LINA: a laboratory inventory system for oligonucleotides, microbial strains, and cell lines.

In this article, we present the Laboratory Inventory Network Application (LINA), a software system that assists research laboratories in keeping track of their collections of biologically relevant materials. This open source application uses relational Microsoft Access database technology as a back end and a Microsoft .NET application as a front end. Preconstructed table templates are provided that contain standardized and customizable data fields. As new samples are added to the inventory, each is provided with a unique laboratory identifier, which is assigned automatically and sequentially, allowing rapid retrieval when a given reagent is required. The LINA contains a number of useful search tools including a general search, which allows database searches using up to four user-defined criteria. The LINA represents an easily implemented and useful organizational tool for biological laboratories with large numbers of strains, clones, or other reagents.

Comparison of Euryarchaea strains in the guts and food-soil of the soil-feeding termite Cubitermes fungifaber across different soil types.

Termites are an important component of tropical soil communities and have a significant effect on the structure and nutrient content of soil. Digestion in termites is related to gut structure, gut physicochemical conditions, and gut symbiotic microbiota. Here we describe the use of 16S rRNA gene sequencing and terminal-restriction fragment length polymorphism (T-RFLP) analysis to examine methanogenic archaea (MA) in the guts and food-soil of the soil-feeder Cubitermes fungifaber Sjostedt across a range of soil types. If these MA are strictly vertically inherited, then the MA in guts should be the same in all individuals even if the soils differ across sites. In contrast, gut MA should reflect what is present in soil if populations are merely a reflection of what is ingested as the insects forage. We show clear differences between the euryarchaeal communities in termite guts and in food-soils from five different sites. Analysis of 16S rRNA gene clones indicated little overlap between the gut and soil communities. Gut clones were related to a termite-derived Methanomicrobiales cluster, to Methanobrevibacter and, surprisingly, to the haloalkaliphile Natronococcus. Soil clones clustered with Methanosarcina, Methanomicrococcus, or rice cluster I. T-RFLP analysis indicated that the archaeal communities in the soil samples differed from site to site, whereas those in termite guts were similar between sites. There was some overlap between the gut and soil communities, but these may represent transient populations in either guts or soil. Our data do not support the hypothesis that termite gut MA are derived from their food-soil but also do not support a purely vertical transmission of gut microflora.

The pharmacodynamic characterization of an antisense oligonucleotide against monoamine oxidase-B (MAO-B) in rat brain striatal tissue.

1. The aim of our work was to pharmacodynamically characterize an antisense oligonucleotide sequence (5'-GCC AAA CTT TTG CAT GAC-3') against MAO-B, using qualitative and quantitative analyses as assessment measures. 2. Qualitative analysis using histochemical staining revealed that intracerebroventricular (ICV) administered antisense (100 picomoles twice daily x 3.5 days) eliminated all visibly detectable histochemical staining for MAO-B throughout the striatum 1, 12, and 24 h after the last antisense treatment. 3. Qualitative analysis using RT-PCR of the time course of MAO-B mRNA expression in the rat striatum following ICV administration of the antisense sequence showed that 12-24 h after the last administration there was a dramatic reduction in MAO-B mRNA expression in the striatum. The reverse and scrambled sequences generated no change in MAO-B mRNA at 1 or 24 h after the last treatment. 4. Quantitative analysis using the MAO-B selective substrate 4-dimethylamino-phenethylamine (DMAPEA) showed that the antisense sequence reduced MAO-B activity by more than 40%, which was comparable to a single 2 mg/kg, ip dose of L-deprenyl. 5. Quantitative analysis of neurotransmitter levels 24 h after the last treatment suggested that the antisense sequence did not produce any significant changes in neurotransmitter levels. 6. Potential mechanisms for enhancing the antisense response and the speculated potential of an antisense against MAO-B for studying neurotoxicity, Parkinson's disease, and the aging process are also discussed.

Molecular phylogenetic profiling of prokaryotic communities in guts of termites with different feeding habits.

Termites are an important group of terrestrial insects that harbor an abundant gut microbiota, many of which contribute to digestion, termite nutrition and gas (CH(4), CO(2) and H(2)) emission. With 2200 described species, termites also provide a good model to study relationships between host diet and gut microbial community structure and function. We examined the relationship between diet and gut prokaryotic community profiles in 24 taxonomically and nutritionally diverse species of termites by using nucleic acid probes targeting 16S-like ribosomal RNAs. The relative abundance of domain-specific 16S-like rRNAs recovered from gut extracts varied considerably (ranges: Archaea (0-3%); Bacteria (15-118%)). Although Bacteria were always detectable and the most abundant, differences in domain-level profiles were correlated with termite diet, as evidenced by higher relative abundances of Archaea in guts of soil-feeding termites, compared to those of wood-feeding species in the same family. The oligonucleotide probes also readily distinguished gut communities of wood-feeding taxa in the family Termitidae (higher termites) from those of other wood-feeding termite families (lower termites). The relative abundances of 16S-like archaeal rRNA in guts were positively correlated with rates of methane emission by live termites, and were consistent with previous work linking high relative rates of methanogenesis with the soil (humus)-feeding habit. Probes for methanogenic Archaea detected members of only two families (Methanobacteriaceae and Methanosarcinaceae) in termite guts, and these typically accounted for 60% of the all archaeal probe signal. In four species of termites, Methanosarcinaceae were dominant, a novel observation for animal gut microbial communities, but no clear relationship was apparent between methanogen family profiles and termite diet or taxonomy.

Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays.

To examine the utility and performance of 50mer oligonucleotide (oligonucleotide probe) microarrays, gene-specific oligonucleotide probes were spotted along with PCR probes onto glass microarrays and the performance of each probe type was evaluated. The specificity of oligonucleotide probes was studied using target RNAs that shared various degrees of sequence similarity. Sensitivity was defined as the ability to detect a 3-fold change in mRNA. No significant difference in sensitivity between oligonucleotide probes and PCR probes was observed and both had a minimum reproducible detection limit of approximately 10 mRNA copies/cell. Specificity studies showed that for a given oligonucleotide probe any 'non-target' transcripts (cDNAs) >75% similar over the 50 base target may show cross-hybridization. Thus non-target sequences which have >75-80% sequence similarity with target sequences (within the oligonucleotide probe 50 base target region) will contribute to the overall signal intensity. In addition, if the 50 base target region is marginally similar, it must not include a stretch of complementary sequence >15 contiguous bases. Therefore, knowledge about the target sequence, as well as its similarity to other mRNAs in the target tissue or RNA sample, is required to design successful oligonucleotide probes for quality microarray results. Together these results validate the utility of oligonucleotide probe (50mer) glass microarrays.

Evidence for seeding of beta -amyloid by intracerebral infusion of Alzheimer brain extracts in beta -amyloid precursor protein-transgenic mice.

Many neurodegenerative diseases are associated with the abnormal sequestration of disease-specific proteins in the brain, but the events that initiate this process remain unclear. To determine whether the deposition of the beta-amyloid peptide (Abeta), a key pathological feature of Alzheimer's disease (AD), can be induced in vivo, we infused dilute supernatants of autopsy-derived neocortical homogenates from Alzheimer's patients unilaterally into the hippocampus and neocortex of 3-month-old beta-amyloid precursor protein (betaAPP)-transgenic mice. Up to 4 weeks after the infusion there was no Abeta-deposition in the brain; however, after 5 months, the AD-tissue-injected hemisphere of the transgenic mice had developed profuse Abeta-immunoreactive senile plaques and vascular deposits, some of which were birefringent with Congo Red. There was limited deposition of diffuse Abeta also in the brains of betaAPP-transgenic mice infused with tissue from an age-matched, non-AD brain with mild beta-amyloidosis, but none in mice receiving extract from a young control case. Abeta deposits also were not found in either vehicle-injected or uninjected transgenic mice or in any nontransgenic mice. The results show that cerebral beta-amyloid can be seeded in vivo by a single inoculation of dilute AD brain extract, demonstrating a key pathogenic commonality between beta-amyloidosis and other neurodegenerative diseases involving abnormal protein polymerization. The paradigm can be used to clarify the conditions that initiate in vivo beta-amyloidogenesis in the brain and may yield a more authentic animal model of Alzheimer's disease and other neurodegenerative disorders.

Inhibitors of V-type ATPases, bafilomycin A1 and concanamycin A, protect against beta-amyloid-mediated effects on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction.

The functional viability of cells can be evaluated using a number of different assay determinants. One common assay involves exposing cells to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which is converted intracellularly to a colored formazan precipitate and often used to assess amyloid peptide-induced cytotoxic effects. The MTT assay was employed to evaluate the role of endosomal uptake and lysosomal acidification in amyloid peptide-treated differentiated PC12 cell cultures using selective vacuolar-type (V-type) ATPase inhibitors. The macrolides bafilomycin A1 (BAF) and concanamycin A (CON) block lysosomal acidification through selective inhibition of the V-type ATPase. Treating nerve growth factor-differentiated PC12 cells with nanomolar concentrations of BAF or CON provides complete protection against the effects of beta-amyloid peptides Abeta(1-42), Abeta(1-40), and Abeta(25-35) and of amylin on MTT dye conversion. These macrolides do not inhibit peptide aggregation, act as antioxidants, or inhibit Abeta uptake by cells. Measurements of lysosomal acidification reveal that the concentrations of BAF and CON effective in reversing Abeta-mediated MTT dye conversion also reverse lysosomal pH. These results suggest that lysosomal acidification is necessary for Abeta effects on MTT dye conversion.

Differential expression of group I metabotropic glutamate receptors (mGluRs) in the rat pheochromocytoma cell line PC12: role of nerve growth factor and ras.

Glutamate treatment of PC12 cells has been shown to result in the accumulation of intracellular inositol phosphates suggesting the presence of glutamate metabotropic receptors (mGluRs) positively coupled to phospholipase C. The present study examined the expression of group I mGluRs (mGluR1 and mGluR5) in PC12 cells. Undifferentiated PC12 cells were found to express both mGluR5 mRNA and receptor protein by reverse transcription polymerase chain reaction (RT-PCR) and western blot techniques. However, mGluR1 mRNA was not detected in these cells and western blot analysis showed only faint mGluR1alpha immunoreactivity suggesting a very low level of mGluR1 expression. Nerve growth factor-induced differentiation of PC12 cells resulted in the induction of mGluR1alpha and mGluR1beta mRNA and mGluR1alpha protein. PC12 cells overexpressing dominant negative ras revealed that NGF-induced mGluR1 induction, but not mGluR5 expression, is dependent on ras pathway activation in these cells. These results suggest PC12 cells may be a useful model for investigating the regulation and expression of group I mGluR isoforms and their role in neuronal processes in vitro.

Trimethyltin stimulates protein kinase C translocation through receptor-mediated phospholipase C activation in PC12 cells.

Trimethyltin (TMT) is a potent neurotoxic compound that initiates a delayed neuronal cell death. Previously we have shown that TMT-induced cytotoxicity is associated with protein kinase C (PKC) translocation and activation. The present study investigates the mechanism underlying TMT-stimulated PKC translocation in PC12 cells. TMT exposure led to a rapid increase in intracellular levels of inositol 1,4,5-trisphosphate (IP3), a product of phospholipase C (PLC). This was significantly decreased by pretreating cells with antagonists to either the cholinergic muscarinic receptor (atropine) or the glutamatergic metabotropic receptor [(+)-alpha-methyl-4-carboxyphenylglycine; (+)-MCPG]. Furthermore, the rise in IP3 level was blocked by pretreating cells with a PLC inhibitor (U-73122) or by a combination of atropine and (+)-MCPG. This pretreatment also significantly decreased TMT-stimulated PKC translocation, indicating that TMT-mediated PKC translocation was related to PLC activation, presumably through formation of diacylglycerol, an endogenous activator of PKC and product of PLC. It is interesting that atropine and (+)-MCPG did not provide protection against TMT-induced cytotoxicity in these cells. However, these data suggest that TMT causes the release of cellular constituents that activate G protein-coupled receptors, ultimately leading to PKC translocation.

RAGE-Abeta interactions in the pathophysiology of Alzheimer's disease.

RAGE is a cell surface molecule primarily identified for its capacity to bind advanced glycation end-products and amphoterin. Immunocytochemical studies demonstrated that in Alzheimer's Disease (AD) the expression of RAGE is elevated in neurons close to neuritic plaque beta-amyloid (Abeta) deposits and in the cells of Abeta containing vessels. Cross-linking of surface bound Abeta 1-40 to endothelial cells, yielded a band of 50 kDa identified as RAGE. Using the soluble extracellular domain of recombinant human RAGE, we found that Abeta binds to RAGE with a Kd = 57 +/- 14 nM, a value close to those found for mouse brain endothelial cells and rat cortical neurons. The interaction of Abeta with RAGE in neuronal, endothelial, and RAGE-transfected COS-1 cells induced oxidative stress, as assessed by the TBARS and MTT assays. ELISA demonstrated a 2.5 times increase of RAGE in AD over control brains. Activated microglia also showed elevated expression of RAGE. In the BV-2 microglial cell line, RAGE bound Abeta in dose dependent manner with a Kd of 25 +/- 9 nM. Soluble Abeta induced the migration of microglia along a concentration gradient, while immobilized Abeta arrested this migration. Abeta-RAGE interaction also activated NF-kappaB, resulting in neuronal up-regulation of macrophage-colony stimulating factor (M-CSF) which also induced microglial migration. Taken together, our data suggest that RAGE-Abeta interactions play an important role in the pathophysiology of Alzheimer's Disease.

Critical evaluation of membrane supports for use in quantitative hybridizations.

The quantification of 16S rRNA by oligonucleotide probe hybridization was investigated with MagnaGraph (Micron Separation, Inc. [MSI]), Magna Charge (MSI), Magna (MSI), Immobilon-N (Millipore Corporation), and Nytran (Schleicher & Schuell, Inc.) membranes as supports for nucleic acid immobilization. The levels of detectability provided by the Magna Charge and Immobilon-N membranes were 20 to 50 times better than those obtained with the MagnaGraph, Magna, and Nytran membranes. The variability of the signal response for individual membranes ranged from 10 to 50%, with the Magna Charge and Immobilon-N membranes demonstrating the lowest variability.

Antagonism of cyanide toxicity by isosorbide dinitrate: possible role of nitric oxide.

In a search for improved cyanide antidotes, the efficacy of isosorbide dinitrate (ISDN), was compared with that of the known cyanide antidote, NaNO2. ISDN was as effective as an optimal dose of NaNO2 in protecting mice against cyanide lethality. To study the mechanism involved, the extent of formation of the cyanide scavenger, methemoglobin, in the action of ISDN was determined. ISDN (300 mg/kg, p.o.) increased methemoglobin from 5 to 10% of total hemoglobin, while, in contrast, NaNO2 (100 mg/kg, i.p.) increased methemoglobin levels to 50% of total hemoglobin. Lowering the dose of NaNO2 to 30 mg/kg reduced methemoglobin levels to approximately 10% of total hemoglobin and in turn nearly abolished its antidotal effect. Decreasing methemoglobin to less than control levels using methylene blue failed to abolish cyanide antagonism by ISDN. Thus, methemoglobin formation by ISDN does not account for its antidotal action. Further studies comparing the respiratory depressant effects of cyanide in the presence of ISDN or NaNO2 also indicated that these two antidotes have different mechanisms of action. Efforts to produce tolerance to the antidotal effect of ISDN against cyanide toxicity were unsuccessful. It is suggested that the well-known ability of ISDN to generate nitric oxide may account for the noted cyanide antagonism.

Activation of protein kinase C by trimethyltin: relevance to neurotoxicity.

The differentiated PC12 cell neuronal model was used to determine the effect of trimethyltin (TMT) on protein kinase C (PKC). Cells treated with 5-20 microM TMT showed a partial and sustained PKC translocation within 30 min and persisted over a 24-h period. TMT treatment was accompanied by a low level of PKC down-regulation over 24 h, which was small compared with that produced by phorbol esters. Confocal imaging of differentiated PC12 cells showed that PKC translocates to the plasma membrane and the translocation is blocked by the PKC inhibitor chelerythrine (1 microM). Phorbol myristate-induced PKC down-regulation or inhibition with chelerythrine provided protection against TMT-induced cytotoxicity. It was concluded that TMT-induced PKC translocation and activation contribute to the cytotoxicity of TMT in differentiated PC12 cells.

Diversity within diversity: molecular approaches to studying microbial interactions with insects.

DNA sequence information has greatly augmented the number of characters available for analysis in phylogenetic research. Nowhere is this more evident than in studies of microbial evolution. Ribosomal DNA sequence data has simultaneously permitted the distinction between individual species and the inference of their phylogenetic relationships in many cases where both were formerly impossible. These have contributed to our understanding of the ecology of particular microbe-host interactions and the history of these relationships over evolutionary time. We describe examples from two ends of the ecological spectrum in insect/bacterial associations: one in which bacteria mediate host cytoplasmic incompatibility and parthenogenesis, and the other in which mycetocyte bacteria augment host nutrition. In the former, the pattern of bacterial interaction is general, with the same or closely related strains of the genus Wolbachia associating with a wide range of insect taxa. In the latter, concordance between host and microbe phylogenies suggests cospeciation between bacteria and host, although it is as yet unclear whether this process has involved step-wise, reciprocal coevolution. We conclude with a discussion of how developments in molecular techniques may aid in analyzing more complex interactions between insects and microbes.

Monitoring the enrichment and isolation of sulfate-reducing bacteria by using oligonucleotide hybridization probes designed from environmentally derived 16S rRNA sequences.

A fluorescently labeled version of a population-specific oligonucleotide hybridization probe was used to monitor the enrichment and isolation of a sulfate-reducing bacterium from a multispecies anaerobic bioreactor. The organism was originally identified as a molecular isolate that was phylogenetically related to Desulfovibrio vulgaris by amplification and sequencing of part of its 16S rRNA sequence. The sequence, in turn, was used to design a population-specific probe. The anaerobic medium used for the organism's enrichment and isolation was based on the physiological properties of the its closest relatives as identified by sequence comparisons. Of 30 isolates examined, only 3 hybridized with the probe. Nearly complete 16S rRNA sequences determined for each of these three isolates (i) had no mismatches with the probe target site, (ii) were identical to the amplified partial sequence of about 500 nucleotides and to one another in all other positions, and (iii) were 93.9% similar to that of D. vulgaris. In addition, one isolate chosen for further study (strain PT-2) had a substrate specificity comparable to that of D. vulgaris. These results confirmed that polymerase chain reaction amplification of 16S rRNA sequences from environmental samples can be accurate and can also provide phylogenetic information from which aspects of a population's physiology can be inferred.

Genesis of acetate and methane by gut bacteria of nutritionally diverse termites.

The evolution of different feeding guilds in termites is paralleled by differences in the activity of their gut microbiota. In wood-feeding termites, carbon dioxide-reducing acetogenic bacteria were found to generally outprocess carbon dioxide-reducing methanogenic bacteria for reductant (presumably hydrogen) generated during microbial fermentation in the hindgut. By contrast, acetogenesis from hydrogen and carbon dioxide was of little significance in fungus-growing and soil-feeding termites, which evolved more methane than their wood- and grass-feeding counterparts. Given the large biomass of termites on the earth and especially in the tropics, these findings should help refine global estimates of carbon dioxide reduction in anoxic habitats and the contribution of termite emissions to atmospheric methane concentrations.

Effect of host diet on production of organic acids and methane by cockroach gut bacteria.

The effect of high-fiber diets on microbial populations and processes in cockroach guts was investigated by feeding American cockroaches (Periplaneta americana) milled cereal leaves, milled corn cob, or commercial bran-type breakfast cereal in place of the commonly used laboratory diet of dog chow. The activities and numbers of specific gut bacteria varied significantly with the insect's diet and developmental stage. Acetate and lactate were the principal organic acids present in the gut fluid of adult cockroaches and occurred at concentrations of up to 17 and 8 mM, respectively. These acids were most abundant in the gut fluid of dog chow-fed insects, and the greatest amounts were generally found in the foregut and midgut regions. Foreguts of dog chow-fed cockroaches contained an abundant population of lactic acid bacteria that formed acetate and lactate from endogenous hexoses present in the foregut. When adult cockroaches were fed dog chow amended with antibacterial drugs, (i) the concentrations of acetate, lactate, and total hexoses in gut fluid decreased significantly, (ii) the numbers of lactic acid bacteria in the foregut also decreased significantly, and (iii) the production of acetate and lactate by foregut homogenates was suppressed. It was estimated that acetate and lactate produced by bacteria in the foregut of dog chow-fed adult P. americana could support up to 14% of the insect's respiratory requirement if taken up and used by the animal. When insects were fed high-fiber diets of bran cereal, cereal leaves, or corn cob, bacterial production of acetate and lactate in the foregut diminished.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetonema longum gen. nov. sp. nov., an H2/CO2 acetogenic bacterium from the termite, Pterotermes occidentis.

A previously undescribed, H2-oxidizing CO2-reducing acetogenic bacterium was isolated from gut contents of the wood-feeding termite, Pterotermes occidentis. Cells of representative strain APO-1 were strictly anaerobic. Gram-negative, endospore-forming motile rods which measured 0.30-0.40 x 6-60 microm. Cells were catalase positive, oxidase negative, and had 51.5 mol percent G + C in their DNA. Optimum conditions for growth on H2 + CO2 were at 30-33 degrees C and pH (initial) 7.8, and under these conditions cells formed acetate according to the equation: 4 H2 + 2 CO2----CH3COOH + 2 H2O. Other energy sources supporting good growth of strain APO-1 included glucose, ribose, and various organic acids. Acetate and butyrate were major fermentation products from most organic compounds tested, however propionate, succinate, and 1,2-propanediol were also formed from some substrates. Based on comparative analysis of 16S rRNA nucleotide sequences, strain APO-1 was related, to but distinct from, members of the genus Sporomusa. Moreover, physiological and morphological differences between strain APO-1 and the six known species of Sporomusa were significant. Consequently, it is proposed herewith that a new genus, Acetonema, be established with strain APO-1 as the type strain of the new species, Acetonema longum. A. longum may contribute to the nutrition of P. occidentis by forming acetate, propionate and butyrate, compounds which are important carbon and energy sources for termites.