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J R Connor

Publications and source records attributed to J R Connor.

At least 109 records · Page 6Linked to original sources
PubMed1994

Histological analysis of selected brain regions of hypotransferrinemic mice.

This study utilizes a mutant mouse line (Hp) in which an established essential trophic factor, transferrin (Tf), is deficient due to a splicing defect in the processing of Tf precursor mRNA. As this mouse mutant is new to neurological research, the initial stage of the investigation, histological analysis of the brain and spinal cord, is reported here. Using a number of standard histological stains, such as hematoxylin and eosin, luxol fast blue/cresyl violet and silver staining, we see a decrease in the amount of white matter and neurofilament staining and altered neuronal morphology throughout the brain and spinal cord. Regions in which postnatal development is significant such as the hippocampus and cerebellum are particularly affected in this mutant. The cells of the dentate gyrus and Ammon's horn of the hippocampus are smaller, more densely packed and the normal orderly appearance of the CA3 and CA4 regions of Ammon's horn is disrupted. The cerebellum has a decrease in white matter and the molecular, Purkinje cell and granule cell layers all show decreased silver staining for neurofilament and appear less ordered than normal. The results demonstrate that neurohistological alterations exist in the adult hypotransferrinemic mice despite systemic replacement of transferrin. Furthermore, these data suggest certain brain regions are particularly sensitive to disruption in iron delivery. The results of this initial study indicate the Hp animal may be an interesting model for investigating specific aspects of neural development and has considerable potential for examining the importance of iron regulation in the brain.

Animals↗
PubMed1994

Iron, transferrin, and ferritin in the rat brain during development and aging.

Iron is a universal cofactor for mitochondrial energy generation and supports the growth and differentiation of all cell types. In the CNS, iron is a key component of systems responsible for myelination and the synthesis of several neurotransmitters. In this study the spatial and temporal pattern of iron and its regulatory proteins transferrin and ferritin are quantitatively examined in the rat CNS during the first 3 weeks of postnatal life and in adults and aged animals. The midbrain, the cerebral cortex, and the cerebellum-pons are examined independently. Iron, transferrin, and ferritin concentrations are highest in all three brain regions at birth and decrease in each region to minimum levels during the third postnatal week. The decrease in levels of iron, transferrin, and ferritin is most pronounced in the cerebellum-pons and cortex and least in the midbrain. From postnatal day 17, iron (total iron content) and ferritin levels increase throughout the lifetime of the rat. In contrast, transferrin levels remain fairly constant in each brain region after postnatal day 24. The midbrain region, which includes the iron-rich regions such as the globus pallidus, substantia nigra, and red nucleus, has the least change in iron with development, has the highest level of ferritin during development, and consistently has the highest level of transferrin at all ages. These observations are consistent with reports that iron important for normal motor function.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1994

Iron acquisition and expression of iron regulatory proteins in the developing brain: manipulation by ethanol exposure, iron deprivation and cellular dysfunction.

The World Health Organization considers iron deficiency the number one nutritional disorder in the world. In this review, the normal pattern for iron accumulation and expression of iron regulatory proteins (transferrin and its receptor, and ferritin) in brain during development are examined biochemically and at the cellular and molecular levels. Iron and the iron-regulatory proteins are at their highest postnatal concentration in the brain at birth, decline over the preweaning period and then increase to adult levels. Evidence is presented that in utero exposure to alcohol, iron-deficient diets, and dysfunctional oligodendrocytes can influence the normal pattern for iron accumulation in the brain which sets off a cascade of events that results in loss of regulatory control of iron. Because iron is an essential cofactor in neurotransmitter synthesis and myelination altering iron availability during vulnerable periods of development may have a permanent influence both on iron homeostasis in the brain and motor and cognitive function. At the cellular level, iron-positive cells in the subventricular zone and myelinogenic foci are present as early as postnatal day 3. Disruption of oligodendrocyte maturation is associated with altered expression and cellular accumulation of iron, transferrin and the transferrin receptor in brain. These data indicate that iron delivered via transferrin and its receptor is intrinsically involved in oligodendrocyte maturation and thus plays a critical role in the onset of myelination. In the adult, oligodendrocytes are the predominant iron-regulatory cell in the brain by virtue of their high content of iron, transferrin and ferritin. From these studies we conclude oligodendrocytes may be responsible for iron regulation in the brain at the cellular level and that brain iron regulatory mechanisms are vulnerable to manipulation during postnatal development.

Animals↗
PubMed1993

Ferritin, transferrin, and iron in selected regions of the adult and aged rat brain.

Iron is necessary for normal neural function but it must be stringently regulated to avoid iron-induced oxidative injury. The regulation of systemic iron is through the proteins transferrin (iron mobilization) and ferritin (iron sequestration). This study examines the cellular and regional distribution of iron and the iron-related proteins ferritin and transferrin in selected regions of the adult and aged rat brain. This information is a necessary prerequisite to understanding the mechanism by which iron homeostasis is maintained in the brain. The predominant cell type containing ferritin, transferrin, and iron throughout the brain at all ages is the oligodendrocyte. Neurons in most brain regions contain granular iron deposits which become more apparent with age. Ferritin and iron are also present in microglial cells in all brain regions, but are particularly abundant in the hippocampus. These latter cells visibly increase in number in all brain regions as the animal approaches senescence. Another area in which immunostaining is notable is surrounding the III ventricle, where transferrin is found in the choroid plexus and ependyma and ferritin and iron are present in tanycytes. The results of this study indicate an important role for neuroglia in the regulation of iron in the brain and also implies that a transport system may exist for the transfer of iron between the brain and cerebrospinal fluid. In the normal rodent brain, the principal cell of iron regulation is the oligodendrocyte; however, the role of microglial cells in the sequestration and detoxification of iron may be significant, particularly as the animal ages. With age there is an increase in stainable iron in neurons without a concomitant increase in neuronal ferritin immunostaining, suggesting a ferritin independent accumulation of neuronal iron with age.

Aging↗
PubMed1993

Transferrin in the central nervous system of the shiverer mouse myelin mutant.

Transferrin, the iron mobilization protein, and its mRNA are normally present in oligodendrocytes. Previous reports using myelin mutants have shown both a decrease in transferrin protein and mRNA when the oligodendrocyte population is compromised. In this study the shiverer mouse mutant in which the oligodendrocyte population is numerically normal, but has both quantitatively diminished and qualitatively abnormal myelin was used. This animal model was chosen to address the question whether expression of the transferrin message and/or protein correlated more closely to the number of oligodendrocytes (normal) or the amount of myelin (abnormally low). A 1/2 to 2/3 decrease in transferrin protein occurred in all brain regions examined except for the spinal cord in the shiverer group compared to both heterozygous littermates and wild type controls. Levels of transferrin transcripts in the brain are not affected by the shiverer mutation. These results taken with previous reports from this laboratory indicate that the presence of oligodendrocytes is a requirement for normal expression of transferrin mRNA in brain but is not sufficient for normal values of the protein. The level of Tf protein correlates more closely with the amount of myelin present than it does with the numbers of oligodendrocytes present. These data are consistent with previous reports from our laboratory that transferrin accumulation by oligodendrocytes is associated with myelin production by these cells. These data further suggest transferrin mRNA may be constitutively expressed by oligodendrocytes and that the protein expression is regulated at the level of translation.

Animals↗
PubMed1993

Ceruloplasmin levels in the human superior temporal gyrus in aging and Alzheimer's disease.

In the present study we observed a loss in excess of 1/3 of the copper transport and anti-oxidant protein ceruloplasmin in both the gray and white matter from superior temporal gyrus in Alzheimer's diseased brains compared to age-matched controls. A decrease in ceruloplasmin could be reflected in decreased cellular metabolic processes such as the electron transport system and a decrease in the ability of the brain to protect itself from oxidative damage. Both decreased metabolic activity and an increase in oxidative insults are known to be associated with the neurological events in Alzheimer's disease, but the mechanism by which these phenomena occur are unknown. These results coupled with previous reports from this laboratory on iron regulatory proteins in the brain suggests one way in which cellular dysfunction and oxidative stress occurs in AD may be through a loss of ability to maintain a balance of essential metals.

Aged↗
PubMed1993

Iron in the brain.

The location and function of iron in the central nervous system are reviewed with particular emphasis on human biology. Iron is distributed to different cell types in the brain in a heterogeneous fashion through the action of transferrin, transferrin receptors, and the metabolic needs of those cells. The function of this iron and its storage is documented in states of growth and development as well as during pathological states associated with aging. The information relating this biology to current observations of attention deficits in iron-deficient humans is also reviewed.

Adult↗
PubMed1992

Iron regulation in the brain: histochemical, biochemical, and molecular considerations.

Despite recognition that iron is important for normal neurological function, the proteins involved in maintaining iron homeostasis within the brain have until recently received little attention. In the past few years, studies aimed at determining both general and cellular control of iron in the brain have increased. Histological studies indicate that maintenance of iron homeostasis in the brain is the responsibility of neuroglia and possibly the choroid plexus. Transferrin, the iron transport protein, has been found predominantly in oligodendrocytes in the brain and in myelinating Schwann cells in the peripheral nervous system. The messenger RNA for transferrin is located in the brain in oligodendrocytes and the choroid plexus. Most of the transferrin protein and transcript expression in the brain is dependent on the presence of a mature population of oligodendrocytes. Transferrin is also involved in the transport of iron across the blood-brain barrier via transferrin receptors on brain capillary endothelial cells. The transferrin receptor is also present on cells within the brain. Ferritin, the iron storage protein, and iron are found in the brain in oligodendrocytes and microglia. Additional cells in which iron and ferritin are found are tanycytes, which are associated with the third ventricle. This latter observation raises interesting possibilities regarding the transport of iron from cerebrospinal fluid into the brain. The high iron requirement of the brain coupled with the high susceptibility of the brain to iron-generated peroxidative damage requires stringent regulation of the availability of iron. Consequently, the iron regulatory proteins are central to understanding mechanisms controlling iron-dependent activity at the cellular level, as well as protection of the brain from oxidative damage. The behavior of brain iron regulatory proteins will be a significant factor in future studies of the neurological diseases resulting from brain iron imbalance. We review the contributions of our laboratory to this field over the past 6 years, discuss current projects, and suggest future directions for study.

Animals↗
PubMed1992

Elevated cAMP is required for stimulation of eicosanoid synthesis by interleukin 1 and bradykinin in BALB/c 3T3 fibroblasts.

In Swiss 3T3 murine fibroblasts, interleukin 1 (IL-1) and bradykinin stimulate prostaglandin E2 (PGE2) synthesis. However, in the present study, we found that neither agonist stimulated PGE2 synthesis in BALB/c 3T3 murine fibroblasts, this in spite of expression of similar numbers of receptors for each agonist compared to Swiss 3T3 cells. When BALB/c 3T3 cells were preincubated with cAMP analogs, both IL-1 and bradykinin stimulated PGE2 synthesis to levels similar to those observed in Swiss 3T3 cells. Similarly, when the cells were preincubated with forskolin, which activates the catalytic subunit of adenylate cyclase directly, or NECA, which stimulates cellular cAMP accumulation by activating adenosine receptors, IL-1 and bradykinin stimulated PGE2 synthesis. Rp-cAMPS, an inhibitor of cAMP-dependent protein kinase, blocked the ability of cAMP or NECA to render cells responsive to IL-1 and bradykinin. In basal BALB/c 3T3 cells, bradykinin and IL-1 stimulated arachidonate release in the absence of cAMP, but little conversion of released arachidonate to PGE2 occurred. cAMP, forskolin, and NECA all increased cyclooxygenase activity in the cells. SV-T2 is a clonal line originating from BALB/c 3T3 transformed with SV-40. In these cells, IL-1 and bradykinin stimulated PGE2 synthesis despite basal intracellular cAMP concentrations similar to BALB/c, and cAMP only modestly potentiated the response. In summary, cyclooxygenase expression appears to be regulated by cAMP in BALB/c 3T3 cells, and SV-40 transformation results in increased cyclooxygenase expression, apparently independent of cAMP.

3T3 Cells↗
PubMed1992

A histochemical study of iron, transferrin, and ferritin in Alzheimer's diseased brains.

Immunohistochemical and histochemical staining were performed on Alzheimer's diseased brain tissue obtained at autopsy. The iron-regulatory proteins transferrin and ferritin as well as iron are, in general, found predominantly in oligodendrocytes similar to that previously reported for normal brain tissue. However, in the vicinity of senile plaques, the staining pattern is altered for both proteins and iron. Transferrin is homogenously distributed around the senile plaques and is apparently extracellular. In addition, transferrin is found in astrocytes in the cerebral cortical white matter of the Alzheimer's tissue rather than its normal distribution in oligodendrocytes. A robust ferritin immunoreaction accompanies senile plaques and many blood vessels in the Alzheimer's brain tissue. Although many ferritin-positive oligodendrocytes are present in the Alzheimer's tissue, most of the ferritin-containing cells associated with senile plaques and blood vessels are microglia. Iron can also be demonstrated in the senile plaques. The iron reaction product is observed both diffusely in proximity of the plaques and in cells associated with the plaques. These data strongly suggest a disruption in brain iron homeostasis in Alzheimer's disease as demonstrated by alterations in the normal cellular distribution of iron and the proteins responsible for iron regulation. These data will contribute to understanding both the potential for oxidative damage and the potential for metal neurotoxicity in Alzheimer's disease.

Alzheimer Disease↗
PubMed1992

Regional distribution of iron and iron-regulatory proteins in the brain in aging and Alzheimer's disease.

It is well established that iron, which is of considerable importance for normal neurological function, is highly regulated in all organ systems. However, until recently, iron regulation in the nervous system has received little attention. This study quantitatively compares the levels of the major iron-regulatory proteins, transferrin and ferritin, and iron itself in three cerebral cortical regions of the human brain from material collected at autopsy. Three groups were studied: 1) normal adult (under 65 yr of age), 2) aged (greater than 65), and 3) Alzheimer's disease. Normally, transferrin is more abundant in white matter than in gray matter. Ferritin is approximately 10x more abundant than transferrin throughout the brain regions examined and is evenly distributed, as is iron, in the gray and white matter. In Alzheimer's disease transferrin is consistently decreased particularly in the white matter of the various cerebral cortical regions examined whereas the iron and ferritin changes are inconsistent. The observations in this study are consistent with our general hypothesis that iron homeostasis is disrupted in the aging brain and the alterations in iron-regulatory proteins are exacerbated in Alzheimer's disease. The decrease in transferrin levels could indicate a decreased mobility and subsequent utilization of iron in the brain. Such a decrease in iron availability could play a significant role in neuronal degeneration and increased peroxidative damage known to occur in Alzheimer's disease.

Adult↗
PubMed1992

Transferrin receptor expression in myelin deficient (md) rats.

The question of iron regulation in the brain is the subject of increasing interest as the evidence continues to accumulate that a loss of brain iron homeostasis plays a significant role in some neurodegenerative diseases. Most cells acquire iron through a specific receptor mediated process involving transferrin, the iron mobilization protein. It appears that in the brain, endothelial cells, neurons, and oligodendrocytes express the transferrin receptor. This study uses a strain of rats (myelin deficient, md) in which oligodendrocytes fail to mature, and examines the consequences of this genetic defect on the expression of the transferrin receptor in the brain. The affinity of transferrin for its receptor is similar between the cerebral cortex and cerebellum in both the normal and myelin deficient rats (Kd = 7.8-10.6 nM). The transferrin receptor density is normally 2-3 times higher in the cerebellum than in the cerebral cortex. In the myelin deficient rat strain, the density of the transferrin receptor is decreased in both the cerebrum (56%) and cerebellum (70%) compared to the littermate control animals. Because oligodendrocytes are the only cell type affected in this mutant, the results suggest that these cells are responsible for a considerable amount of the transferrin receptors that are expressed in the brain (excluding the endothelial cell contributions). These observations are consistent with the existing literature stating that oligodendrocytes are responsible for the majority of transferrin and transferrin mRNA which is expressed in the brain, and support the working hypothesis that imbalances in brain iron homeostasis, particularly during development, are associated with myelin disorders.

Animals↗
PubMed1992

NPC 15669 inhibits the reversed passive Arthus reaction in rats by blocking neutrophil recruitment.

NPC 15669, N-carboxy-L-leucine,N-[(2,7-dimethylfluoren-9-yl)methyl]ester, has been shown to inhibit several inflammatory reactions that depend upon recruitment of neutrophils into the primary lesion. In the present study we examined the effects of NPC 15669 in the reversed passive Arthus reaction, an inflammatory reaction occurring in the skin of rats in response to intracutaneous injection of antigen followed by intravenous administration of antibody. In this model, immune complex formation activates complement, resulting in rapid recruitment of neutrophils to the site, which releases free radicals and proteases that damage capillaries, resulting in plasma leak. NPC 15669 inhibited the increased capillary permeability occurring in the reversed passive Arthus reaction in a dose-dependent manner, with an ED50 of 4 mg/kg. The agent similarly inhibited the recruitment of radiolabeled neutrophils as well as the accumulation of myeloperoxidase, a neutrophil marker. NPC 15669 in vitro inhibited the adherence of formyl-L-Met-L-Leu-L-Phe- or human recombinant C5a-activated neutrophils to endothelium, with IC50 values of 15 to 30 microM (ca. 4-9 micrograms/ml). Measurement of plasma NPC 15669 showed that at the ED50 dose, the average circulating concentration of drug was 5 micrograms/ml, consistent with the hypothesis that NPC 15669 exerts its anti-inflammatory effects by inhibiting neutrophil adherence to endothelium and recruitment into the inflammatory lesion.

Animals↗
PubMed1992

2,6-Diamino-N-([1-(1-oxotridecyl)-2-piperidinyl] methyl)hexanamide (NPC 15437): a novel inhibitor of protein kinase C interacting at the regulatory domain.

NPC 15437 is a prototype member of a new class of synthetically derived protein kinase C (PKC) inhibitors. PKC activity and binding of phorbol ester to the enzyme were inhibited by NPC 15437, with IC50 values of 19 +/- 2 microM and 23 +/- 4 microM, respectively. No inhibition of cAMP-dependent or calcium/calmodulin-dependent protein kinases was observed at concentrations of NPC 15437 up to 300 microM. To investigate the mechanism by which NPC 15437 exerts its effects, a kinetic analysis of the inhibition with respect to three activators of the enzyme, phosphatidylserine, calcium, and phorbol ester, was performed. NPC 15437 was a competitive inhibitor of the activation of PKC by phorbol ester (Ki = 5 +/- 3 microM). Stimulation of PKC alpha by phosphatidylserine was competitively inhibited by NPC 15437 (Ki = 12 +/- 4 microM). The inhibition was mixed with respect to activation by calcium. These results suggest that NPC 15437 is a selective inhibitor of PKC, interacting at the regulatory region of the enzyme. NPC 15437 inhibited PKC in intact cells, dose-dependently antagonizing the phorbol ester-induced phosphorylation of a 47-kDa protein in human platelets.

Animals↗
PubMed1991

NPC 15437 interacts with the C1 domain of protein kinase C. An analysis using mutant PKC constructs.

We recently demonstrated that 2,6,diamino-N-[( 1-(oxotridecyl)-2-piperidinyl]methyl)-hexanamide (NPC 15437) is a selective inhibitor of PKC interacting at the regulatory domain of the enzyme. To further investigate the interaction of NPC 15437 with PKC we expressed a series of cDNAs encoding mutant PKC molecules in COS7 cells. NPC 15437 had no effect on the protein kinase activity of mutants lacking the N-terminal region of the C1 domain. Further, NPC 15437 was a competitive inhibitor of the activation of PKC alpha by phorbol ester and attenuated the binding of phorbol ester to the enzyme in intact cells. The present study demonstrates that mutant enzyme constructs can be used to localize the site of interaction of NPC 15437 with PKC to residues 12-42, which encodes the pseudosubstrate binding domain and part of the first cysteine-rich repeat sequence.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

N-(fluorenyl-9-methoxycarbonyl) amino acids, a class of antiinflammatory agents with a different mechanism of action.

Several members of a series of N-(fluorenyl-9-methoxycarbonyl) amino acids were found to possess a broad spectrum of antiinflammatory activity. The compounds were active against oxazolone dermatitis in mice and adjuvant arthritis in rats, models in which activated T lymphocytes are implicated. The compounds also inhibited T-lymphocyte activation in vitro, assessed by using the mixed lymphocyte reaction. The compounds inhibited the reversed passive Arthus reaction in rats and arachidonic acid-induced dermatitis in mice, models in which leukocyte infiltration is responsible for the inflammatory reaction. More complete evaluation was made of one compound, N-(fluorenyl-9-methoxycarbonyl)leucine (NPC 15199). On histologic examination after arachidonic acid administration, NPC 15199 was found to block recruitment of neutrophils into the inflammatory site. The compound was not a general myelotoxin. Prolonged treatment of animals did not alter bone-marrow progenitor number or the numbers of circulating white blood cells. Further, several white cell functions were not inhibited in vitro, including neutrophil respiratory burst and macrophage phagocytosis. NPC 15199 was effective in blocking antigen arthritis in rabbits and was effective in a therapeutic protocol, reversing oxazolone edema. These studies suggest that N-(fluorenyl-9-methoxycarbonyl) amino acids may be valuable therapeutic agents for inflammatory diseases.

Amino Acids↗
PubMed1991

2,6-Diamino-N-([1-oxotridecyl)-2-piperidinyl]methyl)hexanamide (NPC 15437): a selective inhibitor of protein kinase C.

NPC 15437 inhibited protein kinase C (PKC) activity and [3H]phorbol 12,13-dibutyrate (PDBu) binding to the enzyme in a concentration-dependent manner (IC50 values, 19 +/- 2 microM and 23 +/- 4 microM, respectively). No inhibition of cAMP-dependent protein kinase A (PKA) or calcium/calmodulin-dependent myosin light chain kinase (MLCK) was observed. A detailed kinetic analysis of the interaction of NPC 15437 and a homogeneous preparation of PKC-alpha revealed a competitive type of inhibition with respect to activation of the enzyme by both phorbol 12-myristate 13-acetate (PMA) (Ki = 5 +/- 3 microM) and phosphatidylserine (PS) (Ki = 12 +/- 4 microM). Mixed inhibition (predominantly of the non-competitive type), with respect to activation of the enzyme by calcium, was also observed. These studies indicate that NPC 15437 is a selective inhibitor of PKC, interacting at the regulatory region of the molecule. NPC 15437 inhibited phorbol ester-induced ear edema in mouse (IC50 = 175 micrograms/ear) demonstrating the ability of NPC 15437 to inhibit PKC-mediated activity in intact cells.

Animals↗