation. The role of fluoride here is crucial in that not only does it protect enamel per se but also its presence in solution means that rather insoluble fluoridated species can form very easily, encouraging redeposition. The role of organic material clearly needs further investigation, but there is the real possibility of both inhibition of repair and facilitation of redeposition. For the future, delivering fluoride deep into the lesion would appear to offer the prospect of improved repair. This would entail a delivery vehicle which solved the problem of fluoride uptake by apatite at the tooth surface. Elucidation of the role of organic material may also reveal putative mechanisms for encouraging repair and/or protecting the enamel mineral.

Animals↗