An unusual complementation in non-excitable mutants in Paramecium

A non-excitable behavioural mutant, d4-662, was previously characterized as the fourth pawn locus mutant pwD in Paramecium tetraurelia. We now provide data demonstrating that d4-662 is in fact controlled by a pwB allele that has the unusual feature of complementing other pwB alleles in heterozygous F1 progeny. Neither the cytoplasm nor the nucleoplasm of d4-662 cured the mutational defects of pwB and in the reverse combination of d4-662 and pwB, the result was the same. On the other hand, pwA, another non-excitable mutant, was cured upon cross-injection with d4-662 and mutants carrying trichocyst non-discharge marker genes were also cured. This evidence suggests that d4-662 is a new mutant belonging to pwB, and would be better designated as pwB662. Extensive crossbreeding analyses, however, showed an unusual genetic relationship between d4-662 and pwB (pwB95 or pwB96). When d4-662 was crossed with pwB mutants, many progeny expressing wild-type phenotype or mixed clones of wild-type and pawn cells were obtained in the F1. Less than 12.5% expressed the pawn phenotype. The appearance of wild-type progeny in this F1 strongly suggests that an inter-allelic interaction between pwB662 and other pwB alleles may occur during development of the macronucleus.     

SOCS-1 can suppress CD3zeta- and Syk-mediated NF-AT activation in a non-lymphoid cell line

To elucidate T cell antigen receptor (TCR) signaling leading to activation nuclear factor of activated T cells (NF-AT), we reconstituted TCR signaling to activate NF-AT in a non-lymphoid cell line, 293T. We demonstrated that co-expression of CD8/zeta and Syk were necessary for NF-AT activation in 293T. This NF-AT response was completely inhibited by the addition of cyclosporin A or FK506, but markedly enhanced by the additional expression of Tec protein tyrosine kinase. We also show that the cytokine signaling suppressor, suppressor of cytokine signaling 1, potently inhibited this response by interacting with Syk and immunoreceptor tyrosine-based activation motifs in CD8/zeta. These results imply that this novel system may provide a useful tool to delineate or identify the regulatory molecules for CD3zeta/Syk-mediated NF-AT activation.     

Brachioplasty technique with the use of molds

Over previous years many surgical techniques have been developed to correct lipodystrophy and brachial skin laxity while providing a natural, discreet contour and symmetrical scars, a goal that has not always been achieved. In this study, the authors classify the alterations of the arm into three degrees and propose a different surgical treatment, to be used alone or associated with another procedure, for each classification. They developed a new technique that uses a mold to mark the incision in an italic double S-shape. From 1996 to 1998, 20 patients who underwent this surgery showed symmetrical and smaller scars with better results and minimal complications.     

Antidiabetogenic activity of oleanolic acid glycosides from medicinal foodstuffs

Oleanolic acid glycosides from several medicinal foodstuffs were found to show potent inhibitory activity on the increase of serum glucose levels in oral glucose-loaded rats. By examination of the structure-activity relationships, the 3-O-glucuronide moiety and the 28-carboxyl group in oleanolic acid glycosides were required to exert the hypoglycemic activity. Oleanolic acid glycosides were found to have neither insulin-like nor insulin-releasing activity, but they inhibited gastric emptying and glucose-uptake in the small intestine. Investigation of the mode of action revealed that the inhibition of gastric emptying was mediated by capsaicin-sensitive sensory nerves and the central nervous system. Furthermore, oleanolic acid glycosides were suggested to suppress the gastric emptying by stimulating the release and/or production of dopamine to act through dopamine2 receptors, which in turn causes the release of prostaglandins.     

Decrease in rice allergenic proteins of polished rice grains by incubating with a miso solution

The effect of miso on allergenic proteins in rice seeds was investigated. When polished rice grains were incubated at 37 degrees C for 30-120 min with a 10% miso solution, but not with heat-treated miso or 1% NaCl, the amount of soluble proteins extracted from the rice grains with 1 M NaCl markedly decreased. SDS-PAGE, immunoblotting and densitometric analyses of these soluble proteins and insoluble proteins indicate that 26 kDa globulin and 14-16 kDa allergens in the grains were decreased to 15-60% during incubation with the miso solution, especially soybean-koji miso, without any large change in the content of major insoluble proteins.     

Alcohol absorption inhibitors from bay leaf (Laurus nobilis): structure-requirements of sesquiterpenes for the activity

Through a bioassay-guided separation using inhibitory activity on blood ethanol elevation in oral ethanol-loaded rat, various sesquiterpenes having an alpha-methylene-gamma-butyrolactone moiety, costunolide (1), dehydrocostus lactone (2), zaluzanin D (3), reynosin (4), santamarine (5), 3alpha-acetoxyeudesma-1,4(15),11(13)-trien-12,6alpha-+ ++olide (6) and 3-oxoeudesma-1,4,11(13)-trien-12,6alpha-olide (7), were isolated as the active principle from the leaves of Laurus nobilis (bay leaf, laurel). In order to characterize the structure requirement for the activity, several reduction products (2a-2d) and amino acid adducts (2e, 2f) of the alpha-methylene-gamma-butyrolactone moiety were synthesized from 2 and the inhibitory activities of these sesquiterpenes, together with alpha-methylene-gamma-butyrolactone (12) and its related compounds (13-16), were examined. These results indicated that the gamma-butyrolactone or gamma-butyrolactol moiety having alpha-methylene or alpha-methyl group was essential for the inhibitory activity on ethanol absorption. Since 1, 2 and 12 showed no significant effect on glucose absorption, these sesquiterpenes appeared to selectively inhibit ethanol absorption. In addition, the acute toxicities of 1 and 2 in a single oral administration were found to be lower than that of 12.     

Edit, cut and paste in the nicotinic acetylcholine receptor gene family of Drosophila melanogaster

Nicotinic acetylcholine receptors (nAChRs) are important for fast synaptic cholinergic transmission. They are targets of drugs/chemicals for human and animal health as well as for pest control. With the advent of genome sequencing, entire nAChR gene families have now been described for vertebrates and invertebrates. Mostly, these are extensive with a large number of distinct subunits, making possible many nAChR subtypes differing in transmitter affinity, channel conductance, ion selectivity, desensitization, modulation and pharmacology. The smallest nAChR gene family to date is that of the fruit fly, Drosophila melanogaster, with only 10 members. This apparently compact family belies its true diversity as 4 of the 10 subunits show alternative splicing. Also, using Drosophila, A-to-I pre-mRNA editing has been demonstrated for the first time in nAChRs. Such is the extent of this variation, that one subunit alone (Dalpha6) can potentially generate far more isoforms than seen in entire gene families from other species. We present here three-dimensional models constructed for insect nAChRs, which show that many variations introduced by alternative splicing and RNA editing may influence receptor function.     

Localization of soluble beta-carbonic anhydrase in the marine diatom Phaeodactylum tricornutum. Sorting to the chloroplast and cluster formation on the girdle lamellae

A β-carbonic anhydrase (CA) in the marine diatom Phaeodactylum tricornutum (PtCA1) is encoded by the nuclear genome. This enzyme was previously found to be important for the operation of photosynthesis with a high affinity for dissolved inorganic carbon. A cDNA sequence that encodes PtCA1 (ptca1) was shown to possess a presequence of 138 bp (pre138), which encodes an N-terminal sequence of 46 amino acids (Pre46AA) that does not exist in the mature PtCA1. In this study, pre138 was ligated with the enhanced green fluorescent protein (GFP) gene (egfp), and introduced into P. tricornutum by microprojectile bombardment. Subsequently, the expressed Pre46AA-GFP fusion was shown to be localized in the chloroplast stroma, whereas the expressed GFP without Pre46AA was localized in the cytoplasm. Insertion of the DNA sequence, encoding a mature region of ptca1 (mptca1) between pre138 and egfp, resulted in the formation of particles with concentrated GFP fluorescence in the stroma of P. tricornutum. These particles, 0.3 to 3.0 μm in size, were shown to be distinct from the mitochondria and localized on the surface of the putative girdle lamella. The attachment of the initial one-half of the pre138 to the mptca1-egfp fusion caused the expressed GFP fusion to accumulate in areas surrounding the chloroplast, presumably due to the presence of the endoplasmic reticulum signal encoded by the initial half-sequence and to the absence of the chloroplast transit sequence. These results indicate that PtCA1 is targeted to the stroma by the bipartite sequences of Pre46AA and that the observed GFP particles are formed specifically in the stroma due to the function of the mptca1.     

Inhibitory mechanisms of 1-(3-C-ethynyl-beta-D-ribopentofuranosyl)uracil (EUrd) on RNA synthesis

1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)uracil (EUrd) is an antimetabolite that strongly inhibits RNA synthesis and shows a broad antitumor activity in vitro and in vivo. In mouse mammary tumor FM3A cells, EUrd is sequentially phosphorylated to its 5'-triphosphate, EUTP, a major metabolite, and the RNA synthesis is inhibited proportionally to its intracellular accumulation. To study the inhibitory mechanisms of EUrd on RNA synthesis, we have performed the kinetic analysis of EUTP on RNA polymerization using isolated nuclei RNA synthesis was inhibited competitively by EUTP. The inhibition constant, Ki was much lower than the Km value of UTP (Ki value of EUTP, 84 nM; Km value of UTP, 13 microM), indicating that the high affinity of EUTP could contribute to the specific inhibition of RNA synthesis. As a result of RNA synthesis inhibition, EUrd, but not ara-C, induced shrinkage of nucleoli, which are the main sites for RNA synthesis in FM3A cells. Thus, the strong affinity of EUTP to RNA polymerase and specific inhibition of RNA synthesis could contribute to its antitumor effect. EUrd is expected to be a new antitumor drug, possessing a strong inhibitory effect on the synthesis of RNA.     

Synthesis and biological activity of cyclic ADP-carbocyclic-ribose analogs: structure-activity relationship and conformational analysis of N-1-carbocyclic-ribose moiety

Several cyclic ADP-carbocyclic-ribose analogs 3-10 modified in the N-1-carbocyclic-ribose moiety were synthesized. Their Ca2+-releasing activity was estimated in sea urchin eggs to show that the 3"-deoxy analog 6 shows 5 times more potent activity than cADPcR, but the 2",3"-didieoxy-2",3"-unsunsaturated analog 3 has very weak activity. We also calculated their stable conformation and found that 3 and 6 were significantly different in their stable conformation.