DNA based diagnostic for the quantification of sugarcane root DNA in the field.

Plant root systems play many key roles including nutrient and water uptake, interface with soil microorganisms and resistance to lodging. As for other crops, large and systematic studies of sugarcane root systems have always been hampered by the opaque and solid nature of the soil. In recent years, methods for efficient extraction of DNA from soil and for species-specific DNA amplification have been developed. Such tools could have potential to greatly improve root phenotyping and health diagnostic capability in sugarcane. In this paper, we present a fast, specific and efficient method for the quantification of sugarcane live root cells in soil samples. Previous studies were typically based on mass and length, so we established a calibration to convert root DNA quantity to live root mass. This diagnostic was validated on field samples and used to investigate the fate of the root system after harvest prior to regrowth of the ratoon crop. Two weeks after harvest, the sugarcane roots from the previous crop were still viable. This raises the question of the role that the root system of the harvested crop plays in the performance of the next crop and demonstrates how this test can be used to answer research questions.

An overview of internet biosurveillance.

Internet biosurveillance utilizes unstructured data from diverse web-based sources to provide early warning and situational awareness of public health threats. The scope of source coverage ranges from local media in the vernacular to international media in widely read languages. Internet biosurveillance is a timely modality that is available to government and public health officials, healthcare workers, and the public and private sector, serving as a real-time complementary approach to traditional indicator-based public health disease surveillance methods. Internet biosurveillance also supports the broader activity of epidemic intelligence. This overview covers the current state of the field of Internet biosurveillance, and provides a perspective on the future of the field.

Event-based biosurveillance of respiratory disease in Mexico, 2007-2009: connection to the 2009 influenza A(H1N1) pandemic?

The emergence of the 2009 pandemic influenza A(H1N1) virus in North America and its subsequent global spread highlights the public health need for early warning of infectious disease outbreaks. Event-based biosurveillance, based on local- and regional-level Internet media reports, is one approach to early warning as well as to situational awareness. This study analyses media reports in Mexico collected by the Argus biosurveillance system between 1 October 2007 and 31 May 2009. Results from Mexico are compared with the United States and Canadian media reports obtained from the HealthMap system. A significant increase in reporting frequency of respiratory disease in Mexico during the 2008-9 influenza season relative to that of 2007-8 was observed (p<0.0001). The timing of events, based on media reports, suggests that respiratory disease was prevalent in parts of Mexico, and was reported as unusual, much earlier than the microbiological identification of the pandemic virus. Such observations suggest that abnormal respiratory disease frequency and severity was occurring in Mexico throughout the winter of 2008-2009, though its connection to the emergence of the 2009 pandemic influenza A(H1N1) virus remains unclear.

In vitro studies of amyloid beta-protein fibril assembly and toxicity provide clues to the aetiology of Flemish variant (Ala692-->Gly) Alzheimer's disease.

In a Flemish kindred, an Ala(692)-->Gly amino acid substitution in the amyloid beta-protein precursor (AbetaPP) causes a form of early-onset Alzheimer's disease (AD) which displays prominent amyloid angiopathy and unusually large senile plaque cores. The mechanistic basis of this Flemish form of AD is unknown. Previous in vitro studies of amyloid beta-protein (Abeta) production in HEK-293 cells transfected with cDNA encoding Flemish AbetaPP have shown that full-length [Abeta(1-40)] and truncated [Abeta(5-40) and Abeta(11-40)] forms of Abeta are produced. In an effort to determine how these peptides might contribute to the pathogenesis of the Flemish disease, comparative biophysical and neurotoxicity studies were performed on wild-type and Flemish Abeta(1-40), Abeta(5-40) and Abeta(11-40). The results revealed that the Flemish amino acid substitution increased the solubility of each form of peptide, decreased the rate of formation of thioflavin-T-positive assemblies, and increased the SDS-stability of peptide oligomers. Although the kinetics of peptide assembly were altered by the Ala(21)-->Gly substitution, all three Flemish variants formed fibrils, as did the wild-type peptides. Importantly, toxicity studies using cultured primary rat cortical cells showed that the Flemish assemblies were as potent a neurotoxin as were the wild-type assemblies. Our results are consistent with a pathogenetic process in which conformational changes in Abeta induced by the Ala(21)-->Gly substitution would facilitate peptide adherence to the vascular endothelium, creating nidi for amyloid growth. Increased peptide solubility and assembly stability would favour formation of larger deposits and inhibit their elimination. In addition, increased concentrations of neurotoxic assemblies would accelerate neuronal injury and death.

A de novo designed helix-turn-helix peptide forms nontoxic amyloid fibrils.

We report here that a monomeric de novo designed alpha-helix-turn-alpha-helix peptide, alpha t alpha, when incubated at 37 degrees C in an aqueous buffer at neutral pH, forms nonbranching, protease resistant fibrils that are 6-10 nm in diameter. These fibrils are rich in beta-sheet and bind the amyloidophilic dye Congo red. alpha t alpha fibrils thus display the morphologic, structural, and tinctorial properties of authentic amyloid fibrils. Surprisingly, unlike fibrils formed by peptides such as the amyloid beta-protein or the islet amyloid polypeptide, alpha t alpha fibrils were not toxic to cultured rat primary cortical neurons or PC12 cells. These results suggest that the potential to form fibrils under physiologic conditions is not limited to those proteins associated with amyloidoses and that fibril formation alone is not predictive of cytotoxic activity.

An improved method of preparing the amyloid beta-protein for fibrillogenesis and neurotoxicity experiments.

Synthetic amyloid beta-protein (A beta) is used widely to study fibril formation and the physiologic effects of low molecular weight and fibrillar forms of the peptide on cells in culture or in experimental animals. Not infrequently, conflicting results have arisen in these studies, in part due to variation in the starting conformation and assembly state of A beta. To avoid these problems, we sought a simple, reliable means of preparing A beta for experimental use. We found that solvation of synthetic peptide with sodium hydroxide (A beta x NaOH), followed by lyophilization, produced stocks with superior solubility and fibrillogenesis characteristics. Solubilization of the pretreated material with neutral buffers resulted in a pH transition from approximately 10.5 to neutral, avoiding the isoelectric point of A beta (pI approximately 5.5), at which A beta precipitation and aggregation propensity are maximal. Relative to trifluoroacetate (A beta x TFA) or hydrochloric acid (A beta x HCl) salts of A beta, yields of "low molecular weight A beta" (monomers and/or dimers) were improved significantly by NaOH pretreatment. Time-dependent changes in circular dichroism spectra and Congo red dye-binding showed that A beta x NaOH formed fibrils more readily than did the other A beta preparations and that these fibrils were equally neurotoxic. NaOH pretreatment thus offers advantages for the preparation of A beta for biophysical and physiologic studies.

Protofibrillar intermediates of amyloid beta-protein induce acute electrophysiological changes and progressive neurotoxicity in cortical neurons.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is thought to be caused in part by the age-related accumulation of amyloid beta-protein (Abeta). The presence of neuritic plaques containing abundant Abeta-derived amyloid fibrils in AD brain tissue supports the concept that fibril accumulation per se underlies neuronal dysfunction in AD. Recent observations have begun to challenge this assumption by suggesting that earlier Abeta assemblies formed during the process of fibrillogenesis may also play a role in AD pathogenesis. Here, we present the novel finding that protofibrils (PF), metastable intermediates in amyloid fibril formation, can alter the electrical activity of neurons and cause neuronal loss. Both low molecular weight Abeta (LMW Abeta) and PF reproducibly induced toxicity in mixed brain cultures in a time- and concentration-dependent manner. No increase in fibril formation during the course of the experiments was observed by either Congo red binding or electron microscopy, suggesting that the neurotoxicity of LMW Abeta and PF cannot be explained by conversion to fibrils. Importantly, protofibrils, but not LMW Abeta, produced a rapid increase in EPSPs, action potentials, and membrane depolarizations. These data suggest that PF have inherent biological activity similar to that of mature fibrils. Our results raise the possibility that the preclinical and early clinical progression of AD is driven in part by the accumulation of specific Abeta assembly intermediates formed during the process of fibrillogenesis.

Amyloid beta-protein fibrillogenesis. Structure and biological activity of protofibrillar intermediates.

Alzheimer's disease is characterized by extensive cerebral amyloid deposition. Amyloid deposits associated with damaged neuropil and blood vessels contain abundant fibrils formed by the amyloid beta-protein (Abeta). Fibrils, both in vitro and in vivo, are neurotoxic. For this reason, substantial effort has been expended to develop therapeutic approaches to control Abeta production and amyloidogenesis. Achievement of the latter goal is facilitated by a rigorous mechanistic understanding of the fibrillogenesis process. Recently, we discovered a novel intermediate in the pathway of Abeta fibril formation, the amyloid protofibril (Walsh, D. M., Lomakin, A., Benedek, G. B., Condron, M. M., and Teplow, D. B. (1997) J. Biol. Chem. 272, 22364-22372). We report here results of studies of the assembly, structure, and biological activity of these polymers. We find that protofibrils: 1) are in equilibrium with low molecular weight Abeta (monomeric or dimeric); 2) have a secondary structure characteristic of amyloid fibrils; 3) appear as beaded chains in rotary shadowed preparations examined electron microscopically; 4) give rise to mature amyloid-like fibrils; and 5) affect the normal metabolism of cultured neurons. The implications of these results for the development of therapies for Alzheimer's disease and for our understanding of fibril assembly are discussed.

Attenuation of cortical neuronal apoptosis by gangliosides.

Addition of the natural gangliosides monosialoganglioside (GM1), disialoganglioside, trisialoganglioside, or tetrasialoganglioside in the range of 10 to 100 microM, but not asialoganglioside lacking the sialic acid moiety, attenuated cortical neuronal apoptosis induced by serum deprivation, ionomycin, or cyclosporin A but not by protein kinase inhibitors (staurosporine, genistein, lavendustin A, or herbimycin A). Coaddition of 100 nM wortmannin, a selective inhibitor of phosphatidylinositol 3-kinase, but not 1 microM Go6976, a selective protein kinase C inhibitor, blocked the neuroprotective effect of GM1. In contrast to its antiapoptotic effect, GM1 at up to 200 microM did not attenuate cortical neuronal necrosis induced by exposure to the excitotoxins N-methyl-D-aspartate or kainate. Furthermore, GM1 increased the necrosis induced by oxidative stress (addition of Fe(2+) or buthionine sulfoximine). These data suggest that neuroprotective effects of natural gangliosides may preferentially reflect reduction of neuronal apoptosis rather than necrosis, and be mediated through mechanisms involving activation of phosphatidylinositol 3-kinase.

Expression of the calcium-binding protein, parvalbumin, in cultured cortical neurons using a HSV-1 vector system enhances NMDA neurotoxicity.

Calcium-binding proteins (CaBPs) are a family of proteins having a unique distribution in the brain and are thought to be important in buffering intracellular calcium. Glutamate neurotoxicity is a process by which the over-activation of glutamate receptors can cause the influx of excessive extracellular calcium and neuronal cell death. It has been proposed that neurons containing CaBP may be more resistant to glutamate neurotoxicity due to their increased ability to buffer calcium. Using a herpes simplex virus-1 (HSV-1) vector system we packaged the CaBP gene, parvalbumin, or the marker gene, beta-galactosidase (beta-gal), correctly in viron particles, which were found upon infection to express mRNA specific to these vectors. PC12 and neocortical cultures showed strong immunohistochemical staining for either beta-gal or parv. The cortical cultures stained positively for endogenous glutamate decarboxylase, a marker for GABAergic neurons, but not for endogenous parvalbumin, indicating that parvalbumin was being expressed ectopically from the HSV-1 vector. Interestingly, the expression of parvalbumin increased cortical culture's susceptibility to N-methyl-D-aspartate-induced neurotoxicity. This increase in neurotoxicity was not due to the wild-type virus or the helper virus which accompanies the packaging of these vectors. We speculate that the ectopic expression of parvalbumin in cortical cultures may be increasing glutamate release which in turn increases cell death.

Glutamate receptor-induced 45Ca2+ accumulation in cortical cell culture correlates with subsequent neuronal degeneration.

Murine neuronal and glial cell cultures exposed briefly to glutamate accumulated large amounts of 45Ca2+ from the extracellular medium during the exposure. Most of the accumulation likely reflected influx into neurons, as little accumulation was observed in similarly treated glial cultures. When the concentration of glutamate was varied between 10 and 1000 microM, or exposure duration was varied between 0 and 10 min, the amount of 45Ca2+ accumulation correlated closely with the amount of neuronal death 24 hr later. Both 45Ca2+ accumulation and cell death could be attenuated in a dose-dependent manner by the competitive NMDA antagonist D-aminophosphonovalerate or the noncompetitive antagonist dextrorphan, with IC50 values of approximately 100 microM and 15 microM, respectively. In contrast, neither 45Ca2+ accumulation nor cell death was blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in the presence of high glycine. With brief exposure, high concentrations of AMPA, kainate, or K+ produced much less death or 45Ca2+ accumulation than produced by glutamate, especially if 10 microM MK-801 was included in the exposure medium to block NMDA receptor activation. Kainate- or AMPA-induced 45Ca2+ accumulation or neuronal cell death was blocked with CNQX. However, high K(+)-triggered 45Ca2+ accumulation was only partially blocked with CNQX plus MK-801, consistent with mediation by voltage-gated Ca2+ channels. In addition to measuring the accumulation of 45Ca2+ occurring during agonist exposure, we also assessed accumulation during the 30 min immediately following completion of a 3-5 min exposure to 500 microM NMDA.(ABSTRACT TRUNCATED AT 250 WORDS)

AMPA receptor activation potentiates zinc neurotoxicity.

Extracellular Zn2+ attenuates NMDA receptor-mediated neurotoxicity and increases AMPA receptor-mediated toxicity. Known electrophysiological effects of Zn2+ predict only the former. We considered the possibility that the latter rather reflects AMPA potentiation of Zn2+ toxicity, perhaps mediated by neuronal depolarization and Zn2+ entry through voltage-gated Ca2+ channels. High K+ or kainate also potentiated Zn2+ toxicity, and AMPA plus Zn2+ toxicity was attenuated by raising extracellular Ca2+, or by Ca2+ channel blockers. AMPA plus Zn2+ exposure induced an increase in fluorescence from neurons loaded with the Zn(2+)-sensitive dye TS-Q and increased subsequent 45Ca2+ accumulation. The ability of AMPA receptor activation to potentiate Zn2+ toxicity may be relevant to neuronal death associated with intense activation of glutamatergic pathways.

Oxygen or glucose deprivation-induced neuronal injury in cortical cell cultures is reduced by tetanus toxin.

We examined glutamate-mediated neurotoxicity in cortical cell cultures pretreated with 1-5 micrograms/ml tetanus toxin to attenuate the Ca(2+)-dependent release of neurotransmitters. Efficacy of the tetanus toxin pretreatment was suggested by blockade of electrical burst activity induced by Mg2+ removal and by reduction of glutamate efflux induced by high K+. Tetanus toxin reduced neuronal injury produced by brief exposure to elevated extracellular K+ or to glutamate, situations in which release of endogenous excitatory neurotransmitter is likely to play a role. Furthermore, although glutamate efflux evoked by anoxic conditions may occur largely via Ca(2+)-independent transport, tetanus toxin attenuated both glutamate efflux and neuronal injury following combined oxygen and glucose deprivation. With prolonged exposure periods, the neuroprotective efficacy of tetanus toxin was comparable to that of NMDA receptor antagonists. Presynaptic inhibition of Ca(2+)-dependent glutamate release may be a valuable approach to attenuating hypoxic-ischemic brain injury.

21-Aminosteroids attenuate excitotoxic neuronal injury in cortical cell cultures.

We studied the protective efficacy of novel 21-aminosteroids against several forms of neuronal injury in murine cortical cell cultures. Concentrations of 200 nM to 20 microM partially attenuated the damage induced by glucose deprivation, combined oxygen-glucose deprivation, or exposure to NMDA; maximal protection was less than that produced by NMDA antagonists, but the combination of a 21-aminosteroid plus an NMDA antagonist produced a greater benefit than either drug alone. 21-Aminosteroid addition did not attenuate NMDA-induced whole-cell current, but did block almost all of the damage induced by exposure to iron, a protective action consistent with inhibition of free radical-mediated lipid peroxidation. Lipid peroxidation may be a downstream event mediating a portion of the injury triggered by excess stimulation of NMDA receptors.

Folate interactions with cerebral G proteins.

Intracerebral folate injections produce convulsions and brain lesions, folic acid itself and tetrahydrofolate being more potent toxins than 5-methyltetrahydrofolate, the primary folate of mammalian extracellular fluids. Folates are known to excite neurons, by unknown mechanisms Folates stimulate GTP binding and GTPase activity in slime molds. We observed folate stimulation of GTP gamma S binding and inhibition of high affinity GTPase activity in rat brain membranes. Three fold stimulation of GTP gamma S binding was observed in cerebellar membranes treated with 50 microM FA. Folic acid (FA), dihydrofolate (DHF) and tetrahydrofolate (THF) were much more potent than 5-methyltetrahydrofolate in this regard. The effect varies between brain regions and was greatest in cerebellar and hippocampal membranes. Folates inhibit GTPase activity, with DHF and FA being the most potent and maximum inhibition being to 33% of control values. We find high affinity guanine nucleotide sensitive binding of [3H]FA in cerebellar membranes, another response typical of G protein coupled membrane receptors. Folates were also shown to stimulate the release of [3H]GDP from brain membranes. These effects are seen in washed brain membranes and can not be explained by any known folate metabolic or coenzyme functions. They resemble the effects of cholera toxin, except for their reversibility. They may be relevant to known folate neuroexcitant effects of folates.

The calcium channel blocker nifedipine attenuates slow excitatory amino acid neurotoxicity.

High concentrations of potent N-methyl-D-aspartate (NMDA) agonists can trigger degeneration of cultured mouse cortical neurons after an exposure of only a few minutes; in contrast, selective non-NMDA agonists or low levels of NMDA agonists require exposures of several hours to induce comparable damage. The dihydropyridine calcium channel antagonist nifedipine was used to test whether this slow neurotoxicity is mediated by a calcium influx through voltage-gated channels. Nifedipine had little effect on the widespread neuronal degeneration induced by brief exposure to high concentrations of NMDA but substantially attenuated the neurotoxicity produced by 24-hour exposure to submaximal concentrations of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate, kainate, or quinolinate. Calcium ion influx through dihydropyridine-sensitive, voltage-dependent calcium channels may be an important step in the neuronal injury induced by the prolonged activation of NMDA or non-NMDA glutamate receptors.