Mitochondrial DNA COI characterization of Helicoverpa armigera (Lepidoptera: Noctuidae) from Paraguay and Uruguay.

Since its detection in Brazil in 2013, the Old World cotton bollworm Helicoverpa armigera has been reported in Argentina, Paraguay, and Bolivia. Here we present evidence extending the South American range of H. armigera to Uruguay, using polymerase chain reaction and sequencing of the partial mitochondrial DNA (mtDNA) cytochrome oxidase I region. Molecular characterization of this gene region from individuals from Paraguay also supports previous morphological identification of H. armigera in Paraguay. Shared mtDNA haplotypes in H. armigera from Brazil, Uruguay, and Paraguay were identified. Additional surveying of populations in this region will be imperative to better monitor and understand factors that are underpinning its presence and successful adaptation in these South American regions. We discuss our findings with respect to the development of resistance pest management strategies of this invasive insect pest in a predominantly monoculture soybean crop landscape in the Southern Cone region.

New and evolving concepts in the neurotoxicology of lead.

Lead (Pb) is a xenobiotic metal with no known essential function in cellular growth, proliferation, or signaling. Decades of research characterizing the toxicology of Pb have shown it to be a potent neurotoxicant, especially during nervous system development. New concepts in the neurotoxicology of Pb include advances in understanding the mechanisms and cellular specificity of Pb. Experimental studies have shown that stress can significantly alter the effects of Pb, effects that could potentially be mediated through alterations in the interactions of glucocorticoids with the mesocorticolimbic dopamine system of the brain. Elevated stress, with corresponding elevated glucocorticoid levels, has been postulated to contribute to the increased levels of many diseases and dysfunctions in low socioeconomic status populations. Cellular models of learning and memory have been utilized to investigate the potential mechanisms of Pb-induced cognitive deficits. Examination of long-term potentiation in the rodent hippocampus has revealed Pb-induced increases in threshold, decreases in magnitude, and shorter retention times of synaptic plasticity. Structural plasticity in the form of adult neurogenesis in the hippocampus is also impacted by Pb exposure. The action of Pb on glutamate release, NMDA receptor function, or structural plasticity may underlie perturbations in synaptic plasticity and contribute to learning impairments. In addition to providing insight into potential mechanisms of Pb-induced cognitive deficits, cellular models offer an opportunity to investigate direct effects of Pb on isolated biological substrates. A target of interest is the 78-kDa molecular chaperone glucose-regulated protein (GRP78). GRP78 chaperones the secretion of the cytokine interleukin-6 (IL-6) by astrocytes. In vitro evidence shows that Pb strongly binds to GRP78, induces GRP78 aggregation, and blocks IL-6 secretion in astroglial cells. These findings provide evidence for a significant chaperone deficiency in Pb-exposed astrocytes in culture. In the long term, chaperone deficiency could underlie protein conformational diseases such as Alzheimer's Disease (AD). Lead exposure in early life has been implicated in subsequent progression of amyloidogenesis in rodents during old age. This exposure resulted in an increase in proteins associated with AD pathology viz., beta-amyloid precursor protein (beta-APP), and beta-amyloid (Abeta). These four new lines of research comprise compelling evidence that exposures to Pb have adverse effects on the nervous system, that environmental factors increase nervous system susceptibility to Pb, and that exposures in early life may cause neurodegeneration in later life.

Substituted tetraazaacenaphthylenes as potent CRF1 receptor antagonists for the treatment of depression and anxiety.

Two isomers of the hexahydro-tetraazaacenaphthylene templates (1 and 2) are presented as novel, potent, and selective corticotropin releasing factor-1 (CRF1) receptor antagonists. In this paper, we report the affinity and SAR of a series of compounds, as well as pharmacokinetic characterization of a chosen set. The anxiolitic activity of a selected example (2ba) in the rat pup vocalization model is also presented.

Neurotoxicity of polycyclic aromatic hydrocarbons and simple chemical mixtures.

Polycyclic aromatic hydrocarbons (PAHs) are a major class of environmental pollutants. These chemicals are the products of incomplete combustion and are present in every compartment of the environment. While the carcinogenic potential of these chemicals has been investigated in numerous studies, very little is known about the potential of these chemicals to produce damage to neural cells. The objective of this study was to investigate the toxicity of several model PAHs and binary mixtures of these chemicals in neural cells. Chemicals tested included benzo[a]pyrene (BaP), chrysene, anthracene, and pentachlorophenol (PCP). Four end points, including amino acid incorporation, total protein, total cell count, and viable cells (trypan dye exclusion), were measured in SY5Y human neuroblastoma cells and C6 rat glioma cells. The most sensitive measure of PAH toxicity in neural cells was amino acid incorporation into proteins. BaP was the most toxic of all PAHs tested, and anthracene failed to produce a toxic response at any concentration tested. Without metabolic activation, BaP induced a significant cytotoxic response at a concentration of 30 microM. With activation (0.25% S9), BaP induced a response at concentration levels of 3 microM and 30 microM. Minimal toxicity was observed with chrysene at the highest concentration tested, and anthracene failed to produce a toxic response at any concentration tested. With mixtures of PAHs the majority of samples induced additive responses. The minimum concentration required to induce a significant response was reduced for the mixture of chrysene and BaP when compared to BaP alone. In addition, PCP appeared to increase the inhibition of acetylcholinesterase by mipafox. The data suggest that PAHs are capable of producing damage to neural cells only at concentrations that are near their solubility limits.

Induction of 78 kD glucose-regulated protein (GRP78) expression and redox-regulated transcription factor activity by lead and mercury in C6 rat glioma cells.

Lead (Pb) and mercury (Hg) are widespread environmental contaminants that induce prominent neural toxicity. Although the brain is not the major Pb and Hg depot in the body, these metals preferentially accumulate in astroglia to exert toxic effects. In this study, we examined the effects of Pb acetate and HgCl(2) on the expression of GRP78, a molecular chaperone in the endoplasmic reticulum (ER) that may provide cytoprotection in response to cellular stresses in the C6 rat glioma cell line. We also evaluated the DNA binding activities of several redox-regulated transcription factors in metal-treated cells. Our results showed that mRNA levels of GRP78 were up-regulated by Pb and Hg at 0.1 and 1 micro M, but down-regulated at higher concentrations (10 micro M). GRP78 protein levels increased in a concentration- and time-dependent manner in Pb and/or Hg-treated cells. Pb increased protein binding to the GST- Upsilon a antioxidant/electrophile response element (ARE/EpRE) and to the NF- kappaB consensus binding sequence of the cytomegalovirus 2 (CMB2) promoter, but decreased protein binding to the Ha-ras ARE/EpRE or to the c-fos 12-O-tetradecanoyl-phorbol-13-acetate (TPA) response element (TRE). In contrast, Hg activated DNA binding by all redox-regulated transcription factors. These studies shed some light on the molecular mechanisms of Pb and Hg toxicity in C6 rat glioma cells and suggest that GRP78 and oxidative stress may participate in the neurotoxic response to these metals.

An integrating sphere to measure CD from difficult samples

Integrating spheres are widely used with UV-Vis and occasionally with infrared spectrophotometers to measure different types of samples, either in transmission mode (scattered transmission accessories) or in total/diffuse reflectance mode. We built a prototype sphere of the demountable type, which fits easily the sample compartment of a commercial CD spectropolarimeter, requiring neither any alignment nor the use of a dedicated photomultiplier. Samples can be inserted either at the sphere entrance (for scattered transmission mode) or in the center of the sphere (for total reflectance experiments). Selected experimental data are presented to evaluate sphere efficiency, its wavelength range and results with a single sample in different forms. Copyright 2000 Wiley-Liss, Inc.

Lead targets GRP78, a molecular chaperone, in C6 rat glioma cells.

Exposure to potentially neurotoxic levels of lead (Pb) occurs in about 9% of American children under 6 years of age. Astroglia in the brain serve as a Pb depot, sequestering Pb and preventing its contact with the more sensitive neurons. Astroglia have the capacity to adapt to Pb exposure, and as such are able to tolerate relatively high intracellular Pb accumulation. This tolerance mechanism has yet to be defined in biochemical terms. In the present study, we present evidence that glucose-regulated protein (GRP78), a molecular chaperone in the ER, participates directly or indirectly in the tolerance mechanism. Exposure of cultured C6 rat glioma cells, an astroglia-like cell line, to 1 microM Pb acetate for 1 week raised the intracellular levels of two proteins, one of which was identified by sequence analysis as GRP78. GRP78 accumulation started within 1 day and progressed with time of exposure. Studies in vitro showed that GRP78 bound tightly to affinity columns with Pb(2+) as the affinity ligand and bound weakly when either Zn(2+) or Ni(2+) replaced the Pb(2+). The reduced form of GSH and BSA did not compete with GRP78 to chelate Pb(2+). However, the heavy metal binding domain (HMB) of Menkes protein competed with GRP78 for chelating Pb(2+). The data provide evidence that GRP78 may be a component of the Pb tolerance mechanism through its direct interaction with Pb(2+). Its increased synthesis could be part of the adaptive response to Pb exposure.

Reliable assay of extreme enantiomeric purity values by a new circular dichroism based HPLC detection system.

A new sensitive, selective, and versatile circular dichroism (CD)-based HPLC detection system was used for the validation of the enantiomeric purity assay in the quality control of chiral drugs upon nonchiral stationary phases. The precision and the accuracy of the method were checked for selected samples showing values of the anisotropy factor on the order of 10(-1) to 10(-4). Very high accuracy has been obtained also in the case of extreme enantiomeric purity values (/=99% e.p.) and of a low anisotropy factor (g = 2 x 10(-4)) compound. The high selectivity of this detection system allows a selective monitoring of analytes in complex mixtures and makes the baseline stable.

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters hippocampal astroglia-neuronal gap junctional communication.

Halogenated aromatic hydrocarbons (HAHs) such as dibenzo-p-dioxins are known to alter cognitive function. However, the cellular basis of this disruption is not well understood. One possible deleterious effect of exposure to HAHs could be on gap junctional intercellular communication (GJIC) between neurons and astroglia in the brain. As such, this study examined the effects of the highly toxic prototypic HAH, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on GJIC in rat hippocampal primary cell culture. Initial measurements of fluorescence recovery after photobleaching (gap-FRAP) showed dye transfer between astroglia and neurons. N-octanol, a lipophilic alcohol known to uncouple cells by decreasing the open probability of gap junctional channels blocked astroglial-neuronal (A-N) communication as well as astroglial-astroglial (A-A) communication. TCDD initially downregulated GJIC between neurons and astroglia of treatment, but had no effect on astroglial cell pairs. These results indicate the presence of GJIC between neurons and astroglia in culture and demonstrate different sensitivities of gap junction responses to TCDD in homologous and heterologous cell pairs. The finding that 2,3,7,8-TCDD disrupts GJIC through A-N but not A-A channels may have important implications for impaired brain function resulting from developmental exposure to TCDD.

Bridging the gap between in vitro and in vivo models for neurotoxicology.

In vitro systems are widely used for investigation of neurotoxicant-induced perturbations of cellular functions. A variety of systems exist that demonstrate certain similarities to neurotoxicant-induced events in the intact animal are discussed, including single-cell types, systems that consider endpoints relevant in toxicology, and systems that consider heterogeneous cell interactions. Relationships between the in vitro and in vivo systems are examined in which ethanol, lead, polychlorinated biphenyl compounds, and organophosphate insecticides are examples. Situations in which the in vitro systems have been used to advantage are provided, along with cautions associated with their use.

Blood flow in the internal carotid and middle cerebral arteries: effects of continuous oral conjugated equine estrogens administration with monthly progestogen supplementation on postmenopausal women.

OBJECTIVE: This study was designed in order to evaluate the effect of conjugated equine estrogens (CEE) on internal carotid and middle cerebral artery blood flow in postmenopausal women. DESIGN: Thirty-four healthy postmenopausal women with intact uteri were randomly divided into two groups of 17 subjects each. The first group was treated for 24 weeks with continuous CEE medication (0.625 mg daily) and cyclical supplementations of 5 mg/day of medrogestone acetate, given on the last 12 days of every 4-week period (Prempak, Wyeth, Italy). The second group received no treatment. The pulsatility indices (PI) of both the internal carotid artery and middle cerebral artery were measured. RESULTS: In the treated group, the PI of the interior carotid artery and MCA was reduced from respectively 0.736 (0.016) and 0.745 (0.009) at baseline, to 0.669 (0.021) and 0.670 (0.011) after 24 weeks (p = 0.01); in the control group, the PI values remained unchanged. The between-group difference for both arteries was significant (p < 0.01). CONCLUSIONS: The administration of CEE with cyclical medrogestone supplementation to postmenopausal women induces a statistically significant reduction in the PI of cerebral arteries.

Differential ability of astroglia and neuronal cells to accumulate lead: dependence on cell type and on degree of differentiation.

The apparent ability of astroglia to serve as a lead (Pb) sink in the mature brain may result from either their strategic location, between the blood-brain barrier and neurons, or from intrinsic differences between the ability of astroglia and neurons to accumulate this metal. This phenomenon may be dependent on the degree of cell differentiation. In order to address the latter possibility, Pb accumulation was compared among the following cell culture models: (1) mature and immature rat astroglia, (2) undifferentiated SY5Y human neuroblastoma cells and SY5Y cells differentiated with nerve growth factor, (3) immature rat astroglia grown in differently conditioned media, some of which induce partial differentiation, and (4) rat astroglia and SY5Y cells in co-culture. Astroglial cultures, prepared from 1-day-old rat cerebral hemispheres, were exposed to 1 microM Pb after either 14 (immature) or 21 (mature) days in culture. Pb content of the cells was measured by atomic absorption spectroscopy. Immature astroglia took up less Pb when glutathione (GSH) was added to the medium, suggesting that GSH may regulate Pb uptake in these cells. Undifferentiated neuroblastoma cells accumulated more Pb than did the differentiated ones. Astroglia accumulated up to 24 times more Pb than did neuronal cells. This ability was enhanced by exposure to conditioned medium from a neuroblastoma cell line, but not by endothelial cell-conditioned medium, although this medium induced the expression of a glutamate-activated Ca2+ response. Our findings are in agreement with in vivo studies, and thus validate the use of these cell-culture models for future studies on differential mechanisms of Pb uptake.

Effect of lead exposure and accumulation on copper homeostasis in cultured C6 rat glioma cells.

C6 rat glioma cells resemble rat astroglia in culture in that both cell types accumulate lead (Pb) intracellularly from the medium. As such, C6 cells are a model for Pb accumulation by the brain. In this study, an increase in intracellular Pb accumulation induced by p-chloromercuribenzoate (PCMB) after exposure to 10 microM Pb acetate suggests a role for sulfhydryl groups in Pb retention. Stimulation of Pb accumulation by nifedipine suggests the entry of Pb into these cells by a novel path. Most of the intracellular Pb from exposure for 7 days to 1 microM Pb was associated with high-molecular weight components in cytosol. Pb exposure increased the abundance of three proteins with the following characteristics on two-dimensional gels: 81 kDa with pI of 5.6, 81 kDa with pI of 4. 9, and 71 kDa with pI of 5.6. The levels of five other proteins, ranging in size from 37-41 kDa with pIs of 6.0-6.8 declined. Exposed C6 cells accumulated copper (Cu) intracellularly, and Cu accumulation after Pb exposure was shown by kinetic analysis with 67Cu to result from an increased uptake and a decreased efflux for Cu. Pb-exposed cells also showed increased Cu binding to membranes, which is consistent with the increase of Cu uptake. These data indicate that intracellular Pb interacts with high molecular weight proteins in C6 cells, and exposure also alters membrane transport properties for copper.

Multiple forms of the Menkes Cu-ATPase.

The 5' region of MNK cDNAs has a 45 bp insert terminating at the 5'end with an AGATG sequence. The ATG in the sequence is in-frame with the ATG downstream identified by Vulpe et al (1993) as a translation start site for MNK mRNA. Inserts of 192 bp and 45 bp have been found in the 5' region of MNK mRNAs from BeWo cells, Caco-2 cells and normal human fibroblasts. Extensions to the 5' end of these mRNAs could foretell a modified N-termini in certain forms of the Menkes Cu-ATPase. These modified H2N-terminal extensions are postulated to be targeting signals for post-translational processing and cellular localization. In this report, we provide evidence that the primary Menkes transcript in non-Menkes cells undergoes post-transcriptional splicing that gives rise to multiple transcripts. The data suggest that the Menkes gene is a copper locus that codes for more than one form of the Menkes Cu-ATPase and one of these forms could be a small Cu transport protein.

Proinflammatory and regulatory cytokine levels in AIDS cachexia.

The serum concentrations of inflammatory (Interleukin-1 beta, Tumor necrosis alpha, Interleukin-6) and regulatory cytokines (Interleukin twelve) have been studied in ten AIDS cachectic patients and compared to a control group. A cytokine imbalance, and peculiarly a significant increase in proinflammatory cytokines (Interleukin-1, Interleukin-6, Tumor necrosis Factor alpha) and a decrease in regulatory cytokines such as Interleukin-12 were found. A significant correlation resulted between weight loss and Interleukin-1 beta and 6. A negative correlation between Interleukin-1 and 12 was noted, indicating that this last cytokine has an important regulatory role also in advanced state of the disease.

Copper efflux from murine microvascular cells requires expression of the menkes disease Cu-ATPase.

Previously, we showed that the transport of Cu by PC12 pheochromocytoma cells and C6 glioma cells correlated with the expression of a Cu-transporting ATPase (Atp7a) that has been linked to Menkes disease. Here, we show that cerebrovascular endothelial (CVE) cells that comprise the blood-brain barrier (BBB) also express the gene for the Cu-ATPase. By using reverse transcription-polymerase chain reaction (RT-PCR) and primers designed from mouse Atp7a cDNA, we amplified a 925-bp and a 760-bp cDNA fragment from two extreme regions of Atp7a mRNA from murine CVE cells; 777 bp of the 925-bp fragment and 677 bp of the 760-bp fragment had a 99.7 and 100% sequence homology, respectively, with mouse Atp7a cDNA. The 777-bp sequences covered the heavy metal binding (Hmb) domain and the 677-bp fragment coded for residues at the -COOH terminus of Atp7a. A functional analysis showed that Cu efflux was blocked by the sulfhydryl reagent p-chloromercuribenzoate (p-CMB), a potential inhibitor of Atp7a function. This study provides strong evidence that a Cu-ATPase in the BBB controls the penetration of Cu into the brain and that lesions to the Cu-ATPase in CVE cells are a primary cause of low brain Cu levels in Menkes disease.

Functional analysis of copper homeostasis in cell culture models: a new perspective on internal copper transport.

The movement of copper ions across membrane barriers of vital organs and tissues is a priority topic in nutrition and one for which there continues to be little understanding of the mechanism. Reports of membrane-bound, copper-transporting adenosine triphosphatases (Cu-ATPases) selective for copper ions have brought new focus to the problem and prompted fresh ideas. Using a cell culture model approach, we attempted to learn whether transport into and out of cells depends on a Cu-ATPase. Measurement of transport kinetics in fibroblasts, brain glial cells, neuroblastoma cells, and placental cells showed differences in the rates of copper uptake and response to sulfhydryl reagents. BeWo cells, a human choriocarcinoma placental cell line, behaved as did Menkes fibroblasts by avidly absorbing copper but not releasing copper to the immediate environment. Further tests showed that BeWo cells did not express the transcript for the membrane-bound Cu-ATPase that has been identified with Menkes syndrome. Transcript induction, however, was achieved by growing BeWo cells on porous filters that allowed apical and basolateral surfaces to form. With transcript expression, the cells showed a capacity to release copper into the medium. BeWo cells also synthesized a form of ceruloplasmin whose structure differed from that of the plasma protein and hence may be a product of a different gene. BeWo cells may also express the gene for Wilson disease, thus linking Menkes and Wilson proteins to maternal delivery of copper. We constructed a model in which both ATPases work in concert in a vesicle-based transport mechanism. The vesicle model may help us understand the transport of copper across the placenta and all cells in general.