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Biomedical subjects

E J Miller

Publications and source records attributed to E J Miller.

At least 73 records · Page 4Linked to original sources
PubMed1991

Use of antibodies in the study of protein structure and function in lung diseases.

Proteins normally fold in a variety of three-dimensional structures. This variety of forms accounts for a multiplicity of functions. One of the major undertakings of modern biochemistry is to determine the structure of a given protein and in doing so learn about its function. Much of the effort in the field of protein chemistry is currently focused on defining areas or domains within the molecule that are responsible for its function. Antibodies specific for functional domains on a protein have been powerful tools in these studies and have provided a great deal of the current knowledge related to the location and function of well-defined structural areas within the protein molecules. The use of antibodies in the study of proteins has considerably advanced our knowledge of both the structure of proteins and their interaction with other molecules. In reviewing the use of antibodies for structure and/or function analysis, we have tried not only to review the techniques and applications involving antibodies but also to show how useful antibody reagents may be designed and prepared. In this review, some of the newer uses of antibodies in modern biology will be described, and a few illustrations of each will be provided.

Amino Acid Sequence↗
PubMed1990

A second fibronectin-binding region is present in collagen alpha chains.

The interactions of plasma fibronectin with alpha chains or cyanogen bromide fragments of collagen types I and II have been studied using a variety of techniques. Affinity chromatography of cyanogen bromide-cleaved type II collagen on immobilized fibronectin revealed the binding of cyanogen bromide fragment CB12 in addition to the previously characterized CB10. Using fluorescence polarization, we analyzed the interaction between the collagen peptides and fluorescein isothiocyanate-labeled 42-kDa gelatin-binding fragment of fibronectin in solution. Dissociation constants for the binding of CB10 and CB12 to the fibronectin fragment were calculated as 0.38 and 0.94 microM, respectively, indicating a lower affinity for the uncharacterized site. However, as with CB10, CB12 was able to compete effectively with the intact alpha chain for bindinng to fibronectin. Additionally, both CB10 and CB12 absorbed to tissue culture surfaces were each able to support fibronectin-dependent cell adhesion. Finally, the regions of alpha 2(I) homologous to CB12 and CB10 were found to be active in fibronectin binding, demonstrating the presence of two fibronectin-binding regions in this collagen chain.

Amino Acid Sequence↗
PubMed1990

Amino acid analysis of collagen hydrolysates by reverse-phase high-performance liquid chromatography of 9-fluorenylmethyl chloroformate derivatives.

A recently described procedure for amino acid analyses has been modified and adapted for use in quantitating the unique mixture of products commonly found in hydrolysates of the collagens. The method involves precolumn derivatization of hydrolysates with 9-fluorenylmethyl chloroformate (FMOC-CL), chromatographic separation of the derivatives and excess reagent on a reverse-phase column, and quantitation based on the fluorescent properties of the derivatives. The method takes advantage of the ease with which stable derivatives are formed with the FMOC reagent. Using a ternary gradient system, a complete amino acid analysis with good resolution of all components can be performed within 35 min. The sensitivity of the method is comparable to levels attained by other derivatives and the fluorescence response of each derivative is linear over the total range of 1-800 pmol. Given these parameters, the method allows complete amino acid analyses to be performed on 100 ng of collagen corresponding to a single picomole of a collagen chain (Mr 100,000).

Amino Acids↗
PubMed1990

Craniofacial osseous restoration with osteoinductive proteins in a collagenous delivery system.

Implants fabricated from mixtures of extracted bovine bone proteins and highly purified human type I collagen have been tested for efficacy in the restoration of osseous defects. Experimental implants composed of osteoinductive proteins and collagen, and control implants composed of collagen alone were placed in 15 x 15 mm cranial defects in New Zealand white rabbits. Implants harvested at 10, 12 and 14 week intervals were subjected to histological evaluation as well as quantitative analysis utilizing a computer morphometric planimeter. Defects filled with control implants displayed highly variable repair with a mean of 31% bony healing. Experimental implants were consistently effective in inducing significant bony regeneration with a mean of 91% repair within the time range of the study. In light of these results, it is apparent that an osteoinductive-collagenous implant system represents an efficacious treatment modality in osseous reconstruction and augmentation.

Animals↗
PubMed1990

Liberation of a neutrophil enzyme-releasing peptide from the surface of human alveolar macrophages.

The human alveolar macrophage product, enzyme-releasing peptide (ERP), has a molecular mass of 8,000 Da, and releases azurophilic and specific granule constituents from neutrophils. A murine monoclonal anti-ERP antibody (12E10H), previously used to show a lack of antigenic identity between ERP and C5a, interleukin 1, tumor necrosis factor, and gamma-interferon, showed no cross-reactivity with interleukin 8. 12E10H and a fluorescein-labeled second antibody were used to visualize ERP on the macrophage surface. ERP was removed from alveolar macrophages by a 3-min incubation with 5 X 10(-7) M bovine pancreatic trypsin at 37 degrees C. The washed trypsinized cells could readhere to plastic and exclude trypan blue. Dilution of the trypsin-derived ERP released myeloperoxidase from cytochalasin-B-treated neutrophils dose dependently. The enzyme-releasing ability of the trypsin-derived material was removed by immunoprecipitation using antibody 12E10H bound to Staphylococcal protein A Sepharose 4B. The estimated molecular mass of the trypsin-derived ERP (by molecular sieve chromatography on HPLC) was approximately 8,500 Da. Other proteases (plasmin, thrombin, and cathepsin G) also released ERP from the cell surface, but the ERP was not an active secretagogue for neutrophils. However, macrophages cultured with protease inhibitors did not show decreased ERP accumulation in the medium. Our data indicate that ERP exists on the surface of human alveolar macrophages and can be released by proteases found within the lung environment in some disease states.

Antibodies, Monoclonal↗
PubMed1990

Retroperitoneal accessory spleen.

We report a lower abdominal mass found to be a retroperitoneal accessory spleen. The vascular supply originated from retroperitoneal vessels independent of the splenic, testicular, and spermatic vessels.

Adult↗
PubMed1990

Pseudohypertestosteronemia during danazol therapy. A case report.

Several steroid radioimmunoassay kits, particularly testosterone, cross-react with danazol, producing false elevations of serum steroid levels. A patient taking danazol simultaneously developed signs of virilization and an apparent 15-fold elevation in her serum testosterone. Discontinuation of danazol resulted in a prompt return to physical and biochemical normality.

Adult↗
PubMed1989

Immune response of patients with congenital coagulation disorders to hepatitis B vaccine: suboptimal response and human immunodeficiency virus infection.

Seventy-eight patients with congenital coagulation disorders were treated with hepatitis B vaccine either subcutaneously or intradermally. All the children (eight vaccinated subcutaneously and eight vaccinated intradermally) responded. Seventeen of 19 (90%) anti-HIV-negative adults vaccinated subcutaneously and 14/25 (56%) anti-HIV-negative adults vaccinated intradermally showed an immune response. At 24 months, the anti-HBs level was greater than 10 IU/l in all children vaccinated subcutaneously, 83% of children vaccinated intradermally, 77% of adults vaccinated subcutaneously, and 55% of adults vaccinated intradermally. Eight of 15 (53%) adult patients who were anti-HIV positive were also anti-HBc positive before vaccination and 6/8 (75%) failed to produce an amnestic response to vaccine. Subcutaneous vaccination with regular monitoring of anti-HBs levels and appropriate boosting is recommended.

Adolescent↗
PubMed1989

Characterization of mammalian type IX collagen fragments from limited pepsin digests of a transplantable swarm rat chondrosarcoma.

Collagenous fragments from type IX molecules have been solubilized by limited pepsin proteolysis of a transplantable rat chondrosarcoma and isolated by selective salt precipitation. Chromatography of the solubilized precipitate on CM-cellulose under nondenaturing conditions yielded three fractions. When examined by polarimetry, the material in all three fractions revealed native collagen helical structure with melting points which ranged from 31-37 degrees C. When the fractions were denatured and rechromatographed on a column of agarose beads, the most acidic fraction eluted as 13-kDa polypeptides with and without prior reduction and alkylation. In contrast, the second and third fractions eluted as 100-kDa and 30-kDa polypeptides prior to reduction, but on reduction and alkylation produced reducible products of 34 kDa and 10 kDa, respectively. The general compositional features of the three fractions closely resemble comparable collagenous fragments of type IX collagen from other species. The denaturation products of the 13-kDa nonreducible, the 30-kDa reducible, and the 100-kDa reducible fractions were sequentially purified by CM-cellulose and reversed-phase chromatography to resolve the chain constituents. The isolated 10-kDa, 13-kDa, and 34-kDa chains were cleaved with CNBr, and the cleavage products identified by gel-permeation chromatography. Two 13-kDa polypeptides, 13K2 and 13K3, which did not contain any methionyl residues and were not cleaved with CNBr, were digested with trypsin, and the peptide digests were resolved by reversed-phase chromatography. Comparisons of the CNBr and tryptic cleavage products demonstrate that the three major collagenous fragments are composed of three unique polypeptides. A partial amino acid sequence of an 8-kDa CNBr peptide derived from a purified 10-kDa peptide (10K1) matches identically the amino acid sequence derived from a cDNA sequence in the rat alpha 1(IX) chain (Kimura et al., 1989). These studies, then, present convenient procedures useful in the isolation of mammalian type IX collagen fragments and describe features of the rat molecule, indicating that it is similar to the avian counterpart with respect to chain composition and general molecular structure.

Amino Acid Sequence↗
PubMed1989

The natural history of human immunodeficiency virus infection in a haemophilic cohort.

112 haemophilic patients infected with HIV were followed up with clinical and laboratory assessment between 1 December 1979 and 30 November 1988. Sixty-six (59%) of the patients developed HIV-related clinical symptoms and 22 (20%) developed AIDS. Twenty (18%) of the patients developed p24 antigenaemia. Amongst the 59 patients whose date of seroconversion could be estimated the calculated 8-year cumulative incidence of AIDS was 40% (symptoms 73%). For the whole cohort of 112 patients, the median slope of linear regression of the absolute T4 lymphocyte count was steeper for those with AIDS (-0.113 x 10(9)/l per year) than for those without AIDS (-0.054 x 10(9)/l per year) (P less than 0.02). While 15 cases of AIDS developed during 58 patient-years of follow up after falling below a T4 lymphocyte count of 0.2 x 10(9)/l, only two cases occurred during 450 patient-years before reaching this count. Thus the decline of the T4 lymphocyte count to 0.2 x 10(9)/l may be an appropriate additional end-point for the assessment of new treatments for asymptomatic patients infected with HIV.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Comparison of three tracers for detecting lung epithelial injury in anesthetized sheep.

We compared the ability of three aerosolized tracers to discriminate among control, lung inflation with a positive end expired pressure of 10 cmH2O, lung vascular hypertension and edema without lung injury, and lung edema with lung injury due to intravenous oleic acid. The tracers were 99mTc-diethylenetriaminepentaacetate (99mTc-DTPA, mol wt 492), 99mTc-human serum albumin (99mTc-ALB, mol wt 69,000), and 99mTc-aggregated albumin (99mTc-AGG ALB, mol wt 383,000). 99mTc-DTPA clearance measurements were not able to discriminate lung injury from lung inflation. The 99mTc-AGG ALB clearance rate was unchanged by lung inflation and increased slightly with lung injury. The 99mTc-ALB clearance rate (0.06 +/- 0.02%/min) was unchanged by lung inflation (0.09 +/- 0.02%/min, P greater than 0.05) or 4 h of hypertension without injury (0.09 +/- 0.04%/min, P greater than 0.05). Deposition of 99mTc-ALB within 15 min of the administration of the oleic acid increased the clearance rate to 0.19 +/- 0.06%/min, which correlated well with the postmortem lung water volume (r = 0.92, P less than 0.01). This did not occur when there was a 60-min delay in the deposition of 99mTc-ALB. We conclude that 99mTc-ALB is the best indicator for studying the effects of lung epithelial injury on protein and fluid transport into and out of the air spaces of the lungs in a minimally invasive manner.

Animals↗
PubMed1989

Search for drugs that may reduce the load of neutrophil azurophilic granule enzymes in the lungs of patients with emphysema.

Neutrophil elastase and myeloperoxidase probably play an important role in the development of pulmonary emphysema. We have analyzed drugs from the major classes of agents that alter neutrophil function to determine if there are drugs in use today that can reduce the load of neutrophil elastase or myeloperoxidase in the lungs of smokers. Eleven representative drugs were tested for their ability to inhibit chemotaxis and degranulation. None of the drugs inhibited chemotaxis in a dose-response fashion at concentrations achievable in human plasma. Sulfinpyrazone, phenylbutazone, and auranofin completely inhibited the release of azurophilic granules (myeloperoxidase) and tertiary granules (beta-D-glucuronidase) when formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) was used as the stimulant, and inhibited azurophilic granule release by 69%, 19%, and 64% respectively, but not tertiary granule release when macrophage-conditioned media was used as the stimulus. In conclusion, none of the drugs tested are inhibitors of chemotaxis; however, three are excellent inhibitors of azurophilic granule enzyme release. Of these three, sulfinpyrazone, a drug that is not currently used clinically for its antiinflammatory effects, is the least toxic and should be considered as a potential drug to reduce the elastase and myeloperoxidase load in the lungs of smokers who are developing emphysema.

Anti-Inflammatory Agents↗
PubMed1988

A review of matrix-induced osteogenesis with special reference to its potential use in cranio-facial surgery.

Current data suggests that purified osteoinductive protein packaged into a collagenous delivery vehicle represents a viable alternative to restoring osseous defects with conventional implant materials. The proteins responsible for matrix-induced osteogenesis are the same as those that initiate and complete osseous repair of fractures. Consequently, the process of matrix-induced osteogenesis produced by implantation of the osteoinductive protein mimics the naturally occurring phenomenon of reparative osteogenesis. The collagenous delivery vehicle apparently serves the same role as the inactive collagenous bone matrix which is left after removal of the osteoinductive proteins from demineralized bone and, is required if osteogenesis is to occur. This collagenous delivery vehicle apparently serves a scaffold for the migration into, proliferation, and subsequent differentiation of cells responsible for osteogenesis. In addition, the original form of the collagenous implant dictates the dimensions of the resulting bony tissue. Finally, it is possible to generate an osteoinductive response in many mammals probably including man with purified xenogenic osteoinductive proteins. In conclusion, there is currently much research investigating the phenomenon of matrix-induced osteogenesis and its potential clinical use. In the past few years, there has been a sharp increase in the number of manuscripts published in this area of connective tissue research. Consequently, if the current trend continues, clinicians in the near future can look forward to seeing this material being developed into a convenient "off the shelf" bone replacement system which stimulates host bone formation and repair of cranio-facial osseous defects.

Animals↗
PubMed1988

Autoimmunity to collagen in adult periodontal disease.

Although it has been documented that exogenous antigens of microbial origin are involved in the induction of the local inflammatory responses in human adult periodontitis (AP), endogenous antigens may contribute to the chronicity of this common disease. In this study, we used the enzyme-linked immunospot (ELISPOT) test to enumerate antibody-secreting cells to human collagen Types I-VI by cells isolated from the gingivae and peripheral blood of AP patients. Analyses of dissociated cells from gingivae of 39 AP patients revealed the presence of high numbers of cells that secrete antibodies to Type I collagen, and to a lesser extent, Type III. Although the majority of such cells produced specific antibodies of the IgG class, IgA- and IgM- anti-collagen -secreting cells were also detected. When compared to the total antibody-producing cells, the numbers of cells forming specific antibodies to collagen Type I were surprisingly high. In contrast, anti-collagen antibody-producing cells were rarely detected in the peripheral blood of patients with adult periodontal disease and only low levels of anti-collagen antibodies were present in the serum. The finding of local production of anti-collagen antibodies in AP suggests that autoimmunity may contribute to the pathogenesis of this common disease.

Antibody-Producing Cells↗
PubMed1988

Domain structure and functional analysis of the carboxyl-terminal polyacidic sequence of the RAD6 protein of Saccharomyces cerevisiae.

The RAD6 gene of Saccharomyces cerevisiae, which is required for normal tolerance of DNA damage and for sporulation, encodes a 172-residue protein whose 23 carboxyl-terminal residues are almost all acidic. We show that this polyacidic sequence appends to RAD6 protein as a polyanionic tail and that its function in vivo does not require stoichiometry of length. RAD6 protein was purified to near homogeneity from a yeast strain carrying a RAD6 overproducing plasmid. Approximately the first 150 residues of RAD6 protein composed a structural domain that was resistant to proteinase K and had a Stokes radius typical of a globular protein of its calculated mass. The carboxyl-terminal polyacidic sequence was sensitive to proteinase K, and it endowed RAD6 protein with an aberrantly large Stokes radius that indicates an asymmetric shape. We deduce that RAD6 protein is monomeric and comprises a globular domain with a freely extending polyacidic tail. We tested the phenotypic effects of partial or complete deletion of the polyacidic sequence, demonstrating the presence of the shortened proteins in the cell by using antibody to RAD6 protein. Removal of the entire polyacidic sequence severely reduced sporulation but only slightly affected survival after UV irradiation or UV-induced mutagenesis. Strains with deletions of all but the first 4 or 15 residues of the polyacidic sequence were phenotypically almost wild type or wild type, respectively. We conclude that the intrinsic activity of RAD6 protein resides in the globular domain, that the polyacidic sequence has a stimulatory or modifying role evident primarily in sporulation, and that only a short section apparently functions as effectively as the entire polyacidic sequence.

Alleles↗