Comparison of dendritic cell-mediated immune responses among canine malignant cells

Dendritic cell (DC) vaccination is one of the most attractive immunotherapies for malignancies in dogs. To examine the differences in DC-mediated immune responses from different types of malignancies in dogs, we vaccinated dogs using autologous DCs pulsed with keyhole limpet hemocyanin (KLH) and cell lysate prepared from squamous cell carcinoma SCC2/88 (SCC-KLH-DC), histiocytic sarcoma CHS-5 (CHS-KLH-DC), or B cell leukemia GL-1 (GL-KLH-DC) in vitro. In vivo inductions of immune responses against these tumor cells were compared by the delayed-type hypersensitivity (DTH) skin test. The DTH response against SCC2/88 cells were observed in dogs vaccinated with autologous SCC-KLH-DC, while the response was undetectable against CHS-5 and GL-1 cells in dogs vaccinated with autologous CHS-KLH-DC and GL-KLH-DC. Skin biopsies taken from DTH challenge sites were then examined for immunohistochemistry, and recruitment of CD8 and CD4 T cells was detected at the site where SCC2/88 cells were inoculated in dogs vaccinated with SCC-KLH-DC. By contrast, neither CD8 nor CD4 T cell infiltration was found at the DTH challenge site in the dogs vaccinated with CHS-KLH-DC or GL-KLH-DC. These findings may reflect that the efficacy of immune induction by DC vaccination varies among tumor types and that immune responses could be inducible in squamous cell carcinoma. Our results encouraged further investigation of therapeutic vaccination for dogs with advanced squamous cell carcinoma in clinical trials.     

Stem water transport of Lithocarpus edulis, an evergreen oak with radial-porous wood

The stem water conducting system of an evergreen broad-leaved oak, Lithocarpus edulis (Mak.) Nakai, was investigated. Evergreen broad-leaved oaks (Lithocarpus, Castanopsis, Cyclobalanopsis, Quercus) belonging to the Quercoideae are a major component of Asian monsoon forests, and are characterized by the possession of radial-porous wood. A characteristic of radial-porous wood is the development of aggregate rays between radially oriented files of vessels. We measured the distribution of vessel lumen diameters in a stem cross section and calculated the theoretical water conductivity of the wood. The radial profile of the heat pulse velocity (HPV) was measured for an intact whole tree under field conditions and compared with the theoretical distribution of water conductivity. Soft X-ray photographs of frozen stem sections indicated that most of the vessel lumina were filled with water, including those of vessels more than 20 years old. Even when vessels were relatively wide (lumen diameters > 100 microm), cavitation was negligible. The rate of water uptake from the cut stem base correlated closely with HPV (r = 0.96), and HPV closely reflected the mean volume flow per stem sectional area (SFVS) around the sensor probes. However, the ray tissue sharply inhibited heat transfer, and the positioning of the probes strongly affected the absolute value of HPV. It was also found that HPV more closely reflected the mean sap flow velocity in the vessels than did SFVS.     

Molecular and antigenic similarities of the fimbrial major components between Porphyromonas gulae and P. gingivalis

Porphyromonas gulae is black-pigmented anaerobic bacteria associated with canine periodontitis. There is little information available about the specific identify and relative occurrence of pigmented anaerobes in companion animals. Our aim was to clarify the factor involved in the adherence and colonization of the organism in the oral cavity. Fimbrial protein was purified from P. gulae ATCC 51700. The molecular mass of this protein was approximately 41kDa as estimated by SDS-PAGE. An antibody against 41-kDa fimbrial protein from P. gingivalis ATCC 33277 reacted with fimbrillin of P. gulae ATCC 51700. Immunogold electron microscopy revealed that the anti-41kDa fimbrial serum bound to fimbria on the cell surface of P. gulae ATCC 51700. Thus, fimbrial protein of P. gulae ATCC 51700 had the same size and antigenicity as 41-kDa fimbriae of P. gingivalis ATCC 33277. The nucleotide sequence of the fimA gene from P. gulae ATCC 51700 showed 94% homology with that of P. gingivalis ATCC 33277. Moreover, the deduced amino acid sequences have 96.8% identity. P. gulae has adherent ability to gingival epithelial cells. The properties of P. gulae fimbriae are similar to those of P. gingivalis fimbriae. We suggest that the surface structure of P. gulae may play a role in the colonization of this organism in periodontal pockets in companion animals.     

Azimuthal variations of sap flux density within Japanese cypress xylem trunks and their effects on tree transpiration estimates

Sap flow techniques are practical tools for estimating tree transpiration. Though many previous studies using sap flow techniques did not consider azimuthal variations of sap flux density (F _d) on xylem trunk to estimate tree transpiration, a few studies reported that ignoring the azimuthal variations in F _d could cause large errors in tree transpiration estimates for some tree species. Therefore, examining azimuthal variations in F _d for major plantation tree species is critical for estimating tree transpiration. Using the thermal dissipation method, we examined azimuthal variations in F _d in six trees of Japanese cypress Chamaecyparis obtusa (Sieb. et Zucc.) Endl., which is one of the most common plantation tree species in Japan. We recorded considerable variations among F _d at four different azimuthal directions. The F _d value for one aspect was more than 100% larger than those for the other aspects. We calculated differences between tree transpiration estimates based on F _d for one to three azimuthal directions and those based on F _d for four aspects. The differences relative to tree transpiration estimates based on F _d for four aspects were typically 30, 20, and 10% in accordance with the F _d for one, two, and three measurement aspects, respectively. This finding indicates that ignoring azimuthal variations could cause large errors in tree transpiration estimates for Japanese cypress.     

Absorption spectra of humic acids

For the purpose of explaining the forming process of soil humic acids, the author determined the absorption spectra of various humic acids. From soils and peats which were pretreated with 5% HCl at 70°C for 30 minutes or from those which were not, humic acids were extracted by treating with 0.5% NaOH at boiling temperature for 30 minutes. In these humic acids, the one which is extracted after acid pretreatment is provisionally designated as SrL humic acid and the other as L humic acid. The supernatant alkaline solutions obtained by centrifuging the above mentioned extracts were acidified with hydrochloric acid, and precipitated humic acids were filtered and washed with water until Cl' free. Humic acids were dissolved in 0.1% NaOH and ultrafiltered using collodion membrane. The filtrates were acidified with hydrochloric acid and humic acids were collected by centrifuging, transferred on the filter paper, washed with dilute hydrochloric acid and water successively, then air-dried and pulverized.     

On artificial humic acids

I. Absorption Spectra of Some Artificial Humic Acids

It is a well known fact that numerous dark brown or black and amorphous substances cap be produced from organic compounds by purely chemical processes. Some of them are called artificial humic acids, and have been used as a model of soil humic acids by many workers. But it is doubtful whether such artificial humic acids are considered to be a model of soil humic acids, even if there can be found any similarities in their properties. However it may be an useful method for the study of humus formation to compare artificial humic acids with soil humic acids, and to make efforts to find a good model or to prepare artificial humic acids comparable to soil humic acids under laboratory conditions. For this purpose, first of all, the author prepared artificial humic acids from glucose, hydroquinone and lignin, and their absorption spectra were determined.     

The chemical nature of the green fraction of P type humic acid

P type humic acid has absorption bands near 615, 570 and 450mµ in the visible region, and is a common component of many soils in north temperate regions (1, 2, 3). Its unusual spectral characteristics are due to a green fraction (Pg), which can be separated from the humic acid dissolved in aqueous alkali by chromatography on Sephadex or cellulose powder, and is precipitated as a reddish-brown substance on acidification (2). In aqueous alkali, it has λmax at 613, 568, 448, 430 (infl.) and 281mµ. The distribution of Pg in different soils is probably related to the presence of fungal sclerotia. The highest content of Pg in p type humic acid is about 10%. We have now obtained evidence that the characteristic colour of Pg is due to the presence of a derivative of 4,9 - dihydroxyperylene - 3,10 - quinone.     

Studies on the humus forms of forest soils

Samples described in the previous paper were analyzed for humus composition by the method of Kumada el al,, elementary composition of humic acids, nitrogen distribution among humic acid, fulvic acid, and humin, and organic matter composition by the modified Waksman method. The samples obtained by physical fractionation from each horizon of Higashiyama soil were as follows: f₁ and f₂ from the L layer, f₁, f₂ and f₃ from the F layer, f₁ f₂, sand, silt, and clay fractions from the H-A and A horizons.

With the progress of decomposition, the following tendencies were rather clearly observed.

The extraction ratio of soluble humus, amounts of humic acid and fulvic acid, and PQ, value tended to increase with some exceptions. The degree of humification of humic acid proceeded. Most humic acids belonged to the Rp type, but those of the clay fractions belonged to the B type.

As for the elementary composition of humic acid, transitional changes from the Lf₁ to the clay fraction of the A horizon were observed. But differences in elementary composition among humic acids were far less, compared with those among whole fractions.

Nitrogen contents in humic, fulvic, and humin fractions increased with the progress of decomposition and humiliation, and the largest relative increase was found in fulvic acid nitrogen.

According to the modified Waksman's method, the amounts of residues and protein increased, while the total amounts of each extract, except for the HCl extract, and the amounts of sugars and starch, phenolic substances, hemicelluloses and pectin, and cellulose decreased. Sugars and starch comprised only a small portion of the hot water extract, and polyphenols substances comparable to sugars and starch were also found in the extract. Hemicelluloses and pectin accounted for only about one-half of the HCl extract. Several characteristic differences in the elementary composition of extracts and residues were found.

Pheopigments existed in benzene-ethanol extracts and their amounts seemed to decrease from Lf₁ to Ff₂.     

The reduction mechanism of manganese in paddy soils

Hydroquinone method manganese (soluble in pH 7.0, 1 N-ammonium acetate solution containing 0.2 percent hydroquinone) and microbially active manganese (soluble in pH 7.0, 1 M-magnesium sulfate solution after flooding soils with or without Chinese milk vetch for 12 or 20 days respectively at 30°C) of 22 paddy soils were determined. The amounts of manganese reduced with sodium oxalate under acid conditions (oxalate method manganese (a) and (b), the former was determined under more rigorous conditions than the latter) were also determined and compared with hydroquinone method manganese and microbially active manganese.

Their levels of many soil samples representing soil groups were also determined to examine the dlfferences In amounts of active manganese among soil groups. The results obtained are as follows.

The relationship between microbially and chemically active manganese. 1) The amounts of microbially active manganese in soils were 48 to 68 mg Mn per 100 g oven-dried soil and these were increased by the addition of Chinese milk vetch. 2) The amounts of hydroquinone method manganese were less than microbially active manganese, and the amounts of oxalate method manganese (b) were larger than microbially active manganese. The amounts of oxalate method manganese (a) were the largest of all the types of manganese. 3) There were high correlations between the amounts of various types of active manganese described in 2). The levels of microbially and chemically active manganese. 1) The amounts of microbially active manganese lay between the amounts of chemically active manganese determined by the hydroqulnone method and by the oxalate method (b) In all soil samples representing soil groups. High correlations were found between these types of active manganese. 2) The hydroquinone method was considered to be unsuitable for quantitatively determining the amounts of chemicallY active manganese in soils of high organic matter content. 3) In both cases of microbially active manganese and chemically active manganese, tha widest range and the largest amount determined were both observed in strongly gley soila. The averages of theae types of active manganese were high in strongly gley soils, pea, and muck soils, and black soils. The differences among soil groups were smalle1 than the differences among soil samples, and little tendency was observed in the differences among soil groups.

From these findings described above it is suggested that the oxalate methoo (b) is more appropriate than the hydroqulnone method for determining chemically active manganese as an index of microbially active manganese.     

Studies on the humus forms of forest soils

The results of elementary analysis of each fraction separated physically from each horizon of five forest soils were described.

The biggest fraction was Lf₂ (fresh litter) in L layers, Ff₂ (>2 mm) or Ff₂ (2-1 mm) is F layers and Hf₄ in H layers.

Of all the changes in various parameters which accompanied decomposition and humification, the change in C/N ratio was the most obvious; the C/N ratio for plant remains easily decreased to 25 or 30 during decomposition, but any further decrease in this value occurred with difficulty. It was specifically pointed out that Hf₇ and Hf₂ were fairly different materials from the other fractions.

As for A horizons, both carbon and nitrogen contents increased, and CIN ratios decreased in the order of sand, silt and clay fractions.