Biomedical subjects
Publications and source records attributed to S Buck.
Sheep, mice, cattle, goats and pigs have all been cloned by transfer of a donor cell nucleus into an enucleated ovum, and now we add the successful cloning of a cat (Felis domesticus) to this list. However, this cloning technology may not be readily extendable to other mammalian species if our understanding of their reproductive processes is limited or if there are species-specific obstacles.
When normal-lived Ra strain Drosophila were indirectly selected for longevity, they gave rise to long-lived La strain animals with lower oxidized protein and lipid levels that were temporally coincident with higher antioxidant activities. We wanted to determine whether it was possible to create long-lived animals by a direct selection for increased antioxidant activities. Using the same Ra strain, we selected them over 24 generations for increased resistance to paraquat. Selection was successful: the paraquat-resistant flies had a fourfold increase in their LT(50) (mean lethal time) values. Their extended longevity pattern differs from that of the La strain. The paraquat-resistant animals also have a lower level of antioxidant activity, an increased total P450 enzyme activity level, an altered pattern of energy metabolism, and a significantly lower developmental viability. We interpret these findings as suggesting that similar stress response phenotypes may be generated by different molecular mechanisms, some of which may generate very different types of extended longevity phenotypes.
The expression of epsilon- and gamma-globin mRNA and protein has been determined in three Old World monkey species (Macaca mulatta, Macaca nemestrina, and Cercopithecus aethiops). Using RT-PCR with primers for epsilon- and gamma-globin, both mRNAs were detected in early fetal stages, whereas at 128 days (85% of full term), only gamma was expressed. High-performance liquid chromatography was used for separation and quantitation, and matrix-assisted laser desorption/ionization mass spectrometry was used for identification of globin polypeptides. An alpha-globin polymorphism was observed in all of the species examined. During fetal life, gamma-globin was the predominant expressed beta-type globin. The red blood cells of infants still contained substantial amounts of gamma-globin, which declined to negligible levels in 14 weeks as beta-globin expression reached adult values. The ratio of gamma1- to gamma2-globins (equivalent to Ggamma/Agamma in humans) was approximately 2.5, similar to the Ggamma/Agamma ratio observed in humans. Thus, gamma-globin gene expression in these Old World monkeys species has three features in common with human expression: expression of both duplicated gamma genes, the relative preponderance of gamma1 over gamma2 expression, and the delay of the switch from gamma- to beta-globin until the perinatal period. Thus, the catarrhines seem to share a common pattern of developmental switching in the beta-globin gene cluster, which is distinct from the timing of expression in either prosimians or the New World monkeys. Our results indicate that an Old World monkey, such as Rhesus, could serve as a model organism (resembling humans) for experimentally investigating globin gene expression patterns during the embryonic, fetal, and postnatal stages.
Early during apoptosis, there is a reduction in mitochondrial transmembrane potential (MTP) and externalization of phosphatidylserine (PS) in cell membrane prior to eventual cell death. Flow cytometric detection techniques targeting these changes, reduction of DiOC(6)(3) uptake upon the collapse of MTP and annexin V binding to PS have been successfully used to detect apoptotic cells. These methods have given comparable results when cell lines were used. We compared the two different techniques, DiOC(6)(3) uptake and Annexin V-propidium iodide co-labeling in the quantification of cytarabine, vincristine and daunorubicin induced apoptosis on three leukemia cell lines (HL-60, CEM, U937), and bone marrow blasts from 26 children with acute myeloid leukemia, 14 with T cell acute lymphoblastic leukemia. Anti-Fas-induced apoptosis in culture-grown peripheral blood T lymphocytes on 18 samples from 9 children with non-malignant conditions were also studied by these techniques. Our results showed that there is a correlation (P < 0. 05) between the apoptosis rates measured by these two techniques for drug-induced apoptosis in myeloid and lymphoid blasts, and for anti-Fas mAb-induced apoptosis in T lymphocytes. This data suggests that reduction of the MTP and PS externalization may be common to many apoptotic pathways and techniques targeting either of these changes may be used in quantification of apoptosis in different clinical samples.
Some years ago we applied simultaneously an identical regime of selection for late-life reproduction to several normal-lived sister lines (Ra and Rb) so as to produce several selected long-lived sister lines (La and Lb). The long-lived La and Lb sister lines had statistically identical longevity phenotypes and paraquat resistance phenotypes; however, we noticed some statistically different responses of the two strains at the biochemical level. Extensive work with the La strain showed that transcriptional alterations in antioxidant gene expression are robustly associated with its extended longevity. We decided to critically test the assumption of phenotypic equivalence by subjecting the Lb strain to the same series of molecular assays as was the La strain. The two sister strains are characterized by significantly different mechanisms and patterns of antioxidant gene expression, antioxidant enzyme activity, and oxidative damage. We find that the Lb strain appears to depend on the transcriptional activation of different genes than does the La strain, and on a post-translational up-regulation of at least one other antioxidant defense gene. The phenotypic equivalence observed at the organism level need not hold at the molecular genetic level. This finding suggests that there is more than one molecular mechanism by which antioxidant defense genes can bring about an increased resistance to oxidative stress. The theoretical and empirical implications of these findings are discussed.
Patterns of antioxidant gene expression and of oxidative damage were measured throughout the adult life span of a selected long-lived strain (La) of Drosophila melanogaster and compared to that of their normal-lived progenitor strain (Ra). Extended longevity in the La strain is correlated with enhanced antioxidant defense system gene expression, accumulation of CuZnSOD protein, and an increase in ADS enzyme activities. Extended longevity is strongly associated with a significantly increased resistance to oxidative stress. Reverse-selecting this long-lived strain for shortened longevity (RevLa strain) yields a significant decrease in longevity accompanied by reversion to normal levels of its antioxidant defense system gene expression patterns and antioxidant enzyme patterns. The significant effects of forward and reverse selection in these strains seem limited to the ADS enzymes; 11 other enzymes with primarily metabolic functions show no obvious effect of selection on their activity levels whereas six other enzymes postulated to play a role in flux control may actually be involved in NADPH reoxidation and thus support the enhanced activities of the ADS enzymes. Thus, alterations in the longevity of these Drosophila strains are directly correlated with corresponding alterations in; 1) the mRNA levels of certain antioxidant defense system genes; 2) the protein level of at least one antioxidant defense system gene; 3) the activity levels of the corresponding antioxidant defense system enzymes, and 4) the ability of the organism to resist the biological damage arising from oxidative stress.
Inherent resistance of myeloblasts to vincristine (VCR) has been related to the activity of myeloperoxidase (MPO) which can degrade VCR in the presence of hydrogen peroxide (H2O2). We investigated the relationship between VCR degradation and hypochlorous acid (HOCl) generation from the reaction of H2O2 with chlorine (Cl) as catalyzed by MPO. A cell-free system, three human leukemia cell lines (CEM/CCRF, HL-60, U937) and 15 bone marrow samples from children with acute myeloid leukemia (AML) were studied. VCR cytotoxicity was evaluated by MTT assay and by quantitative measurement of apoptosis. In vitro levels of VCR in cell-free systems were measured by high performance liquid chromatography (HPLC), and intracellular HOCl levels by oxidation of 5-thio-2-nitrobenzoic acid with the accompanying decrease in the absorbency at 412 nm. VCR was degraded by increasing concentrations of HOCl in cell-free systems and this activity was inhibited by taurine, which is known to block HOCl activity. This finding was confirmed by the VCR cytotoxicity studies on cell lines. The HOCl-producing myeloblasts from patients were resistant to VCR. In five samples out of eight HOCl was also detected extracellularly. These results suggest that oxidation by HOCl may be the final step in VCR degradation catalyzed by MPO through its action on intracellular H2O2 and Cl. Leukemia (2000) 14, 47-51.
It has proven relatively easy to select normal-lived strains of Drosophila for extended longevity in the laboratory. Long-lived strains have not been observed in the wild as yet. Of the various life-history traits that have been investigated for their role in modulating the evolution of extended longevity, none have yet shown a consistent or convincing relationship. Other than developmental time, the traits usually investigated in this regard are those associated with the adult phase of the life cycle. We assayed developmental timing and viability in six pairs of normal- and long-lived strains, four pairs of which are from previously described strains and two pairs of which are new strains that have been independently and recently selected. We find that the life-history trait most obviously associated with all our long-lived strains is a significantly reduced developmental viability, with the long-lived strains' having as much as twice the developmental lethality as do any of the normal-lived strains. The long-lived strains also pupate closer to the food, a behavior known to decrease fitness. Thus the reduced fitness of the long-lived strains appears to be due to both physiological and behavioral factors and may well explain why long lived strains are not usually found in the wild. The extension of longevity involves costs as well as benefits that, in this case, are borne by different individuals.
Dental pulp is richly innervated with neurons containing calcitonin gene-related peptide (CGRP) or substance P (SP). Prior studies have demonstrated that inflammation alters these pulpal neuropeptides. In this study, we used a radioimmunoassay to evaluate the specificity of this response and the contribution of axonal transport. Rat mandibular molars were exposed and immunoreactive CGRP (iCGRP) and immunoreactive SP were measured. At 7 to 14 days after exposure, both pulpal iCGRP (73%) and immunoreactive SP (135%) displayed peak increases above control levels. This response was somatotopically restricted, with no changes observed in contralateral (intact) molars, or in ipsilateral mandibular molars after exposure of maxillary molars. Transection of the inferior alveolar nerve on day 13 significantly reduced pulpal levels of iCGRP on day 14. Collectively, these studies indicate that pulpal inflammation evokes a selective alteration in neuropeptide levels, due at least in part to alterations in transport or synthesis of neuropeptides in the trigeminal ganglion.
PURPOSE: We compared the antimicrobial activity of a new multi-purpose disinfection solution (OPTI-FREE EXPRESS with ALDOX) to a 3% hydrogen peroxide disinfecting system. The antimicrobial ingredients in the new solution are polyquaternium-1 and myristamidopropyl dimethylamine. METHODS: The solutions were tested for antimicrobial activity against Staphylococcus sp.., Pseudomonas aeruginosa, Serratia marcescens, Candida albicans, Fusarium solani, and two Acanthamoeba species and were evaluated according to the primary criteria of the FDA and ISO contact lens disinfection procedures. RESULTS AND CONCLUSIONS: OPTI-FREE EXPRESS with ALDOX (EXPRESS MPDS) Multi-Purpose Disinfecting Solution provided a multiple spectrum of antimicrobial activity and met the FDA and ISO primary Stand Alone Test criteria for disinfection of contact lenses. EXPRESS MPDS showed disinfection activity in the range of a 3% hydrogen peroxide system. Unlike the 3% hydrogen peroxide system, it retarded contamination during storage.
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AIMS: To evaluate factors affecting Heidelberg retinal flowmeter (HRF) measurements of retinal and optic nerve head blood flow in human subjects. METHODS: The angle of incidence between laser beam and fundus, and camera distance from the eye, were evaluated for their effect upon measures of blood volume, velocity, and flow in a single 100 x 100 x 400 microns volume of temporal peripapillary retinal tissue in normal volunteers. Both intra and intersession reproducibility of these measures were studied. Intersession data were obtained by taking one image per week for 4 weeks. Finally, the intersession haemodynamic data were examined in the entire image (640 x 2560 x 400 microns), using histograms of pixel by pixel blood flow. RESULTS: Measures of blood volume, velocity, and flow from a single anatomical site were unaffected by laser beam to fundus angle of incidence (n = 12). As camera distance from the eye was increased (from 2 to 5 to 7 cm), flow measurements showed increasing individual changes, despite unaltered measured vessel lengths and constant overall mean flow (n = 14). The coefficient of variation for two intersession images of optic nerve head blood flow averaged 7% (n = 20); in contrast, the 4 week intersession coefficient of variation averaged 30% (n = 15). Intersession reproducibility was increased by using flow histograms from the entire image: the coefficients of variation averaged 16% for total flow and 17% for flow in the pixel of median flow. CONCLUSION: HRF measures of flow are independent of the laser beam to fundus angle of the incidence and dependent upon camera distance from the eye. Intersession reproducibility is best using pixel by pixel analysis of the entire image.
The relationships between dihydrofolate reductase (DHFR) levels or methotrexate membrane transport and acute lymphoblastic leukemia (ALL) immunophenotype were evaluated on 51 T-cell and 44 B-precursor ALL specimens from 90 pediatric ALL patients at diagnosis and relapse, using a fluorescent methotrexate analog (PT430) and flow cytometry assay (Matherly et al, Blood 85:500, 1995). For T-cell ALL, 35 of 45 (78%) of newly diagnosed patients' specimens exhibited elevated DHFR relative to DHFR levels in ALL blasts from methotrexate-responsive patients. For 30 of 45 diagnostic T-ALL specimens, DHFR expression was heterogeneous, with up to 3 separate subpopulations covering a 44-fold range; the DHFR-overproducing fractions comprised 10% to 88% of the total blasts. Elevated DHFR was less common in B-precursor ALL at diagnosis, being detected in only 17 of 36 specimens (47%); 11 of these samples exhibited DHFR heterogeneity. Median maximal DHFR levels were fourfold higher in T-cell than B-precursor ALL at diagnosis. Within a particular phenotypic group, there were no correlations between DHFR levels and patient prognostic features, including age, sex, chromosomal abnormalities, white blood cell counts (WBCs), and percentage of S-phase. However, for B-precursor ALL, there was a notably higher fraction of African-American than white patients with elevated DHFR. For patients (both phenotypes) with low WBCs (<50,000/ microL), event-free survival times were significantly shorter for those expressing DHFR above a threshold level than for patients expressing DHFR below this level (P < .016); this relationship was not seen for patients with high WBCs (>50,000/microL). Elevated DHFR was detected in 11 of 14 relapse specimens (5 of 6 T-cell and 6 of 8 B-precursor). Two of five paired relapse specimens (both T-cell) from patients treated with methotrexate exhibited increased DHFR levels over those at diagnosis (2.5- to 5-fold); the fraction of DHFR-overproducing blasts was also increased in 4 of 5 paired relapse specimens (2 B-precursor and 2 T-cell). In contrast to the variations in DHFR, highly impaired methotrexate transport was detected in only 6 of 95 ALL specimens, including both diagnosis and relapse. There was no correlation between phenotype and impaired transport. These data provide further rationale for the use of mechanistically based prognostic factors to complement known biologic or disease-based prognostic indicators in the design of ALL therapy including methotrexate, particularly with patients presenting with low WBCs. The finding of a markedly increased frequency of DHFR overexpression in T-cell over B-precursor ALL suggests that this difference is associated with the poorer prognosis of patients with T-cell ALL treated with standard-dose antimetabolite therapy and implies that higher-dose methotrexate (> or = 1 g/m2) during consolidation therapy may be useful in the treatment of this disease.
A pre-gamma-globin species was identified by high performance liquid chromatography in platyrrhine primates. Although pre-gamma-globin has not been observed in human hemoglobin, its identification in platyrrhine hemoglobin was facilitated by the functional inactivation of one of the duplicated gamma-globin genes in platyrrhines, which simplified the high performance liquid chromatography elution pattern. Part, but not all, of the pre-gamma was glutathionyl gamma 2-globin, and matrix-assisted laser desorption/ionization mass spectrometry was used to demonstrate that the glutathionyl residue was located on cysteine 93. As this residue is invariant in primates, it is predicted that the formation of glutathionyl gamma-globin will be seen in all primate hemoglobins under appropriate conditions.
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Reverse phase chromatography of the globin chains of adult, newborn, and fetal erythrocytes from three species of New World monkeys (Cebus apella, Aotus azarae, and Callithrix jacchus) representing three of the seven platyrrhine clades showed that gamma-globin expression was fetal in these animals. The globins were identified by a combination of chemical sequencing and mass spectrometric analysis. Since gamma-globin expression is fetal in the other major simian branch, the catarrhines, but embryonic in prosimian primates and nonprimate placental mammals, the evolution of fetal recruitment can now be assigned to the period between the simian-prosimian divergence (55 million years ago) and the platyrrhine-catarrhine divergence (35 million years ago). The gamma-globin gene underwent tandem duplication during the same evolutionary epoch, in accord with a model that suggests that the downstream duplicated gamma-gene (gamma2) was free to acquire the mutations necessary for fetal recruitment. Mass spectrometric analysis of tryptic digests of the gamma-globins verified the amino acid sequences deduced from genomic sequencing. Detailed analysis of high performance liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry data showed that gamma2-globin in Cebus was expressed to a far greater extent than gamma1-globin, supporting inferences drawn from a study of the promoter sequences. A "pre-gamma"-globin was observed in C. apella and shown to be primarily the glutathionyl adduct. The other species, A. azarae and C. jacchus, also express only one gamma-globin polypeptide. This work provides biochemical evidence of an evolutionary trend in the platyrrhines to alter the duplicated gamma-globin gene locus so that only one gamma-globin polypeptide is expressed.
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Bisphosphonates (BPs) are used for the treatment of both benign and malignant diseases characterized by increased bone resorption. Because of their potential nephrotoxicity, currently available BPs have to be administered by slow intravenous infusion, with conventional doses requiring an infusion time of at least 2 h. In the present investigation, we evaluated the safety and efficacy of the new BP ibandronate as administered by intravenous bolus injection. On day 0, 15 normocalcemic breast cancer patients with bone metastases were treated with 3 mg of ibandronate injected intravenously over 60-120 s. Ibandronate treatment led to significant decreases in serum levels of calcium (p < 0.0001) and phosphate (p < 0.0001) and to subsequent increases in serum concentrations of parathyroid hormone (p <0.0001) and calcitriol (p <0.0001). Moreover, there was a significant reduction in the urinary excretion of calcium (p <0.0001), pyridinoline (p <0.001), and deoxypyridinoline (p < 0.0001). Three serious adverse events were observed: vomiting (WHO grade 3), pulmonary infection (WHO grade 2), and deterioration of a pre-existing impaired glucose tolerance (WHO grade 3). Only vomiting appeared to be related to administration of the drug. The most frequent nonserious adverse events were 10 cases of transient clinically asymptomatic hypocalcemia and 8 cases of asymptomatic hypophosphatemia. Serum levels of creatinine and urea nitrogen did not increase, nor did creatinine clearance deteriorate. When tested with the dipstick method, proteinuria was present in five (33%) patients prior to ibandronate treatment (median protein concentration, 30 mg/dl). Following the BP injection, seven (47%) patients showed slight (highest protein concentration, 30 mg/dl) transient proteinuria at at least one time point, of which six cases appeared in conjunction with leucocyturia and three with microhematuria. Side effects specific to aminosubstituted BPs (fever, reduction in white blood cell counts, and lymphocyte counts) were not seen in these 15 patients. In conclusion, a single intravenous injection of 3 mg of ibandronate significantly inhibited osteoclast activity as reflected by the decrease in serum calcium and in urinary parameters of bone resorption. Serum creatinine levels and estimates of creatinine clearance were not affected by therapy. However, before repeated bolus injections of ibandronate at this dosage can be recommended for further clinical trials, whether a relationship exists between the transient pathological urinary findings and injected ibandronate needs to be determined.