Five carboxin-resistant mutants exhibited various responses to carboxin and related fungicides

Five carboxin-resistant mutants from Aspergillus oryzae were characterized by the sensitivities of their mycelial growth and succinate dehydrogenase (SDH) activity to carboxin and three related fungicides. Despite a significant resistance to carboxin, exhibited by all the mutants, their patterns of sensitivity to the other fungicides was distinct. This provides clues to the molecular interaction between SDH and these fungicides.     

Electron microscopic examination of uncultured soil-dwelling bacteria

Bacteria living in soil collected from a rice paddy in Fukuoka, Japan, were examined by electron microscopy using a freeze-substitution fixation method. Most of the observed bacteria could be categorized, based on the structure of the cell envelope and overall morphology, into one of five groups: (i) bacterial spore; (ii) Gram-positive type; (iii) Gram-negative type; (iv) Mycobacterium like; and (v) Archaea like. However, a few of the bacteria could not be readily categorized into one of these groups because they had unique cell wall structures, basically resembling those of Gram-negative bacteria, but with the layer corresponding to the peptidoglycan layer in Gram-negative bacteria being extremely thick, like that of the cortex of a bacterial spore. The characteristic morphological features found in many of these uncultured, soil-dwelling cells were the nucleoid being in a condensed state and the cytoplasm being shrunken. We were able to produce similar morphologies in vitro using a Salmonella sp. by culturing under low-temperature, low-nutrient conditions, similar to those found in some natural environments. These unusual morphologies are therefore hypothesized to be characteristic of bacteria in resting or dormant stages.     

Detection of eggs in the living white grub beetle Dasylepida ishigakiensis (Coleoptera: Scarabaeidae) by magnetic resonance imaging

The use of magnetic resonance imaging (MRI) as a tool for in vivo detection of eggs in living Dasylepida ishigakiensis Niijima et Kinoshita, a major pest of sugarcane, was explored using females with an ovary at different developmental stages. MRI measurements of beetles were performed at 13 °C to avoid motion artifacts on the MR images. Spin–lattice relaxation time-weighted images allowed the observation of eggs at short acquisition times (2 min, 8 s). By comparing MR images with dissection data, criteria for determining mature eggs in MR images were a clear circular or ellipsoidal shape surrounded by a relatively bright rim and a size typically larger than 1.3 mm in the minor axis. Although small oocytes could not be detected, females with a developed or undeveloped ovary could be clearly distinguished based on MR images. The possibility of confusing the digestive tract as eggs in a female with a less developed ovary can be eliminated using a proton density weighted image.     

Immunization of A4galt-deficient mice with glycosphingolipids from renal cell cancers resulted in the generation of anti-sulfoglycolipid monoclonal antibodies

In this study, we immunized Gb3/CD77 synthase gene (A4galt) knockout (KO) mice with glycosphingolipids (GSLs) extracted from 3 renal cell cancer (RCC) cell lines to raise monoclonal antibodies (mAbs) reactive with globo-series GSLs specifically expressed in RCCs. Although a number of mAbs reactive with globo-series GSLs were generated, they reacted with both RCC cell lines and normal kidney cells. When we analyzed recognized antigens by mAbs that were specifically reactive with RCC, but not with normal kidney cells at least on the cell surface, many of them turned out to be reactive with sulfoglycolipids. Eight out of 11 RCC-specific mAbs were reactive with SM2 alone, and the other 3 mAbs were more broadly reactive with sulfated glycolipids, i.e. SM3 and SM4 as well as SM2. In the immunohistochemistry, these anti-sulfoglycolipids mAbs showed RCC-specific reaction, with no or minimal reaction with adjacent normal tissues. Thus, immunization of A4galt KO mice with RCC-derived GSLs resulted in the generation of anti sulfated GSL mAbs, and these mAbs may be applicable for the therapeutics for RCC patients.     

Subunit proximity in the H+-translocating NADH-quinone oxidoreductase probed by zero-length cross-linking

The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of 14 different subunits (designated Nqo1-14), seven of which are located in the membrane domain and the other seven in the peripheral domain. It has been previously reported that membrane domain subunit Nqo7 (ND3) directly interacts with peripheral subunit Nqo6 (PSST) by using a cross-linker, m-maleimidobenzoyl-N-hydrosuccinimide ester, and heterologous expression [Di Bernardo, S., and Yagi, T. (2001) FEBS Lett. 508, 385-388]. To further explore the near-neighbor relationship of the subunits, a zero-length cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), and the Paracoccus membranes were used, and the cross-linked products were examined with antibodies specific to subunits Nqo1-11. The Nqo6 subunit was cross-linked to subunit Nqo9 (TYKY). In addition, a ternary product of Nqo3 (75k), Nqo6, and Nqo7 and binary products of Nqo3 and Nqo6 and of Nqo6 and Nqo7 were observed, but a binary product of Nqo3 and Nqo7 was not detected. The Nqo4 (49k) subunit was found to be associated with the Nqo7 subunit. Furthermore, Paracoccus subunits Nqo3, Nqo6, and Nqo7 were heterologously coexpressed in Escherichia coli, and EDC cross-linking experiments were carried out using the E. coli membranes expressing these three subunits. The results were the same as those obtained with Paracoccus membranes. On the basis of the data, subunit arrangements of NDH-1 were discussed.