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Biomedical subjects

J Green

Publications and source records attributed to J Green.

At least 307 records · Page 17Linked to original sources
PubMed1995

Solution structure of the C-terminal SH2 domain of the human tyrosine kinase Syk complexed with a phosphotyrosine pentapeptide.

BACKGROUND: Recruitment of the intracellular tyrosine kinase Syk to activated immune-response receptors is a critical early step in intracellular signaling. In mast cells, Syk specifically associates with doubly phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) that are found within the IgE receptor. The mechanism by which Syk recognizes these motifs is not fully understood. Both Syk SH2 (Src homology 2) domains are required for high-affinity binding to these motifs, but the C-terminal SH2 domain (Syk-C) can function independently and can bind, in isolation, to the tyrosine-phosphorylated IgE receptor in vitro. In order to improve understanding of the cellular function of Syk, we have determined the solution structure of Syk-C complexed with a phosphotyrosine peptide derived from the gamma subunit of the IgE receptor. RESULTS: The Syk-C:peptide structure is compared with liganded structures of both the SH2 domain of Src and the C-terminal SH2 domain of ZAP-70 (the 70 kDa zeta-associated protein). The topologies of these domains are similar, although significant differences occur in the loop regions. In the Syk-C structure, the phosphotyrosine and leucine residues of the peptide ligand interact with pockets on the protein, and the intervening residues are extended. CONCLUSIONS: Syk-C resembles other SH2 domains in its peptide-binding interactions and overall topology, a result that is consistent with its ability to function as an independent SH2 domain in vitro. This result suggests that Syk-C plays a unique role in the intact Syk protein. The determinants of the binding affinity and selectivity of Syk-C may reside in the least-conserved structural elements that comprise the phosphotyrosine- and leucine-binding sites. These structural features can be exploited for the design of Syk-selective SH2 antagonists for the treatment of allergic disorders and asthma.

Amino Acid Sequence↗
PubMed1995

Molecular basis for interaction of the protein tyrosine kinase ZAP-70 with the T-cell receptor.

The crystal structure of the tandem SH2 domains of human ZAP-70 in complex with a peptide derived from the zeta-subunit of the T-cell receptor reveals an unanticipated interaction between the two domains. A coiled coil of alpha-helices connects the two SH2 domains, producing an interface that constitutes one of the two critical phosphotyrosine binding sites. These and other unique features provide the molecular basis for highly selective association of ZAP-70 with the T-cell receptor.

Amino Acid Sequence↗
PubMed1995

Primary care in the accident and emergency department: I. Prospective identification of patients.

OBJECTIVE: To compare patient characteristics and consultation activities for attenders at accident and emergency departments assessed by nurse triage as presenting with "primary care" or "accident and emergency" type problems. DESIGN: One year prospective study. SETTING: A busy, inner city accident and emergency department in south London. SUBJECTS: Of the 5658 patients treated for new problems during a stratified random sample of 204 three hour sessions between 10 am and 9 pm during June 1989 to May 1990, all "primary care" (2065 patients) and a 10% random sample of "accident and emergency" (291 patients) were included in the analysis. MAIN OUTCOME MEASURES: Patient's age, sex, duration of presenting problem, diagnosis, laboratory and radiographic investigations, treatments, and referrals. RESULTS: 40.9% of attenders with new problems were classified by triage as presenting with "primary care" problems (95% confidence interval 39.6% to 42.2%). Primary care attenders were more likely than accident and emergency patients to be young adults, to have symptoms with a duration of longer than 24 hours, and to present problems not related to injury (all P < 0.001). Accident and emergency patients were considerably more likely to be referred to on call teams and to be admitted. Even so, 9.7% of primary care patients were referred to on call teams and a further 8.9% were referred to the fracture clinic or advised to return to the accident and emergency department for follow up. CONCLUSION: Accident and emergency triage can be developed to identify patients with problems that are more likely to be of a primary care type, and these patients are less likely to receive an investigation, minor surgical procedure, or referral. Many patients in this category, however, receive interventions likely to support their decision to attend accident and emergency rather than general practice. This may reflect limitations in the sensitivity of triage practice or a clinical approach of junior medical staff that includes a propensity to intervene.

Adolescent↗
PubMed1995

Primary care in the accident and emergency department: II. Comparison of general practitioners and hospital doctors.

OBJECTIVE: To compare the process and outcome of "primary care" consultations undertaken by senior house officers, registrars, and general practitioners in an accident and emergency department. DESIGN: Prospective, controlled intervention study. SETTING: A busy, inner city accident and emergency department in south London. SUBJECTS: Patients treated during a stratified random sample of 419 three hour sessions between June 1989 and May 1990 assessed at nurse triage as presenting with problems that could be treated in a primary care setting. 1702 of these patients were treated by sessionally employed local general practitioners, 2382 by senior house officers, and 557 by registrars. MAIN OUTCOME MEASURES: Process variables: laboratory and radiographic investigations, prescriptions, and referrals; outcome variables: results of investigations. RESULTS: Primary care consultations made by accident and emergency medical staff resulted in greater utilisation of investigative, outpatient, and specialist services than those made by general practitioners. For example, the odds ratios for patients receiving radiography were 2.78 (95% confidence interval 2.32 to 3.34) for senior house officer v general practitioner consultations and 2.37 (1.84 to 3.06) for registrars v general practitioners. For referral to hospital specialist on call teams or outpatient departments v discharge to the community the odds ratios were 2.88 (2.39 to 3.47) for senior house officers v general practitioners and 2.57 (1.98 to 3.35) for registrars v general practitioners. CONCLUSION: Employing general practitioners in accident and emergency departments to manage patients with primary care needs seems to result in reduced rates of investigations, prescriptions, and referrals. This suggests important benefits in terms of resource utilisation, but the impact on patient outcome and satisfaction needs to be considered further.

Adolescent↗
PubMed1995

Purification and properties of the lipoate protein ligase of Escherichia coli.

Lipoate is an essential component of the 2-oxoacid dehydrogenase complexes and the glycine-cleavage system of Escherichia coli. It is attached to specific lysine residues in the lipoyl domains of the E2p (lipoate acetyltransferase) subunit of the pyruvate dehydrogenase complex by a Mg(2+)- and ATP-dependent lipoate protein ligase (LPL). LPL was purified from wild-type E. coli, where its abundance is extremely low (< 10 molecules per cell) and from a genetically amplified source. The purified enzyme is a monomeric protein (M(r) 38,000) which forms irregular clusters of needle-like crystals. It is stable at -20 degrees C, but slowly oxidizes to an inactive form containing at least one intramolecular disulphide bond at 4 degrees C. The inactive form could be re-activated by reducing agents or by an as-yet unidentified component (reactivation factor) which is resolved from LPL at the final stage of purification. The pI is 5.80, and the Km values for ATP, Mg2+ and DL-lipoate were determined. Selenolipoate and 6-thio-octanoate were alternative but poorer substrates. Lipoylation was reversibly inhibited by the 6- and 8-seleno-octanoates and 8-thio-octanoate, which reacted with the six cysteine thiol groups of LPL. LPL was inactivated by Cu2+ ions in a process that involved the formation of inter- and intra-molecular disulphide bonds. Studies with lplA mutants lacking LPL activity indicated that E. coli possesses another distinct lipoylation system, although no such activity could be detected in vitro.

Amino Acid Sequence↗
PubMed1995

The gene encoding bovine pregnancy-associated glycoprotein-1, an inactive member of the aspartic proteinase family.

Bovine pregnancy-associated glycoprotein 1 (bPAG1) is a member of the aspartic proteinase family. It becomes detectable in maternal serum soon after implantation and is produced specifically in the invasive binucleate cells of the placenta. As a result of a key mutation within its catalytic center, bPAG1 appears to be proteolytically inactive. Its gene consists of nine exons (size range 99-281 bp) and eight introns (87-1800 bp) organized in a manner very similar to those of proteolytically active mammalian aspartic proteinases. The transcription start point (tsp) is located 53 or 54 bp upstream from the start codon (ATG) and 19 bp downstream from a 5'-TATATAA sequence. Southern blot analyses have indicated the presence of two bPAG1 genes. By screening with an antiserum raised against bPAG1, a less common cDNA with 91% sequence identity to the bPAG1 transcript has been isolated from a placental cDNA library and presumably represents the second gene. At least eight other genes with sequences that hybridize relatively weakly to the bPAG1 probe are present in the bovine genome. Despite the similarities in the transcribed portion of the genes encoding PAG1, pepsinogen and other mammalian aspartic proteinases, the sequences upstream from the tsp of bPAG1 are unique.

Amino Acid Sequence↗
PubMed1995

Measurement of the binding of tyrosyl phosphopeptides to SH2 domains: a reappraisal.

Src homology 2 (SH2) domain-mediated interactions with phosphotyrosine residues are critical in many intracellular signal transduction pathways. Attempts to understand the determinants of specificity and selectivity of these interactions have prompted many binding studies that have used several techniques. Some discrepancies, in both the absolute and relative values of the dissociation constants for particular interactions, are apparent. To establish the correct dissociation constants and to understand the origin of these differences, we have analyzed three previously determined interactions using the techniques of surface plasmon resonance and isothermal titration calorimetry. We find that the binding of SH2 domains to phosphopeptides is weaker than generally presumed. A phosphopeptide based on the hamster polyoma middle tumor antigen interacts with the SH2 domain from Src with an equilibrium dissociation constant (Kd) of 600 nM; a phosphopeptide based on one binding site from the platelet-derived growth factor receptor binds to the N-terminal SH2 domain of the 1-phosphatidylinositol 3-kinase p85 subunit with a Kd of 300 nM; and a phosphopeptide based on the C terminus of Lck binds to the SH2 domain of Lck with a Kd of 4 microM. In addition, we demonstrate that avidity effects that result from the dimerization of glutathione S-transferase fusion proteins with SH2 domains could be responsible for overestimates of affinities for these interactions previously studied by surface plasmon resonance.

Amino Acid Sequence↗
PubMed1995

Detection and characterisation of bisegmented double-stranded RNA viruses (picobirnaviruses) in human faecal specimens.

The prevalence of picobirnaviruses (PBVs) in human stools was investigated by polyacrylamide gel electrophoresis (PAGE) analysis of 832 faecal specimens collected between 1982 and 1993 from patients in various clinical groups. Similar prevalences (9-13%) were detected in patients with or without gastroenteritis and throughout the age range of 3 to > 65 years. Two methods for the extraction of nucleic acid, a phenol/chloroform method and a guanidinium thiocynate (GTC)/silica method, were compared. Detection of PBVs by PAGE was three times more sensitive following RNA extraction by the GTC/silica method. Characterisation of three strains was carried out. Segment sizes ranged from 1.625 to 1.95 kilo base pairs (Kbp) and 2.2 to 2.5 Kbp for the fast and slow migrating bands, respectively. The nuclic acid was shown to be double-stranded RNA (dsRNA) by nuclease digestion. PBV-like particles were detected by electron microscopy in two PAGE-positive stools. Virion diameters ranged from 35 to 41 nm and a buoyant density of 1.38-1.4 g/ml in caesium chloride (CsCl) was demonstrated. These findings suggest that PBVs are widespread in humans in the United Kingdom. However, no disease association could be demonstrated.

Adolescent↗
PubMed1995

Broadly reactive reverse transcriptase polymerase chain reaction for the diagnosis of SRSV-associated gastroenteritis.

A limitation to date of reverse transcriptase polymerase chain reactions (RT-PCRs) for the detection of small, round structured viruses (SRSVs) has been that they have detected only a narrow range of SRSVs due to the marked genomic diversity among strains. A total of 331 faecal samples collected from 136 separate incidents of gastroenteritis occurring in the UK between 1992 and 1994 were examined by RT-PCR employing a single primer pair (N1/E3). SRSV RNA was detected in samples from 93 of 101 (91%) incidents shown to be SRSV-associated by electron microscopy (EM) and in 5 of 35 (14%) SRSV-negative incidents. Amplification products were tested by Southern blot hybridisation with a pool of four digoxigenin (DIG)-labelled oligonucleotides derived from genomic sequence data of SRSV SPIEM types UK 1 to 4. Products from approximately 5% of amplified strains did not hybridise. The N1/E3 primer pair were shown to be SRSV-specific by their failure to amplify other faecal viruses including other human caliciviruses with typical calicivirus morphology. Hybridisation of PCR products with the individual oligonucleotides relating to SRSV SPIEM types UK 1-4 was investigated: 1 of 60 (1.7%) reacted with the UK1 probe, 2/60 (3.4%) reacted with the UK2 probe, 51/60 (85%) with the UK3 probe, and 27/60 (45%) reacted with the UK4 probe. All PCR products that hybridised with the UK4 probe hybridised with the UK3 probe; 6 (10%) failed to hybridise. Identification of this primer pair facilitates routine diagnosis of SRSV infection by RT-PCR and offers the potential for direct detection in food and environmental samples.

Animals↗
PubMed1995

Production of interleukin-6 by osteoblastic cells is independent of medium inorganic phosphate.

A state of severe bone loss is often observed in patients and animals suffering from phosphate (Pi) depletion. Conversely, Pi surfeit may have an anabolic effect on bone and may antagonize bone resorption. To study whether Pi has a direct effect on the production of the bone-resorbing interleukin-6 (IL-6) by osteoblasts, we cultured MC3T3-E1, UMR-106, and isolated rat calvaria cells in media containing varying concentrations of Pi (0-3 mM) and measured the production of IL-6 released into the media. IL-6 production was steady with time and was stimulated by parathyroid hormone, 1,25-dihydroxyvitamin D3, and interleukin-1 alpha. However, IL-6 production did not change with varying Pi concentrations. We concluded that the IL-6 production by osteoblastic cells is independent of the medium Pi.

Animals↗
PubMed1995

Glycoproteins of the aspartyl proteinase gene family secreted by the developing placenta.

Pregnancy in cattle and sheep can be diagnosed by the presence of placentally-derived antigens (pregnancy-associated glycoproteins or PAG-1) in maternal serum soon after implantation begins at about Day 20 following conception. Molecular cloning of their cDNA has revealed that PAG-1 belong to the aspartic proteinase gene family and have about 50% amino acid sequence identity to pepsin. However, critical amino acid substitutions at the active site regions suggest that both bovine and ovine PAG-1 are enzymatically inactive. PAG-1 expression has been shown by in situ hybridization and immunocytochemistry to be localized to the trophoblast binucleate cells, which invade maternal uterine endometrium during implantation. The glycoproteins are concentrated in dense cytoplasmic granules that are discharged after the binucleate cells have migrated to the maternal side of the placental barrier. We suggest, therefore, that the PAG-1 might have an endocrine function either as carriers of other bioactive peptides or by acting as hormones themselves. Recently screening of placental libraries with nucleic acid probes has identified additional cDNA that are very abundant and code for polypeptides (PAG-2 and PAG-3) related to, but antigenically and structurally distinct from PAG-1 described above. These molecules have sequences of amino acids at their catalytic centers that are consistent with their being potentially functional proteinases but their role during pregnancy, like that of PAG-1, is unclear.

Amino Acid Sequence↗
PubMed1995

Evolution of spinal bone loss and biochemical markers of bone remodeling after menopause in normal women.

The main objective of this study was to describe longitudinal patterns of spinal bone loss in normal women who undergo a natural menopause. The second objective was to determine if a proportion of women suffer excessively rapid postmenopausal bone loss from the spine. If this was the case it was the aim to devise a means of predicting the woman at excess risk; but if all women lost bone at similar rates, the aim was to document changing loss rates over the first 5-8 postmenopausal years. Responding women in six suburban general practices recalled for cervical smears who had their last menstrual period 9- 36 months previously were invited to participate in a longitudinal study of bone loss and the biochemical markers plasma osteocalcin and urinary hydroxyproline. Sixty-four subjects agreed to participate, a response rate of 80%. In the ensuing 5 years, six received hormone replacement therapy and are not reported on. The main outcome measures were rates of spinal bone loss over 5 years, measured by dual photon absorptiometry, and radial bone loss over the first 2 years measured to quantitative computed tomography. Spinal bone loss was similar between individuals, with 94% of the variability in the data being accounted for by a statistical model that assumed parallel rates of bone loss. A less restrictive model allowing women to have different rates of spinal bone loss accounted for 12% more of the remaining variance in the data than the previous model. However, rates of radial bone loss were more dissimilar between women than rates of spinal loss. The results of the biochemical data collected serially showed that the plasma osteocalcin rose slowly to a plateau at 5 years postmenopause; in contrast, the hydroxyproline fell progressively with time over the whole period of study. These results were interpreted as being consistent with diminishing rates of bone destruction which gradually reequilibrated with bone formation as time passed after menopause.

Absorptiometry, Photon↗
PubMed1995

Detection of a picobirnavirus associated with Cryptosporidium positive stools from humans.

A picobirnavirus with an atypical genome profile was detected by polyacrylamide gel electrophoresis (PAGE) in 37% (20/54) of human faecal samples also containing oocysts of Cryptosporidium typical of C. parvum. This virus shares many of the characteristics of the previously described picobirnaviruses, but has a significantly smaller genome (1.75 and 1.55 Kbp).

Animals↗
PubMed1995

The hemifusion intermediate and its conversion to complete fusion: regulation by membrane composition.

To fuse, membranes must bend. The energy of each lipid monolayer with respect to bending is minimized at the spontaneous curvature of the monolayer. Two lipids known to promote opposite spontaneous curvatures, lysophosphatidylcholine and arachidonic acid, were added to different sides of planar phospholipid membranes. Lysophosphatidylcholine added to the contacting monolayers of fusing membranes inhibited the hemifusion we observed between lipid vesicles and planar membranes. In contrast, fusion pore formation depended upon the distal monolayer of the planar membrane; lysophosphatidylcholine promoted and arachidonic acid inhibited. Thus, the intermediates of hemifusion and fusion pores in phospholipid membranes involve different membrane monolayers and may have opposite net curvatures, Biological fusion may proceed through similar intermediates.

Arachidonic Acid↗
PubMed1995

Cat shedding of Fel d I is not reduced by washings, Allerpet-C spray, or acepromazine.

BACKGROUND: No published studies have compared the effectiveness of several treatments proposed to reduce cat allergenicity. Cat washing studies demonstrating efficacy involved very small sample sizes or infrequent washings. Allerpet-C (Allerpet, Inc., New York, N.Y.), a widely advertised topical spray, and acepromazine, a tranquilizer advocated as efficacious in subsedating doses, have never been scientifically studied. OBJECTIVE: We compared the effects of cat washing, Allerpet-C spray, and acepromazine with that of no treatment on the shedding of the primary cat allergen, Felis domesticus I by cats. METHODS: In a blinded, comparative, controlled study, we measured the amounts of Fel d I shed during an 8-week treatment period with a sample of 24 female mongrel cats randomly assigned to four groups; one group received weekly distilled water washings, one received weekly Allerpet-C spray applications, one received daily oral acepromazine, and one had no treatment (control). Thirty-minute, twice-weekly air samples were collected from each cat with a laminated plastic-acrylic chamber and air sampler. RESULTS: One-sample, two-sided t tests comparing baseline to final-week measurements revealed no significant change in Fel d I within each group (mean change +/- SD: washing; 487.6 +/- 1896.4 mU per 30 minutes, p = 0.63; Allerpet-C spray, 429.2 +/- 871.6 mU per 30 minutes, p = 0.46 acepromazine; -620.6 +/- 1031.2, p = 0.52 per 30 minutes). Furthermore, analysis of covariance revealed no significant change in Fel d I levels between groups (p = 0.72). CONCLUSIONS: Out data do not show significant reductions in Fel d I shedding as a result of any of these treatments. Therefore we cannot recommend them to patients allergic to cats.

Acepromazine↗