Capsules and cassettes: genetic organization of the capsule locus of Streptococcus pneumoniae.
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Animals↗
Biomedical subjects
T Kelly
Publications and source records attributed to T Kelly.
A potential marker protease of invasiveness, seprase, is localized on invadopodia of human malignant melanoma cells.
Seprase, a large, gelatin-degrading membrane-protease complex, is expressed at the invasive front of malignant melanoma cells on invadopodia, and its surface expression contributes to the invasive phenotype. An in vitro assay was used to determine the matrix-degrading activity of four malignant human melanoma cell lines. The lines differ in matrix-degrading activity with LOX > RPM17951 > A375 > SKMEL28. The seprase and Gelatinase A activities of these cell lines were also investigated. Seprase and active gelatinase A are found in cell membranes of LOX and RPM17951 cells but not those of SKMEL28 cells. Experiments using anti-seprase monoclonal antibodies in conjunction with a cell fractionation technique indicate that seprase consists of M(r) 97,000 polypeptides and is enriched on the ventral membrane of LOX in contact with planar extracellular matrix substratum. Confocal microscopy further substantiates our biochemical findings that seprase, as well as Gelatinase A, is localized on invadopodia membranes with a 6-fold increase of seprase and 4-fold increase of Gelatinase A intensity over the level expressed on dorsal membranes. In addition, LOX cells expressing higher levels of seprase at the cell surface, as selected by fluorescence-activated cell sorting, are significantly more degradative than LOX cells with lower seprase expression. Taken together, our data show a concordance between seprase and Gelatinase A expression on the cell surface at invadopodia and the matrix-degrading activity of human malignant melanoma cells. Seprase and major secreted proteases may act in concert to degrade components of the extracellular matrix during invasion.
Biomarkers, Tumor↗
Invadopodia promote proteolysis of a wide variety of extracellular matrix proteins.
Chicken embryo fibroblasts (CEF) transformed by Rous sarcoma virus invade the extracellular matrix (ECM) using plasma membrane protrusions, termed invadopodia, that contact and dissolve the matrix. Normal cells neither form invadopodia nor degrade the ECM. Here we show that cells expressing invadopodia degrade and enter into a fibronectin-rich matrix produced by normal fibroblasts. Within 6 h after seeding onto the matrix, the invasive cells create an area devoid of matrix fibrils surrounding the cell body. Proteolysis mediates this matrix clearing because sevenfold more radiolabeled matrix is released into the growth media by the transformed cells relative to the normal cells. In addition to this assembled matrix, transformed cells were grown on thin layers of purified ECM proteins, revealing that invadopodia can degrade fibronectin, collagen type I, collagen type IV, and laminin. A 160 kDa protease that is extracted from transformed cells by Triton X-114 partitions into the detergent phase and is prominent in ventral plasma membranes that contact the ECM suggesting that it is a membrane associated protease.
Animals↗
Membrane proteases as potential diagnostic and therapeutic targets for breast malignancy.
Metastasizing cancer cells can invade the extracellular matrix using plasma membrane protrusions, termed invadopodia, that contact and dissolve the matrix. Various membrane associated proteases localized on the invadopodial membranes are responsible for the extracellular matrix degradation. Work from our laboratory shows that secreted proteases including Gelatinase A, and high molecular weight integral membrane proteases are associated with cell surface invadopodia. Three cell types, including chicken embryonic cells transformed by Rous sarcoma virus, human malignant melanoma cell line LOX, and human breast carcinoma cell line MDA-MB-231, retain the invasive phenotype in vitro, express invadopodia, degrade and enter into a fibronectin-rich collagenous matrix. We suggest that invadopodium-associated proteases are ideal targets for the diagnosis and treatment of cancer as their presence in association with primary tumors may signal increased metastatic potential. An approach toward the development of new prognostic markers for breast malignancy involved production of monoclonal antibodies directed against membrane proteases in a mixture of glycoproteins. Double immunofluorescent technique using a known invadopodium marker is designed to select specific monoclonal antibodies colocalizing at the invasion front, on invadopodia of cancer cells. Membrane protease accessibility at the cell surface can therefore be exploited for therapeutic advances by the development of specific antibodies and inhibitors that block their activities, and by the use of monoclonal antibodies to target cytotoxic molecules to micrometastases. Also, this same accessibility may potentially be used to detect surface proteases on micrometastases or to detect components shed by micrometastases in serum.
Antigens, Neoplasm↗
Dual-energy X-ray absorptiometry for measuring total bone mineral content in the rat: study of accuracy and precision.
Sequential studies of osteopenic bone disease in small animals require the availability of non-invasive, accurate and precise methods to assess bone mineral content (BMC) and bone mineral density (BMD). Dual-energy X-ray absorptiometry (DXA), which is currently used in humans for this purpose, can also be applied to small animals by means of adapted software. Precision and accuracy of DXA was evaluated in 10 rats weighing 50-265 g. The rats were anesthetized with a mixture of ketamine-xylazine administrated intraperitoneally. Each rat was scanned six times consecutively in the antero-posterior incidence after repositioning using the rat whole-body software for determination of whole-body BMC and BMD (Hologic QDR 1000, software version 5.52). Scan duration was 10-20 min depending on rat size. After the last measurement, rats were sacrificed and soft tissues were removed by dermestid beetles. Skeletons were then scanned in vitro (ultra high resolution software, version 4.47). Bones were subsequently ashed and dissolved in hydrochloric acid and total body calcium directly assayed by atomic absorption spectrophotometry (TBCa[chem]). Total body calcium was also calculated from the DXA whole-body in vivo measurement (TBCa[DXA]) and from the ultra high resolution measurement (TBCa[UH]) under the assumption that calcium accounts for 40.5% of the BMC expressed as hydroxyapatite. Precision error for whole-body BMC and BMD (mean +/- S.D.) was 1.3% and 1.5%, respectively. Simple regression analysis between TBCa[DXA] or TBCa[UH] and TBCa[chem] revealed tight correlations (n = 0.991 and 0.996, respectively), with slopes and intercepts which were significantly different from 1 and 0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Absorptiometry, Photon↗
Effect of genetic switching of capsular type on virulence of Streptococcus pneumoniae.
To assess the role of capsular serotypes in the virulence of Streptococcus pneumoniae, we have constructed isogenic derivatives differing only in the type of capsule expressed. Strains of types 2, 5, and 6B were converted to type 3 by transformation and selection for an erythromycin resistance marker linked to the type 3 capsule locus. Characterization studies revealed that these type 3 derivatives were indistinguishable from the type 2, type 5, and type 6B parental strains in terms of restriction enzyme fragment pattern and expression of pneumococcal surface protein A (PspA). Expression of the type 3 capsule did not alter the mouse virulence of the similarly virulent type 2 strain. However, alteration of capsule type had a profound effect on the virulence of the type 5 and type 6B derivatives. The highly virulent type 5 strain was essentially avirulent when expressing the type 3 capsule. Conversely, the 50% lethal dose of the relatively avirulent type 6B strain was reduced > 100-fold when the type 3 capsule was expressed. Thus, the serotype of capsule expressed has a major effect on virulence, and this effect is dependent upon the genetic background of the recipient strain.
Animals↗
Risk factors for suicidal behavior in borderline personality disorder.
OBJECTIVE: This study identified potential risk factors for suicidal behavior in patients with borderline personality disorder defined by the Diagnostic Interview for Borderline Patients and by DSM-III-R criteria for patients who did and did not attempt suicide. METHOD: Histories of suicide attempts and attempt characteristics were obtained by Schedule for Affective Disorders and Schizophrenia interviews from 84 patients with borderline personality disorder and were related to severity of borderline pathology, diagnostic comorbidity, and state and trait symptoms. RESULTS: There were 61 patients with a lifetime history of suicide attempts (72.6%), with an average of 3.39 (SD = 2.87) attempts per patient. Attempters were significantly older than nonattempters, with more impulse actions, antisocial personality disorder comorbidity, and state depression. State depression was significantly less severe in patients who had attempted suicide in the present episode (or past year) than in patients who had attempted suicide only in the past. A comorbid diagnosis of major depression, alcoholism, or drug use disorder did not distinguish attempters from nonattempters. Suicide attempt in the present episode was best predicted by the number of prior lifetime attempts. A highly serious intent to commit suicide was predicted by the number of lifetime attempts and subjective depression, while a low intent was predicted by a mixed subtype of borderline personality disorder plus schizotypal personality disorder and paranoid ideation. A high degree of medical lethality was predicted by number of lifetime attempts, older age, and hysteroid dysphoria, while low lethality attempts were associated with high degrees of anger. CONCLUSIONS: Risk factors for suicidal behavior in patients with borderline personality disorder include older age, prior suicide attempts, antisocial personality, impulsive actions, and depressive moods but not comorbid affective disorder, alcoholism, or drug use disorders.
Adult↗
Depressed neutrophil chemotaxis in children suffering blunt trauma.
OBJECTIVE: Impaired neutrophil (PMN) function, due in part to release of immature PMNs into the circulation, contributes to the increased rate of infection observed in adults suffering blunt trauma. The objective of this study was to determine whether similar events occur in children. METHODS: We assessed PMN chemotaxis and PMN maturation in 25 children (7 young children and 18 adolescents) and 25 adults 1 to 9 days after suffering blunt trauma, and in healthy adult control subjects. PMN chemotaxis was determined using a standard micropore filter assay, whereas PMN maturation was determined with 31D8, a novel monoclonal antibody that binds to mature PMNs more avidly than immature PMNs and band forms. RESULTS: In patients suffering blunt trauma, mean PMN chemotactic values were similar among children (44.6 +/- 2.3 microns) and adults (41.3 +/- 2.1 microns) and both were significantly less than among healthy adults (53.5 +/- 2.4 microns, P < .0005). PMN chemotactic values increased significantly in the 9 days after trauma for both children and adults (F = 13.8, df = 1, P < .0002). Mean PMN 31D8 binding among children with trauma (92.5 +/- 5.2) was significantly less than among healthy adults (117.6 +/- 5.4, P < .0009). CONCLUSIONS: Impairment in PMN chemotaxis occurs in children after blunt trauma and is due in part to release of immature PMNs into the circulation.
Adolescent↗
Binding and localization of M(r) 72,000 matrix metalloproteinase at cell surface invadopodia.
Degradation (turnover) of collagenous matrix occurs on the surface of specialized membrane extensions termed "invadopodia," which are sites of cell invasion into the extracellular matrix. Here we show the localization of the M(r) 72,000 type IV collagenase of the matrix metalloproteinase family at invadopodia. When added exogenously, latent M(r) 72,000 collagenase binds to invadopodia of chicken embryo fibroblasts transformed by Rous sarcoma virus, whereupon the bound collagenase loses its propeptide. The collagenase binds to a component contained within the detergent extract of transformed cells, and increased levels of the active M(r) 62,000 form of the collagenase are seen here. Such an association is not detected in the detergent extract derived from normal cells. Using a recently developed cell fractionation procedure to collect cell surfaces enriched in invadopodia, we show that the M(r) 72,000 collagenase associates with the invadopodial fraction and active forms of the enzyme become immobilized on the collagenous surface. Thus, invadopodia direct intense localized degradation of the extracellular matrix by concentrating active membrane-associated collagenases at sites of cellular invasion.
Amino Acid Sequence↗
Purification of a novel glycosylated ferritin from horse heart.
We have previously shown that mRNA coding for ferritin L subunit is present on both cytosolic ribosomes and endoplasmic reticulum-bound ribosomes in rat heart tissue [Campbell et al. (1989) Arch Biochem Biophys 273:89-98]; from this we infer that heart tissue is capable of making a secreted ferritin. We now report the purification from horse heart, of a ferritin that specifically binds to Concanavalin A-Sepharose and is immunologically cross-reactive with antibodies raised against both horse cellular ferritin and horse serum ferritin. Where cellular ferritin is 10 nm in diameter and contains primarily 21-kDa subunits (as determined by gel exclusion chromatography and electron microscopy), the glycosylated heart ferritin is smaller with diameters of 3-5 nm. Antisera raised against serum ferritin cross-reacted with the glycosylated heart ferritin did but did not show significant cross-reactivity with cellular ferritin thus indicating that serum ferritin and glycosylated heart ferritin have antigenic determinants which may not be present on cellular ferritin. The glycosylated ferritin also differs from cellular ferritin in subunit composition, with subunits of 66, 60.5, 53.5, 43.5, and 29.5 kDa, as shown by SDS-PAGE and Western blot analysis. Interestingly, ferritin purified from horse serum contains subunits of similar size.
Animals↗
Sleep inertia: best time not to wake up?
Sleep inertia is a brief period of inferior task performance and/or disorientation immediately after sudden awakening from sleep. Normally sleep inertia lasts < 5 min and has no serious impact on conducting routine jobs. This preliminary study examined whether there are best and worst times to wake up stemming from circadian effects on sleep inertia. Since the process of falling asleep is strongly influenced by circadian time, the reverse process of awakening could be similarly affected. A group of nine subjects stayed awake for a 64-h continuous work period, except for 20-min sleep periods (naps) every 6 h. Another group of 10 subjects stayed awake for 64 h without any sleep. The differences between these two groups in performance degradation are expected to show sleep inertia on the background of sleep deprivation. Sleep inertia was measured with Baddeley's logical reasoning task, which started within 1 min of awakening and lasted for 5 min. There appeared to be no specific circadian time when sleep inertia is either maximal or minimal. An extreme form of sleep inertia was observed, when the process of waking up during the period of the circadian body temperature trough became so traumatic that it created "sleep (nap) aversion." The findings lead to the conclusion that there are no advantages realized on sleep inertia by waking up from sleep at specific times of day.
Body Temperature↗
An in vitro study of the quality of root fillings in teeth obturated by lateral condensation of gutta-percha or Thermafil obturators.
The aim of this in vitro study was to assess the quality of root canal obturation and degree of linear apical dye penetration in teeth root filled with either laterally condensed gutta-percha or Thermafil obturators. A total of 150 teeth with single roots were included in the study. All canals were prepared using a standard step-back technique with anticurvature filing. Radiographs taken of the teeth to show the maximum degree of canal curvature were then exposed and the angle, radius and position of curvature determined. This information about the canals, together with their working length and diameter at end-point, was used to divide the teeth into two experimental and two control groups. A total of eight teeth were excluded because of technical difficulties, 65 were filled with Thermafil obturators, 63 were filled with laterally condensed gutta-percha whilst 14 teeth remained unfilled and acted as controls. Following obturation, the teeth were radiographed in two planes and the technical quality of fill assessed on a four-point scale. All access cavities were then sealed and the teeth immersed in dye for 48 h before being split longitudinally. Linear dye penetration via the apical foramen was then assessed. Canal obturation with Thermafil obturators (0.7 min) was significantly quicker (P < 0.001) than lateral condensation (6.4 min). Apical extrusion of sealer and gutta-percha occurred significantly more often with Thermafil obturators but there was little difference in the technical quality of the fillings and no significant difference in dye penetration. Under the conditions of this study. Thermafil obturators proved a satisfactory alternative to lateral condensation of gutta-percha.
Chi-Square Distribution↗
Intussusception presenting as encephalopathy.
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Consciousness Disorders↗
Equipment leasing: are you overlooking what may be your best financing option?
In this era of increasingly scarce resources, it is imperative that decision makers choose alternatives that are the most efficient and effective use of the resources available. In terms of equipment acquisition, this means not only selecting the right equipment but also choosing the best method of financing the acquisition. Leasing is one alternative that may be the most efficient and effective use of capital. Health information managers should have a basic understanding of the factors to be considered when evaluating the lease vs. purchase alternative. In the competition for increasingly scarce resources, knowing how to present alternatives to the decision makers can make the difference between denial or approval of equipment acquisitions for the health information department.
Capital Expenditures↗
Minimum qualifications for directors: DNA-based genetic-testing laboratories. DNA Testing Subcommittee, Quality Assurance Committee, Council of Regional Networks for Genetic Services.
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DNA↗
The human homologous pairing protein HPP-1 is specifically stimulated by the cognate single-stranded binding protein hRP-A.
Homologous pairing and strand exchange of DNA are catalyzed by the human homologous pairing protein HPP-1 in a magnesium-dependent, ATP-independent reaction that requires homologous DNA substrates and stoichiometric quantities of HPP-1. Here we show that the addition of the purified human single-strand binding (SSB) protein hRP-A to the reaction mixture stimulates the rate of homologous pairing 70-fold and reduces the amount of HPP-1 required for the reaction at least 10-fold. The identification of hRP-A as a stimulatory factor of HPP-1-catalyzed reaction was facilitated by its recognition as a member of a high molecular weight complex of recombination components. Neither the Escherichia coli SSB protein, bacteriophage T4 gene 32 protein, nor the highly conserved Saccharomyces cerevisiae yRP-A SSB protein could substitute for hRP-A in this stimulation. Because only the cognate SSB was capable of stimulating HPP-1, these results suggest that eukaryotes depend on unique and specific interactions between DNA recombination components.
Blotting, Western↗
Rapid one step urine test for human chorionic gonadotrophin in evaluating suspected complications of early pregnancy.
OBJECTIVE: To determine the ability of a sensitive one step urine test to detect human chorionic gonadotrophin in women with suspected complications of early pregnancy. DESIGN: Test on women presenting to accident and emergency department with gynaecological problems over six months. Results were validated using a quantitative assay for human chorionic gonadotrophin in serum and urine. SETTING: Accident and emergency department and gynaecology wards of a university teaching hospital. SUBJECTS: 130 unselected women. MAIN OUTCOME MEASURES: Detection of human chorionic gonadotrophin by one step test, presence of ectopic pregnancy, and results of quantitative analysis of chorionic gonadotrophin in serum and urine. RESULTS: 79 women had a positive urine test result and 51 a negative result. All 12 women with ectopic pregnancy had a positive test result, although urinary concentration varied from 191 IU/l to 47,800 IU/l. Only one woman, who had a faintly positive result, was found not to be pregnant on subsequent examination. The sensitivity and negative predictive values of the urine test were 100% respectively. 33 women were sent home from the accident and emergency department with normal clinical findings after a negative urine test result. All these women had undetectable concentrations of chorionic gonadotrophin in matched samples of urine and serum. CONCLUSIONS: A simple, rapid one step test for chorionic gonadotrophin should be available for the initial evaluation of emergency gynaecological problems. The additional cost of the test is offset by not admitting those patients whose clinical findings are normal and who have a negative urine test result and by reducing the number of women requiring quantitative assays of chorionic gonadotrophin.
Abortion, Threatened↗
Gas-liquid chromatographic analysis of cellular fatty acids for identification of gram-negative anaerobic bacilli.
A commercially available, computer-assisted microbial identification system (MIS) employs gas-liquid chromatographic analysis of cellular fatty acids for bacterial identification. MIS was compared with conventional identification systems. Of 225 gram-negative anaerobes tested, MIS identified 72.4% of the strains to the species level, 88.9% to the appropriate group, and 93.3% to the correct genus.
Bacteriological Techniques↗