Morphologically distinct types of amyloid plaques point the way to a better understanding of Alzheimer's disease pathogenesis.

The details of the sequence of pathological events leading to neuron death in Alzheimer's disease (AD) are not known. Even the formation of amyloid plaques, one of the major histopathological hallmarks of AD, is not clearly understood; both the origin of the amyloid and the means of its deposition remain unclear. It is still widely considered, however, that amyloid plaques undergo gradual growth in the interstitial space of the brain via continual extracellular deposition of amyloid beta peptides at "seeding sites," and that these growing plaques encroach progressively on neurons and their axons and dendritic processes, eventually leading to neuronal death. Actually, histopathological evidence to support this mechanism is sparse and of uncertain validity. The fact that the amyloid deposits in AD brains that are collectively referred to as plaques are of multiple types and that each seems to have a different origin often is overlooked. We have shown experimentally that many of the so-called "diffuse amyloid plaques," which lack associated inflammatory cells, are either the result of leaks of amyloid from blood vessels at focal sites of blood-brain barrier breaches or are artifacts resulting from grazing sections through the margins of dense core plaques. In addition, we have provided experimental evidence that neuronal death via necrosis leaves a residue that takes the form of a spheroid "cloud" of amyloid, released by cell lysis, surrounding a dense core that often contains neuronal nuclear material. Support for a neuronal origin for these "dense core amyloid plaques" includes their ability to attract inflammatory cells (microglia and immigrant macrophages) and that they contain nuclear and cytoplasmic components that are somewhat resistant to proteolysis by lysosomes released during neuronal cell lysis. We discuss here the clinical and therapeutic importance of recognizing that amyloid deposition occurs both within neurons (intracellular) and in the interstitial (extracellular) space of the brain. For dense core plaques, we propose that the latter location largely follows from the former. This scenario suggests that blocking intraneuronal amyloid deposition should be a primary therapeutic target. This strategy also would be effective for blocking the gradual compromise of neuronal function resulting from this intraneuronal deposition, and the eventual death and lysis of these amyloid-burdened neurons that leads to amyloid release and the appearance of dense core amyloid plaques in the interstitium of AD brains.

Simultaneous PCNA and TUNEL labeling for testicular toxicity evaluation suggests that detection of apoptosis may be more sensitive than proliferation.

We previously reported a sensitive, quantitative immunohistochemical assay using formalin fixed, paraffin embedded rat testicular tissues to assess the degree of proliferation-related toxicity. An indexing scheme was devised based on the percentage of PCNA-positive cells positioned as a single layer along the basement membrane at the perimeter of similarly staged seminiferous tubules (PCNA index). We observed significant decreases in the PCNA index in testes of rats treated with an experimental compound that has been shown to produce testicular histopathology. This relatively simple assay provided a more quantitative and sensitive assessment of early testicular toxicity. A separate investigation of the rates of apoptosis in adjacent serial sections of affected rat seminiferous tubules showed that the incidence of apoptosis increased as the rate of proliferation of spermatogonial cells in the tubules decreased. Therefore, we developed a simultaneous PCNA immunohistochemical and TUNEL histochemical assay not only to reduce preparation and analysis time but also to allow determination of the relation between effects of various compounds producing testicular toxicity on the two cellular processes within the same tissue section. We show that an experimental compound known to cause testicular toxicity produced a concurrent reduction of proliferation and increase in apoptosis in seminiferous tubules. In dose-response studies, we show that increased apoptosis was apparent at lower doses that did not show a significant decrease in PCNA, thus indicating the greater sensitivity of the TUNEL indexing assay to detect early evidence of toxicity. Detailed analyses show the presence of TUNEL-positive cells in tubules with normal PCNA labeling, which suggests that an effect on apoptosis occurs prior to significant changes in cell proliferation in the meiotic pathway for this particular testicular toxicant. This single assay employing the simultaneous dual labeling of apoptosis and proliferation has potential utility for detecting early testicular toxicity of experimental compounds in preclinical development and shedding light on potential cellular mechanisms for toxicity, which should help identify compounds with reduced testicular toxicity.

PCNA indexing as a preclinical immunohistochemical biomarker for testicular toxicity.

It is well known that toxicants such as cyclophosphamide and ethanol can have deleterious effects on normal spermatogenesis. End points such as testis weight and sperm counts have been used widely to assess gross structural and functional changes in testes resulting from toxicant exposure. Histopathological assessments are more sensitive measures of testicular health, but generally they are neither quantitative nor sensitive enough to detect early toxicity. Recently, immunolabeling cells with proliferating cell nuclear antigen (PCNA) has been used to identify proliferating spermatogonia; however, there have been no systematic attempts to quantify these changes. We have developed a sensitive, reliable and quantitative assay using immunohistochemistry on formalin fixed, paraffin embedded rat testes to assess the degree of proliferation-related toxicity. An indexing scheme was derived based on the determination of radially positioned PCNA-positive cells within similarly staged seminiferous tubules presenting a single layer of PCNA-positive cells along the basement membrane of the basal tubular compartment. An average of 60 tubules in the testes were counted per animal. Our results show significant decreases in the PCNA index in rats treated with an experimental compound that has been shown to produce testicular histopathology. The analysis provides a quick, reliable, sensitive, and quantitative means for assessing early testicular toxicity. The assay has potential utility as an in vivo biomarker for detecting early testicular toxicity of experimental compounds in preclinical development as well as for refining follow-up compounds with reduced testicular toxicity.

MAP-2 immunolabeling can distinguish diffuse from dense-core amyloid plaques in brains with Alzheimer's disease.

Alzheimer's disease (AD) neuropathology is characterized by the presence of diffuse and dense-core (neuritic) amyloid plaques in specific areas of the brain. The origin of these plaques and the relationship between them is poorly understood. Current methods to identify clearly these types of plaques in the AD brains are largely dependent upon morphological characteristics. Dense-core amyloid plaques in the entorhinal cortex and hippocampus of AD brains might arise from the lysis of neurons overburdened by excessive intracellular deposition of amyloid beta1-42 (Abeta42) peptide. The local release of active lysosomal enzymes, which persist within these plaques, might degrade most of the released intracellular proteins, leaving behind only those that are resistant to proteolytic digestion, such as ubiquitin, tau, neurofilament proteins and amyloid. To test the possibility that proteins that are sensitive to proteolysis may be degraded selectively in plaques, we used immunohistochemistry to examine the distribution of microtubule-associated protein-2 (MAP-2), a protein localized primarily in neuronal dendrites and known to be sensitive to proteolysis. Uniform MAP-2 immunolabeling was detected throughout the somatodendritic compartment of neurons in age-matched control cortical brain tissues as well as throughout areas of Abeta42-positive diffuse plaques in AD brains. In contrast, analysis of serial sections revealed that MAP-2 was absent from Abeta42-positive dense-core plaques in AD brains. Our results indicate that this differential MAP-2 immunolabeling pattern among plaques may be employed as a reliable and sensitive method to distinguish dense-core plaques from diffuse plaques within AD brain tissue. Furthermore, this biochemical distinction indicates that dense-core and diffuse plaques are formed by different mechanisms.

Intracellular accumulation of beta-amyloid(1-42) in neurons is facilitated by the alpha 7 nicotinic acetylcholine receptor in Alzheimer's disease.

Amyloid beta(1-42), a major component of amyloid plaques, binds with exceptionally high affinity to the alpha 7 nicotinic acetylcholine receptor and accumulates intracellularly in neurons of Alzheimer's disease brains. In this study, we investigated the possibility that this binding plays a key role in facilitating intraneuronal accumulation of amyloid beta(1-42). Consecutive section immunohistochemistry and digital imaging were used to reveal the spatial relationship between amyloid beta(1-42) and the alpha 7 receptor in affected neurons of Alzheimer's disease brains. Results showed that neurons containing substantial intracellular accumulations of amyloid beta(1-42) invariably express relatively high levels of the alpha 7 receptor. Furthermore, this receptor is highly co-localized with amyloid beta(1-42) within neurons of Alzheimer's disease brains. To experimentally test the possibility that the binding interaction between exogenous amyloid beta(1-42) and the alpha 7 receptor facilitates internalization and intracellular accumulation of amyloid beta(1-42) in Alzheimer's disease brains, we studied the fate of exogenous amyloid beta(1-42) and its interaction with the alpha 7 receptor in vitro using cultured, transfected neuroblastoma cells that express elevated levels of this receptor. Transfected cells exhibited rapid binding, internalization and accumulation of exogenous amyloid beta(1-42), but not amyloid beta(1-40). Furthermore, the rate and extent of amyloid beta(1-42) internalization was related directly to the alpha 7 receptor protein level, since (1) the rate of amyloid beta(1-42) accumulation was much lower in untransfected cells that express much lower levels of this receptor and (2) internalization was effectively blocked by alpha-bungarotoxin, an alpha 7 receptor antagonist. As in neurons of Alzheimer's disease brains, the alpha 7 receptor in transfected cells was precisely co-localized with amyloid beta(1-42) in prominent intracellular aggregates. Internalization of amyloid beta(1-42) in transfected cells was blocked by phenylarsine oxide, an inhibitor of endocytosis. We suggest that the intraneuronal accumulation of amyloid beta(1-42) in Alzheimer's disease brains occurs predominantly in neurons that express the alpha 7 receptor. In addition, internalization of amyloid beta(1-42) may be facilitated by the high-affinity binding of amyloid beta(1-42) to the alpha 7 receptor on neuronal cell surfaces, followed by endocytosis of the resulting complex. This provides a plausible explanation for the selective vulnerability of neurons expressing the alpha 7 receptor in Alzheimer's disease brains and for the fact that amyloid beta(1-42) is the dominant amyloid beta peptide species in intracellular accumulations and amyloid plaques.

A novel calcium-regulated membrane guanylate cyclase transduction system in the olfactory neuroepithelium.

This report defines the identity of a calcium-regulated membrane guanylate cyclase transduction system in the cilia of olfactory sensory neurons, which is the site of odorant transduction. The membrane fraction of the neuroepithelial layer of the rat exhibited Ca(2+)-dependent guanylate cyclase activity, which was eliminated by the addition of EGTA. This indicated that the cyclase did not represent a rod outer segment guanylate cyclase (ROS-GC), which is inhibited by free Ca(2+). This interpretation was supported by studies with the Ca(2+) binding proteins, GCAPs (guanylate cyclase activating proteins), which stimulate photoreceptor ROS-GC in the absence of Ca(2+). They did not stimulate the olfactory neuroepithelial membrane guanylate cyclase. The olfactory neuroepithelium contained a Ca(2+) binding protein, neurocalcin, which stimulated the cyclase in a Ca(2+)-dependent fashion. The cyclase was cloned from the neuroepithelium and was found to be identical in structure to that of the previously cloned cyclase termed GC-D. The cyclase was expressed in a heterologous cell system, and was reconstituted with its Ca(2+)-dependent activity in the presence of recombinant neurocalcin. The reconstituted cyclase mimicked the native enzyme. Immunocytochemical studies showed that the guanylate cyclase coexists with neurocalcin in the apical region of the cilia. Deletion analysis showed that the neurocalcin-regulated domain resides at the C-terminal region of the cyclase. The findings establish the biochemical, molecular, and functional identity of a novel Ca(2+)-dependent membrane guanylate cyclase transduction system in the cilia of the olfactory epithelium, suggesting a mechanism of the olfactory neuroepithelial guanylate cyclase regulation fundamentally distinct from the phototransduction-linked ROS-GC.

Colon epithelial cell death in 2,4,6-trinitrobenzenesulfonic acid-induced colitis is associated with increased inducible nitric-oxide synthase expression and peroxynitrite production.

Peroxynitrite, derived from the reaction of nitric oxide (NO(.)) with superoxide (O(2)), is a potent nitrating and oxidizing agent that can induce apoptosis in a variety of different cell types. In the present study, we investigated the possible role of peroxynitrite as a mediator of colon epithelial cell death in rat colitis. Rat colon inflammation was induced by intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) and rats were sacrificed 24 h after TNBS administration. Expression of inducible nitric-oxide synthase (iNOS) was detected by reverse transcription-polymerase chain reaction and immunohistochemistry. The enzymatic activities of Ca(2+)-independent iNOS and Ca(2+)-dependent constitutive nitric-oxide synthase were determined biochemically. Evidence of peroxynitrite-mediated cell injury was detected by immunostaining of nitrotyrosine. Apoptosis was examined by in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and DNA gel electrophoresis. To evaluate the specific contribution of peroxynitrite to the observed cell injury, a selective iNOS inhibitor, L-N(G)-[1-iminoethyl]lysine (L-NIL), was administered after TNBS induction. Morphological examination and analysis of TUNEL/cytokeratin double immunofluorescence revealed significant apoptosis in mucosal epithelial cells. Nitrotyrosine was colocalized with TUNEL, strongly demonstrating the association of peroxynitrite with the apoptotic death of colon epithelial cells. The administration of L-NIL reduced iNOS activity in 24-h lesions by 92% and also significantly attenuated both nitrotyrosine staining and apoptotic cell counts in the colon epithelium. These results strongly suggest that local elevated level of peroxynitrite produced from increased iNOS expression and activity is a major contributor to colon epithelial apoptosis during colon inflammation.

A novel human hexameric DNA helicase: expression, purification and characterization.

We have cloned, expressed and purified a hexameric human DNA helicase (hHcsA) from HeLa cells. Sequence analysis demonstrated that the hHcsA has strong sequence homology with DNA helicase genes from Saccharomyces cerevisiae and Caenorhabditis elegans, indicating that this gene appears to be well conserved from yeast to human. The hHcsA gene was cloned and expressed in Escherichia coli and purified to homogeneity. The expressed protein had a subunit molecular mass of 116 kDa and analysis of its native molecular mass by size exclusion chromatography suggested that hHcsA is a hexameric protein. The hHcsA protein had a strong DNA-dependent ATPase activity that was stimulated >/=5-fold by single-stranded DNA (ssDNA). Human hHcsA unwinds duplex DNA and analysis of the polarity of translocation demonstrated that the polarity of DNA unwinding was in a 5'-->3' direction. The helicase activity was stimulated by human and yeast replication protein A, but not significantly by E.coli ssDNA-binding protein. We have analyzed expression levels of the hHcsA gene in HeLa cells during various phases of the cell cycle using in situ hybridization analysis. Our results indicated that the expression of the hHcsA gene, as evidenced from the mRNA levels, is cell cycle-dependent. The maximal level of hHcsA expression was observed in late G(1)/early S phase, suggesting a possible role for this protein during S phase and in DNA synthesis.

Negatively calcium-modulated membrane guanylate cyclase signaling system in the rat olfactory bulb.

The mechanism by which the individual odor signals are translated into the perception of smell in the brain is unknown. The signal processing occurs in the olfactory system which has three major components: olfactory neuroepithelium, olfactory bulb, and olfactory cortex. The neuroepithelial layer is composed of ciliated sensory neurons interspersed among supportive cells. The sensory neurons are the sites of odor transduction, a process that converts the odor signal into an electrical signal. The electrical signal is subsequently received by the neurons of the olfactory bulb, which process the signal and then relay it to the olfactory cortex in the brain. Apart from information about certain biochemical steps of odor transduction, there is almost no knowledge about the means by which the olfactory bulb and cortical neurons process this information. Through biochemical, functional, and immunohistochemical approaches, this study shows the presence of a Ca(2+)-modulated membrane guanylate cyclase (mGC) transduction system in the bulb portion of the olfactory system. The mGC is ROS-GC1. This is coexpressed with its specific modulator, guanylate cyclase activating protein type 1 (GCAP1), in the mitral cells. Thus, a new facet of the Ca(2+)-modulated GCAP1--ROS-GC1 signaling system, which, until now, was believed to be unique to phototransduction, has been revealed. The findings suggest a novel role for this system in the polarization and depolarization phenomena of mitral cells and also contradict the existing belief that no mGC besides GC-D exists in the olfactory neurons.

Pattern of cytokine and adhesion molecule mRNA in hapten-induced relapsing colon inflammation in the rat.

We examined the mRNA expression of cytokines, chemokines, integrins, and selectins in colon lesions of rat colitis with a semi-quantitative RT-PCR assay. Rat colitis was induced by trinitrobenzene sulfonic acid (TNBS) in 50% ethanol. Within 24 h, an acute inflammation occurred with hyperemia, edema, necrosis and an intense infiltration of granulocytes in the mucosa. The lesion proceeded into a T-lymphocyte/monocyte-driven chronic inflammation for two weeks and healed in 6 weeks. An acute inflammation recurred at the same site when the recovered animals were systemically injected with TNBS. We isolated RNA from colon tissue at 24 h, 1, 2, 4, 6 weeks after TNBS treatment and from the relapsed animals. The mRNA for cytokines IL-1beta, IL-6, IL-10 and the chemokines CINC, MIP-1alpha, MCP-1 were significantly (P < 0.05) elevated and persisted for 2 weeks, decreased in 6 weeks and increased again during relapse. IFN-gamma mRNA stayed at control levels initially, but increased dramatically in the second weeks of chronic inflammation as well as in relapse. The mRNA levels of adhesion molecules, ICAM-1, VCAM-1, the mucosal homing integrin beta7 as well as P- and E-selectin were greatly enhanced between 1 and 3 weeks. The data showed that the chronically inflamed tissue expresses a time-dependent changing pattern of TH1 cytokines and adhesion molecules that maintain the infiltration and activation of inflammatory cells and tissue injury.

Evidence that neurones accumulating amyloid can undergo lysis to form amyloid plaques in Alzheimer's disease.

AIMS: Amyloid has recently been shown to accumulate intracellularly in the brains of patients with Alzheimer's disease (AD), yet amyloid plaques are generally thought to arise from gradual extracellular amyloid deposition. We have investigated the possibility of a link between these two apparently conflicting observations. METHODS AND RESULTS: Immunohistochemistry and digital image analysis was used to examine the detailed localization of beta-amyloid(42) (A beta 42), a major component of amyloid plaques, in the entorhinal cortex and hippocampus of AD brains. A beta 42 first selectively accumulates in the perikaryon of pyramidal cells as discrete, granules that appear to be cathepsin D-positive, suggesting that they may represent lysosomes or lysosome-derived structures. AD brain regions abundantly populated with pyramidal neurones exhibiting excessive A beta 42 accumulations also contained evidence of neuronal lysis. Lysis of these A beta 42-burdened neurones apparently resulted in a local, radial dispersion of their cytoplasmic contents, including A beta 42 and lysosomal enzymes, into the surrounding extracellular space. A nuclear remnant was found at the dense core of many amyloid plaques, strengthening the idea that each amyloid plaque represents the end product of a single neuronal cell lysis. The inverse relationship between the amyloid plaque density and pyramidal cell density in the AD brain regions also supports this possibility, as does the close correlation between plaque size and the size of local pyramidal cells. CONCLUSIONS: Our findings suggest that excessive intracellular accumulation of A beta 42-positive material in pyramidal cells can result in cell lysis, and that cell lysis is an important source of amyloid plaques and neuronal loss in AD brains.

Telomere associations in interphase nuclei: possible role in maintenance of interphase chromosome topology.

The relative sizes of individual telomeres in cultured human cells under conditions of cell cycling, replicative quiescence, cell transformation and immortalization were determined using quantitative fluorescence in situ hybridization (Q-FISH) with a telomere-specific peptide nucleic acid (PNA) probe. Results obtained from analysis of telomere length profiles (TLPs), which display the distribution of relative telomere lengths for individual cells, confirmed telomere length heterogeneity at the single cell level and proportional shortening of telomere length during replicative aging of virus-transformed cells. TLPs also revealed that some telomeric ends of chromosomes are so closely juxtaposed within interphase nuclei that their fluorescent signals appear as a single spot. These telomeric associations (TAs) were far more prevalent in interphase nuclei of noncycling normal and virus-transformed cells than in their cycling counterparts. The number of interphase TAs per nucleus observed in late-passage E6/E7-transformed cells did not increase during progression to crisis, suggesting that telomere shortening does not increase the frequency of interphase TAs. Furthermore, interphase TAs were rarely observed in rapidly cycling, telomerase-positive, immortalized cells that exhibit somewhat shortened, but stabilized, telomere length through the activity of telomerase. Our overall results suggest that the number of interphase TAs is dependent more on whether or not cells are cycling than on telomere length, with TAs being most prominent in the nuclei of replicatively quiescent cells in which nonrandom (even preferred) chromosome spatial arrangements have been observed. We propose that interphase TAs may play a role in the generation and/or maintenance of nuclear architecture and chromosome positional stability in interphase nuclei, especially in cells with a prolonged G(1)/G(0) phase and possibly in terminally differentiated cells.

Full amnesty could encourage provider self-disclosure.

The Federal government's current policy toward healthcare providers that voluntarily disclose improprieties has been ineffective because it offers no guarantee of immunity from prosecution. To be successful, a self-disclosure program must offer real incentives to providers to come forward. The government's self-disclosure programs with respect to tax, environmental, and antitrust laws provide models for an effective amnesty program. The success of these three programs, and particularly of the antitrust program, suggests that healthcare providers would be encouraged to come forward and disclose improprieties if, under certain specific conditions, the OIG and DOJ offered a guarantee of full amnesty to the entity and its officers, directors, and employees.

Rod outer segment membrane guanylate cyclase type 1-linked stimulatory and inhibitory calcium signaling systems in the pineal gland: biochemical, molecular, and immunohistochemical evidence.

Recent evidence indicates the presence of a novel alpha(2D/A)-adrenergic receptor (alpha(2D/A)-AR) linked membrane guanylate cyclase signal transduction system in the pineal gland. This system operates via a Ca(2+)-driven rod outer segment membrane guanylate cyclase (ROS-GC). In the present study, this transduction system has been characterized via molecular, immunohistochemical, and biochemical approaches. The two main components of the system are ROS-GC1 and its Ca(2+) regulator, S100B. Both components coexist in pinealocytes where the signaling component alpha(2D/A)-AR also resides. The presence of ROS-GC2 was not detected in the pineal gland. Thus, transduction components involved in processing alpha(2D/A)-AR-mediated signals are Ca(2+), S100B, and ROS-GC1. During this investigation, an intriguing observation was made. In certain pinealocytes, ROS-GC1 coexisted with its other Ca(2+) modulator, guanylate cyclase activating protein type 1 (GCAP1). In these pinealocytes, S100B was not present. The other GCAP protein, GCAP2, which is also a known modulator of ROS-GC in photoreceptors, was not present in the pineal gland. The results establish the identity of an alpha(2D/A)-AR-linked ROS-GC1 transduction system in pinealocytes. Furthermore, the findings show that ROS-GC1, in a separate subpopulation of pinealocytes, is associated with an opposite Ca(2+) signaling pathway, which is similar to phototransduction in retina. Thus, like photoreceptors, pinealocytes sense both positive and negative Ca(2+) signals, where ROS-GC1 plays a pivotal role; however, unlike photoreceptors, the pinealocyte is devoid of the ROS-GC2/GCAP2 signal transduction system.

Chromosomes exhibit preferential positioning in nuclei of quiescent human cells.

The relative spatial positioning of chromosomes 7, 8, 16, X and Y was examined in nuclei of quiescent (noncycling) diploid and triploid human fibroblasts using fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes and digital imaging. In quiescent diploid cells, interhomolog distances and chromosome homolog position maps revealed a nonrandom, preferential topology for chromosomes 7, 8 and 16, whereas chromosome X approximated a more random distribution. Variations in the orientation of nuclei on the culture substratum tended to hinder detection of an ordered chromosome topology at interphase by biasing homolog position maps towards random distributions. Using two chromosome X homologs as reference points in triploid cells (karyotype = 69, XXY), the intranuclear location of chromosome Y was found to be predictable within remarkably narrow spatial limits. Dual-FISH with various combinations of chromosome-specific DNA probes and contrasting fluorochromes was used to identify adjacent chromosomes in mitotic rosettes and test whether they are similarly positioned in interphase nuclei. From among the combinations tested, chromosomes 8 and 11 were found to be closely apposed in most mitotic rosettes and interphase nuclei. Overall, results suggest the existence of an ordered interphase chromosome topology in quiescent human cells in which at least some chromosome homologs exhibit a preferred relative intranuclear location that may correspond to the observed spatial order of chromosomes in rosettes of mitotic cells.

NO modulates P-selectin and ICAM-1 mRNA expression and hemodynamic alterations in hepatic I/R.

The anti-inflammatory role of nitric oxide (NO) was studied in a model of hepatic ischemia-reperfusion (I/R) in rats. Male Fischer rats were subjected to 30 min of no-flow ischemia of the left and median lobes of the liver, and animals were examined for a 4-h period of reperfusion. The animals were divided into the following groups: control-vehicle; I/R-vehicle; I/R-Nomega-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg iv, 10 min before reperfusion); sham control-L-NAME, and I/R-S-nitroso-N-acetyl-penicillamine (SNAP, 25 micromol/kg iv, 10 min before reperfusion, followed by 20 micromol. kg-1. h-1 in 1.0 ml saline infused for 4 h). Results showed that mean arterial blood pressure was significantly increased in the sham control-L-NAME or I/R-L-NAME groups compared with either the I/R-vehicle or I/R-SNAP groups. However, cardiac index (CI) and stroke volume index (SVI) were markedly decreased, and systemic vascular resistance index (SVRI) was dramatically increased. Interestingly, the CI and SVI in rats treated with SNAP were markedly improved over that of the I/R group. Plasma nitrate and nitrite levels were significantly decreased in the I/R-L-NAME group; however, superoxide generation in the ischemic lobes and plasma alanine aminotransferase activity were higher compared with I/R-SNAP rats. The L-NAME-induced enhancement of hepatic injury in rats with I/R may be due in part to neutrophil infiltration, which was significantly increased compared with animals subjected to I/R or I/R-SNAP. The mechanism of L-NAME-enhanced neutrophil infiltration may be due to the fact that the ratios of P-selectin and intercellular adhesion molecule 1 (ICAM-1) mRNA to glyceraldehyde-3-phosphate dehydrogenase mRNA extracted from the ischemic lobes of I/R-L-NAME rats were significantly increased when compared with the I/R-SNAP group. These results suggest that 1) endogenous NO reduces the SVRI and permits an increased CI and SVI; 2) exogenous NO further improves CI and SVI; and 3) endogenous, but not exogenous, NO decreases P-selectin and ICAM-1 mRNA expression, thereby reducing polymorphonuclear neutrophil-dependent reperfusion tissue injury.

Inhibition of nitric oxide synthase attenuates peroxynitrite generation, but augments neutrophil accumulation in hepatic ischemia-reperfusion in rats.

The role of nitric oxide (NO) and peroxynitrite in the process of neutrophil adhesion and infiltration was investigated in a model of hepatic ischemia-reperfusion. Male Fischer rats were subjected to 30 min of hepatic no-flow ischemia followed by 4 h of reperfusion (I/R). I/R induced liver injury as evidenced by a 13.7-fold increase in plasma alanine aminotransferase activity. Induction of liver injury was associated with an increase in neutrophil accumulation in ischemic lobes of livers [215 +/- 27 polymorphonuclear neutrophil leukocytes/50 high-power field (HPF), P < .05 compared with sham control] and 8-fold augmentation of inducible NO synthase (NOS) activity. However, NO levels in the liver decreased; this decrease may be caused by peroxynitrite formation by the reaction of NO with superoxide. Sections of ischemic lobes of the liver tissue of I/R animals exhibited marked immunoreactivity with anti-nitrotyrosine antibody, which indicates the presence of nitrotyrosine. Administration of Nw-nitro-L-arginine methyl ester (10 mg/kg i.v. before reperfusion) attenuated total and inducible NOS activity in both ischemic and nonischemic lobes of liver, and reduced NO levels in plasma and liver. However, NOS inhibition aggravated liver injury as alanine aminotransferase increased by 61% compared with rats subjected to reperfusion injury. Neutrophil accumulation was enhanced in ischemic (436 +/- 48/50 HPF, P < .05 compared with I/R animal) and nonischemic lobes of livers (34 +/- 3.2/50 HPF, P < .05 compared with sham control). NOS inhibition also attenuated immunohistochemically detected nitrotyrosine formation, but increased superoxide production in the liver. The NO-dependent regulation of neutrophil accumulation in the liver may be linked closely to P-selectin and intracellular adhesion molecule-1 expression because inhibition of NOS resulted in significant increases in gene expression of these two adhesion molecules (determined by reverse transcription-polymerase chain reaction analysis). These results suggest that NO is important in attenuating neutrophil accumulation and liver damage in ischemia-reperfusion injury. Inhibition of NOS activity reduces peroxynitrite formation but aggravates liver injury and increases neutrophil accumulation, which suggests that the anti-inflammatory function of NO is more important than the cytotoxic potential of peroxynitrite in acute inflammation.

Augmentation of nitric oxide, superoxide, and peroxynitrite production during cerebral ischemia and reperfusion in the rat.

The effect of ischemia produced by bilateral occlusion of the common carotid arteries (30 min) followed by 4 hours of reperfusion on total and inducible nitric oxide synthase (NOS) activity and the production of nitric oxide (NO), superoxide and peroxynitrite in the cerebral hemispheres was determined in the rat. Compared to sham-operated controls, cerebral ischemia-reperfusion resulted in a significant increase in total and inducible NOS activity and a significant increase in the production of NO and superoxide in the cerebral hemispheres. The level of NO in the plasma and the peripheral leukocyte count were also significantly increased. Immunohistochemical staining for nitrotyrosine (a marker of peroxynitrite production) showed that ischemia-reperfusion resulted in increased synthesis of cerebral peroxynitrite. Administration of the irreversible NOS inhibitor, Nomega-nitro-L-arginine (L-NA), increased superoxide levels in the brain and significantly reduced plasma NO. Total and inducible NOS activity as well as NO and immunoreactive nitrotyrosine, in the cerebral hemispheres were reduced with L-NA administration. The number of leukocytes in the plasma was unaffected by administration of L-NA. These findings suggest that cerebral ischemia-reperfusion causes increased production of reactive oxygen species in the cerebral hemispheres and that the production of peroxynitrite, and not superoxide, may be dependent upon the availability of NO.

Models to elucidate the regulation of adrenal cell death.

Hypophysectomy-induced apoptosis in the rat adrenal gland is slow (not apparent until 12-24 h) and in-situ, 3'-end labeling (ISEL) of DNA strand breaks is restricted to a subpopulation of zona reticularis cells. In addition, it is completely blocked by ACTH. By contrast, apoptosis in the intact rat adrenal gland, cultured in the absence of trophic support, is extensive and rapid. Culture-triggered apoptosis (as determined by oligonucleosome formation) is attenuated by ACTH and is largely restricted to the zonae fasciculata and reticularis even at 3 h (as determined by ISEL and DAPI cytochemistry). Thus, this organ culture system may help elucidate factors that can acutely regulate adrenocortical cell survival. Quartered glands have a nearly 2-fold increase in oligonucleosome formation compared to intact glands at 3 h and are resistant to the antiapoptotic action of ACTH. In contrast, ACTH-induced corticosterone secretion is not attenuated. Angiotensin II (Ang II) enhances culture-triggered apoptosis, and its apoptotic action is attenuated by ACTH. These observations suggest that 1) acute hormonal modulation of apoptosis may require some level of gross adrenal structural integrity, and 2) ACTH and Ang II act in an antagonistic fashion to regulate adrenocortical apoptosis. The apoptotic effect of Ang II may be mediated via the type 2 receptor.

Chromosome spatial order in human cells: evidence for early origin and faithful propagation.

We have investigated the origin and nature of chromosome spatial order in human cells by analyzing and comparing chromosome distribution patterns of normal cells with cells showing specific chromosome numerical anomalies known to arise early in development. Results show that all chromosomes in normal diploid cells, triploid cells and in cells exhibiting nondisjunction trisomy 21 are incorporated into a single, radial array (rosette) throughout mitosis. Analysis of cells using fluorescence in situ hybridization, digital imaging and computer-assisted image analysis suggests that chromosomes within rosettes are segregated into tandemly linked "haploid sets" containing 23 chromosomes each. In cells exhibiting nondisjunction trisomy 21, the distribution of chromosome 21 homologs in rosettes was such that two of the three homologs were closely juxtaposed, a pattern consistent with our current understanding of the mechanism of chromosomal nondisjunction. Rosettes of cells derived from triploid individuals contained chromosomes segregated into three, tandemly linked haploid sets in which chromosome spatial order was preserved, but with chromosome positional order in one haploid set inverted with respect to the other two sets. The spatial separation of homologs in triploid cells was chromosome specific, providing evidence that chromosomes occupy preferred positions within the haploid sets. Since both triploidy and nondisjunction trisomy 21 are chromosome numerical anomalies that arise extremely early in development (e.g., during meiosis or during the first few mitoses), our results support the idea that normal and abnormal chromosome distribution patterns in mitotic human cells are established early in development, and are propagated faithfully by mitosis throughout development and into adult life. Furthermore, our observations suggest that segregation of chromosome homologs into two haploid sets in normal diploid cells is a remnant of fertilization and, in normal diploid cells, reflects segregation of maternal and paternal chromosomes.