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Biomedical subjects

L C Walker

Publications and source records attributed to L C Walker.

At least 55 records · Page 3Linked to original sources
PubMed1995

Cellular and molecular biology of Alzheimer's disease and animal models.

Extraordinary progress has been made in understanding the neuronal populations at risk in AD and some of the mechanisms of cytoskeletal pathology; however, most recent research has focused on the mechanisms of amyloidogenesis and the processing of APP and A beta in in vitro and in vivo systems, including aged nonhuman primates and transgenic mice. Over the next several years, we anticipate that transgenic mice recapitulating many of the features of AD will be produced and that these animals will be very useful for defining the etiologies of this disease, for delineating mechanisms of cell dysfunction and death, and for testing novel therapies. Finally, because several other genetic risk factors, including apoE and chromosome 19 and a locus on chromosome 14, have been identified, it is anticipated that future studies will focus on the role of the products of these genes on the cytoskeleton and APP processing, which will represent exciting and challenging new areas of research.

Aged↗
PubMed1994

Minimum structural requirements for minoxidil inhibition of lysyl hydroxylase in cultured fibroblasts.

The structural features of minoxidil contributing to its inhibitory effect on lysyl hydroxylase in cultured fibroblasts were investigated. Minoxidil analogs in which the pyrimidine ring (two nitrogens) was replaced with a pyridine ring (one nitrogen) or a sym-triazine ring (three nitrogens) suppressed lysyl hydroxylase activity without affecting prolyl hydroxylase activity, as did an analog in which the piperidine ring was replaced with an N,N-diethyl group. By contrast, minoxidil analogs bearing an N-monoalkyl (ethyl or butyl) group in place of the piperidine substituent failed to suppress lysyl hydroxylase activity. The results indicate that nitroxides of pyridine and triazine, in addition to pyrimidine, having an ortho amino group can act as specific inhibitors of lysyl hydroxylase in the cell. The minimum structural requirement for inhibitory activity appears to be an organic moiety containing a tertiary nitrogen para to the nitroxide oxygen, a condition that is best fulfilled by the piperidine ring in minoxidil. Hydroxylation of minoxidil at the 3 or 4 position of the piperidine ring had no impact on its ability to inhibit the post-translational hydroxylation of lysine during collagen biosynthesis. Fibroblasts treated with minoxidil, 3'-hydroxyminoxidil, or 4'-hydroxyminoxidil synthesized a collagen specifically deficient in hydroxylysine by approximately 70%, which completely accounted for the diminished lysyl hydroxylase activity. The percentage of total proteins synthesized as collagen was reduced minimally by minoxidil but not by 3'- or 4'-hydroxyminoxidil. The studies offer a potential means for therapeutic intervention of excessive collagen deposition during fibrosis, using minoxidil or preferably its hydroxy derivatives to limit the supply of hydroxylysine for collagen crosslink formation.

Cell Line↗
PubMed1994

Regulation of lysyl oxidase mRNA in dermal fibroblasts from normal donors and patients with inherited connective tissue disorders.

Lysyl oxidase (LO) is an extracellular copper-dependent enzyme that catalyzes the initial reaction in the formation of lysine or hydroxylysine-derived crosslinks during collagen biosynthesis. We have isolated a cDNA for human LO from skin fibroblast poly(A+)RNA by PCR using primers based on the recently published sequence of human LO. This cDNA probe detects a major mRNA of 4.2 kb on Northern blots of RNA from normal fibroblasts. The level of LO mRNA was not significantly affected by cell density or by ascorbate treatment. Treatment of skin fibroblasts with hydralazine (50 microM), which increases the mRNAs for both the alpha and the beta subunits of prolyl hydroxylase (PH) and the mRNAs for lysyl hydroxylase, also increased LO mRNA by fourfold over a 72-h time course. In contrast, hydralazine dramatically decreased the mRNAs for alpha 1(I) collagen. Administration of minoxidil (500 microM), which specifically decreases LH activity without affecting PH activity or collagen biosynthesis in skin fibroblasts, stimulated the level of LO mRNA. Neither the administration of penicillamine (100 microM), which interferes with collagen cross-linking, nor the administration of beta-aminopropionitrile, which is a strong irreversible inhibitor of LO, to fibroblasts significantly changed the levels of LO mRNA over a 72-h time course. However, bleomycin (0.6 microgram/ml) significantly decreased the 4.2-kb LO mRNA in contrast to the levels of the alpha 1(I) collagen mRNAs, which were unchanged. No significant change was observed in the steady-state levels of LO mRNAs in fibroblasts isolated from patients with certain connective tissue disorders, including Marfan syndrome, Menkes disease, cutis laxa, and pseudoxanthoma elasticum.

Aminopropionitrile↗
PubMed1994

Age-related deposition of glia-associated fibrillar material in brains of C57BL/6 mice.

With advancing age, clusters of unusual granules appear in the brains of C57BL/6 (B6) mice. At the light, confocal laser and electron microscopic levels, the granules represent aggregations of fibrillar material often associated with astrocytes. The fibrillar material is largely free of normal organelles and has been located within astrocytic somata and processes, although in many cases the material is found in the neuropil and is surrounded by a discontinuous membrane. The deposits occur predominantly in hippocampus, but also in piriform cortex, cerebellum and less frequently in some other brain regions. They become evident about six months of age and increase markedly in both number and size thereafter. Incidence of the deposits varies greatly among inbred mouse strains. At six to 12 months of age, granules are abundant in male and female B6, and are absent in BALB/c, CBA, DBA/2 and A mice. In hybrid strains with a B6 background the deposits are also present and thus appear to manifest dominant genetic heritability. Similar granular structures have been described in adult brains of the senescence accelerated mouse and have been noted, albeit very rarely, in aged mice from other strains. While immunostaining of the granules with several polyclonal antisera was found by preabsorption with antigens to be non-specific, immunolabeling with monoclonal antibodies to heparan sulfate proteoglycan core protein and to laminin suggest these or related molecules as components of the fibrillar material. The presence of glycosaminoglycans is supported by staining with periodic acid-Schiff and Gomori's methenamine silver methods. The functional significance of the murine deposits is not yet clear. The deposits do not represent senile plaques with beta-amyloid deposition, but they might mimic the deposition of extracellular matrix molecules that is hypothesized to be a precursor condition for plaque formation and cerebral amyloidosis. Furthermore, the genetic differences in the incidence of the fibrillar deposits has potential to model aspects of familial neurodegenerative diseases.

Aging↗
PubMed1994

Labeling of cerebral amyloid in vivo with a monoclonal antibody.

We assessed the ability of a murine monoclonal antibody to bind selectively to beta-amyloid in the brains of living nonhuman primates. To circumvent the blood-brain barrier, we injected unlabeled antibody 10D5 (murine whole IgG1 and/or Fab fragments) into the cerebrospinal fluid of the cisterna magna in three aged monkeys. A control animal was given an intracisternal injection of nonimmune mouse whole IgG plus Fab. Twenty-four hours later, the animals were perfused and prepared for immunohistochemical detection of bound murine immunoglobulin in brain. All three experimental animals showed selective binding of 10D5 to approximately 5-15% of amyloid deposits in cerebral cortex, primarily near the cortical surface. There was no labeling in the control animal. In vivo-labeled deposits were confirmed to be beta-amyloid by electron microscopy and by in vitro immunohistochemistry in adjacent sections. The animals tolerated the injection well, although some polymorphonuclear leukocytes infiltrated portions of the subarachnoid space and superficial neocortex. These results provide the first demonstration that it may be feasible to selectively direct a tagged monoclonal antibody to beta-amyloid in the brain for therapeutic or diagnostic purposes. With enhancement of labeling efficiency, the method also may be useful for studying the progression of beta-amyloidosis in experimental animals using emission tomography.

Amyloid beta-Peptides↗
PubMed1993

Vasopressin and oxytocin gene expression in the human hypothalamus.

We studied the distribution of messenger ribonucleic acids coding for vasopressin and oxytocin in the human hypothalamus by means of hybridization histochemistry. Numerous large and medium-sized neurons contain vasopressin messenger ribonucleic acid in the paraventricular nucleus, supraoptic nucleus, and accessory magnocellular nucleus. Small, lightly labeled vasopressin neurons also were detected in the suprachiasmatic nucleus. In addition, a relatively sparse band of mostly ovoid, medium-sized vasopressin neurons mingle with unlabeled neurons of the lateral hypothalamic area; these cells extend dorsoventrally from the region ventral to the stria terminalis to the ventrolateral hypothalamus, sometimes transgressing the boundaries of nearby nuclei. We did not detect vasopressin gene expression in neurons of the bed nucleus of the stria terminalis proper, although some of the dorsal-most labeled neurons of the lateral hypothalamus extend into the region of the caudal bed nucleus. Some lateral hypothalamic neurons also encroach upon other extrahypothalamic structures, such as the zona incerta. The nucleus basalis of Meynert complex was, with only rare exceptions, devoid of cells containing vasopressin messenger ribonucleic acid. Oxytocin messenger ribonucleic acid is found in the supraoptic nucleus, paraventricular nucleus, accessory magnocellular nucleus and, less frequently, in neurons of the lateral hypothalamus. In the hypothalamic magnocellular nuclei, oxytocin neurons are somewhat smaller than vasopressin neurons. Vasopressin cells outnumber oxytocin cells in the supraoptic nucleus, but their numbers are comparable in the paraventricular nucleus. As with vasopressin neurons, lateral hypothalamic oxytocin cells loosely span several diencephalic nuclei and encroach occasionally upon adjacent regions. These results confirm that the organization of vasopressin and oxytocin neurons in the human hypothalamus is largely comparable to that in nonhuman species and demonstrate the utility of hybridization histochemistry for elucidating the chemoarchitecture of the human brain.

Adolescent↗
PubMed1993

The age of biosenescence and the incidence of cerebral beta-amyloidosis in aged captive rhesus monkeys.

Autopsy surveillance of 186 rhesus monkeys aged 20 to 36 years revealed that development of major geriatric diseases such as emphysema, coronary sclerosis, and cancer increased rapidly after the age of 25 years, and nearly 70% of the monkeys in each cohort group died by 30 years. According to our 12-year longitudinal survey, the age of biosenescence in captive rhesus monkeys begins around 25 years and the maximum longevity is 36 years. The incidence of cerebral beta-amyloidosis associated with plaque formation and cerebral angiopathy was observed in 51 brains of rhesus monkeys aged 25 to 36 years. Lesions were found in 31 of 51 aged brains (60%) and 6 monkeys over 34 years of age were all severely affected. Despite the size of the plaque, nearly all of them showed immunopositive beta-amyloid. Cerebral angiopathy coexisted in 10 of 31 plaque-positive brains. The basal prefrontal gyrus was the most common site and contained the highest density of plaques, followed by the amygdala region. The amyloid in the liver, spleen, adrenal and pancreatic islets in visceral amyloidosis showed no positivity to the beta-amyloid demonstrated in the brain. As in aged human brains, the incidence of age-dependent cerebral beta-amyloidosis in captive rhesus monkeys showed great individual variation.

Aging↗
PubMed1993

Localization of a laminin-binding protein in brain.

A 110,000 mol.wt laminin-binding protein from newborn mouse brain recognizes a neurite promoting laminin A chain site and is related to the beta-amyloid precursor protein. In the present study, we examined the expression of 110,000 mol.wt laminin-binding protein in brains of adult mice, rats, and non-human primates. Essentially identical immunoreactivities were observed across species with distinct staining of cortical pyramidal neurons with apical dendrites, cerebellar basket cell axons, hippocampal mossy fibers, and fine labeling of processes throughout the brain. Colocalization of immunoreactivities to 110,000 mol.wt laminin-binding protein and to laminin in neurons of the adult rat brain was observed. Electron microscopy demonstrated that 110,000 mol.wt laminin-binding protein-like immunoreactivity is intracellular and is possibly associated with the neuronal cytoskeleton. Western blot analysis revealed that anti-110,000 mol.wt laminin-binding protein also recognizes a 140,000 mol.wt protein in the pellet, in addition to the 110,000 mol.wt protein in the Triton soluble extract. Antibody fractions specific to the two reactive protein species (110,000 mol.wt and 140,000 mol.wt) exhibited cross-reactivity on immunoblots and revealed similar immunohistochemical staining in adult brain. Results suggest a significant interaction between laminin-like molecules and 110,000 mol.wt laminin-binding protein-like molecules in normal brain function, in response to CNS injury and possibly in the pathogenesis of Alzheimer's disease.

Amino Acid Sequence↗
PubMed1993

Age-dependent impairment of mitochondrial function in primate brain.

It has been hypothesized that some of the functional impairments associated with aging are the result of increasing oxidative damage to mitochondrial DNA that produces defects in oxidative phosphorylation. To test this hypothesis, we examined the enzymes that catalyze oxidative phosphorylation in crude mitochondrial preparations from frontoparietal cortex of 20 rhesus monkeys (5-34 years old). Samples were assayed for complex I, complex II-III, complex IV, complex V, and citrate synthase activities. When enzyme activities were corrected for citrate synthase activities (to account for variable degrees of mitochondrial enrichment), linear regression analysis demonstrated a significant negative correlation of the activities of complex I (p < 0.002) and complex IV (p < 0.03) with age but no significant change in complex II-III or complex V activities. Relative to animals 6.9 +/- 0.9 years old (n = 7), the citrate synthase-corrected activity of complex I was reduced by 17% in animals 22.5 +/- 0.9 years old (n = 6) (p < 0.05) and by 22% in animals 30.7 +/- 0.9 years old (n = 7) (p < 0.01). Similar age-related reductions in the activities of complexes I and IV were obtained when enzyme activities were corrected for complex II-III activity. These findings show an age-associated progressive impairment of mitochondrial complex I and complex IV activities in cerebral cortices of primates.

Aging↗
PubMed1992

Basal forebrain neurons and memory: a biochemical, histological, and behavioral study of differential vulnerability to ibotenate and quisqualate.

The differential vulnerability of basal forebrain cells to ibotenate (IBO) or quisqualate (QUIS) was investigated in rats. IBO was also coinjected with cystine (CYS) or zinc (Zn). Cortical choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) activity, neurotensin receptors, and high-affinity choline uptake sites were quantified in conjunction with radioimmunoassays for neurotensin, substance P, and somatostatin; immunocytochemistry for neurotensin-, somatostatin-, Leu-enkephalin-, and ChAT-positive cells; and in situ hybridization histochemistry of somatostatin, substance P, and enkephalin mRNAs. Compared with the performance of controls, continuous alternation performance in a T maze of IBO+Zn or IBO+CYS rats was better than that of IBO rats, whereas the performance of QUIS rats was unimpaired. Of those neurotransmitter systems examined, only ChAT-immunoreactive cells were vulnerable to IBO or QUIS. However, cholinergic cell loss did not correlate with impaired performance.

Animals↗
PubMed1992

Characterization of a partial cDNA for lysyl hydroxylase from human skin fibroblasts; lysyl hydroxylase mRNAs are regulated differently by minoxidil derivatives and hydralazine.

Lysyl hydroxylase (LH) is an essential enzyme in collagen biosynthesis that catalyzes the formation of hydroxylysine required for intermolecular crosslinking of collagen. We have isolated a partial (2.2-kb) cDNA for LH from human skin fibroblasts using PCR. DNA sequencing revealed 72% homology of the human coding sequence with the chick LH sequence at the nucleotide level and 76% homology predicted at the amino acid level. The LH cDNA hybridized strongly with two mRNA species of 2.4 and 3.4 kb on Northern blots of normal fibroblast RNA. Administration of minoxidil decreased both mRNA species without affecting levels of the mRNAs for the beta subunit of prolyl 4-hydroxylase (PH) or alpha 1(I) collagen. Two derivatives of minoxidil (3' hydroxyminoxidil and 4' hydroxyminoxidil) produced similar decreases in LH mRNAs. In contrast hydralazine increased the mRNAs for LH in parallel with its previously reported effect on the mRNA for the beta subunit of PH. This effect is accompanied by virtual elimination of the alpha 1(I) collagen mRNAs. These results on the action of minoxidil and hydralazine at the pretranslational level correlate well with their previously reported effect on enzyme activity and collagen biosynthesis and indicate that changes in steady-state mRNA levels can account directly for changes at the protein level. Moreover, the unique action of minoxidil in specifically decreasing LH mRNAs contrasts with the less specific stimulatory effects of hydralazine and suggests that these pharmaceuticals are regulating expression of LH at a pretranslational level by different mechanisms.

Base Sequence↗
PubMed1991

Galanin mRNA in the nucleus basalis of Meynert complex of baboons and humans.

Galanin, a 29-amino acid peptide, has been shown by immunocytochemistry to occur in most large acetylcholinergic neurons of the complex that includes the nucleus basalis of Meynert and the nucleus of the diagonal band of Broca in nonhuman primates. In contrast, several studies have reported that most large neurons of the human nucleus basalis of Meynert complex appear to lack galanin immunoreactivity. We investigated this apparent species-difference by hybridization histochemistry for galanin messenger ribonucleic acid (mRNA) in humans and baboons. The results confirm previous immunocytochemical data; very few large neurons of the nucleus basalis of Meynert complex in humans contained detectable galanin messenger RNA, whereas most such cells in baboons were labeled by the oligodeoxynucleotide probe. The few labeled neurons in humans were primarily medial or ventral to the main body of the nucleus basalis of Meynert and corresponded in location to a minor population of relatively intensely labeled cells in baboons. These findings indicate that the indetectability of immunoreactive galanin in most cells of the nucleus basalis of Meynert complex in humans is due to a paucity or an absence of galanin messenger RNA and not to differences in posttranslational processing or transport of the peptide. Inasmuch as the probe labeled neurons in several other nuclei of both species, it is unlikely that differences in galanin messenger RNA sequences underlie the species-related disparity in hybridization in the nucleus basalis of Meynert complex. The indetectability of galanin messenger RNA in most cells of the human nucleus basalis of Meynert complex indicates that the expression of the galanin gene is regulated by as yet unidentified influences that differ in human and nonhuman primates. The varying phenotypes of galanin in primates suggest potentially important species-differences in the function of galanin in neurons of the nucleus basalis of Meynert complex.

Adolescent↗
PubMed1991

Antiidiotypic responses to immunization with anti-Leu 3a in human immunodeficiency virus-seropositive individuals.

Anti-idiotypic antibodies to anti-CD4 monoclonal antibodies (MAbs) have been reported to bind to the human immunodeficiency virus (HIV) envelope glycoprotein gp120. To establish whether HIV-infected patients can mount an anti-idiotypic response to murine MAb, four individuals with symptoms of AIDS-related complex (ARC) were immunized over 10 weeks with six intramuscular injections of anti-Leu 3a (1 mg). Despite their immunocompromised state, all patients made anti-constant region and anti-idiotypic antibodies, although the amplitude of the responses varied between individuals. No significant toxic or allergic reactions were seen, and there were no changes in clinical status during the 6 months after immunization. No increase in serum HIV-neutralization titers was seen, and purified anti-idiotypic antibodies did not bind gp120.

Adult↗