Biomedical subjects

D J Allen

Publications and source records attributed to D J Allen.

At least 37 records · Page 2Linked to original sources

PubMed1997

Immunological control of murine gammaherpesvirus infection is independent of perforin.

Perforin-mediated cytotoxic T cell killing has been suggested to be of importance in the control of noncytopathic virus infections, based on studies with lymphocytic choriomeningitis virus (LCMV). We examined the role of perforin in a mouse model of gammaherpesvirus infection using transgenic perforin-deficient mice. Previous work from this laboratory has shown that CD8 T cells are essential for the resolution of the acute lung infection and control of latently infected B cells in murine gamma-herpesvirus 68 infection. The absence of perforin did not significantly affect the kinetics of either the lytic lung infection or the latent spleen infection. Lymphocytes from both perforin-deficient and control mice secreted comparable levels of IFN-gamma, IL-10 and IL-6. In addition, lymphocytes from both strains had similar levels of CD3epsilon-dependent cytotoxic activity in the spleen, draining lymph nodes and bronchoalveolar lavage. These data indicate that the lack of perforin has little affect on the ability of mice to control an experimental gammaherpesvirus infection.

Animals↗

PubMed1997

Pathological changes in the spleens of gamma interferon receptor-deficient mice infected with murine gammaherpesvirus: a role for CD8 T cells.

Murine gammaherpesvirus is a natural rodent pathogen which causes a primary infection in the lungs and establishes a persistent infection in B lymphocytes. During the primary infection, large amounts of gamma interferon (IFN-gamma) are produced by spleen, mediastinal, and cervical lymph node cells. To investigate the role of IFN-gamma in control of the virus infection, mice lacking the cellular receptor for IFN-gamma (IFN-gamma R-/- mice) were infected with murine gammaherpesvirus 68 (MHV68). IFN-gamma R-/- mice showed no difference from wild-type mice in the titers of infectious virus in the lungs or in the rate of clearance of the lung infection. In the spleen, however, clear differences were observed. By 14 days postinfection, spleens from IFN-gamma R-/- mice were pale, shrunken, and fibrous. Histological examination showed that there was an early (day 10) infiltration of granulocytes followed by widespread destruction of splenic architecture (days 14 to 17). A marked decrease in the number of splenic B cells and CD4+ and CD8+ T cells occurred. These changes were accompanied by a 10- to 100-fold greater load of latently infected cells in IFN-gamma R-/- mice than in wild-type mice at 14 to 17 days postinfection, but this was reduced to the levels found in wild-type mice by 21 days postinfection. Treatment of the mice with the antiviral drug 2'-deoxyl-5-ethyl-beta-4'-thiouridine from 6 days postinfection did not prevent the occurrence of these changes. The changes were, however, completely reversed by depletion of CD8+ T cells prior to and during the primary infection. Depletion of CD4+ T cells also reversed the major pathological and virological changes, although in this case there was evidence of some histological changes. Thus, the lack of IFN-gamma receptor had profound consequences in spleens of MHV68-infected mice. The possible mechanisms involved in these changes are discussed.

Animals↗

PubMed1996

Absence of splenic latency in murine gammaherpesvirus 68-infected B cell-deficient mice.

Murine gammaherpesvirus 68 (MHV-68) is a natural pathogen of mice which causes an acute lung infection and establishes a latent infection in B lymphocytes. In this paper we describe the infection in transgenic B cell-deficient (muMT) mice, to determine whether a latent infection can be established in a mouse lacking circulating B lymphocytes. Little difference was observed in the acute lung infection, although there was a slight delay in virus clearance in the muMT mice. This indicates that antiviral antibody is of little importance in the resolution of the lung infection. Neither free nor latent virus could be detected in the spleen in the muMT mice. In addition, these mice did not develop MHV-68-induced splenomegaly. These data suggest that within the lymphoid compartment B lymphocytes are the sole reservoir for MHV-68 infection in vivo, confirming earlier work which identified B cells as the site of latent infection based on cell fractionation studies. In addition, our study shows that CD4-driven lymphocyte expansion leading to splenomegaly is dependent on the presence of MHV-68-infected B cells in the spleen. Although no free virus was detected (using conventional biological assays) in the lung after the resolution of the acute infection, MHV-68 genome was detected in the lungs of both control and muMT mice by PCR analysis. This suggests that cells in the lung may act as a reservoir of latent virus which is independent of the B lymphocyte infection.

Animals↗

PubMed1996

Murine gammaherpesvirus-induced splenomegaly: a critical role for CD4 T cells.

Murine gammaherpesvirus (MHV-68) causes an acute respiratory infection followed by a latent infection in B lymphocytes. In the first 2-3 weeks after infection mice develop a marked splenomegaly, where the spleen cell number increases by 2-3 fold. Cytofluorimetric analysis during splenomegaly revealed an increase in numbers of B lymphocytes and of both CD4+ and CD8+ T lymphocytes. The largest increase relative to uninfected spleens was in the CD8+ population. The number of latently infected cells in the spleen peaked at day 10 post-intraperitoneal infection, then declined to 1/10(6)-1/10(7) cells per spleen. Depletion of CD4+ T lymphocytes prevented the splenomegaly and greatly reduced the peak infective centre level, while having no effect on the long-term of latently infected cells. Given the similarity between MHV-68-induced splenomegaly and Epstein-Barr virus-induced infectious mononucleosis, these data highlight the usefulness of MHV-68 as a mouse model for the study of gammaherpesvirus immunology and pathobiology.

Animals↗

PubMed1994

Expression of immunoglobulin heavy chain-ricin A chain fusions in mammalian cells.

Mammalian cell lines were transfected with antibody heavy (H) chain-ricin A chain gene fusions in attempts to assemble a recombinant immunotoxin. We found that a light chain-secreting mouse plasmacytoma cell line can be transfected stably with such a chimaeric gene, but only if the ricin A chain portion is disarmed by genetic means prior to transfection; if not, stable transfection appears to select for genetic inactivation of the transfected gene. Co-expression of an antibody heavy chain-ricin A chain fusion with light chain in non-lymphoid cells results in cell death. We conclude that the ricin A chain moiety retains biological activity precluding the expression of biologically active antibody-ricin A chain fusion proteins in mammalian cells.

Amino Acid Sequence↗

PubMed1994

Scanning electron microscopy study of endocardial regeneration in bovine pericardial patch-grafts implanted in the canine heart.

The pattern of endocardial regeneration was studied in bovine parietal pericardial patch-grafts implanted in canine hearts. The grafts consisted of fibrous tissue without a cellular lining. They were implanted with either the thoracic or the cardiac surface facing the lumen of the canine ventricle to evaluate the effect on endocardial regeneration. The grafts were retrieved 7, 21, 45 and 60 days after implantation and were examined using scanning electron microscopy. At 7 days, both the thoracic and the cardiac aspect exhibited connective tissue fibers, focally covered by fibrin, platelets and blood cells. The cardiac aspect showed finer and more highly intermingled filamentous fibers than the thoracic aspect. At 21-60 days, the thoracic surface displayed a continuous network of connective fibers with a few blood cells and isolated groups of spindle-shaped cells resembling fibroblasts. At 21-60 days, the cardiac surface showed a diffuse growth of cells on the connective fiber substratum. Regenerating cells first lined the periphery of the grafts (21 days) and then proliferated towards the centrum (45-60 days). These cells varied in size and shape, were mostly closely packed, exhibited numerous microvilli or longer cytoplasmic projections, and resembled regenerating endothelial cells and mature endocardial cells. The topographic arrangement of the new lining cells suggests that they were the result of a per continuitatem regeneration (endothelial re-endothelialization) and that they they originated from the healthy endocardium of the host surrounding the implantation site. The arrangement of the connective fibers, finer on the cardiac than on the thoracic aspect, probably facilitated the development of a cellular lining.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Laser etching of enamel for direct bonding.

The application of laser irradiation to etch dental enamel in preparation for direct bonding of orthodontic appliances has been studied. Forty extracted human teeth were divided into four groups of 10 teeth. Within each group, five teeth were subjected to a 30 sec acid etch of the buccal enamel surface; the other five in each group were etched with a laser. Four power settings on the laser etching unit were used: 80mJ, 1W, 2W and 3W. After etching, brackets were adhered to the prepared buccal enamel surfaces with composite resin. Shear bond strength was tested 7 days later. The findings showed that an acceptable shear bond strength, viz. > or = 0.6 kg/mm, could be achieved at laser power settings of 1 to 3W but not at the lowest setting (80 mJ). However, the mean shear bond strengths obtained with laser treatment of the enamel at 80mJ, 1W and 2W were lower (p < 0.01 or 0.001) than that achieved with acid etching.

Acid Etching, Dental↗

PubMed1993

Thermal effects associated with the Nd/YAG dental laser.

The heat produced at the dentinal pulpal wall opposite the irradiation site was measured during etching of dental enamel with an Nd:YAG laser in preparation for direct bonding of orthodontic appliances. Forty extracted human teeth were randomly divided into four groups of 10 teeth. Within each group, the buccal surfaces of 5 teeth and the lingual surfaces of the other 5 teeth were laser treated for 12 sec. Irradiation was performed with a commercial Nd:YAG laser at the power settings of 80mJ, 1W, 2W and 3W. Prior to irradiation, an occlusal access preparation was made into the pulp in order to facilitate the placement of a thermocouple for measurement of temperature changes at the dentinal pulpal wall opposite the irradiation site. The thermocouple was held against the dentinal pulpal wall and the resulting temperature changes were recorded. Heating effects at the dentinal pulpal wall on both buccal and lingual surfaces showed an increase in heat as a function of the increase in power output from the laser unit (p < 0.01). The temperatures measured at power levels 1-3W appeared to be of sufficient magnitude to cause at least localized pulpal inflammation and possible irreversible damage to the pulp tissue immediately opposite the site of laser irradiation.

Acid Etching, Dental↗

PubMed1992

Effect of impurities on estradiol crystallization in a sustained-release implant.

During the development of a silicone rubber implant for the delivery of estradiol 17-beta some batches of implants made from a certain lot of commercial estradiol inexplicably developed surface crystals of estradiol after several days of storage. An impurity profile was obtained for 28 lots of estradiol by a newly developed HPLC method. One or more impurities may have had a role in the spontaneous crystal growth on the surface of the implants, because the one lot of estradiol that initially had surface crystals on aging produced acceptable implants after multiple recrystallizations. Attempts to isolate suspected impurities for characterization were unsuccessful. During the manufacture of the implants, temperatures sufficient to melt the estradiol (mp, 173-179 degrees C) were used. It was expected that, upon implant cooling, melted impure estradiol would form a thermodynamically more active (i.e., noncrystalline) physical form. This metastable form could have migrated to the implant surface, where ambient conditions favored crystallization. Because melted estradiol of a higher purity tended to crystallize more readily, it was less likely to form a glass upon cooling. The phenomenon of surface crystallization was limited to one lot of estradiol with the highest level of impurities. Data from differential scanning calorimetry studies supported this conclusion.

Calorimetry, Differential Scanning↗

PubMed1992

In vivo evaluation of an electroenzymatic glucose sensor implanted in subcutaneous tissue.

Cleanroom processing techniques have been used to mass-produce flexible, electroenzymatic glucose sensors designed for implantation in subcutaneous tissue. In vitro characterization studies have shown the sensor's performance to be acceptable. Initial in vivo studies were conducted with the sensor implanted in the subcutaneous tissue of rabbits. Sensors implanted in the subcutaneous tissue of normal human subjects showed an excellent correlation between glucose concentrations measured by the sensor and capillary finger sticks measured with a commercial analyzer.

Animals↗

PubMed1991

Interaction of DNA with the Klenow fragment of DNA polymerase I studied by time-resolved fluorescence spectroscopy.

The interaction of a fluorescent duplex DNA oligomer with the Klenow fragment of DNA polymerase I from Escherichia coli has been studied in solution by using time-resolved fluorescence spectroscopy. An aminonaphthalenesulfonate (dansyl) fluorescent probe was linked by a propyl chain to a C5-modified uridine base located at a specific site in the primer strand of the DNA oligomer. The fluorescent oligomer bound tightly to the Klenow fragment (KD = 7.9 nM), and the probe's position within the DNA-protein complex was varied by stepwise elongation of the primer strand upon addition of the appropriate deoxynucleoside triphosphates. The decay of the total fluorescence intensity and the polarization anisotropy were measured with a picosecond laser and a time-correlated single photon counting system. The fluorescence lifetimes, the correlation time for internal rotation, and the angular range of internal rotation varied according to the probe's position within the DNA-protein complex. These results showed that five or six bases of the primer strand upstream of the 3' terminus were in contact with the protein and that within this contact region there were differences in the degree of solvent accessibility and the closeness of contact. Further, a minor binding mode of the DNA-protein complex was identified, on the basis of heterogeneity of the probe environment observed when the probe was positioned seven bases upstream from the primer 3' terminus, which resulted in a distinctive "dip and rise" in the anisotropy decay. Experiments with an epoxy-terminated DNA oligomer and a site-directed mutant protein established that the labeled DNA was binding at the polymerase active site (major form) and at the spatially distinct 3'----5' exonuclease active site (minor form). The abundance of each of these distinct binding modes of the DNA-protein complex was estimated under solution conditions by analyzing the anisotropy decay of the dansyl probe. About 12% of the labeled DNA was bound at the 3'----5' exonuclease site. This method should be useful for investigating the editing mechanism of this important enzyme.

Base Sequence↗

PubMed1990

Interaction of Escherichia coli DNA polymerase I with azidoDNA and fluorescent DNA probes: identification of protein-DNA contacts.

The synthesis of an azidoDNA duplex and its use to photolabel DNA polymerases have been previously described (Gibson & Benkovic, 1987). We now present detailed experiments utilizing this azidoDNA photoprobe as a substrate for Escherichia coli DNA polymerase I (Klenow fragment) and the photoaffinity labeling of the protein. The azidoDNA duplex is an efficient substrate for both the polymerase and 3'----5' exonuclease activities of the enzyme. However, the hydrolytic degradation of the azido-bearing base is dramatically impaired. On the basis of the ability of these duplexes to photolabel the enzyme, we have determined that the protein contacts between five and seven bases of duplex DNA. Incubation of azidoDNA with the Klenow fragment in the presence of magnesium results in the in situ formation of a template-primer with the azido-bearing base bound at the polymerase catalytic site of the enzyme. Photolysis of this complex followed by proteolytic digestion and isolation of DNA-labeled peptides results in the identification of a single residue modified by the photoreactive DNA substrate. We identify Tyr766 as the modified amino acid and thus localize the catalytic site for polymerization in the protein. A mansyl-labeled DNA duplex has been prepared as a fluorescent probe of protein structure. This has been utilized to determine the location of the primer terminus when bound to the Klenow fragment. When the duplex contains five unpaired bases in the primer strand of the duplex, the primer terminus resides predominantly at the exonuclease catalytic site of the enzyme. Removal of the mismatched bases by the exonuclease activity of the enzyme yields a binary complex with the primer terminus now bound predominantly at the polymerase active site. Data are presented which suggest that the rate-limiting step in the exonuclease activity of the enzyme is translocation of the primer terminus from polymerase to exonuclease catalytic sites.

Amino Acid Sequence↗

PubMed1990

Comparison of computerized tomography with magnetic resonance imaging (MRI) in the evaluation of encephalic ventricular volume.

The volume of the encephalic ventricles was determined from computerized tomographic (CT) and magnetic resonance imaging (MRI) scans of seven subjects without apparent pathology and three subjects with enlarged ventricles. Since there are many conditions in which the encephalic ventricles become enlarged such as Alzheimer's disease and hydrocephalus, accurate measurement of these structures provides (1) a valuable and safe means of aiding in the diagnosis of such conditions and (2) important follow-up information on affected patients. This paper presents the data obtained from the second phase of a three phase study. The first phase demonstrated the possibility of measuring fluid filled spaces by MRI in three phantom preparations (small, medium, and large "ventricles"). The results were compared with those obtained from the computerized tomography (CT) scans of the same preparations. This phase of the study compares the volumes obtained from CT scans with those obtained from MRI scans of the same individuals. The volumetric calculations were done with the aid of a Calcomp 9000 digital analyser programmed to compensate for the scale factor and slice thickness of the images. The results obtained from the MRI scans correlated closely with those obtained from the CT scans of the same subjects. The third and final phase of the project is the development of an MRI volumetric data base for the encephalic ventricles using a larger number of subjects.

Cerebral Ventricles↗

PubMed1990

Evaluation of encephalic ventricular volume from the magnetic resonance imaging scans of thirty-eight human subjects.

Accurate volume determination of the encephalic ventricles is of importance in several clinical conditions, including Alzheimer's presenile dementia, schizophrenia, and benign intracranial hypertension. Previous studies have investigated the accuracy with which magnetic resonance imaging (MRI) can be used in clinical practice to evaluate the encephalic ventricles. However, adequate evaluation of pathological conditions depends on a sufficient amount of morphometric data from normal subjects. To begin establishing this data base for "normal" subjects, we evaluated the MRI scans of 38 subjects found to have no apparent pathology and calculated the ventricular volume in each case by using methods previously developed in our laboratory. The results were then compared with published volumes determined from studies that used either ventricular casts or computerized tomographic scans. The average total ventricular volume for all 38 subjects was 17.4 cm3, while that for males was 16.3 cm3 and that for females was 18.0 cm3. A small but significant correlation was found between age of subject and ventricular volume, with ventricular size increasing with age.

Cerebral Ventricles↗

PubMed1989

Resonance energy transfer measurements between substrate binding sites within the large (Klenow) fragment of Escherichia coli DNA polymerase I.

Resonance energy transfer was used to determine separation distances between fluorescent derivatives of substrates for Klenow fragment and a unique sulfhydryl, cysteine 907, on the enzyme. Fluorescent derivatives of duplex DNA, deoxynucleotide triphosphates (dNTP), and deoxynucleotide monophosphates (dNMP), modified with aminonaphthalenesulfonates (ANS), served as energy-transfer donors to the fluorophore used to modify cysteine 907, 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD). The labeling of cysteine 907 with NBD caused no decrease in the enzyme's polymerase activity, suggesting that the probe did not significantly alter the conformation of the enzyme. The efficiency of singlet-singlet resonance energy transfer was determined from the quantum yield of the donor in the presence and absence of acceptor. By Förster's theory, the measured distances between cysteine 907 and binding sites for duplex DNA, dNTP, and dNMP were 25-39, 19-28, and 17-26 A, respectively. As the fluorophores, attached to the substrates via a tether arm, are separated from the substrates by approximately 12 A, the distances measured between binding sites are subject to this uncertainty. To measure the separation between binding sites for duplex DNA and dNMP, and to reduce the uncertainty introduced by the tether arm, two experiments were carried out. In the first, duplex DNA was labeled with the acceptor fluorophore NBD and used with the donor ANS-modified dNMP to yield a measured distance separating these two sites of 19-28 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1989

Fluorescent oligonucleotides and deoxynucleotide triphosphates: preparation and their interaction with the large (Klenow) fragment of Escherichia coli DNA polymerase I.

Fluorescent derivatives of short oligonucleotides of defined sequence were prepared by the incorporation of 5-(propylamino)uridine via current phosphoramidite chemistry, followed by derivatization of the propylamine function with mansyl chloride. These oligomers, annealed to complementary oligomers, yielded short duplex DNA fluorescently labeled at a specific base. The fluorescence emission from this labeled duplex increases upon binding to the Klenow fragment of DNA polymerase I (KF) at specific positions within the duplex DNA. By varying the position of the label within the duplex DNA and observing the emission, points of strong enzyme-DNA interactions were elucidated. A similar fluorescent derivative of a deoxynucleoside triphosphate (dNTP), 5-[[[[[[(5- sulfonaphthalenyl)amino]ethyl]amino]carbonyl]- methyl]thio]-2'-deoxyuridine 5'-triphosphate (AEDANS-S-dUTP), was synthesized, whose emission also was increased upon binding to KF. The change in emission intensities between unbound and bound substrates enabled the measurements of KDs for the DNA and dNTP derivative, which were found to be 0.15 nM and 2.9 microM, respectively. Stopped-flow measurements on these species yielded association and dissociation rates for each. Anisotropy measurements of the labeled base at various positions in the duplex yielded values that support the measurements made by observing the emission intensities.

Base Sequence↗

PubMed1989

DNA substrate structural requirements for the exonuclease and polymerase activities of procaryotic and phage DNA polymerases.

A DNA duplex covalently cross-linked between specific bases has been prepared. This and similar duplexes are substrates for the polymerase and exonuclease activities of the Klenow fragment of Escherichia coli DNA polymerase I and T4 and T7 DNA polymerases. The action of Klenow fragment on these duplexes indicates that the polymerase site does not require that the DNA duplex undergo strand separation for activity, whereas the exonuclease site requires that at least four base pairs of the primer strand must melt out for the exonucleolytic removal of nucleotides from the primer terminus. The exonucleolytic action of T4 and T7 DNA polymerases requires that only two and three bases respectively melt out for excision of nucleotides from the primer terminus. Klenow fragment and T4 DNA polymerase are able to polymerize onto duplexes incapable of strand separation, whereas T7 DNA polymerase seems to require that the primer terminus be at least three bases from the cross-linked base pair. A DNA duplex with a biotin covalently linked to a specific base has been prepared. In the presence of the biotin binding protein avidin, the exonucleolytic activity of Klenow fragment requires that the primer terminus be at least 15 base pairs downstream from the base with the biotin-avidin complex. On the other hand, the polymerase activity of Klenow fragment required that the primer terminus be at least six base pairs downstream from the base with the biotin-avidin complex. These results suggest that the polymerase and exonuclease sites of Klenow are physically separate in solution and exhibit different substrate structural requirements for activity.

Avidin↗

PubMed1988

A surface ultrastructure study of ganglia and digital mucous cysts.

Three digital mucous cysts and three dorsal wrist ganglia were studied by light and scanning electron microscopy. Both exhibited the same findings, namely a porous network of collagenous fibers forming the wall and a fibrillated inner surface composed of a random arrangement of collagen fibers. An intermittent thin membrane was observed on the inner surface, but no evidence of a cellular lining was apparent. This anatomic study lends support to a shared cause.

Cysts↗