Isolation of a fission yeast mutant that is sensitive to valproic acid and defective in the gene encoding Ric1, a putative component of Ypt/Rab-specific GEF for Ryh1 GTPase

Valproic acid (VPA) causes various therapeutic and biological effects, but the exact mechanisms underlying these effects, however, remain elusive. To gain insights into the molecular mechanisms of VPA action, we performed in fission yeast a genetic screen for mutants that show VPA hypersensitivity and have identified several membrane-trafficking mutants including vas1-1/vps45 and vas2-1/aps1. Here, we describe the isolation and characterization of vas3-1/ric1-v3, a mutant allele of the ric1 ⁺ gene encoding a fission yeast homolog of the budding yeast Ric1p, a component of Ypt/Rab-specific guanyl-nucleotide exchange factor (GEF). The Rab GTPase Ryh1 knockout (Δryh1) cells and Δric1 cells exhibited similar phenotypes. The double knockout Δric1Δryh1 cells did not display synthetic growth defects. These results are consistent with the notion that Ric1 may be a component of the GEF complex for Ryh1. Overexpression of wild-type Ryh1 and the constitutively active Ryh1Q70L only partially suppressed the phenotypes of ric1-v3 and Δric1 cells, and they failed to localize to the Golgi/endosomes in ric1-v3 and Δric1 cells. Furthermore, we isolated vps15 ⁺ gene, encoding a serine/threonine protein kinase, as a dosage-dependent suppressor of the temperature-sensitive phenotype of ric1-v3 mutant, but not that of Δric1 cells. Our results showed that the ric1-v3 mutant allele has some residual functional activity and suggest that Vps15 plays a role in the regulation of Ric1 function. In conclusion, Ric1 is a putative component of GEF for Ryh1 and might be regulated by Vps15. Further studies are needed to reveal the mechanism underlying the regulation.     

Molecular methods for differentiating grey seal (Halichoerus grypus) and harbour seal (Phoca vitulina) scat

Pinniped dietary information is crucial for understanding marine ecosystems; however, in the North Atlantic, grey and harbour seals haulout concomitantly and their faeces are visually indistinguishable. Therefore, we developed molecular methods to differentiate the species' scat. PCR primers were created that amplify a portion of mitochondrial 16S ribosomal DNA in grey and harbour seals. The samples were amplified and the resulting products were digested using the restriction enzyme Tsp509I, producing diagnostic banding patterns. These tools provide a mechanism by which separate dietary analysis can be achieved for grey and harbour seals at mixed haulouts in New England.     

Anti-initiating activity of 3beta-methoxy-13alpha,14alpha-epoxyserratan-21beta-ol (PJJ-34) from the stem bark of Picea jezoensis Carr. var. jezoensis

Ultraviolet light is the major cause of skin cancers in human, and several effects of ultraviolet light B (UVB) are thought to contribute to skin photocarcinogenesis. 3Beta-methoxy-13alpha,14alpha-epoxyserratan-21beta-ol (PJJ-34; 1) isolated from Picea jezoensis Carr. var. jezoensis showed the strongest antitumor-promoting activity among naturally occurring triterpenoids in the in vivo two-stage mouse skin-carcinogenesis test. To investigate the anti-initiating activity, we further studied mouse models initiated with ultraviolet-B (UVB) and promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA). Oral administration of the PJJ-34 (1) during a period before and after the three times of UVB irradiation led to a remarkable effect: oral administration of a 0.0025% solution of 1 only to the test group, which started one week before and ended one week after the irradiation, showed less than half papillomas, and inhibition of tumor incidence and tumor multiplicity in comparison to the control group. Therefore, it was recognized that PJJ-34 (1) showed strong anti-initiating activity as well as anti-promoting activity. After all, 1 seems to be useful as cancer-chemopreventive agent.     

Increased leptin may be involved in the short life span of ayu (Plecoglossus altivelis)

It is well known that ayu (Plecoglossus altivelis) die after spawning and have a life span of only 1 year. The determinants for such a short life span are probably connected with spawning and related changes in hormonal homeostases. One of these changes is that the ayu's feeding activity decreases both during and after spawning. We investigated the relationships among leptin, one of the regulators of food intake, and two other major hormones, 17 beta-estradiol and prolactin (PRL). Ir-leptin levels were significantly higher during spawning, and were associated with a decrease in appetite. Ir-leptin levels were also synchronized with levels of 17 beta-estradiol and PRL-like protein. Therefore, one possible explanation for the decrease in appetite during ayu spawning is that the elevation of 17 beta-estradiol homeostasis induced the secretion of Ir-leptin. The inability to decrease leptin to the basal levels because of high estrogen after spawning could be in part responsible for the short life span of ayu.     

Head-head coordination is required for the processive motion of cytoplasmic dynein, an AAA+ molecular motor

Cytoplasmic dynein is an AAA(+)-type molecular motor whose major components are two identical heavy chains containing six AAA(+) modules in tandem. It moves along a single microtubule in multiple steps which are accompanied with multiple ATP hydrolysis. This processive sliding is crucial for cargo transports in vivo. To examine how cytoplasmic dynein exhibits this processivity, we performed in vitro motility assays of two-headed full-length or truncated single-headed heavy chains. The results indicated that four to five molecules of the single-headed heavy chain were required for continuous microtubule sliding, while approximately one molecule of the two-headed full-length heavy chain was enough for the continuous sliding. The ratio of the stroking time to the total ATPase cycle time, which is a quantitative indicator of the processivity, was approximately 0.2 for the single-headed heavy chain, while it was approximately 0.6 for the full-length molecule. When two single-headed heavy chains were artificially linked by a coiled-coil of myosin, the processivity was restored. These results suggest that the two heads of a single cytoplasmic dynein communicate with each other to take processive steps along a microtubule.     

Recognition of oxidized thymine base on the single-stranded DNA by replication protein A

Replication protein A (RAP) is a eukaryotic single-stranded DNA binding protein involved in DNA replication, repair, and recombination. Recent studies indicate that RPA preferentially binds the damaged sites rather than the undamaged sites. Therefore, RPA is thought to be a member ofrepair factories or a sensor of lesion on DNA. To obtain further information of behavior of RPA against the oxidized lesion, we studied the binding affinity of RPA for the single-stranded DNA containing 5-formyluracil, a major lesion of thymine base yielded by the oxidation, using several synthetic oligonucleotides. The affinity of RPA for oligonucleotides was determined by gel shift assay. Results suggest that the surrounding sequence of 5-formyluracil may affect the affinity for RPA, and that the 5-formyluracil on the purine stretch but not the pyrimidine stretch increases the affinity for RPA. Results of affinity labeling experiment of RPA with the oligonucleotides containing 5-formyluracil indicate that RPA1 subunit may directly recognize and bind to the 5-formyluracil on the single-stranded DNA.     

The influence of dietary vitamin C and E supplementation on the physiological response of pirarucu, Arapaima gigas, in net culture

This study evaluated the efficacy of dietary vitamin C (ascorbic acid or AA), vitamin E (alpha-tocopherol or alpha-T), and C+E supplementation on the blood parameters of Arapaima gigas grown in net cages for 45 days. Four treatments were tested: control (commercial feed); C800; E500 and C+E (800+500) with supplementation of 800 mg AA kg(-1), 500 mg alpha-T kg(-1) and 800+500 mg AA+alpha-T kg(-1), respectively. Hematocrit (Ht), red blood cells (RBC), and hemoglobin concentration (Hb) (oxidative status indicators), thrombocytes and leukocytes (immunological indicators), plasma protein and glucose were evaluated. Fish fed vitamin C and C+E supplemented diets showed greater weight gain and survival. Dietary vitamin C and C+E diet supplementation resulted in increased Ht, Hb, RBC, MCHC, total leukocytes, total proteins, thrombocytes and eosinophils compared to the control and alpha-T. The alpha-tocopherol-supplemented diet reduced the number of total thrombocytes, lymphocytes and neutrophils and increased glucose and eosinophils relatively to the control. In general, leukocytes and thrombocytes were good indicators of the efficiency of vitamin on the defense mechanism of the A. gigas reared in cages. Results indicate that high alpha-T diet supplementation provides no benefit for the maintenance of the oxidative or the immunological status of A. gigas. However, it was demonstrated that high dietary AA improves A. gigas immunological status. Red blood cell indices and immune system indicators showed no synergistic effect between the vitamins after supplementing the A. gigas diet with alpha-T+AA.     

Low growth ability of recent influenza clinical isolates in MDCK cells is due to their low receptor binding affinities

Madin Darby canine kidney (MDCK) cells have generally been used to isolate influenza viruses from patients. However, in recent years, most fresh isolates of the H3N2 subtype have shown poor growth in MDCK cell cultures. Such low-growth viruses were often converted to high-growth viruses after several passages through MDCK cell cultures. In the present study, viruses were found to lose a potential glycosylation site near the receptor-binding pocket of hemagglutinin (HA), at the same time as they acquired the high-growth property. The growth curves of viruses in MDCK cell cultures revealed that multi-cycle replication did not function well in the low-growth viruses. However, the production of progeny viruses within a single cycle of growth did not differ much between the low- and high-growth viruses. The high-growth viruses showed higher infection efficiency in MDCK cell cultures than the low-growth viruses. The HA genes of both low- and high-growth viruses were separately cloned into the SV40 vector to compare their receptor binding affinities. The HA of high-growth viruses showed a much higher receptor binding affinity than that of low-growth viruses, when assayed by hemadsorption and the release kinetics of erythrocytes with bacterial neuraminidase. Reverse genetics studies demonstrated that HA was a crucial determinant for multi-cycle replication in MDCK cell cultures. Taken together, these results demonstrate that inefficient multi-cycle growth of fresh isolates is due to their low receptor binding affinities.     

Phylogenetic relationships among Puccinia hemerocallidis, P. funkiae, and P. patriniae (Uredinales) inferred from ITS sequence data

Puccinia hemerocallidis and P. funkiae resemble each other morphologically; however, they are biologically and taxonomically distinct, with telia of the former being restricted to species of Hemerocallis and the latter to Hosta species. However, both fungi share a macrocyclic and heteroecious life cycle with Patrinia villosa as the spermogonial and aecial host. An additional microcyclic rust fungus, P. patriniae, is also known on P. villosa. This microcyclic fungus is similar to the two macrocyclic fungi in its telial structure and teliospore morphology. These similarities in morphology and host relationships suggest the three fungi may also share a close evolutionary relationship. To determine the phylogenetic relationships of the three species, a portion of the nuclear ribosomal DNA repeat encoding the ITS and 5.8S subunit regions was amplified by PCR, sequenced, and analyzed. The resulting phylogenetic trees showed that P. hemerocallidis and P. funkiae share a recent common ancestor and that P. patriniae is closely allied with P. hemerocallidis. The results suggest a possible evolutionary derivation of microcyclic P. patriniae from macrocyclic heteroecious P. hemerocallidis, which fits the evolutionary interpretation of correlated species known as Tranzschel's law.