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Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP-8) to stability and collagenolytic activity by alanine scanning mutagenesis.

Analysis of the hinge region of neutrophil collagenase by alanine scanning mutagenesis revealed that this sequence motif has a pronounced effect on the stability and collagenolytic activity of the active enzyme. The mutagenesis of the amino acid residues in the P1' position of the two autoproteolytically cleaved peptide bonds (Leu243 and Ile248) to Ala showed that the mutant enzymes were more resistant to autoproteolysis. However, these mutants were not completely stable and autoproteolysis occurred mainly at the Ala239-Ile240 peptide bond and the half-life of the active enzyme was increased by 50%. In contrast, mutagenesis of Pro247 --> Ala (P1 of the minor cleavage site Pro247-Ile248) lead to increased susceptibility of the enzyme to autoproteolysis. However, when the other P1 position Gly242 was altered to Ala no effect on stability was observed. The analysis of the ability of the mutant active enzymes to hydrolyse 14C-type I collagen was assessed and our results demonstrate that the hinge sequence motif of neutrophil collagenase is important for collagenolytic activity. The alteration of the Gly242-Leu-Ser-Ser-Asn-Pro-Ile-Gln-Pro247 sequence motif to Gly242-Ala-Ala-Ala-Ala-Pro-Ala-Ala-Pro247 showed that the collagenolytic activity was reduced by 68.4%. In addition, mutagenesis of the downstream sequence motif Pro247-Thr-Gly-Pro-Ser-Thr-Pro-Lys-Pro258 to Pro247-Ala-Ala-Pro-Ala-Ala-Pro-Ala-Pro258 had an even more marked effect on the collagenolytic activity, which was reduced by 87.4%. When the Pro residues in the hinge motif (Pro247, Pro250, Pro253 and Pro256) were altered to Ala the collagenolytic activity dropped to 1.5% of the value observed for wild-type enzyme.

Alanine↗

Stability and change in adult intelligence: 1. Analysis of longitudinal covariance structures.

We address two questions of central interest in adult intellectual development: the equivalence of psychometric tests' measurement properties at different ages, and the stability of individual differences in intelligence over time. We performed a series of longitudinal factor analyses using the LISREL program to model longitudinal data from Schaie's Seattle Longitudinal Study. The results indicate complete invariance in the loadings of five subtests of Thurstone's Primary Mental Abilities battery on a general intelligence factor. Individual differences in general intelligence were highly stable over 14-year epochs, with standardized factor correlations averaging about .9 between adjacent 7-year testing intervals. These results indicate that most individuals in this relatively select longitudinal sample maintained their relative ordering in intelligence.

Adult↗

Effects of time, initial composition, and stabilizing agents on the results of canine cerebrospinal fluid analysis.

BACKGROUND: Cerebrospinal fluid (CSF) is considered highly labile, but not all samples are analyzed immediately. Changes in the composition of CSF could potentially affect diagnostic test results and thus influence decisions about patient management. There has been little scientific inquiry into how variables such as time, initial composition, and storage conditions affect results of standard laboratory analysis of CSF. OBJECTIVES: The objectives of this study were to determine the effects of time, protein concentration, and presence or absence of exogenous stabilizing agents on standard CSF analysis results. METHODS: Thirty abnormal CSF samples from 26 dogs were evaluated. Samples were divided into aliquots comprising different treatment groups and stored at 4 degrees C. Total nucleated cell count (TNCC), differential cell count (DCC), and cell morphology were evaluated for all groups; protein concentration was measured for selected groups. Unaltered aliquots were analyzed immediately (T0Hr) and at 2, 4, 8, 12, 24, and 48 hours (T2Hr-T48Hr); aliquots with added fetal calf serum (FCS) or hydroxyethyl starch (hetastarch) were analyzed at T48Hr. RESULTS: Significant time-dependent changes were observed in DCC in unaltered samples. Mononuclear cells deteriorated more rapidly than did neutrophils. Based on microscopic examination and subjective scoring of cell morphology, cells were consistently more degenerate by T24Hr compared with T0Hr. Samples with protein concentrations > or =50 mg/dL were less susceptible to cell deterioration than those with lower protein concentrations. Adding either FCS or hetastarch improved sample stability. CONCLUSIONS: Delayed analysis of canine CSF by 4-8 hours is unlikely to alter diagnostic interpretation, especially for samples with protein concentrations > or =50 mg/dL. The likelihood of misinterpretation is higher for samples with low cellularity or low protein concentration. We provide specific recommendations for adding FCS or hetastarch to samples that will not be analyzed within 1 hour.

Animals↗

Eliciting patients' preferences for cigarette and alcohol cessation: an application of conjoint analysis.

The strength and stability of preferences for quitting cigarettes versus alcohol in a population of dual users undergoing treatment was examined using conjoint analysis. Patients at a Veteran's Administration substance abuse treatment center ranked nine vignettes from most to least preferred at baseline and 4 weeks later. The vignettes, using a full factorial design, described health states associated with three levels of substance use. We regressed vignette rankings on the levels of smoking and drinking. A larger regression coefficient indicated a stronger preference for quitting. At baseline and follow-up, the group placed more preference on quitting alcohol than cigarettes (coefficients of 2.23 and 2.35 for alcohol cessation and.51 and.73 for smoking cessation). Some subjects preferred smoking to quitting at baseline (23.9%) and follow-up (23.5%). Over time, 29.4% and 35.3% increased their preference for tobacco and alcohol cessation, while 41.2% and 17.6% decreased their preference for cigarette and alcohol cessation. Preferences for stopping alcohol were stronger than for stopping cigarettes, and many preferences changed after a treatment program.

Alcohol Drinking↗

Pitavastatin, Procollagen Pathways, and Plaque Stabilization in Patients With HIV: A Secondary Analysis of the REPRIEVE Randomized Clinical Trial.

IMPORTANCE: In a mechanistic substudy of the Randomized Trial to Prevent Vascular Events in HIV (REPRIEVE) randomized clinical trial, pitavastatin reduced noncalcified plaque (NCP) volume, but specific protein and gene pathways contributing to changes in coronary plaque remain unknown. OBJECTIVE: To use targeted discovery proteomics and transcriptomics approaches to interrogate biological pathways beyond low-density lipoprotein cholesterol (LDL-C), relating statin outcomes to reduce NCP volume and promote plaque stabilization among people with HIV (PWH). DESIGN, SETTING, AND PARTICIPANTS: This was a post hoc analysis of the double-blind, placebo-controlled, REPRIEVE randomized clinical trial. Participants underwent coronary computed tomography angiography (CTA), plasma protein analysis, and transcriptomic analysis at baseline and 2-year follow-up. The trial enrolled PWH from April 2015 to February 2018 at 31 US research sites. PWH without known cardiovascular diseases taking antiretroviral therapy and with low to moderate 10-year cardiovascular risk were eligible. Data analyses were conducted from October 2023 to February 2024. INTERVENTION: Oral pitavastatin calcium, 4 mg per day. MAIN OUTCOMES AND MEASURES: Relative change in plasma proteomics, transcriptomics, and noncalcified plaque volume among those receiving treatment vs placebo. RESULTS: Among 558 individuals (mean [SD] age, 51 [6] years; 455 male [82%]) included in the proteomics assessment, 272 (48.7%) received pitavastatin and 286 (51.3%) received placebo. After adjusting for false discovery rates, pitavastatin increased abundance of procollagen C-endopeptidase enhancer 1 (PCOLCE), neuropilin 1 (NRP-1), major histocompatibility complex class I polypeptide-related sequence A (MIC-A) and B (MIC-B), and decreased abundance of tissue factor pathway inhibitor (TFPI), tumor necrosis factor ligand superfamily member 10 (TRAIL), angiopoietin-related protein 3 (ANGPTL3), and mannose-binding protein C (MBL2). Among these proteins, the association of pitavastatin with PCOLCE (a rate-limiting enzyme of collagen deposition) was greatest, with an effect size of 24.3% (95% CI, 18.0%-30.8%; P&#x2009;<&#x2009;.001). In a transcriptomic analysis, individual collagen genes and collagen gene sets showed increased expression. Among the 195 individuals with plaque at baseline (88 [45.1%] taking pitavastatin, 107 [54.9%] taking placebo), changes in NCP volume were most strongly associated with changes in PCOLCE (%change NCP volume/log2-fold change&#x2009;=&#x2009;-31.9%; 95% CI, -42.9% to -18.7%; P&#x2009;<&#x2009;.001), independent of changes in LDL-C level. Increases in PCOLCE related most strongly to change in the fibro-fatty (<130 Hounsfield units) component of NCP (%change fibro-fatty volume/log2-fold change&#x2009;=&#x2009;-38.5%; 95% CI, -58.1% to -9.7%; P&#x2009;=&#x2009;.01) with a directionally opposite, although nonsignificant, increase in calcified plaque (%change calcified volume/log2-fold change&#x2009;=&#x2009;34.4%; 95% CI, -7.9% to 96.2%; P&#x2009;=&#x2009;.12). CONCLUSIONS AND RELEVANCE: Results of this secondary analysis of the REPRIEVE randomized clinical trial suggest that PCOLCE may be associated with the atherosclerotic plaque stabilization effects of statins by promoting collagen deposition in the extracellular matrix transforming vulnerable plaque phenotypes to more stable coronary lesions. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02344290.

Humans↗

Temporal stability of psychophysiological stress profiles: a re-analysis using intraclass correlation coefficients.

This is a re-analysis of data from a previous study which examined the temporal stability of three psychophysiological responses [frontal electromyographic activity (EMG), hand surface temperature, and heart rate]. Each response was recorded on 64 subjects over four sessions, each of which consisted of a 20-min, adaptation period, a baseline condition, and two stressors (one cognitive, the other physical). Rather than using Pearson product-moment correlations, as nearly all psychophysiological test-retest reliability studies have, we have now analyzed the data using intraclass correlation coefficients. This type of correlation allows one to incorporate more than two test-retest values on the same subjects. Analysis indicated that, with the exception of EMG during the physical stressor, the absolute values of the responses had quite significant reliability (.70 or greater). Treating the responses as relative measures (percent change from baseline or simple change scores from baseline) produced smaller and frequently less stable coefficients. It is concluded that statistical estimates of psychophysiological response reliability are functions of the design and particular reliability analysis employed.

Adolescent↗

Simulations of foot stability during gait characteristic of ankle dorsiflexor weakness in the elderly.

Falls are common among the elderly and often cause injuries. They most frequently occut during walking and are associated with the chronic deterioration in neuromuscular and sensory systems, as well as with ankle dorsiflexor muscular weakness and lowered endurance of these muscles to fatigue. In the present study, a three-dimensional (3-D) finite element model of the structure of the foot was utilized to determine the effects of ankle dorsiflexor muscle weakness on the structural stability of the foot and, consequently, on the risk of falls during gait. The medial-lateral tendency of instability of the foot during gait in such conditions of weakness was analyzed by means of this model to identify the most important muscles used in controlling foot stability in affected individuals. The values of the eccentricity of the center of pressure under the heel during foot placement were used to indicate the degree of foot stability. The computational analysis indicated that it is the tibialis anterior muscle's weakness that dramatically decreases foot stability. Clinical investigation is now needed to correlate the significance of tibialis anterior muscle weakness with other known risk factors affecting the tendency to falls among the elderly, e.g., deterioration of sensory abilities. Rehabilitation practitioners and physical therapists may apply the present analytic approach to evaluate the stability of a foot before treatment and compare the predicted with the actual therapeutic results in terms of optimization of foot-ground pressure.

Accidental Falls↗

Causes of stability of aggression from early childhood to adolescence: a longitudinal genetic analysis in Dutch twins.

This study investigated the contribution of genetic and environmental influences on the stability of aggressive behavior from early childhood to adolescence. Two developmental models, the simplex model and the common factor model, were tested to study the underlying processes of stability and change. Measures of aggressive behavior (AGG) were obtained from maternal CBCL data as part of a large ongoing longitudinal study of the Netherlands Twin Registers (NTR) and included data from 6488 three-year-old twin pairs, 5475 seven-year-old twin pairs, 2983 ten-year-old twin pairs, and 1509 twelve-year-old twin pairs. AGG showed moderate to high stability during childhood. The stability coefficients ranged from 0.41 to 0.77 across varying intervals. Averaged across boys and girls, genetic factors accounted for approximately 65% of the total stability. Longitudinal genetic analysis indicated a simplex model for genetic effects, which suggests a dynamic development process consisting of transmission of existing genetic effects interacting with new genetic influences. This is especially true at age 7, when the influence of new genetic factors was large. Shared environmental factors accounted for approximately 25% of phenotypic stability, and it seemed that a stable set of the same shared environmental factors underlay the development of AGG. Nonshared environmental factors, when important, are age specific. Sex-specific differences for stability were identified. For boys, genetic influences were greater, whereas for girls shared environmental factors were more important. These data support the idea that both genetic and environmental influences play a role in the stability of AGG from age 3 to 12.

Aggression↗

Spinal stabilization of vertebral column tumors.

An analysis of indications, techniques, results of stabilization and decompression of 100 consecutive spinal tumour cases was carried out. Localized metastatic disease is best operated anteriorly. Primary malignancies are best treated with en bloc resection. Pain relief in metastatic disease is achieved by rigid stabilization. The unstable spine secondary to benign or malignant disease often requires stabilization for alleviation of pain; 132 stabilization procedures were performed in 100 patients. There were nine benign and 91 malignant tumors including 71 metastatic. Indications for stabilization were pathological fracture or following decompression. Anterior approaches including implant stabilization were used in those with metastatic disease limited to one to two levels or where significant kyphosis existed. Posterolateral decompression with Luque rod stabilization was indicated where disease was more widespread. In metastatic disease acrylic cement was used both anteriorly and posteriorly together with implant stabilization. Eighty-one percent had good to excellent relief of pain; 68 patients had neurological deficits. Significant neurological return was achieved in 40% of posterior decompressions and 71% of anterior decompressions in metastatic disease. All patients with benign tumors have solid fusions. In malignant disease the use of cement provided stability without loss of fixation in 87 of 91 procedures. Complications were 4% infection and failure of two Harrington rods without wiring, one Luque rod and two anterior constructs. The average longevity of patients treated for metastatic disease was 11.3 months.

Equipment Failure↗

Optimization and stability boundaries for the synchronization of semiconductor lasers with external optical feedback.

We perform a stability and optimization analysis for the synchronization of unidirectionally coupled external-cavity semiconductor lasers. Using rigorous stability criteria, we qualitatively derive the boundaries of the high-quality synchronization basin. The underlying influence of Hopf bifurcations on the stability of the synchronization manifold is also investigated.

Journal Article↗

Coronary plaque classification with intravascular ultrasound radiofrequency data analysis.

BACKGROUND: Atherosclerotic plaque stability is related to histological composition. However, current diagnostic tools do not allow adequate in vivo identification and characterization of plaques. Spectral analysis of backscattered intravascular ultrasound (IVUS) data has potential for real-time in vivo plaque classification. METHODS AND RESULTS: Eighty-eight plaques from 51 left anterior descending coronary arteries were imaged ex vivo at physiological pressure with the use of 30-MHz IVUS transducers. After IVUS imaging, the arteries were pressure-fixed and corresponding histology was collected in matched images. Regions of interest, selected from histology, were 101 fibrous, 56 fibrolipidic, 50 calcified, and 70 calcified-necrotic regions. Classification schemes for model building were computed for autoregressive and classic Fourier spectra by using 75% of the data. The remaining data were used for validation. Autoregressive classification schemes performed better than those from classic Fourier spectra with accuracies of 90.4% for fibrous, 92.8% for fibrolipidic, 90.9% for calcified, and 89.5% for calcified-necrotic regions in the training data set and 79.7%, 81.2%, 92.8%, and 85.5% in the test data, respectively. Tissue maps were reconstructed with the use of accurate predictions of plaque composition from the autoregressive classification scheme. CONCLUSIONS: Coronary plaque composition can be predicted through the use of IVUS radiofrequency data analysis. Autoregressive classification schemes performed better than classic Fourier methods. These techniques allow real-time analysis of IVUS data, enabling in vivo plaque characterization.

Algorithms↗

The absence of constitutive and induced expression of critical cell-adhesion molecules on human cardiac myocytes. Its role in transplant rejection.

Fetal human cardiac myocytes (FHCM) and a cell line derived from FHCM, termed W1, constitutively express low levels of MHC class I antigens and significant levels of ICAM-1 (CD54), and LFA-3 (CD58) but do not express LFA-1 alpha (CD11a), LFA-1 beta (CD18), GMP-140 (CD62), BB1-B7, VCAM-1, and ELAM-1. In vitro incubation of FHCM or the W-1 cell line for varying periods with varying concentrations of IFN-gamma, TNF-alpha, Poly IC, LPS, IL-alpha, IL-1 beta, PMA, PDBu, and supernatant fluids from Con A-activated PBMC or allogeneic MLR cultures failed to induce cell adhesion molecules (CAMs) or costimulatory molecules that are not constitutively expressed on these cells except for MHC class II antigens. In addition, IFN-gamma, Con A, and MLR supernatant fluids (in order of biological activity) not only induced MHC class II antigens but also markedly increased the mean density of expression per cell of MHC class I and ICAM-1. Analysis of the stability of MHC class I/II molecules using agents like brefeldin-A and Western blot analysis of MHC class II molecules suggest that these ligands are very stably expressed on myocytes. Our previous studies have documented the failure of MHC-expressing FHCM to induce an alloproliferative response. The results of the present studies show that this failure is not secondary to the absence of ICAM-1 or LFA-3 or the presence of unstable MHC molecules but is most likely due to the absence of other CAMs/costimulatory molecules that are critically required for inducing allogeneic activation.

Cell Adhesion Molecules↗

Spectral analysis of heart rate variability during isoflurane anesthesia.

The autonomic nervous system is an important neural control system for maintaining cardiovascular stability in humans. Analysis of heart rate variations may provide important clinical information on the influence of anesthesia on the autonomic nervous system and the central nervous system. Therefore, the effects of 1.0, 1.5, and 2.0 minimum alveolar concentrations of isoflurane anesthesia on beat-to-beat heart rate variations were studied in ten patients (ASA Physical Status 1). Spectral analysis was used to determine the intensity of the variations. For each power spectrum, the frequency components were identified as follows: 1) the parasympathetically mediated respiratory component (0.15-0.4 Hz) and 2) both parasympathetically and sympathetically mediated components (0.04-0.15 Hz). The latter was subdivided into the low-frequency component (0.04-0.09 Hz) of vasomotor origin and the mid-frequency component (0.09-0.15 Hz) of baroreceptor origin. Marked reductions in the power of heart rate variations, at all frequencies, were found during isoflurane anesthesia, indicating isoflurane decreased total autonomic nervous system activity. Isoflurane decreased the high-frequency and mid-frequency components in a concentration-dependent manner. The low-frequency component increased transiently at 1.5 minimum alveolar concentrations concomitant with the burst suppression in the electroencephalogram. The ratio of mid-frequency to high-frequency components did not change significantly during isoflurane anesthesia compared with the awake period. These frequency characteristics of heart rate variations during isoflurane anesthesia suggest there are dose-related decreases in autonomic nervous system activity in both the vagus and the cardiac sympathetic nerves.

Adult↗

Improved performance of the second generation alpha 1-AGP columns: applications to the routine assay of plasma levels of alfuzosin hydrochloride.

Described is a direct enantioselective separation of the enantiomers of alfuzosin hydrochloride on the second generation alpha 1-AGP column which offers improved efficiency, shorter analysis, and improved stability with respect to the first generation columns. The method has been applied to the analysis of drug substance in rat plasma. This highly efficient extraction method and the use of fluorimetric detection result in selective and sensitive determination of the enantiomers. The analytical validation parameters demonstrate the applicability of this method to pharmacokinetic and metabolic studies.

Animals↗

Quantification of the influence of HPrSer46P on CcpA-cre interaction.

Carbon catabolite repression (CCR) of the Bacillus megateriumxyl operon is dependent on the catabolite responsive element cre, the catabolite control protein (CcpA) and the histidine-containing phosphocarrier protein phosphorylated at the serine 46 residue (HPrSer46P). The latter is formed in the presence of glucose and mediates CCR via CcpA. We present evidence for the presence of HPrSer46P in a ternary complex with CcpA and cre. We also demonstrate increased stability of this complex compared to the CcpA-cre complex by electrophoretic mobility shift analysis (EMSA). This stabilization by HPrSer46P is the same for the xyl cre and an improved cre. Thus, HPrSer46P is a co-repressor for CcpA. In addition, surface plasmon resonance (SPR) experiments yielded binding constants of CcpA and the CcpA-HPrSer46P complex with cre. HPrSer46P stimulated CcpA binding to cre 50-fold. The binding constant is 4.9(+/- 0.5) x 10(6) M(-1). Non-phosphorylated HPr did not affect the complex formation between CcpA and cre. Previously proposed effects by glucose-6-phosphate, fructose-1,6-diphosphate and NADP on CcpA-cre or CcpA-HPrSer46P-cre formation were not found in EMSA and SPR experiments.

Amino Acid Substitution↗

On models of quantitative genetic variability: a stabilizing selection-balance model.

A model of stabilizing selection on a multilocus character is proposed that allows the maintenance of stable allelic polymorphism and linkage disequilibrium. The model is a generalization of Lerner's model of homeostasis in which heterozygotes are less susceptible to environmental variation and hence are superior to homozygotes under phenotypic stabilizing selection. The analysis is carried out for weak selection with a quadratic-deviation model for the stabilizing selection. The stationary state is characterized by unequal allele frequencies, unequal proportions of complementary gametes, and a reduction of the genetic (and phenotypic) variance by the linkage disequilibrium. The model is compared with Mather's polygenic balance theory, with models that include mutation-selection balance, and others that have been proposed to study the role of linkage disequilibrium in quantitative inheritance.

Alleles↗

Methodological considerations for implementation of lymphocyte subset analysis in a clinical reference laboratory.

As the diagnostic utility of lymphocyte subset analysis has been recognized in the clinical research laboratory, a wide variety of reagents and cell preparation, staining and analysis methods have also been described. Methods that are perfectly suitable for analysis of smaller sample numbers in the biological or clinical research setting are not always appropriate and/or applicable in the setting of a high volume clinical reference laboratory. We describe here some of the specific considerations involved in choosing a method for flow cytometric analysis which minimizes sample preparation and data analysis time while maximizing sample stability, viability, and reproducibility. Monoclonal T- and B-cell reagents from three manufacturers were found to give equivalent results for a reference population of healthy individuals. This was true whether direct or indirect immunofluorescence staining was used and whether cells were prepared by Ficoll-Hypaque fractionation (FH) or by lysis of whole blood. When B cells were enumerated using a polyclonal anti-immunoglobulin reagent, less cytophilic immunoglobulin staining was present after lysis than after FH preparation. However, both preparation methods required additional incubation at 37 degrees C to obtain results concordant with monoclonal B-cell reagents. Standard reagents were chosen on the basis of maximum positive/negative separation and the availability of appropriate negative controls. The effects of collection medium and storage conditions on sample stability and reproducibility of subset analysis were also assessed. Specimens collected in heparin and stored at room temperature in buffered medium gave reproducible results for 3 days after specimen collection, using either FH or lysis as the preparation method. General strategies for instrument optimization, quality control, and biohazard containment are also discussed.

Antibodies, Monoclonal↗

Colony-stimulating factor gene expression in human acute myeloblastic leukemia cells is posttranscriptionally regulated.

Normal human myeloid cells require certain colony stimulating factors (CSFs) for growth and differentiation. These CSFs are normally produced exogenously by accessory cells. However, human acute myeloid leukemia cells have been found, in certain instances, to have constitutive, endogenous production of one or more of these CSFs. In order to address the molecular basis of this apparently anomalous production of CSFs, we studied granulocyte-, monocyte-, and granulocyte/monocyte colony stimulating factor gene expression, gene structure, transcription rate, message stability, and message inducibility in human acute myeloid leukemia samples. CSF gene expression by Northern analysis was variable from no transcripts detectable to transcripts present for all three CSFs. No CSF gene structure abnormalities were detected by Southern blot analysis. Nuclear run-on assays found ongoing and relatively uniform CSF gene transcription irrespective of the levels of CSF expression detected by Northern analysis. However, the stability of the CSF transcripts appeared to correlate with the level detected on Northern blots. Despite the apparent similarity in the regulation of these CSFs to that seen in mature monocytes, the inducibility of the expression of these CSFs was found to differ significantly from that seen in these mature myeloid cells. Taken together, these results suggest that the expression of CSF genes in human myeloid leukemias is primarily mediated through posttranscriptional mechanisms. These mechanisms are separate for each of the CSFs and apparently different from that found in normal blood monocytes.

Blotting, Northern↗