Biomedical subjects
Publications and source records attributed to T Melvin.
The emission of CdSe quantum dots linked to the 5'-end of a DNA sequence is efficiently quenched by hybridisation with a complementary DNA strand with a gold nanoparticle attached at the 3'-end; contact of the quantum dot and gold nanoparticle occurs.
The covalent attachment of DNA oligonucleotides onto crystalline silicon (100) surfaces, in patterns with submicron features, in a straightforward, two-step process is presented. UV light exposure of a hydrogen-terminated silicon (100) surface coated with alkenes functionalized with N-hydroxysuccinimide ester groups resulted in the covalent attachment of the alkene as a monolayer on the surface. Submicron-scale patterning of surfaces was achieved by illumination with an interference pattern obtained by the transmission of 248 nm excimer laser light through a phase mask. The N-hydroxysuccinimide ester surface acted as a template for the subsequent covalent attachment of aminohexyl-modified DNA oligonucleotides. Oligonucleotide patterns, with feature sizes of 500 nm, were reliably produced over large areas. The patterned surfaces were characterized with atomic force microscopy, scanning electron microscopy, epifluorescence microscopy and ellipsometry. Complementary oligonucleotides were hybridized to the surface-attached oligonucleotides with a density of 7 x 10(12) DNA oligonucleotides per square centimetre. The method will offer much potential for the creation of nano- and micro-scale DNA biosensor devices in silicon.
"Light-up" probes, icosa-alpha-thymidylate-thiazole orange conjugates, for the in situ time-resolved detection of messenger ribonucleic acid (mRNA) in living cells are evaluated. Upon annealing with polyA in aqueous solutions, the icosa-alpha-thymidylate-thiazole orange conjugates were shown to be up to 15 times more fluorescent. Microinjection of these probes into adherent fibroblasts resulted in high yields of hybridization and fluorescent signals. Incubation of cells in the presence of these probes resulted in facile internalization of the probe and similar painting of the messenger RNA in the nuclear and cytosolic regions.
Exposure of an aqueous, aerated solution (pH 7) of a double-stranded DNA to 193 nm light, of sufficient energy to ionise DNA, leads to selective, non-random modification at guanine in the form of frank single-strand break (ssb) and base modifications, revealed by treatment with either Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg), Escherichia coli endonuclease III (Nth) or hot piperidine treatment. There is a similar neighbouring base sequence dependence for Fpg- and Nth-sensitive damage as that previously reported for both hot alkali-labile damage and prompt ssb. Low yields of photoproducts, namely pyrimidine dimers, are also revealed using the enzyme T4 endonuclease V (T4 endo V). Although irradiation of DNA with 193 nm light causes photoionisation of all the nucleic acid bases, these results indicate that guanine is the predominant site for localisation of the oxidative damage. These findings are consistent with migration of the radical cation to 'target' damage at guanine sites.
Irradiation of aqueous solutions of plasmid DNA (pUC18) at pH 7.6 with 193 nm laser light results in low yields of prompt single strand breakage (air-saturated sample phi ssh = [1.5 +/- 0.1] x 10(4), argon-saturated sample phi ssh = [0.9 +/- 0.1] x 10(4). Treatment of the irradiated DNA samples with Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) protein results in an approximate 20-fold increase in the yield of single strand break-age (air-saturated sample phi fpg = [33.1 +/- 3.1] x 10(-4), argon-saturated sample phi fpg = [23.8 +/- 2.6] x 10(-4). This result indicates that 193 nm light induces other modification(s) (most likely of the purine moieties) that are 20 times more abundant than prompt strand breakage within the DNA matrix.
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The photoreaction of 2-methyl-1,4-naphthoquinone (MQ, menadione) with DNA and polynucleotides in argon-saturated aqueous solution (pH 7) was studied. Results from laser flash photolysis experiments indicate that triplet quinone reacts with DNA and polyA but not detectably with polyU by one-electron oxidation of the bases of the nucleic acid with formation of the radical anion of the quinone. Irradiation of argon-saturated solutions containing MQ and DNA or polynucleotides (polyU, polyA, polyG or polyC) with 334 nm light leads to an increase in molecular weight for single-stranded DNA, polyA and to a much less extent for polyU. This finding indicates crosslink formation with quantum yields in the range of 10(-5)-10(-3).
Irradiation of DNA with 193 nm light results in monophotonic photoionization, with the formation of a base radical cation and a hydrated electron (phi PI = 0.048-0.065). Although > 50% of the photoionization events initially occur at guanine in DNA, migration of the "hole" from the other bases to guanine occurs to yield predominantly its radical cation or its deprotonated form. From sequence analysis, the data reveal that 193 nm light induces single strand breaks (ssb) in double-stranded DNA preferential 3' to a guanine residue. However, it has previously been reported that 193 nm light yields very low yields of ssb (< 2% of the yield of e-aq). The distribution of these ssb at guanine is nonrandom, showing a dependence on the neighboring base moiety. The efficiency of ssb formation at nonguanine sites is estimated to be at least one order of magnitude lower. The preferred cleavage at guanine is consistent with migration and localization of the electron loss center at guanine. It is argued that singlet oxygen and the photoionized phosphate group of the sugar moiety are not major precursors to ssb. At present, the mechanisms of strand breakage are not known although a guanine radical or one of its products remain potential precursors.
The following study investigates the pathways involved in the induction of single strand breaks (ssb) in various samples of single stranded (ss) DNA (calf thymus, Micrococcus lysodeikticus, Clostridium perfringens) with differing nucleic acid base composition. The time scale for the induction of ssb was determined from changes in the light scattering intensity following pulse irradiation of aqueous solutions containing these ssDNA samples at pH7.8 under either aerated or deaerated conditions. The induction of ssb under these conditions is predominantly by the hydroxyl radical and shows various kinetically distinct components. The immediate ssb (t < 0.02 s) account for approximately 40-60% of the total yield of ssb. The majority of these ssb are suggested to arise from the 'common' initial attack of the hydroxyl radicals at the sugar phosphate backbone for each of the three DNA samples. Furthermore, slower components for ssb formation (t > 0.02 s) were observed and are suggested to occur through base radical mediated H-atom abstraction from the sugar moiety. The half lives for formation of the majority of ssb, formed through this base radical-mediated H-atom abstraction(s), are in the range of 20-43 ms. The yields of these 'base-mediated' ssb vary markedly (under both aerobic and anaerobic conditions) and reflect the base composition of the DNA sample. It is suggested from these studies that the OH-induced base radicals of guanine/cytosine are more effective precursors for strand breakage than those from adenine/thymine in ssDNA.
Nephrotic syndrome is defined as proteinuria sufficiently severe to result in hypoalbuminaemia, oedema and hyperlipidaemia. The early modern history of this illness was characterised by the serendipitous development of renal biopsy technique at approximately the same time as the use of corticosteroids for nephrotic syndrome. The coincidence of these events set the stage for evaluating therapeutic response to corticosteroids and cytotoxic agents in relation to renal histology and ultimate clinical outcome. The International Study of Kidney Disease in Children (ISKDC) was initiated in the 1960s as a multicentre study examining these relationships in children. Over the next decade this study, as well as contributions from other investigators, helped define optimum therapy for these children. It was determined that a child with nephrotic syndrome under the age of 6 years, who did not present with hypertension, azotaemia, hypocomplementaemia or signs of systemic illness, had an approximately 85% chance of responding to corticosteroid therapy. If only those children who had minimal change histology on biopsy were considered, 94% responded. The original regimen which is still used today, was 60 mg/m2 bsa/day prednisone administered on a 3 times per day dosage schedule for 4 weeks, followed by an additional 4 weeks of therapy at a dose of 40 mg/m2 bsa given as a single oral dose every other day. Of those who respond roughly one-third will have no relapses, while almost half will have frequent relapses (greater than or equal to 2 in 6 months) and the rest will have infrequent relapses. Patients in relapse are treated as at presentation but are usually converted to the 40 mg/m2 bsa dose when the urine has been free of protein for 3 days, and are then tapered off or maintained on this dose for several weeks, depending on the individual's history of relapses and incidence of side effects from corticosteroids. For those children who are suffering frequent relapses and severe corticosteroid side effects (e.g. growth failure, morbid obesity, aseptic necrosis of bone), cytotoxic agents were identified as providing long term remission. After inducing remission with conventional corticosteroid dosages, cyclophosphamide is administered at a dose of 2 mg/kg/day given as a single dose for 8 weeks. This regimen was shown to lead to approximately 70% of patients being in remission 2 years after completion of this course of therapy. Chlorambucil given at a dose of 0.2 mg/kg/day as a single oral dose has been equally efficacious.(ABSTRACT TRUNCATED AT 400 WORDS)
Three cases of nephrologic disease characterized clinically by nephrotic syndrome and histologically with mesangial sclerosis associated with Wilms' tumor are documented. Two children were born with ambiguous genitalia and an XY chromosome constitution. A third patient had a 46,XY karyotype and developed a Wilms' tumor but had a normal female phonotype. The evaluation and current management of this syndrome are discussed.
Thirty-one pediatric patients with acute renal allograft rejection were treated with the monoclonal antibody OKT3. In 24 cases, increased doses of steroids followed by a polyclonal antithymocyte globulin were ineffective in reversing the rejection episode. Twenty-eight patients completed the prescribed minimum 10-day treatment course, with effective rejection reversal in 22. Three patients failed to complete the course of therapy: one because of leukopenia that developed after the first dose, one because of a clotted graft, and another because of symptomatic cytomegalovirus infection. The overall success rate of OKT3 for rejection reversal was 74%; however, 55% of recipients had rebound rejection, and 85% of patients had detectable anti-OKT3 antibodies after completion of the course of therapy. Ten patients were treated with a second course of OKT3, and in eight of these patients, rejection was at least temporarily reversed. The starting dose of OKT3 for second-course therapy was the same as that used during first-course therapy, but in five cases the dose was increased during the course because of inadequate therapeutic response. Seven of these patients lost their grafts a mean of 6.5 months after completion of second-course therapy. We looked for anti-OKT3 antibody in nine recipients after completion of a second treatment course and found it in all nine. Our observations regarding a second treatment course with this monoclonal antibody preparation suggest that although rejection reversal may be observed, ultimate graft survival is poor and anti-OKT3 antibody formation is enhanced.
We analyzed the results obtained with protocols for immunosuppression of pediatric recipients of haploidentical living-related renal transplants. In the donor-specific transfusion group transfusion of blood products obtained from the prospective organ donor was performed before transplantation, and at transplantation maintenance immunosuppression of azathioprine and prednisone was begun. In the cyclosporine group donor-specific transfusion was not used, and maintenance immuno-suppression of cyclosporine and azathioprine was begun 1 week before transplantation, with the addition of prednisone at transplantation. Of 24 donor-specific transfusion recipients 3 had circulating cytotoxic antibodies to the prospective donor for an incidence of 12%. There was no significant difference between groups with respect to 1-year actual patient and graft survival (100 and 89 versus 100 and 86%, respectively), 1-year mean serum creatinine level (1.1 versus 1.2 mg./dl.), rejection treatments per patient (2.5 versus 2.6) and total days hospitalized during year 1 after transplantation (27 versus 18), with donor-specific transfusion data presented first. Initial hospitalization was significantly shorter (10 versus 16 days, p less than 0.05) and the incidence of rejection crises within 3 months was significantly less (68 versus 94%, p less than 0.05) in the cyclosporine group. We believe that cyclosporine and azathioprine pre-treatment of pediatric recipients of haploidentical living-related renal transplants with the addition of prednisone at transplantation is preferable to a donor-specific transfusion protocol because there is no risk of recipient sensitization to the prospective donor, and patient and graft survival is not adversely affected.
The photochemistry, photophysics and hydrolytic stability of three substituted aroyl azides were investigated. The azides were selected as model compounds for potential photolabeling agents based on the extreme electrophilic reactivity of aroylnitrenes. Each of the azides studied is a derivative of benzoyl azide containing an acyl group to act as an internal triplet sensitizer and an additional functional group useful for linking the azide to a site-selecting probe molecule. The findings show that benzoyl azides containing the three substituents in a 1,3,5-pattern might be useful for labeling of lipophilic molecules.
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A subgroup of patients with steroid-responsive minimal change nephrotic syndrome (MCNS) is classified as steroid-dependent because of repeated relapses of proteinuria during prednisone taper, or within four weeks of withdrawal of prednisone. The long-term outcome of this subgroup of patients has not been documented. Thirteen patients with onset of steroid-dependent MCNS (biopsy-proven) in childhood have been followed for 10-22 years (mean 15.6 years); 8 patients have been in stable remission for 2-13 years (mean 5.5 years); 3 remain steroid-dependent 12, 18 and 22 years after onset; and 2 had steroid-responsive relapses after 5 and 8 years of remission. In 3 patients steroid-dependent MCNS remitted without cytotoxic therapy 7-11 years after onset. Cyclophosphamide was administered to the remaining 10 patients: 8 had prolonged remissions (1.1-13 years, mean 4.3 years) and 2 relapsed shortly after cyclophosphamide therapy. Of the 8 patients who had prolonged remissions after cyclophosphamide, 3 have had no relapses 5-13 years after therapy. In 5 of these 8 patients steroid-dependency recurred 1.1-3 years after cyclophosphamide and a second course was given, resulting in remissions of 1.4-8 years in 3 patients, change to a frequently relapsing pattern in 1 patient and remission of 3 months in one patient who then became steroid-dependent again. We conclude that the long-term outcome of steroid-dependent MCNS is favorable. Cyclophosphamide is useful in the management of these patients and a prolonged course of alternate-day prednisone therapy after cyclophosphamide may be helpful in maintaining remission.
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Serum amyloid P component (SAP) is a normal plasma glycoprotein immunochemically indistinguishable from amyloid P, a glycoprotein found in all tissue amyloid deposits. SAP has also been shown to be a constituent of normal glomerular basement membrane (GBM). In this study the authors discovered a unique association between SAP and Goodpasture (GP) antigen. In those patients whose GBM lack GP antigen (those with Alport-type hereditary nephritis) SAP is also uniformly absent. Although the relationship between these two components is unknown, this association may provide clues to the abnormality of GBM in Alport-type hereditary nephritis.