Proteobacteria and Bacteroidetes are major phyla of filterable bacteria passing through 0.22 μm pore size membrane filter,in Lake Sanaru,Hamamatsu, Japan

141 filterable bacteria that passed through a 0.22 μm pore size filter were isolated from Lake Sanaru in Hamamatsu, Japan. These belonged to Proteobacteria, Bacteroidetes, Firmicutes, or Actinobacteria among which the first two phyla comprised the majority of the isolates. 48 isolates (12 taxa) are candidates assignable to new bacterial species or genera of Proteobacteria or Bacteroidetes.     

Genetic analysis of Japanese and American specimens of <Emphasis Type="Italic">Scirpus hattorianus</Emphasis> suggests its introduction from North America

Scirpus hattorianus is a possible alien species in Japan, and a clarification of its unclear taxonomy is required to reveal its origin. It is not known whether the plants initially described from Japan represent the same species distributed in North America. To clarify the origin of the species, we attempted to sequence old specimens collected about 80 years ago using newly designed primer pairs specific for short sequences, including the variable sites. Chloroplast sequences of ndhF were compared among Japanese and North American S. hattorianus, and the closely related species, S. atrovirens, S. flaccidifolius, and S. georgianus. We succeeded in sequencing all samples, and two haplotypes were detected in S. hattorianus: one was unique to the species and the other, detected from specimens potentially collected from the same population as the types, was shared by both North American S. hattorianus and two closely related species, S. atrovirens and S. flaccidifolius. Our results suggest that Japanese S. hattorianus is an alien species that was introduced from North America at least twice.     

T Cell-Dependent Activation of Macrophages and Enhancement of Their Phagocytic Activity in the Lungs of Mice Inoculated with Heat-Killed Cryptococcus neoformans: Involvement of IFN-γ and Its Protective Effect against Cryptococcal Infection

Previous investigations have demonstrated that macrophages play a critical role in the first-line cellular defense mechanism against infection with Cryptococcus neoformans. In the present study, to elucidate the way in which anticryptococcal activity of macrophages is regulated at the site of infection, pulmonary intraparenchymal macrophages were directly analyzed for expression of their surface molecules and their phagocytic activities against the organism, and the effects of depletion of T cells and endogenous IFN-γ in vivo on these parameters were examined. In the lungs of mice intratracheally inoculated with heat-killed C. neoformans, macrophages were activated, as indicated by augmented expression of MHC class II, intercellular adhesion molecule-1 (ICAM-1) and Fc receptor (FcR), and about two-thirds of macrophages were found to have ingested an average of 3.77 ± 0.12 yeast cells per macrophage. In mice depleted of both CD4⁺ and CD8⁺ T cells by injecting the specific monoclonal antibodies (mAbs) or anti-IFN-γ mAb, not only augmentation of the expression of macrophage activation markers but also phagocytosis of C. neoformans was significantly reduced. These results suggest that anticryptococcal activity of macrophages is regulated by IFN-γ endogenously produced by T cells. Additionally, treatment with IFN-γ were shown to significantly prolong the survival time of mice infected with viable C. neoformans. Additionally, preimmunization with heat-killed C. neoformans significantly prolonged the survival time of mice which received the following infection.     

Transient expression of β-glucuronidase in plastids of various plant cells and tissues delivered by a pneumatic particle gun

Chloroplast expression plasmids pTRBCL-GUS (tobaccorbcL promoter-gusA-tobaccorbcL terminator) and pHHU3004 (spinach ‘x gene’ promoter-gusA-spinachrbcL terminator) and a control nuclear expression plasmid pBI221 (CaMV 35S promoter-gusA-NOS terminator) were introduced separately into cultured cells and tissues of tobacco andArabidopsis thaliana, as well as into cultured cells of the lower land plants liverwort and hornwort by a pneumatic particle gun. The pTRBCL-GUS and pHHU3004 plasmids produced many blue spots in the BY-2 cells and the roots ofArabidopsis thaliana, but not in any of the green cells or tissues. The results suggest that the pTRBCL-GUS and pHHU3004 plasmids are expressed more in proplastids and amyloplasts than in chloroplasts. GUS activities of the BY-2 cells bombarded with pTRBCL-GUS and pHHU3004 were insensitive to α-amanitin treatment (10 and 50 μg/ml), while that of the cells with pBI221 greatly decreased by the same treatment. Hence, it is likely that the pTRBCL-GUS and pHHU3004 plasmids were substantially expressed in the proplastids.     

Impaired spermatogenesis in the Japanese eel, Anguilla japonica: Possibility of the existence of factors that regulate entry of germ cells into meiosis

In the cultivated male Japanese eel, spermatogonia are the only germ cells present in the testis. Weekly injections of human chorionic gonadotropin (HCG) can induce complete spermatogenesis from proliferation of spermatogonia to spermiogenesis. In some cases, however, HCG injection fails to induce complete spermatogenesis. Testicular morphological observations revealed that HCG-injected eels could be classified into three types based on their testicular conditions. Type 1 eels had a well-developed testis and the milt could be acquired by hand-stripping. In type 2 eels, spermatogenesis was also induced by HCG injection, but testicular size was remarkably smaller than that of type 1 eels, and the milt could not be hand-stripped. At the end of the experiment, type 2 fish had only spermatogonia and a small amount of spermatozoa, but no spermatocytes or spermatids, in their testis. Type 3 eels had thready testis, which did not develop any germ cells during the experimental period. These results suggest that, despite elevations of plasma 11–ketotestosterone levels, HCG injections were not successful in inducing the completion of spermatogenesis in type 2 and type 3 eels. In most spermatogonia of type 2 eels, meiosis was not induced by HCG injections. Furthermore, only few mitotic divisions had occurred as evidenced by the presence of 2³ to 2⁶ late type B spermatogonia in most cysts. This suggests that spermatogonial stem cells undergo four or five, and occasionally six, mitotic divisions before the interruption of spermatogenesis in type 2 eels. It is proposed that those numbers of mitotic divisions are related to a mediator that regulates entry of spermatogonia of the Japanese eel into meiosis.     

Nucleotide Sequence of a 28-kb Mouse Genomic Region Comprising the Imprinted Igf2 Gene

The mouse insulin-like growth factor II gene (Igf2) is physicallylinked to the insulin II gene (Ins2) and both are subject totissue-specific genomic imprinting. The paternal-specific expressionof Igf2 has been associated with hypermethylation of some CpGsites in the 5' flanking region and in the body of the gene.As a first step in analyzing the structural features of thisimprinted locus, we here report the complete nucleotide sequenceof Igf2, including all introns and the intergenic region adjacentto Ins2. This 28-kb segment of mouse chromosome 7 exhibits 80%overall identity with the corresponding rat sequence and hasa high GC content of 52%. In addition to the known CpG islandwithin the second Igf2 promoter, another island was identifiedapproximately 2 kb 5' to the first exon. Other features of thislocus include a 35-fold tandem repeat of an 11-bp sequence thatoverlaps Igf2 pseudo-exon 2, and a B2 repeat element in theintergenic region between Ins2 and Igf2. The GC-richness andthe presence of CpG islands associated with tandem repeats arecommon features of imprinted genes and thus may play a rolein the imprinting mechanism.     

Nested Deletions from a Fixed Site as an Aid to Nucleotide Sequencing: an in vitro System Using Tn3 Transposase

We have previously constructed a cloning/sequencing vector,with an in vivo system capable of creating nested deletionsfrom the end of transposon Tn3, which is useful for sequencinglarge DNAs. Here we report an in vitro system which uses anammonium sulfate fraction of extract from E. coli cells harboringa Tn3 transposase overproducer plasmid to generate nested deletions.A key feature of the procedure is exhaustive digestion of thereaction products with a restriction enzyme that cleaves onlybetween the Tn3 "right" terminus and the cloned fragment. Thisstep reduces the noise level due to mechanisms other than deletionsfrom the Tn3 terminus, and facilitates detection and isolationof the desired deletion products. This system enables us tosave at least 2 days' time when obtaining the necessary deletionscompared with the in vivo system.     

Effect of oxygen-enriched aeration on regeneration of rice (Oryza sativa L.) cell culture

The effect of the oxygen concentration in the aeration gas on regeneration from rice cells in bioreactor cultures was investigated. The efficiency of regeneration in cultures aerated with over 40% oxygen was higher than that in a flask culture. In the case of a culture in which the dissolved oxygen(DO) was saturated by aeration with air, the efficiency of regeneration was less than the half that of cultures aerated with 40% oxygen. In cultures with the DO levels controlled at 8,10 and 12 mg/, the efficiency of regeneration was highest at 12 mg/. In the oxygenenriched cultures, although cell aggregation was observed and the color of plantlets was relatively pale, more than 90% of them grew into healthy plants.Abbreviations DO dissolved oxygen - 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2-(N-morpholino) ethanesulfonic acid - rpm revolution per minute - NAA 1-naphthaleneacetic acid - vvm volume per volume per minute