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Biomedical subjects

R A Graves

Publications and source records attributed to R A Graves.

At least 19 recordsLinked to original sources
PubMed2005

Radioprotection in mice following oral delivery of amifostine nanoparticles.

PURPOSE: Amifostine (Ethyol) is an approved cytoprotective agent prescribed to reduce certain side-effects in the chemotherapy of ovarian or non-small cell lung cancer, or in radiation treatment of head-and-neck cancer. The usefulness of this drug is further hampered, because it is not effective when given orally. The objective of this part of the project was to evaluate the radioprotective efficacy of orally active amifostine nanoparticles. MATERIALS AND METHODS: Radioprotective efficacy was evaluated by measuring the ability of the amifostine nanoparticles (equivalent to 500 mg/Kg) to inhibit whole-body gamma irradiation -induced injury in mice. All mice received acute whole-body gamma irradiation from a Cesium-137 source and the radioprotective efficacy of the formulation was determined by measuring 30-day survival at 9 Gy, bone marrow hemopoeitic progenitor cell survival at 9 Gy and 8 Gy, and intestinal crypt cell survival at 11 Gy. RESULTS: Thirty-day survival, hemopoietic progenitor cell survival, as well as the jejunal crypt cell survival were all significantly enhanced when the mice were treated orally with the amifostine nanoparticles 1 h prior to irradiation. CONCLUSIONS: These results clearly and unequivocally demonstrate that the amifostine nanoparticles developed in our laboratory provides significant protection from acute whole-body gamma irradiation injury in mice.

Administration, Oral↗
PubMed2002

Development of biodegradable microcapsules as carrier for oral controlled delivery of amifostine.

The primary objective of this project was to develop a biodegradable, orally active controlled-release formulation of amifostine. Development of such a formulation will mark an important advancement in the areas of chemoprotection and radioprotection. Biodegradable microcapsules of amifostine were prepared using poly(lactide/glycolide) (PLGA 50:50). The microcapsules were prepared by solvent evaporation technique. Amifostine-loaded microcapsules were evaluated for particle size, surface morphology, thermal characteristics, and drug release. Particle size and surface morphology were determined using scanning electron microscopy (SEM). Thermal characterization was conducted using differential scanning calorimetry (DSC). In vitro release study was performed at 37 degrees C using phosphate buffer (pH 7.4). Amifostine release was calculated by measuring the amount of drug remaining within the microcapsules at a specific sampling time. The amount of amifostine in the samples was determined by high-performance liquid chromatography (HPLC) using an electrochemical detector. The yield of microcapsules was 75%. Scanning electron microscopy pictures revealed that the particles were nearly spherical and smooth with an average size of 54 microm. Differential scanning calorimetry thermograms showed that microcapsules loaded with amifostine have a glass transition at 39.4 degrees C, and the melting endotherm of amifostine was absent. The absence of a melting endotherm for amifostine was an indication that amifostine was not in the crystalline state in the microcapsules, but rather in the form of a solid solution in PLGA. Approximately 50% amifostine was released during the first 6 hr of the in vitro release study. The drug, however, continued to release over the observed period of 12 hr during which 92% amifostine was released.

Administration, Oral↗
PubMed2001

Porous biodegradable microparticles for delivery of pentamidine.

The primary objective of this study was to develop a method for the preparation of porous biodegradable controlled release formulation of poly(lactide/glycolide) (PLGA). The model drug used for this study was pentamidine. Scanning electron microscopy pictures showed that these microparticles are highly porous and spherical in shape. A comparison of particle size reveals a similar median particle size (54-68 microm) in all six batches. The particles are all smaller than 90 microm. Differential scanning calorimetry thermograms revealed that pentamidine was mostly present in the crystalline form in the microparticles and did not dissolve in PLGA. The efficiency of encapsulation of pentamidine was higher than 58% in all six batches. The amount of drug released from these microparticles was at least 12% within the first 60 min. At least 50% of the total drug was released within the first 4 h. Drug release from these microparticles continued for up to 12 h. This faster drug dissolution was due to the highly porous surface. This highly porous surface will allow large molecules to release at a much faster rate than the regular microcapsules/microspheres.

Biodegradation, Environmental↗
PubMed2000

Expression of human alpha 2-adrenergic receptors in adipose tissue of beta 3-adrenergic receptor-deficient mice promotes diet-induced obesity.

Catecholamines play an important role in controlling white adipose tissue function and development. beta- and alpha 2-adrenergic receptors (ARs) couple positively and negatively, respectively, to adenylyl cyclase and are co-expressed in human adipocytes. Previous studies have demonstrated increased adipocyte alpha 2/beta-AR balance in obesity, and it has been proposed that increased alpha 2-ARs in adipose tissue with or without decreased beta-ARs may contribute mechanistically to the development of increased fat mass. To critically test this hypothesis, adipocyte alpha 2/beta-AR balance was genetically manipulated in mice. Human alpha 2A-ARs were transgenically expressed in the adipose tissue of mice that were either homozygous (-/-) or heterozygous (+/-) for a disrupted beta 3-AR allele. Mice expressing alpha 2-ARs in fat, in the absence of beta 3-ARs (beta 3-AR -/- background), developed high fat diet-induced obesity. Strikingly, this effect was due entirely to adipocyte hyperplasia and required the presence of alpha2-ARs, the absence of beta 3-ARs, and a high fat diet. Of note, obese alpha 2-transgenic beta 3 -/- mice failed to develop insulin resistance, which may reflect the fact that expanded fat mass was due to adipocyte hyperplasia and not adipocyte hypertrophy. In summary, we have demonstrated that increased alpha 2/beta-AR balance in adipocytes promotes obesity by stimulating adipocyte hyperplasia. This study also demonstrates one way in which two genes (alpha 2 and beta 3-AR) and diet interact to influence fat mass.

Adipose Tissue↗
PubMed1998

Transcranial Doppler, MRA, and MRI as a screening examination for cerebrovascular disease in patients with sickle cell anemia: an 8-year study.

OBJECTIVE: The authors previously reported five transcranial Doppler ultrasonography (TCD) findings as significant in detecting clinical cerebrovascular disease in a 4-year study in patients with sickle cell disease. This is a follow-up to evaluate the validity of the original findings over another 4-year period during which the study population doubled. A clinical follow-up of the original asymptomatic sickle cell patients with positive TCD, MRA, and MRI was also made. MATERIALS AND METHODS: Over an 8-year period TCD, MRI, and MRA were prospectively performed in 90 sickle cell patients who were clinically asymptomatic for stroke and in 27 sickle cell patients with clinical stroke. RESULTS: Of the 4 out of original 46 control patients in 1992 who had positive MRA and TCD, 3 have subsequently had clinical stroke. None of the 9 original patients with positive TCD and positive MRI but negative MRA have developed stroke. All five original TCD indicators of disease were still significant (P < 0.05) for detecting clinical disease: maximum velocity in ophthalmic artery (OA) > 35 cm/s, mean velocity in middle cerebral artery (MCA) > 170 cm/s, resistive index (RI) in OA < 50, velocity in OA greater than in MCA, and velocity in posterior cerebral (PCA), vertebral, or basilar arteries greater than in MCA. An RI of < 60 in the DA was also now found to be significant [corrected]. Four additional factors were also significant: turbulence, PCA or ACA without MCA, RI < 30, and maximum velocity in MCA > 200 cm/s. CONCLUSION: Positive MRA with a positive TCD in an asymptomatic patient in long-term follow-up suggests a trend for developing clinical stroke. A 4- to 8-year follow-up of nine patients with positive TCD, positive MRI, but not positive MRA did not show development of clinical stroke. Nine Doppler findings are significant in screening for clinically symptomatic vascular disease in sickle cell patients. It is recommended that children with sickle cell disease be screened for cerebrovascular disease with TCD. If one or two indicators of abnormality are present, MRA is recommended. If the MRA is positive, the patient may be considered for transfusion therapy or other treatment for prevention of stroke.

Adolescent↗
PubMed1997

Troglitazone action is independent of adipose tissue.

We have investigated the antidiabetic action of troglitazone in aP2/DTA mice, whose white and brown fat was virtually eliminated by fat-specific expression of diphtheria toxin A chain. aP2/DTA mice had markedly suppressed serum leptin levels and were hyperphagic, but did not gain excess weight. aP2/DTA mice fed a control diet were hyperlipidemic, hyperglycemic, and had hyperinsulinemia indicative of insulin-resistant diabetes. Treatment with troglitazone alleviated the hyperglycemia, normalized the tolerance to intraperitoneally injected glucose, and significantly decreased elevated insulin levels. Troglitazone also markedly decreased the serum levels of cholesterol, triglycerides, and free fatty acids both in wild-type and aP2/DTA mice. The decrease in serum triglycerides in aP2/DTA mice was due to a marked reduction in VLDL- and LDL-associated triglyceride. In skeletal muscle, triglyceride levels were decreased in aP2/DTA mice compared with controls, but glycogen levels were increased. Troglitazone treatment decreased skeletal muscle, but not hepatic triglyceride and increased hepatic and muscle glycogen content in wild-type mice. Troglitazone decreased muscle glycogen content in aP2/DTA mice without affecting muscle triglyceride levels. The levels of peroxisomal proliferator-activated receptor gamma mRNA in liver increased slightly in aP2/DTA mice and were not changed by troglitazone treatment. The results demonstrate that insulin resistance and diabetes can occur in animals without significant adipose deposits. Furthermore, troglitazone can alter glucose and lipid metabolism independent of its effects on adipose tissue.

Adipose Tissue↗
PubMed1997

Transgenic mice overexpressing the beta 1-adrenergic receptor in adipose tissue are resistant to obesity.

The ratio of alpha- to beta-receptors is thought to regulate the lipolytic index of adipose depots. To determine whether increasing the activity of the beta 1-adrenergic receptor (AR) in adipose tissue would affect the lipolytic rate or the development of this tissue, we used the enhancer-promoter region of the adipocyte lipid-binding protein (aP2) gene to direct expression of the human beta 1 AR cDNA to adipose tissue. Expression of the transgene was seen only in brown and white adipose tissue. Adipocytes from transgenic mice were more responsive to beta AR agonists than were adipocytes from nontransgenic mice, both in terms of cAMP production and lipolytic rates. Transgenic animals were partially resistant to diet-induced obesity. They had smaller adipose tissue depots than their nontransgenic littermates, reflecting decreased lipid accumulation in their adipocytes. In addition to increasing the lipolytic rate, overexpression of the beta 1 AR induced the abundant appearance of brown fat cells in subcutaneous white adipose tissue. These results demonstrate that the beta 1 AR is involved in both stimulation of lipolysis and the proliferation of brown fat cells in the context of the whole organism. Moreover, it appears that it is the overall beta AR activity, rather than the particular subtype, that controls these phenomena.

Adenylyl Cyclases↗
PubMed1996

Activation of the nuclear receptor peroxisome proliferator-activated receptor gamma promotes brown adipocyte differentiation.

Brown adipose tissue (BAT) functions in non-shivering and diet-induced thermogenesis via its capacity for uncoupled mitochondrial respiration. BAT dysfunction in rodents is associated with severe defects in energy homeostasis, resulting in obesity and hyperglycemia. Here, we report that the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma), a prostaglandin-activated transcription factor recently implicated as a central regulator of white adipose tissue differentiation, also regulates brown adipocyte function. PPARgamma is abundantly expressed in both embryonic and adult BAT. Treatment of CD-1 rats with the PPARgamma-selective ligand BRL49653, an anti-diabetic drug of the thiazolidinedione class, results in marked increases in the mass of interscapular BAT. In vitro, BRL49653 induces the terminal differentiation of the brown preadipocyte cell line HIB-1B as judged by both changes in cell morphology and expression of uncoupling protein and other adipocyte-specific mRNAs. These data demonstrate that PPARgamma is a key regulatory factor in brown adipocytes and suggest that PPARgamma functions not only in the storage of excess energy in white adipose tissue but also in its dissipation in BAT.

Adipocytes↗
PubMed1996

Differentiation-dependent expression of the brown adipocyte uncoupling protein gene: regulation by peroxisome proliferator-activated receptor gamma.

Uncoupling protein (UCP) is expressed only in brown adipocytes and is responsible for the unique thermogenic properties of this cell type. The novel brown preadipocyte cell line, HIB-1B, expresses UCP in a strictly differentiation-dependent manner. Transgenic mice studies have shown that a region from kb -2.8 to -1.0 of the marine UCP gene is required for brown adipocyte-specific expression. Subsequent analysis identified a potent 220-bp enhancer from kb -2.5 to -2.3. We show that this enhancer is active only in differentiated HIB-1B adipocytes, and we identify a peroxisome proliferator-activated receptor gamma (PPARgamma) response element, referred to as UCP regulatory element 1 (URE1), within the enhancer. URE1 has differentiation-dependent enhancing activity in HIB-1B cells and is required for enhancer action, since mutations of URE1 that block protein binding abolish enhancer activity. We also show that PPAR gamma antibodies block binding to URE1 of nuclear extracts from cultured brown adipocytes and from the brown adipose tissue of cold-exposed mice. Protein binding to URE1 increases substantially during differentiation of HIB-1B preadipocytes, and PPAR-gamma mRNA levels increase correspondingly. Although forced expression of PPAR gamma and retinoid X receptor alpha activates the enhancer in HIB-1B preadipocytes, these receptors are not capable of activating the enhancer in NIH 3T3 fibroblasts. Our results show that PPAR gamma is a regulator of the differentiation-dependent expression of UCP and suggest that there are additional factors in HIB-1B cells required for brown adipocyte-specific UCP expression.

3T3 Cells↗
PubMed1994

Adipocyte-specific transcription factor ARF6 is a heterodimeric complex of two nuclear hormone receptors, PPAR gamma and RXR alpha.

Previously, we identified a novel transcription factor, ARF6, as a key regulator of the tissue-specific adipocyte P2 (aP2) enhancer. In order to identify the proteins which comprise the adipocyte ARF6 complex, we have purified this DNA binding activity from a cultured adipocyte cell line. We have developed a system for growth and differentiation of HIB-1B brown adipocytes in suspension culture that facilitates the production of large quantities of adipocyte nuclear extract. ARF6 was purified from HIB-1B nuclear extract by a combination of conventional and sequence-specific DNA affinity chromotography. Chemical sequencing and mass spectral analysis of tryptic peptides derived from the purified polypeptides identifies the ARF6 complex as a heterodimer of the retinoid X receptor alpha (RXR alpha) and the murine peroxisome proliferator activated receptor gamma (PPAR gamma). Of the known PPAR gamma isoforms, PPAR gamma is the predominant form expressed in adipose tissue. These results suggest that PPAR gamma 2 serves a unique function among PPAR family members as an important regulator of adipocyte-specific gene expression.

Adipocytes↗
PubMed1994

mPPAR gamma 2: tissue-specific regulator of an adipocyte enhancer.

Previously, we have isolated and characterized an enhancer from the 5'-flanking region of the adipocyte P2 (aP2) gene that directs high-level adipocyte-specific gene expression in both cultured cells and transgenic mice. The key regulator of this enhancer is a cell type-restricted nuclear factor termed ARF6. Target sequences for ARF6 in the aP2 enhancer exhibit homology to a direct repeat of hormone response elements (HREs) spaced by one nucleotide; this motif (DR-1) has been demonstrated previously to be the preferred binding site for heterodimers of the retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor (PPAR). We have cloned a novel member of the peroxisome proliferator-activated receptor family designated mPPAR gamma 2, and we demonstrate that a heterodimeric complex of mPPAR gamma 2 and RXR alpha constitute a functional ARF6 complex. Expression of mPPAR gamma 2 is induced very early during the differentiation of several cultured adipocyte cell lines and is strikingly adipose-specific in vivo. mPPAR gamma 2 and RXR alpha form heterodimers on ARF6-binding sites in vitro, and antiserum to RXR alpha specifically inhibits ARF6 activity in adipocyte nuclear extracts. Moreover, forced expression of mPPAR gamma 2 and RXR alpha activates the adipocyte-specific aP2 enhancer in cultured fibroblasts, and this activation is potentiated by peroxisome proliferators, fatty acids, and 9-cis retinoic acid. These results identify mPPAR gamma 2 as the first adipocyte-specific transcription factor and suggest mechanisms whereby fatty acids, peroxisome proliferators, 9-cis retinoic acid, and other lipids may regulate adipocyte gene expression and differentiation.

3T3 Cells↗
PubMed1993

Targeted expression of a toxin gene to adipose tissue: transgenic mice resistant to obesity.

Obesity is characterized by increased adipose tissue mass and is often accompanied by a number of other disorders, such as diabetes, hypertension, and hyperlipidemia. To investigate the interrelationship between excessive adipose tissue mass and these associated disorders, we have attempted to reduce adiposity via targeted expression of an attenuated diphtheria toxin A chain to adipose tissue, using the 5' regulatory region of the adipocyte P2 (aP2) gene. Transgenic mice with high levels of toxin expression developed chylous ascites and died shortly after birth. Transgenic mice expressing lower levels of the transgene had normal adiposity and survived to adulthood; however, they showed a complete resistance to chemically induced obesity. Nevertheless, these animals developed hyperlipidemia equal to or greater than their nontransgenic obese littermates. Moreover, MSG-treated transgenic females were fertile, unlike their obese nontransgenic littermates. These data demonstrate the feasibility of gentle manipulation of adiposity and allow a functional dissection of obesity and its metabolic sequelae.

Adipose Tissue↗
PubMed1993

ADD1: a novel helix-loop-helix transcription factor associated with adipocyte determination and differentiation.

DNA-binding proteins containing the basic helix-loop-helix (bHLH) domain have been implicated in lineage determination and the regulation of specific gene expression in a number of cell types. By oligonucleotide screening of an adipocyte cDNA expression library, we have identified a novel member of the bHLH-leucine zipper transcription factor family designated ADD1. ADD1 mRNA is expressed predominantly in brown adipose tissue in vivo and is regulated during both determination and differentiation of cultured adipocyte cell lines. ADD1 can function as a sequence-specific transcriptional activator in that it stimulates expression of a chloramphenicol acetyltransferase vector containing multiple ADD1 binding sequences but is unable to activate the myosin light-chain enhancer, which contains multiple binding sites for another bHLH factor, MyoD. ADD1 can also activate transcription through a binding site present in the 5'-flanking region of the fatty acid synthetase gene which is expressed in a differentiation-dependent manner in adipose cells. These data suggest that ADD1 plays a role in the regulation of determination- and differentiation-specific gene expression in adipocytes.

Adipose Tissue↗
PubMed1992

Hibernoma formation in transgenic mice and isolation of a brown adipocyte cell line expressing the uncoupling protein gene.

Transgenic mice were produced containing the adipocyte-specific regulatory region from the adipocyte P2 (aP2) gene linked to the simian virus 40 transforming genes. Most of the transgenic mice developed brown fat tumors (hibernomas) in their interscapular brown adipose tissue. Hibernoma formation was noticeable in some of the mice as early as 1 day after birth and most of the mice developed very large tumors by 1 month of age. All of the tumor tissue expressed the brown fat-specific uncoupling protein (UCP) gene as well as the aP2 gene. Several of the tumors have been used to establish cultured cell lines and at least one of these lines can be induced to differentiate into brown adipocytes. The cultured adipocytes express mRNA for UCP upon stimulation with N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, norepinephrine, isoproterenol or D7114, a beta 3 adrenergic agonist. Thus, regulation of the key thermogenic gene UCP can now be studied in an established cell line.

Adipose Tissue, Brown↗
PubMed1992

Identification of a fat cell enhancer: analysis of requirements for adipose tissue-specific gene expression.

The molecular basis for adipose-specific gene expression is not known. To approach the problem of adipocyte gene expression, we have analyzed in detail the capacity of the 5'-flanking region of the adipocyte P2 (aP2) gene to direct cell-type specific gene expression. Although the proximal promoter containing AP-1 and C/EBP binding sites is capable of directing differentiation-dependent gene expression in cultured adipocytes, these constructs are essentially inactive in the tissues of transgenic mice. We found that -5.4 kb of the 5'-flanking region were required to direct heterologous gene (chloramphenicol acetyl transferase; CAT) expression to the adipose tissue of transgenic mice. By deletion analysis, we identified a 520 bp enhancer at -5.4 kb of the aP2 gene. We show that this enhancer can direct high levels of gene expression specifically to the adipose tissue of transgenic mice. This enhancer also functions in a differentiation-dependent manner in cultured adipocytes and cannot be transactivated in preadipocytes by C/EBP. Molecular analysis indicates that several cis- and trans- acting acting elements, though not C/EBP, contribute to the specificity and potency of this enhancer.

Adipose Tissue↗
PubMed1992

Analysis of a tissue-specific enhancer: ARF6 regulates adipogenic gene expression.

The molecular basis of adipocyte-specific gene expression is not well understood. We have previously identified a 518-bp enhancer from the adipocyte P2 gene that stimulates adipose-specific gene expression in both cultured cells and transgenic mice. In this analysis of the enhancer, we have defined and characterized a 122-bp DNA fragment that directs differentiation-dependent gene expression in cultured preadipocytes and adipocytes. Several cis-acting elements have been identified and shown by mutational analysis to be important for full enhancer activity. One pair of sequences, ARE2 and ARE4, binds a nuclear factor (ARF2) present in extracts derived from many cell types. Multiple copies of these elements stimulate gene expression from a minimal promoter in preadipocytes, adipocytes, and several other cultured cell lines. A second pair of elements, ARE6 and ARE7, binds a separate factor (ARF6) that is detected only in nuclear extracts derived from adipocytes. The ability of multimers of ARE6 or ARE7 to stimulate promoter activity is strictly adipocyte specific. Mutations in the ARE6 sequence greatly reduce the activity of the 518-bp enhancer. These data demonstrate that several cis- and trans-acting components contribute to the activity of the adipocyte P2 enhancer and suggest that ARF6, a novel differentiation-dependent factor, may be a key regulator of adipogenic gene expression.

3T3 Cells↗