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Apolipoprotein AI is the major protein component of high-density lipoprotein (HDL) particles in plasma. Inflammatory cytokines such as TNF and IL-1β repress the production of ApoA-I from hepatocytes and increase the expression of serum amyloid A (SAA), which becomes the major protein component of HDL in this context. Consequently, lipid-poor ApoA-I is rapidly catabolized in the liver and the kidney. These findings could be meaningful if ApoA-I, in addition to its proimmune features, had anti-inflammatory potential. In this way, removal of ApoA-I could intensify the inflammatory response, resulting in a more robust effect. Recent studies have suggested that serum apolipoprotein A1 maybe a predictor and prognostic biomarker in acute ischemic stroke in humans. To understand the impact of ApoA-I in stroke, we have begun to analyze the role of ApoA-I in mouse models of stroke. Mice with reduced or deficient in ApoA-I and subjected to ischemia and reperfusion injury or permanent ischemia, had increased inflammatory responses, larger infarct volumes, and poorer outcomes than wildtype mice. Mice transgenic for the human ApoA-I gene showed a level of protection not seen in the wildtype mice when given an ischemic injury. Administration of AAV human ApoA-I increased HDL levels in deficient mice and provided protection against ischemic injury. Additionally, drugs to enhance ApoA-I expression showed protection against strokes in the mouse models. These studies, along with subsequent investigations will provide a better understanding and potential therapeutic approaches in the field of stroke.

•A 3-month high-sodium diet causes pyknosis and deep staining in hippocampal neurons of APP/PS1.•In APP/PS1 with high-sodium diet we observed a lower vascular density (p<0.001) in both hippocampal and cortical areas.•As a result of 3-month high-sodium diet cerebral perfusion of APP/PS1 was not increased markedly (p = 0.3), though it was increased in wild-type controls (p = 0.022).

Transcranial laser therapy (TLT) using a near-infrared energy laser system was tested in the 2x Tg amyloid precursor protein (APP) mouse model of Alzheimer's Disease (AD). TLT was administered 3 times/week at escalating doses, starting at 3 months of age, and was compared to a control group which received no laser treatment. Treatment sessions were continued for a total of six months. The brains were examined for amyloid plaque burden, A peptides (A1-40and A1-42), APP cleavage products (sAPP, CTF) and mitochondrial activity. Administration of TLT was associated with a significant, dose-dependent reduction in amyloid load as indicated by the numbers of A plaques. Levels of A1-40and A1-42levels were likewise reduced in a dose-dependent fashion. All TLT doses produced an increase in brain sAPP and a decrease in CTF levels consistent with an increase in -secretase activity and a decrease in -secretase activity. In addition, TLT increased ATP levels and oxygen utilization in treated animals suggesting improved mitochondrial function. These studies suggest that TLT is a potential candidate for treatment of AD.

Complement is implicated in the inflammatory response and the secondary neuronal damage that occurs after traumatic spinal cord injury (SCI). Complement can be activated by the classical, lectin, or alternative pathways, all of which share a common terminal pathway that culminates in formation of the cytolytic membrane attack complex (MAC). Here, we investigated the role of the alternative and terminal complement pathways in SCI. Mice deficient in the alternative pathway protein factor B (fB) were protected from traumatic SCI in terms of reduced tissue damage and demyelination, reduced inflammatory cell infiltrate, and improved functional recovery. In a clinically relevant paradigm, treatment of mice with an anti-fB mAb resulted in similarly improved outcomes. These improvements were associated with decreased C3 and fB deposition. On the other hand, deficiency of CD59, an inhibitor of the membrane attack complex, resulted in significantly increased injury and impaired functional recovery compared to wild-type mice. Increased injury in CD59-deficient mice was associated with increased MAC deposition, while levels of C3 and fB were unaffected. These data indicate key roles for the alternative and terminal complement pathways in the pathophysiology of SCI. Considering a previous study demonstrating an important role for the classical pathway in promoting SCI, it is likely that the alternative pathway plays a critical role in amplifying classical pathway initiated complement activation.

It is well established that the extracellular deposition of amyloid β (Aβ) peptide plays a central role in the development of Alzheimer's disease (AD). Therefore, either preventing the accumulation of Aβ peptide in the brain or accelerating its clearance may slow the rate of AD onset. Neprilysin (NEP) is the dominant Aβ peptide-degrading enzyme in the brain; NEP becomes inactivated and down-regulated during both the early stages of AD and aging. In this study, we investigated the effect of human (h)NEP gene transfer to the brain in a mouse model of AD before the development of amyloid plaques, and assessed how this treatment modality affected the accumulation of Aβ peptide and associated pathogenetic changes (eg, inflammation, oxidative stress, and memory impairment). Overexpression of hNEP for 4 months in young APP/ΔPS1 double-transgenic mice resulted in reduction in Aβ peptide levels, attenuation of amyloid load, oxidative stress, and inflammation, and improved spatial orientation. Moreover, the overall reduction in amyloidosis and associated pathogenetic changes in the brain resulted in decreased memory impairment by ∼50%. These data suggest that restoring NEP levels in the brain at the early stages of AD is an effective strategy to prevent or attenuate disease progression.

The Atlantic bottlenose dolphin (Tursiops truncatus), a marine mammal found off the Atlantic coast, has become the focus of considerable attention because of an increasing number of mortality events witnessed in this species over the last several years along the southeastern United States. Assessment of the impact of environmental stressors on bottlenose dolphins (BND) has been difficult because of the protected status of these marine mammals. The studies presented herein focused on establishing epidermal cell cultures and cell lines as tools for the in vitro evaluation of environmental stressors on BND skin. Epidermal cell cultures were established from skin samples obtained from Atlantic BND and subjected to karyotype analysis. These cultures were further characterized using immunohistochemical methods demonstrating expression of cytokeratins. By two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), we observed that the proteomic profile of BND skin tissue samples shared distinct similarities with that of skin-derived cultures. Epidermal cell cultures were transfected with a plasmid encoding the SV40 small t- and large T-antigens, as well as the neomycin-resistance gene. Five neomycin-resistant clones were isolated and expanded, and all of them proliferated at a faster rate than nontransfected BND epidermal cultures, which exhibited signs of senescence. Cell lysates prepared from two transfected clones were shown to express, by Western blot analysis, both SV40 tumor antigens. These experimental results are consistent with the concept that transfected clones expressing SV40 tumor antigens represent immortalized BND cell lines. Epidermal cell lines derived from Tursiops truncatuswill provide a unique tool for studying key features of the interaction occurring between dolphins and the environment in which they live at their most crucial interface: the skin. © 2005 Wiley-Liss, Inc.

Alzheimer's disease (AD) is a progressive senile dementia characterized by deposition of a 4 kDa peptide of 39-42 residues known as amyloid beta-peptide (Aβ) in the form of senile plaques and the microtubule associated protein tau as paired helical filaments. Genetic studies have identified mutations in the Aβ precursor protein (APP) as the key triggers for the pathogenesis of AD. Other genes such as presenilins 1 and 2 (PS1 / 2) and apolipoprotein E (APOE) also play a critical role in increased Aβ deposition. Several biochemical and molecular studies using transfected cultured cells and transgenic animals point to mechanisms by which Aβ is generated and aggregated to trigger the neurodegeneration that may cause AD. Three important enzymes collectively known as 'secretases' participate in APP processing leading to the generation of either Aβ or non-amyloid proteins. However, the mechanisms of neurotoxicity of Aβ and the role of APP function in AD remain important unanswered questions. Although early studies recognized the loss of cholesterol and other lipids in the brain, these findings have been poorly connected with AD pathogenesis, despite the identification of the ε4 allele of APOE as a major risk factor in AD. The recent finding that cholesterol can modulate the yield of potentially toxic Aβ has boosted research on its role in AD. Consequently, several cholesterol-reducing drugs are currently being evaluated for the treatment of AD. The present review summarizes our current understanding of the relationship of AD pathogenesis with cholesterol, lipids and other genetic and environmental risk factors.

Scavenger receptor class B type I (SR-BI) delivers cholesterol ester from HDL to cells via a selective uptake mechanism, whereby lipid is transferred from the core of the particle without concomitant degradation of the protein moiety. The precise metabolic fate of HDL particles after selective lipid uptake is not known. To characterize SR-BI-mediated HDL processing in vivo, we expressed high levels of this receptor in livers of apoA-I(-/-) mice by adenoviral vector gene transfer, and then injected the mice with a bolus of human HDL(2) traced with (125)I-dilactitol tyramine. HDL recovered from apoA-I(-/-) mice over-expressing SR-BI was significantly smaller than HDL recovered from control mice as measured by non-denaturing gel electrophoresis. When injected into C57BL/6 mice, these HDL "remnants" were rapidly converted to HDL(2)-sized lipoprotein particles, and were cleared from the plasma at a rate similar to HDL(2). In assays in cultured cells, HDL remnants did not stimulate ATP-binding cassette transporter A1-dependent cholesterol efflux. When mixed with mouse plasma ex vivo, HDL remnants rapidly converted to larger HDL particles. These studies identify a previously ill-defined pathway in HDL metabolism, whereby SR-BI generates small, dense HDL particles that are rapidly remodeled in plasma. This remodeling pathway may represent a process that is important in determining the rate of apoA-I catabolism and HDL-mediated reverse cholesterol transport.

Scavenger receptor BI (SR-BI) is a multi-ligand lipoprotein receptor that mediates selective lipid uptake from HDL, and plays a central role in hepatic HDL metabolism. In this report, we investigated the extent to which SR-BI selective lipid uptake contributes to LDL metabolism. As has been reported for human LDL, mouse SR-BI expressed in transfected cells mediated selective lipid uptake from mouse LDL. However, LDL-cholesteryl oleoyl ester (CE) transfer relative to LDL-CE bound to the cell surface (fractional transfer) was approximately 18-fold lower compared with HDL-CE. Adenoviral vector-mediated SR-BI overexpression in livers of human apoB transgenic mice ( approximately 10-fold increased expression) reduced plasma HDL-cholesterol (HDL-C) and apolipoprotein (apo)A-I concentrations to nearly undetectable levels 3 days after adenovirus infusion. Increased hepatic SR-BI expression resulted in only a modest depletion in LDL-C that was restricted to large LDL particles, and no change in steady-state concentrations of human apoB. Kinetic studies showed a 19% increase in the clearance rate of LDL-CE in mice with increased SR-BI expression, but no change in LDL apolipoprotein clearance. Quantification of hepatic uptake of LDL-CE and LDL-apolipoprotein showed selective uptake of LDL-CE in livers of human apo B transgenic mice. However, such uptake was not significantly increased in mice over-expressing SR-BI. We conclude that SR-BI-mediated selective uptake from LDL plays a minor role in LDL metabolism in vivo.

Background: Amyloid diseases are characterized by the tissue deposition of extracellular proteinaceous material, which results in organ dysfunction and death. Colocalization of heparan sulfate (HS) proteoglycans to amyloid deposits suggests that they may be an early event in amyloid formation and play an important role in fibril formation. Structural analysis has demonstrated that HS interacts with amyloidogenic proteins resulting in structural changes that allow for an increase in β-sheet content, possibly enhancing fibrillogenesis. Recent studies have shown that small-molecule anionic sulfonates or sulfates can arrest inflammation-associated (AA) amyloid induction. Materials and Methods: In the present study, we examine the effect of low-molecular-weight heparins (LMWHs) on the development of amyloid in the mouse model of AA amyloid. In addition, in vitrofibril formation assays were performed to determine the effect of LMWHs on fibrillogenesis. Results: Injection of mice with clinically relevant doses of LMWHs (enoxaparin and dalteparin) demonstrated a reduction in AA amyloid deposition. These compounds were capable of arresting the progression of AA amyloid and eventually resulting in regression of the amyloid deposits. In vitroanalysis indicated that LMWHs prevented AA and Aβ peptide fibril formation by impeding the structural changes necessary for fibril formation. Conclusions: Our findings suggest that the LMWHs may provide beneficial effects against the development of amyloidoses, including Alzheimer’s disease.

Numerous studies indicate a role for oxidative stress in the neuronal degeneration and cell death that occur during ischemia–reperfusion injury. Recent data suggest that inhibition of the proteasome may be a means by which oxidative stress mediates neuronal cell death. In the current study, the authors demonstrate that there is a time-dependent decrease in proteasome activity, which is not associated with decreased expression of proteasome subunits, after cerebral ischemia–reperfusion injury. To determine the role of oxidative stress in mediating proteasome inhibition, ischemia–reperfusion studies were conducted in mice that either overexpressed the antioxidant enzyme glutathione peroxidase [GPX 1(+)], or were devoid of glutathione peroxidase activity (GPX −/−). After ischemia–reperfusion, GPX 1(+) mice displayed decreased infarct size, attenuated neurologic impairment, and reduced levels of proteasome inhibition compared with either GPX −/− or wild type mice. In addition, GPX 1(+) mice displayed lower levels of 4-hydroxynonenal-modified proteasome subunits after ischemia–reperfusion injury. Together, these data indicate that proteasome inhibition occurs during cerebral ischemia–reperfusion injury and is mediated, at least in part, by oxidative stress.

The lack of sensitive and relatively non-invasive measures has hampered monitoring the clinical course of spontaneously developing colitis in IL-2-deficient (−/−) mice. We selected (i) to study the correlation of the acute phase plasma proteins serum amyloid A (SAA) and serum amyloid P component (SAP) levels with colonic disease and (ii) to characterize the amyloidosis in the IL-2−/−animals. IL-2−/−mice exhibited increasing severity of gross intestinal inflammation with age, confined to the distal colon. Histologically, the colonic disease score increased serially in IL-2−/−animals. Wild-type mice showed no activity, while 16-week-old IL-2+/−animals had minimal colitis with small ulcers and lamina propria inflammatory infiltrate. Periportal hepatitis was present and positive Congo red staining indicated amyloidosis of the liver and spleen in 16 week IL-2−/−mice. SAA immunostaining in the liver and spleen was increased in the 8 week and 16 week IL-2−/−and 16 week IL-2+/−animals indicating AA amyloid deposits. Plasma SAA and SAP levels were markedly elevated, and generally preceded the onset of colitis and reflected its severity. Northern analysis showed markedly increased SAA expression in the liver and intestine of IL-2−/−and intestine of IL-2+/−16-week-old animals. Increased intestinal expression of SAA3 (lamina propria macrophages) indicates local inflammation in IL-2+/−animals at 16 weeks. Treatment of 3-week-old animals with systemic IL-2 or IL-1 receptor antagonist (IL-1ra) delayed inflammation, postponed the increase in SAA levels and minimized disease onset. These results further demonstrate that IL-2 plays a significant role in normal immune responses in the body and that plasma SAA levels both reflect colonic disease severity and may indicate subclinical disease in both IL-2−/−and IL-2+/−mice. Furthermore. The mechanism of IL-2-deficient induced colitis appears to be mediated in part through the increase in IL-1. In addition, the IL-2−/−mouse of spontaneous enterocolitis may provide a unique system for studying spontaneously developing AA amyloidosis.

Although degeneration of lower motor neurons is the most striking abnormality in amyotrophic lateral sclerosis (ALS), more subtle alterations may occur in the brain. Mutations in copper/zinc superoxide dismutase (Cu/Zn-SOD) are responsible for some cases of inherited ALS, and expression of mutant Cu/Zn-SOD in transgenic mice results in progressive motor neuron loss and a clinical phenotype similar to that of ALS patients. It is now reported that Cu/Zn-SOD mutant mice exhibit increased vulnerability to focal ischemic brain injury after transient occlusion of the middle cerebral artery. Levels of glucose and glutamate transport in cerebral cortex synaptic terminals were markedly decreased, and levels of membrane lipid peroxidation were increased in Cu/Zn-SOD mutant mice compared to nontransgenic mice. These findings demonstrate that mutant Cu/Zn-SOD may endanger brain neurons by a mechanism involving impairment of glucose and glutamate transporters. Moreover, our data demonstrate a direct adverse effect of the mutant enzyme on synaptic function.

High-density lipoprotein (HDL) exists within the brain and is highly vulnerable to oxidative modifications. The focus of the present study was to determine the effect of HDL and oxidized HDL (oxHDL) upon neurons, astrocytes, and microglia. Administration of highly oxidized HDL, but not native, minimally, or moderately modified HDL resulted in a dose- and time-dependent increase in oxidative stress and death of cultured rat embryonic neurons. Astrocyte and microglia cultures treated with highly oxidized HDL displayed increased reactive oxygen species formation but no toxicity. Application of oxHDL exacerbated oxidative stress and neuron death induced by β-amyloid peptide. Studies using pharmacological inhibitors implicate the involvement of calcium and reactive oxygen species in oxHDL-induced neuronal loss. Neural cells expressing increased levels of BCL-2 had decreased levels of oxidative stress and neuron death following exposure to oxHDL. Together, these data demonstrate that oxHDL increases oxidative stress in neurons, astrocytes, and microglia which ultimately culminate in neuron death.

Introduction:Lipoprotein(a) [Lp(a)] is a unique lipoprotein that has emerged as an independent risk factor for developing vascular disease and stroke.Hypothesis:We hypothesize that SR-BI is critical to the regulation of Lp(a) metabolism and that mutations in SR-BI contribute to the pathogenesis of CVD and stroke in relationship to Lp(a).Methods:Transgenic mice carrying the human Lp(a) [hLp(a)] on the SR-BI (mSR-BI) deficient mouse background, Lp(a) transgenic mice on the human SR-BI (hSR-BI) with and without the mSR-BI, and Lp(a) transgenic mice on the mutant hSR-BI with and without mSR-BI were used to determine the impact on Lp(a) catabolism and atherosclerosis.Results:We determined the levels of Lp(a) in wildtype and SR-BI KO mice, lipid levels and profiles and oxidized state of the Lp(a). In addition, studies on the impact of SR-BI and Lp(a) on atherosclerosis were examined. We showed that Lp(a) was elevated in SR-BI deficient mice and Lp(a) was reduced in hSR-BI tg mice, compared to wildtype SR-BI mice. Lp(a) mice with the mutant hSR-BI protein, showed significantly elevated Lp(a) levels. When Lp(a) levels were increased, Lp(a) was highly oxidized, lipid profiles were altered and mice develop significant atherosclerosis. We also showed that Lp(a) mice showed a higher incidence of hemorrhagic strokes and had worse outcomes following ischemic strokes.Conclusions:These studies will lay the ground work for both basic and translational studies to understand the impact of Lp(a) in CVD and stroke.

Specific proteins of the apolipoprotein serum amyloid (apoSAA) family that are synthesized in large quantities during the acute, early phase of inflammation can serve as the proteinaceous precursors for amyloid fibrils. To model fibrillogenesis in such inflammatory diseases, we have used electron microscopy and X-ray diffraction to examine the structures formed by synthetic peptides corresponding in sequence to the 11 amino-terminal amino acids of murine apoSAA1, apoSAAcej, and apoSAA2 and to the 15 amino-terminal amino acids of apoSAA2. This region is reported to be the major fibrillogenic determinant of apoSAA isoforms. Both in 1 mM Tris buffer and in 35% acetonitrile, 0.1% trifluoracetic acid (ACN/TFA), all of the peptides formed macromolecular assemblies consisting of twisted, ≈40- to 60-Å-thick ribbons, which varied in width from around 40–70 Å (for 11-mer apoSAA2 in Tris) up to 900 Å (for the other peptides). X-ray diffraction patterns recorded from lyophilized peptides, vapor-hydrated samples, and solubilized/dried samples showed hydrogen bonding and intersheet reflections typical of a β-pleated sheet conformation. The coherent lengths measured from the breadths of the X-ray reflections indicated that with hydration the growth of the assemblies in the intersheet stacking direction was comparable to that in the hydrogen-bonding direction, and analysis of oriented samples showed that the β-strands were oriented perpendicular to both the long axis and the face of the assemblies. These X-ray results are consistent with the ribbon- or plate-like morphology of the individual aggregates and emphasize the polymorphic nature of amyloidogenic peptides. Our findings demonstrate that X-ray diffraction measurements on vapor-hydrated or solubilized/dried versus lyophilized, amyloidogenic peptides are a good indicator of their fibrillogenic potential. For example, from the highest to the lowest potential, the peptides examined here were ranked as: Aβ1-28 > Aβ1-40 > apoSAA1 ≈ apoSAAcej > apoSAA2 > Aβ17-42. Experiments in which the three different 11-mer apoSAA isoforms were solubilized in ACN/TFA and then combined as binary mixtures showed that the ribbon morphology was not affected but that the extent of hydrogen bonding in the assemblies was substantially reduced. Our observations on thein vitroassembly of apoSAA analogs emphasize that amyloid fibril formation and morphology depend on primary sequence, length of polypeptide chain, the presence of additional fibrillogenic polypeptides, and solvent conditions.

Ischemic and excitotoxic insults to the brain induce rapid production of tumor necrosis factor-α (TNF), but the role of TNF in neuronal responses to brain injury are unclear. Two different TNF receptors (p55 and p75) are expressed in neurons and glia, To understand the role of TNF in brain injury, we generated mice that lack p55, p75, or both receptors, We report that neuronal damage after focal cerebral ischemia—reperfusion is significantly increased in mice lacking p55 receptors (85 ± 7 mm3infarct volume; mean ± SD) compared with wild-type mice (70 ± 8 mm3) and mice lacking p75 receptors (72 ± 6 mm3). Moreover, mice lacking p55 receptors exhibited increased degeneration of CA3 hippocampal neurons after administration of the excitotoxin kainic acid compared with wild-type mice and mice lacking p75 receptors. When taken together with recent data showing that TNF can prevent apoptosis of cultured neurons exposed to oxidative and metabolic insults, our findings suggest that TNF plays a neuroprotective role after acute brain insults.

Serum amyloid A (SAA) proteins are one of the most inducible acute-phase reactants and are precursors of secondary amyloidosis. In the mouse, SAA1 and SAA2 are induced in approximately equal quantities in response to amyloid induction models. These two isotypes differ in only 9 of 103 amino acid residues; however, only SAA2 is selectively deposited into amyloid fibrils. SAA expression in the CE/J mouse species is an exception in that gene duplication did not occur and the CE/J variant is a hybrid molecule sharing features of SAA1 and SAA2. However, even though it is more closely related to SAA2 it is not deposited as amyloid fibrils. We have developed an adenoviral vector system to overexpress SAA proteins in cell culture to determine the ability of these proteins to form amyloid fibrils, and to study the structural features in relation to amyloid formation. Both the SAA2 and CE/J SAA proteins were synthesized in large quantities and purified to homogeneity. Electron microscopic analysis of the SAA proteins revealed that the SAA2 protein was capable of forming amyloid fibrils, whereas the CE/J SAA was incapable. Radiolabelled SAAs were associated with normal or acute-phase high-density lipoproteins (HDLs); we examined them for their clearance from the circulation. In normal mice, SAA2 had a half-life of 70 min and CE/J SAA had a half-life of 120 min; however, in amyloid mice 50% of the SAA2 cleared in 55 min, compared with 135 min for the CE/J protein. When the SAA proteins were associated with acute-phase HDLs, SAA2 clearance was decreased to 60 min in normal mice compared with 30 min in amyloidogenic mice. Both normal and acute-phase HDLs were capable of depositing SAA2 into preformed amyloid fibrils, whereas the CE/J protein did not become associated with amyloid fibrils. This established approach opens the doors for large-scale SAA production and for the examination of specific amino acids involved in the fibrillogenic capability of the SAA2 molecule in vitro and in vivo.

The c-fosproto-oncogene is activated by transient cerebral ischemia. This activation may signify a specific genetic response to ischemia affecting tolerance to ischemia and ultimate cell survival. Hyperglycemia, which enhances brain injury from transient ischemia, was studied for its effects on this gene system in gerbils by measuring c-fosmRNA 2 h after 20 min of bilateral carotid artery occlusion. Brain c-fosmRNA was increased by ischemia (11.7 ± 5.0, p≤ 0.05, fold increase) compared to nonischemic controls (1.0 ± 1.3). Pretreatment with 1 g/kg of glucose partially reduced postischemic c-fosmRNA (6.3 ± 1.6, p≤ 0.05) while 4 g/kg of glucose completely suppressed postischemic c-fosexpression (0.7 ± 0.3, p≤ 0.05). These data indicate that hyperglycemia suppresses normal postischemic gene expression and suggest the possibility that such suppression is a predictor or even a contributor to hyperglycemia-enhanced ischemic brain damage.

Matrix metalloproteinases and their inhibitors regulate follicular connective tissue remodeling associated with ovulation. We examined the control of metalloproteinase inhibitor activity and gene expression by various treatments in cultured rat granulosa cells and intact whole ovaries. Cells were isolated from preovulatory follicles of immature eCG-primed rats and cultured with various treatments for 24 h. Metalloproteinase inhibitor activity was measured in the media. The addition of LH or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) increased inhibitor activity 2.5- and 2.6-fold above that in control cultures, respectively. Cycloheximide treatment blocked basal and LH- and TPA-stimulated inhibitor activity, suggesting that the increase in granulosa cell inhibitor activity resulted from de novo protein synthesis. Indomethacin, a prostaglandin synthase inhibitor, had no effect on basal or LH-induced granulosa cell inhibitor activity. Addition of the antiestrogen tamoxifen citrate or an aromatase inhibitor, 10-propargylestr−4-ene−3,17-dione, did not affect basal or LH-stimulated inhibitor activity, implying that estrogen is not involved in the signal transduction pathway leading to increased inhibitor activity. Northern analysis demonstrated the presence of mRNA for a tissue-derived metalloproteinase inhibitor, TIMP-1, which increased with LH stimulation, in rat granulosa cells. Similarly, an hCG stimulus increased TIMP-1 mRNA in periovulatory ovaries to the highest levels prior to ovulation. Neither cycloheximide nor indomethacin altered hCG-stimulated TIMP mRNA levels in periovulatory ovaries. The present study demonstrates that the LH- and TPA-induced increase in inhibitor activity resulted from de novo protein synthesis; however, de novo protein synthesis does not appear necessary for the increase in TIMP mRNA. Additionally, neither prostaglandins nor estrogen appears to play a role in signaling metalloproteinase inhibitor production. This study indicates that TIMP is hormonally controlled by LH and that TIMP may act as an important regulator of ovarian connective tissue remodeling during follicular rupture and corpus luteum formation.

THE basis of aging in the Aplysia nervous system is unknown. We now report age–related changes in mRNA expression of phe–met–arg–phe–NH2(FMRFamide) and egg–laying hormone (ELH) in the abdominal ganglion, a part of the CNS, in young, mature and old Aplysia. Northern blot analysis revealed two mRNA species of 1.4 and 3.2kb for RMRFamide and a single mRNA species of 0.8kb for ELH. FMRFamide mRNA level increased 1.5–fold from young to mature and then decreased 3–fold in old animals. ELH mRNA gradually increased between young and mature animals and then escalated 25–fold in old animals. Age differentially affected the mRNA of these two peptides, which may contribute to behavioral changes previously reported.

Amyloid β-peptide (Aβ) is a major fibrillar component of neuritic plaques in Alzheimer's disease (AD) brains and is related to the pathogenesis of the disease. We hypothesized that amyloid formation could be inhibited by peptides homologous to Aβ (position 17-21) with a similar degree of hydrophobicity, but with a very low propensity to adopt a β-sheet conformation by incorporating proline residues (anti-β-sheet peptides or β-sheet inhibitors). An 11-residue peptide with these characteristics binds to Aβ, inhibits Aβ fibril formation and partially disaggregates preformed fibrilsin vitro.Shorter anti-β-sheet peptides and analogs containing D-amino acids are also able to inhibit Aβ fibrillogenesis. The latter are more resistant to proteolytic degradation and may serve as a starting point to design more efficient peptides derivatives to inhibit amyloidogenesis in vivo.

Protein tyrosine phosphorylation plays an important role in the regulation of neuronal function. We examined the effects of inhibition of tyrosine phosphorylation on ischemic neuronal damage in the CA1region of the hippocampus. In the gerbil hippocampus, genistein and lavendustin A, tyrosine kinase inhibitors, were administered 30 min before initiation of 5-min ischemia and reperfusion. Both genistein and lavendustin A blocked tyrosine phosphorylation and prevented delayed neuronal death (DND). However, genistin, an inactive analogue of genistein, did not block DND. Genistein was dose-dependent in the inhibition of DND after ischemia and reperfusion. Administration of genistein 5 to 10 min after ischemia and reperfusion was ineffective in blocking DND in the CA1region of the hippocampus. The tyrosine kinase inhibitors selectively blocked the phosphorylation of microtubule-associated protein (MAP)-2 kinase following ischemia and reperfusion injury. These results suggest that tyrosine phosphorylation in the ischemic brain is important for neuronal injury and that MAP-2 kinase may play a role in the onset of delayed neuronal death.

Polyamines are derived from ornithine by the actions of ornithine decarboxylase (ODC), which is the rate-limiting step in this pathway. Polyamines play a role in cell growth, neoplasia, differentiation, and response to injury. We have shown that transient cerebral ischemia gives rise to increased ODC mRNA and enzyme activity in the gerbil brain. ODC and polyamines are thought to be important in the generation of edema and the neuronal cell loss associated with cerebral ischemia. To test this theory, we examined the ODC activity, putrescine levels, and neuronal density in the CA1region of the hippocampus following ischemia and reperfusion injury in the absence and presence of an inhibitor of ODC activity, α-difluoromethylornithine (DFMO). Pretreatment of animals with DFMO resulted in attenuation of the ODC activity following 5 min of ischemia and 4 h of reperfusion. In addition, DFMO prevented the increase in polyamine levels, as determined by measurement of putrescine in the ischemic brain. These alterations were not due to changes in ODC mRNA level. Further analysis revealed that DFMO treatment blocked the delayed neuronal cell death in the CA1region of the hippocampus that accompanies ischemia and reperfusion injury. Administration of DFMO resulted in a dose-dependent beneficial effect upon neuronal cell survival. These results suggest that ODC enzyme activity and the production of polyamines play a significant role in the response of the brain to ischemic injury.

Ornithine decarboxylase (ODC) is the rate-limiting enzyme that catalyzes the synthesis of polyamines from ornithine and is thought to be involved in the cellular response to growth, differentiation, and stress. Previous studies have demonstrated that transient cerebral ischemia results in an increase in ODC activity and polyamine synthesis. We have used the Mongolian gerbil as a model system to test the hypothesis that the cellular response to ischemia induces a distinct pattern of ODC gene expression. Our results indicate that transient ischemia, induced by bilateral carotid occlusion, elevates ODC mRNA within 1–4 h after reperfusion, which correlates with increased ODC activity and polyamine synthesis. Increased ODC mRNA can be detected in the forebrain, striatum, hippocampus, and midbrain but not the cerebellum, which is not subject to ischemic injury. In contrast, c-fosmRNA increased by 15 min after reperfusion and actin mRNA did not demonstrate alterations in level after ischemia. Pentobarbital prevented the increase in ODC mRNA, whereas the glutamate antagonist MK-801 had no effect on the elevation of ODC gene expression after ischemia. We conclude that the ischemia-induced increase in ODC enzyme activity may be attributed in part to transcriptional activation of the ODC gene.

Brain injury, as occurs in stroke or head trauma, induces a dramatic increase in levels of tumor necrosis factor–α (TNF), but its role in brain injury response is unknown. We generated mice genetically deficient in TNF receptors (TNFR–KO) to determine the role of TNF in brain cell injury responses. Damage to neurons caused by focal cerebral ischemia and epileptic seizures was exacerbated in TNFR–KO mice, indicating that TNF serves a neuroprotective function. Oxidative stress was Increased and levels of an antioxidant enzyme reduced in brain cells of TNFR–KO mice, indicating that TNF protects neurons by stimulating antioxidant pathways. Injury–induced microglial activation was suppressed in TNFR–KO mice, demonstrating a key role for TNF in injury–induced immune response. Drugs that target TNF signaling pathways may prove beneficial in treating stroke and traumatic brain injury.

The serum amyloid A (SAA) proteins are a polymorphic family of apolipoproteins associated with high-density lipoproteins (HDL). Three distinct subfamilies have been identified: (i) a cytokine-induced acute phase subfamily that is hepatically produced and can become the major apolipoprotein on HDL (SAA1, SAA2); (ii) a peripherally produced acute phase SAA3 that is only a minor HDL apolipoprotein; and (iii) a constitutive subfamily (SAA4) that is a minor normal HDL apolipoprotein comprising more than 90% of the SAA during homeostasis. Here we define the structure of theSaa4gene. Similar to other Saa family members, it has four exons and three introns. It is 4588 bp long from the transcription start site to the end of the 3′-untranslated region and is approximately 20% larger than otherSaagenes. We have locatedSaa411 kb upstream fromSaa3and 5 kb downstream fromSaa1,with the pseudogene approximately 1 kb from the 5′ end ofSaa4. Saa4has the same orientation as most otherSaafamily members, with onlySaa2having an opposing orientation. These data promote our understanding of the evolution of theSaafamily. They enhance our ability to develop the mouse as a transgenic and gene deletion model to advance the understanding of the function of these apolipoproteins.

The mechanisms leading to oxidative stress and cellular dysfunction during stroke are not well understood. To test the hypothesis that transient cerebral artery occlusion (MCAo) in mice results in the generation of oxidized phospholipids (oxPLs) that contribute to neuronal cell death and glial activation. Both in vitroand in vivocerebral ischemia and reperfusion injury (IRI) resulted in the elevation of specific oxPLs. Neuronal cell death was determined in the presence of oxPLs and the natural oxPL E06 antibody protected the cells from the toxic effects. IRI in mice gave rise to increased immunoreactivity of oxPLs in the brain. E06 reduced inflammatory markers in the brain following IRI, including iba-1, GFAP and inflammatory cytokines. In addition, oxPLs gave rise to M1 and Mox microglial phenotypes which was reversed in the presence of E06 and elicited a more M2 phenotype. Nrf2 deficient mice show increased infarct volumes and microglia from Nrf2-/-mice show a reduction in Mox gene expression, and E06 protects both mice and cells from the Nrf2 deficit. Cerebral IRI generates oxPLs which triggers neuronal cell loss and inflammation and inactivation of oxPLs in vivo reduces infarct volume and improves outcomes.