Comments on "Dosimetry of interstitial brachytherapy sources: recommendations of the AAPM Radiation Therapy Committee Task Group No. 43" [Med. Phys. 22, 209-234 (1995)].
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Brachytherapy↗
Biomedical subjects
D Jones
Publications and source records attributed to D Jones.
Characterization of a 17-kilodalton antigen of Bartonella henselae reactive with sera from patients with cat scratch disease.
A library of Bartonella (Rochalimaea) henselae DNA was constructed in the cloning vector lambda ZAPII and screened for expression of antigenic proteins by using a pool of sera from patients who had been diagnosed with cat scratch disease (CSD) and had antibodies to Bartonella spp., as determined by indirect fluorescent-antibody (IFA) assay. Ten immunoreactive phages were subcloned as recombinant plasmids by in vivo excision. All 10 recombinants expressed a protein of approximately 17 kDa when they were examined by immunoblot with the pool of human sera. Restriction endonuclease digestion of each recombinant plasmid indicated seven profiles, suggesting that cloning bias was not the reason for repeated isolation of clones expressing the 17-kDa antigen. The gene coding for the 17-kDa antigen was sequenced and shown to code for an open reading frame of 148 amino acids with a predicted molecular mass of 16,893 Da. The amino terminus of the deduced amino acid sequence was hydrophobic in nature and similar in size and composition to signal peptides found in gram-negative bacteria. The remainder of the deduced amino acid sequence was more hydrophilic and may represent surface-exposed epitopes. Further subcloning of the 17-kDa antigen as a biotinylated fusion protein in the expression vector PinPoint Xa-2 resulted in a 30-kDa protein that was highly reactive on immunoblots with individual serum samples from patients with CSD. The agreement between reactivity with the 30-kDa fusion protein on immunoblot analysis and the results obtained by IFA assay was 92% for IFA-positive sera and 88% for IFA-negative sera. The recombinant-expressed 17-kDa protein should be of value as an antigen for serologic diagnosis of CSD and Bartonella infections and warrants further study in attempts to develop a subunit vaccine to prevent long-term Bartonella infection in cats and the potential for further spread of these organisms to humans.
Amino Acid Sequence↗
Characterization of a novel 350-kilodalton nuclear phosphoprotein that is specifically involved in mitotic-phase progression.
A gene assigned to human chromosome 1q32-41 encodes a novel protein of 3,113 amino acids containing an internal tandem repeat of 177 amino acids. The protein, which we have named "mitosin," was identified by direct binding to purified retinoblastoma protein in vitro with a region distantly related to the retinoblastoma protein-binding site of E2F-1. Mitosin is expressed throughout S, G2, and M phases of the cell cycle but is absent in G1. Its localization is dramatically reorganized from a rather homogeneous nuclear distribution in S phase to paired dots at the kinetochore/centromere region, to the spindle apparatus, and then to the midbody during M-phase progression. This spatial reorganization coincides closely with the temporal phosphorylation patterns of mitosin. Overexpression of N-terminally truncated mutants blocks cell cycle progression mainly at G2/M. These results suggest that mitosin may play an important role in mitotic-phase progression.
Amino Acid Sequence↗
Determination of the gene order of the three loci CD2, NGFB, and NRAS at human chromosome band 1p13 and refinement of their localisation at the subband level by fluorescence in situ hybridisation.
The three loci NRAS, NGFB, and CD2 map to human chromosome band 1p13. Using fluorescence in situ hybridisation (FISH) to simultaneously DAPI-banded metaphase chromosomes, we have further refined the localisation of these three genes to specific subbands. NRAS localises to subband 1p13.2 and CD2 and NGFB to 1p13.1. Also, with the use of multicolour FISH, we have determined the order and orientation of the three loci in relation to the centromere. The order is cen-CD2-NGFB-NRAS.
Antigens, CD↗
An estimate of the margin required when defining blocks around the prostate.
The portal films of 54 consecutive patients treated for primary prostate cancer have been compared to the simulation films. The systematic and random uncertainty in the set-up, defined by the couch movement required to move the patient to the simulated position, was determined to be 1.6 mm UP (SD 3.3 mm), 0.3 mm RT (SD 2.6 mm) and 1.3 mm IN (SD 2.4 mm). The area of fields defined on simulation films was compared with that on portal films to determine the error in block production which was -0.7 mm (SD = 0.9 mm). Five sources of uncertainty in the radiotherapy have been identified, three occur before and two during the course of treatment. A method for combining these uncertainties is proposed and used on the data obtained in this study. This provides estimates of the margin required when drawing blocks so that the minimum dose to the target is 95% of the prescription in 95% of treatments. The block margins are not uniform and range from 21 mm, when drawing the block outline to the posterior on a lateral film, to 13 m when drawing laterally on an anterior film.
Humans↗
Paradoxical embolism. An underrecognized problem.
Despite reports of the clinical presentations and devastating consequences of paradoxical embolus (PDE) for more than a century, this diagnosis continues to be frequently missed. Because the prevalence of patent foramen ovale (PFO) is 27 to 35% in the normal population and the presence of deep vein thrombosis or pulmonary embolus may not be clinically obvious, a high suspicion for PDE is needed in the event of unexplained arterial occlusion. While contrast echocardiography and transcranial Doppler ultrasound have facilitated clinical recognition of PDE, the optimum approach to diagnosis requires clarification. Primary therapy for patients with PDE is anticoagulation, with thrombolytics considered in carefully selected individuals, but there is little published information regarding long-term treatment and outcomes. Prevention remains essential whenever possible. It is not yet defined whether prophylactic treatment of persons with recognized predispositions to PDE (eg, PFO and pulmonary hypertension) is beneficial.
Adult↗
Refrigeration in the hospital.
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Air Pollutants↗
A prolonged intracoronary infusion of urokinase for chronic total occlusions: case reviews.
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Aged↗
The response of mouse skin to fractionated doses of fast neutrons (66 MeVp-Be) with variable interfraction and overall treatment times.
PURPOSE: Beam availability for neutron therapy at the National Accelerator Centre at Faure, South Africa is such that treatment fractions are given at irregularly spaced intervals. Such treatment scheduling may not be optimal. METHODS: Investigations were made using the acute skin reaction of the mouse foot to determine the effect of different numbers of regularly and irregularly spaced fractions of p(66)/Be neutrons. Assessment of results was both by average skin reaction and by ED50 values for the incidence of moist desquamation as established by probit analysis. RESULTS: Different numbers of fractions (between 6 and 11) and different times between fractions did not appear to affect the mouse foot response significantly when fractionation was completed within 11 days, i.e. before repopulation began to have an effect. When overall treatment times were longer than 11 days, the mouse foot responses to 6 and 9 fractions with variable interfraction times were similar, provided the overall treatment times were the same and the fractions were at least 24 h apart. The alpha/beta ratio was 87 +/- 27 (SE) Gy for the early response of the BALB/c mouse foot skin to p(66)/Be neutrons. CONCLUSIONS: The response of mouse skin to fractionated p(66)/Be neutrons was independent of fraction number or interfraction time, provided that the overall treatment time was the same.
Animals↗
Susceptibility to tumors induced in mice by ethylnitrosourea is independent of retinoblastoma gene dosage.
The retinoblastoma gene (RB) is a classical tumor suppressor. Several studies have shown that RB dosage is important in determining biological effects. To explore the effect of RB dosage on susceptibility to cancer, three groups of congenic C57BL/6 mice, each of which expresses a different amount of Rb protein from one, two, or three alleles, were treated at postnatal day 12 with a single 60-mg/kg body weight i.p. dose of the DNA-alkylating agent N-ethyl-N'-nitrosourea (ENU). Mice heterozygous for the RB gene developed characteristic pituitary tumors with nearly complete penetrance, whether or not they were treated with ENU. Tumors initiated earlier or progressed more rapidly, however, in ENU-treated mice. Furthermore, although mice treated with ENU had a higher incidence of several nonpituitary tumors compared with untreated controls, no significant differences in the incidence of these tumors were found between wild-type mice (mRB+/+), mice carrying only one normal RB allele and deficient in Rb protein expression (mRB+/-), and mice overexpressing Rb protein from two normal murine RB alleles and a human RB transgene (mRB+/+, hRB+/-). These studies underscore the tissue and mechanistic specificity of tumor predisposition caused by an inherited 50% reduction in RB dosage and indicate that most ENU-induced tumors occur independent of RB inactivation. Nonetheless, they suggest that certain point mutations induced by ENU may participate in the sequence of molecular steps involved in progression of tumor-prone, RB-deficient cells to the fully malignant state.
Animals↗
Quantitative mapping of cytochrome oxidase activity in the central auditory system of the gerbil: a study with calibrated activity standards and metal-intensified histochemistry.
The objective was to obtain detailed topographic determinations of cytochrome oxidase activity in the gerbil central auditory system at the light microscopic level. Quantitative techniques were developed using (1) tissue standards calibrated to express histochemical measures as actual enzyme activity units, (2) densitometry and image analysis of histochemical reaction product formation, (3) spectrophotometry of cytochrome oxidase activity, and (4) a cobalt-intensified staining procedure compatible with autoradiography and other techniques requiring fresh-frozen brains without perfusion-fixation. Linear relationships between incubation time, section thickness, and activity of dissected brain regions, with their reaction product measured densitometrically were determined. Auditory structures with the high activities showed about 8 times the labeling intensity of the white matter or control sections inhibited with cyanide, glutaraldehyde, or heat. This indicated the high sensitivity of the method without loss of specificity. Specific activity for each of the 18 auditory structures measured were all above the units measured for whole brain homogenates, supporting the notion that basal levels of oxidative metabolism are greater for the auditory system. There was a progressive decrement in activity from brain stem to forebrain auditory structures. The more peripheral nuclei also showed a higher proportion of somatic as compared to neuropil reactivity. In contrast, auditory midbrain and thalamocortical regions were characterized primarily by neuropil reactivity. Comparison of intrinsic patterns of activity with morphological schemes to subdivide nuclei, showed a good correspondence with classical subdivisions derived from Golgi studies. The reported activities may provide a base of normative data in the gerbil for subsequent studies of central auditory functions. The method presented fulfilled established quantitative criteria and provided a more sensitive approach for regional mapping studies of brain cytochrome oxidase activity.
Animals↗
Isolation, cloning, and characterization of new chitinase stored in active form in chitin-lined venom reservoir.
A 52-kDa protein was purified from the venom gland of an endoparasitic wasp Chelonus sp. near curvimaculatus. The acidic protein (pl 4.8-5.0) was purified, and the NH2-terminal amino acid sequence was determined by direct protein sequencing. A vector-anchored primer and a degenerate primer derived from an 8-amino acid region of the NH2 terminus were used in anchored polymerase chain reaction to generate a probe for screening a cDNA library prepared from mRNA from the venom gland of this wasp. A positive clone of 1596 nucleotides was isolated, sequenced, and found to contain an open reading frame encoding a protein of 483 amino acids. The amino acid sequence encoded in the cDNA clone at the amino terminus was identical to the sequence determined from the purified 52-kDa protein. Comparison of the inferred protein sequence against all of the sequences in the GenBank data base revealed a high degree of similarity to a 62-kDa insect chitinase that is expressed at each molt for the purpose of digesting the chitinous insect cuticle. Two regions that are conserved in a group of chitinases from insects, bacteria and fungi, were also conserved in the wasp 52-kDa protein. The region between amino acid residues 382 and 464 in the 52-kDa wasp chitinase markedly differed in primary sequence from the corresponding region in the 62-kDa insect chitinase, although this region in both the proteins is predicted to have a similar secondary structure predominantly of coils and turns. This region may impart unique structural properties to the 52-kDa chitinase that enables it to exist in an active form in a chitin-lined reservoir without exerting an autotoxic effect to digest the cuticular storage reservoir. Chitinolytic activity occurring in the total venom gland extract was kinetically indistinguishable from that observed for the purified 52-kDa protein, including the substrate concentrations at which maximal velocity and substrate inhibition were reached. The kinetic properties of the 52-kDa wasp chitinase were quite distinct from Serratia marcescens chitinase for both catalytic rate and substrate inhibition.
Amino Acid Sequence↗
Integration of multiple chicken retroviruses into multiple chicken herpesviruses: herpesviral gD as a common target of integration.
Integration of two different avian retroviruses, reticuloendotheliosis virus (REV) and avian leukosis virus (ALV), into the genome of two different avian herpesviruses, the herpesvirus of turkeys (HVT) and Marek's disease virus (MDV), was investigated. Integration events occurred by the fourth and sixth in vitro passage of cells coinfected with REV/HVT and ALV/MDV, respectively. In order to further characterize the integration events, integrated proviruses and surrounding herpesviral genetic material were cloned and analyzed. In the REV/HVT coinfection experiment, one of the three unique integrated proviruses was found to have integrated into the HVT gD gene, resulting in disruption of the coding region of this gene. The two additional unique integrations were localized to the UL and IRL border regions of HVT, two previously described common sites of REV integration into MDV. Interestingly, one of the integrated proviruses in the HVT genome appeared to be full length, was infectious when transfected into CEF cells, and therefore could potentially function to produce infectious REV from an HVT infectious platform. In the ALV/MDV coinfection experiment, one of two unique integrated proviruses was found to have integrated into the gD gene, resulting in disruption of the coding region of this gene. The second unique integration site was in the polyadenylation site of the SORF2 gene at the boundary of the IRS and US, once again a common site of REV integration into MDV. These results demonstrate that the U/IR-TR border regions of herpesviruses are common sites of retroviral integration. In addition gD, in the US region of the herpesvirus, is a common site of retroviral integration in multiple herpesvirus, indicating a possible selective advantage for disruption of this gene in the in vitro growth of a herpesvirus. Finally, this is the first instance of a full-length provirus found integrated into a herpesvirus genome, indicating that a retrovirus could alter its route of infection by being carried in a herpesvirus genome.
Amino Acid Sequence↗
Purification of 14-3-3 protein and analysis of isoforms in chicken brain.
14-3-3 proteins are apparently ubiquitous eukaryotic proteins that comprise a large number of isoforms. We have used specific antibodies raised against each mammalian isoform to probe for 14-3-3 isoforms in adult hen brains. The results suggest that there is a remarkable degree of similarity in primary structure (at least in the regions containing the epitopes). Reverse-phase HPLC of the purified avian 14-3-3 proteins indicates a high overall degree of similarity in sequence and levels of expression of each isoform that are remarkably similar to their mammalian counterparts.
14-3-3 Proteins↗
Triplex formation by the human Ha-ras promoter inhibits Sp1 binding and in vitro transcription.
The central role of the ras oncogenes in the pathogenesis of a wide variety of human malignancies is well established. Toward developing specific transcriptional inhibitors of the human Ha-ras oncogene, we have designed oligonucleotides to target a region of the Ha-ras promoter (-8 to -28) which contains two of the three Sp1 binding sites essential for transcriptional activity. Gel mobility analysis and DNase I footprinting demonstrate that an oligonucleotide (HR21ap) forms a sequence-specific triple helix with its target site in an antiparallel orientation with respect to the purine-rich duplex strand through predominantly G*G:C triplets. Within the Ha-ras promoter, HR21ap binds exclusively to the proximal target Sp1 sites over a similar nontarget distal sequence which, like the target, contains a consensus Sp1 site. Protein binding assays demonstrate that triplex formation by HR21ap inhibits Sp1 binding to the Ha-ras promoter. Moreover, oligonucleotide-directed triplex formation arrests Ha-ras promoter-dependent transcription in vitro. The results presented here suggest that triplex formation by the Ha-ras promoter targeted oligonucleotide may provide a means to specifically inhibit transcription of this oncogene in vivo.
Base Sequence↗
Characterization of 14-3-3 proteins in adrenal chromaffin cells and demonstration of isoform-specific phospholipid binding.
Isoform-specific antisera were used to examine which 14-3-3 isoforms were present in bovine adrenal chromaffin cells. The eta, tau and sigma isoforms were not detectable, and the epsilon isoform was present at only low levels. 14-3-3 isoforms were readily detected with antisera against the beta, gamma and zeta isoforms. The latter isoforms were found to leak from digitonin-permeabilized chromaffin cells, as expected for cytosolic proteins, but a proportion of each isoform was retained. In subcellular fractionation studies isoforms recognized by the beta and zeta antisera were found in the cytosol and Triton-insoluble cytoskeletal fractions, while the gamma isoform was found in cytosol and also in microsomal and chromaffin granule membrane fractions. The gamma 14-3-3 protein associated with granule membranes was partially removed by a high-salt/carbonate wash, and the membranes could bind further gamma from cytosol or from a purified brain 14-3-3 protein mixture. The binding of gamma 14-3-3 was not Ca(2+)-dependent, nor was it affected by phorbol ester, GTP analogues or cyclic AMP. Using pure phospholipid vesicles it was found that gamma and also epsilon 14-3-3 proteins bound directly to phospholipids. Little binding of brain beta, eta or zeta to phospholipid vesicles was detected. Brain 14-3-3 proteins were also able to aggregate phospholipid vesicles. Recombinant 14-3-3 isoforms (tau and the Xenopus protein) were able to stimulate Ca(2+)-dependent exocytosis in digitonin-permeabilized chromaffin cells. The Xenopus proteins lacks part of the extreme N-terminus, indicating that this domain is not essential for function in exocytosis.
14-3-3 Proteins↗
Mechanism of inhibition of protein kinase C by 14-3-3 isoforms. 14-3-3 isoforms do not have phospholipase A2 activity.
The ability of individual members of the 14-3-3 protein family to inhibit protein kinase C (PKC) has been studied by using a synthetic peptide based on the specific 80 kDa substrate for PKC (MARCKS protein) in two different assay systems. Recombinant 14-3-3 and isoforms renatured by a novel method after separation by reverse-phase h.p.l.c. were studied. The detailed effects of diacylglycerol and the phorbol ester phorbol 12-myristate 13-acetate on the inhibition were also investigated. This suggests that one of the sites of interaction of 14-3-3 may be the cysteine-rich (C1) domain in PKC. Since a region in secreted phospholipase A2 (PLA2) shares similarity with this domain, the ability of 14-3-3 to interact with mammalian PLA2 was studied. Cytosolic PLA2 has some similarity to the C2 region of PKC, and the effect of 14-3-3 on this class of PLA2 was also analysed. In contrast with a previous report, no PLA2 activity was found in brain 14-3-3, nor in any of the recombinant proteins tested. These include zeta 14-3-3 isoform, on which the original observation was made.
14-3-3 Proteins↗
IL-2 reverses human T cell unresponsiveness induced by thymic epithelium in SCID-hu mice.
In this study we investigated the mechanism responsible for the unresponsiveness of thymus-reactive T cells obtained from severe combined immunodeficient (SCID) mice constructed with human fetal liver (FL) stem cells from donor A and an allogeneic human fetal thymus (FT) from donor B (A/B SCID-hu mice). These A/B SCID-hu mice have a human thymus containing B cells, macrophages, and dendritic cells from FL donor A but thymic epithelial cells from FT donor B. The repertoire of human T cells developing in this chimeric thymus is depleted of T cells specific for the HLA Ags of the FL donor, whereas T cells reactive against the HLA Ags expressed by the FT donor are still present. However, these thymocytes failed to proliferate and expressed low levels of the activation markers CD25, CD71, and HLA-DR after stimulation with the EBV-LCL of the FT donor in primary MLRs. This unresponsiveness could be completely reversed by IL-2. Restoration of T cell responsiveness was Ag specific and a unique property of IL-2. The T cells produced very low levels of IL-2 when stimulated with the HLA Ags of FT donor B, whereas they secreted normal levels of IL-2 after activation by third party alloantigens. Low IL-2 production was also observed at the clonal level. CD4+ T cell clones from A/B SCID-hu mice, specific for the HLA Ags of B, produced significantly less IL-2 and granulocyte macrophage-CSF than control CD4+ T cell clones. However, these T cell clones synthesized normal levels of IL-2 and granulocyte macrophage-CSF after stimulation with combinations of PMA/Calo or PMA/anti-CD3 mAb. Thus, T cells that differentiate in a chimeric thymus containing allogeneic host thymic epithelium are rendered tolerant to the HLA Ags expressed by the thymic epithelial cells. This tolerance results in the presence of T cells that do not proliferate properly after Ag-specific stimulation. This lack of proliferation is primarily related to their inability to produce sufficient levels of IL-2 and can be restored by exogenous IL-2.
Animals↗