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Biomedical subjects

A M Gonzalez

Publications and source records attributed to A M Gonzalez.

At least 37 records · Page 2Linked to original sources
PubMed1999

Fibroblast growth factor 2 retargeted adenovirus has redirected cellular tropism: evidence for reduced toxicity and enhanced antitumor activity in mice.

Adenovirus (Ad) have been used as vectors to deliver genes to a wide variety of tissues. Despite achieving high expression levels in vivo, Ad vectors display normal tissue toxicity, transient expression, and antivector immune responses that limit therapeutic potential. To circumvent these problems, several retargeting strategies to abrogate native tropism and redirect Ad uptake through defined receptors have been attempted. Despite success in cell culture, in vivo results have generally not shown sufficient selectivity for target tissues. We have previously identified (C. K. Goldman et al., Cancer Res., 57: 1447-1451, 1997) the fibroblast growth factor (FGF) ligand and receptor families as conferring sufficient specificity and binding affinity to be useful for targeting DNA in vivo. In the present studies, we retargeted Ad using basic FGF (FGF2) as a targeting ligand. Cellular uptake is redirected through high-affinity FGF receptors (FGFRs) and not the more ubiquitous lower-affinity Ad receptors. Initial in vitro experiments demonstrated a 10- to 100-fold increase in gene expression in numerous FGFR positive (FGFR+) cell lines using FGF2-Ad when compared with Ad. To determine whether increased selectivity could be detected in vivo, FGF2-Ad was administered i.v. to normal mice. FGF2-Ad demonstrates markedly decreased hepatic toxicity and liver transgene expression compared with Ad treatment. Importantly, FGF2-Ad encoding the herpes simplex virus thymidine kinase (TK) gene transduces Ad-resistant FGFR+ tumor cells both ex vivo and in vivo, which results in substantially enhanced survival (180-260%) when the prodrug ganciclovir is administered. Because FGFRs are up-regulated on many types of malignant or injured cells, this broadly useful method to redirect native Ad tropism and to increase the potency of gene expression may offer significant therapeutic advantages.

Adenoviridae↗
PubMed1999

D2S, D2L, D3, and D4 dopamine receptors couple to a voltage-dependent potassium current in N18TG2 x mesencephalon hybrid cell (MES-23.5) via distinct G proteins.

We utilized the approach of stably expressing different dopamine (DA) receptors into identified cell lines in an attempt to better understand the coupling of these receptors to membrane ion channels via second messenger systems. Recently, we examined the N18TG2 x mesencephalon (MES-23.5) cell line that is phenotypically similar to mesencephalic dopamine-containing neurons. Whole-cell voltage-clamp methods were used to investigate a voltage-dependent K+ current present in these cells. Untransfected MES-23.5 cells displayed a voltage-dependent slow-onset, slowly inactivating outward current which was not altered by bath application of either the D2 DA receptor agonist quinpirole (QUIN; 10-100 microM) or the D1 DA receptor agonist SKF38393, indicating that these cells were devoid of DA receptors. The K+ current studied was activated upon depolarization from a holding potential of -60 mV to a level more positive than -20 mV and was observed to be sensitive to bath application of tetraethylammonium. When MES-23.5 cells were transfected to stably express the D2S, D2L, D3, and D4 receptors, the same current was observed. In cells expressing D2L, D2S, and D3 receptors, application of the DA receptor agonists QUIN (1-80 microM), 7-hydroxy-dipropylaminoteralin (7-OH-DPAT, 1-80 microM), and dopamine (DA, 1-80 microM), increased the peak outward current by 35-40%. In marked contrast, cells stably expressing the D4 receptor demonstrated a significant DA agonist-induced reduction of the peak K+ current by 40%. For all four receptor subtypes, the D2-like receptor antagonist sulpiride (SUL 5 microM), when coapplied with QUIN (10 microM), totally abolished the change in K+ current normally observed, while coapplication of the D1-like receptor antagonist SCH23390 was without effect. The modulation of K+ current by D2L, D3, and D4 receptor stimulation was prevented by pretreatment of the cells with pertussis toxin (PTX, 500 ng/ml for 4 h). In addition, the intracellular application of a polyclonal antibody which specifically recognizes Goalpha completely blocked the ability of D2L, D3, and D4 receptors to modulate outward K+ currents. In contrast, the intracellular application of an antibody directed against Goalpha was without effect, whereas intracellular application of an antibody recognizing Gsalpha abolished the ability of the D2S receptor to enhance K+ current. These findings demonstrate that different members of the D2 DA receptor family may couple in a given cell to a common effector in dramatically different ways.

Animals↗
PubMed1999

Interactions of the novel antipsychotic aripiprazole (OPC-14597) with dopamine and serotonin receptor subtypes.

OPC-14597 {aripiprazole; 7-(-4(4-(2,3-dichlorophenyl)-1-piperazinyl) butyloxy)-3,4-dihydro-2(1H)-quinolinone} is a novel candidate antipsychotic that has high affinity for striatal dopamine D2-like receptors, but causes few extrapyramidal effects. These studies characterized the molecular pharmacology of OPC-14597, DM-1451 (its major rodent metabolite), and the related quinolinone derivative OPC-4392 at each of the cloned dopamine receptors, and at serotonin 5HT6 and 5HT7 receptors. All three compounds exhibited highest affinity for D2L and D2S receptors relative to the other cloned receptors examined. Both OPC-4392 and OPC-14597 demonstrated dual agonist/antagonist actions at D2L receptors, although the metabolite DM-1451 behaved as a pure antagonist. These data suggest that clinical atypicality can occur with drugs that exhibit selectivity for D2L/D2S rather than D3 or D4 receptors, and raise the possibility that the unusual profile of OPC-14597 in vivo (presynaptic agonist and postsynaptic antagonist) may reflect different functional consequences of this compound interacting with a single dopamine receptor subtype (D2) in distinct cellular locales.

Animals↗
PubMed1998

Establishment of epitope-defined monoclonal antibodies with specificity for fibroblast growth factor receptor types 1 and 2.

The development of specific antibody probes for characterizing the expression of the family of 4 fibroblast growth factor receptor (FGFR) types has been difficult because of their close homology to each other and high degree of evolutionary conservation. Of the existing anti-FGFR monoclonal antibodies (MAbs), there are few that are useful for staining paraffin-embedded tissues. We have raised MAbs against human FGFR1 and FGFR2 in both rats and mice using bacterial recombinant receptor fusion proteins as immunogens. We used peptide epitope mapping to characterize the immune sera and the selected MAbs. Immunized animals were selected that displayed the broadest reactivity against epitopes unique to the immunizing receptor type. We produced FGFR1 specific MAbs that bind epitopes in immunoglobulin domain I (Ig-I) and FGFR2 specific MAbs that bind epitopes in Ig-I, Ig-II, and the acid box. The specificity of the antibodies was demonstrated by ELISA and immunoblot analysis of purified recombinant FGFR1 and FGFR2 extracellular domains produced both in E. coli and in eucaryotic cells. Based on the lack of epitope homology, these MAbs would not be expected to cross-react with FGFR3 or FGFR4. We isolated MAbs that bound to paraffin embedded tissue and immunoblots of recombinant receptor. These epitope-defined MAbs can distinguish between members of the FGF receptor family and should be useful as tools for assessing FGF receptor expression in a variety of normal and diseased tissues.

Amino Acid Sequence↗
PubMed1998

Teaching residents to care for culturally diverse populations.

The authors discuss the growing need for primary care residents to learn how to care for patients of many cultural backgrounds. To effectively learn the needed skills, residents must incorporate insights from areas outside medicine. The authors focus on three such areas: cultural competency, public health, and community-oriented primary care. Regarding cultural competency, the authors make clear that on the one hand, physicians must be trained to be sensitive to cultural differences and patterns, but on the other, they cannot be expected to know the many cultures of their patients in depth. They discuss the Core Curriculum Guidelines on Culturally Sensitive and Competent Health Care created by the Society of Teachers of Family Medicine. Regarding community-oriented primary care (COPC), a process introduced from Europe in 1982, the authors state that one of its key elements is to provide accessible care to diverse and often underserved populations. However, various factors have kept COPC, and the federally funded community health centers that address the concerns of COPC, from having the widespread effects they could have. Regarding public health, the authors review the various services and orientations of public health and show how these help foster care for diverse populations. The authors then briefly describe their own residency program and its work with diverse populations. They conclude by emphasizing the importance for residents of learning the principles and practices embodied in cultural competency, public health, and COPC in order to effectively communicate with their patients.

Attitude to Health↗
PubMed1998

The pulmonary hypertensive fawn-hooded rat has a normal serotonin transporter coding sequence.

The coding sequence of the serotonin transporter gene was compared in two strains of rat-the Wistar and the fawn-hooded rat (FHR). The FHR has an inherited platelet storage-pool deficiency and a widespread impairment of serotonin storage. It is also susceptible to systemic and pulmonary hypertension. The FHR provides a model to study the genetics in human systemic and pulmonary hypertension. We measured platelet function in these two strains by measuring incorporation of radiolabeled serotonin into a platelet suspension and found significant differences in serotonin uptake and release. The coding sequence for the serotonin transporter in the FHR has yet to be reported. No differences were found in the predicted amino acid sequence between these two strains of rat, either in the platelet or the lung samples or when compared with the published sequence of the brown rat. We conclude that differences in the primary structure of the serotonin transporter gene do not account for the altered serotonin storage in the FHR strain.

Amino Acid Sequence↗
PubMed1997

Pulmonary hypertension, family and environment.

The focus of interest on pulmonary hypertension (PH) has come through recent therapeutic advances which prolong and improve quality of life. From this interest, recent observations form family studies indicate that a gene located on chromosome 2 is associated with PH. Environmental factors, the use of an anorectic agent, dexfenfluramine, has been associated with a risk of developing primary PH. A link with hypoxic induced PH has emerged in that a strain of rat, the fawn-hooded rat (FHR) which has a susceptibility to developing PH with mild hypoxia has an inherited storage defect to serotonin. Dexfenfluramine, by inhibiting the serotonin transporter, produces a similar pattern of disturbance of biogenic amine metabolism. This observation provides some insight into potential mechanisms for this complex disorder.

Animals↗
PubMed1996

Targeting DNA to cells with basic fibroblast growth factor (FGF2).

Ligand-mediated targeting of DNA was validated by condensing a plasmid DNA encoding the beta-galactosidase (beta-gal) gene with a basic fibroblast growth factor (FGF2) that was first chemically conjugated to polylysine (K). The conditions that gave optimal binding of this FGF2 to DNA also generated the highest level of beta-gal expression when added to FGF2 target cells like COS-1, 3T3, baby hamster kidney (BHK), or endothelial cells. This beta-gal activity increased in a time- and dose-dependent manner and was dependent on the inclusion of FGF2 in the complex. FGF receptor specificity was demonstrated by competition of the complex with FGF2 and heparin, and by the failure of cytochrome c or histone H1 to mimic the gene-targeting effects of FGF2. The expression of beta-gal was also endosome dependent because chloroquine increased beta-gal expression 8-fold and endosome disruptive peptides increased expression of beta-gal 26-fold. Taken together these findings establish that DNA can be introduced into cells through the high affinity FGF receptor complex, and while its efficiency will require significant enhancements to achieve sustained and elevated transgene expression, the possibility that the technique could be used to deliver DNAs encoding cytotoxic molecules is discussed.

3T3 Cells↗
PubMed1996

Interleukin-1 receptor antagonist allele (IL1RN*2) associated with nephropathy in diabetes mellitus.

We have previously found association between an allele of the interleukin-1 (IL-1) receptor antagonist gene (IL1RN) and several inflammatory diseases, where IL-1 has been implicated in the inflammatory mechanism. We have now, therefore, tested the association of this specific allele (IL1RN*2) with complications of diabetes which have an inflammatory tissue component. We have tested the allele frequency of IL1RN*2 in 128 patients with insulin-dependent and 125 with non-insulin-dependent diabetes mellitus (NIDDM). There was a significant association between carriage of IL1RN*2 and diabetic nephropathy (P<0.001, Pcorrected<0.0012). The association was significant in both types of diabetes, but the observed increase was highest in NIDDM, rising to double the control levels. It appears that IL1RN*2 is a novel genetic marker of severity of inflammatory complications of diseases rather than a marker of disease susceptibility. If the DNA polymorphism is associated with altered gene function, new therapeutic interventions may be possible.

Adult↗
PubMed1996

Distribution of fibroblast growth factor (FGF)-2 and FGF receptor-1 messenger RNA expression and protein presence in the mid-trimester human fetus.

Fibroblast growth factors (FGF) are known to have key roles in embryonic growth and morphogenesis, but their presence and contributions to fetal development are unclear. In particular, little information exists as to the relevance of FGF and their specific receptors to human fetal development. We studied the anatomical distribution of messenger RNA encoding FGF-2 and one of its high affinity receptors, FGFR1, using in situ hybridization in a variety of human fetal tissues in early second trimester. Corresponding protein distributions were determined by immunohistochemistry. Both FGF-2 and FGFR1 mRNA and proteins were found to be present in every organ and tissue examined, but with defined cellular localizations. In skeletal muscle, both FGF-2 and FGFR1 mRNA and peptides were present in differentiated fibers, and both co-localized to proliferating chondrocytes of the epiphyseal growth plate. FGF-2 and FGFR1 mRNA and peptides were also present within cardiac or gastrointestinal smooth muscle. Within the gastrointestinal tract FGF-2 mRNA and peptide were located in the submucosal tissue, whereas FGFR1 was expressed within the overlying mucosa. Similarly, in skin, FGF-2 was expressed within the dermis whereas FGFR1 mRNA and peptide were most apparent in the stratum germinativum of the epidermis. In kidney and lung, FGFR1 mRNA was located in the tubular and alveolar epithelia respectively, whereas FGF-2 was expressed in both epithelial and mesenchymal cell populations. Both growth factor and receptor were widespread in both neuroblasts and glioblasts in the cerebral cortex of the brain. Immunoreactivity for FGF-2 and FGFR1 was seen in all vascular endothelial cells of major vessels and capillaries. Within the skin, kidney, lung, and intestine FGF-2 immunoreactivity was found in basement membranes underlying epithelia, and was associated with the extracellular matrix and plasma membranes of many cell types. The results show that FGF-2 and one of its receptors are widely expressed anatomically in the mid-trimester human fetus.

Blood Vessels↗
PubMed1995

A comprehensive analysis of the distribution of FGF-2 and FGFR1 in the rat brain.

We have examined the cellular distribution of both FGF-2 and FGFR1 immunoreactivity and their mRNAs throughout the normal adult rat brain in order to reconcile numerous disparate findings in the published literature. The results confirm a widespread distribution of FGF-2 and FGFR1 in the rat brain, and different regions express distinct patterns of FGF-2 and FGFR1 mRNA and protein: neuronal and non-neuronal cells show different subcellular distributions that vary according to the area where they are located. The intensity of the staining and hybridization also varies according to the loci examined and the cell type involved. Astrocytes contain the highest levels of FGF-2 and FGFR1 mRNAs, and characteristically, possess high levels of immunoreactive FGF-2 within the nucleus. Amongst non-neuronal cells, oligodendrocytes do not synthesize or contain significant levels of FGF-2 immunoreactivity however, they do express FGFR1 mRNA. In these cells, immunoreactive FGFR1 is mainly associated with the myelin sheaths of neuronal fibers. In ventricular systems, ependymal cells synthesize and contain immunoreactive FGFR1. In contrast, only cells lining the lateral wall of the IIIrd ventricle express FGF-2 mRNA. Subependymal cells contain high levels of both FGF-2 and FGFR1 immunoreactivity. Neurons express low levels of FGF-2 mRNA and immunoreactive FGF-2 is localized predominantly to the perikaryon. However, selected populations of neurons, such as CA2 field of the hippocampus, show high levels of FGF-2 mRNA, in which the nucleus is strongly immunopositive. Similarly, high levels of FGFR1 mRNA are localized to select populations of neurons (e.g. amygdala). FGFR1 immunoreactivity is mainly associated with myelinated fiber tracts (e.g. striatum), and some neurons show immunoreactivity in the perikaryon (e.g. hippocampus), the nucleus (e.g. mesencephalic trigeminal nucleus), or in axonal projections (e.g. hypothalamus). Remarkably, in many of the areas studied, FGF-2 and FGFR1 mRNA and/or their translated protein do not co-localize in neurons (e.g. neo-cortices) or even in the same regions of the brain (e.g. substantia nigra). In other instances, mRNAs for both FGF-2 and FGFR1 colocalize (e.g. supraoptic nucleus). The brain, in contrast to peripheral tissues, contains high levels of FGF-2 and actively expresses its gene under normal physiological conditions. The highly specific anatomical distribution of immunoreactive FGF-2 in neuronal and non-neuronal brain cells, supports the notion that it plays a multifunctional role in the CNS under normal physiology. By correlating the localization and the synthesis of FGF-2 and one of its high affinity receptors, FGFR1, in the CNS, it should be possible to obtain a better understanding of the roles of FGF-2 in normal and pathological conditions.

Animals↗
PubMed1995

Dopamine receptor agonist potencies for inhibition of cell firing correlate with dopamine D3 receptor binding affinities.

The potencies for in vivo inhibition of substantia nigra pars compacta dopamine single cell firing were determined for apomorphine, BHT 920, N-0923, (+/-)-7-hydroxy-dipropylaminotetralin (7-OH-DPAT), (+)-3-(3-hydroxyphenyl)-N-propylpiperidine (3-PPP), pramipexole, quinelorane, quinpirole, RU 24926, U-86170, and U-91356. Significant correlation was obtained between the potencies of these 11 highly efficacious dopamine receptor agonists and the in vitro binding affinities at dopamine D3 receptors, but not at dopamine D2L receptors. These results support a functional role for the dopamine D3 receptor subtype in the autoreceptor-mediated regulation of dopamine cell activity, while a role for dopamine D2 receptors awaits further analysis. In addition, the results demonstrate the limitations of using currently available dopamine receptor agonists to delineate relative in vivo roles for the dopamine D2 and D3 receptor subtypes.

Aminoquinolines↗
PubMed1995

[3H]7-OH-DPAT is capable of labeling dopamine D2 as well as D3 receptors.

The binding of [3H](+)-7-hydroxy-2-(N,N-di-n-propylamino)tetralin ([3H]7-OH-DPAT) to dopamine D2 and D3 receptors expressed in Chinese hamster ovary (CHO) cells was investigated and compared with [3H]methylspiperone. [3H]7-OH-DPAT labeled the D3 receptor in the CHO cells in a guanine nucleotide-insensitive fashion and exhibited a Kd of about 0.5 nM. In the presence of MgCl2. [3H]7-OH-DPAT was also found to label the D2 receptor in CHO cells with high affinity (3.6 nM). The binding of [3H]7-OH-DPAT to the D2 receptor was sensitive to guanine nucleotides suggesting occupancy of a high affinity G protein-coupled state of the receptor. These results suggest that caution should be exercised when using [3H]7-OH-DPAT to label the dopamine D3 receptor in brain tissues.

Animals↗
PubMed1994

Storage, metabolism, and processing of 125I-fibroblast growth factor-2 after intracerebral injection.

Basic fibroblast growth factor (FGF-2) is a potent trophic agent for both neuronal and non-neuronal cells of the mammalian CNS. It can enhance survival and neurite outgrowth of a variety of neuronal types in vitro and in vivo, and recently has been shown to stimulate neuroblast proliferation in culture. To determine the most effective means of introducing FGF-2 into the brain, and to further our understanding of the behavior of exogenous FGF-2 following intracerebral injection, we examined the diffusion and degradation of 125I-FGF-2 following intraventricular or intraparenchymal injection. SDS-PAGE and autoradiography show that when radiolabelled FGF-2 is injected into the parenchyma of the rat brain, it remains at the site of injection where it is detectable for several days. During this time, it is slowly metabolized to 2 specific heparin-binding metabolic fragments that are virtually identical to the ones described for its metabolism by neurons and astrocytes in vitro. Microscopic examination and autoradiography of these tissue sections show that within these areas, FGF-2 diffuses throughout the site of injection. Initially, it migrates along adjacent fiber tracts, binds to specific cells and to basement membranes of the microvasculature, but later on it remains associated to basement membranes and non-neuronal cells. Based on its slow clearance and slow rate metabolic degradation, this FGF-2 is presumed to be in a sequestered form and to have limited activity. In contrast, the intraventricular injection of 125I-FGF leads to a rapid clearance, with some binding to ependymal cells lining the ventricles and little translocation into the parenchyma.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Effects of transforming growth factor beta 1 on scar production in the injured central nervous system of the rat.

In the central nervous system (CNS), nerve regeneration after traumatic injury fails. The formation of a dense fibrous scar is thought to restrict in part the growth of axonal projections, providing one of the many reasons that complete lesions of neural pathways in the adult mammalian CNS are rarely followed by significant functional recovery. In order to determine which mechanisms mediate scar formation in the CNS and to investigate whether they can be modulated in vivo, we have attempted to define the potential role of trophic factors. Our previous studies have shown the focal elevation of transforming growth factor beta 1 (TGF beta 1) expression in lesioned CNS tissue. In the studies described here, we demonstrate that TGF beta 1 participates in the scarring response in the rat brain. First, the elevated protein levels of TGF beta 1 are localized to specific populations of injury-responsive cells in the traumatized CNS. Furthermore, the injection of TGF beta 1 into the brains of injured rats causes a dramatic increase in the scarring response. Conversely, when neutralizing TGF beta 1 antibodies are administered, the deposition of fibrous scar tissue and the formation of a limiting glial membrane that borders the lesion is significantly attenuated, thus establishing a role for the endogenous growth factor in regulation of the non-glial component of the scar. In implicating TGF beta 1 in the scarring response in the CNS, the potential use for TGF beta 1 antagonists as inhibitors of scar formation in the injured mammalian CNS is self-evident.

Animals↗
PubMed1994

Comparative analysis of the genetic associations of HLA-DR3 and tumour necrosis factor alpha with human IDDM.

Insulin-dependent diabetes mellitus (IDDM) is associated with class II molecules of the MHC on chromosome 6, in particular HLA-DR and -DQ alleles, but a pathogenic role for TNF-alpha in the class III region of the MHC has also been implied. We therefore tested whether there was any independent association between a biallelic TNF polymorphism and IDDM. The TNF2 allele was present in 61 of 114 (54%) IDDM patients compared to 101 of 253 (40%) control subjects (odds ratio 1.73; p < 0.02). Stratification analysis in individuals matched for HLA-DR3 revealed, however, that this association was not independent of HLA-DR3 and is most likely to be a result of linkage disequilibrium between these alleles.

Adult↗
PubMed1994

Synaptic vesicular monoamine transporter expression: distribution and pharmacologic profile.

The human vesicular monoamine transporter (hSVMT) cDNA predicts a protein of 515 amino acids that shares 92% amino acid identity with the rat cDNA. Northern analyses reveal expression of 4.3 kb SVMT mRNAs in rat hypothalamus, midbrain and brainstem, a 3 kb hSVMT mRNA in human brainstem and a 4.8 kb hSVMT mRNA in human hypothalamus. In situ hybridization documents significant SVMT expression in human nigra compacta neurons and in rat hypothalamic neurons whose distribution patterns are identical to those previously reported to display histaminergic markers. COS cell hSVMT expression yielded nanomolar affinities for tetrabenazine and reserpine, micromolar affinities for haloperidol, GBR12909, serotonin, mazindol, nomifensin and d-amphetamine, while dopamine, epinephrine, norepinephrine and histamine each displayed millimolar affinities. These observations extend the pharmacological characterization of hSVMT and studies of its distribution, and indicate likely physiological roles for SVMT in packaging monoamine transmitters including histamine.

Animals↗