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Biomedical subjects

J Kaplan

Publications and source records attributed to J Kaplan.

At least 19 recordsLinked to original sources
PubMed1998

Defects in the yeast high affinity iron transport system result in increased metal sensitivity because of the increased expression of transporters with a broad transition metal specificity.

Yeast with defects in vacuolar pH show increased sensitivity to high concentrations of transition metals. This sensitivity has been presumed to result from defective metal storage. We demonstrate that mutations that result in a defective high affinity iron transport system, such as a deletion in the surface ferroxidase FET3, also result in increased metal sensitivity independent of vacuolar function. Multiple copies of transition metal transporter resistance genes, such as COT1 or ZRC1, do not reduce the metal sensitivity of fet3 mutations. Increased metal sensitivity is because of an increased cellular accumulation of transition metals resulting from the increased activity of low affinity iron transporters, such as FET4, that mediates the transport of other transition metals. In cells lacking a high affinity iron transport system, the increased transition metal uptake can be prevented by increased extracellular iron. These results suggest that vacuolar function may not be required for transition metal sequestration.

Base Sequence↗
PubMed1998

Site-directed mutagenesis of the yeast multicopper oxidase Fet3p.

High affinity iron transport in yeast is mediated by two proteins, Fet3p and Ftr1p. The multicopper oxidase Fet3p is thought to convert extracellular ferrous iron to ferric iron, which then crosses the plasma membrane through the permease Ftr1p. Fet3p is capable of oxidizing other substrates, such as p-phenylenediamine, and there is still a question of whether it is the ferroxidase activity that is essential for iron transport. Fet3p is also required for Ftr1p localization to the cell surface, making it difficult to prove a direct role for Fet3p oxidase in high affinity iron transport. In an attempt to generate Fet3p specifically lacking ferroxidase activity, we used site-directed mutagenesis to alter residues within Fet3p that had been suggested to impart iron oxidase activity. These substitutions resulted in either a loss or retention of both p-phenylenediamine and ferroxidase activities, indicating that the ability of Fet3p to act as a ferroxidase involves other amino acids. Inactive Fet3p, however, did mediate Ftr1p localization to the cell surface but did not mediate high affinity iron transport. These observations indicate that the ferroxidase activity of Fet3p is intrinsically required for high affinity iron transport.

Amino Acid Sequence↗
PubMed1998

Complete exon-intron structure of the retina-specific ATP binding transporter gene (ABCR) allows the identification of novel mutations underlying Stargardt disease.

Stargardt disease, an autosomal recessive macular dystrophy of childhood, leading to severe visual impairment, is caused by mutations in the retina-specific ATP binding transporter gene (ABCR). Previously, the ABCR cDNA and part of the exon-intron structure were described. We have determined the complete ABCR exon-intron structure by exon-exon PCR. The ABCR gene encompasses 50 exons, 29 of which are first described here with their corresponding intron-exon boundaries. The discovery of a splicing mutation (571: 2A-->G) and missense mutations in the newly identified exons (R18W, R212C) gives additional support to the broad allelic heterogeneity of Stargardt disease.

ATP-Binding Cassette Transporters↗
PubMed1998

Exclusion of five subunits of cGMP phosphodiesterase in Leber's congenital amaurosis.

Leber's congenital amaurosis (LCA) is the earliest and most severe of all inherited retinal dystrophies. Recently, we mapped an LCA gene to chromosome 17p13.1 (LCA1) and ascribed the disease to mutations of the retinal guanylate cyclase (ret GC) gene in a subset of families of North African ancestry. Owing to the genetic heterogeneity of LCA and considering that LCA1 results from an impaired production of cGMP in the retina (with permanent closure of cGMP-gated cation channels), we hypothesized that the activation of the cGMP phosphodiesterase (PDE) could trigger the disease by lowering the intracellular cGMP level in the retina. The rod and cone cGMP-PDE inhibitory subunits were regarded therefore as candidate genes in LCA. Here, we report the exclusion of five rod and cone cGMP-PDE subunits in LCA families unlinked to chromosome 17p13.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1998

The epsilon4 allele of the apolipoprotein E gene as a potential protective factor for exudative age-related macular degeneration.

PURPOSE: Apolipoprotein E (ApoE) is a polymorphic protein that plays a central part in plasma metabolism of lipids and in central nervous system lipid homeostasis. Our purpose was to evaluate the potential role of ApoE polymorphism in the occurrence of exudative age-related macular degeneration associated with drusen, which contain lipids. METHODS: We analyzed apolipoprotein E genotypes in 116 unrelated patients with exudative age-related macular degeneration in one eye and hard drusen (n = 39) or soft drusen (n = 77) in the other eye, and compared the results with those of age-matched and sex-matched control subjects (n = 168). Apolipoprotein E alleles were detected by a ploymerase chain reaction-based method. RESULTS: A lower frequency of the epsilon4 allele carriers was observed in the exudative age-related macular degeneration group compared with control subjects (12.1% vs 28.6%, respectively; P < .0009). The epsilon4 allele was less frequent in the age-related macular degeneration group compared with control subjects (0.073 vs 0.149, respectively; P < .006). This decreased frequency of the epsilon4 allele was mainly observed in the soft drusen subgroup compared with control subjects (0.045 vs 0.149, respectively; P < .0009). CONCLUSION: This lower relative frequency of the epsilon4 allele supports the hypothesis that the ApoE gene is a genetic protective factor identified in age-related macular degeneration.

Aged↗
PubMed1998

Macular dystrophy, diabetes, and deafness associated with a large mitochondrial DNA deletion.

PURPOSE: To report the mitochondrial DNA in a 17-year-old patient with diabetes, deafness, cataract, and maculopathy. METHOD S: Ophthalmologic examination, fluorescein angiography, and electroretinogram were performed. Detection of deletion was analyzed by polymerase chain reaction and Southern blot, and screening for the A3243G mitochondrial DNA mutation was performed. RESULTS: A short fragment of approximately 8.5 kb corresponding to deleted mitochondrial DNA was detected. The A3243G mitochondrial DNA mutation was not found. CONCLUSIONS: A 7-kb heteroplasmic deletion of the mitochondrial genome was found in this patient. No mitochondrial DNA deletion has been reported previously in association with macular dystrophy.

Adolescent↗
PubMed1998

Endothelial injury and leukocyte adherence in Häutchen preparations from coronary arteries and aorta of cynomolgus monkeys.

The aim was to describe the production of en face preparations (Häutchens) of the endothelium from the aorta and the coronary arteries of cynomolgus monkeys (Macaca fascicularis), to describe the morphology of the endothelium and to estimate endothelial injury. The present study describes, for the first time, the application of this technique to the coronary arteries. Numerous leukocytes (peroxidase-positive and -negative cells) were observed on the endothelium and a positive correlation between the numbers of adherent leukocytes and injured endothelial cells was found. A relatively leukocyte-independent measure of endothelial injury in the aorta was obtained by counting areas with a restricted number of leukocytes. The results indicated an even distribution and a relatively low frequency of injured endothelial cells along the arterial tree. The percentage of injured endothelial cells in LAD and LCX was 0.18 and 0.16, and in non-branched and circumostial areas of the descending thoracic aorta the corresponding figures were 0.22 and 0.28. There were, on average, 344 and 236 adherent leukocytes/mm2 in the non-branched and circumostial areas of the descending thoracic aorta, respectively, compared to 13 in the LAD and 23 in the LCX. Proportionally more peroxidase-positive leukocytes (possibly granulocytes) were found on the coronary endothelium than on the aortic endothelium, and the peroxidase-positive cells were preferentially located around small artery branches in the coronary arteries. The foregoing data provide an estimate of the distribution and frequency of endothelial injury and adherent leukocytes at pathobiologically relevant arterial sites in unmanipulated cynomolgus monkeys, an animal frequently used in atherosclerosis research.

Animals↗
PubMed1998

Psychosocial stress causes endothelial injury in cynomolgus monkeys via beta1-adrenoceptor activation.

Current evidence links psychosocial factors to exacerbation of diet-induced atherosclerosis in monkeys via activation of the sympathetic nervous system. However, it is uncertain whether these factors can potentiate initial lesion formation, and do so even in the absence of dietary provocation, and whether any such effects can be prevented by beta-adrenergic blockade. As endothelial injury has been considered an initiating event in atherogenesis, we studied the effect of psychosocial stress on endothelial integrity in 48 adult male cynomolgus monkeys (Macaca fascicularis). All animals were housed in 12 social groups of four monkeys each for 11 weeks. The monkeys in half of the groups were exposed to a socially unstable ('stressed') condition for 72 h and received saline (n = 8), a lipophilic beta1-blocker (metoprolol, 0.30 mg/kg per h; n = 8), or hydrophillic beta1-blocker (atenolol, 0.15 mg/kg per h; n = 8). The remaining six social groups were assigned to the socially stable (non-stressed) condition; for 72 h these animals all remained in their social groups and were similarly treated with saline (n = 8), metoprolol (n = 8), or atenolol (n = 8). The frequency of IgG-positive (injured) endothelial cells was estimated on en face (Häutchen) preparations from the thoracic aorta and coronary arteries. Psychosocial stress caused a significant increase in the number of injured endothelial cells in the circumostial areas of the descending thoracic aorta in the placebo group (0.3 vs. 0.8%, P < 0.02), an effect that had not been demonstrated previously. Moreover, beta-blockade significantly (P < 0.01) inhibited the stress effect, with no differences between the two beta-blocking agents. The number of injured endothelial cells in the non-branched portions of the aorta and coronary arteries were low and indistinguishable among groups; irregularities in the size and location of branching points in the coronary arteries precluded analysis of these sites. This study demonstrated that psychosocial stress induces endothelial injury, and that this effect is mediated via beta1-adrenoceptor activation.

Adult↗
PubMed1998

Cell line characterization and authentication.

Research and development involving the use of cell lines require precise knowledge of the purity and species of origin of the cell lines used. This can only be assured by periodic monitoring of cultured cell lines for possible contamination by other cells and for characteristics that authenticate the cell line identity. In the absence of such monitoring, inter- and intraspecies cell line contaminations are likely to occur in the laboratories of unsuspecting investigators and can result in the generation of mistaken conclusions with an attendant loss of investigators' time, effort, and resources. This chapter provides a history and an overview of the methods that have been developed for cell line authentication, the type of information each of these different methods provides, and how synthesis of that information can be used to characterize a cell line and confirm its identity. An effective cell line monitoring strategy is described that involves testing for a combination of genetic markers, including cell membrane species antigens, isoenzymes, chromosomes, and DNA fingerprints, and use of databases for each marker system to compare the results obtained with a test cell culture with results from an extensive panel of previously tested cell lines.

Animals↗
PubMed1998

Iron and copper transport in yeast and its relevance to human disease.

Recent progress in the field of copper and iron metabolism has resulted from a convergence of human and yeast genetics. The mechanisms of iron and copper transport are remarkably conserved between yeast and humans. Studies of the yeast homologs of human disease genes involved in metal homeostasis have shed light on the pathophysiology of these disorders.

Biological Transport, Active↗
PubMed1998

Phase I study of paclitaxel (taxol) and granulocyte colony stimulating factor (G-CSF) in patients with unresectable malignancy.

A phase I trial of a 24-hour infusion of paclitaxel was conducted to identify the maximum tolerated dose of paclitaxel with granulocyte colony-stimulating factor (G-CSF) in patients with unresectable malignancy previously untreated with chemotherapy. Nineteen patients with metastatic melanoma or non-small cell lung cancer were treated with paclitaxel administered at 250, 300, 400 mg/m2 every 3 weeks. G-CSF, 5 microg/kg was given as a daily subcutaneous injection 24 hours after the completion of the infusion. Dose limiting myelosuppression and peripheral neuropathy was observed at 400 mg/m2 and 350 mg/m2. Paclitaxel can be safely administered as a 24-hour infusion at 300 mg/m2 with G-CSF. Further studies of paclitaxel and G-CSF are recommended to determine a dose-response relationship in sensitive tumors.

Adult↗
PubMed1998

IL-2-activated murine newborn liver NK cells enhance engraftment of hematopoietic stem cells in MHC-mismatched recipients.

To explore the modulatory effects of IL-2-activated NK cells on hematopoietic stem cell (HSC) engraftment further, we used fresh newborn liver cells (NLC) and IL-2-activated newborn liver cells (ANLC) as combined sources, respectively, of transplanted HSC and IL-2-activated NK cells free of contaminating CD3+ T cells. As previously found with adult IL-2-activated spleen cells, NLC cultured with IL-2 for 7 days exhibited lymphokine-activated killer (LAK) activity, veto activity, and natural suppressor activity, and enhanced both short-term and long-term stem cell engraftment by intact co-injected syngeneic and allogeneic NLC in totally MHC-mismatched lethally irradiated recipients. However, unlike adult IL-2-stimulated adult spleen cells, IL-2-activated NLC lacked CD3+ T cells and failed to induce lethal GVHD. FACS analysis and cell sorting experiments showed that the cells in ANLC which enhanced short-term HSC engraftment belonged to the relatively immature CD3-NK1.1-2B4+ NK cell subset. By contrast, cells belonging to the more mature CD3-NK1.1+2B4+ NK cell subset showed no HSC-enhancing effects. Identification and isolation in humans of similar NK cell enhancers of HSC could lead to a new approach to improving stem cell engraftment in MHC-mismatched recipients without increasing the risk of GVHD.

Animals↗
PubMed1998

Zinc may regulate serum leptin concentrations in humans.

OBJECTIVE: Leptin, the product of the ob gene, plays a key role in a feedback loop that maintains energy balance by signaling the state of energy stores to the brain and by influencing the regulation of appetite and energy metabolism. Zinc also plays an important role in appetite regulation. Thus, we evaluated the relationship between zinc status and the leptin system in humans. METHODS: We studied nine healthy men with marginal zinc deficiency, induced by dietary means, before and after zinc supplementation. RESULTS: Zinc restriction decreased leptin levels while zinc supplementation of zinc-depleted subjects increased circulating leptin levels. In addition, zinc supplementation increased IL-2 and TNF-alpha production that could be responsible for the observed increase in leptin concentrations. CONCLUSIONS: Zinc may influence serum leptin levels, possibly by increasing the production of IL-2 and TNF-alpha.

Adult↗
PubMed1998

Mutations in Emery-Dreifuss muscular dystrophy and their effects on emerin protein expression.

Seventeen families with Emery-Dreifuss muscular dystrophy (EDMD) have been studied both by DNA sequencing and by emerin protein expression. Fourteen had mutations in the X-linked emerin gene, while three showed evidence of autosomal inheritance. Twelve of the 14 emerin mutations caused early termination of translation. An in-frame deletion of six amino acids from the C-terminal transmembrane helix caused almost complete absence of emerin from muscle with no localization to the nuclear membrane, although mRNA levels were normal. This shows that mutant emerin proteins are unstable if they are unable to integrate into a membrane. A 22 bp deletion in the promoter region was expected to result in reduced emerin production, but normal amounts of emerin of normal size were found in leucocytes and lymphoblastoid cell lines. This shows that DNA analysis is necessary to exclude emerin mutations in suspected X-linked EDMD. Emerin levels in female carriers often deviated from the expected 50% and this was due, in at least two families, to skewed emerin mRNA expression from the normal and mutated alleles. In one family with a novel deletion of the last three exons of the emerin gene, a carrier had a cardiomyopathy and very low emerin levels (<5% of normal) due to skewed X-inactivation. In the three autosomal cases of EDMD, emerin was normal on western blots of blood cells, which suggests that autosomal EDMD is not caused by indirect reduction of emerin levels.

Adolescent↗
PubMed1998

Mutations in the retinal guanylate cyclase (RETGC-1) gene in dominant cone-rod dystrophy.

The dominant cone-rod dystrophy gene CORD6 has previously been mapped to within an 8 cM interval on chromosome 17p12-p13. The retinal-specific guanylate cyclase gene (RETGC-1), which maps to within this genetic interval and previously was implicated in Leber's congenital amaurosis, was screened for mutations within this family and in a panel of small families and individuals with various cone and cone- rod dystrophy phenotypes. A missense mutation (E837D) was identified in affected members of the CORD6 family, as well as a second missense mutation (R838C) in three other families with dominant cone-rod dystrophy. RETGC-1 is only the fourth gene to be implicated in cone-rod dystrophy and this is the first report of dominant mutations in this gene.

Adolescent↗
PubMed1998

Relation between literacy, race, and stage of presentation among low-income patients with prostate cancer.

PURPOSE: Diagnosis of advanced prostate cancer is a major health problem, especially among low-income men. Opportunities vary for early detection of prostate cancer for low-income black and white men because of financial, cultural, and social factors. In this study, we evaluated the association of poor literacy skills with higher rates of presentation of advanced stages of prostate cancer among low-income black and white men who received care in equal-access medical systems. PATIENTS AND METHODS: Literacy and stage at diagnosis of prostate cancer were evaluated in 212 low-income men who received medical care in Shreveport, LA, and Chicago, IL. The patients' literacy was assessed with the Rapid Estimate of Adult Literacy in Medicine (REALM), an individually administered reading screening test designed specifically for use in the medical setting. Logistic regression models were used to evaluate predictors of metastatic disease at presentation as a function of patient age, race, literacy, and city. RESULTS: Whereas black men were almost twice as likely to present with stage D prostate cancer (49.5% v 35.9%; P < .05), they were significantly more likely to have literacy levels less than sixth grade (52.3% v 8.7%; P < .001). However, after adjustment for differences in literacy, age, and city, race was not a significant predictor of advanced-stage prostate cancer. CONCLUSION: Low literacy may be an overlooked but significant barrier to the diagnosis of early-stage prostate cancer among low-income white and black men. The development of culturally sensitive, low-literacy educational materials may improve patient awareness of prostate cancer and improve the frequency of diagnosis of early-stage cancer.

Adult↗