Molecular cloning and characterization of cortical rod protein in the giant freshwater prawn Macrobrachium rosenbergii, a species not forming cortical rod structures in the oocytes

The formation of cortical rod structures is a characteristic of fully mature oocytes in penaeid prawns, but such structures are absent from oocytes of giant freshwater prawn Macrobrachium rosenbergii. In the present study, we first demonstrated the presence of a 30-kDa protein, which was immunologically related to kuruma prawn cortical rod protein (CRP), in the ovary of giant freshwater prawn, and subsequently purified this protein. Furthermore, a cDNA encoding the CRP-like protein was isolated. Based on the high homology (98%) in the amino acid sequence with kuruma prawn CRP, the 30-kDa protein has been identified as a CRP homologue of giant freshwater prawn, designated mrCRP. The RT-PCR analysis revealed that mrCRP mRNA was present in the ovary from a prawn with a gonadosomatic index (GSI) of 0.2. Western blot analysis revealed the presence of a CRP-immunoreactive band of 30kDa in the ovary with GSI of 1.6. By immunocytochemistry, CRP-immunopositive signals were detected in the ovary with GSI of 0.9, that had started to accumulate considerable amounts of vitellins and lipids in the peripheral cytoplasm. With progress of vitellogenesis, mrCRP was apparently accumulated in the mature oocytes, although it was not detectable, presumably because a relatively small amount of mrCRP was masked with large amounts of vitellin and lipids. In giant freshwater prawn without forming cortical rod structures, our findings indicate that the oocytes produce mrCRP, a homologue of CRP found in penaeid prawns.     

Lycopene, tomatoes, and coronary heart disease

Tomato and its major antioxidant component lycopene have recently been focused as important antioxidant nutrients because of their ability to reduce reactive oxygen species and to provide health benefits. Most of the studies were undertaken to determine the usefulness of lycopene against cancer and cardiovascular diseases. Epidemiological studies, however, yielded conflicting results. This study was undertaken to compare cardioprotective abilities of tomato juice and lycopene. Rats were gavaged either tomato juice or lycopene for 3 weeks. At the end of 3 weeks, isolated hearts were subjected to 30 min ischemia followed by 2 h of reperfusion. Both tomato juice and lycopene reduced the extent of lipid peroxidation; but only tomato juice, but not lycopene, improved post-ischemic ventricular function, and reduced myocardial infarct size and cardiomyocyte apoptosis. The results indicated for the first time that tomato juice, but not lycopene, possesses cardioprotective ability.     

Efficiency of peptide nucleic acid-directed PCR clamping and its application in the investigation of natural diets of the Japanese eel leptocephali

Polymerase chain reaction (PCR)-clamping using blocking primer and DNA-analogs, such as peptide nucleotide acid (PNA), may be used to selectively amplify target DNA for molecular diet analysis. We investigated PCR-clamping efficiency by studying PNA position and mismatch with complementary DNA by designing PNAs at five different positions on the nuclear rDNA internal transcribed spacer 1 of the Japanese eel Anguilla japonica in association with intra-specific nucleotide substitutions. All five PNAs were observed to efficiently inhibit amplification of a fully complementary DNA template. One mismatch between PNA and template DNA inhibited amplification of the template DNA, while two or more mismatches did not. DNA samples extracted from dorsal muscle and intestine of eight wild-caught leptochephalus larvae were subjected to this analysis, followed by cloning, nucleotide sequence analysis, and database homology search. Among 12 sequence types obtained from the intestine sample, six were identified as fungi. No sequence similarities were found in the database for the remaining six types, which were not related to one another. These results, in conjunction with our laboratory observations on larval feeding, suggest that eel leptocephali may not be dependent upon living plankton for their food source.     

Light- and diffusion-potential-induced shift of carotenoid spectrum in reconstituted vesicles of Rhodopseudomonas sphaeroides

Proteoliposomes were reconstituted from detergent-solubilized pigment · protein complexes of chromatophores of Rhodopseudomonas sphaeroides and soybean phospholipids. The reconstituted vesicles showed a photooxidation of reaction center bacteriochlorophyll and a light-induced spectral shift of carotenoid to longer wavelengths. The red shift similar to that in intact cells or chromatophores, indicates the generation of local fields in the membrane of proteoliposomes. When inside-positive membrane potential was induced by adding valinomycin and potassium salt, a shift of carotenoid spectrum to shorter wavelengths was observed. Therefore, the reconstituted vesicles, at least in the major part of population, produced the light-induced local field in the same direction as in intact cells, which is inside negative. Sidedness of the membrane structure and the direction of electric field formation in reconstituted vesicles were opposite to those in chromatophores (inside-out vesicles).     

A pitfall in diagnosis of human prion diseases using detection of protease-resistant prion protein in urine. Contamination with bacterial outer membrane proteins

Because a definite diagnosis of prion diseases relies on the detection of the abnormal isoform of prion protein (PrPSc), it has been urgently necessary to establish a non-invasive diagnostic test to detect PrPSc in human prion diseases. To evaluate diagnostic usefulness and reliability of the detection of protease-resistant prion protein in urine, we extensively analyzed proteinase K (PK)-resistant proteins in patients affected with prion diseases and control subjects by Western blot, a coupled liquid chromatography and mass spectrometry analysis, and N-terminal sequence analysis. The PK-resistant signal migrating around 32 kDa previously reported by Shaked et al. (Shaked, G. M., Shaked, Y., Kariv-Inbal, Z., Halimi, M., Avraham, I., and Gabizon, R. (2001) J. Biol. Chem. 276, 31479-31482) was not observed in this study. Instead, discrete protein bands with an apparent molecular mass of approximately 37 kDa were detected in the urine of many patients affected with prion diseases and two diseased controls. Although these proteins also gave strong signals in the Western blot using a variety of anti-PrP antibodies as a primary antibody, we found that the signals were still detectable by incubation of secondary antibodies alone, i.e. in the absence of the primary anti-PrP antibodies. Mass spectrometry and N-terminal protein sequencing analysis revealed that the majority of the PK-resistant 37-kDa proteins in the urine of patients were outer membrane proteins (OMPs) of the Enterobacterial species. OMPs isolated from these bacteria were resistant to PK and the PK-resistant OMPs from the Enterobacterial species migrated around 37 kDa on SDS-PAGE. Furthermore, nonspecific binding of OMPs to antibodies could be mistaken for PrPSc. These findings caution that bacterial contamination can affect the immunological detection of prion protein. Therefore, the presence of Enterobacterial species should be excluded in the immunological tests for PrPSc in clinical samples, in particular, urine.     

Evolutionary change in a pine wilt system following the invasion of Japan by the pinewood nematode, <Emphasis Type="Italic">Bursaphelenchus</Emphasis><Emphasis Type="Italic">xylophilus</Emphasis>

Pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causative agent of pine wilt disease (PWD) of pine trees and is transmitted by cerambycid beetles belonging to the genus Monochamus. PWN is believed to have been introduced into Japan from North America at the beginning of the 20th century. In this article, we first provide an outline of the PWD system and the range expansion of PWN in Japan and then review the literature, focusing on the virulence of PWN. Virulence is a heritable trait in PWN, with high virulence being closely related to a high rate of reproduction and within-tree dispersal. When two PWN isolates with different virulence levels are inoculated into pine seedlings, the more virulent nematodes always dominate in dead seedlings. In a laboratory setting, many more virulent nematodes board the insect vectors than avirulent ones. The age at which vectors transmit the most abundant PWNs to pine twigs changes during the course of a PWD epidemic. However, the relation between virulence and transmission of PWN remains as yet relatively unknown. Such information would enable ecologists to predict the evolution of the PWD system. In this review we also compare ecological traits between the PWN and the avirulent congener, B. mucronatus.     

Membrane stabilization of vitamin E; interactions of alpha-tocopherol with phospholipids in bilayer liposomes

13C Spin-lattice relaxation times (T1) of 13C-labeled alpha-tocopherol in three kinds of liposomes varying in their contents of arachidoyl residues have been measured by 13C-NMR spectroscopy. On the basis of T1 values, it is proved that the segmental motion of isoprenoid side chain of alpha-tocopherol tends to increase with an increase in the distance from the chromanol moiety, and that three methyl groups attached on the aromatic ring, have some affinity to unsaturated fatty acid residues rather than those of the isoprenoid side chain. These results are incompatible with the hypothesis of Diplock et al. (1) which 4'a- and 8'a-methyl groups of isoprenoid side chain are fitted in the Z-pockets of arachidoyl chain of polyunsaturated lipids in membrane.     

Interspecific and sexual differences in riverine distribution of tropical eels Anguilla spp.

A total of 261 individuals of the four tropical eel species, Anguilla celebesensis, Anguilla marmorata, Anguilla bicolor pacifica and Anguilla interioris, were collected from 12 locations around Sulawesi Island, Indonesia, to gain knowledge about the riverine distribution of tropical eels. Anguilla marmorata was predominant in the lower reaches of Poso River (94·4% of total eel catch in the sampling area), Poso Lake (93·3%), three small inlet rivers of Tomini Bay (100%) and Laa River (92·3%). Anguilla celebesensis occurred frequently in the inlet rivers of Poso Lake (63·5%). Anguilla bicolor pacifica and Anguilla interioris were rare (1.5 and 0.4%, respectively). Otolith Sr:Ca ratio electron‐probe micro analysis (EPMA) for individual migratory histories revealed that 15 A. celebesensis caught in Poso Lake and its inlet rivers were categorized into 14 river eels (Sr:Ca < 2·5) showing upstream migration seemingly at their elver stage and only one sea eel (Sr:Ca ≥ 6·0) that stayed in the marine habitat for the majority of its life after recruiting to Sulawesi Island before its late upstream migration. In A. marmorata, 19 examined eels from Poso Lake and its inlet rivers were all river eels, while 17 eels from the lower reaches of Poso River were two river eels, six sea eels and nine estuarine eels (2·5 ≤ Sr:Ca < 6·0) that mostly lived in the brackish water. The sex ratio of A. celebesensis was highly skewed towards a dominance of females (99%). In A. marmorata, females were predominant in Poso Lake (95·2%), its inlet rivers (94·7%) and Laa River (100%), while males were more frequent in the lower reaches of Poso River (76·5%) and small inlet rivers of Tomini Bay (94·1%). These results indicate that the riverine distribution pattern of tropical eels differs among species and between sexes.     

Chemical screening and development of novel gibberellin mimics

Gibberellin (GA) plays versatile roles in the regulation of plant growth and development and therefore is widely used as a regulator in agriculture. We performed a chemical library screening and identified a chemical, named 67D, as a stimulator of seed germination that was suppressed by paclobutrazol (PAC), a GA biosynthesis inhibitor. In vitro binding assays indicated that 67D binds to the GID1 receptor. Further studies on the structure–activity relationship identified a chemical, named chemical 6, that strongly promoted seed germination suppressed by PAC. Chemical 6 was further confirmed to promote the degradation of RGA (for repressor of ga1-3), a DELLA protein, and suppress the expression levels of GA3ox1 in the same manner as GA does. 67D and its analogs are supposed to be agonists of GID1 and are expected to be utilized in agriculture and basic research as an alternative to GA.