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A comparison of the avidin-biotin-peroxidase complex (ABC) and peroxidase-anti-peroxidase (PAP) immunocytochemical techniques for demonstrating Sendai virus infection in fixed tissue specimens.

Mice were infected with Sendai virus and killed 8 days later. Lungs were removed and perfused with ethanol, 10% neutral formalin, Bouin's, B-5, or Zenker's fixatives. Tissues were dehydrated, embedded in paraffin, sectioned and stained for the presence of Sendai virus using the avidin-biotin-peroxidase-complex (ABC) and peroxidase antiperoxidase (PAP) immunocytochemical techniques. Results of these techniques were compared. The ABC technique was more sensitive than the PAP. Sendai antigen was demonstrated by the ABC technique in lung tissue fixed with any fixative, whereas antigen could be demonstrated with consistency only in ethanol-fixed lung by the PAP technique. Trypsin treatment of lung prior to immunoperoxidase treatment failed to enhance staining with either technique and actually caused a decrease in staining in ethanol, B-5 and Zenker's-fixed specimens.

Animals↗

Immunological determinants of proteoglycans. Antibodies against the unsaturated oligosaccharide products of chondroitinase ABC-digested cartilage proteoglycans.

Bovine nasal cartilage proteoglycan monomer which had been digested with chondroitinase ABC to form the keratan sulfate-protein core was injected into rabbits. High titer antiserum was obtained as judged by the binding of 125I-labeled keratan sulfate-protein core. Native proteoglycan did not inhibit the interaction of the antiserum with labeled keratan sulfate-protein core. However, the disaccharide obtained from chondroitin 4-sulfate by the action of chondroitinase ABC, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose, inhibited the interaction 50% at a concentration of 500 microM. The corresponding 6-sulfated and nonsulfated disaccharides at the same concentration gave 15% and 10% inhibition, respectively. Chondroitinase-digested proteoglycan from Swarm rat chondrosarcoma was strongly inhibitory, while the hyaluronidase-digested chondrosarcoma proteoglycan exhibited no detectable inhibition. Evidently, the antiserum raised against chondroitinase ABC-digested bovine nasal cartilage proteoglycan contains antibodies which recognize the unsaturated uronic acid residue linked to N-acetylgalactosamine 4-sulfate. These antibodies will be valuable for identifying and quantitating chondroitin 4-sulfate-containing proteoglycans in tissues.

Animals↗

[Change in the turnover of HLA ABC molecules in circulating T lymphocytes in mycosis fungoides].

The turnover of HLA ABC molecules at T and B lymphocyte surface was analyzed in five cases with mycosis fungoides and six healthy controls. The patients had only skin lesions and are staged from T1 to T3 and No, Bo and Mo. The turnover was analyzed by mean of the decrease of the lysis by complement on sensitisized cells with anti HLA ABC antibodies that were incubated for progressive times at 37 degrees C. The results show a largest turnover for the HLA ABC molecules all T cells surface from mycosis fungoides that was significant (PWilcoxon less than 0.01). The turnover of B cells surface was not different from mycosis fungoide and healthy controls. The observed phenomenon was not specific for mycosis fungoide or T cells because previously has been shown in others lympho-proliferative and autoimmune diseases. The authors suggest that the analyzed T lymphocyte, morphologically normal is functionally altered and the behavious could be a metabolic phenotypic marker for the T cell before his coming at skin lesions.

Adult↗

Aspiration biopsy cytology (ABC). its use in diagnosis of lesions of the prostate gland.

Aspiration biopsy cytology (ABC) specimens were obtained from 540 patients by transrectal biopsy because of suspicious lesions or metastases from occult carcinoma. Although poorly differentiated prostatic carcinoma had previously presented few diagnostic problems by ABC, cells from well-differentiated adenocarcinoma had been diagnosed with a low degree of accuracy. To improve recognition of tumor cells, earlier specimens that had yielded false-negative results were rescreened and additional diagnostic criteria observed. By means of these newly supplemented criteria of malignancy, a positive diagnosis was made in 92% of our 40 most recently verified malignant neoplasms, in contrast with 78% in a previous study. Aspirates from 55 men with treated carcinoma were examined for recurrence. Clinical and ABC findings were correlated. Although glands palpated as minuscule or very large correlated precisely with absence or presence of recurrence, in the rest both size and firmness were not necessarily proportional to tumor activity. Thus, fine-needle biopsy has permitted the addition of an accurate, simple study for early evaluation of the recurrence of malignant neoplasm.

Adenocarcinoma↗

"AEIOU: the ABC's" of conversion from laparoscopic to open cholecystectomy.

BACKGROUND AND OBJECTIVES: To examine and classify the reasons for conversion and the points at which laparoscopic cholecystectomies are converted to open procedures and whether these change over time. METHODS: This is a retrospective study of all patients undergoing cholecystectomy from June 1, 1990 to June 30, 1995. Reasons for conversion were classified using the "AEIOU:ABC" system developed for this study and conversion points were assigned chronologically. RESULTS: The "AEIOU:ABC" classification system was utilized. The most common reasons for conversion were: acute inflammation N = 61 (26.1%); adhesions N = 51 (21.8%); and organ system pathology N = 39 (16.7%). The most common conversion points were; after visualization of the peritoneal cavity but prior to dissection of the cystic structures N = 103 (44.0%); dissection of the cystic structures N = 58 (24.8%); initial laparoscopy N = 36 (15.4%). When the reasons for conversion were evaluated for changes over time there was no statistically significant change for the total group or any individual surgeon. Conversion points did not change with increasing operative experience. CONCLUSION: The "AEIOU:ABC" classification system is a simple, effective and easy to use system for classifying the myriad of reasons for conversion. The system needs to be validated prospectively not only for laparoscopic cholecystectomy but for possible application to other laparoscopic procedures.

Cholecystectomy↗

Intraocular pressure and outflow facility are unchanged following acute and chronic intracameral chondroitinase ABC and hyaluronidase in monkeys.

We determined the effect of chronic and acute loss of glycosaminoglycans from the aqueous outflow pathway on intraocular pressure (IOP) and outflow facility in the subhuman primate eye. For the study of the chronic effects of the GAGases, cynomolgus monkeys received intracameral injections of hyaluronidase (Streptomyces, 5 or 50 units, n=2) or chondroitinase ABC (0.05 or 0.25 units, n=2) biweekly for 8 months (4 months for each dose). IOP was measured at 3, 7, 10 and 14 days after each injection. Outflow facility (2-level constant pressure) was determined at 2, 4, 6, and 8 months. Monkeys were killed 6 days after the last injection. The changes in the distribution of hyaluronic acid and sulfated proteoglycans in the outflow pathway were examined using substrate-specific histochemical techniques. The acute effects of these enzymes on outflow facilities (30 min or 2 hr after enzyme) were determined in another group of animals (n=4 for each time enzyme-1). IOP and outflow facility were unchanged compared to controls (heat inactivated enzyme) at any time in the chronically or acutely treated monkeys. Hyaluronic acid staining was absent in the outflow pathways of eyes treated chronically with hyaluronidase compared with control eyes, while collagen-associated sulfated proteoglycans were decreased but not completely removed by the chronic chondroitinase ABC treatment. Chronic loss of these glycosaminoglycans from the trabecular meshwork does not appear to contribute to the IOP elevation and decrease in outflow facility that accompanies open-angle glaucoma. Most importantly, no increase in outflow facility was found with acute hyaluronidase or chondroitinase treatment.

Animals↗

Acute and long-lasting changes in extracellular-matrix chondroitin-sulphate proteoglycans induced by injection of chondroitinase ABC in the adult rat brain.

Lattice-like perineuronal accumulations of extracellular-matrix proteoglycans have been shown to develop during postnatal maturation and to persist throughout life as perineuronal nets (PNs) in many brain regions. However, the dynamics of their reorganization in adults are as yet unknown. The aim of the present study was to examine the capability of PNs for reconstitution after experimental destruction and to search for possible consequences of extracellular-matrix degradation for neurons and glial cells. The changes were induced by single intracortical injections of Proteus vulgaris chondroitinase ABC and studied after postinjection periods of 1 day to 5 months. The N-acetylgalactosamine-binding Wisteria floribunda agglutinin (WFA), an antibody against chondroitin-sulphate proteoglycans, three antibodies recognizing initial chondroitin or chondroitin-sulphate moieties ('stubs') of proteoglycan core proteins, an antibody against the hyaluronan-binding protein component of versican, and biotinylated hyaluronectin, which binds to hyaluronan, were used as cytochemical markers. One day postinjection, the WFA-binding sites and hyaluronan were shown to be almost completely removed within a circumscribed digestion zone. The staining of different core-protein components revealed only fragments of PNs. These changes were found to be partly compensated 4 weeks after injection of chondroitinase ABC. After 8 and 12 weeks postinjection, the cytochemical and structural characteristics as well as the area-specific distribution patterns of PNs were progressively reconstituted. At 5 months postinjection, they could not be distinguished from those in untreated tissue. In contrast to such transient changes, a diffuse chondroitin-sulphate proteoglycan immunoreactivity persisted in the neuropil. Loss of neurons or alterations of their structure as well as reactions of glial cells were not observed. We conclude from this study that PNs, enzymatically destroyed in the adult rat brain, can be completely reconstituted, but the restoration of their extracellular-matrix components needs several months.

Age Factors↗

Cartilage degradation by hyaluronate lyase and chondroitin ABC lyase: a MALDI-TOF mass spectrometric study.

Matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI-TOF MS) has been used to investigate degradation products of two selected polysaccharides of cartilage (chondroitin sulfate and hyaluronic acid). Testicular hyaluronate lyase and chondroitin ABC lyase were used for enzymic digestion of both polysaccharides as well as of cartilage specimens. Polysaccharide solutions and cartilage supernatants were assayed by positive and negative MALDI-TOF MS. Especially chondroitin ABC lyase produced high amounts of digestion products (unsaturated di- and tetrasaccharides) from polysaccharides as well as from cartilage, clearly monitored by MALDI-TOF MS. It is concluded that MALDI-TOF MS provides a precise and fast tool for the determination of oligosaccharides since no previous derivatization is required.

Animals↗

Chondroitinase ABC enhances axonal regrowth through Schwann cell-seeded guidance channels after spinal cord injury.

Grafting of Schwann cell-seeded channels into hemisected adult rat thoracic spinal cords has been tested as a strategy to bridge the injured cord. Despite success in guiding axonal growth into the graft, regeneration across the distal graft-host interface into the host spinal cord was limited. We hypothesized that chondroitin sulfate (CS) glycoforms deposited at the gliotic front of the interface constitute a molecular barrier to axonal growth into the host cord. Because CS glycoforms deposited by purified astrocytes in vitro were removable by digestion with chondroitinase ABC, we attempted to achieve likewise by infusion of the enzyme to the host side of the interface. By 1 month post-treatment, significant numbers of regenerating axons crossed an interface that was subdued in macrophage/microglia reaction and decreased in CS-immunopositivity. The axons extended as far into the caudal cord as 5 mm, in contrast to nil in vehicle-infused controls. Fascicular organizations of axon-Schwann cell units within the regenerated tissue cable were better-preserved in enzyme-treated cords than in vehicle-infused controls. We conclude that CS glycoforms deposited during gliosis at the distal graft-host interface could be cleared by the in vivo action of chondroitinase ABC to improve prospects of axonal regeneration into the host spinal cord.

Animals↗

Chondroitinase ABC promotes axonal regeneration of Clarke's neurons after spinal cord injury.

We examined whether enzymatic digestion of chondroitin sulfate (CS) promoted the axonal regeneration of neurons in Clarke's nucleus (CN) into a peripheral nerve (PN) graft following injury of the spinal cord. After hemisection at T11, a segment of PN graft was implanted at the lesion site. Either vehicle, brain-derived neurotrophic factor (BDNF) or chondroitinase ABC was applied at the implantation site. The postoperative survival period was 4 weeks. Treatment with vehicle or BDNF did not promote the axonal regeneration of CN neurons into the PN graft. Application of 2.5 unit/ml chondroitinase ABC resulted in a significant increase (12.8%) in the number of regenerated CN neurons. Double labeling with Fluoro-Gold and NADPH-diaphorase histochemistry showed that the regenerated CN neurons did not express nitric oxide synthase (NOS). Our results suggest that CS is inhibitory to the regeneration of CN neurons following injury of the spinal cord.

Animals↗

Effects of hyaluronan lyase, hyaluronidase, and chondroitin ABC lyase on mammalian vitreous gel.

PURPOSE: To determine the effects of enzymes on mammalian vitreous gel and to thus infer the structural roles of hyaluronan and chondroitin sulfate in the gel. METHODS: The wet weights of bovine vitreous gels were compared before and after incubation with Streptomyces hyaluronan lyase, chondroitin ABC lyase, testicular hyaluronidase, or buffer alone. The extent of hyaluronan depolymerization was determined by chromatography and that of chondroitin sulfate depolymerization by western blot analysis. RESULTS: After digestion with Streptomyces hyaluronan lyase (30 U/gel), the gel wet weight was the same as that of controls (incubated with buffer alone) despite 94% of the hyaluronan having been depolymerized; when digested with 100 U/gel, the gel wet weight decreased (to 57% of original wet weight versus 86% for controls, P = < 0.001) and hyaluronan was completely depolymerized. Chondroitin ABC lyase digestion (0.2 U/gel) resulted in a slight reduction in gel wet weight (90% versus 96%, P = < 0.001) and depolymerization of 88% of the hyaluronan; the presence of fully digested chondroitin sulfate chains was established. Digestions with 100 and 500 U/gel of testicular hyaluronidase resulted in a decrease (P = < 0.001, both cases) in gel wet weight (53% versus 82%, 100 U/gel; 57%, versus 86%, 500 U/gel) with 75% and 97% hyaluronan depolymerization, respectively. CONCLUSIONS: Depolymerization of all vitreous hyaluronan and of chondroitin sulfate resulted in gel wet weight reduction but not gel destruction. Digestion with 30 U/gel of Streptomyces hyaluronan lyase revealed a small pool (6%) of relatively enzyme-resistant hyaluronan that specifically contributed toward maintaining gel wet weight.

Animals↗

Evaluation of candesartan cilexetil in black patients with systemic hypertension: the ABC Trial.

To assess the efficacy and tolerability of the angiotensin-II receptor blocker candesartan cilexetil, the Association of Black Cardiologists (ABC) coordinated a 12-week, multicenter, double-blind, placebo-controlled clinical trial in 304 black adults with a sitting diastolic blood pressure (DBP) of 91 to 105 mmHg, inclusive. The goal of the ABC Trial was to address previous reports suggesting that drugs that block the renin-angiotensin-aldosterone system may not be effective in controlling BP in hypertensive black individuals. Patients were randomized to receive candesartan cilexetil 16 mg (n = 156) or placebo (n = 148), once daily after a 4- to 5-week single-blind, placebo run-in period. Candesartan cilexetil and placebo doses were doubled if the trough (24 +/- 3 hours after dose) sitting DBP was 90 mmHg or higher after 4 weeks. If the DBP was 90 mmHg or higher at week 8, hydrochlorothiazide 12.5 mg was added to both placebo or candesartan cilexetil 32 mg. Candesartan cilexetil was significantly more effective than placebo in reducing trough sitting systolic BP (SBP) and DBP at week 8 (6.4/5.1 mmHg versus 1.3/2.7 mmHg) and at week 12 (9.3/7.5 mmHg versus 5.7/5.2 mmHg). Hydrochlorothiazide was added in 27 and 50% of patients in the candesartan cilexetil and placebo groups, respectively. Control and responder rates were consistently higher in the candesartan cilexetil treatment group at weeks 8 and 12. Discontinuation and adverse event rates were similar in the two groups. Candesartan cilexetil once daily was effective in reducing BP in a substantial proportion of an exclusively black hypertensive population. The combination of candesartan cilexetil plus hydrochlorothiazide demonstrated additional efficacy while maintaining an excellent tolerability profile.

Adolescent↗

Effects of intracameral injection of chondroitinase ABC in vivo.

A glycosaminoglycan-degrading enzyme, chondroitinase ABC, was introduced into the anterior chamber of three cynomolgus monkeys. Following the injection, the intraocular pressure decreased in the experimental eyes. Depending on the monkey, the intraocular pressure was lowered for 5, 7, or 14 days before it returned to the normal level. Repeated injections produced a similar response. Structurally, the intertrabecular spaces appeared widened, and marked ballooning of the juxtacanalicular tissue was observed. The outer trabecular beams and the inner wall of Schlemm's canal were greatly disorganized. Considerable loss of the juxtacanalicular tissue was noted even 2 months after the enzyme was injected into the anterior chamber. These observations suggest that chondroitinase ABC digested the trabecular glycosaminoglycans, triggering intraocular pressure reduction and causing disorganization of the extracellular matrices of the disorganized trabecular beams, especially in areas near Schlemm's canal.

Animals↗

Coordinated downregulation of the antigen presentation machinery and HLA class I/beta2-microglobulin complex is responsible for HLA-ABC loss in bladder cancer.

Downregulation of MHC class I expression is a widespread phenomenon used by tumor cells to escape antitumor T-cell-mediated immune responses. These alterations may play a role in the clinical course of the disease. The aim of our study was to investigate the molecular mechanism underlying the absence of HLA-class I molecule expression in bladder cancer cells. Microdissected tumor tissues were characterized by real-time quantitative PCR for the expression of HLA-ABC, beta2-microglobulin and the members of the antigen processing machinery (APM) of HLA class I molecules (LMP2, LMP7, TAP1, TAP2 and tapasin). Our results showed that irreversible HLA loss by mutations in the beta2-microglobulin gene was not the cause of low HLA class I expression in bladder cancers. In contrast, we observed a coordinated transcription downregulation of HLA-ABC and beta2-microglobulin and APM genes in microdissected tumor tissue derived from bladder carcinomas. This mechanism may represent a major factor for the downregulation of HLA class I expression and in the subsequent direct recognition of cancer cells by cytolytic T lymphocytes. Because this regulatory mechanism is frequently reversible by IFN-gamma treatment, we conclude that HLA class I expression should be a major consideration for immunotherapeutic purposes in patients with bladder cancer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

ABC transporters Cdr1p, Cdr2p and Cdr3p of a human pathogen Candida albicans are general phospholipid translocators.

We have used fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-tagged phospholipid analogues, NBD-PE (phosphatidylethanolamine), NBD-PC (phosphatidylcholine) and NBD-PS (phosphatidylserine), to demonstrate that Cdr1p and its other homologues, Cdr2p and Cdr3p, belonging to the ATP-binding cassette (ABC) superfamily behave as general phospholipid translocators. Interestingly, CDR1 and CDR2, whose overexpression leads to azole resistance in C. albicans, elicit in-to-out transbilayer phospholipid movement, while CDR3, which is not involved in drug resistance, carries out-to-in translocation of phospholipids between the two monolayers of plasma membrane. Cdr1p, Cdr2p and Cdr3p could be further distinguished on the basis of their sensitivities to different inhibitors. For example, the in-to-out activity associated with Cdr1p and Cdr2p is energy-dependent and sensitive to sulphydryl blocking agents such as N-ethylmaleimide (NEM) and cytoskeleton disrupting agent cytochalasin E, while Cdr3p-associated out-to-in activity is energy-dependent but insensitive to NEM and cytochalasin E. We found that certain drugs, such as fluconazole, cycloheximide and miconazole, to which Cdr1p confers resistance could also affect in-to-out transbilayer movement of NBD-PE, while the same drugs had no effect on Cdr3p-mediated out-to-in translocation of NBD-PE. The ineffectiveness of these drugs to affect Cdr3p mediated out-to-in phospholipid translocation further confirms the inherent difference in the directionality of phospholipid translocation between these pumps. Notwithstanding the role of some of the Cdrps in drug resistance, this study clearly demonstrates that these ABC transporters of C. albicans are phospholipid translocators and this function could represent one of the physiological functions of such large family of proteins.

ATP-Binding Cassette Transporters↗

Evaluation of clinical and environmental anti-estrogens with human estrogen receptor expressed in Saccharomyces cerevisiae: a novel role for ABC-cassette transporters in mediating anti-estrogenic activity.

The effectiveness of anti-estrogens in treating estrogen-dependent diseases is limited by the acquired resistance of some diseases to anti-estrogens. This effect could occur by the export of anti-estrogens by cell membrane transport proteins. To study this phenomenon we have expressed human estrogen receptor (hER) and an estrogen-sensitive reporter in wild-type yeast and two transport-defective strains. In the wild-type strain, the most effective anti-estrogen was nafoxidine. 4-Hydroxy tamoxifen and clomiphene were inactive whereas tamoxifen had significant inhibitory activity in the wild-type strain. Using a strain missing the ABC-cassette transporter Snq2, clomiphene had anti-estrogenic activity. 4-Hydroxy tamoxifen had anti-estrogenic activity only in yeast lacking the transporter Pdr5. Whole cell binding assays indicated that 4-hydroxy tamoxifen is exported by Pdr5. Environmental chemicals such as polychlorinated biphenyls function as partial estrogens and anti-estrogens in yeast. In the absence of Pdr5 or Snq2, the estrogenic activity of 4-hydroxy, 2',4',6'-trichloro biphenyl (3-PCB) was substantially reduced in comparison to its activity in the wild-type strain. Interestingly, the antiestrogenic activity of 3-PCB was equivalent in the wild-type and transporter-defective strains. Our results suggest a novel role for ABC-cassette transporters in regulating the activity of clinical and environmental anti-estrogens.

ATP-Binding Cassette Transporters↗

Another link between phospholipid transmembrane migration and ABC transporter gene family, inferred from a rare inherited disorder of phosphatidylserine externalization.

The mechanisms involved in the maintenance or loss of the asymmetric distribution of phospholipids in the cell plasma membrane remain mysterious. In the yeast Saccharomyces cerevisiae, the transmembrane migration of certain phospholipids is controlled by transcription regulators of various ATP-binding cassette (ABC) transporters. The P-glycoprotein membrane transporters encoded by the multidrug resistance (MDR) genes, members of the ABC protein family, act as lipid translocases in mammalian cells. We report here the lack of expression of MDR genes in lymphoblasts derived from the B cells of a patient with an inherited Scott syndrome, characterized by impaired transmembrane migration of procoagulant phosphatidylserine and hemorrhagic complications. From microsatellite analysis of 7q21.1 and functional assessment, the most likely explanation accounting for Scott phenotype is a mutation in an unlinked gene coding for a regulatory protein necessary for the expression of MDR genes. Because phosphatidylserine externalization is also one of the hallmarks of cells undergoing apoptosis, these observations are suggestive of a relationship between basic processes such as multidrug transport, apoptosis and procoagulant phospholipid exposure.

ATP Binding Cassette Transporter, Subfamily B↗

Genomic organization of the human cholesterol-responsive ABC transporter ABCA7: tandem linkage with the minor histocompatibility antigen HA-1 gene.

We have recently cloned a novel cholesterol-responsive ABC transporter, designated ABCA7, which is predominantly expressed in human leukocytes. Here we report the structure of the human ABCA7 gene. The ABCA7 gene spans a region of approximately 32 kb and comprises 46 exons. Its putative promoter sequence contains potential binding sites for transcription factors with roles in hematopoiesis and cholesterol metabolism. Surprisingly, sequence analysis of the ABCA7 3' gene flanking region revealed that the terminal exon of ABCA7 borders immediately on the 5' end of the coding region of the recently identified human minor histocompatibility antigen HA-1. We demonstrate that the coding regions of ABCA7 and HA-1 are physically separated by a 1.7-kb intergene region. Subsequent genomic structure analysis showed that the HA-1 gene consists of 23 exons which extend across a 16-kb genomic region. Our results provide evidence that the genes for the human minor histocompatibility antigen HA-1 and the ABC transporter ABCA7 are arranged in a head-to-tail array and that both genes localize to a common locus of approximately 48 kb size on chromosome 19p13.3.

ATP-Binding Cassette Transporters↗