On the potential significance of the enzymatic activity of mite allergens to immunogenicity. Clues to structure and function revealed by molecular characterization.
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Allergens↗
Biomedical subjects
C Robinson
Publications and source records attributed to C Robinson.
Incidence of hereditary non-polyposis colorectal cancer in a population-based study of 1137 consecutive cases of colorectal cancer.
BACKGROUND: Previous reports have indicated that 5-13 per cent of colorectal cancer is hereditary. However, the proportion of cases arising as a result of mutations in the hereditary non-polyposis colorectal cancer (HNPCC) genes remains to be determined. METHODS: This study is a part prospective, part retrospective review of all cases of colorectal cancer from a district hospital over 14 years. Some 1137 consecutive patients with colorectal cancer were questioned about their family history of cancer and details were logged on a database. For the past 4 years each case has been re-evaluated where possible. RESULTS: Some 118 patients indicated initially that they had a first-degree relative with colorectal cancer, but on re-evaluation there were significant discrepancies. Only three cases (0.3 per cent) occurred in families which strictly fulfilled the criteria for HNPCC and there were no cases of familial adenomatous polyposis. A total of 16 patients (1.4 per cent) fulfilled looser criteria for HNPCC. CONCLUSION: This population-based study has shown a lower frequency of familial bowel cancer than previous studies and may reflect a lower incidence of inherited mutations in the HNPCC DNA mismatch repair genes than is currently accepted.
Adult↗
Voltage-activated proton current in eosinophils from human blood.
1. The resting membrane potential of freshly purified normodense human eosinophils bathed in and dialysed with quasi-physiological solutions was -63 +/- 2 mV (n = 100). 2. In voltage-clamp mode with quasi-physiological internal and external solutions, voltage steps from the holding potential of -60 mV to levels positive to +20 mV resulted in development of a quasi-instantaneous outward current and a slowly developing outward current. The instantaneous current was absent when the cells were bathed in and dialysed with K(+)-free solution. 3. The slow outward current persisted following simultaneous replacement of K+, Na+ and most of the Cl- with largely impermeant ions (tetraethylammonium, N-methyl-D-glucamine and methanesulphonate) and was augmented when the cell was dialysed with a solution of increased buffering capacity for protons. The observed reversal potential of the current closely followed the hydrogen equilibrium potential over a wide range of internal-external pH combinations, indicating that the conductance underlying the slow outward current was highly selective for H+ ions. 4. Acidification of the pipette solution (increasing [H+]i) augmented the outward H+ current and shifted its activation range negatively, whilst acidification of the external solution had the opposite effect. The voltage dependence of the current is modulated by the transmembrane pH gradient so the only outward current could be activated. However, when the outward current was activated by a voltage step, rapid acidification of external solution produced an inward H+ current which rapidly deactivated. 5. The proton current was reversibly inhibited in a voltage-dependent manner by extracellular application of Zn2+. The apparent dissociation constants were 8 nM (at +40 mV), 36 nM (at +70 mV) and 200 nM (at +100 mV). 6. The proton current was augmented by exposure to 10 microM arachidonic acid. This augmentation consisted of a shift of the voltage dependence of activation to more negative potentials and enhancement of maximum conductance (gH,max). The proton current recorded in eosinophils was significantly augmented under conditions of elevated cytosolic free calcium concentration ([Ca2+]i). The threshold level of [Ca2+]i associated with this effect lay between 0.1 and 1 microM and was not measurably affected by cytosolic acidification. 7. Eosinophils from human blood possess a voltage-dependent H+ conductance (gH) which normally allows protons to move outwards only; raising [Ca2+]i was associated with augmentation of gH and intracellular acidification or arachidonate shifted its activation range negatively towards physiological potentials.
Adult↗
An Arabidopsis thaliana cDNA encoding PS II-X, a 4.1 kDa component of photosystem II: a bipartite presequence mediates SecA/delta pH-independent targeting into thylakoids.
Higher plant photosystem II preparations contain a 4.1 kDa polypeptide (subunit X) associated with the oxygen-evolving core complex. We describe the isolation of a cDNA encoding PS II-X from Arabidopsis thaliana, in which the C-terminal region is highly homologous to partially sequenced PS II-X from wheat and spinach. The mature protein of 42 residues is preceded by a 74-residue, bipartite presequence similar to those involved in the targeting of nuclear-encoded thylakoid lumen proteins, although hydrophobicity analysis indicates the presence of a single transmembrane span in the mature protein. Moreover, import of pre-PS II-X into the thylakoid membrane of isolated chloroplasts is unaffected by inhibitors of either the Sec- or delta pH-dependent thylakoidal protein translocases, suggesting a spontaneous insertion mechanism. PS II-X appears to be encoded as a mature protein by the plastid genome in the chlorophyll a+c- containing alga, Odontella sinensis. We thus propose that the thylakoid transfer signal of Arabidopsis pre-PS II-X represents a recent acquisition, in phylogenetic terms, compared with signals of Sec-dependent lumenal proteins.
Amino Acid Sequence↗
Cell-cycle regulation of B-Myb protein expression: specific phosphorylation during the S phase of the cell cycle.
Previous studies revealed that transcription of B-Myb, which encodes a transcription factor related to the c-Myb proto-oncoprotein, is cell-cycle regulated by an E2F transcription factor-mediated repression mechanism operating in G0/G1. To begin to determine the consequences of transcriptional regulation on B-Myb function, we report here further studies of B-Myb protein expression in the cell cycle. We found that G0-arrest of serum-deprived mouse fibroblasts was achieved without significant reduction in B-Myb levels, moreover, over-expression of B-Myb in stably transfected cells did not prevent their entry into G0. Following serum-induction of arrested fibroblasts, B-Myb abundance increased as cells entered S phase to levels significantly greater than found in cycling cells. This was accompanied by the appearance of a novel phosphorylated form of B-Myb (112 kDa) of distinctly lower electrophoretic mobility than B-Myb present in G1 (110 kDa). The 112 kDa species was S phase-specific even in transfected cells overexpressing B-Myb. Consistent with modification in the S phase of the cell cycle, preliminary evidence suggested that a cyclin A/cdk2, but not cyclin E/cdk2 or cyclin D1/cdk4, complex could induce a similar electrophoretic mobility change in baculovirus-specified B-Myb. These findings show that B-Myb expression may be subject to two levels of control during the cell cycle, transcription and protein phosphorylation.
3T3 Cells↗
Ricin A chain fused to a chloroplast-targeting signal is unfolded on the chloroplast surface prior to import across the envelope membranes.
The initial stages of chloroplast protein import involve the binding of precursor proteins to surface-bound receptors prior to translocation across the envelope membranes in a partially folded conformation. We have analyzed the unfolding process by examining the conformation of a construct, comprising the presequence of a chloroplast protein linked to ricin A chain, before and after binding to the chloroplast surface. We show that the presequence is highly susceptible to proteolysis in solution, probably reflecting a lack of tertiary structure, whereas the A chain passenger protein is resistant to extremely high concentrations of protease, unless deliberately unfolded using denaturant. The A chain moiety is furthermore active, indicating that the presence of the presequence does not prevent formation of a tightly folded, native state. In contrast, receptor-bound p33KRA (fusion protein comprising the 33-kDa presequence plus 22 residues of mature protein, linked to the A chain of ricin) is quantitatively digested by protease concentrations that have little effect on the A chain in solution. We conclude that protein unfolding can take place on the chloroplast surface in the absence of translocation and without the aid of soluble factors.
Chloroplasts↗
Identification of a peptide methionine sulphoxide reductase gene in an oleosin promoter from Brassica napus.
A bidirectional promoter can be defined operationally as a short segment of DNA that regulates divergent transcription. In an attempt to investigate whether the intergenic region between the oleosin and a second open reading frame (ORFII) in Brassica napus (L.) is a divergent promoter, and also to characterize the ORFII, cDNA clones homologous to ORFII were isolated from a leaf cDNA library. A representative cDNA (clone D) of one of the two classes identified was identical, in DNA sequence, to the genomic ORFII. The second representative cDNA (clone O) was 97% identical at the nucleotide level to the genomic ORFII. The predicted amino acid sequence of the cDNA clones each exhibit homology with the peptide methionine sulphoxide reductase (PMSR) of Escherichia coli. The gene structure of ORFII was elucidated and the relative positions of the oleosin, ORFII, and the intergenic promoter region were determined. This confirms that the B. napus oleosin-ORFII intergenic region has divergent promoter activity. Consequently this is the first such plant nuclear divergent promoter identified. RFLP-mapping results showed that all four ORFII genes are linked to four of the six copies of the oleosin genes. This suggested that the bidirectional promoter locus is conserved within the B. napus genome. The ORFII gene product is targeted to the chloroplast, which is consistent with previous data indicating the presence of PMSR activity in the chloroplast. The over-expressed recombinant fusion protein (minus the transitpeptide) showed the capability to reduce peptide methionine sulphoxide residues in vitro, indicating PMSR activity. This study demonstrates that ORFII is transcribed and encodes a plant PMSR, and is the first example of the isolation of a eukaryotic PMSR gene.
Amino Acid Sequence↗
Alanine germination receptors of Bacillus subtilis.
The alanine-stimulated spore germination responses of Bacillus subtilis 168 have been dissected by combining physiological and genetical approaches. From the analyses the authors infer that there are three classes of alanine response. Two of the responses are mediated via the GerA proteins, with and without germinal adjuncts, the third is mediated via the GerB proteins and obligately requires adjuncts.
Alanine↗
Augmentation by eosinophils of gelatinase activity in the airway mucosa: comparative effects as a putative mediator of epithelial injury.
1. We have studied the release of gelatin-degrading enzymes from isolated sheets of bronchial mucosa in the presence and absence of eosinophils. 2. Isolated sheets of bovine bronchial mucosa released gelatin-degrading activity in similar amounts from both the apical and basolateral aspects of the tissue. Gelatinolytic activity could not be increased by treatment of the mucosal sheets with calcium ionophore, A23187. 3. The activity of the released gelatinases could be inhibited by chelation of divalent cations or by the matrix metalloproteinase inhibitors, BB-94 and BB-250. However, inhibitors of serine proteinases, or of cysteine proteinases were without effect. In zymography, major bands of gelatin-degrading activity consistent with gelatinases A and B were identified. 4. Addition of guinea-pig eosinophils to the basolateral aspect of bronchial mucosa for 60 min resulted in an increase in the gelatinolytic activity of the conditioned medium, irrespective of whether the eosinophils were stimulated with ionophore A23187 or not. However, only ionophore-stimulated eosinophils reacted to produce sufficient tissue damage to increase the transepithelial flux of serum albumin. 5. Purified eosinophils were a poor source of gelatinolytic activity, indicating that when interacting with the bronchial mucosa their effect is to increase the apparent release and/or activation of gelatinases derived from the airway mucosa. 6. After organomercurial activation, recombinant human progelatinase A increased the permeability of the bronchial mucosa to mannitol. However, the activity of enzyme and duration of exposure required to do this were greater than the amounts of gelatinase activity detected during eosinophil-mediated injury. Sheets of airway mucosa were also resistant to injury evoked by high concentrations of hydrogen peroxide or plasmin. 7. Collectively, these results suggest that if gelatinases are involved in eosinophil-mediated injury and repair of the bronchial mucosa, they require other mediators to act in concert to bring about outright epithelial cell detachment. This does not preclude the possibility that gelatinases are crucial in rendering the airway mucosa hyperfragile.
Animals↗
Human gingival crevicular fluid keratin at healthy, chronic gingivitis and chronic adult periodontitis sites.
The present study was designed to determine, in a cross-sectional study, whether there was any relationship between the keratin-positive material in gingival crevicular fluid and the clinical periodontal status. Keratins were selected as putative indicators of degradation of epithelial cells cytoskeletal proteins. Keratin positive material was determined by enzyme-linked immunosorbent assay in 42 subjects exhibiting clinical sites of health, chronic gingivitis and chronic periodontitis. The concentration of keratin in parotid saliva was also measured for each subject. Keratin concentration in gingival crevicular fluid samples was significantly greater at sites exhibiting signs of gingivitis and periodontitis compared with healthy sites. No differences were detected between sites exhibiting gingivitis and periodontitis. No differences were found between the 3 groups for the saliva keratin-positive material which was significantly less than that detected in gingival crevicular fluid. These results suggest that gingival crevicular fluid keratin concentration may serve as a marker of gingival damage.
Adult↗
Fluoride distribution in human dental calculus obtained from different sites on the tooth surface.
We examined the site specificity of fluoride (F) distribution in human dental calculus. Teeth with supra- and subgingival calculus were obtained from patients who resided in non-fluoridated areas in Japan and China. Sequential layers of the dental calculus (30 microns thick) were abraded by an abrasive micro-sampling technique and fluoride and phosphorus in the powdered samples were analyzed. Fluoride concentrations were highest in the outer, lowest in the middle and intermediate in the inner layers of dental calculus in general. In the outermost layers fluoride concentrations were highest in calculus found near the tooth cervix both in supra- and subgingival calculus. Fluoride concentrations decreased markedly toward the apical region in subgingival calculus, while it did not change toward the incisal or occlusal region in supragingival calculus. In the inner layers, fluoride concentrations in both supra- and subgingival calculus were not affected by position on the teeth. Fluoride concentrations in subgingival calculus near the apex were lower than in supragingival calculus near the incisal or occlusal region. It was concluded that the fluoride concentrations differ in different regions of dental calculus, probably due to their different mechanisms of formation.
Calcium Phosphates↗
Hyperleptinemia: an early sign of juvenile obesity. Relations to body fat depots and insulin concentrations.
Leptin, the OB gene product, is an adipocyte-derived circulating protein. In several rodent models of obesity, such as the db/db mice, fa/fa rats, and ventromedial hypothalamus-lesioned mice, as well as adult obese subjects, leptin mRNA expression and the circulating levels are elevated, suggesting resistance to its action. However, it is unknown whether the rise in leptin concentration occurs early in the natural evolution of human obesity or is a chronic adaptation to the obese state. Moreover, whether the distribution of body fat (i.e., visceral vs. subcutaneous abdominal fat) influences circulating leptin levels has not been assessed. We have determined in a group of obese and nonobese children and young adults whether leptin levels 1) are increased early in the development of obesity, 2) are related to a specific fat depot measured by magnetic resonance imaging, 3) vary during hyperinsulinemic, euglycemic, and hyperglycemic clamp studies, and 4) are different in males vs. females. In the basal state, leptin levels were elevated in obese children. Children and adults demonstrated a strong positive correlation between leptin concentrations and the subcutaneous fat depot (r = 0.84, P < 0.001). Surprisingly, a weaker correlation was found with visceral fat mass (r = 0.59, P = 0.001). Leptin levels remained unchanged under both euglycemic and hyperglycemic hyperinsulinemic conditions in both obese and nonobese subjects. A pronounced effect of gender on leptin levels was also observed. We conclude that, early in the development of juvenile obesity, leptin concentrations are elevated and are more closely linked to subcutaneous than visceral fat mass. Acute increases in insulin concentrations do not affect circulating leptin levels.
Adipose Tissue↗
Fluoride concentrations and distribution in premolars of children from low and optimal fluoride areas.
We have compared the fluoride (F) concentrations from the enamel surface to the dentino-enamel junction (DEJ), and through dentine to the dentino-pulpal junction (DPJ) in premolars extracted from school children in Chemnitz (former Karl-Marx-Stadt), Germany (F: 1.0 ppm in the water supply), Erfurt, Germany (F: 0.2 ppm in the water supply) and Nagoya, Japan (F: 0.1 ppm in the water supply). In teeth from children in Cheminitz, Erfurt and Nagoya, the profiles of F distribution using an abrasive microsampling technique revealed high F concentrations in the enamel surface, with a substantial decrease towards a plateau in the interior. In dentine the F concentrations were higher than in enamel, and also decreased to a plateau from the DEJ, thereafter increasing considerably towards the DPJ. F concentrations at any depth in the enamel and dentine of teeth from Chemnitz were 2-3 times higher than those in Erfurt and Nagoya. There was no significant difference in F concentrations or distributions between Erfurt and Nagoya. Close to the DEJ in both enamel and dentine as well as the enamel surface and the DPJ side of dentine, higher F concentrations were observed in Chemnitz compared with Erfurt and Nagoya.
Adolescent↗
The chemical composition of tooth enamel in recessive dystrophic epidermolysis bullosa: significance with respect to dental caries.
Previous reports have linked the prevalence of tooth abnormalities with high caries experience in the different types of epidermolysis bullosa (EB). However, it is not known to what extent the apparent susceptibility to enamel caries is due to disease-related altered enamel chemistry in these cases. The aim of this study was to characterize the enamel of teeth from patients suffering from recessive epidermolysis bullosa dystrophica (rEBD) in terms of its mineral content, carbonate content, protein content, and amino acid composition. The results showed that dental enamel from these patients was essentially normal in terms of its chemistry. It is therefore concluded that the high caries experience in recessive dystrophic epidermolysis bullosa patients is probably related to other factors, such as compromised oral hygiene and prolonged oral clearance due to extensive oral soft tissue damage and a cariogenic diet.
Amino Acids↗
Crystal growth in dental enamel: the role of amelogenins and albumin.
Amelogenin-mineral interactions were investigated using an in vitro binding approach. Rat incisor enamel matrix proteins (mainly amelogenins) were dissolved in synthetic enamel fluid and allowed to equilibrate with deproteinised developing enamel crystals. The results showed that amlogenin proteins of 21, 23, 24, 26 and 27-kDa (corresponding to nascent and partially degraded amelogenins) were associated with the crystals whilst the lower Mr amelogenins (< 21 KDa) remained free in the synthetic enamel fluid. These data suggest the nascent and partially degraded amelogenins may interact with developing enamel crystals and could influence their growth. Albumin-mineral interactions were investigated by extracting developing rat incisor enamel with synthetic enamel fluid. Insoluble material (including the enamel crystals) was then further extracted with 0.1 M phosphate buffer (pH 7.4) to desorb any mineral bound proteins. Western blotting using anti-albumin antibodies showed that almost all of the albumin from the secretory stage enamel and a significant proportion of the albumin present in early transition stage was extractable in the synthetic enamel fluid. However, synthetic enamel fluid did not extract albumin from late transition or maturation stage tissue, which could only be removed following further extraction with phosphate buffer. Albumin degradation was apparent during the transition and maturation stages, where it is degraded and ultimately removed. This binding pattern may be related to amelogenin degradation and removal during the transition stage, permitting albumin access to the previously obscured crystal surfaces. That the secretory stage matrix appears to "protect" secretory stage crystals from albumin may be an important consideration in the aetiology of enamel hypoplasias (i.e. incomplete crystal growth) and when using dissociative extraction procedures for the identification of mineral bound proteins.
Albumins↗
The rat amelogenin gene--some aspects of evolution and expression.
This study presents data to support the hypothesis that a major portion of the coding sequence of the amelogenin gene may have arisen by tandem duplication of internal sequences which as a consequence has introduced several additional potential RNA splice acceptor sites into the sequence. This duplication of splice sites has led to an increase in the heterogeneity of amelogenin forms found in developing enamel. By screening a rat enamel organ cDNA library for alternatively spliced products, it appears that as much as 20% of the amelogenin mRNA molecules may be alternatively spliced forms.
Alternative Splicing↗
Predicting the risk of bacteremia in childen with fever and neutropenia.
PURPOSE: We sought to identify factors assessable at the time of admission for fever and neutropenia that predict bacteremia in children with cancer. PATIENTS AND METHODS: One hundred fifteen consecutive episodes of fever and absolute neutrophil count (ANC) less than 500/microliter in 72 children with cancer were studied prospectively to determine the risk of bacteremia using data assessable at the time of presentation. After exploratory analysis identified admission temperature and absolute monocyte count (AMoC) as the strongest predictive factors, recursive partitioning was used to determine cutpoints for these variables that resulted in discrimination between episodes associated with a lower or higher risk of bacteremia. RESULTS: There were 24 episodes of bacteremia (21% of episodes). Episodes were grouped using the cutpoints for AMoC and temperature: 17% were classified as low risk for bacteremia (AMoC > or = 100/microliter), 65% as intermediate risk (AMoC < 100/microliter and temperature < 39.0 degrees C), and 18% as high risk (AMoC < 100/microliter and temperature > or = 39.0 degrees C). No episodes classified as low risk were associated with bacteremia; 19% of intermediate-risk and 48% of high-risk episodes were associated with bacteremia. The odds ratio of bacteremia for the high-risk versus the intermediate-risk group is 4.4 (95% confidence interval, 1.6 to 12.9). The risk classification was validated using data from 57 different episodes of fever and neutropenia treated in the same hospital. CONCLUSION: Three levels of risk for bacteremia are defined using the AMoC and temperature at the time of admission for fever and neutropenia. Trials now should be conducted to test whether these factors may be used to assign some children to less intensive or outpatient antibiotic therapy at the time of presentation with fever and neutropenia.
Adolescent↗
Sec-dependent thylakoid protein translocation. Delta pH requirement is dictated by passenger protein and ATP concentration.
A Sec-type system is responsible for the translocation of a subset of proteins across the thylakoid membrane in higher plant chloroplasts. Previous studies have suggested that the thylakoidal delta pH plays a minor role in this translocation mechanism, but we show here that it can be essential for the translocation process, depending on the identity of the passenger protein and the concentration of ATP. Studies using chimeric proteins show that, whereas the presequence dictates the translocation pathway, the delta pH requirement is dictated exclusively by the passenger protein; some passenger proteins are virtually delta pH-independent whereas others are absolutely dependent. delta pH requirement is not related to charge characteristics of the passenger proteins, ruling out an electrophoretic effect. Analysis of the 33-kDa photosystem II protein reveals an inverse relationship between delta pH requirement and ATP concentration; import into isolated thylakoids is inhibited 14-fold by nigericin at moderate ATP concentrations, and totally inhibited when the ATP concentration is reduced to 2 microM. The results indicate that the roles of the delta pH and ATP overlap and suggest that the delta pH may be obligatory when the passenger protein is abnormally difficult to translocate, possibly due to the folding of the polypeptide chain. We compare the energetics of this system with those of prokaryotic systems from which the chloroplast system is believed to have evolved.
Adenosine Triphosphate↗