Purification and characterization of pig lung carbonyl reductase.

A pyrazole-sensitive carbonyl reductase from pig lung was purified to homogeneity by electrophoretic criteria. Chemical cross-linking study suggested that the native enzyme is a tetramer with a Mr of 103,000, consisting of apparent identical subunits of Mr 24,000. The enzyme reduced aliphatic and aromatic carbonyl compounds with NADPH as a preferable cofactor to NADH and catalyzed the oxidation of secondary alcohols and the aldehyde dismutation in the presence of NAD(P)+. Immunohistochemical study with the antibodies against the enzyme revealed that the enzyme was localized in the ciliated cells, nonciliated bronchiolar cells, Type II alveolar pneumocytes, and the epithelial cells of the ducts of the bronchial glands in the pig lung. In addition to the properties and distribution, the pig lung enzyme was immunochemically similar to the pulmonary enzymes in the guinea pig and mouse. However, the pig enzyme showed the following unusual features. (1) The enzyme exhibited an equatorial specificity in the reduction of 3-ketosteroids; the 4-pro-S hydrogen of NADPH was transferred to the carbonyl carbon atom of 5 alpha- and 5 beta-androstanes, and the respective reduced products were identified as 3 beta- and 3 alpha-hydroxysteroids. (2) Although the NADPH-linked reduction of carbonyl compounds apparently obeyed the Michaelis-Menten kinetics at pH 6.0, the double-reciprocal plots of the velocity vs concentrations of the carbonyl substrates were convex at pH higher than 6.5. The Hill coefficients and [S]0.5 values for the substrates decreased as the pH for reaction increased. The results suggest that the pig enzyme exhibits negative cooperativity with respect to the carbonyl substrates and that the hydrogen ion acts as an allosteric effector abolishing the negative interaction.     

A pathway through interferon-gamma is the main pathway for induction of nitric oxide upon stimulation with bacterial lipopolysaccharide in mouse peritoneal cells.

Production of nitric oxide (NO) in response to bacterial lipopolysaccharide (LPS) was investigated using cultures of mouse peritoneal exudate cells (PEC) and the macrophage cell line RAW264.7. In the presence of anti-(interferon-gamma) (IFN-gamma), NO production was markedly suppressed in the PEC culture but not in the RAW264.7 culture. In the PEC culture, LPS induced both IFN-gamma production and activation of IFN response factor-1, which leads to the gene expression of inducible NO synthase, but neither was induced in the culture of RAW264.7 cells. In addition to anti-(IFN-gamma), antibodies against interleukin (IL)-12 and IL-18 showed a suppressive effect on LPS-induced NO production in the PEC culture, and these antibodies in synergy showed strong suppression. Stimulation of the PEC culture with IL-12 or IL-18 induced production of IFN-gamma and NO, and these cytokines, in combination, exhibited marked synergism. Stimulation of the culture with IFN-gamma induced production of NO, but not IL-12. The macrophage population in the PEC, prepared as adherent cells, responded well to LPS for IL-12 production, but weakly for production of IFN-gamma and NO. The macrophages also responded well to IFN-gamma for NO production. For production of IFN-gamma by stimulation with LPS or IL-12 + IL-18, nonadherent cells were required in the PEC culture. Considering these results overall, the indirect pathway, through the production of intermediates (such as IFN-gamma-inducing cytokines and IFN-gamma) by the cooperation of macrophages with nonadherent cells, was revealed to play the main role in the LPS-induced NO production pathway, as opposed to the direct pathway requiring only a macrophage population.     

Rank and age related feeding strategy observed through field experiments in the Koshima group of Japanese macaques

The feeding process of Japanese monkeys on soy beans which were scattered over a sandy beach on Koshima islet was studied. Younger monkeys were able to pick up more beans when 8 kg of beans were divided and given two times (“two times feeding”) than when the whole amount (8 kg) of beans was given at one time (“one time feeding”). The effect of saturation of the food intake capacity in younger monkeys at the first feeding in “two times feeding” did not appear at the second feeding one hour later. The minutely intake of soy beans (feeding speed) for each age class was analyzed. The decline of feeding speed in adult females after the peak in “one time feeding” was not related to the decline in density of beans on the ground, and this decline was caused by saturation of their food intake capacity. Adult females were divided into four classes according to their dominance rank order: high, lower-high, higher-low, and low classes. The total amount of intake in “one time feeding” was far larger in the high class than in any of the other classes. The total amount of feeding in the first feeding of “two times feeding” increased in accordance with rise in the dominance rank class, and there was no relation to rank and total feeding amount in the second feeding of “two times feeding.” Differences existed in the process of feeding between the rank classes. The feeding speed of the low class was as high as that of the high class on the curve of minutely intake, while the low class stopped feeding much earlier than the high class. The lower-high class displayed a low feeding speed, and stopped feeding the latest. The order of the duration to stay and to feed in the feeding area was lower-high > high > higher-low > low, and this order did not change under the three different feeding conditions, “one time feeding,” and the first and second feedings of “two times feeding.” Adolescent females tended to stay the longest duration in the feeding area among all age classes. Both the lower-high class females and adolescent females had an unstable social status in the Koshima group, and their social status affected their feeding behaviors. The feeding behaviors were similar in attitude depending on social status, and are considered to be maintained for a fairly long time. The feeding strategy of the lower-high class, in staying a longer duration in the artificial feeding area, and departing later, may be effective under the artificial feeding conditions, but it may be a bad strategy in a natural habitat where the food is not so clumped as in artificial feeding, and where choice of other food patches is possible. The above results agree well with previous reports for the Koshima group, indicating that the rank of the lower-high class females was unstable (Mori et al., 1989), and that their reproductive success was low (Watanabe et al., 1992).     

Purification and properties of thiamine-binding proteins from sesame seed

Three thiamine-binding proteins of 17-19 kDa (STBP-I, II, and III) were purified from sesame seed (Sesamum indicum L.). Each of the proteins was composed of two subunits of equal molecular mass and each subunit consisted of a large polypeptide and a small polypeptide linked by a disulfide bond(s). They were rich in glutamic acid (or glutamine) and arginine. Their binding activities were optimal at neutral pH. They bound specifically free thiamine but not thiamine phosphates. STBP-I had higher affinity for thiamine than STBP-II or STBP-III. STBP-II and STBP-III bound one molecule of thiamine per molecule, and STBP-I bound 0.5 molecule. The amino acid composition and structure of the STPBs were similar to those of 2S storage proteins.     

Induced single fertilization in maize

 Bicellular pollen with one vegetative nucleus and one diploid arrested generative cell (”monospermic” pollen) was induced by trifluralin treatment of diploid maize plants at 7–9 days before flowering. The arrested generative cell (seemingly a diploid sperm cell) fused with the central cell of diploid plants and produced shriveled endosperm resembling that of a 2n×4n cross in maize. Dual pollination experiments with a purple embryo marker revealed single fertilization events in which the union of one sperm cell with the egg occurs but there is no union of a second sperm cell with the central cell. Singly fertilized ovules survived at least 4 days. Furthermore, many viable triploid plants were obtained. This technique therefore appears to have the potential for manipulating ploidy level in crops and may become useful in investigating fertilization mechanisms of angiosperms. Received: 1 October 1996 / Revision accepted: 8 January 1997     

Immunohistochemistry of Grandry corpuscles in the oral mucosa of the duck bill: a light- and electron-microscopic study

Grandry corpuscles in the oral mucosa of the upper bill of the duck were immunohistochemically studied using antisera against calcitonin gene-related peptide (CGRP), galanin, methionine-enkephalin, neuropeptide Y (NPY), somatostatin, substance P (SP) and vasoactive intestinal peptide (VIP). Grandry corpuscles in the lamina propria selectively showed only SP-like immunoreactivity. Herbst corpuscles distributed near Grandry corpuscles were negative to all antisera applied. Although immunoreactive products in the Grandry corpuscles were found as granules in the peripheral cytoplasm of the Grandry cell, the axon terminals and satellite cells exhibited no reactivity. In pre-embedding electron-microscopic sections, SP-like immunoreactive products visualized with 3,3-diaminobezidine were localized in the granules of Grandry cells, but no labeling was observed in the cytoplasmic matrix or cell organelles. Electron-immunocytochemical labeling with colloidal gold by the post-embedding method clearly demonstrated that the SP antigen was localized only in the granules. It is presumed that Grandry cells have a secretory function. However, the function and the method of release of the SP contained in the observed granules remains obscure. Some CGRP-, NPY-, SP- and VIP-like-immunoreactive nerve fibers with varicosities associated with blood vessels and nerve fiber bundles of various sizes were observed in the lamina propria, but no such fibers penetrated into the intraepitherial layer. Nerve fibers positive for SP and VIP were also found in the interlobular connective tissue of the palatine glands. Some SP-positive neurons were detected in the vicinity of the palatine glands.     

In situ CO2 gas-exchange in fruits of a tropical tree,Durio zibethinus Murray

We examined the in situ CO₂ gas-exchange of fruits of a tropical tree, Durio zibethinus Murray, growing in an experimental field station of the Universiti Pertanian Malaysia. Day and night dark respiration rates were exponentially related to air temperature. The temperature dependent dark respiration rate showed a clockwise loop as time progressed from morning to night, and the rate was higher in the daytime than at night. The gross photosynthetic rate was estimated by summing the rates of daytime dark respiration and net photosynthesis. Photosynthetic CO₂ refixation, which is defined as the ratio of gross photosynthetic rate to dark respiration rate in the daytime, ranged between 15 and 45%. The photosynthetic CO₂ refixation increased rapidly as the temperature increased in the lower range of air temperature T _c (T _c <28.5 °C), while it decreased gradually as the temperature increased in the higher range (T _c 28.5 °C). Light dependence of photosynthetic CO₂ refixation was approximated by a hyperbolic formula, where light saturation was achieved at 100 mol m^–2 s^–1 and the asymptotic CO₂ refixation was determined to be 37.4%. The estimated gross photosynthesis and dark respiration per day were 1.15 and 4.90 g CO₂ fruit^–1, respectively. Thus the CO₂ refixation reduced the respiration loss per day by 23%. The effect of fruit size on night respiration rate satisfied a power function, where the exponent was larger than unity.