Biomedical subjects

J Cotton

Publications and source records attributed to J Cotton.

At least 37 records · Page 2Linked to original sources

PubMed1997

RCN survey of gynaecology services after the NHS reforms.

This paper presents the results of a survey conducted by the Royal College of Nursing (RCN) Gynaecology Nursing Forum. In late 1994/early 1995, a questionnaire was used to collect data from qualified nurses providing gynaecology hospital services throughout the UK. The questionnaire gathered information about the extent and impact of service reconfigurations following the 1989 NHS reforms (DoH 1989). The article describes these and recommends how specialist nurses can capitalise on the changes in the best interests of their patients.

Female↗

PubMed1997

A potassium-channel toxin from the sea anemone Bunodosoma granulifera, an inhibitor for Kv1 channels. Revision of the amino acid sequence, disulfide-bridge assignment, chemical synthesis, and biological activity.

The potassium channel toxin secreted by the sea anemone Bunodosoma granulifera (BgK) is a 37-amino-acid peptide containing three disulfide bridges. Because a synthetic peptide corresponding to the reported sequence of BgK was found not to fold properly, the sequence was determined again. The new sequence differed from the previous one in the C-terminal tetrapeptide, which contains two cysteines involved in disulfide bridging. The revised sequence is: V C R D W F K E T A C R H A K S L G N C R T S Q K Y R A N C A K T C E L C. The toxin BgK was synthesized according to the new sequence and folded successfully. Disulfide bridges were assigned by peptide mapping on both natural and synthetic forms to be between Cys2-Cys37, Cys11-Cys30 and Cys20-Cys34. The toxin contains a C-terminal free carboxylate as shown by comparing the native toxin with two synthetic peptides containing the C-terminus in either the carboxylate or carboxamido form. Synthetic BgK inhibits binding of 125I-alpha-dendrotoxin to rat brain synaptosomal membranes, similarly to natural BgK (nanomolar range). No activity was observed on maxi-K+ channels incorporated into planar lipid bilayers. The ability of BgK to block voltage-dependent K+ channels was determined from recordings of whole cell currents in Xenopus oocytes injected with cRNA encoding three cloned Kv1 channels (Kv1.1, Kv1.2, Kv1.3) and one Kv3 (Kv3.1) channel. The Shaker-related Kv1 channels are equally affected by BgK, while the Shaw-related channel Kv3.1 is insensitive up to 0.125 microM toxin. Indeed, half blockage of the current through the three Kv1 channels tested occurred in the same concentration range (Kd = 6 nM for Kv1.1, 15 nM for Kv1.2, 10 nM for Kv1.3). The specificity of BgK for the Shaker-related K+ channels indicates that BgK is able to discriminate a large group of neuronal Kv1 channels in situ. The sequence, the disulfide bridge pattern, the secondary structure and the biological activity of BgK demonstrated that the sea anemone toxins, i.e. BgK, ShK and Kaliseptine, constitute novel molecular probes useful for investigating K+ channel properties.

Amino Acid Sequence↗

PubMed1997

On the convergent evolution of animal toxins. Conservation of a diad of functional residues in potassium channel-blocking toxins with unrelated structures.

BgK is a K+ channel-blocking toxin from the sea anemone Bunodosoma granulifera. It is a 37-residue protein that adopts a novel fold, as determined by NMR and modeling. An alanine-scanning-based analysis revealed the functional importance of five residues, which include a critical lysine and an aromatic residue separated by 6.6 +/- 1.0 A. The same diad is found in the three known homologous toxins from sea anemones. More strikingly, a similar functional diad is present in all K+ channel-blocking toxins from scorpions, although these toxins adopt a distinct scaffold. Moreover, the functional diads of potassium channel-blocking toxins from sea anemone and scorpions superimpose in the three-dimensional structures. Therefore, toxins that have unrelated structures but similar functions possess conserved key functional residues, organized in an identical topology, suggesting a convergent functional evolution for these small proteins.

Amino Acid Sequence↗

PubMed1997

Ultraviolet B-radiation dose influences the induction of apoptosis and p53 in human keratinocytes.

p53 is a tumor suppressor gene that has been implicated in a number of important cellular processes, including DNA repair and apoptosis. Genomic damage in human keratinocytes caused by ultraviolet B (UVB) irradiation has been shown to induce both apoptosis and p53 expression. We have previously observed that p53 expression in cultured normal human keratinocytes is predominantly perinuclear; however, exposure of cells to UVB radiation induces a major shift of p53 expression to the nucleus. Using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling, internucleosomal DNA ladders and flow cytometry, we correlated observed changes in p53 expression with the induction of apoptosis at low, intermediate and high doses of UVB radiation. High doses of UVB radiation induced cells to undergo apoptosis, whereas UVB radiation at low doses did not induce apoptosis but appeared to stimulate repair of the DNA damage induced by UVB radiation. Intermediate doses of UVB radiation induced a heterogeneous population of cells to undergo either DNA repair or apoptosis. The level of UVB radiation dose also influenced the induced cellular localization of p53. These observed differences in p53 cellular localization correlated with the induction of DNA repair or apoptosis. In cells undergoing apoptosis, p53 protein was found within the blebs of the degenerating nuclei. Our data give support to increasing evidence that p53 may play a role in both the repair of UV-radiation-induced DNA damage and the induction of apoptosis, and may function as a central control checkpoint in response to UVB-radiation-induced DNA damage.

Apoptosis↗

PubMed1996

Determination of the C-terminal form of an anemone toxin using capillary electrophoresis and mass spectrometry.

To identify the form of the C-terminal amino acid of a sea anemone toxin, the native protein was compared with two synthetic proteins comprising the same sequence and a free or an amide C-terminal form. Using electrospray ionization-mass spectrometry, capillary electrophoresis and the coupling of both techniques, we assigned the C-terminus of the native protein to be in the free carboxyl form.

Animals↗

PubMed1996

Functional-lesion investigation of developmental stuttering with positron emission tomography.

Positron emission tomographic (PET) H2(15)O measurements of resting-state regional cerebral blood flow (CBF) were obtained in 29 right-handed men, 10 of whom stuttered and 19 of whom did not. PET images were analyzed by sampling 74 regions of interest (ROIs), 37 per hemisphere. ROI placement was guided both physiologically and anatomically. Physiological ROI placement was based on speech motor activations. Anatomical ROIs were positioned by reference to a stereotactic, neurosurgical atlas with positions confirmed and finely adjusted by co-registered magnetic-resonance images (MRIs). For all subjects, PET and MR images were normal to visual inspection. Highly significant (p < 0.0001) between-region and between-hemisphere effects were found for both groups, as have been previously reported for normal subjects, but no significant between-group differences were found for any regional CBF values. Analysis by a laterality index found a weakly significant between-groups effect (p = 0.04) that was isolated to five regions, four of which are implicated in speech or hearing. However, these regional laterality effects showed no consistent directionality, nor did these regions have absolute differences in regional blood flow between groups. Present findings do not support recent suggestions that developmental stuttering is associated with abnormalities of brain blood flow at rest. Rather, our findings indicate an essentially normal functional brain terrain with a small number of minor differences in hemispheric symmetry.

Adult↗

PubMed1996

Intensive linkage mapping in a wasp (Bracon hebetor) and a mosquito (Aedes aegypti) with single-strand conformation polymorphism analysis of random amplified polymorphic DNA markers.

The use of random amplified polymorphic DNA from the polymerase chain reaction (RAPD-PCR) allows efficient construction of saturated linkage maps. However, when analyzed by agarose gel electrophoresis, most RAPD-PCR markers segregate as dominant alleles, reducing the amount of linkage information obtained. We describe the use of single strand conformation polymorphism (SSCP) analysis of RAPD markers to generate linkage maps in a haplodiploid parasitic wasp Bracon (Habrobracon) hebetor and a diploid mosquito. Aedes aegypti. RAPD-SSCP analysis revealed segregation of codominant alleles at markers that appeared to segregate as dominant (band presence/band absence) markers or appeared invariant on agarose gels. Our SSCP protocol uses silver staining to detect DNA fractionated on large thin polyacrylamide gels and reveals more polymorphic markers than agarose gel electrophoresis. In B. hebetor, 79 markers were mapped with 12 RAPD primers in six weeks; in A aygpti, 94 markers were mapped with 10 RAPD primers in five weeks. Forty-five percent of markers segregated as codominant loci in B. hebetor, while 11% segregated as codominant loci in A. aegypti. SSCP analysis of RAPD-PCR markers offers a rapid and inexpensive means of constructing intensive linkage maps of many species.

Aedes↗

PubMed1996

Histologic evaluation of preauricular and postauricular human skin after high-energy, short-pulse carbon dioxide laser.

BACKGROUND: Despite its growing use in dermatalogic surgery, the effects of high-energy, short-pulse carbon dioxide laser on human skin have not been well documented. OBJECTIVES: To study the histologic effects of this high-energy, short-pulse CO2 laser on human skin and to compare these changes with the effects of standard chemexfoliation procedures. OBSERVATIONS: Twenty-four hours after laser administration, there was extensive epidermal necrosis and coagulative change in the superficial papillary dermis. With increasing doses of laser energy, there was a statistically significant increase in the depth of dermal wounding (P<.001 for days 1 and 3, F-test). Reepithelialization occurred in most specimens by day 3. By day 90, most specimens showed a subepidermal dermal repair zone consisting of compact new collagen fibers overlying collagen with evidence of solar elastosis. CONCLUSION: This high-energy, short-pulse CO2 laser produces morphologic changes similar to those seen with medium-depth chemical peels. This laser can ablate skin precisely and bloodlessly with little interference in the wound healing process, suggesting that it may serve as an alternative treatment for photoaged skin.

Analysis of Variance↗

PubMed1995

Chemical synthesis, structural and functional characterisation of noxiustoxin, a powerful blocker of lymphocyte voltage-dependent K+ channels.

Two forms of the Centrudoides noxius scorpion noxiustoxin, containing an amidated and an acid C-terminus, were synthesized on a solid support by using Fmoc-chemistry and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) coupling. Comparison of the two synthetic forms with the native toxin by tryptic mapping and CD spectroscopy shows that noxiustoxin possesses an amidated C-terminus and the same fold as all short scorpion toxins. Patch-clamp assays on B lymphocytes demonstrate that noxiustoxin inhibits the voltage-dependent K+ channels with 2 nM affinity, but does not affect the Ca(2+)-activated K+ channels. This toxin, because of its high affinity and specificity for voltage-gated K+ channel, may provide a powerful tool in the investigation of the role(s) of these channels in the T and B lymphocyte activation and proliferation.

Amino Acid Sequence↗

PubMed1995

Immunogenicity of T epitope-containing cyclic peptides. Increasing neutralizing antibody responses by introducing fine chemical changes.

We showed previously that the disulfide-containing T peptide 24-41 C from a highly structured snake toxin elicits, in a free state, Abs that neutralize the toxin, and only a turn structure commonly exists in 24-41 C and the corresponding toxin region. To tentatively increase the neutralizing capacity of antipeptide Abs, we 1) replaced Gly-40 by an aminoisobutyric moiety (24-41 Aib), 2) substituted the half cystines 24 and 41 by penicillamine moieties (24-41 Pen), and 3) introduced an amide bond between the epsilon NH2 of Lys-27 and the gamma-COOH of Glu-38 (24-41 K-E). A solution ELISA made with antitoxin Abs revealed that 24-41 Pen is more antigenic than 24-41 Aib and 24-41 C, which are more antigenic than 24-41 K-E, suggesting that the conformation of 24-41 Pen is most closely related to the corresponding region in the native toxin. The peptides 24-41 Pen, 24-41 Aib, and 24-41 C stimulate T cells from BALB/c mice, whereas 24-41 K-E has lost this property and thereby fails to elicit Abs. Finally, anti-24-41 Pen Abs are more potent at neutralizing the native toxin than anti-24-41 C Abs, which are more potent than anti-24-41 Aib Abs. The efficacy of anti-24-41 Pen Abs was similar to that of a toxin specific mAb. Therefore, introduction of appropriate constraints makes it possible to improve the neutralizing Ab response raised by a synthetic peptide. Such observations should be of interest for the design of efficient synthetic vaccines.

Amino Acid Sequence↗

PubMed1995

Immunogenicity of a disulphide-containing neurotoxin: presentation to T-cells requires a reduction step.

It is known that production in a host of antibodies against a protein is associated with various molecular events. These include the stimulation of specific T-lymphocytes, a step that implies the processing of the protein into peptides by various endosomal/lysosomal enzymes, such as cathepsins. Strikingly, however, we observed in vitro that cathepsins B and D have no degrading effect on toxin alpha from Naja nigricollis, a curaremimetic toxin of 61 amino acids and four disulphides. In sharp contrast, the enzymes exert an efficient cleavage of the toxin polypeptide chain once the toxin disulphides are reduced. We also found that the fully reduced toxin and the native toxin were presented with comparable efficiency to two different T-hybridomas by antigen-presenting cells (APC). Together, the data suggest that presentation of toxin fragments to T-cells requires a reduction step of toxin disulphides and, in agreement with previous findings, that this step may be achieved by APC. We wish to suggest that this phenomenon may commonly occur for any toxic proteins that contain disulphides.

Amino Acid Sequence↗

PubMed1995

Probing immunogenicity of a T cell epitope by L-alanine and D-amino acid scanning.

All residues of the I-Ed restricted fragment 24-36 of a snake toxin were individually changed into L-alanine and the corresponding D-enantiomer. Four analogs substituted with L-Ala at positions 25;30, 31 and 33, and nine analogs substituted with a D-residue along the stretch 25-33 lost most (position 28) or all their capacity to stimulate a toxin-specific T hybridoma. None of these analogs stimulated splenocytes from mice immunized with the peptide 24-36. Only the L-A31 and D-W29 modified analogs could prime a T cell response which, however, showed no cross-reactivity with the native peptide, demonstrating that T cell response selectivity can be deeply modified by mutation or configuration inversion of a single residue. Our data suggest that (i) the region 25-33 is the core of the T epitope that binds to I-Ed, and (ii) Y25 R30 and R33 contribute to the peptide binding by anchoring into pockets of I-Ed. In agreement with T cell priming observations, only the L-A31 and D-W29 modified analogs elicited strong antibody responses, just like the peptide 24-36, whereas nearly all other analogs were less immunogenic. All but the L-Ala30 and L-Ala33 modified analogs were recognized by a 24-36 specific antiserum as well as the native peptide. Altogether, our results show that substitution by D-amino acid in a peptide could be particularly well-suited for either minimizing the risk of hypersensitivity or designing peptidic vaccines.

Alanine↗

PubMed1995

Flow cytometric DNA analysis of extramammary Paget's disease of the vulva.

Vulvar extramammary Paget's disease (EMPD) is an uncommon disease entity that occurs predominantly in postmenopausal white women. The clinical behavior of this neoplasm is extremely variable, reflecting the various histological patterns that have been reported with this lesion. Flow cytometry has been used as a method of obtaining prognostic information about a number of gynecological neoplasms, yet to date there have been no flow cytometric studies performed on this unusual neoplasm. We performed flow cytometric analysis of 14 cases of paraffin-embedded vulvar EMPD in patients ranging from 45 to 86 years of age. We correlated histological features and clinical recurrence risk with DNA analysis. Although we were unable to show a statistically significant correlation between DNA ploidy or S-phase and time to recurrence, we did show a statistically significant correlation between DNA ploidy and histological features. Aneuploidy appears to be associated with in situ sweat gland adenocarcinoma, invasive carcinoma, and lymphatic invasion. These results suggest that Paget's cells with aneuploid DNA stem lines may be associated with the potential for aggressive biologic behavior.

Aged↗

PubMed1994

Boc-Cys(Npys)-OH (BCNP): an appropriate reagent for the identification of T cell epitopes in cystine and/or cysteine-containing proteins.

Some T cell epitopes become inactive when their thiols are blocked with various irreversible reagents (Régnier-Vigouroux, 1988; Maillère, 1992; Maillère et al., 1993). Blocking protein and peptide thiols with BCNP (Boc-Cys(Npys)-OH) constitutes a most appropriate strategy when searching for thiol-containing T cell epitopes. Free cysteines can thus be readily transformed into disulphide-like moieties which not only resist undesirable oxidative reactions but which also remain susceptible to reduction by antigen presenting cells, a prerequisite for the activity of thiol-dependent T cell epitopes. We describe the use of this reagent in a study of the intact disulphide-rich protein, toxin alpha from Naja nigricollis, and also two disulphide-containing toxin fragments.

Amino Acid Sequence↗

PubMed1994

Interaction of protein ligands with receptor fragments. On the residues of curaremimetic toxins that recognize fragments 128-142 and 185-199 of the alpha-subunit of the nicotinic acetylcholine receptor.

Using a solid-phase assay, we found that 3H-labeled alpha Cobtx from Naja naja siamensis, a long-chain curaremimetic toxin, and 3H-labelled toxin alpha from Naja nigricollis, a short-chain toxin both bind specifically but with substantially different affinities (Kd = 4 x 10(-7) M and 50 x 10(-6) M) to fragment 185-199 (T alpha 185-199) of the alpha-subunit of the acetylcholine receptor (AcChoR) from Torpedo marmorata. Then we show that monoderivatizations of residues common to both long-chain and short-chain toxins (Tyr-25, Lys-27, Trp-29, and Lys-53) or to long-chain toxins only (Cys-30 and Cys-34) do not affect the binding of the toxins to T alpha 185-199, suggesting that none of these invariant residues in implicated in the recognition of this AcChoR region. alpha Cobtx and toxin alpha bind to the fragment 128-142 (T alpha 128-142) with more similar affinities (Kd = 3 x 10(-7) M and 1.4 x 10(-6) M) and their binding is dramatically affected by the single abolition of the positive charge of Lys-53, an invariant residue that contributes to AcChoR recognition. Therefore, the data indicate that Lys-53 more specifically recognizes the 128-142 region of AcChoR. Other monoderivatizations have no effect on toxin binding. The approach described in this paper may be of great help to identify toxin residues that establish direct contact with receptor fragments.

Amino Acid Sequence↗

PubMed1994

Goodpasture's syndrome in childhood: treatment with plasmapheresis and immunosuppression.

Goodpasture's syndrome rarely affects children. Therefore, we present our experience in a young boy whose pulmonary hemorrhage was dramatically resolved by three plasma exchanges. We believe the hemorrhage was caused primarily by acute capillaritis. He received cytoxan and steroids and a series of plasma exchanges which removed/suppressed his anti-glomerular basement membrane (anti-GBM) antibody production. However, after a year, his renal function did not return, and he required renal transplantation and continues to do well.

Anti-Glomerular Basement Membrane Disease↗

PubMed1994

Terminal deoxynucleotidyl transferase staining in acute leukemia and normal bone marrow in routinely processed paraffin sections.

Terminal deoxynucleotidyl transferase (TdT) is a nuclear protein widely used as a marker for the diagnosis and classification of acute leukemia. The usual methods for detecting TdT require smears, imprints, or cryostat sections of unfixed tissue. A polyclonal rabbit anti-TdT serum was used to immunostain 54 routinely processed bone marrow sections from patients with acute leukemic disorders, using a recently described antigen-unmasking technique based on microwave oven heating. The specificity of this method of TdT analysis was confirmed by comparing the results obtained with conventional TdT analysis by indirect immunofluorescence. Terminal deoxynucleotidyl transferase reactivity was also evaluated in 44 nonmalignant and normal bone marrow specimens. All cases that were TdT-positive by immunofluorescence (41 of 42 "pre-B" and T-cell acute lymphoblastic leukemia, 2 of 5 acute myeloid leukemia, and 1 of 5 chronic myeloid leukemia in blast crisis) were also positive in paraffin sections. The percentage fluorescence positivity correlated with the percentage of immunoperoxidase stained cells in 44 of 45 cases. The remaining nonneoplastic and normal bone marrow biopsy specimens were TdT-negative. These results show that TdT immunoperoxidase staining of conventionally processed bone marrow specimens can be readily achieved by the use of a simple antigen-unmasking technique and may provide useful diagnostic information particularly in cases in which fresh tissue samples are unavailable.

Acute Disease↗

PubMed1993

Role of thiols in the presentation of a snake toxin to murine T cells.

We isolated and characterized two T hybridomas specific for a highly stable snake toxic protein. One hybridoma, called T1C9, is I-E(d)-restricted and stimulated by both the native and reduced and carboxymethylated (RCM) toxins and by synthetic fragments containing the region 24-36. The other hybridoma, called T1B2, is I-A(d)-restricted and stimulated by the native toxin, only. Neither the RCM toxin nor any of the initial synthetic peptides used in our study could stimulate it. We show that this lack of effect is associated with the presence, in the epitope-containing fragment, of irreversible blocking groups on cysteine residues. Indeed, when the fragment 32-49 has its cysteines involved in either intra-(32-49SS) or mixed disulfides, a stimulation of T1B2 was observed. Fixed APC do not present native toxin to either hybridomas but present RCM toxin to T1C9. Strikingly, fixed APC present the peptide 32-49SS to T1B2; however, we show that this is possible only because the peptide disulfide is reduced. The thiol dependence of this epitope suggests that the native toxin can stimulate T1B2 only after disulfide reduction. This reaction may constitute a major step during the processing of the toxin and more generally of any disulfide-containing Ag.

Amino Acid Sequence↗