Anaerobic degradation of papermill sludge in a two-phase digester containing rumen microorganisms and colonized polyurethane foam

Summary The use of reticulated polyurethane foam as a support material for the immobilization of methanogenic associations and its application to the anaerobic treatment of fine particulate solid wastes was investigated. The colonization of polyurethane support particles in a continuous upflow reactor fed on a mixture of acetate, propionate and butyrate, was both rapid and dense. The combination of rumen microorganisms and colonized support particles in a two-phase digester resulted in an efficient anaerobic decomposition of papermill sludge.     

Characterization of the interaction of lambda exonuclease with the ends of DNA.

Lambda exonuclease processively degrades one strand of double-stranded DNA (dsDNA) in the 5"-3" direction. To understand the mechanism through which this enzyme generates high processivity we are analyzing the first step in the reaction, namely the interaction of lambda exonuclease with the ends of substrate DNA. Endonuclease mapping of lambda exonuclease bound to DNA has shown that the enzyme protects approximately 13-14 bp on dsDNA, and no nucleo-tides on the single-stranded tail of the DNA product. We have developed a rapid fluorescence-based assay using 2-aminopurine and measured the steady-state rate constants for different end-structures of DNA. The relative k(cat)for 5" ends decreases in the order 5" recessed > blunt >> 5" overhang. However, k(cat)/K(m)remains relatively constant for these different structures suggesting they are all used equally efficiently as substrates. From these data we propose that a single-stranded 5" overhang end can bind non-productively to the enzyme and the non-hydrolyzed strand is required to aid in the proper alignment of the 5" end. We have also measured the length-dependence of the steady-state rate para-meters and find that they are consistent with a high degree of processivity.     

Airway cellularity, lipid laden macrophages and microbiology of gastric juice and airways in children with reflux oesophagitis

Background and methods

Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR.

Results

We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 × 10⁵ viral copies/ml in nasal lavage and 1.88 × 10⁵ viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD.

Conclusion

HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely.     

Olfactory contribution to Fos expression during mating in inexperienced male hamsters

Male hamsters are very dependent on chemosensory cues for normal mating behavior. We have previously reported that central, vomeronasal pathways are intensely and selectively activated during mating or pheromonal stimulation. The contribution of main olfactory sensory input to the patterns of c-fos activation was investigated in this study. Sexually inexperienced male hamsters were either made anosmic by intranasal infusion of zinc sulfate or remained intact. Fos protein immunoreactivity was analyzed in main olfactory and vomeronasal pathways of the zinc sulfate-treated, anosmic animals after mating with receptive females for 45 min, and compared with Fos patterns seen in intact mating animals, some of which have been described in a previous publication. The zinc sulfate-treated anosmic males described here all mated when given access to receptive females. Whether mated or unstimulated, anosmic males had little or no Fos expression in main olfactory pathways; significantly less even than in unstimulated intact animals. Mating did not increase Fos expression in main olfactory pathways of intact animals over that of unstimulated intact controls. However, Fos expression in central vomeronasal pathways was significantly higher in mating anosmic males, as in intact males, compared with appropriate non-mating controls. Fos expression was significantly different between intact and zinc sulfate-treated anosmic mating males in only one area studied. The rostral anterior medial amygdala, known to receive a small olfactory terminal field, had significantly lower Fos expression in zinc sulfate-treated anosmic males that mated when compared with intact-mating animals. Thus, functional main olfactory input to the rostral vomeronasal amygdala can be demonstrated but does not appear to be critical for mating behavior in previously inexperienced male hamsters with intact vomeronasal organs. Other main olfactory input appears to have a negligible contribution to Fos-patterns in such animals.      

Simian virus 40 origin- and T-antigen-dependent DNA replication with Drosophila factors in vitro.

DNA replication of double-stranded simian virus 40 (SV40) origin-containing plasmids, which has been previously thought to be a species-specific process that occurs only with factors derived from primate cells, is catalyzed with an extract derived from embryos of the fruit fly Drosophila melanogaster. This reaction is dependent upon both large T antigen, the SV40-encoded replication initiator protein and DNA helicase, and a functional T-antigen binding site at the origin of DNA replication. The efficiency of replication with extracts derived from Drosophila embryos is approximately 10% of that observed with extracts prepared from human 293 cells. This activity is not a unique property of embryonic extracts, as cytoplasmic extracts from Drosophila tissue culture cells also support T-antigen-mediated replication of SV40 DNA. By using highly purified proteins, DNA synthesis is initiated by Drosophila polymerase alpha-primase in a T-antigen-dependent manner in the presence of Drosophila replication protein A (RP-A; also known as single-stranded DNA-binding protein), but neither human RP-A nor Escherichia coli single-stranded DNA-binding protein could substitute for Drosophila RP-A. In reciprocal experiments, however, Drosophila RP-A was able to substitute for human RP-A in reactions carried out with human polymerase alpha-primase. These results collectively indicate that many of the specific functional interactions among T antigen, polymerase alpha-primase, and RP-A are conserved from primates to Drosophila species. Moreover, the observation that SV40 DNA replication can be performed with Drosophila factors provides a useful assay for the study of bidirectional DNA replication in Drosophila species in the context of a complete replication reaction.     

Intermediate filaments in nervous tissues

Intermediate filaments have been isolated from rabbit intradural spinal nerve roots by the axonal flotation method. This method was modified to avoid exposure of axons to low ionic strength medium. The purified filaments are morphologically 75-80 percent pure. The gel electrophoretogram shows four major bands migrating at 200,000, 145,000, 68,000, and 60,000 daltons, respectively. A similar preparation from rabbit brain shows four major polypeptides with mol wt of 200,000 145,000, 68,000, and 51,000 daltons. These results indicate that the neurofilament is composed of a triplet of polypepetides with mol wt of 200,000, 145,000, and 68,000 daltons. The 51,000-dalton band that appears in brain filament preparations as the major polypeptide seems to be of glial origin. The significance of the 60,000- dalton band in the nerve root filament preparation is unclear at this time. Antibodies raised against two of the triplet proteins isolated from calf brain localize by immunofluorescence to neurons in central and peripheral nerve. On the other hand, an antibody to the 51,000-dalton polypeptide gives only glial staining in the brain, and very weak peripheral nerve staining. Prolonged exposure of axons to low ionic strength medium solubilizes almost all of the triplet polypeptides, leaving behind only the 51,000- dalton component. This would indicate that the neurofilament is soluble at low ionic strength, whereas the glial filament is not. These results indicate that neurofilaments and glial filaments are composed of different polypeptides and have different solubility characteristics.     

Functional Neural Correlates of Attentional Deficits in Amnestic Mild Cognitive Impairment

Although amnestic mild cognitive impairment (aMCI; often considered a prodromal phase of Alzheimer’s disease, AD) is most recognized by its implications for decline in memory function, research suggests that deficits in attention are present early in aMCI and may be predictive of progression to AD. The present study used functional magnetic resonance imaging to examine differences in the brain during the attention network test between 8 individuals with aMCI and 8 neurologically healthy, demographically matched controls. While there were no significant behavioral differences between groups for the alerting and orienting functions, patients with aMCI showed more activity in neural regions typically associated with the networks subserving these functions (e.g., temporoparietal junction and posterior parietal regions, respectively). More importantly, there were both behavioral (i.e., greater conflict effect) and corresponding neural deficits in executive control (e.g., less activation in the prefrontal and anterior cingulate cortices). Although based on a small number of patients, our findings suggest that deficits of attention, especially the executive control of attention, may significantly contribute to the behavioral and cognitive deficits of aMCI.     

Intraspecific DNA divergence in Drosophila: a study on parthenogenetic D. mercatorum

Drosophila mercatorum is a species that can give rise to totally homozygous parthenogenetic strains. Using the technique of DNA-DNA hybridization, we have assessed the overall single-copy DNA differences among three independently derived strains that represent three independent genomes. Among strains, the average difference between homoduplex and heteroduplex median melting temperatures is 1.3 degrees C. This represents greater than or equal to 1.3% base-pair mismatch. Normalized percent of reassociation indicates further genetic differences, probably reflecting insertion/deletion differences and/or regions of the genome that are highly variable. This overall intraspecific genetic variation is higher than generally is thought to exist but is consistent with growing evidence of extensive DNA diversity within species of invertebrates. High intraspecific DNA variation may be correlated with rapid phyletic rates of evolution. Because of this high level of variation, the technique of DNA-DNA hybridization may be used to study intraspecific variation in invertebrates but is limited in its usefulness for higher systematic studies.      

Periodontal microflora of HIV infected patients with periodontitis

The aim of this study was to determine the microbial profile of periodontal lesions in HIV seropositive patients and to compare it with rapidly progressing periodontal lesions in systemically healthy patients. The subgingival microflora of 20 CDC II, 20 CDC III, 20 CDC IV/V and 20 systemically healthy patients with rapidly progressing periodontitis was examined. Four sites with greatest probing depth in each patient were selected for microbiological sampling. The samples were cultured aerobically and anaerobically for bacterial isolation using selective and non-selective media. Isolates were characterized to species level by conventional biochemical tests and various identification kits. The microflora of periodontitis lesions within the three stages of the HIV infection was similar to that of progressing periodontitis in systemically healthy adults including Campylobacter rectus, Capnocytophaga spp., Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Selenomonas spp. and Peptostreptococcus micros. However, HIV seropositive periodontitis lesions harboured a range of exogenous pathogens rarely associated with common types of periodontitis including Staphylococcus aureus, Enterobacter cloaca, Pseudomonas aeruginosa, Candida albicans, Enterococcus faecalis, Enterococcus avium, Clostridium difficile, Aspergillus fumigatus, Klebsiella pneumoniae and Mycoplasma incognitum. The lack of immune effector and regulatory cells in HIV infected patients could in fact explain the increase of some opportunistic pathogens and the characteristic and rapidly progressing nature of the periodontal disease in these patients.