Successful stenting in endobronchial Wegener's granulomatosis.
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Adult↗
Biomedical subjects
C Shah
Publications and source records attributed to C Shah.
Ontogeny and cellular localization of SRY transcripts in the human testes and its detection in spermatozoa.
The sex-determining region on the Y (SRY) gene is unequivocally designated as the testis-determining factor in mammals; however, its roles beyond sex determination, if any, have been hitherto unknown. To determine whether SRY has any roles beyond sex determination, herein the expression of SRY mRNA was investigated in the midtrimester human fetal, infantile and adult testes as well as in ejaculated spermatozoa. High levels of SRY transcripts were in situ localized to the Sertoli cells of the developing testis at 9 weeks of gestation, and the expression persisted at comparable levels throughout the midtrimester (until 22 weeks) and also in the testis of an infant at 3 months of age. The germ cells and other somatic cells in the testes of fetuses and the infant were negative for SRY expression. The mRNA for SRY was detected in the spermatogenic cells, particularly the spermatogonia and the round spermatids; the expression was negligible in the meiotic stages. A single transcript of approximately 1.2 kb was detected in the adult testes and isolated spermatogonial cells. In the adult testis, in situ hybridization (ISH) studies revealed a switch in the cellular localization of SRY transcripts. SRY transcripts were also demonstrable by RT-PCR of RNA from ejaculated human spermatozoa. ISH revealed the presence of SRY transcripts in the midpiece of 50% of ejaculated sperm. These results suggest that SRY may have extensive roles in male reproductive physiology, such as maturation of fetal testis, spermatogenesis, sperm maturation and early embryonic development.
Adolescent↗
High-throughput production of Pyrococcus furiosus proteins: considerations for metalloproteins.
Free-living prokaryotic organisms contain all of the proteins required for the basic biochemical processes of life. As part of the Southeastern Collaboratory for Structural Genomics (SECSG), Pyrococcus furiosus is being used as a model system for developing a high-throughput protein expression and purification protocol. Its 1.9 million basepair genome encodes approximately 2200 putative proteins, less than 25% of which show similarity to any structurally characterized protein in the Protein Data Bank. The overall goal of the structural genomics initiative is to determine, in total, all existing protein folds. The immediate objective of this work is to obtain recombinant forms of all P. furiosus proteins in their functional states for structural determination. Proteins successfully produced by overexpression in another organism such as the bacterium Escherichia coli typically contain a single subunit, are soluble and do not contain (complex) cofactors. Analyses of the P. furiosus genome suggest that perhaps only a quarter of the genes encode proteins that would fall into this category. The hypothesis is that lack of the appropriate cofactor or of the partner protein(s) necessary to form a complex are major reasons why many recombinant proteins are insoluble. This work describes development of the production pipeline with attention to prediction and incorporation of cofactors.
Cloning, Molecular↗
Properties of microtubules assembled from mammalian tubulin synthesized in Escherichia coli.
When isolated from tissues, the alpha beta-dimeric protein tubulin consists of multiple isoforms which originate from the expression and subsequent posttranslational modification of multiple polypeptide sequences. Microtubules studied in vitro consist of mixtures of these isoforms. It is therefore not known whether dimers composed of single sequences of alpha- and beta-tubulin can polymerize to form microtubules, or whether posttranslational modifications may be necessary for microtubule assembly. To initiate investigation of these questions, rabbit reticulocyte lysate, which contains the cytoplasmic chaperonin CCT and its cofactors, was employed to prepare substantial quantities (tens of micrograms) of active tubulin by in vitro folding of mouse alpha- and beta-tubulins recombinantly synthesized in E. coli. This recombinant tubulin is composed of only a single alpha-chain and a single beta-chain. When analyzed after folding by isoelectric focusing, each chain yielded only one band, indicating that neither was detectably posttranslationally modified in the course of the folding reaction. When subjected to assembly-promoting conditions, this tubulin formed microtubules without the addition of any exogenous protein. Electron microscopy showed them to be of normal morphology. Analysis of their protein composition showed that they are composed nearly entirely of recombinant tubulin. These results demonstrate that the naturally occurring mixtures of isoforms are not strictly required for the formation of microtubules. They also open a route to other studies, both biomedical and structural, of fully defined tubulin in vitro.
Animals↗
Functional IL-2 receptor beta (CD122) and gamma (CD132) chains are expressed by fibroblast-like synoviocytes: activation by IL-2 stimulates monocyte chemoattractant protein-1 production.
The expression of the IL-2R alpha-, beta-, and gamma-chains, CD25, CD122, and CD132, respectively, was investigated on fibroblast-like synoviocytes (FLS) and dermal fibroblasts (DF). Both protein and mRNA for CD122 and CD132 were observed but there was no evidence of CD25 expression. Quantification of the Ag binding sites for CD122 showed that FLS expressed 4 times more receptor molecules than DF. The functional capability of these receptors was confirmed by the production of monocyte chemoattractant protein-1 (MCP-1) in direct response to stimulation by IL-2, which could be inhibited by neutralizing anti-CD122 mAb. Both rheumatoid arthritis (RA) and osteoarthritis (OA) FLS and DF spontaneously produced MCP-1 in culture over a similar range of concentrations. However, RA and OA FLS produced significantly greater levels of MCP-1 following stimulation by IL-2 and IL-1 beta; RA FLS produced significantly more MCP-1 than OA FLS. Addition of exogenous IL-2 caused a slight, but significant, decrease in MCP-1 production by DF. The addition of neutralizing anti-CD122 mAb to FLS cultures partially, but significantly, reduced the IL-2-induced MCP-1 secretion, but did not effect either the spontaneous or IL-1 beta-induced secretion of MCP-1. Increased tyrosine phosphorylation was observed in FLS lysates following 30-min incubation with IL-2. In conclusion, in the inflamed synovium, as activated T cells migrate through the sublining and lining layer, T cell-derived IL-2 may activate FLS to secrete MCP-1, thus recruiting macrophages into the rheumatoid synovium and perpetuating inflammation.
Antibodies, Blocking↗
The use of fibrin glue in refractory coagulopathic epistaxis.
Epistaxis can be particularly difficult to treat when a patient has a coagulopathy, even if the area bleeding is visible. Where routine measures fail to arrest the bleeding (e.g., nasal cautery and packing), the application of fibrin glue is successful in stopping the bleeding without having to address the underlying coagulopathy. Ten patients with epistaxis secondary to an underlying coagulopathy were treated by local application of fibrin glue to the area bleeding. The bleeding stopped immediately in all patients when the fibrin glue was applied. No patients suffered complications. Fibrin glue application should be considered as a cost-effective means of controlling epistaxis from an identifiable area in the nasal cavity in patients with a coagulopathy.
Cost-Benefit Analysis↗
Role of a home care team in paediatric day-case tonsillectomy.
The feasibility of paediatric day-case tonsillectomy (PDCT) depends on its safety and acceptance by parents and patients. The purpose of our retrospective study of paediatric day-case tonsillectomy was to review the role of the home care team (HCT) in improving the safety and acceptance of the procedure. Between January 1997 and June 1999, 352 consecutive children underwent day-case tonsillectomy. The notes and HCT assessment sheets were reviewed for telephone calls made by HCT or by parents, home visit by HCT, types of complication and their outcome. The primary haemorrhage rate was 0.6 per cent. The effective day-case rate was 97 per cent. The unplanned admission rate was three per cent. The HCT visited about 25 per cent of patients at home. We conclude that paediatric day-case tonsillectomy is associated with high morbidity and considerable parental anxiety that can be dealt with by timely reassurance, support and advice by a dedicated HCT.
Adenoidectomy↗
Extramedullary plasmacytoma of the submandibular gland.
Extramedullary plasmacytoma in the submandibular region is rare and on initial evaluation must be distinguished from multiple myeloma. The diagnostic evaluation includes appropriate radiological and pathological studies including immunohistochemistry. We report a case of extramedullary plasmacytoma in the submandibular gland. A review of the literature suggests that it has a good prognosis, if multiple myeloma is excluded. This patient was treated by surgical excision followed by radiotherapy.
Adult↗
Prefrontal regions play a predominant role in imposing an attentional 'set': evidence from fMRI.
fMRI was used to determine whether prefrontal regions play a predominant role in imposing an attentional 'set' that drives selection of task-relevant information. While monitoring for an atypical item, individuals viewed Stroop stimuli that were either colored words or colored objects. Attentional demands were varied, being greater when the stimuli contained two distinct and incongruent sources of information about the task-relevant attribute (e.g., when attending to color, seeing the word 'blue' in red ink) as compared to only one source (e.g., seeing the word 'late' in red ink). Prefrontal but not anterior cingulate regions exhibited greater activation on incongruent than neutral trials, suggesting that prefrontal cortex has a major role in imposing an attentional 'set'. In addition, we found that prefrontal activation is most likely to occur when that attentional set is difficult to impose.
Attention↗
Separation of tubulin isoforms by isoelectric focusing in immobilized pH gradient gels.
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Animals↗
Complement receptor 1 red cell expression is not controlled by the In(Lu) gene.
BACKGROUND: The In(Lu) gene reportedly suppresses several blood group antigens that are not part of the Lutheran system, including the high-incidence antigens of the Knops blood group system. Because complement receptor 1 (CR1), which is known to carry the Knops system antigens, has a red cell (RBC) expression polymorphism, the role of In(Lu) in the expression of the Knops system antigens was reinvestigated. STUDY DESIGN AND METHODS: Blood samples from nine donors having the Lu(a-b-) phenotype were obtained and immediately phenotyped for Lu(b), Kn(a), McC(a), Sl(a), and Yk(a). The samples were also tested for Lu(a), P1, and AnWj. Immunoblots were performed to study both the CR1 and Lutheran glycoproteins from these donors. RBC expression of CR1 was quantified with an enzyme-linked immunosorbent assay, and the genetic inheritance of the high-expression (H) or low-expression (L) allele for CR1 was determined by Southern blot. RESULTS: Lu(b) was demonstrable only by absorption and elution techniques on all nine samples; however, the high-incidence Knops system antigens were readily detectable by hemagglutination. Two Lu(a-b-) donors (sibs) demonstrated weak Lutheran glycoprotein bands of 78 and 85 kDa on immunoblots, while the other seven Lu(a-b-) samples had no detectable glycoprotein. All donors had CR1*1, and one donor also had CR1*2 on immunoblot. Only one donor was homozygous for the L allele, and all had RBC copy numbers of CR1 within the normal range. CONCLUSIONS: Nine donors with the Lu(a-b-) phenotype showed suppression of the Lutheran system antigens but normal expression of CR1 glycoprotein and the Knops system blood group antigens. This suggests that the genes that suppress Lutheran system antigens do not suppress CR1 or its related blood group antigens.
Alleles↗
Correlation of orbital computed tomographic findings with office probing and irrigation in 17 patients after successful and failed dacryocystorhinostomy.
PURPOSE: To investigate the role of orbital computed tomography (CT) in the evaluation of patients after dacryocystorhinostomy (DCR). METHODS: Computed tomography scans of patients who underwent both successful and failed DCR were examined to determine the relationship of the soft-tissue changes to the surgical ostium, and the findings were correlated with postoperative results of office probing and irrigation. RESULTS: After a successful DCR (n = 7), a patent fistulous tract was confirmed by office probing and irrigation. A characteristic "Y-on-its-side" configuration of the soft tissue was noted on a CT scan. Total occlusion of the osteotomy by soft tissue on probing and irrigation corresponded to a mucocelelike soft-tissue density with a central lucency and soft-tissue obstruction (n = 2). Total occlusion of the osteotomy by inadequate excision of bone and adjacent soft tissue, visualized by CT imaging, was confirmed by palpation of bone by office probing (n = 1). Partial obstruction on probing and irrigation was associated on a CT scan with an increased soft-tissue density (n = 4) and bone in the region of the osteotomy (n = 3). CONCLUSIONS: Computed tomography allows visualization of a patent fistula after successful DCR as well as soft-tissue or bone obstruction of the surgically created fistula after failed DCR. When combined with probing and irrigation, CT helps to formulate a surgical plan after failed DCR.
Anti-Bacterial Agents↗
Applications for the new electrochemiluminescent (ECL) and biosensor technologies.
Biosensor and electrochemiluminescent (ECL) assays are replacing enzyme-linked immunosorbent assays (ELISAs) at Schering-Plough as immunoassays of choice to monitor cytokine levels and detect anti-cytokine antibody responses during cytokine therapy. These new assays provide increased sensitivity and a better correlation with biological assays. Biosensor assays using the BIACORE 2000 (BIACORE, Uppsala, Sweden) are being adopted to support preclinical and clinical trials for the detection of antibodies capable of binding to IL-10 and IL-4. Significant advantages when using a biosensor assay are that real-time and label-free detection permit increased throughput and direct detection of binding interactions which enables detection of low affinity antibodies that are not detected by ELISA. The ECL assays using the ORIGEN Analyser (IGEN, Gaithersburg, MD) that we have implemented to replace existing ELISAs for quantification of serum IL-10 and serum interferon alfa levels are more sensitive and less subject to matrix effects. Data obtained during the validation of these assays are described.
Antibodies↗
Left ventricular diastolic function after coronary artery bypass grafting: a correlative study with three different myocardial protection techniques.
BACKGROUND: This study was designed to examine the effect of myocardial protection on diastolic function after cardiac operations. METHODS: Subjects were patients with normal preoperative diastolic function who were scheduled for coronary artery bypass grafting. Group I received anterograde cardioplegia; group II received anterograde and retrograde cardioplegia; and group III was protected with ventricular fibrillation and intermittent aortic crossclamping. Operations were performed with mild hypothermia and ventricular venting through the left superior pulmonary vein in all cases. Left ventricular diastolic function was evaluated with pulsed-wave Doppler transesophageal echocardiography (samples at the mitral valve leaflet: four-chamber view) and left superior pulmonary vein flow velocity. The flow patterns were stored on videotape and sent to an independent investigator for analysis. Left ventricular ejection fraction was calculated with transesophageal echocardiography (short-axis view, two-dimensional and M-mode). RESULTS: Left ventricular diastolic function, as measured by the ratio between the peak velocities during early filling and atrial contraction and by systolic diastolic superior pulmonary venous flow ratio, was significantly impaired in all three groups 5 minutes after discontinuation of cardiopulmonary bypass. At 1 hour after operation, these values had returned to control levels only in group III. There was an increased incidence of supraventricular arrhythmias in group III. There were no significant hemodynamic differences among the three groups. CONCLUSIONS: Left ventricular diastolic function was severely impaired after cardiopulmonary bypass. The degree of impairment depended on the myocardial protection used. The impairment in diastolic function was less when ventricular fibrillation and intermittent aortic crossclamping were used, and greater when anterograde and retrograde cardioplegia were used.
Adult↗
Direct listing for adult tonsillectomy.
Health care economic efficiency is important. Adult tonsillectomy is a common operation. The decision to operate is made at an out-patient consultation and is based on information in the general practitioner's (GP) referral letter. This study aims to define what proportion of adult tonsillectomies are suitable for listing with an out-patient consultation (direct listing). GPs were informed of the indications (six episodes of tonsillitis in the last year) and contraindications (tonsillectomy for another reason and general ill health) for direct listing. All referrals for 1 year were screened by an otolaryngologist and suitable patients were listed directly. Patients were then reviewed in the anaesthetic pre-admission clinic and inappropriate listings were cancelled and reasons noted. Of all patients, 50% were suitable for direct listing. Of these 3% were later deemed inappropriate. None of the cancellations were due to a flaw in the direct listing process. This would result in savings to the NHS of 2 million pounds per annum.
Adult↗
A host-guest set of triple-helical peptides: stability of Gly-X-Y triplets containing common nonpolar residues.
Host-guest peptide sets have been useful in evaluating the propensity of different amino acids to adopt an alpha-helical or beta-sheet form, and this concept is applied here to the triple-helical conformation. A set of host-guest peptides of the form acetyl-(Gly-Pro-Hyp)3-Gly-X-Y-(Gly-Pro-Hyp)4-Gly-GlyCONH2 was designed to evaluate the contribution of an isolated Gly-X-Y triplet to triple-helix stability in a defined environment. Peptides were synthesized to include guest triplets with the X and Y positions occupied by the most common nonpolar residues found in collagen: Pro (X position) and Hyp (Y position); Ala; Leu, the most frequent hydrophobic residue; and Phe, the only commonly occurring aromatic residue. The guest triplets of the 12 peptides synthesized represent 35% of the sequence found in the alpha 1 chain of type I collagen. All peptides formed stable triple-helical structures, and the peptides showed a range of thermal stabilities (Tm = 21-44 degrees C), depending on the identity of the guest triplet. Thermodynamic calculations indicate these peptides have a range of free energy values (delta delta G = 9 kcal/mol) and suggest that favorable entropy is the dominant factor in increased stability. Replacement of Ala by Leu in the X position did not affect the thermal stability, while an Ala to Leu change in the Y position was destabilizing. These data provide experimental evidence that hydrophobic residues do not stabilize the triple helical conformation. Although Leu and Phe are found almost exclusively in the X position in collagens, peptides with Leu and Phe in the Y position formed stable triple-helices. This supports the hypothesis that the X positional preference of these residues relates to their increased potential for intermolecular hydrophobic interactions rather than their destabilization of the triple-helical molecule. These studies establish the utility of host-guest peptides in defining a scale of triple-helix propensities and in clarifying the interactions stabilizing the triple-helical conformation.
Amino Acid Sequence↗
Unusual respiratory variation in right ventricular pressure following balloon pulmonary valvuloplasty in a pregnant patient.
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Adult↗
Stability of invertase in reverse micelles.
The stability of invertase was studied under various conditions, including at 75 degrees C, in presence of stabilizers (sorbitol and glycerol) at 75 degrees C, and in the presence of denaturants (urea and trichloroacetic acid) at 37 degrees C in reverse micelles. Stability of the invertase in reverse micelles was found to be improved over that of the enzyme in bulk aqueous solution. Sorbitol could enhance enzyme stability as it does in the bulk aqueous system. The stabilizing effect of glycerol was reduced in reverse micelles. The denaturation pattern of urea remains unaltered. However, the denaturation effect of trichloroacetic acid has been reduced in reverse micelles.
Enzyme Stability↗