Ygr125w/Vsb1-dependent accumulation of basic amino acids into vacuoles of Saccharomyces cerevisiae.

The Ygr125w was previously identified as a vacuolar membrane protein by a proteomic analysis. We found that vacuolar levels of basic amino acids drastically decreased in ygr125wΔ cells. Since N- or C-terminally tagged Ygr125w was not functional, an expression plasmid of YGR125w with HA3-tag inserted in its N-terminal hydrophilic region was constructed. Introduction of this plasmid into ygr125w∆ cells restored the vacuolar levels of basic amino acids. We successfully detected the uptake activity of arginine by the vacuolar membrane vesicles depending on HA3-YGR125w expression. A conserved aspartate residue in the predicted first transmembrane helix (D223) was indispensable for the accumulation of basic amino acids. YGR125w has been recently reported as a gene involved in vacuolar storage of arginine; and it is designated as VSB1. Taken together, our findings indicate that Ygr125w/Vsb1 contributes to the uptake of arginine into vacuoles and vacuolar compartmentalization of basic amino acids.

Control of intracellular localization and function of Cx43 by SEMA3F.

Connexin genes are considered to form a family of tumor-suppressor genes. However, the mechanism of connexin-mediated growth control is not well understood. We now provide several lines of evidence which suggest that SEMA3F, a member of the class 3 semaphorin family, which is also reported to be a tumor suppressor, controls the intracellular localization and function of connexin 43 (Cx43). We employed a series of rat liver epithelial cell lines, among which we previously found that the level of expression of malignant phenotypes (IAR20 < IAR27E < IAR6-1 < IAR27F) is inversely related to that of gap junctional intercellular communication (GJIC). When we immunostained SEMA3F and Cx43 in these cell lines, the extent of immunostaining in the plasma membrane of both proteins decreased in the order of IAR20 > IAR27E > IAR6-1 > IAR27F, suggesting a close relationship between Cx43 and SEMA3F. Further studies revealed a partial colocalization of SEMA3F and Cx43 in the plasma membrane of IAR20 cells. We also found that both SEMA3F and Cx43 moved from the cytoplasm to the plasma membrane in a mouse papilloma cell line when E-cadherin became functional after transferring the cells from low- to high-calcium conditions. When SEMA3F gene expression was inhibited by siRNA in IAR20 cells, Cx43 localization in the plasma membrane and GJIC ability were reduced. Moreover, we found that SEMA3F binds with the cytoplasmic loop domain of Cx43, employing the yeast two-hybrid complementation and screening assays. Taken together, these results strongly suggest that SEMA3F directly associates with Cx43 and controls its intracellular localization and function.

Role of the cytoplasmic loop domain of Cx43 in its intracellular localization and function: possible interaction with cadherin.

We have previously shown that intracellular trafficking and function of connexin (Cx) 26 and Cx43 are controlled by E-cadherin. In the present study, we attempted to determine which part of Cx43 is involved in this control mechanism. Since Cx26 has a very short C terminus in the cytoplasm, we hypothesized that the C-terminal domain may not be important for this process and, indeed, found that green fluorescence protein (GFP)-tagged Cx43DeltaC (deleted from the codon 239) moved to the plasma membrane both in P3/22(E), a mouse papilloma cell line which expresses E-cadherin, and HeLa cells only at high calcium culture conditions. We then found that the GFP-tagged Cx43(CL 26)DeltaC mutant, in which the cytoplasmic loop domain of Cx43 was exchanged with that of Cx26, remains in the cytoplasm in HeLa, HeLaCx43 and P3/22(E) cells, suggesting the importance of the cytoplasmic loop domain. In order to determine which part of the cytoplasmic domain plays a key role, we introduced four deletion mutations (deletion of codons 101-111 [mutant D1], 120-130 [D2], 131-137 [D3] or 146-159 [D4]) to the GFP-tagged Cx43DeltaC gene. When these mutants were transfected into HeLa cells, D1 and D4 mutants were localized in the cytoplasm, while D2 and D3 were found in the plasma membrane only in high Ca(2+) medium. However, none of these four mutants recovered gap junctional intercellular communication (GJIC). On the other hand, when these mutants were transfected into HeLaCx43 and P3/22(E) cells (which express functional Cx43), D1, D2 and D3, but not D4, moved to the plasma membrane and colocalized with endogenous Cx43 in high Ca(2+) medium; all of these mutants showed a dominant negative effect on GJIC in HeLaCx43 cells. Further deletion studies indicated that the critical amino acids involved in this intracellular trafficking of Cx43 lie between codons 100 and 102.

[Four cases of pollen-food allergy syndrome suspected the cross reactivity including latex].

BACKGROUND: Generally it is recognized that the occurrence of fruit allergy is attributed not to the sensitization of itself but to the cross reactivity with pollens or latex. But the relationship as to the sensitization between pollen and latex is obscure. So we aimed to investigate the relation of sensitization among pollens, fruits and latex. METHODS: We tried to examine latex-specific IgE titer and practice skin prick test of latex for the patients of pollen-food allergy syndrome. RESULTS: It was confirmed that some patients of pollen-food allergy syndrome showed positive reactions against both specific IgE and skin prick test of latex, though they could tolerate latex products in their daily lives. We present here four patients of such clinical courses concretely. CONCLUSION: The patients of pollen-food allergy syndrome should be practiced examination about latex allergy, even if they can use latex products without any symptoms. And more if positive results are obtained, additional examination such as immunoblot and IgE RAST inhibition test are recommended to practice in order to clarify the unresolved problems, such as 1) which factor is the major allergen to cause cross-reactivity among these three factors? 2) what occurs if patients of same clinical courses with our cases continue to use latex products? Further investigation will be indispensable to resolve these problems in the future.

An Efficient Acylation of Tertiary Alcohols with Isopropenyl Acetate Mediated by an Oxime Ester and Cp(2)Sm(thf)(2).

An efficient method for the acylation of tertiary alcohols with isopropenyl acetate (1) by the use of an oxime and Cp(2)Sm(thf)(2) as catalyst was developed. Thus, various types of tertiary alcohols could be acylated with 1 in the presence of a catalytic amount of cyclohexanone oxime acetate (2) and Cp(2)Sm(thf)(2) under mild conditions to form the corresponding acetates in excellent yields. Acid-sensitive terpene alcohols such as linalool were successfully acetylated by the present method to give acetyl linalool in quantitative yield. This method enables an alternative acylation of tertiary alcohols under acid-free conditions.

Acylation of Alcohols and Amines with Vinyl Acetates Catalyzed by Cp(2)Sm(thf)(2).

Cp(2)Sm(thf)(2) was found to be an efficient catalyst for the acylation of alcohols and amines with esters under mild conditions. In the present acylation, vinyl and isopropenyl acetates served as good acylating agents. Thus, a variety of alcohols and amines underwent acylation with vinyl and isopropenyl acetates in the presence of Cp(2)Sm(thf)(2) to give the corresponding esters and amides in good to excellent yields. This catalytic acylation of alcohols and amines offers an additional useful method by the use of various esters, instead of acid anhydrides and acid chlorides, as acylating agents under very mild conditions.