Proteases in prostate development, function, and pathology.
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Androgens↗
Biomedical subjects
M J Wilson
Publications and source records attributed to M J Wilson.
Cathepsin B in angiogenesis of human prostate: an immunohistochemical and immunoelectron microscopic analysis.
BACKGROUND: Angiogenesis (or neovascularization) is required for the growth of solid organ tumors and precedes invasion of the adjacent stroma by neoplastic cells. We investigated the relative density and distribution of cathepsin B (CB) immunostained microvessels (i.e., small blood vessels and capillaries) in benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), and prostatic adenocarcinoma (CAP) by immunocytochemical localization of an antibody directed against a cathepsin B-derived synthetic peptide (Syn-CB). METHODS: We studied 16 formalin-fixed, prostatectomy specimens that were embedded in paraffin/paraplast for histological examination by hematoxylin and eosin and immuno-localization of the Syn-CB antibody. Selected paraformaldehyde-fixed specimens were embedded in K4M Lowicryl or LRWhite resins. We localized the antibody in thin sections using immunoelectron microscopy techniques. RESULTS: Eight patients had BPH [4 patients with BPH alone, 2 with BPH and PIN, and 2 with BPH and CAP]. Ten cancer cases included one with Gleason histologic score 4, two with score 6, four with score 7, and three with score 8. In CAP cases, Gleason score 6 and 7 tumors had more microvessels than the score 4 or 8 tumors. In both BPH and CAP cases, the antibody was localized chiefly in the endothelial cells of microvessels, but occasionally in ductal and glandular epithelial cells. Ultrastructurally, CB-immunoreactive gold particles were markedly increased at the luminal and basal plasma membrane surfaces and folds of endothelial cells in neoplastic prostate, but not in the endothelial cells of BPH. Furthermore, the presence of CB localizing gold particles in collagen and smooth muscle fibers near the microvessels indicated leakage of the enzyme in prostatic stroma of neoplastic prostate. Similar leakage was not observed in BPH. Morphometric analysis showed that the relative density of microvessels increased two to three times in cancer patients when compared to patients with BPH alone. Our study also indicated that BPH associated with PIN or CAP had an increased density of microvessels when compared to BPH alone. CONCLUSIONS: Our study showed that the relative density and distribution of microvessels are the most important features of neovascularization in prostatic tumors. The relative density of microvessels increased in PIN and CAP when compared to BPH alone. Although the localization of CB is associated with lysosomes of endothelial cells in both BPH and CAP, there is a greater association of CB with the plasma membranes of endothelial cells in CAP than BPH. Immunoelectron microscopy provided evidence that CB might be involved in dissolution of basement membranes in neoplastic tumors during angiogenesis. CB localization has the potential of defining a role for this protease in degradation of extracellular matrix constituents during early steps of angiogenesis.
Adenocarcinoma↗
Immunohistochemical localization of cathepsin B in neoplastic human prostate.
Cathepsin B (CB) has been shown to degrade extracellular matrix (ECM) proteins, and has been reported to be involved in invasion and metastasis of several types of solid organ tumors in human and animals, but CB has not been studied in human prostate cancer (CAP). Our objective was to determine the CB protein immunostaining pattern in CAP and to correlate the immunostaining with the degree of malignancy as reflected in the Gleason grading system. We used two types of CB antibodies (namely, monospecific, polyclonal antibodies to human liver CB prepared in rabbits, and polyclonal antibody produced in sheep) to establish CB localization patterns in neoplastic prostate. Our analysis showed a heterogeneous CB immunostaining pattern in the neoplastic human prostate. CB immunostaining occurred in many, but not all, of the neoplastic columnar/cuboidal cells of acini and isolated cells, i.e., in small ragged glands and clusters (groups) of invasive cells in the prostatic stroma. We have shown that, in general, there was a positive correlation of the intensity of CB immunostaining with the Gleason histologic score (or Gleason grade sum) tumors, i.e., from the lowest scores through score 8, but many of the tumors with scores 9 and 10 showed little CB immunostaining. Our study indicated that the increased CB immunostaining in the Gleason grade sum 5-8 tumors may be associated with increased degradation of ECM, but not in 9 and 10 despite the fact that the latter tumors are more malignant clinically. In well-differentiated tumors, fewer CB immunostaining cells were present than the moderately-differentiated tumors. In other words, most of the stromal invasion of the prostatic ECM occurred in tumors of Gleason grade sums 5-8. We suggest that CB immunostaining might be a useful method to assess stromal invasion of prostatic carcinoma, especially in the higher grade tumors.
Animals↗
Comparison of objective threshold estimation procedures for 40-Hz auditory evoked potentials.
OBJECTIVE: Objective threshold estimation is possible using statistics such as magnitude-squared coherence (MSC) and responses such as the 40-Hz auditory evoked potential (AEP). The purpose of this paper is to compare three general methods of threshold estimation using MSC. DESIGN: Using 500-Hz amplitude-modulated tones and 40-Hz AEPs from 10 paid-volunteer young adults with normal hearing, we compared three MSC-based threshold estimation methods--extrapolation, intensity series, and adaptive. RESULTS: The extrapolation method yielded low thresholds but unacceptable variability. The intensity series method was insensitive and time-consuming. Two adaptive methods displayed good performance in terms of precision and sensitivity. The first employed an MSC criterion with a 13.5% false positive rate (per trial), but achieved overall false positive rates under 5% after 5 to 7 runs, requiring 30 to 40 minutes test time (a run is a single set of ascending or descending trials, terminated by a reversal). The second, which performed better overall, included only a single run, but collected more data at intensities near threshold than at higher intensities; test time could be further reduced (from 22 to 18 minutes) by using a stopping rule based on initial MSC and noise power estimates at each test intensity. If speed is paramount, a simple single-run method achieved threshold estimates in about 9 minutes, starting at 40 dB nHL, with acceptable precision but less sensitivity. CONCLUSIONS: A single-run adaptive method, with or without collection of additional data near threshold, yielded the best results, in terms of statistical performance and data collection time.
Adult↗
Restriction fragment length polymorphism analysis of PCR-amplified 16S ribosomal DNA of human Capnocytophaga.
The confusion in the taxonomic status of the genus Capnocytophaga has made identification of strains and studies on the role of this genus in infectious diseases equivocal. In this study 33 strains of Capnocytophaga including reference strains and various clinical isolates, were studied using RFLP analysis of 16S ribosomal RNA genes. The 16S ribosomal RNA (rRNA) gene sequences from whole cell suspensions and isolated genomic DNA samples were amplified by the polymerase chain reaction (PCR) using eubacterial specific primers. PCR products were purified and characterized by single digestions with 12 restriction endonucleases. Five of these, BanI, CfoI, HaeIII, HphI and RsaII were found to discriminate reproducibly between strains, and restriction patterns (ribotypes) produced by these were analysed to clarify the classification of Capnocytophaga strains. Dendrograms inferring similarities were derived from these data by the UPGMA method. This analysis produced three major clusters of strains, each of which was associated with a previously proposed species type strain: C. gingivalis, C. sputigena and C. ochracea. The results support the division of Capnocytophaga into three species and demonstrate that, despite the heterogeneity of this genus, the modified ribotyping method provides a simple, rapid and reproducible way to identify Capnocytophaga strains.
Bacterial Typing Techniques↗
Objective versus human observer detection of 40-Hz auditory-evoked potentials.
Magnitude-squared coherence (MSC) was used to detect 40-Hz auditory-evoked potentials (AEPs) in a two-alternative forced-choice experiment. MSC performance was found to be better than that of ten human observers. Experienced observers did not perform better than naive observers. In a "yes-no" experiment, observers demonstrated large differences in criterion, with false-positive rates of 2%-20%. Observers also performed better when AEPs were presented in a 50-ms window (two response cycles) than when presented in a 205-ms window (eight response cycles), although total test time was the same; MSC performed equally well in both cases. MSC provides a useful and objective adjunct to visual detection of 40-Hz AEPs.
Adult↗
Molecular and biochemical characterization of a protective 40-kilodalton antigen from Corynebacterium pseudotuberculosis.
A 40-kDa protein from Corynebacterium pseudotuberculosis has been previously identified as a protective antigen against ovine caseous lymphadenitis. From genomic DNA libraries of C. pseudotuberculosis, we have cloned and sequenced the 40-kDa protein gene, which was found to contain an open reading frame of 1,137 bp encoding a protein of 379 amino acids. No significant homology with previously published DNA or amino acid sequence data was found in databases, suggesting that this is a novel protein. Recombinant 40-kDa protein was overexpressed as a fusion protein to 15% of total cell proteins in Escherichia coli. Biochemical analysis of native and recombinant 40-kDa proteins has revealed associated proteolytic activity, which was shown to be of the serine protease type through the use of specific inhibitors. We suggest that this novel protective antigen be termed corynebacterial protease 40 (CP40).
Amino Acid Sequence↗
Identification of Candida species by PCR and restriction fragment length polymorphism analysis of intergenic spacer regions of ribosomal DNA.
The PCR was used to amplify a targeted region of the ribosomal DNA from 84 Candida isolates. Unique product sizes were obtained for Candida guilliermondii, Candida (Torulopsis) glabrata, and Candida pseudotropicalis. Isolates of Candida albicans, Candida tropicalis, Candida stellatoidea, Candida parapsilosis, and Candida krusei could be identified following restriction digestion of the PCR products.
Base Sequence↗
X linked fatal infantile cardiomyopathy maps to Xq28 and is possibly allelic to Barth syndrome.
A number of families with X linked dilated cardiomyopathy with onset in infancy or childhood have now been described, with varying clinical and biochemical features. Of these, one condition, Barth syndrome (BTHS), can be diagnosed clinically by the characteristic associated features of skeletal myopathy, short stature, and neutropenia, but not all of these features are always present. Molecular genetic studies have delineated the gene for BTHS, which maps to distal Xq28, from the gene for so called X linked dilated cardiomyopathy (XLCM), a teenage onset dilated cardiomyopathy, recently mapped to the 5' portion of the dystrophin locus at Xp21. We report a large family in which male infants have died with congenital dilated cardiomyopathy, and there is a strong family history of unexplained death in infant males over at least four generations. Death always occurred in early infancy, without development of the characteristic features associated with Barth syndrome. Molecular analysis localised the gene in this family to Xq28 with lod scores of 2.3 at theta = 0.0 with dinucleotide repeat markers, p26 and p39, near DXS15 and at F8C. The proximal limit to the localisation of the gene in this family is defined by a recombinant at DXS296, while the distal limit could not be differentiated from the telomere. This localisation is consistent with a hypothesis of allelic and clinical heterogeneity at the BTHS locus in Xq28.
Alleles↗
Desbuquois syndrome complicated by obstructive sleep apnoea and cervical kyphosis.
We present two children with Desbuquois syndrome and characteristic facial appearance, and musculoskeletal and radiological findings. Two new complications of this syndrome are obstructive sleep apnea and severe cervical kyphosis.
Abnormalities, Multiple↗
Objective detection of 40 Hz auditory evoked potentials: phase coherence vs. magnitude-squared coherence.
The relative performance of phase coherence (PC) and magnitude-squared coherence (MSC) for detection of steady-state evoked potentials was studied using 40 Hz auditory evoked potentials (AEPs) in 10 normal human subjects. In addition, simulation experiments were carried out to determine the effects of signal amplitude and phase variability on detection performance. All simulations showed MSC performance to be better than PC performance, with further improvements when MSC was supplemented with weighted averaging. However, human 40 Hz AEP data showed essentially identical detection performance for PC and MSC, with or without weighted averaging. These data support a "phase aggregation" model (at least near threshold) over the more usual model in which an AEP signal is added to a stationary noise. Human data collected under "no-stimulus" conditions agree well with theoretical distributions for both PC and MSC. For equal test time, long analysis periods (with less averaging) yielded equal performance to short analysis periods (with more averaging), for both PC and MSC.
Acoustic Stimulation↗
Phase weighting: a method to improve objective detection of steady-state evoked potentials.
Objective response detection statistics such as magnitude-squared coherence (MSC) reflect the degree to which subaverage phases are clustered or dispersed, but not their agreement with an expected, or target, phase. Using signal-plus-noise simulations and human 40-Hz auditory evoked potentials, we tested MSC performance with and without phase weighting, in which MSC values were multiplied by weights related to the phase error between measured phase and target phase. Phase weighting improved MSC performance for both simulated and 40-Hz auditory evoked potential data. However, the improvement was greater for the simulations, probably because target phase was precisely known.
Acoustic Stimulation↗
Identification of a novel antigen from Corynebacterium pseudotuberculosis that protects sheep against caseous lymphadenitis.
A 40-kDa protein antigen from Corynebacterium pseudotuberculosis has been identified by application of a strategy that employs locally derived antibody-secreting cells (ASC). ASC probes generated by culture of ASC obtained from the lymph node draining the site of infection showed a specificity restricted to a 40-kDa antigen. Analysis of immunoblots with sequential serum samples taken from sheep during the course of experimental primary infection with C. pseudotuberculosis also revealed the 40-kDa antigen as an early immunodominant antigen. Sheep vaccinated with two 100-micrograms doses of a 40-kDa antigen preparation in aluminium hydroxide adjuvant were protected against infection with C. pseudotuberculosis, with an 82% reduction in the proportion of infected sheep and a 98% reduction in lung lesions. Sera from vaccinated sheep exhibited a strong response only to the 40-kDa antigen on immunoblots. These results strongly suggest that the 40-kDa antigen plays a major role in immunity to caseous lymphadenitis.
Animals↗
Identifying hearing loss in the intensive care nursery: a 20-year summary.
The outcome of a study on hearing loss in graduates of one third-level and two second-level intensive care nurseries (ICN) is reported. The goals were to identify, test, and fit hearing aids on those who need them. Initial hearing-threshold estimates were obtained by auditory brainstem response (ABR) analyses at the time of discharge from the hospital; the mean percentage of failures to respond to 30-dB nHL clicks (in one or both ears) was 19.8 percent for the third-level ICN graduates (N = 4374), and 12.0 percent for the second-level graduates (N = 1527). About half of those with hearing loss at the initial Test were returned for a ReTest 6-20 weeks later, at which time 48.7 percent of the third-level and 44.0 percent of the second-level group were judged normal because they delivered 25-dB click ABRs bilaterally. Following conventional audiological work-ups of the ones with unilateral or bilateral hearing loss, hearing aids were fitted to 92 in the third-level group (2.1% of those Tested) and 22 of the second-level group (1.4%). Not quite half of these fittings occurred within 1 year of the hospital Test that initially diagnosed the loss.
Acoustic Stimulation↗
Hyperglycemia induces a loss of phospholipid asymmetry in human erythrocytes.
Phospholipid asymmetry in biological membranes is maintained by an aminophospholipid-specific Mg(2+)-ATPase that transports PS and PE from the outer to the inner monolayer. Recent evidence indicates that a loss of phospholipid asymmetry occurs in erythrocytes from diabetic individuals, resulting in the appearance of PS in the membrane outer leaflet. We show that hyperglycemic treatment of normal erythrocytes duplicates this effect. Erythrocytes incubated for 18-24 h in the presence of glucose were assayed for PS transport and transmembrane phospholipid asymmetry. Phospholipid asymmetry in erythrocytes treated with high concentrations of glucose (> 5 mM) showed a time-dependent (t1/2 approximately 12 h) and concentration-dependent (half-maximal concentration approximately 7.5 mM) increase in the accessibility of PS and PE, and a decrease in the accessibility of SM and PC, to exogenous phospholipases. After an 18 h incubation with 20 mM glucose, 40% of the endogenous PS and PE was found in the outer monolayer concomitant with a decrease in the outer monolayer content of SM (from 80% to 50%) and PC (from 75% to 65%). These values are consistent with an almost complete transbilayer scrambling of erythrocyte phospholipids. The loss of PS asymmetry was verified using an assay based on the activation of the prothrombinase complex. The observed loss of asymmetry is not due to inhibition of PS transport or glucose-induced Ca2+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Triphosphate↗
Localization of a biotinylated cathepsin B oligonucleotide probe in human prostate including invasive cells and invasive edges by in situ hybridization.
The cysteine endopeptidase cathepsin B (CB) can degrade basement membrane (BM) proteins (such as laminin, type IV collagen, and fibronectin) at both acid and neutral pHs suggesting that CB has a role in tumor invasion and distant metastasis. The distribution and intensity of CB protein localization vary in normal prostate, benign prostatic hyperplasia (BPH), and neoplastic prostate. These considerations have led us to examine whether the distribution of CB localization in malignant and normal cells is due to storage or active synthesis of CB. In the present study, we examined the localization patterns of CB at the mRNA level in normal prostate, BPH, and well to moderately differentiated neoplastic prostate, focusing on invasive groups of cells and invasive edges of malignant tumors. We used a 25-base biotinylated oligonucleotide CB cDNA "sense" probe to localize CB message in prostate samples obtained from radical prostatectomies. We have determined that CB is actively synthesized by the epithelia of normal, hyperplastic, and neoplastic prostate including some invasive cells in the invasive edges. In both normal and BPH, CB mRNA was localized predominantly in acinar basal cells with some localization in cuboidal/columnar cells. In contrast, in neoplastic prostate, CB mRNA was localized predominantly in columnar cells and in groups of invasive cells and invasive edges. Thus, in malignant prostate the predominant cell types expressing CB differed from those of the normal prostate and BPH. Analysis of CB mRNA localizations indicated a heterogeneity in staining distribution in prostate cancer with some invasive groups of cells and invasive edges exhibiting CB mRNA and others exhibiting little or no reaction products.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Sequence↗
Gelatinolytic and caseinolytic proteinase activities in human prostatic secretions.
Seminal fluid contains a number of proteinase activities, many of which are secreted by the prostate gland. Our objective was to determine proteinase activities in human prostatic secretions which can degrade gelatin and/or casein. Prostatic secretions were collected by prostate massage from men with benign prostatic hyperplasia prior to surgery to relieve obstruction. Significant proteinase activities towards gelatin of about 81, 86, 94, 111, 115 and 163 Kd as well as less active forms of 23, 36, 38, 132, 137, and 148 Kd were detected using protein substrate-polyacrylamide gel zymography. In addition, Ca2+ stimulated activities of approximately 64, 66, 71 and 76 Kd; however, EDTA and EGTA inhibited all activities but the 23, 36 and 38 Kd forms (these were inhibited by benzamidine and epsilon-amino caproic acid). This suggests that the gelatinolytic activities of 64 Kd and greater were metalloproteinases and those of 23, 36, and 38 Kd were serine proteinases. Significant caseinolytic activities of 22, 25, 35, 37, 57, 90, 96, 102 and 116 Kd were found as well as several less active forms and a 12 Kd activity stimulated by Ca2+. Caseinolytic activities of 12, 14, 16, 96, 102, 116, and 126 Kd were inhibited by EDTA and EGTA indicating they are metalloproteinases. The 35, 37, 57 and 58 Kd caseinolytic activities were inhibited by benzamidine, and the 57 and 58 Kd forms by epsilon-aminocaproic acid suggesting they were serine proteinases. There was considerable variability among individuals in the molecular forms of proteinase activity expressed as well as the level of their activity. A significant decrease in the frequency of expression of the 132 Kd gelatinolytic activity was found in secretions from men with atypia or adenocarcinoma, as compared with men with benign prostatic hyperplasia alone. Our results show that human prostatic secretion contains a variety of proteinase activities. The expression of the 132 Kd gelatinolytic activity could prove useful in further evaluation of neoplastic prostatic disease.
Caseins↗
Design of a californium-based epithermal neutron beam for neutron capture therapy.
The potential of the spontaneously fissioning isotope, 252Cf, to provide epithermal neutrons for use in boron neutron capture therapy (BNCT) has been investigated using Monte Carlo simulation. The Monte Carlo code MCNP was used to design an assembly composed of a 26 cm long, 11 cm radius cylindrical D2O moderator followed by a 64 cm long Al filter. Lithium filters are placed between the moderator and the filter and between the Al and the patient. A reflector surrounding the moderator/filter assembly is required in order to maintain adequate therapy flux at the patient position. An ellipsoidal phantom composed of skull- and brain-equivalent material was used to determine the dosimetric effect of this beam. It was found that both advantage depths and advantage ratios compare very favourably with reactor and accelerator epithermal neutron sources. The dose rate obtainable, on the other hand, is 4.1 RBE cGy min-1, based on a very large (1.0 g) source of 252Cf. This dose rate is two to five times lower than those provided by existing reactor beams and can be viewed as a drawback of using 252Cf as a neutron source. Radioisotope sources, however, do offer the advantage of in-hospital installation.
Californium↗