Biomedical subjects

L C Walker

Publications and source records attributed to L C Walker.

At least 37 records · Page 2Linked to original sources

PubMed1999

Cerebrovascular amyloidosis: experimental analysis in vitro and in vivo.

With advancing age, the likelihood of beta-amyloid deposition in the cerebral vasculature increases, particularly in individuals with Alzheimer's disease. The beta-amyloid typically accumulates in the basal lamina of the arteriolar tunica media, and frequently extends into the adjacent neuropil. Cerebrovascular beta-amyloid increases the risk of hemorrhagic stroke, and may also play a role in the pathogenesis of AD. Genetic variations have been identified that are causative or risk factors for cerebrovascular beta-amyloid, including particular mutations in the genes for beta-amyloid precursor protein, presenilins 1 and 2, and possibly cystatin C, as well as polymorphisms in apolipoprotein E. Cerebrovascular amyloidosis is now being studied in a variety of in vitro and in vivo models, including cultured vascular smooth muscle cells, transgenic mice, and aged animals such as nonhuman primates. Methods for delivering agents selectively to vascular amyloid in living subjects are now being developed, and these techniques are paving the way to the development of diagnostic tools and therapies for cerebrovascular amyloidosis.

Amyloidosis↗

PubMed1998

Apolipoprotein E4 promotes incipient Alzheimer pathology in the elderly.

To evaluate the influence of the apolipoprotein E (ApoE) epsilon4 allele on the age at which Alzheimer-like lesions appear in the brain, we analyzed the degree of cerebral beta-amyloidosis and neurofibrillary tangle formation in the hippocampal formation and adjacent cortical areas 28, 27, and 36 of persons who had died between the ages of 50 and 93 years and who had shown no signs of clinical dementia. The occurrence of the three common polymorphisms of the ApoE gene in this sample of 147 routine autopsy cases from eastern Germany was comparable to previously reported values in European and North American populations: ApoEepsilon2/2, 0.7%; ApoEepsilon2/3, 14.3%; ApoEepsilon2/4, 4.1%; ApoEepsilon3/3, 56.5%; ApoEepsilon3/4, 22.4%; and ApoEepsilon4/4, 2.0%. Nondemented persons carrying the ApoEepsilon4 allele were significantly more likely to have senile plaques, diffuse amyloid deposits, cerebrovascular amyloid, and neurofibrillary tangles than were those lacking E4. Comparing the two largest ApoE subgroups, ApoEepsilon3/3 and ApoEepsilon3/4, the relative increase in the occurrence of beta-amyloid in the epsilon3/4 group was evident by the mid-60s, with the relative increase in neurofibrillary tangles in this group emerging slightly earlier. The ApoEepsilon2 allele appears to delay the appearance of the lesions somewhat. We conclude that ApoEepsilon4 promotes the early appearance of beta-amyloid and neurofibrillary tangles in the elderly and that the increased frequency of these lesions is related to the higher risk of Alzheimer disease in persons bearing the ApoEepsilon4 allele.

Aged↗

PubMed1998

beta-Amyloid precursor protein and ss-amyloid peptide immunoreactivity in the rat brain after middle cerebral artery occlusion: effect of age.

BACKGROUND AND PURPOSE: Previous studies have shown that the ss-amyloid precursor protein (ssAPP) is upregulated after cerebral ischemia and that the ss-amyloid (Ass) fragment may be toxic to brain cells. Although stroke in humans usually afflicts the elderly, most experimental studies on the nature of cerebral ischemia have used young animals. To test the hypothesis that the upregulation and/or persistence of amyloidogenic proteins is exacerbated in aged rats after cerebral ischemic stroke, we studied the expression of ssAPP and its proteolytic product Ass in the brains of young and old rats 7 days after temporary cerebral ischemia. METHODS: Focal cerebral ischemia was produced by reversible occlusion of the right middle cerebral artery in 3- and 20-month-old male Sprague-Dawley rats. After 1 week, brains were removed and immunostaining was performed for ssAPP, Ass, and ED1 for macrophages and glial fibrillary acidic protein (GFAP). RESULTS: Histological staining revealed that the degree of necrotic cavitation in the infarct core was relatively less in aged rats than in young rats, suggesting a slower pace of degenerative change and/or tissue removal in older animals. ssAPP immunoreactivity was robustly increased, primarily in macrophage-like, ED1-positive cells in the infarct core and in the penumbra of both young and aged animals. Ass immunoreactivity was evident in GFAP-positive astrocytic somata and processes, and also in clusters of small spherical structures in the penumbra. These Ass-immunoreactive minispheres were more numerous in aged rats than in young rats. CONCLUSIONS: The presence of ssAPP and Ass immunoreactivity in the infarct core and penumbra indicates that cerebral ischemia promotes conditions that are favorable to the focal accumulation of ssAPP and its proteolytic fragments, especially in the aged brain.

Aging↗

PubMed1997

A common duplication in the lysyl hydroxylase gene of patients with Ehlers Danlos syndrome type VI results in preferential stimulation of lysyl hydroxylase activity and mRNA by hydralazine.

Patients with Ehlers Danlos Syndrome type VI (EDS VI) are biochemically characterized by a deficiency of lysyl hydroxylase (LH), an enzyme that hydroxylates lysine residues required in the formation of stable crosslinks in collagen biosynthesis. Recently, in 19% of 35 EDS VI families, a duplication of seven exons in the LH gene has been identified as a common cause of EDS VI. We have observed that in fibroblasts from patients with this duplication mutation, administration of hydralazine, an iron-chelating agent, and ascorbate, a cofactor for LH activity, stimulates LH activity and its mRNA significantly more than in other EDS VI patients who do not have this duplication. Administration of these reagents, either singly or in combination, to fibroblasts from five patients homozygous for the duplication stimulated the low basal level of LH activity (<20% of normal) by five- to sixfold (hydralazine +/- ascorbate) and by twofold (ascorbate alone) at 72 h. This paralleled the increase in the steady-state level of mRNA for LH measured in similarly treated fibroblasts from four of these patients. In contrast, the activity of LH in fibroblasts from six other EDS VI patients and the mRNA from four of these patients who did not have the duplication were increased only three- to fourfold by hydralazine +/- ascorbate. The mechanism for the preferential stimulation of LH activity and mRNA by hydralazine in the EDS VI cells carrying the duplication is unknown, but it could be attributed to the presence of, for example, an enhancer sequence within the duplicated region of the LH gene.

2,2'-Dipyridyl↗

PubMed1997

Animal models of cerebral beta-amyloid angiopathy.

Cerebral amyloid angiopathy (CAA) is a significant risk factor for hemorrhagic stroke in the elderly, and occurs as a sporadic disorder, as a frequent component of Alzheimer's disease, and in several rare, hereditary conditions. The most common type of amyloid found in the vasculature of the brain is beta-amyloid (A beta), the same peptide that occurs in senile plaques. A paucity of animal models has hindered the experimental analysis of CAA. Several transgenic mouse models of cerebral beta-amyloidosis have now been reported, but only one appears to develop significant cerebrovascular amyloid. However, well-characterized models of naturally occurring CAA, particularly aged dogs and non-human primates, have contributed unique insights into the biology of vascular amyloid in recent years. Some non-human primate species have a predilection for developing CAA; the squirrel monkey (Saimiri sciureus), for example, is particularly likely to manifest beta-amyloid deposition in the cerebral blood vessels with age, whereas the rhesus monkey (Macaca mulatta) develops more abundant parenchymal amyloid. These animals have been used to test in vivo beta-amyloid labeling strategies with monoclonal antibodies and radiolabeled A beta. Species-differences in the predominant site of A beta deposition also can be exploited to evaluate factors that direct amyloid selectively to a particular tissue compartment of the brain. For example, the cysteine protease inhibitor, cystatin C, in squirrel monkeys has an amino acid substitution that is similar to the mutant substitution found in some humans with a hereditary form of cystatin C amyloid angiopathy, possibly explaining the predisposition of squirrel monkeys to CAA. The existing animal models have shown considerable utility in deciphering the pathobiology of CAA, and in testing strategies that could be used to diagnose and treat this disorder in humans.

Alzheimer Disease↗

PubMed1997

Empirical assessment of synapse numbers in primate neocortex.

Reliable methods are needed to assess the impact of synaptic loss on brain function. In this empirical study we demonstrate a novel and efficient method using immunocytochemistry (ICC) and modern stereological techniques to quantify synapses in neocortex of adult primates (Macaca fascicularis). Systematic-uniform-random sections through forebrain from two 10-year-old monkeys were immunostained for estimation of synaptophysin-immunoreactive (synaptophysin-IR) presynaptic boutons (synapses). Adjacent sections were stained with cresyl violet for estimation of total number of neuronal cell bodies. The unbiased Cavalieri method was used to estimate total forebrain and neocortical volumes to a high level of precision (coefficient of error (CE) < or = 0.10)). Synapse-to-neuron ratios varied from 860 in frontal cortex to 2300 in parietal-temporal cortex. The combination of Cavalieri and optical disector methods provided a direct means of estimating approximately 1.25 trillion (x 10(12)) total synaptophysin-immunopositive boutons and approximately 1.01 billion (x 10(9)) cell bodies in neocortex, with low CEs (0.12). Time required to make precise estimates of total neocortical and forebrain volumes and total numbers of synapses and neurons in neocortex was approximately 2-3 h per case from stained sections. The approach is a direct and efficient technique to quantify total synapse and neuron numbers within a defined brain structure.

Animals↗

PubMed1997

Characterization of amyloid beta protein species in cerebral amyloid angiopathy of a squirrel monkey by immunocytochemistry and enzyme-linked immunosorbent assay.

We analyzed the composition of amyloid beta protein (A beta) species in cerebral amyloid angiopathy (CAA) of an aged squirrel monkey. Immunocytochemistry demonstrated that the cerebral cortex contained no lesions other than widespread CAA with A beta40 as its apparent major component. However, enzyme-linked immunosorbent assay revealed that A beta42(43) predominated over A beta40 in a formic acid-extracted cortical fraction. These findings suggest possible underestimation of A beta42(43) levels in some previous immunocytochemical investigations.

Aging↗

PubMed1997

Relationship between airway ion transport and a mild pulmonary disease mutation in CFTR.

Patients with cystic fibrosis (CF) display defects in airway ion transport, but the influence of airway transport phenotype on improved prognosis is not known. We studied airway bioelectric properties in five CF patients with the rare A455E mutation that is associated with mild pulmonary disease. We also evaluated five patients possessing premature truncation mutations (G542X and R553X) for which an association with mild pulmonary disease has not been as well established. We found no evidence in vivo that a mild lung disease mutation in the CF transmembrane regulator gene (CFTR) led to correction or partial correction of: (1) unstimulated Cl- secretion; (2) beta-agonist-activated Cl- secretion; (3) basal sodium reabsorption; or (4) amiloride-sensitive airway sodium transport. Early phase therapeutic trials in CF, including human gene transfer trials, rely heavily on improvements in airway potential difference to identify promising interventions and an improved prognosis. Based on our findings in a naturally occurring group of CF patients with an improved pulmonary prognosis (A455E), one can argue that marked clinical benefit might be possible without any improvement whatsoever in airway bioelectric phenotype. Moreover, if genetic modifiers exist that influence the severity of a particular CFTR mutation (e.g., A455E), these may be independent of human airway Cl-secretion in vivo, since we detected minimal Cl--secretory responses in patients with A455E.

Adolescent↗

PubMed1997

Ehlers-Danlos syndrome type VI results from a nonsense mutation and a splice site-mediated exon-skipping mutation in the lysyl hydroxylase gene.

We have characterized a patient with Ehlers-Danlos syndrome type VI as a compound heterozygote for the lysyl hydroxylase (LH) gene, with a pathogenetic mutation in each allele contributing to the very low levels of mRNA and LH activity in his fibroblasts. Amplification of full-length LH cDNAs resulted in normal-sized (2.9-kb) and shortened (2.8-kb) transcripts indicative of two populations of alleles. One allele contained a paternally inherited C1557 to G transition that coded for a premature stop codon (Y511X) and introduced an Nhe I restriction site in exon 14 of the LH gene. The mutation in the other allele was an exon 5 deletion that produced the shortened polymerase chain reaction transcript and generated a premature stop codon at the beginning of exon 7. Sequencing of genomic DNAs spanning exon 5 showed a mutation in the consensus donor splice site at the beginning of intron 5 (gt-->at) in both the proband and his mother. Via reverse transcriptase-polymerase chain reaction, the parents' fibroblasts showed a disproportionately lower level of each mutant allele compared to their normal alleles. This study suggests that the decreased transcription of the LH gene, which may be attributed to the presence of the nonsense mutations, accounts for the LH deficiency, and consequently, this patient's clinical phenotype of Ehlers-Danlos syndrome type VI.

Alleles↗

PubMed1997

Cerebral lipid deposition in aged apolipoprotein-E-deficient mice.

To assess the influence of age and diet on cerebral pathology in mice lacking apolipoprotein E (apoE), four male apoE knockout mice (epsilon -/-), and five male wild-type (epsilon +/+) littermate controls were placed on a high-fat/high-cholesterol diet for 7 weeks beginning at 17 months of age. All four aged knockout mice developed xanthomatous lesions in the brain consisting mostly of crystalline cholesterol clefts, lipid globules, and foam cells. Smaller xanthomas were confined mainly to the choroid plexus and ventral fornix in the roof of the third ventricle, occasionally extending subpially along the choroidal fissure and into the adjacent parenchyma. More advanced xanthomas disrupted adjoining neural tissue in the fornix, hippocampus, and dorsal diencephalon; in one case, over 60% of one telencephalic hemisphere, including nearly the entire neocortex, was obliterated by the lesion. No xanthomas were observed in aged wild-type controls fed the high-fat/high-cholesterol diet. Brains from 42 additional animals, fed only conventional chow, were examined; 3 of 15 aged (15- to 23-month-old) apoE knockout mice developed small choroidal xanthomas. In contrast, no lesions were observed in five young (2- to 4-month-old) apoE knockout mice or in any wild-type controls between the ages of 2 and 23 months. Our findings indicate that disorders of lipid metabolism can induce significant pathological changes in the central nervous system of aged apoE knockout mice, particularly those on a high-fat/high-cholesterol diet. It may be fruitful to seek potential interactions between genetic factors and diet in modulating the risk of Alzheimer's disease and other neurodegenerative disorders in aged humans.

Aging↗

PubMed1996

Intra-arterial infusion of [125I]A beta 1-40 labels amyloid deposits in the aged primate brain in vivo.

Alzheimer's disease is characterized by extracellular amyloid deposits in the brain at both vascular sites (cerebrovascular amyloid, CVA) and within the neuropil (plaques). In the present study we demonstrated that brain amyloid of aged non-human primates is efficiently detected by [125I]A beta in vitro, and assessed the detection of that amyloid in vivo by intravascular infusion of [125I]A beta. Aged squirrel monkeys (Saimiri sciureus) were anesthetized and infused intra-arterially with [125I]A beta, and sacrificed 2 h later. Analysis of the anterior frontal and temporal cortices by autoradiography demonstrated that [125I]A beta was deposited on CVA and that essentially every amyloid deposit which could be detected with thioflavin S or anti-A beta antibodies was also labeled by [125I]A beta. These experiments suggest that intravascular infusion of radiolabeled A beta can be used to detect and image amyloid deposits in the human AD brain.

Aging↗

PubMed1996

Cerebrovascular amyloidosis in squirrel monkeys and rhesus monkeys: apolipoprotein E genotype.

Some neuropathological changes characteristic of aging and Alzheimer's disease (AD) in humans are present also in senescent non-human primates. The human apoE4 allele is associated with an increased risk of developing late-onset familial and sporadic AD. We found that rhesus monkeys and three subspecies of squirrel monkeys are homozygous for apoE phenotype with arginine at positions 112 and 158 as in human apoE4. However, in both species threonine replaces arginine at position 61 of human apoE. It was previously shown that arginine 61 was critical in determining apoE4 lipoprotein distribution in humans.

Alzheimer Disease↗

PubMed1996

Similarities in the age-related hippocampal deposition of periodic acid-schiff-positive granules in the senescence-accelerated mouse P8 and C57BL/6 mouse strains.

With advancing age clusters of abnormal granules positive for periodic acid-Schiff appear in the hippocampus of C57BL/6 (B6) mice and the senescence-accelerated mouse (SAM) P8. The granules can also be visualized with a polyclonal antibody to a 110,000 mol. wt laminin-binding protein and stain specifically with a monoclonal antibody to heparan sulfate proteoglycan. The present study used light and electron-microscopic analysis to compare the staining and morphological properties of these granules in SAM P8 hippocampus with those in B6 hippocampus at different ages. The results of the light-microscopic analysis revealed that granules in SAM P8 and B6 had similar morphology, staining characteristics and distribution patterns, and appeared to have a close association with astrocytic process. The onset of granules in SAM P8 mice (at two to three months of age) was earlier than that observed in B6 mice (at four to six months of age), but the maximum incidence was similar in both strains. Electron-microscopic analysis revealed that the granules in SAM P8 and B6 mice also had a very similar ultrastructure. Granules in both strains were surrounded by a discontinuous membrane and contained mostly crystalline-like, degenerated material. The successive ultrastructural changes from the exterior to interior of the granules suggest that the degenerative process was initiated outside the granules and that degenerative structures migrate inward. Astrocytes and heparan sulfate proteoglycan are closely associated with beta-amyloid deposits in Alzheimer's disease. The presence of astrocyte-associated heparan sulfate proteoglycan-positive material in aged SAM P8 and B6 mice might model age-related alterations in glia function possibly involved in human cerebral amyloidogenesis.

Aging↗

PubMed1996

The expression of a functional, secreted human lysyl hydroxylase in a baculovirus system.

This study reports the expression of functional human lysyl hydroxylase (LH), a post-translational modifying enzyme that catalyzes the hydroxylation of the lysine residues essential for cross-linking in collagen biosynthesis. We have developed a novel baculovirus system for the expression of LH, a protein that exists normally within the lumen of the endoplasmic reticulum, using a powerful baculovirus signal sequence for secretion. The supernatant from Sf9 cells infected with the viral recombinant showed significant LH activity that increased linearly with supernatant concentration, whereas there was no detectable LH activity in the cell pellet. Silver staining of the fractions purified from the active supernatant by concanavalin A Sepharose chromatography and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrated an 85-kDa protein (the expected size of the LH subunit) that was most prominent in those fractions with the highest LH activity. N-terminal amino acid sequencing verified that the N-terminal primary structure of this 85-kDa protein was identical to human LH. Moreover, the activity of the expressed protein was shown to be dependent on the presence of Fe++, ascorbate, and alpha-ketoglutarate, three essential cofactors for LH activity. We have therefore successfully developed a novel expression system that produces functional human LH and enables this normally nonsecretory enzyme to be secreted, facilitating its separation from the intracellular proteins of insect cells. Future applications should allow characterization of the LH active site by crystallographic studies and site-directed mutagenesis for structure-function comparison.

Amino Acid Sequence↗

PubMed1996

Cystatin C. Icelandic-like mutation in an animal model of cerebrovascular beta-amyloidosis.

BACKGROUND AND PURPOSE: Cerebral amyloid angiopathy (CAA) occurs as a sporadic disorder in aged humans, as a frequent component of Alzheimer's disease, or in hereditary cerebral hemorrhage with amyloidosis (HCHWA). The primary histological locus of cerebral amyloid deposition varies in aged humans and in different species of nonhuman primates. In aged rhesus monkeys, amyloid deposition occurs most frequently in senile plaques, whereas in aged squirrel monkeys CAA is more common. We hypothesized that the preponderance of CAA in squirrel monkeys is related to a species-specific amino acid change in cystatin C, a cysteine protease inhibitor, similar to the Leu68Gln substitution found in the amyloid protein of Icelandic patients with HCHWA-I, also termed hereditary cystatin C amyloid angiopathy. METHODS: We performed immunohistochemical analyses of brain sections of aged squirrel and rhesus monkeys with anti-amyloid-beta and anti-cystatin C antibodies and sequenced the cystatin C cDNA of these monkeys. RESULTS: Cerebral amyloid in aged squirrel and rhesus monkeys, previously shown to be immunoreactive with anti-amyloid-beta anti-bodies, reacts also with antibodies to cystatin C. While the predicted amino acid sequence in rhesus monkeys differs from the human sequence by four residues, that of the squirrel monkeys has seven additional amino acid substitutions, one of which is Leu68Met. CONCLUSIONS: The presence of a mutation in squirrel monkeys similar to the one found in HCHWA-I suggests that alterations in cystatin C may influence the likelihood that amyloid will be deposited in the walls of cerebral blood vessels. These observations support the utilization of the monkeys as models to study CAA.

Amino Acid Sequence↗

PubMed1995

The mRNA and the activity of lysyl hydroxylase are up-regulated by the administration of ascorbate and hydralazine to human skin fibroblasts from a patient with Ehlers-Danlos syndrome type VI.

Lysyl hydroxylase (LH) catalyzes the formation of hydroxylysine required for the intermolecular cross-linking of collagen, which is an essential step in collagen biosynthesis. Dermal fibroblasts from patients with Ehlers-Danlos Syndrome type VI (EDS VI), an inherited connective tissue disorder, express decreased levels of LH activity. In the present study we have shown that both the mRNA and the enzyme activity of LH in skin fibroblasts from one EDS VI patient (AT750), a compound heterozygote for the LH gene, are increased by administration of ascorbate and hydralazine, either individually or in combination. Although the AT750 cells express only 24% of the LH activity found in normal cells, a similar fold increase in activities in both EDS VI and normal cells was observed following treatment with ascorbate (1.5- to 2-fold) and hydralazine (2- to 4-fold), which paralleled the increase in their steady state LH mRNAs. Ascorbate increased total collagen production by 2-fold from a similar basal level of collagen synthesis in each cell type. This was confirmed by protein gel analysis which showed increases in pro alpha 1(I), pro alpha 2(I), and pro alpha 1(III) collagen chains in both normal and EDS VI cells. This ascorbate-mediated increase of alpha 1(I) collagen resulted from increased mRNAs for alpha 1(I) collagen in both cell types. Hydralazine treatment, with or without ascorbate, severely decreased the alpha 1(I) collagen mRNAs in fibroblasts from both AT750 and the normal donor; total collagen synthesis was similarly reduced. This study shows that LH activity, which is severely deficient in fibroblasts from an EDS VI patient, can be upregulated by administration of ascorbate and hydralazine as a result of the increased mRNA for LH, suggesting that the mechanism for the regulation of the LH gene is functioning normally in this patient.

Ascorbic Acid↗

PubMed1995

Opioid precursor gene expression in the human hypothalamus.

Using in situ hybridization histochemistry, we studied the distribution of neurons that express preproopiomelanocortin (pre-POMC), preprodynorphin (pre-PDYN), and preproenkephalin (pre-PENK) gene transcripts within the human hypothalamus and surrounding structures. Of the three opioid systems, pre-POMC neurons have the most restricted distribution. Pre-POMC cells are most numerous in the infundibular nucleus and retrochiasmatic area of the mediobasal hypothalamus; a few labeled cells are present within the boundaries of the ventromedial nucleus and infundibular stalk. Pre-POMC message was not found in the limited samples of structures adjacent to the hypothalamus. In contrast to neurons that express pre-POMC, neurons expressing pre-PDYN and pre-PENK are more widely represented throughout the hypothalamus and extrahypothalamic structures. However, pre-PDYN and pre-PENK cells differ from one another in distribution. Pre-PDYN message is especially abundant in neurons of the tuberal and mammillary regions, with a distinct population of labeled cells in the premammillary nucleus and dorsal posterior hypothalamus. Pre-PDYN gene expression also is found in neurons of the dorsomedial nucleus, ventromedial nucleus, caudal magnocellular portion of the paraventricular nucleus, dorsolateral supraoptic nucleus, tuberomammillary nucleus, caudal lateral hypothalamus, and retrochiasmatic area. In structures immediately adjacent to the hypothalamus, pre-PDYN neurons were observed in the caudate nucleus, putamen, cortical nucleus of the amygdala, and bed nucleus of the stria terminalis. Pre-PENK neurons occur in varying numbers in all hypothalamic nuclei except the mammillary bodies. The chiasmatic region is particularly rich in pre-PENK neurons, with the highest packing density in the intermediate nucleus [the intermediate nucleus (Braak and Braak [1987] Anat. Embryol. 176:315-330) has also been termed the sexually dimorphic nucleus of the preoptic area (SDA-POA; Swaab and Fliers [1985] Science 228:1112-1115) or the interstitial nucleus of the anterior hypothalamus 1 (Allen et al. [1989] J. Neurosci. 9:497-506)], dorsal suprachiasmatic nucleus, medial preoptic area, and rostral lateral hypothalamic area. Pre-PENK neurons are numerous in the infundibular nucleus, ventromedial nucleus, dorsomedial nucleus, caudal parvicellular portion of the paraventricular nucleus, tuberomammillary nucleus, lateral hypothalamus, and retrochiasmatic area. Only a few lightly labeled cells were found in the periphery of the supraoptic nucleus and lateral tuberal nucleus. In areas adjacent to the hypothalamus, cells that contain pre-PENK message occur in the nucleus basalis of Meynert, central nucleus of amygdala, bed nucleus of the stria terminalis, caudate nucleus, and putamen.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗

PubMed1995

Cationic lipids for reporter gene and CFTR transfer to rat pulmonary epithelium.

Increasing evidence indicates that cationic liposomes are capable of safely transferring foreign genes to pulmonary epithelium in vitro and in vivo. To transfer reporter genes and the cystic fibrosis transmembrane conductance regulator (CFTR) to mammalian respiratory epithelium we used two cationic lipid formulations: N-[1-(2,3-dioleoyloxy)propyl] N,N,N-triethylammonium chloride (DOTMA), and 1,2-dimyristyloxy-propyl-3-dimethylhydroxyethylammonium bromide (DMRIE) at a 1:1 molar ratio with dioleoyl phosphatidylethanolamine (DOPE). Lipid-DNA conjugates containing either CFTR or LacZ were instilled directly into the airways of Sprague-Dawley rats. Rats treated with LacZ cDNA in vivo demonstrated expression in 30-50% of the large and medium-sized airways, with some airways showing high efficiency gene transfer and expression (in the most proximal airways, 70-80% of surface epithelial cells were positive for expression of a nuclear targeted LacZ). While control and LacZ treated tracheas mounted in Ussing chambers showed minimal stimulation of transepithelial chloride (Cl)-currents by cAMP (suggesting low levels of endogenous rat CFTR activity), tracheas taken from animals receiving CFTR exhibited significant forskolin-stimulated currents at 72 h after gene transfer. Human CFTR gene expression was also detected by polymerase chain reaction (PCR) analysis of reverse transcribed lung RNA. These results, together with previous studies using lipid-mediated gene transfer in mice, help confirm the potential for cationic lipid-mediated gene transfer in the gene therapy of cystic fibrosis in humans.

Animals↗