Stability of fungal biomass continuously fed with tannic acid in a non-sterile moving-packed bed reactor.

A number of bacteria and fungi are known to degrade tannins. In this study, the efficiency of the white-rot fungus, Bjerkandera adusta MUT 2295, was evaluated for the treatment of a synthetic solution prepared with tannic acid. Tests were performed in continuously fed, bench-scale, packed-bed reactors, operated under non-sterile conditions with biomass immobilized within PolyUrethane Foam cubes (PUFs). The main parameters monitored to evaluate the process efficiency were: soluble Chemical Oxygen Demand (sCOD), Total Organic Carbon (TOC) removal, and activities. of Tannase and Lignin Peroxidase. At the end of the process, additional parameters were evaluated, including the increase of fungal dry weight and the presence of ergosterol. The reactor was operative for 210 days, with maximum sCOD and TOC removal of 81% and 73%, respectively. The reduction of sCOD and TOC were positively correlated with the detection of Tannase and Lignin Peroxidase (LiP) activities. Increases in biomass within the PUF cubes was associated with increases in ergosterol concentrations. This study proved that the fungal-based system tested was efficient for the degradation of tannic acid over a period of time, and under non-sterile conditions.

Reassessment of the transhydrogenase/malate shunt pathway in Clostridium thermocellum ATCC 27405 through kinetic characterization of malic enzyme and malate dehydrogenase.

Clostridium thermocellum produces ethanol as one of its major end products from direct fermentation of cellulosic biomass. Therefore, it is viewed as an attractive model for the production of biofuels via consolidated bioprocessing. However, a better understanding of the metabolic pathways, along with their putative regulation, could lead to improved strategies for increasing the production of ethanol. In the absence of an annotated pyruvate kinase in the genome, alternate means of generating pyruvate have been sought. Previous proteomic and transcriptomic work detected high levels of a malate dehydrogenase and malic enzyme, which may be used as part of a malate shunt for the generation of pyruvate from phosphoenolpyruvate. The purification and characterization of the malate dehydrogenase and malic enzyme are described in order to elucidate their putative roles in malate shunt and their potential role in C. thermocellum metabolism. The malate dehydrogenase catalyzed the reduction of oxaloacetate to malate utilizing NADH or NADPH with a kcat of 45.8 s(-1) or 14.9 s(-1), respectively, resulting in a 12-fold increase in catalytic efficiency when using NADH over NADPH. The malic enzyme displayed reversible malate decarboxylation activity with a kcat of 520.8 s(-1). The malic enzyme used NADP(+) as a cofactor along with NH4 (+) and Mn(2+) as activators. Pyrophosphate was found to be a potent inhibitor of malic enzyme activity, with a Ki of 0.036 mM. We propose a putative regulatory mechanism of the malate shunt by pyrophosphate and NH4 (+) based on the characterization of the malate dehydrogenase and malic enzyme.

Effect of cellulose as co-substrate on old landfill leachate treatment using white-rot fungi.

Conventional wastewater treatment technologies are ineffective for remediation of old LandFill Leachate (LFL), and innovative approaches to achieve satisfactory removal of this recalcitrant fraction are needed. This study focused on old LFL treatment with a selected fungal strain, Bjerkandera adusta MUT 2295, through batch and continuous tests, using packed-bed bioreactors under non-sterile conditions. To optimize the process performance, diverse types of co-substrates were used, including milled cellulose from beverage cups waste material. Extracellular enzyme production was assayed, in batch tests, as a function of a) cellulose concentration, b) leachate initial Chemical Oxygen Demand (COD) and Soluble COD (sCOD), and c) co-substrate type. Bioreactors were dosed with an initial start-up of glucose (Rg) or cellulose (Rc). An additional glucose dosage was provided in both reactors, leading to significant performance increases. The highest COD and sCOD removals were i) 63% and 53% in Rg and ii) 54% and 51% in Rc.

Detection of hepatitis A virus (HAV) in oysters (Crassostrea gigas).

Because shellfish (oysters, clams, and mussels) are filter-feeders, pathogens become concentrated within them, and human consumption of raw, or under-cooked shellfish can result in disease outbreaks. Identification of hepatitis A virus (HAV) in shellfish has been difficult for several reasons: the concentration of virions in shellfish tissues are very low, detection methods based on in vitro propagation are unreliable, recovery of virions from shellfish tissues is inefficient, and PCR inhibitors in shellfish tissues limit the success of RT-PCR. These facts underlie difficulties in determining cause and effect relationships between hepatitis A outbreaks and detection of HAV contamination in shellfish samples. We have developed a reliable and highly sensitive method for detection of HAV in oyster tissues at low levels (0.001 FFU/ml-fluorescent focus units per milliliter). Our method combines dissection of the gastrointestinal oyster tract, organic extraction before PEG precipitation, and RNA extraction with Trizol LS, followed by RT-PCR and hybridization using a digoxigenin-labeled HAV cDNA probe. Our results will benefit both public health officials concerned about hepatitis A infections caused by consumption of HAV-contaminated oysters and shellfish producers who require reliable methods for quality control of commercial oyster production.

Detection of p53 mutations in benign and dysplastic nevi.

We have examined melanocytic cells derived directly from fresh biopsy tissue for the presence of p53 mutations. Using selective media that permits growth of melanocytes and inhibits growth of fibroblasts and keratinocytes, we established short-term, primary cultures of melanocytes from skin biopsies of common acquired nevi, dysplastic nevi, and from metastatic melanoma. Using PCR-single-stranded conformational polymorphism analysis, we have detected p53 mutations in 2 of 11 benign compound nevi and 2 of 5 dysplastic nevi. All nevi positive for p53 mutations were derived from patients who previously had cutaneous moles and three of the four had a family and/or personal history of melanoma.

Cloning and localization of the repressor gene (c) of the Mu-like transposable phage D108.

We have localized the D108 thermosensitive (cts) repressor gene to a region of DNA approx. 600 base pairs (bp) in length by sub-cloning an RsaI restriction endonuclease fragment (bp 200 to bp 802 from the left-end of the D108 genome). We determined that the gene product from this fragment appears to be the same size (19 kDa) as that expressed from clones containing larger fragments of D108 DNA. Results from in vitro gel electrophoresis band-retardation and in vivo immunity assays show that the sub-cloned repressor appears to be fully functional.

Rapid evolution in the Nebria gregaria group (Coleoptera: Carabidae) and the paleogeography of the Queen Charlotte Islands.

Morphological differentiation in the ground beetles of the Nebria gregaria group, found on the Queen Charlotte Islands, has been used as support for the glacial refugium proposed for the northwest coast of North America. Two members of this species group, N. charlottae and N. louiseae, are restricted to cobble beaches in this archipelago. A third, N. haida, is found only in alpine regions of the archipelago and the adjacent mainland. The remaining two species of the gregaria group, N. lituyae and N. gregaria, show highly restricted distributions in the mountains of the Alaska panhandle and on the beaches of the Aleutian Islands, respectively. To determine the relationships of the five species, we conducted phylogenetic analyses on nucleotide sequence data obtained from five regions of the mitochondrial DNA. In total, 1835 bp were analyzed. The results suggest that one species, N. lituyae, does not belong in the gregaria group, and that only seven mutations separated the two most divergent of the four remaining species. We also conducted random amplified polymorphic DNA fingerprinting analyses on genomic DNA extracted from the five species. Analyses of genetic diversity revealed a lack of molecular differentiation among the Queen Charlotte species, suggesting that these populations may be postglacial in origin and that together N. gregaria, N. charlottae, N. louiseae, and N. haida might represent local variations of a single species. These results are consistent with conclusions derived for the morphological and genetical differentiation among Gasterosteus populations in the archipelago.

Ocular findings in Kenny's syndrome.

In 1966, Kenny described two patients with an unusual congenital syndrome including dwarfism, thickened long bone cortex, transient hypocalcemia, and normal intelligence. These and other patients previously were incorrectly described as "myopic". Ocular findings in four subjects ranged from uncomplicated nanophthalmos with hyperopia to extreme pseudopapilledema, vascular tortuosity, and mucular crowding. Postmortem findings from one patient showed calcium deposits demonstrable only by special histochemical stains that were distributed uniquely in the cornea. This distribution differed greatly from the pattern seen in band keratopathy. Retinal calcification was also an unusual feature. Because one patient exhibited a pseudodoubling of the optic papilla, the literature was reviewed. We conclude that no convincing case of true doubling of the optic nerve has been described. Ophthalmologists should be alert for undiagnosed electrolyte abnormalities, especially hypocalcemia, in patients with Kenney's syndrome.

Retinal detachment in a battered infant.

A 12-week-old battered infant boy had bilateral retinal detachments. The retina in the left eye was considered inoperable, but retinal detachment surgery was performed in the right eye. This retina was reattached and limited vision was restored. The history, the location of the retinal pathology, and the absence of a lens coloboma helped differentiate this result of physical abuse from a congenital retinal detachment.

Limitations in the use of SSCP analysis.

Since its introduction in 1989, analyses by SSCPA of DNA sequences containing mutations by SSCPA have increased dramatically. While many workers have recognised the utility of the technique, few have examined its limitations. In this paper we report studies using part of the lacI gene from E. coli to measure assay variables. When assay conditions are carefully controlled, the assay is very reproducible. The position and type of mutation have little effect on detection efficiency and changes in sequences 176 and 354 bp in length are detected with comparable efficiencies. Overall detection efficiency is > 90% under most conditions. However, local heating due to excessive power levels, can introduce anomalies.

Improved polymerase fidelity in PCR-SSCPA.

Polymerase fidelity is important in any application of the polymerase chain reaction (PCR). In single-strand conformation polymorphism analysis (SSCPA) where one may be looking for a small number of altered DNA strands in the presence of thousands of unchanged strands it is critical. We have examined the effect of PCR conditions, product purification and SSCP analysis on the measured error rates with 4 thermostable polymerases (Taq, Vent, Pyrostase and Pfu). Error rates have been calculated by densitometric scanning of SSCPA gel images. Using PCR conditions which maximize fidelity and eliminating products which include large additions or deletions we have achieved error rates of 10(-5) to 10(-6). Such low rates heighten the probability that relatively infrequent mutations will be identified. Further, the densitometric scanning of gel images provides a useful modification of conventional SSCPA which facilitates such identification.

Comparison of Doppler US and CT angiography for evaluation of renal artery stenosis.

RATIONALE AND OBJECTIVES: The authors compared Doppler ultrasound (US) with computed tomographic (CT) angiography in the evaluation of stenosis of the main renal artery. MATERIALS AND METHODS: Fifty-six patients who had undergone conventional angiography of the renal arteries participated in a prospective comparison of Doppler US (45 patients) and CT angiography (52 patients). US evaluation included both the main renal artery and segmental renal arteries. RESULTS: There were 27 main renal arteries with at least 50% stenosis in 20 patients. In 36 patients, there was no significant stenosis. All cases of main renal artery stenosis detected with Doppler US of the segmental arteries were also identified with Doppler US of the main renal artery. The by-artery sensitivity (63%) of US of the main renal artery was greater than that (33%) of US of the segmental arteries. CT angiography was more sensitive (96%) than Doppler US (63%) in the detection of stenosis, but the specificity of CT (88%) was similar to that of US (89%). The difference in the area under the receiver operating characteristic curve (AUC) between CT (AUC = 0.94) and US (AUC = 0.82) was statistically significant (P = .038). CONCLUSION: Doppler US of the main renal artery is more sensitive than Doppler US of segmental arteries in the detection of stenosis. CT angiography is more accurate than Doppler US in the evaluation of renal artery stenosis.

Regulation of repressor and early gene expression in Mu-like transposable bacteriophage D108.

The temperate, transposable bacteriophages D108 and Mu are highly homologous, but differ in their lef-end regulatory regions. We have previously cloned the gene encoding the D108 thermo-sensitive (cts) repressor under the control of the lactUV5 promoter. In this work, we report that crude protein extracts containing highly-expressed D108 repressor protect a 77 bp region of DNA, located between 863 bp and 940 bp from the D108 lef--end, from both exonuclease III and DNase I hydrolysis. Nucleotide sequence analysis of this region reveals that is also contains DNA sequences homologous to the consensus DNA-binding site of the Escherichia coli protein, Integration Host Factor (IHF). Crude protein extracts containing highly-expressed IHF specifically bind to, and retard the migration of, DNA fragments containing the D108 regulatory region, and the DNA sequence which IHF protects from DNase I cleave lies directly within the D108 repressor binding region. There are two apparent repressor-specific S1 nuclease-resistant RNA suggests that transcription from the early region promoter, Pe may initiate at or about 1000 bp from the left-end of the D108 genome. Thus though, D108 and Mu utilize three analogous proteins (repressor, ner, and IHF) and the same apparent promoters for early gene regulation and the lytic/lysogenic decision, the organization of these regulatory components is apparently different, suggesting different mechanisms of control of gene expression.

Spodoptera littoralis type B nucleopolyhedrovirus infection of a grasshopper cell line.

We determined that the type B nucleopolyhedrovirus of the Egyptian cottonworm, Spodoptera littoralis (SpliNPV), can infect a cell line derived from a grasshopper. We compared the infectivity of SpliNPV in two lepidopteran cell lines (Sf9 and Md210) and in a cell line (MSE4) derived from the western migratory grasshopper, Melanoplus sanguinipes (Orthoptera: Acrididae). Both Sf9 and MSE4 cells were permissive for SpliNPV replication and supported production of viable progeny. Md210 cells were nonpermissive for SpliNPV, and although the virus entered into these cells, they supported neither viral replication nor production of viable progeny. Infection of MSE4 cells with SpliNPV resulted in cytopathic effects within 48 h post infection and complete destruction of the cells within 5 days. Both virions and polyhedra were detected within virus-infected MSE4 cells by transmission electron microscopy. Extracellular virions were detected in the culture medium and were infectious to Sf9 cells, indicating that the MSE4 cells supported production of viable virus progeny.

Identification, transcription and sequence analysis of the Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) DNA polymerase gene.

Sequence analysis of a 6.4 kb DNA region from the Spodoptera littoralis multinucleocapsid nucleopolyhedrovirus (SpliNPV) revealed a large open reading frame (ORF) encoding a predicted polypeptide of 998 amino acid (aa) residues with a molecular mass of 114.93 kDa, located between 47.2-52.3 m.u. on the SpliNPV genome. Comparative sequence analyses demonstrated that the ORF encodes a DNA polymerase gene (dnapol) that contains conserved exonuclease domains and DNA polymerase motifs found in many prokaryotic, eukaryotic, and viral replicative DNA polymerases. A second ORF, ORF138, located between the lef-3 and dnapol, encodes a 138 aa polypetide that is homologous to ORF66 of the Autographa californica MNPV (AcMNPV). SpliNPV DNA polymerase shares an overall aa sequence identity of 39% with that of AcMNPV. A 3.0 kb SpliNPV dnapol-specific transcript was detected initially at 2 hpi and became abundant 48 hpi by Northern blot analysis. The transcription initiation site was mapped to an NPV early promoter element, ACGT. 3' RACE demonstrated that the SpliNPV dnapol transcript terminated at the polyadenylation signal AATAAA. Sequence analysis suggested that the SpliNPV dnapol and the dnapol of the NPV of S. litura (SpltNPV) are closely related.

Sensitive two-stage PCR of p53 genomic DNA exons 5-9.

To assess the status of the tumor suppressor gene p53 from samples with low levels (sub-nanogram amounts) of genomic DNA (e.g., from exfoliated cells, skin, small biopsies, mucosa), a technique based on two successive rounds of PCR was developed. In the first round, a 1.84-kb fragment spanning exons 5-9 was generated using a "touchdown" protocol. After purification by spun-column chromatography, this fragment was used as a template for the amplification of the individual exons 5-9 with inner primer sets specific for the adjacent intronic regions. Using this nested primer approach, several micrograms of each individual exon were obtained starting from as little as 62.5 pg of total genomic DNA. This material proved ideal for future analyses by single-strand conformational polymorphism (SSCP) analysis and DNA sequencing.

Variations in mitochondrial DNA and gene transcription in freezing-tolerant larvae of Eurosta solidaginis (Diptera: Tephritidae) and Gynaephora groenlandica (Lepidoptera: Lymantriidae).

Respiration, mitochondrial (mt)DNA content, and mitochondrial-specific RNA expression in fat body cells from active and cold-adapted larvae of the goldenrod gall fly, Eurosta solidaginis, and the Arctic woolly bear caterpillar, Gynaephora groenlandica, were compared. Reduced amounts of mtDNA were observed in cold-adapted larvae of both E. solidaginis and G. groenlandica collected in fall or winter, compared with summer-collected larvae. mtDNA increased to levels similar to those of summer-collected larvae after incubation at 10 degrees C or 15 degrees C for 5 h. Mitochondrial-specific RNAs (COI and 16S) were observed in fat body cells of both active and cold-adapted E. solidaginis larvae. Our results suggest that mitochondrial proteins required for respiration may be restored rapidly from stable RNAs present in overwintering larvae.

Characterization of Spodoptera littoralis type B nucleopolyhedrovirus infection in selected insect cell lines.

We have examined Spodoptera littoralis type B nucleopolyhedrovirus (SpliNPV) infections in CLS79, Sf9, and Se1 cells derived from lepidopteran insects of the genus Spodoptera (Family: Noctuidae), Ld652Y cells from Lymantria dispar (Family: Lymantriidae), and Md210 cells from Malacosoma disstria (Family: Lasiocampidae). CLS79, Sf9, and Se1 cells were permissive for SpliNPV infection as these cell lines supported complete viral DNA replication, virus-specific transcription, and production of viable progeny. Neither Ld652Y nor Md210 cells supported production of viable SpliNPV progeny. Ld652Y cells supported limited viral DNA replication and displayed reduced and delayed transcription of viral-specific RNAs. Md210 did not support viral DNA replication and displayed dramatically reduced transcription of viral-specific RNAs. We used transient expression assays as an indirect measure of the translation of SpliNPV early gene products in Sf9, Ld652Y, and Md210 cells. While transactivation of viral promoter-mediated luciferase expression occurred in SpliNPV-infected Ld652Y cells, little to no transactivation activity was detected in SpliNPV-infected Md210 cells. Our data indicated that the block to productive SpliNPV infection in Ld652Y and Md210 cells may be at the level of viral RNA transcription and further suggested that host factors play an important role in productive SpliNPV infection.