Spatially distributed morphological characteristics of macropores in forest soils of Hitachi Ohta Experimental Watershed, Japan

Morphological characteristics of macropores in forest soil profiles were investigated at Hitachi Ohta Experimental Watershed in Japan. Nine individual profiles at different locations (various spatial scales in a catchment) and twenty profiles at one site (a small spatial scale) were excavated to the bedrock to investigate density, origin, diameter, direction, and gradient of macropores. Macropore densities in a soil profile ranged from 3.5 to 29.1 per m and from 5.4 to 75.1 per m², respectively. Subsurface erosion, root channels, and interactions between subsurface erosion and root channels accounted for 36.9, 36.5, and 19.0%, of the described macropores. The mean macropore diameter in organic-rich soil layer (17–20 mm) was larger than in the B horizon (11–14 mm) at both spatial scales. The dominant gradients of all macropores in the organic-rich soil layer and B horizon were at negative oblique angles. Approximately 90% of the macropores in the organic-rich soil layer and approximately 80% of the macropores in the B horizon fell within the range between −50 and 50 degree planar direction. Subsurface flow and root systems are believed to play important roles in determining the morphological characteristics of macropores. These characteristics appear to have variable influences in different soil horizons rather than at different spatial scales. A part of this paper was presented at the 103th (1992) and 105th (1994) Annual Meetings of Japanese Forestry Society.     

Classification of the genus Bacillus based on MALDI-TOF MS analysis of ribosomal proteins coded in S10 and spc operons

A rapid bacterial identification method by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) using ribosomal proteins coded in S10 and spc operons as biomarkers, named the S10-GERMS (the S10-spc-alpha operon gene encoded ribosomal protein mass spectrum) method, was applied for the genus Bacillus a Gram-positive bacterium. The S10-GERMS method could successfully distinguish the difference between B. subtilis subsp. subtilis NBRC 13719(T) and B. subtilis subsp. spizizenii NBRC 101239(T) because of the mass difference of 2 ribosomal subunit proteins, despite the difference of only 2 bases in the 16S rRNA gene between them. The 8 selected reliable and reproducible ribosomal subunit proteins without disturbance of S/N level on MALDI-TOF MS analysis, S10, S14, S19, L18, L22, L24, L29, and L30, coded in S10 and spc operons were significantly useful biomarkers for rapid bacterial classification at species and strain levels by the S10-GERMS method of genus Bacillus strains without purification of ribosomal proteins.     

The functional effect of Gly209 and Ile213 substitutions on lysozyme activity of family 19 chitinase encoded by cyanophage Ma-LMM01

ORF69 in the cyanophage infecting Microcystis aeruginosa, Ma-LMM01, shows homology to the family 19 chitinases where the catalytic domain has structural similarity to lysozyme. Chitinases hydrolyze chitin, a β-1, 4-linked monopolymer of N-acetylglucosamine (GlcNAc); whereas lysozymes hydrolyzes peptidoglycan, alternating β-1, 4-linked copolymers of N-acetylmuramic acid (MurNAc) and GlcNAc. Using amino acid sequence comparison to ORF69, the putative sugar binding residues, Gln162 and Lys165, from the barley chitinase (the model enzyme for the family 19 chitinases) corresponding to subsites −4 and −3 were found to be replaced with Gly209 and Ile213, respectively, in ORF69. To analyze their contribution to substrate binding affinity, ORF69 was cloned into Escherichia coli; and two mutant proteins G209Q and I213K were prepared using site-directed mutagenesis. The wild-type gene product (gp69) showed both lysozyme and chitinase activities. In contrast, the I213K mutant showed a decrease (70%) in lysozyme activity and a significant increase (50%) in chitinase activity; whereas, the G209Q mutant almost completely abolished both enzyme activities. The data suggest the Ile213 residue is involved in recognizing the substrate MurNAc; and Gly209 has significant contribution in chitinase and lysozyme activities for the wild-type gp69.     

Analysis of cross-reactivity of five new chicken monoclonal antibodies which recognize the apical complex of Eimeria using confocal laser immunofluorescence assay

For Apicomplexa (members) the host cell invasion is realized with the help of the organelles located at the apical tip of parasites. In this research paper the characterization of five chicken monoclonal antibodies (mabs) produced against Eimeria acervulina sporozoites is described. All mabs reacted with molecules belonging to the apical complex of chicken Eimeria sporozoites. On immunofluorescence assay (IFA) one mab, 8E-1, recognized an apical tip molecule present on all chicken Eimeria sporozoites, two mabs (8D-2 and HE-4) recognized an antigen present on the apical tip of the same two Eimeria species (E. acervulina and E. brunetti), another mab (5D-11) recognized an antigen present on the apical tip of other two species (E. acervulina and E. maxima) while one mab (8C-3) identified antigens present on the sporozoites and sporocysts wall of only E. acervulina. Besides the apical tip antigens, two mabs (HE-4 and 8D-2) recognized some proteins located in the anterior half of the sporozoites. Collectively, these mabs proved that the apical complex of chicken Eimeria sporozoites share one or more antigens that are expected to play a role in host cell recognition and invasion.     

Application of DGGE analysis to the study of bacterial community structure in plant roots and in nonrhizosphere soil

To analyze the structure of bacterial communities in spinach roots and in the nonrhizosphere soil, we used PeR-amplified 16S rRNA gene fragments separated by denaturing gradient gel electrophoresis (DGGE). DGGE revealed a large number of band patterns, which were ascribed to various bacterial species composing each of the bacterial communities. The pattern from the roots was less complex than that from the soil. It is considered that DGGE analysis is suitable for studies of bacterial community structure in soil-plant ecosystems.     

Identification and characterization of rhizobacteria isolated by enrichment culture on spinach roots

Rhizobacteria can be used for biological control and environmental restoration. In this study, we performed enrichment culture of rhizobacteria, identified isolates, and investigated the physiological properties of the bacterial isolates. Five bacteria differing in their colony morphology were isolated from spinach roots as enriched rhizobacteria. Four isolates were identified by sequencing of 16S rDNA as β and γ-Proteobacteria; 16S rDNA sequencing was not completed on one isolate. Based on microscopic observation, we determined that at least two types of bacteria differing in their morphology co-existed in this isolate, and that it may not be possible to culture the two types separately. Based on tests of substrate utilization, we could not find the characteristics that were common to the isolates. One of the five isolates was inoculated into non-sterile soil, and we examined its root-colonizing ability. The test strain which was not detected in the non-rhizosphere soil, accounted for about 20% of the total bacteria on the roots. These results suggested that enrichment culture might be useful for isolating bacteria with a high root-colonizing ability     

Differential immunolocalization between lingual antimicrobial peptide and lactoferrin in mammary gland of dairy cows

Lingual antimicrobial peptide (LAP), one of the β-defensins in bovines, and lactoferrin (LF) are synthesized in mammary epithelium and have bactericidal and bacteriostatic functions. However, it is not known whether they have similar expression patterns. Therefore, the present study was undertaken to compare (1) immunolocalization of LAP and LF in the mammary gland and (2) changes in concentration of these two components in milk after lipopolysaccharide (LPS) challenge. Bovine mammary tissues without LPS challenge were collected and their sections were immunostained with antibodies to LAP or LF. Milk from our previous study was collected every hour up to 12h and twice daily from d 1 to 7 after LPS challenge (the day of infusion was considered as d 0). These milk samples were measured for LAP but not LF in our previous report. Therefore, concentration of LF was measured by enzyme immunoassay in the present study. Epithelial cells of some alveoli showed immunopositive reaction for LF, but negative for LAP. Conversely, some alveoli were LAP positive in their epithelial cells but LF negative. Many alveoli had immunoreactions for neither LAP nor LF. The concentration of LAP in milk was elevated significantly at 3h after LPS infusion compared with pre-infusion values and remained at a high level until 12h. However, LF concentration in milk remained low at d 0 and increased at d 2. These results suggest that LAP and LF were mostly differentially localized in the alveolar epithelium in mammary glands. The different spatial expressions between them may be associated with their different temporal expression mechanisms.