Glucose 1-phosphate increases active transport of calcium in intestine

Active calcium transport in intestine is essential for serum calcium homeostasis as well as for bone formation. It is well recognized that vitamin D is a major, if not sole, stimulator of intestinal calcium transport activity in mammals. Besides vitamin D, endogenous glucose 1-phosphate (G1P) affects calcium transport activity in some microorganisms. In this study, we investigated whether G1P affects intestinal calcium transport activity in mammals as well. Of several glycolytic intermediates, G1P was the sole sugar compound in stimulating intestinal calcium uptake in Caco-2 cells. G1P stimulated net calcium influx and expression of calbindin D9K protein in rat intestine, through an active transport mechanism. Calcium uptake in G1P-supplemented rats was greater than that in the control rats fed a diet containing adequate vitamin D3. Bone mineral density (BMD) of aged rat femoral metaphysis and diaphysis was also increased by feeding the G1P diet. G1P did not affect serum levels of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] at all. These results suggest that exogenously applied G1P stimulates active transport of calcium in intestine, independent of vitamin D, leading to an increase of BMD.     

Glucose-6-phosphate dehydrogenase cytochemistry using a copper ferrocyanide method and its application to rapidly frozen cells

We describe an improved copper ferrocyanide-based method for cytochemical detection of glucose-6-phosphate dehydrogenase (G6PD), which was used to localize the enzyme within the ultrastructure of rat hepatocytes and adrenocortical cells. With this method, glutaraldehyde fixation and the addition of exogenous electron carriers (for example, phenazine methosulfate) to the cytochemical reaction medium were essential. Copper ferrocyanide reaction product showing the distribution of G6PD was readily recognized at the light microscopic level as Hatchett’s brown staining and at the electron microscopic level as electron-dense deposits. Within stained regions, enzyme cytochemical G6PD activity was found to be associated with ribosome-like structures. Because G6PD is a soluble, cytosolic enzyme, its displacement or extraction may occur during conventional fixation. We, therefore, combined a rapid-freezing technique with G6PD enzyme cytochemistry. The resultant rapid-freezing enzyme cytochemistry enabled us to show the subcellular distribution of G6PD in a more life-like state; the localization of G6PD in rapidly frozen cells was in substantial agreement with that in conventionally fixed cells. Accepted: 14 July 1999     

Intra‐trophic isotopic discrimination of 15N/14N for amino acids in autotrophs: Implications for nitrogen dynamics in ecological studies

The differential discrimination of nitrogen isotopes (¹⁵N/¹⁴N) within amino acids in consumers and their diets has been routinely used to estimate organismal tropic position (TP). Analogous isotopic discrimination can occur within plants, particularly in organs lacking chloroplasts. Such discrimination likely arises from the catabolic deamination of amino acids, resulting in a numerical elevation of estimated TP, within newly synthesized biomass. To investigate this phenomenon, we examined the ¹⁵N/¹⁴N of amino acids (δ¹⁵N_AA) in spring leaves and flowers from eight deciduous and two annual plants. These plants were classified on the basis of their time of bloom, plants that bloomed when their leaves were absent (Type I) versus plants that bloomed while leaves were already present (Type II). Based on the δ¹⁵N_AA values from leaves, both plant types occupied comparable and ecologically realistic mean TPs (=1.0 ± 0.1, mean ± 1σ). However, the estimated TPs of flowers varied significantly (Type I: 2.2 ± 0.2; Type II: 1.0 ± 0.1). We hypothesize that these results can be interpreted by the following sequence of events: (1) Type I floral biomass is synthesized in absence of active photosynthesis; (2) the catabolic deamination of amino acids in particular, leaves behind ¹⁵N in the residual pool of amino acids; and (3) the incorporation of these ¹⁵N‐enriched amino acids within the biomass of Type I flowers results in the numerical elevation of the TPs. In contrast, the actively photosynthesizing Type II leaves energetically sustain the synthesis of Type II flower biomass, precluding any reliance on catabolic deamination of amino acids. Amino acids within Type II flowers are therefore isotopically comparable to the Type II leaves. These findings demonstrate the idiosyncratic nature of the δ¹⁵N_AA values within autotrophic organs and have implications for interpreting trophic hierarchies using primary producers and their consumers.     

Molecular cloning and preliminary expression analysis of banded dogfish (Triakis scyllia) TNF decoy receptor 3 (TNFRSF6B)

Decoy receptor 3 (DcR3), a member of TNF receptor superfamily, is a soluble receptor without death domain and cytoplasmic domain, and secreted by cells and binds with FasL, LIGHT and TL1A. The principal function of DcR3 is the inhibition of apoptosis by the binding cytotoxic ligands. Expression of DcR3 has been reported in a wide array of normal human tissues as well as tumors and tumor cell lines. Recently, DcR3 was reported to modulate a variety of immune responses in mammals. TNFR or DcR3 has been identified in some teleost fishes. However, DcR3 is not reported in cartilaginous fish which is the lowest vertebrate possessing the adaptive immune system. Here we identified DcR3 cDNA in shark (Trsc-DcR3) from an SSH library prepared from peripheral white blood cells stimulated with PMA. Four cysteine-rich domains (CRDs) in common with TNF receptor family members are present in the Trsc-DcR3 sequence. The deduced amino acid sequence of Trsc-DcR3 showed highest identity with the chicken (50.4%), followed by human (46.8%) and rainbow trout (36.5%) DcR3. In a phylogenetic tree of known TNFRSF sequences, the Trsc-DcR3 grouped with the chicken and human DcR3. Trsc-DcR3 mRNA was detected strongly in the gill, moderately in the brain, and weakly in the kidney, thymus and leydig. These data strongly suggest that the gene encoding Trsc-DcR3 in banded dogfish is a homolog of the human gene. mRNA expression of Trsc-DcR3 in the thymus and leydig suggests that DcR3 may act as a modulator in the immune system even at the phylogenetic level of cartilaginous fish.     

Salinity affects the photoacclimation of <Emphasis Type="Italic">Chlamydomonas raudensis</Emphasis> Ettl UWO241

Chlamydomonas raudensis Ettl UWO241, a natural variant of C. raudensis, is deficient in state transitions. Its habitat, the deepest layer of Lake Bonney in Antarctica, features low irradiance, low temperature, and high salinity. Although psychrophily and low-light acclimation of this green alga has been described, very little information is available on the effect of salinity. Here, we demonstrate that this psychrophile is halotolerant, not halophilic, and it shows energy redistribution between photosystem I and II based on energy spillover under low-salt conditions. Furthermore, we revealed that C. raudensis exhibits higher non-photochemical quenching in comparison with the mesophile Chlamydomonas reinhardtii, when grown with low-salt, which is due to the lower proton conductivity across the thylakoid membrane. Significance of the C. raudensis UWO241 traits found in the low salinity culture are implicated with their natural habitats, including the high salinity and extremely stable light environments.     

Overexpression and purification of rat peroxisomal membrane protein 22, PMP22, in Pichia pastoris

Peroxisomal membrane protein 22, PMP22, is an integral membrane protein that has four putative transmembrane-spanning regions. First reported as a major component of rat liver peroxisomal membranes and suggested to be involved in the metabolism of reactive oxygen species, its function and structure are still unknown owing to a lack of biochemical and structural experiments. Here we report the overproduction and purification of rat PMP22 (rPMP22) with the use of a methylotrophic yeast, Pichia pastoris, as a host. rPMP22 was localized not to peroxisomal membranes but to membrane compartments, such as the nuclear envelope. Highly pure rPMP22 was obtained in two steps. Several physicochemical assays indicated that the purified preparation should retain its functional structure. Furthermore, fed-batch fermentation yielded 90 mg of rPMP22 protein from 4L of culture. This is the first report to demonstrate the overproduction of a recombinant rPMP22 in the membrane compartments of P. pastoris.     

Identification of a common microdeletion cluster in 7q21.3 subband among patients with myeloid leukemia and myelodysplastic syndrome

Monosomy 7 and interstitial deletions in the long arm of chromosome 7 (−7/7q−) is a common nonrandom chromosomal abnormality found frequently in myeloid disorders including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and juvenile myelomonocytic leukemia (JMML). Using a short probe-based microarray comparative genomic hybridization (mCGH) technology, we identified a common microdeletion cluster in 7q21.3 subband, which is adjacent to ‘hot deletion region’ thus far identified by conventional methods. This common microdeletion cluster contains three poorly characterized genes; Samd9, Samd9L, and a putative gene LOC253012, which we named Miki. Gene copy number assessment of three genes by real-time PCR revealed heterozygous deletion of these three genes in adult patients with AML and MDS at high frequency, in addition to JMML patients. Miki locates to mitotic spindles and centrosomes and downregulation of Miki by RNA interference induced abnormalities in mitosis and nuclear morphology, similar to myelodysplasia. In addition, a recent report indicated Samd9 as a tumor suppressor. These findings indicate the usefulness of the short probe-based CGH to detect microdeletions. The three genes located to 7q21.3 would be candidates for myeloid tumor-suppressor genes on 7q.     

Inhibition of acetylcholinesterase by metabolites of copper pyrithione (CuPT) and its possible involvement in vertebral deformity of a CuPT-exposed marine teleostean fish

In a previous study, we demonstrated that exposure to an antifouling biocide, copper pyrithione (CuPT), early during life induced vertebral deformity in the larvae of a marine fish, the mummichog (Fundulus heteroclitus). Skeletal deformities may be caused by inhibition by of acetylcholiensterase (AChE) activity, and to elucidate the mechanism underlying the CuPT-associated vertebral deformity, we first examined whether CuPT, zinc pyrithione (ZnPT), and their degradation products could inhibit AChE activity in the fish. Two of the degradation products, 2,2′-dipyridyldisulfide [(PS)₂] and 2,2′-dithiobispyridine-N-oxide [(PT)₂], but neither CuPT nor ZnPT, exhibited prominent AChE-inhibiting activity. Secondly, thin-layer chromatography revealed that mummichog hepatic microsomes metabolized CuPT to produce (PS)₂ in a microsome-dependent manner. The AChE inhibition induced in CuPT-exposed fish is likely due to (PS)₂ that was produced through metabolism of acquired CuPT. (PS)₂ may cause therefore skeletal deformity in CuPT-exposed fish by means of its neuromuscular blocking properties, through a mechanism similar to that proposed for animals exposed to organophosphorous pesticides.     

Larval development of bigmouth manefish Caristius macropus (Perciformes: Caristiidae) from the western North Pacific

Larvae of bigmouth manefish Caristius macropus are described and illustrated on the basis of seven specimens (4.2–10.5 mm in body length) from the Kuroshio waters (0–60 m depth) and the transition waters (surface) between the Kuroshio and Oyashio fronts of the western North Pacific. The present larvae of C. macropus are distinguished from those of Paracaristius maderensis that inhabit the North Pacific by having 39–40 myomeres, 34 dorsal-fin rays, and 22 anal-fin rays. The present study, along with previous studies of the early life stages of caristiids, shows that larvae of the family may be defined by the following characters: body elongate in preflexion stage but becoming deep bodied and hatchet shaped after notochord flexion; anus located near vertical through base of pectoral fin; head large, without spination or serration; a distinct vertical band on the posterior tail throughout the larval stages, and two bands gradually appearing on the tail and trunk during the flexion and postflexion stages; and melanophores present around the notochord tip by the flexion stage. Adult C. macropus are found in the subarctic and temperate waters of the North Pacific; however, the present study and other occurrences of early life stages of the species probably indicate that C. macropus may spawn over a wide area in the North Pacific.     

Involvement of vesicular trafficking system in membrane targeting of the progeny influenza virus genome

The genome of influenza type A virus consists of single-stranded RNAs of negative polarity. Progeny viral RNA (vRNA) replicated in the nucleus is nuclear-exported, and finally transported to the budding site beneath the plasma membrane. However, the precise process of the membrane targeting of vRNA is unclear, although viral proteins and cytoskeleton are thought to play roles. Here, we have visualized the translocation process of progeny vRNA using fluorescence in situ hybridization method. Our results provide an evidence of the involvement of vesicular trafficking in membrane targeting of progeny vRNA independent of that of viral membrane proteins.