Colloidal Gold Cytochemistry of Endo-1,4-beta-Glucanase, 1,4-beta-D-Glucan Cellobiohydrolase, and Endo-1,4-beta-Xylanase: Ultrastructure of Sound and Decayed Birch Wood

Colloidal gold coupled to endo-1,4-beta-glucanase II (EG II) and 1,4-beta-D-glucan cellobiohydrolase I (CBH I), isolated from Trichoderma reesei (QM9414), and endo-1,4-beta-xylanase from Aureobasium pullulans (NRRLY-2311-1) was used successfully to determine the ultrastructural localization of cellulose and xylan in sound birch wood. In addition, these enzyme-gold complexes demonstrated the distribution of cellulose and xylan after decay by three white rot fungi, Phanerochaete chrysosporium, Phellinus pini, and Trametes versicolor, and one brown rot fungus, Fomitopis pinicola. Transverse sections of sound wood showed that EG II was localized primarily on the S(1) layer of the secondary wall, whereas CBH I labeled all layers of the secondary wall. Oblique sections showed a high concentration of gold labeling, using EG II or CBH I. Preference for the sides of the microfibrillar structure was observed for both EG II and CBH I, whereas only CBH I had a specificity for the cut ends of microfibrils. Labeling with the xylanase-gold complex occurred primarily in the inner regions of the S(2) layer, S(1), and the middle lamella. In contrast, little labeling occurred in the middle lamella with EG II or CBH I. Intercellular regions within the cell corners of the middle lamella were less electron dense and labeled positively when EG II- and xylanase-gold were used. Wood decayed by P. chrysosporium or P. pini was delignified, and extensive degradation of the middle lamella was evident. The remaining secondary walls labeled with EG II and CBH I, but little labeling was found with the xylanase-gold complex. Wood decayed by T. versicolor was nonselective, and erosion of all cell wall layers was apparent. Remaining wall layers near sites of erosion labeled with both EG II and CBH I. Erosion troughs that reached the S(1) layer or the middle lamella had less xylanase-gold labeling in the adjacent cell wall that remained. Brown-rotted wood had very low levels of gold particles present in sections treated with EG II or xylanase. Labeling with CBH I had the lowest concentrations in the S(2) layer near cell lumina and corresponded to sites with the most extensive degradation.     

Detection of Lignin Peroxidase and Xylanase by Immunocytochemical Labeling in Wood Decayed by Basidiomycetes

The white rot fungi used in this study caused two different forms of degradation. Phanerochaete chrysosporium, strain BKM-F-1767, and Phellinus pini caused a preferential removal of lignin from birch wood, whereas Trametes (Coriolus) versicolor caused a nonselective attack of all cell wall components. Use of polyclonal antisera to H8 lignin peroxidase and monoclonal antisera to H2 lignin peroxidase followed by immunogold labeling with protein A-gold or protein G-gold, respectively, showed lignin peroxidase extra-and intracellularly to fungal hyphae and within the delignified cell walls after 12 weeks of laboratory decay. Lignin peroxidase was localized at sites within the cell wall where electron-dense areas of the lignified cell wall layers remained. In wood decayed by Trametes versicolor, lignin peroxidase was located primarily along the surface of eroded cell walls. No lignin peroxidase was evident in brown-rotted wood, but slight labeling occurred within hyphal cells. Use of polyclonal antisera to xylanase followed by immunogold labeling showed intense labeling on fungal hyphae and surrounding slime layers and within the woody cell wall, where evidence of degradation was apparent. Colloidal-gold-labeled xylanase was prevalent in wood decayed by all fungi used in this study. Areas of the wood with early stages of cell wall decay had the greatest concentration of gold particles, while little labeling occurred in cells in advanced stages of decay by brown or white rot fungi.     

The structure and evolution of the spider monkey delta-globin gene

We have isolated the delta-globin gene of the New-World spider monkey, Ateles geoffroyi, and compared its nucleotide sequence with those of other primate delta- and beta-globin genes. Among primate delta-globin genes, the rate of nonsynonymous substitutions is much less than the rate of synonymous substitutions. This suggests that primate delta- globin genes may remain under evolutionary conservation, perhaps because hemoglobin A2 has an as yet unknown physiological importance.      

Delignification of Wood Chips and Pulps by Using Natural and Synthetic Porphyrins: Models of Fungal Decay

Kraft pulps, prepared from softwoods, and small chips of birch wood were treated with heme and tert-butyl hydroperoxide in aqueous solutions at reflux temperature. Analyses of treated pulps showed decreases in kappa number (a measure of lignin content) from about 36 to less than 2, with concomitant increases in brightness (80% increase in the better samples). Analyses of treated wood chips revealed selective delignification and removal of hemicelluloses. After 48 h of treatment, lignin losses from the wood chips approached 40%, and xylose/mannose (hemicellulose) losses approached 70%, while glucose (cellulose) losses were less than 10%. Examination of delignified chips by transmission electron microscopy showed that the removal of lignin occurred in a manner virtually indistinguishable from that seen after decay by white rot fungi. Various metalloporphyrins, which act as biomimetic catalysts, were compared to horseradish peroxidase and fungal manganese peroxidase in their abilities to oxidize syringaldazine in an organic solvent, dioxane. The metalloporphyrins and peroxidases behaved similarly, and it appeared that the activities of the peroxidases resulted from the extraction of heme into the organic phase, rather than from the activities of the enzymes themselves. We concluded that heme-tert-butyl hydroperoxide systems in the absence of a protein carrier mimic the decay of lignified tissues by white rot fungi.     

Pathogenicity of Bursaphelenchus xylophilus on Pines in Minnesota and Wisconsin

The pinewood nematode, Bursaphelenchus xylophilus, was inoculated into established native jack and red pines (Pinus banksiana and P. resinosa) and exotic Austrian pine (P. nigra) in Minnesota and Wisconsin forests during summer 1981. The nematode isolates did not kill established nonstressed pine trees growing in the forest. However, the same nematode isolates killed pine seedlings under greenhouse conditions. Girdling the main stem of some trees to induce stress resulted in the death of the majority of inoculated and noninoculated branches of Austrian and jack pines, but no branch death was observed on red pine. Greater numbers of nematodes were extracted from branches of inoculated, girdled trees than from nongirdled trees. The mean number of nematodes extracted from branches of inoculated, nongirdled trees was 0.3 - 14 nematodes per gram of wood.     

Biochemical pathways in prokaryotes can be traced backward through evolutionary time

For the first time, a credible prokaryotic phylogenetic tree is being assembled by Woese and others using quantitative sequence analysis of oligonucleotides in the highly conservative rRNA. This provides an evolutionary scale against which the evolutionary steps that led to the arrangement and regulation of contemporary biochemical pathways can be measured. This paper presents an emerging evolutionary picture of aromatic amino acid biosynthesis within a large superfamily assemblage of prokaryotes that is sufficiently developed to illustrate a new perspective that will be applicable to many other biochemical pathways.      

The immunopathology of experimental allergic encephalomyelitis. I. Quantitative analysis of inflammatory cells in situ

Acute experimental allergic encephalomyelitis (EAE) is a T cell-mediated, neurologic disease that is under immunogenetic control. We systematically analyzed the quantity and distribution of T cells, B cells, and macrophages in the central nervous system (CNS) of susceptible and resistant guinea (GP) with a panel of seven monoclonal antibodies by using the avidin-biotin complex (ABC) immunoperoxidase technique and alpha-naphthyl-butyrate esterase (ANBE) staining. Adult EAE-susceptible strain 13 GP immunized with isogeneic spinal cord homogenate (SC) or with myelin basic protein (MBP) developed clinical signs (paralysis, weight loss, etc.) in 2 to 3 wk. T cells were present in all CNS inflammatory foci and comprised 44% of the perivascular mononuclear cells. T cells diffusely infiltrated the neuropil away from inflammatory cell aggregates. These T cells were judged to be extravascular by the lack of an associated identifiable vessel in counter-stained sections, and by their persistence following exhaustive perfusion of the brains. In routine sections, mononuclear cells could be detected only in perivascular aggregates. IgM+ B cells comprised 9% of the perivascular infiltrates and did not diffusely infiltrate the parenchyma. ANBE+ macrophages comprised the remaining 47% of the identified perivascular cells. SC- and MBP-immunized GP showed equivalent numbers of inflammatory foci, T cells, and macrophages, but SC-immunized GP had more IgM+ cells in the meninges and choroid plexus (p less than 0.001, p less than 0.02, respectively). Virtually all cells in perivascular locations were Ia+. Ia+ mononuclear cells were also present in the neuropil. EAE-resistant strain 2 GP immunized with SC developed no clinical signs. These GP had fewer perivascular foci than strain 13 GP but, when present, the cellular composition, including the density of diffuse parenchymal T cell infiltrates, was indistinguishable. Significantly fewer parenchymal mononuclear cells in the strain 2 GP, however, displayed Ia, both in perivascular and diffuse infiltrates (p less than 0.001). We conclude that T cell migration into the CNS parenchyma is a characteristic feature of acute EAE in the GP, but that T cells can occur in this pattern without clinical signs of disease. The two features that distinguish susceptible and resistant strains were the frequency of perivascular infiltrates and the expression of Ia on parenchymal mononuclear cells, which probably reflects their enhanced immunologic activation in situ.     

The evolutionary history of the amylase multigene family in Drosophila pseudoobscura

In Drosophila pseudoobscura, the amylase (Amy) multigene family is contained within a series of inversions, or gene arrangements, on the third chromosome. The Standard (ST), Santa Cruz (SC), and Tree Line (TL) inversions are central to the phylogeny of arrangements, and have clusters of other arrangements derived from them. The gene arrangements belonging to each of these three clusters have a characteristic number of Amy genes, ranging from three in ST to two in SC to one in TL. This distribution pattern can reflect a history of either duplications or deletions, although the data available in the past did not permit a decision between these alternatives. We provide unambiguous evidence that three Amy genes were present before the divergence of the ST, SC, and TL arrangements. Thus, the current status of the Amy multigene family is the result of deletions in the TL and SC arrangements, which created three new pseudogenes: TL Amy2-psi, TL Amy3-psi, and SC Amy3- psi. Analysis of pseudogene sequences revealed that, in the SC and ST arrangements, pseudogene evolution has been retarded, most likely due to the homogenization effect of gene conversion. Finally, by determining the original copy number, we have reconstructed the evolutionary history of the Amy multigene family and linked it with the evolution of the central gene arrangements.      

USE OF SENSORY EVALUATION FOR MEASURING DEODORIZING EFFECTS OF AN AIR CONDITIONER

The application of sensory methodology for measuring deodorizing effect of an air conditioner equipped with electric plasma was introduced. Deodorizing effect was measured using chemical and sensory methods at different time (0, 30 and 60 min) and mode (control, blowing and cooling) of an air conditioner. Smoke from a roll of cigarette in a closed room was used as a source of odor and the concentrations of acetic acid and ammonia were measured as odorous chemical components. As one of the sensory methods triangle test was used and as a first step to obtain deodorizing effects by triangle test, the threshold of each panelist was obtained as the log dilution ratio of odor concentration at which the difference from odorless air was detected. The odor concentration at each time and mode was calculated using the threshold of the panel and the deodorizing effect was obtained on the basis of the odor concentration. In addition to a triangle test, scaling methods such as category scaling or magnitude estimation were used to measure deodorizing effect of an air conditioner. Deodorizing effects by scaling methods were calculated based on odor intensity with time at each mode. The regression analysis was done between the efficacy of deodorizing effect by sensory test and those by acetic acid and ammonia, the R² values of the regression equations for triangle test, category scale, and magnitude estimation were 0.84, 0.72 and 0.69, respectively. Deodorizing effect by triangle test explained the decrease of acetic acid and ammonia better than those by category scaling or magnitude estimation while high cost and time consuming labor involved in triangle tests reduced the merit. The results of this study demonstrated that various sensory methods could be used to measure deodorizing effect of air conditioners and further researches on fast and reliable methods are needed to establish the official procedures.