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Biomedical subjects

N G Abraham

Publications and source records attributed to N G Abraham.

At least 55 records · Page 3Linked to original sources
PubMed1998

Gene transfer of alpha interferon into hematopoietic stem cells.

Gene transfer or gene therapy has advantages in the treatment of a variety of disorders due to its selective expression within specific mammalian cells. IFN-alpha has been used in the management of leukemia, and gene transfer of the IFN-alpha gene into hematopoietic progenitor cells may have great potential for the treatment of chronic myelogenous leukemia (CML). Therefore, we examined the ability of adenovirus (Ad)-IFN-alpha gene construct to transfect normal bone marrow hematopoietic CD34+ stem cells and the production of IFN-alpha protein by these cells. Ad-cytomegalovirus (CMV) promoter-driven IFN-alpha at multiple doses was assessed to transfect highly purified CD34+ cells in liquid culture. Optimal transduction of CD34+ cells with the AdCMV-IFN-alpha construct was achieved using 120 plaque forming units (pfu). Flow cytometric determinations revealed that there was no significant difference in CD34+ cell viability for the 8 or 12-h transfection periods. Immunoassay of IFN-alpha produced by CD34+ cells shows that IFN-alpha levels increased several fold in transfected cells and this was not seen in CD34+ cells transfected with the heme oxygenase gene (HO-1). These in vitro data suggest that adenovirus-mediated gene transfer of IFN-alpha into hematopoietic stem cells can be achieved and that the IFN-alpha protein is produced by viable CD34 progenitor cells.

Adenoviridae↗
PubMed1998

Retinal pigment epithelial cell-based gene therapy against hemoglobin toxicity.

To determine if overexpression of the human heme oxygenase (HO-1) protects retinal pigment (RPE) cells from hemoglobin toxicity, a human RPE cell line was infected by an adenoviral vector containing the HO-1 (Ad-HO-1) gene or transfected with a plasmid containing the cytomegalovirus promoter and HO-1 cDNA (pRc/CMV-HO-1) complexed to cationic liposomes. Phase contrast microscopy and acid phosphatase activity were examined to insure homogeneity of the cell line. Mitochondrial cytochrome and microsomal heme content were measured in both transduced and control cells. RPE cells were then challenged with hemoglobin and their viability estimated. We determined that cells transfected with Ad HO-1 overexpressed HO-1 compared to control cells: HO-1 mRNA levels were increased 3-fold within 3 days, decreasing in 7 days. In addition, we permanently transfected RPE cells with HO-1 gene. Transfected cell clones selected for neomycin resistance had elevated levels of HO activity 3-fold higher than control. Transfected cells exposed to hemoglobin had a survival rate of 93%; non-transfected cells had a 65-75% rate of survival. Transfected cells overexpressing HO-1 proved highly viable when challenged with hemoglobin. HO-1 appears to be an important component of the cellular anti-oxidant defense mechanisms against hemoglobin toxicity. However, the choice of transient or permanent expression of HO-1 against hemoglobin toxicity and hemorrhage needs to be further evaluated.

Adenoviridae↗
PubMed1997

Heme oxygenase induction with attenuation of experimentally induced corneal inflammation.

Heme oxygenase (HO), by catabolizing heme to bile pigments, down-regulates cellular levels of heme and hemeproteins; certain of the latter, i.e. cytochrome P450s, generate pro-inflammatory products from endogenous substrates. Two HO isozymes, the products of distinct genes, have been described; HO-1 is the inducible one, whereas HO-2 is believed to be constitutively expressed. We studied the inducing effects of several metal compounds [CoCl2, SnCl2, ZnCl2, heme, and cobalt protoporphyrin (CoPP)] on HO-1 mRNA content and enzyme activity in cultures of rabbit corneal epithelial (RCE) cells; these metal compounds are known to induce HO in other tissues. Additionally, we studied HO-1 expression in an experimental model of ocular inflammation produced in rabbit corneas by extended contact lens wear, and the relation of HO expression to the induced inflammatory process. SnCl2 added to RCE cells in vitro produced marked time- and concentration-dependent increases in HO-1 mRNA and HO-1 enzyme activity; CoCl2, ZnCl2, and CoPP were inducers of HO as well, though to a lesser degree than SnCl2. Corneas treated for 6 days with contact lenses impregnated with SnCl2 displayed substantially less corneal inflammation, swelling, and new vessel invasion than did controls; attenuation of ocular inflammation was paralleled by SnCl2-induced increases in HO mRNA and HO activity in corneal epithelial cells from treated eyes. It is suggested that amelioration of the inflammatory response produced by extended contact lens wear is due, in part, to the induction of high levels of HO-1 activity by SnCl2, which results in diminished production of pro-inflammatory mediators generated through heme-dependent metabolic processes. Regulation of HO activity in this manner may have clinical applications.

Animals↗
PubMed1997

Zinc porphyrins: potent inhibitors of hematopoieses in animal and human bone marrow.

The effects of selected heme analogues on heme oxygenase activity in tissues and on human and rabbit bone marrow hematopoietic colony growth were examined. Zinc protoporphyrin (ZnPP) and zinc mesoporphyrin (ZnMP), at concentrations ranging between 1 and 20 microM, produced significant inhibition of human and rabbit bone marrow erythroid (CFU-E, BFU-E) and myeloid (CFU-GM) colony growth. The growth inhibition produced by ZnPP or ZnMP was not overcome with exposure of cultures to elevated levels of the growth factors erythropoietin and granulocyte-macrophage colony stimulating factor. In contrast, tin protoporphyrin and tin mesoporphyrin did not display any significant bone marrow toxicity when used at similar concentrations. In other studies, differential effects of tin mesoporphyrin and ZnMP administered intravenously on kidney heme oxygenase were demonstrated. Chromium mesoporphyrin administered intravenously proved lethal to animals. These results indicate that exposure of bone marrow to ZnPP and ZnMP can be deleterious to hematopoietic cells and raise the possibility that ZnPP, which is endogenously formed and found in high concentration in red blood cells in lead-poisoned children, may itself participate in the bone marrow toxicity produced by this metal.

Animals↗
PubMed1997

Adenovirus mediated alpha interferon (IFN-alpha) gene transfer into CD34+ cells and CML mononuclear cells.

Gene transfer or gene therapy has advantages in the treatment of a variety of disorders due to its selective expression within specific mammalian cells. Interferon-alpha (IFN-alpha) has been used in the management of leukemia but its diverse adverse activities with multiple potential side effects, possibly unrelated to therapeutic targets, may negatively influence the ability of IFN-alpha to treat this disorder. Therefore, we examined the ability of adenovirus (Ad)-IFN-alpha gene construct to transfect normal (CD34+ cells) and chronic myelogenous leukemia (CML) bone marrow mononuclear cells (BMMNC) and the transient overexpression of IFN-alpha in these cells. Ad-cytomegalovirus promoter driven IFN-alpha (AdCMV-IFN-alpha) at multiple doses was assessed to transfect highly purified CD34+ cells in liquid culture, and optimal transduction of CD34+ cells was achieved using 120 plaque forming units. Flow cytometric determinations revealed that there was no significant difference in cell viability for the 4 h or 24 h transfection periods. Immunoassay of IFN-alpha produced by CD34+ cells shows that IFN-alpha levels increased several fold in transfected cells. Transient expression of the IFN-alpha gene did not suppress proliferation of CD34+ progenitors as indicated by BFU-E or colony forming units-granulocyte-macrophage (CFU-GM) growth. Reverse transcriptase/polymerase chain reaction analysis of RNA from CD34+ harvested CFU-GM progenitor cells demonstrated transient IFN-alpha mRNA expression. Similarly, CML BMMNC were transfected with AdCMV-IFN-alpha under similar conditions as described for CD34+ cells. BMMNC cells exposed to adenovirus for 24 h and 48 h were found to express IFN-alpha at a substantial level. This in vitro data suggest that Ad-mediated gene transfer of IFN-alpha into hematopoietic stem cells can be achieved and that the IFN-alpha gene can be translated into its specific mRNA in CD34 progenitor cells.

Adenoviridae↗
PubMed1997

Diminished heme oxygenase-1 mRNA expression in RPE cells from diabetic donors as quantitated by competitive RT/PCR.

PURPOSE: The RPE is essential in the maintenance of retinal vasculature homeostasis, since increased cellular expression of heme oxygenase-1 (HO-1) has been implicated as a defense mechanism against oxidative stress. This study was done in an effort to determine the levels of the stress protein (32 kD), HO-1, in retinal pigment epithelium (RPE) cells obtained from diabetic and normal eyes. METHODS: We measured the levels of HO-1 in the RPE from eight normal and six diabetic donor eyes. For comparison, HO-1 levels were assayed in RPE cells from four donor eyes with long-standing hypertension. Heme oxygenase-1 mRNA copy number was determined by competitive RT/PCR on various ex vivo samples and on RPE cultured from the same donors. Total RNA (1-200 ng) was reverse-transcribed and then amplified by PCR in the same tube as an internal standard obtained by deleting a 50 bp restriction site from the native HO-1 gene. RESULTS: Relative to the RPE obtained from control donor eyes, RPE from diabetic donors exhibited significantly decreased levels of HO-1 mRNA. In contrast, no significant difference in the levels of HO-1 mRNA was observed in RPE samples derived from hypertensive donor eyes. The diabetic group showed a range of 340-450 HO-1 mRNA copies/ng of total RNA, as compared to 425-8,000 HO-1 mRNA copies/ng of total RNA in RPE from normal donors and 460-7605 copies/ ng in hypertensive donor eyes. CONCLUSIONS: This study represents initial studies exploring the quantitative expression of heme oxygenase in the RPE human eyes. The decreased expression of HO-1, a stress/heat shock protein, may in the RPE contribute to the vulnerability of the neuroretina to significant metabolic alterations encountered in the diabetic state. This was a limited study; additional screening from different donor eyes will be done in order to establish the relationship between RPE, HO-1 expression and eye diseases in which oxidative stress is believed to be a determinant in the pathophysiology.

Diabetes Mellitus↗
PubMed1997

Effect of blood substitute, recombinant hemoglobin, on in vivo hematopoietic recovery from AZT toxicity.

We determined the in vivo ability of infused human recombinant hemoglobin 1.1 (hr-Hb) and erythropoietin to rescue the hematopoietic activity from the suppressive effects of AZT in normal and in a murine model of AIDS (MAIDS) mice. Mice were fed with AZT for 8 weeks with or without treatment in the last 4 weeks by administering various concentrations of hr-Hb and/or erythropoietin (Epo). Blood parameters, body weight (BW) and erythroid burst-forming units (BFU-E) for all mice were determined. AZT-treated normal and MAIDS mice showed a significant decrease in hematocrit (64 and 78.1%), hemoglobin (27.2 and 45.5%), BW (17.5 and 35.5%), number of white (66.9 and 42.1%) and red blood cells (65.5 and 38%), and the number of BFU-E (73 and 59%), whereas the AZT-treated normal and MAIDS mice that received hr-Hb (5 mg/kg BW/day) and/or Epo (2 U/mouse/day) showed significant alleviation of AZT cytotoxicity. This was evident by the recovery in all blood indices examined, the number of BFU-E and the BW of mice treated. BFU-E recovery in MAIDS (97%) was greater than that in normal mice (63%) as compared to their controls. hr-Hb produced a similar response as the combination, however recovery was slightly better with the latter in some hematological parameters. Higher concentrations of hr-Hb (10-15 mg) did not result in a more significant increase in most blood indices. Our results indicate that infusion with hr-Hb can alleviate AZT toxicity in normal and in immunodeficient mice, and that hr-Hb may be clinically useful in preventing severe bone marrow depression brought about by various drugs or agents such as AZT.

Animals↗
PubMed1997

Overexpression of the heme oxygenase gene in renal cell carcinoma.

Heme oxygenase (HO) activity has been implicated in the regulation of renal function and cell growth in normal and disease states. Expression of HO genes has been shown to regulate important hemoprotein(s) such as cytochrome P450. In the present study, HO activity was measured in samples of human adenocarcinoma, juxtatumor, and normal renal tissues. The samples were histologically examined to verify the malignant and normal nature. HO activity was 4-fold higher in the adenocarcinoma than in either normal or juxtatumor tissues. We designed a reverse transcriptase-polymerase chain reaction (RT-PCR) method to assess the presence of HO-1 and HO-2 mRNA in biopsy samples of various human renal tissues. Total RNA from renal samples was reverse transcribed and amplified simultaneously by PCR using specific primers for HO-1 and HO-2. Results show that both HO-1 and HO-2 mRNAs were expressed in all renal tissues examined and that HO-1 appeared to be amplified more than HO-2. Northern blot analysis revealed that HO-1 mRNA was elevated by several-fold in adenocarcinoma compared with juxtatumor or normal tissues. In contrast, no differences in HO-2 mRNA levels were observed using either RT-PCR or Northern blot. Cytochrome P450 arachidonic acid epoxygenase and omega-hydroxylase activities were markedly reduced in the tumor tissues, whereas, in the juxtatumor tissue, cytochrome P450 omega-hydroxylase activity was significantly increased. Northern blot analysis using cytochrome P450 cDNA probe 4A2 cDNA for the omega-hydroxylase gene family revealed that mRNA levels for omega-hydroxylase transcripts were significantly decreased in the adenocarcinoma compared with juxtatumor. The decrease in cytochrome P450 4All mRNA levels correlated with a decrease in the arachidonic acid omega-hydroxylation metabolite, 20-HETE. The production of 20-HETE was significantly higher in juxtatumor in agreement with omega-hydroxylase mRNA. Higher levels of HO-1 may be a contributing factor for the undetectable levels of cytochrome P450 arachidonic acid metabolites, 20-HETE, in the adenocarcinoma. Our results suggest that increased generation of mitogenic activities by omega-hydroxylase and 20-HETE in the juxtatumor may be a contributing factor in the development and growth of neoplastic tissues, and the induction of HO in the tumor tissue may be an attempt to limit oxidative injury caused by the cytochrome P450 metabolites and other oxidative stress.

Blotting, Northern↗
PubMed1997

Heme induces the expression of adhesion molecules ICAM-1, VCAM-1, and E selectin in vascular endothelial cells.

Heme is an important immunostimulating agent and oxidative factor contributing to endothelial cell activation. To investigate the mechanism of heme-induced endothelial cell activation, we analyzed the effect of heme and the inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), on the expression of the heme-degrading stress protein, heme oxygenase (HO), and adhesion molecules in human umbilical vein endothelial cells (HUVEC). Indirect immunofluorescence double labeling studies demonstrated a simultaneous increase of ICAM-1 and HO-1 after exposure of cells to heme for 24 hr. Co-expression of HO-1 and ICAM-1 was also demonstrated in TNF-alpha-exposed cells. Dot blot immunoassay and quantitative analysis by ELISA demonstrated that heme treatment for 24 hr caused a 2-fold increase in ICAM-1 expression (P < 0.002) compared with quiescent cells, while in cells stimulated by TNF-alpha for 24 hr ICAM-1 gene expression increased by 5-fold. Moreover, heme exposure also resulted in a marked increase in VCAM-1 and E selectin expression (three and four times over control levels, respectively). On the other hand, TNF-alpha treatment showed similar expression levels for VCAM-1 and E selectin, compared with stimulation by heme (100 microM). The level of HO activity in endothelial cells exposed to heme or TNF-alpha was increased from 24.7 +/- 5.7 pmol bilirubin/mg protein/min in control to 70.0 +/- 9.5 and 36.7 +/- 3.1 pmol bilirubin/mg protein/min in heme- and TNF-alpha-stimulated cells, respectively. These results suggest that upregulation of ICAM-1, VCAM-1, and E selectin expression is associated with oxidative stress induced by hemoglobin/heme and that HO-1 may play a modulating role via its ability to degrade heme to a substance with antioxidant properties.

Cell Adhesion Molecules↗
PubMed1996

Quantitative measurement of heme oxygenase-1 in the human renal adenocarcinoma.

Heme oxygenase (HO-1) is the rate-limiting enzyme in heme catabolism. HO-1, a stress protein, has been suggested to be involved in defense mechanisms against agents that may induce oxidative stress. It has been proposed that renal HO gene expression regulates important hemoprotein(s) such as cytochrome P450 and may be essential to maintain homeostasis in the kidney. Because accurate assessment of HO-1 mRNA in normal and disease states in kidney were not available due to the limited number of cells, we developed a system to quantitate human HO-1 mRNA in samples limited in cell number and/or mRNA copies. Total RNA from human kidney was used to establish this technique; it was reverse-transcribed and then amplified by polymerase chain reaction (PCR) in a tube also containing an internal standard obtained by deleting 50 bp from the original human HO-1 gene. This allowed us to use the same primers for both the sample and internal standard. After amplification, templates were resolved by acrylamide gel electrophoresis and quantitated either by densitometry or radioactivity counted from the bands excised from the gel. When the internal standard is present in the reaction mixture, the ratio of amplified sample vs. the standard template is proportional to the amount of sample RNA, and it is therefore possible to calculate the number of specific mRNA molecules. We have used this approach to quantitate the number of HO-1 mRNA molecules in adenocarcinoma cells. Results show that reverse transcription (RT)/PCR methods were able to determine the number of HO-1 mRNA copies in biopsy samples of human adenocarcinoma cells.

Adenocarcinoma↗
PubMed1996

c-fos protooncogene transcription can be modulated by oligonucleotide-mediated formation of triplex structures in vitro.

A homopurine.homopyrimidine sequence of the c-fos promoter was chosen as a target for a triple helix oligonucleotide. Eight DNA oligonucleotides that ranged from 14 to 31 bp were shown to form a triple helix with three sequences within the c-fos promoter region. Reactive derivatives of homopyrimidine oligonucleotides bearing the 5'- or 3'-terminal DNA alkylation aromatic 2-chloroethylamino group were also synthesized. It was concluded, based on the physical properties of the DNA oligonucleotide complex, that the oligonucleotide forms a colinear triplex with the duplex binding sites. We investigated in detail, using electrophoretic mobility and footprinting protection, whether such oligonucleotide.DNA complexes are of benefit in designing high-affinity probes for a natural DNA sequence in the mouse c-fos gene. Our results demonstrate that four different DNA targets within the c-fos promoter region can form triplex structures with synthetic oligonucleotides in a sequence-specific manner. Moreover, in vitro modifications of the retinoblastoma-gene-product-binding site of the c-fos promoter at position -83 in front of the cAMP/cAMP-responsive element binding site and fos-binding site 3/activator-protein-2-like (FBS3/AP-2-like) site at position -431 by triple helix forming oligonucleotides cause dramatic suppression of fos-chloramphenicol acetyltransferase activity in endothelial cells. These results provide a basis for the development of a specific oligonucleotide target forming triplex-DNA complex, and emphasize the importance of a target forming triplex as a basis for control of gene expression and cell proliferation.

Base Sequence↗
PubMed1996

The role of NF-kappaB in the angiogenic response of coronary microvessel endothelial cells.

The activation of nuclear factor (NF)-kappaB by 12(R)-hydroxyeicosatrienoic acid [12(R)-HETrE], an arachidonic acid metabolite with potent stereospecific proinflammatory and angiogenic properties, was examined and its role in the angiogenic response was determined in capillary endothelial cells derived from coronary microvessels. Electrophoretic mobility-shift assay of nuclear protein extracts from cells treated with 12(R)-HETrE demonstrated a rapid and stereospecific time- and concentration-dependent increase in the binding activity of NF-kappaB, which was inhibitable by the antioxidants N-acetylcysteine, butylated hydroxyanisole, and pyrrolidine dithiocarbamate and was partially attenuated by the protein kinase C inhibitors, staurosporine and calphostin C. Neither 12(S)-HETrE nor other related eicosanoids--e.g., 12(R)-HETE, 12(S)-HETE, and leukotriene B4--stimulated the activation of NF-kappaB relative to 12(R)-HETrE, substantiating the claim for a specific receptor-mediated mechanism. 12(R)-HETrE stimulated the formation of capillary-like cords of microvessel endothelial cells distinguishable from a control; this effect was comparable to that observed with basic fibroblast growth factor (bFGF). Inhibition of NF-kappaB activation resulted in inhibition of capillary-like formation of endothelial cells treated with 12(R)-HETrE by 80% but did not affect growth observed with bFGF. It is suggested that 12(R)-HETrE's angiogenic activity involves the activation of NF-kappaB, possibly via protein kinase C stimulation and the generation of reactive oxygen intermediates for downstream signaling.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1996

Downregulation of the human heme oxygenase gene by glucocorticoids and identification of 56b regulatory elements.

Several human heme oxygenase-1 promoter-driven chloramphenicol acetyltransferase constructs were examined in order to analyze promoter activity of the heme oxygenase-1 gene in microvessel endothelial cells. Heme oxygenase promoter activity was up-regulated by interleukin-6. This induction was shown to be down-regulated by glucocorticoids. Chloramphenicol acetyltransferase assays revealed that the promoter region (56 base pair) between -180 and -120 was responsible for up-regulation by growth factors, as well as for glucocorticoid-directed down-regulation. The same DNA fragments was shown to bind nuclear factor(s) from endothelial cells treated with dexamethasone. Formation of DNA protein complexes peaked after a 6-hour treatment. The DNA fragment was found to contain a sequence recognized by the STAT 3/acute phase response factor.

Acute-Phase Reaction↗
PubMed1996

Specific inhibition of c-fos proto-oncogene expression by triple-helix-forming oligonucleotides.

The promoter region of the c-fos oncogene 5' flanking sequence contains enhancer elements crucial for binding nuclear factors that regulate transcription following cell proliferation and differentiation. Single-stranded deoxyoligonucleotides were chosen for modulation of c-fos protooncogene expression because of their high-affinity binding to specific nucleotide sequences. We designed two oligonucleotides that form a triple-helix complex on the retinoblastoma gene product-responsible element of the c-fos oncogene. Modification of the DNA triplex with dimethyl sulfate and affinity cleaving assays demonstrate that the predicted oligonucleotides form a DNA triplex structure with the c-fos promoter in a sequence-specific manner. Tumorigenic and non-tumorigenic fibroblasts were transiently transfected with fos-CAT plasmid modified with alkylating triplex-forming oligonucleotide reagents. A dramatic depression of CAT activity was found when the cross-linked triple helix complex at the retinoblastoma gene product-related site of the c-fos promoter was used. These experiments suggest that transcription of individual genes can be selectively modulated in cell culture by sequence specific triplex formation in regulatory enhancer sequences.

Alkylating Agents↗
PubMed1996

Dual role of heme oxygenase in epithelial cell injury: contrasting effects of short-term and long-term exposure to oxidant stress.

This study examined the role of heme oxygenase (HO) in the acquisition of resistance to hydrogen peroxide (H2O2) and hemin toxicity by renal epithelial cells (BSC-1). BSC-1 cells adapted by long-term exposure to H2O2 exhibited a twofold increase in basal HO activity and expression of HO-1 mRNA as compared with their wild-type counterparts. Exposure of both adapted and wild-type BSC-1 cells to H2O2 induced HO-1 mRNA. When cells were exposed to H202 for 24 hours, cell viability was reduced; however, an inhibitor of HO activity, Zn 2,4-bis-glycol protoporphyrin IX, improved cell viability. In a similar manner, ZnDBG completely overcame the reduction in cell viability brought about by 1 hour of hemin treatment. In addition, cells preexposed to hemin for 24 hours maintained a high level of HO mRNA and acquired resistance to further challenge with H2O2. Hemin treatment per se was associated with a detectable reduction in BSC-1 cell viability; however, the effect of hemin was not additive to the cytotoxicity of hydrogen peroxide, suggesting a common pathway of cell injury. In conclusion, two interrelated stressors, H2O2 and hemin, produced a stimulation of HO-1, and this was associated with a reduction in the viability of BSC-1 cells. Long-term exposure (24 hours) to both stressors resulted in the acquisition of some resistance to a further acute challenge of oxidant stress in BSC-1 cells.

Adaptation, Physiological↗
PubMed1996

Cytochrome P450 4A expression and arachidonic acid omega-hydroxylation in the kidney of the spontaneously hypertensive rat.

20-Hydroxyeicosatetraenoic acid (20-HETE) is a major arachidonate metabolite in the kidney of the spontaneously hypertensive rat (SHR). The increase in its synthesis has been associated with the elevation of blood pressure in the SHR. The omega-hydroxylation of arachidonic acid is an activity associated with members of the CYP4A gene family which, in the rat, comprises three major isoforms: 4A1, 4A2 and 4A3. 20-HETE displays potent and diverse biological activities which can affect pro- and anti-hypertensive mechanisms dependent upon where, when and by which isoform it has been produced. Therefore, it is important to identify and characterize its biosynthetic system. We compared CYP4A mRNA and protein expression to patterns of 20-HETE synthesis in the SHR kidney. The reverse transcription/polymerase chain reaction (RT/PCR) technique was used to amplify CYP4A mRNA in microdissected nephron segments. Southern blot hybridization of PCR products obtained from nephron segments with the CYP4A1 cDNA probe demonstrated strong signals in S2 and S3 segments of the proximal tubule. Immunoblots of nephron segments using a polyclonal anti-rat liver CYP4A1 antibody which cross-reacts with CYP4A2 and CYP4A3, and 14C-arachidonic acid metabolism, confirmed that arachidonic acid omega-hydroxylation, i.e., 14C-20HETE formation, and CYP4A proteins were also localized mainly in the S2 and S3 segments. Correlation also existed between the age-dependent increase in arachidonate omega-hydroxylation in the kidney and CYP4A mRNA levels as measured by Northern hybridization of total RNA using the CYP4A1 cDNA probe. Immunoblot analysis revealed that at 7 weeks, where 20-HETE production is at its maximum, all three proteins are expressed. CYP4A3 and 4A1 immunoreactive proteins appeared at 3 weeks, showed maximum levels at 5 and 7 weeks, respectively, and gradually decreased to lower levels at 13 and 20 weeks, whereas CYP4A2 levels were undetectable at 3, 5 and 7 weeks but appeared at 13-20 weeks. Additional immunoblots indicated that renal cortical CYP4A1 protein levels were higher in SHR compared to Sprague-Dawley and Wistar-Kyoto rats. The increased levels of CYP4A1-immunoreactive band at 7 weeks corresponded to the maximal activity of arachidonate omega-hydroxylation. Thus, CYP4A1 might play a significant role in contributing to the increased cortical/proximal production of 20-HETE seen in 7-week-old SHR. However, given the high homology among members of the CYP4A gene family and the lack of specific tools to discern among these isoforms, additional studies have to be carried out to substantiate our findings.

Aging↗
PubMed1996

Transfer of human adenine deaminase gene into murine hematopoietic stem cells: sequential study of spleen colony-forming units from bone marrow of living mice and the requirement of the microenvironment.

Irradiated female mice were reconstituted with male hematopoietic stem cells (HSCs) retrovirally marked with human adenine deaminase (hADA) complimentary DNA. HSCs were incubated with interleukin-6 and stem cell factor before coculture with GP+E86-producing cells. Bone marrow HSCs were infused intravenously to irradiated mice and spleen colony-forming units (CFU-S) were evaluated for hADA marked clones by Southern blot analysis. 45 of 54 CFU-S were marked by the hADA gene sequence with multiple copies integrated per genome. Oligoclonal hematopoiesis evolved over time with 1-2 clones demonstrated 5-11 months after reconstitution. Comparable results were obtained with embryonic fetal liver HSCs. Incubation of bone marrow HSCs with adherent stromal cells rather than growth factors produced less efficient gene transfer, and polyclonal hematopoiesis was not observed. Donor origin was established by the Y chromosome probe. These results support the clonal succession model of hematopoiesis.

Aminohydrolases↗