Mouse CD8+ CD122+ T cells with intermediate TCR increasing with age provide a source of early IFN-gamma production

Although CD8+ IL-2Rbeta (CD122)+ T cells with intermediate TCR reportedly develop extrathymically, their functions still remain largely unknown. In the present study, we characterized the function of CD8+ CD122+ T cells with intermediate TCR of C57BL/6 mice. The proportion of CD8+ CD122+ T cells in splenocytes gradually increased with age, whereas CD8+ IL-2Rbeta-negative or -low (CD122-) T cells conversely decreased. The IFN-gamma production from splenocytes stimulated with immobilized anti-CD3 Ab in vitro increased with age, whereas the IL-4 production decreased. When sorted CD8+ CD122+ T cells were stimulated in vitro by the anti-CD3 Ab, they promptly produced a much larger amount of IFN-gamma than did CD8+ CD122- T cells or CD4+ T cells, whereas only CD4+ T cells produced IL-4. The depletion of CD8+ CD122+ T cells from whole splenocytes greatly decreased the CD3-stimulated IFN-gamma production and increased the IL-4 production, whereas the addition of sorted CD8+ CD122+ T cells to CD8+ CD122+ T cell-depleted splenocytes restored the IFN-gamma production and partially decreased IL-4 production. It is of interest that CD8+ CD122+ T cells stimulated CD4+ T cells to produce IFN-gamma. The CD3-stimulated IFN-gamma production from each T cell subset was augmented by macrophages. Furthermore, CD3-stimulated CD8+ CD122+ T cells produced an even greater amount of IFN-gamma than did liver NK1.1+ T cells and also showed antitumor cytotoxicity. These results show that CD8+ CD122+ T cells may thus be an important source of early IFN-gamma production and are suggested to be involved in the immunological changes with aging.     

Chromosome mapping of RNF16 and rnf16, human, mouse and rat genes coding for testis RING finger protein (terf), a member of the RING finger family

RNF16 (ring finger protein 16; alias terf), a member of the RING finger family, has been shown to be exclusively expressed in the testis. Human RNF16 is located at 1q42 based on PCR-assisted analysis of both a human/rodent mono-chromosomal hybrid cell panel and a radiation hybrid-mapping panel. On the other hand, chromosomal mapping of the RNF16 gene by fluorescence in situ hybridization reveals that mouse Rnf16 is located at 11B1.2-B1.3 and rat Rnf16 at 10q22. These results provide additional evidence that the mouse 11B region displays conserved linkage homology with the rat 10q22 region, whereas in the case of RNF16, this homology is only conserved among rodents, distinct from the 1q42 region of the human genome.     

Differences in the floral anthocyanin content of red petunias and Petunia exserta

In order to resolve a conflict between previous papers regarding the floral anthocyanins of red flowers of Petunia exserta, a naturally occurring species, the HPLC profile of this species was compared with that of commercial red garden petunias. Both HPLC profiles extremely superficially resemble each other in terms of relative amounts and retention times of the major anthocyanins. However, co-elution on HPLC of the mixed sample resulted in clear separation of the components. Three major anthocyanins in red petunias were determined to be cyanidin 3-sophoroside, cyanidin 3-glucoside and peonidin 3-glucoside, which exhibited similar behaviors on HPLC to delphinidin 3-glucoside. delphinidin-3-rutinoside and petunidin 3-rutinoside, respectively, the major floral anthocyanins of P. exserta.     

Photometric Application of the Gram Stain Method To Characterize Natural Bacterial Populations in Aquatic Environments

The Gram stain method was applied to the photometric characterization of aquatic bacterial populations with a charge-coupled device camera and an image analyzer. Escherichia coli and Bacillus subtilis were used as standards of typical gram-negative and gram-positive bacteria, respectively. A mounting agent to obtain clear images of Gram-stained bacteria on Nuclepore membrane filters was developed. The bacterial stainability by the Gram stain was indicated by the Gram stain index (GSI), which was applicable not only to the dichotomous classification of bacteria but also to the characterization of cell wall structure. The GSI spectra of natural bacterial populations in water with various levels of eutrophication showed a distinct profile, suggesting possible staining specificity that indicates the presence of a particular bacterial population in the aquatic environment.Gram’s method is the most important and fundamental orthodox method for bacterial identification. It classifies bacteria into two groups, gram-negative and gram-positive. The mechanism of Gram staining is based on the fundamental structural and chemical attributes of bacterial cell walls. The cell walls of gram-positive bacteria have a high percentage of peptidoglycan, while those of gram-negative bacteria have only a thin peptidoglycan layer (1–3, 6). In Gram’s method, an insoluble dye-iodine complex is formed inside bacterial cells and is extracted by alcohol from gram-negative but not gram-positive bacteria (6, 12, 16). There are taxonomically gram-variable species, but some cells of gram-negative or gram-positive species may show gram-variable characteristics due to environmental stress, such as unsuitable nutrients, temperature, pH, or electrolytes (3).Functional differences between gram-positive and gram-negative cell walls have been studied with special emphasis on nutrient uptake from the ambient environment. Gram-negative bacteria have a periplasmic space between the lipopolysaccharide layer and the plasma membrane. In this space, binding proteins initially attach to nutrients and take them to a membrane carrier. Gram-positive bacteria lack the periplasmic space and are believed to have no binding proteins (9). Therefore, nutrient uptake from the environment is easier for gram-negative bacteria than for gram-positive bacteria. Because of this difference, the population density of gram-negative bacteria in more oligotrophic environments could be higher than that of gram-positive bacteria (20).Gram staining is commonly used only to reflect cell wall structure. If Gram staining characterizes not only simple taxonomical dichotomy but also multiple biological functions, it may also be used to correlate bacterial cell wall structure with related physiological responses to the environment. In particular, Gram staining could supply ecological information on natural bacterial populations that are difficult to culture by the present technology.Membrane filter methods are widely used for microscopy in aquatic microbiology because of the low population densities of bacteria in many aquatic environments (4, 11, 16). However, these methods sometimes have problems associated with microscopic observations, causing unclear images of bacterial cells on Nuclepore filters when used with the conventional mounting medium (immersion oil; refractive index [nd] = 1.514). Hence, a suitable mounting agent must be applied to obtain precise image analyses of Gram-stained bacteria on Nuclepore filters.In this study, we have established a distinct method to characterize photometric Gram stain images; it involves the Gram stain index (GSI) for specifying natural bacterial populations in various aquatic environments. For this purpose, we have standardized the GSI of typical gram-negative and gram-positive bacteria by using Escherichia coli and Bacillus subtilis, respectively, and compared these GSI values to those of natural bacterial populations of several freshwater environments. The natural waters we investigated were Hyoutaro-ike pond, Matsumi-ike bog, and Lake Kasumigaura, which are oligotrophic, mesotrophic, and eutrophic water bodies, respectively, as previously determined (8, 10, 13, 18, 22, 23).     

Microorganisms and plant of Autonomous Biological Systems (ABS) samples.

Distribution of microorganisms and cellular structure of an Autonomous Biological Systems (ABS) were studied with a special attention to the effect of space environments. Viable cell densities measured by the direct fluorescence microscopic method were in the order of 10(5) cells/ml for fractions 1 (upper suspension) and 2 (lower suspension), and 10(6) cells/ml for fraction 3 (sediments). These values were 10 to 100 times larger than the values obtained by the classical colony forming unit method. No difference between flight and ground samples was observed in the vertical distribution of viable microorganisms when fractionation and analysis were carried out after recovery. Intracellular distribution of chloroplasts in higher green plants, Ceratophyllum demersum, of flight samples was disturbed after 10 days of flight (24hrs/day light on). After 4 months of flight (Mir/STS-79/81) with 24 hrs light on, Ceratophyllum demersum was completely disintegrated. On the other hand, in the second 4-months-flight experiment with 16 hrs/day light on, Ceratophyllum demersum was only slightly deteriorated.     

High-efficiency cloning of Arabidopsis full-length cDNA by biotinylated CAP trapper

Full-length cDNAs are essential for functional analysis of plant genes. We constructed high-content, full-length cDNA libraries from Arabidopsis thaliana plants based on chemical introduction of a biotin group into the diol residue of the CAP structure of eukaryotic mRNA, followed by RNase I treatment, to select full-length cDNA. More than 90% of the total clones obtained were of full length; recombinant clones were obtained with high efficiency (2.2 × 10⁶/9 μg starting mRNA). Sequence analysis of 111 randomly picked clones indicated that 32 isolated cDNA groups were derived from novel genes in the A. thaliana genome.     

Treatment of Irradiated Mice with High-Dose Ascorbic Acid Reduced Lethality

Ascorbic acid is an effective antioxidant and free radical scavenger. Therefore, it is expected that ascorbic acid should act as a radioprotectant. We investigated the effects of post-radiation treatment with ascorbic acid on mouse survival. Mice received whole body irradiation (WBI) followed by intraperitoneal administration of ascorbic acid. Administration of 3 g/kg of ascorbic acid immediately after exposure significantly increased mouse survival after WBI at 7 to 8 Gy. However, administration of less than 3 g/kg of ascorbic acid was ineffective, and 4 or more g/kg was harmful to the mice. Post-exposure treatment with 3 g/kg of ascorbic acid reduced radiation-induced apoptosis in bone marrow cells and restored hematopoietic function. Treatment with ascorbic acid (3 g/kg) up to 24 h (1, 6, 12, or 24 h) after WBI at 7.5 Gy effectively improved mouse survival; however, treatments beyond 36 h were ineffective. Two treatments with ascorbic acid (1.5 g/kg × 2, immediately and 24 h after radiation, 3 g/kg in total) also improved mouse survival after WBI at 7.5 Gy, accompanied with suppression of radiation-induced free radical metabolites. In conclusion, administration of high-dose ascorbic acid might reduce radiation lethality in mice even after exposure.     

Inhibition of the Prostaglandin Transporter PGT Lowers Blood Pressure in Hypertensive Rats and Mice

Inhibiting the synthesis of endogenous prostaglandins with nonsteroidal anti-inflammatory drugs exacerbates arterial hypertension. We hypothesized that the converse, i.e., raising the level of endogenous prostaglandins, might have anti-hypertensive effects. To accomplish this, we focused on inhibiting the prostaglandin transporter PGT (SLCO2A1), which is the obligatory first step in the inactivation of several common PGs. We first examined the role of PGT in controlling arterial blood pressure blood pressure using anesthetized rats. The high-affinity PGT inhibitor T26A sensitized the ability of exogenous PGE₂ to lower blood pressure, confirming both inhibition of PGT by T26A and the vasodepressor action of PGE₂ T26A administered alone to anesthetized rats dose-dependently lowered blood pressure, and did so to a greater degree in spontaneously hypertensive rats than in Wistar-Kyoto control rats. In mice, T26A added chronically to the drinking water increased the urinary excretion and plasma concentration of PGE₂ over several days, confirming that T26A is orally active in antagonizing PGT. T26A given orally to hypertensive mice normalized blood pressure. T26A increased urinary sodium excretion in mice and, when added to the medium bathing isolated mouse aortas, T26A increased the net release of PGE₂ induced by arachidonic acid, inhibited serotonin-induced vasoconstriction, and potentiated vasodilation induced by exogenous PGE₂. We conclude that pharmacologically inhibiting PGT-mediated prostaglandin metabolism lowers blood pressure, probably by prostaglandin-induced natriuresis and vasodilation. PGT is a novel therapeutic target for treating hypertension.     

Genetic structure of the critically endangered Red‐headed Wood Pigeon Columba janthina nitens and its implications for the management of threatened island populations

The Red‐headed Wood Pigeon Columba janthina nitens is endemic to the Ogasawara Islands, an oceanic island chain located 1000 km south of the main islands of Japan. The subspecies is at high risk of extinction because of its small population size and restricted habitat range. We undertook genetic analyses of this pigeon using sequences of a portion of the mitochondrial control region and five microsatellite markers to estimate the genetic characteristics of two wild populations from the Bonin and Volcano Islands, as well as one captive breeding population. The genetic diversity of the wild individuals was exceptionally low in both the mitochondria (nucleotide diversity = 0.00105) and at the microsatellite (3.2 alleles per locus and H_E = 0.12) loci. Higher numbers of microsatellite genotypes were observed in the Volcano Islands population than in the Bonin Islands population, which may be because of the relatively low impact of human disturbance. The most common mitochondrial haplotypes and microsatellite alleles observed in the two wild populations were completely fixed in the captive population. Our results suggest that the genetic diversity of the captive population needs to be increased. However, introduction of a wild individual into a captive population can lead to a decreased genetic diversity in the wild population and therefore should be done with caution. The genetic differentiation between the Bonin and the Volcano island groups was low, and the populations of the two island groups should be regarded as a single evolutionarily significant unit. However, special consideration is required for habitat conservation in the Volcano Islands, which may be functioning as a sanctuary for the Red‐headed Wood Pigeon. For the long‐term conservation of threatened bird species that live on remote oceanic islands, determination of management units considering gene flow caused by their flying capacity and maintenance of genetically suitable wild and captive populations are essential.     

Extreme rapid warming yields high functional survivals of vitrified 8-cell mouse embryos even when suspended in a half-strength vitrification solution and cooled at moderate rates to −196 °C

To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to do so is to subject them to procedures that convert cell water into a non-crystalline glass. Current belief is that to achieve this vitrification, cells must be suspended in very high concentrations of glass-inducing solutes (i.e., ?6 molal) and cooled at very high rates (i.e., ?1000 °C/min). We report here that both these beliefs are incorrect with respect to the vitrification of 8-cell mouse embryos. In this study, precompaction 8-cell embryos were vitrified in several dilutions of EAFS10/10 using various cooling rates and warming rates. Survival was based on morphology, osmotic functionality, and on the ability to develop to expanded blastocysts. With a warming rate of 117,500 °C/min, the percentages of embryos vitrified in 1×, 0.75×, and 0.5× EAFS that developed to blastocysts were 93%, 92%, and 83%, respectively. And the percentages of morphological survivors that developed to expanded blastocysts were 100%, 92%, and 97%, respectively. Even when the solute concentration of the EAFS was reduced to 33% of normal, we obtained 40% functional survival of these 8-cell embryos.