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Biomedical subjects

L M Isola

Publications and source records attributed to L M Isola.

At least 19 recordsLinked to original sources
PubMed1998

Retroviral gene transfer into cord blood stem/progenitor cells using purified vector stocks.

Cord blood (CB) progenitor/stem cells (P/SC) are ideal targets for early gene therapy in individuals prenatally diagnosed with genetic disorders. Most retroviral transduction protocols were developed using adult peripheral blood stem cells (PBSC) and bone marrow (BM). Less is known about retroviral transduction of CB P/SC. We examined how timing, multiplicity of infection (MOI), and polycations in the transduction media affect transduction efficiency. Rates of transduction were determined in recently isolated CD34+ enriched CB cells and in colonies derived after various times in liquid cultures (LC). CB mononuclear cells (MNC) were separated by ficoll-hypaque centrifugation and enriched for CD34+ cells. Purity was assessed by flow cytometry. Transduction were performed with clinical-grade retroviral stocks at MOIs of 1-20. Transduction was performed with fetal bovine serum (FBS) or autologous plasma, IL-3, GM-CSF, IL-6, and SCF. The retroviral vector contained LacZ and neomycin resistance (neo) reporter genes. Transduction was determined by X-gal stain and by PCR amplification of the reporter genes. No drug selection was used. Twenty-five experiments were done. CB volumes ranged from 35-150 ml. MNC and CD34+ cell counts ranges were: 0.14-840 x 10(6) and 0.1-4.2 x 10(6), respectively. Transduction efficiency in liquid cultures ranged from 4-63%. Higher rates were seen using MOI > or = 10, 2 microg/ml polybrene, and 10% autologous CB plasma. In colonies, transduction rates were 63 to 72% by PCR and 32% by X-gal staining. In LTC-IC derived colonies, transduction was 7% by PCR. Short incubations of CD34+ CB cells with purified retroviral stocks, polybrene, and autologous sera result in high transduction rates of committed progenitors and moderately low efficiencies of transduction of LTC-IC in the absence of drug selection.

Antigens, CD34↗
PubMed1997

Uptake of long chain free fatty acids is selectively up-regulated in adipocytes of Zucker rats with genetic obesity and non-insulin-dependent diabetes mellitus.

To examine whether fatty acid transport is abnormal in obesity, the kinetics of [3H]oleate uptake by hepatocytes, cardiac myocytes, and adipocytes from adult male Wistar (+/+), Zucker lean (fa/+) and fatty (fa/fa), and Zucker diabetic fatty (ZDF) rats were studied. A tissue-specific increase in oleate uptake was found in fa/fa and ZDF adipocytes, in which the Vmax was increased 9-fold (p < 0.005) and 13-fold (p < 0.001), respectively. This increase greatly exceeded the 2-fold increase in the surface area of adipocytes from obese animals, and did not result from trans-stimulation secondary to increased lipolysis. Adipocyte tumor necrosis factor-alpha mRNA levels, assayed by Northern hybridization, increased in the order +/+ < fa/fa < ZDF. Oleate uptake was also studied in adipocytes from 20-24-day-old male +/+, fa/+, and fa/fa weanlings. These animals were not obese, and had equivalent plasma fatty acid and glucose levels. Tumor necrosis factor-alpha mRNA levels in +/+ and fa/fa cells also were similar. Nevertheless, Vmax was increased 2.9-fold (p < 0.005) in fa/fa compared +/+ cells. These studies indicate 1) that regulation of fatty acid uptake is tissue-specific and 2) that up-regulation of adipocyte fatty acid uptake is an early event in Zucker fa/fa rats. These findings are independent of the role of any particular fatty acid transporter. Adipocyte mRNA levels of three putative transporters, mitochondrial aspartate aminotransferase, fatty acid translocase, and fatty acid transporting protein (FATP) were also determined; mitochondrial aspartate aminotransferase and FATP mRNAs correlated strongly with fatty acid uptake.

Adipocytes↗
PubMed1997

A pilot study of allogeneic bone marrow transplantation using related donors stimulated with G-CSF.

Growth factor administration to donors prior to bone marrow (BM) harvesting results in an enrichment of the graft for myeloid precursors. In animals, growth factor-primed BM has a higher repopulating ability than untreated BM. Ten patients received an HLA-identical sibling, allogeneic transplant using granulocyte colony-stimulating factor (G-CSF)-stimulated BM. Stimulation consisted of G-CSF at 10 microg/kg/day for 2 days prior to harvest. Patients were transplanted for various benign and malignant hematological conditions. The GVHD prophylaxis consisted of cyclosporine, methotrexate and/or prednisone. Compared to untreated historical control BM, stimulated BM infusions contained similar number of nucleated cells (mean +/- s.d.: 3.5 +/- 1.5 vs 4.0 +/- 0.9 x 10(8)/kg), CD34+ cells (mean +/- s.d.: 7.5 +/- 3.0 vs 9.4 +/- 6.7 x 10(6)/kg), and CD3+ cells (mean +/- s.d.: 129 +/- 30 vs 190 +/- 59 x 10(6)/kg) but higher numbers of granulocyte-macrophage colony-forming units (mean +/- s.d.: 20 +/- 12 vs 96 +/- 34 x 10(4)/kg). Patients receiving stimulated BM had prompt and stable engraftment of white cells and platelets. On average they attained an ANC of > or = 1 x 10(9)/l 9 days earlier and a platelet count of > or = 20 x 10(9)/l 6 days earlier than historical controls receiving unstimulated HLA-identical sibling BM. Hospitalization was shortened by a mean of 10 days and transfusion requirements were modest. None of the patients developed severe GVHD or disease relapse. Two patients died of severe VOD post-BMT and thus were unevaluable for platelet engraftment. A third patient died of TTP on day 76 post-BMT. Seven patients are alive and well 49-585 days post-BMT. Stimulated BM may provide a valuable alternative to allogeneic BM and PBSC transplants. Ideal stimulation regimens need to be investigated.

Adolescent↗
PubMed1995

3T3 fibroblasts transfected with a cDNA for mitochondrial aspartate aminotransferase express plasma membrane fatty acid-binding protein and saturable fatty acid uptake.

To explore the relationship between mitochondrial aspartate aminotransferase (mAspAT; EC 2.6.1.1) and plasma membrane fatty acid-binding protein (FABPpm) and their role in cellular fatty acid uptake, 3T3 fibroblasts were cotransfected with plasmid pMAAT2, containing a full-length mAspAT cDNA downstream of a Zn(2+)-inducible metallothionein promoter, and pFR400, which conveys methotrexate resistance. Transfectants were selected in methotrexate, cloned, and exposed to increasing methotrexate concentrations to induce gene amplification. Stably transfected clones were characterized by Southern blotting; those with highest copy numbers of pFR400 alone (pFR400) or pFR400 and pMAAT2 (pFR400/pMAAT2) were expanded for further study. [3H]Oleate uptake was measured in medium containing 500 microM bovine serum albumin and 125-1000 microM total oleate (unbound oleate, 18-420 nM) and consisted of saturable and nonsaturable components. pFR400/pMAAT2 cells exhibited no increase in the rate constant for nonsaturable oleate uptake or in the uptake rate of [14C]octanoate under any conditions. By contrast, Vmax (fmol/sec per 50,000 cells) of the saturable oleate uptake component increased 3.5-fold in pFR400/pMAAT2 cells compared to pFR400, with a further 3.2-fold increase in the presence of Zn2+. Zn2+ had no effect in pFR400 controls (P > 0.5). The overall increase in Vmax between pFR400 and pFR400/pMAAT2 in the presence of Zn2+ was 10.4-fold (P < 0.01) and was highly correlated (r = 0.99) with expression of FABPpm in plasma membranes as determined by Western blotting. Neither untransfected 3T3 nor pFR400 cells expressed cell surface FABPpm detectable by immunofluorescence. By contrast, plasma membrane immunofluorescence was detected in pFR400/pMAAT2 cells, especially if cultured in 100 microM Zn2+. The data support the dual hypotheses that mAspAT and FABPpm are identical and mediate saturable long-chain free fatty acid uptake.

3T3 Cells↗
PubMed1995

Mitochondrial aspartate aminotransferase expressed on the surface of 3T3-L1 adipocytes mediates saturable fatty acid uptake.

Physicochemical studies have suggested that the 43-kDa plasma membrane fatty acid binding protein (FABPpm) is closely related to the mitochondrial isoform of aspartate aminotransferase (mAspAT). In the present studies, mAspAT was not detected immunohistochemically or by immunoblotting in plasma membranes of proliferating 3T3-L1 fibroblasts. During controlled differentiation to an adipocyte phenotype, mAspAT became detectable by the second day of confluent growth, prior to accumulation of visible lipid droplets, and was strongly expressed in 8-day differentiated 3T3-L1 adipocytes. The pattern of expression paralleled the previously reported expression both of FABPpm and of the Vmax for saturable uptake of long chain free fatty acids. As with anti-FABPpm, antibodies to mAspAT selectively inhibited the uptake of [3H]-oleate in 3T3-L1 adipocytes but not in fibroblasts, while having no effect on uptake of either 2-deoxyglucose or the medium chain fatty acid octanoate. Preabsorption of anti-FABPpm with mAspAT, or of anti-mAspAT with FABPpm, abolished immunopositivity in immunohistochemical and immunoblotting studies, as well as the ability of either antibody to inhibit [3H]-oleate uptake. These studies provide strong biologic evidence for the identity of FABPpm and mAspAT, and for the hypothesis that FABPpm/mAspAT mediates the uptake of long chain free fatty acids.

3T3 Cells↗
PubMed1994

The hepatocellular uptake of free fatty acids is selectively preserved during starvation.

BACKGROUND/AIMS: The liver loses protein during fasting. This study sought to determine if hepatic protein loss during fasting selectively preserves functions important to survival such as uptake of fatty acids, which are major energy substrates in that condition. METHODS: Initial [3H]oleate uptake and efflux rates in hepatocytes from starved (for 48 hours) and fed male rats were measured in media containing 250 mumol/L albumin at oleate/albumin ratios of 0.2:1-2:1. Uptake rates of sulfobromophthalein, taurocholate, and glucose were also determined. RESULTS: Initial oleate uptake rate was saturable with respect to unbound oleate concentration. Maximum initial velocity expressed per cell number did not differ between fasted and fed animals, but measured cell volume and estimated surface area were decreased in starved vs. fed hepatocytes (921 +/- 21 vs. 1623 +/- 58 microns2, respectively; P < 0.001). Consequently, when expressed per surface area, maximum initial velocity was greater in starved cells (17 +/- 3 vs. 10 +/- 2 [pmol.min-1.micron2] x 10(-7); P < 0.02). Expressed similarly, oleate efflux was also greater from starved hepatocytes and was inhibited by an antibody to plasma membrane fatty acid binding protein (FABPpm). FABPpm concentration per unit area of plasma membrane also increased in starved hepatocytes (P < 0.05). By contrast, uptake rates of sulfobromophthalein, taurocholate, and glucose by starved hepatocytes were decreased when expressed per cell number and unchanged per unit area. CONCLUSIONS: During fasting, the hepatocellular uptake mechanism for oleate is selectively preserved compared with those for sulfobromophthalein, taurocholate, or glucose.

Animals↗
PubMed1994

Recent studies of the cellular uptake of long chain free fatty acids.

Over the last 12 years, we and others have demonstrated that fatty acid uptake is not simply a passive, diffusive process, but an interesting, facilitated mechanism the regulation of which has specific implications for disease. This work also tells another tale. Our earliest experiments were called "squirt studies" by our technician because they involved "squirting" a bolus of labelled metabolite intravenously into one limb of the subject and drawing blood samples from another. These studies were conducted in patients, and later in intact animals. From these we moved progressively to investigations in isolated perfused organs, isolated cells, and sub-cellular organelles and membrane vesicles; to the chemistry of membrane proteins, and most recently, to studies combining recombinant DNA technology with cellular biologic studies of controlled differentiation in tissue culture (Figure 5). The work has been not only a continuing process of scientific investigation but, for the investigators, an on-going process of self-education.

Animals↗
PubMed1992

Characteristics of oleate binding to liver plasma membranes and its uptake by isolated hepatocytes.

To clarify mechanisms of hepatic free fatty acid uptake, [3H]oleate uptake by isolated rat hepatocytes was studied, using solutions of 150 microM bovine serum albumin at oleate:albumin molar ratios of 0.033-6.7:1. Oleate partitioning between liver plasma membranes and albumin was also studied, and used to ascertain the membrane binding function for oleate. The experimental uptake curve was complex, but could be resolved by computer fitting into a sum of two components, one a saturable and the second a linear function of the unbound oleate concentration. The saturable component comprises > 90% of total oleate uptake when the oleate:albumin molar ratio is < 2.5, but < 50% when this ratio is > 5. Membrane binding also consisted of a sum of a saturable and a linear component. By comparison of the computer-fitted uptake and binding functions, separate rate constants for the transfer into the cell of the saturably and non-saturably bound oleate were estimated to be 0.7 s-1 and 0.05 s-1, respectively. The former is compatible with a specific, protein-mediated process. It is 15-times greater than the corresponding rate constant for transfer of non-saturably bound oleate into the cell, which in turn is similar to reported rates of non-specific 'flip-flop' of fatty acids across lipid bilayers. The observed kinetics are not consistent with models in which uptake occurs principally from the albumin-bound pool of oleate, or solely from the oleate which has partitioned passively into the lipid bilayer of the plasma membrane.

Animals↗
PubMed1991

Transgenic animals: a new era in developmental biology and medicine.

In the relatively short period since its development, the technique for the production of transgenic animals by pronuclear microinjection, as established by Gordon et al. (1980), has resulted in studies that have clearly had a profound effect on our basic understanding of mammalian development and genetics, and upon our outlook for genetic engineering. The question now is: What remains to be done? Clearly, a greater understanding of the mechanism of integration could lead to more efficient transfer of genes and lessen the problem of rearrangement of host DNA. A better characterization of enhancers and their mode(s) of action would allow design of molecules with highly specific patterns of gene expression, as well as novel distributions of expression. When these goals are met, it may well become scientifically feasible to transfer genes into the human germline. Whether such an undertaking is advisable or even desirable is an issue requiring open discussion, but it is an issue that is clearly distinct from the question of technical feasibility. The most rational approach to this profound question lies in the continued pursuit of knowledge relating to transgenic technology, rather than self-imposed ignorance. Even if never used as a medical therapy, transfer of genes into the germline will reveal what is certainly one of nature's greatest secrets: the nature of the interaction of genes in the development of a multicellular eukaryotic organism. Through the contributions to this volume and in the years ahead, we will find ourselves in a position to unravel the details of the awesome and fascinating phenomenon that is mammalian development.

Animals↗
PubMed1990

Enzymatic amplification of a Y chromosome repeat in a single blastomere allows identification of the sex of preimplantation mouse embryos.

The polymerase chain reaction (PCR) technique has been adapted to identify the sex of preimplantation mouse embryos rapidly. PCR was used to amplify a specific repeated DNA sequence on the Y chromosome from a single isolated blastomere in under 12 hr. The remainder of the biopsied embryo was then transferred to a pseudopregnant female and carried to term. Using this technique, 72% of embryos can be classed as potentially either male or female. Transfers of such embryos have produced pregnancies with 8/8 fetuses (100%) being of the predicted sex. Variations of the technique have demonstrated certain limitations to the present procedure as well as indicated possible strategies for improvement of the assay. The PCR technique may have wide application in the genetic analysis of preimplantation embryos.

Animals↗
PubMed1990

Plasma membrane fatty acid-binding protein and mitochondrial glutamic-oxaloacetic transaminase of rat liver are related.

The hepatic plasma membrane fatty acid-binding protein (h-FABPPM) and the mitochondrial isoenzyme of glutamic-oxaloacetic transaminase (mGOT) of rat liver have similar amino acid compositions and identical amino acid sequences for residues 3-24. Both proteins migrate with an apparent molecular mass of 43 kDa on SDS/polyacrylamide gel electrophoresis, have a similar pattern of basic charge isomers on isoelectric focusing, are eluted similarly from four different high-performance liquid chromatographic columns, have absorption maxima at 435 nm under acid conditions and 354 nm at pH 8.3, and bind oleate with a Ka approximately 1.2-1.4 x 10(7) M-1. Sinusoidally enriched liver plasma membranes and purified h-FABPPM have GOT enzymatic activity; the relative specific activities (units/mg) of the membranes and purified protein suggest that h-FABPPM constitutes 1-2% of plasma membrane protein in the rat hepatocyte. Monospecific rabbit antiserum against h-FABPPM reacts on Western blotting with mGOT, and vice versa. Antisera against both proteins produce plasma membrane immunofluorescence in rat hepatocytes and selectively inhibit the hepatocellular uptake of [3H]oleate but not that of [35S]sulfobromophthalein or [14C]taurocholate. The inhibition of oleate uptake produced by anti-h-FABPPM can be eliminated by preincubation of the antiserum with mGOT; similarly, the plasma membrane immunofluorescence produced by either antiserum can be eliminated by preincubation with the other antigen. These data suggest that h-FABPPM and mGOT are closely related.

Amino Acid Sequence↗
PubMed1989

Expression of a methotrexate resistant dihydrofolate reductase gene in transgenic mice.

We have previously demonstrated systemic resistance to methotrexate (MTX) in transgenic mice carrying a foreign, mutant dihydrofolate reductase (DHFR, E.C. 1.5.1.3) gene. The new gene was introduced as a cDNA cloned into an expression vector driven by the simian virus 40 (SV40) early promoter. Previous physiologic studies suggested that transgenic mice tolerated drug doses invariably lethal to controls on the basis of gastrointestinal (GI) resistance to MTX. In the present study we evaluated foreign gene expression at the RNA level in the three major sites of MTX toxicity: intestine, liver, and bone marrow. The transgene was transcriptionally active in small bowel, and levels of expression were high in animals tolerating the largest doses of MTX. The gene was also expressed in the liver in some pedigrees, but was not detected in hemopoietic tissues of any of the pedigrees tested. Our studies correlate the site of expression of a drug resistant dhfr gene with an altered physiologic response to MTX, and demonstrate that transgenic mice can be used as a test system for expression of genes considered for use in somatic gene therapy.

Animals↗
PubMed1988

Anemia in a line of transgenic mice carrying a mutant dihydrofolate reductase gene.

Several lines of transgenic mice were produced by pronuclear injection of a full-length cDNA encoding a mutant dihydrofolate reductase (DHFR, E.C. 1.5.1.3). The mutation causes altered enzyme kinetics for folate reduction as well as low affinity for methotrexate (MTX). One line of mice carrying the plasmid displays a moderate-to-severe anemia that is evident in fetuses and newborn mice and that moderates with age. RNA studies revealed high levels of transcription of the mutant gene in the fetal and adult liver, and low or absent expression in adult bone marrow. Transcription of the mutant gene was not found in the fetal liver of other pedigrees examined. The data thus suggest that expression of this mutant gene in the main hematopoietic organ of the fetus adversely affects erythropoiesis by altering the cellular environment for erythroid differentiation, and that translocation of the site of hematopoiesis to bone marrow, where the foreign gene is not expressed, leads to normalization of red cell production.

Anemia↗
PubMed1986

Systemic resistance to methotrexate in transgenic mice carrying a mutant dihydrofolate reductase gene.

A full-length cDNA coding for a mutant dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3), cloned from a mouse fibroblast cell line grown in high concentrations of methotrexate (MTX), was microinjected into mouse embryos to produce transgenic mice. The DHFR cDNA product is 270-fold more resistant to MTX than the wild-type enzyme. Seventeen transgenic mouse lines, identified by Southern blotting of tail or spleen DNA, carried between 1 and 400 copies of the foreign gene per cell. Eight lines have thus far been tested for resistance to MTX. Control mice were treated until death; MTX was withdrawn from transgenic mice when a cumulative MTX dose uniformly fatal for controls was reached. The major site of MTX toxicity was the gastrointestinal tract, with death of controls resulting from fluid and weight loss. Transgenic animals were relatively resistant to these symptoms and tolerated significantly more MTX than control animals. These results show that genes conferring resistance to chemotherapeutic agents can, after transfer into intact organisms, produce systemic drug resistance.

Animals↗