Development and characterization of a collagen degradation assay to assess purified collagenase used in islet isolation.

OBJECTIVES: A collagen degradation activity (CDA) assay was developed to improve the biochemical characterization of purified collagenase used for islet isolation. MATERIALS AND METHODS: Purified class I collagenase (CI) or class II collagenase (CII) from Clostridium histolyticum cultures were used in all experiments. The CDA assay was performed by incubating 50 microg/mL of FITC fibrils with CI or CII for 60 minutes at 35 degrees C. The correlation of the molecular species of the enzyme to CDA was determined by fractionating CI:CII mixtures over an anion exchange column. Individual fractions were analyzed for A280, CDA activity, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to correlate chromatographic analysis of these enzyme mixtures to the molecular species of collagenase effective in collagen degradation. RESULTS: CI has approximately 6 to 17 x higher specific activity than CII in this assay. Assays of different individual fractions recovered after anion exchange chromatography showed that the CDA of collagenase was dependent on the molecular species of the enzyme. Only intact CII and CI with molecular weights >or=100 kDa could degrade collagen fibrils. CONCLUSIONS: This assay provides a more reliable assessment of the functional activity of collagenase enzymes than peptide substrates currently used today. Fractionation of purified collagenase mixtures by anion exchange chromatography followed by analysis of individual fractions by SDS-PAGE and CDA assays will provide a powerful tool to analyze the molecular species of CI and CII required for islet isolation.

Histiocytosis X.

To clarify salient issues pertaining to histiocytosis X--a syndrome that includes Letterer-Siwe disease, Hand-Schuller-Christian disease, and eosinophilic granuloma--the authors review the epidemiologic data and the histologic, morphologic, and clinical bases for diagnosis and prognosis. Histiocytes are defined and their possible histogenesis outlined, and Langerhans cells, which may be a leading element in active lesions, are characterized. The authors outline hypothetic pathogenetic schema, which they recommend be tested by recently developed immunologic and genetic means, since histiocytosis X, at least in its disseminated form, remains an unpredictable disease for which there is no proven effective therapy.

Avidin-biotin amplification procedures for the detection of human terminal deoxynucleotidyl transferase in cell smears.

The authors have developed fluorescent avidin and avidin-peroxidase assays to detect human terminal deoxynucleotidyl transferase (TdT) in cell smears. These assays used specifically purified rabbit anti-calf TdT antibody, biotinylated goat anti-rabbit IgG antibody, and either fluorescein isothiocyanate-avidin or avidin-biotinylated horseradish peroxidase complex. The use of phosphate-buffered saline rather than TRIS-buffered saline or borate-buffered saline was essential to obtain optimal results in the fluorescent avidin assay. Fixation of cells with either 0.5% paraformaldehyde or 10% neutral buffered formalin was found to be superior to either no fixation or fixation with cold methanol or cold 0.1% glutaraldehyde in ethanol. The sensitivities of the fluorescent avidin and avidin-peroxidase assays were shown to be identical, based on staining intensities and percentages of positive cells when both assays were performed on cells from 14 patients with acute lymphoblastic leukemia (ALL), and 5 patients with lymphoblastic lymphoma (LL).

Characterization of human prostatic acid phosphatase radioiodinated by four different procedures.

Prostatic acid phosphatase was labeled by four different iodination procedures: chloramine-T, insoluble or soluble lactoperoxidase, and Bolton-Hunter. Enzyme labeled by the chloramine-T procedure had suboptimal precipitation characteristics in immunoassays, with 36 to 67% of the tracer precipitated in antibody excess. Analysis of the tracer on 7.5% sodium dodecyl sulfate polyacrylamide gel showed that as much as 30% of the radioactivity migrated away from the protein within 6 minutes after the electrophoretic run was begun. The nature of this fast migrating radioiodinated component was not determined. The failure to obtain a tracer labeled by the chloramine-T procedure with optimal immunoprecipitation characteristics led to investigation of alternate radioiodination procedures. Insoluble or soluble lactoperoxidase was less effective than chloramine-T in producing a tracer suitable for radioimmunoassay. In contrast, the Bolton-Hunter procedure produced a tracer that had optimal precipitation characteristics; 88 to 91% of the tracer was precipitated in antibody excess. The tracer was further purified by affinity chromatography to minimize the possibility of other protein contaminants compromising the specificity of a radioimmunoassay for the enzyme.

Human prostatic acid phosphatase: purification, characterization, and optimization of conditions for radioimmunoassay.

Prostatic acid phosphatase was isolated from benign hypertrophic prostate tissue by ammonium sulfate precipitation and affinity chromatography procedures. The purified enzyme was characterized by two-dimensional gel electrophoresis and shown to have a cluster of protein spots with an apparent molecular weight of 48 000 at pI 5.9 to 6.3 in 9 mol/l urea. The specific activity of the purified enzyme was 723 and 659 U/mg protein with alpha-naphthyl phosphate at 30 degrees C and para-nitrophenyl phosphate at 37 degrees C respectively. An antibody to the purified enzyme was raised in rabbits and used in a radioimmunoassay (RIA). The use of a phosphate buffer, pH 6.6, and iodination of prostatic acid phosphatase (PAP) by the Bolton-Hunter procedure improved the precision of the assay when compared to RIA's using a phosphate buffer, pH 7.0 or 7.3, or PAP iodinated by a chloramine-T procedure. The former RIA displaced 50% of the tracer at 2 micrograms of enzyme per liter of serum. The between-run coefficient of variation for 11 assays ranged from 3.9-7.7% with serum at 1.3 to 5.6 micrograms PAP/l.

Ontogeny of postnatal hyoid and larynx descent in humans.

Postnatal descent of the hyoid and larynx relative to the palate and mandible, which occurs uniquely in humans, is an anatomical prerequisite for quantal speech. This study tested the hypothesis that spatial constraints related to deglutition impose greater restrictions on the rate and degree of hyo-laryngeal descent than do adaptations for vocalization. Ontogenetic data on changes in the size and shape of the pharynx, the vocal tract, and the spatial positions of the larynx, hyoid, mandible and hard palate relative to each other and to the oral cavity were obtained for 15 males and 13 females from a longitudinal series of lateral radiographs (the Denver Growth Study) taken between the ages of 1 month and 14 years. To establish growth patterns, nine linear dimensions of the pharynx and 15 different pharyngeal and vocal-tract proportions were regressed against percentage growth. The results demonstrate that certain aspects of vocal-tract shape change markedly during ontogeny, especially in the first postnatal year and during the adolescent growth spurt. The ratio of pharynx height to oral cavity length (which is important for speech) decreases significantly (P<0.001) from 1.5 to 1.0 between birth and 6-8 years, after which it remains stable. In contrast, regression analyses indicated that superoinferior spatial relations between the positions of the vocal folds, the hyoid body, the mandible and the hard palate do not change significantly throughout the entire postnatal growth period (P<0.05). Sexual dimorphism in pharyngeal shape and size before the age of 14 years is very limited. The results suggest that the descent of the hyoid and larynx relative to the mandible is constrained by muscle function related to deglutition, highlighting the different functional roles of the hyoid during speech and oral transport.

Issues for quality assurance in clinical flow cytometry.

An increasing number of clinical laboratories are using flow cytometry to analyze cells stained with fluorescent antibodies or dyes. Many articles discuss the analytical performance of immunofluorescent assays and DNA staining procedures. However, quality assurance transcends the performance of analytical methods and includes preanalytic, analytic, and postanalytic phases of the test procedure. This review focuses on preanalytic and analytic factors that affect the results of flow cytometric analysis of cells stained with fluorescent antibody or dyes specific for nucleic acid. The article reviews the controls used to assess instrument performance followed by a discussion of biological variables, and reagent, methodological, and instrumental factors that may affect the interpretation of these results.

Interference in immunoenzymometric assays caused by IgM anti-mouse IgG antibodies.

Serum samples from a female patient gave falsely elevated results in nine of ten immunoenzymometric assays (IEMAs) that used mouse monoclonal antibodies specific for human chorionic gonadotropin (n = 8), thyrotropin, or creatine kinase-MB isozyme. In contrast, normal results were obtained in five radioimmunoassays that used either mouse monoclonal antibodies or antisera specific for human chorionic gonadotropin (n = 3) or thyrotropin (n = 2). Incubation of her serum with IgG from different species or with F(ab)'2 from mouse IgG prior to IEMA showed that the interference was markedly inhibited by mouse IgG, indicating an antibody specific for the Fc portion of mouse IgG. The interfering activity was bound to and eluted from a column containing Protein A-Sepharose CL-4B. Fractionation of the eluted protein over another column containing Sephacryl S300 showed the activity was enriched in the first protein peak, which contained predominantly IgM. A model is proposed to explain how IgM anti-mouse IgG antibody selectively interferes in IEMAs that use mouse monoclonal antibodies.

Characterization and functional assessment of Clostridium histolyticum class I (C1) collagenases and the synergistic degradation of native collagen in enzyme mixtures containing class II (C2) collagenase.

OBJECTIVES: Clostridium histolyticum expresses two classes of collagenases, C1 and C2. However, degradation of these enzymes by proteases during the fermentation or purification process may lead to numerous molecular forms that lead to inconsistent release of islets from human pancreata. This report defines the amino acid sequence of the truncated forms of C1 (C1b or C1c) that contain a single collagen-binding domain (CBD) and investigates the synergy between the different forms of C1 collagenase and C2 to degrade native collagen. METHODS: Highly purified collagenase isoforms were purified from C. histolyticum culture supernatants using established column chromatography techniques and analyzed using high-pressure liquid chromatograph (HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). The collagen-degrading activity (CDA) assay was used to investigate the synergy between different collagenase molecular forms. RESULTS: MS was used to confirm the sequence of full-length C2 and C1 from the reported gene sequence. These results were correlated with the molecular weights observed on the SDS- PAGE and elution after analytical anion-exchange HPLC. HPLC peaks designated as C1b and C1c were both confirmed to be C1 lacking the terminal CBD. The only difference being the cleavage site leading to a 12 amino acid difference between the two forms. A non-additive synergy in CDA relative to activity of individual collagenases was observed for C2 with each of the three C1 molecular forms. The C1 molecular forms did not display this synergy in the absence of C2. CONCLUSIONS: These observations support earlier reports that suggest the two collagenases bind to different portions of the collagen and have different specificities to cut native collagen. Although the implications of this are not yet understood, they are fundamental in advancing the understanding of how collagenases work together along with the neutral protease to breakdown the extracellular matrix for islet isolation.

Tissue dissociation enzyme neutral protease assessment.

Neutral proteases, essential components of purified tissue dissociation enzymes required for successful human islet isolation, show variable activities and effects of substrate on their activities. Initially we used a spectrophotometric endpoint assay with azocasein substrate to measure neutral protease activity. After critical review of the results, we observed these data to be inconsistent and not correlating expected differences in specific activities between thermolysin and Bacillus polymyxa proteases. This observation led to the development of a fluorescent microplate assay using fluorescein isothyocyanate-conjugated bovine serum albumin (FITC-BSA) as the substrate. This simpler, more flexible method offered a homogeneous, kinetic enzyme assay allowing determination of steady state reaction rates of sample replicates at various dilutions. The assay had a linear range of 4- to 8-fold and interassay coefficients of variation for B polymyxa protease and thermolysin of <9% and <15%, respectively, which were lower than those using the spectrophotometric endpoint assay, namely, 54% and 36%, respectively. This format allowed for incorporation of enzyme inhibitors, as illustrated by addition of sulfhydryl protease inhibitors, which, consistent with earlier reports, strongly indicated that the main contaminant in purified collagenase preparations was clostripain. Determination of the specific activities for several purified neutral proteases showed that the B polymyxa and Clostridium histolyticum proteases had approximately 40% and 15% specific activities, respectively, of those obtained with purified thermolysin, indicating the different characteristics of neutral protease enzymes for cell isolation procedures.

Anthropoid cranial base architecture and scaling relationships.

This paper examines how various measures of basicranial length and cranial base angulation affect the relationship between basicranial flexion and relative brain size in anthropoids, including Homo sapiens. Most recent studies support the "spatial packing" hypothesis, that basicranial flexion in haplorhines maximizes braincase volume relative to basicranial length. However, a few studies find the basicranium is less flexed in H. sapiens than expected for other anthropoids, suggesting that other factors contribute to variation in hominin basicranial flexion. The measure of relative brain size used to test the spatial packing hypothesis, the Index of Relative Encephalization (IRE), is calculated with basicranial length (BL) in its denominator, so that shorter BL and larger brain size potentially inflate H. sapiens IREs. To investigate this problem, the lengths of midline cranial floor sections were scaled relative to the cube root of endocranial volume in 157 specimens from 18 anthropoid species. Results indicate that the posterior cranial base and planum sphenoideum are significantly shorter in H. sapiens than in other anthropoids, accounting for higher IREs. Including the cribriform plate in BL, advisable in studies using anthropoids, affects whether H. sapiens differs from other anthropoids for basicranial flexion vs. IRE. However, despite a shorter BL and elevated IRE, H. sapiens does not deviate significantly from the anthropoid relationship between basicranial flexion and relative brain size for two cranial base angles. Because different measures of cranial base angulation change how H. sapiens falls along the anthropoid regression line, it remains equivocal whether the basicranium is less flexed in H. sapiens than in other anthropoids when compared to relative brain size.

Posterior maxillary (PM) plane and anterior cranial architecture in primates.

This study tests several hypotheses of integration between the cranial base and face in primates. After reviewing the definition and anatomical basis for the posterior maxillary (PM) plane, which demarcates the back of the midface at its junction with the sphenoid, we demonstrate how the PM plane can be identified accurately on radiographs, and confirm that it maintains a 90 degrees angle relative to the Neutral Horizontal Axis of the orbits in all primates. In addition, we use the PM plane to test Dabelow's (1929) hypothesis that the orbits and anterior cranial base are more highly integrated in anthropoids than in strepsirrhines, and we test the hypothesis that the midline anterior cranial base (planum sphenoideum) and anterior cranial floor (planum sphenoideum plus cribriform plate) in primates are highly correlated with each other relative to the PM plane. The mean angle between the anterior cranial base and the PM plane does not differ significantly from 90 degrees in anthropoids, but differs significantly in strepsirrhines. The anterior cranial base and anterior cranial floor, however, correlate well with each other relative to the PM plane in both suborders of primates, independent of orbital orientation and configuration. The PM plane, anterior cranial base, and anterior cranial floor, therefore, form an integrated structural complex, a "facial block," whose orientation relative to the posterior cranial base influences craniofacial shape among anthropoids in which orbital orientation influences the orientation of the anterior cranial base. One such effect is that increases in cranial base flexion shorten the antero-posterior length of the nasopharynx.

The ontogeny of cranial base angulation in humans and chimpanzees and its implications for reconstructing pharyngeal dimensions.

This paper examines differences in the processes by which the cranial base flexes in humans and extends in chimpanzees. In addition, we test the extent to which one can use comparisons of cranial base angles in humans and non-human primates to predict vocal tract dimensions. Four internal cranial base angles and one external cranial base angle were measured in a longitudinal sample of Homo sapiens and a cross-sectional sample of Pan troglodytes. These data show that the processes of cranial base angulation differ substantially in these species. While the human cranial base flexes postnatally in a rapid growth trajectory that is complete by two years, the cranial base in P. troglodytes extends postnatally in a more prolonged skeletal growth trajectory. These comparisons also demonstrate that the rate of cranial base angulation is comparable for different measures, but that angles which incorporate different anterior cranial base measurements correlate poorly. We also examined ontogenetic relationships between internal and external cranial base angles and vocal tract growth in humans to test the hypothesis that cranial base angulation influences pharyngeal dimensions and can, therefore, be used to estimate vocal tract proportions in fossil hominids. Our results indicate that internal and external cranial base angles are independent of the horizontal and vertical dimensions of the vocal tract. Instead, a combination of mandibular and palatal landmarks can be used to predict dimensions of the vocal tract in H. sapiens. The developmental contrasts in cranial base angulation between humans and non-human primates may have important implications for testing hypotheses about the relationship between cranial base flexion and other craniofacial dimensions in hominid evolution.

A micromodification of the CH50 test for the classical pathway of complement.

A micromodification of the CH50 assay of Kabat and Mayer is described in which the total reaction volume has been reduced five-fold. This assay utilizes sheep red blood cells pooled from multiple animals rather than from a single animal to avoid screening procedures of red blood cells from individual sheep. Analysis of natural hemolysin activity in normal serum revealed a correlation with elevated CH50 titers. However, this correlation does not appear to effect the clinical interpretation of the results.

Three immunoassay methods evaluated for quantifying prealbumin (transthyretin) in serum.

We developed an immunoturbidimetric assay for prealbumin on the Cobas Bio centrifugal analyzer and compared results from this assay with those from a rate nephelometric assay (Beckman Instruments Inc.) and a radial immunodiffusion kit (Behring Diagnostics). All three assays were evaluated for precision, linearity, and correlation to each other for analysis of sera from pediatric patients. All assays gave similar results for patients' samples. Values were higher by the radial immunodiffusion assay than by the other two methods, which gave similar results for the same specimens. We conclude that the immunoturbidimetric and rate nephelometric assay for prealbumin are acceptable alternatives for quantifying prealbumin in serum and also have a faster turnaround time than radial immunodiffusion.

Isolation and characterization of electrophoretic variants of human prostatic acid phosphatase.

Prostatic acid phosphatase (EC 3.1.3.2) purified from benign hypertrophic prostate tissue was fractionated by preparative slab isoelectric focusing over a pH gradient of 3.16 to 7.16. Twenty-two of 29 fractions contained enzyme activity. We further examined each active fraction by determining the Michaelis-Menten constant and specific activity. The protein concentration used in the latter determination was estimated either spectrophotometrically or immunochemically by three different radioimmunoassays for the enzyme. Determination of specific activities for each fraction directly correlated enzyme activity with an immunochemical determination, which indicated the immunochemical relationships among different molecular species of the enzyme. We found that the Michaelis-Menten constants for the isolated fractions were similar to the Km value for purified, unfractionated enzyme. Most fractions analyzed by each immunoassay had similar specific activities; the few fractions with discrepant specific activities were found at either end of the pH gradient. The similarity in specific activities among the fractions indicates that RIAs involving polyclonal antisera detect all of the electrophoretic variants of the enzyme.